Therapeutic use of cell-free fat extract for non-alcoholic steatohepatitis

Abstract

The present invention relates to the use of a cell-free fat extract for the treatment of non-alcoholic steatohepatitis. Specifically, the present invention provides a use of the cell-free fat extract for preparing a composition or formulation for preventing and / or treating non-alcoholic steatohepatitis. The cell-free fat extract of the present invention has excellent therapeutic effects on non-alcoholic steatohepatitis.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to the therapeutic use of a cell-free fat extract on non-alcoholic steatohepatitis. Background Art

[0002] Nonalcoholic steatohepatitis (NASH) refers to fatty liver inflammation caused by factors other than alcohol, in which lipids accumulate in the hepatocytes of the liver, often accompanied by inflammation and fibrosis. Nonalcoholic steatohepatitis is a developmental form of nonalcoholic fatty liver disease, accompanied by the appearance of fatty degeneration caused by inflammation and liver cell damage. NASH can lead to liver fibrosis, cirrhosis, liver failure and the development of liver tumors. Although a large amount of research has been done on the pathological mechanism of nonalcoholic steatohepatitis, its pathogenesis has not yet been clearly elucidated. The existing technology also lacks drugs that are effective in treating nonalcoholic steatohepatitis.

[0003] Therefore, there is a need in the art to develop a drug that can effectively treat non-alcoholic steatohepatitis. Summary of the Invention

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

[0005] In a first aspect, the present invention provides a use of a cell-free fat extract for preparing a composition or preparation, wherein the composition or preparation is used for one or more uses selected from the following group: (i) preventing and / or treating non-alcoholic steatohepatitis.

[0006] In another preferred embodiment, the non-alcoholic steatohepatitis is selected from the group consisting of acute non-alcoholic steatohepatitis, chronic non-alcoholic steatohepatitis, or a combination thereof.

[0007] In another preferred embodiment, the prevention and / or treatment of non-alcoholic steatohepatitis comprises one or more methods selected from the following groups for prevention and / or treatment:

[0008] (a) Improve hepatocyte fatty degeneration;

[0009] (b) improving hepatocellular ballooning; and / or

[0010] (b) Improved hepatic inflammatory cell infiltration.

[0011] In another preferred embodiment, the acellular fat extract is an acellular fat extract extracted from fat of human or non-human mammals.

[0012] 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.

[0013] In another preferred embodiment, the composition or preparation includes a pharmaceutical composition or preparation, a food composition or preparation, a health product composition or preparation, or a dietary supplement.

[0014] In another preferred embodiment, the composition or preparation further comprises a pharmaceutically, food, health product or dietary acceptable carrier.

[0015] In another preferred embodiment, the composition or preparation is in the form of an oral preparation, an external preparation or an injection preparation.

[0016] In another preferred embodiment, the injection preparation is an intravenous injection or an intramuscular injection.

[0017] 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, an emulsion, an ointment, a cream, a paste, a cake, a powder, a patch, etc.

[0018] In another preferred embodiment, the composition or preparation is in the form of powder, granules, capsules, injections, tinctures, oral solutions, tablets or lozenges.

[0019] In another preferred embodiment, the composition or preparation is administered externally, topically, or by subcutaneous injection.

[0020] In another preferred embodiment, the cell-free fat extract does not contain cells and does not contain lipid droplets.

[0021] In another preferred embodiment, the lipid droplets are oil droplets released after the adipocytes are broken.

[0022] In another preferred embodiment, the “free from fat droplets” means that in the cell-free 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%.

[0023] In another preferred embodiment, the cells are selected from the group consisting of endothelial cells, adipose stem cells, macrophages, and stromal cells.

[0024] 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.

[0025] In another preferred embodiment, the cell-free fat extract is a naturally obtained nano fat extract without any added ingredients.

[0026] In another preferred embodiment, the “additive-free” means that except for the rinsing step, no solution, solvent, small molecule, chemical agent, or biological additive is added during the preparation of the fat extract.

[0027] In another preferred embodiment, the cell-free fat extract is prepared by emulsifying adipose tissue and then centrifuging it.

[0028] In another preferred embodiment, the cell-free fat extract contains one or more components selected from the following group: IGF-1, BDNF, GDNF, TGF-β1, HGF, bFGF, VEGF, TGF-β1, PDGF, EGF, NT-3, GH, G-CSF, or a combination thereof.

[0029] In another preferred embodiment, the cell-free 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.

[0030] In another preferred embodiment, the acellular fat extract is acellular fat extract.

