WO2022191341A1 - Pharmaceutical composition comprising stem cell-derived exosome for palliating or treating arthritis - Google Patents
Pharmaceutical composition comprising stem cell-derived exosome for palliating or treating arthritis Download PDFInfo
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- WO2022191341A1 WO2022191341A1 PCT/KR2021/002853 KR2021002853W WO2022191341A1 WO 2022191341 A1 WO2022191341 A1 WO 2022191341A1 KR 2021002853 W KR2021002853 W KR 2021002853W WO 2022191341 A1 WO2022191341 A1 WO 2022191341A1
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- pharmaceutical composition
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- stem cells
- exosomes
- arthritis
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Images
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
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
Definitions
- the present invention relates to a pharmaceutical composition for alleviating or treating arthritis.
- rheumatoid arthritis is caused by inflammation due to autoimmunity, and in degenerative arthritis, that is, osteoarthritis, degeneration such as aging occurs in chondrocytes constituting the joint, interleukin-1 and tumor
- MMP matrix metalloproteinase
- arthritis is further exacerbated by the synthesis of more MMPs due to the production of nitric oxide by inflammatory cytokines and the production of self-amplifying cytokines by the produced nitric oxide, which promotes the decomposition of the joint matrix.
- inflammatory cytokines increase the production of prostaglandin E2, a lipid metabolite, to induce an inflammatory response in arthritis.
- Osteoarthritis also called degenerative arthritis, is a type of arthritis that occurs mainly in old age without a specific organic cause. Osteoarthritis is mainly caused by damage to the bones and ligaments constituting the joint due to gradual damage or degenerative changes in the articular cartilage.
- exosomes derived from stem cells not only prevent cartilage erosion due to inflammation by alleviating and treating inflammation generated in cartilage, but also effectively alleviating inflammatory responses in surrounding tissues such as synovial membranes, thereby increasing the therapeutic effect of arthritis. It was found that the present invention was completed.
- compositions for relieving or treating arthritis comprising exosomes derived from stem cells as an active ingredient.
- Embodiment 1 A pharmaceutical composition for relieving or treating arthritis, comprising an exosome derived from proliferating adipose derived stem cells as an active ingredient; Use of exosomes derived from proliferating adipose-derived stem cells for the manufacture of medicines for alleviating or treating arthritis; Or a method for alleviating or treating arthritis, comprising administering to a subject a composition comprising an exosome derived from proliferating adipose-derived stem cells as an active ingredient.
- Embodiment 2 The pharmaceutical composition of Embodiment 1, wherein the adipose-derived stem cells are human adipose-derived stem cells; purpose; or how.
- Embodiment 3 The pharmaceutical composition according to any one of the preceding embodiments, wherein the arthritis is selected from osteoarthritis and rheumatoid arthritis; purpose; or how.
- Embodiment 4 The pharmaceutical composition according to any one of the preceding embodiments, wherein the exosomes are derived from stem cells proliferating in passages 4 to 6; purpose; or how.
- Embodiment 5 The pharmaceutical composition according to any one of the preceding embodiments, wherein the exosome is derived from stem cells proliferating in passage 5; purpose; or how.
- Embodiment 6 The pharmaceutical composition according to any one of the preceding embodiments, wherein the exosome comprises an anti-inflammatory cytokine; purpose; or how.
- Embodiment 7 The pharmaceutical composition according to any one of the preceding embodiments, wherein the anti-inflammatory cytokine is at least one selected from TIMP-1 and TIMP-2; purpose; or how.
- Embodiment 8 An injection for alleviating or treating arthritis comprising the pharmaceutical composition according to any one of the preceding embodiments.
- One aspect of the present invention is to provide a pharmaceutical composition for relieving or treating arthritis, comprising an exosome derived from proliferating adipose derived stem cells as an active ingredient.
- stem cell used in the present invention refers to not only self-renewal ability, but also when an appropriate signal is given as needed under the influence of the environment in which the cell is located, it is Cells with the ability to differentiate into branch cells.
- the stem cells of the present invention may be autologous or allogeneic stem cells, and may be derived from any type of animal, including humans and non-human mammals.
- the stem cells may be derived from the patient's autologous adipose tissue.
- adipose-derived stem cell is a stem cell derived from adipose tissue, and adipose tissue has good conditions for harvesting stem cells because it is easy to collect a large amount of tissue. , adipose-derived stem cells show stable growth and proliferation in culture and can differentiate into various cells when differentiation is induced.
- exosome refers to a membrane-structured vesicle secreted from various types of cells, and has a strong antitumor immune response including major histocompatibility and heat shock proteins, which are important immunologically important proteins. It is known to play a variety of roles, including binding to other cells and tissues and delivering membrane components, proteins, and RNA. Therefore, exosomes containing various genetic information, proteins, and growth factors related to inflammation relief can be used as a composition for treating arthritis.
- the exosomes derived from adipose-derived stem cells of the present invention contain anti-inflammatory cytokines, and thus can play an important role in alleviating and treating arthritis, such as osteoarthritis and rheumatoid arthritis, specifically TIMP- 1 (tissue inhibitors of metalloproteinases-1), TIMP-2, IL-1ra (interleukin-1 receptor antagonist), IL-4 (interluekin-4), IL-10 (Interleukin-10), TGF- ⁇ (transforming growth factor) - ⁇ ) and the like, and these cytokines have a role of inducing inhibition of inflammatory responses and inducing inhibition of inflammatory cytokines.
- TIMP- 1 tissue inhibitors of metalloproteinases-1
- TIMP-2 tissue inhibitors of metalloproteinases-1
- IL-1ra interleukin-1 receptor antagonist
- IL-4 interluekin-4
- IL-10 Interleukin-10
- TGF- ⁇ transforming growth factor
- the exosomes containing the anti-inflammatory cytokine may be derived from stem cells proliferating in subculture, preferably from stem cells proliferating in passage 4 to 6 times, more preferably in passage 5 times. may be derived from When extracting exosomes from stem cells proliferating in the passages 4 to 6, compared with the exosomes extracted from passages 3 or 7, the critical significance of significantly increasing the content of anti-inflammatory cytokines have.
