Therapeutic use of acellular fat extract for lung diseases

Abstract

The present invention relates to a therapeutic use of an acellular fat extract for lung diseases. Specifically, the present invention provides a use of an acellular fat extract for preparing a composition or preparation, wherein the composition or preparation is used to prevent and / or treat pulmonary fibrosis. The acellular fat extract of the present invention has an excellent therapeutic effect on pulmonary fibrosis.

Description

Technical Field

[0001] The invention relates to the field of medicines, and in particular to the therapeutic use of acellular fat extract on lung diseases. Background Art

[0002] Pulmonary fibrosis (PF) is a chronic progressive fibrotic disease caused by multiple factors such as smoking, viral infection, environmental pollution, genetic susceptibility and drugs. It is the most common interstitial lung disease and one of the most serious diseases of the respiratory system. Its pathological characteristics are mainly diffuse inflammatory cell infiltration, fibroblast proliferation, abnormal deposition of extracellular matrix, and severe destruction of alveolar structure. The condition of PF patients often develops progressively, and most patients in the late stage suffer from cardiopulmonary failure and death. At present, the pathogenesis of PF is still unclear, and there is a lack of effective therapeutic drugs in clinical practice.

[0003] Therefore, there is a need in the art to develop a drug that can effectively treat pulmonary fibrosis. Summary of the invention

[0004] The present invention aims to provide a use of a cell-free fat extract in preventing and / or treating pulmonary fibrosis.

[0005] In a first aspect, the present invention provides a use of a cell-free fat extract for preparing a composition or a preparation for preventing and / or treating pulmonary fibrosis.

[0006] In another preferred embodiment, the pulmonary fibrosis includes pulmonary fibrosis caused by cytotoxic antibiotics.

[0007] In another preferred embodiment, the pulmonary fibrosis includes pulmonary fibrosis caused by anti-tumor drugs.

[0008] In another preferred embodiment, the anti-tumor drug includes a chemotherapeutic drug.

[0009] In another preferred embodiment, the anti-tumor drug includes a cytotoxic drug.

[0010] In another preferred embodiment, the anti-tumor drug includes cytotoxic antibiotics.

[0011] In another preferred embodiment, the cytotoxic antibiotic includes bleomycin.

[0012] In another preferred embodiment, the anti-tumor drug includes bleomycin.

[0013] In another preferred embodiment, the pulmonary fibrosis is pulmonary fibrosis induced or caused by bleomycin.

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

[0015] (i) reduce lung weight index;

[0016] (ii) improving inflammatory cell infiltration in the peribronchial, bronchial interstitial and / or alveolar regions of the lungs;

[0017] (ii) improving peribronchial and / or bronchointerstitial fibrosis in the lungs; and / or

[0018] (ii) Improve peribronchial edema in the lungs.

[0019] In another preferred embodiment, the acellular fat extract is an acellular fat extract extracted from fat of human or non-human mammals.

[0020] In another preferred embodiment, the non-human mammal is a monkey, an ape, a cow, a pig, a dog, a sheep, a mouse or a rabbit.

[0021] In another preferred embodiment, the composition or preparation includes a pharmaceutical composition or preparation, a food composition or preparation, a health care product composition or preparation or a dietary supplement.

[0022] In another preferred embodiment, the composition or preparation further comprises a pharmaceutically, food, health product or dietary acceptable carrier.

[0023] In another preferred embodiment, the composition or preparation is in the form of an oral preparation, an external preparation or an injection preparation.

[0024] In another preferred embodiment, the injection preparation is an intravenous injection preparation.

[0025] In another preferred embodiment, the composition or preparation is administered externally, topically, or by subcutaneous injection.

[0026] In another preferred embodiment, the cell-free fat extract contains no cells and no lipid droplets.

[0027] In another preferred embodiment, the lipid droplets are oil droplets released after the adipocytes are broken.

[0028] In another preferred embodiment, the “free of fat droplets” means that in the acellular fat extract, the volume of oil droplets accounts for less than 1% of the total liquid, preferably less than 0.5%, and more preferably less than 0.1%.

[0029] In another preferred embodiment, the cells are selected from the group consisting of endothelial cells, adipose stem cells, macrophages, and stromal cells.

[0030] In another preferred embodiment, the “cell-free” means that the average number of cells in 1 ml of cell-free fat extract is ≤1, preferably ≤0.5, more preferably ≤0.1, or 0.

[0031] In another preferred embodiment, the cell-free fat extract is a naturally obtained nano fat extract without any added ingredients.

[0032] In another preferred embodiment, the “additive-free” means that except for the rinsing step, no solution, solvent, small molecule, chemical agent, and biological additive are added during the preparation of the fat extract.

[0033] In another preferred embodiment, the cell-free fat extract is prepared by emulsifying adipose tissue and then centrifuging it.

[0034] In another preferred embodiment, the cell-free fat extract contains one or more components selected from the following group: IGF-1, BDNF, GDNF, TGF-β, HGF, bFGF, VEGF, TGF-β1, HGF, PDGF, EGF, NT-3, GH, G-CSF, or a combination thereof.

[0035] In another preferred embodiment, the acellular fat extract contains but is not limited to one or more components selected from the following group: IGF-1, BDNF, GDNF, bFGF, VEGF, TGF-β1, HGF, PDGF, or a combination thereof.

[0036] In another preferred embodiment, the acellular fat extract is acellular fat extract.

[0037] In another preferred embodiment, in the cell-free fat extract, the concentration of IGF-1 is 5000-30000pg / ml, preferably 6000-20000pg / ml, more preferably 7000-15000pg / ml, more preferably 8000-12000pg / ml, more preferably 9000-11000pg / ml, more preferably 9500-10500pg / ml.