[0031] In another preferred embodiment, in the cell-free fat extract, the concentration of IGF-1 is 5000-30000 pg / ml, preferably 6000-20000 pg / ml, more preferably 7000-15000 pg / ml, more preferably 8000-12000 pg / ml, more preferably 9000-11000 pg / ml, and more preferably 9500-10500 pg / ml.

[0032] In another preferred embodiment, in the cell-free fat extract, the concentration of BDNF is 800-5000 pg / ml, preferably 1000-4000 pg / ml, more preferably 1200-2500 pg / ml, more preferably 1400-2000 pg / ml, more preferably 1600-2000 pg / ml, more preferably 1700-1850 pg / ml.

[0033] In another preferred embodiment, in the cell-free fat extract, the concentration of GDNF is 800-5000 pg / ml, preferably 1000-4000 pg / ml, more preferably 1200-2500 pg / ml, more preferably 1400-2000 pg / ml, more preferably 1600-2000 pg / ml, more preferably 1700-1900 pg / ml.

[0034] In another preferred embodiment, in the cell-free fat extract, the concentration of bFGF is 50-600 pg / ml, preferably 100-500 pg / ml, more preferably 120-400 pg / ml, more preferably 150-300 pg / ml, more preferably 200-280 pg / ml, more preferably 220-260 pg / ml.

[0035] In another preferred embodiment, in the cell-free fat extract, the concentration of VEGF is 50-500 pg / ml, preferably 100-400 pg / ml, more preferably 120-300 pg / ml, more preferably 150-250 pg / ml, more preferably 170-230 pg / ml, more preferably 190-210 pg / ml.

[0036] In another preferred embodiment, in the cell-free fat extract, the concentration of TGF-β1 is 200-3000 pg / ml, preferably 400-2000 pg / ml, more preferably 600-1500 pg / ml, more preferably 800-1200 pg / ml, more preferably 800-1100 pg / ml, more preferably 900-1000 pg / ml.

[0037] In another preferred embodiment, in the cell-free fat extract, the concentration of HGF is 200-3000 pg / ml, preferably 400-2000 pg / ml, more preferably 600-1500 pg / ml, more preferably 600-1200 pg / ml, more preferably 800-1000 pg / ml, more preferably 850-950 pg / ml.

[0038] In another preferred embodiment, in the cell-free fat extract, the concentration of PDGF is 50-600 pg / ml, preferably 80-400 pg / ml, more preferably 100-300 pg / ml, more preferably 140-220 pg / ml, more preferably 160-200 pg / ml, more preferably 170-190 pg / ml.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] In another preferred embodiment, the cell-free fat extract is prepared by the following method:

[0047] (1) providing a fat tissue raw material, crushing the fat tissue raw material, and rinsing it (e.g., with physiological saline) to obtain rinsed fat tissue;

[0048] (2) centrifuging the rinsed adipose tissue to obtain a layered mixture;

[0049] (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);

[0050] (4) emulsifying the intermediate layer to obtain an emulsified fat mixture (also called nanofat);

[0051] (5) centrifuging the emulsified fat mixture to obtain an intermediate liquid layer, which is the primary fat extract; and

[0052] (6) Filtering and sterilizing the primary fat extract to obtain a cell-free fat extract.

[0053] In a second aspect, the present invention provides a method for preparing a cell-free fat extract, the method comprising the steps of:

[0054] (1) providing a fat tissue raw material, crushing the fat tissue raw material, and rinsing it (e.g., with physiological saline) to obtain rinsed fat tissue;

[0055] (2) centrifuging the rinsed adipose tissue to obtain a layered mixture;

[0056] (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);

[0057] (4) emulsifying the intermediate layer to obtain an emulsified fat mixture (also called nanofat);

[0058] (5) centrifuging the emulsified fat mixture to obtain an intermediate liquid layer, which is the primary fat extract; and

[0059] (6) Filtering and sterilizing the primary fat extract to obtain a cell-free fat extract.

[0060] In another preferred embodiment, the cell-free fat extract is as described in the first aspect of the present invention.

[0061] 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.

[0062] 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.

[0063] In another preferred embodiment, the centrifugation temperature is 2-6°C.

[0064] In another preferred embodiment, in the step (4), the emulsification is mechanical emulsification.

[0065] 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).

[0066] In another preferred embodiment, the blowing method is to connect two 10 ml injection syringes to a three-way pipe and push and blow repeatedly at a uniform speed.

