WO2021249561A1 - 无细胞脂肪提取液对肺部疾病的治疗用途 - Google Patents

无细胞脂肪提取液对肺部疾病的治疗用途 Download PDF

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WO2021249561A1
WO2021249561A1 PCT/CN2021/099865 CN2021099865W WO2021249561A1 WO 2021249561 A1 WO2021249561 A1 WO 2021249561A1 CN 2021099865 W CN2021099865 W CN 2021099865W WO 2021249561 A1 WO2021249561 A1 WO 2021249561A1
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fat
fat extract
acellular
extract
another preferred
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PCT/CN2021/099865
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English (en)
French (fr)
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张文杰
于子优
侯曦凡
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上海萨美细胞技术有限公司
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Priority to US18/001,259 priority Critical patent/US20230248778A1/en
Publication of WO2021249561A1 publication Critical patent/WO2021249561A1/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
    • 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
    • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0653—Adipocytes; Adipose tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/02—Atmosphere, e.g. low oxygen conditions

Definitions

  • the invention relates to the field of medicine, in particular to the therapeutic use of a cell-free fat extract for lung diseases.
  • Acute lung injury refers to the damage of alveolar epithelial cells, pulmonary interstitial capillary damage, destruction of alveolar and capillary barriers, etc. caused by various factors, which cause edema and inflammation of the alveoli and lung interstitium.
  • Cell infiltration is a clinically manifested disease with dyspnea and hypoxemia. It is estimated that it accounts for about 10% of the number of inpatients in intensive care units worldwide, and the mortality rate can reach more than 40%.
  • Acute respiratory distress syndrome (acute respiratory distress syndrome, ARDS) is the most severe stage of acute lung injury. Acute hypoxic respiratory failure caused by acute diffuse damage to the lung parenchyma. The clinical manifestations are progressive dyspnea and stubbornness. Characterized by sexual hypoxemia. All along, ALI/ARDS
  • ALI/ARDS Due to its high morbidity, high fatality rate and lack of treatment methods, the current clinical treatment of ALI/ARDS is mainly through mechanical ventilation for respiratory support, glucocorticoids, and pulmonary vasodilators. However, the current treatment methods are difficult Effectively reduce the mortality of ALI/ARDS, there is still a lack of effective treatment drugs.
  • the purpose of the present invention is to provide a use of acellular fat extract in the treatment of lung diseases such as ALI/ARDS.
  • an acellular fat extract for the preparation of a composition or preparation, and the composition or preparation is used for one or more uses selected from the following group: (i) prevention and /Or treatment of acute respiratory distress syndrome and/or acute lung injury; (ii) prevention and/or treatment of hypoxemia; (iii) improvement of lung tissue inflammation; (iv) improvement of lung tissue damage; (v) prevention and/ Or treat systemic inflammatory response syndrome; (vi) prevent and/or treat multiple organ failure.
  • the prevention and/or treatment of acute respiratory distress syndrome and/or acute lung injury includes one or more methods selected from the following group for prevention and/or treatment:
  • the prevention and/or treatment of hypoxemia includes increasing blood oxygen content.
  • said increasing blood oxygen content includes increasing blood oxygen partial pressure and/or increasing blood oxygen saturation.
  • the improvement of lung inflammation includes reducing the infiltration of inflammatory cells in the lung.
  • the inflammatory cells are selected from the group consisting of leukocytes, neutrophils, lymphocytes, monocytes, or a combination thereof.
  • the improvement of lung tissue damage includes improvement in one or more ways selected from the following group:
  • the patient with hypoxemia suffers from acute respiratory distress syndrome and/or acute lung injury.
  • the patient with lung tissue inflammation suffers from acute respiratory distress syndrome and/or acute lung injury.
  • the patient with lung tissue injury suffers from acute respiratory distress syndrome and/or acute lung injury.
  • 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, rat or rabbit.
  • the composition or preparation includes a pharmaceutical composition or preparation, a food composition or preparation, a health care product composition or preparation, or a dietary supplement.
  • composition or preparation further includes a pharmaceutically, food, health care product or diet acceptable carrier.
  • 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 preparation.
  • composition or preparation is administered by topical, topical, or subcutaneous injection.
  • the acellular fat extract does not contain cells and does not contain lipid droplets.
  • the lipid droplets are oil droplets released after fat cells are broken.
  • the "not containing lipid droplets" means that in the cell-free fat extract, the percentage of oil droplets in the total liquid is less than 1%, preferably less than 0.5%, more preferably less than 0.1%.
  • 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 acellular 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 added components.
  • the "additive-free” refers to that, except for the rinsing step, no solution, solvent, small molecule, chemical agent, or biological additive is added during the preparation process of the fat extract.
  • the fat extract is prepared by centrifuging the fat tissue after emulsification.
  • the fat extract contains but is not limited to one or more components selected from the following group: growth factors IGF-1, BDNF, GDNF, TGF- ⁇ , HGF, bFGF, VEGF, PDGF, EGF, NT-3, GH, G-CSF, or a combination thereof.
  • the cell-free fat extract contains one or more components selected from the following group: IGF-1, BDNF, GDNF, TGF- ⁇ , HGF, bFGF, VEGF, TGF- ⁇ 1 , HGF, 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 following group: IGF-1, BDNF, GDNF, bFGF, VEGF, TGF- ⁇ 1, HGF, PDGF, or a combination thereof.
  • the concentration of IGF-1 is 5000-30000 pg/ml, preferably 6000-20000 pg/ml, more preferably 7000-15000 pg/ml , More preferably 8000-12000pg/ml, more preferably 9000-11000pg/ml, more preferably 9500-10500pg/ml.
  • the concentration of the BDNF is 800-5000pg/ml, preferably 1000-4000pg/ml, more preferably 1200-2500pg/ml, more Preferably 1400-2000pg/ml, more preferably 1600-2000pg/ml, more preferably 1700-1850pg/ml.
