CN114470000A

CN114470000A - Application of cell-free fat extract in treating nonalcoholic steatohepatitis

Info

Publication number: CN114470000A
Application number: CN202011255879.6A
Authority: CN
Inventors: 张文杰; 侯曦凡
Original assignee: Shanghai Seme Cell Technology Co Ltd
Current assignee: Shanghai Seme Cell Technology Co Ltd
Priority date: 2020-11-11
Filing date: 2020-11-11
Publication date: 2022-05-13
Also published as: WO2022100400A1

Abstract

The invention relates to a use of a cell-free fat extract in treating nonalcoholic steatohepatitis. Specifically, the invention provides application of a cell-free fat extract in preparing a composition or a preparation, wherein the composition or the preparation is used for preventing and/or treating nonalcoholic steatohepatitis. The cell-free fat extract of the present invention has an excellent therapeutic effect on nonalcoholic steatohepatitis.

Description

Application of cell-free fat extract in treating nonalcoholic steatohepatitis

Technical Field

The invention relates to the field of medicines, in particular to a treatment application of a cell-free fat extracting solution to nonalcoholic steatohepatitis.

Background

Nonalcoholic steatohepatitis (NASH) refers to steatohepatitis caused by factors other than alcohol, and a phenomenon in which lipid is accumulated in liver cells in the liver, often accompanied by inflammation and fibrosis. Non-alcoholic steatohepatitis is a progressive form of non-alcoholic fatty liver disease, and the phenomenon of steatosis accompanied by inflammation and hepatocyte injury occurs. NASH can lead to liver fibrosis, cirrhosis, liver failure and the development of liver tumors. Although the pathological mechanism of nonalcoholic steatohepatitis has been studied in a large amount, the mechanism of occurrence has not yet been clarified definitely. The prior art also lacks drugs for effectively treating the nonalcoholic steatohepatitis.

Therefore, there is a need in the art to develop a drug that can effectively treat nonalcoholic steatohepatitis.

Disclosure of Invention

The invention aims to provide application of a cell-free fat extract in the aspect of preventing and/or treating nonalcoholic steatohepatitis.

In a first aspect of the invention, there is provided the use of a cell-free fat extract for the preparation of a composition or formulation for one or more uses selected from the group consisting of: (i) preventing and/or treating nonalcoholic steatohepatitis.

In another preferred embodiment, the nonalcoholic steatohepatitis is selected from the group consisting of: acute nonalcoholic steatohepatitis, chronic nonalcoholic steatohepatitis, or a combination thereof.

In another preferred embodiment, said preventing and/or treating nonalcoholic steatohepatitis comprises preventing and/or treating one or more selected from the group consisting of:

(a) improving the fatty degeneration of liver cells;

(b) improving balloon-like degeneration of hepatocytes; and/or

(b) Improving the infiltration of the hepatitis cells.

In another preferred embodiment, the cell-free fat extract is obtained by extracting fat from human or non-human mammal.

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

In another preferred embodiment, the composition or formulation comprises a pharmaceutical composition or formulation, a food composition or formulation, a nutraceutical composition or formulation, or a dietary supplement.

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

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

In another preferred embodiment, the injection preparation is intravenous injection or intramuscular injection.

In another preferred embodiment, the composition or formulation is in the form of a solid dosage form, a semi-solid dosage form, or a liquid dosage form, such as a solution, gel, cream, lotion, ointment, cream, paste, cake, powder, patch, and the like.

In another preferred embodiment, the composition or the preparation is in the form of powder, granules, capsules, injection, tincture, oral liquid, tablets or buccal tablets.

In another preferred embodiment, the composition or formulation is administered by topical, or subcutaneous injection.

In another preferred embodiment, the cell-free fat extract is cell-free and free of lipid droplets.

In another preferred embodiment, the fat droplets are oil droplets released after the fat cells are disrupted.

In another preferred embodiment, the term "free of fat droplets" means that the percentage of oil droplets in the cell-free fat extract is less than 1%, preferably less than 0.5%, more preferably less than 0.1% by volume of the total liquid.

In another preferred embodiment, the cell is selected from the group consisting of: endothelial cells, adipose-derived stem cells, macrophage cells, and stromal cells.

