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WO2022170940A1 - 无细胞脂肪提取物用于改善衰老和促进皮肤年轻化 - Google Patents

无细胞脂肪提取物用于改善衰老和促进皮肤年轻化 Download PDF

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Publication number
WO2022170940A1
WO2022170940A1 PCT/CN2022/073020 CN2022073020W WO2022170940A1 WO 2022170940 A1 WO2022170940 A1 WO 2022170940A1 CN 2022073020 W CN2022073020 W CN 2022073020W WO 2022170940 A1 WO2022170940 A1 WO 2022170940A1
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Prior art keywords
skin
cell
preparation
extract
another preferred
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PCT/CN2022/073020
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English (en)
French (fr)
Inventor
张文杰
蔡宜佐
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上海萨美细胞技术有限公司
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Publication of WO2022170940A1 publication Critical patent/WO2022170940A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/35—Fat tissue; Adipocytes; Stromal cells; Connective tissues
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/92—Oils, fats or waxes; Derivatives thereof, e.g. hydrogenation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/16—Emollients or protectives, e.g. against radiation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/18—Antioxidants, e.g. antiradicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • 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.
  • 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.
  • non-surgical treatment 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.
  • laser therapy can produce thermal effects on tissues and stimulate local collagen synthesis through selective photothermal action, thereby achieving the effect of treating periorbital aging.
  • 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.
  • the skin aging includes periorbital skin aging.
  • described skin aging includes one or more manifestations selected from the following group:
  • the wrinkles include fine lines and/or coarse lines.
  • the improvement of skin aging and/or the promotion of skin rejuvenation comprises one or more methods selected from the following group:
  • the improving skin aging and/or promoting skin rejuvenation has one or more characteristics selected from the group consisting of:
  • the cell-free fat extract is a cell-free fat extract prepared from human or non-human mammalian fat.
  • the non-human mammal is monkey, orangutan, cow, pig, dog, sheep, mouse or rabbit.
  • compositions or preparations include pharmaceutical compositions or preparations, food compositions or preparations, health product compositions or preparations, or dietary supplements.
  • composition or preparation further includes a pharmaceutically, food, health product or dietary acceptable carrier.
  • composition or preparation further includes other drugs for improving skin aging and/or promoting skin rejuvenation.
  • 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
  • the skin filling material is selected from the group consisting of hyaluronic acid, collagen, or a combination thereof.
  • the anti-wrinkle drug includes botulinum toxin.
  • the ingredients for nourishing the skin include vitamins.
  • 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.
  • the dosage form of the composition or preparation is an oral preparation, an external preparation or an injection preparation.
  • the injection preparation is an intravenous injection or an intramuscular injection.
  • the injection preparation is an intradermal injection preparation.
  • the injection preparation is an intradermal injection preparation of periorbital skin.
  • 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.
  • the dosage form of the composition or preparation is powder, granule, capsule, injection, tincture, oral liquid, tablet or lozenge.
  • composition or preparation is administered externally, topically, or by injection.
  • the injection preparation is an intradermal injection preparation.
  • the injection preparation is an intradermal injection preparation of periorbital skin.
  • the cell-free fat extract does not contain cells and does not contain lipid droplets.
  • the lipid droplets are oil droplets released after fat cells are disrupted.
  • 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%.
  • the cells are selected from the group consisting of endothelial cells, adipose stem cells, macrophages, and stromal cells.
  • 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.
  • the cell-free fat extract is a naturally-obtained nano-fat extract without added components.
  • 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.
  • the cell-free adipose extract is prepared by centrifuging adipose tissue after emulsification.
  • the cell-free fat extract contains one or more components selected from the group consisting of IGF-1, BDNF, GDNF, TGF- ⁇ 1, HGF, bFGF, VEGF, TGF- ⁇ 1 , PDGF, EGF, NT-3, GH, G-CSF, or a combination thereof.
  • 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- ⁇ 1, HGF , PDGF, or a combination thereof.
  • the cell-free fat extract is a 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the concentration of TGF- ⁇ 1 is 200-3000pg/ml, preferably 400-2000pg/ml, more preferably 600-1500pg/ml , more preferably 800-1200pg/ml, more preferably 800-1100pg/ml, more preferably 900-1000pg/ml.
  • 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.
  • 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.
  • 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, Optimally 1-1.5:1.
  • the weight ratio of TGF- ⁇ 1 to VEGF is 1-20:1, preferably 1-15:1, more preferably 1-10:1, more preferably 2-8: 1, 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.
  • 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 cell-free fat extract is prepared by the following method:
  • Emulsifying the intermediate layer to obtain an emulsified fat mixture also referred to as nano-fat
  • a second aspect of the present invention provides a method for preparing a cell-free fat extract, the method comprising the steps of:
  • Emulsifying the intermediate layer to obtain an emulsified fat mixture also referred to as nano-fat
  • the cell-free fat extract is as described in the first aspect of the present invention.
  • the centrifugation is performed at 800-2500g, preferably 800-2000g, more preferably 1000-1500g, and most preferably 1100-1300g.
  • the centrifugation time is 1-15 minutes, preferably 1-10 minutes, more preferably 1-8 minutes, and optimally 1-5 minutes.
  • the temperature of the centrifugation is 2-6°C.
  • the emulsification is mechanical emulsification.
  • 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.
  • 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.
  • the emulsification is a method of crushing by a tissue homogenizer.
  • the emulsified fat mixture is further frozen and then thawed.
  • the thawed mixture is used for centrifugation after thawing after freezing.
  • the freezing temperature is -50°C to -120°C, preferably -60°C to -100°C, more preferably -70°C to -90°C.
  • the thawing temperature is 20-40°C, preferably 25-40°C, more preferably 37°C.
  • the number of cycles of thawing after freezing is 1-5 times (preferably 1, 2, 3 or 4 times).
  • 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.
  • the centrifugation is performed at 800-2500g, preferably 800-2000g, more preferably 1000-1500g, and most preferably 1100-1300g.
  • the centrifugation time is 1-15 minutes, preferably 1-10 minutes, more preferably 2-8 minutes, and optimally 3-7 minutes.
  • the temperature of the centrifugation is 2-6°C.
  • the first layer, the second layer, the third layer and the fourth layer are arranged in order from top to bottom.
  • the intermediate liquid layer is a transparent or substantially transparent layer.
  • the filtration and sterilization are performed through a filter (eg, a 0.22 ⁇ m microporous membrane).
  • a filter eg, a 0.22 ⁇ m microporous membrane
  • the filter is a microporous membrane filter.
  • the pore size of the microporous filter membrane is 0.05-0.8 ⁇ m, preferably 0.1-0.5 ⁇ m, more preferably 0.1-0.4 ⁇ m, more preferably 0.15-0.3 ⁇ m, more preferably 0.2-0.25 ⁇ m, optimally 0.22 ⁇ m.
  • 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 ⁇ m filter.
  • the step (6) further includes sub-packaging the fat extract to form a sub-packaged product.
  • the subpackaged extract can be stored at -20°C for later 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) 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.
  • the composition is a pharmaceutical composition, a food composition, a health product composition or a dietary supplement.
  • the dosage form of the composition or preparation is an oral preparation, an external preparation or an injection preparation.
  • the dosage form of the composition or preparation is powder, granule, capsule, injection, tincture, oral liquid, tablet or lozenge.
  • the injection is an intravenous injection or an intramuscular injection.
  • the injection preparation is an intradermal injection preparation.
  • the injection preparation is an intradermal injection preparation of periorbital skin.
  • 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.
  • 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.
  • the subject is a human or a non-human mammal.
  • the non-human mammals include rodents, such as rats and mice.
  • the administration mode is oral administration, topical administration or injection administration.
  • the injection is intradermal injection.
  • the injection is intradermal injection of the periorbital skin.
  • the dose administered by injection is 0.05-0.2 mL of acellular fat extract/1 cm of skin.
  • 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.
  • 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 are the 3rd, 6th, and 6th time after the treatment, respectively. and December.
  • 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 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”.
  • IGF-1 insulin-like growth factors-1
  • BDNF brain-derived neurotrophic factor
  • GDNF glial cellline-derived neurotrophic factor
  • bFGF basic fibroblast growth factor
  • VEGF vascular endothelial growth factor
  • TGF- ⁇ 1 is referred to as transforming growth factor- ⁇ 1.
