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WO2019103381A1 - Utilisation d'une composition comprenant des exosomes dérivés de cellules souches en tant que principe actif pour le renforcement et l'amélioration fonctionnelle de la barrière cutanée - Google Patents

Utilisation d'une composition comprenant des exosomes dérivés de cellules souches en tant que principe actif pour le renforcement et l'amélioration fonctionnelle de la barrière cutanée Download PDF

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Publication number
WO2019103381A1
WO2019103381A1 PCT/KR2018/013751 KR2018013751W WO2019103381A1 WO 2019103381 A1 WO2019103381 A1 WO 2019103381A1 KR 2018013751 W KR2018013751 W KR 2018013751W WO 2019103381 A1 WO2019103381 A1 WO 2019103381A1
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Prior art keywords
skin
skin barrier
composition
improving
function
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Ceased
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PCT/KR2018/013751
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English (en)
Korean (ko)
Inventor
이용원
조병성
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Exocobio Inc
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Exocobio Inc
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Priority claimed from KR1020180071805A external-priority patent/KR20190060646A/ko
Application filed by Exocobio Inc filed Critical Exocobio Inc
Priority to EP18880438.9A priority Critical patent/EP3695830A4/fr
Priority to JP2020528409A priority patent/JP7102021B2/ja
Priority to CN201880075523.9A priority patent/CN111432799A/zh
Publication of WO2019103381A1 publication Critical patent/WO2019103381A1/fr
Priority to US16/878,850 priority patent/US11529370B2/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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 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
    • 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 the use of a composition comprising stem cell-derived exosome as an active ingredient for enhancing skin barrier function and improving function.
  • the present invention also relates to a pharmaceutical composition for improving skin barrier or function, an external preparation for skin and a cosmetic composition comprising the composition.
  • the skin consists of three layers of epidermis, dermis and subcutaneous fat tissue.
  • the epidermis is divided into the stratum corneum, the transparent layer, the granular layer, and the basal layer from the outside to the inside.
  • the stratum corneum which is the outermost layer of the epidermis, is the most important structure of skin barrier function, consisting of flattened corneocytes and SC intercellular lipid.
  • skin barrier function consisting of flattened corneocytes and SC intercellular lipid.
  • lipids such as sphingolipids, phospholipids, cholesterol sulfate and neutral lipids between the keratinocytes of the skin stratum corneum. These lipids exist between keratinocytes, It acts as a skin barrier to prevent evaporation of water and to protect the skin from external irritation or pollution.
  • a multi-lamella lipid layer or a multi-lamella structure formed by intercellular lipids such as ceramides, cholesterol, and fatty acids synthesized by keratinocytes prevents excessive loss of skin moisture, It acts as a skin barrier to prevent allergens and harmful substances from penetrating into the skin.
  • Ceramide is an N-acylated sphingoid compound that is amide-bonded to a sphingoid base called sphingosine, phytosphingosine and sphinganine.
  • the keratinocyte envelope is composed of protein films such as involucrin, loricrin, and filaggrin, and hydroxyceramide in the ceramide binds strongly to these proteins, Thereby making the structure of the semiconductor device robust.
  • ceramides and hydroxyceramides form skin barriers in the multilamellar lipid layer and contribute to the strengthening of the protein skin barrier structure, thus increasing the production of ceramide, hydroxyceramide and / or sphingoblast base, increasing enzyme activity involved in their synthesis , And enzymatic activity involved in their degradation contribute to strengthen skin barrier.
  • skin barriers may be damaged due to various causes such as skin stress due to various stresses and environmental pollution, frequent cleansing, natural skin aging, mutation of FLG function loss, and the like. If the skin barrier is damaged, chemicals and microorganisms easily penetrate into the skin, which can cause dermatitis and dry skin.
  • a composition or a functional cosmetic for enhancing skin barrier or skin moisturizing has been attracting attention recently in order to preemptively protect, strengthen or improve the skin barrier before damage of the skin barrier occurs.