- the mass (pg/protein ( ⁇ g)) of anti-inflammatory cytokines contained per protein of exosomes derived from stem cells proliferating in passages 4 to 6 was increased in passages 3 or 7 At least 2 times or more, at least 3 times or more, at least 4 times or more, at least 5 times or more, at least 6 times or more, at least 7 times or more, at least 8 times or more, at least 9 times the amount contained in exosomes derived from stem cells that fold or more, at least 10 times or more, at least 20 times or more, at least 30 times or more, at least 40 times or more, at least 50 times or more, at least 60 times or more, at least 70 times or more, at least 80 times or more, at least 90 times or more, or at least It can be 100 times or more.
- the pharmaceutical composition according to the present invention may include one or more pharmaceutically acceptable carriers, excipients or diluents together with a pharmaceutically effective amount of exosomes derived from adipose-derived stem cells.
- the pharmaceutically effective amount means an amount sufficient to improve and treat inflammation occurring in the joints.
- the exosomes derived from adipose-derived stem cells according to the present invention have a concentration of 1 ⁇ 10 7 particles/mL to 1 ⁇ 10 10 particles/kg in the pharmaceutical composition, preferably 5 ⁇ 10 7 particles/mL to 1 ⁇ It may be included at a concentration of 10 9 particles/kg.
- the concentration of the exosomes can be appropriately changed depending on the degree of progression of arthritis, the age, weight, health status, sex, administration route, and treatment period of the patient.
- the "pharmaceutically acceptable” refers to a composition that is physiologically acceptable and does not normally cause gastrointestinal disorders, allergic reactions such as dizziness or similar reactions when administered to humans.
- examples of such carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
- fillers, anti-agglomeration agents, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.
- composition of the present invention can be administered by formulating a unit dosage form suitable for administration in the body of a patient according to a conventional method in the pharmaceutical field, and the preparation is administered once or several times to relieve arthritis. to therapeutically effective dosages.
- Formulations suitable for this purpose include, for parenteral administration, injections such as ampoules for injection, injections such as infusion bags, and the like.
- the injection ampoule may be prepared by mixing with an injection solution immediately before use, and physiological saline, glucose, mannitol, Ringer's solution, etc. may be used as the injection solution.
- one or more pharmaceutically acceptable conventional inert carriers for example, preservatives, analgesics, solubilizers or stabilizers in the case of injections, and the like, in the case of formulations for topical administration It may further include a base (base), excipients, lubricants or preservatives and the like.
- composition or pharmaceutical preparation of the present invention prepared in this way may be administered to mammals such as rats, mice, livestock, and humans by various routes such as parenteral and oral administration, and the administration method is any method commonly used in the art.
- the administration method is not limited thereto, but the exosomes extracted from adipose-derived stem cells may be administered intravenously (intravenous injection) or administered to an arthritis site (local administration; Topical administration).
- the present invention provides an injection for alleviating or treating arthritis, comprising a pharmaceutical composition for alleviating and treating arthritis comprising the adipose-derived stem cell-derived exosome as an active ingredient.
- the injection may further include phosphate-buffered saline (PBS). That is, the injection can be used by supporting the exosomes extracted from adipose-derived stem cells in phosphate buffered saline.
- PBS phosphate-buffered saline
- the injection may include hydrogel instead of phosphate buffered saline.
- the hydrogel may be at least one selected from the group consisting of hyaluronic acid, gelatin, alginate, chitosan, fibrin, elastin, collagen, and methylcellulose, and specifically may be a hyaluronic acid hydrogel, but is not limited thereto.
- the stem cell-derived exosome according to the present invention not only prevents cartilage erosion by alleviating and treating inflammation occurring in the cartilage, but also effectively relieves the inflammatory response in surrounding tissues such as the synovial membrane, thereby fundamentally curing arthritis. .
- Figure 2 shows the content of anti-inflammatory cytokines (TIMP-1 and TIMP-2) contained in the exosomes produced for each passage.
- TRIP-1 inflammatory regulator
- hASCs Primary hASCs were purchased from CEFO Bio Co., Ltd (Seoul, Korea), and growth medium and supplements were purchased from Life Technologies (Carlsbad, CA, USA). Using normal culture medium (Dulbecco Modified Eagle Medium high glucose (DMEM) containing 10% fetal bovine serum and 1% penicillin/streptomycin), hASCs were subcultured 3 to 7 times at 37°C and 5% CO 2 conditions. proliferated. Exosomes were extracted for each passage, specifically, before extracting the exosomes, the proliferated stem cells for each passage were replaced with serum-free DMEM medium and maintained for 24 hours, and then the cell culture supernatant was recovered. Then, exosomes were isolated from the recovered cell culture supernatant by a multiple filtration system based on a tangential flow filtration system (TFF).
- DMEM Dulbecco Modified Eagle Medium high glucose
- the size and shape of the exosomes extracted from the proliferating stem cells were confirmed using a transmission electron microscope and dynamic light scattering, and the exosome membrane using flow cytometry. Surface proteins were identified.
- CD9, CD63, and CD81 known as exosome surface marker proteins, were all expressed in the extracted exosomes using flow cytometry (Fig. 1).
- Representative cytokines secreted in relation to inflammation relief include TIMP-1 (tissue inhibitors of metalloproteinases-1) and TIMP-2. It is known that there is
- BM-MSC-Exo a significant TIMP-1 content was not confirmed, and in the case of UC-MSC-Exo, it was confirmed that it contained TIMP-1, but the TIMP-1 content was significantly higher than that of hASC-Exo. was confirmed to be very low ( FIG. 3 ) (*P ⁇ 0.1).
- MIA-induced rat OA model monosodium iodoacetate (MIA)-induced rat OA model
- exosomes produced in passage 5 were administered intra-articularly every week for 3 weeks from 1 week after MIA injection. 4 weeks after MIA injection, the isolated knee joint was stained with India ink, and the observation results are shown in FIG. 4 .
Abstract
The present invention relates to an exosome-based pharmaceutical composition for palliating and treating arthritis. The exosomes according to the present invention can basically cure arthritis by palliating and treating inflammations occurring in the cartilage to prevent cartilage erosion as well as by effectively alleviating even inflammatory reactions in neighboring tissues such as the synovium.
Description
본 발명은 관절염 완화 또는 치료용 약학적 조성물에 관한 것이다. The present invention relates to a pharmaceutical composition for alleviating or treating arthritis.
