WO2020222566A1 - 분리된 미토콘드리아를 유효성분으로 포함하는 근염 예방 또는 치료용 약학 조성물 - Google Patents
분리된 미토콘드리아를 유효성분으로 포함하는 근염 예방 또는 치료용 약학 조성물 Download PDFInfo
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- WO2020222566A1 WO2020222566A1 PCT/KR2020/005769 KR2020005769W WO2020222566A1 WO 2020222566 A1 WO2020222566 A1 WO 2020222566A1 KR 2020005769 W KR2020005769 W KR 2020005769W WO 2020222566 A1 WO2020222566 A1 WO 2020222566A1
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- mitochondria
- cells
- myositis
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Definitions
- the present invention relates to a pharmaceutical composition for preventing or treating myositis comprising mitochondria as an active ingredient.
- Myositis is a disease in which muscle fibers are damaged due to inflammation in the muscles, and the ability to contract muscles decreases along with pain in the muscles.
- Myositis is divided into dermatomyositis, polymyositis, and inclusion body myositis, among which polymyositis and dermatomyositis are inflammatory myopathies, symptoms of decreased muscle strength in the limbs close to the trunk, increased muscle enzyme levels, increased expression of inflammatory cytokines, abnormal EMG, muscle Abnormalities appear on the biopsy.
- muscle weakness due to polymyositis and dermatomyositis progresses gradually over several weeks or months, but in rare cases, it may progress rapidly. Untreated severe muscle weakness can lead to muscle loss. About 15% to 30% of patients suffering from polymyositis have been reported to be accompanied by malignant tumors, and when dermatomyositis occurs in old age, cancer has been reported.
- Steroids, immunosuppressants or immunomodulators are used for the treatment of polymyositis and dermatomyositis.
- Steroids are the most commonly used drugs for early treatment, and whether or not to use immunosuppressants depends on the reaction to steroid treatment and side effects.
- About 75% of myositis patients are prescribed immunosuppressants in addition to steroids.
- an immunomodulator intravenous immunoglobulin has been proven to be effective in improving not only muscle strength but also signs of muscle biopsy in dermatitis.
- immunosuppressants and immunomodulators are directly involved in the immune system and may have side effects, and the effects of the drugs do not last long, so they must be re-injected at intervals of 6 to 8 weeks.
- a myositis-induced mouse model was used to evaluate the myositis treatment effect of an immunosuppressant against CXCL10 (CXC motif chemokine 10), a chemokine that is increased in expression in the muscle tissue of multiple myositis (Kim et al. , Arthritis Research & Therapy 2014, 16:R126).
- CXCL10 CXC motif chemokine 10
- chemokine 10 a chemokine that is increased in expression in the muscle tissue of multiple myositis
- mitochondria are organelles of eukaryotic cells that are involved in the synthesis and regulation of adenosine triphosphate (ATP), a source of energy in cells.
- ATP adenosine triphosphate
- Mitochondria are involved in various metabolic pathways in vivo, such as cell signaling, cell differentiation, cell death, as well as the control of cell cycle and cell growth.
- an object of the present invention is to provide a pharmaceutical composition for treating myositis and a method of treating myositis using the same.
- an aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of myositis comprising mitochondria as an active ingredient.
- Another aspect of the present invention provides a method for preventing or treating myositis comprising administering the pharmaceutical composition to an individual.
- the pharmaceutical composition containing the mitochondria of the present invention as an active ingredient is administered to an individual suffering from myositis, inflammatory cells infiltrating the muscle cells of the individual can be reduced.
- the pharmaceutical composition of the present invention can effectively reduce the expression of inflammatory cytokines in muscle tissue in which myositis has occurred. Therefore, the pharmaceutical composition according to the present invention can be usefully used in the prevention or treatment of myositis.
- 1 is a view showing the measurement of the amount of ATP synthesis of mitochondria isolated from umbilical cord-derived mesenchymal stem cells.
- FIG. 2 is a diagram showing the measurement of membrane potential activity of mitochondria isolated from umbilical cord-derived mesenchymal stem cells.
- FIG. 3 is a view showing the measurement of active oxygen in mitochondria isolated from umbilical cord-derived mesenchymal stem cells.
- FIG. 4 is a schematic diagram of a first animal experiment plan for confirming the myositis treatment effect according to administration of mitochondria using myositis-induced mice.
- Figure 5 is a photograph of the quadriceps and hamstring muscles of the negative control group, the positive control group, and the experimental group to which foreign mitochondria were administered to confirm the inflammatory cells infiltrating muscle fibers with H & E (Hematoxylin & eosin).
- FIG. 6 is a view in which the quadriceps of the negative control group, the positive control group, and the experimental group to which foreign mitochondria were administered were stained with H&E, and then the number of inflammatory cells infiltrated with muscle fibers was measured and scored.
- FIG. 7 is a diagram showing the blood IL-6 concentration of mice in a normal group, a negative control group, a positive control group, and an experimental group to which mitochondria were administered.
- FIG. 10 is a diagram schematically illustrating a second animal experiment plan for confirming the myositis treatment effect according to the administration of foreign mitochondria using myositis-induced mice.
- 11 is a photograph of the quadriceps muscle of the negative control group, the positive control group, and the experimental group to which foreign mitochondria were administered to confirm the inflammatory cells infiltrated into the muscle fibers with H&E.
- FIG. 12 is a photograph of the muscles of the popliteal region of the negative control group, the positive control group, and the experimental group administered with foreign mitochondria in order to confirm the inflammatory cells infiltrated into the muscle fibers with H&E.
- FIG. 13 is a view obtained by measuring the number of inflammatory cells infiltrated into the muscle fibers after staining the quadriceps muscle of the negative control group, the positive control group, and the experimental group administered with foreign mitochondria with H&E.
- FIG. 14 is a diagram showing the blood IL-1 ⁇ concentration of mice in the normal group, the negative control group, the positive control group, and the experimental group mice to which foreign mitochondria were administered.
- 15 is a diagram showing the serum IL-6 concentration of mice in the normal group, the negative control group, the positive control group, and the experimental group mice to which foreign mitochondria were administered.
- FIG. 16 is a graph showing the concentration of TNF- ⁇ in the blood of mice in a normal group, a negative control group, a positive control group, and an experimental group to which foreign mitochondria were administered.
- FIG. 17 is a diagram showing the expression levels of IL-6 mRNA in muscles of mice in a normal group, a negative control group, a positive control group, and an experimental group to which foreign mitochondria were administered.
- FIG. 18 is a diagram schematically illustrating a 3rd animal experiment plan for confirming the effect of myositis treatment according to the administration of foreign mitochondria using myositis-induced mice.
