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WO2020251245A1 - Composition pharmaceutique, pour le traitement de maladies métaboliques, comprenant des exomoses dérivés du lait en tant que principe actif - Google Patents

Composition pharmaceutique, pour le traitement de maladies métaboliques, comprenant des exomoses dérivés du lait en tant que principe actif Download PDF

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WO2020251245A1
WO2020251245A1 PCT/KR2020/007480 KR2020007480W WO2020251245A1 WO 2020251245 A1 WO2020251245 A1 WO 2020251245A1 KR 2020007480 W KR2020007480 W KR 2020007480W WO 2020251245 A1 WO2020251245 A1 WO 2020251245A1
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milk
exosomes
metabolic diseases
supernatant
treating
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Korean (ko)
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안지영
안근아
김양훈
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Chungbuk National Univiversity CBNU
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Chungbuk National Univiversity CBNU
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Priority to CN202080043177.3A priority Critical patent/CN113993526B/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
    • A61K35/20—Milk; Whey; Colostrum
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/328—Foods, ingredients or supplements having a functional effect on health having effect on glycaemic control and diabetes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/332—Promoters of weight control and weight loss

Definitions

  • the present invention relates to a pharmaceutical composition for the treatment of metabolic diseases comprising an exosome derived from milk as an active ingredient.
  • Diabetes is one of the metabolic diseases in which the concentration of glucose in the blood increases, and is a disease caused by insufficient secretion of insulin or failure to function normally. Diabetes is largely divided into type 1 and type 2, and type 1 diabetes occurs because beta cells of the pancreas are destroyed by autoimmune mechanisms, viruses, and infections, and the secretion of insulin is absolutely insufficient.
  • type 2 diabetes refers to a case in which insulin is not secreted or the secreted insulin does not function properly in the body, so that blood sugar in the body cannot be controlled and rises.
  • diabetes causes microvascular complications such as retina, kidney, and nerves, and macrovascular complications such as stroke, angina, myocardial infarction and peripheral vascular disease.
  • microvascular complications such as retina, kidney, and nerves
  • macrovascular complications such as stroke, angina, myocardial infarction and peripheral vascular disease.
  • Diabetes is treated using drug therapy, exercise therapy, and diet, and insulin medicines and various blood sugar drugs are used depending on the patient's symptoms.
  • diabetes is a complex disease characterized by excessive sugar production in the liver, insulin resistance, and decreased sugar processing capacity in muscle and fat cells, and thus, the induction of various side effects cannot be prevented with only a specific treatment method.
  • drug therapy uses insulin and chemicals, so it can cause side effects and patient tolerance.
  • exosomes are small vesicles having a membrane structure secreted from various types of cells and have a diameter of about 50 to 200 nm. According to recent research results, it has been reported that exosomes originate from specific compartments within cells called multivesicular bodies (MVBs) and are released out of cells, rather than being directly separated from the plasma membrane.
  • MVBs multivesicular bodies
  • Exosomes are substances produced in the metabolic activity of almost all eukaryotic organisms and bacteria, as well as cells such as mast cells, lymphocytes, astrocytes, platelets, neurons, endothelial cells, and epithelial cells. Blood, urine, saliva, It is separated from all body fluids, including breast milk, amniotic fluid, ascites, cerebrospinal fluid, etc.
  • exosomes are recently spotlighted as biological samples useful for diagnosis because they contain various proteins, lipids, nucleic acids, etc. by reflecting the state of cells or cell membranes. This is because each cell-specific component reflects the unique biological function of the cell of origin (donor cell) from which the exosome is derived, so that the size of the exosome isolated from each cell, the composition of proteins and nucleic acids are different. to be.
  • Korean Patent Publication No. 10-2019-0011213 discloses that exosomes extracted from adipose tissue-derived stem cells exhibit bone regeneration promoting and bone density enhancing effects, and thus can be usefully used in the treatment of osteoporosis. have.
  • An object of the present invention is to provide a use of exosomes derived from milk for the treatment of metabolic diseases.