[0067] In another preferred embodiment, in the step (4), the emulsification is performed by crushing with a tissue homogenizer.

[0068] In another preferred embodiment, in the step (5), before the emulsified fat mixture is subjected to centrifugal treatment, the step further includes freezing and then thawing the emulsified fat mixture.

[0069] In another preferred embodiment, after the freezing and thawing treatment, the thawed mixture is used for centrifugation.

[0070] 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.

[0071] In another preferred embodiment, the thawing temperature is 20-40°C, preferably 25-40°C, and more preferably 37°C.

[0072] In another preferred embodiment, the number of cycles of freezing and thawing is 1-5 times (preferably 1, 2, 3 or 4 times).

[0073] In another preferred embodiment, in the step (5), after centrifugation, the emulsified fat mixture is divided 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.

[0074] 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.

[0075] 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.

[0076] In another preferred embodiment, the centrifugation temperature is 2-6°C.

[0077] 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.

[0078] In another preferred embodiment, in the step (5), the intermediate liquid layer is a transparent or substantially transparent layer.

[0079] In another preferred embodiment, in step (6), the filtration bag is capable of removing fat cells from the primary fat extract.

[0080] 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).

[0081] In another preferred embodiment, the filter is a microporous membrane filter.

[0082] 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.

[0083] In another preferred embodiment, in 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).

[0084] In another preferred embodiment, step (6) further comprises packaging the fat extract to form packaged products. (The packaged extract can be stored at -20°C for future use; can be thawed at low temperature (e.g., -4°C) or room temperature and used directly, or can be thawed and stored at low temperature (e.g., 4°C) for a period of time before use).

[0085] 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.

[0086] In a fourth aspect, the present invention provides a composition or preparation comprising (a) the cell-free 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.

[0087] In another preferred embodiment, the composition is a pharmaceutical composition, a food composition, a health product composition or a dietary supplement.

[0088] In another preferred embodiment, the composition or preparation is in the form of an oral preparation, an external preparation or an injection preparation.

[0089] In another preferred embodiment, the composition or preparation is in the form of powder, granules, capsules, injections, tinctures, oral solutions, tablets or lozenges.

[0090] In another preferred embodiment, the injection is an intravenous injection or an intramuscular injection.

[0091] 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, an emulsion, an ointment, a cream, a paste, a cake, a powder, a patch, etc.

[0092] In another preferred embodiment, in the composition or preparation, the mass percentage of the cell-free fat extract is 5 wt %, preferably 1-20 wt %, based on the total weight of the composition or preparation.

[0093] In a fifth aspect, 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.

[0094] In a sixth aspect, the present invention provides a method for preventing and / or treating non-alcoholic steatohepatitis, comprising administering the cell-free fat extract according to the third aspect of the present invention to a subject in need thereof.

[0095] In another preferred embodiment, the subject is a human or non-human mammal.

[0096] In another preferred embodiment, the non-human mammals include rodents, such as rats and mice.

[0097] 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 described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0098] Figure 1 HE staining (100X) of mice in different groups, wherein the semi-quantitative grading results "+", "++" and "+++" indicate increasing degrees of lesions. DETAILED DESCRIPTION

[0099] After extensive and in-depth research, the inventors have developed for the first time a cell-free fat extract that has excellent therapeutic effects on non-alcoholic steatohepatitis. Based on this, the present invention was completed.

[0100] the term

[0101] 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.

[0102] As used herein, the terms "include," "comprise," and "contain" are used interchangeably to encompass not only open definitions but also semi-closed and closed definitions. In other words, the terms encompass "consisting of," "consisting essentially of."

[0103] As used herein, the terms "cell free fat extract", "cell free fat extract" and "CEFFE" are used interchangeably.

[0104] In the present invention, the term "prevention" refers to a method of preventing the onset of a disease and / or its attendant symptoms or protecting a subject from acquiring a disease. As used herein, "prevention" also includes delaying the onset of a disease and / or its attendant symptoms and reducing the risk of a subject acquiring a disease.

[0105] "Treatment" as used herein includes delaying and stopping the progression of the disease, or eliminating the disease, and does not require 100% inhibition, elimination, or reversal. In some embodiments, the composition or pharmaceutical composition of the present invention reduces, inhibits, and / or reverses non-alcoholic steatohepatitis, for example, by at least about 10%, at least about 30%, at least about 50%, or at least about 80%, compared to the levels observed in the absence of the composition, medicine kit, food kit, or health product kit, or combination of active ingredients described herein.

