Pharmaceutical composition for preventing or treating myositis, comprising isolated mitochondria as active ingredient

Abstract

To provide a pharmaceutical composition for preventing or treating myositis.SOLUTION: The present invention relates to a pharmaceutical composition for preventing or treating myositis, comprising mitochondria as an active ingredient. When the pharmaceutical composition of the present invention comprising exogenous mitochondria as an active ingredient is administered to a subject suffering from myositis, inflammatory cells infiltrated into the muscle cells of the subject can be reduced. In addition, the pharmaceutical composition of the present invention effectively inhibits the expression of IL-1β, TNF-α, and IL-6, inflammatory cytokines. Therefore, the pharmaceutical composition according to the present invention can be usefully used for preventing or treating myositis.SELECTED DRAWING: Figure 20

Description

[Technical field]

[0001] The present invention relates to a pharmaceutical composition for preventing or treating myositis, which comprises mitochondria as an active ingredient.

[0002] [Background technology] Myositis is a disease in which inflammation occurs in muscles, muscle fibers are damaged, muscle pain is caused, and the ability of muscles to contract is reduced. Myositis is divided into dermatomyositis, polymyositis, and inclusion body myositis, and polymyositis and dermatomyositis in particular are inflammatory muscle diseases that show symptoms such as muscle weakness in the limbs close to the trunk, increased muscle enzyme levels, increased expression of inflammatory cytokines, abnormal electromyograms, and abnormal muscle biopsies.

[0003] In addition, muscle weakness due to polymyositis and dermatomyositis mostly progresses gradually over weeks to months, but in rare cases it can progress rapidly. Severe muscle weakness, if untreated, can lead to muscle loss. Approximately 15% to 30% of patients with polymyositis have been reported to develop malignant tumors, and when dermatomyositis occurs in elderly people, both conditions are associated with cancer.

[0004] Steroids, immunosuppressants, or immunomodulators are used to treat polymyositis and dermatomyositis. Steroids are the most commonly used drug for early treatment, and the decision to use immunosuppressants depends on the response to steroid treatment and side effects. Approximately 75% of myositis patients are prescribed additional immunosuppressants in addition to steroids. Recently, immunoglobulin for intravenous administration as an immunomodulator has been shown to be effective in improving muscle strength and signs seen in muscle biopsies of dermatomyositis, and is used for dermatomyositis. However, immunosuppressants and immunomodulators have the disadvantages that they have side effects because they directly affect the immune system, and the effects of the drug do not last for a long time, requiring repeated injections of the drug every 6 to 8 weeks.

[0005] In addition, myositis-induced mouse models have been developed recently and are being used to develop drugs to treat myositis. In particular, in 2007, a single administration of recombinant skeletal muscle fast type C protein was shown to be able to induce polymyositis in C57BL / 6 mice, suggesting the possibility of attempting to study disease-specific treatments for myositis (Sugihara T et al., Arthritis Rheum. 2007, 56(4):1304-14). Myositis-induced mouse models have been used to evaluate the effects of immunosuppressants in myositis treatment against CXCL10 (CXC motif chemokine 10), a chemokine whose expression is increased in muscle tissue in polymyositis (Kim et al., Arthritis Research & Therapy 2014, 16:R126).

[0006] Meanwhile, mitochondria are organelles of eukaryotic cells involved in the synthesis and regulation of adenosine triphosphate (ATP), an intracellular energy source. Mitochondria are involved in various metabolic pathways in vivo, such as intracellular signal transduction, cell differentiation, cell death, and the control of the cell cycle and cell proliferation.

[0007] [Detailed Description of the Invention] [Technical issues] Although research into the treatment of myositis is being conducted, the drugs developed have side effects and require regular injections, so no innovative treatment has been developed to date. Therefore, continued research and development of safe and effective drugs for myositis is necessary.

[0008] It is therefore an object of the present invention to provide a pharmaceutical composition for treating myositis and a method for treating myositis using the same.

[0009] [Means to solve the problem] In order to solve the above problems, one aspect of the present invention is to provide a pharmaceutical composition for preventing or treating myositis, which comprises mitochondria as an active ingredient.

[0010] Another aspect of the present invention is to provide a method for preventing or treating myositis comprising the step of administering to a subject a pharmaceutical composition.