  • Another object of the present invention is to provide a use for promoting insulin secretion of exosomes derived from milk.
  • Another object of the present invention is to provide a method for extracting exosomes from milk.
  • the present invention provides a pharmaceutical composition for preventing or treating metabolic diseases, including exosomes derived from milk as an active ingredient.
  • the present invention provides a health functional food for preventing or improving metabolic diseases comprising exosomes derived from milk as an active ingredient.
  • the present invention provides a composition for promoting insulin secretion comprising exosomes derived from milk as an active ingredient.
  • the present invention comprises the steps of treating milk with acetic acid and obtaining a first supernatant; Treating the obtained first supernatant with one or more reagents selected from the group consisting of ethylenediaminetetraacetic acid (EDTA) and hydrogen chloride (HCl) to obtain a second supernatant; And it provides a method for extracting exosomes from milk comprising the step of extracting the exosomes from the obtained secondary supernatant.
  • EDTA ethylenediaminetetraacetic acid
  • HCl hydrogen chloride
  • the present invention provides a method for preventing, improving, or treating metabolic diseases comprising administering to an individual an exosome derived from milk.
  • the present invention provides the use of exosomes derived from milk for use in the manufacture of medicaments for the prevention, amelioration or treatment of metabolic diseases.
  • exosomes derived from milk of the present invention contain mi-155 and mi-375, which are known to have antidiabetic activity, and mRNA for IRS-1 and GLUT-4 proteins, which are factors promoting insulin signaling.
  • mi-155 and mi-375 which are known to have antidiabetic activity
  • mRNA for IRS-1 and GLUT-4 proteins which are factors promoting insulin signaling.
  • 1 is a view showing the results of visual confirmation of exosomes extracted by treating milk with 1, 2, 5 or 10% (v/v) acetic acid with EDTA or PBS.
  • FIG. 2 is a diagram illustrating a result of photographing exosomes extracted from milk with a transmission electron microscope (TEM) (A) and a result of confirming the expression of TSG101 protein, an exosome marker protein, in the extracted exosomes (B).
  • TEM transmission electron microscope
  • 3 is a graph showing the size distribution of the exosomes extracted from milk.
  • Figure 4 is a Ct value (A), standard melting curve (B) and differential melting curve (C) result graphs for confirming the presence of miRNAs of mi-155 and mi-375 in exosomes extracted from milk.
  • FIG. 5 is a diagram showing the results of confirming whether phosphorylation is induced in the AKT protein by treating the pancreatic cancer cell line with insulin.
  • FIG. 6 is a graph showing changes in the expression of mRNA for IRS-1 and GLUT-4 proteins, which are factors that promote insulin signaling by processing exosomes extracted from milk in pancreatic cancer cell lines.
  • FIG. 7 is a graph showing changes in the expression of mRNA for C/EBP ⁇ and PDK4 proteins, which are factors that inhibit insulin signaling by processing exosomes extracted from milk in pancreatic cancer cell lines.
  • the present invention provides a pharmaceutical composition for preventing or treating metabolic diseases comprising exosomes derived from milk as an active ingredient.
  • exosome refers to a membrane-structured vesicle secreted from various types of cells.
  • the exosomes bind to other cells or tissues and perform various functions such as transferring membrane components, proteins, and RNA, and generally have an average diameter of about 50 to 200 nm.
  • exosomes according to the present invention are 50 to 250 nm, 50 to 220 nm, 50 to 200 nm, 50 to 180 nm, 80 to 250 nm, 80 to 220 nm, 80 to 200 nm, 80 to 180 nm, It may have an average diameter of 100 to 250 nm, 100 to 220 nm, 100 to 200 nm or 100 to 180 nm.
  • the exosome may express an exosome marker protein, and specifically, the exosome marker protein may be a TSG101 protein.
  • the TSG101 protein may include all sequences known as TSG101 protein in the art, and may have 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more homology with a known sequence.
  • the TSG101 protein may be a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 7.