[0106] As used herein, "ameliorate" includes preventing, treating, alleviating, reversing, and relieving, among others.

[0107] As used herein, the term "IGF-1" refers to insulin-like growth factor-1.

[0108] As used herein, the term "BDNF" refers to brain-derived neurotrophic factor (BDNF).

[0109] As used herein, the term "GDNF" refers to glial cellline-derived neurotrophic factor.

[0110] As used herein, the term "bFGF" refers to basic fibroblast growth factor.

[0111] As used herein, the term "VEGF" stands for vascular endothelial growth factor.

[0112] As used herein, the term "TGF-β1" stands for transforming growth factor-β1.

[0113] As used herein, the term "HGF" refers to hepatocyte growth factor

[0114] As used herein, the term "PDGF" refers to platelet-derived growth factor

[0115] As used herein, the term "EGF" refers to Epidermal Growth Factor

[0116] As used herein, the term "NT-3" refers to neurotrophins-3.

[0117] As used herein, the term "GH" refers to Growth Hormone.

[0118] As used herein, the term "G-CSF" refers to granulocyte colony stimulating factor.

[0119] Cell free fat extract (CEFFE) and preparation method thereof

[0120] As used herein, the terms "acellular adipose extract of the present invention," "extract of the present invention," "fat extract of the present invention," and the like are used interchangeably to refer to an extract (or extractant) derived from adipose tissue that is prepared without the addition of any solutions, solvents, small molecules, chemicals, or biological additives 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. Furthermore, it should be understood that while it is not necessary to add any additives (or added ingredients) to the extract of the present invention during its preparation, some or a small amount of safe substances (e.g., a small amount of water) that do not negatively or adversely affect the activity of the extract of the present invention may be added.

[0121] In a preferred embodiment of the present invention, the acellular fat extract is acellular fat extract.

[0122] The acellular adipose extract of the present invention may include a variety of cytokines. Representatively, the acellular adipose extract includes one or more of IGF-1, BDNF, GDNF, TGF-β, HGF, bFGF, VEGF, TGF-β1, PDGF, EGF, NT-3, GH, and G-CSF.

[0123] In another preferred embodiment, in the cell-free fat extract, the concentration of IGF-1 is 5000-30000 pg / ml, preferably 6000-20000 pg / ml, more preferably 7000-15000 pg / ml, more preferably 8000-12000 pg / ml, more preferably 9000-11000 pg / ml, and more preferably 9500-10500 pg / ml.

[0124] In another preferred embodiment, in the cell-free fat extract, the concentration of BDNF is 800-5000 pg / ml, preferably 1000-4000 pg / ml, more preferably 1200-2500 pg / ml, more preferably 1400-2000 pg / ml, more preferably 1600-2000 pg / ml, more preferably 1700-1850 pg / ml.

[0125] In another preferred embodiment, in the cell-free fat extract, the concentration of GDNF is 800-5000 pg / ml, preferably 1000-4000 pg / ml, more preferably 1200-2500 pg / ml, more preferably 1400-2000 pg / ml, more preferably 1600-2000 pg / ml, more preferably 1700-1900 pg / ml.

[0126] In another preferred embodiment, in the cell-free fat extract, the concentration of bFGF is 50-600 pg / ml, preferably 100-500 pg / ml, more preferably 120-400 pg / ml, more preferably 150-300 pg / ml, more preferably 200-280 pg / ml, more preferably 220-260 pg / ml.

[0127] In another preferred embodiment, in the cell-free fat extract, the concentration of VEGF is 50-500 pg / ml, preferably 100-400 pg / ml, more preferably 120-300 pg / ml, more preferably 150-250 pg / ml, more preferably 170-230 pg / ml, more preferably 190-210 pg / ml.

[0128] In another preferred embodiment, in the cell-free fat extract, the concentration of TGF-β1 is 200-3000 pg / ml, preferably 400-2000 pg / ml, more preferably 600-1500 pg / ml, more preferably 800-1200 pg / ml, more preferably 800-1100 pg / ml, more preferably 900-1000 pg / ml.

[0129] In another preferred embodiment, in the cell-free fat extract, the concentration of HGF is 200-3000 pg / ml, preferably 400-2000 pg / ml, more preferably 600-1500 pg / ml, more preferably 600-1200 pg / ml, more preferably 800-1000 pg / ml, more preferably 850-950 pg / ml.

