Injectable composition containing isolated mitochondria and its uses

Abstract

The present invention relates to an injectable composition containing isolated mitochondria. More specifically, there is provided an injectable liquid composition containing glycine, sugar, buffer and mitochondria, and a pharmaceutical composition containing the above injectable liquid composition as an active ingredient. The injectable liquid composition according to the present invention prevents or treats mitochondrial-related diseases by enhancing the stability of mitochondria, and prevents and inhibits thrombus formation in the blood of a subject taking the composition, thereby ensuring the safety of a mitochondrial therapeutic agent and maintaining the activity of mitochondria. Therefore, the composition according to the present invention solves the aggregation that may occur when mitochondria are administered in vivo, thereby enabling the safe and effective injection of mitochondria into the body for the prevention and treatment of various diseases caused by mitochondrial dysfunction.

Description

Technical Field

[0001] The present invention relates to an injectable composition containing isolated mitochondria and its uses. More specifically, the present invention relates to an injectable liquid composition containing glycine or glycine oligomers, sugars, buffers, and mitochondria, and a pharmaceutical composition containing the injectable liquid composition as an active ingredient. Background Art

[0002] Mitochondria, as an energy source, play a key role in various physiological processes, such as ATP synthesis, production of reactive oxygen species, and apoptosis. Therefore, mitochondrial damage may lead to various diseases, and most mitochondrial disorders are caused by genetic or acquired mutations occurring in mitochondrial DNA. For example, the function of mitochondria may be altered due to abnormal mitochondrial membrane potential, oxidative stress caused by reactive oxygen species, free radicals, etc., and swelling caused by defects in the oxidative phosphorylation function of generating energy in mitochondria. Examples of such mitochondrial dysfunction have been reported, including mitochondrial genetic diseases, inflammatory diseases (such as rheumatoid arthritis), ischemic diseases, infectious diseases, heart diseases, myopathies, degenerative diseases (such as Parkinson's disease and Alzheimer's disease), the occurrence and metastasis of various cancers, etc.

[0003] In order to treat diseases associated with such mitochondrial dysfunction, recently, more and more attempts have been made to isolate mitochondria from cells or tissues for therapeutic purposes and inject them back into the living body for treatment.

[0004] Due to the increasing research on the therapeutic uses of mitochondria, research on how to maintain the in vitro stability of isolated mitochondria and effectively deliver isolated mitochondria to target cells or tissues is also increasing.

[0005] However, a mitochondrial delivery therapy for mitochondrial-related diseases has not been developed because there are problems of therapeutic range limitations or side effects when injecting isolated mitochondria in vivo.

[0006] Effectively delivering mitochondria to target cells or tissues is an important factor in the effectiveness of mitochondrion-containing drugs for treating mitochondrial-related diseases. It has been reported that the successful delivery of isolated mitochondria depends on the quantity and quality of the mitochondria to be injected and an appropriate delivery route. When the mitochondria are not physically or functionally damaged, the mitochondria can be effectively delivered and absorbed into target cells or tissues.

[0007] Meanwhile, mitochondria can be delivered by local injection and systemic administration. In local injection, mitochondria are directly injected into the target organ, while in systemic administration, mitochondria are injected through blood vessels. The main safety issues associated with intravascular injection of mitochondria are thrombosis and microvascular occlusion. Even when cells such as stem cell therapeutics are administered via blood vessels, blood clots are formed due to the binding of the administered stem cells and platelets, which obstructs blood flow and reduces the oxygen supply to the cells. Heparin, an anticoagulant, is co-administered to reduce and resolve these problems. However, it has been reported that heparin inhibits the intracellular delivery of mitochondria (E.E. Kesner et al.). Therefore, it is difficult to co-administer heparin during the intravascular injection of mitochondria, and there is currently no suitable anticoagulant.

[0008] Therefore, in order to effectively deliver a drug containing isolated mitochondria to target cells or tissues, a special formulation for isolated mitochondria is needed, which can maintain the in vitro safety of the mitochondrial drug and address platelet aggregation or thrombosis, which are side effects that occur during intravascular injection. Summary of the Invention

[0009] Technical problem

[0010] Under such circumstances, the present inventors have made extensive efforts and developed an effective injectable formulation for delivering a drug containing mitochondria to cells or tissues for the treatment of mitochondrial-related diseases. As a result, it has been confirmed that the injectable formulation of the present invention can inhibit platelet aggregation or thrombosis even without using an anticoagulant. In addition, it has been confirmed that the injectable formulation of the present invention not only inhibits the occurrence of mitochondrial aggregation but also does not impair the activity of mitochondria, and it has been confirmed that mitochondria can be stably administered in vivo by injection, thus completing the present invention.

[0011] Technical solution

[0012] To achieve the above object, according to one embodiment, the present invention provides an injectable liquid composition containing glycine, sugar, a buffer, and mitochondria.

[0013] According to another embodiment, the present invention further provides a pharmaceutical composition for preventing or treating mitochondrial-related diseases, the pharmaceutical composition containing the injectable liquid composition as an active ingredient.

[0014] According to another embodiment, the present invention further provides a method for preventing or treating mitochondrial-related diseases, the method comprising administering the injectable liquid composition to a subject.

