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WO2024090922A1 - Composition pharmaceutique pour la prévention ou le traitement de maladies du système nerveux cérébral, comprenant, en tant que principes actifs, une culture de cellules souches et des vésicules extracellulaires isolées de celle-ci - Google Patents

Composition pharmaceutique pour la prévention ou le traitement de maladies du système nerveux cérébral, comprenant, en tant que principes actifs, une culture de cellules souches et des vésicules extracellulaires isolées de celle-ci Download PDF

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WO2024090922A1
WO2024090922A1 PCT/KR2023/016456 KR2023016456W WO2024090922A1 WO 2024090922 A1 WO2024090922 A1 WO 2024090922A1 KR 2023016456 W KR2023016456 W KR 2023016456W WO 2024090922 A1 WO2024090922 A1 WO 2024090922A1
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culture medium
nervous system
stem cells
brain
preventing
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Korean (ko)
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김윤배
최은경
김태명
박동선
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Designed Cells Co ltd
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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
    • A61K35/48—Reproductive organs
    • A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
    • 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
    • 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
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • 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
    • A23V2200/322—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the nervous system or on mental function
    • 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
    • A23V2250/00—Food ingredients
    • A23V2250/20—Natural extracts
    • A23V2250/204—Animal extracts

Definitions

  • the present invention relates to a pharmaceutical composition for the prevention or treatment of brain nervous system diseases, comprising stem cell culture media and extracellular vesicles isolated therefrom as active ingredients.
  • Cerebral palsy is a disorder that includes impaired motor and posture development and is caused by various non-progressive damage to the brain during fetal or infancy. In patients with cerebral palsy, motor impairment is often accompanied by sensory, cognitive, communication, strabismus, visual perception, and behavioral impairments. Cerebral palsy can be caused by a variety of factors, but intrauterine infection or asphyxia during delivery are considered the main causes. It is known that the incidence of cerebral palsy is particularly high in premature babies, and it is well known that respiratory dysfunction is the main cause.
  • cerebral palsy is mainly carried out through rehabilitation treatment including physical, labor, language and psychological therapy, and treatments such as anti-cerebral palsy drugs, botulism injections, orthopedic surgery and orthotics are also used. .
  • rehabilitation treatment including physical, labor, language and psychological therapy, and treatments such as anti-cerebral palsy drugs, botulism injections, orthopedic surgery and orthotics are also used.
  • treatments such as anti-cerebral palsy drugs, botulism injections, orthopedic surgery and orthotics are also used.
  • cerebral palsy there is currently no fundamental treatment method that can actually treat cerebral palsy.
  • Republic of Korea Patent Publication No. 10-2021-0114946 relates to deutetrabenazine for the treatment of dyskinesia in cerebral palsy, and is administered at a dose of about 6 mg/day to about 48 mg/day to cerebral palsy patients in one or two doses. It is disclosed that administering a daily dose of deutetrabenazine at mg/day reduces abnormal involuntary movements in patients associated with dyskinesia in cerebral palsy.
  • the purpose of the present invention is to provide a pharmaceutical composition for the prevention or treatment of brain nervous system diseases containing stem cell culture medium and extracellular vesicles isolated therefrom as active ingredients.
  • the present invention provides a method for preventing or treating cranial nervous system diseases comprising as an active ingredient at least one selected from the group consisting of stem cell culture medium and extracellular vesicles (extracellular vehicles (EV)) isolated therefrom.
  • Pharmaceutical compositions are provided.
  • the present invention provides a health functional food for preventing or improving cranial nervous system diseases containing as an active ingredient at least one selected from the group consisting of stem cell culture medium and extracellular vesicles isolated therefrom.
  • the present invention provides a method for preventing, improving, or treating a brain nervous system disease, comprising administering to an individual at least one selected from the group consisting of a stem cell culture medium and extracellular vesicles isolated therefrom.
  • the present invention provides the use of at least one selected from the group consisting of stem cell culture media and extracellular vesicles isolated therefrom for use in the production of drugs for preventing, improving, or treating cranial nervous system diseases.
  • the stem cell culture medium according to the present invention and the extracellular vesicles isolated therefrom are administered into the body, they move to brain tissue, protect neural stem cells and oligodendrocytes, suppress the expression of inflammatory factors, and inhibit the growth of oligodendrocytes. It can be useful in the treatment of cranial nervous system diseases by promoting differentiation and maturation and improving clinical symptoms in animal models of cerebral palsy.
  • Figure 1 is a photograph (A) showing the results of confirming the expression of exosome markers CD9, CD63, and CD81 in exosome-enriched culture medium (ERCM) of amniotic membrane-derived mesenchymal stem cells obtained in an example of the present invention (A).
  • the result of checking the number is a graph (B) and the result of observing exosomes under a microscope is a photo (C).
  • Figure 2 is a graph showing the results of confirming the expression of growth factors and neurotrophic factors in exosome-rich culture medium of amniotic membrane-derived mesenchymal stem cells obtained in an example of the present invention.