  • the concentration of the GDNF is 800-5000 pg/ml, preferably 1000-4000 pg/ml, more preferably 1200-2500 pg/ml, more Preferably 1400-2000pg/ml, more preferably 1600-2000pg/ml, more preferably 1700-1900pg/ml.
  • the concentration of bFGF is 50-600pg/ml, preferably 100-500pg/ml, more preferably 120-400pg/ml, more It is preferably 150-300pg/ml, more preferably 200-280pg/ml, more preferably 220-260pg/ml.
  • the concentration of the VEGF is 50-500pg/ml, preferably 100-400pg/ml, more preferably 120-300pg/ml, more It is preferably 150-250pg/ml, more preferably 170-230pg/ml, more preferably 190-210pg/ml.
  • the concentration of TGF- ⁇ 1 is 200-3000 pg/ml, preferably 400-2000 pg/ml, more preferably 600-1500 pg/ml , More preferably 800-1200pg/ml, more preferably 800-1100pg/ml, more preferably 900-1000pg/ml.
  • the concentration of HGF is 200-3000 pg/ml, preferably 400-2000 pg/ml, more preferably 600-1500 pg/ml, more It is preferably 600-1200pg/ml, more preferably 800-1000pg/ml, more preferably 850-950pg/ml.
  • the concentration of PDGF is 50-600pg/ml, preferably 80-400pg/ml, more preferably 100-300pg/ml, more Preferably 140-220pg/ml, more preferably 160-200pg/ml, and even more preferably 170-190pg/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, Best 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. Better 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, most preferably 0.7-1.2:1.
  • the acellular fat extract is liquid.
  • the acellular fat extract is prepared by the following method:
  • the second aspect of the present invention provides a method for preparing a cell-free fat extract, the method comprising the steps:
  • 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 1-8 min, and most preferably 1-5 min.
  • the temperature of the centrifugation is 2-6°C.
  • the emulsification is mechanical emulsification.
  • the mechanical emulsification is repeated pipetting (such as pipetting 20-200 times, preferably 20-150 times, more preferably 20-100 times, more preferably 30-50 times) through a syringe for mechanical emulsification. emulsification.
  • the method of pipetting is that two 10ml injection syringes are connected to a three-way pipe and repeatedly pumped at a constant speed.
  • the emulsification is a method of crushing by a tissue homogenizer.
  • the emulsified fat mixture is frozen and then thawed.
  • 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 divided 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, and 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 pack 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 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, best 0.22 ⁇ m.
  • the filtration and sterilization are performed through the first filter that can filter out cells, and then the second filter that can filter out pathogens (such as bacteria).
  • Filter such as 0.22 ⁇ m filter.
  • the step (6) further includes dividing the fat extract to form a divided product.
  • the aliquoted extract can be stored at -20°C until use; it can be used directly after thawing at low temperature (such as -4°C) or normal temperature, or it can be stored at low temperature (such as 4°C) after thawing for a period of time, and then used ).
  • the third aspect of the present invention provides an acellular 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, said composition or preparation comprising (a) the acellular fat extract according to the third aspect of the present invention; and (b) pharmaceutically, food , Health products or dietary acceptable carriers or excipients.
  • the dosage form of the composition or preparation is powder, granule, capsule, injection, tincture, oral liquid, tablet or lozenge.
  • the injection is intravenous injection or intramuscular injection.
  • the dosage form of the composition or preparation is a solid dosage form, a semi-solid dosage form, or a liquid dosage form, such as a solution, gel, cream, lotion, ointment, cream, paste, cake, powder, Patches, etc.
  • the mass percentage of the acellular fat extract is 5 wt%, preferably 1-20 wt%, based on the total weight of the cosmetic composition.
  • the 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 includes the steps of: combining the acellular fat extract according to the third aspect of the present invention with a pharmaceutical It is mixed with carriers or excipients acceptable for food, food, health care products or diet to form a composition or preparation.
  • the sixth aspect of the present invention provides a method of (i) preventing and/or treating acute respiratory distress syndrome and/or acute lung injury; (ii) preventing and/or treating hypoxemia; (iii) improving lung tissue inflammation; (iv) Improving lung tissue damage; (v) preventing and/or treating systemic inflammatory response syndrome; (vi) preventing and/or treating multiple organ failure methods, administered to a subject in need as described in the third aspect of the present invention Of the acellular fat extract.
  • the subject is a human or non-human mammal.
  • the non-human mammals include rodents, such as rats and mice.
  • Figure 1 shows the survival rate of ARDS model rats.
  • Figure 2 shows the arterial partial pressure of oxygen and blood oxygen saturation in model rats (M ⁇ SD, *p ⁇ 0.05, **p ⁇ 0.01).
  • Figure 3 shows the count and classification of inflammatory cells in alveolar lavage fluid (*p ⁇ 0.05, **p ⁇ 0.01).
  • Figure 4 shows HE staining of lung histopathology.
  • the terms “including,” “including,” and “containing” are used interchangeably, and include not only open-ended definitions, but also semi-closed and closed-ended definitions. In other words, the term includes “consisting of” and “consisting essentially of”.
  • ALI acute lung injury
  • acute respiratory distress syndrome acute respiratory distress syndrome
  • ARDS acute respiratory distress syndrome
  • systemic inflammatory response syndrome and “SIRS” are used interchangeably.
  • prevention means a method of preventing the onset of a disease and/or its accompanying symptoms or protecting a subject from acquiring the disease. "Prevention” as used herein also includes delaying the onset of the disease and/or its accompanying symptoms and reducing the subject's risk of getting the disease.
  • the "treatment” in the present invention includes delaying and stopping the progression of the disease, or eliminating the disease, and does not require 100% inhibition, elimination and reversal.
