WO2023008978A1 - Sncg-targeting composition for treating or diagnosing degenerative disease - Google Patents

Sncg-targeting composition for treating or diagnosing degenerative disease Download PDF

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Publication number
WO2023008978A1
WO2023008978A1 PCT/KR2022/011297 KR2022011297W WO2023008978A1 WO 2023008978 A1 WO2023008978 A1 WO 2023008978A1 KR 2022011297 W KR2022011297 W KR 2022011297W WO 2023008978 A1 WO2023008978 A1 WO 2023008978A1
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sncg
expression
degenerative arthritis
gene
pharmaceutical composition
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PCT/KR2022/011297
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French (fr)
Korean (ko)
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양시영
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아주대학교산학협력단
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Priority claimed from KR1020210100767A external-priority patent/KR20230018813A/en
Priority claimed from KR1020210100766A external-priority patent/KR20230018812A/en
Priority claimed from KR1020210101683A external-priority patent/KR20230020593A/en
Application filed by 아주대학교산학협력단 filed Critical 아주대학교산학협력단
Publication of WO2023008978A1 publication Critical patent/WO2023008978A1/en

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    • 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
    • A23L33/13—Nucleic acids or derivatives thereof
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic 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
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9

Definitions

  • the present invention relates to a composition for treating or diagnosing degenerative diseases targeting Synuclein Gamma (SNCG).
  • SNCG Synuclein Gamma
  • Osteoarthritis is a degenerative joint disease mainly caused by inhibition of cartilage ECM synthesis and promotion of cartilage destruction.
  • Many etiologic risk factors and pathophysiological processes associated with aging contribute to the progression of osteoarthritis.
  • Mechanical stress, including joint instability and injury, and aging-related factors that predispose to osteoarthritis are potential causes of degenerative arthritis. These factors are caused by activation of biochemical pathways in chondrocytes, a unique cell type that produces various catabolic and anabolic factors, leading to degradation of ECM (extracellular matrix) by Mmp (Matrix metalloproteinase), dedifferentiation of chondrocytes, and apoptosis.
  • ECM extracellular matrix
  • Mmp Microx metalloproteinase
  • cartilage tissue constituting a joint does not normally regenerate in vivo once damaged.
  • daily activities are restricted along with severe pain, and when chronic, it causes fatal degenerative arthritis, which interferes with normal life or professional activities.
  • rheumatoid arthritis unlike degenerative arthritis caused by the destruction of cartilage cells and cartilage tissue, the progression of the disease by an autoimmune reaction is known to be an important causative factor.
  • Rheumatoid arthritis is a chronic autoimmune disease characterized by inflammation and proliferation of synovial cells, and unlike degenerative arthritis, osteoporosis and bone erosion of bones around joints occur.
  • Rheumatoid arthritis progresses when inflammation of the synovial membrane spreads to the joint capsule, ligament, and tendon, and invades the bone. Therefore, degenerative arthritis and rheumatoid arthritis have completely different causes and progression stages, and treatment methods for them are also different.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • penicillamine steroid hormones
  • TNF inhibitors interleukin inhibitors
  • JAK inhibitors anti-CD related inhibitors suitable for blocking the inflammatory mechanism.
  • applicable NSAID drugs and steroid hormones are used in patients with degenerative arthritis for the purpose of relieving joint pain and inflammation, but since they only relieve symptoms rather than treating the disease itself, they cannot serve as a practical therapeutic agent for degenerative arthritis.
  • degenerative arthritis which is mainly caused by destruction of cartilage cells and cartilage tissue
  • rheumatoid arthritis which is an inflammatory arthritis, in its cause and phenomenon
  • the treatment method for degenerative arthritis is also different from the treatment method for rheumatoid arthritis.
  • Synuclein Gamma is a protein encoded by the SNCG gene and is a synuclein family protein known to cause neurodegenerative diseases. It has been reported that high levels of SNCG are identified in advanced breast cancer, and overexpression of SNCG is associated with breast tumor development. SNCGs are found primarily in the peripheral nervous system (primary sensory neurons, sympathetic neurons, and motor neurons) and in the retina, as well as in the brain, ovaries, tumors, and olfactory epithelium. SNCG is known as a tumor marker and has been found in the retina of Alzheimer's patients. However, until now, it has not been known what function SNCG plays in normal cells, but many studies are being conducted based on its association with neurodegenerative diseases. .
  • Degenerative neurological diseases are diseases that cause degenerative changes in the nerve cells of the central nervous system and cause various symptoms. The disease progresses continuously over the years. Representative neurodegenerative diseases include Parkinson's disease, Alzheimer's disease, degenerative arthritis or amyotrophic lateral sclerosis (ALS).
  • Degenerative arthritis is a type of arthritis, also referred to as osteoarthritis, and refers to arthritis caused by degenerative changes in cartilage and surrounding bones in synovial joints. That is, degenerative arthritis is a disease characterized by gradual loss of articular cartilage, hypertrophy of bones located below the cartilage, bone production at the edge of the joint, and non-specific synovial inflammation. Osteoarthritis is a disease caused by damage to cartilage due to aging or excessive physical pressure (eg, obesity, trauma, etc.). Therefore, degenerative arthritis shows severe pain and movement disorders in joints that receive a lot of weight, that is, knee (knee) joints and hip (hip) joints.