[0130] In another preferred embodiment, in the cell-free fat extract, the concentration of PDGF is 50-600 pg / ml, preferably 80-400 pg / ml, more preferably 100-300 pg / ml, more preferably 140-220 pg / ml, more preferably 160-200 pg / ml, more preferably 170-190 pg / ml.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] Preferably, the cell-free fat extract of the present invention is prepared by the method described in the second aspect of the present invention.

[0139] Typically, the cell-free fat extract of the present invention is prepared by the following method:

[0140] (1) providing a fat tissue raw material, crushing the fat tissue raw material, and rinsing it (e.g., with physiological saline) to obtain rinsed fat tissue;

[0141] (2) centrifuging the rinsed adipose tissue to obtain a layered mixture;

[0142] (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);

[0143] (4) emulsifying the intermediate layer to obtain an emulsified fat mixture (also called nanofat);

[0144] (5) centrifuging the emulsified fat mixture to obtain an intermediate liquid layer, which is the primary fat extract; and

[0145] (6) Filtering and sterilizing the primary fat extract to obtain a cell-free fat extract.

[0146] 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.

[0147] 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.

[0148] In another preferred embodiment, in the step (4), the emulsification is mechanical emulsification.

[0149] 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).

[0150] In another preferred embodiment, the blowing method is to connect two 10 ml injection syringes to a three-way pipe and push and blow repeatedly at a uniform speed.

[0151] In another preferred embodiment, in the step (4), the emulsification is performed by a tissue homogenizer.

[0152] In another preferred embodiment, in the step (5), before the emulsified fat mixture is subjected to centrifugal treatment, the step further includes freezing and then thawing the emulsified fat mixture.

[0153] In another preferred embodiment, after the freezing and thawing treatment, the thawed mixture is used for centrifugation.

[0154] 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.

[0155] In another preferred embodiment, the thawing temperature is 20-40°C, preferably 25-40°C, and more preferably 37°C.

[0156] In another preferred embodiment, the number of cycles of freezing and thawing is 1-5 times (preferably 1, 2, 3 or 4 times).

[0157] In another preferred embodiment, in the step (5), after centrifugation, the emulsified fat mixture is divided 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.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] In another preferred embodiment, in the step (5), the intermediate liquid layer is a transparent or substantially transparent layer.

[0162] In another preferred embodiment, in step (6), the filtration bag is capable of removing fat cells from the primary fat extract.

[0163] 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).

[0164] In another preferred embodiment, the filter is a microporous membrane filter.

[0165] 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.

[0166] In another preferred embodiment, in 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).

[0167] In another preferred embodiment, step (6) further comprises packaging the fat extract to form packaged products. (The packaged extract can be stored at -20°C for future use; can be thawed at low temperature (e.g., -4°C) or room temperature and used directly, or can be thawed and stored at low temperature (e.g., 4°C) for a period of time before use).

[0168] Nonalcoholic steatohepatitis

[0169] Nonalcoholic steatohepatitis (NASH) refers to steatohepatitis caused by factors other than alcohol. It involves the accumulation of lipids in hepatocytes, accompanied by inflammation and fibrosis. NASH is an extreme form of nonalcoholic fatty liver disease, characterized by fatty degeneration accompanied by inflammation and liver cell damage. NASH can lead to advanced liver fibrosis, cirrhosis, liver failure, and liver tumors. Although extensive research has been conducted on the pathogenesis of NASH, its underlying mechanisms remain elusive.

[0170] Composition and administration

[0171] The compositions of the present invention include (but are not limited to): pharmaceutical compositions, food compositions, health care compositions, dietary supplements, etc.

[0172] Typically, the cell-free fat extract of the present invention can be prepared into pharmaceutical compositions, such as tablets, capsules, powders, microgranules, solutions, lozenges, jellies, creams, spirits, suspensions, tinctures, poultices, liniments, lotions, and aerosols. Pharmaceutical compositions can be prepared by commonly known preparation techniques, and suitable pharmaceutical additives can be added to the medicine.

[0173] The composition of the present invention may also include a pharmaceutically, food, health product or dietary acceptable carrier. "Pharmaceutically, food, health 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 compound of the present invention and with each other without significantly reducing the efficacy of the compound. Some examples of pharmaceutically, food, health 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, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0174] 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.

[0175] The composition or preparation of the present invention is in the form of an oral preparation, an external preparation or an injection preparation. Typically, 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 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.

[0176] 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 well known in the art. They may contain opacifying agents.

[0177] 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, isopropyl alcohol, 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 thereof.

[0178] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and flavoring agents.

