WO2019004738A2 - Utilisation d'une composition comprenant un exosome dérivé de cellules souches adipeuses en tant que principe actif pour atténuer la dermatite - Google Patents

Utilisation d'une composition comprenant un exosome dérivé de cellules souches adipeuses en tant que principe actif pour atténuer la dermatite Download PDF

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WO2019004738A2
WO2019004738A2 PCT/KR2018/007326 KR2018007326W WO2019004738A2 WO 2019004738 A2 WO2019004738 A2 WO 2019004738A2 KR 2018007326 W KR2018007326 W KR 2018007326W WO 2019004738 A2 WO2019004738 A2 WO 2019004738A2
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Prior art keywords
skin
dermatitis
exosome
present
exosomes
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PCT/KR2018/007326
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English (en)
Korean (ko)
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WO2019004738A3 (fr
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이용원
조병성
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주식회사 엑소코바이오
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Priority claimed from KR1020180018617A external-priority patent/KR20190003316A/ko
Application filed by 주식회사 엑소코바이오 filed Critical 주식회사 엑소코바이오
Priority to JP2019571619A priority Critical patent/JP6970459B2/ja
Priority to ES18824926T priority patent/ES2978498T3/es
Priority to CN201880054562.0A priority patent/CN111148520B/zh
Priority to EP18824926.2A priority patent/EP3639832B1/fr
Publication of WO2019004738A2 publication Critical patent/WO2019004738A2/fr
Publication of WO2019004738A3 publication Critical patent/WO2019004738A3/fr
Priority to US16/727,739 priority patent/US11612621B2/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin

Definitions

  • the present invention relates to a composition for improving dermatitis in a composition
  • a composition for improving dermatitis in a composition
  • a composition comprising an exosome derived from adipose stem cells as an active ingredient.
  • the present invention also relates to a pharmaceutical composition for preventing, improving, alleviating or treating dermatitis, an external preparation for skin and a cosmetic composition containing the composition.
  • the present invention can obtain a large amount of exosomes derived from adipose stem cells having a high purity and a uniform particle size distribution that can be clinically applicable in the prevention, improvement, alleviation or treatment of dermatitis,
  • This invention relates to a clinically and commercially superior technique capable of providing a large amount of a composition containing an exosome derived from an adipose stem cell, which is excellent in activity, as an effective ingredient at a low cost.
  • the skin of the human body is an organ that physically and chemically protects the body from the outside and performs the biochemical functions necessary for the metabolism of the whole body.
  • inflammatory skin diseases that appear on human skin are called dermatitis.
  • Relatively common dermatitis includes atopic dermatitis, contact dermatitis, and seborrhoic dermatitis.
  • Contact dermatitis is a dermatitis caused by contact with foreign substances. Contact dermatitis is characterized by irritant contact dermatitis and allergic contact dermatitis, depending on the mechanism by which one substance can cause both of these reactions simultaneously. Most of the substances that cause contact dermatitis are organic compounds. When the dermatitis-inducing substance comes into contact with the skin sensitized to these substances, it is known that the memory cell senses it and secretes various chemicals to cause inflammation.
  • Atopic dermatitis is a chronic inflammatory skin disease characterized by itchy and eczematous lesions. According to the results of previous studies, it is known that various factors are involved in the onset of atopic dermatitis. When atopic dermatitis is induced, inflammatory cells such as eosinophil, neutrophil and mast cell are observed in the lesion and immunoglobulin IgE produced from mast cells increases. In addition, IL-4, IL-5, and IL-13, which are inflammatory cytokines, are amplified and amplified by this process.
  • TSLP Thimic stromal lymphopoietin
  • seborrheic dermatitis is a chronic inflammatory skin disease that occurs due to increased activity of sebaceous gland, which occurs in the scalp and face, especially around eyebrows, nose, lips, ears, underarms, chest, groin and the like.
  • Drug remedies developed to date include steroids, antihistamines, and immunosuppressants such as cyclosporin A.
  • these drugs have serious problems such as skin atrophy, vasodilation, pigment desalination, hypersensitivity of injection site, tolerance and neutropenia.
  • these medicines have limitations that can help control symptoms to an adequate level rather than the underlying treatment.
  • Embryonic stem cells or embryonic stem cells derived from embryonic stem cells are excellent in their ability to differentiate and regenerate and have a low rejection rate. However, they can not be applied to clinical practice due to ethical problems, and there is a risk of forming tumors.
  • skin regeneration and treatment methods using adult stem cells have been proposed. However, when adult adult stem cells use an adult adult stem cell, there is a risk of causing graft-versus-host disease. In order to treat using adult stem cells, There is a complicated and costly problem in that stem cells are collected and cultured.
  • exosomes which have intercellular signal transduction
  • researches on its components and functions are actively under way.
  • Extracellular vesicles Cells release various membrane-type vesicles in the extracellular environment, and these release vesicles are commonly referred to as extracellular vesicles (EVs).
  • the extracellular endoplasmic reticulum is sometimes referred to as cell membrane-derived endoplasmic reticulum, ectosomes, shedding vesicles, microparticles, exosomes and, in some cases, differentiated from exosomes.
  • Exosome is an endoplasmic reticulum of several tens to several hundreds of nanometers in size, composed of double lipid membranes identical to the cell membrane structure, and contains proteins, nucleic acids (mRNA, miRNA, etc.) called exosomal cargo inside.
  • Exosomal cargo contains a wide range of signaling factors, which are known to be specific for cell types and differentially regulated by the environment of the secretory cells.
  • Exosome is an intercellular signaling mediator that is secreted by the cell, and various cell signals transmitted through it regulate cell behavior including activation, growth, migration, differentiation, de-differentiation, apoptosis, and necrosis of target cells It is known.
  • Exosomes contain specific genetic material and bioactivity factors depending on the nature and condition of the derived cells.
  • the proliferating stem cell-derived exosomes regulate cell behavior such as cell migration, proliferation and differentiation, and reflect the characteristics of stem cells involved in tissue regeneration (Nature Review Immunology 2002 (2) 569-579).
  • the present inventors have sought to develop a therapeutic agent that is superior in efficacy and safety compared to a conventional dermatitis treating agent in relation to dermatitis accompanied with itching and inflammation. Accordingly, the inventors of the present invention have made intensive studies on the new use of exosomes derived from adipose stem cells, and found that exosome isolated from adipose stem cell culture liquid solves the above-mentioned problems of stem cell itself or stem cell culture solution safety And is effective for preventing, improving, alleviating or treating dermatitis. Thus, the present invention has been completed.
  • An object of the present invention is to provide a composition for improving dermatitis of a composition comprising an exosome derived from adipose stem cells as an active ingredient.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing, improving, alleviating or treating dermatitis, an external preparation for skin and a cosmetic composition containing the composition.
  • the present invention provides a composition for preventing, improving, alleviating or treating dermatitis, which comprises an exosome derived from adipose stem cells as an active ingredient.
  • the present invention can obtain a large amount of exosomes derived from adipose stem cells having a high purity and a uniform particle size distribution that can be clinically applicable in the prevention, improvement, alleviation or treatment of dermatitis,
  • the present invention provides a novel clinical and commercial superior technique which can provide a large amount of a composition containing the exosome derived from an excellent adipose stem cell as an effective ingredient at a low cost.