In another preferred embodiment, the term "cell-free" means that the average number of cells in 1ml of cell-free fat extract is less than or equal to 1, preferably less than or equal to 0.5, more preferably less than or equal to 0.1, or is 0.

In another preferred example, the cell-free fat extract is a naturally obtained nano fat extract without additional ingredients.

In another preferred embodiment, the term "free of added ingredients" means that no solution, solvent, small molecule, chemical, and biological additives are added during the preparation of the fat extract except for the rinsing step.

In another preferred embodiment, the cell-free fat extract is prepared by emulsifying fat tissue and centrifuging.

In another preferred embodiment, the cell-free fat extract comprises one or more components selected from the group consisting of: IGF-1, BDNF, GDNF, TGF-beta 1, HGF, bFGF, VEGF, TGF-beta 1, PDGF, EGF, NT-3, GH, G-CSF, or combinations thereof.

In another preferred embodiment, the one 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- β 1, HGF, PDGF, or combinations thereof.

In another preferred example, the cell-free fat extract is a cell-free fat extract.

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

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

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

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

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

In another preferred embodiment, the concentration of TGF-beta 1 in the cell-free fat extract is 200-3000pg/ml, preferably 400-2000pg/ml, more preferably 600-1500pg/ml, more preferably 800-1200pg/ml, more preferably 800-1100pg/ml, more preferably 900-1000 pg/ml.

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

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

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

in another preferred embodiment, the weight ratio of BDNF to VEGF is 2-20: 1, preferably 4-15: 1, more preferably 6 to 12: 1, optimally 8-9.5: 1.

in another preferred embodiment, the weight ratio of GDNF to VEGF is 2-20: 1, preferably 4-15: 1, more preferably 6 to 12: 1, optimally 8.5-9.5: 1.

in another preferred embodiment, the weight ratio of bFGF to VEGF is 0.2 to 8: 1, preferably 0.5 to 5: 1, more preferably 0.6 to 2: 1, more preferably 0.8 to 1.6: 1, optimally 1-1.5: 1.

in another preferred embodiment, the weight ratio of TGF-beta 1 to VEGF is 1-20: 1, preferably 1 to 15: 1, more preferably 1 to 10: 1, more preferably 2 to 8: 1, more preferably 4 to 6: 1.

in another preferred example, the weight ratio of HGF to VEGF is 1-20: 1, preferably 1-15: 1, more preferably 1 to 10: 1, more preferably 2 to 8: 1, more preferably 4 to 5.5: 1.

in another preferred embodiment, the weight ratio of PDGF to VEGF is 0.1 to 3: 1, preferably 0.2-2: 1, more preferably 0.4 to 1.5: 1, optimally 0.7-1.2: 1.

in another preferred embodiment, the cell-free fat extract is prepared by the following method:

(1) providing an adipose tissue material, disrupting the adipose tissue material, and rinsing (e.g., with physiological saline) to obtain rinsed adipose 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 layered mixture, and collecting the middle layer (i.e., fat layer containing adipocytes);

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

(5) centrifuging the emulsified fat mixture to obtain an intermediate liquid layer, namely a fat primary extract; and

(6) filtering and sterilizing the fat primary extract to obtain a cell-free fat extract.

In a second aspect of the present invention, there is provided a method for preparing a cell-free fat extract, the method comprising the steps of:

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

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

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

In another preferred embodiment, in the step (2), the centrifugation is performed at 2500 g-.

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

In another preferred embodiment, the temperature of the centrifugation is 2-6 ℃.

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

In another preferred embodiment, the mechanical emulsification is mechanical emulsification by repeated beating (e.g. 20-200 times, preferably 20-150 times, more preferably 20-100 times, more preferably 30-50 times) with a syringe.

In another preferred example, the blowing and beating mode is that 210 ml injection syringes are connected with a three-way pipe and repeatedly pushed and beaten at a constant speed.

In another preferred example, in the step (4), the emulsifying is a method of breaking up by a tissue homogenizer.

In another preferred embodiment, in the step (5), before the emulsified fat mixture is processed by centrifugation, the emulsified fat mixture is frozen and then thawed.

