WO2022139439A1 - Pharmaceutical composition for preventing or treating dermatitis, containing multifunctional exosomes derived from progenitor cells - Google Patents
Pharmaceutical composition for preventing or treating dermatitis, containing multifunctional exosomes derived from progenitor cells Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
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- 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
- A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
-
- 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
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95
Definitions
- the present invention relates to a composition for preventing or treating dermatitis using progenitor cell-derived exosomes as an active ingredient, and the composition of the present invention comprises a pharmaceutical composition for preventing, improving, alleviating or treating dermatitis, an ointment, a spray, etc. It relates to a cosmetic composition.
- inflammatory skin diseases Many dermatitis represented by atopic dermatitis are called inflammatory skin diseases. Although the exact pathogenesis of inflammatory skin disease has not been elucidated, it is closely related to the complex dysfunction of immune cells. In addition, histologically, most inflammatory skin diseases show perivascular immune cell infiltration (Ackerman et al., Histologic diagnosis of inflammatory skin diseases, 3rd edition, 2005.).
- Dermatitis treatments developed so far include steroids, antihistamines, and Dupilumab (brand name: Dupixent). Steroids and antihistamines have various side effects and are difficult to use continuously because they are not fundamental treatments for dermatitis.
- Dupixent was released on the market as a monoclonal antibody drug that simultaneously targets the cytokines IL-4 and IL-13, it is a laparoscopic injection. The side effects and various side effects are becoming a problem.
- Dupixent also targets cytokines, it is not a fundamental therapeutic agent that fundamentally modulates cytokine-secreting immune cells.
- cytokines since many drug candidates under clinical research target cytokines like Dupixent, they will face the same problem in the future.
- Exosomes are membrane-enclosed vesicles with a size of 30-150 nm produced by endosomes. Under physiological conditions, cells of all living organisms secrete exosomes. Other extracellular vesicles, such as microvesicals and apoptotic bodies, are similar in size to exosomes, but differ in their biogenesis pathways. The biggest role of exosomes is to transport nucleic acid information such as proteins, lipids, mRNA, microRNA, and long non-coding RNA as a cargo, that is, a material carrier.
- exosomes play an important role in cell-to-cell communication by continuously passing these information back and forth between cells.
- microRNAs are the most important of the substances transported by exosomes, and exosomes are the key to showing physiological or therapeutic efficacy (Zhang et al., Exosomes beyond stem cells for restorative therapy in stroke and neurological injury, Nature reviews neurology, 15, 193-203 (2019)).
- Korean Patent Application Laid-Open Nos. 2020-0084798 and No. 2045188 also disclose a technique for a composition for soothing the skin containing stem cell-derived exosomes as an active ingredient.
- the mesenchymal stem cell-derived exosomes are judged to have limited effects like the mesenchymal stem cells.
- the present invention proceeded to develop a therapeutic agent focusing on the perivascular immune cell infiltration phenomenon seen in most inflammatory skin diseases.
- studies on the regulation of neutrophils and lymphocytes were conducted.
- the ratio of neutrophils to lymphocytes reflects the incidence of atopic dermatitis, and it is reported that the ratio of neutrophils to lymphocytes increases with the increase of the SCORAD (Scoring Atopic Dermatitis) index, which indicates the degree of atopic dermatitis (Jiang et al., Assessment of neutrophil to lymphocyte ratio and platelet to lymphocyte ration in atopic dermatitis patients, Medical science monitor, 23:1340-1346 (2017)).
- the present invention obtained exosomes from progenitor cells that directly inhibit the infiltration of perivascular immune cells and control neutrophils and lymphocytes by a mechanism completely different from the existing dermatitis treatment agents, and the exosomes are different from existing drugs.
- the present invention has been completed by revealing the epochal effect of treating dermatitis.
- an object of the present invention to provide a composition that can prevent or treat dermatitis of a composition comprising a progenitor cell-derived exosome as an active ingredient.
- an object of the present invention is to suppress and remove infiltrated immune cells around blood vessels found in dermatitis to restore the tissue microenvironment and blood vessels, to restore the balance of neutrophils and lymphocytes, and to relieve cellular inflammation fundamentally in dermatitis. It is to provide a composition that can inhibit and alleviate, and can be effectively used for the prevention or treatment of various immune diseases or inflammatory diseases such as dermatitis.
- Another object of the present invention is to provide a pharmaceutical composition for preventing, improving, alleviating or treating dermatitis, an external preparation for skin and a cosmetic composition comprising the exosome.
- Another object of the present invention is to obtain a large amount of sterilized progenitor cell-derived exosomes with high concentration, high purity, and uniform particle size distribution, and to effectively use the obtained stable, safe, and highly active progenitor cell-derived exosomes.
- a pharmaceutical composition for preventing, improving, alleviating or treating dermatitis containing a progenitor cell-derived exosome.
- the progenitor cells are preferably negative for CD24, CD117, and SSEA-1 among surface markers and positive for CD31, CD45RA, CD105, CD146, and TRA-1-60.
- the exosome preferably contains CD44, CD53, and CD98 in addition to CD9, CD63, and CD81 among surface markers.
- the exosome of the present invention passes the progenitor cell culture solution through a filter to filter at least any one of cell debris, wastes, and large particles, sterilizing the filtered culture solution with a sterile filter, and tangentially connecting the sterilized culture solution Separating the exosomes through concentration and diafiltration using a flow filtration method, adding a stabilization buffer to the separated exosomes and diluting them to a starting volume again, and converting the diluted exosomes into a liquid exchange system It can be obtained by performing the step of sterilization by passing through a sterilization filter and storing in a sealed storage container.
- a hollow fiber filter of 10,000 Da, 50,000 Da or 75,000 Da may be used.
- the concentration and diafiltration of the step proceed intermittently or continuously with tangential flow filtration, the concentration is concentrated 10 to 25 times relative to the starting volume, and the diafiltration process is at least 10 times the volume of the concentrated volume. It may include a process of filtration with a buffer having.
- the exosome dilution preferably includes a process of diluting the total volume equal to the starting volume by adding a stabilization buffer of 10 to 25 times to the concentrated and diafiltered volume.
- an external preparation for skin for preventing, improving, alleviating or treating dermatitis comprising a progenitor cell-derived exosome as an active ingredient.
- a cosmetic composition for preventing, improving, alleviating dermatitis comprising a progenitor cell-derived exosome as an active ingredient.
- composition of the present invention inhibits the infiltration of perivascular immune cells commonly found in most dermatitis, restores blood vessels and removes infiltrated immune cells, and regulates neutrophils and lymphocytes, the root causes of inflammation, to reduce the severity of the disease and It can prevent, ameliorate, alleviate or treat dermatitis by reducing the proportion of neutrophils and lymphocytes, and simultaneously inhibiting various inflammatory cytokines.
- composition of the present invention suggests a new mechanism and treatment method that has not been attempted before and cannot be solved by conventional techniques in the R&D approach for treating dermatitis.
- it can be applied to a wide range of dermatitis and effectively suppress and alleviate dermatitis because it directly controls the infiltration of immune cells around blood vessels and immune cells.
- composition of the present invention can be usefully used as a pharmaceutical composition for preventing, improving, alleviating or treating dermatitis, as an external preparation for skin, and as a cosmetic composition.
- the present invention can produce high-concentration, high-purity, uniform particle size and sterilized specific progenitor cell-derived exosomes at a low cost, and the technology can scale-up and GMP application, and the excellent activity of the exosomes of the present invention Since it shows excellent efficacy even at a relatively low concentration compared to the stem cell-derived exosomes reported so far, the commercial use value is very high.
- FIG. 1 is a flowchart illustrating a process for producing a progenitor cell exosome according to an embodiment of the present invention.
- Figure 2 is a graph showing the nanoparticle tracking analysis results showing the particle size distribution of the exosome obtained according to an embodiment of the present invention.
- Figure 3 shows the results of analyzing the exosomes obtained according to an embodiment of the present invention with an electron microscope.
- Figure 5 shows the results of adjusting the neutrophil and lymphocyte ratio through hematological examination after applying the exosome obtained according to one embodiment of the present invention to the rat.
- Figure 6 shows the result of absorbing the exosomes by the skin cells when the exosomes obtained according to an embodiment of the present invention are treated with fluorescence staining to the skin cells.
- Figure 10 shows the result of confirming the degree to which the exosome obtained according to an embodiment of the present invention is absorbed into the human skin.
- FIG. 11 shows a photograph showing that the affected area of dermatitis is remarkably improved after the composition comprising exosomes according to one embodiment of the present invention is applied to human skin.
- the term 'prevention' refers to any process of suppressing, delaying or alleviating the onset of immune diseases or inflammatory diseases, such as dermatitis, due to administration of the pharmaceutical composition of the present invention, should not be construed as meaning
- the term 'improvement' refers to any process in which the symptoms of an immune disease or an inflammatory disease, such as dermatitis, are improved to some extent due to the administration of the pharmaceutical composition of the present invention, and the narrow should not be construed as meaning
- the term 'treatment' refers to all processes in which symptoms of immune diseases or inflammatory diseases, such as dermatitis, are improved or improved due to administration of the pharmaceutical composition of the present invention, and a narrow meaning in which the symptoms of the disease completely disappear should not be construed as
- the present invention provides a technique for mass-producing sterilized exosomes with high concentration, high purity, and uniform particle size that can be clinically applied in preventing, improving, alleviating or treating dermatitis, and a composition comprising the exosomes as an active ingredient, exosomes provides clinically and commercially outstanding breakthrough technology that can provide high-volume, low-cost
- exosome refers to a vesicle composed of a double phospholipid membrane identical to the structure of a cell membrane.
- the present invention suppresses and removes infiltrated immune cells around blood vessels found in dermatitis when applied as a pharmaceutical composition such as an external preparation for skin or an injection as a composition containing, as an active ingredient, specific progenitor cell-derived exosomes, not exosomes previously identified. It restores the tissue microenvironment and blood vessels, restores the balance of neutrophils and lymphocytes, and relieves cellular inflammation to fundamentally suppress and alleviate dermatitis, and has an excellent effect in preventing, improving, alleviating, or treating dermatitis. indicates.
- the exosome composition for treatment of the present invention improves, alleviates, and treats dermatitis by a novel mechanism completely different from all previously known approaches to dermatitis, and it is clearly revealed that these effects are mechanisms and effects that cannot be reached by the prior art.
- HaCaT cells a human skin cell line, were treated with 10% Fetal bovine serum (purchased from ThermoFisher Scientific), 2 mM L-glutamine (purchased from ThermoFisher Scientific), and 1% Antibiotics-antimyotics (purchased from ThermoFisher Scientific). 5% CO 2 in the containing DMEM (purchased from ThermoFisher Scientific) medium, and subcultured at 37 °C conditions.
