WO2023284466A1 - 无细胞脂肪提取物用于治疗卵巢功能不全 - Google Patents

无细胞脂肪提取物用于治疗卵巢功能不全 Download PDF

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WO2023284466A1
WO2023284466A1 PCT/CN2022/098413 CN2022098413W WO2023284466A1 WO 2023284466 A1 WO2023284466 A1 WO 2023284466A1 CN 2022098413 W CN2022098413 W CN 2022098413W WO 2023284466 A1 WO2023284466 A1 WO 2023284466A1
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cell
fat extract
free fat
another preferred
preparation
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PCT/CN2022/098413
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English (en)
French (fr)
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张文杰
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上海萨美细胞技术有限公司
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1808—Epidermal growth factor [EGF] urogastrone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1833—Hepatocyte growth factor; Scatter factor; Tumor cytotoxic factor II
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1841—Transforming growth factor [TGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A61K38/1866—Vascular endothelial growth factor [VEGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/27—Growth hormone [GH] (Somatotropin)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/30—Insulin-like growth factors (Somatomedins), e.g. IGF-1, IGF-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • 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
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • 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 extract for treating ovarian insufficiency.
  • Premature ovarian insufficiency also known as "hypogonadism” refers to acquired hypergonadotropic dysplasia before the age of 40. It is a gradual physiological process for women from fetus to old age. As a gonad organ, the ovary performs the important physiological functions of periodic ovulation and secretion of sex hormones. With the continuous development of chemotherapy regimens, many patients with malignant tumors can survive for a long time or even be cured; however, due to the damage of ovarian function caused by chemotherapy, premature amenorrhea and infertility occur, which affects the quality of life of female patients. Preserving fertility and physiological functions while treating diseases is an urgent problem for female malignant tumor patients to solve.
  • hormone replacement therapy is mainly used at home and abroad, but long-term use leads to an increase in the prevalence of ovarian cancer, which makes the application of this therapy controversial. Therefore, there is no reliable treatment that can help restore ovarian function in women. Therefore, there is a need in the art to develop a drug that can effectively treat ovarian insufficiency.
  • the purpose of the present invention is to provide a use of a cell-free fat extract in preventing and/or treating ovarian insufficiency and/or its complications.
  • the first aspect of the present invention provides the use of a cell-free fat extract for preparing a composition or preparation for preventing and/or treating ovarian insufficiency and/or its complications.
  • the subject of ovarian insufficiency and/or its complications is a human or a non-human mammal.
  • the complications of ovarian insufficiency are selected from the group consisting of secondary amenorrhea, decreased estrogen level, increased gonadotropin level, decreased follicle number, decreased follicle quality, early amenorrhea, infertility, or a combination thereof.
  • the ovarian insufficiency includes ovarian insufficiency caused by chemical damage.
  • the chemical damage includes damage caused by chemotherapy drugs.
  • the chemotherapeutic drugs include chemotherapeutic drugs for preventing and/or treating tumors.
  • the chemotherapy drugs include paclitaxel or doxorubicin.
  • the chemotherapy drug is selected from the group consisting of cyclophosphamide, busulfan, paclitaxel, doxorubicin, or a combination thereof.
  • the prevention and/or treatment of ovarian insufficiency and/or its complications include:
  • the promotion of ovarian function recovery includes one or more features selected from the following group:
  • said improving fertility includes increasing litter size.
  • the cell-free fat extract is a cell-free fat extract prepared from fat in humans or non-human mammals.
  • the non-human mammal is monkey, orangutan, cow, pig, dog, sheep, mouse or rabbit.
  • the composition or preparation includes a pharmaceutical composition or preparation, a food composition or preparation, a health product composition or preparation or a dietary supplement.
  • composition or preparation further includes a pharmaceutically, food, health product or dietary acceptable carrier.
  • composition or preparation further includes other drugs for preventing and/or treating ovarian insufficiency and/or its complications.
  • the other drugs for preventing and/or treating ovarian insufficiency and/or its complications are selected from the group consisting of hormone replacement therapy (HRT), dehydroepiandrosterone (DHEA), clomiphene Wait.
  • HRT hormone replacement therapy
  • DHEA dehydroepiandrosterone
  • clomiphene Wait a group consisting of hormone replacement therapy (HRT), dehydroepiandrosterone (DHEA), clomiphene Wait.