  • Figure 3 shows the results of confirming the degree of distribution within brain tissue according to the administration time of the exosome-rich culture medium of amniotic membrane-derived mesenchymal stem cells obtained in an example of the present invention (A) and the AUC analysis results of the result graph (B) This is a graph representing .
  • Figure 4 is a diagram showing the results of confirming the distribution of the normal culture medium of amniotic membrane-derived mesenchymal stem cells obtained in an example of the present invention in an animal model.
  • Figure 5 is a diagram showing the results of confirming the distribution of the exosome-rich culture medium of amniotic membrane-derived mesenchymal stem cells obtained in an example of the present invention in an animal model.
  • Figure 6 is a graph showing the results confirming the neural stem cell protection effect by LPS treatment of the exosome-rich culture medium of amniotic membrane-derived mesenchymal stem cells obtained in an example of the present invention.
  • Figure 7 is a graph showing the results confirming the neural stem cell protection effect by KCN treatment of the exosome-rich culture medium of amniotic membrane-derived mesenchymal stem cells obtained in an example of the present invention.
  • Figure 8 shows the results of confirming changes in the expression of proteins related to apoptosis or cell differentiation by treatment with LPS (A) or KCN (B) in the exosome-rich culture medium of amniotic membrane-derived mesenchymal stem cells obtained in an example of the present invention. am.
  • Figure 9 shows the results of confirming changes in expression of proteins related to oligodendrocyte differentiation by treatment with LPS (A) or KCN (B) in the exosome-rich culture medium of amniotic membrane-derived mesenchymal stem cells obtained in an example of the present invention. am.
  • Figure 10 is a graph showing the results confirming the protective effect of oligodendrocyte progenitor cells by LPS treatment of exosome-rich culture medium of amniotic membrane-derived mesenchymal stem cells obtained in an example of the present invention.
  • Figure 11 is a graph showing the results confirming the oligodendrocyte protection effect by KCN treatment of the exosome-rich culture medium of amniotic membrane-derived mesenchymal stem cells obtained in an example of the present invention.
  • Figure 12 shows the results of confirming changes in the expression of proteins related to apoptosis or cell differentiation by treatment with LPS (A) or KCN (B) in exosome-rich culture medium of amniotic membrane-derived mesenchymal stem cells obtained in an example of the present invention. am.
  • Figure 13 shows the results of confirming changes in expression of proteins related to oligodendrocyte differentiation by treatment with LPS (A) or KCN (B) in the exosome-rich culture medium of amniotic membrane-derived mesenchymal stem cells obtained in an example of the present invention. It is a drawing.
  • Figure 14 shows the results of confirming changes in expression of proteins related to oligodendrocyte maturation by treatment with LPS (A) or KCN (B) in the exosome-rich culture medium of amniotic membrane-derived mesenchymal stem cells obtained in an example of the present invention. It is a drawing.
  • Figure 15 is a graph showing the results of a cylinder test confirming the effect of treating cerebral palsy by an exosome-rich culture medium of amniotic membrane-derived mesenchymal stem cells obtained in an example of the present invention.
  • Figure 16 is a graph showing the results of confirming the treatment effect of cerebral palsy by the exosome-rich culture medium of amniotic membrane-derived mesenchymal stem cells obtained in an example of the present invention using a rotarod.
  • Figure 17 is a graph showing the results of confirming the effect of treating cerebral palsy by the exosome-rich culture medium of amniotic membrane-derived mesenchymal stem cells obtained in an example of the present invention using a passive avoidance test (A) or a water maze test (B).
  • Figure 18 is a graph showing the results of confirming changes in gene expression of inflammatory factors in the brain tissue of an animal model of cerebral palsy caused by exosome-rich culture medium of amniotic membrane-derived mesenchymal stem cells obtained in an example of the present invention.
  • Figure 19 is a diagram showing the results of confirming changes in the expression of proteins related to apoptosis or cell differentiation in the brain tissue of a cerebral palsy animal model by exosome-rich culture medium of amniotic membrane-derived mesenchymal stem cells obtained in an example of the present invention.
  • Figure 20 is a diagram showing the results of confirming the expression changes of proteins related to oligodendrocyte differentiation in the brain tissue of an animal model of cerebral palsy by exosome-rich culture medium of amniotic membrane-derived mesenchymal stem cells obtained in an example of the present invention.
  • Figure 21 is a diagram showing the results of confirming the expression changes of proteins related to oligodendrocyte maturation in the brain tissue of an animal model of cerebral palsy by exosome-rich culture medium of amniotic membrane-derived mesenchymal stem cells obtained in an example of the present invention.
  • Figures 22 and 23 are diagrams showing the results of confirming changes in the expression of growth factors and neurotrophic factors in the brain tissue of an animal model of cerebral palsy caused by exosome-rich culture medium of amniotic membrane-derived mesenchymal stem cells obtained in an example of the present invention.