- FIG. 19 shows that the number of inflammatory cells stained with H&E (Hematoxylin & eosin) decreased after mitochondrial transplantation of mice induced myositis model.
- H&E Hematoxylin & eosin
- FIG. 20 shows that the histological score was decreased by the scoring method after transplanting the mitochondria to the mouse induced myositis model.
- Fig. 21 shows the reduction of inflammatory cytokine after mitochondria are transplanted into mice induced myositis model.
- FIG. 22 shows that mitochondria activity increased after mitochondria were implanted in mice induced myositis model.
- FIG. 28 is a view comparing the ATP activity of mitochondria isolated from umbilical cord-derived mesenchymal stem cells stored frozen and mitochondria isolated from cultured umbilical cord-derived mesenchymal stem cells.
- 29 is a view comparing the membrane potentials of mitochondria isolated from umbilical cord-derived mesenchymal stem cells stored frozen and mitochondria isolated from cultured umbilical cord-derived mesenchymal stem cells.
- FIG. 30 is a diagram showing the measurement of free radicals in mitochondria isolated from umbilical cord-derived mesenchymal stem cells and mitochondria isolated from cultured umbilical cord-derived mesenchymal stem cells.
- FIG. 31 is a diagram showing the number of mitochondria in a solution containing mitochondria at a concentration of 1 ⁇ g/ml measured using a particle counter (Multisizer 4e, Beckman Coulter).
- FIG. 32 is a diagram showing the number of mitochondria in a solution containing mitochondria at a concentration of 2.5 ⁇ g/ml, measured using a particle counter.
- 33 is a diagram showing the number of mitochondria in a solution containing mitochondria at a concentration of 5 ⁇ g/ml, measured using a particle counter.
- FIG. 34 is a diagram confirming the ability of TNF- ⁇ , IL-1 ⁇ , and IL-6 to inhibit mRNA expression by various types of cell-derived mitochondria in RAW264.7 cells activated with LPS.
- FIG. 35 is an observation of the ability to inhibit the expression of IL-6 mRNA by various types of cell-derived mitochondria in THP-1 cells activated with LPS.
- FIG. 36 shows the ability to inhibit the expression of IL-6 protein by various types of cell-derived mitochondria in THP-1 cells activated with LPS.
- One aspect of the present invention provides a pharmaceutical composition for preventing or treating myositis comprising mitochondria as an active ingredient.
- myositis refers to a disease in which muscle fibers are damaged due to inflammation in the muscles. Specifically, myositis is divided into dermatomyositis, polymyositis and inclusion body myositis, of which polymyositis and dermatomyositis belong to inflammatory myopathies. In the muscle tissue of polymyositis or dermatomyositis, the expression of inflammatory cytokines or chemokines such as CXCL10, IL-1 ⁇ , TNF- ⁇ , and IL-6 is increased.
- inflammatory cytokines or chemokines such as CXCL10, IL-1 ⁇ , TNF- ⁇ , and IL-6 is increased.
- Immunosuppressants or immunomodulators that inhibit CXCL10, IL-1 ⁇ , TNF- ⁇ or IL-6 are being developed as therapeutic agents for myositis.
- an immunosuppressive agent or an immunomodulatory agent there is a problem that side effects appear due to direct involvement in the immune system.
- a myositis-induced mouse model may be used to develop a myositis treatment.
- CFA Complete Freund's adjuvant
- PT pertussis toxin
- the tissues in which inflammation and inflammatory cells are observed in the muscle of a myositis patient are the quadriceps and the popliteal muscles, the quadriceps and the popliteal muscle tissues can also be used in myositis-causing mice.
- active ingredient refers to an ingredient that exhibits activity alone or together with an adjuvant (carrier) that is not active as such.
- metabolomic profiles were analyzed in both the white muscle of the quadriceps and the red muscle of the soleus muscle using CE-TOFMS in the muscle of the C-protein induced myositis mouse model (CIM).
- CCM C-protein induced myositis mouse model
- the ratio of malate and aspartate was decreased, and it was confirmed that mitochondria damage was present.
- inflammation was alleviated and mitochondria damage was restored by a test to confirm the efficacy of several doses of foreign mitochondria injection in the CIM mouse model.
- the mitochondria may be obtained from a mammal, or may be obtained from a human. Specifically, the mitochondria may be isolated from cells or tissues. For example, the mitochondria may be isolated from cells cultured in vitro. In addition, the mitochondria may be obtained from somatic cells, germ cells, blood cells, or stem cells. In addition, the mitochondria may be obtained from platelets. The mitochondria may be normal mitochondria obtained from cells having normal mitochondrial biological activity. In addition, the mitochondria may be cultured in vitro.
- the mitochondria may be obtained from autologous, allogenic or xenogenic.
- autologous mitochondria refers to mitochondria obtained from tissues or cells of the same individual.
- homologous mitochondria refers to mitochondria obtained from individuals belonging to the same species as the individual and having different genotypes for alleles.
- heterogeneous mitochondria refer to mitochondria obtained from individuals belonging to different species from the individual.
- the somatic cells may be muscle cells, hepatocytes, neurons, fibroblasts, epithelial cells, adipocytes, bone cells, white blood cells, lymphocytes, platelets, or mucosal cells.
- the germ cells are cells that undergo meiosis and somatic division, and may be sperm or egg.
- the stem cells may be any one selected from the group consisting of mesenchymal stem cells, adult stem cells, dedifferentiated stem cells, embryonic stem cells, bone marrow stem cells, neural stem cells, limbic stem cells, and tissue-derived stem cells.
- 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.
- the mitochondria when the mitochondria are separated from a specific cell, the mitochondria may be separated through various known methods such as using a specific buffer solution or using a potential difference and a magnetic field.
- the mitochondrial separation can be obtained by crushing and centrifuging cells in terms of maintaining mitochondrial activity.
- culturing cells, and first centrifuging a pharmaceutical composition containing such cells to produce a pellet resuspending the pellet in a buffer solution, homogenizing, and preparing the homogenized solution.
- Second centrifugation may be performed to prepare a supernatant, and the supernatant may be subjected to a third centrifugation to purify mitochondria.
- the first to third centrifugation may be performed at a temperature of 0°C to 10°C, preferably 3°C to 5°C.
- the time during which the centrifugation is performed may be performed for 1 to 50 minutes, and may be appropriately adjusted according to the number of centrifugation and the content of the sample.
- the first centrifugation may be performed at a rate of 100 ⁇ g to 1,000 ⁇ g , or 200 ⁇ g to 700 ⁇ g , or 300 ⁇ g to 450 ⁇ g .