  • a humectant having a property of absorbing moisture or an occlusive moisturizer preventing evaporation of water is used for strengthening or improving the skin barrier. If skin barrier is damaged once, skin barrier is not well restored even if moisturizer is applied, but steroid treatment is prescribed. However, long term use of steroid treatment can cause skin atrophy or capillary enlargement, . Therefore, it is important to protect, strengthen or improve the skin barrier before the skin barrier is damaged.
  • 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.
  • a method for improving or treating skin condition or disease using adult stem cells has been proposed.
  • the use of adult stem cells other than the patient's own adult stem cells may cause graft-versus-host disease, and 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 safe and novel material that is superior in skin barrier enhancement or function improvement effect as compared with the conventional moisturizing agents and steroid therapeutic agents in terms of skin barrier enhancement and function improvement. Accordingly, the present inventors have intensively studied new uses of exosomes derived from stem cells, and found that exosome isolated from a stem cell culture solution can solve the safety problem of the stem cell itself or stem cell culture broth as described above , And is effective for enhancing skin barrier function or improving function, thus completing the present invention.
  • Another object of the present invention is to provide a pharmaceutical composition for enhancing or improving skin barrier, an external preparation for skin and a cosmetic composition comprising the composition.
  • Another object of the present invention is to provide a method for preventing, inhibiting, alleviating, improving or treating skin diseases due to skin barrier function damage using the composition.
  • the present invention provides a composition for enhancing skin barrier or improving function, comprising an exosome derived from a stem cell as an active ingredient.
  • 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 can be varied according to the cell type, the separation method and the measurement method) (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.
  • exosome refers to a vesicle having a nano-sized vesicle structure secreted from the stem cells and released into the extracellular space and having a composition similar to that of exosome (for example, exosome- Pseudo-bezacl).
  • the type of the stem cell is not limited, but may be, for example, a mesenchymal stem cell, for example, a fat, a bone marrow, an umbilical cord or a cord blood derived stem cell, Derived stem cells.
  • the type of the adipose-derived stem cell is not limited as long as it does not cause a risk of infection by a pathogen and does not cause an immune rejection reaction, but it may be preferably a human adipose-derived stem cell.
  • the stem cell-derived exosome used in the present invention is effective for enhancing skin barrier function and improving function, and can be used in a variety of stem cell originated exosomes which are used in the art or can be used in the future Of course it is. Therefore, it should be understood that the exosomes derived from the stem cells isolated according to the separation method of the following Examples should be understood as an example of exosome that can be used in the present invention, and the present invention is not limited thereto.
  • skin barrier enhancement or function improvement means to protect, enhance or improve the skin barrier function of the stratum corneum, and to improve the function or regeneration of dermal or subcutaneous fat tissue It does not mean.
  • skin barrier enhancement or improvement of function is intended to prevent moisture reduction in the stratum corneum of the skin, for example, to improve the moisture reduction index of the stratum corneum such as TEWL (transdermal water loss), increase skin hydration, Enhancement and / or improvement of the skin barrier function of the true horny layer of the skin through the improvement of the skin barrier index such as the increase in the production amount of the corticosteroid, ceramide, dihydroceramide and / or sphingo base.
  • TEWL transdermal water loss
  • the term “ skin moisturizing” means that the homeostasis of the living body is maintained by appropriately controlling moisture loss (moisture evaporation) of the skin and the like. &Quot; Skin trouble " means that the skin turns red due to external stimuli or physical changes, and itching appears, and in the case of severe cases, small protuberance or rash occurs.
  • the composition for enhancing skin barrier function or improving function of the present invention when applied to a pharmaceutical composition, an external preparation for skin or a cosmetic composition, shows a significant effect on enhancing skin barrier or improving function of stem cells derived from stem cells contained as an active ingredient, It is possible to solve the safety problem of the cell itself or the stem cell culture liquid. Therefore, the stem cell-derived exosome contained in the composition for enhancing skin barrier function of the present invention is a completely different mechanism from the conventional limited wrinkle improvement and skin regeneration, It is clear that the efficacy is entirely unpredictable from the prior art.
  • the composition for enhancing skin barrier function or improving function comprises exosome derived from stem cells as an effective ingredient.
