Therapeutic use of cell-free fat extract in arthritis

Abstract

The present invention relates to the therapeutic use of a cell-free fat extract for arthritis. Specifically, the present invention provides the use of a cell-free fat extract for preparing a composition or preparation for one or more uses selected from the following group: (i) preventing and / or treating arthritis; (ii) preventing and / or treating pain; (iii) movement disorders. The cell-free fat extract described in the present invention has excellent therapeutic effects on arthritis and its symptoms of pain and movement disorders.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and particularly to the therapeutic use of cell-free fat extract for arthritis. Background Art

[0002] Arthritis is an inflammatory disease that occurs in the joints and their surrounding tissues of the human body, caused by inflammation, infection, degeneration, trauma or other factors. The clinical manifestations include joint redness, swelling, heat, pain, dysfunction and joint deformity. In severe cases, it can lead to joint disability and affect the quality of life of patients.

[0003] Osteoarthritis (OA) is a type of arthritis that seriously affects the health of patients. Osteoarthritis is a cartilage degenerative disease that starts from articular cartilage and gradually erodes to subchondral bone and surrounding tissues, resulting in focal and erosive joint lesions, thus causing joint pain, movement disorders and deformity. The incidence of OA increases with age and has become the leading cause of disability in middle-aged and elderly people, seriously affecting the work and life of patients. Current treatment methods include conservative treatment and surgical treatment. Intra-articular drug injection, such as injection of sodium hyaluronate, glucocorticoids, etc., in the early stage of the disease course is the mainstream conservative treatment method for relieving OA symptoms. However, there is still a lack of ideal therapeutic drugs, and finding safe and effective biological agents has become an urgent problem to be solved.

[0004] Therefore, there is a need in the art to develop a drug that can effectively treat arthritis. Summary of the Invention

[0005] The object of the present invention is to provide the use of cell-free fat extract in the prevention and / or treatment of arthritis.

[0006] In the first aspect of the present invention, there is provided the use of a cell-free fat extract for preparing a composition or preparation, and the composition or preparation is used for one or more of the following uses: (i) preventing and / or treating arthritis; (ii) preventing and / or treating pain; (iii) movement disorder.

[0007] In another preferred embodiment, the arthritis includes osteoarthritis.

[0008] In another preferred embodiment, the arthritis includes drug-induced osteoarthritis.

[0009] In another preferred embodiment, the arthritis includes degenerative osteoarthritis.

[0010] In another preferred embodiment, the arthritis includes cartilage degenerative osteoarthritis. In another preferred embodiment, the osteoarthritis includes arthritis at weight-bearing joints and joints with more activity.

[0011] In another preferred embodiment, the osteoarthritis is selected from the group consisting of: cervical osteoarthritis, lumbar osteoarthritis, knee osteoarthritis, hip osteoarthritis, or a combination thereof.

[0012] In another preferred embodiment, the osteoarthritis includes knee osteoarthritis. Knee osteoarthritis is a chronic osteoarthropathy mainly characterized by degenerative changes of knee joint cartilage.

[0013] In another preferred embodiment, the pain includes arthritic pain.

[0014] In another preferred embodiment, the activity disorder includes the activity disorder caused by arthritis.

[0015] In another preferred embodiment, the prevention and / or treatment of arthritis is carried out by one or more of the following methods:

[0016] (a) inhibiting joint cartilage fibrosis; and / or

[0017] (b) improving the number of articular chondrocytes.

[0018] In another preferred embodiment, the improvement includes an increase.

[0019] In another preferred embodiment, the acellular fat extract is an acellular fat extract prepared by extracting from fat in human or non-human mammals.

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

[0021] 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.

[0022] In another preferred embodiment, the composition or preparation further includes a pharmaceutically, food-wise, health product or dietarily acceptable carrier.

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

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

[0025] In another preferred embodiment, the dosage form of the composition or preparation is a solid dosage form, a semi-solid dosage form, or a liquid dosage form, such as a solution, gel, cream, lotion, ointment, paste, cake, powder, patch, etc.

[0026] In another preferred embodiment, the dosage form of the composition or preparation is a powder, granule, capsule, injection, tincture, oral liquid, tablet or lozenge.

[0027] In another preferred embodiment, the composition or preparation is administered by topical, local, or subcutaneous injection.

[0028] In another preferred embodiment, the cell-free fat extract does not contain cells and does not contain lipid droplets.

[0029] In another preferred embodiment, the lipid droplets are oil droplets released after the fragmentation of adipocytes.

[0030] In another preferred embodiment, the "does not contain lipid droplets" means that in the cell-free fat extract, the volume percentage of oil droplets in the total liquid is less than 1%, preferably less than 0.5%, more preferably less than 0.1%.

[0031] In another preferred embodiment, the cells are selected from the group consisting of: endothelial cells, adipose stem cells, macrophage blood cells, stromal cells.

[0032] 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.

[0033] In another preferred embodiment, the cell-free fat extract is a naturally obtained nano-fat extract without added ingredients.

[0034] In another preferred embodiment, the "without added ingredients" means that except for the rinsing step, no solutions, solvents, small molecules, chemical agents, and biological additives are added during the preparation of the fat extract.

[0035] In another preferred embodiment, the cell-free fat extract is prepared by emulsifying adipose tissue and then centrifuging.

[0036] In another preferred embodiment, the cell-free fat extract contains one or more components selected from the group consisting of: IGF-1, BDNF, GDNF, TGF-β1, HGF, bFGF, VEGF, TGF-β1, PDGF, EGF, NT-3, GH, G-CSF, or a combination thereof.

[0037] In another preferred embodiment, the cell-free fat extract contains, but is not limited to, one or more components selected from the group consisting of: IGF-1, BDNF, GDNF, bFGF, VEGF, TGF-β1, HGF, PDGF, or a combination thereof.

[0038] In another preferred embodiment, the cell-free fat extract is a cell-free fat extract solution.

[0039] In another preferred example, in the acellular 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 most preferably 9500 - 10500 pg / ml.

[0040] In another preferred example, in the acellular 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, and most preferably 1700 - 1850 pg / ml.

[0041] In another preferred example, in the acellular 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, and most preferably 1700 - 1900 pg / ml.

[0042] In another preferred example, in the acellular 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, and most preferably 220 - 260 pg / ml.

[0043] In another preferred example, in the acellular 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, and most preferably 190 - 210 pg / ml.