류마티스 관절염은 자가면역에 의한 염증이 원인인 것으로 이해되고 있으며, 퇴행성 관절염, 즉 골관절염은 관절을 구성하는 연골세포(chondrocytes)에 노화 등의 퇴행이 발생하고, 인터루킨-1(interleukin-1) 및 종양괴사인자-α(tumor necrosis factor-α) 등의 염증성 사이토카인이 생성됨에 따라, 관절기질을 분해하는 기질 금속단백질 분해효소 (matrix metalloproteinase; MMP)의 합성 및 활성이 관절세포에서 증가됨으로 인해 관절조직이 파괴됨으로써 유발되는 질병이다. It is understood that rheumatoid arthritis is caused by inflammation due to autoimmunity, and in degenerative arthritis, that is, osteoarthritis, degeneration such as aging occurs in chondrocytes constituting the joint, interleukin-1 and tumor As inflammatory cytokines such as tumor necrosis factor-α are produced, the synthesis and activity of matrix metalloproteinase (MMP) that decomposes the joint matrix is increased in joint cells. It is a disease caused by the destruction of
또한, 관절염은 염증성 사이토카인에 의한 일산화질소의 생성과, 생성된 일산화질소에 의한 자가 증폭적인 사이토카인의 생성으로 인해 더욱 많은 MMP의 합성이 유발되어 관절기질의 분해가 촉진됨으로써 더욱 악화된다. 이와 동시에, 염증성 사이토카인은 지질 대사산물인 프로스타글란딘 E2의 생성을 증가시켜 관절염에서의 염증반응을 유발시킨다. 골관절염은 퇴행성 관절염으로도 명명되는 관절염의 일종으로서, 특정한 기질적 원인이 없이 주로 노년에 많이 발생되며 만성적으로 진행되면 관절구조의 변형으로 보행 장애 등의 운동 장애를 수반한다. 골관절염은 주로 관절연골(cartilage)의 점진적인 손상이나 퇴행성 변화로 인해 관절을 이루는 뼈와 인대 등에 손상이 일어나서 염증이 발생된다. 골관절염이 진행될 때 TNF-α, IL-1 등의 염증성 사이토카인(cytokine)의 생성이 증가하고 콜라게네이즈(collagenase), 스트로멜라이신(stromelysin) 등과 같은 MMPs의 분비가 증가되어 관절연골의 파괴가 이루어지게 된다. 또한 MMPs는 IL-1, TNF-α 등을 유도시키게 되는데 이로 인하여 근육, 건, 인대 등과 같은 조직에도 영향을 끼쳐 심한 염증을 일으킨다.In addition, arthritis is further exacerbated by the synthesis of more MMPs due to the production of nitric oxide by inflammatory cytokines and the production of self-amplifying cytokines by the produced nitric oxide, which promotes the decomposition of the joint matrix. At the same time, inflammatory cytokines increase the production of prostaglandin E2, a lipid metabolite, to induce an inflammatory response in arthritis. Osteoarthritis, also called degenerative arthritis, is a type of arthritis that occurs mainly in old age without a specific organic cause. Osteoarthritis is mainly caused by damage to the bones and ligaments constituting the joint due to gradual damage or degenerative changes in the articular cartilage. When osteoarthritis progresses, the production of inflammatory cytokines such as TNF-α and IL-1 increases, and the secretion of MMPs such as collagenase and stromelysin increases, leading to the destruction of articular cartilage. will be done In addition, MMPs induce IL-1 and TNF-α, which in turn affects tissues such as muscles, tendons, and ligaments, causing severe inflammation.
본 명세서 전체에 걸쳐 다수의 문헌이 참조되고 그 인용이 표시되어 있다. 인용된 문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다. Numerous references are referenced throughout this specification and citations thereof are indicated. The disclosures of the cited documents are hereby incorporated by reference in their entirety to more clearly set forth the content of the present invention and the level of the art to which the present invention pertains.
본 발명자들은 줄기세포로부터 유래한 엑소좀이 연골에 발생한 염증을 완화 및 치료하여 염증에 의한 연골 침식을 방지할 뿐만 아니라, 활막(synovium)과 같은 주변 조직 내의 염증 반응까지도 효과적으로 완화시켜 관절염 치료 효과가 있음을 밝혀내어 본 발명을 완성하였다.The present inventors found that exosomes derived from stem cells not only prevent cartilage erosion due to inflammation by alleviating and treating inflammation generated in cartilage, but also effectively alleviating inflammatory responses in surrounding tissues such as synovial membranes, thereby increasing the therapeutic effect of arthritis. It was found that the present invention was completed.
따라서, 본 발명의 목적은 줄기세포로부터 유래한 엑소좀을 유효성분으로 포함하는, 관절염 완화 또는 치료용 약학적 조성물을 제공하는 데 있다. Accordingly, it is an object of the present invention to provide a pharmaceutical composition for relieving or treating arthritis, comprising exosomes derived from stem cells as an active ingredient.
더 상세하게는 본 발명의 목적은 다음의 구현예들을 제공하는 데 있다.More particularly, it is an object of the present invention to provide the following embodiments.
구현예 1. 증식하는 지방유래 줄기세포(adipose derived stem cells)로부터 유래한 엑소좀을 유효성분으로 포함하는, 관절염 완화 또는 치료용 약학적 조성물; 증식하는 지방유래 줄기세포로부터 유래한 엑소좀의 관절염 완화 또는 치료용 의약품의 제조를 위한 용도; 또는 증식하는 지방유래 줄기세포로부터 유래한 엑소좀을 유효성분으로 포함하는 조성물을 대상체에 투여하는 것을 포함하는, 관절염 완화 또는 치료 방법. Embodiment 1. A pharmaceutical composition for relieving or treating arthritis, comprising an exosome derived from proliferating adipose derived stem cells as an active ingredient; Use of exosomes derived from proliferating adipose-derived stem cells for the manufacture of medicines for alleviating or treating arthritis; Or a method for alleviating or treating arthritis, comprising administering to a subject a composition comprising an exosome derived from proliferating adipose-derived stem cells as an active ingredient.
구현예 2. 구현예 1에 있어서, 상기 지방유래 줄기세포는 인간 지방유래 줄기세포인 것인 약학적 조성물; 용도; 또는 방법. Embodiment 2. The pharmaceutical composition of Embodiment 1, wherein the adipose-derived stem cells are human adipose-derived stem cells; purpose; or how.
