WO2022170940A1

WO2022170940A1 - Cell-free fat extract for use in improving aging and promoting skin rejuvenation

Info

Publication number: WO2022170940A1
Application number: PCT/CN2022/073020
Authority: WO
Inventors: 张文杰; 蔡宜佐
Original assignee: 上海萨美细胞技术有限公司
Priority date: 2021-02-10
Filing date: 2022-01-20
Publication date: 2022-08-18
Also published as: CN114903920A

Abstract

Provided are a cell-free fat extract, and a use thereof in improving aging and promoting skin rejuvenation. The cell-free fat extract may be used for preparing a composition or preparation, and the composition or preparation is used for improving skin aging and/or promoting skin rejuvenation.

Description

Cell-Free Fat Extracts for Aging Improvement and Skin Rejuvenation

technical field

The invention relates to the field of medicine, in particular to the use of cell-free fat extracts for improving aging and promoting skin rejuvenation.

Background technique

Skin aging is an inevitable process. The early manifestations of skin aging are mainly reflected in periorbital aging, manifested as periorbital wrinkles, skin sagging, decreased elasticity and pigmentation. The current treatment of skin aging (such as periorbital aging) mainly includes surgical treatment and non-surgical treatment. However, due to the problems of long recovery period and scarring in surgical treatment, the research focus is on non-surgical treatment.

Non-surgical treatment includes physical therapy and biological therapy. The former is mainly laser therapy, which can produce thermal effects on tissues and stimulate local collagen synthesis through selective photothermal action, thereby achieving the effect of treating periorbital aging. However, in clinical applications Among them, it was found that the laser effect lasted for a short time, and it usually returned to the preoperative state soon after surgery, and there was no clear laser treatment plan for periorbital rejuvenation. There is a lack of an effective drug for the treatment of skin aging in the prior art, and the development of an effective drug for the treatment of skin aging has become a hot research topic today.

Therefore, there is a need in the art to develop a drug that can effectively improve skin aging and promote skin rejuvenation.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide the use of a cell-free fat extract in improving skin aging and/or promoting skin rejuvenation.

A first aspect of the present invention provides the use of a cell-free fat extract for preparing a composition or formulation for improving skin aging and/or promoting skin rejuvenation.

In another preferred embodiment, the skin aging includes periorbital skin aging.

In another preferred embodiment, described skin aging includes one or more manifestations selected from the following group:

(a) an increase in the number of skin wrinkles;

(b) increased skin roughness;

(c) reduced skin smoothness;

(d) reduced skin elasticity;

(e) loose skin; and/or

(f) Dehydration of the skin.

In another preferred embodiment, the wrinkles include fine lines and/or coarse lines.

In another preferred embodiment, the improvement of skin aging and/or the promotion of skin rejuvenation comprises one or more methods selected from the following group:

(i) reduce the number of skin wrinkles;

(ii) reduce skin roughness;

(iii) improve the smoothness of the skin;

(iv) increase skin elasticity;

(v) reduce skin moisture loss; and/or

(vi) Improve the water retention capacity of the skin.

In another preferred embodiment, the improving skin aging and/or promoting skin rejuvenation has one or more characteristics selected from the group consisting of:

(i) skin roughness parameters decreased by more than 20% after 12 months of follow-up;

(ii) The total elasticity value R2 increased by more than 20% after 12 months of follow-up;

(iii) After 12 months of follow-up, the R5 of the net elasticity value increased by more than 60%;

(iv) The elasticity value R7 increased by more than 30% after 12 months of follow-up;

(v) Transepidermal water loss did not exceed 40% after 12 months of follow-up.

In another preferred embodiment, the cell-free fat extract is a cell-free fat extract prepared from human or non-human mammalian fat.

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

In another preferred example, the compositions or preparations include pharmaceutical compositions or preparations, food compositions or preparations, health product compositions or preparations, or dietary supplements.

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

In another preferred embodiment, the composition or preparation further includes other drugs for improving skin aging and/or promoting skin rejuvenation.

In another preferred example, the other drugs for improving skin aging and/or promoting skin rejuvenation are selected from the group consisting of skin fillers, anti-wrinkle drugs, and ingredients for nourishing skin

In another preferred embodiment, the skin filling material is selected from the group consisting of hyaluronic acid, collagen, or a combination thereof.

In another preferred embodiment, the anti-wrinkle drug includes botulinum toxin.

