WO2021143912A1

WO2021143912A1 - Therapeutic action of cell-free fat extract on fatty liver and complications thereof

Info

Publication number: WO2021143912A1
Application number: PCT/CN2021/072553
Authority: WO
Inventors: 王斌; 赵莉
Original assignee: 上海萨美细胞技术有限公司
Priority date: 2020-01-16
Filing date: 2021-01-18
Publication date: 2021-07-22
Also published as: CN113116929A

Abstract

The present invention relates to a therapeutic action of cell-free fat extract on fatty liver and complications thereof. In particular, the present invention provides a use of the cell-free fat extract for preparing a composition or a preparation, which is used for preventing and/or treating fatty liver and/or complications thereof. The cell-free fat extract of the present invention has an excellent therapeutic effect on fatty liver and/or complications thereof.

Description

Therapeutic effect of acellular fat extract on fatty liver and its complications

Technical field

The invention belongs to the field of biotechnology, and specifically relates to the therapeutic effect of a cell-free fat extract on fatty liver and its complications.

Background technique

Fatty liver disease is one of the common chronic liver diseases, which can develop into hepatitis (NASH), liver cirrhosis, liver cancer, etc. as the disease progresses. It has become a widespread global disease affecting public health in the past decade, but it is still lacking in ideals. To find safe and effective biological agents has become an urgent problem to be solved.

Therefore, there is an urgent need to develop a drug that can safely and effectively treat fatty liver.

Summary of the invention

The purpose of the present invention is to provide a cell-free fat extract for safe and effective treatment of fatty liver and/or its complications.

In the first aspect of the present invention, there is provided a use of an acellular fat extract for preparing a composition or preparation for preventing and/or treating fatty liver and/or its complications.

In another preferred embodiment, the fatty liver is alcoholic or non-alcoholic fatty liver.

In another preferred embodiment, the fatty liver is fatty liver caused by a high-fat diet or obesity.

In another preferred embodiment, the fatty liver is obese fatty liver.

In another preferred embodiment, the fatty liver is fatty liver caused by lipid metabolism disorder.

In another preferred embodiment, the lipid metabolism disorder is a lipid metabolism disorder in the blood and/or a lipid metabolism disorder in the liver.

In another preferred embodiment, the lipid metabolism disorder is hyperlipidemia.

In another preferred embodiment, the lipid metabolism disorder is selected from the group consisting of high triglycerides, high cholesterol, low high and low density lipoprotein, high low density lipoprotein, or a combination thereof.

In another preferred embodiment, the hyperlipidemia is selected from the group consisting of high triglycerides, high cholesterol, low high and low density lipoprotein, high low density lipoprotein, or a combination thereof.

In another preferred embodiment, the prevention and/or treatment of fatty liver and/or its complications includes:

(i) Inhibit, alleviate and/or reverse the growth of fatty liver;

(ii) Reduce the weight of fatty liver;

(iii) Improve fatty degeneration of liver tissue; and/or

(iv) Inhibiting and/improving liver fat inflammation.

In another preferred embodiment, the prevention and/or treatment of fatty liver and/or its complications includes (i) inhibiting, alleviating and/or reversing the growth of fatty liver.

In another preferred embodiment, the prevention and/or treatment of fatty liver and/or its complications includes (ii) reducing the weight of fatty liver.

In another preferred embodiment, the prevention and/or treatment of fatty liver and/or its complications includes (iii) improving fatty degeneration of liver tissue.

In another preferred embodiment, the prevention and/or treatment of fatty liver and/or its complications includes (iv) inhibiting and/improving liver fatty inflammation.

In another preferred example, the improvement of fatty degeneration of liver tissue includes:

(a) Inhibit and/or reduce liver fat vacuoles.

In another preferred embodiment, the complications are selected from the group consisting of liver fibrosis, liver cirrhosis, liver cancer, diabetes, cardiovascular disease, kidney disease, colorectal cancer, endocrine disease

In another preferred embodiment, the diabetes is type 2 diabetes.