[0015] Beneficial effect

[0016] The injectable liquid composition containing mitochondria for preventing or treating diseases related to mitochondrial dysfunction according to the present invention not only maintains the membrane potential and stability of mitochondria by inhibiting aggregation when using mitochondria after separating them from cells or tissues, but also ensures the safety of mitochondrial injection by inhibiting thrombus formation in subjects receiving mitochondrial injection. Since the composition according to the present invention can prevent thrombocytopenia and aggregation that may occur when isolated allogeneic or autologous mitochondria are administered in vivo, it is expected to significantly improve the commercial availability of mitochondrial injection by enabling the widespread application of isolated mitochondria in the treatment of various diseases caused by mitochondrial deterioration or mitochondrial dysfunction. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The following description with reference to the drawings can more specifically understand exemplary embodiments, wherein:

[0018] Figure 1 Images showing the phenomenon of self-coagulation of isolated mitochondria were presented, and the degree of aggregation of mitochondria isolated from human umbilical cord-derived mesenchymal stem cells (UC-hMSC) and Chinese hamster ovary-derived cells (CHO) in the SHE storage solution of the present invention (specifically, physiological NaCl solution (saline), used as an injection, as a control) was confirmed;

[0019] Figure 2a Images showing platelet aggregation induced by mitochondria isolated from various cells in vitro were presented, and platelet aggregation induced by mitochondria obtained from platelets isolated from human umbilical cord-derived mesenchymal stem cells (UC-hMSC), human bone marrow-derived mesenchymal stem cells (BM-hMSC), rat skeletal muscle-derived cell line (L6), or whole blood was confirmed (specifically, CTR refers to the control without mitochondrial treatment);

[0020] Figure 2b Images showing that platelet aggregation induced by mitochondria isolated from human umbilical cord-derived mesenchymal stem cells (UC-hMSC) stored in saline or SHE storage solution was confirmed. Specifically, it was confirmed that platelet aggregation was partially inhibited during treatment with heparin (H) as an anticoagulant;

[0021] Figure 3 Images showing the inhibitory effect of anticoagulants on platelet aggregation were presented, and it was confirmed that when platelets isolated from whole blood were treated with heparin and mitochondria simultaneously, platelet aggregation induced by mitochondria was inhibited as the concentration of heparin increased;

[0022] Figure 4 A graph showing the change in the number of platelets according to the mitochondrial vascular injection preparation is presented, in which when mitochondria isolated from human umbilical cord-derived mesenchymal stem cells (UC-hMSC) are intravenously injected into a mouse model (specifically, MT (mitochondria) represents mitochondria, TTB represents Tris-trehalose buffer solution, and SHE represents sucrose-HEPES-EGTA buffer solution), the change in the number of platelets in the blood is compared according to the composition of the injection solution and the presence or absence of an anticoagulant;

[0023] Figure 5 A graph showing the effect of the glycine concentration in the composition of the present invention on the mitochondrial activity level is presented, in which the change in the fluorescence value of a mitochondrial membrane potential marker is measured after mitochondria are stored in a solution containing different concentrations of glycine for 24 hours (specifically, TMRE refers to tetramethyrhodamine ethyl ester, a mitochondrial membrane potential marker);

[0024] Figure 6 An image showing the effect of glycine contained in the composition of the present invention in inhibiting platelet aggregation induced by mitochondria is presented, in which it is confirmed that when the glycine concentration in the composition of the present invention is 25 mM or higher, platelet aggregation induced by mitochondria can be inhibited;

[0025] Figure 7 An image and a table are presented, which show the inhibitory effect of the glycine-containing mitochondrial composition preparation of the present invention on platelet aggregation, in which it is confirmed that in the glycine-containing composition of the present invention, mitochondria do not cause platelet aggregation as compared with the composition without glycine addition; and

[0026] Figure 8 A table showing the inhibitory effect of the mitochondrial composition preparation of the present invention containing glycine or its oligomer on platelet aggregation, in which it is confirmed that in the glycine oligomer-containing composition of the present invention, mitochondria do not cause platelet aggregation as compared with the composition without glycine addition. Detailed Description of the Invention

[0027] Hereinafter, specific embodiments will be described in detail with reference to the accompanying drawings.

[0028] Liquid Injectable Composition Containing Mitochondria

[0029] In one aspect, the present invention provides a liquid injectable composition containing glycine or its oligomer, a sugar, a buffer, and mitochondria.

[0030] As used herein, the term "mitochondria" refers to the organelles of eukaryotic cells that are involved in the synthesis and regulation of adenosine triphosphate (ATP), which serves as an energy source within the cell, and is related to the control of various metabolic pathways in the body, such as cell signaling, cell differentiation, apoptosis, the cell cycle, and cell growth. Therefore, it has been reported that the deterioration or dysfunction of mitochondria caused by genetic, environmental, or unknown reasons is associated with the occurrence of various diseases, such as mitochondrial-related genetic diseases, inflammatory diseases (e.g., rheumatoid arthritis), ischemic diseases, infectious diseases, heart diseases, muscle diseases, degenerative diseases (such as Parkinson's disease and Alzheimer's disease), various cancers, and cancer metastasis, etc.

[0031] The injectable liquid composition of the present invention is a composition for preventing or treating diseases related to mitochondrial function. When mitochondria are administered by injection, it can inhibit thrombosis caused by mitochondrial aggregation, thrombocytopenia, and aggregation, etc., maintain and / or enhance the stability of mitochondria, and also stably maintain the activity of mitochondria.

[0032] The injectable liquid composition of the present invention may contain glycine or its oligomers, sugars, buffers, and mitochondria.

[0033] The glycine oligomer refers to an oligomer formed by the binding of one or more glycines. The glycine oligomer may be composed of 2 to 20 glycines. Specifically, the glycine oligomer may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 glycines. In addition, the glycine oligomer may contain 2 to 10 or 3 to 5 glycines. In one embodiment, the glycine oligomer may be a glycine dimer or a glycine trimer.