In another preferred example, the ingredients for nourishing the skin include vitamins.

In another preferred embodiment, the vitamin component is selected from the group consisting of vitamin B1, vitamin B2, vitamin B12, vitamin C, vitamin D, vitamin E, or a combination thereof.

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 injection preparation is an intravenous injection or an intramuscular injection.

In another preferred embodiment, the injection preparation is an intradermal injection preparation.

In another preferred embodiment, the injection preparation is an intradermal injection preparation of periorbital skin.

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

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 embodiment, the composition or preparation is administered externally, topically, or by 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 disrupted.

In another preferred embodiment, the "does not contain lipid droplets" means that in the cell-free fat extract, the volume of oil droplets accounts for less than 1% of the total liquid, preferably less than 0.5%, 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 embodiment, the "cell-free" refers to the average number of cells in 1 ml of cell-free fat extract ≤1, preferably ≤0.5, more preferably ≤0.1, or 0.

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

In another preferred example, the "no added components" means that, except for the rinsing step, no solution, solvent, small molecule, chemical agent, and biological additive are added during the preparation of the fat extract.

In another preferred embodiment, the cell-free adipose extract is prepared by centrifuging adipose tissue after emulsification.

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

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

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

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

In another preferred example, 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 1400-2000 pg/ml, more preferably 1600-2000 pg/ml, more preferably 1700-1850 pg/ml.

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

In another preferred example, in the cell-free fat extract, the concentration of the bFGF is 50-600pg/ml, preferably 100-500pg/ml, more preferably 120-400pg/ml, more Preferably 150-300 pg/ml, more preferably 200-280 pg/ml, more preferably 220-260 pg/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 Preferably 150-250 pg/ml, more preferably 170-230 pg/ml, more preferably 190-210 pg/ml.

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

In another preferred example, 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 140-220 pg/ml, more preferably 160-200 pg/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 40-60:1, 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 embodiment, the weight ratio of GDNF to VEGF is 2-20:1, preferably 4-15:1, more preferably 6-12:1, and most preferably 8.5-9.5:1.

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, Optimally 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, preferably 4-6:1.

In another preferred embodiment, the weight ratio of HGF to VEGF is 1-20:1, preferably 1-15:1, more preferably 1-10:1, more preferably 2-8:1, 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, and most preferably 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 raw material, crushing the adipose tissue raw material, and rinsing (such as with physiological saline), thereby obtaining rinsed adipose tissue;

(2) centrifuging the rinsed 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 (ie, the fat layer containing adipocytes);

(4) Emulsifying the intermediate layer to obtain an emulsified fat mixture (also referred to as nano-fat);

(5) subjecting the emulsified fat mixture to centrifugation to obtain an intermediate liquid layer, i.e., the initial fat extract; and

(6) Filtration and sterilization of the primary fat extract to obtain a cell-free fat extract.

A second aspect of the present invention provides 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;

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

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 minutes, preferably 1-10 minutes, more preferably 1-8 minutes, and optimally 1-5 minutes.

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

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

In another preferred embodiment, the mechanical emulsification is performed mechanically by repeated blowing through a syringe (eg 20-200 times, preferably 20-150 times, more preferably 20-100 times, more preferably 30-50 times). emulsification.

In another preferred example, the method of blowing and beating is that two 10ml injection syringes are connected with a tee tube to repeatedly push and beat at a constant speed.

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

In another preferred example, in the step (5), before the emulsified fat mixture is subjected to centrifugal treatment, the emulsified fat mixture is further frozen and then thawed.

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

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 embodiment, 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 layered into four layers, the first layer is an oil layer, the second layer is a residual adipose tissue layer, and the third layer is a liquid layer layer (ie, the middle liquid layer), and the fourth layer is the cell/tissue debris sedimentation 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 minutes, preferably 1-10 minutes, more preferably 2-8 minutes, and optimally 3-7 minutes.

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 bag can remove the adipocytes in the primary fat extract.

In another preferred example, in the step (6), the filtration and sterilization are performed through a filter (eg, 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 0.2-0.25 μm, optimally 0.22 μm.

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

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

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

The fourth aspect of the present invention provides a composition or preparation for improving skin aging and/or promoting skin rejuvenation, the composition or preparation comprising (a) the acellular fat according to the third aspect of the present invention an extract; and (b) a pharmaceutically, food, nutraceutical or dietary acceptable carrier or excipient.