[0044] In another preferred example, in the acellular 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, and most preferably 900 - 1000 pg / ml.

[0045] In another preferred example, in the acellular fat extract, the concentration of the 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, and most preferably 850 - 950 pg / ml.

[0046] In another preferred example, in the acellular fat extract, the concentration of the 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, and most preferably 170 - 190 pg / ml.

[0047] In another preferred example, 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.

[0048] In another preferred example, 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.

[0049] In another preferred example, 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.

[0050] In another preferred example, 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.

[0051] In another preferred example, 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.

[0052] In another preferred example, 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.

[0053] In another preferred example, 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.

[0054] In another preferred example, the acellular fat extract is prepared by the following method:

[0055] (1) Provide a raw material of adipose tissue, crush the raw material of adipose tissue, and perform rinsing (such as with physiological saline), so as to obtain the rinsed adipose tissue;

[0056] (2) Centrifuge the rinsed adipose tissue to obtain a stratified mixture;

[0057] (3) For the stratified mixture, remove the upper oil layer and the lower water layer, and collect the middle layer (i.e., the fat layer containing adipocytes);

[0058] (4) Emulsify the middle layer to obtain an emulsified fat mixture (also called nano fat);

[0059] (5) Centrifuge the emulsified fat mixture to obtain an intermediate liquid layer, which is the crude fat extract; and

[0060] (6) Filter and sterilize the crude fat extract to obtain the acellular fat extract.

[0061] In the second aspect of the present invention, there is provided a method for preparing an acellular fat extract, and the method includes the steps:

[0062] (1) Provide a raw material of adipose tissue, crush the raw material of adipose tissue, and perform rinsing (such as with physiological saline), so as to obtain the rinsed adipose tissue;

[0063] (2) Centrifuge the rinsed adipose tissue to obtain a stratified mixture;

[0064] (3) For the stratified mixture, remove the upper oil layer and the lower water layer, and collect the middle layer (i.e., the fat layer containing adipocytes);

[0065] (4) Emulsify the middle layer to obtain an emulsified fat mixture (also called nano fat);

[0066] (5) Centrifuge the emulsified fat mixture to obtain an intermediate liquid layer, which is the crude fat extract; and

[0067] (6) Filter and sterilize the crude fat extract to obtain the acellular fat extract.

[0068] In another preferred example, the acellular fat extract is as described in the first aspect of the present invention.

[0069] In another preferred embodiment, in step (2), the centrifugation is carried out at 800 - 2500 g, preferably 800 - 2000 g, more preferably 1000 - 1500 g, and most preferably 1100 - 1300 g.

[0070] In another preferred embodiment, in step (2), the centrifugation time is 1 - 15 min, preferably 1 - 10 min, more preferably 1 - 8 min, and most preferably 1 - 5 min.

[0071] In another preferred embodiment, the temperature of the centrifugation is 2 - 6 °C.

[0072] In another preferred embodiment, in step (4), the emulsification is mechanical emulsification.

[0073] In another preferred embodiment, the mechanical emulsification is carried out by repeatedly pipetting with a syringe (such as pipetting 20 - 200 times, preferably 20 - 150 times, more preferably 20 - 100 times, and more preferably 30 - 50 times).

[0074] In another preferred embodiment, the pipetting method is to repeatedly and uniformly push and pull with two 10 - ml syringe barrels connected by a three - way tube.

[0075] In another preferred embodiment, in step (4), the emulsification is by the method of disrupting with a tissue homogenizer.

[0076] In another preferred embodiment, in step (5), before subjecting the emulsified fat mixture to centrifugation, it further includes freezing and then thawing the emulsified fat mixture.

[0077] In another preferred embodiment, after the freezing - and - thawing treatment, the thawed mixture is used for centrifugation.

[0078] 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.

[0079] In another preferred embodiment, the thawing temperature is 20 - 40 °C, preferably 25 - 40 °C, and more preferably 37 °C.

[0080] In another preferred embodiment, the number of freezing - and - thawing cycles is 1 - 5 times (preferably 1, 2, 3, or 4 times).

[0081] In another preferred embodiment, in step (5), after centrifugation, the emulsified fat mixture is layered into 4 layers. The first layer is the oil layer, the second layer is the residual fat tissue layer, the third layer is the liquid layer (i.e., the middle liquid layer), and the fourth layer is the cell / tissue debris precipitation layer.

[0082] In another preferred embodiment, in the step (5), the centrifugation is carried out at 800 - 2500 g, preferably 800 - 2000 g, more preferably 1000 - 1500 g, and most preferably 1100 - 1300 g.

[0083] 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.

[0084] In another preferred embodiment, the temperature of the centrifugation is 2 - 6 °C.

[0085] 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.

[0086] In another preferred embodiment, in the step (5), the intermediate liquid layer is a transparent or substantially transparent layer.

[0087] In another preferred embodiment, in the step (6), the filter pack can remove fat cells from the crude fat extract.

[0088] In another preferred embodiment, in the step (6), the filtration and sterilization are carried out through a filter (such as a 0.22 μm microporous membrane).

[0089] In another preferred embodiment, the filter is a microporous membrane filter.

[0090] In another preferred embodiment, the pore size of the microporous 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.

[0091] In another preferred embodiment, in the step (6), the filtration and sterilization are carried out first 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).

[0092] In another preferred embodiment, in the step (6), it further includes sub-packaging the fat extract to form a sub-packaged product. (The sub-packaged extract can be stored at -20 °C for future use; it can be directly used after thawing at low temperature (such as -4 °C) or at room temperature, or stored at low temperature (such as 4 °C) for a period of time after thawing and then used).

[0093] In the third aspect of the present invention, there is provided a cell-free fat extract, which is obtained by the method as described in the second aspect of the present invention.

[0094] In a fourth aspect of the present invention, there is provided a composition or preparation, which comprises (a) a 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.

[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 dosage form of the composition or preparation is an oral preparation, a topical preparation or an injection preparation.

[0097] In another preferred embodiment, the dosage form of the composition or preparation is powder, granule, capsule, injection, tincture, oral liquid, tablet or buccal tablet.

[0098] In another preferred embodiment, the injection is an intravenous injection or an intramuscular injection.

[0099] In another preferred embodiment, the dosage form of the composition or preparation is a solid dosage form, a semi-solid dosage form or a liquid dosage form, such as solution, gel, cream, emulsion, paste, ointment, cream, paste, cake, powder, patch, etc.

[0100] 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.