[0034] The glycine or its oligomers may be present in the injectable liquid composition at a concentration of 1 mM or higher. In addition, glycine or its oligomers are preferably present in the injectable liquid composition at a concentration of 15 mM or higher. Specifically, the glycine or its oligomers may be present at a concentration of 15 mM to 150 mM, 17 mM to 130 mM, 20 mM to 120 mM, 22 mM to 110 mM, or 25 mM to 100 mM. Specifically, the glycine or its oligomers may be present at approximately 1 mM, approximately 5 mM, approximately 10 mM, approximately 15 mM, approximately 20 mM, approximately 25 mM, approximately 30 mM, approximately 35 mM, approximately 40 mM, approximately 45 mM, approximately 50 mM, approximately 55 mM, approximately 60 mM, approximately 65 mM, approximately 70 mM, approximately 75 mM, approximately 80 mM, approximately 85 mM, or approximately 90 mM.

[0035] In addition, the glycine can be used together with one or more amino acids selected from the group consisting of histidine, isoleucine, leucine, lysine acetate, methionine, phenylalanine, threonine, tryptophan, valine, alanine, arginine, aspartic acid, cysteine, glutamic acid, proline, serine, and tyrosine, but is not limited thereto.

[0036] In addition, the glycine can be used together with glycine monomers and glycine oligomers. In one embodiment, the glycine monomers and glycine dimers can be used together in a liquid composition for injection. In addition, glycine monomers and glycine trimers can be used together in a liquid composition for injection. In addition, glycine dimers and glycine trimers can be used together in a liquid composition for injection. In addition, glycine monomers, glycine dimers, and glycine trimers can be used together in a liquid composition for injection.

[0037] The sugar contained in the liquid composition for injection of the present invention can be one or more selected from the group consisting of sucrose, trehalose, mannitol, sorbitol, glucose, fructose, mannose, maltose, lactose, isomaltose, dextran, and dextrin, but is not limited thereto. Specifically, the sugar can be trehalose, mannitol, or sucrose. Preferably, the sugar can be trehalose.

[0038] The buffer solution contained in the liquid composition for injection of the present invention can be selected from the group consisting of Tris buffer solution, hydroxyethyl piperazine ethane sulfonic acid (HEPES) buffer solution, 3-(N-morpholine) propanesulfonic acid (MOPS) buffer solution, and buffer solutions containing acetate or phosphate, but is not limited thereto. Preferably, the buffer solution can be an injectable Tris buffer solution.

[0039] The pH value of the buffer solution can be in the range of about 6.3 to about 9.5, but is not limited thereto. In addition, the pH can be in the range of about 7.0 to about 7.8, or about 7.2 to about 7.6. Preferably, the pH can be in the range of about 7.3 to about 7.5.

[0040] In addition, the buffer solution can be present in the liquid composition for injection at a concentration of 5 mM to 50 mM, 8 mM to 40 mM, 10 mM to 35 mM, 13 mM to 30 mM, or 15 mM to 25 mM, but is not limited thereto.

[0041] The liquid composition for injection of the present invention can be administered parenterally. In this case, parenteral administration can be carried out by methods such as vascular administration, subcutaneous administration, mucosal administration, intramuscular administration, joint administration, ocular administration, etc. In one embodiment of the present invention, the composition can preferably be administered by intravenous injection.

[0042] In addition, the osmolarity of the liquid composition for injection of the present invention can be in the range of 200 mOsm to 400 mOsm, 230 mOsm to 380 mOsm, 250 mOsm to 350 mOsm, 260 mOsm to 320 mOsm, 270 mOsm to 330 mOsm or 280 mOsm to 300 mOsm.

[0043] The osmolarity within the above range contributes to long-term storage at a temperature of 2°C to 8°C or higher, while making the composition suitable for parenteral administration (e.g., intravenous, intramuscular or subcutaneous injection) without causing side effects to the subject.

[0044] As used herein, the term "osmolarity" refers to the number of moles of solute that creates the osmotic pressure in a solution per kilogram of solvent, and the osmolarity is determined by measuring the freezing point depression of a sample using an osmometer.

[0045] The subject can be a mammal, such as a human, dog, cow, horse, pig, sheep, goat, cat, mouse, rabbit and rat, but is not limited thereto, and preferably a human.

[0046] In addition, the liquid composition for injection may further contain a chelating agent. The chelating agent can be one or more selected from the group consisting of injectable grade ethylene glycol tetraacetic acid (EGTA), ethylenediaminetetraacetic acid (EDTA) and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), but is not limited thereto.

[0047] After obtaining the mitochondria contained in the liquid composition for injection, the chelating agent can remove the damage caused by ion leakage.

[0048] In the liquid composition for injection of the present invention, the mitochondria may be mitochondria obtained from eukaryotes, or may be mitochondria obtained from mammals or humans. Specifically, the mitochondria may be mitochondria isolated from cells or tissues. For example, the mitochondria may be mitochondria obtained from somatic cells, germ cells or stem cells, or may be mitochondria isolated from blood cells or platelets. In addition, the mitochondria may be used after disruption and isolation following concentration of the tissue or cells, or the mitochondria may be mitochondria isolated from a tissue or cell sample that has been thawed after cryopreservation, thawed and disrupted.

[0049] Specifically, the somatic cells may be muscle cells, liver cells, neurons, fibroblasts, epithelial cells, adipocytes, osteocytes, leukocytes, lymphocytes, platelets or mucosal cells.

[0050] In addition, the stem cells are undifferentiated cells with the potential to differentiate into various types of tissue cells, and 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, but are not limited thereto. Specifically, the mesenchymal stem cells may be obtained from any one of the group consisting of umbilical cord, cord blood, bone marrow, adipose tissue, muscle, nerve, skin, synovial fluid, testis, amnion and placenta.

[0051] In addition, the mitochondria may be mitochondria isolated from cells or tissues cultured in vitro, or may be mitochondria isolated from a sample thawed after cryopreservation.