- a method of culturing progenitor cells to obtain exosomes belonging to the present invention is as follows. Progenitor cells were resuspended in Advanced DMEM medium containing 20% fetal bovine serum (FBS) (purchased from Thermo Fisher Scientific), inoculated in a cell culture dish, and then cultured under 5% CO 2 , 37°C conditions.
- FBS fetal bovine serum
- the exosomes contained in the culture medium have a high concentration and a uniform particle size distribution, so it is easy to separate the exosomes by a tangential flow filtration system, and no additives such as chemicals or small molecule substances are added.
- the culture medium was pumped at a rate of 20ml/min and filtered through a filter (using Sartopore 2 XLG SartoScale 25 (0.8 ⁇ m, 0.2 ⁇ m)) to remove impurities such as cell debris, wastes and large particles, and sterilization was carried out.
- the filter-sterilized culture solution was immediately stored frozen or stored in a -20°C freezer and then thawed, and then exosomes were separated using a tangential flow filtration device (TFF).
- TMF tangential flow filtration device
- concentration and diafiltration were performed by a tangential flow filtration method.
- a hollow fiber filter purchased from Repligen was used as a filter for the tangential flow filtration method.
- various materials and molecular weight cutoff can be selected according to the properties of the exosomes and the purpose of filtration. Exosomes were selectively separated by the selected molecular weight limit, and all particles, proteins, lipids, nucleic acids, and low molecular weight compounds smaller than the selected molecular weight limit were removed.
- mPES Modified Polyethersulfone
- PS Polysulfone
- PES Polyethersulfone
- ME Mated Cellulose Ester
- 1x phosphate buffered saline 1xPBS, free of calcium and magnesium, purchased from Corning
- a stabilization buffer 10 was added to 25 times so that the volume of the exosomes was the same as the starting volume.
- exosomes isolated by the method of the present invention are finally sterilized by filtration with a filter (Sartopore 2 XLG SartoScale 25 (0.8 ⁇ m, 0.2 ⁇ m), and then completely sterile through the port (purchased from Thermo Fisher Scientific) of the disposable assembly airtight container immediately following. was transported in an airtight container.
- a filter Stemopore 2 XLG SartoScale 25 (0.8 ⁇ m, 0.2 ⁇ m)
- the exosomes of the present invention When looking at the size distribution of the exosomes of the present invention, since they are intensively distributed on an average of about 90-100 nm, when the exosomes are used as a dermatitis treatment agent, the skin penetration is excellent and the skin has an extremely high absorption capacity for the exosomes.
- composition containing the exosomes obtained according to the separation method of one embodiment of the present invention is applied to the skin of a normal person for 1 hour to induce inflammation, and then to the normal skin, inflamed skin and inflamed skin to the skin tissue treated with the exosomes.
- the results of performing a biopsy on the Korean peninsula are shown in FIG. 4 , and through the biopsy results of FIG. 4 , the skin tissue after 1 hour treatment with exosomes on the inflamed skin recovered the perivascular immune cell infiltration and infiltrated immunity compared to the inflammatory skin tissue. It can be seen that the cells are removed and blood vessels are restored.
- the external skin preparation or cosmetic composition containing the exosomes obtained according to the separation method of one embodiment of the present invention as an active ingredient as confirmed through the clinical tests as described above, infiltration of peripheral immune cells around blood vessels occurring in most dermatitis It can exhibit preventive, ameliorating, alleviating or therapeutic effects on the symptoms.
- the hair of the cervical region was removed to about 6cmx6cm using an epilator (Max 45, purchased from MOSER), and the area of 5cmx5cm was determined as the transdermal administration site.
- the exosomes were applied to the administration site using a disposable syringe, and the control material was applied, and then an Elizabeth collar (neck collar) was attached to the neck and removed after 6 hours of application of the exosomes.
- the exosomes of the present invention and the control material were administered by 300ul once a day for 13 weeks.
- the rats treated with the exosomes of the present invention and the control material were fasted for at least 18 hours before autopsy, and then anesthetized with isoflurane, an inhaled anesthetic, on the day of the autopsy, and blood was collected from the abdominal aorta.
- blood was collected from the abdominal aorta.
- a hemocytometer purchased from SYSMEX. Referring to FIG. 5, which is the result of measuring the white blood cell percentage, it can be seen that the ratio of neutrophils to lymphocytes in the experimental group is significantly lower in both male and female rats than in the control group.
- the composition of the present invention reduces the ratio of neutrophils to lymphocytes by reducing neutrophils causing dermatitis and increasing lymphocytes.
- the pharmaceutical composition containing the exosome of the present invention can be usefully used as a pharmaceutical composition, external preparation for skin and cosmetic composition for preventing, improving, alleviating or treating dermatitis to inhibit the activity or involvement of immune cells related to inflammation.
- the exosomes were fluorescently stained with DiD cell labeling solution (purchased from ThermoFisher Scientific).
- HaCaT cells were suspended in DMEM medium containing 10% fetal bovine serum and 2mM L-glutamine, and the cells were dispensed in a culture dish to have a density of 30-40%. The next day, the exosomes stained with DiD fluorescence were treated and cultured for 2 to 4 hours. After incubation, they were fixed with 4% PFA, treated with a fluorescent dye, bisbenzimide (Bisbenzimide, Hoechst 33342, purchased from ThermoFisher Scientific), and analyzed with a fluorescence microscope.
- Bisbenzimide Bisbenzimide, Hoechst 33342, purchased from ThermoFisher Scientific
- FIG. 6 shows the results of absorbing exosomes by skin cells when the exosomes obtained according to an embodiment of the present invention are fluorescently stained and treated with skin cells, and the red dots in FIG. It indicates that the moth was absorbed effectively, and the blue color indicates the cell nucleus of the skin cell.
- HaCaT cells were suspended in DMEM medium containing 10% fetal bovine serum and 2mM L-glutamine, and the cells were dispensed in a culture dish to have a density of 30-40%. The next day, after inducing an inflammatory reaction with a fresh medium containing LPS, the exosomes of the present invention were treated and cultured for 24 hours.
- the cell lysate and the culture supernatant were taken and the changes in the exosomes present therein were measured with a nanoparticle tracking analyzer, and iNOS (purchased from Abcam), IL-6, TNF ⁇ , TSLP (ThermoFisher) Changes in TARC (purchased from Scientific) and TARC (purchased from Sigma Aldrich) were measured with an ELISA kit.
- Figure 7 shows a change in the secretion of exosomes secreted by inflammatory skin cells by the exosomes obtained according to an embodiment of the present invention
- Figure 8 is a decrease in iNOS formation by the exosomes obtained according to an embodiment of the present invention Showing the results of confirming the effect
- FIGS. 9a to 9d show the amount of change in (a) IL-6, (b) TNF ⁇ , (c) TSLP, (d) TARC by the exosome obtained according to an embodiment of the present invention Shows the results confirmed by the ELISA kit.
- the control on the x-axis is a normal skin cell negative control group
- LPS is a positive control that induced skin cell inflammatory response with LPS
- LPS+exosome is an experimental group treated with exosomes after induced skin cell inflammatory response with LPS.
- the y-axis represents the concentration of each substance.
- exosomes of the present invention reduce the production of IL-6, TNF ⁇ , TSLP, and TARC cytokines induced by LPS.
- the exosome of the present invention has effective activity for preventing, improving, alleviating or treating dermatitis and reducing the inflammatory response, so the exosome of the present invention is clinically useful for preventing, improving, alleviating or treating dermatitis. It has been shown that it can be used as an active ingredient in a pharmaceutical composition.
- Vybrant DiD cell-labeling solution purchased from ThermoFisher Sicentific
- 5ul of 1mM DiD was mixed with 1ml of the exosome of the present invention and reacted at 37°C for 20 minutes while blocking light.
- 9ml of DMEM (purchased from ThermoFisher Sicentific) medium and 5ml of Total exosome isolation reagent purchased from ThermoFisher Sicentific were mixed and sufficiently reacted, precipitated, and then resuspended in 1ml of 1-fold phosphate buffered saline (purchased from Corning) was used.
- the DiD-exosome was applied to the outer surface of the human skin tissue (purchased from Goma Biotech) in which the DiD-exosome was fixed to a Franz diffusion cell (purchased from BNC Tech) that can be tested for transdermal absorption, and the inside was filled with 1xPBS. To prevent drying of the applied DiD-exosome, it was sealed with foil and allowed to react for 24 hours to allow the DiD-exosome to be absorbed into the skin.
- the liquid on the outer surface of the human skin tissue was absorbed with a wiper (Kimwipes), washed thoroughly with 1x phosphate buffered saline, and after completely removing moisture from the human skin tissue, quickly frozen in a liquid nitrogen tank for 10 minutes did The stratum corneum was removed with a mass of rapidly frozen human skin tissue, and the human skin tissue from which the stratum corneum was removed was observed under a fluorescence microscope. 10 is shown. Through the results of Figure 10, it is shown that the exosomes of the present invention can effectively pass through the human skin barrier and be deeply delivered to the epidermis and dermis. Therefore, the external preparation or cosmetic composition for skin containing the exosomes as an active ingredient is expected to act effectively in preventing, improving, alleviating or treating dermatitis.
- composition containing exosomes obtained according to the method of one embodiment of the present invention was applied to the affected areas (neck, arm, leg, etc.) of 5 severe atopic patients twice a day for 4 to 8 weeks, The condition of the affected area before and after application of the composition of the present invention is shown.
- composition comprising exosomes according to one embodiment of the present invention
- severe itchiness of patients is remarkably relieved, and skin lesions due to dermatitis are remarkably improved.
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Abstract
The present invention relates to a pharmaceutical composition for preventing or treating dermatitis, containing exosomes derived from progenitor cells. The composition of the present invention has excellent effects on the prevention or treatment of dermatitis with respect to visual lesions, histological examination, cytokines and the like. In particular, the composition of the present invention inhibits and removes infiltrated immune cells around blood vessels to restore tissue microenvironment and blood vessels, recovers the balance of neutrophils and lymphocytes and mitigates cell inflammation, and thus fundamentally inhibits and alleviates dermatitis, and is effectively usable in the prevention or treatment of various immune diseases or inflammatory diseases such as dermatitis.
Description
본 발명은 전구세포 유래 엑소좀을 유효성분으로 하는 피부염의 예방 또는 치료용 조성물에 관한 것으로서, 본 발명의 상기 조성물은 피부염의 예방, 개선, 완화 또는 치료용 약학 조성물, 연고, 스프레이 등 피부외용제 및 화장료 조성물에 관한 것이다.The present invention relates to a composition for preventing or treating dermatitis using progenitor cell-derived exosomes as an active ingredient, and the composition of the present invention comprises a pharmaceutical composition for preventing, improving, alleviating or treating dermatitis, an ointment, a spray, etc. It relates to a cosmetic composition.
