WO2021096089A1 - Nouvelle composition comprenant des exosomes dérivés de cellules souches et du polydésoxyribonucléotide en tant que principes actifs - Google Patents

Nouvelle composition comprenant des exosomes dérivés de cellules souches et du polydésoxyribonucléotide en tant que principes actifs Download PDF

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WO2021096089A1
WO2021096089A1 PCT/KR2020/014418 KR2020014418W WO2021096089A1 WO 2021096089 A1 WO2021096089 A1 WO 2021096089A1 KR 2020014418 W KR2020014418 W KR 2020014418W WO 2021096089 A1 WO2021096089 A1 WO 2021096089A1
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skin
exosomes
stem cell
cosmetic composition
mammal
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PCT/KR2020/014418
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Korean (ko)
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조병성
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주식회사 엑소코바이오
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • 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
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • 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
    • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
    • 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
    • A61Q19/08—Anti-ageing preparations

Definitions

  • the present invention relates to a new composition comprising stem cell-derived exosomes and polydeoxyribonucleotides as active ingredients, and more particularly, by containing a combination of stem cell-derived exosomes and polydeoxyribonucleotides as active ingredients. It relates to a new composition capable of enhancing the effect of whitening, skin regeneration, wound healing, wound healing promotion, skin wrinkle improvement and/or skin elasticity improvement.
  • the present invention relates to a cosmetic composition for improving skin wrinkles, improving skin elasticity or skin regeneration, a cosmetic composition for whitening, and a pharmaceutical composition for skin regeneration, wound healing or promoting wound healing, a quasi-drug or skin external preparation comprising the composition.
  • Substances known to be effective in improving skin wrinkles include adenosine and retinoic acid, but adenosine has little efficacy in clinical practice, and retinoic acid cannot be used in women of childbearing potential and has side effects such as erythema.
  • Human skin color is determined by the concentration and distribution of melanin inside the skin. Melanin is converted from tyrosine to dopa (DOPA) and dopaquinone by tyrosinase and then synthesized through a non-enzymatic oxidation reaction. When melanin is produced in excess, pigmentation occurs and spots, spots, and freckles appear on the face, neck, and arms, making the appearance unpleasant. Skin whitening agents are being developed to improve spots, freckles, and dark skin tone, but it is difficult to develop skin whitening agents that have excellent whitening effects and no side effects. For example, skin whitening agents that selectively attack melanocytes that produce melanin have excellent whitening effects, but have side effects of skin toxicity.
  • Extracellular vesicles are sometimes called cell membrane-derived vesicles, ectosomes, shedding vesicles, microparticles, exosomes, etc., and in some cases, they are used separately from exosomes.
  • Exosomes are vesicles having a size of tens to hundreds of nanometers consisting of a double phospholipid membrane that has the same structure as a cell membrane, and contains proteins and nucleic acids (mRNA, miRNA, etc.) called exosomes cargo (cargo).
  • Exosomal cargo contains a wide range of signaling factors, and these signaling factors are known to be specific to the cell type and are regulated differently depending on the environment of the secretory cell.
  • Exosomes are intercellular signaling mediators secreted by cells, and various cellular signals transmitted through them regulate cellular behavior including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. Is known.
  • Exosomes contain specific genetic material and bioactive factors depending on the nature and state of the derived cell. In the case of proliferating stem cell-derived exosomes, cell behaviors such as cell migration, proliferation and differentiation are regulated, and the characteristics of stem cells related to tissue regeneration are reflected (Nature Review Immunology 2002 (2) 569-579).
  • exosomes which are called cell avatars, contain bioactive factors such as growth factors, similar to cells, and serve as a carrier that carries bioactive factors between cells and cells, that is, the communication between cells and cells.
  • Exosomes are known not only to be released from animal cells such as stem cells, immune cells, fibroblasts, and cancer cells, but also from cells of various organisms such as plants, bacteria, fungi, and algae. .
  • Korean Patent Laid-Open Publication No. 10-2019-0114620 discloses a cosmetic composition containing a human fat stem cell culture medium extract and a polydeoxyribonucleotide (PDRN), but the human fat stem cell culture medium is secreted as cells grow. Since it contains components such as waste products, antibiotics added to prevent contamination, and animal-derived serum, it is highly likely to be exposed to various risks when used on skin or wounds.