  • the composition of the present invention is compared with the level observed in the absence of the acellular fat extract, composition, kit, food box or health care product box, or active ingredient combination of the present invention.
  • the pharmaceutical composition reduces, inhibits, and/or reverses diabetes, for example, at least about 10%, at least about 30%, at least about 50%, or at least about 80%.
  • improvement includes prevention, treatment, alleviation, reversal and alleviation, and so on.
  • IGF-1 insulin-like growth factors-1 (insulin-like growth factors-1).
  • BDNF brain-derived neurotrophic factor
  • GDNF glialcellline-derived neurotrophic factor
  • bFGF basic fibroblast growth factor
  • VEGF vascular endothelial growth factor
  • TGF- ⁇ 1 is called transforming growth factor- ⁇ 1 (transforming growth factor- ⁇ 1).
  • HGF hepatocyte growth factor
  • PDGF platelet-derived growth factor
  • EGF Epidermal Growth Factor
  • NT-3 neurotrophins-3 (neurotrophins-3).
  • GH Growth Hormone
  • G-CSF granulocyte colony stimulating factor
  • CEFFE Cell free fat extract
  • the terms "the cell-free fat extract of the present invention”, “the extract of the present invention”, “the fat extract of the present invention” and the like are used interchangeably and refer to the preparation process of the fat extract (except for the rinsing step). ) Extracts (or extracts) derived from adipose tissue prepared without adding any solutions, solvents, small molecules, chemical agents, and biological additives. A typical method of preparing the extract of the present invention is as described above in the second aspect of the present invention.
  • the acellular fat extract of the present invention can be derived from human adipose tissue. It is purified from nano fat by removing oil and cell/extracellular matrix after centrifugation. It is a cell-free, easy to prepare, and rich in various Kind of growth factor liquid.
  • the acellular fat extract is acellular fat extract.
  • the acellular fat extract of the present invention may include a variety of 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.
  • IGF-1 IGF-1, BDNF, GDNF, TGF- ⁇ , HGF, bFGF, VEGF, TGF- ⁇ 1, PDGF, EGF, NT-3, GH and G-CSF.
  • the cell-free fat extract contains but is not limited to one or more components selected from the following group: IGF-1, BDNF, GDNF, bFGF, VEGF, TGF- ⁇ 1, HGF, PDGF, or a combination thereof.
  • the concentration of IGF-1 is 5000-30000 pg/ml, preferably 6000-20000 pg/ml, more preferably 7000-15000 pg/ml , More preferably 8000-12000pg/ml, more preferably 9000-11000pg/ml, more preferably 9500-10500pg/ml.
  • the concentration of the BDNF is 800-5000pg/ml, preferably 1000-4000pg/ml, more preferably 1200-2500pg/ml, more Preferably 1400-2000pg/ml, more preferably 1600-2000pg/ml, more preferably 1700-1850pg/ml.
  • the concentration of the GDNF is 800-5000 pg/ml, preferably 1000-4000 pg/ml, more preferably 1200-2500 pg/ml, more Preferably 1400-2000pg/ml, more preferably 1600-2000pg/ml, more preferably 1700-1900pg/ml.
  • the concentration of bFGF is 50-600pg/ml, preferably 100-500pg/ml, more preferably 120-400pg/ml, more It is preferably 150-300pg/ml, more preferably 200-280pg/ml, more preferably 220-260pg/ml.
  • the concentration of the VEGF is 50-500pg/ml, preferably 100-400pg/ml, more preferably 120-300pg/ml, more It is preferably 150-250pg/ml, more preferably 170-230pg/ml, more preferably 190-210pg/ml.
  • the concentration of TGF- ⁇ 1 is 200-3000 pg/ml, preferably 400-2000 pg/ml, more preferably 600-1500 pg/ml , More preferably 800-1200pg/ml, more preferably 800-1100pg/ml, more preferably 900-1000pg/ml.
  • the concentration of HGF is 200-3000 pg/ml, preferably 400-2000 pg/ml, more preferably 600-1500 pg/ml, more It is preferably 600-1200pg/ml, more preferably 800-1000pg/ml, more preferably 850-950pg/ml.
  • the concentration of PDGF is 50-600pg/ml, preferably 80-400pg/ml, more preferably 100-300pg/ml, more Preferably 140-220pg/ml, more preferably 160-200pg/ml, and even more preferably 170-190pg/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, Best 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. Better 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, most preferably 0.7-1.2:1.
  • the cell-free fat extract of the present invention is prepared by the method described 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, and most preferably 1100-1300g.
  • the centrifugation time is 1-15 min, preferably 1-10 min, more preferably 1-8 min, and most preferably 1-5 min.
  • the emulsification is mechanical emulsification.
  • the mechanical emulsification is repeated pipetting (such as pipetting 20-200 times, preferably 20-150 times, more preferably 20-100 times, more preferably 30-50 times) through a syringe for mechanical emulsification. emulsification.
  • the method of pipetting is that two 10ml injection syringes are connected to a three-way pipe and repeatedly pumped at a constant speed.
  • the emulsification is a method of crushing by a tissue homogenizer.
  • the emulsified fat mixture is frozen and then thawed.
  • 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 divided 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, and 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 pack 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 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, best 0.22 ⁇ m.
  • the filtration and sterilization are firstly passed through the first filter that can filter out cells, and then passed through the second filter that can filter out pathogens (such as bacteria).
  • Filter such as 0.22 ⁇ m filter.
  • the step (6) further includes dividing the fat extract to form a divided product.
  • the aliquoted extract can be stored at -20°C until use; it can be used directly after thawing at low temperature (such as -4°C) or normal temperature, or it can be stored at low temperature (such as 4°C) after thawing for a period of time, and then used ).