  • HGF Hepatocyte Growth Factor
  • PDGF Platelet derived growth factor
  • EGF Epidermal Growth Factor
  • NT-3 As used in the text, the term "NT-3" is referred to as neurotrophins-3.
  • GH Growth Hormone
  • G-CSF granulocyte colony stimulating factor
  • CEFFE Cell free fat extract
  • 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.
  • 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.
  • the cell-free fat extract is a cell-free fat extract.
  • the cell-free adipose extract of the present invention various cytokines may be included.
  • the cell-free adipose extract comprises IGF-1, BDNF, GDNF, TGF- ⁇ , HGF, bFGF, VEGF, TGF- ⁇ 1, PDGF, EGF, NT-3, GH and G-CSF one or more.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the concentration of TGF- ⁇ 1 is 200-3000pg/ml, preferably 400-2000pg/ml, more preferably 600-1500pg/ml , more preferably 800-1200pg/ml, more preferably 800-1100pg/ml, more preferably 900-1000pg/ml.
  • 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.
  • 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.
  • 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, Optimally 1-1.5:1.
  • the weight ratio of TGF- ⁇ 1 to VEGF is 1-20:1, preferably 1-15:1, more preferably 1-10:1, more preferably 2-8: 1, 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.
  • 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 cell-free fat extract of the present invention is prepared by the method as described in the second aspect of the present invention.
  • the cell-free fat extracts of the present invention are prepared by the following methods:
  • Emulsifying the intermediate layer to obtain an emulsified fat mixture also referred to as nano-fat
  • the centrifugation is performed at 800-2500g, preferably 800-2000g, more preferably 1000-1500g, and most preferably 1100-1300g.
  • the centrifugation time is 1-15 minutes, preferably 1-10 minutes, more preferably 1-8 minutes, and optimally 1-5 minutes.
  • the emulsification is mechanical emulsification.
  • 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.
  • the blowing method is to repeatedly push and beat at a constant speed with two 10ml injection syringes connected to a tee tube.
  • the emulsification is a method of crushing by a tissue homogenizer.
  • the emulsified fat mixture is further frozen and then thawed.
  • the thawed mixture is used for centrifugation after thawing after freezing.
  • the freezing temperature is -50°C to -120°C, preferably -60°C to -100°C, more preferably -70°C to -90°C.
  • the thawing temperature is 20-40°C, preferably 25-40°C, more preferably 37°C.
  • the number of cycles of thawing after freezing is 1-5 times (preferably 1, 2, 3 or 4 times).
  • 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.
  • the centrifugation is performed at 800-2500g, preferably 800-2000g, more preferably 1000-1500g, and most preferably 1100-1300g.
  • the centrifugation time is 1-15 minutes, preferably 1-10 minutes, more preferably 2-8 minutes, and optimally 3-7 minutes.
  • the first layer, the second layer, the third layer and the fourth layer are arranged in order from top to bottom.
  • the intermediate liquid layer is a transparent or substantially transparent layer.
  • the filter bag in the step (6), can remove the adipocytes in the primary fat extract.
  • the filtration and sterilization are performed through a filter (eg, a 0.22 ⁇ m microporous membrane).
  • a filter eg, a 0.22 ⁇ m microporous membrane
  • the filter is a microporous membrane filter.
  • the pore size of the microporous filter membrane is 0.05-0.8 ⁇ m, preferably 0.1-0.5 ⁇ m, more preferably 0.1-0.4 ⁇ m, more preferably 0.15-0.3 ⁇ m, more preferably 0.2-0.25 ⁇ m, optimally 0.22 ⁇ m.
  • 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 ⁇ m filter.
  • the step (6) further includes sub-packaging the fat extract to form a sub-packaged product.
  • the subpackaged extract can be stored at -20°C for later 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) for a period of time after thawing, and then used ).
  • 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.
  • the term “improving” includes prevention and/or treatment.
  • 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 includes delaying and stopping the progression of the disease, or eliminating the disease, and does not require 100% inhibition, elimination and reversal.
  • 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%.
  • the aging skin site is not particularly limited.
  • the skin aging includes periorbital skin aging.
  • described skin aging includes one or more manifestations selected from the following group:
  • the wrinkles include fine lines and/or coarse lines.
  • the improvement of skin aging and/or the promotion of skin rejuvenation comprises one or more methods selected from the group consisting of:
  • 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.
  • the subject is a human or a non-human mammal.
  • the non-human mammals include rodents, such as rats and mice.
  • the administration mode is oral administration, topical administration or injection administration.
  • the injection is intradermal injection.
  • the injection is intradermal injection of the periorbital skin.
  • compositions described in the present invention include (but are not limited to): pharmaceutical compositions, food compositions, health care compositions, dietary supplements, and the like.
  • 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.
  • 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.
  • 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.
  • cellulose and its derivatives such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.
  • gelatin such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, 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.
  • 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.
  • solid dosage forms for oral administration or administration include capsules, tablets, pills, powders and granules.
  • 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 sodium citrate
  • 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.
  • 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.
  • compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the daily dose is usually 0.1 to 1000 mg, preferably 1 to 600 mg, more preferably 2 to 300 mg.
  • 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 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.
  • 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 .
  • 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.
  • the middle layer ie, the fat layer containing adipocytes
  • 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.
  • the content of cytokines including IGF-1, BDNF, GDNF, bFGF, VEGF, TGF- ⁇ 1, 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- ⁇ 1 (954.5 pg/ml), HGF (898.4 pg/ml) and PDGF (179.9 pg/ml).
  • 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.
  • treatment was performed with a total of 5 injections, followed by follow-up at 3, 6, and 12 months after the end of treatment.
  • the Vivoscan skin detector (CK, Germany) was used to detect the three points of the infraorbital inside and outside.
  • SEr periorbital skin roughness parameters
  • SEsm skin smoothness parameters
  • 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.
  • 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.
  • TEWL transepidermal water loss
  • 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.
  • 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).
  • 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- ⁇ ; 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).
  • 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).
  • 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.
  • 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.