[0052] In addition, the mitochondria may be mitochondria obtained from autologous, allogenic or xenogenic species. Specifically, autologous mitochondria refer to mitochondria obtained from tissues or cells of the same subject. In addition, allogenic mitochondria refer to mitochondria obtained from subjects belonging to the same species but having different allelic genotypes. In addition, xenogenic mitochondria refer to mitochondria obtained from subjects belonging to different species.

[0053] Meanwhile, in the case of isolating mitochondria from specific cells, for example, mitochondria can be isolated by various improved methods including various known methods, such as using a specific buffer solution or potential difference and magnetic field.

[0054] From the perspective of maintaining mitochondrial activity, mitochondrial isolation can be carried out by disrupting cells and centrifugation. In a specific embodiment, the isolation can be carried out in the following steps: culturing cells and performing a first centrifugation on the cell-containing composition to produce a pellet; resuspending the pellet in a buffer solution and homogenizing the resulting product; performing a second centrifugation on the homogenized solution to prepare a supernatant; and performing a third centrifugation on the supernatant to purify the mitochondria. Specifically, from the aspect of maintaining cell activity, it is preferable to adjust the time for performing the second centrifugation to be shorter than the times for performing the first and third centrifugations. The centrifugation speed can increase from the first centrifugation to the third centrifugation.

[0055] Specifically, the first to third centrifugations can be carried out at a temperature of 0°C to 10°C, preferably at a temperature of 3°C to 5°C. In addition, the centrifugation can be carried out for 1 to 50 minutes, and the centrifugation time can be appropriately adjusted according to the number of centrifugations, sample content, etc.

[0056] In addition, the first centrifugation can be carried out at a speed of 100×g to 1000×g, 200×g to 700×g, or 300×g to 450×g. In addition, the second centrifugation can be carried out at a speed of 1×g to 2000×g, 25×g to 1800×g, or 500×g to 1600×g. In addition, the third centrifugation can be carried out at a speed of 100×g to 20000×g, 500×g to 18000×g, or 800×g to 15000×g.

[0057] The liquid composition for injection of the present invention can be stored in a container selected from the group consisting of vials, cartridges, syringes, and autoinjectors.

[0058] In addition, the container storing the liquid composition for injection of the present invention can be stored at room temperature, a refrigerated temperature of 2°C to 8°C, or a temperature of 25°C to 40°C until the composition is administered to a subject in need of treatment.

[0059] The subject can be a mammal, such as a human, dog, cow, horse, pig, sheep, goat, cat, mouse, rabbit, or rat, but is not limited thereto, and preferably a human.

[0060] The liquid composition for injection can be administered parenterally, such as by intravenous administration, subcutaneous administration, mucosal administration, intramuscular administration, ocular administration, or intraperitoneal administration, but is not limited thereto, using an 18G to 32G needle in a volume of 5 mL or less, 3 mL or less, or 2 mL or less.

[0061] Use of the liquid composition for injection containing mitochondria

[0062] On the other hand, the present invention provides a pharmaceutical composition for preventing or treating a disease, which comprises an injectable liquid composition containing isolated mitochondria. Specifically, the use of the pharmaceutical composition can be for preventing or treating mitochondrial-related diseases.

[0063] As used herein, the term "mitochondrial-related diseases" generally refers to diseases caused by genetic, environmental, or unknown reasons resulting in deterioration of mitochondrial function or mitochondrial dysfunction. For example, these diseases include genetic diseases (such as Leber hereditary optic neuropathy (LHON), Leigh syndrome, Myoclonic epilepsy associated with ragged-red fibers (MERRF), mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke (MELAS), etc.); degenerative neurological diseases (such as Parkinson's disease and Alzheimer's disease); metabolic diseases (such as diabetes and obesity); and inflammatory diseases (such as sepsis, rheumatoid arthritis, etc.).

[0064] The pharmaceutical composition of the present invention contains isolated mitochondria as an active ingredient, and can prevent or treat any one disease selected from the group consisting of sepsis (KR 10-2019-0050017A), cancer (KR 10-12126199B1), heart disease, rheumatoid arthritis (KR102019-0094124A), ischemic diseases (KR 10~2019277B1), infectious diseases (KR 10-2018-0054523A), Parkinson's disease, Alzheimer's disease, and muscle diseases (KR 10-2019-0090754A), but is not limited thereto.

[0065] Specifically, the cancer can be gastric cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, lymphoma, etc., but is not limited thereto.

[0066] In addition, the ischemic disease can be severe limb ischemia, ischemic stroke, ischemic heart disease, ischemic colitis, etc., but is not limited thereto. Specifically, the ischemic disease can be a disease caused by mitochondrial abnormalities, which includes all ischemic cell disorders caused by deterioration of mitochondrial function.

[0067] In addition, the infectious disease may be hepatitis B, hepatitis C, human papilloma virus (HPV) infection, cytomegalovirus infection, viral respiratory disease, influenza virus infection, etc., but is not limited thereto.

[0068] In addition, the muscle disease may be MELAS syndrome, MERRF syndrome, Kearns-Sayre syndrome, myopathy, encephalomyopathy, myasthenia, myasthenia gravis, amyotrophic lateral sclerosis, muscular dystrophy, muscle atrophy, hypomyotonia, muscle weakness, muscle stiffness, etc., but is not limited thereto. Specifically, the muscle disease may include any muscle cell disorder caused by reduced mitochondrial function.

[0069] As used herein, the term "prevention" refers to any act of inhibiting the occurrence of a mitochondrial-related disease or delaying the onset of a mitochondrial-related disease by administering the pharmaceutical composition. As used herein, the term "treatment" refers to any action of improving or beneficially altering the symptoms of a mitochondrial-related disease by administering the pharmaceutical composition.

[0070] The pharmaceutical composition may comprise a therapeutically effective amount or a pharmaceutically effective amount.