  • the exosome is characterized by reducing transepidermal water loss (TEWL) and increasing skin hydration.
  • TEWL transepidermal water loss
  • the exosome is characterized by increasing the amount of at least one of ceramide, dihydroceramid or sphingoid base produced in the skin.
  • the exosome may increase the amount of at least one of C16 ceramide, C18 ceramide, C20 ceramide, C22 ceramide, C24 ceramide, or C24: 1 ceramide, and the amount of total ceramide produced.
  • the exosome can increase the amount of at least one of C16 dihydroceramide, C18 dihydroceramide, C22 dihydroceramide, C24 dihydroceramide, or C24: 1 dihydroceramide and the amount of total dihydroceramide produced have.
  • the exosomes may increase the amount of at least one of S1P (Sphingosine-1-phosphate) or sphingosine.
  • the exosome increases the activity of SPHK1, a sphingosine kinase in the skin, and decomposes Sphingosine 1-phosphate (S1P)
  • S1P Sphingosine 1-phosphate
  • the exosome can reduce the expression or production of TSLP (Thymic stromal lymphopoietin), IL-4 and IL-13 in the skin.
  • TSLP Thimic stromal lymphopoietin
  • the composition for enhancing skin barrier function or improving function may be a pharmaceutical composition.
  • the pharmaceutical composition may be formulated as an injection.
  • the composition for enhancing or improving skin barrier may be a cosmetic composition or an external preparation for skin.
  • the cosmetic composition may be a cream or lotion.
  • composition for enhancing or improving skin barrier properties of the present invention can be effectively used for preventing, inhibiting, alleviating or improving skin barrier function damage caused by various causes.
  • the skin barrier strengthening function improving composition according to 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 parenteral administration includes transdermal administration, intraperitoneal administration, intravenous administration, intraarterial administration, intramuscular administration, intramuscular administration, Subcutaneous administration, intradermal administration, topical administration, rectal administration, and the like.
  • parenteral administration includes transdermal administration, intraperitoneal administration, intravenous administration, intraarterial administration, intramuscular administration, intramuscular administration, Subcutaneous administration, intradermal 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
  • 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 for skin barrier strengthening or function improvement is prepared with an external preparation for skin and / or a cosmetic composition
  • a composition commonly used in cosmetics or external preparation for skin For example, a moisturizing agent, an antioxidant, an oily 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 can be appropriately added as needed.
  • the external preparation for skin and / or cosmetic composition according to one embodiment of the present invention may contain, in addition to exosomes derived from stem cells, a skin barrier strengthening function or a skin barrier function which has been conventionally used as far as it does not impair its action
  • An improving agent and / or a moisturizing agent may be mixed together and 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 type of the hydrogel is not limited, but it is preferably a hydrogel obtained by dispersing the gel 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 strengthening skin barrier, improving function and / or skin moisturizing.
  • Cosmetic formulations can be prepared into any formulation conventionally produced in the art have. For example, it is possible to use 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, But are not limited to, soaps, shampoos, surfactant-containing cleansers, bath salts, body lotions, body creams, body oils, body essences, body cleansers, hair dyeing agents, hair tonics and the like.
  • 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 controlling the condition of mammalian skin by enhancing skin barrier or improving function except for therapeutic use by using the composition for enhancing or improving the skin barrier.
  • conditioning of the skin means improvement of the condition of the skin and / or prevention of the condition of the skin
  • improvement of the condition of the skin means visual and / Or a tactile perceptible positive change.
  • improving the condition of the skin may be improved skin moisturization, improving skin smoothness, preventing dry skin, preventing skin troubles, reducing redness of skin, and the like.
  • the cosmetic method of one embodiment of the present invention comprises the steps of: (a) applying a composition for skin barrier enhancement or function improvement directly to the skin of a mammal; (b) applying the composition for skin barrier enhancement or function improvement, Contacting or attaching the patch, mask pack or mask sheet to the skin of the mammal, or sequentially advancing (a) and (b) above.
  • step (a) lotion or cream may be used as the composition for enhancing the skin barrier or improving the function of the skin barrier.