  • exosomes refers to an endoplasmic reticulum of several tens to several hundred nanometers (preferably about 30 to 200 nm) in size, consisting of double lipid membranes identical in structure to the cell membrane
  • the particle size of the exosome may be variable depending on the type of stem cells to be separated, the method of isolation and the method of measurement) (Vasiliy S. Chernyshev et al., "Size and shape characterization of hydrated and desiccated exosomes", Anal Bioanal Chem, ) DOI 10.1007 / s00216-015-8535-3).
  • Exosomes contain proteins called exosomal cargo (cargo), nucleic acids (mRNA, miRNA, etc.).
  • Exosomal cargo contains a wide range of signaling factors, which are known to be specific for cell types and differentially regulated by the environment of the secretory cells.
  • Exosome is an intercellular signaling mediator that is secreted by the cell, and various cell signals transmitted through it regulate cell behavior including activation, growth, migration, differentiation, de-differentiation, apoptosis, and necrosis of target cells It is known.
  • iontophoresis refers to a method in which an ionized active ingredient is allowed to permeate through the skin by an electrical repulsive force by applying a minute current to the skin to which the effective substance is applied, .
  • the iontophoresis used in one embodiment of the present invention is a method in which a current flows from an external power source to an electrode patch on the skin to introduce minute current into the skin, A method in which minute current is introduced, a method in which minute current is introduced into skin through a patch equipped with a reversed electrodialysis means for generating current through a difference in ion concentration between a high concentration electrolyte solution and a low concentration electrolyte solution, and the like .
  • the present invention is not limited thereto, and it is needless to say that various types of iontophoresis can be used.
  • exosomes derived from adipose stem cells Limited wrinkle improvement and skin regeneration effects using exosomes derived from adipose stem cells have been reported. Although exosomes derived from neural stem cells, which are not adipose stem cells, have been reported to be effective in the treatment of brain damage and inflammatory diseases due to rejection of stem cell transplantation, the use of exosomes isolated from the adipose- Treatment of dermatitis, etc. " has not been known.
  • Adipose-derived stem cells derived from fat can be obtained in large quantities by simple procedures such as liposuction, and about 40 times more stem cells than bone marrow, umbilical cord or umbilical cord blood, It is difficult to economically mass-separate exosomes having a high purity and uniform particle size distribution from the adipose-derived stem cell culture liquid because the fat is abundant in the fat, such as cellular debris, waste products, proteins and macromolecules. Therefore, in terms of economics, there were technical barriers to massive separation of exosomes having a high purity particle size distribution from the adipose stem cell culture fluid.
  • the exosome derived from adipose stem cells contained as an active ingredient has a significant effect on the prevention, improvement, alleviation or treatment of dermatitis,
  • composition for preventing, improving, alleviating or treating dermatitis contains exosome derived from adipose stem cells as an active ingredient.
  • the exosome can be obtained by performing the following steps: (a) adding trehalose to the adipose stem cell culture, (b) adding the trehalose (C) separating the exosomes from the filtered adipose stem cell culture liquid using TFF (Tangential Flow Filtration), and (d) separating the exosomes with diafiltration ) Was added to the buffer solution to be used for the separation of the exosomes, and the desalted and exfoliated diafiltration was performed using TFF (Tangential Flow Filtration) using the buffer solution to which the trehalose was added. ).
  • trehalose is added to the buffer solution used for desalting and diafiltration in the step (d), whereby the exosome having a uniform particle size distribution and high purity can be effectively (See Figs. 6A to 6E).
  • trehalose in the present invention imparts a function of efficiently separating exosomes from impurities such as cellular debris, waste products, proteins and macromolecules.
  • desalination and buffer exchange can be performed continuously or intermittently. Desalting and buffer exchange can be carried out using a buffer solution having a volume of at least 4 times, preferably 6 times to 10 times, more preferably 12 times the starting volume.
  • a TFF filter having a molecular weight cutoff (MWCO) of 100,000 Da (Dalton), 300,000 Da, 500,000 Da or 750,000 Da, or a 0.05 ⁇ TFF filter may be used for TFF.
  • the step (c) may further include a step of concentrating the solution to a volume of 1/100 to 1/25 using TFF (Tangential Flow Filtration).
  • the filtered adipose stem cell culture fluid may be subjected to sonication before TFF.
  • the exosome is characterized by decreasing the amount of expression of IL-4 and IL-31 in skin tissue or skin cells. Additionally, the exosomes may reduce the expression level of at least one of IL-23 or TNF-a in skin tissue or skin cells.
  • the exosome can reduce the level of blood IgE and the number of blood leukocytes and eosinophils.
  • the exosome can reduce the number of mast cells, CD86 + cells and CD206 + cells in the dermal tissue.
  • the exosome is characterized by reducing TEWL and increasing skin hydration.
  • the exosome is characterized by reducing TNF- ⁇ mRNA expression, IL-6 mRNA expression, and IL-1 ⁇ mRNA expression in an in vitro assay.
  • the type of adipose stem cells is not limited as long as it does not cause the risk of infection by a pathogen and does not cause an immune rejection reaction, but may be preferably stem cells derived from human adipose.
  • composition of one embodiment of the present invention can be effectively used for preventing, improving, alleviating or treating various dermatoses accompanied by itch, and is preferably used for the treatment of contact dermatitis, irritant contact dermatitis, allergic contact dermatitis, phototoxic and photoallergic contact dermatitis, It can be used for atopic dermatitis, acne or eczema, and more preferably for atopic dermatitis, seborrheic dermatitis, autoimmune dermatitis, autoimmune progesterone dermatitis, ulcerative dermatitis, acne or eczema.
  • composition of one embodiment of the present invention may be prepared from a pharmaceutical composition.
  • the pharmaceutical composition according to one embodiment of the present invention may be various oral or parenteral formulations.
  • the pharmaceutical composition of one embodiment of the present invention may include a pharmaceutically acceptable carrier, excipient or diluent.
  • the carrier, excipient and diluent include lactose, dextrose, trehalose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium carbonate, calcium silicate, cellulose , Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. .
  • composition according to one embodiment of the present invention may be administered orally or parenterally in the form of powders, pills, tablets, capsules, suspensions, emulsions, syrups, granules, elixirs, aerosols, Or in the form of sterile injectable solutions.
  • Administration of the pharmaceutical composition according to one embodiment of the present invention means introduction of a predetermined substance into a patient by any appropriate method, and the administration route of the pharmaceutical composition may be administered through any ordinary route so long as the drug can reach the target tissue .
  • the pharmaceutical composition of one embodiment of the present invention may be administered orally or parenterally, and examples of the parenteral administration include transdermal administration, intraperitoneal administration, intravenous administration, intraarterial administration, intramuscular administration, subcutaneous administration, Administration, topical administration, rectal administration, and the like.
  • the pharmaceutical composition of one embodiment of the present invention may be administered by any device capable of moving the active substance to a target tissue or cell.
  • An effective amount of the pharmaceutical composition of one embodiment of the present invention means an amount required for administration in order to expect the therapeutic effect of the disease.
  • the preparation for parenteral administration of the pharmaceutical composition of one embodiment of the present invention may be a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized preparation, or a left-over preparation.
  • the preparation for parenteral administration of the pharmaceutical composition of one embodiment of the present invention may also be prepared by injection.
  • the injection agent of one embodiment of the present invention may be, but not limited to, an aqueous injection agent, a non-aqueous injection agent, an aqueous suspension injection agent, a non-aqueous suspension injection agent, or a solid injection agent used by dissolving or suspending.
