Therapeutic use of acellular fat extract for lung diseases

Abstract

The present invention relates to the therapeutic use of acellular fat extract for lung diseases. Specifically, the present invention provides a use of acellular fat extract for preparing a composition or preparation, wherein the composition or preparation is used for one or more uses selected from the following groups: (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. 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 damage, systemic inflammatory response syndrome and multiple organ failure.

Description

Technical Field

[0001] The invention relates to the field of medicines, and in particular to the therapeutic use of acellular fat extract on lung diseases. Background Art

[0002] Acute lung injury (ALI) refers to a disease in which alveolar epithelial cells are damaged, interstitial capillaries are damaged, and the alveolar and capillary barriers are destroyed due to various factors, resulting in edema and inflammatory cell infiltration of the alveoli and interstitium. Clinically, it is characterized by dyspnea and hypoxemia. It is estimated to account for about 10% of the number of hospitalizations in intensive care units worldwide, and the mortality rate can reach more than 40%. Acute respiratory distress syndrome (ARDS) is the most serious stage of acute lung injury, which is acute hypoxic respiratory failure caused by acute diffuse damage to the lung parenchyma. The clinical manifestations are characterized by progressive dyspnea and refractory hypoxemia. ALI / ARDS has always been

[0003] Due to its high morbidity, high mortality rate and lack of treatment methods, the current clinical treatment of ALI / ARDS mainly relies on mechanical ventilation for respiratory support and treatments such as glucocorticoids and pulmonary vasodilators. However, current treatment methods are difficult to effectively reduce the mortality rate of ALI / ARDS, and there is still a lack of effective therapeutic drugs.

[0004] Therefore, there is a need in the art to develop a drug that can effectively treat ALI / ARDS. Summary of the invention

[0005] The purpose of the present invention is to provide a use of a cell-free fat extract in treating lung diseases such as ALI / ARDS.

[0006] In a first aspect, the present invention provides a use of a cell-free fat extract for preparing a composition or a preparation, wherein the composition or preparation is used for one or more uses selected from the following groups: (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; and (vi) preventing and / or treating multiple organ failure.

[0007] In another preferred embodiment, the prevention and / or treatment of acute respiratory distress syndrome and / or acute lung injury comprises one or more methods selected from the following groups for prevention and / or treatment:

[0008] (i) Increase blood oxygen levels;

[0009] (ii) improving lung tissue inflammation;

[0010] (iii) improving lung tissue damage; and / or

[0011] (iv) Improve respiratory function.

[0012] In another preferred embodiment, the prevention and / or treatment of hypoxemia includes increasing blood oxygen content.

[0013] In another preferred embodiment, the increasing of blood oxygen content includes increasing blood oxygen partial pressure and / or increasing blood oxygen saturation.

[0014] In another preferred embodiment, the improvement of lung tissue inflammation includes reducing inflammatory cell infiltration in the lungs.

[0015] In another preferred embodiment, the inflammatory cells are selected from the group consisting of leukocytes, neutrophils, lymphocytes, monocytes, or a combination thereof.

[0016] In another preferred embodiment, the improvement of lung tissue damage comprises one or more methods selected from the following group:

[0017] (iv-1) Reduce inflammatory cell infiltration in the lungs;

[0018] (iv-2) inhibiting the accumulation of macrophages in alveoli;

[0019] (iv-3) inhibiting alveolar wall thickening;

[0020] (iv-4) inhibiting alveolar and / or peri-alveolar vascular bleeding; and / or

[0021] (iv-5) Inhibit the deposition of fibrinoid substances in the alveoli.

[0022] In another preferred embodiment, the hypoxemic patient suffers from acute respiratory distress syndrome and / or acute lung injury.

[0023] In another preferred embodiment, the patient with lung tissue inflammation suffers from acute respiratory distress syndrome and / or acute lung injury.

[0024] In another preferred embodiment, the patient with lung tissue damage suffers from acute respiratory distress syndrome and / or acute lung injury.

[0025] In another preferred embodiment, the acellular fat extract is an acellular fat extract extracted from fat of human or non-human mammals.

[0026] In another preferred embodiment, the non-human mammal is a monkey, an ape, a cow, a pig, a dog, a sheep, a mouse or a rabbit.

[0027] In another preferred embodiment, the composition or preparation includes a pharmaceutical composition or preparation.

[0028] In another preferred embodiment, the composition or preparation further comprises a pharmaceutically acceptable carrier.

[0029] In another preferred embodiment, the composition or preparation is in the form of an oral preparation, an external preparation or an injection preparation.

[0030] In another preferred embodiment, the injection preparation is an intravenous injection preparation.

[0031] In another preferred embodiment, the composition or preparation is administered externally, topically, or by subcutaneous injection.

[0032] In another preferred embodiment, the cell-free fat extract contains no cells and no lipid droplets.

[0033] In another preferred embodiment, the lipid droplets are oil droplets released after the adipocytes are broken.

[0034] In another preferred embodiment, the “free of fat droplets” means that in the acellular fat extract, the volume of oil droplets accounts for less than 1% of the total liquid, preferably less than 0.5%, and more preferably less than 0.1%.