  • Another embodiment of the present invention provides a method of preventing, inhibiting, alleviating, ameliorating, or treating a skin disorder due to skin barrier function impairment comprising the step of administering to a mammal a therapeutically effective amount of the above pharmaceutical composition do.
  • the mammal may be a human, a dog, a cat, a rodent, a horse, a cow, a monkey, or a pig.
  • composition for enhancing or improving skin barrier properties of the present invention improves objective indicators related to skin barrier protection, enhancement or function improvement.
  • the composition for enhancing or improving the skin barrier of the present invention can improve the moisture reduction index of the stratum corneum such as TEWL (transdermal water loss) and increase the skin hydration.
  • composition for enhancing or improving skin barrier properties of the present invention is useful for increasing the content of ceramide, dihydroceramid and / or sphingoid base, increasing enzyme activity involved in synthesis thereof, and decreasing enzyme activity involved in degradation thereof Show efficacy.
  • composition for enhancing or improving the skin barrier of the present invention reduces TSLP, IL-4 and IL-13, which are closely related to skin barrier damage, and inhibits a vicious cycle in which lipids and proteins contributing to skin barrier are reduced, It can contribute to restoration of barrier function.
  • composition for enhancing or improving the skin barrier of the present invention can be usefully used as a pharmaceutical composition for enhancing skin barrier or improving function, an external preparation for skin, and a cosmetic composition.
  • FIG. 1 shows the results of physical property analysis of exosomes obtained according to one embodiment of the present invention.
  • Figure 1A shows the particle size distribution and number of particles by TRPS (tunable resistive pulse sensing) analysis.
  • Figure 1B shows particle size distribution and particle number by NTA (nanoparticle tracking analysis) analysis.
  • Fig. 1C shows particle images by transmission electron microscopy (TEM) according to magnification.
  • TEM transmission electron microscopy
  • Figure 1D shows the Western blot results of exosomes obtained according to one embodiment of the present invention.
  • RTI ID 0.0 > 1E < / RTI > shows flow cytometric analysis results for CD63 and CD81 in marker assays for exosomes obtained according to one embodiment of the present invention.
  • FIG. 2 shows the result of confirming the absence of cytotoxicity after treatment of exosome according to one embodiment of the present invention with human skin fibroblast HS68 cells.
  • FIG. 3 shows a real-time PCR result in which the expression level of TNF- ⁇ , IL-6, IL-1 ⁇ and iNOS mRNA induced by LPS was decreased when RAW 264.7 cells were treated with LPS and the present exosome FIG. 3
  • FIG. 4 shows experimental results of examining the effect of reducing exogenous NO formation, which is a type of inflammatory reaction, by exoose according to one embodiment of the present invention.
  • PBS is phosphate-buffered saline
  • DEX is dexamethasone
  • EXO is exosome
  • CM is conditioned medium
  • CM-EXO is adipocyte- (Exosome-depeleted conditioned media).
  • FIG. 5 shows experimental results on the reduction effect of TNF-.alpha., An inflammatory cytokine, by exosome according to one embodiment of the present invention.
  • PBS represents phosphate-buffered saline
  • DEX represents dexamethasone
  • each numeral represents exosome throughput ( ⁇ g / mL).
  • FIG. 6 shows experimental results comparing the NO formation reduction effect by the separated exosome and the NO formation reduction effect by the exo-species isolated by the conventional precipitation method (PPT) according to one embodiment of the present invention .
  • FIG. 6A is a result of NTA analysis of exosomes isolated by the conventional precipitation method
  • FIG. 6B 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. 7 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 has been induced.
  • TEWL transepidermal water loss
  • FIG. 8 is a graph showing that skin hydration is increased dose-dependently on exosomes as a result of treatment with exosomes according to one embodiment of the present invention in mice in which skin barrier damage has been induced.
  • FIG. 9 shows that body weight of mice suffering from skin barrier damage was decreased due to side effects in the dexamethasone treatment group, whereas in the experimental group treated with exosome according to one embodiment of the present invention, .
  • FIGS. 10A to 10G are graphs showing increase in content of ceramide in the skin of mice treated with exosomes according to one embodiment of the present invention.