[0101] In a fifth aspect of the present invention, there is provided a method for preparing the composition or preparation as described in the fourth aspect of the present invention, the method comprising the step 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 the composition or preparation.

[0102] In a sixth aspect of the present invention, there is provided a method for (i) preventing and / or treating arthritis; (ii) preventing and / or treating pain; and / or (iii) treating movement disorders, by administering to a subject in need the cell-free fat extract as described in the third aspect of the present invention.

[0103] In another preferred embodiment, the subject is a human or a non-human mammal.

[0104] In another preferred embodiment, the non-human mammal includes rodents, such as rats and mice.

[0105] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] Figure 1 Changes in the body weights of animals in each group over time after drug administration.

[0107] Figure 2 The paw withdrawal pressure values of rats before and after establishing the model.

[0108] Figure 3 The paw withdrawal pressure values of rats in each group before and after drug administration.

[0109] Figure 4 The absolute value of the difference in the pressure of both feet of rats before and after establishing the model.

[0110] Figure 5 The absolute value of the difference in the pressure of both feet of rats in each group before and after drug administration.

[0111] Figure 6 The HE staining results (200×) of rats in different groups.

[0112] Figure 7 The Safranin O-fast green staining results (200×) of rats in different groups. Detailed implementation mode

[0113] Through extensive and in-depth research, the present inventor has developed for the first time that cell-free fat extract has excellent therapeutic effects on arthritis and its symptoms of pain and movement disorders. Based on this, the present invention has been completed.

[0114] Terms

[0115] Unless otherwise defined, the meanings of all technical and scientific terms used herein are the same as those commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0116] As used herein, the terms "comprising", "including" and "containing" can be used interchangeably, and include not only open definitions, but also semi-closed and closed definitions. In other words, the said terms include "consisting of" and "consisting essentially of".

[0117] As used herein, the terms "cell-free fat extract", "Cell free fat extract" and "CEFFE" can be used interchangeably.

[0118] 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. "Prevention" as used herein also includes delaying the onset of a disease and / or its attendant symptoms and reducing the risk of the subject getting the disease.

[0119] "Treatment" as used herein includes delaying and arresting the progression of a disease, or eliminating the disease, and does not require 100% inhibition, eradication, and reversal. In some embodiments, the compositions or pharmaceutical compositions of the present invention alleviate, inhibit, and / or reverse diabetes, 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 compositions, kits, food kits, or health care product kits, or combinations of active ingredients of the present invention.

[0120] As used herein, "ameliorate" includes prevent, treat, relieve, reverse, and mitigate, and the like.

[0121] As used herein, the term "IGF-1" refers to insulin-like growth factors-1.

[0122] As used herein, the term "BDNF" refers to brain-derived neurotrophic factor (BDNF).

[0123] As used herein, the term "GDNF" refers to glial cell line-derived neurotrophic factor.

[0124] As used herein, the term "bFGF" refers to basic fibroblast growth factor.

[0125] As used herein, the term "VEGF" refers to vascular endothelial growth factor.

[0126] As used herein, the term "TGF-β1" refers to transforming growth factor-β1.

[0127] As used herein, the term "HGF" refers to hepatocyte growth factor

[0128] As used herein, the term "PDGF" refers to Platelet derived growth factor

[0129] As used herein, the term "EGF" refers to Epidermal Growth Factor

[0130] As used in the text, the term "NT-3" refers to neurotrophins-3.

[0131] As used in the text, the term "GH" refers to Growth Hormone.

[0132] As used in the text, the term "G-CSF" refers to granulocyte colony stimulating factor.

[0133] Cell free fat extract (CEFFE) and its preparation method

[0134] As used herein, the terms "cell free 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 extract solution) derived from adipose tissue prepared without adding any solution, solvent, small molecule, chemical agent, and biological additive during the preparation process of the fat extract (except for the rinsing step). A typical method for preparing the extract of the present invention is as described in the second aspect of the present invention above. In addition, it should be understood that although no additives (or added components) need to be added during the preparation of the extract of the present invention, some or a small amount of safe substances that have no negative or adverse effects on the activity of the extract of the present invention (such as a small amount of water) can also be added.

[0135] In a preferred embodiment of the present invention, the cell free fat extract is a cell free fat extract solution.

[0136] The cell free fat extract of the present invention may include a variety of cytokines. Representatively, the cell free fat 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.

[0137] 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, more preferably 9500 - 10500 pg / ml.

[0138] In another preferred example, in the acellular adipose extract, the concentration of BDNF is 800 - 5000 pg / ml, preferably 1000 - 4000 pg / ml, more preferably 1200 - 2500 pg / ml, still more preferably 1400 - 2000 pg / ml, even more preferably 1600 - 2000 pg / ml, and most preferably 1700 - 1850 pg / ml.

[0139] In another preferred example, in the acellular adipose extract, the concentration of GDNF is 800 - 5000 pg / ml, preferably 1000 - 4000 pg / ml, more preferably 1200 - 2500 pg / ml, still more preferably 1400 - 2000 pg / ml, even more preferably 1600 - 2000 pg / ml, and most preferably 1700 - 1900 pg / ml.

[0140] In another preferred example, in the acellular adipose extract, the concentration of bFGF is 50 - 600 pg / ml, preferably 100 - 500 pg / ml, more preferably 120 - 400 pg / ml, still more preferably 150 - 300 pg / ml, even more preferably 200 - 280 pg / ml, and most preferably 220 - 260 pg / ml.

[0141] In another preferred example, in the acellular adipose extract, the concentration of VEGF is 50 - 500 pg / ml, preferably 100 - 400 pg / ml, more preferably 120 - 300 pg / ml, still more preferably 150 - 250 pg / ml, even more preferably 170 - 230 pg / ml, and most preferably 190 - 210 pg / ml.

[0142] In another preferred example, in the acellular adipose extract, the concentration of TGF-β1 is 200 - 3000 pg / ml, preferably 400 - 2000 pg / ml, more preferably 600 - 1500 pg / ml, still more preferably 800 - 1200 pg / ml, even more preferably 800 - 1100 pg / ml, and most preferably 900 - 1000 pg / ml.

[0143] In another preferred example, in the acellular adipose extract, the concentration of HGF is 200 - 3000 pg / ml, preferably 400 - 2000 pg / ml, more preferably 600 - 1500 pg / ml, still more preferably 600 - 1200 pg / ml, even more preferably 800 - 1000 pg / ml, and most preferably 850 - 950 pg / ml.