  • the dosage form of the composition or preparation is oral preparation, external preparation or injection preparation.
  • the injection preparation is an intravenous injection or an intramuscular injection.
  • the dosage form of the composition or preparation is solid dosage form, semi-solid dosage form, or liquid dosage form, such as solution, gel, cream, emulsion, ointment, cream, paste, cake, powder, patch 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, locally, or by injection.
  • the cell-free fat extract does not contain cells and lipid droplets.
  • the lipid droplets are oil droplets released after fat cells are broken.
  • the "free of lipid droplets" means that in the cell-free fat extract, the volume of oil droplets accounts for less than 1%, 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" means that the average number of cells in 1 ml of cell-free fat extract is ⁇ 1, preferably ⁇ 0.5, more preferably ⁇ 0.1, or 0.
  • the cell-free fat extract is a naturally obtained nano-fat extract without additives.
  • the "without added ingredients” means that except for the rinsing step, no solutions, solvents, small molecules, chemical agents, and biological additives are added during the preparation of the fat extract.
  • the cell-free fat extract is prepared by emulsifying adipose tissue and then centrifuging.
  • 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 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 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 Preferably 1400-2000 pg/ml, more preferably 1600-2000 pg/ml, more preferably 1700-1900 pg/ml.
  • the concentration of bFGF is 50-600pg/ml, preferably 100-500pg/ml, more preferably 120-400pg/ml, more preferably Preferably 150-300 pg/ml, more preferably 200-280 pg/ml, more preferably 220-260 pg/ml.
  • the concentration of VEGF is 50-500pg/ml, preferably 100-400pg/ml, more preferably 120-300pg/ml, more preferably 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 HGF is 200-3000pg/ml, preferably 400-2000pg/ml, more preferably 600-1500pg/ml, more preferably 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 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, 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, 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, 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.
  • 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:
  • the present invention provides a method for preparing a cell-free fat extract, the method comprising the steps of:
  • 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, most preferably 1100-1300g.
  • the centrifugation time is 1-15 min, preferably 1-10 min, more preferably 1-8 min, most preferably 1-5 min.
  • the centrifugation temperature is 2-6°C.
  • the emulsification is mechanical emulsification.
  • the mechanical emulsification is carried out mechanically by repeated blowing (such as blowing 20-200 times, preferably 20-150 times, more preferably 20-100 times, more preferably 30-50 times) with a syringe. emulsification.
  • the blowing method is to repeatedly push and beat at a constant speed with two 10ml injection syringes connected to a three-way tube.
  • the emulsification is crushed by a tissue homogenizer.
  • step (5) before the emulsified fat mixture is centrifuged, it further includes freezing and thawing the emulsified fat mixture.
  • the thawed mixture is used for centrifugation.
  • 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 4 layers, the first layer is an oil layer, the second layer is a residual fat tissue layer, and the third layer is a liquid layer (that is, the middle liquid layer), 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 min, preferably 1-10 min, more preferably 2-8 min, most preferably 3-7 min.
  • the centrifugation temperature is 2-6°C.
  • the first layer, the second layer, the third layer and the fourth layer are arranged sequentially 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 fat cells in the initial fat extract.
  • the filtration and sterilization are performed through a filter (such as a 0.22 ⁇ m microporous membrane).
  • the filter is a microporous membrane filter.
  • the pore size of the microporous 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 first passed through a first filter that can filter out cells, and then through a second filter that can filter out pathogens (such as bacteria). filter (such as a 0.22 ⁇ m filter).
  • the step (6) further includes subpackaging the fat extract to form subpackaged products.
  • 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 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 prepared by the method as described in the second aspect of the present invention.
  • the fourth aspect of the present invention provides a composition or preparation, said composition or preparation comprising (a) the cell-free fat extract as described in the third aspect of the present invention; and (b) pharmaceutical, food, Health products or dietary acceptable carriers or excipients.
  • 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 oral preparation, external preparation or 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 dosage form of the composition or preparation is solid dosage form, semi-solid dosage form, or liquid dosage form, such as solution, gel, cream, emulsion, ointment, cream, paste, cake, powder, patch 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 as described in the fourth aspect of the present invention, and the method includes the steps of: combining the cell-free fat extract as described in the third aspect of the present invention with pharmaceutical On the food, on the food, on the health care product or on the dietary acceptable carrier or excipient, thus form the composition or preparation.