  • Injection agents of one embodiment of the present invention may be administered orally or parenterally depending on the type thereof, for example, distilled water for injection, vegetable oil (for example, peanut oil, sesame oil, camellia oil, etc.), monoglyceride, diglyceride, propylene glycol, camphor, benzoic acid estradiol, , Arsenobenzazole sodium, or streptomycin sulfate, and may optionally contain stabilizers or preservatives.
  • the compounding ratio of the pharmaceutical composition according to one embodiment of the present invention can be appropriately selected depending on the kind, quantity and form of the additional components as described above.
  • the pharmaceutical composition of the present invention may contain about 0.1 to 99% by weight, preferably about 10 to 90% by weight.
  • a suitable dose of the pharmaceutical composition of one embodiment of the present invention may be appropriately determined depending on the kind of disease of the patient, the severity of the disease, the type of the formulation, the formulation method, the age, sex, weight, health condition, diet, Can be adjusted according to the method.
  • the pharmaceutical composition of one embodiment of the present invention when the pharmaceutical composition of one embodiment of the present invention is administered to an adult, it may be administered at a dose of 0.001 mg / kg to 100 mg / kg per day for 1 to several times.
  • composition of one embodiment of the present invention is prepared with an external preparation for skin and / or a cosmetic composition
  • the components commonly used in cosmetics or external skin preparations for example, moisturizers, antioxidants , An oil component, an ultraviolet absorber, an emulsifier, a surfactant, a thickener, alcohols, a powder component, a colorant, an aqueous component, water, various skin nutrients, and the like.
  • the external preparation for skin may be prepared by mixing together conventionally used dermatitis remedy and / or moisturizing agent in addition to exosome derived from adipose stem cells to the extent that the action thereof (suppression of dermatitis and itching) Can be used.
  • the exosome of the present invention may be supported on or mixed with at least one of hydrogel, hyaluronic acid, hyaluronic acid salt (for example, sodium hyaluronate), or hyaluronic acid gel.
  • the kind of the hydrogel is not limited, but it may be a hydrogel obtained by dispersing a gelated polymer in a polyhydric alcohol.
  • the gel polymer is at least one selected from the group consisting of pluronic, purified agar, agarose, gellan gum, alginic acid, carrageenan, cacao gum, xanthan gum, galactomannan, glucomannan, pectin, cellulose, guar gum and locust bean gum
  • the polyhydric alcohol may be at least one selected from the group consisting of ethylene glycol, propylene glycol, 1,3-butylene glycol, isobutylene glycol, dipropylene glycol, sorbitol, xylitol and glycerin.
  • the external preparation for skin and / or the cosmetic composition according to one embodiment of the present invention can be used in the form of, for example, patches, mask packs, mask sheets, creams, tonics, ointments, suspensions, emulsions, pastes, lotions, , Mist, foundation, powder, oil paper, and the like.
  • the external preparation for skin and / or cosmetic composition may be applied or deposited on at least one surface of the patch, mask pack, or mask sheet.
  • the external preparation for skin is made of a cosmetic composition
  • a cosmetic composition it is used for the purpose of preventing, improving, alleviating, or normalizing dermatitis.
  • Cosmetic formulations can be prepared in any formulations conventionally produced in the art .
  • a lotion, a mask pack, a mask sheet, a softener a nutritional lotion, a convergent lotion, a nutritional cream, a massage cream, an eye cream, a cleansing cream, an essence, an essence, a cleansing lotion, a cleansing foam
  • the external preparation for skin and / or cosmetic composition includes components commonly used in external preparations for skin and / or cosmetics and includes conventional additives such as antioxidants, stabilizers, solubilizers, vitamins, And may include a carrier. Further, in the respective formulations for the external preparation for skin and / or cosmetic composition, the other ingredients can be mixed and selected without difficulty by the person skilled in the art depending on the kind of the external preparation for skin and / or cosmetic composition or purpose of use.
  • Another embodiment of the present invention provides a cosmetic method for regulating the condition of mammalian skin, except for therapeutic use, using the above cosmetic composition.
  • conditioning of the skin means improvement of the condition of the skin and / or prevention of the condition of the skin, and improvement of the condition of the skin means visual and / Or a tactile perceptible positive change.
  • the cosmetic method of one embodiment of the present invention comprises the steps of (a) applying the cosmetic composition directly onto the skin of a mammal, (b) applying the patch, mask pack or mask sheet coated or deposited with the cosmetic composition to the skin , Or sequentially advancing the above (a) and (b).
  • the cosmetic method of one embodiment of the present invention may further comprise performing iontophoresis by flowing a microcurrent to the skin of the mammal to which the cosmetic composition is applied.
  • the cosmetic method of one embodiment of the present invention may further comprise contacting or attaching the iontophoresis device to the skin of the mammal.
  • the iontophoresis device is composed of a flexible battery, a lithium ion secondary battery, an alkaline battery, a dry battery, a mercury battery, a lithium battery, a nickel-cadmium battery, A mask pack, or a mask sheet on which the at least one battery is mounted.
  • Another embodiment of the present invention provides a method of preventing, ameliorating, alleviating or treating dermatitis, comprising administering to the mammal a therapeutically effective amount of the above pharmaceutical composition.
  • the mammal may be a human, a dog, a cat, a rodent, a horse, a cow, a monkey, or a pig.
  • composition of the present invention reduces the production of various inflammatory cytokines and inflammation-related factors that cause dermatitis and inhibits the activity or involvement of inflammation-related immune cells, thereby preventing, ameliorating, alleviating or treating dermatitis.
  • composition of the present invention can protect and enhance the skin barrier by reducing moisture evaporation of the stratum corneum, improving skin moisturization, and preventing, improving, alleviating or treating dermatitis through the protection and strengthening of the skin barrier.
  • compositions of the present invention act on multimeric cytokine targets that cause dermatitis and thus can be applied broadly to dermatitis caused by various causes and effectively inhibit and alleviate dermatitis.
  • the composition of the present invention can control cytokine targets with different expression patterns depending on the degree of mild, moderate, and severe disease of dermatitis, and thus can be applied to prevent, improve, alleviate or treat mild, moderate or severe dermatitis It is expected.
  • the composition of the present invention is expected to be applicable to the prevention, improvement, alleviation or treatment of acute and chronic dermatitis since the expression patterns of cytokines having different expression patterns can be controlled according to the onset patterns of acute and chronic dermatitis.
  • composition of the present invention can be usefully used as a pharmaceutical composition for preventing, improving, alleviating or treating dermatitis, external preparation for skin and cosmetic composition.
  • the present invention can obtain a large amount of exosome derived from adipose stem cells at a low cost and having a high purity and a uniform particle size distribution. Therefore, the present invention can provide a large amount of a composition containing an exosome derived from adipose stem cells excellent in functional activity as an effective ingredient at low cost.
  • the composition of the present invention is scale-upable and is also suitable for GMP (Good Manufacturing Practice).
  • FIG. 1 is a flow chart illustrating a process of separating and purifying exosomes in a method for producing exosomes from an adipose stem cell culture solution according to one embodiment of the present invention.
  • FIG. 2 shows a result of measuring the relative amount of protein contained in a solution for each step of preparing exosome from an adipose stem cell culture solution according to one embodiment of the present invention.