구현예 3. 선행하는 구현예들 중 어느 하나에 있어서, 상기 관절염은 골관절염(osteoarthritis) 및 류마티스 관절염(rheumatoid arthritis)으로부터 선택되는 것인 약학적 조성물; 용도; 또는 방법. Embodiment 3. The pharmaceutical composition according to any one of the preceding embodiments, wherein the arthritis is selected from osteoarthritis and rheumatoid arthritis; purpose; or how.
구현예 4. 선행하는 구현예들 중 어느 하나에 있어서, 상기 엑소좀은 4회 내지 6회 계대 배양에서 증식하는 줄기세포로부터 유래한 것인 약학적 조성물; 용도; 또는 방법. Embodiment 4. The pharmaceutical composition according to any one of the preceding embodiments, wherein the exosomes are derived from stem cells proliferating in passages 4 to 6; purpose; or how.
구현예 5. 선행하는 구현예들 중 어느 하나에 있어서, 상기 엑소좀은 5회 계대 배양에서 증식하는 줄기세포로부터 유래한 것인 약학적 조성물; 용도; 또는 방법. Embodiment 5. The pharmaceutical composition according to any one of the preceding embodiments, wherein the exosome is derived from stem cells proliferating in passage 5; purpose; or how.
구현예 6. 선행하는 구현예들 중 어느 하나에 있어서, 상기 엑소좀은 항염증성 사이토카인을 포함하는 것인 약학적 조성물; 용도; 또는 방법.Embodiment 6. The pharmaceutical composition according to any one of the preceding embodiments, wherein the exosome comprises an anti-inflammatory cytokine; purpose; or how.
구현예 7. 선행하는 구현예들 중 어느 하나에 있어서, 상기 항염증성 사이토카인은 TIMP-1 및 TIMP-2로부터 선택되는 1 이상인 것인 약학적 조성물; 용도; 또는 방법.Embodiment 7. The pharmaceutical composition according to any one of the preceding embodiments, wherein the anti-inflammatory cytokine is at least one selected from TIMP-1 and TIMP-2; purpose; or how.
구현예 8. 선행하는 구현예들 중 어느 하나에 따른 약학적 조성물을 포함하는 관절염 완화 또는 치료용 주사제.Embodiment 8. An injection for alleviating or treating arthritis comprising the pharmaceutical composition according to any one of the preceding embodiments.
본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다. Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.
본 발명의 하나의 관점은 증식하는 지방유래 줄기세포(adipose derived stem cells)로부터 유래한 엑소좀을 유효성분으로 포함하는, 관절염 완화 또는 치료용 약학적 조성물을 제공하는 것이다.One aspect of the present invention is to provide a pharmaceutical composition for relieving or treating arthritis, comprising an exosome derived from proliferating adipose derived stem cells as an active ingredient.
본 발명에서 사용된 용어 "줄기세포(stem cell)"란 자기 복제 능력뿐만 아니라, 세포가 위치하는 환경의 영향을 받아 필요에 따라 적절한 신호가 주어질 경우, 다중분화능(multi potency)의 특성에 의해 여러 가지 세포로 분화할 수 있는 특성이 있는 세포이다.The term "stem cell" used in the present invention refers to not only self-renewal ability, but also when an appropriate signal is given as needed under the influence of the environment in which the cell is located, it is Cells with the ability to differentiate into branch cells.
본 발명의 줄기세포는 자가 또는 동종 유래 줄기세포일 수 있으며, 인간 및 비인간 포유류를 포함한 임의 유형의 동물 유래일 수 있다. 바람직하게는 상기 줄기세포는 환자의 자가 지방 조직으로부터 유래한 것일 수 있다.The stem cells of the present invention may be autologous or allogeneic stem cells, and may be derived from any type of animal, including humans and non-human mammals. Preferably, the stem cells may be derived from the patient's autologous adipose tissue.
본 발명에서 사용된 용어 "지방유래 줄기세포(adipose-derived stem cell)"란 지방조직에서 유래하는 줄기세포로, 지방조직은 많은 양의 조직 채취가 용이하여 줄기세포를 수확하는데 좋은 조건을 가지고 있으며, 지방 유래 줄기세포는 배양 시 안정적인 성장과 증식을 보여주고 분화를 유도하였을 때 다양한 세포로의 분화가 가능하다. As used herein, the term "adipose-derived stem cell" is a stem cell derived from adipose tissue, and adipose tissue has good conditions for harvesting stem cells because it is easy to collect a large amount of tissue. , adipose-derived stem cells show stable growth and proliferation in culture and can differentiate into various cells when differentiation is induced.
본 발명에서 사용된 용어, "엑소좀 (exosome)"이란 여러 종류의 세포들로부터 분비되는 막 구조의 소낭체로, 면역학적으로 중요한 단백질인 주조직적합체와 열충격 단백질을 포함하여 강력한 항종양 면역 반응을 유도하며, 다른 세포 및 조직에 결합하여 막 구성요소, 단백질, RNA를 전달하는 등 다양한 역할을 하는 것으로 알려져 있다. 그러므로 염증 완화에 관련된 다양한 유전정보, 단백질, 성장인자를 함유하고 있는 엑소좀을 이용하여 관절염 치료제의 조성물로 활용할 수 있다. As used herein, the term "exosome" refers to a membrane-structured vesicle secreted from various types of cells, and has a strong antitumor immune response including major histocompatibility and heat shock proteins, which are important immunologically important proteins. It is known to play a variety of roles, including binding to other cells and tissues and delivering membrane components, proteins, and RNA. Therefore, exosomes containing various genetic information, proteins, and growth factors related to inflammation relief can be used as a composition for treating arthritis.
본 발명의 지방유래 줄기세포로부터 유래한 엑소좀은 항염증성 사이토카인를 함유하고 있어, 골관절염(osteoarthritis) 및 류마티스 관절염(rheumatoid arthritis)과 같은 관절염 완화 및 치료에서 중요한 역할을 할 수 있는데, 구체적으로 TIMP-1 (tissue inhibitors of metalloproteinases-1), TIMP-2, IL-1ra (interleukin-1 receptor antagonist), IL-4 (interluekin-4), IL-10 (Interleukin-10), TGF-β (transforming growth factor-β) 등을 포함하며, 이들 사이토카인은 염증반응 억제를 유도하고 염증성 사이토카인을 억제하도록 유도시키는 역할을 가지고 있다.The exosomes derived from adipose-derived stem cells of the present invention contain anti-inflammatory cytokines, and thus can play an important role in alleviating and treating arthritis, such as osteoarthritis and rheumatoid arthritis, specifically TIMP- 1 (tissue inhibitors of metalloproteinases-1), TIMP-2, IL-1ra (interleukin-1 receptor antagonist), IL-4 (interluekin-4), IL-10 (Interleukin-10), TGF-β (transforming growth factor) -β) and the like, and these cytokines have a role of inducing inhibition of inflammatory responses and inducing inhibition of inflammatory cytokines.