In another preferred embodiment, the composition or preparation also includes other drugs for preventing and/or treating fatty liver and/or its complications.

In another preferred embodiment, the other drugs for preventing and/or treating fatty liver and/or its complications are selected from the following group: liver protection drugs, insulin sensitizers, lipid-lowering drugs, weight loss drugs, or combinations thereof .

In another preferred embodiment, the liver protection drug is selected from the group consisting of polyene phosphatidylcholine, glutathione (GSH), silymarin (Ben), S-adenosylmethionine (methionine), Bicyclic alcohol, diammonium glycyrrhizinate, ursodeoxycholic acid (UDCA), or a combination thereof.

In another preferred embodiment, the insulin sensitizer is selected from the group consisting of metformin, peroxisome proliferation factor activated receptor (PPARs) agonists, PPARγ agonists, thiazolidinediones (TZDs), PPARα /δ agonist, glucagon-like peptide-1 (GLP-1) analog, or a combination thereof.

In another preferred embodiment, the lipid-lowering drug is selected from the following group: statins (such as atorvastatin, rosuvastatin), fibrates (such as clofibrate, ribet, bezafibrate) , Aluminum clofibrate and difibrate), or a combination thereof.

In another preferred embodiment, the lipid-lowering drug is selected from the group consisting of orlistat, lorcaserin, phentermine-topiramate compound preparation, naltrexone, bupropion, liraglutide, Or a combination.

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

In another preferred embodiment, the composition or preparation further includes a pharmaceutically, food, health care product or diet acceptable carrier.

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

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

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

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

In another preferred example, the "not containing lipid droplets" means that in the cell-free fat extract, the percentage of oil droplets in the total liquid is less than 1%, preferably less than 0.5%, more preferably less than 0.1%.

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

In another preferred example, the "cell-free" means that the average number of cells in 1 ml of acellular fat extract is ≤1, preferably ≤0.5, more preferably ≤0.1, or 0.

In another preferred embodiment, the cell-free fat extract is a naturally obtained nano-fat extract without added components.

In another preferred example, the "additive-free" means that no solution, solvent, small molecule, chemical agent, or biological additive is added during the preparation process of the fat extract except for the rinsing step.

In another preferred embodiment, the fat extract is prepared by centrifugation after emulsifying adipose tissue.

In another preferred embodiment, the fat extract contains but is not limited to one or more components selected from the following group: growth factors IGF-1, BDNF, GDNF, TGF-β, HGF, bFGF, VEGF, PDGF, EGF, NT-3, GH, G-CSF, or a combination thereof.

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

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

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

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

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

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

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

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

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

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

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, most preferably 45-55: 1.

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

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.

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, Best 1-1.5:1.

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. Better 4-6:1.

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

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, most preferably 0.7-1.2:1.

In another preferred embodiment, the acellular fat extract is liquid.

In another preferred embodiment, the acellular fat extract is prepared by the following method:

(1) Provide an adipose tissue raw material, cut the adipose tissue raw material, and rinse (such as using physiological saline) to obtain rinsed adipose tissue;

(2) Centrifuging the washed adipose 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 intermediate layer to obtain an emulsified fat mixture (also called nano fat);

(5) Centrifuging the emulsified fat mixture to obtain an intermediate liquid layer, which is the initial fat extract; and

(6) Filtering and sterilizing the initial fat extract to obtain a cell-free fat extract.

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

(2) Centrifuging the washed adipose tissue to obtain a layered mixture;

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

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

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

In another preferred example, the mechanical emulsification is performed by repeated pipetting (such as pipetting 20-200 times, preferably 20-150 times, more preferably 20-100 times, more preferably 30-50 times) through a syringe. emulsification.

In another preferred example, the method of pipetting is that two 10ml injection syringes are connected to a three-way pipe and repeatedly pumped at a constant speed.