[0038] In another preferred embodiment, in the cell-free fat extract, the concentration of BDNF is 800-5000pg / ml, preferably 1000-4000pg / ml, more preferably 1200-2500pg / ml, more preferably 1400-2000pg / ml, more preferably 1600-2000pg / ml, more preferably 1700-1850pg / ml.

[0039] In another preferred embodiment, in the cell-free fat extract, the concentration of GDNF is 800-5000pg / ml, preferably 1000-4000pg / ml, more preferably 1200-2500pg / ml, more preferably 1400-2000pg / ml, more preferably 1600-2000pg / ml, more preferably 1700-1900pg / ml.

[0040] In another preferred embodiment, in the cell-free fat extract, the concentration of bFGF is 50-600pg / ml, preferably 100-500pg / ml, more preferably 120-400pg / ml, more preferably 150-300pg / ml, more preferably 200-280pg / ml, more preferably 220-260pg / ml.

[0041] In another preferred embodiment, in the cell-free fat extract, the concentration of VEGF is 50-500pg / ml, preferably 100-400pg / ml, more preferably 120-300pg / ml, more preferably 150-250pg / ml, more preferably 170-230pg / ml, more preferably 190-210pg / ml.

[0042] In another preferred embodiment, in the cell-free fat extract, the concentration of TGF-β1 is 200-3000pg / ml, preferably 400-2000pg / ml, more preferably 600-1500pg / ml, more preferably 800-1200pg / ml, more preferably 800-1100pg / ml, more preferably 900-1000pg / ml.

[0043] In another preferred embodiment, in the cell-free fat extract, the concentration of HGF is 200-3000pg / ml, preferably 400-2000pg / ml, more preferably 600-1500pg / ml, more preferably 600-1200pg / ml, more preferably 800-1000pg / ml, more preferably 850-950pg / ml.

[0044] In another preferred embodiment, in the cell-free fat extract, the concentration of PDGF is 50-600pg / ml, preferably 80-400pg / ml, more preferably 100-300pg / ml, more preferably 140-220pg / ml, more preferably 160-200pg / ml, more preferably 170-190pg / ml.

[0045] In another preferred embodiment, the weight ratio of IGF-1 to VEGF is 20-100:1, preferably 30-70:1, more preferably 40-60:1, and most preferably 45-55:1.

[0046] In another preferred embodiment, the weight ratio of BDNF to VEGF is 2-20:1, preferably 4-15:1, more preferably 6-12:1, and most preferably 8-9.5:1.

[0047] In another preferred embodiment, the weight ratio of GDNF to VEGF is 2-20:1, preferably 4-15:1, more preferably 6-12:1, and most preferably 8.5-9.5:1.

[0048] In another preferred embodiment, the weight ratio of bFGF to VEGF is 0.2-8:1, preferably 0.5-5:1, more preferably 0.6-2:1, more preferably 0.8-1.6:1, and most preferably 1-1.5:1.

[0049] In another preferred embodiment, the weight ratio of TGF-β1 to VEGF is 1-20:1, preferably 1-15:1, more preferably 1-10:1, more preferably 2-8:1, and more preferably 4-6:1.

[0050] In another preferred embodiment, the weight ratio of HGF to VEGF is 1-20:1, preferably 1-15:1, more preferably 1-10:1, more preferably 2-8:1, and more preferably 4-5.5:1.

[0051] In another preferred embodiment, the weight ratio of PDGF to VEGF is 0.1-3:1, preferably 0.2-2:1, more preferably 0.4-1.5:1, and most preferably 0.7-1.2:1.

[0052] In another preferred embodiment, the cell-free fat extract is prepared by the following method:

[0053] (1) providing a fat tissue raw material, breaking the fat tissue raw material, and rinsing it (e.g., using physiological saline) to obtain rinsed fat tissue;

[0054] (2) centrifuging the rinsed adipose tissue to obtain a layered mixture;

[0055] (3) removing the upper oil layer and the lower water layer from the stratified mixture, and collecting the middle layer (i.e., the fat layer containing fat cells);

[0056] (4) emulsifying the intermediate layer to obtain an emulsified fat mixture (also called nano fat);

[0057] (5) centrifuging the emulsified fat mixture to obtain an intermediate liquid layer, i.e., a primary fat extract; and

[0058] (6) Filtering and sterilizing the primary fat extract to obtain a cell-free fat extract.

[0059] In a second aspect, the present invention provides a method for preparing a cell-free fat extract, the method comprising the steps of:

[0060] (1) providing a fat tissue raw material, breaking the fat tissue raw material, and rinsing it (e.g., using physiological saline) to obtain rinsed fat tissue;

[0061] (2) centrifuging the rinsed adipose tissue to obtain a layered mixture;

[0062] (3) removing the upper oil layer and the lower water layer from the stratified mixture, and collecting the middle layer (i.e., the fat layer containing fat cells);

[0063] (4) emulsifying the intermediate layer to obtain an emulsified fat mixture (also called nano fat);

[0064] (5) centrifuging the emulsified fat mixture to obtain an intermediate liquid layer, i.e., a primary fat extract; and

[0065] (6) Filtering and sterilizing the primary fat extract to obtain a cell-free fat extract.

[0066] In another preferred embodiment, the cell-free fat extract is as described in the first aspect of the present invention.

[0067] In another preferred embodiment, in the step (2), the centrifugation is performed at 800-2500 g, preferably 800-2000 g, more preferably 1000-1500 g, and most preferably 1100-1300 g.

[0068] In another preferred embodiment, in the step (2), the centrifugation time is 1-15 min, preferably 1-10 min, more preferably 1-8 min, and most preferably 1-5 min.