  • FIGS. 11A to 11F are graphs showing the increase in the amount of dihydroceramide in the skin as a result of treatment of exosome according to one embodiment of the present invention in mice in which skin barrier damage has been induced.
  • FIG. 12 is a graph showing an increase in the content of sphingoid bases in the skin as a result of treatment with exosomes according to one embodiment of the present invention in mice in which skin barrier damage has been induced.
  • FIG. 12A shows the increase in the content of S1P (Sphingosine-1-phosphate)
  • FIG. 12B shows the increase in the content of sphingosine-1-phosphate.
  • FIG. 13 is a graph showing that the activity of SPHK1 is increased and the activity of S1P lyase is reduced in the skin as a result of treatment of exosome according to one embodiment of the present invention in mice in which skin barrier damage has been induced.
  • FIG. 14A is a photograph of a tissue section taken after H & E staining of dorsal skin tissue in each experimental group
  • FIG. 14B is a graph comparing ear thicknesses.
  • FIG. 15 is a photograph of a tissue section taken after staining skin tissue with toluidine blue for each experimental group.
  • FIG. 16 is a photograph showing the size of mouse spine in each experimental group.
  • FIG. 17 is a graph showing an ELISA result showing that the TSLP level in the skin was reduced in the mouse treated with exosomes according to one embodiment of the present invention in a mouse in which skin barrier damage was induced.
  • FIG. 18 is a graph showing an ELISA result showing reduction of IL-4 levels in skin as a result of treating exosomes according to one embodiment of the present invention in mice in which skin barrier damage was induced.
  • FIG. 19 is a graph showing an ELISA result showing that IL-13 levels in the skin were reduced in the mice treated with exosomes according to one embodiment of the present invention in mice in which skin barrier damage was induced.
  • FIG. 20 shows that when the skin barrier is damaged, TSLP, IL-4, and IL-13 are increased and Th2 type cytokines (IL-4 and IL-13) decrease the lipids and proteins that contribute to the skin barrier, And a repetition of a vicious circle is made.
  • 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.
  • 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.
  • NTA nanoparticle tracking analysis
  • TRPS tunable resistive pulse sensing
  • FIG. 1D 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. 1E shows the presence of CD63 and CD81 markers as a result of analysis using flow cytometry 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.
  • exosomes derived from stem cells used in the present invention are not limited to the exosomes of the above-mentioned embodiments, and it is possible to use various stem cell-derived exosomes which are used in the art or can be used in the future Of course. It is to be understood that the exosomes derived from the stem cells isolated according to the above embodiments are to be understood as an example of exosomes derived from stem cells which can be used in the present invention, and the present invention is not limited thereto.
  • 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 the cells were mixed with 80 ⁇ 90% confluency 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 comparative group was based on the number of cells cultured in the normal cell culture medium not treated with exosome, and it was confirmed that the exosome-induced cytotoxicity did not appear within the tested concentration range (FIG. 2).
  • 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 can prevent, inhibit, alleviate or ameliorate the damage of the skin barrier function due to the inflammatory reaction.
  • the composition for enhancing the skin barrier or function of the present invention can prevent skin barrier It is expected to be useful for protection, enhancement or improvement of functions.
  • 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. 6A) isolated by the conventional precipitation method has a low uniformity of the particle size distribution as compared with the exosome (see FIG. 6B) isolated and purified by the TFF method of one embodiment of the present invention, Respectively. Also, as shown in FIG.
  • 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 for enhancing the skin barrier and improving function of the present invention, which contains the stem cell-derived exosome having excellent functional activity as an active ingredient, is excellent in damage to the skin barrier function It is far superior to the prior art in terms of the effect of preventing, inhibiting, alleviating or restoring the disease.
  • performance or functional activity e. G., Uniformity of particle size distribution, inhibition of NO production, Reduction of inflammatory response, etc.
  • Example 7 Animal model causing skin barrier damage
  • Oxazolone was used to establish animal models to confirm skin barrier enhancement or function improvement. 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 in which the skin barrier function is impaired by inducing an increase in the pH of the stratum corneum (Journal of Investigative Dermatology (2008) 128 (1), 79-86).
