Pharmaceutical composition for preventing or treating hearing loss or tinnitus, comprising mitochondria as active ingredients

Abstract

A pharmaceutical composition for preventing or treating hearing loss or tinnitus, comprising mitochondria as an active ingredient, is provided. The pharmaceutical composition has the effect of preventing apoptosis of inner ear cells due to damage and inhibiting hearing loss, and therefore can effectively reduce or treat hearing loss or tinnitus.

Description

[Technical field]

[0001]

[0001] The present disclosure relates to a pharmaceutical composition for preventing or treating hearing loss or tinnitus, comprising mitochondria as an active ingredient. [Background technology]

[0002]

[0002] The pinna and ear canal are classified as the outer ear, the eardrum and ossicles as the middle ear, and the cochlea and auditory nerve as the inner ear. Sound is acoustic energy that is conducted through the pinna and ear canal to vibrate the eardrum, which generates mechanical energy that is conducted to the ossicles. The stapes, the last bone of the eardrum, is connected to the cochlea and transfers the conducted energy to the lymph fluid in the cochlea. The energy transferred to the lymph fluid causes lymph fluid waves, which stimulate the hair cells of the cochlea. The movement of the hair cells causes ionic changes, so that neurotransmitters are transferred to the auditory nerve attached to the hair cells, and the sound energy is transferred from the auditory nerve to the brain in the form of electrical energy.

[0003]

[0003] When the external and middle ears, which are the organs that conduct sound, are affected by diseases such as inflammation, in most cases they can be restored through treatment or surgery, and the resulting hearing impairment can be restored through treatment. This type of hearing loss is called conductive hearing loss. On the other hand, sensorineural hearing loss refers to hearing loss caused by problems with the cochlea, which is the organ that detects sound, the auditory nerve, which conducts sound in the form of electrical energy, and the brain, which plays a comprehensive role in identifying and understanding sound.

[0004]

[0004] Among the body's sensory organs, the hearing organ is the most basic sense for communication, which is one of the most important senses for acquiring language and knowledge and for living a human life in society. Most hearing loss belongs to sensorineural hearing loss, which has no treatment other than prevention to date. Once hearing loss occurs, auxiliary means such as hearing aids are used or mechanical devices are implanted. Furthermore, hearing loss can be accompanied by tinnitus.

[0005]

[0005] Korean Patent Application No. 10-2008-0044810 discloses that an extract of Scutellaria baicalensis has a significant effect on preventing hearing damage in an animal model of noise-induced hearing loss. In addition, antioxidants, NMDA (N-methyl-D-aspartic acid) antagonists, apoptosis inhibitors, growth factors, etc., have been studied as effective substances for preventing or treating hearing loss. However, these substances have shown limitations in progressing to clinical trials.

[0006]

[0006] Meanwhile, mitochondria are organelles essential for the survival of eukaryotic cells, involved in the synthesis and control of adenosine triphosphate (ATP) as an energy source. Mitochondria are important organelles involved in various metabolic pathways in vivo, such as cell signaling, cell differentiation and cell death, as well as the control of cell cycle and cell proliferation. There is no study reporting that a pharmaceutical composition containing mitochondria as an active ingredient may be related to the treatment of hearing loss or tinnitus. Summary of the Invention [Problem to be solved by the invention]

[0007]

[0007] In order to solve the above-mentioned problems, the present inventors have developed a method for fundamentally treating hearing loss by developing a composition for treating hearing loss, which comprises isolated mitochondria as an active ingredient for treating hearing loss or tinnitus. [Means for solving the problem]

[0008]

[0008] In one aspect, the present disclosure provides a pharmaceutical composition for preventing or treating hearing loss or tinnitus, comprising isolated mitochondria as an active ingredient. Effect of the Invention

[0009]

[0009] A pharmaceutical composition for preventing or treating hearing loss or tinnitus, which contains mitochondria as an active ingredient, has the effect of suppressing inner ear cell death caused by damage and, as a result, the effect of suppressing hearing impairment, and by these effects effectively alleviates or treats hearing loss or tinnitus. [Brief description of the drawings]

[0010] [Figure 1]

[0010] FIG. 1 is a graph showing changes in viability of mouse inner ear cells following treatment with antibiotics (gentamicin and kanamycin), anticancer drug (cisplatin) or hydrogen peroxide. [Diagram 2]

[0011] 1 is a graph showing changes in viability of mouse inner ear cells damaged by an antibiotic (gentamicin) following treatment with mitochondria. [Diagram 3]

[0012] 1 is a graph showing the change in viability of mouse inner ear cells damaged by an antibiotic (kanamycin) following treatment with mitochondria. [Figure 4]

[0013] FIG. 1 is a graph showing changes in reactive oxygen species generated from antibiotic (gentamicin) damaged mouse inner ear cells following treatment with mitochondria. [Diagram 5]

[0014] FIG. 1 is a graph showing changes in reactive oxygen species generated from antibiotic (kanamycin) damaged mouse inner ear cells following treatment with mitochondria. [Figure 6]

[0015] 1 is a graph showing the degree of hearing recovery in a noise-induced hearing loss model after treatment with mitochondria. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011]

[0016] Therapeutic agent for treating hearing loss comprising isolated mitochondria as an active ingredient

[0017] In one aspect, the present disclosure provides a pharmaceutical composition for preventing or treating hearing loss or tinnitus, comprising isolated mitochondria as an active ingredient.