  • PDRN polydeoxyribonucleotide
  • the inventors of the present invention have been continuing intensive research on new application fields of exosomes and technology that can increase the physiological activity of exosomes through grafting with medical or cosmetic technology.
  • Stem cell-derived exosomes and polydeoxyribonucleotides When combined, it was confirmed that the effect of whitening, skin regeneration, wound healing, wound healing promotion, skin wrinkle improvement and/or skin elasticity improvement can be effectively enhanced, thereby completing the present invention.
  • An object of the present invention is to enhance the efficacy of whitening, skin regeneration, wound healing, wound healing promotion, skin wrinkle improvement and/or skin elasticity improvement by containing stem cell-derived exosomes and polydeoxyribonucleotides as active ingredients. It is to provide a new composition.
  • Another object of the present invention is to provide a cosmetic composition for improving skin wrinkles, improving skin elasticity or regenerating skin, and a cosmetic composition for whitening, including the composition.
  • Another object of the present invention is to provide a pharmaceutical composition for skin regeneration, wound healing, or wound healing promotion, a quasi-drug or a skin external preparation comprising the composition.
  • Another object of the present invention is to provide a cosmetic method or a treatment method for whitening, skin regeneration, wound healing, wound healing promotion, skin wrinkle improvement and/or skin elasticity improvement using the above composition.
  • the present invention is a composition for enhancing the efficacy of whitening, skin regeneration, wound healing, wound healing promotion, skin wrinkle improvement and/or skin elasticity improvement, stem cell-derived exosomes and polydeoxyribo It provides a composition comprising a combination with a nucleotide as an active ingredient.
  • extracellular vesicles generally encompasses membrane-derived vesicles (membrane vesicles), ectosomes, shedding vesicles, microparticles, or equivalents thereof. It is used in the sense of saying. Depending on the separation environment, conditions, and methods, extracellular vesicles may have the same meaning as exosomes, and may have the same or similar size as exosomes, but may have a meaning including nanovesicles that do not have the composition of exosomes.
  • exosomes refers to vesicles having a size of tens to hundreds of nanometers (preferably about 30 to 200 nm) composed of a double phospholipid membrane identical to the structure of the cell membrane (however, the separation target is The particle size of the exosomes may vary depending on the cell type, separation method, and measurement method. (Vasiliy S. Chernyshev et al., "Size and shape characterization of hydrated and desiccated exosomes", Anal Bioanal Chem, (2015) DOI 10.1007/s00216-015-8535-3). Exosomes contain proteins and nucleic acids (mRNA, miRNA, etc.) called exosome cargo.
  • Exosomal cargo contains a wide range of signaling factors, and these signaling factors are known to be specific to the cell type and are regulated differently depending on the environment of the secretory cell.
  • Exosomes are intercellular signaling mediators secreted by cells, and various cellular signals transmitted through them regulate cellular behavior including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. Is known.
  • exosome has a nano-sized vesicle structure secreted from stem cells and released into the extracellular space, and has a composition similar to that of exosomes (eg, exosomes- It means to include all similar vesicles).
  • the type of stem cells is not limited, but as an example that does not limit the present invention, embryonic stem cells, induced pluripotent stem cells (iPSCs), adult stem cells, embryonic stem cells-derived mesenchymal stem cells , Or mesenchymal stem cells derived from induced pluripotent stem cells.
  • the adult stem cells are at least one adult selected from the group consisting of mesenchymal stem cells, human tissue-derived mesenchymal stromal cells, human tissue-derived mesenchymal stem cells, and multipotent stem cells. It can be a stem cell.
  • the mesenchymal stem cells may be mesenchymal stem cells derived from one or more tissues selected from the group consisting of umbilical cord, cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, Wharton jelly, and placenta.
  • the adult stem cells may be mesenchymal stem cells, for example, stem cells derived from fat, bone marrow, umbilical cord or umbilical cord blood, and more preferably stem cells derived from fat.
  • the type of the stem cell is not limited as long as there is no risk of infection by a pathogen and does not cause an immune rejection reaction, but may be preferably human-derived stem cells, more preferably human adipose tissue-derived stem cells.