  • the ratio of the total amount of protein in each step was expressed as a relative ratio of total protein amount to the total stem cell culture solution.
  • the experimental results show the results obtained in two different batches, respectively.
  • FIG. 3 shows the results of measuring the productivity and purity of exosomes obtained according to one embodiment of the present invention.
  • the productivity of exosomes was calculated as "the number of particles of exosome obtained per mL of stem cell culture (CM)" and the purity of exosome was calculated as "the number of particles of exosome per microgram of protein unit contained in the final fraction" Respectively.
  • the experimental results show the results obtained in five different batches.
  • 4A to 4E show results of physical property analysis of exosomes obtained according to one embodiment of the present invention.
  • 4A shows particle size distribution and number of particles by TRPS (tunable resistive pulse sensing) analysis.
  • Figure 4B shows particle size distribution and number of particles by NTA (nanoparticle tracking analysis) analysis.
  • 4C shows particle images by transmission electron microscopy (TEM) according to magnification.
  • 4D shows the Western blot results of exosomes obtained according to one embodiment of the present invention.
  • &Quot; Figure 4E shows flow cytometric analysis results for CD63 and CD81 in marker assays for exosomes obtained according to one embodiment of the present invention.
  • Figures 5A-5C show the results of NTA analysis on particle size distribution showing that a uniform and high-purity exosome is obtained with trehalose addition. As the amount of trehalose added increases, a particle size distribution having a single peak can be obtained.
  • 6A to 6C show results of NTA analysis showing particle size distribution according to whether trehalose was added in the process of producing exosome according to one embodiment of the present invention.
  • 6A shows a case where trehalose is added throughout the production process
  • Fig. 6B shows a case where trehalose is added after the cell culture solution is stored in a frozen state
  • the results are shown without adding os.
  • 6D shows the results of comparing relative productivity and relative concentration of exosomes isolated by the method of FIGS. 6A to 6C.
  • Figure 6E shows the mean size of exosomes isolated by the method of Figures 6A-6C.
  • FIG. 8 shows experimental results of confirming the effect of reducing exogenous NO formation, which is a kind of inflammatory reaction, by exoose according to one embodiment of the present invention.
  • PBS is phosphate-buffered saline
  • DEX is dexamethasone
  • EXO is exosomes
  • CM is conditioned medium
  • CM-EXO is exosomally removed adipose-derived adipose stem cells (Exosome-depeleted conditioned media).
  • FIG. 9 shows experimental results of confirming the effect of reducing exogenous TNF-.alpha., An inflammatory cytokine, by exoose according to one embodiment of the present invention.
  • PBS represents phosphate-buffered saline
  • DEX represents dexamethasone
  • each numeral represents exosome throughput ( ⁇ g / mL).
  • FIGS. 10A to 10C are graphs showing experimental results comparing the effect of reducing NO formation by the separated exosome and the reducing effect of NO formation by the exoose isolated by the conventional precipitation method (PPT) according to one embodiment of the present invention / RTI >
  • FIG. 10A is a result of NTA analysis of exosomes separated by a conventional precipitation method
  • FIG. 10B is a result of NTA analysis of exosomes isolated and purified by the method according to one embodiment of the present invention
  • the degree of reduction of NO formation was expressed as a relative ratio (%) to the degree of NO formation reduction by the positive control dexamethasone (Dex).
  • FIG. 11 shows the results of confirming that atopic symptoms are alleviated as a result of treating the exosome according to one embodiment of the present invention in an atopy-induced mouse (dermatitis-inducing animal model 1).
  • FIG. 12 shows a real-time PCR result in which the amount of inflammatory cytokines IL-4 and IL-31 mRNA in the skin damaged area sample after treating the dermatitis-inducing animal model 1 according to an embodiment of the present invention was examined Graph.
  • FIG. 13 shows the result of treatment of exosome according to one embodiment of the present invention in a mouse in which atopy has been induced (dermatitis-inducing animal model 2) to confirm that atopy symptoms are relieved dose-dependently on exosomes.
  • FIG. 14 is a graph comparing the results of FIG. 13 with numerical values.
  • FIG. 15 is a graph showing changes in the amounts of inflammatory cytokines IL-4, IL-31, TNF-a and IL-23 mRNA in skin damage area samples after treatment with exoemia according to one embodiment of the present invention in dermatitis- And a real-time PCR result.
  • FIG. 17 shows fluorescence intensity measurement results of exosomes stained with PKH67.
  • FIG. 18 is a fluorescence microscope image showing the degree of the fluorescence-stained exosome of the present invention transferred into the pig skin tissue.
  • FIG. It is an image of a fluorescence microscope obtained by diluting a fluorescently-stained exosome of the present invention in a buffer solution and applying it to the surface of a pig skin after a certain period of time.
  • FIG. 19 is a graph showing a fluorescence microscope image obtained by confirming the extent to which the exosome of the present invention is fluorescently transferred into a mouse skin tissue, and a total fluorescence intensity obtained by measuring the fluorescence intensity on each image from the fluorescence microscope image to be.
  • the upper part of FIG. 19 is an image of a confocal fluorescence microscope obtained after a certain time has elapsed after application of the fluoresceinized exosome of the present invention to the surface of a mouse skin by diluting it in a buffer solution.
  • FIG. 20 shows the result of confirming the absence of cytotoxicity after treating exosome according to one embodiment of the present invention to human skin fibroblast HS68 cells.
  • FIG. 21 is a graph showing the results of an ionophore treatment in which a composition containing an exosome according to an embodiment of the present invention is applied to human skin (circles) and then a microcurrent is applied to the skin As a result, it is a photograph showing that the erythema of the skin (the affected part) which has severe dermatitis is remarkably improved.
  • FIG. 22 is a photograph showing that atopic symptoms are significantly improved as a result of subcutaneous injection of exosomes according to one embodiment of the present invention to a Shetland cichlids suffering from naturally occurring severe atopic dermatitis.
  • FIG. 23 is a graph showing that the transepidermal water loss (TEWL) of the exosomes is dose-dependently reduced as a result of treatment with exosomes according to one embodiment of the present invention in mice in which skin barrier damage causing dermatitis is caused .
  • TEWL transepidermal water loss
  • 24 is a graph showing that skin hydration is increased in a dose-dependent manner in exosomes as a result of treatment with exosomes according to one embodiment of the present invention, to be.
  • FIG. 25 is a graph showing a result of measuring body weight of mice suffering from skin barrier damage that causes dermatitis. As shown in FIG. 25, in the dexamethasone-treated group, body weight was decreased due to adverse effects. In the experimental group treated with exosomes according to one embodiment of the present invention, Is almost not decreased.
  • Figure 26 shows that when the exosomes of the present invention were co-treated with LPS and IFN-y for THP-1 derived macrophages obtained after differentiating THP-1 monocytes into macrophages, proinflammatory cytokines Lt; / RTI > mRNA expression of IL-6, which is a human IL-6.
  • FIGS. 27 to 29 show that the THP-1-derived macrophages obtained by differentiating THP-1 monocytes into macrophages were pre-treated with the present exosomes and incubated for a predetermined time, and then treated with LPS and IFN- ⁇ Time PCR results confirming that the expression levels of IL-6, IL-1 [beta] and TNF- [alpha] mRNAs, which are proinflammatory cytokines, are decreased.
  • Mouse macrophage line RAW 264.7 was purchased from Korean Cell Line Bank and cultured.