  • the exosomes may include miRNAs (microRNAs) known to exhibit antidiabetic activity.
  • the miRNA may be any one or more selected from the group consisting of mi-155 and mi-375, and more specifically mi-155 and mi-375.
  • the mi-155 may be a polynucleotide composed of the nucleotide sequence of SEQ ID NO: 8
  • mi-375 may be a polynucleotide composed of the nucleotide sequence of SEQ ID NO: 9.
  • the exosome may increase the expression of a factor that promotes the insulin signaling mechanism and at the same time inhibit the expression of a factor that inhibits the insulin signaling mechanism. Therefore, the exosomes according to the present invention can be used for the prevention or treatment of metabolic diseases.
  • the metabolic disease may be obesity, diabetes, or a diabetic complication
  • the diabetic complication may be diabetic retinopathy, diabetic cataract, diabetic nephropathy, diabetic neuropathy, diabetic foot ulcer or diabetic vascular complication. .
  • Exosomes according to the invention can be obtained by a method comprising the following steps:
  • EDTA ethylenediaminetetraacetic acid
  • HCl hydrogen chloride
  • milk and acetic acid are 1:5 to 150, 1:5 to 120, 1:5 to 110, 1:5 to 80, 1:5 to 60, 1:5 to 30, 1:5 to 15, 1 It may be added in a volume ratio of :8 to 150, 1:8 to 120, 1:8 to 110, 1:8 to 80, 1:8 to 60, 1:8 to 30, or 1:8 to 15.
  • the pharmaceutical composition according to the present invention may contain 10 to 95% by weight of exosomes derived from milk, which is an active ingredient, based on the total weight of the composition.
  • the pharmaceutical composition of the present invention may further include one or more active ingredients exhibiting the same or similar functions in addition to the active ingredients.
  • the pharmaceutical composition of the present invention may contain a carrier, a diluent, an excipient, or a mixture thereof commonly used in biological preparations.
  • Any pharmaceutically acceptable carrier can be used as long as it is suitable for delivering the composition in vivo.
  • the carrier is Merck Index, 13th ed., Merck & Co. Inc., saline, sterile water, Ringer's solution, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture thereof.
  • conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed.
  • diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be added.
  • the composition of the present invention may be formulated as an oral or parenteral formulation.
  • Oral formulations may include solid formulations and liquid formulations.
  • the solid preparation may be a tablet, a pill, a powder, a granule, a capsule, or a troche, and the solid preparation may be prepared by adding at least one excipient to the composition.
  • the excipient may be starch, calcium carbonate, sucrose, lactose, gelatin, or a mixture thereof.
  • the solid preparation may contain a lubricant, examples of which include magnesium stearate and talc.
  • the liquid formulation may be a suspension, a liquid formulation, an emulsion or a syrup. At this time, the liquid formulation may contain excipients such as wetting agents, sweetening agents, fragrances, preservatives, and the like.
  • the parenteral preparations may include injections, suppositories, powders for respiratory inhalation, aerosols for sprays, powders and creams.
  • the injection may include a sterilized aqueous solution, a non-aqueous solvent, a suspension solvent, an emulsion, and the like.
  • the non-aqueous solvent or suspension solvent vegetable oils such as propylene glycol, polyethylene glycol, and olive oil, or injectable esters such as ethyl oleate may be used.
  • the present invention provides a health functional food for preventing or improving metabolic diseases comprising exosomes derived from milk as an active ingredient.
  • the exosomes derived from milk included as an active ingredient in the health functional food may have the characteristics as described above.
  • the exosomes are 50 to 250 nm, 50 to 220 nm, 50 to 200 nm, 50 to 180 nm, 80 to 250 nm, 80 to 220 nm, 80 to 200 nm, 80 to 180 nm, 100 to 250 Nm, 100 to 220 nm, 100 to 200 nm, or 100 to 180 nm.
  • the exosome may express an exosome marker protein, and specifically, the exosome marker protein may be a TSG101 protein.