In another preferred embodiment, in the step (4), the emulsification is a method of crushing by a tissue homogenizer.

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

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

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

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

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

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

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

In another preferred example, 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.

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

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 pack can remove the fat cells in the initial fat extract.

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

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

In another preferred example, the pore size of the microporous filter membrane is 0.05-0.8 μm, preferably 0.1-0.5 μm, more preferably 0.1-0.4 μm, more preferably 0.15-0.3 μm, more preferably 0.2-0.25μm, best 0.22μm.

In another preferred example, in the step (6), the filtration and sterilization are firstly passed through the first filter that can filter out cells, and then passed through the second filter that can filter out pathogens (such as bacteria). Filter (such as 0.22μm filter).

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

The third aspect of the present invention provides a composition or preparation comprising (a) the acellular fat extract according to the second aspect of the present invention; and/or (b) pharmacy, food, health products or dietary Accepted carrier or excipient.

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

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

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, cream, paste, cake, powder, Patches and so on.

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

The fourth aspect of the present invention provides a method for preparing a composition or preparation, the method comprising the steps of: combining the acellular fat extract described in the second aspect of the present invention with pharmacy, food, health products or diet The acceptable carriers or excipients are mixed to form a composition or preparation.

The fifth aspect of the present invention provides a method for preventing and/or treating fatty liver and/or its complications, the method comprising the steps of: administering the acellular fat extract of the second aspect of the present invention to a subject in need .

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

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

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as the embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, I will not repeat them one by one here.

Description of the drawings

Figure 1 shows the wet liver weights of mice in the CHOW group, PBS buffer group and CEFFE treatment group.

Figure 2 shows the histological staining of the liver of mice in the CHOW group, PBS buffer group and CEFFE treatment group.

Figure 3 shows the void area in the liver of mice in the CHOW group, PBS buffer group and CEFFE treatment group.

Detailed ways

After extensive and in-depth research, the inventors developed for the first time an acellular fat extract that can effectively treat fatty liver and/or its complications. In the present invention, animal experiments have proved that the acellular fat extract of the present invention can effectively prevent and/or treat fatty liver and/or its complications. The present invention has been completed on this basis.

the term

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

As used herein, the term "containing" or "including (including)" can be open, semi-closed, and closed. In other words, the term also includes "substantially consisting of" or "consisting of".

As used herein, the terms "fatty liver and/or its complications" and "fatty liver and/or fatty liver complications" are used interchangeably.

As used in the text, the term "IGF-1" is called insulin-like growth factors-1 (insulin-like growth factors-1).

As used in the text, the term "BDNF" is called brain-derived neurotrophic factor (BDNF).

As used in the text, the term "GDNF" is referred to as glialcellline-derived neurotrophic factor.

As used in the text, the term "bFGF" is called basic fibroblast growth factor (basic fibroblast growth factor).

As used in the text, the term "VEGF" is called vascular endothelial growth factor (vascular endothelial growth factor).

As used in the text, the term "TGF-β1" is called transforming growth factor-β1 (transforming growth factor-β1).

As used in the text, the term "HGF" is called hepatocyte growth factor

As used in the text, the term "PDGF" is called platelet-derived growth factor (Platelet-derived growth factor)

As used in the text, the term "EGF" is called Epidermal Growth Factor (Epidermal Growth Factor)

As used in the text, the term "NT-3" is called neurotrophins-3 (neurotrophins-3).

As used in the text, the term "GH" is called Growth Hormone.

As used in the text, the term "G-CSF" is called granulocyte colony stimulating factor (granulocyte colony stimulating factor).