  • the sixth aspect of the present invention provides a method for preventing and/or treating ovarian insufficiency and/or its complications, administering the cell-free fat extract as described in the third aspect of the present invention to the subject in need.
  • the subject is a human or a non-human mammal.
  • the non-human mammal includes rodents, such as rats and mice.
  • the administration is oral, external or injection.
  • FIG 1 shows the effect of CEFFE on E2 hormone levels in POI mice.
  • FIG. 2 shows the effect of CEFFE on AMH hormone levels in POI mice.
  • FIG. 3 shows the effect of CEFFE on FSH hormone levels in POI mice.
  • Figure 4 shows the histological detection results of the ovaries of POI mice before and after CEFFE treatment.
  • Figure 4A POI for 2 weeks
  • Figure 4B POI+CF treatment for 2 weeks
  • Figure 4C POI for 4 weeks
  • Figure 4D POI+CF treatment for 4 weeks
  • Figure 4E normal ovary.
  • the present inventors After extensive and intensive research, the present inventors have firstly developed a cell-free fat extract that is excellent in the treatment of ovarian insufficiency and/or its complications (especially ovarian insufficiency and/or its complications caused by chemical damage) effect.
  • the terms “comprising”, “comprising” 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 (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
  • Cell free fat extract Cell free fat extract (Cell free fat extract, CEFFE/CF) and preparation method thereof
  • the terms "cell-free fat extract of the present invention”, “extract of the present invention”, “fat extract of the present invention” etc. are used interchangeably and refer to )
  • 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 need to add any additives (or added ingredients) in the preparation process, some or a small amount of safe substances (such as a small amount) that have no negative or adverse effects on the activity of the extract of the present invention can 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 fraction after centrifugation, and is a cell-free, easy to prepare, rich in various A liquid of growth factors.
  • the cell-free fat extract is a cell-free fat extract.
  • the cell-free fat extract of the present invention may contain various cytokines.
  • the cell-free fat extract includes IGF-1, BDNF, GDNF, TGF- ⁇ , HGF, bFGF, VEGF, TGF- ⁇ 1, PDGF, EGF, NT-3, GH and G-CSF one or more.
  • 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.
  • the concentration of BDNF is 800-5000pg/ml, preferably 1000-4000pg/ml, more preferably 1200-2500pg/ml, more preferably 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 Preferably 1400-2000 pg/ml, more preferably 1600-2000 pg/ml, more preferably 1700-1900 pg/ml.
  • the concentration of bFGF is 50-600pg/ml, preferably 100-500pg/ml, more preferably 120-400pg/ml, more preferably Preferably 150-300 pg/ml, more preferably 200-280 pg/ml, more preferably 220-260 pg/ml.
  • the concentration of VEGF is 50-500pg/ml, preferably 100-400pg/ml, more preferably 120-300pg/ml, more preferably 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 HGF is 200-3000pg/ml, preferably 400-2000pg/ml, more preferably 600-1500pg/ml, more preferably 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 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, 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, 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, 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.
  • 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 above in the second aspect of the present invention.
  • the cell-free fat extract of the present invention is prepared by the following method:
  • the centrifugation is performed at 800-2500g, preferably 800-2000g, more preferably 1000-1500g, most preferably 1100-1300g.
  • the centrifugation time is 1-15 min, preferably 1-10 min, more preferably 1-8 min, most preferably 1-5 min.
  • the emulsification is mechanical emulsification.
  • the mechanical emulsification is carried out mechanically by repeated blowing (such as blowing 20-200 times, preferably 20-150 times, more preferably 20-100 times, more preferably 30-50 times) with a syringe. emulsification.
  • the blowing method is to repeatedly push and beat at a constant speed with two 10ml injection syringes connected to a three-way tube.
  • the emulsification is crushed by a tissue homogenizer.
  • step (5) before the emulsified fat mixture is processed by centrifugation, it also includes freezing and thawing the emulsified fat mixture.
  • the thawed mixture is used for centrifugation.
  • 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 4 layers, the first layer is an oil layer, the second layer is a residual fat tissue layer, and the third layer is a liquid layer (that is, the middle liquid layer), and the fourth layer is the cell/tissue debris sedimentation layer.
  • the centrifugation is carried out at 800-2500g, preferably 800-2000g, more preferably 1000-1500g, most preferably 1100-1300g.