  • the acellular fat extract of the present invention can effectively prevent and/or treat acute respiratory distress syndrome, acute lung injury, hypoxemia, lung tissue inflammation, lung tissue injury, systemic inflammatory response syndrome and/or multiple organs Functional failure.
  • the acellular fat extract of the present invention includes one or more uses selected from the following group: (i) prevention and/or treatment of acute respiratory distress syndrome and/or acute lung injury; (ii) Prevent and/or treat hypoxemia; (iii) improve lung tissue inflammation; (iv) improve lung tissue damage; (v) prevent and/or treat systemic inflammatory response syndrome; and/or (vi) prevent and/or Treat multiple organ failure.
  • the prevention and/or treatment of acute respiratory distress syndrome and/or acute lung injury includes one or more methods selected from the following group to prevent and/or treat:
  • the prevention and/or treatment of hypoxemia includes increasing blood oxygen content.
  • said increasing blood oxygen content includes increasing blood oxygen partial pressure and/or increasing blood oxygen saturation.
  • the improvement of lung inflammation includes reducing the infiltration of inflammatory cells in the lung.
  • the inflammatory cells include (but are not limited to): white blood cells, neutrophils, lymphocytes, monocytes, or a combination thereof.
  • the improvement of lung tissue damage includes improvement in one or more ways selected from the following group:
  • the patient with hypoxemia suffers from acute respiratory distress syndrome and/or acute lung injury.
  • the patient suffering from lung tissue inflammation suffers from acute respiratory distress syndrome and/or acute lung injury.
  • the patient with lung tissue injury suffers from acute respiratory distress syndrome and/or acute lung injury.
  • the present invention also provides a method of (i) preventing and/or treating acute respiratory distress syndrome and/or acute lung injury; (ii) preventing and/or treating hypoxemia; (iii) improving lung tissue inflammation; (iv) Improve lung tissue damage; (v) prevent and/or treat systemic inflammatory response syndrome; and/or (vi) prevent and/or treat multiple organ failure methods, the method comprising the steps of: administering the present invention to a subject in need The acellular fat extract.
  • the subject is a human or non-human mammal.
  • the non-human mammals include rodents, such as rats and mice.
  • composition of the present invention includes (but is not limited to): pharmaceutical composition, food composition, health care composition, dietary supplement and the like.
  • the acellular fat extract of the present invention can be prepared into pharmaceutical compositions, such as tablets, capsules, powders, microparticles, solutions, lozenges, jellies, cream preparations, glutinous agents, suspensions, Dosage forms such as tinctures, poultices, liniments, lotions, and aerosols.
  • the pharmaceutical composition can be prepared by generally known preparation techniques, and suitable pharmaceutical additives can be added to the drug.
  • composition of the present invention may also include a pharmaceutically, food, health care product or dietary acceptable carrier.
  • “Pharmaceutically, food, health product or dietary acceptable carrier” refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity And sufficiently low toxicity.
  • “Compatibility” here means that the components in the composition can be blended with the compound of the present invention and between them without significantly reducing the efficacy of the compound.
  • acceptable carriers for pharmaceutically, food, health care products or diets are cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.) , Gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol) Etc.), emulsifiers (e.g. ), wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.
  • cellulose and its derivatives such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.
  • Gelatin talc
  • solid lubricants such as stearic acid
  • the administration method of the composition of the present invention is not particularly limited. Representative administration methods include (but are not limited to): oral, parenteral (intravenous, intramuscular), topical administration, and oral administration and injection administration are preferred.
  • Solid dosage forms for oral administration or administration include capsules, tablets, pills, powders and granules.
  • the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or mixed with the following ingredients: (a) fillers or compatibilizers, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (c) humectants, For example, glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) Absorption accelerators, for example, quaternary amine compounds; (g) wetting agents, such as cetyl alcohol and
  • Solid dosage forms such as tablets, sugar pills, capsules, pills and granules can be prepared with coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers.
  • Liquid dosage forms for oral administration or administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures.
  • the liquid dosage form 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.
  • composition may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfumes.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfumes.
  • the suspension may contain suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • suspending agents for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • composition for parenteral injection may contain physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
  • the dosage forms of the compound of the present invention for topical application or administration include ointments, powders, patches, sprays, and inhalants.
  • the active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required if necessary.
  • the acellular fat extract of the present invention can be administered or administered alone, or in combination with other drugs for preventing and/or treating fatty liver and/or its complications.
  • a safe and effective amount of the acellular fat extract of the present invention is applied to human or non-human animals (such as rats, mice, dogs, cats, cows, chickens, ducks, etc.) in need of treatment, wherein
  • the current dose is the effective dose that is acceptable in pharmacy, food or health care products.
  • safe and effective amount refers to an amount that produces function or activity on humans and/or animals and can be accepted by humans and/or animals. Those of ordinary skill in the art should understand that the "safe and effective amount” may vary depending on the form of the pharmaceutical composition, the route of administration, the excipients of the drug used, the severity of the disease, and the combination with other drugs. It's different.
  • the daily dose is usually 0.1 to 1000 mg, preferably 1 to 600 mg, and more preferably 2 to 300 mg.
  • the specific dosage should also consider factors such as the route of administration, the patient's health status, etc., which are all within the skill range of a skilled physician.
  • the present invention finds for the first time that the acellular fat extract can effectively prevent and/or treat acute respiratory distress syndrome, acute lung injury, hypoxemia, lung tissue inflammation, lung tissue injury, systemic inflammatory response syndrome and multiple organ failure.
  • Fat is obtained by volunteers with informed consent.
  • the preparation method of acellular adipose tissue extract is as follows:
  • 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 swelling fluid is anesthetized, a 3mm liposuction cannula with a large side hole (2mmx7mm) is connected to a 20mL syringe, and the obtained fat is sucked radially under artificial negative pressure. After the swelling fluid is removed, normal saline is used Rinse 3 times.