  • stem cell-derived exosomes used in the present invention have whitening, skin regeneration, wound healing, wound healing promotion, skin wrinkle improvement and/or skin elasticity improvement effects and do not cause adverse effects on the human body, they are used in the art or in the future. It goes without saying that various stem cell-derived exosomes that can be used can be used. Therefore, it should be understood that the stem cell-derived exosomes isolated according to the separation method of the following examples should be understood as an example of the exosomes that can be used in the present invention, and the present invention is not limited thereto.
  • polydeoxyribonucleotide is a material containing a deoxyribonucleotide polymer, and is used to mean a DNA polymer fragment complex or a DNA polymer fragment mixture unless otherwise defined.
  • Polydeoxyribonucleotides are generally prepared by extracting from salmon semen, and in addition, may be prepared by extracting from semen, testis, milt and/or eggs of fish such as salmon, trout, herring, and tuna.
  • polydeoxyribonucleotides can be prepared by extraction from spermatozoa and high heat treatment.
  • skin elasticity refers to a characteristic in which the skin deformed by an external force easily returns to its original shape when the corresponding external force is removed.
  • skin wrinkles refers to fine lines caused by deterioration of the skin, and may be caused by genes, a decrease in collagen and elastin present in the skin dermis, and external environments.
  • skin wrinkle improvement refers to inhibiting or inhibiting the generation of wrinkles on the skin, or alleviating wrinkles that have already been generated.
  • whitening as used herein includes increasing the brightness of the skin whose brightness has decreased due to an excessive amount of pigments such as melanin, or maintaining the brightness of the skin at a certain level.
  • a composition comprising a combination of a stem cell-derived exosome and a polydeoxyribonucleotide of the present invention as an active ingredient may be a cosmetic composition, a pharmaceutical composition, a quasi-drug, or a skin external preparation.
  • the present invention provides a cosmetic composition for improving skin wrinkles, improving skin elasticity, or regenerating skin, comprising a combination of stem cell-derived exosomes and polydeoxyribonucleotides as an active ingredient.
  • a cosmetic composition for whitening comprising a combination of stem cell-derived exosomes and polydeoxyribonucleotides as an active ingredient.
  • the cosmetic composition of one embodiment of the present invention may be, for example, a cream or lotion.
  • the present invention provides a pharmaceutical composition for promoting skin regeneration, wound healing, or wound healing, comprising a combination of a stem cell-derived exosome and a polydeoxyribonucleotide as an active ingredient, a quasi-drug or a skin external preparation.
  • the pharmaceutical composition may be in the form of injections, injections, sprays, liquids or patches, and the quasi-drugs or external skin preparations may be liquids, ointments, creams, sprays, patches, gels, or aerosols. I can.
  • composition of one embodiment of the present invention when used as a pharmaceutical composition, it 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, gum acacia, alginate, gelatin, calcium phosphate, calcium carbonate, calcium silicate, cellulose , Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, etc., but are not limited thereto.
  • the effective amount of the pharmaceutical composition of one embodiment of the present invention means an amount required for administration in order to expect skin regeneration, wound healing, and/or wound healing promoting effect.
  • the blending ratio of the pharmaceutical composition according to an embodiment of the present invention may be appropriately selected according to the type, amount, or form of the additional ingredients as described above.
  • the pharmaceutical composition of the present invention may be included in about 0.1 to 99% by weight, preferably about 10 to 90% by weight.
  • the appropriate dosage of the pharmaceutical composition of one embodiment of the present invention may be adjusted according to the severity of the disease, the type of the formulation, the formulation method, the patient's age, sex, weight, health condition, diet, excretion rate, administration time, and administration method. I can.
  • administering the pharmaceutical composition of one embodiment of the present invention to an adult it may be administered in one to several times in a dose of 0.001 mg/kg to 100 mg/kg per day.
  • composition of one embodiment of the present invention is prepared as a quasi-drug, external skin preparation and/or cosmetic composition
  • it is usually used in a quasi-drug, external skin preparation and/or cosmetic composition within the scope of not impairing the effect of the present invention.
  • Ingredients for example, moisturizers, antioxidants, oily ingredients, ultraviolet absorbers, emulsifiers, surfactants, thickeners, alcohols, powder ingredients, colorants, aqueous ingredients, water, various skin nutrients, etc. can be appropriately blended as needed.