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

In another preferred embodiment, the freezing temperature is from-50 ℃ to-120 ℃, preferably from-60 ℃ to-100 ℃, more preferably from-70 ℃ to-90 ℃.

In another preferred embodiment, the thawing temperature is 20-40 deg.C, preferably 25-40 deg.C, more preferably 37 deg.C.

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

In another preferred example, in the step (5), after centrifugation, the emulsified fat mixture is layered into 4 layers, the first layer is an oil layer, the second layer is a residual fat tissue layer, the third layer is a liquid layer (i.e. an intermediate liquid layer), and the fourth layer is a cell/tissue debris precipitation layer.

In another preferred embodiment, in the step (5), the centrifugation is performed at 2500 g-.

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

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

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

In another preferred example, in the step (6), the filter bag can remove fat cells in the fat primary extract.

In another preferred embodiment, in the step (6), the filtration and sterilization are performed by a filter (e.g., a 0.22 μm microporous membrane).

In another preferred embodiment, the filter is a microfiltration membrane filter.

In another preferred embodiment, the pore size of the microfiltration membrane is 0.05 to 0.8. mu.m, preferably 0.1 to 0.5. mu.m, more preferably 0.1 to 0.4. mu.m, more preferably 0.15 to 0.3. mu.m, more preferably 0.2 to 0.25. mu.m, most preferably 0.22. mu.m.

In another preferred embodiment, in the step (6), the filtration and sterilization are performed by passing through a first cell-rejecting filter and then a second cell-rejecting filter (e.g., a 0.22 μm filter).

In another preferred example, the step (6) further comprises subpackaging the fat extract to form a subpackaged product. (the sub-packaged extract can be stored at-20 deg.C for use, or thawed at low temperature (such as-4 deg.C) or normal temperature for use directly, or thawed and stored at low temperature (such as 4 deg.C) for a period of time for use).

In a third aspect of the invention, there is provided a cell-free fat extract obtained by the method according to the second aspect of the invention.

In a fourth aspect of the invention, there is provided a composition or formulation comprising (a) a cell-free fat extract according to the third aspect of the invention; and (b) a pharmaceutically, food, nutraceutical, or dietetically acceptable carrier or excipient.

In another preferred embodiment, the composition is a pharmaceutical composition, a food composition, a nutraceutical composition or a dietary supplement.

In another preferred embodiment, the injection is intravenous injection or intramuscular injection.

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

In a fifth aspect of the invention, there is provided a method of preparing a composition or formulation according to the fourth aspect of the invention, said method comprising the steps of: mixing the cell-free fat extract according to the third aspect of the present invention with a pharmaceutically, food, nutraceutical or dietetically acceptable carrier or excipient to form a composition or formulation.

In a sixth aspect of the present invention, there is provided a method for preventing and/or treating nonalcoholic steatohepatitis by administering the cell-free fat extract according to the third aspect of the present invention to a subject in need thereof.

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

In another preferred embodiment, the non-human mammal includes a rodent, such as a rat, a mouse.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

Fig. 1 is a HE staining (100X) of mice of different groups, wherein the semi-quantitative ranking results "+", "+ +" and "+ + + + + + +" indicate increasing lesion size in order.

Detailed Description

The present inventors have made extensive and intensive studies and have for the first time developed a cell-free fat extract having an excellent therapeutic effect on nonalcoholic steatohepatitis. The present invention has been completed on the basis of this finding.

Term(s) for

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.

As used herein, the terms "comprising," "including," and "containing" are used interchangeably and include not only open-ended definitions, but also semi-closed and closed-ended definitions. In other words, the term includes "consisting of … …", "consisting essentially of … …".

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

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, "preventing" also includes delaying the onset of a disease and/or its attendant symptoms and reducing the risk of acquiring a disease in a subject.

"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 invention reduces, inhibits and/or reverses non-alcoholic steatohepatitis, e.g., by at least about 10%, at least about 30%, at least about 50%, or at least about 80%, as compared to the levels observed in the absence of the composition, kit, food or nutraceutical kit, active ingredient combination of the invention.

As used herein, "improving" includes preventing, treating, alleviating, reversing, alleviating, and the like.

As used herein, the term "IGF-1" is referred to as insulin-like growth factor 1(insulin-like growth factors-1).