[0179] In addition to the active ingredients, suspensions may contain suspending agents such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0180] Compositions for parenteral injection may comprise 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.

[0181] Dosage forms for topical or topical administration of the 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.

[0182] The cell-free fat extract of the present invention can be administered or administrated alone, or in combination with other drugs for preventing and / or treating non-alcoholic fatty liver disease and / or its complications.

[0183] When administering 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.) in need of treatment, wherein the dosage during administration is an effective dosage that is acceptable for pharmaceutical use, food use, or health supplements. 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 skilled in the art will 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 60 kg person, the daily dosage is generally 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 patient's health status, all of which are within the skill of a skilled physician.

[0184] The main advantages of the present invention include:

[0185] The present invention discovers for the first time that the cell-free fat extract has an excellent therapeutic effect on non-alcoholic steatohepatitis.

[0186] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0187] Example 1

[0188] 1. Experimental Methods

[0189] Preparation of cell-free fat extract (CEFFE)

[0190] Fat was obtained from volunteers with informed consent. The preparation method of acellular adipose tissue extract is as follows:

[0191] (1) Adipose tissue was obtained from 6 healthy women undergoing conventional liposuction, with an average age of 31 years (range, 24-36 years). After local anesthesia with injection of tumescent fluid, a 3 mm liposuction cannula with a large side hole (2 mm x 7 mm) was connected to a 20 mL syringe. 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 normal saline.

[0192] (2) The washed adipose tissue was placed in a centrifuge tube and centrifuged at 1200 g for 3 minutes at 4°C to obtain a layered mixture.

[0193] (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.

[0194] (4) The intermediate layer was mechanically emulsified by repeatedly pushing and hitting the intermediate layer 30 times at a constant speed using two 10 ml syringes connected to a three-way tube, and a mechanically emulsified fat mixture (also called nano fat) was obtained.

[0195] (5) The mechanically emulsified fat mixture is placed in a -80°C freezer 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 four layers, the first layer being an oil layer, the second layer being a residual fat tissue layer, the third layer being a liquid layer, and the fourth layer being a cell / tissue fragment precipitation layer. The oil layer and the residual fat tissue layer are removed, and the liquid layer is aspirated. During the aspiration process, contamination of the cell / tissue fragment precipitation layer is avoided, thereby obtaining a primary fat extract.

[0196] (6) The obtained primary fat extract was sterilized by filtration 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 aliquoted and frozen at -20°C and thawed at 4°C before use.

[0197] The cell-free adipose extracts were then assayed for cytokine levels using ELISA kits, including IGF-1, BDNF, GDNF, bFGF, VEGF, TGF-β1, 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), and PDGF (179.9 pg / ml).

[0198] 1.2 Establishment of mouse nonalcoholic steatohepatitis (NASH) model, grouping, and drug administration

[0199] All the mice that passed the quarantine were screened and randomly divided into 5 groups based on the principle of similar weight, with 8 mice in each group. The animal grouping and dosage of each group are shown in Table 1 below:

[0200] Table 1Table 1Dosage of rats in different groups

[0201]

[0202] Modeling method: All animals were fed SPF-grade mouse feed during the test period. After grouping, the normal control group mice were fed SPF-grade mouse feed, and the mice in the other groups were fed MCD (Methionine and Choline Deficient L-Amino Acid Diet) feed to start non-alcoholic fatty liver disease modeling until the end of the experiment.

[0203] Dosing Method and Frequency: Dosing began at the time of animal modeling and was administered twice weekly for 4 weeks, for a total of 8 doses (Day 1, Day 4, Day 8, Day 11, Day 15, Day 18, Day 22, and Day 25). All doses were administered via tail vein injection. The normal control group received no treatment, the model control group received saline as a negative control, and the CEFFE group received the corresponding dose of CEFFE solution. Day 1 is the first day of dosing, Day 2 is the second day, and so on.

[0204] 1.3 Pathological tissue observation

[0205] After liver perfusion, the left lobe of the liver was fixed in 10% neutral formalin according to the sampling method described above. The liver was sectioned and stained with HE to observe liver histopathological changes. The main observation indicators were hepatocyte fatty degeneration, hepatocyte ballooning degeneration, and the degree of inflammatory cell infiltration. Semi-quantitative grading was performed according to the degree of lesions, and NAS scoring was performed. The NAS scoring criteria are shown in Table 2 below:

[0206] Table 2 NAS scoring criteria

[0207]

[0208] 1.7 Data Statistical Analysis

[0209] Measurement data were expressed as mean ± standard deviation. Dynamic measurement data such as body weight were compared between groups using two-way analysis of variance using Graphpad Prism 5.0 software. If there were differences (P < 0.05), the Bonferroni post test was used to compare the normal control group with the model control group, and between the model control group and CEFFE.