- the second centrifugation may be performed at a rate of 1 ⁇ g to 2,000 ⁇ g , or 25 ⁇ g to 1,800 ⁇ g , or 500 ⁇ g to 1,600 ⁇ g .
- the third centrifugation may be performed at a rate of 100 ⁇ g to 20,000 ⁇ g , or 500 ⁇ g to 18,000 ⁇ g , or 800 ⁇ g to 15,000 ⁇ g .
- the separated mitochondria can be quantified by quantifying protein.
- the isolated mitochondria may be quantified through a BCA (bicinchoninic acid assay) assay.
- the mitochondria in the pharmaceutical composition may be contained in a concentration of 0.1 ⁇ g/ml to 1,000 ⁇ g/ml, 1 ⁇ g/ml to 750 ⁇ g/ml, or 25 ⁇ g/ml to 500 ⁇ g/ml. In one embodiment of the present invention, concentrations of 25 ⁇ g/ml, 50 ⁇ g/ml, and 100 ⁇ g/ml were used.
- the number of the separated mitochondria can be measured through a particle counter (Multisizer 4e, Beckman Coulter), and referring to a paper written by James D. McCully ( J Vis Exp . 2014; (91): 51682.)
- the number of mitochondria may be as shown in Table 1 below.
- Amount of isolated mitochondria ( ⁇ g) Number of mitochondria Concentration ( ⁇ g/mL) 0.01 2.16 ⁇ 10 5 ⁇ 0.01 ⁇ 10 5 0.1
- Example 9 of the present invention as a result of measuring the number of mitochondria using a particle counter of 1 ⁇ g/ml, 2.5 ⁇ g/ml and 5 ⁇ g/ml, 1.96 ⁇ 10 6 ⁇ 0.98 ⁇ 10 6 , 5.97 ⁇ The measurements were 10 6 ⁇ 0.19 ⁇ 10 6 and 1.01 ⁇ 10 7 ⁇ 0.32 ⁇ 10 7 pieces.
- the number of mitochondria at a concentration of 10 ⁇ g/ml is 2.16 ⁇ 10 7 ⁇ 0.08 ⁇ 10 7 and when multiplied by twice the number of mitochondria at a concentration of 5 ⁇ g/ml, 2.02 ⁇ 10 7 ⁇ 0.64 ⁇ It was confirmed to have a similar number range of 10 7 pieces.
- the mitochondria in the pharmaceutical composition may be included in an amount of 1 ⁇ 10 5 number of mitochondria/ml to 5 ⁇ 10 9 number of mitochondria/ml.
- the mitochondria in the pharmaceutical composition are 1 ⁇ 10 5 number of mitochondria/ml to 5 ⁇ 10 9 number of mitochondria/ml, 2 ⁇ 10 5 number of mitochondria/ml to 2 ⁇ 10 9 number of mitochondria/ml, 5 ⁇ 10 5 Number of mitochondria/ml to 1 ⁇ 10 9 Number of mitochondria/ml, 1 ⁇ 10 6 Number of mitochondria/ml to 5 ⁇ 10 8 Number of mitochondria/ml, 2 ⁇ 10 6 Number of mitochondria/ml to 2 ⁇ 10 8 Number of mitochondria/ml, It may be included in an amount of 5 ⁇ 10 6 number of mitochondria/ml to 1 ⁇ 10 8 number of mitochondria/ml or 1 ⁇ 10 7 number of mitochondria/ml to 5 ⁇ 10 7 number of mitochondria/ml.
- the therapeutically effective dose of mitochondria included in the pharmaceutical composition may be 3 ⁇ 10 5 mitochondria number/kg to 1.5 ⁇ 10 10 mitochondria number/kg once based on the body weight of the subject to be administered.
- the therapeutically effective dose of mitochondria in the pharmaceutical composition is 3 ⁇ 10 5 mitochondria number/kg to 1.5 ⁇ 10 10 mitochondria number/kg, 6 ⁇ 10 5 mitochondria number/kg once based on the body weight of the subject to be administered.
- the pharmaceutical composition may be administered once to 10 times, 3 to 8 times, or 5 to 6 times, preferably 5 times.
- the administration interval may be 1 to 7 days or 2 to 5 days, preferably 3 days.
- the pharmaceutical composition according to the present invention can be administered to humans or other mammals suffering from myositis or suffering from such disease or disease.
- the pharmaceutical composition may be an injection that can be administered intravenously, intramuscularly or subcutaneously, and preferably may be an injection formulation.
- the pharmaceutical composition according to the present invention is a very stable injection product physically and chemically by adjusting the pH using a buffer solution such as an acid aqueous solution or phosphate that can be used as an injection in order to secure product stability according to the distribution of injection formulations. Can be manufactured.
- a buffer solution such as an acid aqueous solution or phosphate that can be used as an injection in order to secure product stability according to the distribution of injection formulations. Can be manufactured.
- the pharmaceutical composition of the present invention may contain water for injection.
- the water for injection refers to distilled water prepared to dissolve a solid injection or dilute a water-soluble injection.
- the pharmaceutical composition of the present invention may include a stabilizer or a solubilizer.
- the stabilizer may be pyrosulfite, citric acid, or ethylenediaminetetraacetic acid
- the solubility aid is hydrochloric acid, acetic acid, sodium hydroxide, sodium hydrogen carbonate, sodium carbonate or potassium hydroxide.
- the present invention provides a method for preventing or treating myositis comprising administering the pharmaceutical composition described above to an individual.
- the individual may be a mammal, preferably a human.
- the administration may be intra-venous, intra-muscular, or intra-dermal administration.
- the pharmaceutical composition according to the present invention can supply foreign mitochondria having normal activity to the vein of an individual suffering from myositis, thereby increasing the activity of cells with reduced mitochondrial function or useful for regeneration of cells with abnormal mitochondrial function, It can be used for the prevention or treatment of myositis.
- mitochondria for the prevention or treatment of myositis. Details on mitochondria and myositis are as described above.
- Alpha-MEM Alpha-MEM (Alpha-Minimum Essential) containing 10% (v/v) fetal bovine serum (FBS, Gibco), 100 ⁇ g/ml streptomycin and 100 U/ml ampicillin from human umbilical cord-derived mesenchymal stem cells Medium) and cultured for 72 hours. After the culture was completed, it was washed twice using DPBS (Dulbecco's phosphate buffered saline, Gibco). Washed cells were treated with 0.25% (v/v) Trypsin-EDTA (TE, Gibco) to obtain cells.
- DPBS Dulbecco's phosphate buffered saline
- the obtained cells were recovered with a concentration of 1 ⁇ 10 7 cells/ml using a hemocytometer in order to separate mitochondria.