[0012]

[0018] As used herein, the term "mitochondria" is a double-membrane-bound organelle found in most eukaryotic organisms that produces the majority of intracellular adenosine triphosphate (ATP).

[0013]

[0019] As used herein, the term "isolated mitochondria" refers to mitochondria obtained from autologous, allogeneic or xenogeneic sources.

[0014]

[0020] As used herein, the term "autologous mitochondria" refers to mitochondria obtained from plasma, tissue, bone marrow, or cells of the same subject. Additionally, the term "allogeneic mitochondria" refers to mitochondria obtained from plasma, tissue, bone marrow, platelets, or cells of a subject belonging to the same species but having a different genotype for an allele. Additionally, the term "xenogeneic mitochondria" refers to mitochondria obtained from plasma, tissue, bone marrow, or cells of a subject belonging to a different species.

[0015]

[0021] The subject may be a mammal, preferably a human.

[0016]

[0022] The mitochondria may be isolated from the cells of a subject. The mitochondria may be obtained from autologous or heterologous cells cultured in vitro. The cells may have normal biological activity.

[0017]

[0023] As used herein, the term "cell" refers to a structural or functional unit of a living organism that consists of a cytoplasm surrounded by a cell membrane and that contains biomolecules such as proteins and nucleic acids. The cell may refer to a cell that contains mitochondria inside the cell membrane.

[0018]

[0024] Mitochondria may also be used by concentrating and disrupting tissue or cells and then isolating the mitochondria, or mitochondria may be isolated from tissue or cell samples that have been cryopreserved and then thawed and disrupted.

[0019]

[0025] The mitochondria may be intact, disrupted, or a combination thereof, in one embodiment, even if the mitochondria are in a disrupted form, they may still exhibit a pharmacological effect as long as they retain mitochondrial activity.

[0020]

[0026] In one embodiment, the cell may be any one selected from the group consisting of stem cells, somatic cells, germ cells, and platelets.

[0021]

[0027] As used herein, the term "stem cell" refers to an undifferentiated cell that has the ability to differentiate into various types of tissue cells. The stem cell 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, and tissue-derived stem cells.

[0022]

[0028] The mesenchymal stem cells may be derived from any one selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta. Preferably, the mesenchymal stem cells may be derived from human umbilical cord.

[0023]

[0029] As used herein, the term "somatic cells" refers to cells that constitute an organism, excluding germ cells. The somatic cells may be any one selected from the group consisting of muscle cells, hepatocytes, fibroblasts, epithelial cells, nerve cells, adipocytes, osteocytes, periosteal cells, leukocytes, lymphocytes, and mucosal cells. Preferably, the somatic cells may be obtained from muscle cells or hepatocytes with good mitochondrial activity. Furthermore, the somatic cells may be obtained from autologous or heterologous blood PBMC cells.

[0024]

[0030] As used herein, the term "germ cells" refers to cells that form a zygote during reproduction in organisms that undergo sexual reproduction. Mitochondria can be obtained from autologous or heterologous germ cells. The germ cells can be sperm or eggs.

[0025]

[0031] As used herein, the term "platelet" refers to the solid components in blood that play a key role in blood clotting by assembling fibrin to form clots. Mitochondria can be obtained from autologous or heterologous platelets.

[0026]

[0032] Furthermore, isolated mitochondria can have normal biological activity. In particular, as used herein, mitochondria with normal biological activity can have one or more characteristics from the group consisting of: (i) having membrane potential, (ii) producing mitochondrial ATP, and (iii) removing ROS or reducing the activity of ROS in mitochondria.

[0027]

[0033] In one embodiment, a pharmaceutical composition of the present disclosure comprising isolated mitochondria as an active ingredient has been shown to prevent inner ear cell death when administered to a subject, and thus effectively alleviates or treats hearing loss or tinnitus.

[0028]

[0034] As used herein, the term "tinnitus" refers to a disorder of auditory perception without an actual auditory stimulus. The perceived sound may be a clicking, buzzing, hissing or roaring sound. Tinnitus is commonly associated with hearing loss.

[0029]

[0035] Tinnitus can be caused by hearing loss, otitis media, heart disease, vascular disease, Meniere's disease, brain tumors, acoustic neuroma, migraines, temporomandibular joint disorders, exposure to drugs, head injury, earwax, stress, or depression. Additionally, tinnitus can be associated with hearing loss.