[0071] As used herein, the term "therapeutically effective amount" or "pharmaceutically effective amount" refers to the amount of the composition that is effective in preventing or treating the target disease, and also refers to the amount sufficient to treat the disease with a reasonable benefit / risk ratio applicable to medical treatment and without causing side effects. The level of the effective amount can be determined according to factors including the patient's health condition, disease type, severity, drug activity, drug sensitivity, administration method, administration time, administration route, excretion rate, treatment duration, drugs used in combination or simultaneously, and other factors well known in the medical field.

[0072] In the pharmaceutical composition containing the liquid composition for injection of the present invention, the concentration of the mitochondria can be 0.1 μg / mL to 500 μg / mL, 0.2 μg / mL to 450 μg / mL or 0.5 μg / mL to 400 μg / mL, but is not limited thereto. By including mitochondria at a concentration within the above range, when the pharmaceutical composition is administered, it is easy to adjust the dose of the mitochondria, and thus the degree of improvement of symptoms of mitochondrial-related diseases (such as cancer, inflammatory diseases, neurodegenerative diseases, metabolic diseases, infectious diseases, muscle diseases, heart diseases, ischemic diseases, etc.) in patients can be improved. Specifically, the dose of mitochondria can be quantified by quantifying the membrane proteins of the isolated mitochondria from blood, cells, tissues, etc. Specifically, the isolated mitochondria can be quantified by the Bradford protein assay (James D. McCully, J. Vis. Exp. 2014;(91):51682).

[0073] Specifically, as the pharmaceutical composition of the present invention, the mitochondria can be administered once in an amount of 0.01 mg / kg to 5 mg / kg, 0.1 mg / kg to 4 mg / kg or 0.25 mg / kg to 2.5 mg / kg based on the body weight of the subject, but is not limited thereto. That is, from the aspect of cell activity, most preferably, as the pharmaceutical composition, the mitochondria are administered in the above range based on the body weight of the subject suffering from mitochondrial-related diseases.

[0074] In addition, the pharmaceutical composition can be administered 1 to 10 times, 3 to 8 times or 5 to 6 times, preferably 5 times. Specifically, the dosing interval can be 1 to 7 days or 2 to 5 days, preferably at an interval of 3 days.

[0075] As used herein, the term "administer" refers to the process of delivering a specific material into a subject by any suitable method, as long as the composition can reach the target tissue, and the administration route of the composition can be administered by any general route. Preferably, it can be parenteral administration, such as intraperitoneal administration, intravascular administration, intramuscular administration, mucosal administration, subcutaneous administration, intradermal administration and ocular administration, etc., but is not limited thereto.

[0076] In addition, the pharmaceutical composition of the present invention can be administered to humans or other mammals who may be suffering from mitochondrial-related diseases or are suffering from such diseases.

[0077] Therefore, the pharmaceutical composition of the present invention can be prepared into a physically and chemically very stable injection by adjusting the pH using a buffer solution available for injections, so as to ensure the stability of the product according to the dispensing of the injection prescription.

[0078] Specifically, the pharmaceutical composition of the present invention may contain water for injection. The water for injection is distilled water, which is prepared for dissolving solid injections or diluting water-soluble injections, and it can be glucose injection, xylitol injection, D-mannitol injection, fructose injection, physiological saline, dextran 40 injection, dextran 70 injection, amino acid injection, Ringer's solution, lactated Ringer's solution, or Tris, HEPES, citric acid, phosphate buffer, sodium dihydrogen phosphate-citric acid buffer, etc. with a pH value ranging from 3.5 to 8.

[0079] In addition, the pharmaceutical composition of the present invention may contain stabilizers or solubilizers. For example, the stabilizer can be pyrosulfite or ethylene diamineteteaacetic acid, and the solubilizer can be hydrochloric acid, acetic acid, potassium hydroxide, potassium bicarbonate, potassium carbonate, or Tris.

[0080] In a specific embodiment, the pharmaceutical composition may contain a mixed preservation solution (e.g., Trehalose-Tris-Glycine (TTG) solution), and can be a commonly used pharmaceutically acceptable pharmaceutical preparation. As additives to the pharmaceutical composition, antioxidants, ATP, and magnesium, etc. that can effectively maintain and activate mitochondrial function can be contained.

[0081] On the other hand, the present invention provides the use of the pharmaceutical composition for preventing or treating mitochondrial-related diseases. Then, the pharmaceutical composition can be the liquid composition for injection. In this regard, the liquid composition for injection, mitochondrial-related diseases, prevention, and treatment are as described above.

[0082] On the other hand, the present invention provides a method for preventing or treating a disease, including administering the above-mentioned pharmaceutical composition to a subject.

[0083] Then, the liquid composition for injection, administration, dosage, prevention, and treatment are as described above. The subject can be a patient suffering from a certain disease or an individual who may suffer from the disease. The subject can be a mammal, preferably a human.

[0084] In addition, the liquid composition for injection can be an injection that can be administered through blood vessels or an injection that can be administered locally.

[0085] The disease is a mitochondrial-related disease, and the specific disease is as described above. Specifically, the administration can be carried out by intravenous, intramuscular or intradermal administration. By doing so, the pharmaceutical composition of the present invention can directly provide isolated mitochondria with normal activity to the lesion where the disease occurs, thereby can be used to increase the activity of cells with deteriorated mitochondrial function or to regenerate cells with mitochondrial dysfunction, and can be used for the prevention or treatment of the above-mentioned mitochondrial-related diseases.

[0086] On the other hand, the present invention provides the use of the injectable liquid composition in the preparation of a drug for the prevention or treatment of mitochondrial-related diseases. In this regard, the pharmaceutical composition, mitochondrial-related diseases, prevention and treatment are as described above.

[0087] Embodiment of the invention

[0088] Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are for illustrative purposes of the present invention, and the scope of the present invention should not be limited to these examples.

