WO2020262971A1 - Composition for preventing or treating diseases caused by mitochondrial dysfunction - Google Patents

Composition for preventing or treating diseases caused by mitochondrial dysfunction Download PDF

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WO2020262971A1
WO2020262971A1 PCT/KR2020/008247 KR2020008247W WO2020262971A1 WO 2020262971 A1 WO2020262971 A1 WO 2020262971A1 KR 2020008247 W KR2020008247 W KR 2020008247W WO 2020262971 A1 WO2020262971 A1 WO 2020262971A1
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disease
mitophagy
palmatin
activity
mitochondrial
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French (fr)
Korean (ko)
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윤진호
김동현
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동아대학교 산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/4833—Physical analysis of biological material of solid biological material, e.g. tissue samples, cell cultures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to a composition for preventing or treating diseases caused by mitochondrial dysfunction, comprising palmatine as an active ingredient.
  • Mitophagy is an intracellular degradation mechanism that removes damaged or unnecessary mitochondria.When mitochondrial damage occurs, it is surrounded by a membrane to form an autophagosome and fuse it with a lysosome to selectively select damaged mitochondria. It has been reported to play a role in removing. It is known that the activity of mitophagy is important in regulating mitochondrial function and maintaining tissue function in various cells including nerve cells.
  • CCCP and FCCP are mitochondrial membrane potential inhibitors (uncouplers) that depolarize the mitochondrial membrane potential, and rotenone acts as a Complex I inhibitor. They directly induce mitochondrial damage, thereby inducing mitophagy activity, which is a mechanism for removing damaged mitochondria, but because they are highly toxic to cells, they cannot be used as drugs for promoting mitophagy activity.
  • Another object of the present invention is (a) treating a test substance to cells expressing mitokeima (mt-Keima); And (b) measuring the activity of mitophagy (mitophagy); mitochondrial dysfunction (mitochondrial dysfunction) comprising a screening method for a disease caused by the treatment of.
  • mitokeima mitokeima
  • mitophagy mitophagy
  • mitochondrial dysfunction mitochondrial dysfunction
  • Another object of the present invention is to provide a method for promoting mitophagy activity comprising the step of treating mitochondrial dysfunction cells with palmatine in vitro .
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cognitive dysfunction comprising palmatine as an active ingredient.
  • Another object of the present invention is to provide a food composition for preventing or improving cognitive dysfunction comprising palmatine as an active ingredient.
  • Another object of the present invention is to provide a method for preventing or treating diseases caused by mitochondrial dysfunction, comprising administering to an individual a pharmaceutically effective amount of the pharmaceutical composition.
  • Another object of the present invention is to provide a method for preventing or treating cognitive dysfunction, comprising administering to an individual a pharmaceutically effective amount of the pharmaceutical composition.
  • the present invention provides a composition for preventing or treating diseases caused by mitochondrial dysfunction, including palmatine as an active ingredient.
  • the present invention comprises the steps of: (a) treating a test substance on cells expressing mt-Keima; And (b) measuring the activity of mitophagy (mitophagy); provides a method for screening a therapeutic agent for diseases caused by mitochondrial dysfunction comprising.
  • the present invention provides a method for promoting mitophagy activity, including the step of treating mitochondrial dysfunction cells with palmatin in vitro .
  • the present invention provides a pharmaceutical composition for preventing or treating cognitive dysfunction comprising palmatin (palamtine) as an active ingredient.
  • the present invention provides a food composition for preventing or improving cognitive dysfunction comprising palmatine (palamtine) as an active ingredient.
  • the present invention provides a method for preventing or treating diseases caused by mitochondrial dysfunction, comprising administering to an individual a pharmaceutically effective amount of the above pharmaceutical composition.
  • the present invention provides a method for preventing or treating cognitive dysfunction, comprising administering to an individual the above pharmaceutical composition of a pharmaceutically effective yarn.
  • composition according to the present invention has an effect of promoting the activity of mitophagy, and thus can be usefully used in the treatment of diseases caused by mitochondrial dysfunction.
  • 1A shows a method of measuring mitophagy activity by flow cytometry.
  • Figure 1B is a graph showing mitophagy activity by (Flow cytometry) by flow cytometry.
  • 2A is a fluorescence measurement of mitophagy activity by confocal microscopy.
  • 2B is a diagram confirming the activity of mitophagy by confocal microscopy.
  • 2C is a diagram showing an equation for measuring the activity of mitophagy by confocal microscopy.
  • 3A is a result of analyzing the activity of mitophagy by palmatin in human lung epithelial cell lines by flow cytometry.
  • 3B is a result of analyzing the activity of mitophagy by palmatin in a human lung epithelial cell line with a confocal microscope.
  • 3C is a result of analyzing the amount of mitochondria by palmatin in a human lung epithelial cell line.
  • 4A is a result of analyzing the activity of mitophagy by palmatin in HeLa cells (HeLa-Parkin) expressing Parkin by flow cytometry.
  • 4B is a result of analyzing the activity of mitophagy by palmatin in HeLa cells (HeLa-Parkin) expressing Parkin with a confocal microscope.
  • 5A is a result of analyzing the activity of mitophagy according to the treatment concentration of palmatin.
  • 5B is a result of analyzing the activity of mitophagy according to the treatment time of palmatin.
  • 6A is a diagram showing the fluorescence expression pattern of mitochondria by treatment with palmatin in Hela cell lines expressing mitochondria, ER and Goli fluorescent markers with a confocal microscope.
  • Figure 6B is a schematic diagram of the mitochondria, ER and Hela cell lines expressing Goli fluorescent markers, treated with palmatin, to quantify the fluorescence expression pattern of mitochondria.
  • 7A is a result of analyzing the mitochondrial membrane potential according to palmatin treatment by TMRM assay.
  • 7C is a result of analyzing the activity of mitophagy according to the treatment of palmatin.
  • 7D is a result of analysis of changes in mitochondrial active oxygen according to NAC (N-Acetylcysteine) treatment and palmatin treatment by MitoSOX assay.
  • 7E is a result of analysis of mitochondrial free radicals according to the treatment of palmatin by DCF-DA assay.
  • 9A is a result of analyzing the amount of mitochondria using NAO fluorescent dye after treatment with palmatin.
  • 9B is a result of analyzing the mRNA expression level of PGC-1 ⁇ , a regulator of mitochondrial biosynthesis, through RT-PCR after treatment with palmatin.
  • 9C is a result of analyzing the mRNA expression level of NRF1, a regulator of mitochondrial biosynthesis, through RT-PCR after palmatin treatment.
  • 9D is a result of analyzing the mRNA expression level of TFAM, a regulator of mitochondrial biosynthesis, through RT-PCR after palmatin treatment.
  • Figure 10A shows the expression of mitochondrial proteins Mfn2 and COX2 proteins by Western blot when palmatin was treated in a PINK1-deficient MEF cell line.
  • FIG. 10B is a graph confirming that when palmatin was treated in a PINK1-deficient MEF cell line, free radicals decreased.
  • 10C is a diagram confirming that the mitochondrial respiration rate increased when palmatin was treated in the PINK1 deficient MEF cell line.
  • FIG. 11 is a diagram illustrating the effect of improving movement by administering palmatin to a Drosophila animal model of amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • FIG. 12 is a diagram showing the effect of improving exercise by administering palmatin to an animal model of A ⁇ overexpressing Drosophila, which is Alzha and is an animal model.
  • the present invention provides a composition for preventing or treating diseases caused by mitochondrial dysfunction, comprising palmatine as an active ingredient.
  • Palmatin (palmatine, CAS Number: 3486-67-7), which is a compound according to the present invention, refers to a compound represented by the following formula (1).
  • the palmatin may promote the activity of mitophagy (mitophagy).
  • palmatin may increase mitochondrial cellular respiration.
  • mitochondria of the present invention is an intracellular decomposition mechanism that removes damaged or unnecessary mitochondria. When damage to the mitochondria occurs, an autophagosome is formed and fused with lysosomes to selectively select the damaged mitochondria. Can be disassembled and removed.
  • the palmatine may be administered at a concentration of 0.1 to 200 ⁇ M, preferably may be a concentration of 1 to 100 ⁇ M, more preferably a concentration of 20 to 100 ⁇ M It may be, more preferably, it may be a concentration of 20 to 80 ⁇ M, but is not limited thereto.
  • composition according to the present invention may further comprise a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means exhibiting properties that are not toxic to cells or humans exposed to the composition.
  • the carrier may be used without limitation as long as it is known in the art such as a buffering agent, a preservative, a painless agent, a solubilizing agent, an isotonic agent, a stabilizer, a base agent, an excipient, and a lubricant.
  • composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. have. Further, it may be used in the form of an ointment, lotion, spray, patch, cream, powder, suspension, gel or gel for external skin.
  • Carriers, excipients and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils.
  • diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient, such as starch, calcium carbonate, in the Cycotria rubra extract, It is prepared by mixing sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.
  • Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. have.
  • Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories.
  • non-aqueous solvent and suspending agent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used.
  • injectable ester such as ethyl oleate
  • a base for suppositories witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like can be used.
  • composition of the present invention is administered in a pharmaceutically effective amount.
  • administration means introducing a predetermined substance to an individual by an appropriate method, and the route of administration of the composition may be administered through any general route as long as it can reach the target tissue.
  • Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration may be administered, but are not limited thereto.
  • the term "individual” refers to all animals including humans, such as mice, mice, and domestic animals. Preferably, it may be a mammal including a human.
  • pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not cause side effects, and the effective dose level is the sex, age, and Weight, health status, type of disease, severity, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration, and rate of excretion, duration of treatment, factors including drugs used in combination or concurrently, and other fields of medicine. It can be readily determined by a person skilled in the art according to well-known factors. Administration may be administered once a day at the recommended dosage, or may be divided several times.
  • the disease due to the mitochondrial dysfunction may be selected from the group consisting of degenerative diseases, cancer, allergic diseases, and cognitive dysfunction.
  • the disease due to mitochondrial dysfunction is MELAS syndrome (mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes), ALS (amyotrophic lateral sclerosis, ALS), and Charcot Marie's disease (Charcot Marie Tooth disease, CMT) may be any one selected from the group consisting of.
  • the degenerative disease is Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, multiple sclerosis, and immune system abnormalities. It may be one or more selected from the group consisting of brain insufficiency, progressive neurodegenerative disease, metabolic brain disease, Niemann-Pick disease, cerebral ischemia, and dementia due to cerebral hemorrhage, and preferably Parkinson's disease. However, it is not limited thereto.
  • the cancer is colon cancer, CTH-producing tumor, acute lymphocytic or lymphoblastic leukemia, acute or chronic lymphocytic leukemia, acute non-lymphocyte leukemia, bladder cancer, brain tumor, breast cancer, cervical cancer , Chronic myelogenous leukemia, bowel cancer, T-zone lymphoma, endometriosis, esophageal cancer, gall bladder cancer, Ewing's sarcoma, head and neck cancer, tongue cancer, Hopkins' lymphoma, Caposis' sarcoma, kidney cancer, liver cancer, lung cancer, mesothelioma, Multiple myeloma, neuroblastoma, non-Hopekin's lymphoma, osteosarcoma, ovarian cancer, neuroblastoma, mammary cancer, cervical cancer, prostate cancer, pancreatic cancer, penis cancer, retinoblastoma, skin cancer, gastric cancer, thyroid cancer, uterine cancer, testicular cancer
  • the allergic disease is atopic dermatitis, allergic dermatitis, contact dermatitis, urticaria, itching, insect allergy, food allergy, drug allergy, edema, Hypersensitivity (anaphylaxis), allergic rhinitis (allergic rhinitis), asthma (asthma) and allergic conjunctivitis (allergic conjunctivitis) may be one or more selected from, but is not limited thereto.