  • the exosomes may include miRNAs (microRNAs) known to exhibit antidiabetic activity.
  • miRNA may be any one or more selected from the group consisting of mi-155 and mi-375, and more specifically mi-155 and mi-375.
  • the metabolic disease may be obesity, diabetes, or a diabetic complication
  • the diabetic complication may be diabetic retinopathy, diabetic cataract, diabetic nephropathy, diabetic neuropathy, diabetic foot ulcer or diabetic vascular complication.
  • the present invention provides a composition for promoting insulin secretion comprising exosomes derived from milk as an active ingredient.
  • the exosomes derived from milk included as an active ingredient in the composition for promoting insulin secretion may have the characteristics as described above.
  • the exosomes are 50 to 250 nm, 50 to 220 nm, 50 to 200 nm, 50 to 180 nm, 80 to 250 nm, 80 to 220 nm, 80 to 200 nm, 80 to 180 nm, 100 to 250 Nm, 100 to 220 nm, 100 to 200 nm, or 100 to 180 nm.
  • the exosome may express an exosome marker protein, and specifically, the exosome marker protein may be a TSG101 protein.
  • the exosomes may include miRNAs (microRNAs) known to exhibit antidiabetic activity.
  • the miRNA may be any one or more selected from the group consisting of mi-155 and mi-375, and more specifically mi-155 and mi-375. Accordingly, the exosome may increase the expression of a factor that promotes the insulin signaling mechanism and at the same time inhibit the expression of a factor that inhibits the insulin signaling mechanism.
  • the present invention provides a step of treating milk with acetic acid and obtaining a first supernatant; Treating the obtained first supernatant with any one or more solutions selected from the group consisting of EDTA and HCl and obtaining a second supernatant; And it provides a method for extracting exosomes from milk comprising the step of extracting the exosomes from the obtained secondary supernatant.
  • the method for extracting exosomes from milk according to the present invention provides a step of treating milk with acetic acid and obtaining a first supernatant.
  • acetic acid may be added to remove proteins and fats contained in milk, at this time, acetic acid is 0.5 to 15, 0.5 to 12, 0.5 to 8, 0.5 to 6, 0.5 to 3, 0.5 to 1.5, It may be included in a volume ratio of 0.8 to 15, 0.8 to 12, 0.8 to 8, 0.8 to 6, 0.8 to 3, 0.8 to 1.5% (v/v).
  • the acetic acid-treated milk can be centrifuged to obtain only the first supernatant. The centrifugation may be performed according to conditions known in the art, and may be appropriately modified by a person skilled in the art as necessary.
  • the step may further include filtering the obtained first supernatant to obtain a filtrate.
  • the filtration may be performed according to a method known in the art, and specifically, may be performed with a filter having a pore size of 0.3 to 0.7 ⁇ m.
  • the method for extracting exosomes from milk according to the present invention provides a step of treating the obtained first supernatant with any one or more solutions selected from the group consisting of EDTA and HCl and obtaining a second supernatant.
  • This step may be carried out to remove residues such as casein from milk from which proteins and fats have been removed by treatment with acetic acid.
  • EDTA or HCl may be added in the same amount as the acetic acid.
  • the first supernatant to which EDTA or HCl is added can be centrifuged again to obtain only the second supernatant. The centrifugation may be performed according to conditions known in the art, and may be appropriately modified by a person skilled in the art as necessary.
  • the step may further include filtering the obtained secondary supernatant to obtain a filtrate.
  • the filtration may be performed according to a method known in the art, and specifically, may be performed with a filter having a pore size of 0.1 to 0.5 ⁇ m.
  • milk and acetic acid are 1:5 to 150, 1:5 to 120, 1:5 to 110, 1:5 to 80, 1:5 to 60, 1:5 to 30, 1:5 to 15, 1 It may be added in a volume ratio of :8 to 150, 1:8 to 120, 1:8 to 110, 1:8 to 80, 1:8 to 60, 1:8 to 30, or 1:8 to 15.