  • Figure 24 is a diagram showing the results of confirming the expression of myelin protein in the brain tissue of a cerebral palsy animal model by exosome-rich culture medium of amniotic membrane-derived mesenchymal stem cells obtained in an example of the present invention.
  • Figure 25 is a diagram showing the results of simultaneously confirming the expression of PDGFR and SOX10 proteins in the brain tissue of a cerebral palsy animal model by exosome-rich culture medium of amniotic membrane-derived mesenchymal stem cells obtained in an example of the present invention.
  • Figure 26 is a diagram showing the results of simultaneously confirming the expression of PDGFR and Oligo2 proteins in the brain tissue of a cerebral palsy animal model by exosome-rich culture medium of amniotic membrane-derived mesenchymal stem cells obtained in an example of the present invention.
  • the present invention provides a pharmaceutical composition for the prevention or treatment of brain nervous system diseases containing as an active ingredient at least one selected from the group consisting of stem cell culture medium and extracellular vesicles (extracellular vehicles (EV)) isolated therefrom.
  • stem cell culture medium and extracellular vesicles (extracellular vehicles (EV)) isolated therefrom.
  • extracellular vehicles (EV) extracellular vehicles
  • stem cell refers to a relatively underdeveloped, undifferentiated cell that has the ability to differentiate into cells of a specific tissue.
  • Stem cells can be divided into pluripotent stem cells, multipotent stem cells, or unipotent stem cells based on their differentiation ability.
  • the stem cells can be classified into embryonic stem cells, adult stem cells, or induced pluripotent stem cells (iPSC) produced from human somatic cells, depending on the cell of origin. You can.
  • the stem cells according to the present invention may be adult stem cells.
  • the term, adult stem cells refers to undifferentiated cells that exist in adult tissues or organs, can differentiate into desired cells, and can self-renew.
  • the stem cells according to the present invention may be amniotic membrane-derived adult stem cells.
  • the amniotic membrane-derived adult stem cells may be any one or more selected from the group consisting of amniotic membrane-derived mesenchymal stem cells and epithelial stem cells. .
  • the stem cell culture medium according to the present invention can be obtained by culturing stem cells under low oxygen concentration.
  • the low oxygen concentration may be lower than the average oxygen concentration of about 20% under normal atmosphere.
  • the low oxygen concentration is 10% or less, 0.1 to 10%, 0.1 to 8%, 0.1 to 5%, 0.1 to 4%, 1 to 10%, 1 to 8%, 1 to 5%, 1 to 4%. %, 2 to 10%, 2 to 8%, 2 to 5% or 2 to 4%.
  • the culture is performed for 1 to 80 hours, 1 to 70 hours, 1 to 60 hours, 1 to 50 hours, 10 to 80 hours, 10 to 70 hours, 10 to 60 hours, 10 to 50 hours, 20 to 80 hours, 20 to 70 hours, 20 to 60 hours, 20 to 50 hours, 30 to 80 hours, 30 to 70 hours, 30 to 60 hours, 30 to 50 hours, 40 to 80 hours, 40 to 70 hours, 40 to 60 hours, or It can be performed for 40 to 50 hours.
  • Stem cell culture medium cultured as described above may contain a higher level of exosomes compared to culture medium cultured under conventional conditions. That is, the stem cell culture medium may be an exosome-rich culture medium.
  • exosome-rich conditioned medium refers to a culture medium obtained by culturing stem cells having the characteristics described above. That is, the pharmaceutical composition according to the present invention can achieve the desired effect not only by stem cell culture medium but also by exosomes contained therein.
  • exosome refers to an extracellular vesicle (EV) with a membrane structure secreted from various types of cells.
  • the exosomes perform various functions such as binding to other cells or tissues and delivering membrane components, proteins, and RNA, and may generally have an average diameter of about 50 to 200 nm.
  • the exosomes can be identified using marker proteins contained therein.
  • the marker protein may include all types of marker proteins known in the art.
  • the marker protein may be the CD9 protein.
  • the stem cell culture medium according to the present invention may be filtered or concentrated to remove impurities as needed.
  • methods for isolating exosomes from the culture medium are also known in the art. The method may be appropriately modified and performed by a person skilled in the art as needed.
  • composition according to the present invention may include extracellular vesicles contained in stem cell culture medium having the characteristics described above as an active ingredient.
  • extracellular vehicle refers to a vesicle secreted from a cell and surrounded by a double phospholipid membrane like a cell membrane.
  • the extracellular vesicle may include all components known as extracellular vesicles in the art, and may specifically be one or more selected from the group consisting of exosomes and microvesicles.
  • Extracellular vesicles are known to serve as essential mediators for information exchange between cells, and contain specific proteins, mRNA, miRNA, ncRNA, etc. depending on the nature and state of the cell from which they are derived.