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Abstract

提供了一种无细胞脂肪提取物及其用于改善衰老和促进皮肤年轻化的用途。该无胞脂肪提取物可用于制备组合物或制剂，组合物或制剂用于改善皮肤衰老和/或促进皮肤年轻化。

Description

无细胞脂肪提取物用于改善衰老和促进皮肤年轻化 技术领域
本发明涉及药物领域，具体涉及无细胞脂肪提取物用于改善衰老和促进皮肤年轻化。
背景技术
皮肤衰老是不可避免的过程。皮肤衰老的早期表现，主要体现在眶周的老化，表现为眶周皱纹、皮肤松弛、弹性下降及色素沉着等。目前的治疗皮肤衰老(如眶周老化)方式主要包括手术治疗及非手术治疗，但由于手术治疗存在恢复期较长、疤痕等问题，研究的热点集中于非手术治疗。
非手术治疗包含物理治疗及生物治疗，前者主要为激光治疗，可通过选择性光热作用，对组织产生热效应，刺激局部胶原蛋白合成增加，从而达到治疗眶周老化的效果，然而，在临床应用中，发现激光疗效维持时间较短，通常在术后很快恢复至术前状态，且尚未有明确的眶周年轻化的激光治疗方案。现有技术中缺乏一种有效治疗皮肤衰老的药物，开发一种有效治疗皮肤衰老的药物成为当今研究的热点。
因此，本领域需要开发一种能够有效改善皮肤衰老和促进皮肤年轻化的药物。
发明内容
本发明的目在于提供一种无细胞脂肪提取物在改善皮肤衰老和/或促进皮肤年轻化方面中的用途。
本发明第一方面，提供一种无细胞脂肪提取物的用途，用于制备组合物或制剂，所述组合物或制剂用于改善皮肤衰老和/或促进皮肤年轻化。
在另一优选例中，所述的皮肤衰老包括眶周皮肤衰老。
在另一优选例中，所述的皮肤衰老包括选自下组的一种或多种表现：
(a)皮肤皱纹数量增多；
(b)皮肤粗糙度增加；
(c)皮肤光滑度降低；
(d)皮肤弹性度降低；
(e)皮肤松弛；和/或
(f)皮肤失水。
在另一优选例中，所述的皱纹包括细纹和/或粗纹。
在另一优选例中，所述的改善皮肤衰老和/或促进皮肤年轻化包括选自下组的一种或多种方式进行：
(i)减少皮肤皱纹数量；
(ii)降低皮肤粗糙度；
(iii)提高皮肤的光滑度；
(iv)增加皮肤弹性；
(v)降低皮肤水分流失；和/或
(vi)提高皮肤的保水能力。
在另一优选例中，所述的改善皮肤衰老和/或促进皮肤年轻化具有选自下组的一个或多个特征：
(i)随访12个月后皮肤粗糙度参数降低超过20％；
(ii)随访12个月后总弹性数值R2升高超过20％；
(iii)随访12个月后净弹性值数R5升高超过60％；
(iv)随访12个月后弹性值数R7升高超过30％；
(v)随访12个月后经皮水分流失不超过40％。
在另一优选例中，所述的无细胞脂肪提取物为从人或非人哺乳动物中的脂肪中提取制备获得的无细胞脂肪提取物。
在另一优选例中，所述的非人哺乳动物为猴、猩猩、牛、猪、狗、羊、鼠或兔。
在另一优选例中，所述的组合物或制剂包括药物组合物或制剂、食品组合物或制剂、保健品组合物或制剂或膳食补充剂。
在另一优选例中，所述的组合物或制剂还包括药学上、食品上、保健品或膳食上可接受的载体。
在另一优选例中，所述的组合物或制剂还包括其它改善皮肤衰老和/或促进皮肤年轻化的药物。
在另一优选例中，所述的其它改善皮肤衰老和/或促进皮肤年轻化的药物选自下组：皮肤填充材料、抗皱的药物、滋养皮肤的成分
在另一优选例中，所述的皮肤填充材料选自下组：透明质酸、胶原，或其组合。
在另一优选例中，所述的抗皱的药物包括肉毒杆菌毒素。
在另一优选例中，所述的滋养皮肤的成分包括维生素类成分。
在另一优选例中，所述的维生素类成选自下组：维生素B1、维生素B2、维生素B12、维生素C、维生素D、维生素E，或其组合。
在另一优选例中，所述的组合物或制剂的剂型为口服制剂、外用制剂或注射制剂。
在另一优选例中，所述的注射制剂为静脉注射剂或肌肉注射剂。
在另一优选例中，所述的注射制剂为真皮内注射制剂。
在另一优选例中，所述的注射制剂为眶周皮肤的真皮内注射制剂。
在另一优选例中，所述组合物或制剂的剂型为固体剂型、半固体剂型、或液体剂型，如溶液、凝胶、膏霜、乳液、膏剂、霜剂、糊剂、饼、粉剂、贴剂等。
在另一优选例中，所述组合物或制剂的剂型为粉剂、颗粒剂、胶囊剂、注射剂、酊剂、口服液、片剂或含片。
在另一优选例中，所述的组合物或制剂通过外用、局部、或注射方式施用。
在另一优选例中，所述的注射制剂为真皮内注射制剂。
在另一优选例中，所述的注射制剂为眶周皮肤的真皮内注射制剂。
在另一优选例中，所述无细胞脂肪提取物不含有细胞且不含有脂滴。
在另一优选例中，所述脂滴为脂肪细胞破碎后释放的油滴。
在另一优选例中，所述“不含有脂滴”指所述无细胞脂肪提取物中，油滴体积占总液体百分比小于1％，优选地小于0.5％，更优选地小于0.1％。
在另一优选例中，所述细胞选自下组：内皮细胞、脂肪干细胞、巨噬血细胞、基质细胞。
在另一优选例中，所述“无细胞”指1ml无细胞脂肪提取物中的细胞平均数量≤1 个，优选地≤0.5个，更佳地≤0.1个，或为0个。
在另一优选例中，所述无细胞脂肪提取物为天然获得的无添加成分的纳米脂肪提取物。
在另一优选例中，所述“无添加成分的”指除漂洗步骤外，在所述脂肪提取物的制备过程中未添加任何溶液、溶剂、小分子、化学制剂、和生物添加剂。
在另一优选例中，所述种无细胞脂肪提取物是通过将脂肪组织经过乳化后离心制备获得。
在另一优选例中，所述的无细胞脂肪提取物含有一种或多种选自下组的组分：IGF-1、BDNF、GDNF、TGF-β1、HGF、bFGF、VEGF、TGF-β1、PDGF、EGF、NT-3、GH、G-CSF，或其组合。
在另一优选例中，所述的种无细胞脂肪提取物含有但不限于一种或多种选自下组的组分：IGF-1、BDNF、GDNF、bFGF、VEGF、TGF-β1、HGF、PDGF，或其组合。
在另一优选例中，所述的无细胞脂肪提取物为无细胞脂肪提取液。
在另一优选例中，在所述的无细胞脂肪提取物中，所述的IGF-1的浓度为5000-30000pg/ml，较佳地6000-20000pg/ml，更佳地7000-15000pg/ml，更佳地8000-12000pg/ml，更佳地9000-11000pg/ml，更佳地9500-10500pg/ml。
在另一优选例中，在所述的无细胞脂肪提取物中，所述的BDNF的浓度为800-5000pg/ml，较佳地1000-4000pg/ml，更佳地1200-2500pg/ml，更佳地1400-2000pg/ml，更佳地1600-2000pg/ml，更佳地1700-1850pg/ml。
在另一优选例中，在所述的无细胞脂肪提取物中，所述的GDNF的浓度为800-5000pg/ml，较佳地1000-4000pg/ml，更佳地1200-2500pg/ml，更佳地1400-2000pg/ml，更佳地1600-2000pg/ml，更佳地1700-1900pg/ml。
在另一优选例中，在所述的无细胞脂肪提取物中，所述的bFGF的浓度为50-600pg/ml，较佳地100-500pg/ml，更佳地120-400pg/ml，更佳地150-300pg/ml，更佳地200-280pg/ml，更佳地220-260pg/ml。
在另一优选例中，在所述的无细胞脂肪提取物中，所述的VEGF的浓度为50-500pg/ml，较佳地100-400pg/ml，更佳地120-300pg/ml，更佳地150-250pg/ml， 更佳地170-230pg/ml，更佳地190-210pg/ml。
在另一优选例中，在所述的无细胞脂肪提取物中，所述的TGF-β1的浓度为200-3000pg/ml，较佳地400-2000pg/ml，更佳地600-1500pg/ml，更佳地800-1200pg/ml，更佳地800-1100pg/ml，更佳地900-1000pg/ml。
在另一优选例中，在所述的无细胞脂肪提取物中，所述的HGF的浓度为200-3000pg/ml，较佳地400-2000pg/ml，更佳地600-1500pg/ml，更佳地600-1200pg/ml，更佳地800-1000pg/ml，更佳地850-950pg/ml。
在另一优选例中，在所述的无细胞脂肪提取物中，所述的PDGF的浓度为50-600pg/ml，较佳地80-400pg/ml，更佳地100-300pg/ml，更佳地140-220pg/ml，更佳地160-200pg/ml，更佳地170-190pg/ml。