Cell-free fat extract and preparation method thereof

As used herein, the terms "the cell-free fat extract of the present invention", "the extract of the present invention", "the fat extract of the present invention" and the like are used interchangeably and refer to the preparation process of the fat extract (except for the rinsing step). ) Extracts (or extracts) derived from adipose tissue prepared without adding any solutions, solvents, small molecules, chemical agents, and biological additives. A typical method for preparing the extract of the present invention is as described above in the second aspect of the present invention. In addition, it should be understood that although the extract of the present invention does not need to add any additives (or added ingredients) during the preparation process, 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) water).

The acellular fat extract of the present invention can be derived from human adipose tissue. It is purified from nano fat by removing oil and cell/extracellular matrix after centrifugation. It is a cell-free, easy to prepare, and rich in various types of fat. Kind of growth factor liquid.

In a preferred embodiment of the present invention, the acellular fat extract is an acellular fat extract.

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

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

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

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

(2) Centrifuging the washed adipose tissue to obtain a layered mixture;

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

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

Fatty liver and/or its complications

Fatty liver (fatty liver) refers to the pathological changes of excessive accumulation of fat in liver cells caused by various reasons. It is a common liver pathological change. Normally, normal human liver tissue contains a small amount of fat, such as triglycerides, phospholipids, glycolipids, and cholesterol, and its weight is about 3% to 5% of the liver weight. If the accumulation of fat in the liver is too much, it will exceed the weight of the liver. When 5% or more than 50% of liver cells have fatty degeneration in histology, it can be called fatty liver.

The formation of fatty liver is usually related to factors such as obesity, high-fat diet, and alcohol consumption. These factors lead to excessive accumulation of fat in liver cells, which leads to fatty liver. According to the classification of causes, fatty liver disease can be divided into alcoholic fatty liver disease (Alcoholic fatty liver disease) and non-alcoholic fatty liver disease (Non-alcoholic fatty liver disease, NAFLD). Among them, non-alcoholic fatty liver disease is a clinicopathological syndrome characterized by diffuse hepatocyte bullous steatosis without a history of excessive drinking. As the disease progresses, it can develop into non-alcoholic hepatitis (NASH), liver Cirrhosis, liver cancer, etc.

Representatively, the fatty liver of the present invention includes (but is not limited to) one or more fatty livers of the following group: alcoholic fatty liver, non-alcoholic fatty liver, fatty liver caused by high-fat diet or obesity, fatty liver Fatty liver caused by metabolic disorders.

In a preferred example, fatty liver caused by lipid metabolism disorder is lipid metabolism disorder in the blood and/or lipid metabolism disorder in the liver.

Typically, the lipid metabolism disorder includes (but is not limited to): high triglycerides, high cholesterol, low high and low density lipoprotein, high low density lipoprotein, or a combination thereof.

In another preferred embodiment of the present invention, the fatty liver complications include (but are not limited to): liver fibrosis, liver cirrhosis, liver cancer, diabetes, cardiovascular disease, kidney disease, colorectal cancer, endocrine disease

In another preferred embodiment, the diabetes is type 2 diabetes.

use

The acellular fat extraction of the present invention can be used to prevent and/or treat fatty liver and/or its complications.

As used herein, the term "prevention" refers to a method of preventing the onset of a disease and/or its accompanying symptoms or protecting a subject from acquiring the disease. As used herein, "prevention" also includes delaying the onset of the disease and/or its accompanying symptoms and reducing the risk of a subject's disease.

As used herein, the term "treatment" includes delaying and stopping the progression of the disease, or eliminating the disease, and does not require 100% inhibition, elimination, and reversal. In some embodiments, compared with the level observed in the absence of the acellular fat extraction of the invention, the acellular fat extraction or composition of the invention reduces, inhibits and suppresses fatty liver and/or its complications. /Or reversal, for example, at least about 10%, at least about 30%, at least about 50%, or at least about 80%.

Representatively, the acellular fat extraction described in the present invention prevents and/or treats fatty liver and/or its complications through one or more methods including (but not limited to) the following groups:

(i) Inhibit, alleviate and/or reverse the growth of fatty liver;

(ii) Reduce the weight of fatty liver;

(iii) Improve fatty degeneration of liver tissue; and/or

(iv) Inhibiting and/improving liver fat inflammation.