[0035] In another preferred embodiment, the cells are selected from the group consisting of endothelial cells, adipose stem cells, macrophages, and stromal cells.

[0036] In another preferred embodiment, 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.

[0037] In another preferred embodiment, the cell-free fat extract is a naturally obtained nano fat extract without any added ingredients.

[0038] In another preferred embodiment, the “additive-free” means that except for the rinsing step, no solution, solvent, small molecule, chemical agent, and biological additive are added during the preparation of the fat extract.

[0039] In another preferred embodiment, the fat extract is prepared by emulsifying adipose tissue and then centrifuging it.

[0040] In another preferred embodiment, the fat extract contains but is not limited to one or more components selected from the following group: growth factors IGF-1, BDNF, GDNF, TGF-β, HGF, bFGF, VEGF, PDGF, EGF, NT-3, GH, G-CSF, or a combination thereof.

[0041] In another preferred embodiment, the cell-free fat extract is prepared by the following method:

[0042] (1) providing a fat tissue material, chopping the fat tissue material, and rinsing it (e.g., using physiological saline) to obtain rinsed fat tissue;

[0043] (2) centrifuging the rinsed adipose tissue to obtain a layered mixture;

[0044] (3) removing the upper oil layer and the lower water layer from the stratified mixture, and collecting the middle layer (i.e., the fat layer containing fat cells);

[0045] (4) emulsifying the intermediate layer to obtain an emulsified fat mixture (also called nano fat);

[0046] (5) centrifuging the emulsified fat mixture to obtain an intermediate liquid layer, i.e., a primary fat extract; and

[0047] (6) Filtering and sterilizing the primary fat extract to obtain a cell-free fat extract.

[0048] In a second aspect, the present invention provides a method for preparing a cell-free fat extract, the method comprising the steps of:

[0049] (1) providing a fat tissue material, chopping the fat tissue material, and rinsing it (e.g., using physiological saline) to obtain rinsed fat tissue;

[0050] (2) centrifuging the rinsed adipose tissue to obtain a layered mixture;

[0051] (3) removing the upper oil layer and the lower water layer from the stratified mixture, and collecting the middle layer (i.e., the fat layer containing fat cells);

[0052] (4) emulsifying the intermediate layer to obtain an emulsified fat mixture (also called nano fat);

[0053] (5) centrifuging the emulsified fat mixture to obtain an intermediate liquid layer, i.e., a primary fat extract; and

[0054] (6) Filtering and sterilizing the primary fat extract to obtain a cell-free fat extract.

[0055] In another preferred embodiment, in the step (2), the centrifugation is performed at 800-2500 g, preferably 800-2000 g, more preferably 1000-1500 g, and most preferably 1100-1300 g.

[0056] In another preferred embodiment, in the step (2), the centrifugation time is 1-15 min, preferably 1-10 min, more preferably 1-8 min, and most preferably 1-5 min.

[0057] In another preferred embodiment, the centrifugation temperature is 2-6°C.

[0058] In another preferred embodiment, in the step (4), the emulsification is mechanical emulsification.

[0059] In another preferred embodiment, the mechanical emulsification is performed by repeatedly blowing with a syringe (such as blowing 20-200 times, preferably 20-150 times, more preferably 20-100 times, and more preferably 30-50 times).

[0060] In another preferred embodiment, the blowing method is to connect two 10 ml injection syringes to a three-way tube and push and beat repeatedly at a uniform speed.

[0061] In another preferred embodiment, in the step (4), the emulsification is performed by breaking up the tissue using a tissue homogenizer.

[0062] In another preferred embodiment, in the step (5), before the emulsified fat mixture is subjected to centrifugal treatment, the step further includes freezing and then thawing the emulsified fat mixture.

[0063] In another preferred embodiment, after the freezing and thawing treatment, the thawed mixture is used for centrifugation.

[0064] In another preferred embodiment, the freezing temperature is -50°C to -120°C, preferably -60°C to -100°C, and more preferably -70°C to -90°C.

[0065] In another preferred embodiment, the thawing temperature is 20-40°C, preferably 25-40°C, and more preferably 37°C.

[0066] In another preferred embodiment, the number of cycles of freezing and thawing is 1-5 times (preferably 1, 2, 3 or 4 times).

[0067] In another preferred embodiment, in the step (5), after centrifugation, the emulsified fat mixture is stratified into four layers, the first layer is an oil layer, the second layer is a residual fat tissue layer, the third layer is a liquid layer (i.e., an intermediate liquid layer), and the fourth layer is a cell / tissue fragment precipitation layer.

[0068] In another preferred embodiment, in the step (5), the centrifugation is performed at 800-2500 g, preferably 800-2000 g, more preferably 1000-1500 g, and most preferably 1100-1300 g.

[0069] In another preferred embodiment, in the step (5), the centrifugation time is 1-15 min, preferably 1-10 min, more preferably 2-8 min, and most preferably 3-7 min.