[0011] [Effects of the invention] When the pharmaceutical composition of the present invention, which contains mitochondria as an active ingredient, is administered to a subject suffering from myositis, it can reduce inflammatory cells infiltrating into the muscle cells of the subject. Furthermore, the pharmaceutical composition of the present invention can effectively reduce the expression of inflammatory cytokines in muscle tissue suffering from myositis. Therefore, the pharmaceutical composition of the present invention can be usefully used to prevent or treat myositis. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 shows the measurement of ATP synthesis in mitochondria isolated from umbilical cord-derived mesenchymal stem cells. [Diagram 2] FIG. 1 shows measurements of membrane potential activity in mitochondria isolated from umbilical cord-derived mesenchymal stem cells. [Diagram 3] FIG. 1 shows the measurement of reactive oxygen species in mitochondria isolated from umbilical cord-derived mesenchymal stem cells. [Figure 4] FIG. 1 shows an outline of the primary animal experiment plan for confirming the effect of myositis treatment by administration of mitochondria using myositis-induced mice. [Diagram 5] FIG. 1 shows photographs of quadriceps and hamstring muscles stained with H&E (hematoxylin and eosin) from a negative control group, a positive control group, and an experimental group receiving exogenous mitochondria to identify inflammatory cells that had infiltrated into muscle fibers. [Figure 6] FIG. 1 shows the measurement and scoring of the number of inflammatory cells infiltrating into muscle fibers after H&E staining of quadriceps muscles in the negative control group, positive control group, and experimental group administered exogenous mitochondria. [Figure 7] FIG. 1 shows IL-6 concentrations in mouse blood in a normal group, a negative control group, a positive control group, and an experimental group administered mitochondria. [Figure 8] FIG. 1 shows PET / MRI photographs of mice in a negative control group, a positive control group, and an experimental group administered exogenous mitochondria. [Figure 9] FIG. 1 shows that mitochondrial activity was increased after mitochondrial transplantation in an induced myositis mouse model. [Figure 10] FIG. 1 shows an outline of a secondary animal experiment plan for confirming the effect of myositis treatment by administration of exogenous mitochondria using myositis-induced mice. [Figure 11] FIG. 1 shows photographs of quadriceps muscles stained with H&E in the negative control group, the positive control group, and the experimental group administered exogenous mitochondria to identify inflammatory cells that had infiltrated into muscle fibers. [Figure 12] FIG. 1 shows photographs of hamstring muscles stained with H&E in the negative control group, the positive control group, and the experimental group receiving exogenous mitochondria to identify inflammatory cells that had infiltrated into the muscle fibers. [Figure 13] FIG. 1 shows the measurement and scoring of the number of inflammatory cells infiltrating into muscle fibers after H&E staining of quadriceps muscles in the negative control group, positive control group, and experimental group administered exogenous mitochondria. [Figure 14] FIG. 1 shows the concentration of IL-1β in the blood of mice in a normal group, a negative control group, a positive control group, and an experimental group administered exogenous mitochondria. [Figure 15] FIG. 1 shows IL-6 concentrations in the blood of mice in a normal group, a negative control group, a positive control group, and an experimental group administered exogenous mitochondria. [Figure 16] FIG. 1 shows the TNF-α concentrations in the blood of mice in a normal group, a negative control group, a positive control group, and an experimental group administered exogenous mitochondria. [Figure 17] FIG. 1 shows the expression levels of IL-6 mRNA in mouse muscles in a normal group, a negative control group, a positive control group, and an experimental group administered exogenous mitochondria. [Figure 18]FIG. 1 shows an outline of a tertiary animal experiment plan for confirming the effect of administering exogenous mitochondria to treat myositis using myositis-induced mice. [Figure 19] FIG. 1 confirms that the number of inflammatory cells stained with H&E (hematoxylin and eosin) was reduced following mitochondrial transplantation in an induced myositis mouse model. [Figure 20] FIG. 1 confirms that the histological score according to the scoring system was reduced after mitochondrial transplantation in an induced myositis mouse model. [Figure 21] FIG. 1 shows that the number of inflammatory cytokines was reduced after mitochondrial transplantation in an induced myositis mouse model. [Figure 22] FIG. 1 shows that mitochondrial activity was increased after mitochondrial transplantation in an induced myositis mouse model. [Diagram 23] FIG. 1 shows increases and decreases in total metabolites in skeletal muscle as a result of analysis of muscle profile heatmaps by metabolomic analysis after mitochondrial transplantation in an induced myositis mouse model. [Figure 24] FIG. 13: Metabolomic analysis confirms that after mitochondrial transplantation in a myositis-induced mouse model, the relative quantitative ratios of malate and aspartate were significantly increased and restored to control levels. [Diagram 25] FIG. 13: Metabolomic analysis confirms that after mitochondrial transplantation in a myositis-induced mouse model, the relative quantitative ratios of malate and aspartate were significantly increased and restored to control levels. [Figure 26] FIG. 13: Metabolomic analysis confirms that after mitochondrial transplantation in a myositis-induced mouse model, the relative quantitative ratios of malate and aspartate were significantly increased and restored to control levels. [Figure 27] FIG. 13: Metabolomic analysis confirms that after mitochondrial transplantation in a myositis-induced mouse model, the relative quantitative ratios of malate and aspartate were significantly increased and restored to control levels. [Figure 28] FIG. 1 shows a comparison of ATP activity of mitochondria isolated from cryopreserved mesenchymal stem cells derived from umbilical cord and ATP activity of mitochondria isolated from cultured mesenchymal stem cells derived from umbilical cord. [Figure 29] FIG. 1 shows a comparison of the membrane potential of mitochondria isolated from cryopreserved mesenchymal stem cells derived from umbilical cord with the membrane potential of mitochondria isolated from cultured mesenchymal stem cells derived from umbilical cord. [Diagram 30] FIG. 1 shows the measurement of reactive oxygen species in mitochondria isolated from cryopreserved mesenchymal stem cells derived from umbilical cord and mitochondria isolated from cultured mesenchymal stem cells derived from umbilical cord. [Diagram 31] FIG. 1 shows the measurement of the number of mitochondria in a solution containing a concentration of 1 μg / mL of mitochondria using a particle counter (Multisizer 4e, Beckman Coulter). [Diagram 32] FIG. 1 shows the measurement of the number of mitochondria in a solution containing a concentration of 2.5 μg / mL of mitochondria using a particle counter. [Diagram 33] FIG. 1 shows the measurement of the number of mitochondria in a solution containing a concentration of 5 μg / mL of mitochondria using a particle counter. [Diagram 34] FIG. 1 confirms the ability to inhibit the expression of TNF-α, IL-1β, and IL-6 mRNA by mitochondria from several cell types in LPS-stimulated RAW264.7 cells. [Diagram 35] FIG. 14 shows the ability of mitochondria derived from several types of cells to inhibit IL-6 mRNA expression in LPS-activated THP-1 cells. [Diagram 36] FIG. 1 shows the observation of the ability of mitochondria derived from several types of cells to inhibit IL-6 protein expression in LPS-activated THP-1 cells. [Figure 37] FIG. 1 shows muscle information used in metabolomic analysis.

[0013] [Best Mode for Carrying Out the Invention] The present invention will now be described in detail.

[0014] One aspect of the present invention is to provide a pharmaceutical composition for preventing or treating myositis, which comprises mitochondria as an active ingredient.

[0015] As used herein, the term "myositis" refers to a disease in which muscle inflammation occurs and muscle fibers are damaged. In particular, myositis is divided into dermatomyositis, polymyositis, and inclusion body myositis, among which polymyositis and dermatomyositis belong to inflammatory muscle diseases. In muscle tissue of polymyositis or dermatomyositis, the expression of inflammatory cytokines or chemokines such as CXCL10, IL-1β, TNF-α, and IL-6 is increased. Immunosuppressants or immunomodulators that inhibit CXCL10, IL-1β, TNF-α, or IL-6 have been developed as therapeutic agents for myositis. However, in the case of immunosuppressants or immunomodulators, there is a problem that side effects occur due to direct involvement in the immune system.

[0016] In addition, myositis-induced mouse models can be used to develop drugs to treat myositis. Myositis-induced mouse models can be created by intradermal injection of CFA (complete Freund's adjuvant) containing C protein fragments and heat-killed Mycobacterium butyricum, and intraperitoneal injection of PT (pertussis toxin). In this case, CD8 T cells may migrate from 7 days after drug injection and infiltrate into muscles, causing inflammation. In this case, the tissues in which inflammation and inflammatory cells are observed in the muscles of myositis patients are the quadriceps and hamstring muscles, so quadriceps and hamstring muscle tissues may also be used in myositis-induced mice.

[0017] Unless otherwise specified in this specification, the term "active ingredient" refers to an ingredient that exhibits activity alone or in combination with an adjuvant (carrier) that has no activity by itself.

[0018] As shown in the experimental example, the metabolic profiles of both quadriceps, white muscle, and soleus, red muscle were analyzed using CE-TOFMS in the muscles of a C-protein-induced myositis mouse model (CIM). In the myositis-induced group, the ratio of malate to aspartate was decreased, and based on the above, mitochondrial damage was confirmed. As a result of the experiment, a test to confirm the efficacy of exogenous mitochondrial injection at several doses confirmed that inflammation was improved and mitochondrial damage was restored in the CIM mouse model.

[0019] The mitochondria may be obtained from a mammal or from a human. In particular, the mitochondria may be isolated from a cell or tissue. For example, the mitochondria may be isolated from cells cultured in vitro. Furthermore, the mitochondria may be obtained from somatic cells, germ cells, blood cells, or stem cells. Furthermore, the mitochondria may be obtained from platelets. The mitochondria may be normal mitochondria obtained from cells in which the mitochondrial biological activity is normal. Furthermore, the mitochondria may be cultured in vitro.

[0020] Furthermore, mitochondria may be obtained from autologous, allogeneic, or xenogeneic subjects. In particular, autologous mitochondria refers to mitochondria obtained from tissues or cells of the same subject. Furthermore, allogeneic mitochondria refers to mitochondria obtained from a subject belonging to the same species as the subject, but with a different allelic genotype. Furthermore, heterologous mitochondria refers to mitochondria obtained from a subject belonging to a different species than the subject.

[0021] In particular, the somatic cells may be muscle cells, hepatocytes, nerve cells, fibroblasts, epithelial cells, adipocytes, bone cells, leukocytes, lymphocytes, platelets, or mucosal cells. Furthermore, the germ cells may be cells undergoing meiosis and mitosis, and may be sperm or eggs. Furthermore, the stem cells may be any one selected from the group consisting of mesenchymal stem cells, adult stem cells, induced pluripotent stem cells, embryonic stem cells, bone marrow stem cells, neural stem cells, limbal stem cells, and tissue-derived stem cells. In this case, the mesenchymal stem cells may be any one selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta.

[0022] On the other hand, if mitochondria are isolated from specific cells, they can be isolated by various known methods, for example using specific buffers or using potential differences and magnetic fields.