  • the present invention comprises the steps of: (a) treating a test substance on cells expressing mt-Keima; And (b) measuring the activity of mitophagy (mitophagy); provides a method for screening a therapeutic agent for diseases caused by mitochondrial dysfunction comprising.
  • mitokeima mt-Keima, mitochondria-targeted Keima
  • mitochondria-targeted Keima mitochondria-targeted Keima
  • the mitophagy activity may be measured by flow cytometry or confocal microscopy.
  • test substance when the test substance promotes mitophagy activity, determining that it is a therapeutic agent for a disease due to mitochondrial dysfunction may be additionally included.
  • the present invention provides a method for promoting mitophagy activity, including the step of treating mitochondrial dysfunction cells with palmatin in vitro .
  • the present invention provides a pharmaceutical composition for preventing or treating cognitive dysfunction comprising palmatin (palamtine) as an active ingredient.
  • the cognitive dysfunction is from the group consisting of lethargy, memory loss, forgetfulness, cognitive decline, learning disability, attention loss, depression, hearing loss, painlessness, inactivity and discrimination. It may be one or more selected, but is not limited thereto.
  • the present invention provides a food composition for preventing or improving cognitive dysfunction comprising palmatine (palamtine) as an active ingredient.
  • the present invention provides a method for preventing or treating diseases caused by mitochondrial dysfunction, comprising administering to an individual a pharmaceutically effective amount of the above pharmaceutical composition.
  • the present invention provides a method for preventing or treating cognitive dysfunction, comprising administering to an individual a pharmaceutically effective amount of the pharmaceutical composition.
  • the present invention provides a method for preventing and treating mitochondrial dysfunction or cognitive dysfunction comprising administering to an individual a pharmaceutically effective amount of the pharmaceutical composition.
  • the pharmaceutical composition of the present invention is administered in a therapeutically effective amount or in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type and severity of the subject, age, sex, activity of the drug, and Sensitivity, administration time, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field.
  • the present inventors performed an experiment for producing a cell line expressing mitokema. Briefly, a 100-mm culture dish was prepared by coating it with 0.001% poly-L-lysine for 10 minutes at room temperature. After dispensing 4 x 10 6 293L cells into the coated culture dish, DMEM with 10% FBS added. Incubated for 24 hours in the culture medium. Thereafter, the mitokeima plasmid DNA (pLVX-mt-Keima, 3 ⁇ g) and packaging DNA (psPAX2, 3.75 ⁇ g; and pVSVG, 0.375 ⁇ g) were added and transfection was performed for 8 hours. Thereafter, the culture medium was exchanged with a fresh port culture medium and further cultured for 48 hours, and then a culture medium containing the virus was taken and filtered through a 0.45 ⁇ m filter to prepare a mitokeima lentivirus.
  • pLVX-mt-Keima 3 ⁇ g
  • packaging DNA psPAX2, 3.
  • Cells to be expressed mitokema were prepared by incubating for 24 hours in a 60-mm culture dish, 1 ml of pre-produced mitokema lentivirus and 1 ml of culture solution were mixed, and then added to the cells to be expressed mitokema. Infected for hours. Thereafter, the culture medium was removed, and puromycin (2 ⁇ g/ml) was treated to select only cells into which the virus was introduced.
  • mitokema fluorescence was simultaneously measured by two lasers at 405 nm and 561 nm.
  • Mitophagy activity was calculated as the ratio of pixels that appear red due to high mitophagy activity among all the pixels of the confocal microscope image (FIG. 2).
  • the present inventors screened a library of plant-derived physiologically active substances and discovered palmatin, a structurally similar isoquinoline alkaloid, as a mitophagy activity promoting substance.
  • Example 3 Analysis of the effect of promoting mitophagy activity by palmatin in human lung epithelial cell lines
  • the present inventors conducted an experiment to verify the effect of promoting the activity of mitophagy by palmatin. Briefly, in BEAS-2B cells, a human lung epithelial cell line expressing mitokema, palmatin (400 ⁇ M) or CCCP (10 ⁇ M), known as a mitochondrial membrane potential inhibitor (uncoupler), was applied to 24 cells. After treatment for a period of time, the activity of mitophagy was measured using a flow cytometer or a confocal microscope, or the activity of mitophagy due to the quantitative reduction of mitochondria was measured.
  • a human lung epithelial cell line expressing mitokema, palmatin (400 ⁇ M) or CCCP (10 ⁇ M), known as a mitochondrial membrane potential inhibitor (uncoupler) was applied to 24 cells. After treatment for a period of time, the activity of mitophagy was measured using a flow cytometer or a confocal microscope, or the activity of mitophagy due to the quantitative reduction of mitochondria was measured.
  • Example 4 Analysis of the effect of promoting mitophagy activity by palmatin in cervical cancer cell lines
  • the present inventors conducted an experiment to confirm whether the activity of mitophagy is promoted by palmatin in other cell lines. Briefly, Parkin (E3 ubiquitin ligase)-expressing cervical cancer cell line HeLa cells (HeLa-Parkin) were treated with palmatin or CCCP and then mitophagy activity was analyzed.
  • Parkin E3 ubiquitin ligase
  • HeLa-Parkin cervical cancer cell line HeLa cells
  • Example 5 Analysis of the effect of promoting mitophagy activity according to the treatment concentration and treatment time of palmatin
  • the present inventors conducted an experiment to analyze the effect of promoting mitophagy activity according to the treatment concentration and treatment time of palmatin. Briefly, palmatin was treated for 24 hours at a given concentration (0, 5, 10, 20, 40, 60, 80, 100, 200, 400 and 800 ⁇ M) to BEAS-2B cells expressing mitokema, followed by flow cytometry. Mitophagy activity was measured using an analyzer. In addition, BEAS-2B cells were treated with palmatin (400 ⁇ M) for a given time (0, 3, 6, 12, 18 and 24 hours), and then mitophagy activity was measured using a flow cytometer.
  • the present inventors described a Hela cell line expressing each of the mitochondria (mito-YFP), organelle ER (ER-GFP) and organelle Golgi (Golgi-GFP) fluorescent markers.
  • the fields were treated with palmatin at a concentration of 200 or 400 ⁇ M, and cultured for 24 hours. After incubation, changes in the fluorescence signal of each organelle were analyzed and visualized with a confocal microscope.
  • the present inventors conducted an experiment to determine what changes occur in mitochondrial membrane potential and active oxygen by palmatin.
  • the mitochondrial membrane potential was analyzed by TMRM (tetramethylrhodamine methyl ester) assay, and the mitochondrial reactive oxygen (ROS) was analyzed by MitoSOX assay and DCF-DA assay.
  • TMRM tetramethylrhodamine methyl ester
  • ROS mitochondrial reactive oxygen
  • palmatin increases mitophagy activity by a mechanism different from mitochondrial toxins such as CCCP.
  • AMPK AMP-activated protein kinase
  • the present inventors conducted an experiment to analyze whether the biosynthesis of new mitochondria is promoted after the activation of mitophagy by palmatin occurs. As a result of analyzing the amount of mitochondria using a fluorescent dye of NAO (10-N-nonyl acridine orange), it was confirmed that the amount of mitochondria decreased immediately after palmatin treatment, and then recovered little by little over time (Fig. 9A).
  • Parkinson's disease PINK PTEN-induced kinase 1 deficient MEF (murine embryonic fibroblast) cell line to confirm whether palmatin has an effect of improving mitochondrial function, and function by palmatin.
  • the improvement effect was confirmed.
  • the PINK1 deficient MEF cell line was treated with palmatin at a concentration of 100, 200 or 400 ⁇ M, and then cultured for 24 hours.
  • cells were obtained after cultivation, and the expression of mitochondrial proteins Mfn2 and COX2 proteins was investigated using Western blot.
  • the effect of improving mitochondrial function was analyzed by flow cytometry after mitoSox staining, and the mitochondrial respiration rate was analyzed by XF24 analyzer.
  • the improvement of mitochondrial function was determined by treating the PINK-deficient MEF cell line (PINK KO) with DMSO as a positive control, and treatment with palmatin at 400 ⁇ M for 24 hours as an experimental group, and analyzing mitochondrial free radicals and mitochondrial respiration rate after 4 days. .
  • ALS amyotrophic lateral sclerosis
  • the ALS Drosophila animal model was used to confirm the effect. Specifically, a climbing assay was performed for 25 days by administering 5 mM palmatin to the TDP-4 overexpression model, which is a representative ALS fruit fly animal model.
  • TDP-4 overexpression model which is a representative ALS fruit fly animal model.
  • motility in an Alzheimer's animal model the same experiment was performed using an A ⁇ overexpressing Drosophila animal model having an Alzheimer's phenotype under the same conditions as above.
  • the palmatine of the present invention induces mitophagy to remove dysfunctional mitochondria.
  • the respiration rate of mitochondria was specifically increased, and it showed an effect of improving motility in animal models of degenerative diseases (ALS and Alzheimer's) caused by mitochondrial dysfunction, effectively improving cognitive function, thereby improving degenerative diseases. Confirmed.

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Abstract

The present invention relates to a composition for preventing or treating diseases caused by mitochondrial dysfunction, containing palmatine as an active ingredient, wherein palmatine promotes mitophagy activity, and thus can be effectively used in the treatment of diseases caused by mitochondrial dysfunction.

Description

미토콘드리아 기능이상으로 인한 질환의 예방 또는 치료용 조성물Composition for preventing or treating diseases caused by mitochondrial dysfunction
본 발명은 팔마틴 (palmatine)을 유효성분으로 포함하는 미토콘드리아 기능이상 (mitochondrial dysfunction)으로 인한 질환의 예방 또는 치료용 조성물에 관한 것이다.The present invention relates to a composition for preventing or treating diseases caused by mitochondrial dysfunction, comprising palmatine as an active ingredient.
미토파지 (mitophagy)는 손상되었거나 불필요한 미토콘드리아를 제거하는 세포 내 분해기전으로서, 미토콘드리아 손상이 발생하였을 때 막으로 둘러싸 오토파고좀 (autophagosome)을 형성하고 이를 리소좀 (lysosome)과 융합함으로써 손상된 미토콘드리아를 선택적으로 제거하는 역할을 수행하는 것으로 보고되었다. 이러한 미토파지의 활성은 신경세포를 비롯한 여러 세포들에서 미토콘드리아 기능을 조절하고 조직의 기능을 유지하는데 중요하다는 것이 알려져 있다.Mitophagy is an intracellular degradation mechanism that removes damaged or unnecessary mitochondria.When mitochondrial damage occurs, it is surrounded by a membrane to form an autophagosome and fuse it with a lysosome to selectively select damaged mitochondria. It has been reported to play a role in removing. It is known that the activity of mitophagy is important in regulating mitochondrial function and maintaining tissue function in various cells including nerve cells.
최근 연구에 따르면, 미토파지의 활성 저해는 손상된 미토콘드리아의 축적을 통해 운동신경의 사멸을 야기하여 파킨슨병과 같은 퇴행성 뇌질환을 일으킬 수 있다는 것이 밝혀졌다. 또한, 미토파지 활성의 이상이 파킨슨병, 알츠하이머병, 루게릭병과 같은 퇴행성 뇌질환을 비롯하여, 말초신경병증, 심장질환, 대사질환, 암 등 광범위한 인체질환들과 관련되는 것으로 보고되면서 인체질환에서 미토파지의 역할과 질환치료에의 이용가능성에 대한 연구자들의 관심이 높아지고 있다. According to a recent study, it was found that inhibition of mitophagy activity can lead to degenerative brain diseases such as Parkinson's disease by causing the death of motor nerves through the accumulation of damaged mitochondria. In addition, as it has been reported that abnormalities in mitophagy activity are related to a wide range of human diseases such as degenerative brain diseases such as Parkinson's disease, Alzheimer's disease, Lou Gehrig's disease, peripheral neuropathy, heart disease, metabolic disease, and cancer. There is increasing interest of researchers about the role of the drug and its applicability to disease treatment.