  • the centrifugation time is 1-15 min, preferably 1-10 min, more preferably 2-8 min, most preferably 3-7 min.
  • the first layer, the second layer, the third layer and the fourth layer are arranged sequentially 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 fat cells in the initial fat extract.
  • the filtration and sterilization are performed through a filter (such as a 0.22 ⁇ m microporous membrane).
  • the filter is a microporous membrane filter.
  • the pore size of the microporous 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 first passed through a first filter that can filter out cells, and then through a second filter that can filter out pathogens (such as bacteria).
  • filter such as a 0.22 ⁇ m filter.
  • the step (6) further includes subpackaging the fat extract to form subpackaged products.
  • 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 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 preparing a composition or preparation, and the composition or preparation is used for preventing and/or treating ovarian insufficiency and/or its complications.
  • prevention means 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 in the present invention includes delaying and terminating the progression of the disease, or eliminating the disease, and does not require 100% inhibition, eradication and reversal.
  • a cell-free fat extract of the invention reduces, inhibits and/or reverses ovarian insufficiency, e.g., by at least about 10%, at least About 30%, at least about 50%, or at least about 80%.
  • the present invention also provides a method for preventing and/or treating ovarian insufficiency and/or its complications, and administering the cell-free fat extract as described in the present invention to the object in need.
  • the subject is a human or a non-human mammal.
  • the non-human mammal includes rodents, such as rats and mice.
  • the administration is oral, external or injection.
  • compositions of 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 extract of the present invention can be prepared into pharmaceutical compositions, such as tablets, capsules, powders, granules, solutions, lozenges, jellies, cream preparations, spirits, suspensions, Dosage forms such as tinctures, poultices, liniments, lotions, and aerosols.
  • Pharmaceutical compositions can be prepared by generally known preparation techniques, and appropriate pharmaceutical additives can be added to the drug.
  • composition of the present invention may also include pharmaceutically, food-, health-care or diet-acceptable carriers.
  • “Pharmaceutical, food, health care or dietary acceptable carrier” refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity.
  • “Compatibility” herein means that the components of the composition can be blended with the compound of the present invention and with each other without significantly reducing the efficacy of the compound.
  • acceptable carriers include cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.) , gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol etc.), emulsifiers (such as Tween ), wetting agent (such as sodium lauryl sulfate), coloring agent, flavoring agent, stabilizer, antioxidant, preservative, pyrogen-free water, etc.
  • cellulose and its derivatives such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.
  • gelatin such as talc
  • solid lubricants such as stearic acid, magnesium stearate
  • calcium sulfate such as soybean oil
  • the administration method of the composition of the present invention is not particularly limited, and representative administration methods include (but not limited to): oral, parenteral (intravenous, intramuscular), local administration, and preferred administration methods are oral administration and injection administration.
  • the dosage forms of the compositions or preparations of the present invention are oral preparations, external preparations or injection preparations.
  • solid dosage forms for oral administration or administration include capsules, tablets, pills, powders and granules.
  • the active compound is admixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with (a) fillers or extenders, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, For example, glycerol; (d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow agents, such as paraffin; (f) Absorption accelerator
  • Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shell materials, such as enteric coatings and others 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 used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances, etc.
  • inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylform
  • 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 in addition to the active ingredient, may contain suspending agents, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, mixtures of these substances, and the like.
  • suspending agents for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, 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 vehicles include water, ethanol, polyols, and suitable mixtures thereof.
  • Dosage forms for topical administration or administration of a compound 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 which may be required, if necessary.
  • the cell-free fat extract of the present invention can be administered or administered alone, or combined with other drugs for preventing and/or treating ovarian insufficiency and/or its complications.
  • a safe and effective amount of the cell-free fat extract of the present invention is applied to human or non-human animals (such as rats, mice, dogs, cats, cows, chickens, ducks, etc.) in need of treatment, wherein the administration
  • the hourly dose is the acceptable and considered effective dose in medicine, food or health products.
  • the term "safe and effective amount” refers to an amount that produces functions or activities on humans and/or animals and is acceptable to humans and/or animals. Those of ordinary skill in the art should understand that the "safe and effective amount” may vary depending on the form of the pharmaceutical composition, the route of administration, the adjuvant of the drug used, the severity of the disease, and the combination with other drugs. different.