- the cell line was subjected to the first centrifugation at a rate of 350 ⁇ g for 10 minutes at 4°C.
- the obtained pellet was recovered, resuspended in a buffer solution, and homogenized for 10 to 15 minutes.
- the composition containing the pellets was subjected to a second centrifugation at a rate of 1,100 ⁇ g for 3 minutes at a temperature of 4° C. to obtain a supernatant.
- the supernatant was subjected to a third centrifugation at a rate of 12,000 ⁇ g for 15 minutes at 4° C. to separate mitochondria from the cell line.
- the mitochondria thus obtained were mixed with PBS and then filled into a syringe.
- Alpha-MEM Alpha-MEM (Alpha-Minimum essential) containing 10% fetal bovine serum (FBS, Gibco), 100 ⁇ g/ml streptomycin, and 100 U/ml ampicillin for human umbilical cord-derived mesenchymal stem cells (UC-MSC) medium, Gibco) medium and cultured for 72 hours. After the cultivation of the cells was completed, the cells were washed twice using DPBS. Then, cells were obtained by treatment with 0.25% Trypsin/EDTA. After the cells were resuspended so that the cell concentration became 1 ⁇ 10 7 cells/ml, the first centrifugation was performed at a rate of 350 ⁇ g for 10 minutes at 4°C.
- FBS fetal bovine serum
- U-MSC human umbilical cord-derived mesenchymal stem cells
- the washed cells were resuspended using a mitochondrial separation solution and then crushed using a 1 ml syringe. Subsequently, the cell lysate was centrifuged at 1,500xg for 5 minutes at 4°C to remove impurities, and the supernatant containing mitochondria was recovered. The recovered supernatant was centrifuged at 20,000xg for 5 minutes at 4°C to recover the precipitated mitochondria, and the separated mitochondria were suspended in Tris buffer and used for protein quantification by the BCA method and then used in the experiment.
- BM-MSC Human bone marrow-derived mesenchymal stem cells
- Human fibroblasts (CCD-8LU, ATCC) were inoculated in DMEM (Gibco) medium containing 10% fetal bovine serum (FBS, Gibco), 100 ⁇ g/ml streptomycin and 100 U/ml ampicillin for 72 hours. During incubation.
- DMEM Gibco
- FBS fetal bovine serum
- iPSCs Human dedifferentiated stem cells
- the platelet lysate was centrifuged at 1,500xg for 5 minutes at 4°C to remove impurities, and the supernatant containing mitochondria was recovered.
- the recovered supernatant was centrifuged at 20,000xg for 5 minutes at 4°C to recover the precipitated mitochondria, and the separated mitochondria were suspended in Tris buffer and used for protein quantification and then for the experiment.
- DMEM-High glucose Dulbecco's modified eagle's medium-high glucose
- FBS fetal bovine serum
- L6 cells American Type Culture Collection, ATCC, CRL-1458
- a source cell line derived from rat skeletal muscle. , Gibco medium and cultured for 72 hours.
- JC-1 molecular probes, cat no. 1743159
- the prepared 5 ⁇ g of mitochondria was mixed with 50 ⁇ l of PBS and then dispensed into a 96-well-plate, and 50 ⁇ l of PBS containing no mitochondria as a control group was also dispensed into a 96-well-plate.
- 5 ⁇ g of mitochondria was mixed with 50 ⁇ l of CCCP (R&D systems, CAS 555-60-2), reacted at room temperature for 10 minutes, and then dispensed into a 96-well plate.
- CCCP is an ion carrier of mitochondria and inhibits mitochondrial function by depolarizing the mitochondrial membrane potential.
- the JC-1 dye was treated and reacted to each well to a concentration of 2 ⁇ M, and the absorbance was measured using a fluorescence microplate reader.
- the JC-1 dye is present as a monomer at a low concentration and exhibits green fluorescence, and at a high concentration, the JC-1 dye aggregates to exhibit red fluorescence (Monomer: Ex 485 / Em 530, J-aggregate: Ex 535 nm / Em 590 nm).
- the mitochondrial membrane potential was analyzed by calculating the ratio of the absorbance of the green fluorescence to the absorbance of the red fluorescence.
- the prepared 5 ⁇ g of mitochondria was mixed with 50 ⁇ l of PBS and then dispensed into a 96-well-plate, and 50 ⁇ l of PBS containing no mitochondria as a control group was also dispensed into a 96-well-plate.
- the MitoSOX red indicator dye was mixed with 50 ⁇ l of PBS to make a concentration of 10 ⁇ M, and then treated in each well and reacted for 20 minutes in an incubator under conditions of 37°C and 5% CO 2 .
- the absorbance was measured using a fluorescent microplate reader (Ex 510 nm / Em 580 nm). As a result, it was confirmed that the mitochondrial active oxygen in the mitochondria was low in both the control group and the experimental group (FIG. 3). Through this, it was confirmed that the mitochondria isolated in Preparation Example 1 were not damaged.
- CFA Complete Freund's adjuvant
- PT pertussis toxin
- a group in which the mitochondria isolated in Preparation Example 1 (5 ⁇ g) was administered once intravenously on the 1st or 7th day after myositis induction was set as the experimental group.
- the group in which 100 ⁇ l of PBS was administered intraperitoneally was set as a negative control group
- the group in which 0.8 mg/kg of dexamethasone was administered intraperitoneally from day 1 to day 14 after myositis induction was set as a positive control group. (Fig. 4).
- Example 4.2 Identification of inflammatory infiltrating muscle fibers
- mice of each group of Example 4.1 were sacrificed on the 14th day, and the quadriceps and hamstring muscle tissues were collected, stained with H & E (Hematoxylin & eosin), and then the infiltration of inflammatory cells was observed with an optical microscope.
- H & E Hematoxylin & eosin
- mice of each group were sacrificed, the quadriceps muscle and the popliteal muscle tissues were collected and stained with H&E, and the number of muscle fibers infiltrated with inflammatory cells was evaluated using a scoring system.
- the score measurement method of the score system method is shown in Table 2 below. At this time, the average values of the right and left muscles of the quadriceps and the popliteal muscles were compared.
- Example 4.3 Checking the concentration of cytokines in the blood
- Example 4.4 Inflammatory response confirmation through PET/MRI analysis
- the expression of oxidative phosphorylation complex II in quadriceps was decreased in the negative control group (CIM) compared to the control group (Control) and increased in the experimental group (CIM+Mito day 7) compared to the positive control group (DEXA).
- the expression of TOM20 in soleus was decreased in the negative control group (CIM) compared to the control group (Control), and increased in the experimental group (CIM+Mito day 1, CIM+Mito day 7) compared to the positive control group (DEXA) (Fig. 9 ).