[0070] In another preferred embodiment, in the step (5), the first layer, the second layer, the third layer and the fourth layer are arranged in sequence from top to bottom.

[0071] In another preferred embodiment, in the step (5), the intermediate liquid layer is a transparent or substantially transparent layer.

[0072] In another preferred embodiment, in the step (6), the filtration bag is capable of removing fat cells from the primary fat extract.

[0073] In another preferred embodiment, in the step (6), the filtration and sterilization are performed through a filter (such as a 0.22 μm microporous filter membrane).

[0074] In another preferred embodiment, the filter is a microporous membrane filter.

[0075] In another preferred embodiment, the pore size of the microporous filter membrane is 0.05-0.8 μm, preferably 0.1-0.5 μm, more preferably 0.1-0.4 μm, more preferably 0.15-0.3 μm, more preferably 0.2-0.25 μm, and most preferably 0.22 μm.

[0076] In another preferred embodiment, in the step (6), the filtration and sterilization are first performed through a first filter that can filter out cells, and then through a second filter (such as a 0.22 μm filter) that can filter out pathogens (such as bacteria).

[0077] In another preferred embodiment, the step (6) further comprises packaging the fat extract to form packaged products. (The packaged extract can be stored at -20°C for later use; can be used directly after thawing at low temperature (such as -4°C) or room temperature, or can be stored at low temperature (such as 4°C) for a period of time after thawing before use).

[0078] The third aspect of the present invention provides a cell-free fat extract, which is prepared by the method described in the second aspect of the present invention.

[0079] The fourth aspect of the present invention provides a composition or preparation, comprising (a) the cell-free fat extract as described in the third aspect of the present invention; and (b) a pharmaceutically acceptable carrier or excipient.

[0080] In another preferred embodiment, the composition or preparation is in the form of powder, granules, capsules, injections, tinctures, oral solutions, tablets or lozenges.

[0081] In another preferred embodiment, the injection is an intravenous injection or an intramuscular injection.

[0082] In another preferred embodiment, the dosage form of the composition or preparation is a solid dosage form, a semisolid dosage form, or a liquid dosage form, such as a solution, a gel, a cream, a lotion, an ointment, a cream, a paste, a cake, a powder, a patch, etc.

[0083] In another preferred embodiment, in the composition or preparation, the mass percentage of the acellular fat extract is 5wt%, preferably 1-20wt%, based on the total weight of the cosmetic composition.

[0084] 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, the method comprising the steps of: mixing the cell-free fat extract as described in the third aspect of the present invention with a pharmaceutically acceptable carrier or excipient to form a composition or preparation.

[0085] In a sixth aspect, the present invention provides a method for (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 injury; (v) preventing and / or treating systemic inflammatory response syndrome; and (vi) preventing and / or treating multiple organ failure, by administering the cell-free fat extract as described in the third aspect of the present invention to a subject in need.

[0086] In another preferred embodiment, the subject is a human or non-human mammal.

[0087] In another preferred embodiment, the non-human mammals include rodents, such as rats and mice.

[0088] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 is the survival rate of ARDS model rats.

[0090] Figure 2 Arterial oxygen partial pressure and blood oxygen saturation of model rats (M±SD, *p<0.05, **p<0.01).

[0091] Figure 3 The count and classification of inflammatory cells in bronchoalveolar lavage fluid (*p<0.05, **p<0.01).

[0092] Figure 4 HE staining of lung tissue pathology. DETAILED DESCRIPTION

[0093] After extensive and in-depth research, the inventors have developed for the first time a cell-free fat extract that 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. The present invention was completed on this basis.

[0094] the term

[0095] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0096] As used herein, the terms "include", "comprises", and "comprising" are used interchangeably, and include not only open definitions, but also semi-closed and closed definitions. In other words, the terms include "consisting of", "consisting essentially of".

[0097] In the present invention, the terms "acute lung injury" and "ALI" are used interchangeably.

[0098] In the present invention, the terms "acute respiratory distress syndrome" and "ARDS" are used interchangeably.

[0099] In the present invention, the terms "systemic inflammatory response syndrome" and "SIRS" are used interchangeably.

[0100] In the present invention, the terms "multiple organ failure" and "MODS" are used interchangeably.

[0101] In the present invention, the term "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 a disease and / or its attendant symptoms and reducing the risk of a subject acquiring a disease.

[0102] The "treatment" described in the present invention includes delaying and stopping the progression of the disease, or eliminating the disease, and does not require 100% inhibition, elimination and reversal. In some embodiments, compared with the level observed when the composition, kit, and active ingredient combination described in the present invention are not present, the composition or pharmaceutical composition described in the present invention reduces, inhibits and / or reverses diabetes, for example, by at least about 10%, at least about 30%, at least about 50%, or at least about 80%.

[0103] As used herein, "ameliorating" includes preventing, curing, alleviating, reversing, and alleviating, among others.

