Pharmaceutical composition for alleviating or treating arthritis, comprising exosomes derived from stem cells

Abstract

The present invention relates to a pharmaceutical composition for alleviating and treating arthritis based on exosomes. The exosomes according to the present invention can not only alleviate and treat inflammation occurring in cartilage to prevent cartilage erosion, but also effectively alleviate inflammatory reactions in surrounding tissues such as synovium, thereby fundamentally treating arthritis.

Description

Detailed Description of the Invention

[0001] [Technical field] The present invention relates to pharmaceutical compositions for the alleviation or treatment of arthritis. [Background technology] Rheumatoid arthritis is understood to be caused by autoimmune inflammation. Degenerative arthritis, or osteoarthritis, is a disease induced by the degeneration of chondrocytes, which make up the joints, through aging and other processes. This causes the production of inflammatory cytokines, such as interleukin-1 and tumor necrosis factor-α, which increase the synthesis and activity of matrix metalloproteinases (MMPs), which degrade the joint matrix, in articular cells, resulting in the destruction of joint tissue.

[0002] In addition, arthritis worsens when inflammatory cytokines produce nitric oxide, which in turn amplifies cytokine production, leading to increased synthesis of MMPs and accelerated degradation of the joint matrix. Inflammatory cytokines also increase the production of prostaglandin E2, a lipid metabolite, inducing the inflammatory response in arthritis. Osteoarthritis, also known as degenerative arthritis, is a type of arthritis that frequently occurs in older adults without a specific underlying cause. Chronic progression can lead to movement disorders, including gait disturbances, due to deformation of the joint structure. Osteoarthritis primarily occurs as a result of gradual damage and degenerative changes to the articular cartilage, which damages the bones and ligaments that make up the joint, resulting in inflammation. As osteoarthritis progresses, increased production of inflammatory cytokines, such as TNF-α and IL-1, and increased secretion of MMPs, such as collagenase and stromelysin, leads to the destruction of articular cartilage. MMP also induces IL-1, TNF-α, etc., which affects tissues such as muscles, tendons, and ligaments, causing severe inflammation.

[0003] Numerous documents are referenced and citations are provided throughout this specification, the disclosures of which are hereby incorporated by reference in their entirety to more clearly describe the state of the art to which this invention pertains and the content of the present invention. [Summary of the Invention] [Problem to be solved by the invention] The present invention was completed based on the discovery that exosomes derived from stem cells alleviate and treat inflammation in cartilage, thereby preventing cartilage erosion caused by inflammation and also effectively alleviating inflammatory reactions in surrounding tissues such as the synovium, thereby having a therapeutic effect on arthritis.

[0004] Therefore, an object of the present invention is to provide a pharmaceutical composition for alleviating or treating arthritis, which comprises exosomes derived from stem cells as an active ingredient. More specifically, the present invention aims to provide the following embodiments.

[0005] Example 1. A pharmaceutical composition for alleviating or treating arthritis, comprising exosomes 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 a medicament for alleviating or treating arthritis; or a method for alleviating or treating arthritis, comprising administering to a subject a composition comprising exosomes derived from proliferating adipose-derived stem cells as an active ingredient.

[0006] Embodiment 2. The pharmaceutical composition, use, or method of embodiment 1, wherein the adipose-derived stem cells are human adipose-derived stem cells.

[0007] Embodiment 3. The pharmaceutical composition; use; or method of any one of the preceding embodiments, wherein the arthritis is selected from osteoarthritis and rheumatoid arthritis.

[0008] Embodiment 4. The pharmaceutical composition, use, or method of any one of the preceding embodiments, wherein the exosomes are derived from stem cells proliferated through 4 to 6 passages.

[0009] Embodiment 5. The pharmaceutical composition; use; or method of any one of the preceding embodiments, wherein the exosomes are derived from stem cells expanded for five passages.

[0010] Embodiment 6: The pharmaceutical composition; use; or method of any one of the preceding embodiments, wherein the exosomes comprise an anti-inflammatory cytokine.

[0011] Embodiment 7. The pharmaceutical composition; use; or method of any one of the preceding embodiments, wherein the anti-inflammatory cytokine is one or more selected from TIMP-1 and TIMP-2.

[0012] Embodiment 8. An injectable preparation for alleviating or treating arthritis, comprising a pharmaceutical composition according to any one of the preceding embodiments.

[0013] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, the claims and the drawings. [Means for solving the problem] One aspect of the present invention provides a pharmaceutical composition for alleviating or treating arthritis, comprising exosomes derived from proliferating adipose-derived stem cells as an active ingredient.