아토피 피부염을 대표로 하는 많은 피부염은 염증성 피부질환으로 칭해진다. 염증성 피부질환의 명확한 발병기전은 밝혀지지 않았지만 면역세포들의 복합적인 기능장애와 밀접한 연관성이 있다. 또한, 조직학적으로 염증성 피부질환은 대부분 혈관주위 면역세포 침윤현상을 보인다(Ackerman et al., Histologic diagnosis of inflammatory skin diseases, 3rd edition, 2005.).Many dermatitis represented by atopic dermatitis are called inflammatory skin diseases. Although the exact pathogenesis of inflammatory skin disease has not been elucidated, it is closely related to the complex dysfunction of immune cells. In addition, histologically, most inflammatory skin diseases show perivascular immune cell infiltration (Ackerman et al., Histologic diagnosis of inflammatory skin diseases, 3rd edition, 2005.).
이 외에도 염증성 피부염에서는 면역글로불린 IgE 증가와 사이토카인 iNOS, IL-6, TNFα, TSLP(Thymic stromal lymphopoietin), CCL17(TARC) 등의 증가가 관찰되고 이런 요소들이 피부염의 중증도와 연관되어 있고, 피부염에서 자주 보이는 가려움증, 건조증, 피부 병변의 원인이라고 알려져 있다. In addition, increase in immunoglobulin IgE and increase in cytokines iNOS, IL-6, TNFα, TSLP (Thymic stromal lymphopoietin), and CCL17 (TARC) are observed in inflammatory dermatitis. It is known to be the cause of frequent itching, dryness, and skin lesions.
이러한 연구를 바탕으로, 염증성 피부질환 치료제를 개발하고자 하는 노력들이 있다. 현재까지 개발된 피부염 치료제는 스테로이드, 항히스타민제 및 듀플리맙(Dupilumab, 브랜드명 : 듀피젠트) 등이 있다. 스테로이드와 항히스타민제는 다양한 부작용이 보고되어 있을 뿐만 아니라 피부염에 대한 근본적인 치료제가 아니기에 지속적으로 사용하기가 어려운 문제점이 있다. 듀피젠트는 사이토카인인 IL-4와 IL-13을 동시에 타깃하는 단일클론항체 의약품으로 시중에서 출시되었지만 복강경 주사제로 사용 편이성, 연령 및 아토피 피부염의 중증도에 대한 사용제한, 높은 가격에 대한 부담 및 제한적인 효과와 다양한 부작용이 문제가 되고 있다. Based on these studies, there are efforts to develop a therapeutic agent for inflammatory skin disease. Dermatitis treatments developed so far include steroids, antihistamines, and Dupilumab (brand name: Dupixent). Steroids and antihistamines have various side effects and are difficult to use continuously because they are not fundamental treatments for dermatitis. Although Dupixent was released on the market as a monoclonal antibody drug that simultaneously targets the cytokines IL-4 and IL-13, it is a laparoscopic injection. The side effects and various side effects are becoming a problem.
이와 같이, 현재까지 발굴된 피부염 치료제는 모두 증상을 완화시키는 것에 초점을 맞추고 있으며, 근본적인 치료를 위한 접근방식의 치료제는 현재로는 없다. 듀피젠트도 사이토카인을 표적으로 삼지만 근본적으로 사이토카인을 분비하는 면역세포를 조절하는 근본적인 치료제는 아니다. 또한, 임상연구 중에 있는 많은 치료제 후보물질들이 듀피젠트와 같이 사이토카인이 표적이므로 향후 동일한 문제에 직면하게 될 것이다.As such, all of the dermatitis therapeutics discovered so far focus on alleviating symptoms, and there is currently no therapeutic approach for fundamental treatment. Although Dupixent also targets cytokines, it is not a fundamental therapeutic agent that fundamentally modulates cytokine-secreting immune cells. In addition, since many drug candidates under clinical research target cytokines like Dupixent, they will face the same problem in the future.
한편, 전통적인 치료제 연구와 달리 줄기세포로 피부염 치료제를 개발하고자 하는 노력이 진행되고 있다. 하지만, 최근 중간엽 줄기세포 아토피 피부염 치료제가 임상 3상에서 유효성을 입증하는데 실패하면서 줄기세포 관련 피부염 치료제 연구개발에도 적신호가 켜진 상황이다. On the other hand, unlike traditional treatment research, efforts are being made to develop a treatment for dermatitis using stem cells. However, as the recent mesenchymal stem cell atopic dermatitis treatment failed to prove its effectiveness in phase 3 clinical trials, a red light has been turned on for the research and development of stem cell-related dermatitis treatment.
이에 중간엽 줄기세포 분비인자인 엑소좀 등을 이용한 피부염 치료제 개발도 시도되고 있다. 엑소좀은 엔도솜(endosome)에서 생성되는 크기가 30-150nm되는 막으로 둘러싸인 소낭체이다. 생리학적 조건에서 모든 생물체의 세포는 엑소좀를 분비한다. 미세소포(microvesicals), 세포자멸사 소체(apoptotic bodies) 등 기타 세포 외 소낭체들의 크기도 엑소좀과 비슷하지만 생물발생 경로가 다르다. 엑소좀의 가장 큰 역할은 카고(cargo) 즉 물질운반체로서 단백질, 지질, mRNA, microRNA, long non-coding RNA 등 핵산정보를 운반하는 것이다. 생리학적 조건에서 엑소좀은 지속적으로 세포와 세포사이를 오가면서 이런 정보를 전달하여 세포들의 교류에 중요한 역할을 한다. 특히 엑소좀이 운반하는 물질 중에서 microRNA들이 가장 중요하다는 것이 밝혀졌고 엑소좀이 생리학적 또는 치료 효능을 나타낼 수 있는 핵심이다 (Zhang et al., Exosomes beyond stem cells for restorative therapy in stroke and neurological injury, Nature reviews neurology, 15, 193-203 (2019)). 한국공개특허 제2020-0084798호와 제2045188호 등에도 줄기세포 유래 엑소좀을 유효성분으로 포함하는 피부 진정용 조성물에 관한 기술이 제시되어 있다. 그러나, 엑소좀의 특성은 유래 세포에 따라 결정되기에 중간엽 줄기세포 유래 엑소좀은 중간엽 줄기세포와 같이 그 효과가 제한적일 것으로 판단되고 있다. Therefore, attempts are being made to develop a treatment for dermatitis using exosomes, which are mesenchymal stem cell secretion factors. Exosomes are membrane-enclosed vesicles with a size of 30-150 nm produced by endosomes. Under physiological conditions, cells of all living organisms secrete exosomes. Other extracellular vesicles, such as microvesicals and apoptotic bodies, are similar in size to exosomes, but differ in their biogenesis pathways. The biggest role of exosomes is to transport nucleic acid information such as proteins, lipids, mRNA, microRNA, and long non-coding RNA as a cargo, that is, a material carrier. Under physiological conditions, exosomes play an important role in cell-to-cell communication by continuously passing these information back and forth between cells. In particular, it has been found that microRNAs are the most important of the substances transported by exosomes, and exosomes are the key to showing physiological or therapeutic efficacy (Zhang et al., Exosomes beyond stem cells for restorative therapy in stroke and neurological injury, Nature reviews neurology, 15, 193-203 (2019)). Korean Patent Application Laid-Open Nos. 2020-0084798 and No. 2045188 also disclose a technique for a composition for soothing the skin containing stem cell-derived exosomes as an active ingredient. However, since the characteristics of the exosomes are determined depending on the derived cells, the mesenchymal stem cell-derived exosomes are judged to have limited effects like the mesenchymal stem cells.
본 발명은 대부분의 염증성 피부질환에 보이는 혈관주위 면역세포 침윤현상에 초점을 맞추어 치료제 개발을 진행하였다. 그 중에서도, 호중구와 림프구의 조절에 대한 연구를 진행하였다. 호중구와 림프구 비율은 아토피 피부염의 발병도를 반영하고, 아토피 피부염 정도를 나타내는 SCORAD (Scoring Atopic Dermatitis) index의 증가에 따라 호중구와 림프구 비율이 증가하는 것으로 보고되어 있다(Jiang et al., Assessment of neutrophil to lymphocyte ratio and platelet to lymphocyte ration in atopic dermatitis patients, Medical science monitor, 23:1340-1346(2017)). 또한, 호중구와 림프구 비율은 건선과 밀접한 연관성이 있고, 건선 환자에서 피부질환의 범위가 넓고 중증도가 심할수록 혈액 속 호중구와 림프구 비율이 증가하는 것으로 보고되어 있다(B.B.Sen et al., Neutrophil to lymphocyte ratio as a measure of systemic inflammation in psoriasis, Cutaneous and Ocular Toxicology, 33, 223-227(2014)). The present invention proceeded to develop a therapeutic agent focusing on the perivascular immune cell infiltration phenomenon seen in most inflammatory skin diseases. Among them, studies on the regulation of neutrophils and lymphocytes were conducted. The ratio of neutrophils to lymphocytes reflects the incidence of atopic dermatitis, and it is reported that the ratio of neutrophils to lymphocytes increases with the increase of the SCORAD (Scoring Atopic Dermatitis) index, which indicates the degree of atopic dermatitis (Jiang et al., Assessment of neutrophil to lymphocyte ratio and platelet to lymphocyte ration in atopic dermatitis patients, Medical science monitor, 23:1340-1346 (2017)). In addition, it has been reported that the ratio of neutrophils to lymphocytes is closely related to psoriasis, and the ratio of neutrophils to lymphocytes in the blood increases as the range and severity of skin diseases in psoriasis patients increases (B.B.Sen et al., Neutrophil to lymphocyte) ratio as a measure of systemic inflammation in psoriasis, Cutaneous and Ocular Toxicology, 33, 223-227 (2014)).
결과적으로 본 발명은 기존의 피부염 치료제와는 전혀 다른 기전으로 직접적으로 혈관주위 면역세포 침윤현상을 억제하고 호중구와 림프구를 조절하는 엑소좀을 전구세포로부터 획득하였고, 해당 엑소좀이 현존하는 약물과 달리 근본적으로 면역시스템을 조절하여 피부염을 치료하는 획기적인 효과를 밝힘으로써 본 발명을 완성하였다.As a result, the present invention obtained exosomes from progenitor cells that directly inhibit the infiltration of perivascular immune cells and control neutrophils and lymphocytes by a mechanism completely different from the existing dermatitis treatment agents, and the exosomes are different from existing drugs. By fundamentally regulating the immune system, the present invention has been completed by revealing the epochal effect of treating dermatitis.