[0023] In terms of maintaining mitochondrial activity, the isolation of mitochondria can be obtained by crushing and centrifuging cells.In one embodiment, the isolation can be carried out by the steps of culturing cells, carrying out a first centrifugation of the pharmaceutical composition containing cells to produce a pellet, resuspending the pellet in a buffer solution and homogenizing it, carrying out a second centrifugation of the homogenized solution to produce a supernatant, and carrying out a third centrifugation of the supernatant to purify mitochondria.In this case, in terms of maintaining cell activity, the time of carrying out the second centrifugation is preferably adjusted to be shorter than the time of carrying out the first centrifugation and the third centrifugation, and the speed may increase from the first centrifugation to the third centrifugation.

[0024] In particular, the first to third centrifugations may be performed at a temperature of 0° C. to 10° C., preferably 3° C. to 5° C. Furthermore, the time for performing the centrifugation may be 1 minute to 50 minutes, and can be appropriately adjusted depending on the number of centrifugations, the content of the sample, etc.

[0025] Furthermore, the first centrifugation can be performed at a speed of 100×g to 1,000×g, or 200×g to 700×g, or 300×g to 450×g. Furthermore, the second centrifugation can be performed at a speed of 1×g to 2,000×g, or 25×g to 1,800×g, or 500×g to 1,600×g. Furthermore, the third centrifugation can be performed at a speed of 100×g to 20,000×g, or 500×g to 18,000×g, or 800×g to 15,000×g.

[0026] The isolated mitochondria can be quantified by quantifying protein. In particular, the isolated mitochondria can be quantified by BCA (bicinchoninic acid assay) analysis. In this case, the mitochondria in the pharmaceutical composition may be contained at a concentration of 0.1 μg / mL to 1000 μg / mL, 1 μg / mL to 750 μg / mL, or 25 μg / mL to 500 μg / mL. In one example of the present invention, the mitochondria were used at concentrations of 25 μg / mL, 50 μg / mL, and 100 μg / mL.

[0027] Furthermore, the number of isolated mitochondria may be measured by a particle counter (Multisizer 4e, Beckman Coulter), and the number of mitochondria may be as shown in Table 1 below, referring to the paper written by James D. McCully (J Vis Exp. 2014; (91): 51682).

[0028] [Table 1] [Table 1]

[0029] As shown in Example 9 of the present invention, the number of mitochondria at concentrations of 1 μg / mL, 2.5 μg / mL, and 5 μg / mL was measured using a particle counter, and the number was 1.96 × 10 6 ±0.98×10 6 , 5.97×10 6 ±0.19×10 6 , 1.01×10 7 ±0.32×10 7In comparison with Table 1 above, the number of mitochondria at a concentration of 10 μg / mL was 2.16 × 10 7 ±0.08×10 7 This is calculated by multiplying the number of mitochondria at a concentration of 5 μg / mL by 2, which is 2.02 × 10 7 ±0.64×10 7 In this case, the mitochondria in the pharmaceutical composition are approximately 1×10 5 Mitochondria / mL to 5 x 10 9 In particular, the mitochondria in the pharmaceutical composition may be present in an amount of 1×10 5 Mitochondria / mL ~ 5 x 10 9 Mitochondria / mL, 2 x 10 5 Mitochondria / mL ~ 2 x 10 9 Mitochondria / mL, 5 x 10 5 Mitochondria / mL ~ 1 x 10 9 Mitochondria / mL, 1 x 10 6 Mitochondria / mL ~ 5 x 10 8 Mitochondria / mL, 2 x 10 6 Mitochondria / mL ~ 2 x 10 8 Mitochondria / mL, 5 x 10 6 Mitochondria / mL ~ 1 x 10 8 mitochondria / mL, or 1 x 10 7 Mitochondria / mL ~ 5 x 10 7 The pharmaceutical composition may contain mitochondria at a concentration and content in the ranges described above, so that the dosage of mitochondria upon administration can be easily adjusted, and the degree of improvement of the myositis symptoms of the patient can be further increased.

[0030] In particular, the therapeutically effective amount of mitochondria contained in the pharmaceutical composition is 3×10 5 Mitochondria / kg ~ 1.5 x 10 10 In particular, the therapeutically effective amount of mitochondria contained in the pharmaceutical composition may be 3×10 mitochondria / kg per dose based on the body weight of the subject. 5Mitochondria / kg ~ 1.5 x 10 10 Mitochondria / kg, 6 × 10 5 Mitochondria / kg ~ 6 x 10 9 Mitochondria / kg, 1.5 × 10 6 Mitochondria / kg ~3×10 9 Mitochondria / kg, 3 × 10 6 Mitochondria / kg ~ 1.5 x 10 9 Mitochondria / kg, 6 × 10 6 Mitochondria / kg ~ 6 x 10 8 Mitochondria / kg, 1.5 × 10 7 Mitochondria / kg ~3×10 8 mitochondria / kg or 3 x 10 7 Mitochondria / kg ~ 1.5 x 10 8 In other words, it is most preferable from the viewpoint of cell activity that the pharmaceutical composition containing mitochondria is administered within the above-mentioned mitochondria dosage range based on the body weight of the subject with myositis.

[0031] Furthermore, the pharmaceutical composition can be administered 1 to 10 times, 3 to 8 times, or 5 to 6 times, preferably 5 times, with the administration interval being 1 to 7 days or 2 to 5 days, preferably 3 days.

[0032] Furthermore, the pharmaceutical composition according to the present invention can be administered to a human or other mammal susceptible to or suffering from myositis, and can be an injectable solution, preferably an injectable preparation, that can be administered intravenously, intramuscularly, or subcutaneously.

[0033] Therefore, in order to ensure product stability during distribution of the formulated injection, the pharmaceutical composition according to the present invention can be prepared as an injection that is highly stable physically or chemically by adjusting the pH using an acidic aqueous solution or a buffer solution such as a phosphate that can be used for injection.

[0034] In particular, the pharmaceutical composition of the present invention may include water for injection, which refers to distilled water made for dissolving solid injection liquids or diluting water-soluble injection liquids.

[0035] In addition, the pharmaceutical composition of the present invention may include a stabilizer or solubilizer. For example, the stabilizer may be pyrosulfite, citric acid, or ethylenediaminetetraacetic acid, and the solubilizer may be hydrochloric acid, acetic acid, sodium hydroxide, sodium bicarbonate, sodium carbonate, or potassium hydroxide.

[0036] In another aspect of the invention, the invention provides a method for preventing or treating myositis comprising the step of administering to a subject a pharmaceutical composition as described above, wherein the subject may be a mammal, preferably a human.

[0037] In this case, administration may be intravenous, intramuscular, or intradermal. Thus, the pharmaceutical composition according to the present invention can supply exogenous mitochondria with normal activity to the veins of subjects suffering from myositis, which may be useful for increasing the activity of cells with reduced mitochondrial function or regenerating cells with abnormal mitochondrial function, and can be used for the prevention or treatment of myositis.

[0038] In another aspect of the present invention, the present invention provides a use of isolated mitochondria for preventing or treating myositis. The details of mitochondria and myositis are as described above.

[0039] [Mode for carrying out the invention] In the following, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided to facilitate understanding of the present invention, and the content of the present invention is not limited to the following examples.

[0040] I. Preparation of a Composition Containing Mitochondria Preparation Example 1. Preparation of a composition containing mitochondria isolated from mesenchymal stem cells derived from human umbilical cord I Human umbilical cord-derived mesenchymal stem cells were inoculated into Alpha-MEM (Alpha-Minimum Essential Medium) medium containing 10% (v / v) fetal bovine serum (FBS, Gibco), 100 μg / mL streptomycin, and 100 U / mL ampicillin, and cultured for 72 hours. After the culture was completed, the cells were washed twice with DPBS (Dulbecco's phosphate-buffered saline, Gibco). The washed cells were treated with 0.25% (v / v) trypsin-EDTA (TE, Gibco) to obtain cells.