In another preferred example, the composition is a pharmaceutical composition, a food composition, a health product composition or a dietary supplement.

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

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

The fifth aspect of the present invention provides a method for preparing the composition or preparation according to the fourth aspect of the present invention, the method comprising the steps of: mixing the cell-free fat extract according to the third aspect of the present invention with a pharmaceutical It is mixed with acceptable carriers or excipients on food, health product or diet to form a composition or preparation.

The sixth aspect of the present invention provides a method for improving skin aging and/or promoting skin rejuvenation, by applying the cell-free fat extract according to the third aspect of the present invention to a subject in need.

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

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

In another preferred embodiment, the administration mode is oral administration, topical administration or injection administration.

In another preferred embodiment, the injection is intradermal injection.

In another preferred embodiment, the injection is intradermal injection of the periorbital skin.

In another preferred embodiment, the dose administered by injection is 0.05-0.2 mL of acellular fat extract/1 cm of skin.

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 (eg, the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, it is not repeated here.

Description of drawings

Figure 1 is a photo of the periorbital skin after CEFFE treatment, where Pre-treatment is before treatment, 3M-fellow up, 6M-fellow up and 12M-fellow up are follow-up at 3, 6 and 12 months after treatment, respectively.

Figure 2 shows the change in skin roughness of the periorbital skin after CEFFE treatment, where Pre represents before CEFFE administration, 1st, 2nd, 3rd, 4th and 5th are the 1st, 2nd, 3rd, 4th and 5th CEFFE administration, respectively Medication time, 3m, 6m and 12m were 3, 6 and 12 months after the end of treatment, respectively.

Figure 3 shows the changes of skin elasticity of the periorbital skin after CEFFE treatment, where Pre represents the time before CEFFE administration, 1st, 2nd, 3rd, 4th and 5th are the 1st, 2nd, 3rd, 4th and 5th times of CEFFE administration, respectively , 3m, 6m and 12m were 3, 6 and 12 months after the end of treatment, respectively.

Figure 4 shows the water loss of the periorbital skin after CEFFE treatment. Among them, Pre represents the time before CEFFE administration, 1st, 2nd, 3rd, 4th, and 5th are the 1st, 2nd, 3rd, 4th, and 5th times of CEFFE administration, respectively, and 3m, 6m, and 12m are the 3rd, 6th, and 6th time after the treatment, respectively. and December.

Detailed ways

After extensive and in-depth research, the present inventors have developed for the first time that a cell-free fat extract has an excellent improving effect on skin aging. The research of the examples of the present invention shows that the cell-free fat extract of the present invention has an excellent therapeutic effect on skin aging. 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 one of ordinary skill in the art to which this invention belongs.

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

As used in the text, the term "IGF-1" is referred to as insulin-like growth factors-1.

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

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

As used in the text, the term "bFGF" is referred to as basic fibroblast growth factor.

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

As used herein, the term "TGF-β1" is referred to as transforming growth factor-β1.

As used in the text, the term "HGF" is referred to as Hepatocyte Growth Factor

As used in the text, the term "PDGF" is referred to as Platelet derived growth factor

As used in the text, the term "EGF" is referred to as Epidermal Growth Factor

As used in the text, the term "NT-3" is referred to as neurotrophins-3.

As used in the text, the term "GH" is referred to as Growth Hormone.

As used in the text, the term "G-CSF" is referred to as granulocyte colony stimulating factor.

Cell free fat extract (CEFFE) and preparation method thereof

As used herein, the terms "cell-free adipose extract of the present invention", "extract of the present invention", "fat extract of the present invention" and the like are used interchangeably to refer to the process during the preparation of the fat extract (other than the rinsing step) ) An adipose tissue-derived extract (or extract) prepared without the addition of any solutions, solvents, small molecules, chemicals, 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 have to add any additives (or added components) during the preparation process, some or small amounts of safe substances (such as small amounts) that do not negatively or adversely affect the activity of the extract of the present invention may also be added. water).

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

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

In the cell-free adipose extract of the present invention, various cytokines may be included. Typically, the cell-free adipose extract comprises IGF-1, BDNF, GDNF, TGF-β, HGF, bFGF, VEGF, TGF-β1, PDGF, EGF, NT-3, GH and G-CSF one or more.