  • the method of extracting exosomes from milk according to the present invention provides a step of extracting exosomes from the obtained secondary supernatant.
  • the extraction of exosomes can be performed by obtaining a pellet through centrifugation.
  • the centrifugation may be performed according to conditions known in the art, and may be appropriately modified by a person skilled in the art as necessary.
  • the present invention provides a method for preventing, improving, or treating metabolic diseases comprising administering to an individual an exosome derived from milk.
  • the exosomes derived from milk administered to an individual for the prevention, improvement or treatment of the metabolic disease may have the characteristics as described above.
  • the exosomes are 50 to 250 nm, 50 to 220 nm, 50 to 200 nm, 50 to 180 nm, 80 to 250 nm, 80 to 220 nm, 80 to 200 nm, 80 to 180 nm, 100 to 250 Nm, 100 to 220 nm, 100 to 200 nm, or 100 to 180 nm.
  • the exosome may express an exosome marker protein, and specifically, the exosome marker protein may be a TSG101 protein.
  • the exosomes may include miRNAs (microRNAs) known to exhibit antidiabetic activity.
  • miRNA may be any one or more selected from the group consisting of mi-155 and mi-375, and more specifically mi-155 and mi-375.
  • the metabolic disease may be obesity, diabetes, or a diabetic complication
  • the diabetic complication may be diabetic retinopathy, diabetic cataract, diabetic nephropathy, diabetic neuropathy, diabetic foot ulcer or diabetic vascular complication.
  • the individual may be a mammal, and specifically, may be a human.
  • the administration may be oral or parenteral according to the desired method.
  • Parenteral administration may include intraperitoneal, rectal, subcutaneous, intravenous, intramuscular or intrathoracic injection.
  • the administration may be administered in a pharmaceutically effective amount. This may vary depending on the type of disease, the severity, the activity of the drug, the patient's sensitivity to the drug, the administration time, the administration route, the treatment period, and the drugs used at the same time.
  • the active ingredient according to the present invention may be administered in an amount of 0.0001 to 1,000 mg/kg, specifically 0.001 to 500 mg/kg, and the administration may be once or several times a day.
  • administration may be performed alone or in combination with other therapeutic agents.
  • administration may be sequential or simultaneous.
  • the present invention provides the use of exosomes derived from milk for use in the manufacture of medicaments for the prevention, amelioration or treatment of metabolic diseases.
  • the exosomes derived from milk used in the manufacture of a drug for the prevention, improvement or treatment of the metabolic disease may have the above-described characteristics.
  • the exosomes are 50 to 250 nm, 50 to 220 nm, 50 to 200 nm, 50 to 180 nm, 80 to 250 nm, 80 to 220 nm, 80 to 200 nm, 80 to 180 nm, 100 to 250 Nm, 100 to 220 nm, 100 to 200 nm, or 100 to 180 nm.
  • the exosome may express an exosome marker protein, and specifically, the exosome marker protein may be a TSG101 protein.
  • the exosomes may include miRNAs (microRNAs) known to exhibit antidiabetic activity.
  • miRNA may be any one or more selected from the group consisting of mi-155 and mi-375, and more specifically mi-155 and mi-375.
  • the metabolic disease may be obesity, diabetes, or a diabetic complication
  • the diabetic complication may be diabetic retinopathy, diabetic cataract, diabetic nephropathy, diabetic neuropathy, diabetic foot ulcer or diabetic vascular complication.
  • EDTA ethylenediaminetetraacetic acid
  • PBS phosphate buffer saline
  • Example 1 The shape of the exosomes extracted in Example 1 was analyzed using a transmission electron microscope (TEM) by performing negative staining in a conventional manner.
  • TEM transmission electron microscope
  • the size of the exosome was confirmed using qNano (iZon, Australia). Specifically, the device was calibrated using 200 nm calibration particles, and exosomes were prepared as samples by dilution in an electrolyte buffer with a dilution factor of 1/100. The size of exosomes was measured using the software built into the instrument. As a result, the morphology of the exosomes confirmed by TEM is shown in FIG. 1A, and a graph showing the size distribution of the exosomes is shown in FIG. 2.