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

As used herein, the term "GDNF" is referred to as glial cell-derived neurotrophic factor (glicellline-derived neurotrophic factor).

As used herein, the term "bFGF" refers to basic fibroblast growth factor (basic fibroblast growth factor).

As used herein, the term "VEGF" is referred to as vascular endothelial growth factor (vascular endothelial growth factor).

As used herein, the term "TGF-. beta.1" refers to transforming growth factor-. beta.1 (transforming growth factor-. beta.1).

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

As used herein, the term "PDGF" refers to Platelet-derived growth factor (Platelet derived growth factor)

As used herein, the term "EGF" refers to Epidermal Growth Factor (Epidermal Growth Factor)

As used herein, the term "NT-3" refers to neurotrophic factor 3 (neurotropphins-3).

As used herein, the term "GH" is referred to as Growth Hormone (Growth Hormone).

As used herein, the term "G-CSF" refers to a granulocyte colony stimulating factor (granulocytic colony stimulating factor).

Cell free fat extract (CEFFE) and its preparation method

As used herein, the terms "cell-free fat extract of the present invention", "fat extract of the present invention", and the like, which are used interchangeably, refer to an extract (or extract) derived from adipose tissue prepared without the addition of any solution, solvent, small molecule, chemical, and biological additive during the preparation of the fat extract (except for the rinsing step). A typical method of preparing an extract of the invention is as described above in the second aspect of the invention. Furthermore, it is to be understood that while the extract of the present invention does not require the addition of any additives (or additional ingredients) during the preparation process, some or a small amount of a safe substance (such as a small amount of water) that does not adversely or adversely affect the activity of the extract of the present invention may be added.

In a preferred embodiment of the present invention, the cell-free fat extract is a cell-free fat extract.

The cell-free fat extract according to the present invention may include various cytokines. Typically, the cell-free fat extract comprises one or more of IGF-1, BDNF, GDNF, TGF- β, HGF, bFGF, VEGF, TGF- β 1, PDGF, EGF, NT-3, GH and G-CSF.

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

preferably, the cell-free fat extract according to the present invention is prepared by the method according to the second aspect of the present invention as described above.

Typically, the cell-free fat extract according to the present invention is prepared by the following method:

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

Non-alcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) refers to steatohepatitis caused in addition to alcoholic factors, i.e., a phenomenon in which lipid accumulation occurs in hepatocytes in the liver, and is also accompanied by inflammation and fibrosis phenomena. Non-alcoholic steatohepatitis is an extremely progressive form of non-alcoholic fatty liver disease, and the phenomenon of steatosis accompanied by inflammation and hepatocyte injury occurs. NASH can lead to late stage liver fibrosis, cirrhosis, liver failure and the development of liver tumors. Although the pathological mechanism of nonalcoholic steatohepatitis has been studied in a large amount, the mechanism of occurrence has not yet been clarified definitely.

Compositions and applications

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

Typically, the acellular fat extract of the present invention may be prepared into pharmaceutical compositions such as tablets, capsules, powders, fine granules, solutions, lozenges, jellies, cream formulations, spirits, suspensions, tinctures, poultices, liniments, lotions, and aerosols. The pharmaceutical composition can be prepared by a generally known preparation technique, and a suitable pharmaceutical additive can be added to the drug.

The compositions of the present invention may also include pharmaceutically, comestibly, nutraceutically or dietetically acceptable carriers. "pharmaceutically, food, nutraceutical, or dietetically acceptable carrier" means: one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of intermixing with and with the compounds of the present invention without significantly diminishing the efficacy of the compounds. Examples of acceptable carrier moieties for pharmaceutically, food, nutraceutical or dietetically acceptable carriers are cellulose and its derivatives (e.g. sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g. stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g. soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g. propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (e.g. propylene glycol, glycerol, mannitol, sorbitol, etc.)

) Wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

The mode of administration of the composition of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, parenteral (intravenous, intramuscular), topical, preferred modes of administration are oral and injection.

The composition or the preparation of the invention is in the form of oral preparation, external preparation or 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, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) disintegrating agents, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol 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 capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings and other materials well known in the art. They may comprise opacifying agents.