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

Typically, the cell-free fat extracts of the present invention are prepared by the following methods:

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

In another preferred example, the blowing method is to repeatedly push and beat at a constant speed with two 10ml injection syringes connected to a tee tube.

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

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

use

The present invention provides the use of a cell-free fat extract for the preparation of a composition or formulation for improving skin aging and/or promoting skin rejuvenation.

In the present invention, the term "improving" includes prevention and/or treatment.

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

"Treatment" as used in the present invention includes delaying and stopping the progression of the disease, or eliminating the disease, and does not require 100% inhibition, elimination and reversal. In some embodiments, the cell-free fat extract of the present invention reduces, inhibits and/or reverses skin aging, eg, by at least about 10%, at least about 30%, at least about 50%, or at least about 80%.

In the present invention, the aging skin site is not particularly limited. Preferably, the skin aging includes periorbital skin aging.

In a preferred embodiment of the present invention, described skin aging includes one or more manifestations selected from the following group:

(a) an increase in the number of skin wrinkles;

(b) increased skin roughness;

(c) reduced skin smoothness;

(d) reduced skin elasticity;

(e) loose skin; and/or

(f) Dehydration of the skin.

In a preferred embodiment of the present invention, the improvement of skin aging and/or the promotion of skin rejuvenation comprises one or more methods selected from the group consisting of:

(i) reduce the number of skin wrinkles;

(ii) reduce skin roughness;

(iii) improve the smoothness of the skin;

(iv) increase skin elasticity;

(v) reduce skin moisture loss; and/or

(vi) Improve the water retention capacity of the skin.

The present invention also provides a method of improving skin aging and/or promoting skin rejuvenation by administering the cell-free fat extract of the present invention to a subject in need thereof.

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

In another preferred embodiment, the injection is intradermal injection.

Composition and Administration

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

Typically, the cell-free fat extracts of the present invention can be prepared into pharmaceutical compositions such as tablets, capsules, powders, microparticles, solutions, lozenges, jellies, creams, elixirs, suspensions, Dosage forms such as tinctures, poultices, liniments, lotions, and aerosols. Pharmaceutical compositions can be prepared by generally known preparation techniques, and suitable pharmaceutical additives can be added to the medicament.

The composition of the present invention may also include a pharmaceutically, food, health product or dietary acceptable carrier. "Pharmaceutically, food, nutraceutical or dietary acceptable carrier" means: one or more compatible solid or liquid filler or gel substances, which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. "Compatibility" as used herein means that the components of the composition can be admixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Pharmaceutical, food, health product or dietary acceptable carrier Examples of acceptable carrier moieties 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

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

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, and preferred modes of administration are oral administration and injection. For example, the injection administration is intradermal injection, preferably intradermal injection of the periorbital skin.

The dosage form of the composition or preparation of the present invention is an oral preparation, an external preparation or an injection preparation. Typically, solid dosage forms for oral administration or administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with (a) fillers or compatibilizers, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as, for example, hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, For example, glycerol; (d) disintegrants, such as agar, calcium carbonate, potato 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, dragees, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials well known in the art. They may contain opacifying agents.

Liquid dosage forms for oral administration or administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally employed 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.

Besides these inert diluents, the compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.

In addition to the active ingredient, suspensions may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances 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 non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof. Typically, the injection preparation is an intradermal injection preparation, preferably, the injection preparation is an intradermal injection preparation of the periorbital skin.

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

The cell-free adipose extract of the present invention may 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 cell-free fat extract of the present invention is suitable for human or non-human animals (such as rats, mice, dogs, cats, cows, chickens, ducks, etc.) in need of treatment, wherein the administration The current dose is the effective dose that can be considered as 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 in humans and/or animals and is acceptable to humans and/or animals. Those of ordinary skill in the art should understand that the "safe and effective amount" may vary with 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, etc. different. For example, for a person weighing 60 kg, the daily dose is usually 0.1 to 1000 mg, preferably 1 to 600 mg, more preferably 2 to 300 mg. Of course, the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.

The main advantages of the present invention include:

1. The present invention finds for the first time that the cell-free fat extract has an excellent therapeutic effect on skin aging and promotes skin rejuvenation.