[0104] Cell free fat extract (CEFFE) and preparation method thereof

[0105] As used herein, the terms "acellular fat extract of the present invention", "extract of the present invention", "fat extract of the present invention", etc. are used interchangeably and refer to an extract (or extracting liquid) derived from adipose tissue that is prepared without adding any solution, solvent, small molecule, chemical agent, and biological additive during the fat extract preparation process (except for the rinsing step). A typical method for preparing the extract of the present invention is as described above in the second aspect of the present invention. In addition, it should be understood that although it is not necessary to add any additives (or added ingredients) to the extract of the present invention during the preparation process, some or a small amount of safe substances (such as a small amount of water) that have no negative or adverse effects on the activity of the extract of the present invention can also be added.

[0106] Typically, the cell-free fat extract of the present invention is prepared by the following method:

[0107] (1) providing a fat tissue material, chopping the fat tissue material, and rinsing it (e.g., using physiological saline) to obtain rinsed fat tissue;

[0108] (2) centrifuging the rinsed adipose tissue to obtain a layered mixture;

[0109] (3) removing the upper oil layer and the lower water layer from the stratified mixture, and collecting the middle layer (i.e., the fat layer containing fat cells);

[0110] (4) emulsifying the intermediate layer to obtain an emulsified fat mixture (also called nano fat);

[0111] (5) centrifuging the emulsified fat mixture to obtain an intermediate liquid layer, i.e., a primary fat extract; and

[0112] (6) Filtering and sterilizing the primary fat extract to obtain a cell-free fat extract.

[0113] In another preferred embodiment, in the step (2), the centrifugation is performed at 800-2500 g, preferably 800-2000 g, more preferably 1000-1500 g, and most preferably 1100-1300 g.

[0114] In another preferred embodiment, in the step (2), the centrifugation time is 1-15 min, preferably 1-10 min, more preferably 1-8 min, and most preferably 1-5 min.

[0115] In another preferred embodiment, in the step (4), the emulsification is mechanical emulsification.

[0116] In another preferred embodiment, the mechanical emulsification is performed by repeatedly blowing with a syringe (such as blowing 20-200 times, preferably 20-150 times, more preferably 20-100 times, and more preferably 30-50 times).

[0117] In another preferred embodiment, the blowing method is to connect two 10 ml injection syringes to a three-way tube and push and beat repeatedly at a uniform speed.

[0118] In another preferred embodiment, in the step (4), the emulsification is performed by breaking up the tissue using a tissue homogenizer.

[0119] In another preferred embodiment, in the step (5), before the emulsified fat mixture is subjected to centrifugal treatment, the step further includes freezing and then thawing the emulsified fat mixture.

[0120] In another preferred embodiment, after the freezing and thawing treatment, the thawed mixture is used for centrifugation.

[0121] In another preferred embodiment, the freezing temperature is -50°C to -120°C, preferably -60°C to -100°C, and more preferably -70°C to -90°C.

[0122] In another preferred embodiment, the thawing temperature is 20-40°C, preferably 25-40°C, and more preferably 37°C.

[0123] In another preferred embodiment, the number of cycles of freezing and thawing is 1-5 times (preferably 1, 2, 3 or 4 times).

[0124] In another preferred embodiment, in the step (5), after centrifugation, the emulsified fat mixture is stratified into four layers, the first layer is an oil layer, the second layer is a residual fat tissue layer, the third layer is a liquid layer (i.e., an intermediate liquid layer), and the fourth layer is a cell / tissue fragment precipitation layer.

[0125] In another preferred embodiment, in the step (5), the centrifugation is performed at 800-2500 g, preferably 800-2000 g, more preferably 1000-1500 g, and most preferably 1100-1300 g.

[0126] In another preferred embodiment, in the step (5), the centrifugation time is 1-15 min, preferably 1-10 min, more preferably 2-8 min, and most preferably 3-7 min.

[0127] In another preferred embodiment, in the step (5), the first layer, the second layer, the third layer and the fourth layer are arranged in sequence from top to bottom.

[0128] In another preferred embodiment, in the step (5), the intermediate liquid layer is a transparent or substantially transparent layer.

[0129] In another preferred embodiment, in the step (6), the filtration bag is capable of removing fat cells from the primary fat extract.

[0130] In another preferred embodiment, in the step (6), the filtration and sterilization are performed through a filter (such as a 0.22 μm microporous filter membrane).

[0131] In another preferred embodiment, the filter is a microporous membrane filter.

[0132] In another preferred embodiment, the pore size of the microporous filter membrane is 0.05-0.8 μm, preferably 0.1-0.5 μm, more preferably 0.1-0.4 μm, more preferably 0.15-0.3 μm, more preferably 0.2-0.25 μm, and most preferably 0.22 μm.

[0133] In another preferred embodiment, in the step (6), the filtration and sterilization are first performed through a first filter that can filter out cells, and then through a second filter (such as a 0.22 μm filter) that can filter out pathogens (such as bacteria).

[0134] In another preferred embodiment, the step (6) further comprises packaging the fat extract to form packaged products. (The packaged extract can be stored at -20°C for later use; can be used directly after thawing at low temperature (such as -4°C) or room temperature, or can be stored at low temperature (such as 4°C) for a period of time after thawing before use).