  • 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 group (referred to as “ C " in FIG. 7) that caused skin barrier damage with oxazolone;
  • Exosomal low dose Exosomes prepared in Example 2 were subcutaneously injected (SC: subcutaneous injection) at a dose of 1 ⁇ g per subject for 3 weeks three times a week after inducing skin barrier damage with oxazolone Quot; L ");
  • Exosomes prepared in Example 2 after inducing skin barrier damage with oxazolone were subcutaneously administered for 3 weeks at a dose of 3 ⁇ g per day for 4 weeks (indicated as "M" in FIG. 7 );
  • Exosome caused by damage to skin barrier with oxazolone, exosome prepared in Example 2 was subcutaneously administered at a dose of 10 [mu] g per day for 4 weeks per week (indicated as " H ");
  • dexamethasone an experimental group (positive control) in which 0.03% dexamethasone (purchased from Sigma) dissolved in ethanol after inducing skin barrier damage with oxazolone was administered subcutaneously for 4 weeks three times a week 7 " D ").
  • the average moisture evaporation of the skin was measured using a TEWL measuring device, and the moisture content of the skin was measured using a moisture meter (Corneometer CM825) (Courage-Khazaka eletronic GmbH, Germany) Were measured.
  • the transepidermal water loss (TEWL) of the exosomes was reduced in a dose-dependent manner in the experimental groups (3) to (5) in which the exosome of the embodiment of the present invention was treated as compared to the disease control group That is, a decrease in moisture evaporation amount of the stratum corneum (Fig. 7).
  • a decrease in moisture evaporation amount of the stratum corneum Fig. 7
  • an increase in the skin hydration of the exosomes in a dose-dependent manner was observed in the experimental groups (3) to (5) in which the exosome of Example 1 of the present invention was treated as compared to the disease control group (FIG. 8). Therefore, the composition comprising the exosome of the present invention as an active ingredient can protect and enhance the skin barrier by reducing moisture evaporation of the stratum corneum and improving skin moisturization.
  • mice of Example 7 were sacrificed and skin samples were collected.
  • ceramide, total ceramide, sphingosine, and sphingosine-1-phosphate (S1P) by carbon length was analyzed using LC-MS / MS (API 3200 Triple quadruple mass, AB / SCIEX) as follows.
  • Total lipids were extracted according to literature known in the art (J Invest Dermatol. 2010 Oct, 130 (10): 2472-80).
  • Ceramide, sphingosine, and sphingosine-1-phosphate were derivatized with OPA (o-phthalaldehyde) reagent and quantified using LC-MS / MS system equipped with a fluorescence detector (J Invest Dermatol. 2010 Oct, 130 10: 2472-80; Arch Pharm Res.
  • OPA o-phthalaldehyde
  • the sphingolipid content was expressed as " pmol / g protein ", and protein quantification was performed according to the conventional BCA method.
  • the activity of SPHK1, a sphingosine kinase was analyzed using LC-MS / MS as follows. (PH 7.4) containing 5 mM EDTA, 5 mM EGTA, 3 mM ⁇ -mercaptoethanol, 5% glycerol, protease inhibitor (Sigma-Aldrich) and phosphatase inhibitor (Roche) The lysate was incubated with 10 [mu] l of 200 [mu] M C17-sphingosine (SPHK substrate) (Avanti Polar Lipids).
  • Triton X-100 Triton X-100
  • 3, v / v / v 8:: 4 MeOH HCl.
  • C17-Spinganin-1-phosphate 100 pmol; Avanti Polar Lipids
  • the organic phase separated by the addition of CHCl 3 was dried under vacuum and the dried material was re-dissolved in methanol and analyzed by LC-ESI-MS / MS (API 3200 Triple quadruple mass, AB / SCIEX).
  • SPHK1 activity was expressed as " C17-S1P pmol / mg protein / min ", and protein quantification was performed according to the conventional BCA method.
  • S1P lyase activity was expressed as " pentadecanol alcohols / mg protein / min ", and protein quantification was performed according to the conventional BCA method.