2. The cell-free fat extract of the present invention is a cell-free component that can avoid cell-related problems in clinical applications, including, for example, genetic stability after cell processing, cell viability and viability after injection, The multiple administration and storage of cells, and the immunogenicity of cells when using allogeneic fat, the cell-free fat extract of the present invention has the advantages of higher safety and lower side effects in the preparation and prevention of skin aging .

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples are usually in accordance with conventional conditions, or in accordance with the conditions suggested by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

Example 1

1. Preparation of Cell-Free Fat Extract (CEFFE)

Fat was obtained from volunteers with informed consent. The preparation method of cell-free adipose tissue extract is as follows:

(1) Adipose tissue was obtained from 6 healthy women who underwent conventional liposuction, with an average age of 31 years (24-36 years). After local injection of tumescent fluid anesthesia, a 3mm liposuction cannula with a large lateral hole (2mm x 7mm) was used to connect a 20mL syringe, and radial suction was performed under artificial negative pressure. Rinse 3 times with normal saline.

(2) Take the rinsed adipose tissue, place it in a centrifuge tube, put it in a centrifuge and centrifuge at 1200g for 3 minutes at 4°C 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 (ie, the fat layer containing adipocytes) is collected.

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

(5) The mechanically emulsified fat mixture was placed in a -80°C refrigerator for freezing, and then thawed in a 37°C water bath. After a single freeze-thaw cycle, the thawed fat mixture was centrifuged at 1200g at 4°C for 5 minutes to obtain fractions. The layered mixture is divided into 4 layers, the first layer is the oil layer, the second layer is the residual adipose tissue layer, the third layer is the liquid layer, and the fourth layer is the cell/tissue debris precipitation layer, remove the oil layer and For the residual adipose 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 extraction solution.

(6) Filter and sterilize the obtained fat primary extract through a 0.22 μm filter, thereby sterilizing and removing possible mixed living cells, thereby obtaining cell-free fat extract (CEFFE), which is frozen in aliquots and stored at -20°C , thawed at 4°C when used.

For the prepared cell-free fat extract, the content of cytokines, including IGF-1, BDNF, GDNF, bFGF, VEGF, TGF-β1, HGF and PDGF, was detected by ELISA immunosorbent assay kit. 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 (954.5 pg/ml), HGF (898.4 pg/ml) and PDGF (179.9 pg/ml).

2. Experimental method

2.1 Subjects of administration

Five patients with periorbital aging who had not received any treatment were included. The patients are healthy adults without serious underlying diseases, infectious diseases, mental and psychological disorders. All patients were fully informed and signed informed consent voluntarily.

2.2 Method of administration

The injection was performed 1 hour after the periorbital topical anesthetic was applied. The intradermal drip injection technique was used to inject 0.1 mL of CEFFE every 1 cm from the lateral orbital canthus to the medial canthus, and from the lower eyelid margin to the infraorbital margin from 0.5 cm. Immediately after liposuction and every two weeks thereafter, treatment was performed with a total of 5 injections, followed by follow-up at 3, 6, and 12 months after the end of treatment.

3. Detection and evaluation

3.1 General observation

Before surgery and at each follow-up, the patients will be photographed with a standard camera (Canon 70D, Japan), and the changes of periorbital fine lines will be observed by comparing the photos.

3.2 Objective detection

3.2.1 Skin wrinkles:

In a room with a temperature of 24-25°C and a relative humidity of 40%-60%, the Vivoscan skin detector (CK, Germany) was used to detect the three points of the infraorbital inside and outside. The periorbital skin roughness parameters (SEr) and skin smoothness parameters (SEsm) before surgery and at each follow-up were recorded by software analysis. Taking the pre-CEFFE treatment as the base point, the changes of postoperative periorbital skin roughness parameters were calculated, where n was the number of visits per visit.

3.2.2 Skin elasticity

At the same room temperature and humidity, before surgery and at each follow-up, the skin elasticity of the medial and lateral orbital skin was measured by MPA580 skin measuring instrument (CK, Germany), and the total elasticity value R2, net elasticity value R5 and elasticity were recorded. The data of the value number R7. Also according to the above calculation formula, the change of skin elasticity around the orbit after operation was calculated with the preoperative as the base point.

3.2.3 Skin barrier

Under the same conditions, the water loss of the medial and lateral skin in the periorbital periorbital area will be detected by the water loss test probe Tewameter TM300 instrument (CK, Germany) at each visit, and the data of transepidermal water loss (TEWL (transepidermal waterloss)) value will be recorded. to evaluate skin barrier function. According to the above calculation formula, the changes of the skin barrier around the orbit after surgery were calculated based on the preoperative point.