미토파지 활성 촉진을 통해 질병을 치료하기 위한 미토파지 조절화합물을 발굴하기 위해서는 세포 및 생체 미토파지 변화를 손쉽게 측정할 수 있는 연구시스템이 필수적으로 필요하다. 그러나, 민감하고 정량적으로 미토파지 활성을 측정할 수 있는 실험적 방법의 부재로 미토파지 활성자체를 판독지표 (read-out)로 한 미토파지 조절 화합물의 탐색은 수행된 적이 없었다. 지금까지 널리 사용되고 있는 LC3 기반 검출 방법은 미토파지의 초기단계인 오토파고좀 형성단계만을 측정할 수 있기 때문에 측정감도가 낮고 정량적 측정이 어려운 한계를 지니고 있다. 또한, 몇몇 연구그룹에서 시도된 제어물질 발굴시도도 대부분 PINK-Parkin의 미토콘드리아 이동이나, 미토파지 과정에서 일어나는 미토콘드리아의 분열 (fission) 등 간접적 판독지표들을 사용하여 실제 생리적 조건에서 미토파지 활성을 제어할 수 있는 물질을 발굴하지 못하고 있다.In order to discover mitophagy control compounds for treating diseases by promoting mitophagy activity, a research system that can easily measure changes in cell and biological mitophagy is essential. However, the search for a mitophagy modulating compound using the mitophagy activity itself as a read-out has never been performed due to the lack of an experimental method capable of sensitive and quantitatively measuring mitophagy activity. The LC3-based detection method, which has been widely used so far, can only measure the autophagosome formation stage, which is the initial stage of mitophagy, so it has a low measurement sensitivity and a limitation in quantitative measurement. In addition, most of the attempts to discover control substances that have been attempted by several research groups can control mitophagy activity under actual physiological conditions by using indirect reading indicators such as mitochondrial migration of PINK-Parkin and mitochondrial fission occurring in the mitophagy process. It has not been able to find a material that can be used.
현재 실험적으로 미토파지 활성을 유도하는 방법은 CCCP, FCCP, rotenone 등과 같이 미토콘드리아의 기능이상을 유도하는 소위 '미토콘드리아 독소들 (mitochondrial toxins)'을 처리하는 것이다. CCCP와 FCCP는 미토콘드리아 막전위 저해제 (uncoupler)로서 미토콘드리아 막전위를 탈분극시키며, rotenone은 Complex I 저해제로 작용한다. 이들은 직접적으로 미토콘드리아 손상을 유도함으로써 손상된 미토콘드리아의 제거기전인 미토파지 활성을 유도하지만, 세포에 대한 독성이 강하기 때문에 미토파지 활성 촉진을 위한 약물로는 사용할 수 없는 제한이 있다.Currently, the experimental method to induce mitophagy activity is to treat so-called'mitochondrial toxins' that induce mitochondrial dysfunction, such as CCCP, FCCP, and rotenone. CCCP and FCCP are mitochondrial membrane potential inhibitors (uncouplers) that depolarize the mitochondrial membrane potential, and rotenone acts as a Complex I inhibitor. They directly induce mitochondrial damage, thereby inducing mitophagy activity, which is a mechanism for removing damaged mitochondria, but because they are highly toxic to cells, they cannot be used as drugs for promoting mitophagy activity.
본 발명의 목적은 팔마틴 (palmatine)을 유효성분으로 포함하는 미토콘드리아 기능이상 (mitochondrial dysfunction)으로 인한 질환의 예방 또는 치료용 조성물을 제공하는 것이다. It is an object of the present invention to provide a composition for preventing or treating diseases caused by mitochondrial dysfunction, comprising palmatine as an active ingredient.
본 발명의 또 다른 목적은 (a) 미토케이마 (mt-Keima)를 발현하는 세포에 시험물질을 처리하는 단계; 및 (b) 미토파지 (mitophagy)의 활성을 측정하는 단계;를 포함하는 미토콘드리아 기능이상 (mitochondrial dysfunction)으로 인한 질환 치료제의 스크리닝 방법을 제공하는 것이다. Another object of the present invention is (a) treating a test substance to cells expressing mitokeima (mt-Keima); And (b) measuring the activity of mitophagy (mitophagy); mitochondrial dysfunction (mitochondrial dysfunction) comprising a screening method for a disease caused by the treatment of.
본 발명의 또 다른 목적은 시험관 내 (in vitro)에서 팔마틴 (palmatine)을 미토콘드리아 기능이상 세포에 처리하는 단계를 포함하는 미토파지의 활성을 촉진시키는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for promoting mitophagy activity comprising the step of treating mitochondrial dysfunction cells with palmatine in vitro .
본 발명의 또 다른 목적은 팔마틴(palmatine)을 유효성분으로 포함하는 인지기능 장애 예방 또는 치료용 약학적 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cognitive dysfunction comprising palmatine as an active ingredient.
본 발명의 또 다른 목적은 팔마틴(palmatine)을 유효성분으로 포함하는 인지기능 장애 예방 또는 개선용 식품 조성물을 제공하는 것이다. Another object of the present invention is to provide a food composition for preventing or improving cognitive dysfunction comprising palmatine as an active ingredient.
본 발명의 또 다른 목적은 약학적으로 유효한 양의 상기의 약학적 조성물을 개체에 투여하는 단계를 포함하는, 미토콘드리아 기능이상 (mitochondrial dysfunction)으로 인한 질환의 예방 또는 치료 방법을 제공하는 것이다.Another object of the present invention is to provide a method for preventing or treating diseases caused by mitochondrial dysfunction, comprising administering to an individual a pharmaceutically effective amount of the pharmaceutical composition.
본 발명의 또 다른 목적은 약학적으로 유효한 양의 상기의 약학적 조성물을 개체에 투여하는 단계를 포함하는, 인지기능 장애의 예방 또는 치료 방법을 제공하는 것이다.Another object of the present invention is to provide a method for preventing or treating cognitive dysfunction, comprising administering to an individual a pharmaceutically effective amount of the pharmaceutical composition.
상기 목적을 달성하기 위하여, 본 발명은 팔마틴 (palmatine)을 유효성분으로 포함하는 미토콘드리아 기능이상 (mitochondrial dysfunction)으로 인한 질환의 예방 또는 치료용 조성물을 제공한다. In order to achieve the above object, the present invention provides a composition for preventing or treating diseases caused by mitochondrial dysfunction, including palmatine as an active ingredient.
또한, 본 발명은 (a) 미토케이마 (mt-Keima)를 발현하는 세포에 시험물질을 처리하는 단계; 및 (b) 미토파지 (mitophagy)의 활성을 측정하는 단계;를 포함하는 미토콘드리아 기능이상 (mitochondrial dysfunction)으로 인한 질환 치료제의 스크리닝 방법을 제공한다. In addition, the present invention comprises the steps of: (a) treating a test substance on cells expressing mt-Keima; And (b) measuring the activity of mitophagy (mitophagy); provides a method for screening a therapeutic agent for diseases caused by mitochondrial dysfunction comprising.
또한, 본 발명은 시험관 내 (in vitro)에서 팔마틴 (palamtine)을 미토콘드리아 기능이상 세포에 처리하는 단계를 포함하는 미토파지의 활성을 촉진시키는 방법을 제공한다. In addition, the present invention provides a method for promoting mitophagy activity, including the step of treating mitochondrial dysfunction cells with palmatin in vitro .
또한, 본 발명은 팔마틴 (palamtine)을 유효성분으로 포함하는 인지기능 장애 예방 또는 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating cognitive dysfunction comprising palmatin (palamtine) as an active ingredient.
또한, 본 발명은 팔마틴 (palamtine)을 유효성분으로 포함하는 인지기능 장애 예방 또는 개선용 식품 조성물을 제공한다.In addition, the present invention provides a food composition for preventing or improving cognitive dysfunction comprising palmatine (palamtine) as an active ingredient.
또한, 본 발명은 약학적으로 유효한 양의 상기의 약학적 조성물을 개체에 투여하는 단계를 포함하는, 미토콘드리아 기능이상 (mitochondrial dysfunction)으로 인한 질환의 예방 또는 치료 방법을 제공한다.In addition, the present invention provides a method for preventing or treating diseases caused by mitochondrial dysfunction, comprising administering to an individual a pharmaceutically effective amount of the above pharmaceutical composition.
또한, 본 발명은 약학적으로 유효한 얀의 상기의 약학적 조성물을 개체에 투여하는 단계를 포함하는, 인지기능 장애의 예방 또는 치료 방법을 제공한다.In addition, the present invention provides a method for preventing or treating cognitive dysfunction, comprising administering to an individual the above pharmaceutical composition of a pharmaceutically effective yarn.
본 발명에 따른 조성물은 미토파지의 활성을 촉진하는 효과가 있어, 미토콘드리아 기능이상으로 인한 질환의 치료에 유용하게 사용할 수 있다. The composition according to the present invention has an effect of promoting the activity of mitophagy, and thus can be usefully used in the treatment of diseases caused by mitochondrial dysfunction.
도 1A는 유세포 분석법 (Flow cytometry)에 의한 미토파지 활성 측정 방법을 나타낸 것이다.1A shows a method of measuring mitophagy activity by flow cytometry.
도 1B는 유세포 분석법에 의한 (Flow cytometry)에 의한 미토파지 활성을 그래프로 나타낸 것이다. Figure 1B is a graph showing mitophagy activity by (Flow cytometry) by flow cytometry.
도 2A는 공초점 현미경 (confocal microscopy)에 의한 미토파지의 활성을 형광으로 측정한 것이다.2A is a fluorescence measurement of mitophagy activity by confocal microscopy.
도 2B는 공초점 현미경 (confocal microscopy)에 의한 미토파지의 활성을 확인한 도이다.2B is a diagram confirming the activity of mitophagy by confocal microscopy.
도 2C는 공초점 현미경 (confocal microscopy)에 의한 미토파지의 활성을 측정하는 수학식을 나타낸 도이다.2C is a diagram showing an equation for measuring the activity of mitophagy by confocal microscopy.
도 3A는 인간 폐상피세포주에서 팔마틴에 의한 미토파지의 활성을 유세포 분석법으로 분석한 결과이다.3A is a result of analyzing the activity of mitophagy by palmatin in human lung epithelial cell lines by flow cytometry.
도 3B는은 인간 폐상피세포주에서 팔마틴에 의한 미토파지의 활성을 공초점현미경으로 분석한 결과이다.3B is a result of analyzing the activity of mitophagy by palmatin in a human lung epithelial cell line with a confocal microscope.
도 3C는은 인간 폐상피세포주에서 팔마틴에 의한 미토콘드리아의 양을 분석한 결과이다. 3C is a result of analyzing the amount of mitochondria by palmatin in a human lung epithelial cell line.
도 4A는 Parkin이 발현되는 HeLa 세포 (HeLa-Parkin)에서 팔마틴에 의한 미토파지의 활성을 유세포 분석법으로 분석한 결과이다.4A is a result of analyzing the activity of mitophagy by palmatin in HeLa cells (HeLa-Parkin) expressing Parkin by flow cytometry.