[0041] To isolate mitochondria, the resulting cells were counted at 1 x 10 using a hemocytometer. 7 The cells were collected at a concentration of 10000 cells / mL. The cell line was subjected to a first centrifugation at a speed of 350×g and a temperature of 4° C. for 10 minutes. At this time, the pellet obtained was collected, resuspended in a buffer solution, and subjected to homogenization for 10-15 minutes. The composition containing the pellet was then subjected to a second centrifugation at a speed of 1100×g and a temperature of 4° C. for 3 minutes to obtain a supernatant. The supernatant was subjected to a third centrifugation at a speed of 12,000×g and a temperature of 4° C. for 15 minutes to isolate mitochondria from the cell line. The mitochondria thus obtained were mixed with PBS and then loaded into a syringe.

[0042] Preparation Example 2. Preparation of a composition containing mitochondria isolated from mesenchymal stem cells derived from human umbilical cord II Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) were inoculated into alpha-MEM (alpha minimum essential medium, Gibco) medium containing 10% fetal bovine serum (FBS, Gibco), 100 μg / mL streptomycin, and 100 U / mL ampicillin, and cultured for 72 hours. After the cell culture was completed, the cells were washed twice with DPBS. Then, the cells were obtained by treating with 0.25% trypsin / EDTA. The cell concentration was 1 × 10 7 After resuspending the cells to 100 cells / mL, the cells were subjected to a first centrifugation at a speed of 350×g for 10 minutes at a temperature of 4° C.

[0043] The washed cells were resuspended in mitochondrial isolation solution and then disrupted using a 1 ml syringe. The solution containing the disrupted cells was then centrifuged at 1500×g for 5 minutes at 4°C to remove impurities, and the supernatant containing mitochondria was collected. The collected supernatant was centrifuged at 20000×g for 5 minutes at 4°C to collect precipitated mitochondria, and the isolated mitochondria were suspended in Tris buffer, and protein was quantified by the BCA method before being used in the experiment.

[0044] Preparation Example 3. Preparation of a composition containing mitochondria isolated from human bone marrow-derived mesenchymal stem cells Human bone marrow-derived mesenchymal stem cells (BM-MSCs) were inoculated into DMEM (Gibco) medium containing 10% fetal bovine serum (FBS, Gibco), 100 μg / mL streptomycin, and 100 U / mL ampicillin, and cultured for 72 hours.

[0045] After the cell culture was completed, mitochondria were harvested, quantified as described in Preparation Example 2, and then used in the experiment.

[0046] Preparation Example 4. Preparation of a composition containing mitochondria isolated from human fibroblasts Human fibroblast cells (CCD-8LU, ATCC) were inoculated into DMEM (Gibco) medium containing 10% fetal bovine serum (FBS, Gibco), 100 μg / mL streptomycin, and 100 U / mL ampicillin, and cultured for 72 hours.

[0047] After the cell culture was completed, mitochondria were harvested, quantified as described in Preparation Example 2, and then used in the experiment.

[0048] Preparation Example 5. Preparation of a composition containing mitochondria isolated from human induced pluripotent stem cells Human induced pluripotent stem cells (iPSCs) were used, cultured in TeSR™-E8™ (Stem Cells 05990) medium in cell culture vessels coated with 10 μg / ml vitronectin (Stem Cells 07180).

[0049] After the cell culture was completed, mitochondria were harvested, quantified as described in Preparation Example 2, and then used in the experiment.

[0050] Preparation Example 6. Preparation of a composition containing mitochondria isolated from platelet mitochondria To isolate mitochondria from platelets, porcine whole blood was centrifuged at 500×g for 3 minutes at ambient temperature, and the supernatant containing platelet-rich plasma (PRP) was then collected. The collected supernatant was centrifuged at 1,500×g for 5 minutes, the supernatant was removed, and the precipitate containing platelets was collected. The concentrated platelet precipitate was resuspended using PBS, and then centrifuged at 1,500×g for 5 minutes and washed. The washed platelets were resuspended in mitochondrial isolation solution and disrupted using a 1 ml syringe. The solution containing the disrupted platelets was then centrifuged at 1,500×g for 5 minutes at 4°C to remove impurities, and the supernatant containing mitochondria was collected. The collected supernatant was centrifuged at 20,000×g for 5 minutes at 4°C to collect the precipitated mitochondria, and the isolated mitochondria were suspended in Tris buffer, and the protein was quantified before being used in the experiment.

[0051] Preparation Example 7. Preparation of a composition containing mitochondria isolated from myoblasts derived from rat skeletal muscle L6 cells (American Type Culture Collection, ATCC, CRL-1458), a myoblast cell line derived from rat skeletal muscle, were inoculated into DMEM-high glucose (Dulbecco's modified Eagle's medium-high glucose, Gibco) medium containing 10% fetal bovine serum (FBS, Gibco) and cultured for 72 h.

[0052] After the cell culture was completed, mitochondria were harvested, quantified as described in Preparation Example 2, and then used in the experiment.

[0053] II. Characterization of mitochondria Example 1. Confirmation of mitochondrial ATP synthesis To confirm whether the mitochondria isolated in Preparation Example 1 could synthesize ATP normally, the mitochondrial protein concentration of the isolated mitochondria was quantified by BCA (bicinchoninic acid assay) analysis, and 5 μg of mitochondria was prepared. Then, the amount of ATP was quantified using a CellTiter-Glo luminescence kit (Promega, Madison, WI) according to the manufacturer's manual.

[0054] In particular, as the experimental group, 5 μg of prepared mitochondria was mixed in 100 μL of PBS and then dispensed into a 96-well plate. In addition, as the control group, 100 μL of PBS without mitochondria was dispensed into a 96-well plate. Then, each well was treated with 100 μL of the test solution included in the CellTiter-Glo Luminescence Kit, and then reacted on a stirrer for 2 minutes and mixed thoroughly. Then, it was reacted at ambient temperature for 10 minutes, and then the absorbance was measured at a wavelength of 560 nm using a luminescence microplate reader.

[0055] As a result, it was confirmed that the amount of ATP in the experimental group containing mitochondria was approximately three times that of the control group (Figure 1). From the above, it was confirmed that the mitochondria isolated in Preparation Example 1 were synthesizing ATP normally.

[0056] Example 2. Measurement of mitochondrial membrane potential To measure the membrane potential of the mitochondria isolated in Preparation Example 1, the mitochondrial protein concentration of the isolated mitochondria was quantified by BCA, and 5 μg of mitochondria was prepared. The mitochondrial membrane potential was measured using JC-1 (molecular probes, catalog number 1743159) dye.

[0057] Specifically, as an experimental group, 5 μg of prepared mitochondria was mixed with 50 μL of PBS and dispensed into a 96-well plate. In addition, as a control group, 50 μL of PBS without mitochondria was dispensed into a 96-well plate. In addition, as an additional experimental group, 5 μg of mitochondria was mixed with 50 μL of CCCP (R&D systems, CAS 555-60-2), reacted at room temperature for 10 minutes, and then dispensed into a 96-well plate. In this case, CCCP is a mitochondrial ion transporter that depolarizes the mitochondrial membrane potential, thereby inhibiting mitochondrial function.

[0058] Then, each well was treated with JC-1 dye at a concentration of 2 μM, and after reaction, the absorbance was measured using a fluorescence microplate reader. At this time, the JC-1 dye exists as a monomer and shows green fluorescence at low concentrations, and at high concentrations, the JC-1 dye aggregates and shows red fluorescence (monomer: Ex 485 / Em 530, J-aggregate: Ex 535nm / Em 590nm). The mitochondrial membrane potential was analyzed by calculating the ratio of the absorbance of green fluorescence to the absorbance of red fluorescence.