  • the quasi-drug, skin external preparation, and/or cosmetic composition of one embodiment of the present invention has an action (e.g., whitening, skin regeneration, wound healing, Acceleration of wound healing, improvement of skin wrinkles and/or improvement of skin elasticity, etc.) may be used in combination with conventionally used skin improvement agents, whitening agents, wound treatment agents, and/or moisturizers.
  • an action e.g., whitening, skin regeneration, wound healing, Acceleration of wound healing, improvement of skin wrinkles and/or improvement of skin elasticity, etc.
  • conventionally used skin improvement agents, whitening agents, wound treatment agents, and/or moisturizers may be used in combination with conventionally used skin improvement agents, whitening agents, wound treatment agents, and/or moisturizers.
  • the quasi-drug, skin external preparation and/or cosmetic composition of one embodiment of the present invention is, for example, a patch, a mask pack, a mask sheet, a cream, a tonic, an ointment, a suspension, an emulsion, a paste, a lotion, a gel, an oil, a pack, It can be applied to various forms such as spray, aerosol, mist, foundation, powder, and oil paper.
  • the cosmetic composition of one embodiment of the present invention may be used for the purpose of whitening, improving skin wrinkles, improving skin elasticity, or regenerating skin, and the cosmetic formulation may be prepared in any formulation conventionally prepared in the art.
  • the quasi-drug, external skin preparation and/or cosmetic composition of one embodiment of the present invention includes components commonly used in quasi-drug, external skin preparations and/or cosmetic compositions, such as antioxidants, stabilizers, solubilizers, vitamins, pigments And conventional adjuvants and carriers such as fragrances.
  • components commonly used in quasi-drug, external skin preparations and/or cosmetic compositions such as antioxidants, stabilizers, solubilizers, vitamins, pigments And conventional adjuvants and carriers such as fragrances.
  • other ingredients may be appropriately selected and blended by a person skilled in the art without difficulty depending on the type or purpose of use of the quasi-drug, external skin preparation and/or cosmetic composition. have.
  • Another embodiment of the present invention provides a cosmetic method for controlling the condition of mammalian skin except for treatment by using the cosmetic composition.
  • controlling the condition of the skin means improving the condition of the skin and/or controlling the condition of the skin prophylactically, and improving the condition of the skin means visual and/or feeling of the appearance and feel of the skin tissue. Or it means a positive change that can be perceived tactilely.
  • the improvement of the condition of the skin may be improvement of skin elasticity, improvement of skin wrinkles, skin whitening and/or skin regeneration.
  • the cosmetic method of one embodiment of the present invention includes (a) applying the cosmetic composition directly to the skin of a mammal, (b) applying the patch, mask pack, or mask sheet to which the cosmetic composition is applied or deposited on the skin of the mammal. It includes contacting or attaching to, or sequentially proceeding the above (a) and (b).
  • a lotion or cream may be used as a cosmetic composition.
  • step (c) after step (b), the patch, mask pack, or mask sheet is removed from the skin of the mammal, and the cosmetic composition is applied to the skin of the mammal. It may further include the step of applying.
  • a lotion or cream may be used as a cosmetic composition.
  • the mammal may be a human, a dog, a cat, a rodent, a horse, a cow, a monkey, or a pig.
  • the present invention comprises the step of administering a therapeutically effective amount of the pharmaceutical composition to a mammal, or applying the quasi-drug or the external preparation for skin to the skin or wound site of a mammal, skin regeneration, wound treatment or It provides a method for accelerating wound healing.
  • the mammal may be a human, dog, cat, rodent, horse, cow, monkey, or pig.
  • composition of the present invention contains a combination of stem cell-derived exosomes and polydeoxyribonucleotides as an active ingredient, so that whitening, skin regeneration, wound healing, and more excellent than a composition containing only stem cell-derived exosomes or polydeoxyribonucleotides, It may exhibit the effect of promoting wound healing, improving skin wrinkles and/or improving skin elasticity.
  • FIG. 1 shows the results of analyzing physical properties of stem cell-derived exosomes obtained according to an embodiment of the present invention.
  • Fig. 1A shows the particle size distribution and the number of particles obtained by nanoparticle tracking analysis (NTA).
  • Fig. 1B is a photograph of a particle image taken by TEM (transmitted electron microscopy).