도 4B는 Parkin이 발현되는 HeLa 세포 (HeLa-Parkin)에서 팔마틴에 의한 미토파지의 활성을 공초점 현미경으로 분석한 결과이다.4B is a result of analyzing the activity of mitophagy by palmatin in HeLa cells (HeLa-Parkin) expressing Parkin with a confocal microscope.
도 5A는 팔마틴의 처리농도에 따른 미토파지의 활성을 분석한 결과이다. 5A is a result of analyzing the activity of mitophagy according to the treatment concentration of palmatin.
도 5B는 팔마틴의 처리시간에 따른 미토파지의 활성을 분석한 결과이다. 5B is a result of analyzing the activity of mitophagy according to the treatment time of palmatin.
도 6A는 미토콘드리아, ER 및 Goli 형광 마커를 발현하는 Hela 세포주에서, 팔마틴을 처리하여, 미토콘드리아의 형광 발현 양상을 공초점 현미경으로 확인한 도이다.6A is a diagram showing the fluorescence expression pattern of mitochondria by treatment with palmatin in Hela cell lines expressing mitochondria, ER and Goli fluorescent markers with a confocal microscope.
도 6B는 미토콘드리아, ER 및 Goli 형광 마커를 발현하는 Hela 세포주에서, 팔마틴을 처리하여, 미토콘드리아의 형광 발현 양상을 수치화하여 도식화한 것이다.Figure 6B is a schematic diagram of the mitochondria, ER and Hela cell lines expressing Goli fluorescent markers, treated with palmatin, to quantify the fluorescence expression pattern of mitochondria.
도 7A는 팔마틴의 처리에 따른 미토콘드리아 막전위를 TMRM assay로 분석한 결과이다. 7A is a result of analyzing the mitochondrial membrane potential according to palmatin treatment by TMRM assay.
도 7B는 팔마틴의 처리에 따른 미토콘드리아 활성산소를 MitoSOX assay로 분석한 결과이다. 7B is a result of analyzing mitochondrial active oxygen according to the treatment of palmatin by MitoSOX assay.
도 7C는 팔마틴의 처리에 따른 미토파지의 활성을 분석한 결과이다. 7C is a result of analyzing the activity of mitophagy according to the treatment of palmatin.
도 7D는 NAC (N-Acetylcysteine)처리 및 팔마틴의 처리에 따른 미토콘드리아 활성산소 변화를 MitoSOX assay로 분석한 결과이다. 7D is a result of analysis of changes in mitochondrial active oxygen according to NAC (N-Acetylcysteine) treatment and palmatin treatment by MitoSOX assay.
도 7E는 팔마틴의 처리에 따른 미토콘드리아 활성산소를 DCF-DA assay로 분석한 결과이다. 7E is a result of analysis of mitochondrial free radicals according to the treatment of palmatin by DCF-DA assay.
도 8은 팔마틴 및 AMPK 억제제 (Compound C)를 처리한 후 미토파지의 활성을 분석한 결과이다. 8 is a result of analyzing the activity of mitophagy after treatment with palmatin and an AMPK inhibitor (Compound C).
도 9A는 팔마틴을 처리한 후 NAO 형광 염료를 이용하여 미토콘드리아의 양을 분석한 결과이다. 9A is a result of analyzing the amount of mitochondria using NAO fluorescent dye after treatment with palmatin.
도 9B는 팔마틴을 처리한 후 미토콘드리아 생합성의 조절 인자인 PGC-1α의 mRNA 발현량을 RT-PCR을 통해 분석한 결과이다. 9B is a result of analyzing the mRNA expression level of PGC-1α, a regulator of mitochondrial biosynthesis, through RT-PCR after treatment with palmatin.
도 9C는 팔마틴을 처리한 후 미토콘드리아 생합성의 조절 인자인 NRF1의 mRNA 발현량을 RT-PCR을 통해 분석한 결과이다. 9C is a result of analyzing the mRNA expression level of NRF1, a regulator of mitochondrial biosynthesis, through RT-PCR after palmatin treatment.
도 9D는 팔마틴을 처리한 후 미토콘드리아 생합성의 조절 인자인 TFAM 의 mRNA 발현량을 RT-PCR을 통해 분석한 결과이다.9D is a result of analyzing the mRNA expression level of TFAM, a regulator of mitochondrial biosynthesis, through RT-PCR after palmatin treatment.
도 10A는 PINK1 결핍 MEF 세포주에서, 팔마틴을 처리하였을 때, 미토콘드리아 단백질인 Mfn2 및 COX2 단백질의 발현을 웨스턴 블랏으로 확인한 것이다.Figure 10A shows the expression of mitochondrial proteins Mfn2 and COX2 proteins by Western blot when palmatin was treated in a PINK1-deficient MEF cell line.
도 10B는 PINK1 결핍 MEF 세포주에서, 팔마틴을 처리하였을 때, 활성산소가 감소하는 것을 확인한 그래프이다.FIG. 10B is a graph confirming that when palmatin was treated in a PINK1-deficient MEF cell line, free radicals decreased.
도 10C는 PINK1 결핍 MEF 세포주에서, 팔마틴을 처리하였을 때, 미토콘드리아 호흡율이 증가한 것을 확인한 도이다.10C is a diagram confirming that the mitochondrial respiration rate increased when palmatin was treated in the PINK1 deficient MEF cell line.
도 11은 근위축성 측삭경화증 (Amyotrophic lateral sclerosis, ALS) 초파리 동물모델에 팔마틴을 투여하여, 운동개선 효과를 확인한 도이다.11 is a diagram illustrating the effect of improving movement by administering palmatin to a Drosophila animal model of amyotrophic lateral sclerosis (ALS).
도 12는 알츠하이며 동물모델인 Aβ 과발현 초파리 동물모델에 팔마틴을 투여하여, 운동개선 효과를 확인한 도이다.12 is a diagram showing the effect of improving exercise by administering palmatin to an animal model of Aβ overexpressing Drosophila, which is Alzha and is an animal model.
본 발명은 팔마틴 (palmatine)을 유효성분으로 포함하는 미토콘드리아 기능이상 (mitochondrial dysfunction)으로 인한 질환의 예방 또는 치료용 조성물을 제공한다. The present invention provides a composition for preventing or treating diseases caused by mitochondrial dysfunction, comprising palmatine as an active ingredient.
본 발명에 따른 화합물인 팔마틴 (palmatine, CAS Number: 3486-67-7)은 하기 화학식 1 로 표시되는 화합물을 의미한다. Palmatin (palmatine, CAS Number: 3486-67-7), which is a compound according to the present invention, refers to a compound represented by the following formula (1).
[화학식 1][Formula 1]
Figure PCTKR2020008247-appb-I000001
Figure PCTKR2020008247-appb-I000001
본 발명의 일 실시예에 있어서, 상기 팔마틴은 미토파지 (mitophagy)의 활성을 촉진시키는 것일 수 있다. In one embodiment of the present invention, the palmatin may promote the activity of mitophagy (mitophagy).
본 발명의 일 실시예에 있어서, 팔마틴은 미토콘드리아의 세포호흡율을 증가시키는 것일 수 있다.In one embodiment of the present invention, palmatin may increase mitochondrial cellular respiration.
본 발명의 용어, "미토파지 (mitophagy)"는 손상되었거나 불필요한 미토콘드리아를 제거하는 세포 내 분해기전으로서, 미토콘드리아의 손상이 발생한 경우 오토파고좀(autophagosome)을 형성하고 리소좀과 융합하여 손상된 미토콘드리아를 선택적으로 분해하여 제거할 수 있다. The term "mitophagy" of the present invention is an intracellular decomposition mechanism that removes damaged or unnecessary mitochondria. When damage to the mitochondria occurs, an autophagosome is formed and fused with lysosomes to selectively select the damaged mitochondria. Can be disassembled and removed.
본 발명의 일 실시예에 있어서, 상기 팔마틴은 0.1 내지 200 μM의 농도로 투여되는 것일 수 있고, 바람직하게는 1 내지 100 μM의 농도인 것일 수 있고, 더욱 바람직하게는 20 내지 100 μM의 농도인 것일 수 있고, 더욱 바람직하게는 20 내지 80 μM의 농도인 것일 수 있으나, 이에 제한되는 것은 아니다. In one embodiment of the present invention, the palmatine may be administered at a concentration of 0.1 to 200 μM, preferably may be a concentration of 1 to 100 μM, more preferably a concentration of 20 to 100 μM It may be, more preferably, it may be a concentration of 20 to 80 μM, but is not limited thereto.
본 발명에 따른 조성물은 약학적으로 허용가능한 담체를 추가로 포함할 수 있다. 본 발명에서 용어, "약학적으로 허용가능한"이란 상기 조성물에 노출되는 세포나 인간에게 독성이 없는 특성을 나타내는 것을 의미한다. 상기 담체는 완충제, 보존제, 무통화제, 가용화제, 등장제, 안정화제, 기제, 부형제, 윤활제 등 당업계에 공지된 것이라면 제한 없이 사용할 수 있다. The composition according to the present invention may further comprise a pharmaceutically acceptable carrier. In the present invention, the term "pharmaceutically acceptable" means exhibiting properties that are not toxic to cells or humans exposed to the composition. The carrier may be used without limitation as long as it is known in the art such as a buffering agent, a preservative, a painless agent, a solubilizing agent, an isotonic agent, a stabilizer, a base agent, an excipient, and a lubricant.
또한, 본 발명의 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 나아가, 연고제, 로션제, 스프레이제, 패취제, 크림제, 산제, 현탁제, 겔제 또는 젤의 형태의 피부 외용제의 형태로 사용될 수 있다. 본 발명의 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다.In addition, the composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. have. Further, it may be used in the form of an ointment, lotion, spray, patch, cream, powder, suspension, gel or gel for external skin. Carriers, excipients and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used.
경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 싸이코트리아 루브라 추출물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트 (calcium carbonate), 수크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스티레이트, 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데, 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜 (propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈 (tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient, such as starch, calcium carbonate, in the Cycotria rubra extract, It is prepared by mixing sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. have. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like can be used.
본 발명의 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명의 용어 "투여"란 적절한 방법으로 개체에게 소정의 물질을 도입하는 것을 의미하며 상기 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여 투여될 수 있다. 복강내 투여, 정맥내 투여, 근육내 투여, 피하 투여, 피내 투여, 경구 투여, 국소 투여, 비내 투여, 폐내 투여, 직장내 투여될 수 있으나, 이에 제한되지는 않는다.The composition of the present invention is administered in a pharmaceutically effective amount. The term "administration" of the present invention means introducing a predetermined substance to an individual by an appropriate method, and the route of administration of the composition may be administered through any general route as long as it can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration may be administered, but are not limited thereto.
상기 용어, "개체"란 인간을 포함한 쥐, 생쥐, 가축 등의 모든 동물을 의미한다. 바람직하게는, 인간을 포함한 포유동물일 수 있다.The term "individual" refers to all animals including humans, such as mice, mice, and domestic animals. Preferably, it may be a mammal including a human.
상기 용어, "약학적으로 유효한 양"이란 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분하며 부작용을 일으키지 않을 정도의 양을 의미하며, 유효 용량 수준은 환자의 성별, 연령, 체중, 건강 상태, 질병의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 방법, 투여 시간, 투여 경로, 및 배출 비율, 치료 기간, 배합 또는 동시에 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 당업자에 의해 용이하게 결정될 수 있다. 투여는 상기 권장 투여량을 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다.The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not cause side effects, and the effective dose level is the sex, age, and Weight, health status, type of disease, severity, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration, and rate of excretion, duration of treatment, factors including drugs used in combination or concurrently, and other fields of medicine. It can be readily determined by a person skilled in the art according to well-known factors. Administration may be administered once a day at the recommended dosage, or may be divided several times.