[0030]

[0036] As used herein, the term "hearing loss" refers to a condition characterized by reduced hearing or hearing impairment. Hearing loss may be conductive or sensorineural.

[0031]

[0037] As used herein, the term "conductive hearing loss" refers to hearing loss that occurs when sound waves are not conducted normally due to damage to organs such as the outer ear, eardrum, and middle ear.

[0032]

[0038] As used herein, the term "sensorineural hearing loss" refers to hearing loss caused by abnormalities in the function of the cochlea or abnormalities in the auditory nerve or central nervous system, which conducts auditory stimuli to the brain.

[0033]

[0039] The sensorineural hearing loss may be congenital hearing loss, noise-induced hearing loss, sudden sensorineural hearing loss, infection-induced hearing loss, traumatic hearing loss, presbycusis, ototoxic hearing loss, autoimmune hearing loss, Meniere's disease, or hearing loss due to nerve damage.

[0034]

[0040] As used herein, the term "noise-induced hearing loss" refers to hearing loss caused by damage to the cochlea from external noise.

[0035]

[0041] As used herein, the term "sudden sensorineural hearing loss" refers to a hearing loss of about 30 dB or greater across at least three consecutive hearing test frequencies occurring within about a three day period.

[0036]

[0042] As used herein, the term "infection-induced hearing loss" refers to hearing loss caused by viral infections, such as rubella virus and congenital cytomegalovirus (CMV) infection.

[0037]

[0043] As used herein, the term "traumatic hearing loss" refers to hearing loss resulting from a perilymphatic fistula or pneumolabyrinthine caused by trauma.

[0038]

[0044] As used herein, the term "presbycusis" refers to hearing loss caused by degenerative changes due to aging.

[0039]

[0045] As used herein, the term "autoimmune hearing loss" refers to hearing loss caused by an autoimmune disease.

[0040]

[0046] As used herein, the term "Meniere's disease" refers to a disease characterized by the co-occurrence of symptoms such as spatial disorientation, hearing loss, tinnitus, and a feeling of fullness in the ears.

[0041]

[0047] As used herein, the term "nerve damage-induced hearing loss" refers to hearing loss caused by abnormalities in any of the neural areas ranging from the auditory cells of the cochlea to the areas of the brain that control hearing.

[0042]

[0048] As used herein, the term "ototoxic hearing loss" refers to hearing loss caused by a decrease in inner ear function due to drugs, chemicals, or radiation. In one embodiment, ototoxic hearing loss can be caused by the use of anti-cancer drugs, antibiotics, or radiation. In particular, ototoxic hearing loss can be caused by an increase in the concentration of ROS in inner ear cells due to the use of anti-cancer drugs, antibiotics, or radiation, which causes damage to the inner ear.

[0043]

[0049] As used herein, the term "anti-cancer agent" refers to a drug that inhibits the growth of cancer cells and causes necrosis of cancer cells. Anti-cancer agents can cause neurotoxicity.

[0044]

[0050] The anticancer agent may be one or more selected from the group consisting of taxane anticancer agents, platinum anticancer agents, and vinca alkaloid anticancer agents. Preferably, the anticancer agent may be a platinum anticancer agent.

[0045]

[0051] The taxane anticancer drug may be one or more selected from the group consisting of docetaxel, oraxol, paclitaxel, ditaxel, taxol, and taxotere.

[0046]

[0052] The vinca alkaloid anticancer agent may be one or more selected from the group consisting of vinblastine, vincristine, vindesine, and vinorelbine.

[0047]

[0053] The platinum-based anticancer drug may be one or more selected from the group consisting of cisplatin, carboplatin, and oxaliplatin.

[0048]

[0054] As used herein, the term "antibiotic" refers to a drug that prevents the growth of or kills other microorganisms. Antibiotics may kill or inhibit the growth of bacteria through one or more mechanisms selected from the group consisting of inhibition of cell wall synthesis, alteration of cell wall permeability, inhibition of protein synthesis, inhibition of nucleic acid synthesis, and inhibition of folate synthesis.

[0049]

[0055] The antibiotic may be one or more selected from the group consisting of β-lactams, sulfonamides, aminoglycosides, tetracyclines, glycopeptides, ansamycins, penicillins, macrolides, streptogramins, cephalosporins, and quinolones. Preferably, the antibiotic is an aminoglycoside.

[0050]

[0056] In one embodiment, the antibiotic may be one or more selected from the group consisting of streptomycin, neomycin, kanamycin, gentamicin, tobramycin, amikacin, netilmicin, dibekacin, sisomicin, livodomycin, and paromomycin.