As used herein, the term "reversal" includes the elimination of diseases that have already appeared, and does not require 100% elimination and reversal. In some embodiments, the acellular fat extraction or composition of the invention reverses fatty liver, for example, at least about 10%, at least about 10%, compared to the level observed in the absence of the acellular fat extraction of the invention. 30%, at least about 50%, or at least about 80%.

As used herein, the term "alleviation" includes reducing the development of the disease, alleviating the symptoms of the disease, and the like.

In a preferred embodiment of the present invention, the steatosis of liver tissue can be improved by inhibiting and/or reducing liver fat vacuoles.

The present invention also provides a method for preventing and/or treating fatty liver and/or its complications. The method includes the step of administering the acellular fat extract of the present invention to a desired subject, thereby treating fatty liver.

In a preferred example, the object is a human or non-human animal.

In another preferred embodiment, the non-human animals are rats, mice, dogs, cats, cows, chickens, ducks and the like.

Composition and application

The composition of the present invention includes (but is not limited to): pharmaceutical composition, food composition, health care composition, dietary supplement and the like.

Representatively, the acellular fat extract of the present invention can be prepared into pharmaceutical compositions, such as tablets, capsules, powders, microparticles, solutions, lozenges, jellies, cream preparations, spirits, suspensions, Dosage forms such as tinctures, poultices, liniments, lotions, and aerosols. The pharmaceutical composition can be prepared by generally known preparation techniques, and suitable pharmaceutical additives can be added to the drug.

The composition of the present invention may also include a pharmaceutically, food, health care product or dietary acceptable carrier. "Pharmaceutically, food, health product or dietary acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity And sufficiently low toxicity. "Compatibility" here means that the components in the composition can be blended with the compound of the present invention and between them without significantly reducing the efficacy of the compound. Examples of acceptable carriers for pharmaceutically, food, health care products or diets are cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.) , Gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol) Etc.), emulsifiers (such as Tween

), wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

The composition of the present invention may also include other drugs for preventing and/or treating fatty liver and/or its complications. Preferably, the other drugs for preventing and/or treating fatty liver and/or its complications are selected from the following group: liver protection drugs, insulin sensitizers, lipid lowering drugs, weight loss drugs, or a combination thereof.

Representatively, the liver protection includes (but is not limited to): polyene phosphatidylcholine, glutathione (GSH), silymarin (Ben), S-adenosylmethionine (methionine), bicyclic Alcohol, diammonium glycyrrhizinate, ursodeoxycholic acid (UDCA), or a combination thereof.

Representatively, the insulin sensitizer is selected from the following group: metformin, peroxisome proliferation factor activated receptor (PPARs) agonist, PPARγ agonist thiazolidinediones (TZDs), PPARα/δ agonist Agent, glucagon-like peptide-1 (GLP-1) analog, or a combination thereof.

Representatively, the lipid lowering includes (but is not limited to): statins (such as atorvastatin, rosuvastatin), fibrates (such as clofibrate, liberate, bezafibrate, Aluminum clofibrate and difibrate), or a combination thereof.

Typically, the lipid lowering includes (but is not limited to): orlistat, lorcaserin, phentermine-topiramate compound preparation, naltrexone, bupropion, liraglutide, or Its combination.

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

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 mixed with the following ingredients: (a) fillers or compatibilizers, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (c) humectants, For example, glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) Absorption accelerators, such as quaternary amine compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, hard Calcium fatty acid, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain buffering agents.

Solid dosage forms such as tablets, sugar pills, capsules, pills and granules can be prepared with coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers.

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

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

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

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

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

The acellular fat extract of the present invention can be administered or administered alone, or in combination with other drugs for preventing and/or treating fatty liver and/or its complications.