3.2.4 Adverse reactions

All adverse reactions during and after treatment were recorded, including intraoperative pain, postoperative bruising, redness, itching, dryness, allergy and infection.

4. Statistical analysis

All data were analyzed by SPSS software, using analysis of variance homogeneity (One-Way ANOVA), or Kruskal-Wallis test, if there was a statistical difference (p < 0.05), then compared between groups.

5. Experimental results

5.1 General observation

By comparing the standardized imaging and Vivoscan photos before and after treatment, it was found that the periorbital fine lines began to improve to varying degrees during the third CEFFE treatment, and with the increase of follow-up time, the degree of improvement became more obvious. As a result, the originally small and numerous folds around the orbit gradually flattened, some fine lines disappeared, and some coarse wrinkles were smoothed out or even disappeared. When the follow-up was up to 12 months, the effect was only slightly lower than that in 6 months. The texture is slightly rough, but fine lines and wrinkles are still excellently improved (see Figure 1).

5.2 Objective evaluation

5.2.1 Skin wrinkles

On the photos taken by Vivoscan, further analyze the changes of wrinkles before and after surgery, including SEr%, SEsm%. SEr is the skin roughness parameter, representing the roughness of the skin, the smaller the value, the less rough the skin; SEsm is the skin smoothness parameter, representing the fineness of the skin, the smaller the value, the smoother and finer the skin. During the follow-up, it was found that the objective evaluation indicators of periorbital wrinkles all showed a downward trend, but the effect rebounded slightly at the 3-month follow-up. At subsequent follow-up visits at 6 and 12 months, the best wrinkle improvement was observed, which was maintained until the 12-month follow-up (Figure 2).

5.2.2 Skin elasticity

The elasticity data recorded by the MPA580 skin measuring instrument at each visit was analyzed, including the data of total elasticity value R2, net elasticity value R5 and elasticity value R7. R2 is the overall elasticity degree of the skin. The larger the value, the more representative the skin. The better the elasticity. R5 is the net elasticity of the skin, that is, the ratio of the retracted part of the skin to the part attracted by the negative pressure when the skin is released from the negative pressure. The larger the value, the better the skin retraction ability and the more elastic the skin. R7 is the skin elasticity value, which represents the ratio of the retracted part of the skin to the maximum lifting amount of the skin under the release of negative pressure. The larger the value, the better the skin elasticity. It was observed that the skin elasticity increased slowly after 3 treatments, especially at the 6-month follow-up, and an increase in skin elasticity was still observed at the 12-month follow-up (as shown in Figure 3).

5.2.3 Skin barrier

Reflects skin barrier capacity through transepidermal water loss. After one treatment, the degree of transepidermal water loss began to decrease, with the increase of the number of treatments, the lower the degree of transepidermal water loss, the skin barrier was significantly improved, and the best skin condition was observed at 6 months, while maintaining December (shown in Figure 4), indicating that CEFFE has excellent water retention capacity.

5.2.4 Complications

During the operation, all patients complained of mild tolerable pain during injection, which lasted for about 1.80±1.3 hours. 80% of patients (n=4) developed mild bruising lasting 4.13±3.08 days postoperatively. 20% of patients (n=1) developed redness and swelling after surgery, which recovered within 1 day. No other side effects such as itching, dryness, allergy and infection were observed.

in conclusion

There are various treatments for periorbital aging, including surgical and non-surgical treatments. However, there are problems such as long recovery period and scarring in surgical treatment, which cause the majority of beauty seekers to stay away. Non-surgical treatment includes laser treatment and minimally invasive treatment, but the efficacy is uncertain, so there are few treatment options for patients to choose.