在另一优选例中，所述的IGF-1与VEGF的重量比为20-100：1，较佳地30-70：1，更佳地40-60：1，最佳地45-55：1。
在另一优选例中，所述的BDNF与VEGF的重量比为2-20：1，较佳地4-15：1，更佳地6-12：1，最佳地8-9.5：1。
在另一优选例中，所述的GDNF与VEGF的重量比为2-20：1，较佳地4-15：1，更佳地6-12：1，最佳地8.5-9.5：1。
在另一优选例中，所述的bFGF与VEGF的重量比为0.2-8：1，较佳地0.5-5：1，更佳地0.6-2：1，更佳地0.8-1.6：1，最佳地1-1.5：1。
在另一优选例中，所述的TGF-β1与VEGF的重量比为1-20：1，较佳地1-15：1，更佳地1-10：1，更佳地2-8：1，更佳地4-6：1。
在另一优选例中，所述的HGF与VEGF的重量比为1-20：1，较佳地1-15：1，更佳地1-10：1，更佳地2-8：1，更佳地4-5.5：1。
在另一优选例中，所述的PDGF与VEGF的重量比为0.1-3：1，较佳地0.2-2：1，更佳地0.4-1.5：1，最佳地0.7-1.2：1。
在另一优选例中，所述的无细胞脂肪提取物通过以下方法制备：
(1)提供一脂肪组织原料，将所述脂肪组织原料破碎，并进行漂洗(如用生理盐水)，从而获得经漂洗的脂肪组织；
(2)对所述经漂洗后的脂肪组织进行离心，获得分层的混合物；
(3)对所述分层的混合物，去除上层油层和下层水层，收集中间层(即含脂肪细胞的脂肪层)；
(4)对所述中间层进行乳化，获得乳化的脂肪混合物(也称为纳米脂肪)；
(5)将所述乳化的脂肪混合物通过离心处理，从而获得中间液体层，即为脂肪初提物；和
(6)对所述脂肪初提物进行过滤和除菌，从而获得无细胞的脂肪提取物。
本发明第二方面，提供一种制备无细胞脂肪提取物的方法，所述的方法包括步骤：
(1)提供一脂肪组织原料，将所述脂肪组织原料破碎，并进行漂洗(如用生理盐水)，从而获得经漂洗的脂肪组织；
(2)对所述经漂洗后的脂肪组织进行离心，获得分层的混合物；
(3)对所述分层的混合物，去除上层油层和下层水层，收集中间层(即含脂肪细胞的脂肪层)；
(4)对所述中间层进行乳化，获得乳化的脂肪混合物(也称为纳米脂肪)；
(5)将所述乳化的脂肪混合物通过离心处理，从而获得中间液体层，即为脂肪初提物；和
(6)对所述脂肪初提物进行过滤和除菌，从而获得无细胞的脂肪提取物。
在另一优选例中，所述的无细胞脂肪提取物如本发明第一方面所述。
在另一优选例中，所述的步骤(2)中，所述离心在800-2500g下离心，较佳地800-2000g，更佳地1000-1500g，最佳地1100-1300g。
在另一优选例中，所述的步骤(2)中，所述离心的时间为1-15min，较佳地1-10min，更佳地1-8min，最佳地1-5min。
在另一优选例中，所述的离心的温度为2-6℃。
在另一优选例中，所述的步骤(4)中，所述的乳化为机械乳化。
在另一优选例中，所述机械乳化为经注射器反复吹打(如吹打20-200次，较佳地20-150次，更佳地20-100次，更佳地30-50次)进行机械乳化。
在另一优选例中，所述的吹打的方式为2个10ml注射针筒连接三通管反复匀 速推打。
在另一优选例中，，所述的步骤(4)中，所述乳化为通过组织匀浆机打碎的方法。
在另一优选例中，所述的步骤(5)中，在将所述乳化的脂肪混合物通过离心处理前，还包括对所述乳化的脂肪混合物冷冻后解冻处理。
在另一优选例中，冷冻后解冻处理后，将解冻后的混合物用于离心。
在另一优选例中，所述的冷冻的温度为-50℃至-120℃，较佳地-60℃至-100℃，更佳地-70℃至-90℃。
在另一优选例中，所述的解冻的温度为20-40℃，较佳地25-40℃，更佳地37℃。
在另一优选例中，所述的冷冻后解冻的循环次数为1-5次(优选为1、2、3或4次)。
在另一优选例中，所述的步骤(5)中，离心后，所述乳化的脂肪混合物分层4层，第一层为油层，第二层为残余脂肪组织层，第三层为液体层(即为中间液体层)，第四层为细胞/组织碎片沉淀层。
在另一优选例中，所述的步骤(5)中，所述离心在800-2500g下离心，较佳地800-2000g，更佳地1000-1500g，最佳地1100-1300g。
在另一优选例中，所述的步骤(5)中，所述离心的时间为1-15min，较佳地1-10min，更佳地2-8min，最佳地3-7min。
在另一优选例中，所述的离心的温度为2-6℃。
在另一优选例中，所述的步骤(5)中，第一层、第二层、第三层和第四层从上到下依次排列。
在另一优选例中，所述的步骤(5)中，所述的中间液体层为透明或基本透明层。
在另一优选例中，所述的步骤(6)中，所述的过滤包能够将脂肪初提物中的脂肪细胞除去。
在另一优选例中，所述的步骤(6)中，所述的过滤和除菌是通过滤器(如0.22μm微孔滤膜)进行。
在另一优选例中，所述的过滤器为微孔滤膜过滤器。
在另一优选例中，所述的微孔滤膜的孔径大小为0.05-0.8μm，较佳地0.1-0.5μm，更佳地0.1-0.4μm，更佳地0.15-0.3μm，更佳地0.2-0.25μm，最佳地0.22μm。
在另一优选例中，所述的步骤(6)中，所述的过滤和除菌是先通过可滤去细胞的第一过滤器，然后再通过可滤去病原体(如细菌)的第二滤器(如0.22μm的滤器)进行的。
在另一优选例中，所述的步骤(6)中，还包括对所述脂肪提取物进行分装，形成分装的产品。(所述分装后的提取物可于-20℃保存待用；可低温(如-4℃)或常温解冻后直接使用，或解冻后置于低温(如4℃)保存一段时间，然后使用)。
本发明第三方面，提供一种无细胞脂肪提取物，所述的无细胞脂肪提取物通过如本发明第二方面所述的方法制备获得。
本发明第四方面，提供一种用于改善皮肤衰老和/或促进皮肤年轻化的组合物或制剂，所述的组合物或制剂包含(a)如本发明第三方面所述的无细胞脂肪提取物；和(b)药学上、食品上、保健品或膳食上可接受的载体或赋形剂。
在另一优选例中，所述的组合物为药物组合物、食品组合物、保健品组合物或膳食补充剂。
在另一优选例中，所述的组合物或制剂的剂型为口服制剂、外用制剂或注射制剂。
在另一优选例中，所述组合物或制剂的剂型为粉剂、颗粒剂、胶囊剂、注射剂、酊剂、口服液、片剂或含片。
在另一优选例中，所述的注射剂为静脉注射剂或肌肉注射剂。
在另一优选例中，所述的注射制剂为真皮内注射制剂。
在另一优选例中，所述的注射制剂为眶周皮肤的真皮内注射制剂。
在另一优选例中，所述组合物或制剂的剂型为固体剂型、半固体剂型、或液体剂型，如溶液、凝胶、膏霜、乳液、膏剂、霜剂、糊剂、饼、粉剂、贴剂等。
在另一优选例中，在所述组合物或制剂中，无细胞脂肪提取物的质量百分比为5wt％，较佳地1-20wt％，以合物或制剂的总重量计。
本发明第五方面，提供一种制备如本发明第四方面所述的组合物或制剂的方法，所述的方法包括步骤：将如本发明第三方面所述的无细胞脂肪提取物与药学上、食品上、保健品或膳食上可接受的载体或赋形剂混合，从而形成组合物或制剂。
本发明第六方面，提供一种改善皮肤衰老和/或促进皮肤年轻化的方法，对需要的对象施用如本发明第三方面所述的的无细胞脂肪提取物。
在另一优选例中，所述的对象为人或非人哺乳动物。
在另一优选例中，所述非人哺乳动物包括啮齿动物，如大鼠、小鼠。
在另一优选例中，所述的施用方式为口服、外用或注射施用。
在另一优选例中，所述的注射为真皮内注射。
在另一优选例中，所述的注射为眶周皮肤的真皮内注射。
在另一优选例中，所述注射施用的剂量为0.05-0.2mL无细胞脂肪提取物/1cm皮肤。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1为眶周皮肤经CEFFE治疗后皮肤照片，其中，Pre-treatment为治疗前，3M-fellow up、6M-fellow up和12M-fellow up分别为治疗后的3、6和12月的随访。
图2为眶周皮肤经CEFFE治疗后皮肤粗糙度在变化，其中，Pre代表CEFFE给药治疗前，1st、2nd、3rd、4th和5th分别为第1、2、3、4和5次CEFFE给药时间，3m、6m和12m分别为治疗结束后3、6及12月。
图3为眶周皮肤经CEFFE治疗后皮肤弹性变化，其中，Pre代表CEFFE给药治疗前，1st、2nd、3rd、4th和5th分别为第1、2、3、4和5次CEFFE给药时间，3m、6m和12m分别为治疗结束后3、6及12月。