  • DMEM purchased from ThermoFisher Scientific
  • medium containing 10% fetal bovine serum purchased from ThermoFisher Scientific
  • 1% antibiotic-antimycotics purchased from ThermoFisher Scientific 2 , and 37 ° C.
  • HS68 cells a human dermal fibroblast, were purchased from ATCC and cultured in RPMI 1640 supplemented with 10% fetal bovine serum (purchased from ThermoFisher Scientific) and 1% antibiotic-antimycotics (purchased from ThermoFisher Scientific) The cells were subcultured in DMEM (purchased from ThermoFisher Scientific) medium containing 5% CO 2 at 37 ° C.
  • adipose-derived stem cells were cultured at 5% CO 2 and 37 ° C. Then, the cells were washed with a phosphate-buffered saline (purchased from ThermoFisher Scientific), replaced with serum-free, non-phenol red medium, cultured for 1 to 10 days, and the supernatant .
  • a phosphate-buffered saline purchased from ThermoFisher Scientific
  • Trehalose was added to the culture medium in an amount of 2% by weight in order to obtain an exosome having a uniform particle size distribution and high purity in the process of separating exosome.
  • the culture was filtered with a 0.22 ⁇ m filter to remove impurities such as cellular debris, waste products and large particles.
  • the filtered cultures were immediately separated to isolate exosomes.
  • the filtered culture was stored in a refrigerator (image below 10 ° C) and used for exosome isolation.
  • the filtered culture was frozen in an ultra-low temperature freezer at -60 ° C or lower, and thawed, followed by exosome isolation. Then, the exosomes were separated from the culture medium by using Tangential Flow Filtration (TFF).
  • TMF Tangential Flow Filtration
  • Example 1 a TFF (Tangential Flow Filtration) method was used for separating, concentrating, desalting and diafiltration exosomes from a culture filtrated with a 0.22 ⁇ m filter.
  • TFF method was used to isolate and concentrate the exosomes, sonication of the culture medium to loosen lumps of potential exosomes.
  • a cartridge filter also called a hollow fiber filter (purchased from GE Healthcare) or a cassette filter (purchased from Pall or Sartorius or Merck Millipore) was used.
  • the TFF filter can be selected by a variety of molecular weight cutoffs (MWCO). Exosomes were selectively isolated and concentrated by selected MWCOs, and particles, proteins, lipids, nucleic acids, low molecular weight compounds, etc. smaller than MWCO were removed.
  • MWCO molecular weight cutoffs
  • TFF filters of MWCO 100,000 Da (Dalton), 300,000 Da, or 500,000 Da were used.
  • the culture medium was concentrated to a volume of about 1/100 to 1/25 using the TFF method, and substances smaller than MWCO were removed to separate the exosomes.
  • Separated and concentrated exosomal solutions were further desalted and buffered (diafiltration) using the TFF method.
  • the desalination and buffer exchange are performed by continuous diafiltration or discontinuous diafiltration, and at least 4 times, preferably 6 times to 10 times or more, more preferably, Was performed using a buffer solution having a volume of 12 times or more.
  • 2% by weight of trehalose dissolved in PBS was added to obtain an exosome having a uniform particle size distribution and high purity.
  • 6A to 6E show the effect of obtaining an exosome having a high purity and a uniform particle size distribution with trehalose treatment at a high yield.
  • the amount of protein in the fractions of the isolated exosome, culture medium, and TFF separation process was measured using BCA colorimetry (purchased from ThermoFisher Scientific) or FluoroProfile fluorescence (purchased from Sigma).
  • BCA colorimetry purchased from ThermoFisher Scientific
  • FluoroProfile fluorescence purchased from Sigma.
  • the extent to which the exosome was isolated and concentrated by the TFF method of the present invention and the removal of proteins, lipids, nucleic acids, and low-molecular compounds was monitored by a protein determination method, and the results are shown in FIG. As a result, it was found that the protein present in the culture solution was very effectively removed by the TFF method of one embodiment of the present invention.
  • FIG. 3 shows the results of comparing the productivity and purity in five independent batches when isolating exosomes by the TFF method of one embodiment of the present invention. As a result of analyzing the results obtained from the five independent batches, it was confirmed that the exosome can be separated very stably by the TFF method of one embodiment of the present invention.
  • 5A to 5C show results of NTA analysis of the size distribution of exosome according to whether or not trehalose was added after the exosome was separated by the TFF method.
  • concentration of trehalose was increased to 0% by weight, 1% by weight and 2% by weight (from top to bottom of FIGS. 5A to 5C) and repeated three times.
  • particles having a size of 300 nm or more were identified, while particles having a size of 300 nm or more were reduced by increasing the amount of trehalose, and the size distribution of the exosome was uniformized .
  • FIG. 4D shows the presence of CD9, CD63, CD81 and TSG101 markers as a result of performing Western blotting on isolated exosomes according to the method of one embodiment of the present invention.
  • Anti-CD9 purchased from Abcam
  • anti-CD63 purchasedd from System Biosciences
  • anti-CD81 purchasedd from System Biosciences
  • anti-TSG101 purchasedd from Abcam
  • FIG. 4E shows the presence of CD63 and CD81 markers as a result of analysis using a flow cytometer on exosomes isolated according to the method of one embodiment of the present invention.
  • Human CD63 isolation / detection kit purchased from ThermoFisher Scientific
  • PE-mouse anti-human CD63 PE-Mouse anti markers were stained using the PE-mouse CD63 (purchased from BD) and PE-mouse anti-human CD81 (purchased from BD), and analyzed using a flow cytometer (ACEA Biosciences) Respectively.
  • trehalose is added in the separation and / or purification process using tangential flow filtration to isolate and purify exosomes having high purity and uniform particle size distribution in a high yield in an economical and efficient manner . Also, it can be seen that the processes of the separation method according to one embodiment of the present invention are scalable and suitable for GMP.
  • exosomes were treated by concentration to the cells and the proliferation rate of the cells was confirmed.
  • HS68 cells were suspended in DMEM containing 10% FBS, and then the cells were subcultured to have a confluency of 80 to 90% and cultured in a 5% CO 2 incubator at 37 ° C for 24 hours. After 24 hours, the culture solution was removed, and the cell survival rate was evaluated by culturing the exosome prepared in Example 2 for each concentration and culturing for 24 to 72 hours.
  • WST-1 reagent purchased from Takara
  • MTT reagent purchased from Sigma
  • CellTiter-Glo reagent purchased from Promega
  • Aramar Blue reagent alamarBlue reagent purchased from ThermoFisher Scientific
  • a microplate reader purchased from Molecular Devices
  • the comparison group was based on the number of cells cultured in the normal cell culture medium without the treatment of exosome, and it was confirmed that the exosome-induced cytotoxicity did not appear within the tested concentration range (FIG. 20).
  • RAW 264.7 cells were suspended in DMEM medium containing 10% FBS and dispensed into each well of a multiwell plate to have a confluency of 80-90%.
  • the exosomes of the present invention diluted in a fresh serum-free medium containing LPS (exosome prepared in Example 2) were cultured for 1 to 24 hours at an appropriate concentration.
  • the cultured supernatant was taken and NO and inflammatory cytokines present in the culture were measured to confirm the inflammatory response.
  • the inflammatory response in the culture medium was measured using a NO detection kit (purchased from Intron Bio or Promega).