[0059] As a result, the experimental group containing mitochondria showed high membrane potential activity. On the other hand, the additional experimental group in which mitochondria were treated with CCCP showed low membrane potential activity (Figure 2). From the above, it was confirmed that the mitochondria isolated in Preparation Example 1 showed normal membrane potential activity.

[0060] Example 3. Measurement of mitochondrial reactive oxygen species To confirm damage to the mitochondria isolated in Preparation Example 1, the mitochondrial protein concentration of the isolated mitochondria was quantified by BCA, and 5 μg of mitochondria was prepared. Mitochondria reactive oxygen species (ROS) in the mitochondria were measured using MitoSOX Red indicator dye (Invitrogen, Cat. No. M36008).

[0061] In particular, as an experimental group, 5 μg of prepared mitochondria was mixed with 50 μL of PBS and dispensed into a 96-well plate. Furthermore, as a control group, 50 μL of PBS not containing mitochondria was dispensed into a 96-well plate. Then, MitoSOX Red indicator dye was mixed with 50 μL of PBS to a concentration of 10 μM, and each well was treated with the mixture and reacted for 20 minutes in an incubator under conditions of 37°C and 5% CO2. After the reaction was completed, the absorbance was measured using a fluorescent microplate reader (Ex 510 nm / Em 580 nm). As a result, it was confirmed that the mitochondrial reactive oxygen species in the mitochondria were low in both the control group and the experimental group (Figure 3). From the above, it was confirmed that there was no damage to the mitochondria isolated in Preparation Example 1.

[0062] III. Confirmation of the effect of mitochondria on the treatment of myositis in vivo Example 4. Confirmation of the effect of exogenous mitochondrial administration on the treatment of myositis in a myositis-induced mouse model: Primary experiment Example 4.1. Construction of a myositis-induced mouse model and administration of mitochondria (n=3) CFA (complete Freund's adjuvant) containing 200 μg of C protein fragment and 100 μg of heat-killed Mycobacterium butyricum was injected intradermally into 8-week-old C57BL / 6 female mice, and 2 μg of PT (pertussis toxin) was injected intradermally.

[0063] The experimental group was administered a single intravenous dose of mitochondria isolated in Preparation Example 1 (5 μg) on ​​the 1st or 7th day after myositis induction. In addition, the negative control group was administered 100 μL of PBS intraperitoneally, and the positive control group was administered 0.8 mg / kg of dexamethasone intraperitoneally every day from the 1st to the 14th day after myositis induction (FIG. 4).

[0064] Example 4.2. Identification of muscle fibers infiltrated with inflammation Mice from each group in Example 4.1 were sacrificed on day 14, and tissues from the quadriceps and hamstring muscles were harvested and stained with H&E (hematoxylin and eosin), after which the infiltration of inflammatory cells was observed under an optical microscope.

[0065] As a result, it was confirmed that the number of inflammatory cells infiltrating into muscle fibers was reduced in the positive control group and the experimental group compared to the negative control group (Figure 5). Furthermore, on the 14th day, mice from each group were sacrificed, and tissues from the quadriceps and hamstring muscles were collected and stained with H&E, after which a scoring system was used to evaluate the number of muscle fibers infiltrated by inflammatory cells. The scoring method of the scoring system is shown in Table 2 below. In this case, the average values ​​of the left and right muscles of the quadriceps and hamstring muscles were compared.

[0066] [Table 2] [Table 2]

[0067] As a result, the scores of inflammation-infiltrated muscle fibers were reduced in the positive control group and the experimental group compared to the negative control group (Figure 6).

[0068] Example 4.3. Confirmation of cytokine concentration in blood To confirm the concentration of IL-6 in the blood of normal mice and mice in each group of Example 4.1 on day 14, serum was isolated from the blood of mice in each group, and then IL-6 in the blood was measured using an IL-6 ELISA kit (R&D Systems, MN, USA) according to the manufacturer's manual.

[0069] As a result, it was confirmed that blood IL-6 concentrations increased in the control group, while blood IL-6 concentrations decreased in the positive control group and the experimental group (Figure 7).

[0070] Example 4.4. Confirmation of inflammatory response by PET / MRI analysis First, in order to increase the glucose uptake efficiency in the tissues of the mice in each group in Example 4.1, food was not given for 8 hours before imaging. After 8 hours, 200 uci 18 F-FDG (Cyclotron Room, Department of Nuclear Medicine, Seoul National University Hospital) was injected intravenously into mice, and PET / MR images were taken 1 h later.

[0071] As a result, strong radionuclide signals were observed in the legs of mice in the negative control group. 18 F-FDG radiopharmaceuticals are often selectively taken up by inflammatory cells, macrophages, and therefore inflammatory responses may be easily monitored by nuclear medicine imaging techniques (Figure 8).

[0072] Example 4.5. Identification of changes in expression of mitochondrial oxidative phosphorylation complexes The expression of oxidative phosphorylation complex II in the quadriceps muscle of the negative control group (CIM) was decreased compared to the control group, and the expression of the experimental group (CIM + Mito day 7) was increased compared to the positive control group (DEXA). The expression of TOM20 in the plantaris muscle of the negative control group (CIM) was decreased compared to the control group, and the expression of the experimental group (CIM + Mito day 1, CIM + Mito day 7) was increased compared to the positive control group (DEXA) (Figure 9).

[0073] Considering the experimental results of Examples 4.2 to 4.5 above, a secondary experiment was performed by setting the time point for mitochondrial administration to 7 days after induction of myositis.

[0074] Example 5. Confirmation of the effect of exogenous mitochondrial administration on the treatment of myositis in a myositis-induced mouse model: Secondary experiment Example 5.1. Construction of myositis-induced mouse model and administration of mitochondria (n=10) CFA containing 200 μg of C protein fragment and 100 μg of heat-killed M. butyricum was injected intradermally into 8-week-old C57BL / 6 female mice, and 2 μg of PT was injected intraperitoneally.

[0075] The experimental group was administered a single intravenous dose of mitochondria isolated in Preparation Example 1 (5 μg) on ​​the 7th day after myositis induction. In addition, the negative control group was administered 100 μL of PBS intraperitoneally, and the positive control group was administered 0.8 mg / kg of dexamethasone intraperitoneally every day from the 7th to the 14th day after myositis induction (FIG. 10).

[0076] Example 5.2. Identification of muscle fibers infiltrated with inflammation Mice from each group in Example 5.1 were sacrificed on day 14, and tissues from the quadriceps and hamstring muscles were collected and stained with H&E, after which the infiltration of inflammatory cells was observed using an optical microscope. As a result, it was confirmed that the number of inflammatory cells infiltrating into muscle fibers was reduced in the positive control group and the experimental group compared to the negative control group (Figures 11 and 12).

[0077] Furthermore, on day 14, mice from each group were sacrificed, and tissues from the quadriceps and hamstring muscles were collected and stained with H&E, and a scoring system was used to evaluate the number of muscle fibers infiltrated with inflammatory cells. The scoring system was performed in the same manner as in Example 4.2. In this case, the average scores of the left and right muscles of the quadriceps and hamstring muscles were compared. As a result, the scores of muscle fibers infiltrated with inflammation in the positive control group and the experimental group were significantly reduced compared to the negative control group (Figure 13).