[0135] use

[0136] The cell-free 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 organ failure.

[0137] Typically, the cell-free fat extract of the present invention includes one or more uses selected from the following groups: (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; and / or (vi) preventing and / or treating multiple organ failure.

[0138] In another preferred embodiment, the prevention and / or treatment of acute respiratory distress syndrome and / or acute lung injury comprises one or more methods selected from the following groups for prevention and / or treatment:

[0139] (ii) Increase blood oxygen levels;

[0140] (ii) improving lung tissue inflammation;

[0141] (iii) improving lung tissue damage; and / or

[0142] (iv) Improve respiratory function.

[0143] In another preferred embodiment, the prevention and / or treatment of hypoxemia includes increasing blood oxygen content.

[0144] In another preferred embodiment, the increasing of blood oxygen content includes increasing blood oxygen partial pressure and / or increasing blood oxygen saturation.

[0145] In another preferred embodiment, the improvement of lung tissue inflammation includes reducing inflammatory cell infiltration in the lungs.

[0146] In another preferred embodiment, the inflammatory cells include (but are not limited to): leukocytes, neutrophils, lymphocytes, monocytes, or a combination thereof.

[0147] In another preferred embodiment, the improvement of lung tissue damage comprises one or more methods selected from the following group:

[0148] (iii-1) Reduce inflammatory cell infiltration in the lungs;

[0149] (iii-2) inhibiting the accumulation of macrophages in alveoli;

[0150] (iii-3) inhibiting alveolar wall thickening;

[0151] (iii-4) inhibiting alveolar and / or peri-alveolar vascular bleeding; and / or

[0152] (iii-5) Inhibit the deposition of fibrinoid substances in the alveoli.

[0153] In another preferred embodiment, the hypoxemic patient suffers from acute respiratory distress syndrome and / or acute lung injury.

[0154] In another preferred embodiment, the patient with lung tissue inflammation suffers from acute respiratory distress syndrome and / or acute lung injury.

[0155] In another preferred embodiment, the patient with lung tissue damage suffers from acute respiratory distress syndrome and / or acute lung injury.

[0156] The present invention also provides a method for (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; and / or (vi) preventing and / or treating multiple organ failure, the method comprising the steps of administering the cell-free fat extract of the present invention to a subject in need thereof.

[0157] In another preferred embodiment, the subject is a human or non-human mammal.

[0158] In another preferred embodiment, the non-human mammals include rodents, such as rats and mice.

[0159] Composition and administration

[0160] The compositions of the present invention include (but are not limited to): pharmaceutical compositions, etc.

[0161] Representatively, the acellular fat extract of the present invention can be prepared into a pharmaceutical composition, such as a dosage form of tablets, capsules, powders, microgranules, solutions, lozenges, jellies, cream preparations, spirits, suspensions, tinctures, poultices, liniments, lotions, and aerosols. The pharmaceutical composition can be prepared by a commonly known preparation technique, and suitable pharmaceutical additives can be added to the medicine.

[0162] The composition of the present invention may also include a pharmaceutically acceptable carrier. "Pharmaceutically 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 mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carriers include 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, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0163] There is no particular limitation on the administration of the composition of the present invention. Representative administration methods include (but are not limited to): oral administration, parenteral administration (intravenous administration, intramuscular administration), and topical administration. The preferred administration methods are oral administration and injection administration.

[0164] Solid dosage forms for oral administration or delivery include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0165] Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other materials known in the art. They may contain opacifying agents.

[0166] Liquid dosage forms for oral administration or administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent 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-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

[0167] Besides these inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents, and perfumes.

[0168] In addition to the active ingredients, suspensions may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances.

[0169] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0170] Dosage forms for topical or administrable compounds of the invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0171] The cell-free fat extract of the present invention can be administered alone or in combination with other drugs for preventing and / or treating fatty liver and / or its complications.

[0172] When applying the composition, a safe and effective amount of the acellular fat extract of the present invention is applied to a human or non-human animal (such as rats, mice, dogs, cats, cattle, chickens, ducks, etc.) in need of treatment, wherein the dosage during application is a pharmaceutically acceptable effective dosage. As used herein, the term "safe and effective amount" refers to an amount that produces a function or activity to humans and / or animals and can be accepted by humans and / or animals. It should be understood by those of ordinary skill in the art that the "safe and effective amount" may vary with the form of the pharmaceutical composition, the route of administration, the adjuvants of the drugs used, the severity of the disease, and the combination of other drugs. For example, for a person weighing 60 kg, the daily dosage is usually 0.1 to 1000 mg, preferably 1 to 600 mg, and more preferably 2-300 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the health status of the patient, which are all within the skill range of skilled physicians.

[0173] The main advantages of the present invention include:

[0174] The present invention discovers for the first time that 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.