  • Extracellular vesicles are usually spherical with a diameter of 50 to 200 nm, and specific biomarkers are expressed on their membrane surface, and the biomarkers include integrins, MHC molecules, and cytoskeletal proteins. More specifically, the biomarker may be a tetraspanin protein such as CD9, CD63, and CD81.
  • microvesicle refers to an extracellular vesicle that has a phospholipid bilayer membrane with a diameter of 30 to 1,000 nm and is involved in communication between cells and transports mRNA, miRNA, etc.
  • microvesicles remove misfolding proteins, cytotoxic substances, and metabolic wastes from cells, and changes in the level of these microvesicles can cause diseases such as cancer. Unlike exosomes, they are formed through the process of membrane budding or exocytosis.
  • the cranial nervous system disease may include all diseases caused by abnormalities or disorders in some or all of the brain, spinal cord, cranial nerves, spinal nerves, and autonomic nervous system that constitute the cranial nervous system.
  • the cranial nervous system disease may be a brain damage disease.
  • the brain nervous system disease may be a nerve damage disease.
  • the brain nervous system diseases include cerebral palsy, brain injury, cerebral brain injury, ischemic brain injury, concussion, brain contusion, stroke, cerebral infarction, cerebral hemorrhage, amyotrophic axonal sclerosis, primary axonal sclerosis, multiple sclerosis, nervous system dysfunction, or It may be degenerative ataxia.
  • the pharmaceutical composition according to the present invention may contain 10 to 95% by weight of the active ingredient, the stem cell culture medium, and the extracellular vesicles separated therefrom, based on the total weight of the composition.
  • the pharmaceutical composition of the present invention may further include one or more active ingredients that exhibit the same or similar functions in addition to the above-mentioned effective ingredients.
  • the pharmaceutical composition of the present invention may include carriers, diluents, excipients, or mixtures thereof commonly used in biological products.
  • Any pharmaceutically acceptable carrier can be used as long as it is suitable for delivering the composition into the body.
  • the carrier is Merck Index, 13th ed., Merck & Co. Inc., saline solution, sterile water, Ringer's solution, dextrose solution, maltodextrin solution, glycerol, ethanol, or mixtures thereof.
  • conventional additives such as antioxidants, buffers, bacteriostatic agents, etc. can be added as needed.
  • diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be added.
  • the composition of the present invention may be formulated as an oral preparation or a parenteral preparation.
  • Oral preparations may include solid preparations and liquid preparations.
  • the solid preparation may be a tablet, pill, powder, granule, capsule, or troche, and such 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 mixtures thereof.
  • the solid preparation may contain a lubricant, examples of which include magnesium styrate and talc.
  • the liquid preparation may be a suspension, oral solution, emulsion, or syrup.
  • the liquid formulation may contain excipients such as wetting agents, sweeteners, fragrances, preservatives, etc.
  • the parenteral preparations may include injections, suppositories, powders for respiratory inhalation, aerosol preparations for sprays, powders, eye drops and creams.
  • the injection may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, etc.
  • the non-aqueous solvent or suspension may be propylene glycol, polyethylene glycol, vegetable oil such as olive oil, or injectable ester such as ethyl oleate.
  • the present invention provides a health functional food for preventing or improving cranial nervous system diseases containing as an active ingredient at least one selected from the group consisting of stem cell culture medium and extracellular vesicles isolated therefrom.
  • the stem cell culture medium contained in the health functional food according to the present invention and the extracellular vesicles isolated therefrom may have the characteristics described above.
  • the brain nervous system disease may also have the characteristics described above.
  • the health functional food can be added as is to food with its active ingredient, stem cell culture medium, and extracellular vesicles isolated therefrom, or can be used together with other foods or food ingredients.
  • the content of the active ingredient added may be determined depending on the purpose, and may generally be 0.01 to 90 parts by weight of the total weight of the food.
  • the form and type of the health functional food are not particularly limited.
  • the health functional food may be in the form of tablets, capsules, powders, granules, liquids, and pills.
  • the health functional food may contain various flavors, sweeteners, or natural carbohydrates as additional ingredients.
  • the sweetener may be a natural or synthetic sweetener, and examples of natural sweeteners include thaumatin and stevia extract. Meanwhile, examples of synthetic sweeteners include saccharin and aspartame.
  • the natural carbohydrate may be monosaccharide, disaccharide, polysaccharide, oligosaccharide, sugar alcohol, etc.
  • the health functional food of the present invention contains nutrients, vitamins, electrolytes, flavors, colorants, pexane and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, and preservatives. , glycerin, alcohol, etc. may be further included. These ingredients can be used independently or in combination.
  • the ratio of the additive may be selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.
  • the present invention provides a method for preventing, improving, or treating a brain nervous system disease, comprising administering to an individual at least one selected from the group consisting of a stem cell culture medium and extracellular vesicles isolated therefrom.
  • Stem cell culture medium and extracellular vesicles isolated therefrom used in the prevention, improvement or treatment method according to the present invention may have the characteristics described above.