[0078] Example 5.3. Confirmation of cytokine concentration in blood To confirm the concentrations of IL-1β, IL-6, and TNF-α in the blood of normal mice and mice in each group of Example 5.1 on day 14, serum was isolated from the blood of mice in each group, and then the concentrations of IL-1β, IL-6, and TNF-α in the blood were measured using an IL-1β ELISA kit (R&D Systems, MN, USA), an IL-6 ELISA kit, and a TNF-α ELISA kit (R&D, Systems, MN, USA), respectively, according to the manufacturer's manual.

[0079] As a result, the blood IL-6 concentration was increased in the positive control group compared to the negative control group, whereas the blood IL-6, IL-1β, and TNF-α concentrations were decreased in the experimental group compared to the negative control group (Figures 14, 15, and 16).

[0080] Example 5.4. Confirmation of changes in IL-6 mRNA expression The expression level of IL-6 mRNA was confirmed in the mRNA isolated by RT-qPCR from the muscles of normal mice and mice of each group of Example 5.1 on day 14. In particular, total RNA was isolated from the muscles using TRIzol reagent (Invitrogen), and qPCR was performed using SYBR Green (Perkin Elmer, MA, USA) and 7,500 Fast Real-Time PCR System (Applied Biosystems). The experimental results were normalized to the amount of β-actin mRNA. In this case, the primers used are shown in Table 3 below.

[0081] [Table 3] [Table 3]

[0082] As a result, it was confirmed that the expression of IL-6 mRNA was decreased in the experimental group, whereas it was confirmed that the expression of IL-6 mRNA was not decreased in the positive control group, and the experimental group was more effective in decreasing the expression of IL-6 mRNA than the positive control group (Figure 17).

[0083] Example 5.5. Confirmation of changes in mitochondrial function by metabolomic analysis After inducing myositis animal model (CIM), mitochondrial function was measured in quadriceps and soleus muscles of the control group, negative control group (CIM), positive control group (DEXA), and group transplanted with 5 μg mitochondria by metabolomic analysis based on CE-TOFMS in positive and negative ion modes. For measurement by improving the analytical quality of CE-MS analysis, samples were diluted as shown in FIG.

[0084] As can be seen from the analysis of muscle profile heat maps, it was shown that there was a significant effect on the metabolite profile of skeletal muscle in the negative control group (CIM) compared to the control group (Figure 23). Compared to the positive control group (DEXA), it was confirmed that the metabolite profile of the mitochondrial transplant group was restored similarly to that of the control group. The malate-aspartate shuttle (sometimes shortened, malate shuttle, malate aspartate shuttle-deficient) is a biochemical system that transfers electrons generated during the glycolysis process across the semi-permeable inner membrane of mitochondria to perform oxidative phosphorylation in eukaryotes.

[0085] Mitochondrial dysfunction observed in the myositis model is associated with the malate-aspartate shuttle, and was confirmed by a decrease in the ratio of the relative quantitative values ​​of malate and aspartate. The ratio of the relative quantitative values ​​of malate and aspartate was significantly increased after mitochondrial transplantation compared to the negative control group (CIM) and the positive control group (DEXA), and was confirmed to have recovered to a level similar to that of the control group (Figures 24 to 27).

[0086] In conclusion, the therapeutic effect of transplantation of 5 μg of mitochondria was confirmed in the CIM mouse model.

[0087] Example 6. Confirmation of the effect of exogenous mitochondrial administration on the treatment of myositis in a myositis-induced mouse model: Third experiment Example 6.1. Experimental Method (n=5) CFA (complete Freund's adjuvant) containing 200 μg of C protein fragment and 100 μg of heat-killed Mycobacterium butyricum was injected intradermally into 8-week-old C57BL / 6 female mice, and 2 μg of PT (pertussis toxin) was injected intraperitoneally.

[0088] Seven days after myositis induction, the mitochondria isolated in Preparation Example 1 were administered intravenously once at a dose of 0.2 μg, 1 μg, or 5 μg, as the experimental group. In addition, the group administered 100 μL of PBS intraperitoneally was used as the negative control group, and the group administered dexamethasone (DEXA) intraperitoneally at a dose of 0.8 mg / kg daily from day 7 to day 14 after myositis induction was used as the positive control group (FIG. 18). As an inflammation evaluation method, myositis after H&E staining was scored from 1 to 6 according to the histological severity.

[0089] The expression levels of inflammatory cytokines in mRNA isolated from muscle were observed by RT-qPCR. After mitochondrial transplantation, the mitochondrial activity levels were evaluated by Western blot analysis of the expression of mitochondrial oxidative phosphorylation complexes (OXPHOS complexes). The information of the animal test groups is shown in Table 4 below.

[0090] [Table 4] [Table 4]

[0091] Example 6.2. Identification of muscle fibers infiltrated with inflammation Mice from each group (Table 4) were sacrificed on day 14, and tissues from the quadriceps and hamstring muscles were collected and stained with H&E (hematoxylin and eosin), and then the infiltration of inflammatory cells was observed under an optical microscope. As a result, it was confirmed that the number of inflammatory cells infiltrating into muscle fibers was reduced in the positive control group and the experimental group compared to the negative control group (Figure 19). Furthermore, mice from each group were sacrificed on day 14, and tissues from the quadriceps and hamstring muscles were collected and stained with H&E, and then the number of muscle fibers infiltrated by inflammatory cells was evaluated using a 4-scoring system. The score measurement method for the scoring system is shown in Table 5 below. In this case, the average values ​​of the left and right muscles of the quadriceps and hamstring muscles were compared.

[0092] [Table 5] [Table 5]

[0093] As a result, it was confirmed that the number of inflammatory cells infiltrating into muscle fibers was significantly reduced in the positive control group and the group transplanted with 5 μg of mitochondria, compared to the negative control group (Figure 20).

[0094] Example 6.3. Confirmation of changes in IL-6 and TNF-α mRNA expression The expression levels of inflammatory cytokines IL-6 and TNF-α mRNA were confirmed by RT-qPCR in mRNA isolated from the muscles of mice in the control group, negative control group (CIM), positive control group (DEXA), and experimental group (mitochondrial transplant group). In particular, total RNA was isolated from the muscles using TRIzol reagent (Invitrogen), and qPCR was performed using SYBR Green (Perkin Elmer, MA, USA) and 7,500 Fast Real-Time PCR System (Applied Biosystems). The experimental results were normalized to the amount of β-actin mRNA. In this case, the primers used in RT-qPCR are shown in Table 6 below.

[0095] [Table 6] [Table 6]

[0096] As a result, it was confirmed that IL-6 mRNA expression tended to decrease in the muscles of the mitochondria transplantation group. Furthermore, it was confirmed that TNF-α mRNA expression was significantly decreased in the group transplanted with 5 μg of mitochondria. On the other hand, IL-6 and TNF-α mRNA expression was not decreased in the muscles of the positive control group (DEXA), and it was confirmed that the mitochondria transplantation group was more effective than the positive control group (DEXA) in reducing the expression of IL-6 and TNF-α mRNA (Figure 21).

[0097] Example 6.4. Identification of changes in expression of mitochondrial oxidative phosphorylation complexes Changes in the expression of oxidative phosphorylation complexes in proteins isolated from muscle of mice from the control, negative control (CIM), positive control (DEXA) and experimental (mitochondrial transplant) groups were confirmed by Western blotting (Total OXPHOS Mouse WB Antibody Cocktail, Abcam). It was confirmed that the expression of oxidative phosphorylation complexes I and II was decreased in the negative control (CIM) group compared to the control group, and that the expression of oxidative phosphorylation complexes I and II was increased in the experimental (mitochondrial transplant) group at all doses compared to the positive control (DEXA) group (Figure 22).

[0098] In conclusion, the therapeutic effect and dose-dependence of transplantation of 5 μg of mitochondria was confirmed in the CIM mouse model.