상기 항염증성 사이토카인을 함유하는 엑소좀은 계대 배양에서 증식하는 줄기세포로부터 유래한 것일 수 있는데, 바람직하게는 4회 내지 6회 계대 배양, 더욱 바람직하게는 5회 계대 배양에서 증식하는 줄기세포로부터 유래한 것일 수 있다. 상기 4회 내지 6회 계대 배양에서 증식하는 줄기세포로부터 엑소좀을 추출하는 경우, 3회 또는 7회 계대로부터 추출한 엑소좀과 비교하여, 항염증성 사이토카인의 함유량이 현저하게 상승하는 임계적 의의가 있다.The exosomes containing the anti-inflammatory cytokine may be derived from stem cells proliferating in subculture, preferably from stem cells proliferating in passage 4 to 6 times, more preferably in passage 5 times. may be derived from When extracting exosomes from stem cells proliferating in the passages 4 to 6, compared with the exosomes extracted from passages 3 or 7, the critical significance of significantly increasing the content of anti-inflammatory cytokines have.
구체적으로, 4회 내지 6회 계대 배양에서 증식하는 줄기세포로부터 유래한 엑소좀의 protein 당 함유된 항염증성 사이토카인의 질량(pg/protein(μg))은, 3회 또는 7회 계대 배양에서 증식하는 줄기세포로부터 유래한 엑소좀에 함유된 양의 적어도 2배 이상, 적어도 3배 이상, 적어도 4배 이상, 적어도 5배 이상, 적어도 6배 이상, 적어도 7배 이상, 적어도 8배 이상, 적어도 9배 이상, 적어도 10배 이상, 적어도 20배 이상, 적어도 30배 이상, 적어도 40배 이상, 적어도 50배 이상, 적어도 60배 이상, 적어도 70배 이상, 적어도 80배 이상, 적어도 90배 이상, 또는 적어도 100배 이상일 수 있다.Specifically, the mass (pg/protein (μg)) of anti-inflammatory cytokines contained per protein of exosomes derived from stem cells proliferating in passages 4 to 6 was increased in passages 3 or 7 At least 2 times or more, at least 3 times or more, at least 4 times or more, at least 5 times or more, at least 6 times or more, at least 7 times or more, at least 8 times or more, at least 9 times the amount contained in exosomes derived from stem cells that fold or more, at least 10 times or more, at least 20 times or more, at least 30 times or more, at least 40 times or more, at least 50 times or more, at least 60 times or more, at least 70 times or more, at least 80 times or more, at least 90 times or more, or at least It can be 100 times or more.
본 발명에 따른 약학적 조성물은 지방유래 줄기세포로부터 유래한 엑소좀의 약학적으로 유효한 양과 함께 하나 이상의 약학적으로 허용되는 담체, 부형제 또는 희석제를 포함할 수 있다. The pharmaceutical composition according to the present invention may include one or more pharmaceutically acceptable carriers, excipients or diluents together with a pharmaceutically effective amount of exosomes derived from adipose-derived stem cells.
상기에서 약학적으로 유효한 양이란 관절에 발생한 염증을 개선 및 치료하기에 충분한 양을 의미한다. 본 발명에 따른 지방유래 줄기세포로부터 유래한 엑소좀은 상기 약학적 조성물 중에 1×10
7 particles/mL 내지 1×10
10 particles/㎏의 농도, 바람직하게는 5×10
7 particles/mL 내지 1×10
9 particles/㎏의 농도로 포함될 수 있다. 그러나 상기 엑소좀의 농도는 관절염의 진행 정도, 환자의 연령, 체중, 건강상태, 성별, 투여 경로 및 치료 기간 등에 따라 적절히 변화될 수 있다.In the above, the pharmaceutically effective amount means an amount sufficient to improve and treat inflammation occurring in the joints. The exosomes derived from adipose-derived stem cells according to the present invention have a concentration of 1×10 7 particles/mL to 1×10 10 particles/kg in the pharmaceutical composition, preferably 5×10 7 particles/mL to 1× It may be included at a concentration of 10 9 particles/kg. However, the concentration of the exosomes can be appropriately changed depending on the degree of progression of arthritis, the age, weight, health status, sex, administration route, and treatment period of the patient.
또한, 상기 "약학적으로 허용되는"이란 생리학적으로 허용되고 인간에게 투여될 때, 통상적으로 위장 장애, 현기증과 같은 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 조성물을 의미한다. 상기 담체, 부형제 및 희석제의 예로는, 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 또한, 충진제, 항응집제, 윤활제, 습윤제, 향료, 유화제 및 방부제 등을 추가로 포함할 수 있다.In addition, the "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not normally cause gastrointestinal disorders, allergic reactions such as dizziness or similar reactions when administered to humans. Examples of such carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, fillers, anti-agglomeration agents, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.
또한, 본 발명의 조성물은 약학적 분야에서 통상의 방법에 따라 환자의 신체 내 투여에 적합한 단위투여형의 제제로 제형화시켜 투여할 수 있으며, 상기 제제는 1회 또는 수회 투여에 의해 관절염을 완화 내지 치료할 수 있는 효과적인 투여량을 포함한다. 이러한 목적에 적합한 제형으로는 비경구투여 제제로서 주사용 앰플과 같은 주사제, 주입 백과 같은 주입제, 등이 바람직하다. 상기 주사용 앰플은 사용 직전에 주사액과 혼합 조제할 수 있으며, 주사액으로는 생리 식염수, 포도당, 만니톨, 링거액 등을 사용할 수 있다. In addition, the composition of the present invention can be administered by formulating a unit dosage form suitable for administration in the body of a patient according to a conventional method in the pharmaceutical field, and the preparation is administered once or several times to relieve arthritis. to therapeutically effective dosages. Formulations suitable for this purpose include, for parenteral administration, injections such as ampoules for injection, injections such as infusion bags, and the like. The injection ampoule may be prepared by mixing with an injection solution immediately before use, and physiological saline, glucose, mannitol, Ringer's solution, etc. may be used as the injection solution.