[0069] In another preferred embodiment, the centrifugation temperature is 2-6°C.

[0070] In another preferred embodiment, in the step (4), the emulsification is mechanical emulsification.

[0071] In another preferred embodiment, the mechanical emulsification is performed by repeatedly blowing with a syringe (such as blowing 20-200 times, preferably 20-150 times, more preferably 20-100 times, and more preferably 30-50 times).

[0072] In another preferred embodiment, the blowing method is to connect two 10 ml injection syringes to a three-way tube and push and beat repeatedly at a uniform speed.

[0073] In another preferred embodiment, in the step (4), the emulsification is performed by breaking up the tissue using a tissue homogenizer.

[0074] In another preferred embodiment, in the step (5), before subjecting the emulsified fat mixture to centrifugal treatment, the step further includes subjecting the emulsified fat mixture to freezing and then thawing.

[0075] In another preferred embodiment, after the freezing and thawing treatment, the thawed mixture is used for centrifugation.

[0076] In another preferred embodiment, the freezing temperature is -50°C to -120°C, preferably -60°C to -100°C, and more preferably -70°C to -90°C.

[0077] In another preferred embodiment, the thawing temperature is 20-40°C, preferably 25-40°C, and more preferably 37°C.

[0078] In another preferred embodiment, the number of cycles of freezing and thawing is 1-5 times (preferably 1, 2, 3 or 4 times).

[0079] In another preferred embodiment, in the step (5), after centrifugation, the emulsified fat mixture is stratified into four layers, the first layer is an oil layer, the second layer is a residual fat tissue layer, the third layer is a liquid layer (i.e., an intermediate liquid layer), and the fourth layer is a cell / tissue fragment precipitation layer.

[0080] In another preferred embodiment, in the step (5), the centrifugation is performed at 800-2500 g, preferably 800-2000 g, more preferably 1000-1500 g, and most preferably 1100-1300 g.

[0081] In another preferred embodiment, in the step (5), the centrifugation time is 1-15 min, preferably 1-10 min, more preferably 2-8 min, and most preferably 3-7 min.

[0082] In another preferred embodiment, in the step (5), the first layer, the second layer, the third layer and the fourth layer are arranged in sequence from top to bottom.

[0083] In another preferred embodiment, in the step (5), the intermediate liquid layer is a transparent or substantially transparent layer.

[0084] In another preferred embodiment, in the step (6), the filtration bag is capable of removing fat cells from the primary fat extract.

[0085] In another preferred embodiment, in the step (6), the filtration and sterilization are performed through a filter (such as a 0.22 μm microporous filter membrane).

[0086] In another preferred embodiment, the filter is a microporous membrane filter.

[0087] In another preferred embodiment, the pore size of the microporous filter membrane is 0.05-0.8 μm, preferably 0.1-0.5 μm, more preferably 0.1-0.4 μm, more preferably 0.15-0.3 μm, more preferably 0.2-0.25 μm, and most preferably 0.22 μm.

[0088] In another preferred embodiment, in the step (6), the filtration and sterilization are first performed through a first filter that can filter out cells, and then through a second filter (such as a 0.22 μm filter) that can filter out pathogens (such as bacteria).

[0089] In another preferred embodiment, the step (6) further comprises packaging the fat extract to form packaged products. (The packaged extract can be stored at -20°C for later use; can be used directly after thawing at low temperature (such as -4°C) or room temperature, or can be stored at low temperature (such as 4°C) for a period of time after thawing before use).

[0090] In another preferred embodiment, the composition or preparation is in the form of powder, granules, capsules, injections, tinctures, oral solutions, tablets or lozenges.

[0091] In another preferred embodiment, the injection is an intravenous injection or an intramuscular injection.

[0092] In another preferred embodiment, the dosage form of the composition or preparation is a solid dosage form, a semisolid dosage form, or a liquid dosage form, such as a solution, a gel, a cream, a lotion, an ointment, a cream, a paste, a cake, a powder, a patch, etc.

[0093] The third aspect of the present invention provides a cell-free fat extract, which is prepared by the method described in the second aspect of the present invention.

[0094] In a fourth aspect, the present invention provides a composition or preparation, comprising (a) the acellular fat extract as described in the third aspect of the present invention; and (b) a pharmaceutically, food, health product or dietary acceptable carrier or excipient.

[0095] In another preferred embodiment, the composition is a pharmaceutical composition, a food composition, a health product composition or a dietary supplement.

[0096] In another preferred embodiment, the composition or preparation is in the form of powder, granules, capsules, injections, tinctures, oral solutions, tablets or lozenges.

[0097] In another preferred embodiment, the injection is an intravenous injection or an intramuscular injection.

[0098] In another preferred embodiment, the dosage form of the composition or preparation is a solid dosage form, a semisolid dosage form, or a liquid dosage form, such as a solution, a gel, a cream, a lotion, an ointment, a cream, a paste, a cake, a powder, a patch, etc.

[0099] In another preferred embodiment, in the composition or preparation, the mass percentage of the acellular fat extract is 5 wt %, preferably 1-20 wt %, based on the total weight of the cosmetic composition.

[0100] The fifth aspect of the present invention provides a method for preparing the composition or preparation as described in the fourth aspect of the present invention, the method comprising the steps of: mixing the cell-free fat extract as described in the third aspect of the present invention with a pharmaceutically, food, health product or dietary acceptable carrier or excipient to form a composition or preparation.

[0101] In a sixth aspect, the present invention provides a method for preventing and / or treating pulmonary fibrosis, comprising administering the acellular fat extract as described in the third aspect of the present invention to a subject in need thereof.