[0051]

[0057] In one embodiment, the pharmaceutical composition of the present disclosure comprising isolated mitochondria as active ingredients can reduce the concentration of ROS increased by the use of anti-cancer drugs, antibiotics or radiation, preventing damage to the inner ear, thereby preventing or treating ototoxic hearing loss or tinnitus.

[0052]

[0058] As used herein, the term "treatment" can be used to encompass both therapeutic and prophylactic treatment. Prophylaxis can be used to mean alleviating or reducing a condition or disease in a subject.

[0053]

[0059] As used herein, the term "active ingredient" refers to an ingredient that exhibits activity alone or in combination with an auxiliary agent (carrier) that does not have activity by itself. In one embodiment, isolated mitochondria as an active ingredient can prevent inner ear cell death, increase in ROS, or hearing loss due to damage.

[0054]

[0060] Isolation of mitochondria from specific cells can be performed using various known methods such as using specific buffers or using an electric potential or a magnetic field, etc. Additionally, isolation of mitochondria can include centrifuging and filtering the plasma to remove all cellular components and centrifuging the filtered plasma.

[0055]

[0061] The isolation of mitochondria can be performed by disrupting and centrifuging tissue or cells in order to maintain mitochondrial activity. In one embodiment, the isolation of mitochondria can be performed by culturing cells, performing a first centrifugation of the composition containing the cells to produce a pellet, resuspending the pellet in a buffer and homogenizing it, performing a second centrifugation of the homogenized solution to produce a supernatant, and performing a third centrifugation of the supernatant to purify mitochondria. In order to maintain cell activity, the time for performing the second centrifugation is adjusted to be shorter than the time for performing the first and third centrifugations. The speed can be increased from the first centrifugation to the second centrifugation and the third centrifugation.

[0056]

[0062] Specifically, the first to third centrifugations may be carried out at a temperature of about 0° C. to about 10° C., preferably about 3° C. to about 5° C. The time for which the centrifugation is carried out may be about 1 to 50 minutes, and may be appropriately adjusted depending on the number of centrifugations and the contents of the sample.

[0057]

[0063] The first centrifugation may be performed at a speed of about 100×g to about 1,000×g, about 200×g to about 700×g, or about 300×g to about 450×g. The second centrifugation may be performed at a speed of about 1×g to about 2,000×g, about 25×g to about 1,800×g, or about 500×g to about 1,600×g. The third centrifugation may be performed at a speed of about 100×g to about 20,000×g, about 500×g to about 18,000×g, or about 800×g to about 15,000×g.

[0058]

[0064] Also, pharmaceutically acceptable sugars can be used to stabilize the obtained mitochondria. In particular, pharmaceutically acceptable sugars can be, but are not limited to, sucrose, mannitol, trehalose, etc. Also, pharmaceutically acceptable Tris, HEPES, phosphate, etc. can be used as buffers, but are not limited to these.

[0059]

[0065] On the other hand, after obtaining mitochondria, additives such as chelating agents and antioxidants can be used to remove the damage caused by ion leakage and suppress oxidative stress.Additives can include, but are not limited to, any reagents widely known in the art.For example, additives can be, but are not limited to, EDTA, EGTA, citric acid, glycine, taurine, ATP, etc.

[0060]

[0066] Also, the isolation of mitochondria can be performed by thawing, disrupting and centrifuging frozen cells or tissues in order to maintain mitochondrial activity. The method for obtaining mitochondria can be performed by freezing cells or tissues, thawing cells or tissues and disrupting the thawed cells and tissues.

[0061]

[0067] The freezing temperature is not limited, but may be below -1° C. In detail, the temperature may be about -5° C. to about -200° C., about -15° C. to about -180° C., about -25° C. to about -160° C., about -40° C. to about -140° C., about -55° C. to about -120° C., about -60° C. to about -100° C., or about -70° C. to about -90° C.

[0062]

[0068] Freezing can be performed using liquid nitrogen (LN2) or a refrigerator. In particular, the refrigeration or freezing process can be performed by quick freezing using LN2, or by using a refrigerator such as a refrigerator freezer, a freezer, or a freezer container. Freezing is not particularly limited as long as it can refrigerate or freeze mitochondria.

[0063]

[0069] The freezing time is not particularly limited, so long as the frozen mitochondria have normal activity.

[0064]

[0070] The isolated mitochondria may be quantified by quantifying mitochondrial membrane proteins. In particular, the isolated mitochondria may be quantified using a BCA (bicinchoninic acid) assay. The mitochondria may be included in the pharmaceutical composition at a concentration of 0.1 μg / ml to 1,000 μg / ml, 1 μg / ml to 750 μg / ml, 25 μg / ml to 500 μg / ml, 25 μg / ml to 150 μg / ml, or 25 μg / ml to 100 μg / ml. In one embodiment, the mitochondria are used at a concentration of 25 μg / ml or 50 μg / ml.

[0065]

[0071] Furthermore, the number of isolated mitochondria can be measured using a particle counter (Multisizer 4e, Bechman Coulter).