  • the ELISA kit (purchased from the R & D system) was performed according to the manufacturer's manual to confirm the amount of inflammatory cytokine TNF- ⁇ in the group treated with LPS alone and the group treated with exosomes of the present invention.
  • Dexamethasone (purchased from Sigma) was treated as a positive control.
  • cDNA was prepared from the total RNA obtained from the RAW 264.7 cells treated as described above, and the amount of mRNA change of iNOS, TNF- ⁇ , IL-6 and IL-1 ⁇ was measured using a real-time PCR method.
  • the GAPDH gene was used as a standard gene for quantifying the above genes.
  • the types and sequences of the primers used in the real-time PCR are shown in Table 1 below.
  • the exosome of the present invention has an activity of reducing the inflammatory reaction induced by LPS, which is useful for preventing, improving, alleviating or treating dermatitis, and the exosome of the present invention is useful for preventing, , Can be usefully utilized as an effective ingredient of a composition for alleviation or treatment.
  • exosomes isolated by the conventional precipitation method were prepared in addition to the exosomes obtained by the TFF separation purification of one embodiment of the present invention.
  • the precipitation method was performed according to the protocol of the manufacturer (System Biosciences).
  • the exosome (see Fig. 10A) isolated by the conventional precipitation method has a low uniformity of the particle size distribution and a large particle size distribution (see Fig. 10B) as compared with the exosome (see Fig. 10B) isolated and purified by the TFF method of one embodiment of the present invention Respectively.
  • FIG. 10A The exosome isolated by the conventional precipitation method has a low uniformity of the particle size distribution and a large particle size distribution (see Fig. 10B) as compared with the exosome (see Fig. 10B) isolated and purified by the TFF method of one embodiment of the present invention Respectively.
  • the exosomes obtained according to the separation method of one embodiment of the present invention have improved performance or functional activity (e. G., Uniformity of particle size distribution, inhibition of NO production, Reduction of inflammatory response, etc.), and the composition of the present invention, containing the stem cell-derived exosome having excellent functional activity as an active ingredient, is far superior to the conventional art in terms of preventing, improving, alleviating or treating dermatitis .
  • performance or functional activity e. G., Uniformity of particle size distribution, inhibition of NO production, Reduction of inflammatory response, etc.
  • Example 7 Dermatitis inducing animal model 1
  • mice Male NC / Nga mice (16-18 g, 5 weeks old; purchased from the central laboratory animal) were purchased and used for this experiment through a 7-day adaptation period. The mice that had undergone adaptation were classified into 5 groups as follows after induction of dermatitis.
  • Example 2 Dermatitis induced by house dust mite extract. Exosome prepared in Example 2 was administered intravenously (IV) for 2 weeks three times a week at a dose of 2.8 ⁇ g per subject
  • SC Exposure prepared in Example 2 after inducing dermatitis by house dust mite extract was subcutaneously injected (SC: subcutaneous injection) for 2 weeks three times a week at a dose of 2.8 ⁇ g per individual
  • the pinna portion of the NC / Nga mouse (purchased from the central laboratory animal) was shaved with a razor, and then the hair removal agent was applied in a proper amount to remove hair.
  • the hair removal agent was wiped off and the AD-inducing reagent (house dust mite extract; purchased from BioStir Inc.) was uniformly applied to the pinna area using a micropipette tip. If necessary, 150 ⁇ L of a 4% SDS aqueous solution was uniformly applied to the pinna portion of the pinna using a micropipette tip. Dried using a drier in a cold air, and then dried naturally for about 2-3 hours. Then, the AD-inducing reagent was uniformly applied to a pinna portion using a micro pipette tip. All pretreatments were carried out twice a week, six times a total of three weeks.
  • the skin clinical index was evaluated before starting the administration of exosome prepared in Example 2, and the groups were randomly distributed so that the average score of each group was uniformly distributed according to the ranking score.
  • FIG. 11A are photographs showing atopy symptom relief by atopy-induced mouse treatment (No treatment) and exosome treatment (IV and SC) according to one embodiment of the present invention.
  • FIG. 11B is a graphical representation of the atopic clinical scores of the experimental groups (2) to (5), and it was confirmed that the atopic clinical score was improved in the group treated with exosomes according to one embodiment of the present invention.
  • FIG. 11C is a graph showing relative changes in the thickness of the ear measured in each of the experimental groups (2) to (5) compared to the normal group of (1), wherein the exosome according to one embodiment of the present invention The ear thickness was reduced in one group.
  • dermatitis was induced as in Example 7 and classified into the following 9 groups.
  • Control dermatitis-induced group: a negative control group (referred to as "C" in FIG. 13)
  • SC subcutaneous injection
  • SC, M exosome, medium: exonome prepared in Example 2 after induction of dermatitis by house dust mite extract was subcutaneously injected (SC) three times a week for 4 weeks at a dose of 1.4 ⁇ g per subject
  • SC, H exonome prepared in Example 2 after induction of dermatitis by house dust mite extract was subcutaneously injected (SC) three times a week for 3 weeks at a dose of 10 ⁇ g per subject
  • the dermatitis induction proceeded as in Example 7, and an excess of AD-inducing reagent was applied so that the average skin clinical index was 9 at the time of administration of exosome.
  • the skin clinical index was evaluated before starting the administration of exosome prepared in Example 2, and the groups were randomly distributed so that the average score of each group was uniformly distributed according to the ranking score.
  • FIG. 14A is a graph illustrating the relative improvement of the atopic clinical scores of the experimental groups (2) to (9), wherein the groups treated with exosomes (IV and SC) according to one embodiment of the present invention were dose- And atopic clinical scores were improved.
  • FIG. 14B is a graph showing the thickness of the ear skin tissue measured in each experimental group of (2) to (9) in comparison with the normal group of (1), which is a group treated with exosomes according to one embodiment of the present invention (IV and SC) showed a dose-dependent decrease in the thickness of the ear skin tissue.
  • the ear skin tissue of the sacrificed mice was stained with toluidine blue, and the degree of infiltration of mast cells, which is a kind of inflammatory cells, was measured.
  • 14C is a graph showing the number of mast cells measured in each of the experimental groups (2) to (9) in comparison with the normal group of (1), wherein the group treated with exosomes according to one embodiment of the present invention And SC) showed a dose-dependent decrease in the influx of mast cells.
  • CD86 + cell numbers and CD206 + cell counts were measured after immunohistochemical staining with anti-CD86 antibody and anti-CD206 antibody (Abcam, Cambridge, MA) in ear skin tissue sections of sacrificed mice.
  • 14D and 14E are graphs showing the number of CD86 + cells and the number of CD206 + cells measured in each of the experimental groups (2) to (9) in comparison with the normal group of (1) The number of CD86 + cells and CD206 + cells were decreased in a dose-dependent manner in the group treated with mosses (IV and SC).
  • CDNA was prepared from the total RNA obtained by grinding the tissues of skin injured parts of the sacrificed mice, and the amount of various inflammatory cytokine mRNAs, which is a major cause of dermatitis, was measured using a real time PCR method.
  • GAPDH gene was used as a standard gene for quantifying IL-4, IL-31, IL-23 and TNF-alpha genes.
  • the types and sequences of the primers used in the real-time PCR are shown in Table 2 below.