[0144] In another preferred embodiment, in the acellular adipose 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] Preferably, the acellular adipose extract of the present invention is obtained by the method described in the second aspect of the present invention as above.

[0153] Typically, the acellular adipose extract of the present invention is prepared by the following method:

[0154] (1) Provide a raw material of adipose tissue, crush the raw material of adipose tissue, and perform rinsing (such as with physiological saline) to obtain rinsed adipose tissue;

[0155] (2) Centrifuge the rinsed adipose tissue to obtain a stratified mixture;

[0156] (3) For the stratified mixture, remove the upper oil layer and the lower water layer, and collect the middle layer (i.e., the adipose layer containing adipocytes);

[0157] (4) Emulsify the middle layer to obtain an emulsified adipose mixture (also known as nano-fat);

[0158] (5) Centrifuge the emulsified adipose mixture to obtain an intermediate liquid layer, which is the crude adipose extract; and

[0159] (6) Filter and sterilize the crude adipose extract to obtain a cell-free adipose extract.

[0160] Specifically, the cell-free adipose extract of the present invention

[0161] In another preferred example, in step (2), the centrifugation is carried out at 800 - 2500 g, preferably 800 - 2000 g, more preferably 1000 - 1500 g, and most preferably 1100 - 1300 g.

[0162] In another preferred example, in step (2), the centrifugation time is 1 - 15 min, preferably 1 - 10 min, more preferably 1 - 8 min, and most preferably 1 - 5 min.

[0163] In another preferred example, in step (4), the emulsification is mechanical emulsification.

[0164] In another preferred example, the mechanical emulsification is carried out by repeatedly pipetting with a syringe (such as pipetting 20 - 200 times, preferably 20 - 150 times, more preferably 20 - 100 times, and more preferably 30 - 50 times).

[0165] In another preferred example, the pipetting method is to repeatedly and uniformly push and pull with two 10-ml syringe barrels connected by a three-way tube.

[0166] In another preferred example, in step (4), the emulsification is by the method of crushing with a tissue homogenizer.

[0167] In another preferred example, in step (5), before centrifuging the emulsified adipose mixture, it further includes freezing and then thawing the emulsified adipose mixture.

[0168] In another preferred example, after the freezing and thawing treatment, the thawed mixture is used for centrifugation.

[0169] In another preferred example, the freezing temperature is -50°C to -120°C, preferably -60°C to -100°C, more preferably -70°C to -90°C.

[0170] In another preferred example, the thawing temperature is 20 - 40°C, preferably 25 - 40°C, more preferably 37°C.

[0171] In another preferred example, the number of freeze-thaw cycles is 1 - 5 times (preferably 1, 2, 3, or 4 times).

[0172] In another preferred example, in step (5), after centrifugation, the emulsified fat mixture is stratified into 4 layers. The first layer is the oil layer, the second layer is the residual fat tissue layer, the third layer is the liquid layer (i.e., the middle liquid layer), and the fourth layer is the cell / tissue debris precipitation layer.

[0173] In another preferred example, in step (5), the centrifugation is carried out at 800 - 2500g, preferably 800 - 2000g, more preferably 1000 - 1500g, and most preferably 1100 - 1300g.

[0174] In another preferred example, in step (5), the centrifugation time is 1 - 15 min, preferably 1 - 10 min, more preferably 2 - 8 min, and most preferably 3 - 7 min.

[0175] In another preferred example, in step (5), the first layer, the second layer, the third layer, and the fourth layer are arranged in sequence from top to bottom.

[0176] In another preferred example, in step (5), the middle liquid layer is a transparent or substantially transparent layer.

[0177] In another preferred example, in step (6), the filter package can remove fat cells from the crude fat extract.

[0178] In another preferred example, in step (6), the filtration and sterilization are carried out through a filter (such as a 0.22μm microporous membrane).

[0179] In another preferred example, the filter is a microporous membrane filter.

[0180] In another preferred example, the pore size of the microporous membrane is 0.05 - 0.8μm, preferably 0.1 - 0.5μm, more preferably 0.1 - 0.4μm, more preferably 0.15 - 0.3μm, more preferably 0.2 - 0.25μm, and most preferably 0.22μm.

[0181] In another preferred example, in step (6), the filtration and sterilization are carried out by first passing 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).

[0182] In another preferred example, in step (6), it further includes packaging the fat extract to form a packaged product. (The packaged extract can be stored at -20°C for future use; it can be directly used after thawing at low temperature (such as -4°C) or at room temperature, or stored at low temperature (such as 4°C) for a period of time after thawing and then used).

[0183] Arthritis

[0184] Arthritis refers to an inflammatory disease that occurs in the joints and their surrounding tissues of the human body, caused by inflammation, infection, degeneration, trauma or other factors. The clinical manifestations are redness, swelling, heat, pain, dysfunction and joint deformity of the joints. In severe cases, it leads to joint disability and affects the quality of life of patients.

[0185] Typically, the arthritis described in the present invention is osteoarthritis.

[0186] Osteoarthritis and its symptoms

[0187] Osteoarthritis (OA) is a cartilage degenerative disease that starts from articular cartilage and gradually erodes to subchondral bone and surrounding tissues, leading to focal and erosive joint lesions, thus causing symptoms such as joint pain, joint stiffness, joint swelling, movement disorders and deformity.

[0188] In the present invention, there are many inducing factors for the osteoarthritis, and the etiology is not yet fully clear. It may be related to factors such as advanced age, obesity, drugs, occupational overuse, etc.

[0189] In the present invention, the occurrence site of the osteoarthritis is not particularly limited. For example, it can be weight-bearing joints and joints with more activity, such as arthritis in the cervical vertebrae, lumbar vertebrae, knee joints, hip joints and other parts.

[0190] Compositions and administrations

[0191] The compositions described in the present invention include (but are not limited to): pharmaceutical compositions, food compositions, health care compositions, dietary supplements, etc.

[0192] Typically, the cell-free fat extract of the present invention can be prepared into a pharmaceutical composition, such as dosage forms like tablets, capsules, powders, microparticles, solutions, lozenges, gels, cream preparations, spirits, suspensions, tinctures, poultices, liniments, lotions, and aerosols. The pharmaceutical composition can be prepared by commonly known preparation techniques, and suitable pharmaceutical additives can be added to the drug.