  • the brain nervous system disease may also have the characteristics described above.
  • the subject may be a mammal, and may specifically be a human.
  • the administration may be oral or parenteral depending on the desired method.
  • Parenteral administration may include intraperitoneal, intrarectal, 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, severity, activity of the drug, patient's sensitivity to the drug, administration time, administration route, treatment period, drugs used simultaneously, etc. However, for a desirable effect, the amount of the active ingredient included in the administration may be 0.0001 to 1,000 mg/kg, specifically 0.001 to 500 mg/kg. The administration may be once or several times a day.
  • administration may be administered alone or in combination with other therapeutic agents.
  • administration may be sequential or simultaneous.
  • the present invention provides the use of at least one selected from the group consisting of stem cell culture media and extracellular vesicles isolated therefrom for use in the production of drugs for preventing, improving, or treating cranial nervous system diseases.
  • Stem cell culture medium and extracellular vesicles isolated therefrom used for manufacturing the drug according to the present invention may have the characteristics described above.
  • the brain nervous system disease may also have the characteristics described above.
  • AMMSC Human amniotic mesenchymal stem cells
  • the collected amniotic tissue was treated with collagenase I, an equal amount of culture medium containing 10% fetal bovine serum (FBS) was added, and centrifuged at 1,500 rpm for 10 minutes. The supernatant was removed, the pellet was washed twice, and red blood cells were lysed with RBC lysis buffer. The remaining cells from the lysate were suspended in keratinocyte serum-free medium (SFM, Invitrogen, USA) containing 5% FBS, 100 unit/ml of penicillin, and 100 mg/ml of streptomycin. The suspension was cultured under conditions of 37°C and 5% CO 2 while the culture medium was replaced every 2 to 3 days. By analyzing the cultured stem cells using a flow cytometry system to identify marker genes, it was confirmed that the stem cells were amniotic mesenchymal stem cells.
  • FBS fetal bovine serum
  • the human amniotic mesenchymal stem cells obtained above were cultured as follows to prepare an exosome-rich culture medium.
  • the human amniotic mesenchymal stem cells were placed in a hyper flask (Nunc, USA) and suspended in serum-free medium. It was cultured for 3 days under 5% carbon dioxide and 2% oxygen conditions. The culture medium was taken and filtered using a bottle-top vacuum filter system (0.22 ⁇ m, PES membrane, Corning, USA). The filtered culture medium was concentrated 30 times using Vivaflow-200 (Sartorius, Germany) and freeze-dried to prepare an exosome-rich conditioned medium. Meanwhile, a culture medium in which the human amniotic mesenchymal stem cells and epithelial stem cells were cultured under 20% oxygen was prepared as a normal conditioned medium.
  • exosomes present in the exosome-rich culture medium obtained above were analyzed by confirming the expression of exosome markers CD9, CD63, and CD81.
  • exosomes were isolated from the prepared exosome-rich culture medium by a conventional method.
  • the amount of protein in the isolated exosomes was quantified using a protein DC assay kit (Bio-Rad Laboratories, USA), and 25 ⁇ g was taken and mixed with 6 ⁇ denaturation buffer, and incubated at 95°C for 10 minutes.
  • the protein was denatured by reacting for a while.
  • the denatured protein was subjected to electrophoresis using a 12% SDS-polyacrylamide gel and transferred to an immobilon-P PVDF membrane. To prevent non-specific binding with antibodies, the membrane was pretreated in 5% skim milk.
  • the pretreated PVDF membrane was washed, and primary antibodies against CD9, CD63, or CD81 proteins (1:1000, Abcam, UK) were added and reacted overnight. After the reaction was over, the PVDF membrane was washed with TBS-T buffer containing 0.1% Tween-20, treated with a secondary antibody, HRP-conjugated anti-mouse antibody (1:2000, Abcam, UK), and incubated at room temperature. The reaction was again conducted for 2 hours. After the reaction was over, the PVDF membrane was washed with TBS-T buffer, checked with the WestFemto maximum sensitivity substrate kit, and the resulting image was captured with a ChemiDoc Imager (Bio-Rad Laboratories, USA). was analyzed. As a result, the expression levels of CD9, CD63, and CD81 were photographed and the results are shown in Figure 1A.
  • Exosomes present in the exosome-rich culture medium obtained above were quantitatively confirmed using the NTA system (Nanosight NS300, NanoSight, UK). At this time, all analysis samples were diluted 1:1000 to prepare 1.0 ⁇ 10 8 particles/ml. As a result, the content of exosomes is shown in Figure 1B, and the results of observing exosomes using transmission electron microscopy (TEM) are shown in Figure 1C.
  • exosomes were confirmed in the culture medium of amniotic mesenchymal stem cells cultured under conditions of low oxygen concentration, and specifically contained 4.5 ⁇ 10 10 exosomes/ml. Meanwhile, 9.0 ⁇ 10 8 exosomes/ml were confirmed in the amniotic mesenchymal stem cell culture medium cultured under normal conditions.