  • the group treated with the exosomes of the present invention decreased the mRNA expression levels of IL-4, IL-31, TNF- ⁇ and IL-23 in a dose-dependent manner compared to the control and prednisolone- Related cytokines are a major target for development of dermatitis-related therapeutics. Decreased expression levels for these multiple targets are associated with inhibition and mitigation of dermatitis.
  • IL-4 initiates isotype class switching to IgE and activates eosinophils.
  • IL-31 affects isotype class switching to IgE, inflammatory cells aggregate into the skin, and increased IL-31 is associated with exacerbation of dermatitis.
  • IL-23 differentiates ThO-type T-cells into pathogenic helper T-cells that produce TNF- ⁇ , and TNF- ⁇ is known to be associated with exacerbation of dermatitis when plasma concentrations are high.
  • the exosomes of the present invention act on a variety of inflammatory cytokine targets (i.e. IL-4, IL-31, IL-23, and TNF-a) causing dermatitis, Is expected to be able to effectively and effectively inhibit and alleviate dermatitis.
  • IL-4 inflammatory cytokine targets
  • IL-31 IL-31
  • IL-23 IL-23
  • TNF-a inflammatory cytokine targets
  • IgE immunoglobulin E
  • the number of blood cells was measured using whole blood of sacrificed mice. A certain amount of whole blood cells were centrifuged at 1,000 rpm for 10 minutes using Saito Spin (purchased from ThermoFisher Scientific), slide-dried, and then subjected to Diff-Quik staining. Then, white blood cells and eosinophils was measured. Through these experiments, it was confirmed that the number of leukocytes and eosinophils in blood was reduced in a dose-dependent manner in the group treated with exosome prepared in Example 2 (FIG. 16B and FIG. 16C).
  • the composition of the present invention not only reduces the serum IgE level, which is an inflammatory reaction factor that causes dermatitis, but also decreases the number of blood leukocytes and eosinophils and also produces various inflammatory cytokines and inflammation- And inhibits the activity or involvement of inflammation-related immune cells.
  • it can be usefully used as a pharmaceutical composition for preventing, improving, alleviating or treating dermatitis, an external preparation for skin, and a cosmetic composition.
  • Example 11 Animal model causing skin barrier damage causing dermatitis
  • dermatitis symptoms can be ameliorated, alleviated or treated.
  • Oxazolone is used to establish an animal model in which skin barrier damage caused by dermatitis is caused. Oxazolone causes impairment of skin barrier function when applied to the skin. It causes decrease of moisture of skin stratum corneum caused by increase of transepidermal water loss (TEWL), decrease of expression of loricrin, involucin tin, It is possible to provide an animal model with impaired skin barrier function that causes an increase in the pH of the stratum corneum and causes dermatitis (Journal of Investigative Dermatology (2008) 128 (1), 79-86).
  • TEWL transepidermal water loss
  • loricrin loricrin
  • involucin tin involucin
  • mice Female SKH-1 mice (5 weeks old; purchased from the central laboratory animal) were purchased and used for this experiment through a 7-day adaptation period. The mice that had undergone adaptation were divided into 6 groups as shown below after inducing skin barrier damage.
  • Control skin barrier impairment group: negative control which caused skin barrier damage by oxazolone (denoted by " C " in FIG. 23);
  • Exosomal low dose Exosome prepared in Example 2 after inducing skin barrier damage with oxazolone was subcutaneously injected (SC: subcutaneous injection) at a dose of 1 ⁇ g per subject for 4 weeks (FIG. 23 Quot; L ");
  • Dexamethasone An experimental group (positive control) (100 mg / d) in which 0.03% dexamethasone (purchased from Sigma) dissolved in ethanol after inducing skin barrier damage with oxazolone was subcutaneously administered for 4 weeks three times a week Quot; D ").
  • the average moisture evaporation of the skin was measured using a TEWL measuring instrument and the skin moisture content was measured using a moisture meter (Corneometer CM825) (Courage-Khazaka eletronic GmbH, Germany) .
  • the composition comprising the exosome of the present invention as an active ingredient can reduce moisture evaporation of the stratum corneum and improve skin moisturization, thereby protecting and strengthening the skin barrier. Through the protection and strengthening of the skin barrier, It can alleviate or cure.
  • Example 12 Skin permeability test of exosome of the present invention
  • PKH67 dye (purchased from Sigma) was used to prepare fluorescently stained exosomes. 1 mM PKH67 was diluted in Diluent C (purchased from Sigma) to prepare a 10 ⁇ M PKH67 solution, mixed with an appropriate concentration of exosome solution, and allowed to react at room temperature for 10 minutes in the absence of light. After the reaction, an MW3000 spin column (purchased from ThermoFisher Scientific) was used to remove residual PKH67 dye from the exosome of the present invention (hereinafter abbreviated as "PKH-exosomes”) stained with PKH67.
  • PKH67 which did not react with the exosome of the present invention was removed, and fluorescence of sufficient intensity was detected in PKH-exosomes (FIG. 17).
  • the PKH-exosomes were dispersed in phosphate buffer (PBS) at a proper concentration, for example, a concentration of 1 ⁇ 10 5 to 1 ⁇ 10 9 particles / mL, and then applied to the skin outer surface of the pig skin tissue.
  • PBS phosphate buffer
  • the nonwoven fabric was covered and allowed to react for an appropriate period of time, for example, 30 minutes to 1 hour to allow the PKH-exosomes to be delivered to the subcutaneous tissues.
  • the pig skin tissue was immersed in a 3.7% formaldehyde solution, reacted overnight, and washed three times for 5 minutes each with phosphate buffered saline.
  • the washed pig skin tissue was immersed in a 30% sucrose solution and treated with OCT compound. After washing three times for 5 minutes with phosphate buffer solution, sections were prepared on a longitudinal section using a microtome, and tissue sections were placed on a slide glass. On the other hand, the preparation of the tissue section may be carried out before the tissue is fixed with formaldehyde solution. Fluorescence detected from PKH-exosomes in tissue sections was observed using fluorescence microscopy. In this way, PKH-exosomes transmitted through the epidermis of the pig skin tissue and transferred to the subcutaneous tissues were identified (Fig. 18). As shown in Fig.
  • the exosome of the present invention can effectively pass through the skin barrier and be deeply transferred into the skin tissue, and can be effectively absorbed to the skin. Accordingly, the external preparation for skin or cosmetic composition containing the exosome as an active ingredient is expected to act effectively in preventing, improving, alleviating or treating dermatitis.
  • PKH-exosomes were dispersed in phosphate buffer (PBS) at a proper concentration, for example, 1 ⁇ 10 5 to 1 ⁇ 10 9 particles / mL, and then applied to the outside of the skin tissue of the mice.
  • PBS phosphate buffer
  • the nonwoven fabric was pre-positioned outside the mouse skin tissue, and PKH-exosomes were injected between the nonwoven fabric and skin tissue. Then, the reaction was carried out for 30 minutes to 1 hour.
  • the exosome of the present invention can effectively pass through the skin barrier and be deeply transferred into the skin tissue, and can be effectively absorbed to the skin (FIG. 19).
  • the external preparation for skin or cosmetic composition containing the exosome as an active ingredient is expected to act effectively in preventing, improving, alleviating or treating dermatitis.
  • Example 13 Treatment of composition comprising exosome as an active ingredient for human skin
  • a composition (exosome-containing suspension) containing the exosome obtained according to the separation method of one embodiment of the present invention is applied to the affected part (hand, neck, arm, etc.) of three severe atopic patients for 1 to 2 weeks And then iontophoresis was performed by using a iontophoresis device to flow a microcurrent into the affected part of the composition.