- the second experiment was performed by setting the time point of administration of mitochondria to the 7th day after induction of myositis.
- mice C57BL/6 female 8-week-old mice were injected intradermally with CFA containing 200 ⁇ g of C protein fragment and 100 ⁇ g of heat-treated bacterium, and 2 ⁇ g of PT was intraperitoneally injected.
- a group in which the mitochondria isolated in Preparation Example 1 (5 ⁇ g) was administered once intravenously was set as the experimental group.
- the group in which 100 ⁇ l of PBS was administered intraperitoneally was set as a negative control
- the group in which 0.8 mg/kg of dexamethasone was administered intraperitoneally from day 7 to day 14 after myositis induction was set as a positive control group. (Fig. 10).
- Example 5.2 Identification of inflammatory infiltrating muscle fibers
- mice of each group of Example 5.1 were sacrificed on the 14th day, and the quadriceps and popliteal muscle tissues were collected, stained with H&E, and observed for infiltration of inflammatory cells with an optical microscope. As a result, it was confirmed that the number of inflammatory cells infiltrated into the muscle fibers of the positive control group and the experimental group was decreased compared to the negative control group (FIGS. 11 and 12).
- mice of each group were sacrificed, the quadriceps muscle and the popliteal muscle tissues were collected and stained with H&E, and the number of muscle fibers infiltrated with inflammatory cells was evaluated using a scoring system.
- the score measurement method of the scoring system was performed in the same manner as in Example 4.2. At this time, the average values of the right and left muscles of the quadriceps and the popliteal muscles were compared. As a result, the score of the inflammatory-infiltrated muscle fibers of the positive control group and the experimental group was significantly reduced compared to the negative control group (FIG. 13).
- Example 5.3 Checking the concentration of cytokines in the blood
- IL-6 mRNA expression was confirmed by RT-qPCR in the muscle-separated mRNA of normal mice and mice of each group of Example 5.1 on the 14th day. Specifically, total RNA was isolated from muscle 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 ⁇ -actin mRNA. At this time, the primers used are shown in Table 3 below.
- the negative control group significantly affected the metabolite profile of the skeletal muscle compared to the control group (Fig. 23). It was confirmed that the mitochondrial transplant group recovered similarly to the metabolite profile of the control group compared to the positive control group (DEXA).
- the malate-aspartate shuttle (sometimes simply a malate aspartate shuttle defect) is a biochemical system that translocates the electrons generated during the process across the semipermeable inner membrane of the mitochondria for oxidative phosphorylation in eukaryotes.
- Mitochondrial dysfunction seen in the myositis model is associated with the malate-aspartate shuttle, and it was confirmed by a decrease in the ratio of the relative quantitative value of malic acid and aspartate.
- This ratio of the relative quantitative value of malic acid and aspartate was significantly increased after mitochondrial transplantation compared to the negative control group (CIM) and the positive control group (DEXA), and it was confirmed that recovery was at a level similar to that of the control group (Fig. 24 to Fig. 27).
- CFA Complete Freund's adjuvant
- PT pertussis toxin
- a group administered a single intravenous dose of the mitochondria isolated in Preparation Example 1 was 0.2 ug, 1 ug, and 5 ug was set as the experimental group.
- the group in which 100 ⁇ l of PBS was administered intraperitoneally was set as the negative control group, and the group administered with 0.8 mg/kg of dexamethasone (DEXA) intraperitoneally from day 7 to day 14 after induction of myositis It was set as a positive control group (Fig. 18).
- DEXA dexamethasone
- the level of inflammatory cytokine expression in mRNA isolated from muscle was observed through RT-qPCR. After mitochondrial transplantation, the degree of mitochondrial activity was evaluated by Western blot analysis of the expression of mitochondrial oxidative phosphorylation complexes (OXPHOS complexes). Table 4 shows the animal test group information.
- Example 6.2 Identification of inflammatory infiltrating muscle fibers
- mice of each group were sacrificed on the 14th day to collect quadriceps and hamstring muscle tissues, stained with H&E (Hematoxylin & eosin), and observed infiltration of inflammatory cells with an optical microscope. . As a result, it was confirmed that the number of inflammatory cells infiltrated into the muscle fibers of the positive control group and the experimental group was decreased compared to the negative control group (FIG. 19).
- mice of each group were sacrificed, the quadriceps muscle and the popliteal muscle tissues were collected and stained with H&E, and the number of muscle fibers infiltrated with inflammatory cells was evaluated using a scoring system.
- the score measurement method of the score system method is shown in Table 5 below. At this time, the average values of the right and left muscles of the quadriceps and the popliteal muscles were compared.
- IL-6 and TNF- ⁇ mRNA which are inflammatory cytokines, were measured in the control group (Control), the negative control group (CIM), the positive control group (DEXA), and the experimental group (mitochondrial transplant group) by the muscle of each group of mice. Confirmed through -qPCR. Specifically, total RNA was isolated from muscle using TRIzol reagent (Invitrogen), and qPCR was performed using SYBR Green (Perkin Elmer, MA, USA) and 7500 Fast Real-Time PCR system (Applied Biosystems). The experimental results were normalized to the amount of ⁇ -actin mRNA. At this time, the primers used for RT-qPCR are shown in Table 6 below.
- IL-6 mRNA expression tends to decrease in the muscles of the mitochondrial transplant group.
- TNF- ⁇ mRNA expression was significantly reduced in the mitochondrial 5 ug transplant group.
- the expression of IL-6 and TNF- ⁇ mRNA in the muscles of the positive control group (Dexa) was not decreased, and the mitochondrial transplant group was more effective than the positive control group (Dexa) in terms of the effect of reducing IL-6 and TNF- ⁇ mRNA. It was confirmed (Fig. 21).
- the mitochondria prepared in Preparation Example 1 were administered intravenously to ICR mice once, and then changes in body weight and long-term changes through autopsy were confirmed.
- the experiment was conducted by dividing each 12 male and female 7-week-old ICR mice into 4 groups as shown in Table 7 below.
- the G1 group was administered an excipient.
- the G2 to G4 groups were administered 25 ⁇ g, 50 ⁇ g or 100 ⁇ g of mitochondria, respectively.
- mitochondria were administered in an amount exceeding the approximate lethal dose (ALD).
- the administration site was sterilized with a 70% alcohol cotton, and then an excipient or mitochondria was administered at a rate of 1 ml/min through the caudal vein using a syringe equipped with a 26 gauge injection needle.