When the composition is administered, a safe and effective amount of the acellular fat extract of the present invention is applied to human or non-human animals (such as rats, mice, dogs, cats, cows, chickens, ducks, etc.) in need of treatment, wherein The current dose is the effective dose that is acceptable in pharmacy, food or health care products. As used herein, the term "safe and effective amount" refers to an amount that produces function or activity on humans and/or animals and can be accepted by 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. It's different. For example, for a person with a body weight of 60 kg, the daily dose is usually 0.1 to 1000 mg, preferably 1 to 600 mg, and more preferably 2 to 300 mg. Of course, the specific dosage should also consider factors such as the route of administration and the patient's health status, which are all within the skill range of a skilled physician.

The present invention will be further explained 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 that do not indicate specific conditions in the following examples usually follow the conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

Example 1

1. Experimental method

1.1 Preparation of Cell free fat extract (CEFFE)

Fat is obtained by volunteers with informed consent. The preparation method of acellular adipose tissue extract is as follows:

(1) Take the fat obtained by suction or surgical resection, cut it into pieces and rinse it with normal saline 3 times.

(2) Take the rinsed adipose tissue, place it in a centrifuge tube, put it in a centrifuge and centrifuge at 1200g for 3 minutes to obtain a layered mixture.

(3) For the layered mixture, the upper oil layer and the lower water layer are removed, and the middle layer (that is, the fat layer containing fat cells) is collected.

(4) For the middle layer, use two 10ml injection syringes connected to a three-way tube to repeatedly push 30 times at a constant speed to perform mechanical emulsification and obtain a mechanically emulsified fat mixture (also called nanofat).

(5) Put the mechanically emulsified fat mixture into a refrigerator at -80°C and freeze it in a 37°C water bath. After a single freeze-thaw cycle, centrifuge the thawed fat mixture at 1200g for 5 minutes to obtain a layered Mixture, the layered mixture is divided 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, and the fourth layer is the cell/tissue debris sedimentation layer to remove the oil layer and residual fat The tissue layer, the liquid layer is sucked, and the contamination of the cell/tissue debris sediment layer is avoided during the sucking process, so as to obtain the initial fat extract.

(6) Filter and sterilize the obtained primary fat extract through a 0.22μm filter to sterilize and remove the living cells that may be mixed, thereby obtaining a cell-free fat extract, and store it in aliquots and freeze at -20°C. When using Thaw at 4°C.

For the prepared cell-free fat extract, use an ELISA immunosorbent assay kit to detect the content of cytokines, including IGF-1, BDNF, GDNF, bFGF, VEGF, TGF-β1, HGF, PDGF and other cytokines. The average concentrations of 6 samples were as follows: IGF-1 (9840.6pg/ml), BDNF (1764.5pg/ml), GDNF (1831.9pg/ml), bFGF (242.3pg/ml), VEGF (202.9pg/ml), TGF-β1 (954.5pg/ml), HGF (898.4pg/ml), PDGF (179.9pg/ml).

1.2 Establishment, grouping and administration of mouse fatty liver model

6-week-old C57 mice were purchased from Shanghai Experimental Animal Center. The mouse fatty liver model was induced by a high-fat diet for 4 consecutive weeks to obtain fatty liver model mice. 10 fatty liver model mice were randomly divided into two groups, the PBS buffer group and the CEFFE treatment group, 5 in each group. In addition, 5 normal healthy mice that were not induced by a high-fat diet were used as the normal feeding group (CHOW Group). Fatty liver model mice in the CEFFE treatment group were treated with tail vein injection of CEFFE at a dose of 250 μl. Fatty liver model mice in the PBS buffer group were treated with tail vein injection of PBS buffer. The treatment cycle was 30 days and the drug was administered every four days. The mice in the PBS buffer group and the CEFFE treatment group continued to be given a high-fat diet during the administration period. In addition, the mice in the normal feeding group (CHOW group) were given a normal healthy diet as a blank control.