  • SPHK1 is an enzyme involved in the synthesis of sphingolipids that constitute skin barrier. Therefore, increased activity of SPHK1 means increase of ceramide synthesis and enhancement of skin barrier.
  • S1P lyase is an enzyme that decomposes sphingosine-1-phosphate to inhibit the synthesis of sphingolipid. Therefore, reduction of its activity means reduction of ceramide synthesis inhibition and reinforcement of skin barrier.
  • the composition comprising the exosome as an active ingredient according to one embodiment of the present invention can be used for improving objective indicators related to protection, enhancement and / or improvement of skin barrier function, for example, ceramide, dihydroceramide And increase in the amount of sphingobase produced, increase in enzyme activity involved in their synthesis, and decrease in enzyme activity involved in their degradation. Accordingly, the composition comprising the exosome as an active ingredient of the one embodiment of the present invention can be usefully used as a pharmaceutical composition for skin barrier enhancement or function improvement, an external preparation for skin, and a cosmetic composition.
  • Example 9 Comparison of ear thickness and size of an animal model
  • FIG. 14 (a) is a photograph taken after the dorsal skin tissue of a mouse was stained with H & E
  • FIG. 14 (b) shows the ear thickness measured in each of the experimental groups (2) to (6) Graph. It was confirmed that the ear thickness was reduced in a dose-dependent manner in the experimental group of (3) to (5) in which the exosome of the present invention was treated as compared with the disease control group.
  • FIG. 15 is a photograph of a skin tissue of a sacrificed mouse after staining with toluidine blue.
  • the experimental group (exosome high dose treatment group) (5) treated with exosome according to one embodiment of the present invention Of the infiltration was significantly decreased.
  • treatment with the positive control dexamethasone did not reduce the invasion of mast cells.
  • compositions comprising an exosome of one embodiment of the present invention can prevent, inhibit, alleviate or ameliorate the inflammatory response and thereby the damage of the skin barrier function. Therefore, it is expected that the composition for skin barrier strengthening or function improvement of the present invention is useful for protecting, enhancing or improving the skin barrier function.
  • mice in Example 7 were sacrificed and skin samples were collected.
  • the levels of TSLP (Thymic stromal lymphopoietin), IL-4, and IL-13 in the skin tissues were confirmed by ELISA.
  • the skin tissue samples were cut into 5 mL of ice-cold Ripa buffer (containing 1 ⁇ protease inhibitor), minced and centrifuged at 2,000 rpm for 5 minutes at 4 ° C to remove impurities. Protein was quantitated with BCA kit and 100 ⁇ L (10 ⁇ g protein) was used for ELISA analysis after adjusting protein concentration to 100 ⁇ g / mL with protease inhibitor-containing lipase.
  • TSLP is involved in maturation of antigen presenting cells (dendritic cells and Langerhans cells) and promotes secretion in keratinocytes and mast cells in case of skin barrier damage.
  • antigen presenting cells dendritic cells and Langerhans cells
  • These mature antigen-presenting cells activate Th cells and activated Th cells increase Th2-type cytokines such as IL-4 and IL-13.
  • IL-4 and IL-13 contribute to the skin barrier (E.g., ceramides) and proteins (e.g., filagreen, inbolucrin, and loricrin) to further repel skin barriers (see FIG. 20).
  • the candidate substance involved in the recovery of the skin barrier function reduces the expression and / or production of TSLP, IL-4 and / or IL-13, it can break the above-mentioned vicious circle and contribute to restoration of skin barrier function and enhancement of function .
  • TSLP, IL-4, and IL-13 in the skin tissues of the experimental groups (3) to (5) treated with exosome of one embodiment of the present invention And the level was significantly decreased.
  • the exosome of one embodiment of the present invention significantly decreases TSLP levels in skin tissue, thereby inhibiting the maturation of antigen presenting cells and lowering Th2 activation.
  • the reduction of Th2 activation by treatment with exosomes of the present invention and the reduction of Th2 cytokines such as IL-4 and IL-13 thereby inhibits vicious cycle in which lipids and proteins contributing to skin barrier are inhibited, It can contribute to the restoration of function.