  • iontophoresis was performed by using a iontophoresis device to flow a microcurrent into the affected part of the composition.
  • the external preparation for skin or cosmetic composition containing the exosome obtained as an active ingredient according to the separation method of one embodiment of the present invention can prevent, improve, alleviate or cure dermatitis .
  • Example 14 Treatment of a Composition Containing Exosome as an Active Ingredient for Canine Animals
  • FIG. 22A shows that erythema and inflammation are severe in the abdomen as a photograph of the affected area before administration of the exosome of the present invention.
  • the dermatitis symptoms were significantly improved from the third day after the administration of the exosome of the present invention (Fig. 22B), and the dermatitis almost disappeared on the tenth day after administration (Fig. 22C) and the second week after administration (Fig. 22D).
  • the vitality of the Shetland sheath was significantly increased and the body weight increased to 16 kg. The increase of vitality and the increase of the concentration are thought to be due to the improvement of dermatitis symptom.
  • composition containing the exosome obtained according to the separation method of one embodiment of the present invention can effectively alleviate or improve the dermatitis, as confirmed by the animal test on the canine animal as described above, The effect can be maintained continuously.
  • Example 15 Determination of anti-inflammatory effect using THP-1 monocyte
  • THP-1 monocyte THP-1 human monocyte
  • the anti-inflammatory effect of the exosome of the present invention was confirmed as follows.
  • THP-1 monocytes were cultured in RPMI supplemented with 10% FBS (Fetal Bovine Serum), 1% penicillin-streptomycin and 100 nM Phorbol 12-myristate 13-acetate (purchased from Sigma) 1640 (purchased from ThermoFisher Scientific) medium at 1640 (purchased from ThermoFisher Scientific), and then seeded in a 12-well plate at a density of 6 ⁇ 10 5 cells / mL and cultured in a 5% CO 2 incubator at 37 ° C. for 48 hours to obtain THP-1 Derived macrophage (THP-1 derived macrophage). The differentiated cells were then washed once with PBS and cultured in RPMI 1640 medium without formalin 12-myristate 13-acetate for 24 hours to stabilize the cells.
  • FBS Fetal Bovine Serum
  • penicillin-streptomycin 100 nM Phorbol 12-myristate 13-acetate
  • 1640 purchased from ThermoFisher
  • RPMI 1640 medium without FBS Fetal Bovine Serum
  • 100 ng / mL LPS purchased from Sigma
  • 20 ng / mL IFN -gamma purchased from Peprotech
  • RPMI 1640 medium containing no FBS Fetal Bovine Serum
  • dexamethasone purchased from Sigma
  • dexamethasone purchased from Sigma
  • CDNA was prepared from the RNA isolated from cultured THP-1 derived macrophages, and cDNA was prepared from the pro-inflammatory cytokines IL-6, IL-1 ⁇ And TNF-alpha mRNA expression levels were measured. GAPDH was used as a standard gene for quantifying the genes.
  • the types and sequences of the primers used in the PCR are shown in Table 3 below.
  • the forward primer (5 '- > 3')
  • the reverse primer (5 '- > 3') IL-6 AAGCCAGAGCTGTGCAGATGAGTA (SEQ ID NO: 21) TGTCCTGCAGCCACTGGTTC (SEQ ID NO: 22) IL-1?
  • THP-1 derived macrophages obtained after differentiating THP-1 monocytes into macrophages were treated with the exosomes of the present invention together with LPS and IFN-y (co-treatment)
  • the expression level of IL-6 mRNA, which is a proinflammatory cytokine was decreased as compared with the negative control group.
  • THP-1-derived macrophages obtained after differentiating THP-1 monocytes into macrophages were pretreated with the exosomes of the present invention before LPS and IFN- -treatment), the expression levels of pro-inflammatory cytokines IL-6, IL-1 [beta] and TNF- [alpha] were decreased compared to the negative control.
  • the proinflammatory cytokines IL-6, IL-1 [beta] and TNF- [alpha] increase expression in proinflammatory M1 macrophages and decrease expression in anti-inflammatory M2 macrophages. Therefore, the above results suggest that the exosome of the present invention can inhibit, alleviate and reduce the inflammatory reaction and the dermatitis symptom thereof, and this result strongly supports the animal experimental results discussed above.
  • Example 16 Preparation of cosmetic composition containing exosome of the present invention
  • the cosmetic composition was prepared by suspending the exosomes prepared in Example 2 at the concentration of 1704 ⁇ g / mL with the ingredients listed in Table 4 below. The content of each component is shown in Table 4 below.

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Abstract

La présente invention concerne une composition contenant comme principe actif des exosomes dérivés de cellules souches adipeuses humaines pour prévenir, atténuer ou traiter la dermatite. Agissant sur de multiples cibles de cytokine à l'origine de la dermatite, une composition selon la présente invention peut être appliquée à un large spectre de dermatites attribuées à diverses causes et peut supprimer ou réduire efficacement la dermatite. De plus, la composition de la présente invention peut réduire l'évaporation de l'humidité de la couche cornée et améliorer l'hydratation de la peau pour protéger et renforcer les barrières cutanées, la dermatite pouvant ainsi être évitée, atténuée, réduite ou traitée.
PCT/KR2018/007326 2017-06-30 2018-06-28 Utilisation d'une composition comprenant un exosome dérivé de cellules souches adipeuses en tant que principe actif pour atténuer la dermatite WO2019004738A2 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
JP2019571619A JP6970459B2 (ja) 2017-06-30 2018-06-28 脂肪由来幹細胞由来のエキソソームを有効成分として含む組成物の皮膚炎の改善の用途
ES18824926T ES2978498T3 (es) 2017-06-30 2018-06-28 Utilización de una composición que comprende un exosoma derivado de células madre adiposas como ingrediente eficaz en el alivio de la dermatitis
CN201880054562.0A CN111148520B (zh) 2017-06-30 2018-06-28 包含来源于脂肪来源干细胞的外排体作为有效成分的组合物在改善皮炎中的用途
EP18824926.2A EP3639832B1 (fr) 2017-06-30 2018-06-28 Utilisation d'une composition comprenant un exosome dérivé de cellules souches adipeuses en tant que principe actif pour atténuer la dermatite
US16/727,739 US11612621B2 (en) 2017-06-30 2019-12-26 Use of composition comprising exosome derived from adipose-derived stem cell as effective ingredient in ameliorating dermatitis

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KR10-2017-0083506 2017-06-30
KR20170083506 2017-06-30
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JP2022539228A (ja) * 2019-07-02 2022-09-07 インダストリー－ユニバーシティー コーペレイション ファンデーション ハンヤン ユニバーシティー エリカ キャンパス 植物エクソソームの大量生産方法
JP7274712B2 (ja) 2019-07-02 2023-05-17 インダストリー－ユニバーシティー コーペレイション ファンデーション ハンヤン ユニバーシティー エリカ キャンパス 植物エクソソームの大量生産方法
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JP2023517118A (ja) * 2020-03-13 2023-04-21 メディヘルプライン カンパニー，リミテッド Ｔｌｓｐ抑制および皮膚炎症性疾患の治療または改善のための龍眼肉含有混合生薬抽出物を含む局所用組成物およびその使用
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