[0014] The term "stem cell" as used herein refers to a cell that not only has the ability to self-renew, but also has the ability to differentiate into various cells due to its multipotency when given appropriate signals as needed, in response to the influence of the environment in which the cell is located.

[0015] The stem cells of the present invention may be autologous or allogeneic, and may be derived from any type of animal, including humans and non-human mammals. Preferably, the stem cells are derived from the patient's own adipose tissue.

[0016] The term "adipose-derived stem cells" used in the present invention refers to stem cells derived from adipose tissue. Adipose tissue is an ideal source of stem cells because it is easy to harvest large amounts of tissue. Adipose-derived stem cells exhibit stable growth and proliferation in culture and can differentiate into a variety of cells when induced to differentiate.

[0017] The term "exosome" as used in the present invention refers to a membrane-structured endoplasmic reticulum secreted by various cells, which contains immunologically important proteins such as major histocompatibility proteins and heat shock proteins and is known to play various roles, such as inducing a potent anti-tumor immune response and binding to other cells and tissues to deliver membrane components, proteins, and RNA. Therefore, exosomes containing various genetic information, proteins, and growth factors related to inflammation alleviation can be used as a composition for treating arthritis.

[0018] The exosomes derived from the adipose-derived stem cells of the present invention contain anti-inflammatory cytokines, which can play an important role in the alleviation and treatment of arthritis, such as osteoarthritis and rheumatoid arthritis. Specifically, these cytokines include TIMP-1 (tissue inhibitor of metalloproteinase-1), TIMP-2, IL-1ra (interleukin-1 receptor antagonist), IL-4 (interleukin-4), IL-10 (interleukin-10), and TGF-β (transforming growth factor-β). These cytokines play a role in inducing inhibition of inflammatory responses and inhibiting pro-inflammatory cytokines.

[0019] The exosomes containing the anti-inflammatory cytokines may be derived from stem cells proliferated through subculture, preferably from stem cells proliferated through 4 to 6 subcultures, more preferably from stem cells proliferated through 5 subcultures. When exosomes are extracted from stem cells proliferated through 4 to 6 subcultures, the content of anti-inflammatory cytokines is significantly increased compared to exosomes extracted from stem cells proliferated through 3 or 7 subcultures, which is of critical significance.

[0020] Specifically, the mass of anti-inflammatory cytokines contained per protein (pg / µg protein) in exosomes derived from stem cells expanded through 4 to 6 passages can be at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold the amount contained in exosomes derived from stem cells expanded through 3 or 7 passages. Pharmaceutical compositions according to the present invention may comprise a pharmaceutically effective amount of exosomes derived from adipose-derived stem cells together with one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0021] The term "pharmacologically effective amount" refers to an amount sufficient to improve and treat inflammation occurring in a joint. The exosomes derived from adipose-derived stem cells according to the present invention are present in the pharmaceutical composition in an amount of 1×10 7 Particles / mL~1×10 10 Concentration of particles / mL, preferably 5 x 10 7 Particles / mL~1×10 9 However, the concentration of the exosomes may be appropriately changed depending on the progression of arthritis, the patient's age, weight, health condition, sex, administration route, and treatment period.

[0022] "Pharmaceutically acceptable" means a composition that is physiologically acceptable and does not normally cause allergic or similar reactions, such as gastrointestinal disorders or dizziness, when administered to humans. Examples of the carrier, excipient, and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. The composition may further contain fillers, anti-agglomerating agents, lubricants, wetting agents, flavorings, emulsifiers, preservatives, and the like.

[0023] The compositions of the present invention can be formulated into unit-dose preparations suitable for administration to a patient's body using conventional methods in the pharmaceutical field, and the preparations contain an effective dose that can alleviate or treat arthritis with one or more administrations. Suitable preparations for this purpose include parenteral preparations, such as injections (e.g., injection ampoules) and infusion bags. The injection ampoules are prepared by mixing with an injection solution immediately before use, which can be physiological saline, glucose, mannitol, Ringer's solution, or the like.

[0024] The pharmaceutical preparation may further contain, in addition to the active ingredient, one or more pharmaceutically acceptable conventional inactive carriers, such as a preservative, a soothing agent, a solubilizing agent, or a stabilizer in the case of an injection, or a base, an excipient, a lubricant, or a preservative in the case of a preparation for topical administration.

[0025] The composition or pharmaceutical formulation of the present invention thus prepared can be administered to mammals such as rats, mice, livestock, and humans via various routes, including parenteral and oral administration, and any administration method commonly used in the art can be used, including, but not limited to, oral, rectal, intravenous, intramuscular, subcutaneous, intrauterine dura, or intracerebroventricular injection.