图4为眶周皮肤经CEFFE治疗后皮肤水分流失情况。其中，Pre代表CEFFE给药治疗前，1st、2nd、3rd、4th和5th分别为第1、2、3、4和5次CEFFE给药时间，3m、6m和12m分别为治疗结束后3、6及12月。
具体实施方式
本发明人经过广泛而深入的研究，首次开发了无细胞脂肪提取物对皮肤衰老具有优异的改善作用。本发明实施例研究表明，本发明所述的无细胞脂肪提取液对皮肤衰老具有优异的治疗效果。在此基础上完成了本发明。
术语
除非另有定义，否则本文中所用的所有技术和科学术语的含义与本发明所属领域普通技术人员普遍理解的含义相同。
如本文所用，术语“包括”、“包含”与“含有”可互换使用，不仅包括开放式定义，还包括半封闭式、和封闭式定义。换言之，所述术语包括了“由……构成”、“基本上由……构成”。
如文本所用，术语“IGF-1”称为胰岛素样生长因子1(insulin-like growth factors-1)。
如文本所用，术语“BDNF”称为脑源性神经营养因子(brain-derived neurotrophic factor，BDNF)。
如文本所用，术语“GDNF”称为胶质细胞源性神经营养因子(glialcellline-derivedneurotrophicfactor)。
如文本所用，术语“bFGF”称为碱性成纤维细胞生长因子(basic fibroblast growth factor)。
如文本所用，术语“VEGF”称为血管内皮生长因子(vascular endothelial growth factor)。
如文本所用，术语“TGF-β1”称为转化生长因子-β1(transforming growth factor-β1)。
如文本所用，术语“HGF”称为肝细胞生长因子
如文本所用，术语“PDGF”称为血小板衍生生长因子(Platelet derived growth factor)
如文本所用，术语“EGF”称为表皮细胞生长因子(Epidermal Growth Factor)
如文本所用，术语“NT-3”称为神经营养因子3(neurotrophins-3)。
如文本所用，术语“GH”称为生长激素(Growth Hormone)。
如文本所用，术语“G-CSF”称为粒细胞集落刺激因子(granulocyte colony stimulating factor)。
无细胞脂肪提取物(Cell free fat extract，CEFFE)及其制备方法
如本文所用，术语“本发明的无细胞脂肪提取物”、“本发明提取物”、“本发明的脂肪提取物”等可互换使用，指在脂肪提取物制备过程中(除漂洗步骤外)未添加任何溶液、溶剂、小分子、化学制剂、和生物添加剂所制备的源自脂肪组织的提取物(或提取液)。一种典型的制备本发明提取物的方法如上本发明第二方面中所述。此外，应理解，虽然本发明提取物在制备过程中不必添加任何添加剂(或添加成分)，但是也可以添加一些或少量的对本发明提取物的活性无负面或不利影响的安全的物质(如少量水)。
本发明的无细胞脂肪提取物可来源于人类脂肪组织，它是通过离心后除去油和细胞/细胞外基质部分而从纳米脂肪中提纯出来的，是一种无细胞、易于制备、富含各种生长因子的液体。
在本发明的一个优选例中，所述的无细胞脂肪提取物为无细胞脂肪提取液。
在本发明所述的无细胞脂肪提取物，可以包括多种细胞因子。代表性地，所述的无细胞脂肪提取物包括IGF-1、BDNF、GDNF、TGF-β、HGF、bFGF、VEGF、TGF-β1、PDGF、EGF、NT-3、GH和G-CSF中的一种或多种。
在另一优选例中，在所述的无细胞脂肪提取物中，所述的IGF-1的浓度为5000-30000pg/ml，较佳地6000-20000pg/ml，更佳地7000-15000pg/ml，更佳地8000-12000pg/ml，更佳地9000-11000pg/ml，更佳地9500-10500pg/ml。
在另一优选例中，在所述的无细胞脂肪提取物中，所述的BDNF的浓度为800-5000pg/ml，较佳地1000-4000pg/ml，更佳地1200-2500pg/ml，更佳地1400-2000 pg/ml，更佳地1600-2000pg/ml，更佳地1700-1850pg/ml。
在另一优选例中，在所述的无细胞脂肪提取物中，所述的GDNF的浓度为800-5000pg/ml，较佳地1000-4000pg/ml，更佳地1200-2500pg/ml，更佳地1400-2000pg/ml，更佳地1600-2000pg/ml，更佳地1700-1900pg/ml。
在另一优选例中，在所述的无细胞脂肪提取物中，所述的bFGF的浓度为50-600pg/ml，较佳地100-500pg/ml，更佳地120-400pg/ml，更佳地150-300pg/ml，更佳地200-280pg/ml，更佳地220-260pg/ml。
在另一优选例中，在所述的无细胞脂肪提取物中，所述的VEGF的浓度为50-500pg/ml，较佳地100-400pg/ml，更佳地120-300pg/ml，更佳地150-250pg/ml，更佳地170-230pg/ml，更佳地190-210pg/ml。
在另一优选例中，在所述的无细胞脂肪提取物中，所述的TGF-β1的浓度为200-3000pg/ml，较佳地400-2000pg/ml，更佳地600-1500pg/ml，更佳地800-1200pg/ml，更佳地800-1100pg/ml，更佳地900-1000pg/ml。
在另一优选例中，在所述的无细胞脂肪提取物中，所述的HGF的浓度为200-3000pg/ml，较佳地400-2000pg/ml，更佳地600-1500pg/ml，更佳地600-1200pg/ml，更佳地800-1000pg/ml，更佳地850-950pg/ml。
在另一优选例中，在所述的无细胞脂肪提取物中，所述的PDGF的浓度为50-600pg/ml，较佳地80-400pg/ml，更佳地100-300pg/ml，更佳地140-220pg/ml，更佳地160-200pg/ml，更佳地170-190pg/ml。
在另一优选例中，所述的IGF-1与VEGF的重量比为20-100：1，较佳地30-70：1，更佳地40-60：1，最佳地45-55：1。
在另一优选例中，所述的BDNF与VEGF的重量比为2-20：1，较佳地4-15：1，更佳地6-12：1，最佳地8-9.5：1。
在另一优选例中，所述的GDNF与VEGF的重量比为2-20：1，较佳地4-15：1，更佳地6-12：1，最佳地8.5-9.5：1。
在另一优选例中，所述的bFGF与VEGF的重量比为0.2-8：1，较佳地0.5-5：1，更佳地0.6-2：1，更佳地0.8-1.6：1，最佳地1-1.5：1。
在另一优选例中，所述的TGF-β1与VEGF的重量比为1-20：1，较佳地1-15：1， 更佳地1-10：1，更佳地2-8：1，更佳地4-6：1。
在另一优选例中，所述的HGF与VEGF的重量比为1-20：1，较佳地1-15：1，更佳地1-10：1，更佳地2-8：1，更佳地4-5.5：1。
在另一优选例中，所述的PDGF与VEGF的重量比为0.1-3：1，较佳地0.2-2：1，更佳地0.4-1.5：1，最佳地0.7-1.2：1。
优选地，本发明所述的无细胞脂肪提取物通过如上述本发明第二方面所述的方法制备获得。
代表性地，本发明所述的无细胞脂肪提取物通过以下方法制备：
(1)提供一脂肪组织原料，将所述脂肪组织原料破碎，并进行漂洗(如用生理盐水)，从而获得经漂洗的脂肪组织；
(2)对所述经漂洗后的脂肪组织进行离心，获得分层的混合物；
(3)对所述分层的混合物，去除上层油层和下层水层，收集中间层(即含脂肪细胞的脂肪层)；
(4)对所述中间层进行乳化，获得乳化的脂肪混合物(也称为纳米脂肪)；
(5)将所述乳化的脂肪混合物通过离心处理，从而获得中间液体层，即为脂肪初提物；和
(6)对所述脂肪初提物进行过滤和除菌，从而获得无细胞的脂肪提取物。
在另一优选例中，所述的步骤(2)中，所述离心在800-2500g下离心，较佳地800-2000g，更佳地1000-1500g，最佳地1100-1300g。
在另一优选例中，所述的步骤(2)中，所述离心的时间为1-15min，较佳地1-10min，更佳地1-8min，最佳地1-5min。
在另一优选例中，所述的步骤(4)中，所述的乳化为机械乳化。