  • Parkinson's disease is a degenerative disease of the central nervous system mainly caused by degeneration or arteriosclerotic changes of the diencephalon, and movement disorder is the main symptom. It is caused by a lack of the transmitter dopamine. Parkinson's disease is a chronic, progressive motor neurological disease that occurs when dopamine-producing nerve cells are damaged and dopamine is lacking, resulting in acetylcholine, which was maintained in balance, predominance. This is manifested by trembling tremors, stiffness of the limbs or body, bradykinesia that slows movement, and postural instability that prevents balance.
  • the present invention provides a biomarker composition for early diagnosis of degenerative arthritis by confirming changes in the expression or activity level of SNCG, and a diagnostic kit capable of measuring the expression level of the biomarker, and diagnosing degenerative arthritis using the biomarker It is intended to provide a method and a method for screening a therapeutic agent for degenerative arthritis.
  • compositions for preventing or treating degenerative diseases containing an SNCG expression or activity inhibitor as an active ingredient.
  • the present invention provides a biomarker composition for diagnosing degenerative arthritis comprising a synuclein gamma (SNCG) gene or a protein encoded by the gene.
  • SNCG synuclein gamma
  • the present invention provides a kit for diagnosing degenerative arthritis containing, as an active ingredient, an agent for detecting the expression or activity level of a SNCG gene or a protein encoded by the gene.
  • the present invention provides a method for providing information for diagnosing degenerative arthritis comprising the step of confirming the expression or activity level of a SNCG gene or a protein encoded by the gene from a sample isolated from a specimen.
  • the present invention comprises the steps of processing a candidate substance to chondrocytes isolated from a patient with degenerative arthritis; Checking the expression level of SNCG from the chondrocytes treated with the candidate substance; and a method for screening a treatment for degenerative arthritis, wherein the candidate substance having a reduced expression level of the SNCG compared to a group not treated with the candidate substance is provided as a treatment for degenerative arthritis.
  • the present invention provides a pharmaceutical composition for preventing or treating degenerative arthritis containing an SNCG expression or activity inhibitor as an active ingredient.
  • the present invention provides a health food for preventing or improving degenerative arthritis, which contains a SNCG expression or activity inhibitor as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating SNCG-related diseases, containing a SNCG expression or activity regulator as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating Synuclein Gamma (SNCG) overexpression diseases, comprising a compound selected from AZ-628 or PD-184352 or a pharmaceutically acceptable salt thereof as an active ingredient.
  • SNCG Synuclein Gamma
  • the present invention provides a health functional food composition for preventing or improving Synuclein Gamma (SNCG) overexpression diseases, comprising AZ-628 or PD-184352 as an active ingredient.
  • SNCG Synuclein Gamma
  • SNCG expression and activity increase during the onset of degenerative arthritis, and accordingly, it was confirmed that the expression of Mmp3, Mmp13, and Cox2, which induce cartilage destruction, was increased, and it was confirmed that the expression of the above genes was suppressed by suppressing SNCG expression. Accordingly, SNCG is provided as a biomarker for diagnosing degenerative arthritis, and as a result of depletion of SNCG, the effect of degenerative arthritis is alleviated, the SNCG expression or activity inhibitor is intended to be provided as a therapeutic agent for degenerative arthritis.
  • AZ-628 or PD-184352 a synuclein gamma (SNCG) inhibitor, binds to the C-terminus of the synuclein gamma (SNCG) protein and binds to the degenerative disease-related factors regulated by SNCG, MMP3, MMP13, and COX2.
  • SNCG synuclein gamma
  • FIG. 1 is a result of confirming SNCG expression in degenerative arthritis patients and degenerative arthritis-induced mouse joint tissues
  • FIG. 1A is the result confirmed in human joints
  • FIG. 1B is the result confirmed in mouse joints.
  • FIG. 2 is a result of confirming the correlation between modulation of SNCG expression and degenerative arthritis
  • FIG. 2A confirms that catabolic factors such as Mmp3, Mmp13 and Cox2 are increased when chondrocytes are treated with recombinant SNCG at different concentrations.
  • 2B shows the expression of SNCG, MMP3, MMP13 and COX2 in chondrocytes infected with Ad-C or Ad-SNCG. This is the result of western blot analysis confirming that the expression of Col2 and Sox9 is increased and the expression of Sox9 is decreased at the same time, and FIG. 2C and FIG.
  • 2D are Safranin-O staining, OARSI grade, osteophyte formation, and Ad-C and As a result of confirming cartilage destruction and the development of degenerative arthritis in the mouse knee joint injected with Ad-SNCG into the joint, it was confirmed that cartilage destruction was promoted when SNCG was overexpressed.
  • Figure 3 is a result of confirming that the pathogenesis of degenerative arthritis is reduced in SNCG knockout mice.
  • Figure 3A is a safranin-O staining result confirming cartilage destruction after inducing degenerative arthritis by applying surgical DMM to WT and SNCG knockout mice.
  • Figure 3B is the result of confirming cartilage destruction by OARSI grade, osteophyte formation, and subchondral bone plate thickness.
  • 5 is a result of selecting PD-184352 as a Synuclein Gamma (SNCG) inhibitor by in silico compound screening using the CMap approach.
  • SNCG Synuclein Gamma
  • SNCG Synuclein Gamma
  • AZ-628 binds to the C-terminal of the AZ-628 amino acid sequence treated with glutamine (Q), glutamic acid (E), glycine (G), alanine (A), and lysine (K) (yellow color) indicate
  • AZ-628 binds to the C-terminal of the AZ-628 amino acid sequence treated with glutamine (Q), glutamic acid (E), glycine (G), alanine (A), and lysine (K) (yellow color) indicate
  • the inventors of the present invention confirmed that SNCG expression and activity increased in the early stage of degenerative arthritis, and thus increased expression of Mmp3, Mmp13, and Cox2, which induce cartilage destruction, and found that the expression of the above genes was suppressed by suppressing SNCG expression. As confirmed, the present invention was completed in order to diagnose degenerative arthritis early by providing SNCG as a biomarker for degenerative arthritis and to improve the therapeutic effect of degenerative arthritis by regulating the expression or activity of SNCG.