[0193] 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 gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatible" herein 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 product, or dietary acceptable carriers are cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, 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 wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0194] There is no particular limitation on the administration mode of the composition of the present invention. Representative administration modes include (but are not limited to): oral, parenteral (intravenous, intramuscular), topical administration, and the preferred administration modes are oral administration and injection administration.

[0195] The dosage form of the composition or preparation described in the present invention is an oral preparation, an external preparation or an injection preparation. Representatively, solid dosage forms for oral administration or dosing 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 dibasic calcium phosphate, or is mixed with the following components: (a) fillers or bulking agents, such as starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, such as hydroxypropylmethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (c) humectants, such as glycerol; (d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) dissolution retardants, such as paraffin wax; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain buffering agents.

[0196] Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and casings, such as enteric coatings and other materials well known in the art. They may contain opacifying agents.

[0197] Liquid dosage forms for oral administration or dosing include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3 - butanediol, dimethylformamide, and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, or mixtures of these substances.

[0198] In addition to these inert diluents, the composition may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweetening agents, flavoring agents and fragrances.

[0199] In addition to the active ingredient, the suspension may contain suspending agents, such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum monostearate and agar, or mixtures of these substances.

[0200] The composition for parenteral injection may contain physiologically acceptable sterile aqueous or non - aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for re - dissolving into sterile injectable solutions or dispersions. Suitable aqueous and non - aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0201] Dosage forms of the compounds of the present invention for topical administration or drug delivery include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required if necessary.

[0202] The cell-free fat extract of the present invention can be administered or dosed alone, or in combination with other drugs for preventing and / or treating fatty liver and / or its complications.

[0203] When administering the composition, a safe and effective amount of the cell-free fat extract of the present invention is applied to a human or non-human animal (such as rats, mice, dogs, cats, cows, chickens, ducks, etc.) in need of treatment, and the dosage during administration is an effective dosage acceptable in pharmacy, food or health products. As used herein, the term "safe and effective amount" refers to an amount that produces a function or activity in humans and / or animals and is acceptable to humans and / or animals. Those of ordinary skill in the art should understand that the "safe and effective amount" may vary depending on the form of the pharmaceutical composition, the route of administration, the excipients of the drug used, the severity of the disease, and the combination with other drugs. For example, for a person weighing 60 kg, the daily dosage is usually 0.1-1000 mg, preferably 1-600 mg, more preferably 2-300 mg. Of course, the specific dosage should also consider factors such as the route of administration and the health status of the patient, which are within the scope of the skills of a skilled physician.

[0204] The main advantages of the present invention include:

[0205] The present invention discovers for the first time that the cell-free fat extract has excellent therapeutic effects on arthritis and its symptoms of pain and movement disorders.

[0206] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0207] Example 1

[0208] 1. Experimental method

[0209] 1.1. Preparation of cell-free fat extract (CEFFE)

[0210] Fat was obtained from volunteers under the condition of obtaining informed consent. The preparation method of the cell-free adipose tissue extract is as follows:

[0211] (1) Adipose tissue was obtained from 6 healthy women who underwent conventional liposuction, with an average age of 31 years (24 - 36 years). After local injection of tumescent fluid for anesthesia, a 3mm liposuction cannula with large side holes (2mm x 7mm) was connected to a 20mL syringe, and radial aspiration was performed under artificial negative pressure. The obtained fat was allowed to stand upright and still. After removing the tumescent fluid, it was rinsed 3 times with normal saline.

[0212] (2) The rinsed adipose tissue was placed in a centrifuge tube and centrifuged in a centrifuge at 1200g for 3 minutes at 4°C to obtain a stratified mixture.

[0213] (3) For the stratified mixture, the upper oil layer and the lower water layer were removed, and the middle layer (i.e., the fat layer containing adipocytes) was collected.

[0214] (4) For the middle layer, two 10ml syringe barrels were connected to a three-way tube and repeatedly pushed and pulled evenly 30 times to perform mechanical emulsification, and a mechanically emulsified fat mixture (also known as nano-fat) was obtained.

[0215] (5) The mechanically emulsified fat mixture was placed in an -80°C refrigerator for freezing, and then thawed in a 37°C water bath. After a single freeze-thaw cycle, the thawed fat mixture was centrifuged at 1200g for 5 minutes at 4°C to obtain a stratified mixture. The stratified mixture was divided into 4 layers. The first layer was the oil layer, the second layer was the residual adipose tissue layer, the third layer was the liquid layer, and the fourth layer was the cell / tissue debris precipitation layer. The oil layer and the residual adipose tissue layer were removed, and the liquid layer was aspirated, and contamination from the cell / tissue debris precipitation layer was avoided during the aspiration process, thus obtaining a crude fat extract.

[0216] (6) The obtained crude fat extract was filtered and sterilized through a 0.22μm filter to sterilize and remove any potentially mixed live cells, thus obtaining a cell-free fat extract (CEFFE). It was aliquoted and stored frozen at -20°C, and thawed at 4°C when in use.

[0217] For the prepared cell-free fat extract, an ELISA immunosorbent assay kit was used to detect the cytokine content, including cytokines such as IGF-1, BDNF, GDNF, bFGF, VEGF, TGF-β1, HGF, and PDGF. The average concentrations detected in 6 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).

[0218] 1.2 Establishment, grouping, and administration of the rat osteoarthritis (OA) model

[0219] Sodium iodoacetate (MIA) is one of the compounds used to induce the osteoarthritis (OA) model. Its mechanism of action is to promote the production of reactive oxygen species, induce mitochondrial membrane depolarization, further increase the release of cytochrome C, activate the activity of Caspase3, and lead to chondrocyte apoptosis.

[0220] In this study, the model was established by injecting MIA into the knee joint cavity. The experimental animals were 8-week-old male SD rats. Six rats were randomly selected as the normal control group, and the other rats were used for OA model establishment. After anesthesia with isoflurane, the rats in the model group were injected with 50 μL of 40 mg / mL MIA solution into the left hind limb joint cavity. One week after modeling, 24 rats with significant increases in the bilateral foot pressure difference and significant decreases in the left hind foot VonFrey value were selected and randomly divided into 4 groups of 6 rats each. The dosing regimens for the normal control group and the rats after modeling were as follows:

[0221] Table 1 Dosing regimens for rats in different groups

[0222]

[0223] Note: The rats in the normal control group were not treated throughout the experiment; the rats in the model control group were given 0.9% sodium chloride injection, and the rats in the low-dose CEFFE group, medium-dose CEFFE group, and high-dose CEFFE group were given different doses of cell-free fat extract (CEFFE). The rats in the model control group, low-dose CEFFE group, medium-dose CEFFE group, and high-dose CEFFE group were all administered into the left joint cavity once every 2 weeks (a total of 4 administrations), and the day of administration was designated as Day1 (counted as the 1st week).