  • the daily dosage is usually 0.1-1000 mg, preferably 1-600 mg, more preferably 2-300 mg.
  • factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.
  • the present invention finds for the first time that the cell-free fat extract has excellent preventive and therapeutic effects on ovarian insufficiency and/or its complications.
  • the cell-free fat extract of the present invention is a cell-free component, which can avoid cell-related problems in clinical applications, such as genetic stability after cell processing, cell activity and survival rate after injection, The multiple administration storage of cells, and the immunogenicity of cells when using allogeneic fat, the cell-free fat extract of the present invention has higher safety and The advantage of lower side effects.
  • Adipose tissue was obtained from 6 healthy women who underwent conventional liposuction, with an average age of 31 years (24-36 years). After local injection of tumescent fluid for anesthesia, use a 3mm liposuction cannula with a large side hole (2mm x 7mm) to connect to a 20mL syringe, suck radially under artificial negative pressure, stand the obtained fat upright, remove the tumescent fluid, and use Rinse with saline 3 times.
  • the middle layer ie, the fat layer containing fat cells
  • the mechanically emulsified fat mixture was frozen in a -80°C refrigerator, 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 mixture of layers, the layered mixture is divided into 4 layers, the first layer is the oil layer, the second layer is the residual fat tissue layer, the third layer is the liquid layer, and the fourth layer is the cell/tissue debris sedimentation layer, remove the oil layer and The residual adipose tissue layer is absorbed into the liquid layer, and the pollution of the cell/tissue debris sediment layer is avoided during the absorption process, so as to obtain the primary fat extraction solution.
  • ELISA immunosorbent assay kit was used to detect the content of cytokines, including IGF-1, BDNF, GDNF, bFGF, VEGF, TGF- ⁇ 1, HGF and PDGF and other cytokines.
  • the average concentrations of the 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).
  • CEFFE cell-free fat extract
  • mice were selected for model establishment, and a one-time intraperitoneal injection of cyclophosphamide with a loading dose of 120 mg/kg combined with intradermal injection of 12 mg/kg of busulfan in a total of 0.1 mL (using PBS as the solvent).
  • control group normal mice not injected with chemotherapy drugs were used as objects for experimental observation.
  • Fig. 1 and Fig. 2 illustrate that the levels of E2 (estradiol) hormone and AMH (anti-Müllerian hormone) in mice with ovarian insufficiency caused by chemotherapy are significantly reduced, and after intraperitoneal injection of CEFFE, the level of E2 hormone is significantly improved, and Stability was maintained until the four-week endpoint of the observation period.
  • the difference in hormone levels before and after injection of CEFFE was statistically significant.
  • the results prove that CEFFE can significantly improve the levels of E2 hormone and AMH hormone in chemotherapy-induced ovarian insufficiency mice, which is beneficial to the recovery of ovarian function. ( * P ⁇ 0.05)
  • Fig. 3 shows that the level of FSH (follicle-stimulating hormone) in mice with ovarian insufficiency caused by chemotherapy increases significantly. more stable. The difference in hormone levels before and after injection of CEFFE was statistically significant. The results prove that CEFFE can significantly reduce the level of FSH hormone in chemotherapy-induced ovarian insufficiency mice, which is beneficial to the recovery of ovarian function. ( * P ⁇ 0.05)
  • FIG. 4A In the POI 2w group, a large number of secondary follicles degenerated, atresia, and a small amount of inflammatory cell infiltration were seen (Fig. 4A).
  • the structure of follicles In the POI+CF 2w group, the structure of follicles was basically normal, and the medulla was partially collapsed (Fig. 4B).
  • the POI 4w group In the POI 4w group, the number of primordial follicles decreased, the corpus luteum and more interstitial glands were seen, and ovulation should have just occurred (Fig. 4C).
  • a small amount of inflammatory cell infiltration was seen in the group after POI+CF 4W, and the overall structure was normal (Fig. 4D). The normal ovarian section is shown in Fig. 4E.
  • CEFFE promoted the recovery of ovarian function and structure in ovarian insufficiency caused by chemical injury.
  • CEFFE has an excellent therapeutic effect on ovarian insufficiency and its complications caused by chemical injury.