在另一优选例中，所述机械乳化为经注射器反复吹打(如吹打20-200次，较佳地20-150次，更佳地20-100次，更佳地30-50次)进行机械乳化。
在另一优选例中，所述的吹打的方式为2个10ml注射针筒连接三通管反复匀速推打。
在另一优选例中，所述的步骤(4)中，所述乳化为通过组织匀浆机打碎的方法。
在另一优选例中，所述的步骤(5)中，在将所述乳化的脂肪混合物通过离心处理前，还包括对所述乳化的脂肪混合物冷冻后解冻处理。
在另一优选例中，冷冻后解冻处理后，将解冻后的混合物用于离心。
在另一优选例中，所述的冷冻的温度为-50℃至-120℃，较佳地-60℃至-100℃，更佳地-70℃至-90℃。
在另一优选例中，所述的解冻的温度为20-40℃，较佳地25-40℃，更佳地37℃。
在另一优选例中，所述的冷冻后解冻的循环次数为1-5次(优选为1、2、3或4次)。
在另一优选例中，所述的步骤(5)中，离心后，所述乳化的脂肪混合物分层4层，第一层为油层，第二层为残余脂肪组织层，第三层为液体层(即为中间液体层)，第四层为细胞/组织碎片沉淀层。
在另一优选例中，所述的步骤(5)中，所述离心在800-2500g下离心，较佳地800-2000g，更佳地1000-1500g，最佳地1100-1300g。
在另一优选例中，所述的步骤(5)中，所述离心的时间为1-15min，较佳地1-10min，更佳地2-8min，最佳地3-7min。
在另一优选例中，所述的步骤(5)中，第一层、第二层、第三层和第四层从上到下依次排列。
在另一优选例中，所述的步骤(5)中，所述的中间液体层为透明或基本透明层。
在另一优选例中，所述的步骤(6)中，所述的过滤包能够将脂肪初提物中的脂肪细胞除去。
在另一优选例中，所述的步骤(6)中，所述的过滤和除菌是通过滤器(如0.22μm微孔滤膜)进行。
在另一优选例中，所述的过滤器为微孔滤膜过滤器。
在另一优选例中，所述的微孔滤膜的孔径大小为0.05-0.8μm，较佳地0.1-0.5μm，更佳地0.1-0.4μm，更佳地0.15-0.3μm，更佳地0.2-0.25μm，最佳地0.22μm。
在另一优选例中，所述的步骤(6)中，所述的过滤和除菌是先通过可滤去细胞的第一过滤器，然后再通过可滤去病原体(如细菌)的第二滤器(如0.22μm的滤器)进 行的。
在另一优选例中，所述的步骤(6)中，还包括对所述脂肪提取物进行分装，形成分装的产品。(所述分装后的提取物可于-20℃保存待用；可低温(如-4℃)或常温解冻后直接使用，或解冻后置于低温(如4℃)保存一段时间，然后使用)。
用途
本发明提供一种无细胞脂肪提取物的用途，用于制备组合物或制剂，所述组合物或制剂用于改善皮肤衰老和/或促进皮肤年轻化。
在本发明中，术语“改善”包括预防和/或治疗。
在本发明中，术语“预防”表示预防疾病和/或它的附随症状的发作或者保护对象免于获得疾病的方法。本文中使用的"预防"还包括延迟疾病和/或它的附随症状的发作和降低对象的得病的风险。
本发明所述的“治疗”包括延缓和终止疾病的进展，或消除疾病，并不需要100％抑制、消灭和逆转。在一些实施方案中，与不存在本发明所述的无细胞脂肪提取物观相比，本发明所述无细胞脂肪提取物减轻、抑制和/或逆转了皮肤衰老例如至少约10％、至少约30％、至少约50％、或至少约80％。
在本发明中，所述的衰老的皮肤部位并没有特别的限制，优选地，所述的皮肤衰老包括眶周皮肤衰老。
在本发明一个优选例中，所述的皮肤衰老包括选自下组的一种或多种表现：
(a)皮肤皱纹数量增多；
(b)皮肤粗糙度增加；
(c)皮肤光滑度降低；
(d)皮肤弹性度降低；
(e)皮肤松弛；和/或
(f)皮肤失水。
在另一优选例中，所述的皱纹包括细纹和/或粗纹。
在本发明一个优选例中，所述的改善皮肤衰老和/或促进皮肤年轻化包括选 自下组的一种或多种方式进行：
(i)减少皮肤皱纹数量；
(ii)降低皮肤粗糙度；
(iii)提高皮肤的光滑度；
(iv)增加皮肤弹性；
(v)降低皮肤水分流失；和/或
(vi)提高皮肤的保水能力。
本发明还提供一种改善皮肤衰老和/或促进皮肤年轻化的方法，对需要的对象施用如本发明所述的的无细胞脂肪提取物。
在另一优选例中，所述的对象为人或非人哺乳动物。
在另一优选例中，所述非人哺乳动物包括啮齿动物，如大鼠、小鼠。
在另一优选例中，所述的施用方式为口服、外用或注射施用。
在另一优选例中，所述的注射为真皮内注射。
在另一优选例中，所述的注射为眶周皮肤的真皮内注射。
组合物和施用
本发明所述的组合物包括(但并不限于)：药物组合物、食品组合物、保健组合物、膳食补充剂等。
代表性地，可将本发明的无细胞脂肪提取物制备成药物组合物，诸如片剂、胶囊、粉剂、微粒剂、溶液剂、锭剂、胶冻、乳膏制剂、醑剂、悬液、酊、泥敷剂、搽剂、洗剂、和气雾剂之类的剂型。药物组合物能够由通常已知的制备技术来制备，并且合适的药物添加剂能够被添加到该药物中。
本发明的组合物还可以包括药学上、食品上、保健品或膳食上可接受的载体。“药学上、食品上、保健品或膳食上可接受的载体”指的是：一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明的化合物以及它们之间相互掺和，而不明显降低化合物的药效。药学上、食品上、保健品 或膳食上可接受的载体可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如
Figure PCTCN2022073020-appb-000001
)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。
本发明组合物施用方式没有特别限制，代表性的施用方式包括(但并不限于)：口服、肠胃外(静脉内、肌肉内)、局部施用，优选的施用方式为口服施用和注射施用。例如，所述的注射施用为真皮内注射，较佳地眶周皮肤的真皮内注射。
本发明所述的组合物或制剂的剂型为口服制剂、外用制剂或注射制剂。代表性地，用于口服施用或给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。在这些固体剂型中，活性化合物与至少一种常规惰性赋形剂(或载体)混合，如柠檬酸钠或磷酸二钙，或与下述成分混合：(a)填料或增容剂，例如，淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸；(b)粘合剂，例如，羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和阿拉伯胶；(c)保湿剂，例如，甘油；(d)崩解剂，例如，琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐、和碳酸钠；(e)缓溶剂，例如石蜡；(f)吸收加速剂，例如，季胺化合物；(g)润湿剂，例如鲸蜡醇和单硬脂酸甘油酯；(h)吸附剂，例如，高岭土；和(i)润滑剂，例如，滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠，或其混合物。胶囊剂、片剂和丸剂中，剂型也可包含缓冲剂。
固体剂型如片剂、糖丸、胶囊剂、丸剂和颗粒剂可采用包衣和壳材制备，如肠衣和其它本领域公知的材料。它们可包含不透明剂。
用于口服施用或给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性化合物外，液体剂型可包含本领域中常规采用的惰性稀释剂，如水或其它溶剂，增溶剂和乳化剂，例知，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1，3-丁二醇、二甲基甲酰胺以及油，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。
除了这些惰性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬浮 剂、甜味剂、娇味剂和香料。
除了活性成分外，悬浮液可包含悬浮剂，例如，乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。
用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。代表性地，所述的注射制剂为真皮内注射制剂，较佳地，所述的注射制剂为眶周皮肤的真皮内注射制剂。