Liquid dosage forms for oral administration or administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly employed in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of such materials and the like.

In addition to these inert diluents, the compositions may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active ingredients, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these materials, and the like.

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 nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.

Dosage forms for topical administration or administration of the compounds of the present 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 which may be required if necessary.

The cell-free fat extract of the present invention may be administered or dosed alone or in combination with other agents for preventing and/or treating non-alcoholic fatty liver disease and/or its complications.

The composition is administered in a safe and effective amount of the cell-free fat extract of the present invention to human or non-human animals (e.g., rats, mice, dogs, cats, cows, chickens, ducks, etc.) in need of treatment, wherein the administration is in an amount that is pharmaceutically, dietetically or nutraceutically acceptable as an effective administration dose. The term "safe and effective amount" as used herein, refers to an amount that produces a function or activity in and is acceptable to humans and/or animals. It will be understood by those skilled in the art that the safe and effective amount may vary with the form of the pharmaceutical composition, the route of administration, the excipients used, the severity of the disease, and the combination with other drugs. For example, the daily dose for a human of 60kg body weight is usually 0.1 to 1000mg, preferably 1 to 600mg, more preferably 2 to 300 mg. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

The main advantages of the invention include:

the invention discovers that the cell-free fat extract has excellent treatment effect on the nonalcoholic steatohepatitis for the first time.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.

Example 1

1. Experimental methods

1.1. Preparation of Cell free fat extract (CEFFE)

Fat was obtained by volunteers under informed consent. The preparation method of the cell-free adipose tissue extract comprises the following steps:

(1) adipose tissue was obtained from 6 healthy women of routine liposuction, mean age 31 years (24-36 years). After local injection of tumescent solution for anesthesia, a 20mL syringe was connected using a 3mm liposuction cannula with a large side hole (2 mm. times.7 mm), and then suction was performed radially under artificial negative pressure to stand the obtained fat still upright, and after removal of tumescent solution, the solution was rinsed 3 times with physiological saline.

(2) The rinsed adipose tissue was placed in a centrifuge tube and centrifuged in a centrifuge at 1200g 4 ℃ for 3 minutes to obtain a layered mixture.

(3) The upper oil layer and the lower aqueous layer were removed from the layered mixture, and the middle layer (i.e., the fat layer containing adipocytes) was collected.

(4) And repeatedly pushing the middle layer for 30 times at constant speed by using 210 ml injection syringes connected with a three-way pipe, so as to perform mechanical emulsification, and obtain a mechanically emulsified fat mixture (also called nano fat).

(5) Placing the mechanically emulsified fat mixture into a refrigerator at minus 80 ℃ for freezing, then carrying out water bath thawing at 37 ℃, after single freeze-thaw cycle, centrifuging the thawed fat mixture for 5 minutes at 1200g and 4 ℃ to obtain a layered mixture, wherein the layered mixture is divided into 4 layers in total, the first layer is an oil layer, the second layer is a residual fat tissue layer, the third layer is a liquid layer, the fourth layer is a cell/tissue fragment sediment layer, removing the oil layer and the residual fat tissue layer, sucking the liquid layer, and avoiding the pollution of the cell/tissue fragment sediment layer in the sucking process, thereby obtaining the primary fat extracting solution.

(6) Filtering the obtained fat primary extract with 0.22 μm filter to sterilize and remove possible mixed living cells to obtain cell-free fat extract (CEFFE), freezing at-20 deg.C for storage, and thawing at 4 deg.C for use.

And detecting the content of cell factors including IGF-1, BDNF, GDNF, bFGF, VEGF, TGF-beta 1, HGF, PDGF and other cell factors by using an ELISA immunoadsorption determination kit for the prepared cell-free fat extracting solution. The average concentration of 6 samples tested was as follows: IGF-1(9840.6pg/ml), BDNF (1764.5pg/ml), GDNF (1831.9pg/ml), bFGF (242.3pg/ml), VEGF (202.9pg/ml), TGF-. beta.1 (954.5pg/ml), HGF (898.4pg/ml) and PDGF (179.9 pg/ml).