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Abstract

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预防和/或治疗卵巢功能不全和/或其并发症。所述的无细胞脂肪提取物对卵巢功能不全和/或其并发症具有优异的治疗效果。

Description

无细胞脂肪提取物用于治疗卵巢功能不全 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治疗卵巢功能不全。
背景技术
卵巢功能不全(premature ovarian insufficiency，POI)，又称“卵巢功能低下”，是指40岁以前的获得性高促性腺发育不良。女性从胎儿到老年是渐进的生理过程，卵巢作为性腺器官，行使着周期性排卵与分泌性激素的重要生理功能。随着化疗方案的不断进展，很多恶性肿瘤患者得以长期存活甚至治愈；但由于化疗导致的卵巢功能损害，进而出现提前闭经和不孕，影响了女性患者的生存质量。在治疗疾病的同时能够保留生育及生理功能，是女性恶性肿瘤患者迫切希望解决的问题。目前，国内外主要应用激素替代疗法，但长期应用导致卵巢癌患病率增加，使得该疗法的应用存在较大争议。因此，尚未有可靠的治疗方法可以帮助恢复女性卵巢功能。因此，本领域需要开发一种能够有效治疗卵巢功能不全的药物。
发明内容
本发明的目在于提供一种无细胞脂肪提取物在预防和/或治疗卵巢功能不全和/或其并发症方面中的用途。
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预防和/或治疗卵巢功能不全和/或其并发症。
在另一优选例中，所述的卵巢功能不全和/或其并发症的对象为人或非人哺乳动物。
在另一优选例中，所述的卵巢功能不全并发症选自下组：继发性闭经、雌激素水平下降、促性腺激素水平上升、卵泡数目下降、卵泡质量降低、提前闭经、不孕，或其组合。
在另一优选例中，所述的卵巢功能不全包括化学性损伤引起的卵巢功能不 全。
在另一优选例中，所述的化学性损伤包括化疗药物导致的损伤。
在另一优选例中，所述的化疗药物包括用于预防和/或治疗肿瘤的化疗药物。
在另一优选例中，所述的化疗药物包括紫杉醇或阿霉素。
在另一优选例中，所述的化疗药物选自下组：环磷酰胺、白消安、紫杉醇、阿霉素，或其组合。
在另一优选例中，所述的预防和/或治疗卵巢功能不全和/或其并发症包括：
(i)促进卵巢功能恢复；
(ii)提升生育能力。
在另一优选例中，所述促进卵巢功能恢复包括选自下组的一个或多个特征：
(i)雌二醇激素水平上升；
(ii)抗缪勒氏管激素水平上升；
(iii)促卵泡激素水平下降；
(iv)恢复卵泡结构。
在另一优选例中，所述的提升生育能力包括提高产仔数量。
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预防和/或治疗卵巢功能不全和/或其并发症的药物。
在另一优选例中，所述的其它预防和/或治疗卵巢功能不全和/或其并发症的药物选自下组：激素替代疗法(HRT)、脱氢表雄酮(DHEA)、克罗米芬等。
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，提供一种预防和/或治疗卵巢功能不全和/或其并发症的方法，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。
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显示了CEFFE对POI小鼠的E2激素水平的影响。
图2显示了CEFFE对POI小鼠的AMH激素水平的影响。
图3显示了CEFFE对POI小鼠的FSH激素水平的影响。
图4显示了CEFFE治疗前后POI小鼠的卵巢组织学检测结果。图4A：POI2周；图4B：POI+CF治疗2周；图4C：POI 4周；图4D：POI+CF治疗4周；图4E:正常卵巢。
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卵巢功能不全和/或其并发症(尤其是化学性损伤引起的卵巢功能不全和/或其并发症)具有优异的治疗作用。
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/CF)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1400-2000pg/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。
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预防和/或治疗卵巢功能不全和/或其并发症。
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/或逆转了卵巢功能不全例如至少约10％、至少约30％、至少约50％、或至少约80％。
本发明还提供一种预防和/或治疗卵巢功能不全和/或其并发症的方法，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。
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(如吐温
Figure PCTCN2022098413-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。
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。