[0210] 2. Results

[0211] 2.1 CEFFE treatment alleviates the severity of liver lesions in model mice

[0212] The livers of animals euthanized on D27 after administration were observed under a microscope. Figure 1 As shown, from Figure 1As can be seen, the livers of mice in the model control, low-dose CEFFE, medium-dose CEFFE, and high-dose CEFFE groups all showed varying degrees of model-related hepatocyte steatosis, hepatocyte ballooning, and inflammatory cell infiltration, with hepatocyte ballooning being the predominant lesion. Compared with the model control group, the low-dose CEFFE group showed no significant changes in hepatocyte steatosis, inflammatory cell infiltration, or hepatocyte ballooning. However, the severity of hepatocyte steatosis, hepatocyte ballooning, and inflammatory cell infiltration was reduced in the medium- and high-dose CEFFE groups. NAS scores decreased with increasing CEFFE dose, indicating that CEFFE treatment reduced the severity of liver pathological changes in mice in a dose-dependent manner.

[0213] Figure 1 A. Normal control group, normal liver.

[0214] Figure 1 B model control group: liver hepatocyte fatty degeneration (++), hepatocyte ballooning degeneration (++) and inflammatory cell infiltration (++).

[0215] Figure 1 C. CEFFE low-dose group: hepatocyte fatty degeneration (++), hepatocyte ballooning degeneration (+++) and inflammatory cell infiltration (++).

[0216] Figure 1 D. CEFFE medium-dose group: hepatocyte fatty degeneration (+), hepatocyte ballooning degeneration (++) and inflammatory cell infiltration (+) in the liver.

[0217] Figure 1 E. CEFFE high-dose group: hepatocyte fatty degeneration (+), hepatocyte ballooning degeneration (+) and inflammatory cell infiltration (+) in the liver.

[0218] The summary of drug-related microscopic observation results of animals euthanized on Day 27 after drug administration is shown in Table 3:

[0219] Table 3 Summary of drug-related microscopic observation results of animals euthanized on Day 27 after drug administration

[0220]

[0221] The results of the NAS score of the liver of the animals euthanized on D27 after administration are shown in Table 4:

[0222] Table 4 Statistics of NAS scores of livers of animals euthanized on D27 after drug administration

[0223]

[0224] in conclusion

[0225] The results of this example show that CEFFE has an excellent therapeutic effect on non-alcoholic steatohepatitis.

[0226] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A use of a cell-free fat extract, characterized in that: For preparing a pharmaceutical composition for treating non-alcoholic steatohepatitis; Wherein, 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 nanofat; (5) subjecting the emulsified fat mixture to centrifugation, wherein the emulsified fat mixture is separated into four layers, the first layer being an oil layer, the second layer being a residual fat tissue layer, the third layer being an intermediate liquid layer, and the fourth layer being a cell / tissue debris sedimentation layer, and obtaining the intermediate liquid layer of the third layer, which is the primary fat extract; and (6) filtering and sterilizing the primary fat extract to obtain a cell-free fat extract; The cell-free fat extract contains bFGF, and the concentration of bFGF is 50-600 pg / ml.

2. The use according to claim 1, characterized in that The non-alcoholic steatohepatitis is selected from the group consisting of acute non-alcoholic steatohepatitis, chronic non-alcoholic steatohepatitis, or a combination thereof.

3. The use according to claim 1, characterized in that In step (5), the intermediate liquid layer is a transparent layer.

4. The use according to claim 1, wherein The acellular fat extract further contains one or more components selected from the group consisting of IGF-1, BDNF, GDNF, TGF-β, HGF, VEGF, TGF-β1, HGF, PDGF, EGF, NT-3, GH, G-CSF, or a combination thereof.

5. The use according to claim 4, characterized in that The cell-free fat extract comprises one or more characteristics selected from the group consisting of: In the cell-free fat extract, the concentration of IGF-1 is 5000-30000 pg / ml; In the cell-free fat extract, the concentration of BDNF is 800-5000 pg / ml; In the cell-free fat extract, the concentration of GDNF is 800-5000 pg / ml; In the cell-free fat extract, the concentration of bFGF is 50-600 pg / ml; In the cell-free fat extract, the concentration of VEGF is 50-500 pg / ml; In the cell-free fat extract, the concentration of TGF-β1 is 200-3000 pg / ml; In the cell-free fat extract, the concentration of HGF is 200-3000 pg / ml; In the cell-free fat extract, the concentration of PDGF is 50-600 pg / ml.