  • the present invention may provide a biomarker composition for diagnosing degenerative arthritis comprising a synuclein gamma (SNCG) gene or a protein encoded by the gene.
  • SNCG synuclein gamma
  • the Synuclein Gamma may be UniGene ID Mm.41063.
  • the present invention can provide a kit for diagnosing degenerative arthritis containing, as an active ingredient, an agent for detecting the expression or activity level of a SNCG gene or a protein encoded by the gene.
  • the agent may be selected from the group consisting of a primer or probe that specifically binds to the SNCG gene, an antibody, a peptide, an aptamer, or a compound that specifically binds to the SNCG protein.
  • the present invention can provide a method for providing information for diagnosing degenerative arthritis, which includes the step of confirming the expression or activity level of a SNCG gene or a protein encoded by the gene from a sample isolated from a specimen.
  • the sample may be selected from the group consisting of cells, tissues, blood, serum, urine and saliva.
  • a method of providing information for diagnosing degenerative arthritis may include diagnosing degenerative arthritis when the expression or activity level of the SNCG gene or a protein encoded by the gene is higher than that of a normal control group.
  • the present invention comprises the steps of processing a candidate substance to chondrocytes isolated from a patient with degenerative arthritis; Checking the expression level of SNCG from the chondrocytes treated with the candidate substance; and a method of screening for a treatment for degenerative arthritis, wherein the candidate substance, in which the expression level of the SNCG is reduced compared to a group not treated with the candidate substance, is provided as a treatment for degenerative arthritis.
  • the "biomarker" of the present invention is a substance that can be diagnosed by distinguishing the tissue or cell of a subject suspected of having degenerative arthritis from the tissue or cell of a normal control group, and measures the increase or decrease in the tissue or cell of the subject compared to the normal control group.
  • organic biomolecules such as visible proteins or nucleic acids, lipids, glycolipids, glycoproteins, and the like;
  • diagnosis in a broad sense means to judge the actual condition of a patient's disease in all aspects.
  • the content of judgment is the name of the disease, etiology, type of disease, severity, detailed condition of the disease, and presence or absence of complications.
  • the present invention can provide a pharmaceutical composition for preventing or treating degenerative arthritis containing a synuclein gamma (SNCG) expression or activity inhibitor as an active ingredient.
  • SNCG synuclein gamma
  • the SNCG expression inhibitor is at least one from the group consisting of antisense nucleotides, small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA, and ribozymes that specifically bind to mRNA of a gene or gene promoting SNCG expression may be selected.
  • siRNA small interfering RNA
  • shRNA short hairpin RNA
  • microRNA microRNA
  • ribozymes that specifically bind to mRNA of a gene or gene promoting SNCG expression may be selected.
  • the SNCG activity inhibitor may be one or more selected from the group consisting of proteins that promote SNCG activity or compounds that specifically bind to SNCG proteins, peptides, peptidomimetics, proteins, aptamers, and antibodies.
  • the pharmaceutical composition is any one selected from the group consisting of injections, granules, powders, tablets, pills, capsules, suppositories, gels, suspensions, emulsions, drops or liquids according to conventional methods. formulations can be used.
  • a pharmaceutical composition for preventing or treating degenerative arthritis containing a SNCG expression or activity inhibitor as an active ingredient is suitable carriers, excipients, disintegrants, sweeteners, coating agents, It may further include one or more additives selected from the group consisting of an expanding agent, a lubricant, a flavoring agent, an antioxidant, a buffer, a bacteriostatic agent, a diluent, a dispersing agent, a surfactant, a binder, and a lubricant.
  • carriers, excipients and diluents are 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 oil may be used, and solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules.
  • solid preparations may be prepared by mixing at least one or more excipients, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the composition.
  • excipients for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc.
  • lubricants such as magnesium stearate and talc may also be used.
  • Liquid preparations for oral administration include suspensions, solutions for oral use, emulsions, syrups, and the like, and various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included in addition to commonly used simple diluents such as water and liquid paraffin.
  • Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like.
  • Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents.
  • As a base material of the suppository witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.
  • the pharmaceutical composition is administered in a conventional manner through intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or intradermal routes.
  • a preferable dosage of the SNCG expression or activity inhibitor may vary depending on the condition and weight of the subject, the type and severity of the disease, the type of drug, the route and duration of administration, and may be appropriately selected by a person skilled in the art. According to one embodiment of the present invention, but not limited thereto, the daily dosage may be 0.01 to 200 mg/kg, specifically 0.1 to 200 mg/kg, and more specifically 0.1 to 100 mg/kg. Administration may be administered once a day or divided into several administrations, and the scope of the present invention is not limited thereby.
  • the 'subject' may be a mammal including a human, but is not limited to these examples.
  • the present invention can provide a health food for preventing or improving degenerative arthritis containing an SNCG expression or activity inhibitor as an active ingredient.
  • the health food may be used together with other foods or food additives in addition to the SNCG expression or activity inhibitors, and may be appropriately used according to conventional methods.
  • the mixing amount of the active ingredient may be appropriately determined depending on the purpose of use thereof, for example, prevention, health or therapeutic treatment.