[0224] 1.3 Von Frey test

[0225] Detection index: Paw withdrawal threshold (g).

[0226] Detection time: Once before modeling, 1 week after modeling, and 1 week after each administration.

[0227] Detection method: Place the animal in the test container and let it adapt to the environment for about 10 minutes. Measure the pressure of the left paw retraction of the animal (when the pressure reaches the maximum value of 50 g and the latency lasts until 40 s, and the animal still does not show a paw retraction reaction, to avoid causing tissue damage, manually end the measurement. The paw retraction latency and pressure value are recorded as 40 s and 50 g respectively). Measure each paw 2 - 3 times at intervals of 1 - 3 minutes, and take the average of the two results with similar values. (If the measurement process stops due to the spontaneous movement of the animal, it needs to be re-measured after an interval of 1 - 3 minutes. If it is uncertain, multiple measurements can be taken to calculate the average value to ensure that the paw retraction of the animal is caused by mechanical stimulation).

[0228] Instrument used: Dynamic plantar tactile stimulator (Model: 37450; Manufacturer: Ugo Basile).

[0229] 1.4 Bipedal balance measurement

[0230] Detection index: Bipedal pressure difference (g).

[0231] Detection time: Once before modeling, 1 week after modeling, and 1 week after each administration.

[0232] Detection method: Place the animal in the container. Wait until the animal calms down. When the left and right feet are in the corresponding sensing areas and the numbers showing the weight bearing of the left and right feet remain relatively stable (at least unchanged for 3 s), record the data.

[0233] Instrument used: Bipedal balance tester (Weight bearing asymmetry) (Model: 600MR; Manufacturer:

[0234] IITC life science).

[0235] 1.5 Histopathological examination

[0236] One week after the end of the last administration and after the behavioral detection, euthanize the animals. Rinse the left joint tissue with normal saline, place it in 10% neutral formalin solution for fixation, decalcify and dehydrate, and section (cut 2 slices along the cartilage surface, 5 μm per slice). One slice is stained with HE, and the other slice is stained with Safranin O-fast green staining.

[0237] The HE staining uses a conventional 4 - grading method to grade the microscopic results, which are slightly (+), mildly (++), moderately (+++), and severely (++++), for easy comparison between groups. For the Safranin O-fast green staining method, randomly select 3 fields of view on each slide for scoring. The scoring criteria are shown in Table 2 below:

[0238] Table 2 Scoring criteria for Safranin O-fast green staining method

[0239]

[0240] 1.6 Data statistical analysis

[0241] Measurements are expressed as mean ± standard deviation. All data statistics are performed using SPSS 13.0 statistical software. Homogeneity of variance test is conducted on all data. For data with homogeneity of variance (P>0.05), one-way analysis of variance is performed. For data with differences (P≤0.05), LSD multiple comparison analysis is carried out, and P≤0.05 is considered to have statistical differences; for data with inhomogeneity of variance (P≤0.05), Kruskal-Wallis non-parametric test is performed. For data with differences (P≤0.05), Mann-Whitney pairwise analysis and comparison are carried out, and P≤0.05 is considered to have statistical differences.

[0242] 2 Results

[0243] 2.1 CEFFE treatment had no obvious changes in the general condition of model rats

[0244] After administration, no deaths or near-death situations were observed in each group of animals. At the time of euthanasia at the end of the experiment (Day 54), no obvious abnormalities were observed in gross observation. Before administration, the body weights of each group of animals were similar; after administration, the body weights of each group of animals increased with time, and the body weights at each time point were similar. The changes in body weights of each group are shown in Figure 1 .

[0245] 2.2 CEFFE treatment significantly increased the mechanical pain threshold of model rats

[0246] The changes in the paw withdrawal pressure values of animals before and after modeling are as shown in Figure 2 . From Figure 2 , it can be seen that before modeling, the paw withdrawal pressure values of the model group and the normal control group rats were similar (28.1±5.0 vs 27.8±6.2 g, P>0.05). One week after MIA modeling, the paw withdrawal pressure value of the model group was significantly lower than that of the normal control group (14.2±3.7 vs 32.1±4.1 g, P<0.001), indicating that MIA successfully induced an osteoarthritis model in SD rats.

[0247] The paw withdrawal pressure values of each group of arthritis model rats before and after administration are as shown in Figure 3 . From Figure 3It can be seen that before drug administration, the paw withdrawal pressure values of the rats in the model control group and each dose group of CEFFE were similar, and were significantly lower than those in the normal control group (P<0.001). During the experiment, the paw withdrawal pressure values of the rats in the normal control group fluctuated between 30.0±4.9 and 44.5±5.2 g. After drug administration, the paw withdrawal pressure values of the model control group fluctuated less and were significantly lower than those in the normal control group at each time point (P<0.001). The paw withdrawal pressure values of the low-dose CEFFE group were similar to those of the model control group at each time point except at 1 week after the fourth drug administration (i.e., the eighth week), when they were significantly higher than those of the model control group (26.2±7.5 vs 15.9±4.7 g, P<0.01). The paw withdrawal pressure values of the medium-dose CEFFE group were similar to those of the model control group at each time point except at 1 week after the fourth drug administration (i.e., the eighth week), when they were significantly higher than those of the model control group (25.3±4.3 vs 15.9±4.7 g, P<0.01). The paw withdrawal pressure values of the high-dose CEFFE group were significantly higher than those of the model control group from 1 week after the first drug administration (i.e., the second week) to the end of the experiment (1 week after the fourth drug administration) (P<0.05 - P<0.01). The changing trends of the paw withdrawal pressure values of the animals in each group before and after drug administration are shown in Figure 3

[0248] Therefore, from Figure 3 it can be seen that CEFFE has excellent therapeutic effects on the pain of osteoarthritis.