본 발명의 목적은 전구세포 유래의 엑소좀을 유효성분으로 포함하는 조성물의 피부염 예방 또는 치료할 수 있는 조성물을 제공하는 것을 목적으로 한다. 특히 본 발명의 목적은 피부염에서 발견되는 혈관주위의 침윤된 면역세포를 억제 및 제거하여 조직의 미세환경과 혈관을 복구하고, 호중과 림프구의 평형을 회복시키며, 세포의 염증을 완화하여 근본적으로 피부염을 억제 및 완화시킬 수 있으며, 피부염과 같은 다양한 면역질환 또는 염증 질환의 예방 또는 치료에 효과적으로 사용될 수 있는 조성물을 제공하는데 있다.It is an object of the present invention to provide a composition that can prevent or treat dermatitis of a composition comprising a progenitor cell-derived exosome as an active ingredient. In particular, an object of the present invention is to suppress and remove infiltrated immune cells around blood vessels found in dermatitis to restore the tissue microenvironment and blood vessels, to restore the balance of neutrophils and lymphocytes, and to relieve cellular inflammation fundamentally in dermatitis. It is to provide a composition that can inhibit and alleviate, and can be effectively used for the prevention or treatment of various immune diseases or inflammatory diseases such as dermatitis.
본 발명의 다른 목적은 상기 엑소좀을 포함하는 피부염의 예방, 개선, 완화 또는 치료용 약학 조성물, 피부외용제 및 화장료 조성물을 제공하는데 있다.Another object of the present invention is to provide a pharmaceutical composition for preventing, improving, alleviating or treating dermatitis, an external preparation for skin and a cosmetic composition comprising the exosome.
본 발명의 또 다른 목적은 고농도, 고순도, 입자크기 분포가 균일한 멸균된 전구세포 유래 엑소좀을 대량으로 수득할 수 있고, 이와 같이 수득된 안정적이고 안전하며 활성이 뛰어난 전구세포 유래 엑소좀을 유효성분으로 함유하는 조성물을 제공하는데 있다.Another object of the present invention is to obtain a large amount of sterilized progenitor cell-derived exosomes with high concentration, high purity, and uniform particle size distribution, and to effectively use the obtained stable, safe, and highly active progenitor cell-derived exosomes. To provide a composition containing as an ingredient.
상기 목적을 달성하기 위한 본 발명의 일측면에 따르면, 전구세포 유래 엑소좀을 함유하는 피부염의 예방, 개선, 완화 또는 치료용 약학적 조성물이 제공된다.According to one aspect of the present invention for achieving the above object, there is provided a pharmaceutical composition for preventing, improving, alleviating or treating dermatitis containing a progenitor cell-derived exosome.
여기서, 상기 전구세포는 표면마커 중에서 CD24, CD117, SSEA-1에 대하여 음성이고 CD31, CD45RA, CD105, CD146, TRA-1-60에 대하여 양성인 것이 바람직하다.Here, the progenitor cells are preferably negative for CD24, CD117, and SSEA-1 among surface markers and positive for CD31, CD45RA, CD105, CD146, and TRA-1-60.
또한, 상기 엑소좀은 표면마커 중에서 CD9, CD63, CD81 외에도 CD44, CD53, CD98을 함유하는 것이 바람직하다.In addition, the exosome preferably contains CD44, CD53, and CD98 in addition to CD9, CD63, and CD81 among surface markers.
그리고, 본 발명의 엑소좀은 전구세포 배양액을 필터에 통과시켜 세포 잔해물, 노폐물, 거대 입자 중 적어도 어느 하나를 여과하는 단계, 상기 여과된 배양액을 멸균 필터로 멸균하는 단계, 상기 멸균된 배양액을 접선흐름여과방법을 이용하여 농축과 정용여과과정을 통해 엑소좀을 분리하는 단계, 상기 분리된 엑소좀에 안정화 버퍼를 추가하여 다시 시작 부피가 되도록 희석하는 단계 및 상기 희석된 엑소좀을 액체 교환 시스템으로 멸균 필터에 통과시켜 멸균한 후 밀봉된 저장용기에 저장하는 단계를 수행하여 수득될 수 있다.And, the exosome of the present invention passes the progenitor cell culture solution through a filter to filter at least any one of cell debris, wastes, and large particles, sterilizing the filtered culture solution with a sterile filter, and tangentially connecting the sterilized culture solution Separating the exosomes through concentration and diafiltration using a flow filtration method, adding a stabilization buffer to the separated exosomes and diluting them to a starting volume again, and converting the diluted exosomes into a liquid exchange system It can be obtained by performing the step of sterilization by passing through a sterilization filter and storing in a sealed storage container.
여기서, 접선흐름여과를 위한 분자량 한계치를 얻기 위하여 10,000Da, 50,000Da 또는 75,000Da의 중공사 필터를 사용할 수 있다.Here, in order to obtain a molecular weight limit value for tangential flow filtration, a hollow fiber filter of 10,000 Da, 50,000 Da or 75,000 Da may be used.
그리고, 상기 단계의 농축과 정용여과는 접선흐름여과를 단속 또는 연속적으로 진행하고, 농축은 시작 부피에 대하여 10배 내지 25배 농축하고, 정용여과 과정은 농축된 부피에 대하여 적어도 10배의 부피를 갖는 버퍼로 여과하는 과정을 포함할 수 있다.And, the concentration and diafiltration of the step proceed intermittently or continuously with tangential flow filtration, the concentration is concentrated 10 to 25 times relative to the starting volume, and the diafiltration process is at least 10 times the volume of the concentrated volume. It may include a process of filtration with a buffer having.
또한, 상기 엑소좀 희석은 농축 및 정용여과된 부피에 대하여 10배 내지 25배의 안정화 버퍼를 첨가하여 전체 부피가 시작 부피와 같도록 희석하는 과정을 포함하는 것이 바람직하다.In addition, the exosome dilution preferably includes a process of diluting the total volume equal to the starting volume by adding a stabilization buffer of 10 to 25 times to the concentrated and diafiltered volume.
상기 목적을 달성하기 위한 본 발명의 다른 일측면에 따르면, 전구세포 유래 엑소좀을 유효성분으로 포함하는, 피부염의 예방, 개선, 완화 또는 치료용 피부외용제가 제공된다.According to another aspect of the present invention for achieving the above object, there is provided an external preparation for skin for preventing, improving, alleviating or treating dermatitis, comprising a progenitor cell-derived exosome as an active ingredient.
상기 목적을 달성하기 위한 본 발명의 또 다른 일측면에 따르면, 전구세포 유래 엑소좀을 유효성분으로 포함하는, 피부염의 예방, 개선, 완화용 화장료 조성물이 제공된다.According to another aspect of the present invention for achieving the above object, there is provided a cosmetic composition for preventing, improving, alleviating dermatitis, comprising a progenitor cell-derived exosome as an active ingredient.
본 발명의 조성물은 대부분의 피부염에서 공통으로 발견되는 혈관주위 면역세포 침윤현상을 억제하고, 혈관을 복구하고 침윤된 면역세포를 제거하며, 염증의 근본적인 원인인 호중구와 림프구를 조절하여 질병의 중증도와 연관성을 보이는 호중구와 림프구 비율을 감소시키며, 동시에 다양한 염증성 사이토카인을 억제하여 피부염의 예방, 개선, 완화 또는 치료할 수 있다. The composition of the present invention inhibits the infiltration of perivascular immune cells commonly found in most dermatitis, restores blood vessels and removes infiltrated immune cells, and regulates neutrophils and lymphocytes, the root causes of inflammation, to reduce the severity of the disease and It can prevent, ameliorate, alleviate or treat dermatitis by reducing the proportion of neutrophils and lymphocytes, and simultaneously inhibiting various inflammatory cytokines.
특히, 본 발명의 조성물은 피부염의 치료제 연구개발 접근법에 있어 종래에 시도한 적이 없고 종래의 기술로 해결이 되지 않는 새로운 기전과 치료법을 제시한다. 또한, 혈관주위 면역세포 침윤현상 및 면역세포를 직접 제어하기에 광범위한 피부염에 적용이 가능하고 효과적으로 피부염을 억제 및 완화할 수 있다. In particular, the composition of the present invention suggests a new mechanism and treatment method that has not been attempted before and cannot be solved by conventional techniques in the R&D approach for treating dermatitis. In addition, it can be applied to a wide range of dermatitis and effectively suppress and alleviate dermatitis because it directly controls the infiltration of immune cells around blood vessels and immune cells.
따라서, 본 발명의 조성물은 피부염의 예방, 개선, 완화 또는 치료용 약학조성물, 피부외용제 및 화장료 조성물로서 유용하게 사용될 수 있다. Therefore, the composition of the present invention can be usefully used as a pharmaceutical composition for preventing, improving, alleviating or treating dermatitis, as an external preparation for skin, and as a cosmetic composition.
또한, 본 발명은 고농도, 고순도, 입자크기가 균일하고 멸균된 특이성 전구세포 유래 엑소좀을 저가로 대량으로 생산할 수 있고 해당 기술은 스케일업과 GMP 적용이 가능하며, 본 발명의 엑소좀의 뛰어난 활성으로 현재까지 보고된 줄기세포 유래 엑소좀보다 상대적으로 낮은 농도에서도 뛰어난 효능을 보이기에 상업적으로 활용가치가 굉장히 높다.In addition, the present invention can produce high-concentration, high-purity, uniform particle size and sterilized specific progenitor cell-derived exosomes at a low cost, and the technology can scale-up and GMP application, and the excellent activity of the exosomes of the present invention Since it shows excellent efficacy even at a relatively low concentration compared to the stem cell-derived exosomes reported so far, the commercial use value is very high.
도 1은 본 발명의 일 구체예에 따라 전구세포 엑소좀을 제조하는 과정을 설명하는 흐름도이다.1 is a flowchart illustrating a process for producing a progenitor cell exosome according to an embodiment of the present invention.
도 2는 본 발명의 일 구체예에 따라 얻어진 엑소좀의 입자크기 분포를 나타내는 나노입자 추적 분석결과를 도시한 그래프이다. Figure 2 is a graph showing the nanoparticle tracking analysis results showing the particle size distribution of the exosome obtained according to an embodiment of the present invention.
도 3은 본 발명의 일 구체예에 따라 얻어진 엑소좀을 전자현미경으로 분석한 결과를 나타낸 것이다.Figure 3 shows the results of analyzing the exosomes obtained according to an embodiment of the present invention with an electron microscope.
도 4는 본 발명의 일 구체예에 따라 얻어진 엑소좀을 사람피부에 적용한 후 피부조직 생검 수행 결과를 정상피부 및 염증성 피부와 비교한 것이다.4 is a comparison of the skin tissue biopsy results after applying the exosome obtained according to an embodiment of the present invention to human skin with normal skin and inflammatory skin.