  • the effective dose of the compound contained in the health food may be used according to the effective dose of the therapeutic agent, but may be less than the above range in the case of long-term intake for the purpose of health and hygiene or health control. Since there is no problem in terms of safety, it is certain that the components can be used in an amount greater than the above range.
  • the present invention can provide a pharmaceutical composition for preventing or treating SNCG-related diseases containing a SNCG expression or activity regulator as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating Synuclein Gamma (SNCG) overexpression diseases, comprising a compound represented by Formula 1 or Formula 2 below or a pharmaceutically acceptable salt thereof as an active ingredient.
  • SNCG Synuclein Gamma
  • the pharmaceutically acceptable salt refers to an acid addition salt formed by a pharmaceutically acceptable free acid
  • the pharmaceutically acceptable salt refers to a salt commonly used in the pharmaceutical industry, for example
  • an inorganic ion salt made of calcium, potassium, sodium or magnesium
  • an inorganic acid salt made of hydrochloric acid, nitric acid, phosphoric acid, hydrobromic acid, iodic acid, perchloric acid or sulfuric acid
  • the compound represented by Formula 1 is AZ-628, specifically 3-(2-cyanopropan-2-yl)-N-(4-methyl-3-((3-methyl-4-oxo-3, 4-dihydroquinazolin-6-yl)amino)phenol)benzamide (3-(2-cyanopropan-2-yl)-N-(4-methyl-3-((3-methyl-4-oxo-3 ,4-dihydroquinazolin-6-yl)amino)phenyl)benzamide).
  • the compound inhibits the expression of MMP3, MMP13 and COX2 by binding to the C-terminus of Synuclein Gamma (SNCG) protein.
  • the compound represented by Formula 2 is PD-184352 and is 2-((2-chloro-4-iodophenyl)amino)-N-(cyclopropylmethoxy)-3,4-difluorobenzamide (2- ((2-chloro-4-iodophenyl)amino)-N-(cyclopropylmethoxy)-3,4-difluorobenzamide).
  • the compound inhibits the expression of MMP3, MMP13 and COX2 by binding to the C-terminus of Synuclein Gamma (SNCG) protein.
  • the pharmaceutical composition is to be administered to a patient with a disease caused by overexpression of Synuclein Gamma (SNCG).
  • SNCG Synuclein Gamma
  • the synuclein gamma (SNCG) overexpression disease is degenerative arthritis or Parkinson's disease.
  • the pharmaceutical composition of the present invention is prepared in unit dosage form or multi-dose by formulating using a pharmaceutically acceptable carrier according to a method that can be easily performed by those skilled in the art. It can be prepared by incorporating into a container.
  • the pharmaceutically acceptable carrier is one commonly used in formulation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like.
  • the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components.
  • the content of the additives included in the pharmaceutical composition is not particularly limited and may be appropriately adjusted within the range of content used in conventional formulations.
  • the pharmaceutical composition may be formulated as an aqueous solution, suspension, emulsion, etc. for injection, pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, warning agents, lotions, liniment agents, pasta agents, and cataplasma agents. It may be formulated in the form of one or more external preparations selected from the group consisting of agents.
  • the pharmaceutical composition of the present invention may further contain pharmaceutically acceptable carriers and diluents for formulation.
  • pharmaceutically acceptable carriers and diluents include excipients such as starch, sugar and mannitol, fillers and extenders such as calcium phosphate, cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose, gelatin, alginates, and polyvinyl fibres.
  • binders such as rolidone, etc., lubricants such as talc, calcium stearate, hydrogenated castor oil and polyethylene glycol, disintegrants such as povidone, crospovidone, surfactants such as polysorbates, cetyl alcohol, and glycerol; don't
  • the pharmaceutically acceptable carrier and diluent may be biologically and physiologically compatible with the subject.
  • diluents include, but are not limited to, saline, aqueous buffers, solvents and/or dispersion media.
  • the pharmaceutical composition of the present invention may be administered orally or parenterally (eg, intravenous, subcutaneous, intraperitoneal or topical application) depending on the desired method.
  • parenterally eg, intravenous, subcutaneous, intraperitoneal or topical application
  • it may be formulated into tablets, troches, lozenges, aqueous suspensions, oily suspensions, powders, granules, emulsions, hard capsules, soft capsules, syrups or elixirs.
  • parenteral administration it may be formulated as an injection solution, suppository, powder for respiratory inhalation, aerosol for spray, ointment, powder for application, oil, cream, etc.
  • the dosage of the pharmaceutical composition of the present invention depends on the condition and weight of the patient, age, sex, health condition, dietary constitution specificity, the nature of the preparation, the severity of the disease, the administration time of the composition, the administration method, the administration period or interval, and the excretion rate. , And the range may vary depending on the type of drug, and may be appropriately selected by a person skilled in the art. For example, it may be in the range of about 0.1 to 10,000 mg/kg, but is not limited thereto, and may be divided and administered once or several times a day.
  • the pharmaceutical composition may be administered orally or parenterally (eg, intravenous, subcutaneous, intraperitoneal or topical application) depending on the desired method.
  • the pharmaceutically effective amount and effective dose of the pharmaceutical composition of the present invention may vary depending on the formulation method, administration method, administration time and/or administration route of the pharmaceutical composition, and those skilled in the art can use the purpose Dosages effective for treatment can be easily determined and prescribed. Administration of the pharmaceutical composition of the present invention may be administered once a day, or may be divided into several administrations.