[0249] 2.3 CEFFE treatment significantly reduces the difference in hind limb body weight distribution and alleviates the symptoms of model rats

[0250] The absolute value of the pressure difference between the two feet of the rats before and after modeling is as Figure 4 shown. From Figure 4 it can be seen that before modeling, the absolute values of the pressure differences between the two feet of the modeling group and the normal control group were similar (8±5 vs 9±7 g, P>0.05). One week after MIA modeling, the absolute value of the pressure difference between the two feet of the modeling group was significantly higher than that of the normal control group (66±19 vs 8±5 g, P<0.001), indicating that MIA successfully induced an osteoarthritis model. The changes in the absolute values of the pressure differences between the two feet of the animals before and after modeling are shown in Figure 4 .

[0251] Before drug administration, the absolute values of the pressure differences between the two feet of the model control group and each group of CEFFE were similar and were significantly higher than those of the normal control group (P<0.001). The absolute values of the pressure differences between the two feet of the rats with arthritis models in each group before and after drug administration are as Figure 5 shown, and from Figure 5It can be seen that during the experimental drug administration process, the absolute value of the pressure difference between the two feet in the normal control group fluctuated between 4±2 and 8±6 g. After drug administration, the absolute value of the pressure difference between the two feet at each time point in the model control group was significantly higher than that in the normal control group (P<0.01 - P<0.001). The absolute value of the pressure difference between the two feet in the low-dose CEFFE group was similar to that in the model control group at each time point except at 1 week after the fourth drug administration (i.e., the eighth week), when it was significantly lower than that in the model control group (27±8 vs 55±19 g, P<0.05). The absolute value of the pressure difference between the two feet in the medium-dose CEFFE group was significantly lower than that in the model control group at other time points except at 1 week after the third drug administration (i.e., the sixth week), when it was similar to that in the model control group (P<0.05 - P<0.01). The absolute value of the pressure difference between the two feet in the high-dose CEFFE group was significantly lower than that in the model control group from 1 week after the first drug administration (i.e., the second week) to the end of the experiment (1 week after the fourth drug administration) (P<0.05 - P<0.01).

[0252] The absolute value of the pressure difference between the two feet reflects joint load-bearing. The smaller the difference, the closer the load-bearing is to normal. The absolute value of the pressure difference between the two feet can reflect the improvement of the comprehensive symptoms of arthritis treatment, including joint pain and movement disorders, etc. From Figure 5 it can be seen that CEFFE can improve arthritis and its symptoms such as joint pain and movement disorders.

[0253] 2.4 CEFFE treatment effectively improves the degree of osteoarthritis lesions in model rats

[0254] Under the microscope, mild to moderate articular cartilage fibrosis, mild to severe reduction in the number of articular cartilage cells, mild articular cartilage cell hyperplasia / degeneration, and mild to mild articular cartilage cell degeneration / necrosis / erosion were visible in the knee joints of animals in the model control group; the above pathological changes are all typical lesions of osteoarthritis, indicating successful modeling.

[0255] Mild to moderate articular cartilage fibrosis, mild to moderate reduction in the number of articular cartilage cells, mild to mild articular cartilage cell hyperplasia / degeneration, and mild to mild articular cartilage cell degeneration / necrosis / erosion were visible in the knee joints of animals in the low-, medium-, and high-dose CEFFE groups. Compared with the animals in the model control group, the incidence rate and / or degree of the above lesions in the medium- and high-dose CEFFE groups were reduced, indicating that CEFFE at medium and high doses can improve the degree of osteoarthritis lesions induced by MIA in rats. The results of Safranin O-fast green staining showed that compared with the model control group, the scores of the Safranin O-fast green staining results in the medium- and high-dose CEFFE groups were reduced. The HE staining and Safranin O-fast green staining results of rats in different groups are shown respectively as Figure 6 and Figure 7as shown

[0256] The histological results of HE staining of rats in different groups were as follows:

[0257] Figure 6 A. Normal control group (without any treatment), euthanized on the 54th day (Day54) of the experiment, and no obvious abnormality was found in the cartilage tissue of the knee joint (lower end of the femur).

[0258] Figure 6 B. Model control group (rats with MIA-induced osteoarthritis model injected with normal saline), euthanized on the 54th day (Day54) of the experiment, and moderate articular cartilage fibrosis and severe reduction in the number of articular chondrocytes were visible in the knee joint (lower end of the femur).

[0259] Figure 6 C. CEFFE low-dose group, rats with MIA-induced osteoarthritis model were injected with CEFFE into the joint cavity, euthanized on the 54th day (Day54) of the experiment, and moderate articular cartilage fibrosis and severe reduction in the number of articular chondrocytes were visible in the knee joint (lower end of the femur).

[0260] Figure 6 D. CEFFE medium-dose group, rats with MIA-induced osteoarthritis model were injected with CEFFE into the joint cavity, euthanized on the 54th day (Day54) of the experiment, and mild articular cartilage fibrosis and mild reduction in the number of articular chondrocytes were visible in the knee joint (lower end of the femur).

[0261] Figure 6 E. CEFFE high-dose group, rats with MIA-induced osteoarthritis model were injected with CEFFE into the joint cavity, euthanized on the 54th day (Day54) of the experiment, and mild articular cartilage fibrosis and mild reduction in the number of articular chondrocytes were visible in the knee joint (lower end of the femur).

[0262] Among them, the summary table of the main pathological changes in the HE staining of the knee joints of rats in different groups is shown in Table 3:

[0263] Table 3 Summary table of the main pathological changes in the HE staining of the knee joints of rats in different groups

[0264]

[0265] The histological results of Safranin O-fast green staining of rats in different groups were as follows:

[0266] Figure 7 A. Normal control group, (without any treatment), euthanized on the 54th day (D54) of the experiment, and no obvious abnormality was found in the cartilage tissue of the knee joint (lower end of the femur).

[0267] Figure 7 B. Model control group, (MIA-induced osteoarthritis model rats were given saline injection), euthanized on the 54th day (D54) of the experiment. Moderate articular cartilage fibrosis and severe reduction in the number of articular chondrocytes were visible in the knee joint (lower end of the femur).

[0268] Figure 7 C. CEFFE low-dose group, CEFFE was injected into the joint cavity of MIA-induced osteoarthritis model rats, euthanized on the 54th day (D54) of the experiment. Moderate articular cartilage fibrosis and severe reduction in the number of articular chondrocytes were visible in the knee joint (lower end of the femur).

[0269] Figure 7 D. CEFFE medium-dose group, CEFFE was injected into the joint cavity of MIA-induced osteoarthritis model rats, euthanized on the 54th day (D54) of the experiment. Slight articular cartilage fibrosis and slight reduction in the number of articular chondrocytes were visible in the knee joint (lower end of the femur).