도 5는 본 발명의 일 구체예에 따라 얻어진 엑소좀을 랫드에 적용한 후 혈액학적 검사를 통해 호중구와 림프구 비율이 조절된 결과를 나타낸 것이다.Figure 5 shows the results of adjusting the neutrophil and lymphocyte ratio through hematological examination after applying the exosome obtained according to one embodiment of the present invention to the rat.
도 6는 본 발명의 일 구체예에 따라 얻어진 엑소좀을 형광염색하여 피부세포에 처리하였을 때 피부세포가 엑소좀을 흡수한 결과를 나타낸 것이다.Figure 6 shows the result of absorbing the exosomes by the skin cells when the exosomes obtained according to an embodiment of the present invention are treated with fluorescence staining to the skin cells.
도 7은 본 발명의 일 구체예에 따라 얻어진 엑소좀에 의한 염증성 피부세포가 분비하는 엑소좀의 분비변화를 나타낸 것이다.7 shows a change in the secretion of exosomes secreted by inflammatory skin cells by the exosomes obtained according to an embodiment of the present invention.
도 8은 본 발명의 일 구체예에 따라 얻어진 엑소좀에 의한 iNOS 형성 감소 효과를 확인한 결과를 나타낸 것이다.8 shows the results of confirming the effect of reducing iNOS formation by the exosomes obtained according to an embodiment of the present invention.
도 9은 본 발명의 일 구체예에 따라 얻어진 엑소좀에 의한 (a) IL-6, (b) TNFα, (c) TSLP, (d) TARC의 변화량을 ELISA 키트로 확인한 결과를 나타낸 것이다.9 shows the results of confirming the amount of change in (a) IL-6, (b) TNFα, (c) TSLP, and (d) TARC by the exosomes obtained according to an embodiment of the present invention with an ELISA kit.
도 10은 본 발명의 일 구체예에 따라 얻어진 엑소좀이 인체 피부에 흡수되는 정도를 확인한 결과를 나타낸 것이다.Figure 10 shows the result of confirming the degree to which the exosome obtained according to an embodiment of the present invention is absorbed into the human skin.
도 11은 본 발명의 일 구체예에 다른 엑소좀을 포함하는 조성물을 사람의 피부에 도포한 후 피부염의 환부가 현저히 개선되는 것을 보여주는 사진을 나타낸 것이다.11 shows a photograph showing that the affected area of dermatitis is remarkably improved after the composition comprising exosomes according to one embodiment of the present invention is applied to human skin.
본 발명에 기재된 실시예 및 도면에 도시된 구성은 본 발명의 바람직한 실시예에 불과할 뿐이고, 본 발명의 기술적 사상을 모두 표현하는 것은 아니므로, 본 발명의 권리범위는 본문에 설명된 실시예 및 도면에 의하여 제한되는 것으로 해석되어서는 아니 된다. 즉, 실시예는 다양한 변경이 가능하고 여러 가지 형태를 가질 수 있으므로 본 발명의 권리범위는 기술적 사상을 실현할 수 있는 균등물들을 포함하는 것으로 이해되어야 한다. 또한, 본 발명에서 제시된 목적 또는 효과는 특정 실시예가 이를 전부 포함하여야 한다거나 그러한 효과만을 포함하여야 한다는 의미는 아니므로, 본 발명의 권리범위는 이에 의하여 제한되는 것으로 이해되어서는 아니 될 것이다.The configuration shown in the embodiments and drawings described in the present invention is only a preferred embodiment of the present invention, and does not express all the technical ideas of the present invention, so the scope of the present invention is the embodiment and drawings described in the text should not be construed as being limited by That is, since the embodiment may have various changes and may have various forms, it should be understood that the scope of the present invention includes equivalents capable of realizing the technical idea. In addition, since the object or effect presented in the present invention does not mean that a specific embodiment should include all of them or only such effects, it should not be understood that the scope of the present invention is limited thereby.
여기서 사용되는 모든 용어들은 다르게 정의되지 않는 한, 본 발명이 속하는 분야에서 통상의 지식을 가진 자에 의해 일반적으로 이해되는 것과 동일한 의미를 가진다. 일반적으로 사용되는 사전에 정의되어 있는 용어들은 관련 기술의 문맥상 가지는 의미와 일치하는 것으로 해석되어야 하며, 본 발명에서 명백하게 정의하지 않는 이상적이거나 과도하게 형식적인 의미를 지니는 것으로 해석될 수 없다.All terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs, unless otherwise defined. Terms defined in general used in the dictionary should be interpreted as having the meaning consistent with the context of the related art, and cannot be interpreted as having an ideal or excessively formal meaning not explicitly defined in the present invention.
본 발명에 있어서, ‘예방’이란 용어는 본 발명의 약학조성물의 투여로 인해 면역질환 또는 염증질환, 예컨대 피부염의 발병을 억제 또는 지연시키거나 완화시키는 모든 과정을 나타내며, 질병의 발생을 차단하는 좁은 의미로 해석되어서는 안된다. In the present invention, the term 'prevention' refers to any process of suppressing, delaying or alleviating the onset of immune diseases or inflammatory diseases, such as dermatitis, due to administration of the pharmaceutical composition of the present invention, should not be construed as meaning
본 발명에 있어서, ‘개선’이란 용어는 본 발명의 약학조성물의 투여로 인해 면역질환 또는 염증질환, 예컨대 피부염의 증세가 어느 정도 개선되는 모든 과정을 나타내며, 해당 질환의 증상이 어느 정도 나아지는 좁은 의미로 해석되어서는 안된다.In the present invention, the term 'improvement' refers to any process in which the symptoms of an immune disease or an inflammatory disease, such as dermatitis, are improved to some extent due to the administration of the pharmaceutical composition of the present invention, and the narrow should not be construed as meaning
본 발명에 있어서, ‘치료’란 용어는 본 발명의 약학조성물의 투여로 인해 면역질환이나 염증질환, 예컨대 피부염의 증세가 호전되거나 나아지는 모든 과정을 나태내며, 해당 질환의 증상이 완전히 사라지는 좁은 의미로 해석되어서는 안된다. In the present invention, the term 'treatment' refers to all processes in which symptoms of immune diseases or inflammatory diseases, such as dermatitis, are improved or improved due to administration of the pharmaceutical composition of the present invention, and a narrow meaning in which the symptoms of the disease completely disappear should not be construed as
본 발명은 피부염 예방, 개선, 완화 또는 치료에 있어서 임상적 적용이 가능한 고농도, 고순도, 입자크기가 균일한 멸균된 엑소좀을 대량생산하는 기술과 상기 엑소좀을 유효성분으로 포함하는 조성물을 엑소좀을 저가로 대량으로 제공할 수 있는 임상 및 상업적으로 뛰어난 획기적인 기술을 제공한다. The present invention provides a technique for mass-producing sterilized exosomes with high concentration, high purity, and uniform particle size that can be clinically applied in preventing, improving, alleviating or treating dermatitis, and a composition comprising the exosomes as an active ingredient, exosomes provides clinically and commercially outstanding breakthrough technology that can provide high-volume, low-cost
본 명세서에서 사용된 용어, “엑소좀(exosome)”은 세포막의 구조와 동일한 이중인지질막으로 이루어진 소낭체를 의미한다. As used herein, the term “exosome” refers to a vesicle composed of a double phospholipid membrane identical to the structure of a cell membrane.
피부염의 연구에 따르면, 위에서 언급한 바와 같이, 줄기세포 또는 줄기세포 유래 엑소좀 효능에 대한 연구가 보고되어 있으나, 중간엽 줄기세포 같은 경우 임상 3상에서 효능 입증에 실패하여 줄기세포의 효능에 대해 부정적인 상황이며, 엑소좀의 특성은 유래 세포에 따라 결정되기에 줄기세포의 임상결과를 통해 줄기세포가 분비하는 엑소좀 또한 피부염에 대한 효능이 제한적일 것으로 판단되고 있다. According to the study of dermatitis, as mentioned above, studies on the efficacy of stem cells or stem cell-derived exosomes have been reported. situation, and the characteristics of exosomes are determined depending on the derived cells, so it is judged that the exosomes secreted by stem cells have limited efficacy against dermatitis through the clinical results of stem cells.
본 발명은 기존에 밝혀진 엑소좀이 아닌 특이성 전구세포 유래 엑소좀을 유효성분으로 하는 조성물로서 피부외용제 또는 주사제와 같은 약학조성물로 적용될 때, 피부염에서 발견되는 혈관주위의 침윤된 면역세포를 억제 및 제거하여 조직의 미세환경과 혈관을 복구하고, 호중과 림프구의 평형을 회복시키며, 세포의 염증을 완화하여 근본적으로 피부염을 억제 및 완화시킬 수 있으며 피부염의 예방, 개선, 완화, 또는 치료에 뛰어난 효과를 나타낸다. 따라서, 본 발명의 치료용 엑소좀 조성물은 종래에 알려진 모든 피부염 접근방법과 전혀 다른 새로운 기전으로 피부염을 개선, 완화, 치료하여 이러한 효능은 종래기술로는 전혀 도달할 수 없는 기전과 효과임을 명백히 밝혀둔다.The present invention suppresses and removes infiltrated immune cells around blood vessels found in dermatitis when applied as a pharmaceutical composition such as an external preparation for skin or an injection as a composition containing, as an active ingredient, specific progenitor cell-derived exosomes, not exosomes previously identified. It restores the tissue microenvironment and blood vessels, restores the balance of neutrophils and lymphocytes, and relieves cellular inflammation to fundamentally suppress and alleviate dermatitis, and has an excellent effect in preventing, improving, alleviating, or treating dermatitis. indicates. Therefore, the exosome composition for treatment of the present invention improves, alleviates, and treats dermatitis by a novel mechanism completely different from all previously known approaches to dermatitis, and it is clearly revealed that these effects are mechanisms and effects that cannot be reached by the prior art. put
이하 본 발명을 실시예를 통해 더욱 상세히 설명한다. 단, 하기 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 권리범위를 제한하거나 한정하는 것이 아니다. Hereinafter, the present invention will be described in more detail through examples. However, the following examples are merely illustrative of the content of the present invention and do not limit or limit the scope of the present invention.
(1) 세포의 배양(1) cell culture
인간 피부세포주인 HaCaT 세포를 10% 우태아혈청(Fetal bovine serum, ThermoFisher Scientific에서 구매), 2mM L-글루타민(ThermoFisher Scientific에서 구매) 및 1% 항생제-항진균제(Antibiotics-antimyotics, ThermoFisher Scientific에서 구매)가 함유된 DMEM(ThermoFisher Scientific에서 구매)배지에 5% CO2, 37℃조건에서 계대배양하였다. HaCaT cells, a human skin cell line, were treated with 10% Fetal bovine serum (purchased from ThermoFisher Scientific), 2 mM L-glutamine (purchased from ThermoFisher Scientific), and 1% Antibiotics-antimyotics (purchased from ThermoFisher Scientific). 5% CO 2 in the containing DMEM (purchased from ThermoFisher Scientific) medium, and subcultured at 37 °C conditions.