[0102] In another preferred embodiment, the subject is a human or non-human mammal.

[0103] In another preferred embodiment, the non-human mammals include rodents, such as rats and mice.

[0104] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] Figure 1 The figure shows the effect (mean) of the survival rate (%) of rats with pulmonary fibrosis in different groups.

[0106] Figure 2 The changes in body weight (g) of rats with pulmonary fibrosis in different groups over time (x±SD).

[0107] Figure 3 The lung weight index (%) of rats with pulmonary fibrosis in different groups (x±SD).

[0108] Figure 4 HE staining (200X) of the inflammatory cell infiltration in the lung tissue of rats with pulmonary fibrosis in different groups.

[0109] Figure 5 Masson staining of the degree of lung tissue fibrosis in rats with pulmonary fibrosis in different groups (200X).

[0110] Figure 6 These are the results of histopathological evaluation of lung tissue fibrosis in rats with pulmonary fibrosis in different groups. DETAILED DESCRIPTION

[0111] After extensive and in-depth research, the inventors have developed for the first time that acellular fat extract has excellent therapeutic effects on pulmonary fibrosis, and on this basis, the present invention has been completed.

[0112] the term

[0113] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0114] As used herein, the terms "include", "comprises", and "comprising" are used interchangeably, and include not only open definitions, but also semi-closed and closed definitions. In other words, the terms include "consisting of", "consisting essentially of".

[0115] As used herein, the terms "cell free fat extract", "Cell free fat extract" and "CEFFE" are used interchangeably.

[0116] In the present invention, the term "prevention" means a method of preventing the onset of a disease and / or its attendant symptoms or protecting a subject from acquiring a disease. "Prevention" as used herein also includes delaying the onset of a disease and / or its attendant symptoms and reducing the risk of a subject acquiring a disease.

[0117] The "treatment" described in the present invention includes delaying and stopping the progression of the disease, or eliminating the disease, and does not require 100% inhibition, elimination and reversal. In some embodiments, compared with the level observed when the composition, medicine box, food box or health product box, active ingredient combination described in the present invention is not present, the composition or pharmaceutical composition described in the present invention reduces, inhibits and / or reverses diabetes, for example, by at least about 10%, at least about 30%, at least about 50%, or at least about 80%.

[0118] As used herein, "ameliorating" includes preventing, curing, alleviating, reversing, and alleviating, among others.

[0119] As used herein, the term "IGF-1" stands for insulin-like growth factors-1.

[0120] As used herein, the term "BDNF" refers to brain-derived neurotrophic factor (BDNF).

[0121] As used herein, the term "GDNF" stands for glial cellline-derived neurotrophic factor.

[0122] As used herein, the term "bFGF" stands for basic fibroblast growth factor.

[0123] As used herein, the term "VEGF" stands for vascular endothelial growth factor.

[0124] As used herein, the term "TGF-β" stands for transforming growth factor-β.

[0125] As used herein, the term "HGF" refers to hepatocyte growth factor

[0126] As used herein, the term "PDGF" refers to platelet derived growth factor

[0127] As used herein, the term "EGF" refers to Epidermal Growth Factor

[0128] As used herein, the term "NT-3" refers to neurotrophins-3.

[0129] As used herein, the term "GH" stands for Growth Hormone.

[0130] As used herein, the term "G-CSF" stands for granulocyte colony stimulating factor.

[0131] Cell free fat extract (CEFFE) and preparation method thereof

[0132] As used herein, the terms "acellular fat extract of the present invention", "extract of the present invention", "fat extract of the present invention", etc. are used interchangeably and refer to an extract (or extracting liquid) derived from adipose tissue that is prepared without adding any solution, solvent, small molecule, chemical agent, and biological additive during the fat extract preparation process (except for the rinsing step). A typical method for preparing the extract of the present invention is as described above in the second aspect of the present invention. In addition, it should be understood that although it is not necessary to add any additives (or added ingredients) to the extract of the present invention during the preparation process, some or a small amount of safe substances (such as a small amount of water) that have no negative or adverse effects on the activity of the extract of the present invention can also be added.

[0133] The acellular fat extract of the present invention may include a variety of cytokines. Representatively, the acellular fat extract includes one or more of IGF-1, BDNF, GDNF, TGF-β, HGF, bFGF, VEGF, TGF-β1, HGF, PDGF, EGF, NT-3, GH and G-CSF.

[0134] Preferably, the cell-free fat extract of the present invention is prepared by the method described in the second aspect of the present invention.

[0135] Typically, the cell-free fat extract of the present invention is prepared by the following method:

[0136] (1) providing a fat tissue raw material, breaking the fat tissue raw material, and rinsing it (e.g., using physiological saline) to obtain rinsed fat tissue;

[0137] (2) centrifuging the rinsed adipose tissue to obtain a layered mixture;

[0138] (3) removing the upper oil layer and the lower water layer from the stratified mixture, and collecting the middle layer (i.e., the fat layer containing fat cells);

[0139] (4) emulsifying the intermediate layer to obtain an emulsified fat mixture (also called nano fat);

[0140] (5) centrifuging the emulsified fat mixture to obtain an intermediate liquid layer, i.e., a primary fat extract; and

[0141] (6) Filtering and sterilizing the primary fat extract to obtain a cell-free fat extract.

[0142] Specifically, the cell-free fat extract of the present invention

[0143] In another preferred embodiment, in the step (2), the centrifugation is performed at 800-2500 g, preferably 800-2000 g, more preferably 1000-1500 g, and most preferably 1100-1300 g.

[0144] In another preferred embodiment, in the step (2), the centrifugation time is 1-15 min, preferably 1-10 min, more preferably 1-8 min, and most preferably 1-5 min.