  • Fig. 1C shows the results of Western blot for the positive markers of the stem cell-derived exosomes obtained according to an embodiment of the present invention.
  • Fig. 1D shows the results of Western blot for negative markers of stem cell-derived exosomes obtained according to an embodiment of the present invention.
  • Fig. 1E shows the results of flow cytometry for CD9, CD63 and CD81 in the analysis of markers for stem cell-derived exosomes obtained according to an embodiment of the present invention.
  • Figure 2 shows the result of confirming that there is no cytotoxicity after treatment with the stem cell-derived exosomes according to an embodiment of the present invention on HS68 cells, which are human skin fibroblasts.
  • HS68 cells human dermal fibroblasts, are purchased from ATCC and contain 10% fetal bovine serum (purchased from ThermoFisher Scientific) and 1% antibiotics-antimycotics (purchased from ThermoFisher Scientific). In the containing DMEM (purchased from ThermoFisher Scientific) medium, 5% CO 2 , it was subcultured at 37°C.
  • HDF Human dermal fibroblast
  • fetal bovine serum purchased from ThermoFisher Scientific
  • antibiotics-antimycotics purchased from ThermoFisher Scientific
  • DMEM purchased from ThermoFisher Scientific was subcultured in 5% CO 2 , 37°C conditions.
  • B16F10 cells mouse melanoma cells, were purchased from ATCC, 10% fetal bovine serum (purchased from ThermoFisher Scientific), 1% antibiotics-antimycotics (purchased from ThermoFisher Scientific), DMEM containing L-glutamine (purchased from ThermoFisher Scientific) and sodium pyruvate (purchased from ThermoFisher Scientific), phenol-free red (purchased from ThermoFisher Scientific) medium in 5% CO 2 , and subcultured at 37°C. .
  • Adipose-derived stem cells were cultured under conditions of 5% CO 2 and 37°C according to a cell culture method known in the art to which the present invention pertains. Then, after washing with phosphate-buffered saline (purchased from ThermoFisher Scientific), it was replaced with a serum-free and phenol red medium, and cultured for 1 to 10 days, and the supernatant (hereinafter, culture solution) was recovered. .
  • 2% by weight of trehalose was added to the culture medium in order to obtain exosomes having a uniform particle size distribution and high purity.
  • the culture solution was filtered through a 0.22 ⁇ m filter to remove impurities such as cell debris, waste products, and large particles.
  • the filtered culture solution immediately separated exosomes through a separation process.
  • the filtered culture solution was stored in a refrigerator (image 10° C. or less) and used to separate exosomes.
  • the filtered culture was frozen and stored in a cryogenic freezer at -60°C or lower, then thawed, and then exosome separation was performed. Thereafter, exosomes were separated from the culture medium using a tangential flow filtration (TFF).
  • TMF tangential flow filtration
  • Example 2 Isolation and purification of exosomes by the TFF method
  • exosomes were separated from the culture medium filtered with a 0.22 ⁇ m filter, concentrated, desalted, and buffer exchanged (diafiltration) using a TFF (Tangential Flow Filtration) method.
  • a filter for the TFF method a cartridge filter (aka hollow fiber filter; purchased from GE Healthcare) or a cassette filter (purchased from Pall or Sartorius or Merck Millipore) was used.
  • TFF filters can be selected by various molecular weight cutoffs (MWCO). The exosomes were selectively separated and concentrated by the selected MWCO, and particles, proteins, lipids, nucleic acids, and low-molecular compounds smaller than the MWCO were removed.
  • a TFF filter of MWCO 100,000 Da (Dalton), 300,000 Da, or 500,000 Da was used. While the culture medium was concentrated to a volume of about 1/100 to 1/25 using the TFF method, substances smaller than MWCO were removed to separate exosomes.
  • the separated and concentrated exosome solution was further desalted and buffer exchanged (diafiltration) using the TFF method.
  • desalting and buffer exchange were performed continuously (continuous diafiltration) or discontinuous diafiltration, and at least 4 times, preferably 6 times to 10 times or more, more preferably with respect to the starting volume. It was carried out using a buffer solution having a volume of at least 12 times.
  • 2% by weight of trehalose dissolved in PBS was added to obtain exosomes having a uniform particle size distribution and high purity.