  • the isolated exosomes had a size of approximately 68 nm.
  • the growth factors and neurotrophic factors present in the exosome-rich culture medium obtained above were analyzed as follows. Specifically, fibroblast growth factor (FGF), elongation factor G (EFG), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), and transforming growth factor (TGF- ⁇ ). - ⁇ ), BDNF (brain-derived neurotrophic factor), and PDGF (platelet-derived growth factor) protein expression levels were confirmed using ELISA analysis according to the manufacturer's protocol. At this time, normal culture medium was used as a control, and the expression levels of the identified growth factors and neurotrophic factors are shown in Figure 2.
  • FGF fibroblast growth factor
  • EGF elongation factor G
  • PDGF platelet-derived growth factor
  • IGF insulin-like growth factor
  • VEGF vascular endothelial growth factor
  • TGF- ⁇ transforming growth factor
  • - ⁇ BDNF (brain-derived neurotrophic factor)
  • Sprague-Dawley rats at 9 weeks of gestation were prepared by rearing at a constant temperature of 23 ⁇ 3°C and relative humidity of 50 ⁇ 10% with a 12-hour light/dark cycle. When reared, they were fed a standard rodent diet and water was provided ad libitum. Pregnant rats gave birth through vaginal delivery, and 5 days later, the left common carotid artery was occluded by surgery, placed in a hypoxia incubator (Don Whitley Scientific, UK), and exposed to 8% O 2 and 92% N 2 for 2 hours. . Thereafter, the sutures of the baby rats were removed to induce reperfusion, and after recovery for 30 minutes, 1 mg/kg LPS was injected intraperitoneally to induce inflammation.
  • Exosome-rich culture medium containing 10 ⁇ 10 12 particles was mixed with 2 ⁇ l of ExoGlowTM-Vivo fluorescence dye and reacted at room temperature for 2 hours to determine the presence of exosome-rich culture medium.
  • the exosomes were labeled.
  • the reactant was filtered in a conventional manner using a column filled with Sepharose CL-6B to remove the fluorescent dye that was not labeled in the exosomes.
  • Seven days after birth 4.5 ⁇ 10 10 pn of exosome-rich culture medium was administered to the baby rat in which cerebral palsy was induced in Experimental Example 2-1 through the tail vein. At this time, normal rats were used as a control group.
  • Brain extraction was performed after completely anesthetizing the rat and perfusing the whole body with PBS through the heart for 5 minutes.
  • the fluorescence intensity from the extracted brain was measured at a wavelength in the near-infrared range (excitation: 784 nm, emission: 806 nm), and the measured results are shown in Figure 3A, and the results of AUC analysis using the results are shown in Figure 3B.
  • pictures of the fluorescence intensity were shown in Figures 4 and 5.
  • the F3 cell line a human neural stem cell line
  • DMEM Dulbecco's modified Eagle's medium, Biowest, France
  • penicillin and streptomycin as antibiotics
  • 10% heat-inactivated FBS at 37°C and 5% CO 2 conditions. It was cultured in .
  • the prepared F3 cell line was dispensed into a culture dish with a diameter of 10 cm and cultured for 24 hours, and then the cultured cells were washed with PBS.
  • Washed cells were treated with 10 ⁇ g/ml of lipopolysaccharide (LPS) or potassium cyanide (KCN) at 5 ⁇ M, and simultaneously treated with 10, 30, or 100 ⁇ g/ml of exosome-rich culture medium. At this time, the concentration of exosomes was confirmed using the commonly used BCA method. After culturing the cells for 24 hours, the content of LDH (lactate dehydrogenase) released into the culture medium was confirmed using an LDH assay kit (Promega, USA) according to the manufacturer's protocol. Meanwhile, the mRNA expression levels of inflammatory factors were confirmed by performing qRT-PCR using a conventional method, and the sequences of the primers used are shown in Table 1 below.
  • LDH lactate dehydrogenase
  • Bax protein which is related to apoptosis
  • Bcl-2 a protein that inhibits apoptosis
  • AKT and PI3K proteins related to cell differentiation
  • the protective effect of the exosome-rich culture medium obtained above on oligodendrocyte progenitor cells from damage caused by prenatal factors or hypoxia was confirmed by the following method.
  • the experiment was performed in the same manner as Experiment 3 above, except that the F3.Olig2 cell line was used instead of the F3 cell line.
  • the results of confirming the concentration of LDH and mRNA expression of inflammatory factors are shown in Figures 10 and 11, and the results of confirming the expression of proteins related to apoptosis or differentiation are shown in Figures 12 to 14.
  • the concentration of LDH and the mRNA expression of inflammatory factors which were significantly increased by treatment with LPS or KCN, were suppressed in a treatment concentration-dependent manner in the exosome-rich culture medium.