  • the middle layer that is, the fat layer containing fat cells
  • 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
  • the fourth layer is the cell/tissue debris sedimentation layer.
  • the oil layer and the The remaining fat tissue layer, the liquid layer is sucked, and the contamination of the cell/tissue debris deposit layer is avoided during the sucking process, so as to obtain the initial fat extract.
  • cytokine content including IGF-1, BDNF, GDNF, bFGF, VEGF, TGF- ⁇ 1, HGF, 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 (954.5pg/ml), HGF (898.4pg/ml), PDGF (179.9pg/ml).
  • LPS Lipopolysaccharide
  • LPS lipopolysaccharide
  • each group was given lipopolysaccharide (0.8mg/kg, 400 ⁇ L/kg) by intraperitoneal injection with a disposable microsyringe.
  • the LPS solution Using a small animal anesthesia laryngoscopy, the base of the animal’s tongue is pressed to expose the glottis, and a quantitative amount of lipopolysaccharide will be extracted ( The LPS) solution’s lung micro-liquid nebulizer needle (blunt) is gently inserted into the trachea, and then the piston is quickly pushed to atomize the LPS solution into the lungs, the needle is quickly pulled out, and the animal is removed from the holder.
  • the head Face up and rotate left and right to distribute LPS as evenly as possible in each lung lobe.
  • Administration route tail vein injection for intravenous injection, a disposable sterile syringe is used to extract the dose of each animal, and the tail vein is slowly (approximately 10 to 60 seconds) injected for administration.
  • Dosing frequency and duration The first administration of the modeling reagent (LPS) was started about 1 hour after the animal airway atomization on Day1; the second administration on Day2; the interval between the two administrations was about 24h ( ⁇ 20min).
  • LPS modeling reagent
  • Detection time the day after the second administration (Day3: about 48h after modeling).
  • Rats were anesthetized by intraperitoneal injection of chloral hydrate (350mg/kg, 100mg/mL), the abdominal midline was cut longitudinally, the abdominal aorta was separated, the arterial blood was collected by an arterial blood collection device, and the blood collection device was rubbed in the palm of the hand Move the syringe and turn it upside down for 5 seconds each. None withdraw the blood collection device for blood mixing.
  • Detection method After the arterial blood is collected, gently insert the needle into the blood injection port of the test card, slowly push the blood in, and fill up the sample filling tube. When the blood reaches the sample point, stop the sample and close the lid. The blood will automatically enter the test tube, insert the test card into the blood gas analyzer, and wait for the test result.
  • Detection indicators oxygen partial pressure PO2 (mmHg), carbon dioxide partial pressure PCO2 (mmHg), pH, blood oxygen saturation SO2%.
  • BALF treatment Centrifuge the collected lavage fluid for 20 min at 4°C and about 2000 rpm. The supernatant is stored below -70°C until use, and the pellet is resuspended in 1 mL PBS buffer for white blood cell counting and classification.