[0175] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0176] Example 1

[0177] 1.1. Preparation of cell free fat extract (CEFFE)

[0178] The fat was obtained from volunteers with informed consent. The preparation method of acellular adipose tissue extract is as follows:

[0179] (1) Adipose tissue was obtained from 6 healthy women who underwent conventional liposuction, with an average age of 31 years (range, 24-36 years). After local injection of tumescent fluid for anesthesia, a 3 mm liposuction cannula with a large side hole (2 mm x 7 mm) was connected to a 20 mL syringe and radial suction was performed under artificial negative pressure. The obtained fat was placed upright and still, and after removing the tumescent fluid, it was rinsed three times with saline.

[0180] (2) The washed adipose tissue was placed in a centrifuge tube and centrifuged at 1200 g at 4°C for 3 minutes to obtain a layered mixture.

[0181] (3) For the stratified mixture, the upper oil layer and the lower water layer are removed, and the middle layer (i.e., the fat layer containing fat cells) is collected.

[0182] (4) The intermediate layer is mechanically emulsified by repeatedly pushing and beating at a constant speed 30 times using two 10 ml injection syringes connected to a three-way tube to obtain a mechanically emulsified fat mixture (also called nano fat).

[0183] (5) The mechanically emulsified fat mixture is placed in a -80°C refrigerator for freezing, and then thawed in a 37°C water bath. After a single freeze-thaw cycle, the thawed fat mixture is centrifuged at 1200g and 4°C for 5 minutes to obtain a layered mixture. The layered mixture is divided into 4 layers, the first layer is an oil layer, the second layer is a residual fat tissue layer, the third layer is a liquid layer, and the fourth layer is a cell / tissue fragment precipitation layer. The oil layer and the residual fat tissue layer are removed, and the liquid layer is absorbed. During the absorption process, contamination of the cell / tissue fragment precipitation layer is avoided, thereby obtaining a primary fat extract.

[0184] (6) The obtained primary fat extract is filtered and sterilized through a 0.22 μm filter to sterilize and remove any living cells that may be mixed in, thereby obtaining a cell-free fat extract, which is then packaged and frozen at -20°C and thawed at 4°C before use.

[0185] 1.2 Establishment and grouping of rat ARDS model

[0186] Lipopolysaccharide (LPS) is used to construct an ARDS (acute respiratory distress syndrome) rat model

[0187] Eight-week-old male SD rats were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. 24 animals were randomly divided into 4 groups: control group, low-dose group, medium-dose group, and high-dose group, with 6 rats in each group. All 24 animals were intraperitoneally injected with lipopolysaccharide (LPS), a modeling agent combined with intra-airway atomization. The specific steps are as follows:

[0188] Day 0: Each group was given lipopolysaccharide (0.8 mg / kg, 400 μL / kg) by intraperitoneal injection using a disposable microsyringe according to the animal weight.

[0189] Day 1 (16h±30min after intraperitoneal injection): isoflurane inhalation anesthesia, each group of animals were given lipopolysaccharide (5mg / kg, 1000μL / kg) by intratracheal atomization. The main experimental steps are as follows: the animals were anesthetized by isoflurane inhalation, and then fixed on a rat holder placed at 45°. A small animal anesthesia laryngoscope was used to press the tongue root of the animal to expose the glottis. The lung micro-liquid atomizer needle (blunt) for extracting a quantitative lipopolysaccharide (LPS) solution was gently inserted into the trachea, and then the piston was quickly pushed to atomize the LPS solution into the lungs. The needle was quickly pulled out, and the animal was removed from the holder, with the head facing up and rotated left and right to make the LPS as evenly distributed as possible in each lung lobe.

[0190] 1.3 CEFFE treatment dosage and method

[0191] On Day 1, about 1 hour after the animal was nebulized with the modeling agent (LPS) in the airway, CEFFE treatment or saline control was given according to the dose in Table 1 below.

[0192] Table 1

[0193]

[0194] Administration route: Intravenous injection via tail vein. Use a disposable sterile syringe to extract the drug amount for each animal and slowly (about 10 to 60 seconds) inject it into the tail vein. Administration frequency and duration: The first administration begins about 1 hour after the modeling agent (LPS) is atomized into the airway of the animal on Day 1; the second administration is performed on Day 2; the interval between the two administrations is about 24 hours (±20 minutes).

[0195] 1.4 Survival rate records

[0196] The ratio of the number of surviving animals in each group to the total number of animals in each group during the test period was calculated, and the survival rate (%) = number of surviving animals in each group / total number of animals in each group × 100%.

[0197] 1.5 Blood gas test

[0198] Detection time: the day after the second administration (Day 3: about 48 hours after modeling).

[0199] Sample collection: Rats were anesthetized by intraperitoneal injection of chloral hydrate (350 mg / kg, 100 mg / mL). The abdomen was longitudinally incised at the midline, and the abdominal aorta was isolated. About 0.2 mL of arterial blood was collected using an arterial blood collector. The syringe was rubbed in the palm of the hand and turned upside down for 5 seconds each. The blood collector must not be withdrawn for mixing.