  • exosomes extracted from milk had an average diameter of 109 nm, and exosomes were extracted with an average number of 4 ⁇ 10 11 particles/ml.
  • TSG101 protein an exosome marker protein
  • RIPA buffer was added to exosomes, mixed well, and the cells were lysed while stirring for 30 minutes on ice.
  • the protein contained in the cell lysate was quantified with a BCA protein assay kit (Promega, USA), and then tris-glycine-SDS (tris-glycine-SDS) using 10% acrylamide gel. SDS) was electrophoresed for 70 minutes at 100 V in buffer. The electrophoresed protein band was confirmed through Coomassie blue staining, and transferred to the membrane at 250 mA for 60 minutes.
  • the membrane was pretreated with PBS-T buffer containing 5% (w/v) skim milk for 2 hours, and washed with washing buffer for 1 hour.
  • Anti-TSG101 antibody (ab125011, abcam), which is a primary antibody against TSG101 protein, was added to the washed membrane, followed by reaction at 4°C overnight. After the reaction was completed, the membrane was washed with a washing buffer for 1 hour, and a secondary antibody, goat anti-rabbit IgG (H+L) secondary antibody, HRP (Thermofisher, #65-6120) was added thereto at room temperature for 2 hours. After the reaction, it was washed again with a washing buffer for 1 hour. A photograph of the result confirmed by performing an ECL (Amersham, USA) reaction using the washed membrane is shown in FIG. 2B.
  • miRNA was extracted from exosomes according to the manufacturer's protocol, and adenylic acid polymerization reaction (polyadenylation) was performed on the extracted miRNA by a conventional method. At this time, the reaction was carried out at 37° C. for 30 minutes, and cDNA was synthesized using the polyA-miRNA obtained through the reaction as a template using the TOPscriptTM cDNA synthesis kit (Enzynomics, Korea).
  • the Ct values of mi-155 and mi-375 were 26.5 ⁇ 0.1 and 29.5 ⁇ 0.2, respectively, and the melt curve was confirmed from the results calculated as shown in FIGS. 4B and 4C. . From the above results, it was found that mi-155 and mi-375 were abundantly present in exosomes extracted from milk.
  • pancreatic cells When the pancreatic cells were treated with insulin, it was confirmed by Western blot whether or not AKT (protein kinase B) protein was phosphorylated by positive insulin signaling.
  • AKT protein kinase B
  • pancreatic cancer cell lines (ATCC) were dispensed into a 100 mm cell culture dish, and cultured under conditions of 37°C and 5% CO 2 . After 36 hours of incubation, the cells were washed and a culture medium containing no serum was added to starve the cells for 24 hours. Thereafter, the cells were washed, insulin was added to a concentration of 200 nM, and reacted for 30 minutes. At this time, cells that did not starve cells (C) and cells that were not treated with insulin (0) were used as controls. Western blot was performed in a conventional method using the cells after the reaction.
  • pancreatic cancer cell lines were prepared under the same conditions and methods as described in Experimental Example 1.
  • the starved cells were treated with 10 11 particles of exosomes for 24 hours and reacted for 24 hours.
  • a 200 nM insulin treatment group was used as a positive control.
  • mRNA of a pancreatic cancer cell line was extracted using an mRNA extraction kit (GeneAll Biotechnology, Korea) according to the manufacturer's protocol.
  • cDNA was synthesized using a TOPscriptTM cDNA synthesis kit (Enzynomics, Korea). Specifically, 2 ⁇ l of reverse transcriptase buffer, 1 ⁇ l of reverse transcriptase, 2 ⁇ l of dNTP mixture, 100 ng of template RNA, 1 ⁇ l of oligo dT and 0.5 ⁇ l of RNase inhibitor were mixed, and total A reaction product was prepared by adding sterilized deionized water (DDI water) to a volume of 20 ⁇ l. The reaction was reacted at 42° C. for 5 minutes and 60° C. for 1 hour to synthesize cDNA, and then left at 95° C. for 5 minutes to inactivate.