[0089] Preparation Example 1. Cell Culture

[0090] Human umbilical cord-derived mesenchymal stem cells (UC-hMSC) and human bone marrow-derived mesenchymal stem cells (BM-hMSC) were respectively inoculated into alpha-minimum essential medium (α-MEM, Gibco) containing 10% fetal bovine serum (FBS, Gibco), 100 μg / mL streptomycin and 100 U / mL penicillin, and cultured for 72 hours.

[0091] A Chinese hamster ovary-derived cell line (i.e., CHO cells) was inoculated in Dulbecco's modified eagle medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, Gibco), and cultured for 72 hours.

[0092] L6 cells (American Type Culture Collection, ATCC, CRL-1458), a myoblast cell line derived from rat skeletal muscle, were inoculated into Dulbecco's modified eagle's medium-high glucose (DMEM-high glucose) containing 10% fetal bovine serum (FBS, Gibco), and cultured for 72 hours.

[0093] After the respective culturing of the cells, each cell was washed twice with Dulbecco's phosphate buffered saline (DPBS, Gibco). Thereafter, the cells were obtained by treating with 0.25% trypsin-EDTA (TE). The obtained cells were resuspended to 1×10 7 cells / mL for mitochondrial extraction.

[0094] Preparation Example 2. Obtaining platelets

[0095] Preparation Example 2.1 Isolating platelets from porcine whole blood

[0096] The porcine whole blood was centrifuged at 300×g for 5 minutes to obtain a turbid yellow supernatant containing platelets and plasma. The obtained supernatant was centrifuged at 1500×g for 15 minutes to obtain platelet pellets (granules). The obtained platelet pellets were replaced with an equal volume of Dulbecco's phosphate buffered saline (DPBS) solution, centrifuged at 1500×g for 15 minutes for washing, and then resuspended in an equal volume of fresh DPBS solution.

[0097] Preparation Example 2.2 Isolating platelets from human whole blood

[0098] The human whole blood containing K2-EDTA (eipotassium ethylenediaminetetraacetic acid) was centrifuged at 300×g for 3 minutes to obtain a yellow supernatant. The obtained supernatant was centrifuged at 1500×g for 15 minutes to obtain platelet pellets (granules). The obtained platelet pellets were resuspended in Dulbecco's phosphate-buffered saline (DPBS) solution.

[0099] Example 1. Isolation and storage of mitochondria

[0100] Example 1.1 Isolation of mitochondria

[0101] The corresponding culture containing the cells cultured in Preparation Example 1 was centrifuged at 380×g for 3 minutes and the supernatant was removed. The recovered cells were mixed with DPBS solution and centrifuged at 1100×g for 3 minutes. Specifically, all subsequent processes were carried out under refrigerated temperature conditions, including the solutions used.

[0102] After removing the supernatant and adding the homogenization buffer, the cells were resuspended, transferred to a fresh tube, and disrupted and homogenized physically using a 1 mL syringe. The resulting supernatant was recovered by centrifuging the homogenate obtained by disrupting the cells at 2000×g for 10 minutes at a temperature of about 4 °C. The recovered supernatant was centrifuged again at 2000×g. The supernatant obtained after centrifugation was centrifuged at 12000×g for 10 minutes to recover the mitochondrial pellet.

[0103] The solution in Table 1 was used as the storage buffer for mitochondria, added to the recovered mitochondrial pellet, centrifuged at 12000×g for 10 minutes, and the supernatant was removed. The supernatant was removed without losing the precipitated mitochondria, and the storage buffer for mitochondria was re-added, and then the mixture was centrifuged again at 12000×g to completely remove the supernatant. After completely removing the supernatant, the components for mitochondrial storage in Table 1 below were added to the obtained precipitated mitochondria and resuspended.

[0104] [Table 1]

[0105]

[0106] Example 1.2 Isolation of Mitochondria Using a Stable Preparation

[0107] Mitochondria were isolated from cells using a SHE solution (250 mM sucrose, 20 mM HEPES (pH 7.4), and 2 mM EGTA). Specifically, buffer solutions #1 to #6 (composed of sugar (sucrose, mannitol, or trehalose), buffer (HEPES or Tris), chelating agent (EGTA or EDTA), and amino acid) shown in Table 1 were added to the animal and human cells cultured or being cultured in Preparation Example 1 above, and the cells were resuspended. Then, after disrupting the cells in the same manner as in Example 1.1, mitochondria were obtained from the cells.

[0108] Example 2 Observation of Self-Aggregation of Isolated Mitochondria

[0109] Example 2.1 Staining and Storage of Mitochondria

[0110] The concentration of mitochondria was determined by taking a portion of the isolated mitochondrial solution and measuring the protein concentration using the bicinchoninic acid (BCA) assay. 50 μg of the isolated mitochondria were stained with 100 nM mitochondrial-specific green label MitoTracker Green (Molecular Prove, Eugene, OR) at 4 °C for 10 minutes. 50 μg of mitochondria were treated per 1 mL of each solution, and each resulting product was stored in a confocal dish at 4 °C in solution state. After 16 hours, the degree of self-aggregation of mitochondria was observed in the confocal dish using a confocal laser scanning microscope (Carl Zeiss, Germany). Specifically, the isolated mitochondria were stored in normal saline or SHE solution for testing.

[0111] Example 2.2 Confirmation of Isolated Mitochondrial Aggregation

[0112] To confirm the phenomenon of mitochondrial self-aggregation induced in the storage solution of mitochondria isolated from cells, after isolating mitochondria from human umbilical cord-derived mesenchymal stem cells (UC-hMSC) and Chinese hamster ovary-derived cells (CHO), the isolated mitochondria were stored in SHE storage solution (250 mM sucrose, 20 mM HEPES (pH 7.4), and 2 mM EGTA) and NaCl solution (normal saline, saline), respectively. After 16 hours of storage, the aggregation of the isolated mitochondria themselves was confirmed using a confocal laser scanning microscope.