  • the present invention is a health functional food composition for preventing or improving SNCG (Synuclein Gamma) overexpression disease comprising a compound represented by Formula 1 or Formula 2 and a food additive acceptable in food science
  • the present invention can be generally used as a commonly used food.
  • the food supplement additives include conventional food additives in the art, for example, flavoring agents, flavoring agents, coloring agents, fillers, stabilizers, and the like, and are exemplified below.
  • the health drink composition of the present invention has no particular limitations on the liquid component except for containing the extract as an essential component in the indicated ratio, and may contain various flavors or natural carbohydrates as additional components like conventional beverages.
  • natural carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; polysaccharides such as dextrins, cyclodextrins; and sugar alcohols such as xylitol, sorbitol, and erythritol.
  • natural flavoring agents thaumatin, stevia extract (eg rebaudioside A, glycyrrhizin, etc.)
  • synthetic flavoring agents sacharin, aspartame, etc.
  • composition of the present invention contains various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids , a protective colloidal thickener, a pH adjusting agent, a stabilizer, a preservative, glycerin, alcohol, a carbonating agent used in carbonated beverages, and the like.
  • the compositions of the present invention may contain fruit flesh for preparing natural fruit juice, fruit juice beverages, and vegetable beverages. These components may be used independently or in combination.
  • the food composition of the present invention can be used as a health functional food.
  • health functional food refers to food manufactured and processed using raw materials or ingredients having useful functionalities for the human body in accordance with the Health Functional Food Act, and "functional” refers to food that is not related to the structure and function of the human body. It refers to intake for the purpose of obtaining useful effects for health purposes such as regulating nutrients or physiological functions.
  • the food composition of the present invention may include conventional food additives, and the suitability as the "food additive" is determined according to the general rules of the food additive code approved by the Ministry of Food and Drug Safety and general test methods, etc., unless otherwise specified. It is judged according to the specifications and standards for the item.
  • Items listed in the "Food Additive Code” include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid, natural additives such as dark pigment, licorice extract, crystalline cellulose, goyang pigment, guar gum, and mixed preparations such as sodium L-glutamate preparations, noodle-added alkali preparations, preservative preparations, and tar color preparations.
  • chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid
  • natural additives such as dark pigment, licorice extract, crystalline cellulose, goyang pigment, guar gum
  • mixed preparations such as sodium L-glutamate preparations, noodle-added alkali preparations, preservative preparations, and tar color preparations.
  • the food composition of the present invention can be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills and the like.
  • hard capsules can be prepared by mixing and filling the composition according to the present invention with additives such as excipients in conventional hard capsules
  • soft capsules can be prepared by filling the composition according to the present invention. It can be prepared by mixing with additives such as excipients and filling in a capsule base such as gelatin.
  • the soft capsule may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary.
  • prevention refers to all activities that inhibit or delay a disease by administering the composition according to the present invention.
  • treatment refers to all activities that improve or beneficially change the symptoms of a disease by administering the composition according to the present invention.
  • improvement means any action that improves the bad condition of a disease by administering or ingesting the composition of the present invention to a subject.
  • mice Male C57BL/6 and SNCG ⁇ / ⁇ mice (008843-B6.129P2- SNCG tm1vlb /J, Jackson laboratory, USA) were prepared according to the guidelines of the Institutional Animal Care and Use Committee, which approved all animal procedures at the Laboratory Animal Research Center of Ajou University. maintained according to
  • the degenerative arthritis animal model was performed with male C57BL/6 and SNCG-/- mice (008843-B6.129P2- SNCG tm1vlb /J, Jackson laboratory, USA), and all animal experiments were performed using Institutional approved by Ajou University Laboratory Animal Research Center. It was performed according to the guidelines of the Animal Care and Use Committee. To prepare an animal model for degenerative arthritis, 12-week-old male mice were subjected to Destabilization of Medial Meniscus (DMM) surgery and sacrificed 10 weeks after surgery. In the case of female mice, degenerative arthritis caused by female hormones was excluded from the experiment.
  • DMM Medial Meniscus
  • Adenoviruses for intra-articular injection were purchased from Vector Biolabs (Malvern, USA): Ad-C (1060), Ad-Synuclein Gamma (SNCG) (ADV-223826). Wild-type mice were injected twice weekly with adenovirus (1 x 10 9 PFUs/10 ⁇ L) into the knee joint, and the mice were sacrificed 3 weeks after the first injection.
  • Mouse articular chondrocytes were isolated from the cartilage of ICR mice on day 5 after birth, enzymatically digested with proteolytic enzyme and collagenase, and then mixed with 10% FBS, 100 units/mL of penicillin, and 100ug/mL of streptomycin. It was maintained in supplemented DMEM (Capricorn science GmbH; Hessen, Germany). On day 3, cells (4.25 ⁇ 10 5 cells/well) were infected with adenovirus or treated with recombinant protein. Mlkl+/+ and Mlkl-/- MEFs were maintained in DMEM supplemented with 10% FBS and penicillin-streptomycin.
  • Antibodies were purchased from Abcam (Cambridge, UK; SNCG, COX2, MMP3, MMP13), Col2a1 (Merck, USA), Sox9 (Novus, Littleton, USA) and Erk (610408, Beckton Dickinson, New Jersey, USA). Recombinant SNCG was purchased from Abcam.
  • Cells were lysed in M2 buffer, and mouse tissue was lysed in lysis buffer consisting of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 50 mM NaF, 1% Tween 20, 0.2% NP-40 and protease inhibitors. Equal amounts of cell extracts were separated by SDS-PAGE (6% stacking gel and 10% running gel) and analyzed by immunoblotting.