  • DDI water sterilized deionized water
  • the factors related to insulin signaling are insulin receptor substrate 1 (IRS-1), glucose transporter type 4 (GLUT-4), and C/EBP ⁇ (CCAAT/enhancer).
  • -binding protein ⁇ ) and PDK4 (pyruvate dehydrogenase kinase 4) protein mRNA expression was confirmed.
  • Real-time PCR was repeated a total of 50 times, reacting for 3 minutes at 95°C, 10 seconds at 95°C, 60 seconds at 55°C and 20 seconds at 72°C, and then adjust the Ct value by setting a threshold. After that, the actual real-time amplification amount was confirmed.
  • the primer was used a general-purpose primer generally used in the art.
  • the amplification results were calculated using the Rotor geneQ program, and the calculated results are shown in FIGS. 6 and 7.
  • the mRNA for IRS-1 and GLUT-4 proteins which are factors that promote insulin signaling, significantly increased their expression compared to the positive control group.
  • the expression of the mRNA for C/EBP ⁇ and PDK4 protein which are factors that inhibit insulin signaling, were suppressed.
  • the exosomes according to the present invention can be used in the treatment of diabetes by increasing the signaling of insulin.

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Abstract

L'invention concerne une composition pharmaceutique pour le traitement de maladies métaboliques, comprenant des exosomes dérivés du lait en tant que principe actif. Les exosomes dérivés du lait de la présente invention comprennent plus particulièrement mi-155 et mi-375 connus pour avoir une activité antidiabétique, favoriser l'expression de l'ARNm de protéines IRS-1 et GLUT-4 qui sont des facteurs favorisant la transmission de signaux de l'insuline et inhiber l'expression de l'ARNm de protéines C/EBPβ et PDK4 qui sont des facteurs inhibant la transmission de signaux de l'insuline, et peuvent ainsi être utilisés pour traiter des maladies métaboliques.
PCT/KR2020/007480 2019-06-13 2020-06-10 Composition pharmaceutique, pour le traitement de maladies métaboliques, comprenant des exomoses dérivés du lait en tant que principe actif Ceased WO2020251245A1 (fr)

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KR1020190070090A KR102223138B1 (ko) 2019-06-13 2019-06-13 우유로부터 유래된 엑소좀을 유효성분으로 포함하는 대사성 질환 치료용 약학적 조성물
KR10-2019-0070090 2019-06-13

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113993526A (zh) * 2019-06-13 2022-01-28 忠北大学校产学协力团 包含源自牛奶的外泌体作为有效成分的用于治疗代谢性疾病的药物组合物
WO2025050229A1 (fr) * 2023-09-04 2025-03-13 谛邈生物科技（新加坡）有限公司 Mev et son utilisation dans la préparation d'un médicament pour la prévention et/ou le traitement d'un ulcère gastrique

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102559516B1 (ko) * 2020-08-31 2023-07-26 경희대학교 산학협력단 우유 엑소좀을 포함하는 갈색지방화 유도용 조성물
KR102627823B1 (ko) * 2021-05-11 2024-01-19 충북대학교 산학협력단 우유로부터 유래된 엑소좀을 유효성분으로 포함하는 흉터 생성 억제용 조성물
KR102729797B1 (ko) 2022-01-21 2024-11-14 한국과학기술연구원 우유 유래 엑소좀을 유효성분으로 포함하는 장 건강기능 개선용 조성물 및 그 제조방법