[0113] The results are as Figure 1 shown. Mitochondrial aggregation formed in the saline NaCl solution (normal saline) used as the injection solution. Therefore, for mitochondria, since mitochondrial aggregation occurs when saline containing a high concentration of sodium salt is used as the injection solution, it was found that a stable storage buffer that does not cause aggregation should be used.

[0114] Example 3. Observation of Mitochondria-Induced Platelet Aggregation

[0115] To observe platelet aggregation induced by mitochondria isolated from cells, mitochondria were isolated from human umbilical cord-derived mesenchymal stem cells (UC-hMSC), human bone marrow-derived mesenchymal stem cells (BM-hMSC), rat skeletal muscle-derived cell line (L6), and platelet cells, and stained with mitochondrial-specific deep red label MitoTracker DeepRed.

[0116] Specifically, the platelets resuspended in DPBS solution in Preparation Example 2 were stained with 0.5 μM mitochondrial-specific green label MitoTracker Green at room temperature for 10 minutes, and then washed with DPBS solution.

[0117] After treating 5 μg of the stained mitochondria with 1 mL of platelet solution (10 7 / mL), the resulting mixture was cultured with shaking at 37 °C and 100 rpm for 1 hour. After dispersing 3 μL of the mitochondria-treated platelets on a slide glass, they were covered with a cover glass and observed using a confocal laser scanning microscope (Carl Zeiss, Germany) ( Figure 2a ).

[0118] As a result, all mitochondria isolated from platelets, rat skeletal muscle-derived cell line (L6), human umbilical cord-derived mesenchymal stem cells (UC-hMSC), and human bone marrow-derived mesenchymal stem cells (BM-hMSC) could induce platelet aggregation. It was thus found that when mitochondria are injected into blood vessels, there is an inherent risk of inducing platelet aggregation, thereby causing a decrease in platelet count or blood clot formation. Specifically, the isolated mitochondria were stored in physiological saline for experiments.

[0119] Example 4. Confirmation of the inhibitory effect of mitochondrial storage solvents and anticoagulants on platelet aggregation

[0120] To confirm the inhibitory effect of mitochondrial storage solvents and anticoagulants on platelet aggregation, mitochondria of human umbilical cord-derived mesenchymal stem cells (UC-hMSC) were isolated and stored in physiological saline and SHE solution as storage solvents for 1 hour respectively. The resulting mixtures were treated with platelets isolated from porcine whole blood and then with heparin. After 1 hour, the results were confirmed using a confocal laser scanning microscope.

[0121] As Figure 2b shown, mitochondria derived from human umbilical cord-derived mesenchymal stem cells (UC-hMSC) and stored in physiological saline and SHE solution increased platelet aggregation, and when treated with heparin, the aggregation was partially inhibited. In addition, as Figure 3 shown, it was found that when platelets were treated with mitochondria of human umbilical cord-derived mesenchymal stem cells (UC-hMSC) and stored in SHE solution, as the concentration of heparin as an anticoagulant increased, the platelet aggregation induced by mitochondria was inhibited.

[0122] Example 5. Screening of mitochondrial injection preparation buffers

[0123] When human umbilical cord-derived mesenchymal stem cell (UC-hMSC)-isolated mitochondria were administered once by intravenous injection (50 μg / 0.3 mL) to BALB / c-nu / nu mice, changes in the number of platelets in the blood were compared and evaluated according to the components of the injection solution and the presence / absence of anticoagulants. To confirm the effect of intravascular injection of mitochondria in mice on reducing the number of platelets, the specific experimental design is shown in Table 2 below.

[0124] [Table 2]

[0125] Experimental group Buffer solution applied Remarks PBS Normal saline Excipient control UC-PBS Normal saline UC mitochondria-PBS UC-PBS + heparin Normal saline UC mitochondria–PBS + heparin (10 IU / head) UC-TTB Tris-trehalose UC mitochondria-TTB UC-TTB + heparin Tris-trehalose UC mitochondria-TTB + heparin UC-SHE HEPES-sucrose-EDTA UC mitochondria-SHE

[0126] It was confirmed that when normal saline was used as the component of the injection solution, the number of platelets decreased the most, while the decrease in the number of platelets was less in the injection solution containing sucrose or trehalose. In addition, when heparin was added, the decrease in platelets was restored to some extent, but the decrease in platelets was not completely inhibited ( Figure 4 ).

[0127] Example 6. Effect of glycine concentration on mitochondrial activity

[0128] Among the components for stabilizing mitochondria described in Example 1, an attempt was made to determine the effect of glycine concentration on mitochondrial activity.

[0129] First, mitochondria were isolated by the isolation method described in Example 1 above, and the isolated mitochondria were stained with tetramethylrhodamine ethyl ester (TMRE) as a mitochondrial membrane potential marker and then washed. After washing, the obtained mitochondria were stored in each solution, where the concentration conditions of Solution 2 in Example 1 were set in the same manner, and Tris (20 mM) and trehalose (195 mM), while the glycine concentration was adjusted differently in the range of 0 mM to 150 mM. After storing for 24 hours, the change in fluorescence value was measured ( Figure 5 ).