[0066]

[0072] In one embodiment, mitochondria are present at 1x10 5 mitochondria / ml ~ 9x10 9 The mitochondria may be present in the pharmaceutical composition in an amount of 1x10 mitochondria / ml. 5 pieces / ml~5x10 9 pcs / ml, 2x10 5 pieces / ml~2x10 9 pcs / ml, 5x10 5 pieces / ml~1x10 9 pcs / ml, 1x10 6 pieces / ml~5x10 8pcs / ml, 2x10 6 pieces / ml~2x10 8 pcs / ml, 5x10 6 pieces / ml~1x10 8 pcs / ml or 1x10 7 pieces / ml~5x10 7 The pharmaceutical composition may contain the compound in an amount of 1 / ml.

[0067]

[0073] The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any carrier that is a non-toxic substance suitable for delivery to a patient. Distilled water, alcohol, fat, wax or inert solid may be included as a carrier. Pharmacologically acceptable adjuvants (buffers, dispersants) may also be included in the pharmaceutical composition.

[0068]

[0074] In particular, the pharmaceutical composition may be prepared into a parenteral formulation by a conventional method known in the art, depending on the route of administration, by including a pharma- ceutically acceptable carrier in addition to the active ingredient. "Pharmaceutically acceptable" means that it is not more toxic than the subject to which it is applied (prescribed), and can be tolerated without inhibiting the activity of the active ingredient.

[0069]

[0075] When the pharmaceutical composition of the present disclosure is prepared as a parenteral preparation, the pharmaceutical composition can be formulated in the form of injection, transdermal drug and nasal inhaler by using a suitable carrier according to a method known in the art.When formulated as an injection, a suitable carrier can include sterilized water, ethanol, polyol such as glycerin or propylene glycol, or their mixture.Preferably, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, or sterilized water for injection, or isotonic liquid such as 5% dextrose can be used.

[0070]

[0076] The pharmaceutical composition of the present disclosure may be an injection preparation.Therefore, in order to ensure the product safety for the distribution of injection, the pharmaceutical composition of the present disclosure may be prepared as an injection preparation that is highly stable physically and chemically by adjusting pH using an acid aqueous solution or a buffer solution such as phosphate that can be used for injection.

[0071]

[0077] In particular, the pharmaceutical compositions of the present disclosure may include water for injection.

[0072]

[0078] The water for injection may be glucose injection, xylitol injection, D-mannitol injection, fructose injection, saline, dextran 40 injection, dextran 70 injection, amino acid injection, Ringer's solution, lactated Ringer's solution, or phosphate buffer or sodium dihydrogen phosphate-citrate buffer, with a pH ranging from about 3.5 to about 7.5, made to dissolve solid injectables or to reconstitute water-soluble injectables, is distilled water.

[0073]

[0079] The pharmaceutical composition of the present disclosure may further comprise a stabilizer or solubilizer. For example, the stabilizer may be pyrosulfate or ethylenediaminetetraacetic acid, and the solubilizer may be hydrochloric acid, acetic acid, potassium hydroxide phosphate, potassium bicarbonate, potassium carbonate or Tris. In one embodiment, the pharmaceutical composition may comprise a mixed preservative such as TTG (trehalose-tris-glycine) solution, which may be commonly used in pharma-ceutically acceptable drug formulations.

[0074]

[0080] In particular, the pharmaceutical composition of the present disclosure may include a liquid composition for injection in addition to isolated mitochondria. The isolated mitochondria are as described above. The pharmaceutical composition of the present disclosure including the liquid composition for injection may be used as a composition for preventing or treating diseases related to mitochondrial function. In addition, administration of mitochondria through injection may suppress the formation of blood clots that may be caused by mitochondrial aggregation, reduction and aggregation of platelets, etc., maintain and / or enhance mitochondrial stability, and stably maintain mitochondrial activity.

[0075]

[0081] The liquid composition may include glycine, a sugar and a buffer.

[0076]

[0082] Glycine may be present in the liquid composition for injection at a concentration of about 15 mM or more, specifically, at a concentration of about 15 mM to about 150 mM, about 17 mM to about 130 mM, about 20 mM to about 120 mM, about 22 mM to about 110 mM, or about 25 mM to about 100 mM, but is not limited thereto. Glycine may also 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.

[0077]

[0083] The sugars contained in the liquid composition for injection may be, but are not limited to, one or more selected from the group consisting of sucrose, trehalose, mannitol, sorbitol, glucose, fructose, mannose, maltose, lactose, isomaltose, dextran, and dextrin. In particular, the sugars may be trehalose, mannitol, or sucrose. Preferably, the sugars may be trehalose.