The cell-free fat extract is obtained by further physical purification on the basis of nano-fat in fat derivatives. After testing by Elisa, it was found that it contains a large amount of active growth factors. Such as angiogenic growth factors VEGF, bFGF, TGF-β; neurotrophic factors GDNF and BDNF; In the present study, we directly injected acellular fat extract into the periorbital dermis of patients, and by comparing preoperative and postoperative standardized photographs, we found that the number of infraorbital fine lines was reduced and the skin was neat (Figure 1). At the same time, the objective wrinkle evaluation also confirmed that after multiple treatments, the suborbital skin roughness parameter SEr and skin smoothness parameter SEsm both showed a downward trend, indicating a trend of gradual smoothness and smooth and neat wrinkles (Figure 2). At the second visit, the periorbital wrinkles were significantly reduced and the skin was delicate, while at the third visit, the wrinkles slightly increased and the skin was slightly rough, but with the increase of the number of treatments, the wrinkles gradually decreased and the skin was smoother. After stopping treatment, the effect continued until 12 months of follow-up, and although there was a trend of mild recurrence, it was still improved compared with before treatment. Increased skin elasticity was observed after treatment (Fig. 3), and transdermal water loss improved, and with continued treatment, the degree of skin water loss gradually decreased, and lasted until 12 months of follow-up (Fig. 4). Cell-free fat extract treatment has few complications and no serious side effects. Only mild intraoperative pain and short-term postoperative bruising were observed in the process of improving skin aging, indicating that cell-free fat extract treatment is safe and reliable.

Therefore, the research results of this example show that CEFFE has the effect of anti-aging and promoting skin rejuvenation.

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by 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 a cell-free fat extract, characterized in that it is used to prepare a composition or preparation for improving skin aging and/or promoting skin rejuvenation.
The use of claim 1, wherein the skin aging comprises periorbital skin aging.
The use of claim 1, wherein the skin aging comprises one or more manifestations selected from the group consisting of:

(a) an increase in the number of skin wrinkles;

(b) increased skin roughness;

(c) reduced skin smoothness;

(d) reduced skin elasticity;

(e) loose skin; and/or

(f) Dehydration of the skin.
The use according to claim 1, wherein the improving skin aging and/or promoting skin rejuvenation comprises one or more ways selected from the group consisting of:

(i) reduce the number of skin wrinkles;

(ii) reduce skin roughness;

(iii) improve the smoothness of the skin;

(iv) increase skin elasticity;

(v) reduce skin moisture loss; and/or

(vi) Improve the water retention capacity of the skin.
The use according to claim 1, wherein the composition or preparation comprises a pharmaceutical composition or preparation, a food composition or preparation, a health 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 dosage form of the composition or preparation is an intradermal injection preparation.
The use of claim 1, wherein the cell-free fat extract contains one or more components selected from the group consisting of IGF-1, BDNF, GDNF, TGF-β1, HGF, bFGF , VEGF, TGF-β1, HGF, PDGF, EGF, NT-3, GH, G-CSF, or a combination thereof.
The use of claim 1, wherein the cell-free adipose extract contains, but is not limited to, one or more components selected from the group consisting of IGF-1, BDNF, GDNF, bFGF, VEGF , TGF-β1, HGF, PDGF, or a combination thereof.
The use of claim 8, wherein said 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 9500-10500pg/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 cell-free fat extract, the concentration of GDNF is 800-5000pg/ml, preferably 1000-4000pg/ml, more preferably 1200-2500pg/ml, more preferably 1400-2000pg/ml , more preferably 1600-2000pg/ml, more preferably 1700-1900pg/ml;

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

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 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 cell-free 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 800-1000 pg/ml, more preferably 850-950 pg/ml; and/or

In the cell-free fat extract, the concentration of the 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 of claim 8, wherein said 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 40-60:1, and 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.
The use according to claim 1, wherein the cell-free fat extract is prepared by the following method:

(1) providing an adipose tissue raw material, crushing the adipose tissue raw material, and rinsing (such as with physiological saline), thereby obtaining rinsed adipose tissue;

(2) centrifuging the rinsed 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 (ie, the fat layer containing adipocytes);

(4) Emulsifying the intermediate layer to obtain an emulsified fat mixture (also referred to as nano-fat);

(5) subjecting the emulsified fat mixture to centrifugation to obtain an intermediate liquid layer, i.e., the initial fat extract; and

(6) Filtration and sterilization of the primary fat extract to obtain a cell-free fat extract.
A composition or preparation for improving skin aging and/or promoting skin rejuvenation, characterized in that the composition or preparation comprises:

(a) cell-free adipose extract; and

(b) A pharmaceutically, food, health product or dietary acceptable carrier or excipient.
A method of improving skin aging and/or promoting skin rejuvenation, characterized in that a cell-free adipose extract is administered to a subject in need thereof.
The method of claim 14, wherein the mode of administration is intradermal injection.