[0026] Specifically, although the administration method is not limited thereto, exosomes extracted from adipose-derived stem cells can be administered intravenously (intravenous injection) or administered to the arthritis site (local administration).

[0027] In another aspect, the present invention provides an injectable preparation for alleviating or treating arthritis, which comprises a pharmaceutical composition for alleviating and treating arthritis that contains exosomes derived from the above-mentioned adipose-derived stem cells as an active ingredient.

[0028] The injection may further contain phosphate buffered saline (PBS), i.e., the injection may be used by carrying exosomes extracted from adipose-derived stem cells in phosphate buffered saline.

[0029] The injection may contain a hydrogel instead of phosphate buffered saline, and the hydrogel may be at least one selected from the group consisting of hyaluronic acid, gelatin, alginate, chitosan, fibrin, elastin, collagen, and methylcellulose, specifically, but not limited to, hyaluronic acid hydrogel. [Effects of the invention] The stem cell-derived exosomes according to the present invention not only alleviate and treat inflammation in cartilage to prevent cartilage erosion, but also effectively alleviate inflammatory responses in surrounding tissues such as the synovium, thereby fundamentally treating arthritis. [Brief description of the drawing] [Figure 1] Results of microscopic analysis of exosomes and analysis of exosome surface markers. [Figure 2] The content of anti-inflammatory cytokines (TIMP-1 and TIMP-2) contained in exosomes produced at each passage is shown. [Figure 3] Results of analyzing the inflammatory regulator (TIMP-1) in exosomes produced from human adipose and human umbilical cord-derived stem cells (exosomes produced from human bone marrow-derived stem cells were not shown, as no significant content was confirmed). [Fig. 4] Results of staining and observation of knee joints isolated from an MIA-induced rat osteoarthritis model. [Figure 5] Results of evaluating the efficacy of exosomes on the inflammatory response of the surrounding synovium and IFP in an MIA-induced rat osteoarthritis model. [Mode for Carrying Out the Invention] The present invention will be described in more detail below with reference to examples. It will be obvious to those skilled in the art that these examples are provided merely to more specifically illustrate the present invention and are not intended to limit the scope of the present invention.

[0030] <Example> 1. Extraction of exosomes from proliferating stem cells Primary human adipose-derived stem cells (hASCs) were purchased from CEFO Bio (Seoul, Korea), and growth medium and supplements were purchased from Life Technologies (Carlsbad, CA, USA). hASCs were expanded in general culture medium (Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and 1% penicillin / streptomycin) at 37°C and 5% CO2 for 3 to 7 passages. Exosomes were extracted after each passage. Specifically, before exosome extraction, the expanded stem cells were replaced with serum-free DMEM medium and maintained for 24 hours, after which the cell culture supernatant was collected. Exosomes were then isolated from the collected cell culture supernatant using a multiple filtration system based on a tangential flow filtration (TFF) system.

[0031] 2. Microscopic observation of exosomes and analysis of surface markers The size and pattern of exosomes extracted from proliferating stem cells were confirmed using a transmission electron microscope and a nanoparticle analyzer (Dynamic Light Scattering (DLS)), and the surface proteins of the exosome membrane were identified using flow cytometry.

[0032] As a result, the pattern of the extracted exosomes could be confirmed using a transmission electron microscope, and it was confirmed that each exosome had an average size of approximately 86.46 nm (Figure 1).

[0033] Furthermore, flow cytometry confirmed that CD9, CD63, and CD81, known exosome surface marker proteins, were all expressed in the extracted exosomes (Figure 1).

[0034] 3. Evaluation of anti-inflammatory factors contained in exosomes Representative cytokines secreted to alleviate inflammation include TIMP-1 (tissue inhibitor of metalloproteinase-1) and TIMP-2, which are known to play a role in inducing inhibition of inflammatory responses and inducing inhibition of inflammatory cytokines.

[0035] To confirm the anti-inflammatory factors contained in the exosomes produced at passages 3, 5, and 7, cytokine analysis was performed using each exosome, and the results are shown in Figure 2. The experimental results confirmed that the content of anti-inflammatory cytokines (TIMP-1 and TIMP-2) inside the exosomes of human adipose-derived stem cells was significantly increased in the exosomes produced at passage 5 compared to the exosomes produced at passage 3 or passage 7 (Figure 2).