[0078]

[0084] The buffer contained in the liquid composition for injection may be selected from the group consisting of, but not limited to, Tris buffer, HEPES buffer, MOPS buffer and acetate-containing or phosphate-containing buffer. Preferably, the buffer may be an injectable Tris buffer.

[0079]

[0085] The pH of the buffer is not limited, but may be in the range of about 7.0 to about 7.8, about 7.2 to about 7.6, or about 7.3 to about 7.5.

[0080]

[0086] Furthermore, the buffer may be present in the injectable liquid composition at a concentration of, but not limited to, about 5 mM to about 50 mM, about 8 mM to about 40 mM, about 10 mM to about 35 mM, about 13 mM to about 30 mM, or about 15 mM to about 25 mM.

[0081]

[0087] The liquid composition for injection may have an osmolality of about 200 to about 400 mOsm, about 230 to about 380 mOsm, about 250 to about 350 mOsm, about 260 to about 320 mOsm, about 270 to about 330 mOsm, or about 280 to about 300 mOsm. The osmolality in the above ranges facilitates long-term storage at temperatures of 2° C. to 8° C. or higher, and makes the composition suitable for parenteral administration, such as intravenous, intramuscular, or subcutaneous administration, without causing side effects in the subject.

[0082]

[0088] As used herein, the term "osmolality" refers to the number of moles of solute contributing to the osmotic pressure of a solution per kilogram of solvent. Osmolality is determined by measuring the freezing point depression of a sample using an osmometer.

[0083]

[0089] Additionally, the injectable liquid composition may further comprise a chelating agent.

[0084]

[0090] The chelating agent may be one or more selected from the group consisting of, but not limited to, injectable grade EGTA, EDTA, and BAPTA. The chelating agent may eliminate damage caused by ion leakage after obtaining mitochondria for the injectable liquid composition.

[0085]

[0091] Additionally, the pharmaceutical compositions may contain additives such as antioxidants, ATP, magnesium, etc., that are effective in maintaining mitochondrial function and activity.

[0086]

[0092] The pharmaceutical composition of the present disclosure may be stored in a container selected from the group consisting of a vial, a cartridge, a syringe, and an automatic injection device. Furthermore, the container storing the pharmaceutical composition may be stored at room temperature, a refrigerated temperature of about 2° C. to about 8° C., or a temperature of about 25° C. to about 40° C. until administration to a subject in need of treatment.

[0087]

[0093] The subject may be a mammal, such as, but not limited to, a human, dog, cow, horse, pig, sheep, goat, cat, mouse, rabbit or rat, preferably a human.

[0088]

[0094] The pharmaceutical compositions of the present disclosure are administered in a pharmacologic effective amount. The term "therapeutically effective amount" or "pharmaceutical effective amount" refers to an amount of a compound or composition that is effective to prevent or treat hearing loss, sufficient to treat the disease at a reasonable risk-benefit ratio applicable to medical treatments and without causing side effects. The level of the effective amount can be determined by factors including the patient's health status, the type and severity of the disease, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, combination or simultaneous use of drugs, and other factors well known in the medical art. In one embodiment, a therapeutically effective amount refers to an amount of drug effective to treat or prevent hearing loss.

[0089]

[0095] As used herein, the term "administration" means introducing a formulated substance into a subject by an appropriate method, and the composition may be administered through any common route as long as it can reach the target tissue. The pharmaceutical composition may be administered intratympanically, intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, topically, intranasally, or intrarectally, but is not limited thereto. Intratympanic administration may be by direct injection into the tympanic cavity, or administration through surgery (e.g., myringotomy).

[0090]

[0096] The preferred dosage of the pharmaceutical composition of the present disclosure per administration may be, but is not limited to, about 0.01 mg mitochondria / kg to about 5 mg mitochondria / kg, about 0.1 mg mitochondria / kg to about 4 mg mitochondria / kg, or about 0.25 mg mitochondria / kg to about 2.5 mg mitochondria / kg based on the body weight of the subject to be administered. In terms of cell activity, it is most preferred that the pharmaceutical composition is administered so that the isolated mitochondria are administered within the above-mentioned range based on the body weight of the subject suffering from hearing loss. Furthermore, the pharmaceutical composition may be administered 1 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. Such dosages should not be construed as limiting the scope of the present invention in any manner.

[0091]

[0097] The term "subject" refers to an individual to whom the composition of the present disclosure may be applied (prescribed), and may be a subject suffering from hearing loss. Furthermore, the subject may be a mammal, such as a human, rat, mouse, or livestock, but preferably a human.

[0092]

[0098] In one embodiment, the pharmaceutical composition may further comprise a known agent for preventing or treating hearing loss, and administration of the pharmaceutical composition may be combined with additional hearing loss treatment.

[0093]

[0099] Another aspect of the present disclosure provides a method of treating and / or preventing hearing loss comprising administering isolated mitochondria to a subject.