用于局部施用或给药的本发明化合物的剂型包括软膏剂、散剂、贴剂、喷射剂和吸入剂。活性成分在无菌条件下与生理上可接受的载体及任何防腐剂、缓冲剂，或必要时可能需要的推进剂一起混合。
本发明无细胞脂肪提取物可以单独施用或给药，或者与其它预防和/或治疗脂肪肝和/或其并发症的药物联合施用或给药。
施用组合物时，是将安全有效量的本发明无细胞脂肪提取物适用于需要治疗的人或非人动物(如大鼠、小鼠、狗、猫、牛、鸡、鸭等)，其中施用时剂量为药学上、食品上或保健品上可接受认为的有效给药剂量。如本文所用，术语“安全有效量”，是指对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。本领域的普通技术人员应该理解，所述的“安全有效量”可随着药物组合物的形式、给药途径、所用药物的辅料、疾病的严重程度以及与其他药物联合用药等情况的不同而有所不同。例如，对于60kg体重的人而言，日给药剂量通常为0.1～1000mg，优选1～600mg，更优选为2-300mg。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明的主要优点包括：
1.本发明首次发现无细胞脂肪提取物对皮肤衰老具有优异的治疗效果，促进皮肤年轻化。
2.本发明所述的无细胞脂肪提取物是一种无细胞组分，可以避免临床应用中 与细胞相关的问题，例如包括细胞加工后的遗传稳定性，注射后的细胞活性和存活率，细胞的多次给药储存，以及使用同种异体脂肪时细胞的免疫原性，本发明所述的无细胞脂肪提取物在作为制备防治皮肤衰老中有着较高的安全性和较低副作用的优势。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例1
1.无细胞脂肪提取液(CEFFE)的制备
脂肪由自愿者在获得知情同意的条件下获得。无细胞脂肪组织提取液的制备方法如下：
(1)脂肪组织获取自6名常规脂肪抽吸术的健康女性，平均年龄31岁(24-36岁)。局部注射肿胀液麻醉后，使用具有大侧孔(2mm x 7mm)的3mm吸脂抽脂套管连接20mL注射器，人工负压下放射状抽吸，将获得的脂肪直立静止，去除肿胀液后，用生理盐水漂洗3遍。
(2)取经漂洗后的脂肪组织，置于离心管中，放入离心机中以1200g 4℃离心3分钟后，获得分层的混合物。
(3)对所述分层的混合物，去除上层油层和下层水层，收集中间层(即含脂肪细胞的脂肪层)。
(4)对所述中间层，用2个10ml注射针筒连接三通管反复匀速推打30次，从而进行机械乳化，并获得经机械乳化的脂肪混合物(也称为纳米脂肪)。
(5)将所述经机械乳化的脂肪混合物置入-80℃冰箱冷冻，再进行37℃水浴解冻，单次冻融循环后，将解冻后的脂肪混合物以1200g 4℃离心5分钟，获得分层的混合物，分层的混合物共分为4层，第一层为油层，第二层为残余脂肪组织层，第三层为液体层，第四层为细胞/组织碎片沉淀层，去除油层和残余脂肪组 织层，吸取液体层，吸取过程中避免细胞/组织碎片沉淀层污染，从而得到脂肪初提取液。
(6)将得到的脂肪初提取液经0.22μm滤器过滤除菌，从而灭菌并去除可能混有的活细胞，从而获得无细胞脂肪提取液(CEFFE)，分装冻存于-20℃保存，使用时4℃解冻。
对制备得到的无细胞脂肪提取液，使用ELISA免疫吸附测定试剂盒检测细胞因子含量，包括IGF-1、BDNF、GDNF、bFGF、VEGF、TGF-β1、HGF和PDGF等细胞因子。6例样本检测平均浓度如下：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)和PDGF(179.9pg/ml)。
2.实验方法
2.1给药对象
纳入表现为眶周老化，且未曾接受过任何治疗的患者5例。患者为健康成人，无严重基础疾病、传染病、精神及心理障碍。所有患者均充分知情告知，并自愿签署知情同意书。
2.2给药方法
眶周外敷麻药1小时后，进行注射。采用真皮内点滴式的注射技巧，从眶外眦至内眦处，下睑缘0.5cm至眶下缘处，每1cm注射0.1mL CEFFE。抽脂术后即刻及随后每两周进行治疗，共注射5次，治疗结束后的3、6及12月进行随访(fellow up)。
3.检测及评价
3.1大体观察
术前及每次随访，通过照相机(佳能70D，日本)对患者将进行标准化摄影，对比照片观察眶周细纹的变化。
3.2客观检测
3.2.1皮肤皱纹：
在温度为24-25℃，相对湿度为40％-60％的室内，使用Vivoscan皮肤检测仪(CK，德国)对眶下内中外三点的进行检测。通过软件分析记录术前及每次随访眶周的皮肤粗糙度参数(SEr)、皮肤平滑度参数(SEsm)。以CEFFE治疗前为基点，计算术后眶周的皮肤粗糙度参数的变化情况，其中n为每次就诊的次数。
3.2.2皮肤弹性
同样室温及湿度下，在术前及每次随访时，通过MPA580皮肤测量仪(CK，德国)进行眶周内中外侧皮肤的弹性检测，记录总弹性值数R2、净弹性值数R5及弹性值数R7的数据。同样根据上述计算公式，以术前为基点，计算术后眶周的皮肤弹性的变化情况。
3.2.3皮肤屏障
在同样的条件下，每次就诊将通过水分流失测试探头Tewameter TM300仪(CK，德国)进行眶周内中外侧皮肤的水分流失检测，记录经皮水分流失的数据TEWL(transepidermal waterloss))值，以评价皮肤屏障功能。根据上述计算公式，以术前为基点，计算术后眶周的皮肤屏障的变化情况。
3.2.4不良反应
记录所有治疗过程中及术后发生的不良反应，包括书术中疼痛、术后淤青、红肿、瘙痒、干燥、过敏及感染等。
4.统计学分析
所有数据均以SPSS软件进行分析，采用方差齐性分析(0ne-Way ANOVA)，或者是Kruskal-Wallis检验，若有统计学差异(p＜0.05)，再进行各组间比较。
5.实验结果
5.1大体观察
通过比较治疗前后标准化摄像及Vivoscan的照片，发现第三次CEFFE治疗时眶周细纹不同程度地开始改善，且随着随访时间的增加，改善程度越明显，随访6月时，仍然具有优异的效果，眶周原本细小而多的褶皱，逐渐平坦，部分细纹消失，部分粗大皱纹也得以平整，甚至消失，在随访至12月时，效果较6月时，只是稍 降低，眶周区域皮肤纹理稍粗糙，但细纹及皱纹仍有优异改善(如图1所示)。
5.2客观评价
5.2.1皮肤皱纹
在Vivoscan拍摄的照片上，进一步分析术前后的皱纹变化情况，包括SEr％，SEsm％。SEr为皮肤粗糙度参数，代表皮肤粗糙程度，数值越小皮肤越不粗糙；SEsm为皮肤平滑度参数，代表皮肤细腻程度，数值越小代表皮肤越光滑细腻。随访时发现，眶周的皱纹客观评价指标，均有下降趋势，但在3月随访时效果稍有反弹。而随后在6月以及12月地随访时，可观察到皱纹改善情况最佳，并且可持续维持至12月的随访(图2)。
5.2.2皮肤弹性
对每次就诊时使用MPA580皮肤测量仪记录的弹性数据进行分析，包括总弹性数值R2、净弹性值数R5及弹性值数R7的数据，R2为皮肤总体弹性程度，数值越越大，代表皮肤弹性越好。R5为皮肤净弹性能力，即皮肤在松开负压下，皮肤回缩的部分与负压吸引的部分的比值，数值越大，代表皮肤回缩能力越好，皮肤弹性更加。R7为皮肤弹性数值，代表皮肤在松开负压下，皮肤回缩的部分占皮肤最大拉升量之比，数值越大，代表皮肤弹性越好。观察发现皮肤弹性在3次治疗后缓慢增加，尤其在6月随访时最为明显，并在12月随访时仍能观察到皮肤弹性增加(如图3所示)。
5.2.3皮肤屏障
通过经皮水分流失的情况，反应皮肤屏障能力。在一次治疗后，经皮水分流失的程度开始下降，随着治疗次数的增加，经皮水分流失的程度越低，皮肤屏障明显提升，并且在6月时观察到最佳皮肤状态，同时可维持12月(如图4所示)，表明CEFFE具有优异的保水能力。
5.2.4并发症
术中所有病人均诉注射时有可忍受的轻度疼痛，并持续约1.80±1.3小时候缓 解。80％的患者(n＝4)在术后出现持续4.13±3.08天的轻度淤青。20％的患者(n＝1)在术后出现红肿的情况，在1天内恢复。未见其余副作用如瘙痒、干燥、过敏及感染等副作用。
结论
眶周老化的治疗种类繁多，主要包括手术及非手术类治疗。然而手术治疗存在恢复期长、疤痕等问题，导致广大求美者望而却步。而非手术治疗包含激光治疗及微创治疗等，但疗效存在不确定性，因而可以供患者选择的治疗方案少之又少。