  • SNCG expression was investigated in human and mouse degenerative arthritis intact and damaged cartilage samples.
  • the expression of Mmp3, Mmp13, and Cox2 increased compared to the non-injured sample, and at the same time, it was confirmed that the expression of SNCG was significantly higher (Fig. 1A; human, 1B; mouse).
  • MMP3 and MMP13 are known to play important roles in the pathogenesis of degenerative arthritis, and Cox2 is mainly involved in inflammation and induces cartilage matrix degradation by activating collagenase and aggrecanase.
  • SNCG synuclein gamma
  • chondrocytes were treated with SNCG or overexpression was induced to confirm changes in related factors.
  • Figure 2A when recombinant SNCG (Recombinant SNCG) is treated with chondrocytes, the expression of catabolic factors such as Mmp3, Mmp13 and Cox2 is increased, and the expression of anabolic factors such as Col2 and Sox9 is decreased. It was confirmed by Western blot.
  • SNCG-deficient mice were purchased from Jackson Laboratory (Bar Harbor, ME USA), and degenerative arthritis models were induced by treating SNCG-deficient mice with DMM, which is the most suitable method for developing human degenerative arthritis.
  • cartilage destruction, degenerative arthritis manifestation, and catabolic factor expression were significantly reduced in these mice compared to WT mice.
  • AZ-628 selected in Example 4 is effective as a treatment for degenerative arthritis
  • the effect on the expression of MMP3, MMP13 and COX2 overexpressed by Synuclein Gamma (SNCG) was evaluated.
  • SNCG Synuclein Gamma
  • FIG. 6 as a result of treating AZ-628 to chondrocytes in which SNCG (Synuclein Gamma) overexpression was induced by adenovirus, the expression of MMP3, MMP13, and COX2 induced by SNCG (synuclein gamma) overexpression was 628 was found to decrease with the administered dose.
  • PD-184352 selected in Example 4 is effective as a therapeutic agent for degenerative arthritis, its effect on the expression of MMP3, MMP13 and COX2 overexpressed by Synuclein Gamma (SNCG) was evaluated.
  • SNCG Synuclein Gamma
  • FIG. 7 as a result of treating PD-184352 to chondrocytes in which SNCG (Synuclein Gamma) overexpression was induced by adenovirus, the expression of MMP3, MMP13, and COX2 induced by SNCG (synuclein gamma) overexpression was found to be PD-184352. 184352 has been shown to decrease with the administered dose.

Abstract

The present invention relates to a biomarker composition for diagnosing osteoarthritis, comprising a synuclein gamma (SNCG) gene or a protein encoded by the SNCG gene, and a diagnosis method using same. It was confirmed that upon pathogenesis of osteoarthritis, the expression and activity of SNCG increased, and accordingly, the expression of Mmp3, Mmp13, and Cox2, which induce cartilage destruction, increased and that the expression of the aforementioned genes was inhibited by the inhibition of SNCG expression. Accordingly, the present invention is to provide SNCG as a biomarker for diagnosing osteoarthritis and to provide, with the confirmation of an effect of ameliorating osteoarthritis as a result of the depletion of SNCG expression, an SNCG expression or activity inhibitor as a therapeutic agent for osteoarthritis. The present invention, by confirming that AZ-628 or PD-184352 as an SNCG inhibitor binds to the C-terminus of SNCG protein and inhibits the expression of MMP3, MMP13, and COX2, which are factors related to degenerative diseases and controlled by SNCG, provides the compound as a therapeutic agent for osteoarthritis and Parkinson's disease that are caused by SNCG.

Description

SNCG를 표적으로 하는 퇴행성 질환 치료 또는 진단용 조성물Composition for treating or diagnosing degenerative diseases targeting SNCG
본 발명은 SNCG (Synuclein Gamma)를 표적으로 하는 퇴행성 질환 치료 또는 진단용 조성물에 관한 것이다.The present invention relates to a composition for treating or diagnosing degenerative diseases targeting Synuclein Gamma (SNCG).
퇴행성 관절염 (osteoarthritis, OA)은 주로 연골 형성 (cartilage ECM synthesis) 억제 및 연골조직 파괴 (cartilage destruction) 촉진으로 오는 퇴행성관절 질환이다. 노화와 관련된 많은 병인학적 위험 인자들과 병리생리학적 과정들이 퇴행성관절염의 진행에 기여한다. 관절 불안정성과 손상을 포함한 기계적 스트레스 및 퇴행성관절염에 걸리기 쉽게 하는 노화 관련 인자들이 잠재적인 퇴행성관절염 야기 기작들이다. 이러한 인자들은 다양한 이화 및 동화 인자들을 생산하는 독특한 세포 타입인 연골세포 내 생화학적 경로들의 활성화로 인해 Mmp (Matrix metalloproteinase)에 의한 ECM (extracellular matrix)의 분해, 그리고 연골세포의 탈분화 (dedifferentiation) 및 아팝토시스 (apoptosis)를 통한 ECM 합성의 중단을 초래한다. 특히, 관절을 이루고 있는 연골 조직은 한번 손상되게 되면 정상적으로 생체 내에서 재생이 되지 않는다. 이러한 관절의 연골조직이 손상될 경우 심한 통증과 함께 일상 활동에 제한을 받게 되며, 만성화될 경우 치명적인 퇴행성관절염을 유발하게 되어 정상적인 생활이나 직업적인 활동을 방해하게 된다.Osteoarthritis (OA) is a degenerative joint disease mainly caused by inhibition of cartilage ECM synthesis and promotion of cartilage destruction. Many etiologic risk factors and pathophysiological processes associated with aging contribute to the progression of osteoarthritis. Mechanical stress, including joint instability and injury, and aging-related factors that predispose to osteoarthritis are potential causes of degenerative arthritis. These factors are caused by activation of biochemical pathways in chondrocytes, a unique cell type that produces various catabolic and anabolic factors, leading to degradation of ECM (extracellular matrix) by Mmp (Matrix metalloproteinase), dedifferentiation of chondrocytes, and apoptosis. It results in the cessation of ECM synthesis through apoptosis. In particular, cartilage tissue constituting a joint does not normally regenerate in vivo once damaged. When the cartilage tissue of these joints is damaged, daily activities are restricted along with severe pain, and when chronic, it causes fatal degenerative arthritis, which interferes with normal life or professional activities.