1.2 mouse non-alcoholic steatohepatitis (NASH) model establishment, grouping and administration

All quarantine-qualified mice were screened 40 randomly into 5 groups of 8 mice per group according to the weight approach principle. The groups of animals and the dose administered are shown in table 1 below:

table 1 administration of different groups of rats

The molding mode is as follows: during the test period, all animals are fed with SPF level mouse feed, after grouping, normal control group mice are fed with SPF level mouse feed, and the rest groups of mice are fed with MCD (Methionine and Choline Deficient L-Amino Acid Diet) feed to start non-alcoholic steatohepatitis modeling until the end point of the test.

Administration mode and frequency: animals were modelled and dosed simultaneously, 2 times weekly, 4 weeks for a total of 8 doses (Day1, Day 4, Day 8, Day 11, Day 15, Day 18, Day 22, Day 25). The administration mode is tail vein injection, the normal control group is not treated, the model control group is given physiological saline as a negative control, and the CEFFE group is given CEFFE solution with corresponding dose. Day1 of administration was Day1, Day2 was Day2, and so on.

1.3 pathological tissue Observation

After liver perfusion, according to the above material taking method, the left lobe of the liver is placed in 10% neutral formalin for fixation, sliced, and HE staining is carried out to observe the histopathological change of the liver, wherein the main observation indexes are fatty degeneration of liver cells, balloon-like degeneration of liver cells and inflammatory cell infiltration degree, semi-quantitative grading is carried out according to lesion degree, and NAS grading is carried out. The NAS scoring criteria are shown in table 2 below:

TABLE 2 NAS scoring criteria

1.7 statistical analysis of data

The data are expressed as mean ± standard deviation and the dynamic measurements such as body weight are compared between groups using Graphpad prism 5.0 software with two-way analysis of variance and, if there is a difference (P <0.05), between normal and model control and between model control and CEFFE with Bonferroni post test.

2. Results

2.1 treatment with CEFFE to reduce the extent of liver lesions in model mice

The results of microscopic observation of the livers of the animals euthanized with D27 after administration are shown in FIG. 1, and it can be seen from FIG. 1 that the livers of the mice in the model control group, the CEFFE low dose group, the CEFFE medium dose group and the CEFFE high dose group all have different degrees of hepatosteatosis, hepatoballoon-like degeneration and inflammatory cell infiltration related to model making, and the lesions are mainly hepatoballoon-like degeneration. Compared with a model control group, the hepatic cell steatosis, inflammatory cell infiltration and hepatic cell ballooning degeneration of the CEFFE low-dose group have no obvious change; in CEFFE, the degree of hepatic cell steatosis, hepatic cell balloon-like degeneration and inflammatory cell infiltration lesion of medium and high dose groups is reduced. The NAS score shows a descending trend along with the increase of the administration dose of the CEFFE, and the CEFFE treatment is shown to reduce the pathological change degree of the liver of the mouse and has certain dose dependence.

Figure 1a. normal control group, normal liver.

Figure 1B model control: hepatic steatosis (+), hepatocellular balloon-like degeneration (+ +), and inflammatory cell infiltration (+ +).

Fig. 1c. ceffe low dose group: hepatic steatosis (+ +), hepatocellular balloon-like degeneration (+++), and inflammatory cell infiltration (++).

Dose groups in ceffe: hepatic steatosis (+), hepatic balloon degeneration (+), and inflammatory cell infiltration (+).

Figure 1e. ceffe high dose group: liver cell steatosis (+), hepatocyte ballooning degeneration (+) and inflammatory cell infiltration (+).

The results of microscopic observations related to drug in post-dose Day27 euthanized animals are summarized in table 3:

TABLE 3 summary of microscopic observations relating to drug-related euthanized animals by Day27 post-dose

The results of NAS scoring of the livers of the euthanized D27 animals after dosing are shown in table 4:

TABLE 4 statistics of D27 euthanized animal liver NAS scores after dosing

Conclusion

The results of this example show that CEFFE has an excellent therapeutic effect on nonalcoholic steatohepatitis.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. Use of a cell-free fat extract for the preparation of a composition or formulation for one or more uses selected from the group consisting of: (i) preventing and/or treating nonalcoholic steatohepatitis.

2. The use of claim 1, wherein the nonalcoholic steatohepatitis is selected from the group consisting of: acute nonalcoholic steatohepatitis, chronic nonalcoholic steatohepatitis, or a combination thereof.