본 발명에 속하는 엑소좀을 획득하기 위한 전구세포의 배양방법은 아래와 같다. 전구세포를 20% 우태아혈청(FBS)(Thermo Fisher Scientific에서 구매)을 함유한 Advanced DMEM배지로 재부유시켜 세포배양접시에 접종한 후 5% CO2, 37℃ 조건 하에서 배양하였다. 75% 이상의 세포가 부착된 후, 1배 인산완충생리식염수(1xPBS, 칼슘과 마그네슘이 없음, Corning에서 구매)로 충분히 세척후, 10% 녹아웃 혈청 대체물(Knockout serum replacement), 2mM L-글루타민의 디펩타이드 형태(L-alanyl-L-glutamine dipeptide)를 함유한 DMEM/F-12배지로 교체하여 그 배양액을 회수하였다.A method of culturing progenitor cells to obtain exosomes belonging to the present invention is as follows. Progenitor cells were resuspended in Advanced DMEM medium containing 20% fetal bovine serum (FBS) (purchased from Thermo Fisher Scientific), inoculated in a cell culture dish, and then cultured under 5% CO 2 , 37°C conditions. After more than 75% of cells have adhered, wash thoroughly with 1x phosphate-buffered saline (1xPBS, free of calcium and magnesium, purchased from Corning), and then wash with 10% knockout serum replacement, 2mM L-glutamine The culture medium was recovered by replacing it with DMEM/F-12 medium containing the peptide form (L-alanyl-L-glutamine dipeptide).
(2) 접선흐름여과를 이용한 엑소좀 분리(2) Separation of exosomes using tangential flow filtration
상기 배양액에 함유된 엑소좀은 농도가 높고, 입자크기 분포가 균일하기에 접선흐름여과 시스템으로 엑소좀을 분리하기 용이하고 화학물질 또는 소분자 물질 등 첨가물을 추가하지 않았다. 우선 배양액을 20ml/min의 속도로 펌핑하여 필터(Sartopore 2 XLG SartoScale 25(0.8μm, 0.2μm) 사용)로 여과하여 세포 잔해물, 노폐물 및 거대입자 등 불순물을 제거하고 멸균까지 진행하였다. 여과멸균된 배양액은 즉시 또는 -20℃ 냉동고에 동결보관하였다가 해동시킨 후, 접선흐름여과장치(TFF: Tangential Flow Filtration)를 이용하여 엑소좀을 분리하였다. The exosomes contained in the culture medium have a high concentration and a uniform particle size distribution, so it is easy to separate the exosomes by a tangential flow filtration system, and no additives such as chemicals or small molecule substances are added. First, the culture medium was pumped at a rate of 20ml/min and filtered through a filter (using Sartopore 2 XLG SartoScale 25 (0.8μm, 0.2μm)) to remove impurities such as cell debris, wastes and large particles, and sterilization was carried out. The filter-sterilized culture solution was immediately stored frozen or stored in a -20°C freezer and then thawed, and then exosomes were separated using a tangential flow filtration device (TFF).
상기 여과멸균된 배양액에서 엑소좀을 분리하기 위하여 접선흐름여과방법으로 농축 및 정용여과(diafiltration)를 진행하였다. 접선흐름여과방법을 위한 필터로는 중공사 필터(Repligen에서 구매)를 사용하였다. 중공사 필터는 엑소좀의 성질과 여과 목적에 따라 다양한 재질과 분자량 한계치(Molecular weight cutoff)을 선택할 수 있다. 선택된 분자량 한계치에 의해 선별적으로 엑소좀을 분리하였고 선택된 분자량 한계치보다 작은 입자나 단백질, 지질, 핵산, 저분자 화합물 등은 모두 제거하였다. 엑소좀을 분리하기 위한 중공 섬유로는 mPES(Modified Polyethersulfone), PS(Polysulfone), PES(Polyethersulfone) 또는 ME(Mixed Cellulose Ester)재질을 사용하였고, 분자량 한계치는 10,000Da, 50,000Da 또는 75,000Da를 사용하였다. 접선흐름 여과방법을 이용한 농축 및 정용여과(diafiltration)과정은 단속 또는 연속적으로 수행하였고 농축은 시작 부피에 대하여 10배 내지 25배 농축하고, 정용여과는 농축한 부피에 대하여 적어도 10배, 바람직하게는 12배 내지 15배 이상의 부피를 갖는 완충용액을 이용하여 수행하였다. 완충용액은 1배 인산완충생리식염수(1xPBS, 칼슘과 마그네슘이 없음, Corning에서 구매)를 사용하였고 어떠한 화학물질 또는 저분자 물질도 첨가하지 않았다. 접선흐름여과방법으로 농축과 정용여과를 거친 엑소좀은 안정화 버퍼를 10배 내지 25배 추가하여 엑소좀의 부피가 시작 부피와 같게 되도록 희석하였다.In order to separate the exosomes from the filter-sterilized culture medium, concentration and diafiltration were performed by a tangential flow filtration method. A hollow fiber filter (purchased from Repligen) was used as a filter for the tangential flow filtration method. For the hollow fiber filter, various materials and molecular weight cutoff can be selected according to the properties of the exosomes and the purpose of filtration. Exosomes were selectively separated by the selected molecular weight limit, and all particles, proteins, lipids, nucleic acids, and low molecular weight compounds smaller than the selected molecular weight limit were removed. As hollow fibers for separating exosomes, mPES (Modified Polyethersulfone), PS (Polysulfone), PES (Polyethersulfone) or ME (Mixed Cellulose Ester) materials were used, and the molecular weight limit was 10,000 Da, 50,000 Da or 75,000 Da. did Concentration and diafiltration using the tangential flow filtration method were performed intermittently or continuously, and the concentration was concentrated 10 to 25 times relative to the starting volume, and diafiltration was performed at least 10 times relative to the concentrated volume, preferably This was performed using a buffer solution having a volume of 12 to 15 times or more. As the buffer solution, 1x phosphate buffered saline (1xPBS, free of calcium and magnesium, purchased from Corning) was used, and no chemicals or low molecular weight substances were added. The exosomes subjected to concentration and diafiltration by the tangential flow filtration method were diluted by adding a stabilization buffer 10 to 25 times so that the volume of the exosomes was the same as the starting volume.
본 발명의 방법으로 분리한 엑소좀을 최종적으로 필터(Sartopore 2 XLG SartoScale 25(0.8μm, 0.2μm)로 여과 멸균하고 바로 이어지는 일회용 조립 밀폐용기의 포트(Thermo Fisher Scientific에서 구매)를 통해 완전한 무균상태가 보장된 밀폐용기에 수송하였다. The exosomes isolated by the method of the present invention are finally sterilized by filtration with a filter (Sartopore 2 XLG SartoScale 25 (0.8 μm, 0.2 μm), and then completely sterile through the port (purchased from Thermo Fisher Scientific) of the disposable assembly airtight container immediately following. was transported in an airtight container.
(3) 분리된 엑소좀의 특성 분석(3) Characterization of isolated exosomes
본 발명의 엑소좀의 크기분포와 모양을 확인하기 위하여 나노입자 추적 분석기(NanoSight NS300 제품, Malvern Panalytical에서 구매)와 전자현미경으로 분석하였다. 나노입자 추적 분석기로 크기분포를 분석한 결과는 도 2와 같고, 음성염색(negative staining)법으로 염색하여 분석한 전자현미경 사진결과는 도 3과 같다.In order to confirm the size distribution and shape of the exosomes of the present invention, it was analyzed with a nanoparticle tracking analyzer (NanoSight NS300 product, purchased from Malvern Panalytical) and an electron microscope. The result of analyzing the size distribution with the nanoparticle tracking analyzer is shown in FIG. 2 , and the result of the electron microscope image analyzed by staining with a negative staining method is shown in FIG. 3 .
본 발명의 엑소좀의 크기분포를 볼 때 평균 약 90-100nm에 집중적으로 분포되어 있기에 상기 엑소좀을 피부염 치료제로 사용할 경우 피부 침투력이 뛰어나고 피부가 상기 엑소좀에 대한 흡수능력이 월등히 높다.When looking at the size distribution of the exosomes of the present invention, since they are intensively distributed on an average of about 90-100 nm, when the exosomes are used as a dermatitis treatment agent, the skin penetration is excellent and the skin has an extremely high absorption capacity for the exosomes.
(4) 사람 피부조직 생검을 통한 엑소좀 효과 분석(4) Analysis of the effect of exosomes through human skin tissue biopsy
본 발명의 일 구체예의 분리방법에 따라 수득된 엑소좀을 함유하는 조성물을 정상인의 피부에 염증을 유발하여 1시간 도포한 후 정상피부, 염증성 피부 및 염증성 피부에 상기 엑소좀을 처리한 피부조직에 대한 생검을 실시한 결과가 도 4에 나타나 있으며, 도 4의 생검 결과를 통해 염증성 피부에 엑소좀을 1시간 처리한 후의 피부 조직은 염증성 피부 조직에 비해 혈관주위 면역세포 침윤현상이 회복되고 침윤된 면역세포가 제거되며 혈관이 복구되었음을 알 수 있다.The composition containing the exosomes obtained according to the separation method of one embodiment of the present invention is applied to the skin of a normal person for 1 hour to induce inflammation, and then to the normal skin, inflamed skin and inflamed skin to the skin tissue treated with the exosomes. The results of performing a biopsy on the Korean peninsula are shown in FIG. 4 , and through the biopsy results of FIG. 4 , the skin tissue after 1 hour treatment with exosomes on the inflamed skin recovered the perivascular immune cell infiltration and infiltrated immunity compared to the inflammatory skin tissue. It can be seen that the cells are removed and blood vessels are restored.
따라서, 본 발명의 일 구체예의 분리방법에 따라 수득된 엑소좀을 유효성분으로 함유하는 피부외용제 또는 화장료 조성물은 상기와 같은 임상시험을 통해 확인되는 바와 같이 대부분의 피부염에서 발생하는 혈관주위 면역세포 침윤현상에 대해 예방, 개선, 완화 또는 치료효과를 나타낼 수 있다.Therefore, the external skin preparation or cosmetic composition containing the exosomes obtained according to the separation method of one embodiment of the present invention as an active ingredient, as confirmed through the clinical tests as described above, infiltration of peripheral immune cells around blood vessels occurring in most dermatitis It can exhibit preventive, ameliorating, alleviating or therapeutic effects on the symptoms.