- Example 8 Comparison of properties of mitochondria isolated from umbilical cord-derived stem cells stored frozen and mitochondria isolated from cultured umbilical cord-derived stem cells
- Umbilical cord-derived mesenchymal stem cells were inoculated into Alpha-MEM medium containing 10% (v/v) fetal calf serum (FBS), 100 ⁇ g/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. The obtained cells are resuspended so that the cells become 1 ⁇ 10 7 cells/ml using a hemocytometer, transferred to a cryopreservation container by placing them in a freezing tube, and then frozen at -80°C for 24 hours to freeze liquid nitrogen. Stored in a storage tank. Separation of mitochondria from umbilical cord-derived stem cells stored frozen was separated in the same manner as in Preparation Example 1, and the cultured cell-derived mitochondria isolated in Preparation Example 1 and ATP activity, membrane potential, and mitochondrial reactive oxygen properties were compared.
- FBS fetal calf serum
- TE Trypsin-EDTA
- Example 9 Measurement of the number of mitochondria using a particle counter
- the mitochondria isolated from the human umbilical cord-derived mesenchymal stem cells isolated in Preparation Example 1 were prepared at concentrations of 1 ⁇ g/ml, 2.5 ⁇ g/ml, and 5 ⁇ g/ml, and then particle counters (Multisizer 4e, Beckman Coulter) to measure the number of mitochondria. At this time, it was measured twice for each concentration, and the measurement results are shown in Table 9 and FIGS. 31 to 33 below.
- Example 10 Comparison of anti-inflammatory activity using quantitative real-time polymerization chain reaction by various types of cell-derived mitochondria in RAW264.7 cells
- Example 3 In order to compare and analyze the anti-inflammatory activity of mitochondria obtained from various cells by the method of Example 2, Example 3, Example 4, and Example 7, a cell-based assay using a quantitative real-time polymerization chain reaction method was conducted. .
- RAW264.7 cells a mouse-derived macrophage cell line, were cultured in DMEM medium containing 10% FBS. About 3 ⁇ 10 5 cells/well of cells were inoculated into a 6 well plate and cultured for 24 hours, followed by depletion conditions in DMEM medium from which FBS was removed for about 24 hours.
- LPS salmonella-derived lipopolysaccharide
- UC-MSC bone marrow-derived mesenchymal stem cells
- BM-MSC umbilical cord-derived mesenchymal stem cells
- L6 myoblast rat myoblasts
- CCD-8LU mitochondria obtained from human lung-derived fibroblasts
- RNA extract Trizol reagent, Thermo Fisher Scientific
- RNAase-free distilled water was added, and quantification and purity of the obtained RNA were measured using a spectrophotometer.
- RNAse inhibitor M- By adding MLV reverse transcriptase (Enzynomics, Korea), a cDNA synthesis reaction was performed at 42°C for 60 minutes.
- 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 a final cDNA.
- Changes in the expression of the TNF- ⁇ gene, IL-1 ⁇ gene, and IL-6 gene, which are characteristic genes of the inflammatory response, were observed through quantitative real-time polymerization chain reaction. GAPDH gene was quantified together to correct for differences in expression.
- the nucleotide sequences of the genes used in the quantitative real-time polymerization chain reaction are as described in Table 10 below.
- Example 11 Comparison of anti-inflammatory activity by various types of cell-derived mitochondria in human mononuclear cells (THP-1)
- THP-1 cells Human-derived mononuclear cells, THP-1 cells, were cultured in RPMI medium containing 10% FBS. Cells of 4 ⁇ 10 5 cells/well were inoculated into a 24 well plate and cultured for 15 to 16 hours in RPMI medium containing 1% FBS.
- Salmonella-derived lipopolysaccharide was treated at a concentration of 2 ⁇ g/ml for 6 hours to induce an inflammatory response in the THP-1 cell line. After 6 hours of lipopolysaccharide treatment, mitochondria obtained from each cell were treated and further cultured for 24 hours. At this time, the negative control group was a lipopolysaccharide and a mitochondrial untreated group, and the positive control group was a group treated with a lipopolysaccharide at a concentration of 2 ⁇ g/ml alone.
- a lipopolysaccharide of 2 ⁇ g/ml was treated, and umbilical cord-derived mesenchymal stem cells (UC-MSC) obtained by the method of Examples 2, 4, 5 and 6 after 6 hours.
- U-MSC umbilical cord-derived mesenchymal stem cells
- IPS human dedifferentiated stem cells
- mitochondria obtained from porcine platelets were each treated with 40 ⁇ g.
- cells were used in quantitative real-time polymerization chain reaction method, and culture medium was used in ELISA method.
- RNA extract Trizol reagent, Thermo Fisher Scientific
- 0.1 ml of chloroform was added, stirred for 15 seconds, and then centrifuged at 12,000xg for 10 minutes.
- the separated supernatant was taken, the same volume of isopropyl alcohol was added, centrifuged at 12,000xg for 10 minutes, the supernatant was removed, washed once with 75% ethanol, and dried at room temperature.
- RNA 50 ⁇ l of purified distilled water without RNAase was added, and the quantification and purity of RNA were measured using a spectrophotometer.
- 2 ⁇ g of purified total RNA was subjected to a binding reaction with oligo dT at 70° C. for 5 minutes, and then 10X reverse transcription reaction buffer, 10 mM dNTP, RNAse inhibitor, and M-MLV reverse transcriptase (Enzynomics, Korea) were added. Then, the cDNA synthesis reaction was performed at 42° C. for 60 minutes. After the reaction was heated at 72° C. for 5 minutes to inactivate the reverse transcriptase, RNase H was added to remove single stranded RNA to obtain cDNA.
- a quantitative polymerization chain reaction (quantitative RT-PCR) was performed using the primers shown in Table 11 below to determine whether the expression of cytokines of the pro-inflammatory factors was changed. At this time, the difference in expression was corrected by quantifying it with 18S as a gene for correction.
- IL-6 gene was increased when lipopolysaccharide was treated in human mononuclear cells, THP-1 cells.
- the expression of IL-6 gene induced by lipopolysaccharide was It was confirmed that umbilical cord-derived mesenchymal stem cells, human lung-derived fibroblasts, human dedifferentiated stem cells, and mitochondria obtained from porcine platelets were inhibited to a significant level when treated. Through this, it was confirmed that the mitochondria obtained from various cells showed remarkably excellent anti-inflammatory activity (FIG. 35, * P ⁇ 0.05).
- IL-6 protein was increased when lipopolysaccharide was treated in human mononuclear cells, THP-1 cells, and IL-6 protein induced by lipopolysaccharide was umbilical cord-derived mesenchymal stem.
- Cells, human lung-derived fibroblasts, human dedifferentiated stem cells, and mitochondria obtained from porcine platelets were significantly inhibited when treated, and were consistent with gene expression results. Through this, it was confirmed that mitochondria obtained from various cells exhibited remarkably excellent anti-inflammatory activity (FIG. 36, * P ⁇ 0.05).