  • Bax protein which is associated with apoptosis, significantly increased by treatment with LPS or KCN, was suppressed by exosome-rich culture medium
  • Bcl-2 a protein that inhibits apoptosis, was expressed in exosomes. Expression was increased by treatment with worm-rich cultures.
  • AKT and PI3K proteins related to cell differentiation, was also significantly increased by treatment with exosome-rich culture medium.
  • CNPase (2'3'-cyclic-nucleotide 3'-phosphodiesterase) and MBP (myelin basic protein) proteins, which are proteins related to the maturation of oligodendrocytes, increased upon treatment of LPS or KCN.
  • MBP myelin basic protein
  • an exosome-rich culture medium was administered into the tail vein of a baby rat in which cerebral palsy was induced through the procedure. At this time, in the case of one-time administration, the dose was 1.5 was administered. Then, on the 10th, 20th, 30th or 40th day after birth of the baby rat, the rat was placed in a glass cylinder with a diameter of 21 cm and a length of 34 cm and the front paw was in contact with the wall of the carrier for 3 minutes. If the weight bearing was on the left paw, Normal, if caught on the right side, it was recorded as damaged. From the above results, foot preference (%) was calculated using Equation 1 below, and the results are shown in Figure 15.
  • rats with induced brain damage had a decreased preference for using the forelimb opposite to the brain injury area, but this was significantly recovered due to the administration of exosome-rich culture medium, and both forelimbs were used on the 20th to 40th day after birth. was used in similar proportions.
  • the animal model administered the exosome-rich culture medium was placed on a rod rotating at a constant speed of 12 rpm, and the time it maintained balance on the rod was recorded. Recordings were performed on the 10th, 20th, 30th, or 40th day after birth of the baby rats, and were repeated three times, and the average values are shown in Figure 16.
  • the animal model administered the exosome-rich culture medium was tested once a day for 5 days starting on the 41st day after birth using a passive avoidance box (Med Associated). Specifically, the animal was placed in the bright room of a passive avoidance box divided into a dark room and a bright room, and when the animal moved to the dark room, an electric shock was applied at 1 mA for 2 seconds. Afterwards, in the same test, memory was evaluated by measuring the time the animal stayed in the bright room to confirm the memory caused by the electric shock in the dark room. At this time, a residence time of 300 seconds was evaluated as complete recovery of memory, and the evaluated results are shown in Figure 17A.
  • a passive avoidance box Med Associated
  • rats with induced brain damage had a reduced residence time in the bright room, but this was recovered by administration of exosome-rich culture medium.
  • a water maze test was performed once a day for 5 days starting from day 41 of birth using an animal model administered with an exosome-rich culture medium.
  • a hidden platform device fills a circular tank with water to a depth of 27 cm, maintains the temperature at 22 ⁇ 2°C, and fills the water surface with Styrofoam to cover the refuge platform 2 cm below the water surface. , 10 cm in diameter, 25 cm in height) was installed.
  • a baby rat was placed in a water tank, and the time required to memorize the markers around the tank and find a platform located at a certain location in the tank was measured. As a result, the time required to find the platform is shown in Figure 17B.
  • the brain tissue of the animal model administered the exosome-rich culture medium was sacrificed and obtained after the experiment was completed, and changes in gene expression of inflammatory factors in the obtained brain tissue were confirmed.
  • the experiment was performed in the same manner as Experiment 3 above, except that rat brain tissue was used instead of the F3 cell line, and in this case, the primers listed in Table 2 below were used. As a result, the results of confirming the mRNA expression of inflammatory factors are shown in Figure 18.
  • the mRNA expression of inflammatory factors was significantly increased in the brain tissue of rats induced with cerebral palsy, but this was significantly suppressed by the administration of exosome-rich culture medium.
  • the exosome-rich culture medium was administered three times repeatedly, the mRNA expression of inflammatory factors was suppressed to normal levels.
  • the brain tissue of the animal model administered the exosome-rich culture medium was sacrificed and obtained after the experiment was completed, and changes in protein expression in the obtained brain tissue were confirmed by Western blot.
  • the experiment tested Bax, Bcl-2, p-AKT, p-PI3K, PDGFR A, SOX10, Nkx2.2, Oligo2, NT3 (neurotrophin-3), NGF (nerve growth factor), BDNF, CNTF (ciliary neurotrophic factor), glial cell-derived growth factor (GDNF), PDGF A, platelet-derived growth factor B (PDGF B), CNPase, MBP, or actin protein. It was performed in the same manner as described above. The results of confirming the expression changes of the above proteins are shown in Figures 19 to 23.
  • neurotrophic factors NT3, NGF, BDNF, CNTF, GDNF, PDGF A, and PDGF B proteins were also significantly increased by treatment with exosome-rich culture medium.
  • the brain tissue of the animal model administered the exosome-rich culture medium was sacrificed and obtained after the experiment was completed.