无细胞脂肪提取液是在脂肪衍生物中纳米脂肪的基础上，进一步通过物理提纯方式所获得。经过Elisa检测，发现其内含有大量活性生长因子。如促血管生长因子VEGF，bFGF，TGF-β；神经营养因子GDNF以及BDNF；以及其他生长因子IGF等，并推测其具有促进血管新生、抗炎等作用。在本研究中，我们将无细胞脂肪提取液直接注射至患者眶周真皮内，通过比较术前术后标准化摄影的照片，发现眶下的细纹数量减少，皮肤整洁(图1)。同时客观皱纹评价也证实，在多次治疗后，眶下的皮肤粗造度参数SEr、皮肤光滑度参数SEsm均呈下降趋势，表示呈逐渐光滑，皱纹平坦整洁的趋势(图2)。在第二次就诊时，眶周皱纹明显减少，皮肤细腻，而在第三次就诊时皱纹少许增加，皮肤稍粗糙，但随着治疗次数增加，皱纹逐渐减少，皮肤更加光滑。停止治疗后，效果仍然持续至随访12月，虽然有轻度反复的趋势，但相比起治疗前仍有所改善。治疗后观察到皮肤弹性增高(图3)，经皮失水的情况好转，随着持续的治疗，皮肤失水的程度也逐渐下降，并且持续至随访12月(图4)。无细胞脂肪提取液治疗的并发症少，无严重副作用，在用于改善皮肤衰老的过程中仅观察到轻微的术中疼痛及术后短时间淤青，表明无细胞脂肪提取液治疗安全可靠。
因此，本实施例的研究结果表明CEFFE具有抗皮肤衰老，促进皮肤年轻化的作用。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后， 本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种无细胞脂肪提取物的用途，其特征在于，用于制备组合物或制剂，所述组合物或制剂用于改善皮肤衰老和/或促进皮肤年轻化。
  2. 如权利要求1所述的用途，其特征在于，所述的皮肤衰老包括眶周皮肤衰老。
  3. 如权利要求1所述的用途，其特征在于，所述的皮肤衰老包括选自下组的一种或多种表现：
    (a)皮肤皱纹数量增多；
    (b)皮肤粗糙度增加；
    (c)皮肤光滑度降低；
    (d)皮肤弹性度降低；
    (e)皮肤松弛；和/或
    (f)皮肤失水。
  4. 如权利要求1所述的用途，其特征在于，所述的改善皮肤衰老和/或促进皮肤年轻化包括选自下组的一种或多种方式进行：
    (i)减少皮肤皱纹数量；
    (ii)降低皮肤粗糙度；
    (iii)提高皮肤的光滑度；
    (iv)增加皮肤弹性；
    (v)降低皮肤水分流失；和/或
    (vi)提高皮肤的保水能力。
  5. 如权利要求1所述的用途，其特征在于，所述的组合物或制剂包括药物组合物或制剂、食品组合物或制剂、保健品组合物或制剂或膳食补充剂。
  6. 如权利要求1所述的用途，其特征在于，所述的组合物或制剂的剂型为口服制剂、外用制剂或注射制剂。
  7. 如权利要求1所述的用途，其特征在于，所述的组合物或制剂的剂型为真皮内注射制剂。
  8. 如权利要求1所述的用途，其特征在于，所述的无细胞脂肪提取物含有一种或多种选自下组的组分：IGF-1、BDNF、GDNF、TGF-β1、HGF、bFGF、VEGF、TGF-β1、HGF、PDGF、EGF、NT-3、GH、G-CSF，或其组合。
  9. 如权利要求1所述的用途，其特征在于，所述的种无细胞脂肪提取物含有但不限于一种或多种选自下组的组分：IGF-1、BDNF、GDNF、bFGF、VEGF、TGF-β1、HGF、PDGF，或其组合。
  10. 如权利要求8所述的用途，其特征在于，所示的无细胞脂肪提取物包括选自下组的一种或多种特征：
    在所述的无细胞脂肪提取物中，所述的IGF-1的浓度为5000-30000pg/ml，较佳地6000-20000pg/ml，更佳地7000-15000pg/ml，更佳地8000-12000pg/ml，更佳地9000-11000pg/ml，更佳地9500-10500pg/ml；
    在所述的无细胞脂肪提取物中，所述的BDNF的浓度为800-5000pg/ml，较佳地1000-4000pg/ml，更佳地1200-2500pg/ml，更佳地1400-2000pg/ml，更佳地1600-2000pg/ml，更佳地1700-1850pg/ml；
    在所述的无细胞脂肪提取物中，所述的GDNF的浓度为800-5000pg/ml，较佳地1000-4000pg/ml，更佳地1200-2500pg/ml，更佳地1400-2000pg/ml，更佳地1600-2000pg/ml，更佳地1700-1900pg/ml；
    在所述的无细胞脂肪提取物中，所述的bFGF的浓度为50-600pg/ml，较佳地100-500pg/ml，更佳地120-400pg/ml，更佳地150-300pg/ml，更佳地200-280pg/ml，更佳地220-260pg/ml；
    在所述的无细胞脂肪提取物中，所述的VEGF的浓度为50-500pg/ml，较佳地100-400pg/ml，更佳地120-300pg/ml，更佳地150-250pg/ml，更佳地170-230pg/ml，更佳地190-210pg/ml；
    在所述的无细胞脂肪提取物中，所述的TGF-β1的浓度为200-3000pg/ml，较佳地400-2000pg/ml，更佳地600-1500pg/ml，更佳地800-1200pg/ml，更佳地800-1100pg/ml，更佳地900-1000pg/ml；
    在所述的无细胞脂肪提取物中，所述的HGF的浓度为200-3000pg/ml，较佳地400-2000pg/ml，更佳地600-1500pg/ml，更佳地600-1200pg/ml，更佳地800-1000 pg/ml，更佳地850-950pg/ml；和/或
    在所述的无细胞脂肪提取物中，所述的PDGF的浓度为50-600pg/ml，较佳地80-400pg/ml，更佳地100-300pg/ml，更佳地140-220pg/ml，更佳地160-200pg/ml，更佳地170-190pg/ml。
  11. 如权利要求8所述的用途，其特征在于，所示的无细胞脂肪提取物包括选自下组的一种或多种特征：
    所述的IGF-1与VEGF的重量比为20-100：1，较佳地30-70：1，更佳地40-60：1，最佳地45-55：1；
    所述的BDNF与VEGF的重量比为2-20：1，较佳地4-15：1，更佳地6-12：1，最佳地8-9.5：1；
    所述的GDNF与VEGF的重量比为2-20：1，较佳地4-15：1，更佳地6-12：1，最佳地8.5-9.5：1；
    所述的bFGF与VEGF的重量比为0.2-8：1，较佳地0.5-5：1，更佳地0.6-2：1，更佳地0.8-1.6：1，最佳地1-1.5：1；
    所述的TGF-β1与VEGF的重量比为1-20：1，较佳地1-15：1，更佳地1-10：1，更佳地2-8：1，更佳地4-6：1；
    所述的HGF与VEGF的重量比为1-20：1，较佳地1-15：1，更佳地1-10：1，更佳地2-8：1，更佳地4-5.5：1；和/或
    所述的PDGF与VEGF的重量比为0.1-3：1，较佳地0.2-2：1，更佳地0.4-1.5：1，最佳地0.7-1.2：1。
  12. 如权利要求1所述的用途，其特征在于，所述的无细胞脂肪提取物通过以下方法制备：
    (1)提供一脂肪组织原料，将所述脂肪组织原料破碎，并进行漂洗(如用生理盐水)，从而获得经漂洗的脂肪组织；
    (2)对所述经漂洗后的脂肪组织进行离心，获得分层的混合物；
    (3)对所述分层的混合物，去除上层油层和下层水层，收集中间层(即含脂肪细胞的脂肪层)；
    (4)对所述中间层进行乳化，获得乳化的脂肪混合物(也称为纳米脂肪)；
    (5)将所述乳化的脂肪混合物通过离心处理，从而获得中间液体层，即为脂肪初提物；和
    (6)对所述脂肪初提物进行过滤和除菌，从而获得无细胞的脂肪提取物。
  13. 一种用于改善皮肤衰老和/或促进皮肤年轻化的组合物或制剂，其特征在于，所述的组合物或制剂包含：
    (a)无细胞脂肪提取物；和
    (b)药学上、食品上、保健品或膳食上可接受的载体或赋形剂。
  14. 一种改善皮肤衰老和/或促进皮肤年轻化的方法，其特征在于，对需要的对象施用无细胞脂肪提取物。
  15. 如权利要求14所述的方法，其特征在于，所述施用方式为真皮内注射施用。
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