[0145] In another preferred embodiment, in the step (4), the emulsification is mechanical emulsification.

[0146] In another preferred embodiment, the mechanical emulsification is performed by repeatedly blowing with a syringe (such as blowing 20-200 times, preferably 20-150 times, more preferably 20-100 times, and more preferably 30-50 times).

[0147] In another preferred embodiment, the blowing method is to connect two 10 ml injection syringes to a three-way tube and push and beat repeatedly at a uniform speed.

[0148] In another preferred embodiment, in the step (4), the emulsification is performed by breaking up the tissue using a tissue homogenizer.

[0149] In another preferred embodiment, in the step (5), before subjecting the emulsified fat mixture to centrifugal treatment, the step further includes subjecting the emulsified fat mixture to freezing and then thawing.

[0150] In another preferred embodiment, after the freezing and thawing treatment, the thawed mixture is used for centrifugation.

[0151] In another preferred embodiment, the freezing temperature is -50°C to -120°C, preferably -60°C to -100°C, and more preferably -70°C to -90°C.

[0152] In another preferred embodiment, the thawing temperature is 20-40°C, preferably 25-40°C, and more preferably 37°C.

[0153] In another preferred embodiment, the number of cycles of freezing and thawing is 1-5 times (preferably 1, 2, 3 or 4 times).

[0154] In another preferred embodiment, in the step (5), after centrifugation, the emulsified fat mixture is stratified into four layers, the first layer is an oil layer, the second layer is a residual fat tissue layer, the third layer is a liquid layer (i.e., an intermediate liquid layer), and the fourth layer is a cell / tissue fragment precipitation layer.

[0155] In another preferred embodiment, in the step (5), the centrifugation is performed at 800-2500 g, preferably 800-2000 g, more preferably 1000-1500 g, and most preferably 1100-1300 g.

[0156] In another preferred embodiment, in the step (5), the centrifugation time is 1-15 min, preferably 1-10 min, more preferably 2-8 min, and most preferably 3-7 min.

[0157] In another preferred embodiment, in the step (5), the first layer, the second layer, the third layer and the fourth layer are arranged in sequence from top to bottom.

[0158] In another preferred embodiment, in the step (5), the intermediate liquid layer is a transparent or substantially transparent layer.

[0159] In another preferred embodiment, in the step (6), the filtration bag is capable of removing fat cells from the primary fat extract.

[0160] In another preferred embodiment, in the step (6), the filtration and sterilization are performed through a filter (such as a 0.22 μm microporous filter membrane).

[0161] In another preferred embodiment, the filter is a microporous membrane filter.

[0162] In another preferred embodiment, the pore size of the microporous filter membrane is 0.05-0.8 μm, preferably 0.1-0.5 μm, more preferably 0.1-0.4 μm, more preferably 0.15-0.3 μm, more preferably 0.2-0.25 μm, and most preferably 0.22 μm.

[0163] In another preferred embodiment, in the step (6), the filtration and sterilization are first performed through a first filter that can filter out cells, and then through a second filter (such as a 0.22 μm filter) that can filter out pathogens (such as bacteria).

[0164] In another preferred embodiment, the step (6) further comprises packaging the fat extract to form packaged products. (The packaged extract can be stored at -20°C for later use; can be used directly after thawing at low temperature (such as -4°C) or room temperature, or can be stored at low temperature (such as 4°C) for a period of time after thawing before use).

[0165] Pulmonary fibrosis

[0166] Pulmonary fibrosis (PF) is a disease characterized by diffuse pneumonia and alveolar structural disorder, which eventually leads to pulmonary interstitial fibrosis. It is a serious pathological feature common to a class of clinically known interstitial lung diseases. Interstitial lung diseases can include seven categories: primary in the lungs, associated with systemic rheumatic diseases, caused by drugs or radiation treatments, associated with the environment or occupation, associated with pulmonary vascular disease, alveolar congestion diseases, and hereditary diseases. According to the cause of the disease, it can be divided into two categories: idiopathic and secondary. The common feature is that inflammation caused by various reasons first damages the normal alveolar structure, that is, alveolitis occurs; instead, collagen scar tissue accumulates to repair the damage, that is, fibrosis occurs, causing the lung tissue to gradually lose its normal respiratory function, resulting in symptoms such as dyspnea and hypoxia, and eventually leading to respiratory failure. Pulmonary fibrosis caused by various reasons, especially idiopathic pulmonary fibrosis, has been on the rise in recent years.

[0167] Pulmonary fibrosis can be caused by a variety of factors, such as cytotoxic antibiotics, chemotherapeutic drugs, etc.

[0168] Typically, the pulmonary fibrosis is induced by cytotoxic antibiotics such as bleomycin.

[0169] Typically, the pulmonary fibrosis is induced by anti-tumor drugs (eg, the chemotherapy drug bleomycin).

[0170] Typically, the pulmonary fibrosis is bleomycin-induced or caused pulmonary fibrosis.

[0171] use

[0172] The cell-free fat extract of the present invention can effectively prevent and / or treat pulmonary fibrosis.

[0173] Typically, the prevention and / or treatment of pulmonary fibrosis comprises one or more methods selected from the following groups for prevention and / or treatment:

[0174] (i) reduce lung weight index;

[0175] (ii) improving inflammatory cell infiltration in the peribronchial, bronchial interstitial and / or alveolar regions of the lungs;

[0176] (ii) improving peribronchial and / or bronchointerstitial fibrosis in the lungs; and / or

[0177] (ii) Improve peribronchial edema in the lungs.

[0178] The present invention also provides a method for preventing and / or treating pulmonary fibrosis, the method comprising the step of administering the acellular fat extract of the present invention to a subject in need thereof.