  • Example 11 Preparation of cosmetic composition containing exosome of the present invention
  • the exosome stock solution of 1704 ⁇ g / mL prepared in Example 2 was diluted and mixed with the ingredients listed in Table 2 to suspend the cosmetic composition (lotion).
  • the final cosmetic composition was prepared to contain exosomes at a concentration of 2 x 10 < 4 > particles / mL. The content of each component is shown in Table 2 below.
  • the exosome stock solution of 1704 ⁇ g / mL prepared in Example 2 was diluted and mixed with the ingredients listed in Table 3 to suspend the cosmetic composition (cream).
  • the final cosmetic composition was prepared to contain exosomes at a concentration of 2 x 10 < 4 > particles / mL. The content of each component is shown in Table 3 below.
  • the stock solution of the exosome prepared in Example 2 at a concentration of 1704 ⁇ g / mL was diluted and mixed with the ingredients listed in Table 4 below to suspend.
  • the obtained cosmetic composition was applied or immersed to prepare a mask pack.
  • the exosomes were applied or immersed in a mask pack at a concentration of 4 x 10 < 3 > particles / mL.
  • the content of each component is shown in Table 4 below.

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Abstract

La présente invention concerne une composition pour le renforcement et l'amélioration fonctionnelle de la barrière cutanée qui améliore des indicateurs objectifs associés à la protection, au renforcement et à l'amélioration fonctionnelle de la barrière cutanée. La composition pour le renforcement et l'amélioration fonctionnelle de la barrière cutanée selon la présente invention peut améliorer l'indice de perte d'eau de la couche cornée, par exemple l'indice de perte d'eau transépidermique (TEWL) et renforcer l'hydratation de la peau. De plus, la composition pour le renforcement et l'amélioration fonctionnelle de la barrière cutanée de la présente invention a pour effets d'augmenter les teneurs en céramide, en dihydrocéramide et en bases sphingoïdes, de renforcer l'activité d'une enzyme impliquée dans leur synthèse et de réduire l'activité d'une enzyme impliquée dans leur dégradation. En outre, la composition pour le renforcement ou l'amélioration fonctionnelle de la barrière cutanée de la présente invention réduit la TSLP, l'IL-4 et l'IL-13, qui sont étroitement associées aux lésions de la barrière cutanée, pour bloquer le cercle vicieux de la réduction de la contribution des lipides et des protéines à la barrière cutanée et peut ainsi contribuer à la régénération des fonctions de la barrière cutanée.
PCT/KR2018/013751 2017-11-24 2018-11-13 Utilisation d'une composition comprenant des exosomes dérivés de cellules souches en tant que principe actif pour le renforcement et l'amélioration fonctionnelle de la barrière cutanée Ceased WO2019103381A1 (fr)

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EP18880438.9A EP3695830A4 (fr) 2017-11-24 2018-11-13 Utilisation d'une composition comprenant des exosomes dérivés de cellules souches en tant que principe actif pour le renforcement et l'amélioration fonctionnelle de la barrière cutanée
JP2020528409A JP7102021B2 (ja) 2017-11-24 2018-11-13 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バリアの強化ないし機能改善の用途
CN201880075523.9A CN111432799A (zh) 2017-11-24 2018-11-13 包含干细胞来源外排体作为有效成分的组合物在使皮肤屏障增强和使其功能改善中的用途
US16/878,850 US11529370B2 (en) 2017-11-24 2020-05-20 Use of composition comprising stem cell-derived exosome as effective ingredient in strengthening skin barrier and improving skin barrier function

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KR1020180071805A KR20190060646A (ko) 2017-11-24 2018-06-22 줄기세포 유래의 엑소좀 및/또는 세포외 소포체를 유효성분으로 포함하는 조성물의 피부장벽 강화 내지 기능 개선 용도
KR1020180093873A KR20190060651A (ko) 2017-11-24 2018-08-10 줄기세포 유래의 엑소좀을 유효성분으로 포함하는 조성물의 피부장벽 강화 내지 기능 개선 용도
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