6. The use according to claim 5, characterized in that The cell-free fat extract comprises one or more characteristics selected from the group consisting of: In the cell-free fat extract, the concentration of IGF-1 is 6000-20000 pg / ml; In the cell-free fat extract, the concentration of BDNF is 1000-4000 pg / ml; In the cell-free fat extract, the concentration of GDNF is 1000-4000 pg / ml; In the cell-free fat extract, the concentration of bFGF is 100-500 pg / ml; In the cell-free fat extract, the concentration of VEGF is 100-400 pg / ml; In the cell-free fat extract, the concentration of TGF-β1 is 400-2000 pg / ml; In the cell-free fat extract, the concentration of HGF is 400-2000 pg / ml; In the cell-free fat extract, the concentration of PDGF is 80-400 pg / ml.

7. The use according to claim 5, characterized in that The cell-free fat extract comprises one or more characteristics selected from the group consisting of: In the cell-free fat extract, the concentration of IGF-1 is 7000-15000 pg / ml; In the cell-free fat extract, the concentration of BDNF is 1200-2500 pg / ml; In the cell-free fat extract, the concentration of GDNF is 1200-2500 pg / ml; In the cell-free fat extract, the concentration of bFGF is 120-400 pg / ml; In the cell-free fat extract, the concentration of VEGF is 120-300 pg / ml; In the cell-free fat extract, the concentration of TGF-β1 is 600-1500 pg / ml; In the cell-free fat extract, the concentration of HGF is 600-1500 pg / ml; In the cell-free fat extract, the concentration of PDGF is 100-300 pg / ml.

8. The use according to claim 4, characterized in that The cell-free fat extract comprises one or more characteristics selected from the group consisting of: The weight ratio of IGF-1 to VEGF is 20-100:1; The weight ratio of BDNF to VEGF is 2-20:1; The weight ratio of GDNF to VEGF is 2-20:1; The weight ratio of bFGF to VEGF is 0.2-8:1; The weight ratio of TGF-β1 to VEGF is 1-20:1; The weight ratio of HGF to VEGF is 1-20:1; The weight ratio of PDGF to VEGF is 0.1-3:

1.

9. The use according to claim 8, characterized in that The cell-free fat extract comprises one or more characteristics selected from the group consisting of: The weight ratio of IGF-1 to VEGF is 30-70:1; The weight ratio of BDNF to VEGF is 4-15:1; The weight ratio of GDNF to VEGF is 4-15:1; The weight ratio of bFGF to VEGF is 0.5-5:1; The weight ratio of TGF-β1 to VEGF is 1-10:1; The weight ratio of HGF to VEGF is 1-10:1; The weight ratio of PDGF to VEGF is 0.2-2:

1.

10. The use according to claim 9, characterized in that The cell-free fat extract comprises one or more characteristics selected from the group consisting of: The weight ratio of IGF-1 to VEGF is 40-60:1; The weight ratio of BDNF to VEGF is 6-12:1; The weight ratio of GDNF to VEGF is 6-12:1; The weight ratio of bFGF to VEGF is 0.6-2:1; The weight ratio of TGF-β1 to VEGF is 2-8:1; The weight ratio of HGF to VEGF is 2-8:1; The weight ratio of PDGF to VEGF is 0.4-1.5:

1.

11. The use according to claim 1, characterized in that The cell-free fat extract does not contain cells and does not contain lipid droplets. The term "does not contain lipid droplets" means that the volume of oil droplets in the cell-free fat extract accounts for less than 1% of the total liquid.

12. The use according to claim 1, wherein The cell-free fat extract is a naturally obtained nano fat extract without any added ingredients.

13. The use according to claim 1, wherein In the step (5), before the emulsified fat mixture is subjected to centrifugal treatment, the emulsified fat mixture is further subjected to freezing and then thawing treatment.

14. The use according to claim 1, wherein The dosage form of the pharmaceutical composition is an oral preparation, an external preparation or an injection preparation.

15. The use according to claim 14, characterized in that The injection preparation is an intravenous injection or an intramuscular injection.

16. The use according to claim 14, characterized in that The pharmaceutical composition is administered externally or by subcutaneous injection.

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