[0130] Example 7. Inhibitory effect of glycine on platelet aggregation

[0131] To confirm the inhibitory effect of glycine as an amino acid on platelet aggregation in mitochondria, mitochondria were first isolated from human umbilical cord-derived mesenchymal stem cells (UC-hMSC) and then stained with the mitochondrial-specific deep red marker MitoTracker DeepRed. Thereafter, 1 μg of the stained mitochondria was suspended in a composition solution containing 10 μL of Tris-trehalose buffer (TTB) with glycine concentrations of 0 mM, 1 mM, 5 mM, 10 mM, 25 mM, 50 mM, and 100 mM, refrigerated for 1 hour, and treated with 1 mL of platelet solution (10 7 / mL). After treatment, the resulting product was cultured with shaking at 100 rpm at 37 °C for 1 hour. 3 μL of mitochondria-treated platelets were observed using a confocal laser scanning microscope (Carl Zeiss, Germany) ( Figure 6 ).

[0132] As a result, it was found that mitochondria could not inhibit platelet aggregation at low glycine concentrations, but in the experimental groups with a glycine concentration of 25 mM or higher, aggregation was gradually inhibited. From the above results, it can be inferred that glycine can effectively inhibit mitochondria-induced platelet aggregation.

[0133] Example 8. Effect of a mitochondrial composition preparation containing glycine on platelet aggregation

[0134] To confirm the inhibitory effect of a mitochondrial composition preparation containing glycine as an amino acid on platelet aggregation, mitochondria were first isolated from human umbilical cord-derived mesenchymal stem cells (UC-hMSC) and then stained with the mitochondrial-specific deep red marker MitoTracker DeepRed. Then, 1 μg of the stained mitochondria was suspended in 10 μL of Tris buffer ( Figure 7 the specific compositions of 1, 2, 3, 4, and 5 are shown), and treated with 1 mL of platelet solution (10 7 / mL). After treatment, the resulting product was cultured with shaking at 100 rpm at 37 °C for 1 hour. 3 μL of mitochondria-treated platelets were observed using a confocal laser scanning microscope (Carl Zeiss, Germany) ( Figure 7 ).

[0135] The results confirmed that there were differences in the types of sugars (sucrose, trehalose, and mannitol) in the main components for mitochondrial stability even in the absence of platelet aggregation, and for all three types of sugars, no platelet aggregation was observed in the compositions with glycine added. However, in the compositions without glycine added, mitochondria induced platelet aggregation.

[0136] Example 9. Effect of glycine on human platelet aggregation

[0137] To observe platelet aggregation induced by mitochondria isolated from cells, after isolating mitochondria from human umbilical cord-derived mesenchymal stem cells (UC-hMSCs), they were stained with the mitochondrial-specific deep red marker MitoTracker DeepRed. Specifically, platelets resuspended in DPBS solution in Preparation Example 2 were stained with 0.5 μM of the mitochondrial-specific green marker MitoTracker Green at room temperature for 10 minutes and washed with DPBS solution.

[0138] 1 μg of the stained mitochondria was suspended in 10 μL of Tris-trehalose-glycine (TTG) buffer, Tris-trehalose-glycine-glycine (TTG-G) buffer, or Tris-trehalose-glycine-glycine-glycine (TTG-G-G) buffer, refrigerated for 1 hour, and then treated with 1 mL of platelet solution (10 7 / mL).

[0139] After treatment, the resulting product was cultured with shaking at 100 rpm at 37 °C for 1 hour. After dispersing 3 μL of the mitochondria-treated platelets on a glass slide, they were covered with a coverslip and observed using a confocal laser scanning microscope (Carl Zeiss, Germany) ( Figure 8 ).

[0140] The results confirmed that no platelet aggregation was observed in glycine dimer, glycine trimer, and glycine monomer. Therefore, it was confirmed that glycine oligomers are the main components for mitochondrial stabilization without platelet aggregation.

[0141] Although the injectable composition containing isolated mitochondria and its uses have been described with reference to specific embodiments, it is not limited thereto. Thus, those skilled in the art will readily understand that various modifications and changes can be made thereto without departing from the spirit and scope of the present invention defined by the appended claims.

Claims

1. A liquid composition for injection, which comprises glycine or its oligomer, sugar, buffer and mitochondria; wherein the glycine oligomer is a glycine dimer or a glycine trimer; wherein the concentration of the glycine or its oligomer is 25 mM - 150 mM; wherein the sugar is selected from one or more of the group consisting of sucrose, trehalose and mannitol.

2. The liquid composition for injection according to claim 1, wherein the buffer is selected from the group consisting of Tris buffer, HEPES (hydroxyethyl piperazine ethane sulfonic acid) buffer, MOPS (3-(N-morpholino)propanesulfonic acid) buffer and buffers containing acetate or phosphate.

3. The liquid composition for injection according to claim 2, wherein the buffer is Tris buffer.

4. The liquid composition for injection according to claim 1, wherein the pH of the buffer is 7.0 to 7.

8.

5. The liquid composition for injection according to claim 1, wherein the concentration of the buffer is 5 mM to 50 mM.

6. The liquid composition for injection according to claim 1, wherein the composition has an osmolarity of 200 mOsm to 400 mOsm.

7. The liquid composition for injection according to claim 1, wherein the mitochondria are isolated from eukaryotic cells or tissues.

8. The liquid composition for injection according to claim 7, wherein the cell is any one selected from the group consisting of somatic cells and germ cells of mammals.

9. The liquid composition for injection according to claim 8, wherein the somatic cell is any one selected from the group consisting of stem cells and blood cells.

10. The liquid composition for injection according to claim 9, wherein the blood cell is a platelet.

11. A pharmaceutical composition for preventing or treating mitochondrial dysfunction diseases, which comprises the liquid composition for injection according to any one of claims 1 to 10.

12. The pharmaceutical composition according to claim 11, wherein the disease is a mitochondrial dysfunction disease, which is selected from the group consisting of chronic inflammatory diseases, acute inflammatory diseases, ischemic diseases, neurological diseases, heart diseases, muscle diseases, degenerative diseases, metabolic diseases, fibrotic diseases, joint diseases, eye diseases, hair loss and immune-related diseases.

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