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Abstract
Description
분리한 미토콘드리아의 양 (㎍) | 미토콘드리아 개수 | 농도 (㎍/㎖) |
0.01 | 2.16×105 ± 0.01×105 | 0.1 |
1 | 2.16×107 ± 0.08×107 | 10 |
25 | 0.54×109 ± 0.02×109 | 250 |
50 | 1.08×109 ± 0.04×109 | 500 |
100 | 2.16×109 ± 0.08×109 | 1,000 |
점수 | 평가방식(염증 침윤 근섬유 수) |
1 | 염증 침윤 근섬유 1 개 관찰 |
2 | 염증 침윤 근섬유 2-5 개 관찰 |
3 | 염증 침윤 근섬유 6-15 개 관찰 |
4 | 염증 침윤 근섬유 16-30 개 관찰 |
5 | 염증 침윤 근섬유 30-99 개 관찰 |
6 | 염증 침윤 근섬유 100 개 이상 관찰 |
프라이머 | 서열정보 | 서열번호 |
IL-6-F | TAGTCCTTCCTACCCCAATTTCC | 1 |
IL-6-R | TTGGTCCTTAGCCACTCCTTC | 2 |
그룹 | 개체 수 (n) | CIM 유도 | 목적 |
Control (non-treat) | 5 | x | 대조군 |
Vehicle (CIM) | 5 | O | 음성대조군 |
Dexamethasone | 5 | O | 양성대조군 |
MT 0.2 ug | 5 | O | 실험군 |
MT 1 ug | 5 | O | 실험군 |
MT 5 ug | 5 | O | 실험군 |
총 개체수 | 30 |
점수 | 평가방식(염증 침윤 근섬유 수) |
1 | 염증 침윤 근섬유 1 개 관찰 |
2 | 염증 침윤 근섬유 2-5 개 관찰 |
3 | 염증 침윤 근섬유 6-15 개 관찰 |
4 | 염증 침윤 근섬유 16-30 개 관찰 |
5 | 염증 침윤 근섬유 30-99 개 관찰 |
6 | 염증 침윤 근섬유 100 개 이상 관찰 |
프라이머 | 서열정보 | 서열번호 |
IL-6-F | TAGTCCTTCCTACCCCAATTTCC | 1 |
IL-6-R | TTGGTCCTTAGCCACTCCTTC | 2 |
TNF-α-F | CCCTCACACTCAGATCATCTTCT | 3 |
TNF-α-R | GCTACGACGTGGGCTACAG | 4 |
그룹 | 성별 | 개체 수 | 투여물질 | 투여경로 | 투여량 (㎍/개체) | 투여량 (㎖/개체) | 농도 (㎍/㎖) |
G1 | M/F | 3/3 | 부형제 | IV | - | 0.3 | - |
G2 | M/F | 3/3 | 미토콘드리아 | IV | 25 | 0.3 | 100 |
G3 | M/F | 3/3 | 미토콘드리아 | IV | 50 | 0.3 | 200 |
G4 | M/F | 3/3 | 미토콘드리아 | IV | 100 | 0.3 | 400 |
수컷 체중(g) | 암컷 체중(g) | |||||||
일 | 그룹 | 그룹 | ||||||
G1 | G2 | G3 | G4 | G1 | G2 | G3 | G4 | |
1 | 37.48±1.78 | 37.88±1.11 | 37.94±1.18 | 38.02±1.07 | 29.12±1.36 | 29.09±1.28 | 29.18±0.99 | 29.32±0.93 |
2 | 37.62±1.55 | 38.06±0.55 | 36.46±1.71 | 36.59±1.45 | 29.23±1.48 | 28.96±0.76 | 28.93±0.76 | 28.21±0.96 |
4 | 37.50±1.86 | 37.88±0.66 | 36.61±2.17 | 37.46±1.55 | 28.99±0.96 | 28.97±.61 | 29.54±1.62 | 28.51±1.09 |
8 | 38.49±1.53 | 38.75±1.65 | 37.12±3.09 | 38.96±1.79 | 29.13±0.71 | 29.58±0.27 | 29.90±1.78 | 28.92±1.79 |
15 | 39.21±1.11 | 39.19±1.17 | 37.73±2.85 | 39.98±1.39 | 30.56±0.52 | 30.22±0.45 | 30.82±1.43 | 29.70±1.39 |
N | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 |
1 ㎍/㎖ | 2.5 ㎍/㎖ | 5 ㎍/㎖ | ||||
미토콘드리아 개수/㎖ | 1차 | 2차 | 1차 | 2차 | 1차 | 2차 |
2.66E±06 | 1.25E±06 | 6.11E±06 | 5.83E±06 | 1.24E±07 | 7.83E±06 | |
평균 | 1.96×106±0.98×106 | 5.97×106±0.19×106 | 1.01×107±0.32×107 |
프라이머 | 서열 |
TNF-alpha-S | 5'-TCTCATCAGTTCTATGGCCC-3' (서열번호 5) |
TNF-alpha-AS | 5'-GGGAGTAGACAAGGTACAAC-3' (서열번호 6) |
IL-1beta-F | 5'-AACCTGCTGGTGTGTGACGTTC-3' (서열번호 7) |
IL-1beta-R | 5'-CAGCACGAGGCTTTTTTGTTGT-3' (서열번호 8) |
IL-6-AS | 5'-CTAGGTTTGCCGAGTAGATCT-3' (서열번호 9) |
IL-6-S | 5'-CCAAACTGGATATAATCAGGAAAT -3' (서열번호 10) |
GAPDH-S | 5'-GGTGAAGGTCGGTGTGAAG-3' (서열번호 11) |
GAPDH-AS | 5'-CTCGCTCCTGGAAGATGGTG-3' (서열번호 12) |
primer | 염기서열 |
Human IL-6-S | ccacacagacagccactcac (서열번호 13) |
Human IL-6-AS | tttcaccaggcaagtctcct (서열번호 14) |
Human 18S-S | ctcccacttggataactgtgg (서열번호 15) |
Human 18S-AS | gaccgggttggttttgatct (서열번호 16) |
Claims (12)
- 미토콘드리아를 유효성분으로 포함하는 근염 예방 또는 치료용 약학 조성물.
- 제1항에 있어서,상기 미토콘드리아는 세포 또는 조직으로부터 분리된 것인, 근염 예방 또는 치료용 약학 조성물.
- 제2항에 있어서,상기 미토콘드리아는 체외 배양된 세포부터 분리된 것인, 근염 예방 또는 치료용 약학 조성물.