Among them, the PBS buffer injection group was used as a negative control, and the CHOW group was used as a blank control.

1.3 Statistical analysis

The data was tested by SPSS software by one-way ANOVA, and statistical analysis was performed on the significance of the data differences. The results were expressed as mean±standard deviation.

2. Experimental results

2.1 Mouse liver weight determination

After the mice in each group were sacrificed on the 30th day of treatment, the liver was stripped, and the wet weight of the liver was weighed. The results are shown in Table 1 and Figure 1.

Table 1 Wet liver weight of mice in different experimental groups (Mean±SD, n=5)

实验组组Experimental group	CHOW组CHOW group	PBS缓冲液组PBS buffer group	CEFFE治疗组CEFFE treatment group
Mean±SD(g)Mean±SD(g)	1.35±0.101.35±0.10	2.28±0.272.28±0.27	1.80±0.331.80±0.33

Remarks: CHOW vs.PBS, p value = 0.0002** (<0.01); CHOW vs. CEFFE, p value = 0.0474* (<0.5); PBS vs. CEFFE, p value = 0.0318* (<0.5).

It can be seen from Table 1 and Figure 1 that the wet liver weight of fatty liver mice increased significantly. Compared with the PBS buffer group, the wet weight of the liver in the CEFFE treatment group decreased significantly, indicating that CEFFE has a significant therapeutic effect on fatty liver.

2.2 Histological evaluation

On the 30th day of treatment, the mice were sacrificed, the liver was stripped, part of the liver tissue was immersed in 4% paraformaldehyde, fixed for 24 hours, and then embedded in paraffin. Paraffin sections were routinely performed, H&E stained, and photographed under an optical microscope (3 random fields/specimen ), Image J software calculates the area of the cavitation area, and the results are shown in Table 2 and Figure 2-3.

Table 2 The area of vacuoles in the liver of mice in different experimental groups (Mean±SD, n=3)

实验组test group	CHOW组CHOW group	PBS缓冲液组PBS buffer group	CEFFE治疗组CEFFE treatment group
Mean±SD(μm ²) Mean±SD(μm ² )	2.51±0.892.51±0.89	424.40±62.25424.40±62.25	219.30±52.76219.30±52.76

CHOW vs.PBS, p<0.001**; CHOW vs.CEFFE, p<<0.001*; PBS vs.CEFFE<0.001**.

As can be seen from Table 2, Figure 2 and Figure 3, compared with CHOW, large areas of fatty vacuoles with local inflammation were seen in the PBS buffer group, while the CEFFE treatment group had significantly reduced liver fatty vacuoles and reduced inflammation, indicating that CEFFE It can improve liver steatosis in fatty liver mice and is used to treat fatty liver.

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

Claims

A use of acellular fat extract, which is characterized in that it is used to prepare a composition or preparation, and the composition or preparation is used to prevent and/or treat fatty liver and/or its complications.
The use according to claim 1, wherein the fatty liver is alcoholic or non-alcoholic fatty liver.
The use according to claim 1, wherein the fatty liver is fatty liver caused by a high-fat diet or obesity.
The use according to claim 1, wherein the fatty liver is fatty liver caused by lipid metabolism disorder.
The use according to claim 4, wherein the lipid metabolism disorder is selected from the group consisting of high triglycerides, high cholesterol, low high and low density lipoproteins, high low density lipoproteins, or combination.
The use according to claim 1, wherein the prevention and/or treatment of fatty liver and/or its complications comprises:

(i) Inhibit, alleviate and/or reverse the growth of fatty liver;

(ii) Reduce the weight of fatty liver;