[0270] Figure 7 E. CEFFE high-dose group, CEFFE was injected into the joint cavity of MIA-induced osteoarthritis model rats, euthanized on the 54th day (D54) of the experiment. Slight articular cartilage fibrosis and slight reduction in the number of articular chondrocytes were visible in the knee joint (lower end of the femur).

[0271] Among them, the summary table of the main pathological changes in the safranin O-fast green staining of the knee joints of rats in different groups is shown in Table 4:

[0272] Table 4 Summary table of the main pathological changes in the safranin O-fast green staining of the knee joints of rats in different groups

[0273]

[0274]

[0275] 3 Conclusions

[0276] In this study, CEFFE was injected into the joint cavity to treat MIA-induced osteoarthritis model rats. Behavioral studies showed that CEFFE treatment could effectively increase the mechanical pain threshold of model rats, relieve mechanical pain, reduce the pressure difference between the two feet, alleviate the abnormal hind limb load caused by osteoarthritis, and relieve the symptoms of osteoarthritis; histopathological results also confirmed that CEFFE treatment could effectively reduce the degree of osteoarthritis lesions and reduce cartilage tissue damage. In summary, CEFFE has excellent therapeutic effects on osteoarthritis.

[0277] All documents mentioned in this invention are cited herein by reference as if each individual document was cited by reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. Use of a cell-free fat extract, characterized in that, it is used for preparing a pharmaceutical composition, and the pharmaceutical composition is used for one or more uses selected from the following groups: (i) treating arthritis; (ii) treating pain, where the pain is arthritis pain; (iii) treating movement disorders, where the movement disorders are movement disorders caused by arthritis; wherein, the cell-free fat extract is prepared by the following method: (1) Provide a fat tissue raw material, crush the fat tissue raw material, and perform rinsing to obtain rinsed fat tissue; (2) Centrifuge the rinsed fat tissue to obtain a layered mixture; (3) For the layered mixture, remove the upper oil layer and the lower water layer, and collect the middle layer, that is, the fat layer containing fat cells; (4) Emulsify the middle layer to obtain an emulsified fat mixture, also known as nano-fat; (5) Centrifuge the emulsified fat mixture to obtain an intermediate liquid layer, which is the crude fat extract; and (6) Filter and sterilize the crude fat extract to obtain a cell-free fat extract; moreover, the cell-free fat extract does not contain cells and does not contain lipid droplets; the arthritis is osteoarthritis.

2. The use according to claim 1, characterized in that, in step (2), the centrifugation is carried out at 800 - 2500 g.

3. The use according to claim 1, characterized in that, the osteoarthritis includes degenerative osteoarthritis.

4. The use according to claim 1, characterized in that, in step (4), the emulsification is carried out by the method of breaking with a tissue homogenizer.

5. The use according to claim 1, characterized in that, the osteoarthritis is selected from the following groups: cervical osteoarthritis, lumbar osteoarthritis, knee osteoarthritis, hip osteoarthritis, or a combination thereof.

6. The use according to claim 5, characterized in that, the osteoarthritis is knee osteoarthritis.

7. The use according to claim 1, characterized in that, the cell-free fat extract is a cell-free fat extract prepared by extracting from fat in humans or non-human mammals.

8. The use according to claim 1, characterized in that, in the pharmaceutical composition, the mass percentage of the cell-free fat extract is 1 - 20 wt%, based on the total weight of the pharmaceutical composition.

9. 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.

10. The use according to claim 1, characterized in that, the pharmaceutical composition is administered by external application, topical application, or subcutaneous injection.

11. The use according to claim 1, characterized in that, the "not containing lipid droplets" means that in the cell-free fat extract, the volume percentage of oil droplets in the total liquid is less than 1%; the "cell-free" means that the average number of cells in 1 ml of the cell-free fat extract ≤ 1.

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

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

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

15. The use according to claim 14, characterized in that in the acellular 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; and the concentration of PDGF is 50 - 600 pg / ml.

16. The use according to claim 15, characterized in that in the acellular 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; and the concentration of PDGF is 100 - 300 pg / ml.

17. The use according to claim 15, characterized in that in the acellular 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 1400 - 2000 pg / ml; the concentration of bFGF is 200 - 280 pg / ml; the concentration of VEGF is 150 - 250 pg / ml; the concentration of TGF-β1 is 800 - 1200 pg / ml; The concentration of the HGF is 800 - 1000 pg / ml; and the concentration of the PDGF is 140 - 220 pg / ml.

18. The use according to claim 14, characterized in that the cell-free fat extract has the following characteristics: the weight ratio of the IGF-1 to the VEGF is 20 - 100:1; the weight ratio of the BDNF to the VEGF is 2 - 20:1; the weight ratio of the GDNF to the VEGF is 2 - 20:1; the weight ratio of the bFGF to the VEGF is 0.2 - 8:1; the weight ratio of the TGF-β1 to the VEGF is 1 - 20:1; the weight ratio of the HGF to the VEGF is 1 - 20:1; and the weight ratio of the PDGF to the VEGF is 0.1 - 3:

1.

19. The use according to claim 18, characterized in that the cell-free fat extract has the following characteristics: the weight ratio of the IGF-1 to the VEGF is 30 - 70:1; the weight ratio of the BDNF to the VEGF is 4 - 15:1; the weight ratio of the GDNF to the VEGF is 4 - 15:1; the weight ratio of the bFGF to the VEGF is 0.6 - 2:1; the weight ratio of the TGF-β1 to the VEGF is 1 - 10:1; the weight ratio of the HGF to the VEGF is 1 - 10:1; and the weight ratio of the PDGF to the VEGF is 0.4 - 1.5:

1.

20. The use according to claim 18, characterized in that the cell-free fat extract has the following characteristics: the weight ratio of the IGF-1 to the VEGF is 40 - 60:1; the weight ratio of the BDNF to the VEGF is 6 - 12:1; the weight ratio of the GDNF to the VEGF is 6 - 12:1; the weight ratio of the bFGF to the VEGF is 0.8 - 1.6:1; the weight ratio of the TGF-β1 to the VEGF is 2 - 8:1; the weight ratio of the HGF to the VEGF is 2 - 8:1; and the weight ratio of the PDGF to the VEGF is 0.7 - 1.2:

1.

21. The use according to claim 1, characterized in that in step (5), before subjecting the emulsified fat mixture to centrifugation treatment, it further includes freezing and then thawing the emulsified fat mixture.

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