(5) 랫드를 이용한 엑소좀 효과 분석(5) Analysis of the effect of exosomes using rats
6주령의 암수 랫드 각각 20마리를 아래와 같이 2군으로 분류하였다.Twenty six-week-old male and female rats each were classified into 2 groups as follows.
- 대조군: 정상 대조군- Control: Normal Control
- 실험군: 경배부 피모를 제모기(Max 45, MOSER에서 구매)를 이용하여 약 6cmx6cm로 제모하였고 5cmx5cm 넓이를 경피투여 부위로 결정하였다. 투여부위에 일회용 주사기를 이용하여 엑소좀을 도포하고, 대조군은 대조물질을 도포한 후, 엘리자베스 칼라(넥 칼라)를 경부에 장착하고 엑소좀 도포 6시간 경과 후에 이를 제거하였다. 본 발명의 엑소좀과 대조물질은 300ul씩 1일 1회 13주간 투여하였다.- Experimental group: The hair of the cervical region was removed to about 6cmx6cm using an epilator (Max 45, purchased from MOSER), and the area of 5cmx5cm was determined as the transdermal administration site. The exosomes were applied to the administration site using a disposable syringe, and the control material was applied, and then an Elizabeth collar (neck collar) was attached to the neck and removed after 6 hours of application of the exosomes. The exosomes of the present invention and the control material were administered by 300ul once a day for 13 weeks.
본 발명의 엑소좀과 대조물질을 처리한 랫드에 대하여 부검 전 약 18시간 이상 절식시킨 후, 부검일에 흡입마취제인 아이소플루레인(isoflurane)으로 마취하여 배대동맥으로부터 혈액을 채취하였다. 혈액학적 검사는 채취한 혈액 약 1ml를 EDTA 함유 튜브에 넣은 후, 혈구분석기(SYSMEX에서 구매)로 백혈구 백분율을 측정하였다. 백혈구 백분율을 측정 결과인 도 5를 살펴보면, 수컷과 암컷 랫드 모두 대조군에 비하여 실험군에서 림프구 대비 호중구의 비율이 상당히 낮음을 알 수 있다.The rats treated with the exosomes of the present invention and the control material were fasted for at least 18 hours before autopsy, and then anesthetized with isoflurane, an inhaled anesthetic, on the day of the autopsy, and blood was collected from the abdominal aorta. For hematological examination, about 1 ml of the collected blood was placed in a tube containing EDTA, and the white blood cell percentage was measured with a hemocytometer (purchased from SYSMEX). Referring to FIG. 5, which is the result of measuring the white blood cell percentage, it can be seen that the ratio of neutrophils to lymphocytes in the experimental group is significantly lower in both male and female rats than in the control group.
상기와 같은 결과들로부터 본 발명의 조성물은 피부염의 원인이 되는 호중구를 감소시키고 림프구를 증가시켜 호중와 림프구의 비율을 감소시킨다는 것을 알 수 있다. 본 발명의 엑소좀을 함유하는 약학조성물은 염증 관련 면역세포의 활성 내지는 관여를 저해하기에 피부염의 예방, 개선, 완화 또는 치료용 약학조성물, 피부외용제 및 화장료 조성물로서 유용하게 사용될 수 있다.From the above results, it can be seen that the composition of the present invention reduces the ratio of neutrophils to lymphocytes by reducing neutrophils causing dermatitis and increasing lymphocytes. The pharmaceutical composition containing the exosome of the present invention can be usefully used as a pharmaceutical composition, external preparation for skin and cosmetic composition for preventing, improving, alleviating or treating dermatitis to inhibit the activity or involvement of immune cells related to inflammation.
(6) 본 발명의 엑소좀의 피부세포 흡수 시험(6) skin cell absorption test of exosomes of the present invention
본 발명의 엑소좀을 추적하기 위하여 DiD cell labeling solution(ThermoFisher Scientific에서 구매)을 처리하여 상기 엑소좀을 형광염색하였다.In order to track the exosomes of the present invention, the exosomes were fluorescently stained with DiD cell labeling solution (purchased from ThermoFisher Scientific).
HaCaT 세포를 10% 우태아혈청, 2mM L-글루타민을 포함한 DMEM배지에 현탁시키고 이를 배양접시에 30~40%의 밀집도를 갖도록 분주하였다. 다음날 DiD형광염색한 엑소좀을 2~4시간처리하여 배양하였다. 배양이 끝난 후 4% PFA로 고정시킨 후 형광염색제인 비스벤지마이드(Bisbenzimide, Hoechst 33342, ThermoFisher Scientific에서 구매)로 처리하여 형광현미경으로 분석하였다.HaCaT cells were suspended in DMEM medium containing 10% fetal bovine serum and 2mM L-glutamine, and the cells were dispensed in a culture dish to have a density of 30-40%. The next day, the exosomes stained with DiD fluorescence were treated and cultured for 2 to 4 hours. After incubation, they were fixed with 4% PFA, treated with a fluorescent dye, bisbenzimide (Bisbenzimide, Hoechst 33342, purchased from ThermoFisher Scientific), and analyzed with a fluorescence microscope.
도 6은 본 발명의 일 구체예에 따라 얻어진 엑소좀을 형광염색하여 피부세포에 처리하였을 때 피부세포가 엑소좀을 흡수한 결과를 나타낸 것으로서, 도 6에서 빨간색 점은 피부세포가 본 발명의 엑소좀을 효과적으로 흡수하였다는 것을 나타내고 파란색은 피부세포의 세포핵을 나타낸다.6 shows the results of absorbing exosomes by skin cells when the exosomes obtained according to an embodiment of the present invention are fluorescently stained and treated with skin cells, and the red dots in FIG. It indicates that the moth was absorbed effectively, and the blue color indicates the cell nucleus of the skin cell.
(7) 본 발명의 엑소좀의 항염증 효과 시험(7) Anti-inflammatory effect test of the exosome of the present invention
HaCaT 세포를 10% 우태아혈청, 2mM L-글루타민을 포함한 DMEM배지에 현탁시키고 이를 배양접시에 30~40%의 밀집도를 갖도록 분주하였다. 다음날 LPS가 포함된 새로운 배지로 염증반응을 유발한 후 본 발명의 엑소좀을 24시간동안 처리하여 배양하였다. 배양이 끝난 후 세포파쇄물과 배양상층액을 취하여 그 속에 존재하는 엑소좀의 변화를 나노입자 추적 분석기로 측정하였고, 또한 그 속에 존재하는 iNOS(Abcam에서 구매), IL-6, TNFα, TSLP(ThermoFisher Scientific에서 구매), TARC(Sigma Aldrich에서 구매)의 변화량을 ELISA kit로 측정하였다. HaCaT cells were suspended in DMEM medium containing 10% fetal bovine serum and 2mM L-glutamine, and the cells were dispensed in a culture dish to have a density of 30-40%. The next day, after inducing an inflammatory reaction with a fresh medium containing LPS, the exosomes of the present invention were treated and cultured for 24 hours. After the culture was completed, the cell lysate and the culture supernatant were taken and the changes in the exosomes present therein were measured with a nanoparticle tracking analyzer, and iNOS (purchased from Abcam), IL-6, TNFα, TSLP (ThermoFisher) Changes in TARC (purchased from Scientific) and TARC (purchased from Sigma Aldrich) were measured with an ELISA kit.
도 7은 본 발명의 일 구체예에 따라 얻어진 엑소좀에 의한 염증성 피부세포가 분비하는 엑소좀의 분비변화를 나타낸 것, 도 8은 본 발명의 일 구체예에 따라 얻어진 엑소좀에 의한 iNOS 형성 감소 효과를 확인한 결과를 나타낸 것, 도 9a 내지 9d는 본 발명의 일 구체예에 따라 얻어진 엑소좀에 의한 (a) IL-6, (b) TNFα, (c) TSLP, (d) TARC의 변화량을 ELISA 키트로 확인한 결과를 나타낸 것이다.Figure 7 shows a change in the secretion of exosomes secreted by inflammatory skin cells by the exosomes obtained according to an embodiment of the present invention, Figure 8 is a decrease in iNOS formation by the exosomes obtained according to an embodiment of the present invention Showing the results of confirming the effect, FIGS. 9a to 9d show the amount of change in (a) IL-6, (b) TNFα, (c) TSLP, (d) TARC by the exosome obtained according to an embodiment of the present invention Shows the results confirmed by the ELISA kit.
도 7 내지 9에서 x축의 control은 정상 피부세포 음성대조군, LPS는 LPS로 피부세포 염증반응을 유발한 양성 대조군, LPS+exosome은 LPS로 피부세포 염증반응을 유발한 후 엑소좀을 처리한 실험군을 나타내고, y축은 각 물질의 농도를 나타낸다. 7 to 9, the control on the x-axis is a normal skin cell negative control group, LPS is a positive control that induced skin cell inflammatory response with LPS, and LPS+exosome is an experimental group treated with exosomes after induced skin cell inflammatory response with LPS. and the y-axis represents the concentration of each substance.
도 7에 도시된 바와 같이, 본 발명의 엑소좀은 인간 피부세포인 HaCaT 세포에서 LPS에 의해 유도되는 엑소좀의 분비증가를 효과적으로 감소시키는 것을 확인하였다.As shown in FIG. 7 , it was confirmed that the exosomes of the present invention effectively reduced the increase in secretion of exosomes induced by LPS in HaCaT cells, which are human skin cells.
도 8에 도시된 바와 같이, 본 발명의 엑소좀은 인간 피부세포인 HaCaT 세포에서 LPS에 의해 유도되는 염증반응인 iNOS생성을 효과적으로 감소시킨다는 것을 확인하였다. As shown in FIG. 8, it was confirmed that the exosome of the present invention effectively reduced iNOS production, which is an inflammatory response induced by LPS in HaCaT cells, which are human skin cells.
또한, 도 9와 같이 본 발명의 엑소좀은 LPS에 의해 유도되는 IL-6, TNFα, TSLP, TARC 사이토카인의 생성을 감소시키다는 것을 확인하였다. In addition, as shown in Figure 9, it was confirmed that the exosomes of the present invention reduce the production of IL-6, TNFα, TSLP, and TARC cytokines induced by LPS.
이러한 결과는 본 발명의 엑소좀이 피부염의 예방, 개선, 완화 또는 치료를 위한 효과적인 활성을 가지고 염증반응을 감소시키기에, 본 발명의 엑소좀은 피부염의 예방, 개선, 완화 또는 치료를 위한 임상적 약학조성물의 유효성분으로 활용될 수 있는 것임을 보여주고 있다.These results show that the exosome of the present invention has effective activity for preventing, improving, alleviating or treating dermatitis and reducing the inflammatory response, so the exosome of the present invention is clinically useful for preventing, improving, alleviating or treating dermatitis. It has been shown that it can be used as an active ingredient in a pharmaceutical composition.