3. The use according to claim 1, wherein the prevention and/or treatment of nonalcoholic steatohepatitis comprises prevention and/or treatment in one or more ways selected from the group consisting of:

(a) improving the fatty degeneration of liver cells;

(b) improving balloon-like degeneration of hepatocytes; and/or

(b) Improving the infiltration of the hepatitis cells.

4. The use according to claim 1, wherein the cell-free fat extract comprises one or more components selected from the group consisting of: IGF-1, BDNF, GDNF, TGF-beta, HGF, bFGF, VEGF, TGF-beta 1, HGF, PDGF, EGF, NT-3, GH, G-CSF, or combinations thereof.

5. The use according to claim 4, wherein 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 5000-30000pg/ml, preferably 6000-20000pg/ml, more preferably 7000-15000pg/ml, more preferably 8000-12000pg/ml, more preferably 9000-11000pg/ml, more preferably 9500-10500 pg/ml;

in the cell-free fat extract, the concentration of BDNF is 800-5000pg/ml, preferably 1000-4000pg/ml, more preferably 1200-2500pg/ml, more preferably 1400-2000pg/ml, more preferably 1600-2000pg/ml, more preferably 1700-1850 pg/ml;

in the cell-free fat extract, the concentration of GDNF is 800-;

in the cell-free fat extract, the concentration of bFGF is 50-600pg/ml, preferably 100-500pg/ml, more preferably 120-400pg/ml, more preferably 150-300pg/ml, more preferably 200-280pg/ml, more preferably 220-260 pg/ml;

in the cell-free fat extract, the concentration of VEGF is 50-500pg/ml, preferably 100-400pg/ml, more preferably 120-300pg/ml, more preferably 150-250pg/ml, more preferably 170-230pg/ml, more preferably 190-210 pg/ml;

in the cell-free fat extract, the concentration of TGF-beta 1 is 200-3000pg/ml, preferably 400-2000pg/ml, more preferably 600-1500pg/ml, more preferably 800-1200pg/ml, more preferably 800-1100pg/ml, more preferably 900-1000 pg/ml;

in the cell-free fat extract, the concentration of HGF is 200-3000pg/ml, preferably 400-2000pg/ml, more preferably 600-1500pg/ml, more preferably 600-1200pg/ml, more preferably 800-1000pg/ml, more preferably 850-950 pg/ml; and/or

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

6. The use according to claim 4, wherein 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, preferably 30-70: 1, more preferably 40 to 60: 1, optimally 45-55: 1;

the weight ratio of BDNF to VEGF is 2-20: 1, preferably 4-15: 1, more preferably 6 to 12: 1, optimally 8-9.5: 1;

the weight ratio of GDNF to VEGF is 2-20: 1, preferably 4-15: 1, more preferably 6-12: 1, optimally 8.5-9.5: 1;

the weight ratio of the bFGF to the VEGF is 0.2-8: 1, preferably 0.5 to 5: 1, more preferably 0.6 to 2: 1, more preferably 0.8 to 1.6: 1, optimally 1-1.5: 1;

the weight ratio of TGF-beta 1 to VEGF is 1-20: 1, preferably 1 to 15: 1, more preferably 1 to 10: 1, more preferably 2 to 8: 1, more preferably 4 to 6: 1;

the weight ratio of HGF to VEGF is 1-20: 1, preferably 1 to 15: 1, more preferably 1 to 10: 1, more preferably 2 to 8: 1, more preferably 4 to 5.5: 1; and/or

The weight ratio of PDGF to VEGF is 0.1-3: 1, preferably 0.2-2: 1, more preferably 0.4 to 1.5: 1, optimally 0.7-1.2: 1.

7. the use according to claim 1, wherein the cell-free fat extract is prepared by:

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

8. The use of claim 1, wherein the composition or formulation is in the form of an oral formulation, a topical formulation or an injectable formulation.

9. A cell-free fat extract, wherein the cell-free fat extract is prepared by the following method:

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

10. A method for preventing and/or treating nonalcoholic steatohepatitis, comprising administering the cell-free fat extract according to claim 9 to a subject in need thereof.