[0179] In another preferred embodiment, the subject is a human or non-human mammal.

[0180] In another preferred embodiment, the non-human mammals include rodents, such as rats and mice.

[0181] Composition and administration

[0182] The compositions of the present invention include (but are not limited to): pharmaceutical compositions, food compositions, health care compositions, dietary supplements, and the like.

[0183] Representatively, the acellular fat extract of the present invention can be prepared into a pharmaceutical composition, such as a dosage form of tablets, capsules, powders, microgranules, solutions, lozenges, jellies, cream preparations, spirits, suspensions, tinctures, poultices, liniments, lotions, and aerosols. The pharmaceutical composition can be prepared by a commonly known preparation technique, and suitable pharmaceutical additives can be added to the medicine.

[0184] The composition of the present invention may also include a pharmaceutically, food, health care product or dietary acceptable carrier. "Pharmaceutically, food, health care product or dietary acceptable carrier" 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. "Compatibility" 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, food, health care product or dietary acceptable carriers include 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), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0185] There is no particular limitation on the administration of the composition of the present invention. Representative administration methods include (but are not limited to): oral administration, parenteral administration (intravenous administration, intramuscular administration), and topical administration. The preferred administration methods are oral administration and injection administration.

[0186] The dosage form of the composition or preparation of the present invention is an oral preparation, an external preparation or an injection preparation. Representatively, solid dosage forms for oral administration or administration 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 dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0187] Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other materials known in the art. They may contain opacifying agents.

[0188] Liquid dosage forms for oral administration or administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

[0189] Besides these inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents, and perfumes.

[0190] In addition to the active ingredients, suspensions may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances.

[0191] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0192] Dosage forms for topical or administrable compounds of the invention 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.

[0193] The cell-free fat extract of the present invention can be administered alone or in combination with other drugs for preventing and / or treating fatty liver and / or its complications.

[0194] When applying the composition, a safe and effective amount of the acellular fat extract of the present invention is applied to a human or non-human animal (such as rats, mice, dogs, cats, cattle, chickens, ducks, etc.) that needs treatment, wherein the dosage during application is an effective dosage that is acceptable in medicine, food, or health products. As used herein, the term "safe and effective amount" refers to an amount that produces a function or activity to humans and / or animals and can be accepted by humans and / or animals. It should be understood by those of ordinary skill in the art that the "safe and effective amount" may vary with the form of the pharmaceutical composition, the route of administration, the adjuvants of the drugs used, the severity of the disease, and the combination of other drugs. For example, for a person weighing 60 kg, the daily dosage is usually 0.1 to 1000 mg, preferably 1 to 600 mg, and more preferably 2 to 300 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the health status of the patient, which are all within the skill range of skilled physicians.

[0195] The main advantages of the present invention include:

[0196] The present invention discovers for the first time that the cell-free fat extract has an excellent therapeutic effect on pulmonary fibrosis.

[0197] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0198] Example 1

[0199] 1 Experimental methods

[0200] 1.1. Preparation of cell free fat extract (CEFFE)

[0201] The fat was obtained from volunteers with informed consent. The preparation method of acellular adipose tissue extract is as follows:

[0202] (1) Adipose tissue was obtained from 6 healthy women who underwent conventional liposuction, with an average age of 31 years (range, 24-36 years). After local injection of tumescent fluid for anesthesia, a 3 mm liposuction cannula with a large side hole (2 mm x 7 mm) was connected to a 20 mL syringe and radial suction was performed under artificial negative pressure. The obtained fat was placed upright and still, and after removing the tumescent fluid, it was rinsed three times with saline.

[0203] (2) The washed adipose tissue was placed in a centrifuge tube and centrifuged at 1200 g at 4°C for 3 minutes to obtain a layered mixture.

[0204] (3) For the stratified mixture, the upper oil layer and the lower water layer are removed, and the middle layer (i.e., the fat layer containing fat cells) is collected.

[0205] (4) The intermediate layer is mechanically emulsified by repeatedly pushing and beating at a constant speed 30 times using two 10 ml injection syringes connected to a three-way tube to obtain a mechanically emulsified fat mixture (also called nano fat).

[0206] (5) The mechanically emulsified fat mixture is placed in a -80°C refrigerator for freezing, and then thawed in a 37°C water bath. After a single freeze-thaw cycle, the thawed fat mixture is centrifuged at 1200g and 4°C for 5 minutes to obtain a layered mixture. The layered mixture is divided into 4 layers, the first layer is an oil layer, the second layer is a residual fat tissue layer, the third layer is a liquid layer, and the fourth layer is a cell / tissue fragment precipitation layer. The oil layer and the residual fat tissue layer are removed, and the liquid layer is absorbed. During the absorption process, contamination of the cell / tissue fragment precipitation layer is avoided, thereby obtaining a primary fat extract.

[0207] (6) The obtained primary fat extract was filtered through a 0.22 μm filter to sterilize and remove any living cells that may be mixed in, thereby obtaining a cell-free fat extract (CEFFE), which was then packaged and frozen at -20°C and thawed at 4°C before use.

[0208] The prepared acellular fat extract was tested for growth factor content using an ELISA immunosorbent assay kit, including cytokines such as IGF-1, BDNF, GDNF, bFGF, VEGF, TGF-β, HGF and PDGF. The average concentrations of the six samples were as follows: IGF-1 (9840.6 pg / ml), BDNF (1764.5 pg / ml), GDNF (1831.9 pg / ml), bFGF (242.3 pg / ml), VEGF (202.9 pg / ml), TGF-β1 (954.5 pg / ml), HGF (898.4 pg / ml), PDGF (179.9 pg / ml).