상기 약학적 제제에는 상기 유효성분 외에 하나 또는 그 이상의 약학적으로 허용가능한 통상의 불활성 담체, 예를 들어, 주사제의 경우에는 보존제, 무통화제, 가용화제 또는 안정화제 등을, 국소 투여용 제제의 경우에는 기제 (base), 부형제, 윤활제 또는 보존제 등을 추가로 포함할 수 있다.In the pharmaceutical formulation, in addition to the active ingredient, one or more pharmaceutically acceptable conventional inert carriers, for example, preservatives, analgesics, solubilizers or stabilizers in the case of injections, and the like, in the case of formulations for topical administration It may further include a base (base), excipients, lubricants or preservatives and the like.
이렇게 제조된 본 발명의 조성물 또는 약학적 제제는 쥐, 생쥐, 가축, 인간 등의 포유동물에 비경구, 경구 등의 다양한 경로로 투여될 수 있으며, 투여 방법은 당업계에서 통상적으로 사용하는 모든 방식에 의해 투여될 수 있다. 이에 제한되지는 않으나, 경구, 직장 또는 정맥, 근육, 피하, 자궁내 경막 또는 뇌혈관 내(intracerebroventricular) 주사 등에 의해 투여될 수 있다.The composition or pharmaceutical preparation of the present invention prepared in this way may be administered to mammals such as rats, mice, livestock, and humans by various routes such as parenteral and oral administration, and the administration method is any method commonly used in the art. can be administered by Although not limited thereto, it may be administered by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.
구체적으로, 투여 방법은 이에 제한되지는 않으나, 지방유래 줄기세포로부터 추출된 엑소좀을 정맥 내로 투여(정맥주사; Intravenous injection)하거나, 관절염 부위로 투여(국소 투여; Topical administration) 될 수 있다. Specifically, the administration method is not limited thereto, but the exosomes extracted from adipose-derived stem cells may be administered intravenously (intravenous injection) or administered to an arthritis site (local administration; Topical administration).
다른 하나의 양태로서, 본 발명은 전술한 지방유래 줄기세포로부터 유래한 엑소좀을 유효성분으로 포함하는 관절염 완화 및 치료용 약학적 조성물을 포함하는 관절염 완화 또는 치료용 주사제를 제공한다.As another aspect, the present invention provides an injection for alleviating or treating arthritis, comprising a pharmaceutical composition for alleviating and treating arthritis comprising the adipose-derived stem cell-derived exosome as an active ingredient.
상기 주사제는 인산완충식염수(phosphate-buffered saline, PBS)를 더 포함할 수 있다. 즉, 상기 주사제는 인산완충식염수에 지방 유래 줄기세포로부터 추출된 엑소좀을 담지하여 사용할 수 있다. The injection may further include phosphate-buffered saline (PBS). That is, the injection can be used by supporting the exosomes extracted from adipose-derived stem cells in phosphate buffered saline.
상기 주사제는 인산완충식염수 대신 하이드로젤을 포함할 수도 있다. 상기 하이드로젤은 히알루론산, 젤라틴, 알지네이트, 키토산, 피브린, 엘라스틴, 콜라겐 및 메틸셀룰로오즈로 구성된 군으로부터 선택된 어느 하나 이상일 수 있고, 구체적으로 히알루론산 하이드로젤일 수 있으나, 이에 한정되지 않는다.The injection may include hydrogel instead of phosphate buffered saline. The hydrogel may be at least one selected from the group consisting of hyaluronic acid, gelatin, alginate, chitosan, fibrin, elastin, collagen, and methylcellulose, and specifically may be a hyaluronic acid hydrogel, but is not limited thereto.
본 발명에 따른 줄기세포유래 엑소좀은 연골에 발생한 염증을 완화 및 치료하여 연골 침식을 방지할 뿐만 아니라, 활막(synovium)과 같은 주변 조직 내의 염증 반응까지도 효과적으로 완화시켜 관절염을 근본적으로 치유할 수 있다. The stem cell-derived exosome according to the present invention not only prevents cartilage erosion by alleviating and treating inflammation occurring in the cartilage, but also effectively relieves the inflammatory response in surrounding tissues such as the synovial membrane, thereby fundamentally curing arthritis. .
도 1은 엑소좀의 현미경 분석 및 엑소좀 표면 마커 분석 결과를 나타낸 것이다.1 shows the results of microscopic analysis and exosome surface marker analysis of exosomes.
도 2는 각 계대별로 생산된 엑소좀에 포함된 항염증 사이토카인(TIMP-1 및 TIMP-2)의 함유량을 나타낸 것이다.Figure 2 shows the content of anti-inflammatory cytokines (TIMP-1 and TIMP-2) contained in the exosomes produced for each passage.
도 3은 인간지방과 인간제대유래 줄기세포로부터 생산된 엑소좀의 염증조절인자(TIMP-1)를 분석한 결과이다(인간골수유래 줄기세포로부터 생산된 엑소좀의 경우 유의한 함량이 확인되지 아니하여 표시하지 않음).3 is a result of analyzing the inflammatory regulator (TIMP-1) of exosomes produced from human fat and human umbilical cord-derived stem cells (in the case of exosomes produced from human bone marrow-derived stem cells, a significant content was not confirmed. not indicated).
도 4는 MIA 유도 래트 골관절염 모델에서 분리된 무릎 관절을 염색하고 관찰한 결과를 나타낸 것이다.4 shows the results of staining and observing the knee joint isolated from the MIA-induced rat osteoarthritis model.
도 5는 MIA 유도 래트 골관절염 모델에서 주변 활막 및 IFP의 염증 반응에 대한 엑소좀의 효능을 평가한 결과를 나타낸 것이다.5 shows the results of evaluating the efficacy of the exosomes on the inflammatory response of the peripheral synovial membrane and IFP in the MIA-induced rat osteoarthritis model.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명 하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에게 있어서 자명할 것이다. Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.