- 제2항에 있어서,상기 세포는 체세포, 생식세포, 줄기세포 및 이들의 조합으로 이루어진 군으로부터 선택되는 어느 하나인 것인, 근염 예방 또는 치료용 약학 조성물.
- 제4항에 있어서,상기 체세포가 근육세포, 간세포, 신경세포, 섬유아세포, 상피세포, 지방세포, 골세포, 백혈구, 림프구, 혈소판 또는 점막세포로 및 이들의 조합으로 이루어진 군으로부터 선택되는 어느 하나인 것인, 근염 예방 또는 치료용 약학 조성물.
- 제4항에 있어서,상기 생식세포가 정자, 난자 및 이들의 조합으로 이루어진 군으로부터 선택되는 어느 하나인 것인, 근염 예방 또는 치료용 약학 조성물.
- 제4항에 있어서,상기 줄기세포가 중간엽줄기세포, 성체줄기세포, 역분화줄기세포, 배아줄기세포, 골수줄기세포, 신경줄기세포, 윤부줄기세포, 조직 유래 줄기세포 및 이들의 조합으로 이루어진 군으로부터 선택되는 어느 하나인 것인, 근염 예방 또는 치료용 약학 조성물.
- 제7항에 있어서,상기 중간엽줄기세포가 탯줄, 제대혈, 골수, 지방, 근육, 신경, 피부, 양막, 태반, 활액, 정소, 골막 및 이들의 조합으로 이루어진 군으로부터 선택되는 어느 하나로부터 수득된 것인, 근염 예방 또는 치료용 약학 조성물.
- 제1항에 있어서,상기 약학 조성물에 대하여, 상기 미토콘드리아는 0.1 ㎍/㎖ 내지 1000 ㎍/㎖의 농도로 포함되는, 근염 예방 또는 치료용 약학 조성물.
- 제1항에 있어서,상기 약학 조성물에 대하여, 상기 미토콘드리아는 1×105 내지 5×108 미토콘드리아 개수/㎖의 함량으로 포함되는 것인, 근염 또는 치료용 약학 조성물.
- 제1항 내지 제10항 중 어느 한 항의 약학 조성물을 개체에 투여하는 단계를 포함하는 근염 예방 또는 치료 방법.
- 근염 예방 또는 치료를 위한 분리된 미토콘드리아의 용도.
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EP20798096.2A EP3964218A4 (en) | 2019-04-30 | 2020-04-29 | PHARMACEUTICAL COMPOSITION FOR PREVENTION OR TREATMENT OF MYOSITIS WITH ISOLATED MITOCHONDRIA AS ACTIVE INGREDIENT |
US17/607,850 US20220211754A1 (en) | 2019-04-30 | 2020-04-29 | Pharmaceutical composition for preventing or treating myositis, comprising isolated mitochondria as active ingredient |
CN202080048016.3A CN114051410A (zh) | 2019-04-30 | 2020-04-29 | 包含分离的线粒体为有效成分的预防或治疗肌炎的药物组合物 |
JP2021563663A JP2022530232A (ja) | 2019-04-30 | 2020-04-29 | 活性成分として単離されたミトコンドリアを含む、筋炎を予防又は治療するための医薬組成物 |
AU2020264869A AU2020264869A1 (en) | 2019-04-30 | 2020-04-29 | Pharmaceutical composition for preventing or treating myositis, comprising isolated mitochondria as active ingredient |
CA3138170A CA3138170A1 (en) | 2019-04-30 | 2020-04-29 | Pharmaceutical composition for preventing or treating myositis, comprising isolated mitochondria as active ingredient |
BR112021021804A BR112021021804A2 (pt) | 2019-04-30 | 2020-04-29 | Composição farmacêutica para prevenção ou tratamento de miosite compreendendo mitocôndrias isoladas como ingrediente ativo |
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008137035A1 (en) * | 2007-05-02 | 2008-11-13 | The Mclean Hospital Corporation | Methods and compositions for mitochondrial replacement therapy |
KR20180062387A (ko) * | 2016-11-30 | 2018-06-08 | 차의과학대학교 산학협력단 | 미토콘드리아를 포함하는 근질환 예방 또는 치료용 약학 조성물 |
US20190111016A1 (en) * | 2016-02-26 | 2019-04-18 | The Regents Of The University Of California | Methods of treating muscle and liver disorders |
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EP3540067A4 (en) | 2016-11-14 | 2020-10-28 | Paean Biotechnology Inc. | METHOD OF INTRODUCING EXOGENOUS MITOCHONDRIA INTO CELLS |
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008137035A1 (en) * | 2007-05-02 | 2008-11-13 | The Mclean Hospital Corporation | Methods and compositions for mitochondrial replacement therapy |
US20190111016A1 (en) * | 2016-02-26 | 2019-04-18 | The Regents Of The University Of California | Methods of treating muscle and liver disorders |
KR20180062387A (ko) * | 2016-11-30 | 2018-06-08 | 차의과학대학교 산학협력단 | 미토콘드리아를 포함하는 근질환 예방 또는 치료용 약학 조성물 |
Non-Patent Citations (6)
Title |
---|
A. OLDFORS, A. R. MOSLEMI, L. JONASSON, M. OHLSSON, G. KOLLBERG, C. LINDBERG: "Mitochondrial abnormalities in inclusion-body myositis", NEUROLOGY, vol. 66, no. 1, 24 January 2006 (2006-01-24), pages S49 - S55, XP009531485, ISSN: 0028-3878, DOI: 10.1212/01.wnl.0000192127.63013.8d * |
JAMES D. MCCULLY, J VIS EXP, no. 91, 2014, pages 51682 |
KIM ET AL., ARTHRITIS RESEARCH & THERAPY, vol. 16, 2014, pages R126 |
RYGIEL, K. A. ET AL.: "Mitochondrial and inflammatory changes in sporadic inclusion body myositis", NEUROPATHOLOGY AND APPLIED NEUROBIOLOGY, vol. 41, 2015, pages 288 - 303, XP055756609 * |
See also references of EP3964218A4 |
SUGIHARA T ET AL., ARTHRITIS RHEUM, vol. 56, no. 4, 2007, pages 1304 - 14 |
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KR20200126929A (ko) | 2020-11-09 |
CA3138170A1 (en) | 2020-11-05 |
KR102391020B1 (ko) | 2022-04-26 |
JP2022530232A (ja) | 2022-06-28 |
AU2020264869A1 (en) | 2021-11-18 |
EP3964218A4 (en) | 2022-07-06 |
BR112021021804A2 (pt) | 2022-01-04 |
TW202106314A (zh) | 2021-02-16 |
US20220211754A1 (en) | 2022-07-07 |
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