[0209] 1.2 Establishment of rat pulmonary fibrosis model and grouping

[0210] Thirty male SD rats were randomly divided into a model control group (9 rats), a CEFFE low-dose group (7 rats), a CEFFE medium-dose group (7 rats), and a CEFFE high-dose group (7 rats) according to their body weights before grouping. All 30 animals were given a modeling agent (bleomycin) by intratracheal aerosolization. On Day 1, all animals were given bleomycin (7 mg / kg, 1 mL / kg) by intratracheal aerosolization to establish the model.

[0211] Day 1: The animal was anesthetized by inhalation of isoflurane and then fixed on a rat holder at 45°. A small animal anesthesia laryngoscope was used to press the root of the animal's tongue to expose the glottis. The lung micro-liquid nebulizer needle (blunt) for extracting a quantitative amount of bleomycin solution was gently inserted into the trachea about 1 cm, and then the piston was quickly pushed to atomize the bleomycin solution into the lungs. The needle was quickly pulled out, and the animal was removed from the holder, with the head facing up and rotated left and right to distribute bleomycin as evenly as possible in each lung lobe. The administration volume was retained to the integer. When the administration volume was between two graduation capacity lines, the administration volume was absorbed according to the upper graduation value.

[0212] After all animal models were established, the test article or vehicle control was administered according to Table 1 below.

[0213] Table 1

[0214] Group No. Group Dosing volume (mL / kg) Dosage (mg / kg) 1 Normal saline control group 5 0 2 CEFFE low dose group 2.5 9.28 3 CEFFE medium dose group 5 18.55 4 CEFFE high dose group 15 59.25

[0215] Day 1 to Day 28: Perform routine animal feeding and general clinical observation records.

[0216] Route of administration: intravenous injection

[0217] Dosing frequency and duration: Day 2, Day 6, Day 10, Day 14, Day 18, Day 22, Day 26, once every 4 days, for a total of 7 times.

[0218] Intravenous administration: Based on the animal weight recently weighed, use a disposable sterile syringe to draw the amount of medicine for each animal, and slowly inject it into the tail vein (about 10 to 180 seconds). The administration volume is retained to 1 decimal place. When the administration volume is between two graduation capacity lines, draw the administration volume according to the upper graduation value.

[0219] The animals were weighed on the day of grouping, modeling, drug administration and before euthanasia to evaluate the changes in body weight.

[0220] 1.3 Lung weight index and histopathological examination

[0221] The animals were weighed on the day of grouping, modeling, drug administration, and before euthanasia. All animals were dissected on Day 28, and the tissues were preserved. The lungs, trachea, and bronchi of the animals were observed for abnormalities. The lungs were separated and fat and other tissues were removed. The surface liquid was absorbed with filter paper, and the lung weight was weighed to calculate the lung weight index. Lung weight index (%) = lung weight / body weight × 100%.

[0222] The right lung and bronchioles were fixed in 10% neutral buffered formalin solution, embedded in paraffin, sliced, prepared, and HE stained to evaluate the degree of inflammatory cell infiltration in lung tissue, and Masson staining to evaluate the degree of lung tissue fibrosis. The histopathological evaluation levels are as follows:

[0223] Grade 1: Mild = lesions are barely visible and / or few / the affected area or lesions are small.

[0224] Grade 2: Mild = lesions are easily visible, but only noticeable because of the small number of lesions / small area affected or lesions.

[0225] Grade 3: Moderate = Lesions are clearly present and evaluable in size and / or number.

[0226] Grade 4: Severe = large area of ​​lesion involvement or large lesion size.

[0227] 1.4 Statistical analysis

[0228] Data collection: Data were collected by system generation and manual recording.

[0229] Data analysis: The statistical software SPSS13.0 or GraphPad Prism 5 was used to process the data. All statistical analyses were performed using two-tailed analysis, with the statistical level set at P ≤ 0.05. All data were expressed as "mean ± standard deviation".

[0230] 2. Results

[0231] 2.1 CEFFE treatment improves the survival rate and body weight of rats with pulmonary fibrosis model

[0232] On Day 28, the survival rates of rats in the pulmonary fibrosis model in different groups were as follows: Figure 1 As shown, from Figure 1 It can be seen that the survival rates of animals in the normal saline control group, CEEFE low-dose group, CEEFE medium-dose group, and CEEFE high-dose group were 89%, 100%, 100%, and 100%, respectively. The survival rates of animals in each CEEFE group were higher than those in the model control group, indicating that CEEFE can significantly improve the survival rate of rats with pulmonary fibrosis model.

[0233] Changes of body weight of rats with pulmonary fibrosis model in different groups over time Figure 2 As shown, from Figure 2It can be seen that the mean body weight of animals in each group decreased on Day 6 after modeling, and then the mean body weight of each group increased steadily with the extension of the experimental period; compared with the animals in the model control group, the mean body weight of animals in the CEFFE low-dose, CEFFE medium-dose and CEFFE high-dose groups increased more significantly on Day 18, Day 22, Day 26 and Day 28, with statistically significant differences. Figure 2 The results showed that CEEFE could significantly increase the body weight of rats with pulmonary fibrosis model.

[0234] 2.2 CEFFE treatment reduces lung weight index in rats with pulmonary fibrosis model

[0235] Lung weight index (%) of rats with pulmonary fibrosis in different groups Figure 3 As shown, from Figure 3 It can be seen that compared with the control group, the lung weight index of animals in the CEFFE low-dose group, CEFFE medium-dose group and CEFFE high-dose group was reduced, with statistically significant differences.