  • the obtained brain tissue was perfused with a 10% paraformaldehyde solution and then placed in the same solution and fixed for 48 hours.
  • the fixed brain tissue was cryoprotected for 72 hours using a 30% sucrose solution, and the brain tissue was cut into 10 ⁇ m thick cross sections to obtain samples.
  • the obtained sample was treated with LFB dye, and images were observed and photographed under a microscope, and the results are shown in FIG. 24.
  • the concentration of myelin was significantly decreased due to the induction of cerebral palsy, but this was recovered by administration of exosome-rich culture medium. In particular, when exosome-rich culture medium was administered three times, it recovered to normal levels.
  • brain tissue was cryoprotected by the method described in Experimental Example 8, and a coronal cryosection was obtained by cutting at a thickness of 10 ⁇ m at a position of 1.0 mm from bregma.
  • the obtained fragments were washed with TBS (Tris-buffered saline) buffer and treated with 3% hydrogen peroxide for 5 minutes to inhibit endogenous peroxidase activity.
  • the sections were washed again with TBS buffer and pretreated with 5% BSA.
  • the pretreated sections were treated with a primary antibody (1:300, rabbit polyclonal antibody, Chemicon, USA) that specifically binds to PDGFR and reacted at 4°C overnight.
  • PDGFR protein As shown in Figures 25 and 26, the expression of PDGFR protein was decreased in rats with cerebral palsy, but this was recovered by administration of exosome-rich culture medium. Additionally, PDGFR-positive cells also had high expression of SOX10 and Oligo2 proteins.

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Abstract

La présente invention concerne une composition pharmaceutique pour la prévention ou le traitement de maladies du système nerveux cérébral, comprenant, en tant que principes actifs, une culture de cellules souches et des vésicules extracellulaires isolées à partir de celle-ci. En particulier, lorsqu'ils sont administrés in vivo, la culture de cellules souches et les vésicules extracellulaires isolées à partir de celles-ci, de la présente invention, se déplacent vers des tissus cérébraux de façon à protéger les cellules souches neurales et les oligodendrocytes, à supprimer l'expression de facteurs inflammatoires, à favoriser la différenciation et la maturation des oligodendrocytes et à atténuer les symptômes cliniques dans un modèle animal paralysant cérébral, et peuvent ainsi être efficacement utilisés dans le traitement de maladies du système nerveux cérébral.
PCT/KR2023/016456 2022-10-24 2023-10-23 Composition pharmaceutique pour la prévention ou le traitement de maladies du système nerveux cérébral, comprenant, en tant que principes actifs, une culture de cellules souches et des vésicules extracellulaires isolées de celle-ci Ceased WO2024090922A1 (fr)

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Citations (5)

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Publication number Priority date Publication date Assignee Title
KR20130019356A (ko) * 2011-08-16 2013-02-26 사회복지법인 삼성생명공익재단 줄기세포 유래 미세소포를 포함하는 신경 생성 촉진용 조성물
KR20150069554A (ko) * 2013-12-12 2015-06-23 사회복지법인 삼성생명공익재단 줄기세포 유래 엑소좀을 유효성분으로 포함하는 뇌혈관 질환 치료용 약학적 조성물
WO2018226758A2 (fr) * 2017-06-05 2018-12-13 The Regents Of The University Of California Procédés de production et d'isolement améliorés de vésicules d'origine cellulaire et de traitement d'une inflammation et d'un dommage neurologique
US20190008902A1 (en) * 2015-12-30 2019-01-10 The Regents Of The University Of California Methods for enhanced production and isolation of cell-derived vesicles
KR20200142045A (ko) * 2018-04-10 2020-12-21 브레인스톰 셀 세라퓨틱스 리미티드 세포 타입 특이적인 엑소좀 및 그 용도

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113395966A (zh) 2018-12-13 2021-09-14 奥斯佩克斯医药公司 用于治疗脑瘫中的运动障碍的方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20130019356A (ko) * 2011-08-16 2013-02-26 사회복지법인 삼성생명공익재단 줄기세포 유래 미세소포를 포함하는 신경 생성 촉진용 조성물
KR20150069554A (ko) * 2013-12-12 2015-06-23 사회복지법인 삼성생명공익재단 줄기세포 유래 엑소좀을 유효성분으로 포함하는 뇌혈관 질환 치료용 약학적 조성물
US20190008902A1 (en) * 2015-12-30 2019-01-10 The Regents Of The University Of California Methods for enhanced production and isolation of cell-derived vesicles
WO2018226758A2 (fr) * 2017-06-05 2018-12-13 The Regents Of The University Of California Procédés de production et d'isolement améliorés de vésicules d'origine cellulaire et de traitement d'une inflammation et d'un dommage neurologique
KR20200142045A (ko) * 2018-04-10 2020-12-21 브레인스톰 셀 세라퓨틱스 리미티드 세포 타입 특이적인 엑소좀 및 그 용도

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