본 발명의 일 실시예에 있어서, 상기 미토콘드리아 기능이상으로 인한 질환은 퇴행성 질환, 암, 알러지성 질환 및 인지기능 장애로 이루어진 군에서 선택된 것일 수 있다.In one embodiment of the present invention, the disease due to the mitochondrial dysfunction may be selected from the group consisting of degenerative diseases, cancer, allergic diseases, and cognitive dysfunction.
본 발명의 일 실시예에 있어서, 상기 미토콘드리아 기능이상으로 인한 질환은 MELAS 증후군 (mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes), 루게릭병 (amyotrophic lateral sclerosis, ALS) 및 샤르코 마리 투스 질환 (Charcot Marie Tooth disease, CMT)으로 이루어진 군에서 선택되는 어느 하나인 것일 수 있다. In one embodiment of the present invention, the disease due to mitochondrial dysfunction is MELAS syndrome (mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes), ALS (amyotrophic lateral sclerosis, ALS), and Charcot Marie's disease (Charcot Marie Tooth disease, CMT) may be any one selected from the group consisting of.
본 발명의 일 실시예에 있어서, 상기 퇴행성 질환은 알츠하이머병 (Alzheimer's disease), 파킨슨병 (Parkinson's disease), 루게릭병(amyotrophic lateral sclerosis), 헌팅턴병 (Huntington's Disease), 다발성 경화증(Multiple sclerosis), 면역계이상 뇌기능 부전, 진행성 신경퇴행질환, 대사성 뇌질환, 니만-픽병(Niemann-Pick disease), 뇌 허혈 및 뇌출혈로 인한 치매(dementia)로 이루어진 군으로부터 선택된 1종 이상인 것일 수 있으며, 바람직하게는 파킨슨병이나, 이에 제한되지는 않는다.In one embodiment of the present invention, the degenerative disease is Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, multiple sclerosis, and immune system abnormalities. It may be one or more selected from the group consisting of brain insufficiency, progressive neurodegenerative disease, metabolic brain disease, Niemann-Pick disease, cerebral ischemia, and dementia due to cerebral hemorrhage, and preferably Parkinson's disease. However, it is not limited thereto.
본 발명의 일 실시예에 있어서, 상기 암은 대장암, CTH 생성 종양, 급성 림프구성 또는 림프아구성 백혈병, 급성 또는 만성의 림포구성 백혈병, 급성 비림프구성 백혈병, 방광암, 뇌종양, 유방암, 경관암, 만성 골수성 백혈병, 장암, T-존 림프종, 자궁내막증, 식도암, 담즙 방광암, 에윙스 육종(Ewing's sarcoma), 두 및 목암, 설암, 홉킨스 림프종, 카포시스 육종, 신장암, 간암, 폐암, 중피종, 다발성 골수종, 신경아세포종, 비홉킨 림프종, 골육종, 난소암, 신경아세포종, 유선암, 경관암, 전립선암, 췌장암, 페니스암, 레티노블라스토마, 피부암, 위암, 갑상선암, 자궁암, 고환암, 윌름스 종양, 및 트로포블라스토마로 이루어진 군으로부터 선택된 1종 이상인 것일 수 있으나 이에 제한되지는 않는다.In one embodiment of the present invention, the cancer is colon cancer, CTH-producing tumor, acute lymphocytic or lymphoblastic leukemia, acute or chronic lymphocytic leukemia, acute non-lymphocyte leukemia, bladder cancer, brain tumor, breast cancer, cervical cancer , Chronic myelogenous leukemia, bowel cancer, T-zone lymphoma, endometriosis, esophageal cancer, gall bladder cancer, Ewing's sarcoma, head and neck cancer, tongue cancer, Hopkins' lymphoma, Caposis' sarcoma, kidney cancer, liver cancer, lung cancer, mesothelioma, Multiple myeloma, neuroblastoma, non-Hopekin's lymphoma, osteosarcoma, ovarian cancer, neuroblastoma, mammary cancer, cervical cancer, prostate cancer, pancreatic cancer, penis cancer, retinoblastoma, skin cancer, gastric cancer, thyroid cancer, uterine cancer, testicular cancer, Wilms' tumor, and It may be one or more selected from the group consisting of trophoblastoma, but is not limited thereto.
본 발명의 일 실시예에 따르면, 상기 알러지성 질환은, 아토피성 피부염(atopic dermatitis), 알러지성 피부염(allergic dermatitis), 접촉성 피부염, 두드러기, 가려움증, 곤충 알러지, 식품 알러지, 약품 알러지, 부종, 과민증(anaphylaxis), 알러지성 비염(allergic rhinitis), 천식(asthma) 및 알러지성 결막염(allergic conjunctivitis) 중에서 선택된 1종 이상인 것일 수 있으나 이에 제한되지는 않는다.According to an embodiment of the present invention, the allergic disease is atopic dermatitis, allergic dermatitis, contact dermatitis, urticaria, itching, insect allergy, food allergy, drug allergy, edema, Hypersensitivity (anaphylaxis), allergic rhinitis (allergic rhinitis), asthma (asthma) and allergic conjunctivitis (allergic conjunctivitis) may be one or more selected from, but is not limited thereto.
또한, 본 발명은 (a) 미토케이마 (mt-Keima)를 발현하는 세포에 시험물질을 처리하는 단계; 및 (b) 미토파지 (mitophagy)의 활성을 측정하는 단계;를 포함하는 미토콘드리아 기능이상 (mitochondrial dysfunction)으로 인한 질환 치료제의 스크리닝 방법을 제공한다. In addition, the present invention comprises the steps of: (a) treating a test substance on cells expressing mt-Keima; And (b) measuring the activity of mitophagy (mitophagy); provides a method for screening a therapeutic agent for diseases caused by mitochondrial dysfunction comprising.
본 발명의 용어, "미토케이마(mt-Keima, mitochondria-targeted Keima)"는 pH 의존적으로 형광특성이 변화하는 산호유래 형광단백질 (Katayama H et al, 2011, Chem Biol)을 말한다. The term "mitokeima (mt-Keima, mitochondria-targeted Keima)" of the present invention refers to a coral-derived fluorescent protein whose fluorescence properties change in a pH-dependent manner (Katayama H et al, 2011, Chem Biol).
본 발명의 일 실시예에 있어서, 상기 미토파지의 활성은 유세포분석법 (flow cytometry) 또는 공초점 현미경 (confocal microscopy)에 의하여 측정되는 것일 수 있다. In one embodiment of the present invention, the mitophagy activity may be measured by flow cytometry or confocal microscopy.
본 발명의 일 실시예에 있어서, 상기 시험물질이 미토파지의 활성을 촉진시킨 경우 미토콘드리아 기능이상으로 인한 질환 치료제인 것으로 판단하는 단계를 추가적으로 더 포함하는 것일 수 있다. In one embodiment of the present invention, when the test substance promotes mitophagy activity, determining that it is a therapeutic agent for a disease due to mitochondrial dysfunction may be additionally included.
또한, 본 발명은 시험관 내 (in vitro)에서 팔마틴 (palamtine)을 미토콘드리아 기능이상 세포에 처리하는 단계를 포함하는 미토파지의 활성을 촉진시키는 방법을 제공한다. In addition, the present invention provides a method for promoting mitophagy activity, including the step of treating mitochondrial dysfunction cells with palmatin in vitro .
또한, 본 발명은 팔마틴 (palamtine)을 유효성분으로 포함하는 인지기능 장애 예방 또는 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating cognitive dysfunction comprising palmatin (palamtine) as an active ingredient.
본 발명의 일 실시예에 있어서, 상기 인지기능 장애는, 무기력, 기억력 감퇴, 건망증, 인지력 감퇴, 학습장애, 주의력 감퇴, 우울증, 청력 감퇴, 무통증, 무발한증 및 식별력 감퇴로 구성되는 군으로부터 선택된 1종 이상인 것일 수 있으나 이에 제한되지는 않는다.In one embodiment of the present invention, the cognitive dysfunction is from the group consisting of lethargy, memory loss, forgetfulness, cognitive decline, learning disability, attention loss, depression, hearing loss, painlessness, inactivity and discrimination. It may be one or more selected, but is not limited thereto.
또한, 본 발명은 팔마틴 (palamtine)을 유효성분으로 포함하는 인지기능 장애 예방 또는 개선용 식품 조성물을 제공한다.In addition, the present invention provides a food composition for preventing or improving cognitive dysfunction comprising palmatine (palamtine) as an active ingredient.
또한, 본 발명은 약학적으로 유효한 양의 상기의 약학적 조성물을 개체에 투여하는 단계를 포함하는, 미토콘드리아 기능이상 (mitochondrial dysfunction)으로 인한 질환의 예방 또는 치료 방법을 제공한다.In addition, the present invention provides a method for preventing or treating diseases caused by mitochondrial dysfunction, comprising administering to an individual a pharmaceutically effective amount of the above pharmaceutical composition.
또한, 본 발명은 약학적으로 유효한 양의 상기의 약학적 조성물을 개체에 투여하는 단계를 포함하는, 인지기능 장애의 예방 또는 치료 방법을 제공한다.In addition, the present invention provides a method for preventing or treating cognitive dysfunction, comprising administering to an individual a pharmaceutically effective amount of the pharmaceutical composition.
또한 본 발명은 약학적으로 유효한 양의 상기 약학적 조성물을 개체에 투여하는 단계를 포함하는 미토콘드리아 기능이상 또는 인지기능 장애의 예방 및 치료방법을 제공한다. 본 발명의 약학 조성물은 치료적 유효량 또는 약학으로 유효한 양으로 투여한다. 용어 "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 개체 종류 및 중증도, 연령, 성별, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다.In addition, the present invention provides a method for preventing and treating mitochondrial dysfunction or cognitive dysfunction comprising administering to an individual a pharmaceutically effective amount of the pharmaceutical composition. The pharmaceutical composition of the present invention is administered in a therapeutically effective amount or in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type and severity of the subject, age, sex, activity of the drug, and Sensitivity, administration time, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field.
이하, 본 발명을 실시예를 통하여 더욱 상세히 설명하기로 한다. 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention more specifically, and the scope of the present invention is not limited to these examples.
실시예 1. 미토파지의 활성 측정 방법Example 1. Method for measuring mitophagy activity
1.1. 미토케이마 발현 세포주의 제작 1.1. Construction of mitokeima-expressing cell line
본 발명자들은 미토케이마가 발현된 세포주를 제작하기 위한 실험을 수행하였다. 간단히, 100-mm 배양 접시는 0.001 % poly-L-lysine으로 실온에서 10분 동안 코팅하여 준비하고, 4 x 106 개의 293L 세포를 코팅된 배양접시에 분주한 후, 10 % FBS가 첨가된 DMEM 배양액에서 24시간 동안 배양하였다. 이후, 미토케이마 플라스미드 DNA (pLVX-mt-Keima, 3 μg)와 packaging DNA (psPAX2, 3.75 μg; 및 pVSVG, 0.375 μg)를 넣고 8시간 동안 형질주입 (transfection)을 수행하였다. 이후, 신선항 배양액으로 교환하고 48시간 동안 추가 배양한 후, 바이러스가 포함된 배양액을 취하여 0.45 μm 필터로 걸러 미토케이마 렌티바이러스를 준비하였다. The present inventors performed an experiment for producing a cell line expressing mitokema. Briefly, a 100-mm culture dish was prepared by coating it with 0.001% poly-L-lysine for 10 minutes at room temperature. After dispensing 4 x 10 6 293L cells into the coated culture dish, DMEM with 10% FBS added. Incubated for 24 hours in the culture medium. Thereafter, the mitokeima plasmid DNA (pLVX-mt-Keima, 3 μg) and packaging DNA (psPAX2, 3.75 μg; and pVSVG, 0.375 μg) were added and transfection was performed for 8 hours. Thereafter, the culture medium was exchanged with a fresh port culture medium and further cultured for 48 hours, and then a culture medium containing the virus was taken and filtered through a 0.45 μm filter to prepare a mitokeima lentivirus.