[0036] In addition, we used exosomes produced from human bone marrow-derived stem cells (BM-MSC-Exo; passage 3) and exosomes produced from human umbilical cord-derived stem cells (UC-MSC-Exo; passage 5) as control groups to measure the relative TIMP-1 intensity compared to exosomes produced from human adipose-derived stem cells (hASC-Exo; passage 5). The results showed that BM-MSC-Exo did not contain significant TIMP-1 content, while UC-MSC-Exo contained TIMP-1, but the TIMP-1 content was significantly lower than that of hASC-Exo (Figure 3) (*P<0.1).

[0037] 4. Therapeutic efficacy of hASC-EVs in an MIA-induced OA model Using exosomes produced at passage 5, which were confirmed to have the most excellent anti-inflammatory effect in section 3 above, we evaluated their therapeutic efficacy for arthritis in an MIA (Monosodium Iodoacetate)-induced rat OA model. Specifically, exosomes were administered intra-articularly weekly for three weeks, starting one week after MIA injection. Four weeks after MIA injection, isolated knee joints were stained with Indian ink, and the results are shown in Figure 4.

[0038] The results of the experiment confirmed that the area of ​​cartilage surface eroded by inflammation in the MIA model joints was significantly reduced in the hASC-Exo treatment group compared to the PBS and HA (hyaluronic acid) treatment groups (Figure 4).

[0039] 5. Evaluation of the effect of exosomes on synovial inflammation MIA injection into the knee joint induced not only cartilage degradation but also changes in periarticular tissues, including the perimeniscal synovium and infrapatellar fat pad (IFP). The effects of hASC-Exo on the inflammatory responses of the perimeniscal synovium and IFP were evaluated, and the results are shown in Figure 5.

[0040] In the acute arthritis model, synovial inflammation, including subintimal fibrosis and angiogenesis, was observed in the PBS-treated group. However, the hASC-Exo-treated group prevented fibrous deposition and angiogenesis and maintained the adipocyte-rich pattern of IFPs (Figure 5). In the chronic arthritis model, the PBS group exhibited severe synovial inflammation, including proliferation, extensive subintimal fibrosis and angiogenesis, and cartilage fragments, while the hASC-Exo-treated group exhibited reduced synovial inflammation (Figure 5).

[0041] These results indicate that hASC-Exo not only prevents cartilage degradation in an arthritis model, but also alleviates the inflammatory response of the surrounding synovium and IFP, thereby having a therapeutic effect on arthritis. [Brief explanation of the drawings]

[0042] [Figure 1a] 1 shows the results of microscopic analysis of exosomes and analysis of exosome surface markers. [Figure 1b] 1 shows the results of microscopic analysis of exosomes and analysis of exosome surface markers. [Figure 1c] 1 shows the results of microscopic analysis of exosomes and analysis of exosome surface markers. [Figure 2] This shows the content of anti-inflammatory cytokines (TIMP-1 and TIMP-2) contained in exosomes produced at each passage. [Figure 3] These are the results of analyzing the inflammatory regulator (TIMP-1) in exosomes produced from human adipose and human umbilical cord-derived stem cells (no significant content was confirmed in exosomes produced from human bone marrow-derived stem cells, so the results are not shown). [Figure 4a] This shows the results of staining and observing knee joints isolated from an MIA-induced rat osteoarthritis model. [Figure 4b] This shows the results of staining and observing knee joints isolated from an MIA-induced rat osteoarthritis model. [Figure 5a] These are the results of evaluating the efficacy of exosomes on the inflammatory response of the surrounding synovium and IFP in an MIA-induced rat osteoarthritis model. [Figure 5b] These are the results of evaluating the efficacy of exosomes on the inflammatory response of the surrounding synovium and IFP in an MIA-induced rat osteoarthritis model. [Figure 5c] These are the results of evaluating the efficacy of exosomes on the inflammatory response of the surrounding synovium and IFP in an MIA-induced rat osteoarthritis model.

Claims

1. A method for producing a pharmaceutical composition for alleviating or treating arthritis, arthrofibrosis, and joint angiogenesis, comprising exosomes derived from proliferating adipose-derived stem cells as an active ingredient, The method for producing a pharmaceutical composition, wherein the exosomes are isolated from stem cells that have been proliferated through five subcultures.

2. The method of claim 1 , wherein the adipose-derived stem cells are human adipose-derived stem cells.

3. 2. The method of claim 1, wherein the arthritis is selected from osteoarthritis and rheumatoid arthritis.

4. The method according to claim 1, wherein the exosomes contain an anti-inflammatory cytokine.

5. The method according to claim 4, wherein the anti-inflammatory cytokine is one or more selected from TIMP-1 and TIMP-2.

6. A method for producing an injectable preparation for alleviating or treating arthritis, arthrofibrosis, and joint angiogenesis, comprising producing a pharmaceutical composition by the production method described in any one of claims 1 to 5.

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