  • Leukocyte classification and detection The resuspended lung lavage fluid is counted and classified by an automatic blood cell analyzer.
  • the inflammatory cell count in BALF reflects the inflammation of lung tissue, and the inflammatory cell count and classification of alveolar lavage fluid are shown in Table 3 and Figure 3.
  • WBC white blood cells
  • Neut refers to the number of neutrophils
  • Lymph refers to lymphocytes
  • Mono refers to monocytes
  • SIRS Systemic Inflammatory Response Syndrome

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Abstract

一种无细胞脂肪提取物及其制备方法和用途，用于制备组合物或制剂，组合物或制剂用于选自下组的一种或多种用途：预防和/或治疗急性呼吸窘迫综合征和/或急性肺损伤；预防和/或治疗低氧血症；改善肺组织炎症；改善肺组织损伤；预防和/或治疗全身炎症反应综合征；预防和/或治疗多器官功能衰竭。

Description

无细胞脂肪提取液对肺部疾病的治疗用途 技术领域
本发明涉及药物领域，具体涉及无细胞脂肪提取液对肺部疾病的治疗用途。
背景技术
急性肺损伤(acute lung injure，ALI)是指由各种因素引起的肺泡上皮细胞受损、肺间质毛细血管损伤、肺泡和毛细血管屏障被破坏等，引起肺泡和肺间质的水肿与炎细胞浸润，临床上以呼吸困难和低氧血症为主要表现的一种疾病。据估计在全世界范围内约占重症监护室住院人数的10％，死亡率可达40％以上。急性呼吸窘迫综合征(acute respiratory distress syndrome,ARDS)是急性肺损伤发展至最严重阶段，因肺实质发生急性弥漫性损伤而导致的急性缺氧性呼吸衰竭，临床表现以进行性呼吸困难和顽固性低氧血症为特征。一直以来，ALI/ARDS
因其高发病率、高病死率和治疗方法匮乏而目前，对于ALI/ARDS的临床治疗，主要通过机械通气进行呼吸支持和糖皮质激素、肺血管舒张剂等治疗，然而，目前的治疗手段难以有效降低ALI/ARDS的死亡率，尚缺乏有效的治疗药物。
因此，本领域需要开发一种能够有效治疗ALI/ARDS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治疗ALI/ARDS等肺部疾病方面中的用途。
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选自下组的一种或多种用途：(i)预防和/或治疗急性呼吸窘迫综合征和/或急性肺损伤；(ii)预防和/或治疗低氧血症；(iii)改善肺组织炎症；(iv)改善肺组织损伤；(v)预防和/或治疗全身炎症反应综合征；(vi)预防和/或治疗多器官功能衰竭。
在另一优选例中，所预防和/或治疗急性呼吸窘迫综合征和/或急性肺损伤包 括选自下组的一种或多种方式进行预防和/或治疗：
(i)提高血氧含量；
(ii)改善肺组织炎症；
(iii)改善肺组织损伤；和/或
(iv)改善呼吸功能。
在另一优选例中，所述的预防和/或治疗低氧血症包括提高血氧含量。
在另一优选例中，所述的提高血氧含量包括提高血氧分压和/或提高血氧饱和度。
在另一优选例中，所述的改善肺组织炎症包括减轻肺内炎细胞浸润。
在另一优选例中，所述的炎细胞选自下组：白细胞、中性粒细胞数、淋巴细胞、单核细胞，或其组合。
在另一优选例中，所述的改善肺组织损伤包括选自下组的一种或多种方式进行改善：
(iv-1)减轻肺内炎细胞浸润；
(iv-2)抑制肺泡内巨噬细胞聚集；
(iv-3)抑制肺泡壁增厚；
(iv-4)抑制肺泡和/或肺泡血管周围出血；和/或
(iv-5)抑制肺泡内纤维素样物质沉积。
在另一优选例中，所述低氧血症的患者患有急性呼吸窘迫综合征和/或急性肺损伤。
在另一优选例中，所述肺组织炎症的患者患有急性呼吸窘迫综合征和/或急性肺损伤。
在另一优选例中，所述肺组织损伤的患者患有急性呼吸窘迫综合征和/或急性肺损伤。
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制剂。
在另一优选例中，所述的组合物或制剂通过外用、局部、或皮下注射方式施用。
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，所述的脂肪提取物含有但不限于一种或多种选自下组的组分：生长因子IGF-1、BDNF、GDNF、TGF-β、HGF、bFGF、VEGF、PDGF、EGF、NT-3、GH、G-CSF、或其组合。
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-β、HGF、bFGF、VEGF、TGF-β1、HGF、PDGF、 EGF、NT-3、GH、G-CSF，或其组合。
在另一优选例中，所述的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，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为液体。
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剪碎，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剪碎，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％，以化妆品组合物的总重量计。
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(i)预防和/或治疗急性呼吸窘迫综合征和/或急性肺损伤；(ii)预防和/或治疗低氧血症；(iii)改善肺组织炎症；(iv)改善肺组织损伤；(v)预防和/或治疗全身炎症反应综合征；(vi)预防和/或治疗多器官功能衰竭的方法，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。
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ARDS模型大鼠存活率。
图2为模型大鼠动脉血氧分压与血氧饱和度(M±SD，*p<0.05,**p<0.01)。
图3为肺泡灌洗液炎细胞计数及分类情况(*p<0.05,**p<0.01)。
图4为肺组织病理学HE染色。
具体实施方式
本发明人经过广泛而深入的研究，首次开发了一种能够有效预防和/或治疗急性呼吸窘迫综合征、急性肺损伤、低氧血症、肺组织炎症、肺组织损伤、全身炎症反应综合征和多器官功能衰竭的无细胞脂肪提取物。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”。
在本发明中，术语“急性肺损伤(acute lung injure)”与“ALI”可互换使用。
在本发明中，术语“急性呼吸窘迫综合征(acute respiratory distress syndrome)”与“ARDS”可互换使用。