미토케이마를 발현시키고자 하는 세포는 60-mm 배양 접시에서 24시간 배양하여 준비하였고, 미리 제작한 미토케이마 렌티바이러스 1 ml과 배양액 1 ml을 섞은 후 미토케이마를 발현시키고자 하는 세포에 넣어 24시간 동안 감염시켰다. 이후, 배양액을 제거하고, 푸로마이신(puromycin, 2 μg/ml)을 처리하여 바이러스가 도입된 세포만을 선별하였다. Cells to be expressed mitokema were prepared by incubating for 24 hours in a 60-mm culture dish, 1 ml of pre-produced mitokema lentivirus and 1 ml of culture solution were mixed, and then added to the cells to be expressed mitokema. Infected for hours. Thereafter, the culture medium was removed, and puromycin (2 μg/ml) was treated to select only cells into which the virus was introduced.
1.2. 유세포 분석법 (Flow cytometry)에 의한 미토파지 활성 측정 방법1.2. Method for measuring mitophagy activity by flow cytometry
유세포분석기 (FACS)를 사용한 미토케이마를 발현하는 세포의 미토파지 활성 측정은 기존 논문의 방법에 따라 수행하였다 (J Vis Exp. 2018 Aug 12;(138)). 미토파지의 활성을 분석하기 위하여 미토케이마 형광은 405 nm 및 561 nm의 두 레이저에 의해 동시에 측정하였다. 즉, 각 세포의 405 nm 레이저에 의한 형광신호를 610 ± 10 nm 검출기로 검출한 신호 (BV605)와 동시에 561 nm 레이저에 의한 형광신호를 610 ± 10 nm 검출기로 측정한 신호 (PE-CF594)를 점 도식 (dot plot)으로 표시한 후, 미토파지 활성이 높아 "high" 게이트 내에 위치하는 세포의 비율을 미토파지 활성으로 정의하였다 (도 1).The measurement of mitophagy activity of cells expressing mitokema using flow cytometry (FACS) was performed according to the method of the previous paper (J Vis Exp. 2018 Aug 12;(138)). In order to analyze the activity of mitophagy, mitokema fluorescence was simultaneously measured by two lasers at 405 nm and 561 nm. That is, the signal obtained by detecting the fluorescence signal by the 405 nm laser of each cell with a 610 ± 10 nm detector (BV605) and the signal by measuring the fluorescent signal by the 561 nm laser by the 610 ± 10 nm detector (PE-CF594) After plotting with a dot plot, the proportion of cells located in the "high" gate due to high mitophagy activity was defined as mitophagy activity (FIG. 1).
1.3. 공초점 현미경 (confocal microscopy)에 의한 미토파지의 활성 측정 방법 1.3. Method for measuring mitophagy activity by confocal microscopy
공초점 현미경을 사용한 미토케이마 형광분석에서 488 nm 레이저로부터의 형광신호를 녹색 (green)으로 나태내고, 555 nm 레이저로부터의 형광신호를 적색 (red)으로 나타낸 경우, 정상 미토콘드리아는 녹색으로 나타나고, 미토파지에 의해 리소좀 내로 들어간 미토콘드리아는 적색 반점 (red puncta)으로 나타나게 된다. 미토파지 활성은 공초점 현미경 이미지의 전체 픽셀가운데 미토파지 활성이 높아 적색으로 나타나는 픽셀의 비율을 미토파지 활성으로 계산하였다 (도 2).In mitokeima fluorescence analysis using a confocal microscope, when the fluorescence signal from the 488 nm laser is displayed in green and the fluorescence signal from the 555 nm laser is displayed in red, normal mitochondria appear in green, Mitochondria that enter the lysosome by mitophagy appear as red puncta. Mitophagy activity was calculated as the ratio of pixels that appear red due to high mitophagy activity among all the pixels of the confocal microscope image (FIG. 2).
실시예 2. 미토파지 활성 조절 화합물의 발굴Example 2. Discovery of mitophagy activity modulating compound
본 발명자들은 식물유래 생리활성물질 라이브러리를 스크리닝하여 구조적으로 유사한 이소퀴놀린 알카로이드인 팔마틴 (Palmatine)을 미토파지의 활성 촉진물질로 발굴하였다.The present inventors screened a library of plant-derived physiologically active substances and discovered palmatin, a structurally similar isoquinoline alkaloid, as a mitophagy activity promoting substance.
실시예 3. 인간 폐상피세포주에서 팔마틴에 의한 미토파지의 활성 촉진 효과 분석Example 3. Analysis of the effect of promoting mitophagy activity by palmatin in human lung epithelial cell lines
본 발명자들은 팔마틴에 의한 미토파지의 활성 촉진 효과를 검증하기 위한 실험을 수행하였다. 간단히, 미토케이마를 발현하는 인간 폐상피세포주인 BEAS-2B 세포에 팔마틴 (400 μM) 또는 미토콘드리아 막전위 저해제 (uncoupler)로 알려진 CCCP (10 μM)를 24 시간 동안 처리한 후, 유세포분석기 또는 공초점현미경을 사용하여 미토파지의 활성을 측정하거나 미토콘드리아 양적 감소에 의한 미토파지의 활성을 측정하였다. The present inventors conducted an experiment to verify the effect of promoting the activity of mitophagy by palmatin. Briefly, in BEAS-2B cells, a human lung epithelial cell line expressing mitokema, palmatin (400 μM) or CCCP (10 μM), known as a mitochondrial membrane potential inhibitor (uncoupler), was applied to 24 cells. After treatment for a period of time, the activity of mitophagy was measured using a flow cytometer or a confocal microscope, or the activity of mitophagy due to the quantitative reduction of mitochondria was measured.
그 결과, 유세포분석기를 사용하여 미토파지의 활성을 측정한 경우 팔마틴을 처리한 그룹에서는 대조군(con)에 비해 미토파지의 활성이 현저하게 증가되었음을 확인하였다 (도 3A). 또한, 공초점현미경을 사용하여 미토파지의 활성을 측정한 경우에도 팔마틴을 처리한 그룹에서는 대조군(con)에 비해 미토파지의 활성이 증가되었음을 확인하였다 (도 3B). 미토콘드리아의 양에 의한 미토파지의 활성을 측정한 경우에도 팔마틴을 처리한 경우 대조군(con)에 비해 미토콘드리아의 양이 감소된 것을 확인하였고, 이는 팔마틴에 의해 미토파지의 활성이 증가되었음을 간접적으로 확인하였다(도 3c).As a result, when the activity of mitophagy was measured using a flow cytometer, it was confirmed that the activity of mitophagy was significantly increased in the palmatin-treated group compared to the control group (con) (FIG. 3A ). In addition, even when the activity of mitophagy was measured using a confocal microscope, it was confirmed that the activity of mitophagy was increased in the group treated with palmatin compared to the control group (con) (FIG. 3B ). Even when the mitophage activity by the amount of mitochondria was measured, it was confirmed that the amount of mitochondria decreased compared to the control group (con) when palmatin was treated, which indirectly indicated that the mitophage activity was increased by palmatin. It was confirmed (Fig. 3c).
실시예 4. 자궁경부암 세포주에서 팔마틴에 의한 미토파지의 활성 촉진 효과 분석Example 4. Analysis of the effect of promoting mitophagy activity by palmatin in cervical cancer cell lines
본 발명자들은 다른 세포주에서도 팔마틴에 의하여 미토파지의 활성이 촉진되는지 확인하는 실험을 수행하였다. 간단히, Parkin (E3 ubiquitin ligase)이 발현되는 자궁경부암 세포주인 HeLa 세포 (HeLa-Parkin)에 팔마틴 또는 CCCP를 처리한 후 미토파지의 활성을 분석하였다. The present inventors conducted an experiment to confirm whether the activity of mitophagy is promoted by palmatin in other cell lines. Briefly, Parkin (E3 ubiquitin ligase)-expressing cervical cancer cell line HeLa cells (HeLa-Parkin) were treated with palmatin or CCCP and then mitophagy activity was analyzed.
그 결과, Parkin이 발현되는 HeLa 세포에서는 팔마틴의 처리에 의해 미토파지의 활성이 현저하게 증가됨을 확인하였다 (도 4A). 미토콘드리아의 양을 분석한 결과에서도 팔마틴을 처리한 경우 미토콘드리아의 양이 감소된 것으로 확인되었고, 이는 유세포분석기를 사용하여 미토파지의 활성을 분석한 결과와 마찬가지로 팔마틴을 처리한 경우 미토파지의 활성이 증가됨을 간접적으로 확인하였다 (도 4B). As a result, it was confirmed that in HeLa cells expressing Parkin, mitophagy activity was remarkably increased by treatment with palmatin (FIG. 4A). As a result of analyzing the amount of mitochondria, it was confirmed that the amount of mitochondria decreased when palmatin was treated. This is similar to the result of analyzing the activity of mitophagy using a flow cytometer, and mitophagy activity when palmatin was treated. It was confirmed indirectly that this increase (Fig. 4B).
실시예 5. 팔마틴의 처리농도 및 처리시간에 따른 미토파지의 활성 촉진 효과 분석Example 5. Analysis of the effect of promoting mitophagy activity according to the treatment concentration and treatment time of palmatin
본 발명자들은 팔마틴의 처리농도 및 처리시간에 따른 미토파지의 활성 촉진 효과를 분석하는 실험을 수행하였다. 간단히, 팔마틴은 미토케이마를 발현하는 BEAS-2B 세포에 주어진 농도 (0, 5, 10, 20, 40, 60, 80, 100, 200, 400 및 800 μM)에서 24시간 동안 처리한 후, 유세포분석기을 이용하여 미토파지 활성을 측정하였다. 또한, BEAS-2B 세포에 팔마틴 (400 μM)을 주어진 시간 (0, 3, 6, 12, 18 및 24 시간) 동안 처리한 후, 유세포분석기을 이용하여 미토파지 활성을 측정하였다. The present inventors conducted an experiment to analyze the effect of promoting mitophagy activity according to the treatment concentration and treatment time of palmatin. Briefly, palmatin was treated for 24 hours at a given concentration (0, 5, 10, 20, 40, 60, 80, 100, 200, 400 and 800 μM) to BEAS-2B cells expressing mitokema, followed by flow cytometry. Mitophagy activity was measured using an analyzer. In addition, BEAS-2B cells were treated with palmatin (400 μM) for a given time (0, 3, 6, 12, 18 and 24 hours), and then mitophagy activity was measured using a flow cytometer.
그 결과, 팔마틴의 처리농도가 5 내지 40 μM 에서는 미토파지의 활성에 어떠한 차이가 나타나지 않았고, 80 μM 부터 미토파지의 활성이 증가하였으며, 800 μM 까지 농도의존적으로 증가하였음을 확인하였다 (도 5A). 또한, 팔마틴의 처리시간의 경우 3 시간부터 미토파지의 활성이 증가하였고, 24 시간에 최대로 활성이 증가하였음을 확인하였다 (도 5B).As a result, when the treatment concentration of palmatin was 5 to 40 μM, there was no difference in mitophagy activity, and it was confirmed that the mitophagy activity increased from 80 μM, and concentration-dependently increased to 800 μM (Fig. 5A. ). In addition, in the case of the treatment time of palmatin, the activity of mitophagy increased from 3 hours, and it was confirmed that the activity increased maximally at 24 hours (Fig. 5B).