[0099] IV. Determination of Toxicity and Physical Properties of Mitochondrial-Containing Compositions Example 7. Toxicity experiments In order to confirm that the administration of mitochondria would result in toxicity, the mitochondria prepared in Preparation Example 1 were administered intravenously once to ICR mice, and changes in body weight and organs were then confirmed by autopsy, etc. Twelve male and female 7-week-old ICR mice were divided into four groups as shown in Table 7 below, and the experiment was conducted.

[0100] [Table 7] [Table 7]

[0101] As shown in Table 7 above, the G1 group was administered with vehicle. The G2 to G4 groups were administered with 25 μg, 50 μg, or 100 μg of mitochondria, respectively. At this time, the G4 group was administered with mitochondria at or above the approximate lethal dose (ALD). At this point, the administration site was disinfected with 70% alcohol cotton, and then the vehicle or mitochondria was administered at a rate of 1 mL / min via the tail vein using a syringe equipped with a 26-gauge needle.

[0102] First, all mice were observed for general symptoms at least once a day, and the type and severity of general symptoms, including death, during the breeding period were recorded for each subject. However, on the day of administration, observations were continued until 1 hour after administration, and then observations were conducted at 1-hour intervals for 5 hours. Endangered and dead animals were treated according to the planned necropsy animals. The first day of administration of vehicle or mitochondria was set as day 1.

[0103] As a result of observing the general symptoms, no dead animals were observed in any group during the entire test period, and the abnormal symptoms observed on the first day after mitochondria administration were not observed during the subsequent test period, which is considered to be a temporary change caused by mitochondria. Furthermore, the body weights of all mouse subjects were measured before administration, and on the second, fourth, eighth, and fifteenth days after administration. The measurement results are shown in Table 8 below.

[0104] [Table 8] [Table 8]

[0105] As shown in Table 8, no significant changes in body weight were observed in groups G1 to G4. Furthermore, on day 15, all mouse subjects were anesthetized, and the abdomen was cut open to visually inspect all organs. As a result, no changes in organs were observed in groups G1 to G4.

[0106] These results confirmed that a single intravenous administration of mitochondria under these test conditions in ICR mice showed no toxicity in either males or females at mitochondrial concentrations up to 100 μg / head.

[0107] Example 8. Comparison of the characteristics of mitochondria isolated from cryopreserved stem cells derived from umbilical cord and from cultured stem cells derived from umbilical cord Umbilical cord-derived mesenchymal stem cells were seeded in alpha-MEM medium containing 10% (v / v) fetal bovine serum (FBS), 100 μg / mL streptomycin, and 100 U / mL ampicillin, and cultured for 72 hours. The cultured cells were treated with 0.25% trypsin-EDTA (TE) to obtain cells. A hemocytometer was used to count the number of cells, 1 × 10 7 The resulting cells were resuspended at a concentration of 10000000 cells / mL, placed in a freezing tube, transferred to a cryopreservation container, and then frozen at -80°C for 24 hours and stored in a liquid nitrogen cryopreservation bank. Mitochondria isolated from umbilical cord-derived cryopreserved stem cells were isolated in the same manner as in Preparation Example 1 above, and were compared with mitochondria from cultured cells isolated in Preparation Example 1 in terms of ATP activity, membrane potential, and mitochondrial reactive oxygen species properties.

[0108] As a result, to compare the ATP synthesis capacity between mitochondria isolated from cryopreserved stem cells derived from umbilical cord and mitochondria isolated from cultured stem cells derived from umbilical cord, a substrate (ADDP) was added, and it was confirmed that ATP activity was restored to a similar ratio in both conditions compared to basal energy metabolism. Furthermore, it was confirmed that the membrane potential activity was similar in both conditions, and the generation of mitochondrial reactive oxygen species was also similar (Figures 28-30).

[0109] Example 9. Measuring mitochondrial number using a particle counter Solutions of mitochondria isolated from human umbilical cord-derived mesenchymal stem cells isolated in Preparation Example 1 were prepared at concentrations of 1 μg / mL, 2.5 μg / mL, and 5 μg / mL, and then the number of mitochondria was measured using a particle counter (Multisizer 4e, Beckman Coulter). At this time, measurements were performed twice for each concentration, and the measurement results are shown in Table 9 and Figures 31 to 33 below.

[0110] [Table 9] [Table 9]

[0111] V. Confirmation of the anti-inflammatory effect of mitochondria in vitro Example 10. Comparison of anti-inflammatory activity in RAW264.7 cells by mitochondria from several cell types using quantitative real-time polymerase chain reaction To compare and analyze the anti-inflammatory activity of mitochondria obtained from various cells by the methods of Examples 2, 3, 4, and 7, cell-based analytical experiments were performed using quantitative real-time polymerase chain reaction.

[0112] RAW264.7 cells, a mouse-derived macrophage cell line, were cultured in DMEM medium containing 10% FBS. Approximately 3 × 10 5 Cells / well were seeded into 6-well plates and cultured for 24 hours, followed by starvation in FBS-free DMEM medium for approximately 24 hours.

[0113] After 24 hours, the cells were treated with 1 μg / mL of Salmonella-derived lipopolysaccharide (LPS) for 6 hours to induce an inflammatory response in the macrophage cell line. After 6 hours of lipopolysaccharide treatment, the cells were washed twice with PBS buffer, then treated with mitochondria obtained from each cell, and cultured for another 18 hours. In this case, the negative control group was a group not treated with lipopolysaccharide and mitochondria, and the positive control group was a group treated only with 1 μg / mL of lipopolysaccharide. In addition, the experimental groups were treated with 1 μg / mL of lipopolysaccharide, and after 6 hours, they were treated with 30 μg of mitochondria obtained from bone marrow-derived mesenchymal stem cells (BM-MSCs), umbilical cord-derived mesenchymal stem cells (UC-MSCs), rat myoblasts (L6 myoblasts), and human lung-derived fibroblasts (CCD-8LU), respectively.

[0114] After 18 hours of mitochondrial treatment, the culture medium was removed, PBS buffer was added to the cells to wash them twice, and 0.5 mL of RNA extract (Trizol Reagent, Thermo Fisher Scientific) was added directly, followed by leaving the cells at ambient temperature for 10 minutes. Next, 0.1 mL of chloroform was added, vortexed for 15 seconds, and centrifuged at approximately 12,000 × g for 10 minutes.

[0115] The separated supernatant was taken and the same amount of isopropyl alcohol was added, followed by centrifugation at 12000×g for 10 minutes. The liquid was then removed, washed once with 75% ethanol, and dried at ambient temperature. After drying, approximately 50 μL of RNAase-free purified distilled water was added, and the amount and purity of the obtained RNA were measured using a spectrophotometer.

[0116] To synthesize cDNA using the obtained RNA, 2 μg of purified total RNA was reacted with oligo dT at 70°C for 5 minutes, and then 10× reverse transcription buffer, 10 mM dNTPs, RNAse inhibitor, and M-MLV reverse transcriptase (Enzynomics, Korea) were added, and cDNA synthesis reaction was carried out at 42°C for 60 minutes.

[0117] After the cDNA synthesis reaction was completed, the reverse transcriptase was inactivated by heating at 72°C for 5 minutes, and then RNase H was added to remove single-stranded RNA to obtain the final cDNA. The expression changes of TNF-α, IL-1β, and IL-6 genes, which are characteristic genes of inflammatory responses, were observed by quantitative real-time polymerase chain reaction. The GAPDH gene was quantified together with those genes to calibrate the expression differences. The base sequences of the genes used in the quantitative real-time polymerase chain reaction are listed in Table 10 below.