在本发明中，术语“全身炎症反应综合征”与“SIRS”可互换使用。
在本发明中，术语“多器官功能衰竭”与“MODS”可互换使用。
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、组合物、药盒、食品盒或保健品盒、活性成分组合时观察到的水平相比，本发明所述组合物或药物组合物减轻、抑制和/或逆转了糖尿病例如至少约10％、至少约30％、至少约50％、或至少约80％。
如本文所用，“改善”包括预防、治疗、缓解、逆转和减轻等等。
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，所述的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，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剪碎，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
本发明所述的无细胞脂肪提取物能够有效预防和/或治疗急性呼吸窘迫综合征、急性肺损伤、低氧血症、肺组织炎症、肺组织损伤、全身炎症反应综合征和 /或多器官功能衰竭。
代表性地，本发明所述的无细胞脂肪提取物包括选自下组的一种或多种用途：(i)预防和/或治疗急性呼吸窘迫综合征和/或急性肺损伤；(ii)预防和/或治疗低氧血症；(iii)改善肺组织炎症；(iv)改善肺组织损伤；(v)预防和/或治疗全身炎症反应综合征；和/或(vi)预防和/或治疗多器官功能衰竭。
在另一优选例中，所预防和/或治疗急性呼吸窘迫综合征和/或急性肺损伤包括选自下组的一种或多种方式进行预防和/或治疗：
(ii)提高血氧含量；
(ii)改善肺组织炎症；
(iii)改善肺组织损伤；和/或
(iv)改善呼吸功能。
在另一优选例中，所述的预防和/或治疗低氧血症包括提高血氧含量。
在另一优选例中，所述的提高血氧含量包括提高血氧分压和/或提高血氧饱和度。
在另一优选例中，所述的改善肺组织炎症包括减轻肺内炎细胞浸润。
在另一优选例中，所述的炎细胞包括(但不限于)：白细胞、中性粒细胞数、淋巴细胞、单核细胞，或其组合。
在另一优选例中，所述的改善肺组织损伤包括选自下组的一种或多种方式进行改善：
(iii-1)减轻肺内炎细胞浸润；
(iii-2)抑制肺泡内巨噬细胞聚集；
(iii-3)抑制肺泡壁增厚；
(iii-4)抑制肺泡和/或肺泡血管周围出血；和/或
(iii-5)抑制肺泡内纤维素样物质沉积。
在另一优选例中，所述低氧血症的患者患有急性呼吸窘迫综合征和/或急性肺损伤。
在另一优选例中，所述肺组织炎症的患者患有急性呼吸窘迫综合征和/或急 性肺损伤。
在另一优选例中，所述肺组织损伤的患者患有急性呼吸窘迫综合征和/或急性肺损伤。
本发明还提供一种(i)预防和/或治疗急性呼吸窘迫综合征和/或急性肺损伤；(ii)预防和/或治疗低氧血症；(iii)改善肺组织炎症；(iv)改善肺组织损伤；(v)预防和/或治疗全身炎症反应综合征；和/或(vi)预防和/或治疗多器官功能衰竭的方法，所述方法包括步骤：对需要的对象施用本发明所述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。
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 PCTCN2021099865-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。
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：
本发明首次发现无细胞脂肪提取物能够有效预防和/或治疗急性呼吸窘迫综合征、急性肺损伤、低氧血症、肺组织炎症、肺组织损伤、全身炎症反应综合征和多器官功能衰竭。
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
1.1.无细胞脂肪提取液(Cell free fat extract,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名行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(2mmx7mm)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，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),PDGF(179.9pg/ml)。
1.2大鼠ARDS模型建立及分组
脂多糖(LPS)用于构建ARDS(急性呼吸窘迫综合征)大鼠模型
8周龄雄性SD大鼠购自北京维通利华实验动物技术有限公司。将24只动物随机分为4组：对照组、低剂量组、中剂量组、高剂量组，6只/组。24只动物均腹腔注射联合气道内雾化造模试剂脂多糖(LPS)。具体步骤如下：
Day0：各组根据动物体重,采用一次性微量注射器腹腔注射给予脂多糖(0.8mg/kg，400μL/kg)。
Day1(腹腔注射16h±30min后)：异氟烷吸入麻醉，各组动物气管内雾化给予脂多糖(5mg/kg，1000μL/kg)。主要实验步骤如下：动物异氟烷吸入麻醉，然后固定于呈45°放置的大鼠固定器上，使用小动物麻醉咽喉镜，压住动物舌根部，暴露声门，将抽取定量的脂多糖(LPS)溶液的肺部微型液体雾化器针头(钝性)轻柔的插入气管内，然后快速推动活塞，将LPS溶液雾化进入肺脏，快速拔出针头，从固定器上取下动物，头部朝上，左右旋转，使LPS尽可能的均匀分布于各肺叶。
1.3 CEFFE治疗剂量与方法
Day1动物气道内雾化给予造模试剂(LPS)约1h后，再按照下表1剂量给予CEFFE治疗或生理盐水对照。
表1
Figure PCTCN2021099865-appb-000002
给药途径：尾静脉注射静脉注射给药，采用一次性无菌注射器抽取每只动物的药量，尾静脉缓慢(约10～60秒)注射给药。给药频率及期限：Day1动物气道内雾化给予造模试剂(LPS)约1h后开始首次给药；Day2进行第二次给药；两次给 药间隔约24h(±20min)。
1.4存活率记录
计算试验周期内各组动物存活数与每组总动物数的比值，存活率(％)＝组存活动物数/组总动物数×100％。
1.5血气检测
检测时间：第二次给药结束后次日(Day3：造模后约48h)。
样品采集：大鼠腹腔注射水合氯醛(350mg/kg，100mg/mL)实施麻醉，腹中线纵向切开，分离腹主动脉，采用动脉采血器采集动脉血约0.2mL，采血器于手掌内搓动注射器及上下颠倒各5秒，绝对不能回抽采血器进行血液混匀处理。
检测方法：动脉血采集后，轻柔的将针头插入测试卡的血液注入口中，将血液缓慢推入，并加满样品加注管，当血液到达加样位时，停止加样，扣上盖子，血液会自动进入测试管内，将测试卡插入血气分析仪中，等待检测结果。
检测指标：氧分压PO2(mmHg)、二氧化碳分压PCO2(mmHg)、pH、血氧饱和度SO2％。
1.6肺灌洗液(BALF)检测
Day3所有动物腹腔注射水合氯醛(350mg/kg，100mg/mL)实施麻醉，腹主动脉放血实施安乐死。濒死动物采用异氟烷麻醉后，腹主动脉放血实施安乐死。