(8) 본 발명의 엑소좀의 인체 피부 투과능력 시험(8) Human skin penetration ability test of exosomes of the present invention
본 발명의 엑소좀을 형광염색하기 위하여 Vybrant DiD cell-labeling solution(ThermoFisher Sicentific에서 구매)를 사용하였다. 1mM DiD 5ul를 1ml의 본 발명의 엑소좀과 혼합하여 빛을 차단한 상태로 37℃에서 20분간 반응시켰다. 9ml의 DMEM((ThermoFisher Sicentific에서 구매)배지와 5ml의 Total exosome isolation reagent(ThermoFisher Sicentific에서 구매)를 혼합하여 충분히 반응시킨 후 침전시킨 후 1ml의 1배 인산완충생리식염수(Corning에서 구매)로 재현탁하여 사용하였다.In order to fluorescently stain the exosomes of the present invention, Vybrant DiD cell-labeling solution (purchased from ThermoFisher Sicentific) was used. 5ul of 1mM DiD was mixed with 1ml of the exosome of the present invention and reacted at 37°C for 20 minutes while blocking light. 9ml of DMEM (purchased from ThermoFisher Sicentific) medium and 5ml of Total exosome isolation reagent (purchased from ThermoFisher Sicentific) were mixed and sufficiently reacted, precipitated, and then resuspended in 1ml of 1-fold phosphate buffered saline (purchased from Corning) was used.
경피 흡수를 실험할 수 있는 Franz diffusion cell(비엔씨테크에서 구매)에 상기 DiD-엑소좀을 고정한 인체 피부조직(고마바이오텍에서 구매)의 바깥면에 도포하였고 내부는 1xPBS로 채워주었다. 도포된 DiD-엑소좀의 건조를 막기 위하여 호일로 밀봉하여 24시간동안 반응시켜 DiD-엑소좀이 피부에 흡수되게 하였다. 반응이 종료된 후, 인체 피부조직 바깥면의 액체를 와이퍼(Kimwipes)로 흡수한 후 1배 인산완충생리식염수로 충분히 세척하였고, 인체 피부조직의 수분을 완전히 제거한 후 액체질소탱크에 10분간 급속냉동하였다. 급속냉동된 인체 피부조직을 매스로 각질층을 제거하고 각질층이 제거된 인체 피부조직을 형광현미경으로 관찰하였으며, 본 발명의 일 구체예에 따라 얻어진 엑소좀이 인체 피부에 흡수되는 정도를 확인한 결과가 도 10에 나타나 있다. 도 10의 결과를 통해, 본 발명의 엑소좀은 인체 피부장벽을 효과적으로 통과하여 표피와 진피까지 깊숙이 전달될 수 있다는 것을 보여주고 있다. 따라서, 상기 엑소좀을 유효성분으로 함유하는 피부 외용제 또는 화장료 조성물은 피부염의 예방, 개선, 완화 또는 치료 등에 있어서 효과적으로 작용할 것으로 기대된다. The DiD-exosome was applied to the outer surface of the human skin tissue (purchased from Goma Biotech) in which the DiD-exosome was fixed to a Franz diffusion cell (purchased from BNC Tech) that can be tested for transdermal absorption, and the inside was filled with 1xPBS. To prevent drying of the applied DiD-exosome, it was sealed with foil and allowed to react for 24 hours to allow the DiD-exosome to be absorbed into the skin. After the reaction was completed, the liquid on the outer surface of the human skin tissue was absorbed with a wiper (Kimwipes), washed thoroughly with 1x phosphate buffered saline, and after completely removing moisture from the human skin tissue, quickly frozen in a liquid nitrogen tank for 10 minutes did The stratum corneum was removed with a mass of rapidly frozen human skin tissue, and the human skin tissue from which the stratum corneum was removed was observed under a fluorescence microscope. 10 is shown. Through the results of Figure 10, it is shown that the exosomes of the present invention can effectively pass through the human skin barrier and be deeply delivered to the epidermis and dermis. Therefore, the external preparation or cosmetic composition for skin containing the exosomes as an active ingredient is expected to act effectively in preventing, improving, alleviating or treating dermatitis.
(9) 사람 피부에서 엑소좀을 유효성분으로 포함하는 조성물의 효과 시험(9) Efficacy test of a composition containing exosomes as an active ingredient in human skin
본 발명의 일 구체예의 방법에 따라 수득된 엑소좀을 함유하는 조성물을 중증 아토피 환자 5인의 환부(목, 팔, 다리 등)에 4~8주동안 1일 2회 도포하였으며, 도 11a 및 11b에 본 발명의 조성물 도포 전후의 환부 상태가 나타나 있다.The composition containing exosomes obtained according to the method of one embodiment of the present invention was applied to the affected areas (neck, arm, leg, etc.) of 5 severe atopic patients twice a day for 4 to 8 weeks, The condition of the affected area before and after application of the composition of the present invention is shown.
도 11a 및 11b를 통해, 본 발명의 일 구체예에 다른 엑소좀을 포함하는 조성물을 사람의 피부에 도포한 경우 환자들의 극심한 가려움증이 현저히 완화되었고, 피부염으로 인한 피부병변이 현저히 개선되었음을 알 수 있다.11a and 11b, it can be seen that, when the composition comprising exosomes according to one embodiment of the present invention is applied to human skin, severe itchiness of patients is remarkably relieved, and skin lesions due to dermatitis are remarkably improved. .
Claims (9)
- 전구세포 유래 엑소좀을 함유하는 피부염의 예방, 개선, 완화 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing, improving, alleviating or treating dermatitis containing progenitor cell-derived exosomes.
- 제1항에 있어서, According to claim 1,상기 전구세포는 표면마커 중에서 CD24, CD117 또는 SSEA-1에 대하여 음성이고 CD31, CD45RA, CD105, CD146 또는 TRA-1-60에 대하여 양성인 전구세포로부터 유래한 것을 특징으로 하는 피부염의 예방, 개선, 완화 또는 치료용 약학적 조성물. The progenitor cells are negative for CD24, CD117 or SSEA-1 among surface markers and are derived from progenitor cells that are positive for CD31, CD45RA, CD105, CD146 or TRA-1-60. Prevention, improvement, alleviation of dermatitis or a therapeutic pharmaceutical composition.
- 제1항에 있어서, According to claim 1,상기 엑소좀은 표면마커 중에서 CD9, CD63, CD81 외에도 CD44, CD53 또는 CD98을 함유하고 있는 것을 특징으로 하는 피부염의 예방, 개선, 완화 또는 치료용 약학적 조성물.The exosome is a pharmaceutical composition for preventing, improving, alleviating or treating dermatitis, characterized in that it contains CD44, CD53 or CD98 in addition to CD9, CD63, and CD81 among surface markers.
- 제1항에 있어서, 상기 엑소좀은 하기의 단계들을 수행하여 수득되는 피부염의 예방, 개선, 완화 또는 치료용 약학적 조성물According to claim 1, wherein the exosome is a pharmaceutical composition for preventing, improving, alleviating or treating dermatitis obtained by performing the following stepsa. 전구세포 배양액을 필터에 통과시켜 세포 잔해물, 노폐물, 거대 입자 중 적어도 어느 하나를 여과하는 단계a. Passing the progenitor cell culture solution through a filter to filter at least one of cell debris, wastes, and large particlesb. 상기 여과된 배양액을 멸균 필터로 멸균하는 단계b. Sterilizing the filtered culture solution with a sterile filterc. 상기 멸균된 배양액을 접선흐름여과방법을 이용하여 농축과 정용여과과정을 통해 엑소좀을 분리하는 단계c. Separating the exosomes through concentration and diafiltration of the sterilized culture solution using a tangential flow filtration methodd. 상기 분리된 엑소좀에 안정화 버퍼를 추가하여 다시 시작 부피가 되도록 희석하는 단계d. Dilute to the starting volume again by adding a stabilization buffer to the isolated exosomee. 상기 희석된 엑소좀을 액체 교환 시스템으로 멸균 필터에 통과시켜 멸균한 후 밀봉된 저장용기에 저장하는 단계e. Storing the diluted exosomes in a sealed storage container after sterilization by passing them through a sterile filter with a liquid exchange system
- 제4항에 있어서, 5. The method of claim 4,접선흐름여과를 위한 분자량 한계치를 얻기 위하여 10,000Da, 50,000Da 또는 75,000Da의 중공사 필터를 사용하는 것을 특징으로 하는 피부염의 예방, 개선, 완화 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing, improving, alleviating or treating dermatitis, characterized in that a hollow fiber filter of 10,000Da, 50,000Da or 75,000Da is used to obtain a molecular weight limit value for tangential flow filtration.
- 제4항에 있어서, 5. The method of claim 4,상기 c단계의 농축과 정용여과는 접선흐름여과를 단속 또는 연속적으로 진행하고, 농축은 시작 부피에 대하여 10배 내지 25배 농축하고, 정용여과 과정은 농축된 부피에 대하여 적어도 10배의 부피를 갖는 버퍼로 여과하는 과정을 포함하는 것을 특징으로 하는 피부염의 예방, 개선, 완화 또는 치료용 약학적 조성물.The concentration and diafiltration of step c are performed intermittently or continuously with tangential flow filtration, the concentration is concentrated 10 to 25 times relative to the starting volume, and the diafiltration process is at least 10 times the volume of the concentrated volume. A pharmaceutical composition for preventing, improving, alleviating or treating dermatitis, comprising the process of filtering with a buffer.
- 제4항에 있어서,5. The method of claim 4,상기 엑소좀 희석은 농축 및 정용여과된 부피에 대하여 10배 내지 25배의 안정화 버퍼를 첨가하여 전체 부피가 시작 부피와 같도록 희석하는 과정을 포함하는 것을 특징으로 하는 피부염의 예방, 개선, 완화 또는 치료용 약학적 조성물.The exosome dilution prevents, improves, alleviates or improves dermatitis, characterized in that it includes a process of diluting the total volume to the same as the starting volume by adding a stabilization buffer of 10 to 25 times to the concentrated and diafiltered volume A therapeutic pharmaceutical composition.
- 전구세포 유래 엑소좀을 유효성분으로 포함하는, 피부염의 예방, 개선, 완화 또는 치료용 피부외용제.A skin external preparation for preventing, improving, alleviating or treating dermatitis, comprising progenitor cell-derived exosomes as an active ingredient.
- 전구세포 유래 엑소좀을 유효성분으로 포함하는, 피부염의 예방, 개선, 완화용 화장료 조성물.A cosmetic composition for preventing, improving, alleviating dermatitis, comprising progenitor cell-derived exosomes as an active ingredient.
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