실시예Example
1. 증식하는 줄기세포로부터의 엑소좀 추출1. Extraction of exosomes from proliferating stem cells
1차 hASC(Primary hASCs)는 CEFO Bio Co., Ltd(Seoul, Korea)에서 구입했으며 성장 배지와 보충제는 Life Technologies(Carlsbad, CA, USA)에서 구입하였다. 일반 배양배지(10% fetal bovine serum, 1% penicillin/streptomycin를 함유하는 Dulbecco Modified Eagle Medium high glucose(DMEM))를 사용하여 37℃, 5 % CO
2 조건에서 hASC를 3회 내지 7회 계대 배양하여 증식시켰다. 각 계대(passage)마다 엑소좀을 추출하였는데, 구체적으로 엑소좀을 추출하기 전에 각 계대 별 증식된 줄기세포를 무혈청 DMEM 배지로 교체하여 24시간 동안 유지한 후, 세포 배양 상층액을 회수하였다. 그 다음 접선 흐름 여과 시스템 (TFF)에 기반한 다중 여과 시스템에 의해 상기 회수한 세포 배양 상층액로부터 엑소좀을 분리하였다. Primary hASCs were purchased from CEFO Bio Co., Ltd (Seoul, Korea), and growth medium and supplements were purchased from Life Technologies (Carlsbad, CA, USA). Using normal culture medium (Dulbecco Modified Eagle Medium high glucose (DMEM) containing 10% fetal bovine serum and 1% penicillin/streptomycin), hASCs were subcultured 3 to 7 times at 37°C and 5% CO 2 conditions. proliferated. Exosomes were extracted for each passage, specifically, before extracting the exosomes, the proliferated stem cells for each passage were replaced with serum-free DMEM medium and maintained for 24 hours, and then the cell culture supernatant was recovered. Then, exosomes were isolated from the recovered cell culture supernatant by a multiple filtration system based on a tangential flow filtration system (TFF).
2. 엑소좀의 현미경 관찰 및 표면 마커 분석2. Microscopic observation and surface marker analysis of exosomes
상기 증식하는 줄기세포로부터 추출된 엑소좀을 투과전자현미경(transmission electron microscope)과 나노입자분석기(dynamic light scattering)를 사용하여 크기 및 모양을 확인하고, 유세포 분석기(flow cytometry)를 이용하여 엑소좀 막 표면 단백질을 확인하였다. The size and shape of the exosomes extracted from the proliferating stem cells were confirmed using a transmission electron microscope and dynamic light scattering, and the exosome membrane using flow cytometry. Surface proteins were identified.
그 결과, 추출된 엑소좀의 모양을 투과전자현미경으로 확인할 수 있었으며, 각각의 크기는 평균적으로 약 86.46 nm인 것을 확인하였다(도 1).As a result, the shape of the extracted exosomes could be confirmed with a transmission electron microscope, and it was confirmed that the size of each was about 86.46 nm on average (Fig. 1).
또한, 유세포 분석기를 이용하여 엑소좀 표면 마커 단백질로 알려진 CD9, CD63 및 CD81이 추출된 엑소좀에서 모두 발현됨을 확인하였다(도1).In addition, it was confirmed that CD9, CD63, and CD81, known as exosome surface marker proteins, were all expressed in the extracted exosomes using flow cytometry (Fig. 1).
3. 엑소좀에 함유된 항염증성 인자 평가3. Anti-inflammatory factors contained in exosomes Evaluation
염증 완화에 관련하여 분비되는 대표적인 사이토카인으로서 TIMP-1 (tissue inhibitors of metalloproteinases-1) 및 TIMP-2가 있는데, 이들 사이토카인은 염증반응 억제를 유도하고 염증성 사이토카인을 억제하도록 유도시키는 역할을 가지고 있는 것으로 알려져 있다.Representative cytokines secreted in relation to inflammation relief include TIMP-1 (tissue inhibitors of metalloproteinases-1) and TIMP-2. It is known that there is
상기 passage 3, 5 및 7에서 생산된 엑소좀에 함유된 항염증 인자를 확인하기 위해, 각각의 엑소좀을 사용하여 사이토카인 분석을 실시하고 그 결과를 도 2에 나타내었다. 실험 결과, passage 5에서 생산된 엑소좀이 passage 3 또는 passage 7에서 생산된 엑소좀보다 인간지방유래줄기세포 엑소좀 내부에 항염증 사이토카인(TIMP-1 및 TIMP-2)의 함유량이 크게 향상된 것이 확인되었다(도 2).In order to confirm the anti-inflammatory factors contained in the exosomes produced in passages 3, 5 and 7, cytokine analysis was performed using each exosome, and the results are shown in FIG. 2 . As a result of the experiment, the content of anti-inflammatory cytokines (TIMP-1 and TIMP-2) inside the exosomes of human adipose-derived stem cells was significantly improved in the exosomes produced in passage 5 compared to the exosomes produced in passage 3 or passage 7. was confirmed (FIG. 2).
또한, 비교 대조군으로 인간 골수유래 줄기세포(human Bone Marrow derived mesenchymal stem cells; passage 3)로부터 생산된 엑소좀(BM-MSC-Exo) 및 인간 제대유래 줄기세포(human umbilical cord mesenchymal stem cells)로부터 생산된 엑소좀(UC-MSC-Exo; passage 5)을 사용하여, 상기 인간 지방유래 줄기세포로부터 생산된 엑소좀(hASC-Exo; passage 5)과의 상대적인 TIMP-1 intensity를 측정하였다. 실험 결과, BM-MSC-Exo의 경우 유의한 TIMP-1 함량이 확인되지 아니하였고, UC-MSC-Exo의 경우 TIMP-1을 함유하는 것으로 확인되었으나, hASC-Exo에 비해 TIMP-1 함량이 현저하게 낮은 것으로 확인되었다(도 3) (*P < 0.1).In addition, as a comparative control, exosomes produced from human Bone Marrow derived mesenchymal stem cells (passage 3) (BM-MSC-Exo) and human umbilical cord mesenchymal stem cells produced from Using the exosomes (UC-MSC-Exo; passage 5), the relative TIMP-1 intensity with the exosomes (hASC-Exo; passage 5) produced from the human adipose-derived stem cells was measured. As a result, in the case of BM-MSC-Exo, a significant TIMP-1 content was not confirmed, and in the case of UC-MSC-Exo, it was confirmed that it contained TIMP-1, but the TIMP-1 content was significantly higher than that of hASC-Exo. was confirmed to be very low ( FIG. 3 ) (*P < 0.1).