(iii) Improve fatty degeneration of liver tissue; and/or

(iv) Inhibiting and/improving liver fat inflammation.
The use according to claim 1, wherein the complications are selected from the group consisting of liver fibrosis, cirrhosis, liver cancer, diabetes, cardiovascular disease, kidney disease, colorectal cancer, endocrine disease.
The use according to claim 1, wherein the composition or preparation further includes other drugs for preventing and/or treating fatty liver and/or its complications; preferably, the other preventing and/or The medicine for treating fatty liver and/or its complications is selected from the following group: liver protection medicine, insulin sensitizer, lipid-lowering medicine, weight loss medicine, or a combination thereof.
The use according to claim 8, wherein the hepatoprotective drug is selected from the group consisting of polyene phosphatidylcholine, glutathione, silymarin, S-adenosylmethionine, bicyclic alcohol, Diammonium glycyrrhizinate, ursodeoxycholic acid, or a combination thereof;

The insulin sensitizer is selected from the following group: metformin, peroxisome proliferation factor activated receptor agonist, PPARγ agonist thiazolidinediones, PPARα/δ agonist, glucagon-like peptide-1 Analogues, or combinations thereof;

The lipid-lowering drugs are selected from the following group: statins (such as atorvastatin, rosuvastatin), fibrates (such as clofibrate, liberate, bezafibrate, aluminum clofibrate and Dibet), or a combination thereof; and/or

The lipid-lowering drug is selected from the following group: oserit, liraglutide, or a combination thereof.
The use according to claim 1, wherein the composition or preparation comprises a pharmaceutical composition or preparation, a food composition or preparation, a health care product composition or preparation, or a dietary supplement.
The use according to claim 1, wherein the dosage form of the composition or preparation is an oral preparation, an external preparation or an injection preparation.
The use according to claim 1, wherein the acellular fat extract is prepared by the following method:

(1) Provide an adipose tissue raw material, cut the adipose tissue raw material, and rinse (such as using physiological saline) to obtain rinsed adipose tissue;

(2) Centrifuging the washed adipose 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 intermediate layer to obtain an emulsified fat mixture (also called nano fat);

(5) Centrifuging the emulsified fat mixture to obtain an intermediate liquid layer, which is the initial fat extract; and

(6) Filtering and sterilizing the initial fat extract to obtain a cell-free fat extract.
The use according to claim 1, wherein the acellular fat extract includes one or more characteristics selected from the following group:

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 /ml, more preferably 9000-11000pg/ml, more preferably 9500-10500pg/ml;

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

In the acellular fat extract, the concentration of the GDNF is 800-5000pg/ml, preferably 1000-4000pg/ml, more preferably 1200-2500pg/ml, more preferably 1400-2000pg/ml , Better 1600-2000pg/ml, better 1700-1900pg/ml;

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

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

In the cell-free fat extract, the concentration of TGF-β1 is 200-3000pg/ml, preferably 400-2000pg/ml, more preferably 600-1500pg/ml, more preferably 800-1200pg /ml, more preferably 800-1100pg/ml, more preferably 900-1000pg/ml;

In the acellular fat extract, the concentration of the HGF is 200-3000pg/ml, preferably 400-2000pg/ml, more preferably 600-1500pg/ml, more preferably 600-1200pg/ml , More preferably 800-1000pg/ml, more preferably 850-950pg/ml; 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 170-190pg/ml.
The use according to claim 1, wherein the acellular fat extract includes one or more characteristics selected from the following group:

The weight ratio of IGF-1 to VEGF is 20-100:1, preferably 30-70:1, more preferably 40-60:1, most preferably 45-55:1;

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;

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;

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;

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, more preferably 4- 6:1;

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, more preferably 4-5.5: 1; and/or

The weight ratio of PDGF to VEGF is 0.1-3:1, preferably 0.2-2:1, more preferably 0.4-1.5:1, and most preferably 0.7-1.2:1.
A method for preventing and/or treating fatty liver and/or its complications, characterized in that the method comprises the steps of: administering a cell-free fat extract to a subject in need.