CN117337974B (zh) * 2022-06-29 2025-10-31 逸惟生物科技(北京)有限公司 一种番茄红素制剂及其制备方法
ES2977833A1 (es) 2023-01-17 2024-08-30 Univ Madrid Autonoma Procedimiento para la obtención de exosomas a partir de mazada, exosomas obtenidos y aplicaciones de los mismos
KR20250113951A (ko) * 2024-01-19 2025-07-28 엑소제니끄 주식회사 밀크 엑소좀을 유효성분으로 포함하는 신경 손상 치료용 약학적 조성물

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016056119A (ja) * 2014-09-09 2016-04-21 森永乳業株式会社 アポトーシス抑制剤
EP3192518A1 (fr) * 2014-09-09 2017-07-19 Morinaga Milk Industry Co., Ltd. Agent anti-inflammatoire
WO2018170332A1 (fr) * 2017-03-15 2018-09-20 Nutech Ventures Vésicules extracellulaires et procédés d'utilisation
KR20190011213A (ko) * 2017-07-24 2019-02-01 한양대학교 에리카산학협력단 줄기세포로부터 추출된 엑소좀을 유효성분으로 포함하는 골다공증 예방 또는 치료용 조성물

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101524079B1 (ko) 2014-12-03 2015-06-04 서울대학교산학협력단 엑소솜을 이용한 성체 유래 세포로부터 인슐린 생산세포로의 분화 유도 방법
WO2017131352A1 (fr) * 2016-01-26 2017-08-03 한양대학교 에리카산학협력단 Composition destinée à induire une différentiation en adipocytes beiges comportant un exosome extrait de cellules souches
CN108315440B (zh) * 2018-03-29 2021-02-19 华南农业大学 一种区分水牛和奶牛奶的miRNA标记物及其应用
KR102223138B1 (ko) * 2019-06-13 2021-03-04 충북대학교 산학협력단 우유로부터 유래된 엑소좀을 유효성분으로 포함하는 대사성 질환 치료용 약학적 조성물
IL296959A (en) * 2020-04-03 2022-12-01 Abbott Lab Bovine milk exosome products and methods, nutritional compositions, and therapeutic methods
KR102732209B1 (ko) * 2020-12-24 2024-11-19 순천향대학교 산학협력단 모유 유래 엑소좀 마이크로 rna
WO2023070429A1 (fr) * 2021-10-28 2023-05-04 谛邈生物科技(北京)有限公司 Utilisation d'exosomes de lait dans la préparation d'un vecteur de médicament
CN116036298B (zh) * 2021-10-28 2024-06-04 光武惠文生物科技(北京)有限公司 牛奶外泌体在制备药物载体中的应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016056119A (ja) * 2014-09-09 2016-04-21 森永乳業株式会社 アポトーシス抑制剤
EP3192518A1 (fr) * 2014-09-09 2017-07-19 Morinaga Milk Industry Co., Ltd. Agent anti-inflammatoire
WO2018170332A1 (fr) * 2017-03-15 2018-09-20 Nutech Ventures Vésicules extracellulaires et procédés d'utilisation
KR20190011213A (ko) * 2017-07-24 2019-02-01 한양대학교 에리카산학협력단 줄기세포로부터 추출된 엑소좀을 유효성분으로 포함하는 골다공증 예방 또는 치료용 조성물

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ESSER, NATHALIE, LEGRAND-POELS SYLVIE, PIETTE JACQUES, SCHEEN ANDRÉ J., PAQUOT NICOLAS: "Inflammation as a link between obesity, metabolic syndrome and type 2 diabetes", DIABETES RESEARCH AND CLINICAL PRACTICE, vol. 105, 2014, pages 141 - 150, XP055769520, DOI: 10.1016/j.diabres.2014.04.006 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113993526A (zh) * 2019-06-13 2022-01-28 忠北大学校产学协力团 包含源自牛奶的外泌体作为有效成分的用于治疗代谢性疾病的药物组合物
CN113993526B (zh) * 2019-06-13 2024-06-18 忠北大学校产学协力团 包含源自牛奶的外泌体作为有效成分的用于治疗代谢性疾病的药物组合物
WO2025050229A1 (fr) * 2023-09-04 2025-03-13 谛邈生物科技（新加坡）有限公司 Mev et son utilisation dans la préparation d'un médicament pour la prévention et/ou le traitement d'un ulcère gastrique

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