样本采集：动物安乐死后，剪开颈部和胸部皮肤及组织暴露气管、支气管及肺脏，分离右肺支气管，并结扎。在气管下穿缝合线，并在甲状软骨下适当位置于气管软骨环之间做1/2切口，将气管插管沿切口处向气道内缓慢插入至左侧支气管处，用穿好的缝合线在切口向心方向的适当部位将插管与气管扎紧固定。用注射器吸取3mL的PBS缓冲液缓慢注入，进行肺泡灌洗，反复灌洗3次，每次冲洗停留约10s，收集灌洗液于合适容量的离心管中(不低于2.1mL)。
BALF处理：将收集的灌洗液于4℃，约2000rpm条件下，离心20min。上清液于-70℃以下保存待用，沉淀用1mL PBS缓冲液重悬，用于白细胞计数及分类。
白细胞分类检测：重悬后的肺灌洗液，采用全自动血球分析仪进行白细胞计 数及分类。
1.7组织病理学
各组动物进行解剖，并保存组织。尸检过程中，观察动物肺脏、气管及支气管是否异常。剪取右肺置于10％中性缓冲福尔马林溶液中固定、石蜡包埋、切片、制片、HE染色进行肺组织病理形态学观察，使用标准术语诊断和分类，按4分级法(轻微，轻度，中度，重度)对肺组织进行病理学检测。
1.8统计分析
数据采用SPSS软件进行单因素ANOVA检验，对数据差异显著性进行统计分析，并采用Bonferreni法进行事后检验，结果以平均数±标准差表示。
2.结果
2.1 CEFFE治疗有效提高ARDS大鼠存活率
记录各组大鼠存活情况，ARDS模型大鼠存活率的如图1所示，对照组死亡2只，存活率为67％，CEFFE治疗组未见死亡大鼠，表明CEFFE治疗有效提高了ARDS大鼠存活率。
2.2 CEFFE治疗有效改善ARDS大鼠呼吸功能，减轻低氧血症
动脉血气分析结果如表2和图2所示。
表2 模型大鼠动脉血氧分压PO2与血氧饱和度SO2％(M±SD)
Figure PCTCN2021099865-appb-000003
从表2和图2中可以看出，ADRS模型大鼠PO2明显受损。与对照组和低剂量治 疗组相比，高剂量CEFFE治疗组氧分压PO2和血氧饱和度SO2％显著增高(P<0.05)，表明CEFFE治疗有效改善模型大鼠呼吸功能，减轻低氧血症。
2.3 CEFFE治疗有效减轻ARDS大鼠肺组织炎细胞浸润
BALF中炎细胞计数反应肺组织炎症情况，肺泡灌洗液炎细胞计数及分类情况如表3和图3所示。
表3肺泡灌洗液炎细胞计数及分类情况(M±SD)
Figure PCTCN2021099865-appb-000004
备注：WBC指白细胞；Neut指中性粒细胞数；Lymph指淋巴细胞；Mono指单核细胞；
从表3和图3中可已看出，中剂量和高剂量CEFFE治疗组大鼠BALF中白细胞数目、中性粒细胞数目均显著低于对照组(P<0.05)，且呈剂量依赖性，表明肺内炎细胞浸润明显减轻，CEFFE治疗具有减轻ARDS大鼠肺内炎症的作用。
2.4 CEFFE治疗有效改善ARDS大鼠肺组织损伤
组织病理学HE染色结果如图4所示，其病理组织学半定量评价结果表4所示。
表4 病理组织学半定量评价
Figure PCTCN2021099865-appb-000005
Figure PCTCN2021099865-appb-000006
从表4和图4中可以看出，对照组肺脏及支气管间质/肺泡以中性粒细胞为主的炎细胞浸润、肺泡内巨噬细胞聚集、肺泡壁增厚、肺泡/血管周围出血以及肺泡内纤维素样物质沉积；与模型对照组相比，CEFFE治疗中剂量组和高剂量组炎细胞浸润减轻、肺泡/血管周围出血有所改善，且存在剂量相关性，即高剂量组的改善作用较中剂量组明显；此外，高剂量组在肺泡内纤维素样物质沉积上同样有所改善，这些结果表明CEFFE治疗能够有效减轻ARDS大鼠肺组织损伤。
结论：
全身炎症反应综合征(SIRS)是由感染或损伤引起的一种临床综合征，全 身炎症介质释放引起细胞因子/炎症因子风暴、炎症细胞激活等是其主要的促发因素，引起细胞凋亡、休克、免疫功能失调、脏器受损等。感染、创伤等引发了SIRS，失控的进一步发展为多器官功能衰竭(MODS)，肺脏是这一病程演变过程中最易受损伤的器官，因此，ALI/ARDS是SIRS患者重要的疾病症状。
本实验结果表明，CEFFE治疗能够有效改善ALI/ARDS引起的肺组织损伤，减轻肺内炎症，改善肺功能，纠正低氧血症，降低死亡率。CEEFE对ALI/ARDS的治疗作用提示了其对改善SIRS具有治疗价值，也提示了其对其他呼吸系统疾病的潜在治疗作用。
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选自下组的一种或多种用途：(i)预防和/或治疗急性呼吸窘迫综合征和/或急性肺损伤；(ii)预防和/或治疗低氧血症；(iii)改善肺组织炎症；(iv)改善肺组织损伤；(v)预防和/或治疗全身炎症反应综合征；(vi)预防和/或治疗多器官功能衰竭。
  2. 如权利要求1所述的用途，其特征在于，所预防和/或治疗急性呼吸窘迫综合征和/或急性肺损伤包括选自下组的一种或多种方式进行预防和/或治疗：
    (i)提高血氧含量；
    (ii)改善肺组织炎症；
    (iii)改善肺组织损伤；和/或
    (iv)改善呼吸功能。
  3. 如权利要求1所述的用途，其特征在于，所述的预防和/或治疗低氧血症包括提高血氧含量。
  4. 如权利要求3所述的用途，其特征在于，所述的提高血氧含量包括提高血氧分压和/或提高血氧饱和度。
  5. 如权利要求1所述的用途，其特征在于，所述的改善肺组织炎症包括减轻肺内炎细胞浸润。
  6. 如权利要求1所述的用途，其特征在于，所述的改善肺组织损伤包括选自下组的一种或多种方式进行改善：
    (iv-1)减轻肺内炎细胞浸润；
    (iv-2)抑制肺泡内巨噬细胞聚集；
    (iv-3)抑制肺泡壁增厚；
    (iv-4)抑制肺泡和/或肺泡血管周围出血；和/或
    (iv-5)抑制肺泡内纤维素样物质沉积。
  7. 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剪碎，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。
  8. 如权利要求5所述的用途，其特征在于，所述的炎细胞选自下组：白细胞、中性粒细胞数、淋巴细胞、单核细胞，或其组合。
  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、HGF、bFGF、VEGF、TGF-β1、HGF、PDGF、EGF、NT-3、GH、G-CSF，或其组合。
  11. 如权利要求10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。
  12. 如权利要求10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。
  13. 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剪碎，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。
  14. 一种无细胞脂肪提取物，其特征在于，所述的无细胞脂肪提取物通过如权利要求13所述的方法制备获得。
  15. 一种(i)预防和/或治疗急性呼吸窘迫综合征和/或急性肺损伤；(ii)预防和/或治疗低氧血症；(iii)改善肺组织炎症；(iv)改善肺组织损伤；(v)预防和/或治疗全身炎症反应综合征；和/或(vi)预防和/或治疗多器官功能衰竭的方法，其特征在于，所述方法包括步骤：对需要的对象施用如权利要求13所述的的无细胞脂肪提取物。
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