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/或治疗卵巢功能不全和/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卵巢功能不全和/或其并发症具有 优异的预防和治疗效果。
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防治卵巢功能不全和/或其并发症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.实验方法和结果
考察无细胞脂肪提取液(CEFFE)对因化学性损伤导致卵巢功能不全和其并发症的治疗效果。具体为建立化学药物导致的卵巢功能不全(POI)小鼠模型，应用CEFFE对其进行治疗，通过各项指标观察小鼠卵巢功能的改善程度。
POI组选择C57小鼠进行模型建立，一次性腹腔注射环磷酰胺负荷剂量120mg/kg联合皮内注射白消安12mg/kg共0.1mL(以PBS为溶剂)。对照组以未注射化疗药物的正常小鼠作为对象进行实验观察。
体内实验：确定建立POI小鼠模型成功后，对小鼠进行CEFFE治疗。实验(POI+CF)组选择蛋白含量3.0以上的CEFFE制剂进行腹腔注射，0.1mL/天，连续处理7天。部分小鼠于处理后2周、4周取材卵巢，进行包括性激素水平、卵巢组织学的检测。性激素水平结果如图1、图2、图3所示，卵巢组织学结果如图4所示。
图1和图2说明化疗导致卵巢功能不全的小鼠E2(雌二醇)激素和AMH(抗缪勒氏管激素)水平均大幅下降，经腹腔注射CEFFE后，E2激素水平有明显提高，且稳定维持至观察时间的四周终末点。注射CEFFE前后的激素水平差异具有统计学意义。该结果证明，CEFFE对于化疗导致的卵巢功能不全小鼠E2激素水平和 AMH激素水平具有明显的提升作用，有利于卵巢功能的恢复。( *P<0.05)
图3说明化疗导致卵巢功能不全的小鼠FSH(促卵泡激素)激素水平大幅升高，经腹腔注射CEFFE后，FSH数值有明显下降，且在四周的观察终末点时数值持续下降并逐渐趋于稳定。注射CEFFE前后的激素水平差异具有统计学意义。该结果证明，CEFFE对于化疗导致的卵巢功能不全小鼠FSH激素水平具有明显的降低作用，有利于卵巢功能的恢复。( *P<0.05)
POI 2w组中，可见大量次级卵泡退行、闭锁，少量炎症细胞浸润(图4A)。POI+CF 2w组可见卵泡结构基本正常,髓质部分塌陷(图4B)。POI 4w组中可见原始卵泡数量减少，黄体和较多间质腺，应该刚进行过排卵(图4C)。POI+CF 4W后组中可见少量炎症细胞浸润，整体结构正常(图4D)，正常卵巢切片如图4E所示。
结果发现，CEFFE可促进因化学性损伤导致卵巢功能不全的卵巢功能和结构的恢复。
因此，CEFFE对因化学性损伤导致卵巢功能不全和其并发症具有优异的治疗效果。
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预防和/或治疗卵巢功能不全和/或其并发症。
  2. 如权利要求1所述的用途，其特征在于，所述的卵巢功能不全并发症选自下组：继发性闭经、雌激素水平下降、促性腺激素水平上升、卵泡数目下降、卵泡质量降低、提前闭经、不孕，或其组合。
  3. 如权利要求1所述的用途，其特征在于，所述的卵巢功能不全包括化学性损伤引起的卵巢功能不全。
  4. 如权利要求3所述的用途，其特征在于，所述的化学性损伤包括化疗药物导致的损伤。
  5. 如权利要求4所述的用途，其特征在于，所述的化疗药物选自下组：环磷酰胺、白消安、紫杉醇、阿霉素，或其组合。
  6. 如权利要求1所述的用途，其特征在于，所述的预防和/或治疗卵巢功能不全和/或其并发症包括：
    (i)促进卵巢功能恢复；
    (ii)提升生育能力。
  7. 如权利要求6所述的用途，其特征在于，所述促进卵巢功能恢复包括选自下组的一个或多个特征：
    (i)提高雌二醇激素水平；
    (ii)提高抗缪勒氏管激素水平；
    (iii)降低促卵泡激素水平；
    (iv)促进卵泡结构恢复。
  8. 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。
  9. 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。
  10. 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通过外用、局部、或注射方式施用。
  11. 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。
  12. 如权利要求11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800-1000pg/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。
  13. 如权利要求11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。
  14. 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。
  15. 一种预防和/或治疗卵巢功能不全和/或其并发症的方法，其特征在于，对需要的对象施用无细胞脂肪提取物。
PCT/CN2022/098413 2021-07-14 2022-06-13 无细胞脂肪提取物用于治疗卵巢功能不全 WO2023284466A1 (zh)

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