[0200] Detection method: After collecting arterial blood, gently insert the needle into the blood injection port of the test card, slowly push the blood in, and fill the sample filling tube. When the blood reaches the sample adding position, stop adding the sample, close the lid, and the blood will automatically enter the test tube. Insert the test card into the blood gas analyzer and wait for the test results.

[0201] Detection indicators: oxygen partial pressure PO2 (mmHg), carbon dioxide partial pressure PCO2 (mmHg), pH, blood oxygen saturation SO2%.

[0202] 1.6 BALF testing

[0203] On Day 3, all animals were anesthetized by intraperitoneal injection of chloral hydrate (350 mg / kg, 100 mg / mL) and euthanized by bleeding from the abdominal aorta. Moribund animals were anesthetized with isoflurane and euthanized by bleeding from the abdominal aorta.

[0204] Sample collection: After the animal is euthanized, the skin and tissue of the neck and chest are cut to expose the trachea, bronchi and lungs, and the right lung bronchus is separated and ligated. Sutures are passed under the trachea, and a 1 / 2 incision is made between the tracheal cartilage rings at an appropriate position under the thyroid cartilage. The endotracheal tube is slowly inserted into the airway along the incision to the left bronchus, and the intubation tube is tied and fixed to the trachea at an appropriate position in the centrifugal direction of the incision with the sutures. 3 mL of PBS buffer is drawn with a syringe and slowly injected for alveolar lavage. The lavage is repeated 3 times, and each flushing stays for about 10 seconds. The lavage fluid is collected in a centrifuge tube of appropriate capacity (not less than 2.1 mL).

[0205] BALF treatment: The collected lavage fluid was centrifuged at 4°C and about 2000 rpm for 20 min. The supernatant was stored below -70°C for later use, and the precipitate was resuspended in 1 mL of PBS buffer for white blood cell counting and classification.

[0206] Leukocyte classification test: After resuspending the lung lavage fluid, the leukocyte count and classification are performed using a fully automatic blood cell analyzer.

[0207] 1.7 Histopathology

[0208] Animals in each group were dissected and their tissues preserved. During the autopsy, the lungs, trachea and bronchi of the animals were observed for abnormalities. The right lung was cut and fixed in 10% neutral buffered formalin solution, embedded in paraffin, sliced, prepared, and HE stained for pathological morphology of lung tissue. Standard terminology was used for diagnosis and classification, and pathological examination of lung tissue was performed according to the 4-grade method (mild, mild, moderate, severe).

[0209] 1.8 Statistical analysis

[0210] The data were analyzed by one-way ANOVA test using SPSS software, and the Bonferreni method was used for post hoc test. The results are expressed as mean ± standard deviation.

[0211] 2. Results

[0212] 2.1 CEFFE treatment effectively improves the survival rate of ARDS rats

[0213] The survival status of rats in each group was recorded. The survival rate of ARDS model rats was as follows: Figure 1 As shown, 2 rats died in the control group, with a survival rate of 67%, while no rats died in the CEFFE treatment group, indicating that CEFFE treatment effectively improved the survival rate of ARDS rats.

[0214] 2.2 CEFFE treatment effectively improved the respiratory function and alleviated hypoxemia in ARDS rats

[0215] The results of arterial blood gas analysis are shown in Table 2 and Figure 2 shown.

[0216] Table 2 Arterial oxygen partial pressure PO2 and blood oxygen saturation SO2% (M±SD) of model rats

[0217]

[0218] From Table 2 and Figure 2 It can be seen that PO2 of ADRS model rats was significantly impaired. Compared with the control group and the low-dose treatment group, the oxygen partial pressure PO2 and blood oxygen saturation SO2% in the high-dose CEFFE treatment group were significantly increased (P<0.05), indicating that CEFFE treatment effectively improved the respiratory function of the model rats and alleviated hypoxemia.

[0219] 2.3 CEFFE treatment effectively reduces inflammatory cell infiltration in the lung tissue of ARDS rats

[0220] The inflammatory cell count in BALF reflects the inflammation of lung tissue. The inflammatory cell count and classification of bronchoalveolar lavage fluid are shown in Table 3 and Figure 3 shown.

[0221] Table 3 Inflammatory cell count and classification of bronchoalveolar lavage fluid (M±SD)

[0222]

[0223] Note: WBC refers to white blood cells; Neut refers to the number of neutrophils; Lymph refers to lymphocytes; Mono refers to monocytes;

[0224] From Table 3 and Figure 3It can be seen that the number of white blood cells and neutrophils in BALF of rats in the medium-dose and high-dose CEFFE treatment groups were significantly lower than those in the control group (P<0.05), and were dose-dependent, indicating that the infiltration of inflammatory cells in the lungs was significantly reduced. CEFFE treatment has the effect of reducing inflammation in the lungs of ARDS rats.

[0225] 2.4 CEFFE treatment effectively improves lung tissue damage in ARDS rats

[0226] Histopathological HE staining results are as follows Figure 4 The semi-quantitative evaluation results of pathological histology are shown in Table 4.