[0118] [Table 10] [Table 10]

[0119] As shown in the experimental results, it was found that the expression of TNF-α, IL-1β, and IL-6 genes was increased when RAW264.7 cells, a mouse macrophage cell line, were treated with lipopolysaccharide. Furthermore, it was confirmed that the expression of TNF-α, IL-1β, and IL-6 genes induced by lipopolysaccharide was significantly inhibited when the cells were treated with mitochondria obtained from bone marrow-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, rat myoblasts, and human lung fibroblasts. From the above, it was confirmed that the mitochondria obtained from the cells used in the present invention exhibited significantly excellent anti-inflammatory activity (Figure 34).

[0120] Example 11. Comparison of anti-inflammatory activity by mitochondria derived from several types of cells in human mononuclear cells (THP-1) Human mononuclear cells, THP-1 cells, were cultured in RPMI medium containing 10% FBS. 5 Cells / well were seeded into 24-well plates and cultured in RPMI medium containing 1% FBS for 15–16 h.

[0121] The cells were treated with Salmonella-derived lipopolysaccharide (LPS) at a concentration of 2 μg / mL for 6 hours to induce an inflammatory response in the THP-1 cell line. After 6 hours of lipopolysaccharide treatment, the cells were treated with mitochondria obtained from each cell and further treated for 24 hours. In this case, the negative control group was a group not treated with lipopolysaccharide and mitochondria, and the positive control group was a group treated with lipopolysaccharide at a concentration of 2 μg. In addition, the experimental group was treated with lipopolysaccharide at a concentration of 2 μg / mL, and after 6 hours, it was treated with umbilical cord-derived mesenchymal stem cells (UC-MSC), human lung-derived fibroblasts (CCD-8LU), human induced pluripotent stem cells (IPS), and mitochondria obtained from porcine platelets in an amount of 40 μg each obtained by the methods of Examples 2, 4, 5, and 6. To compare the anti-inflammatory activity after the reaction, the cells were used for quantitative real-time polymerase chain reaction and the culture medium was used for ELISA.

[0122] Example 11.1 Comparison of Anti-inflammatory Activity Using Quantitative Real-Time Polymerase Chain Reaction After that, the culture medium was removed, PBS buffer was added to the cells to wash them twice, and 0.5 mL of RNA extract (Trizol reagent, Thermo Fisher Scientific) was added directly. After standing at ambient temperature for 10 minutes, 0.1 mL of chloroform was added, stirred for 15 seconds, and centrifuged at 12000 × g for 10 minutes. The separated supernatant was taken, and the same amount of isopropyl alcohol was added, centrifuged at 12000 × g for 10 minutes, after which the supernatant was removed, washed once with 75% ethanol, and dried at ambient temperature.

[0123] 50 μL of RNAase-free purified distilled water was added, and the amount and purity of the obtained RNA was measured by a spectrophotometer. To synthesize cDNA, 2 μg of purified total RNA was subjected to a binding reaction with oligo dT for 5 minutes at 70°C, after which 10X reverse transcription buffer, 10 mM dNTPs, RNAse inhibitor, and M-MLV reverse transcriptase (Enzynomics, Korea) were added, and the cDNA synthesis reaction was carried out at 42°C for 60 minutes. After the reaction, the reverse transcriptase was inactivated by heating at 72°C for 5 minutes, and then RNase H was added to remove single-stranded RNA to obtain cDNA.

[0124] Quantitative polymerase chain reaction (quantitative RT-PCR) was performed using the primers shown in Table 11 below to determine whether expression of inflammatory cytokines was altered, where expression differences were corrected for by quantification using 18S as a correction gene.

[0125] [Table 11] [Table 11]

[0126] As shown in the experimental results, when human mononuclear cells, THP-1 cells, were treated with lipopolysaccharide, the expression of IL-6 gene was found to increase. Furthermore, when treated with mitochondria obtained from umbilical cord-derived mesenchymal stem cells, human lung-derived fibroblasts, human induced pluripotent stem cells, and porcine platelets, the expression of IL-6 gene induced by lipopolysaccharide was significantly inhibited. From the above, it was confirmed that mitochondria obtained from various cells exhibited significantly excellent anti-inflammatory activity (Figure 35, *P<0.05).

[0127] Example 11.2. Comparison of anti-inflammatory activity using ELISA method To confirm the expression level of IL-6, an inflammatory cytokine of THP-1 cells, in the obtained supernatant, an experiment was performed using human IL-6 (R&D Systems) according to the manufacturer's manual.

[0128] 100 μL of coating solution was placed in a 96-well plate, reacted overnight at ambient temperature, washed three times, then reacted with reagent dilution solution at ambient temperature for 1 hour, and washed three times. The 10-fold diluted supernatant and standard solution were reacted at ambient temperature for 2 hours, then washed three times, and each well was treated with a labeled antibody (detection antibody) and then reacted at ambient temperature for 2 hours. After washing three times, the streptavidin solution (streptavidin-HRP) was reacted at ambient temperature for 20 minutes, then washed three times, and reacted with a color solution (substrate solution) in a dark room at ambient temperature for 20 minutes, after which the reaction stop solution was added and the absorbance was measured at a wavelength of 450 nm.

[0129] As shown in the experimental results, when human mononuclear cells, THP-1 cells, were treated with lipopolysaccharide, IL-6 protein was increased. Furthermore, when treated with mitochondria obtained from umbilical cord-derived mesenchymal stem cells, human lung-derived fibroblasts, human induced pluripotent stem cells, and porcine platelets, the IL-6 protein induced by lipopolysaccharide was significantly inhibited, which was consistent with the results of gene expression. From the above, it was confirmed that mitochondria obtained from various cells exhibited significantly excellent anti-inflammatory activity (Figure 36, *P<0.05).

Claims

1. A pharmaceutical composition for preventing or treating myositis, comprising mitochondria as an active ingredient.

2. The pharmaceutical composition for preventing or treating myositis according to claim 1, wherein the mitochondria are isolated from cells or tissues.

3. The pharmaceutical composition for preventing or treating myositis according to claim 2, wherein the mitochondria are isolated from in vitro cultured cells.

4. The pharmaceutical composition for preventing or treating myositis according to claim 2, wherein the cells are any one selected from the group consisting of somatic cells, germ cells, stem cells, and combinations thereof.

5. The pharmaceutical composition for preventing or treating myositis according to claim 4, wherein the somatic cells are any one selected from the group consisting of muscle cells, liver cells, nerve cells, fibroblasts, epithelial cells, adipocytes, bone cells, leukocytes, lymphocytes, platelets, or mucosal cells, and combinations thereof.

6. The pharmaceutical composition for preventing or treating myositis according to claim 4, wherein the reproductive cells are any one selected from the group consisting of sperm, eggs, and combinations thereof.

7. The pharmaceutical composition for preventing or treating myositis according to claim 4, wherein the stem cells are any one selected from the group consisting of mesenchymal stem cells, adult stem cells, induced pluripotent stem cells, embryonic stem cells, bone marrow stem cells, neural stem cells, limbal stem cells, tissue-derived stem cells, and combinations thereof.

8. The pharmaceutical composition for preventing or treating myositis according to claim 7, wherein the mesenchymal stem cells are obtained from any one selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, placenta, synovial fluid, testis, periosteum, and combinations thereof.

9. The pharmaceutical composition for preventing or treating myositis according to claim 1, wherein the mitochondria are contained in a concentration of 0.1 μg / mL to 1000 μg / mL in the pharmaceutical composition.

10. The mitochondria are 1×10 ５ ~5×10 ８ A pharmaceutical composition for preventing or treating myositis according to claim 1, comprising a content of mitochondria / mL.

11. A method for preventing or treating myositis, comprising the step of administering to a subject the pharmaceutical composition according to any one of claims 1 to 10.

12. Use of isolated mitochondria for preventing or treating myositis.

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