[0094]

[0100] Additionally, the pharmaceutical composition may further comprise known agents for preventing or treating hearing loss, and administration of the pharmaceutical composition may be combined with additional hearing loss treatments. EXAMPLES

[0095]

[0101] Hereinafter, the present disclosure will be described in more detail by the following examples. However, the following examples are only for the purpose of illustrating the present disclosure, and the scope of the present disclosure is not limited thereto.

[0096]

[0102] Preparation Example 1. Preparation of mitochondria

[0103] 1x10 7 Umbilical cord-derived mesenchymal stem cells stored at a concentration of cells / ml were physically disrupted using pressure, homogenized, and then subjected to a first centrifugation at a speed of 1,100×g. Mitochondria were obtained using differential centrifugation, isolating mitochondria from cells by centrifuging the supernatant at a speed of 12,000×g at 4° C. for 15 minutes.

[0097]

[0104] Example 1. Determination of mouse inner ear cell viability upon treatment with antibiotics or anticancer drugs

[0105] Example 1.1. Culture of Mouse Inner Ear Cells

[0106] Mouse inner ear cells (House Ear Institute-Organ of Corti 1, HEI-OC1) were cultured in DMEM (Dulbecco's modified Eagle's medium, Gibco) medium containing 10% fetal bovine serum (FBS, Gibco), 100 μg / ml streptomycin, and 100 U / ml ampicillin.

[0098]

[0107] Example 1.2. Determination of viability of inner ear cells

[0108] The mouse inner ear cells obtained in Example 1.1 were treated with an antibiotic or an anticancer drug, and then the cell viability was determined. 3 Mouse inner ear cells (House Ear Institute-Organ of Corti 1) (HEI-OC1) were cultured in DMEM medium, and after 16 hours, the cells were treated with gentamicin (2mM, 4mM, 6mM, 8mM, and 10mM), cisplatin (5mM, 10mM, 15mM, 20mM, 25mM, and 50mM), kanamycin (5mM, 10mM, 15mM, and 20mM), or H2O2 (25mM, 50mM, or 100mM) for 24 hours. Cell viability was measured using the WST-1 assay.

[0099]

[0109] As shown in Figure 1, the viability of inner ear cells was decreased in a concentration-dependent manner in all groups treated with gentamicin, cisplatin, kanamycin or H2O2 compared to the group not treated with drugs (Con.). The results imply that hearing loss may be caused by damage to inner ear cells by drugs or reactive oxygen species.

[0100]

[0110] Example 2. Confirmation of reduction in damage caused by antibiotics upon treatment with mitochondria

[0111] Example 2.1. Confirmation of recovery from antibiotic-induced decrease in cell viability

[0112] It was examined whether mitochondrial treatment had an effect on reversing the reduced viability of inner ear cells damaged by the drug treatment of Example 1.2.

[0101]

[0113] For details, see 6x10 3Mitochondrial mouse inner ear cells (House Ear Institute-Organ of Corti 1) (HEI-OC1) were cultured in antibiotic DMEM medium. After 16 hours, the cells were treated with 10 mM gentamicin or 16.2 mM kanamycin for 24 hours, and then treated with mitochondria of Preparation 1 at concentrations of 0 μg / ml, 0.1 μg / ml, 0.2 μg / ml, 0.4 μg / ml, 0.8 μg / ml and 1.2 μg / ml for 24 hours. The viability of the inner ear cells after treatment was measured using the WST-1 assay.

[0102]

[0114] As shown in Figures 2 and 3, it was confirmed that the cell viability was restored when treated with mitochondria compared to the group not treated with mitochondria (0 μg / ml). Furthermore, this restoration effect was achieved in a concentration-dependent manner depending on the mitochondria concentration.

[0103]

[0115] Example 2.2. Confirmation of the effect of suppressing the increase in reactive oxygen species caused by antibiotics

[0116] We investigated whether mitochondrial treatment had an effect on suppressing the increase in reactive oxygen species in inner ear cells damaged by the drug treatment of Example 1.2.

[0104]

[0117] For details, see 6x10 3Inner ear cells (House Ear Institute-Organ of Corti 1) (HEI-OC1) were cultured in DMEM medium, and after 16 hours, the cells were treated with 10 mM gentamicin or 20 mM kanamycin. After 24 hours of treatment, the cells were treated with mitochondria. The gentamicin-treated group was treated with mitochondria of Preparation Example 1 at concentrations of 0 μg / ml, 0.1 μg / ml, 0.2 μg / ml, 0.4 μg / ml, 0.8 μg / ml, 1.2 μg / ml, and 1.8 μg / ml, respectively. The kanamycin-treated group was treated with mitochondria of Preparation Example 1 at concentrations of 0 μg / ml and 0.4 μg / ml, respectively. After 24 hours, the treated medium was stained with DCF-DA (Invitrogen), a staining reagent specific to intracellular reactive oxygen species, for 1 hour, and then reactive oxygen species were measured using a plate reader (Biotek, Synergy HTX). In addition, the number of cells was corrected after staining with Hoechst33432 (Invitrogen), a staining reagent specific to nuclei.