  • the separated exosomes were measured for particle size and concentration by nanoparticle tracking analysis (NTA; purchased from Malvern).
  • NTA nanoparticle tracking analysis
  • TEM transmission electron microscopy
  • 1C is a result of performing Western blot on the positive markers of exosomes isolated according to the separation method of an embodiment of the present invention, confirming the presence of CD63, CD9, CD81, Alix, and TSG101 markers.
  • Anti-CD63, anti-CD9, anti-CD81, anti-Alix and anti-TSG101 were used as antibodies for each marker, respectively.
  • 1D is a result of performing Western blot on the negative marker of exosomes isolated according to the separation method of an embodiment of the present invention.
  • Anti-GM130 and anti-calnexin were used as antibodies for each marker, respectively.
  • GM130 and Calnexin are negative markers that should not be present in exosomes when characterizing exosomes.
  • FIG. 1D GM130 and Calnexin were found to be present in the lysate of adipocytes, but it was confirmed that they were not present in the exosomes isolated according to the isolation method of one embodiment of the present invention. Accordingly, when synthesizing the results of FIGS. 1C and 1D, it can be seen that exosomes isolated according to the separation method of an embodiment of the present invention are exosomes satisfying the characteristic analysis of positive and negative markers.
  • FIG. 1E shows the presence of CD9, CD63 and CD81 markers as a result of analyzing exosomes isolated according to the isolation method of one embodiment of the present invention using a flow cytometer.
  • an exosome-human CD81 separation/detection kit purchased from ThermoFisher Scientific
  • PE-mouse anti-human CD9 PE-Mouse anti -human CD9
  • PE-mouse anti-human CD63 PE-Mouse anti-human CD63
  • PE-mouse anti-human CD81 PE-mouse anti-human CD81
  • HS68 cells which are human skin fibroblasts
  • the cells were treated with exosomes at different concentrations, and the proliferation rate of the cells was confirmed.
  • the HS68 cells were suspended in DMEM containing 10% FBS, they were dispensed to have a confluency of 80 to 90%, and cultured in a 37°C, 5% CO 2 incubator for 24 hours. After 24 hours, the culture medium was removed, and the exosomes prepared in Example 2 were treated according to concentration, and the cell viability was evaluated while incubating for 24 to 72 hours.
  • the comparative group was based on the number of cells cultured in a general cell culture medium that was not treated with exosomes, and it was confirmed that cytotoxicity by the exosomes of the present invention did not appear within the tested concentration range (FIG. 2).
  • Example 5 Preparation of a combination of stem cell-derived exosomes and polydeoxyribonucleotides
  • Stem cell-derived exosomes prepared as in Example 2 were mixed with polydeoxyribonucleotide (product name: "Lablue", manufactured by ExoCobio Co., Ltd.) and reacted at room temperature for 15 minutes (hereinafter, "PDRN-Exo A little mixed solution (PDRN+EXO)").
  • the polydeoxyribonucleotide was manufactured by Exocobio Co., Ltd. (Seoul, Korea), but is not limited thereto, and various polydeoxyribonucleotides commercially available. Can be used.
  • the concentration of stem cell-derived exosomes in the composition may be adjusted to, for example, approximately 1 to 10,000 ⁇ g/mL in protein concentration.
  • HDF human skin fibroblasts
  • Negative control group an experimental group treated with a scratch-round only serum-free medium
  • PDRN Polydeoxyribonucleotide
  • Stem cell-derived exosomes (EXO): Experimental group in which the stem cell-derived exosomes prepared in Example 2 were diluted in serum-free medium and treated with scratch-ound (final treatment concentration: stem cell-derived exosomes 20 ⁇ g/mL );
  • PDRN-exosomal mixture (PDRN + EXO): Experimental group prepared in Example 5 by diluting the PDRN-exosomal mixture prepared in Example 5 in a serum-free medium and treated with scratch-ound (final treatment concentration: 100 ⁇ g of polydeoxyribonucleotides /mL + stem cell-derived exosomes 20 ⁇ g/mL).
  • Each of the experimental groups was treated with Scratch-Wound, and then HDF was incubated for 12 hours at 5% CO 2 and 37°C.