[0236] 2.3 CEFFE treatment improves lung inflammation in model rats

[0237] During this experiment, intra-airway aerosolized bleomycin induced pulmonary fibrosis, and microscopic observations showed inflammatory cell infiltration mainly consisting of mononuclear cells, neutrophils, and macrophages around the bronchial / interstitial / alveoli, bronchial / interstitial fibrosis, and bronchial edema. Alveolar hemorrhage, pulmonary embolism, and alveolar dilatation were partially observed, among which inflammatory cell infiltration mainly consisting of mononuclear cells, neutrophils, and macrophages around the bronchial / interstitial / alveoli, bronchial / interstitial fibrosis, and bronchial edema were common lesions in the model control group.

[0238] HE staining of inflammatory cell infiltration in lung tissue of rats with pulmonary fibrosis in different groups Figure 4 As shown, Masson staining of the degree of lung fibrosis in rats with pulmonary fibrosis in different groups is shown in Figure 5 The histopathological evaluation results of lung fibrosis in rats with pulmonary fibrosis in different groups are shown in Figure 6 Compared with the saline control group, each CEFFE treatment group had a significant improvement in the infiltration of inflammatory cells around bronchial / interstitial / alveolar areas, fibrosis around bronchial / interstitial areas, and edema around bronchial areas. The low- and medium-dose CEFFE groups had a certain dose relationship and the improvement was better than that of the high-dose group.

[0239] in conclusion

[0240] In the examples, a rat pulmonary fibrosis model was established by administering bleomycin by intratracheal atomization, and CEFFE was injected intravenously for treatment. It was confirmed that CEFFE could effectively improve the survival rate and body weight of the model rats, reduce the lung weight index, and alleviate inflammation and damage of the alveolar tissue, indicating that CEFFE treatment could effectively improve the pulmonary fibrosis and systemic condition of the model rats, indicating that CEFFE has an excellent therapeutic effect on pulmonary fibrosis.

[0241] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. 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 claims attached to this application.

Claims

1. A use of a cell-free fat extract, characterized in that: Used for preparing a pharmaceutical composition, wherein the pharmaceutical composition is used for treating pulmonary fibrosis, wherein the pulmonary fibrosis is bleomycin-induced or caused pulmonary fibrosis; The acellular fat extract contains the following components: IGF-1, BDNF, GDNF, bFGF, VEGF, TGF-β, HGF and PDGF; the acellular fat extract does not contain cells and does not contain lipid droplets; Furthermore, the cell-free fat extract is prepared by the following method: (1) providing a fat tissue raw material, crushing the fat tissue raw material, and rinsing the fat tissue raw material to obtain rinsed fat tissue; (2) centrifuging the rinsed adipose tissue to obtain a layered mixture; (3) removing the upper oil layer and the lower water layer from the stratified mixture, and collecting the middle layer, i.e., the fat layer containing fat cells; (4) emulsifying the intermediate layer to obtain an emulsified fat mixture, also known as nano fat; (5) centrifuging the emulsified fat mixture to obtain an intermediate liquid layer, i.e., a primary fat extract; and (6) Filtering and sterilizing the primary fat extract to obtain a cell-free fat extract.

2. The use according to claim 1, characterized in that The term "free of fat droplets" means that the volume of oil droplets in the acellular fat extract accounts for less than 1% of the total liquid; The term "cell-free" means that the average number of cells in 1 ml of cell-free fat extract is ≤ 1.

3. The use according to claim 1, characterized in that The term "free of fat droplets" means that the volume of oil droplets in the acellular fat extract accounts for less than 0.5% of the total liquid; The term "cell-free" means that the average number of cells in 1 ml of cell-free fat extract is ≤ 0.

5.

4. The use according to claim 1, characterized in that The term "free of fat droplets" means that the volume of oil droplets in the acellular fat extract accounts for less than 0.1% of the total liquid; The term "cell-free" means that the average number of cells in 1 ml of cell-free fat extract is 0.

5. The use according to claim 1, characterized in that In the cell-free fat extract, the concentration of IGF-1 is 5000-30000 pg / ml; The concentration of BDNF is 800-5000 pg / ml; The concentration of GDNF is 800-5000 pg / ml; The concentration of bFGF is 50-600 pg / ml; The concentration of VEGF is 50-500 pg / ml; The concentration of TGF-β1 is 200-3000 pg / ml; The concentration of HGF is 200-3000 pg / ml; The concentration of PDGF is 50-600 pg / ml.

6. The use according to claim 1, characterized in that In the cell-free fat extract, the concentration of IGF-1 is 7000-15000 pg / ml; The concentration of BDNF is 1200-2500 pg / ml; The concentration of GDNF is 1200-2500 pg / ml; The concentration of bFGF is 120-400 pg / ml; The concentration of VEGF is 120-300 pg / ml; The concentration of TGF-β1 is 600-1500 pg / ml; The concentration of HGF is 600-1500 pg / ml; The concentration of PDGF is 100-300 pg / ml.

7. The use according to claim 1, characterized in that In the cell-free fat extract, the concentration of IGF-1 is 9000-11000 pg / ml; The concentration of BDNF is 1600-2000 pg / ml; The concentration of GDNF is 1600-2000 pg / ml; The concentration of bFGF is 200-280 pg / ml; The concentration of VEGF is 170-230 pg / ml; The concentration of TGF-β1 is 800-1100 pg / ml; The concentration of HGF is 800-1000 pg / ml; The concentration of PDGF is 160-200 pg / ml.

8. The use according to claim 1, characterized in that The dosage form of the pharmaceutical composition is an oral preparation, an external preparation or an injection preparation.

9. The use according to claim 8, characterized in that The injection preparation is an intravenous injection preparation.

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