[0105]

[0118] As shown in Figures 4 and 5, it was confirmed that the rate of increase in reactive oxygen species was lower in the mitochondria-treated group compared to the group not treated with mitochondria (0 μg / ml). It was also confirmed that the intracellular reactive oxygen species increased by drug treatment were reduced to normal levels by mitochondria treatment.

[0106]

[0119] Example 3. Confirmation of the hearing recovery effect of mitochondrial treatment in an animal model of noise-induced hearing loss

[0120] An experimental group in which hearing loss was induced by noise and a control group in which hearing loss was not induced were prepared using 12-week-old C57BL6 female mice. In detail, 12-week-old C57BL6 female mice in the experimental group were exposed to a sound of 120 dB for 2 hours. In addition, 20 μg of mitochondria obtained in Preparation Example 1 was injected twice into the tail vein before and after the 2-hour exposure to the sound of 120 dB. In the control group, hearing loss was not induced and the control group was not treated with mitochondria. Auditory brainstem responses (ABRs) were measured in the control and experimental groups 1, 4, 7, and 14 days after the injection.

[0107]

[0121] As shown in Figure 6, mice with noise-induced hearing loss did not respond to sounds between 20 and 70 dB, but the experimental group administered mitochondria responded to sounds between 30 and 70 dB, just like the control group. These results indicate that mitochondria have the effect of restoring hearing from noise-induced hearing loss.

Claims

1. A pharmaceutical composition for preventing or treating hearing loss or tinnitus, comprising isolated mitochondria as active ingredients.

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

3. 3. The pharmaceutical composition for preventing or treating hearing loss or tinnitus according to claim 2, wherein the cells are somatic cells, germ cells, stem cells, blood cells, or combinations thereof.

4. The pharmaceutical composition for preventing or treating hearing loss or tinnitus according to claim 3 , wherein the stem cells are mesenchymal stem cells, induced pluripotent stem cells, embryonic stem cells, or a combination thereof.

5. 5. The pharmaceutical composition for preventing or treating hearing loss or tinnitus according to claim 4, wherein the mesenchymal stem cells are derived from umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, placenta, synovial fluid, testis, periosteum, or a combination thereof.

6. 2. The pharmaceutical composition for preventing or treating hearing loss or tinnitus according to claim 1, wherein the isolated mitochondria have normal activity.

7. The pharmaceutical composition for preventing or treating hearing loss or tinnitus according to claim 1, wherein the hearing loss is conductive hearing loss or sensorineural hearing loss.

8. The pharmaceutical composition for preventing or treating hearing loss or tinnitus according to claim 7, wherein the sensorineural hearing loss is noise-induced hearing loss, sudden sensorineural hearing loss, infection-induced hearing loss, traumatic hearing loss, presbycusis, ototoxic hearing loss, autoimmune hearing loss, Meniere's disease, or hearing loss due to nerve damage.

9. The pharmaceutical composition for preventing or treating hearing loss or tinnitus according to claim 8, wherein the ototoxic hearing loss is caused by the use of anticancer drugs, antibiotics or radiation.

10. The pharmaceutical composition for preventing or treating hearing loss or tinnitus according to claim 9, wherein the anticancer agent is one or more selected from the group consisting of taxane anticancer agents, platinum anticancer agents, and vinca alkaloid anticancer agents.

11. The pharmaceutical composition for preventing or treating hearing loss or tinnitus according to claim 10, wherein the anticancer drug is a platinum-based anticancer drug.

12. The pharmaceutical composition for preventing or treating hearing loss or tinnitus according to claim 11, wherein the platinum-based anticancer drug is one or more selected from the group consisting of cisplatin, carboplatin and oxaliplatin.

13. 10. The pharmaceutical composition for preventing or treating hearing loss or tinnitus according to claim 9, wherein the antibiotic is one or more selected from the group consisting of streptomycin, neomycin, kanamycin, gentamicin, tobramycin, amikacin, netilmicin, dibekacin, sisomicin, ribodomycin and paromomycin.

14. 2. The pharmaceutical composition for preventing or treating hearing loss or tinnitus according to claim 1, wherein the isolated mitochondria prevent inner ear cell death.

15. 2. The pharmaceutical composition for preventing or treating hearing loss or tinnitus according to claim 1, wherein the isolated mitochondria are contained in the pharmaceutical composition at a concentration of 0.1 μg / ml to 1,000 μg / ml.

16. 13. A method for preventing or treating hearing loss or tinnitus, comprising administering to a subject the pharmaceutical composition of claim 1.

17. 13. Use of the pharmaceutical composition of claim 1 for preventing or treating hearing loss or tinnitus.

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