실시예 6. 팔마틴에 의한 미토파지 특이적 유도활성 확인Example 6. Confirmation of mitophagy-specific induction activity by palmatin
본 발명자들은 팔마틴에 의한 미토파지 특이적 유도활성을 확인하기 위하여, 미토콘드리아(mito-YFP), 세포소기관 ER(ER-GFP) 및 세포소기관 Golgi(Golgi-GFP) 형광마커를 각각 발현하는 Hela 세포주들에 팔마틴을 200 또는 400 μM 농도로 처리하여, 24시간 배양하였다. 배양 후 각 세포소기관 형광신호의 변화를 공초점 현미경으로 분석하여 가시화 하였다.In order to confirm the mitophagy-specific induction activity by palmatine, the present inventors described a Hela cell line expressing each of the mitochondria (mito-YFP), organelle ER (ER-GFP) and organelle Golgi (Golgi-GFP) fluorescent markers. The fields were treated with palmatin at a concentration of 200 or 400 μM, and cultured for 24 hours. After incubation, changes in the fluorescence signal of each organelle were analyzed and visualized with a confocal microscope.
그 결과 도 6에 나타낸 바와 같이, 팔마틴의 농도가 증가할수록 미토콘드리아의 형광이 유의적으로 감소하는 것을 확인 하였으며, ER 및 Golgi에서는 형광의 변화가 없는 것을 확인하여, 팔마틴이 다른 세포소기관에는 영향을 끼치지 않으면서, 미토파지에 특이적인 활성화를 유도하는 것을 확인하였다.As a result, as shown in Figure 6, it was confirmed that the fluorescence of mitochondria significantly decreased as the concentration of palmatin increased, and it was confirmed that there was no change in fluorescence in ER and Golgi, and palmatin affected other organelles. It was confirmed that it induces mitophagy-specific activation without causing
실시예 7. 팔마틴에 의한 미토파지 활성화에서 미토콘드리아 막전위 및 활성산소 변화 분석Example 7. Analysis of changes in mitochondrial membrane potential and active oxygen in mitophagy activation by palmatin
본 발명자들은 팔마틴에 의해 미토콘드리아의 막전위 및 활성산소에 어떠한 변화가 일어나는지 확인하는 실험을 수행하였다. 미토콘드리아의 막전위(mitochondrial membrane potential)는 TMRM (tetramethylrhodamine methyl ester) 어세이로 분석되었고, 미토콘드리아의 활성산소(ROS)는 MitoSOX 어세이 및 DCF-DA 어세이를 통해 분석되었다. The present inventors conducted an experiment to determine what changes occur in mitochondrial membrane potential and active oxygen by palmatin. The mitochondrial membrane potential was analyzed by TMRM (tetramethylrhodamine methyl ester) assay, and the mitochondrial reactive oxygen (ROS) was analyzed by MitoSOX assay and DCF-DA assay.
그 결과, 팔마틴을 처리한 경우 CCCP를 처리한 경우에 비해 미토콘드리아의 막전위에 큰 차이가 나타나지 않았다 (도 7A). 활성산소(ROS)의 경우에도 CCCP를 처리한 그룹에서는 현저하게 증가되었으나, 팔마틴을 처리한 그룹에서는 큰 차이가 나타나지 않았다 (도 7B). As a result, when palmatin was treated, there was no significant difference in the mitochondrial membrane potential compared to the CCCP treatment (FIG. 7A ). In the case of active oxygen (ROS), it was significantly increased in the CCCP-treated group, but there was no significant difference in the palmatin-treated group (FIG. 7B).
특히, 항산화제인 NAC (N-Acetylcysteine)를 처리한 경우 CCCP 에 의한 미토파지의 활성은 억제된 반면, 팔마틴에 의한 미토파지의 활성은 항산화제의 처리와 관계없이 유지되는 것을 확인하였다 (도 7C). 또한, NAC를 처리한 경우 CCCP에 의해 증가된 ROS가 현저하게 감소되었으나, 팔마틴에서는 ROS가 크게 감소되지 않았다 (도 7D 및 7E). In particular, it was confirmed that when treatment with the antioxidant NAC (N-Acetylcysteine) inhibited the activity of mitophagy by CCCP, the activity of mitophagy by palmatin was maintained regardless of the treatment of the antioxidant (Fig. 7C). ). In addition, when NAC was treated, the ROS increased by CCCP was significantly reduced, but the ROS was not significantly reduced in Palmatin (FIGS. 7D and 7E ).
상기 결과에 따르면, 팔마틴은 CCCP와 같은 미토콘드리아의 독소들과 다른 메커니즘에 의해 미토파지의 활성을 증가시키는 것으로 확인되었다. According to the above results, it was confirmed that palmatin increases mitophagy activity by a mechanism different from mitochondrial toxins such as CCCP.
실시예 8. 팔마틴에 의한 미토파지 활성화에서 AMPK (AMP-activated protein kinase) 역할 규명Example 8. Identification of the role of AMPK (AMP-activated protein kinase) in mitophagy activation by palmatin
AMPK (AMP-activated protein kinase)의 활성화는 오토파지 및 미토파지의 개시 단계에서 중요한 것으로 보고되고 있다. 이에 본 발명자들은 팔마틴이 AMPK에 의존적으로 미토파지의 활성을 촉진하는지 확인하는 실험을 수행하였다. 간단히, 미토케이마를 발현하는 BEAS-2B 세포에 팔마틴 (400 μM)을 여러 다른 농도(2.5, 5 및 10 μM)의 AMPK 억제제 (inhibitor)인 Compound C 와 함께 3시간 동안 처리한 후, 유세포분석기를 이용하여 미토파지의 활성을 분석하였다. Activation of AMPK (AMP-activated protein kinase) is reported to be important in the initiation stage of autophagy and mitophagy. Accordingly, the present inventors conducted an experiment to confirm whether palmatin promotes mitophagy activity dependent on AMPK. Briefly, MITOKEMA-expressing BEAS-2B cells were treated with palmatin (400 μM) at various concentrations (2.5, 5 and 10 μM) with Compound C, an AMPK inhibitor, for 3 hours, followed by flow cytometry. Was used to analyze the activity of mitophagy.
그 결과, 2.5 및 5 μM의 Compund C가 처리된 경우에는 어떠한 효과가 나타나지 않았으나, 10 μM의 Compund C가 처리된 경우 팔마틴에 의해 증가된 미토파지의 활성이 감소되었음을 확인하였다 (도 8). 상기 결과로부터, 팔마틴은 AMPK에 의존적으로 미토파지의 활성을 촉진한다는 것을 확인하였다. As a result, when 2.5 and 5 μM of Compund C were treated, no effect was observed, but when 10 μM of Compund C was treated, it was confirmed that the increased mitophagy activity by palmatin decreased (FIG. 8 ). From the above results, it was confirmed that palmatin promotes mitophagy activity dependent on AMPK.
실시예 9. 팔마틴에 의한 미토콘드리아 생합성 촉진Example 9. Promoting mitochondrial biosynthesis by palmatin
본 발명자들은 팔마틴에 의한 미토파지의 활성 촉진이 일어난 후에 새로운 미토콘드리아의 생합성이 촉진되는지 분석하는 실험을 수행하였다. NAO (10-N-nonyl acridine orange)인 형광 염료를 이용하여 미토콘드리아의 양을 분석한 결과, 팔마틴을 처리한 직후에는 미토콘드리아의 양이 감소하였다가 시간이 지남에 따라 조금씩 회복됨을 확인하였다 (도 9A). The present inventors conducted an experiment to analyze whether the biosynthesis of new mitochondria is promoted after the activation of mitophagy by palmatin occurs. As a result of analyzing the amount of mitochondria using a fluorescent dye of NAO (10-N-nonyl acridine orange), it was confirmed that the amount of mitochondria decreased immediately after palmatin treatment, and then recovered little by little over time (Fig. 9A).
또한, 미토콘드리아 생합성에 중요한 조절 인자로 알려진 PGC-1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha), NRF1 (nuclear respiratory factor 1) 및 TFAM (mitochondrial transcription factor A)의 mRNA 발현량을 RT-PCR을 이용하여 확인한 결과, PGC-1α, NRF1 및 TFAM의 mRNA는 팔마틴을 처리한 후 48 시간에서 유의미하게 증가하였음을 확인하였다 (도 9B, 9C 및 9D). In addition, the mRNA expression levels of PGC-1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha), NRF1 (nuclear respiratory factor 1), and TFAM (mitochondrial transcription factor A), which are known as important regulatory factors for mitochondrial biosynthesis, were measured by RT-PCR. As a result of confirming using, it was confirmed that the mRNA of PGC-1α, NRF1 and TFAM significantly increased at 48 hours after treatment with palmatin (FIGS. 9B, 9C and 9D).
상기 결과로부터, 팔마틴은 미토콘드리아의 생합성을 유도한다는 것을 확인하였다. From the above results, it was confirmed that palmatin induces mitochondrial biosynthesis.
실시예 10. 팔마틴의 파킨슨병 개선 효과 확인Example 10. Confirmation of the effect of Palmatin on improving Parkinson's disease
본 발명자들은 파킨슨병 모델에서, 팔마틴이 미토콘드리아의 기능개선 효과를 가지는지 확인하기 위하여, 파킨슨병 PINK(PTEN-induced kinase 1) 결핍 MEF(murine embryonic fibroblast)세포주를 사용하여, 팔마틴에 의한 기능개선 효과를 확인하였다. 구체적으로, PINK1 결손 MEF세포주에 팔마틴을 100, 200 또는 400 μM 농도로 처리한 후 24시간 동안 배양하였다. 팔마틴에 의한 미토파지 증가를 검증하기 위하여 배양 후 세포를 수득하여, 미토콘드리아 단백질인 Mfn2 및 COX2 단백질의 발현을 웨스턴블랏을 이용하여 조사하였다. 또한 미토콘드리아 기능개선 효과는, 미토콘드리아 기능이상의 지표인 활성산소 증가를 mitoSox 염색 후 유세포 분석기로 분석하고, 미토콘드리아 호흡율을 XF24 analyser로 분석하였다. 미토콘드리아 기능개선은 PINK 결핍 MEF 세포주(PINK KO)에 양성 대조군으로, DMSO를 처리하고, 실험군으로 팔마틴을 400 μM농도로 24시간 처리하고, 4일후 미토콘드리아 활성산소 및 미토콘드리아의 호흡율을 분석하여 결정하였다.In the Parkinson's disease model, the present inventors used a Parkinson's disease PINK (PTEN-induced kinase 1) deficient MEF (murine embryonic fibroblast) cell line to confirm whether palmatin has an effect of improving mitochondrial function, and function by palmatin. The improvement effect was confirmed. Specifically, the PINK1 deficient MEF cell line was treated with palmatin at a concentration of 100, 200 or 400 μM, and then cultured for 24 hours. In order to verify the increase in mitophagy caused by palmatin, cells were obtained after cultivation, and the expression of mitochondrial proteins Mfn2 and COX2 proteins was investigated using Western blot. In addition, the effect of improving mitochondrial function was analyzed by flow cytometry after mitoSox staining, and the mitochondrial respiration rate was analyzed by XF24 analyzer. The improvement of mitochondrial function was determined by treating the PINK-deficient MEF cell line (PINK KO) with DMSO as a positive control, and treatment with palmatin at 400 μM for 24 hours as an experimental group, and analyzing mitochondrial free radicals and mitochondrial respiration rate after 4 days. .