  • composition comprising the combination of the stem cell-derived exosomes and polydeoxyribonucleotides of the present invention as an active ingredient may be useful for skin regeneration, wound healing, promoting wound healing, improving skin wrinkles and/or improving skin elasticity. It is expected.
  • the whitening effect of the combination of stem cell-derived exosomes and polydeoxyribonucleotides was confirmed through the degree of inhibition of melanin production in mouse melanoma cells.
  • the melanoma cells are cells derived from mouse melanoma, and are cells that secrete a black pigment called melanin. Melanoma cells were dispensed into 48-well plates by 7,000 cells per unit area, and then cultured for 24 hours at 5% CO 2 and 37°C.
  • Control a culture medium mixed with ⁇ -MSH (melanin synthesis stimulator), but an experimental group treated with melanoma cells;
  • Arbutin an experimental group in which 1 mM arbutin, a positive control, was diluted in a culture medium mixed with ⁇ -MSH and treated on melanoma cells;
  • Polydeoxyribonucleotide (PDRN): Experimental group in which polydeoxyribonucleotide was diluted in a culture medium mixed with ⁇ -MSH and treated on melanoma cells (final treatment concentration: polydeoxyribonucleotide 600 ⁇ g/mL );
  • Stem cell-derived exosomes (EXO): Experimental group in which the stem cell-derived exosomes prepared in Example 2 were diluted in a culture medium mixed with ⁇ -MSH and treated on melanoma cells (final treatment concentration: stem cell-derived exo Some 6 mg/mL);
  • PDRN-exosomal mixture (PDRN + EXO): Experimental group in which the PDRN-exosomal mixture prepared in Example 5 was diluted in a culture medium mixed with ⁇ -MSH and treated on melanoma cells (final treatment concentration: Polyde Oxyribonucleotide 600 ⁇ g/mL + stem cell-derived exosomes 6 mg/mL).
  • Each of the experimental groups was treated with melanoma cells, and then the melanoma cells were cultured for 48 hours under conditions of 5% CO 2 and 37°C. Thereafter, the culture solution was recovered and the melanoma cells were washed using a washing solution (PBS; purchased from ThermoFisher).
  • PBS washing solution
  • the recovered culture medium and CCK-8 assay reagent (Dojindo) were mixed, incubated for 1 hour and 30 minutes at 37°C and 5% CO 2 , and then the supernatant was transferred to a 96-well plate and absorbance at 405 nm was measured. I did.
  • the washed melanoma cells were treated with 1 N NaOH (purchased from MerckMillipore) mixed with 10% DMSO, and the plate was sealed at 80°C. By heating for 30 minutes, melanin in the melanoma cells was extracted. The extracted melanin was calculated by measuring the absorbance at 405 nm.
  • a composition comprising a combination of a stem cell-derived exosome and a polydeoxyribonucleotide of the present invention as an active ingredient has a whitening effect, and can be usefully used as an active ingredient of a cosmetic composition for whitening.

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Abstract

La présente invention concerne une composition pour augmenter les effets du blanchiment, de la régénération de la peau, de la cicatrisation des plaies, de la favorisation de la cicatrisation des plaies, de la réduction des rides de la peau et/ou de l'amélioration de l'élasticité de la peau, la composition comprenant, en tant que principe actif, une combinaison d'un exosome dérivé de cellules souches et d'un polydésoxyribonucléotide. La composition de la présente invention comprend, en tant que principe actif, une combinaison d'un exosome dérivé de cellules souches et d'un polydésoxyribonucléotide, et peut ainsi présenter les effets du blanchiment, de la régénération de la peau, de la cicatrisation des plaies, de la favorisation de la cicatrisation des plaies, de la réduction des rides de la peau et/ou de l'amélioration de l'élasticité de la peau, qui sont supérieurs aux effets d'une composition comprenant uniquement un exosome dérivé de cellules souches ou un polydésoxyribonucléotide.
PCT/KR2020/014418 2019-11-15 2020-10-21 Nouvelle composition comprenant des exosomes dérivés de cellules souches et du polydésoxyribonucléotide en tant que principes actifs WO2021096089A1 (fr)

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KR1020200103024A KR102260525B1 (ko) 2019-11-15 2020-08-18 줄기세포 유래 엑소좀과 폴리데옥시리보뉴클레오티드를 유효성분으로 포함하는 새로운 조성물

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