[0227] Table 4 Semi-quantitative evaluation of pathological histology

[0228]

[0229]

[0230] From Table 4 and Figure 4 It can be seen that the control group showed inflammatory cell infiltration mainly composed of neutrophils in the lungs and bronchial interstitium / alveoli, macrophage aggregation in the alveoli, thickening of the alveolar wall, alveolar / perivascular hemorrhage, and fibrinoid deposition in the alveoli; compared with the model control group, the inflammatory cell infiltration in the medium-dose and high-dose groups of CEFFE treatment was alleviated, and the alveolar / perivascular hemorrhage was improved, and there was a dose-related effect, that is, the improvement in the high-dose group was more obvious than that in the medium-dose group; in addition, the high-dose group also showed improvement in the deposition of fibrinoid material in the alveoli. These results indicate that CEFFE treatment can effectively reduce lung tissue damage in ARDS rats.

[0231] in conclusion:

[0232] Systemic inflammatory response syndrome (SIRS) is a clinical syndrome caused by infection or injury. The main triggering factors are the release of systemic inflammatory mediators, which causes cytokine / inflammatory factor storm and inflammatory cell activation, causing cell apoptosis, shock, immune dysfunction, organ damage, etc. Infection, trauma, etc. trigger SIRS, which further develops into multiple organ failure (MODS) when out of control. The lungs are the most vulnerable organ to damage during this course of disease evolution. Therefore, ALI / ARDS is an important disease symptom of SIRS patients.

[0233] The results of this experiment show that CEFFE treatment can effectively improve lung tissue damage caused by ALI / ARDS, reduce lung inflammation, improve lung function, correct hypoxemia, and reduce mortality. The therapeutic effect of CEEFE on ALI / ARDS suggests that it has therapeutic value in improving SIRS and also suggests its potential therapeutic effect on other respiratory diseases.

[0234] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A use of a cell-free fat extract, characterized in that: Used for preparing a pharmaceutical composition or preparation for preventing and / or treating acute respiratory distress syndrome and / or acute lung injury; The cell-free fat extract is prepared by the following method: (1) providing a fat tissue raw material, cutting the fat tissue raw material into pieces, and rinsing the pieces to obtain rinsed fat tissue; (2) centrifuging the rinsed adipose tissue to obtain a layered mixture; (3) removing the upper oil layer and the lower water layer from the stratified mixture, and collecting the middle layer, i.e., the fat layer containing fat cells; (4) emulsifying the intermediate layer to obtain an emulsified fat mixture; (5) centrifuging the emulsified fat mixture to obtain an intermediate liquid layer, i.e., a primary fat extract; and (6) filtering and sterilizing the primary fat extract to obtain a cell-free fat extract; In the step (5), after centrifugation, the emulsified fat mixture is separated into four layers, the first layer is an oil layer, the second layer is a residual fat tissue layer, the third layer is a liquid layer, i.e., an intermediate liquid layer, and the fourth layer is a cell / tissue fragment precipitation layer; Furthermore, the cell-free fat extract does not contain cells and does not contain fat droplets, and the "does not contain fat droplets" means that the volume of oil droplets in the cell-free fat extract accounts for less than 1% of the total liquid; The term "cell-free" means that the average number of cells in 1 ml of cell-free fat extract is ≤ 1; The acellular fat extract is prepared by extracting fat from human or non-human mammals.

2. The use according to claim 1, characterized in that The pharmaceutical composition or preparation also includes a pharmaceutically acceptable carrier.

3. The use according to claim 1, characterized in that The term "free of lipid droplets" means that the volume of lipid droplets in the cell-free fat extract accounts for less than 0.5% of the total liquid; The term "cell-free" means that the average number of cells in 1 ml of cell-free fat extract is ≤ 0.

5.

4. The use according to claim 1, characterized in that The term "free of lipid droplets" means that the volume of lipid droplets in the cell-free fat extract accounts for less than 0.1% of the total liquid; The term "cell-free" means that the average number of cells in 1 ml of cell-free fat extract is 0.

5. The use according to claim 1, characterized in that The fat extract contains components selected from the following group: growth factors IGF-1, BDNF, GDNF, TGF-β, HGF, bFGF, VEGF, PDGF, EGF, NT-3, GH, G-CSF, or a combination thereof.

6. The use according to claim 1, characterized in that The dosage form of the composition or preparation is an injection preparation.

7. The use according to claim 6, characterized in that The injection preparation is an intravenous injection preparation.

8. The use according to claim 1, characterized in that The cell-free fat extract is a naturally obtained nano fat extract without any added ingredients; The term "additive-free" means that, except for the rinsing step, no solution, solvent, small molecule, chemical agent, or biological additive is added during the preparation of the fat extract.

9. The use according to claim 1, characterized in that In the step (4), the emulsification is mechanical emulsification; the mechanical emulsification is performed by repeatedly blowing with a syringe.

10. The use according to claim 1, characterized in that In the step (4), the emulsification is performed by breaking up the tissue using a tissue homogenizer.

11. The use according to claim 1, characterized in that In the step (5), before the emulsified fat mixture is centrifuged, the emulsified fat mixture is further subjected to a freezing and then thawing process.

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