그 결과, 도 10A에 나타낸 바와 같이, 팔마틴을 처리하면, 농도 의존적으로 미토콘드리아 단백질인 Mfn2 및 COX2의 발현이 감소하는 것을 확인하여, 미토파지의 활성이 증가한 것을 확인하였다.As a result, as shown in Fig. 10A, it was confirmed that, when palmatin was treated, the expression of the mitochondrial proteins Mfn2 and COX2 decreased in a concentration-dependent manner, and the mitophagy activity increased.
또한, mitoSox로 염색한 결과, PINK1 결핍 MEF 세포주(PINK1 KO, DMSO)가 정상 세포주(WT)보다 활성산소가 증가되어 있음을 확인하였으며, 팔마틴을 처리하였을 때, 활성산소가, 정상세포주와 유사한 수준으로 감소하는 것을 확인하였다(도 10B).In addition, as a result of staining with mitoSox, it was confirmed that the PINK1-deficient MEF cell line (PINK1 KO, DMSO) had an increased free radical than that of the normal cell line (WT), and when palmatin was treated, the active oxygen was similar to that of the normal cell line. It was confirmed that it decreased to the level (FIG. 10B).
미토콘드리아의 호흡율을 분석한 결과, 정상세포주(WT)와 비교하여, PINK1 결핍 MEF 세포주(PINK1 KO)에서 미토콘드리아의 호흡율이 현저히 감소되어 있는 것을 확인하였으며, 팔마틴을 처리하면 미토콘드리아의 호흡율이 유의하게 증가한 것을 확인하였다(도 10C).As a result of analyzing the mitochondrial respiration rate, it was confirmed that the mitochondrial respiration rate was significantly reduced in the PINK1-deficient MEF cell line (PINK1 KO) compared to the normal cell line (WT).When palmatin was treated, the mitochondrial respiration rate was significantly increased. It was confirmed (Fig. 10C).
실시예 11. 팔마틴의 퇴행성 질환 동물모델에서의 운동성 개선 확인Example 11. Confirmation of improvement of motility in animal models of degenerative diseases of palmatin
퇴행성 질환 중 근위축성측상 경화증(amyotrophic lateral sclerosis, ALS) 및 알츠하이머에 대한 팔마틴의 효과를 확인하기 위하여, ALS 초파리 동물모델을 이용하여, 그 효과를 확인하였다. 구체적으로, 대표적인 ALS 초파리 동물모델인 TDP-4 과발현 모델에, 팔마틴을 5 mM을 투여하여, 25일간 climbing assay를 시행하였다. 또한, 알츠하이머 동물모델에서의 운동성을 평가하기 위하여, 상기와 동일한 조건에서, 알츠하이머 표현형을 가지는 Aβ 과발현 초파리 동물모델을 사용하여 동일한 실험을 수행하였다.In order to confirm the effect of palmatin on amyotrophic lateral sclerosis (ALS) and Alzheimer's among degenerative diseases, the ALS Drosophila animal model was used to confirm the effect. Specifically, a climbing assay was performed for 25 days by administering 5 mM palmatin to the TDP-4 overexpression model, which is a representative ALS fruit fly animal model. In addition, in order to evaluate motility in an Alzheimer's animal model, the same experiment was performed using an Aβ overexpressing Drosophila animal model having an Alzheimer's phenotype under the same conditions as above.
그 결과, 도 11 및 도 12에 나타낸 바와 같이, 팔마틴을 처리하면, 대조군과 비교하여, ALS 및 알츠하이머 질병에서, 운동성이 개선되는 것을 확인하였다.As a result, as shown in FIGS. 11 and 12, it was confirmed that, when palmatin was treated, motility was improved in ALS and Alzheimer's disease, compared to the control group.
따라서, 본 발명의 팔마틴은, 미토파지를 유도하여, 기능 이상 미토콘드리아를 제거하는 것을 확인하였다. 또한 구체적으로 미토콘드리아의 호흡율은 증가시키는 것을 확인하였으며, 미토콘드리아의 기능이상으로 유발된 퇴행성 질환 동물모델(ALS 및 알츠하이머)에서 운동성을 개선시키는 효과를 나타내어, 효과적으로 인지기능을 개선시킴으로서 퇴행성 질환을 개선시키는 것을 확인하였다.Therefore, it was confirmed that the palmatine of the present invention induces mitophagy to remove dysfunctional mitochondria. In addition, it was confirmed that the respiration rate of mitochondria was specifically increased, and it showed an effect of improving motility in animal models of degenerative diseases (ALS and Alzheimer's) caused by mitochondrial dysfunction, effectively improving cognitive function, thereby improving degenerative diseases. Confirmed.

Claims (17)

  1. 팔마틴(palamtine)을 유효성분으로 포함하는 미토콘드리아 기능이상 (mitochondrial dysfunction)으로 인한 질환의 예방 또는 치료용 조성물.A composition for preventing or treating diseases caused by mitochondrial dysfunction, comprising palmatin as an active ingredient.
  2. 제 1 항에 있어서, The method of claim 1,
    상기 팔마틴은 미토파지 (mitophagy)의 활성을 촉진시키는 것을 특징으로 하는 조성물. The composition characterized in that the palmatin promotes the activity of mitophagy (mitophagy).
  3. 제 1 항에 있어서, The method of claim 1,
    상기 팔마틴은 미토콘드리아의 세포호흡율을 증가시키는 것을 특징으로 하는 조성물. The composition characterized in that the palmatin increases the cellular respiration rate of mitochondria.
  4. 제 1 항에 있어서, The method of claim 1,
    상기 미토콘드리아 기능이상으로 인한 질환은 퇴행성 질환인 것을 특징으로 하는 조성물. The composition characterized in that the disease due to the mitochondrial dysfunction is a degenerative disease.
  5. 제 4 항에 있어서, The method of claim 4,
    상기 퇴행성 질환은 알츠하이머병 (Alzheimer's disease), 파킨슨병 (Parkinson's disease), 루게릭병(amyotrophic lateral sclerosis), 헌팅턴병 (Huntington's Disease), 다발성 경화증(Multiple sclerosis), 면역계이상 뇌기능 부전, 진행성 신경퇴행질환, 대사성 뇌질환, 니만-픽병(Niemann-Pick disease), 뇌 허혈 및 뇌출혈로 인한 치매(dementia)로 이루어지는 그룹에서 선택되는 어느 하나인 것을 특징으로 하는 조성물. The degenerative diseases include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, multiple sclerosis, immune system abnormal brain dysfunction, progressive neurodegenerative disease, A composition, characterized in that it is any one selected from the group consisting of metabolic brain disease, Niemann-Pick disease, brain ischemia, and dementia caused by cerebral hemorrhage.
  6. 제 1 항에 있어서, The method of claim 1,
    상기 미토콘드리아 기능이상으로 인한 질환은 MELAS 증후군 (mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes), 루게릭병 (amyotrophic lateral sclerosis, ALS) 및 샤르코 마리 투스 질환 (Charcot Marie Tooth disease, CMT)으로 이루어진 군에서 선택되는 어느 하나인 것을 특징으로 하는 조성물. Diseases caused by the mitochondrial dysfunction were in the group consisting of MELAS syndrome (mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes), amyotrophic lateral sclerosis (ALS) and Charcot Marie Tooth disease (CMT). Composition, characterized in that any one selected.
  7. (a) 미토케이마 (mt-Keima)를 발현하는 세포에 시험물질을 처리하는 단계; 및 (a) treating cells expressing mitokeima (mt-Keima) with a test substance; And
    (b) 미토파지 (mitophagy)의 활성을 측정하는 단계;를 포함하는 미토콘드리아 기능이상 (mitochondrial dysfunction)으로 인한 질환 치료제의 스크리닝 방법.(B) measuring the activity of mitophagy (mitophagy); mitochondrial dysfunction (mitochondrial dysfunction) comprising a screening method for the treatment of diseases caused by.
  8. 제 7 항에 있어서, The method of claim 7,
    상기 미토파지의 활성은 유세포분석법 (flow cytometry) 또는 공초점 현미경 (confocal microscopy)에 의하여 측정되는 것을 특징으로 하는 방법. The method of claim 1, wherein the mitophagy activity is measured by flow cytometry or confocal microscopy.
  9. 제 7 항에 있어서, The method of claim 7,
    상기 시험물질이 미토파지의 활성을 촉진시킨 경우 미토콘드리아 기능이상으로 인한 질환 치료제인 것으로 판단하는 단계를 추가적으로 더 포함하는 것을 특징으로 하는 방법. The method further comprising the step of determining that the test substance is a therapeutic agent for a disease due to mitochondrial dysfunction when the test substance promotes mitophagy activity.
  10. 제 7 항에 있어서, The method of claim 7,
    상기 미토콘드리아 기능이상으로 인한 질환은 퇴행성 질환인 것을 특징으로 하는 방법. The method characterized in that the disease caused by the mitochondrial dysfunction is a degenerative disease.
  11. 제 10 항에 있어서, The method of claim 10,
    상기 퇴행성 질환은 알츠하이머병 (Alzheimer's disease), 파킨슨병 (Parkinson's disease), 루게릭병(amyotrophic lateral sclerosis), 헌팅턴병 (Huntington's Disease), 다발성 경화증(Multiple sclerosis), 면역계이상 뇌기능 부전, 진행성 신경퇴행질환, 대사성 뇌질환, 니만-픽병(Niemann-Pick disease), 뇌 허혈 및 뇌출혈로 인한 치매(dementia)로 이루어지는 그룹에서 선택되는 어느 하나인 것을 특징으로 하는 방법. The degenerative diseases include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, multiple sclerosis, immune system abnormal brain dysfunction, progressive neurodegenerative disease, Metabolic brain disease, Niemann-Pick disease (Niemann-Pick disease), brain ischemia and dementia caused by cerebral hemorrhage (dementia) any one selected from the group consisting of.
  12. 제 7 항에 있어서, The method of claim 7,
    상기 미토콘드리아 기능이상으로 인한 질환은 MELAS 증후군 (mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes), 루게릭병 (amyotrophic lateral sclerosis, ALS) 및 샤르코 마리 투스 질환 (Charcot Marie Tooth disease, CMT)으로 이루어진 군에서 선택되는 어느 하나인 것을 특징으로 하는 방법. Diseases due to the mitochondrial dysfunction were in the group consisting of MELAS syndrome (mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes), amyotrophic lateral sclerosis (ALS), and Charcot Marie Tooth disease (CMT). Method, characterized in that any one selected.
  13. 시험관 내 (in vitro)에서 팔마틴(palamtine)을 미토콘드리아 기능이상 세포에 처리하는 단계를 포함하는 미토파지의 활성을 촉진시키는 방법.A method of promoting mitophagy activity comprising the step of treating mitochondrial dysfunction cells with palmatin in vitro .
  14. 팔마틴(palamtine)을 유효성분으로 포함하는 인지기능 장애 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating cognitive dysfunction comprising palmatin as an active ingredient.
  15. 팔마틴(palamtine)을 유효성분으로 포함하는 인지기능 장애 예방 또는 개선용 식품 조성물.A food composition for preventing or improving cognitive dysfunction comprising palmatin as an active ingredient.
  16. 약학적으로 유효한 양의 제1항의 약학적 조성물을 개체에 투여하는 단계를 포함하는, 미토콘드리아 기능이상 (mitochondrial dysfunction)으로 인한 질환의 예방 또는 치료 방법.A method for preventing or treating diseases caused by mitochondrial dysfunction, comprising administering to a subject a pharmaceutically effective amount of the pharmaceutical composition of claim 1.
  17. 약학적으로 유효한 양의 제 14항의 약학적 조성물을 개체에 투여하는 단계를 포함하는, 인지기능 장애의 예방 또는 치료 방법.A method for preventing or treating cognitive dysfunction comprising administering to an individual the pharmaceutical composition of claim 14 in a pharmaceutically effective amount.
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