반면, 류마티스관절염 (rheumatoid arthritis, RA)의 경우, 연골세포 및 연골조직 파괴로 인해 유발되는 퇴행성 관절염과는 달리, 자가면역반응에 의한 질환의 진행이 중요한 원인 인자로 알려져 있다. 류마티스 관절염은 활막 세포의 염증과 증식을 특징으로 하는, 만성 자가면역 질환 (autoimmune diseases)으로서, 퇴행성관절염과 달리 관절 주위 뼈의 골다공증 및 골미란 등이 발생한다. 류마티스 관절염은 활막 (synovial membrane)의 염증이 관절막 (joint capsule)과 인대 (ligament), 건 (tendon)으로 퍼지고, 뼈로 침범하여 진행되게 된다. 따라서, 퇴행성관절염과 류미티스 관절염은 그 발명 원인 및 진행단계가 전혀 상이하며, 이에 대한 치료 방법도 상이하다.On the other hand, in the case of rheumatoid arthritis (RA), unlike degenerative arthritis caused by the destruction of cartilage cells and cartilage tissue, the progression of the disease by an autoimmune reaction is known to be an important causative factor. Rheumatoid arthritis is a chronic autoimmune disease characterized by inflammation and proliferation of synovial cells, and unlike degenerative arthritis, osteoporosis and bone erosion of bones around joints occur. Rheumatoid arthritis progresses when inflammation of the synovial membrane spreads to the joint capsule, ligament, and tendon, and invades the bone. Therefore, degenerative arthritis and rheumatoid arthritis have completely different causes and progression stages, and treatment methods for them are also different.
지금까지 알려진 류마티스관절염 치료제로는 염증 기전을 차단하기 위해 적당한 비스테로이드성 항염증 약물(NSAIDs), 페니실라민, 스테로이드성 호르몬, TNF 억제제, 인터류킨 억제제, JAK 억제제, 항-CD 관련 억제제 등이 이에 해당한다. 관절 통증 및 염증 완화 목적으로 NSAID 약물 및 스테로이드성 호르몬이 퇴행성관절염 환자에 사용되고 있지만, 이는 질병 자체를 치료하기 보다는 증상만을 완화시키므로 실질적인 퇴행성관절염 치료제로서의 역할은 될 수 없다. 뿐만 아니라, 연골세포 및 연골조직 파괴에 의해 주로 발생하는 퇴행성관절염은 그 발병 원인과 현상이 염증성 관절염인, 류마티스관절염과는 엄연히 다르기 때문에, 퇴행성관절염 치료방법 역시 류마티스관절염 치료방법과는 다를 수밖에 없다.So far, known therapeutic agents for rheumatoid arthritis include non-steroidal anti-inflammatory drugs (NSAIDs), penicillamine, steroid hormones, TNF inhibitors, interleukin inhibitors, JAK inhibitors, and anti-CD related inhibitors suitable for blocking the inflammatory mechanism. applicable NSAID drugs and steroid hormones are used in patients with degenerative arthritis for the purpose of relieving joint pain and inflammation, but since they only relieve symptoms rather than treating the disease itself, they cannot serve as a practical therapeutic agent for degenerative arthritis. In addition, since degenerative arthritis, which is mainly caused by destruction of cartilage cells and cartilage tissue, is distinctly different from rheumatoid arthritis, which is an inflammatory arthritis, in its cause and phenomenon, the treatment method for degenerative arthritis is also different from the treatment method for rheumatoid arthritis.
따라서, 퇴행성 관절염의 진행을 막거나 늦추기 위해서는 퇴행성관절염을 조기에 진단할 필요가 있으나, 현재까지는 퇴행성관절염을 진단하는 방법으로 정형외과의 병원내진 또는 X-ray와 같은 방법 등 만이 알려져 있는 실정이다.Therefore, in order to prevent or slow down the progression of degenerative arthritis, it is necessary to diagnose degenerative arthritis at an early stage, but until now, as a method of diagnosing degenerative arthritis, only methods such as orthopedic hospital visits or X-rays have been known.