4. MIA 유도 OA 모델에서 hASC-EV의 치료 효능4. Therapeutic efficacy of hASC-EV in MIA-induced OA model
상기 3에서 항염증 효과가 가장 우수한 것으로 확인된 passage 5에서 생산된 엑소좀을 사용하여 MIA 유도 래트 OA 모델(monosodium iodoacetate (MIA)-induced rat OA model)에서의 관절염 치료 효능을 평가하였다. 구체적으로, 엑소좀을 MIA 주입 후 1 주일부터 3 주 동안 매주 관절 내에 투여하였다. MIA 주사 4 주 후, 분리된 무릎 관절을 인도 잉크로 염색하였으며 관찰 결과를 도 4에 나타내었다.Arthritis treatment efficacy in the MIA-induced rat OA model (monosodium iodoacetate (MIA)-induced rat OA model) was evaluated using the exosomes produced in passage 5, which was confirmed to have the best anti-inflammatory effect in 3 above. Specifically, exosomes were administered intra-articularly every week for 3 weeks from 1 week after MIA injection. 4 weeks after MIA injection, the isolated knee joint was stained with India ink, and the observation results are shown in FIG. 4 .
실험 결과, hASC-EXO 처리 그룹은 MIA 모델의 관절에서 PBS 및 HA 처리 그룹에 비해, 염증에 의하여 침식된 연골 표면의 면적이 크게 감소했음이 확인되었다(도 4). As a result of the experiment, it was confirmed that the area of the cartilage surface eroded by inflammation was significantly reduced in the hASC-EXO-treated group compared to the PBS and HA-treated groups in the joints of the MIA model ( FIG. 4 ).
5. 활막 염증에 대한 엑소좀의 영향 평가5. Evaluation of the effect of exosomes on synovial inflammation
무릎 관절(knee joint)의 MIA 주입은 연골 저하(cartilage degradation)뿐만 아니라 주변 활막(perimeniscal synovium) 및 슬개 아래 지방 패드(infrapatellar fat pad; IFP)를 포함한 관절 주변 조직의 변화를 유도하였다. 이러한 주변 활막 및 IFP의 염증 반응에 대한 hASC-EXO의 효과를 평가하고 그 결과를 도 5에 나타내었다.MIA injection of the knee joint induced cartilage degradation as well as changes in the tissues surrounding the joint, including the perimeniscal synovium and the infrapatellar fat pad (IFP). The effect of hASC-EXO on the inflammatory response of the surrounding synovial membrane and IFP was evaluated, and the results are shown in FIG. 5 .
급성 관절염 모델에서는 PBS 처리 군에서 내막하 섬유증 및 혈관 신생을 포함한 활막 염증이 관찰되었다. 그러나 hASC-EXO 투여군에서는 섬유성 침착 및 혈관 형성을 방지하고 IFP의 지방 세포가 풍부한 모양을 유지하는 것이 확인되었다(도 5). 또한, 만성 관절염 모델의 PBS 그룹에서는 증식, 광범위한 내막하 섬유증과 혈관 형성 및 연골 단편을 포함한 심각한 활막 염증이 발견된 반면, hASC-EXO 투여군은 활막 염증이 감소되는 것으로 확인되었다(도 5). In the acute arthritis model, synovial inflammation including subintimal fibrosis and angiogenesis was observed in the PBS-treated group. However, it was confirmed that in the hASC-EXO-administered group, fibrotic deposition and angiogenesis were prevented and the IFP adipocyte-rich shape was maintained ( FIG. 5 ). In addition, in the PBS group of the chronic arthritis model, severe synovial inflammation including proliferation, extensive subintimal fibrosis and angiogenesis and cartilage fragmentation was found, whereas in the hASC-EXO-administered group, synovial inflammation was reduced ( FIG. 5 ).
이러한 결과는 hASC-EXO가 관절염 모델에서 연골 저하를 방지할 뿐만 아니라, 주변 활막 및 IFP의 염증 반응을 완화시켜 관절염 치료 효과가 있음을 나타낸다.These results indicate that hASC-EXO not only prevents cartilage degradation in the arthritis model, but also relieves the inflammatory response of the surrounding synovial membrane and IFP, thereby having an arthritis treatment effect.
Claims (8)
- 증식하는 지방유래 줄기세포(adipose derived stem cells)로부터 유래한 엑소좀을 유효성분으로 포함하는, 관절염 완화 또는 치료용 약학적 조성물. A pharmaceutical composition for relieving or treating arthritis, comprising, as an active ingredient, an exosome derived from proliferating adipose derived stem cells (adipose derived stem cells).
- 제1항에 있어서, 상기 지방유래 줄기세포는 인간 지방유래 줄기세포인 것인 약학적 조성물.The pharmaceutical composition according to claim 1, wherein the adipose-derived stem cells are human adipose-derived stem cells.
- 제1항에 있어서, 상기 관절염은 골관절염(osteoarthritis) 및 류마티스 관절염(rheumatoid arthritis)으로부터 선택되는 것인 약학적 조성물.The pharmaceutical composition according to claim 1, wherein the arthritis is selected from osteoarthritis and rheumatoid arthritis.
- 제1항에 있어서, 상기 엑소좀은 4회 내지 6회 계대 배양에서 증식하는 줄기세포로부터 유래한 것인 약학적 조성물.The pharmaceutical composition according to claim 1, wherein the exosomes are derived from stem cells proliferating in passages 4 to 6 times.
- 제1항에 있어서, 상기 엑소좀은 5회 계대 배양에서 증식하는 줄기세포로부터 유래한 것인 약학적 조성물.The pharmaceutical composition according to claim 1, wherein the exosomes are derived from stem cells proliferating in passage 5.
- 제1항에 있어서, 상기 엑소좀은 항염증성 사이토카인을 포함하는 것인 약학적 조성물.The pharmaceutical composition according to claim 1, wherein the exosome comprises an anti-inflammatory cytokine.
- 제6항에 있어서, 상기 항염증성 사이토카인은 TIMP-1 및 TIMP-2로부터 선택되는 1 이상인 것인 약학적 조성물.The pharmaceutical composition according to claim 6, wherein the anti-inflammatory cytokine is at least one selected from TIMP-1 and TIMP-2.
- 제1항 내지 제7항 중 어느 하나의 항에 따른 약학적 조성물을 포함하는 관절염 완화 또는 치료용 주사제.An injection for relieving or treating arthritis comprising the pharmaceutical composition according to any one of claims 1 to 7.
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| KR20190066885A (en) * | 2017-12-06 | 2019-06-14 | 주식회사 디자인셀 | Composition for treating or preventing arthritis comprising culture solution of stem cell-derived exosome |
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