또한, SNCG(Synuclein Gamma)는 SNCG 유전자에 의해 암호화되는 단백질로, 신경 퇴행성 질환의 병인으로 알려진 synuclein 계열의 단백질이다. 진행성 유방암에서 높은 수분으로 SNCG가 확인되며, SNCG의 과발현은 유방 종양 발생에 관련이 있다고 보고된 바 있다. SNCG는 주로 말초 신경계(일차 감각 뉴런, 교감 뉴런, 운동 뉴런)와 망막에서 발견되며, 뇌, 난소, 종양 및 후각 상피에서도 발견된다. SNCG는 종양의 마커로도 알려져 있고, 알츠하이머 환자의 망막에서도 발견된 바 있으나, 현재까지 SNCG가 정상 세포에서 어떠한 기능을 하는지 알려진 바 없으나, 퇴행성 신경 질환과 관련이 있음을 토대로 많은 연구가 진행되고 있다.In addition, Synuclein Gamma (SNCG) is a protein encoded by the SNCG gene and is a synuclein family protein known to cause neurodegenerative diseases. It has been reported that high levels of SNCG are identified in advanced breast cancer, and overexpression of SNCG is associated with breast tumor development. SNCGs are found primarily in the peripheral nervous system (primary sensory neurons, sympathetic neurons, and motor neurons) and in the retina, as well as in the brain, ovaries, tumors, and olfactory epithelium. SNCG is known as a tumor marker and has been found in the retina of Alzheimer's patients. However, until now, it has not been known what function SNCG plays in normal cells, but many studies are being conducted based on its association with neurodegenerative diseases. .
퇴행성 신경질환이란 중추신경계의 신경세포에 퇴행성 변화가 나타나면서 여러 가지 증상을 유발하는 질환들로서, 대부분 발생 원인이 알려져 있지 않으며, 질병의 발병이 서서히 시작하고, 일단 발병하면 사망할 때까지 수년 혹은 수십년에 걸쳐 지속적으로 병이 진행한다. 대표적인 퇴행성 신경질환으로는 파킨슨병, 알츠하이머병, 퇴행성 관절염 또는 근위축성측삭경화증(ALS) 등을 들 수 있다.Degenerative neurological diseases are diseases that cause degenerative changes in the nerve cells of the central nervous system and cause various symptoms. The disease progresses continuously over the years. Representative neurodegenerative diseases include Parkinson's disease, Alzheimer's disease, degenerative arthritis or amyotrophic lateral sclerosis (ALS).
퇴행성 관절염(degenerative arthritis)은 골관절염(osteoarthritis)로도 지칭되는 관절염의 일종으로서, 윤활관절에서 연골과 주위골에 퇴행성 변화가 나타나서 생기는 관절염을 말한다. 즉, 퇴행성 관절염은 관절 연골의 점차적인 소실과 더불어 연골 하방에 위치한 뼈의 비대, 관절 가장자리 부위의 골 생성, 및 비특이적인 활막 염증을 특징으로 하는 질환이다. 퇴행성 관절염은 노화나 과도한 물리적 압박(예를 들어, 비만, 외상 등)에 의해서 연골이 손상되어 발생하는 질환이다. 따라서, 퇴행성 관절염은 체중을 많이 받는 관절, 즉, 무릎(슬)관절, 엉덩이 (고)관절 등에 심한 통증과 운동 장애를 나타내며, 장기간 방치할 경우에는 관절의 변형까지 초래하게 된다.Degenerative arthritis is a type of arthritis, also referred to as osteoarthritis, and refers to arthritis caused by degenerative changes in cartilage and surrounding bones in synovial joints. That is, degenerative arthritis is a disease characterized by gradual loss of articular cartilage, hypertrophy of bones located below the cartilage, bone production at the edge of the joint, and non-specific synovial inflammation. Osteoarthritis is a disease caused by damage to cartilage due to aging or excessive physical pressure (eg, obesity, trauma, etc.). Therefore, degenerative arthritis shows severe pain and movement disorders in joints that receive a lot of weight, that is, knee (knee) joints and hip (hip) joints.
파킨슨병(Parkinson's disease)은 간뇌의 변성 또는 동맥경화적인 변화를 주로 한 중추신경계의 퇴행성 질환으로, 운동장애가 주증상이며, 정상인의 뇌에서 흑색질이라는 부위의 신경세포들이 변성되고 이 곳에서 만들어지는 신경전달 물질인 도파민이 결핍되어 초래된다. 파킨슨병은 도파민(dopamine)을 만드는 신경세포가 손상되어 도파민이 부족해지면서 균형을 유지하던 아세틸콜린이 우세하여 나타나는 만성, 진행성 운동신경계 질환이며, 이러한 질환의 주요증상은 손이나 팔, 다리, 얼굴 등이 떨리는 진전이나 팔다리나 몸이 뻣뻣해지는 경직, 움직임이 느려지는 운동완서, 균형을 잡지 못하는 자세 불안정 등으로 나타난다.Parkinson's disease is a degenerative disease of the central nervous system mainly caused by degeneration or arteriosclerotic changes of the diencephalon, and movement disorder is the main symptom. It is caused by a lack of the transmitter dopamine. Parkinson's disease is a chronic, progressive motor neurological disease that occurs when dopamine-producing nerve cells are damaged and dopamine is lacking, resulting in acetylcholine, which was maintained in balance, predominance. This is manifested by trembling tremors, stiffness of the limbs or body, bradykinesia that slows movement, and postural instability that prevents balance.
본 발명은 SNCG의 발현 또는 활성 수준 변화를 확인하여 퇴행성 관절염을 조기에 진단하기 위한 바이오마커 조성물 및 상기 바이오마커를 발현 수준을 측정할 수 있는 진단키트를 제공하며, 상기 바이오마커를 이용한 퇴행성 관절염 진단방법, 퇴행성 관절염 치료제 스크리닝 방법을 제공하고자 한다. The present invention provides a biomarker composition for early diagnosis of degenerative arthritis by confirming changes in the expression or activity level of SNCG, and a diagnostic kit capable of measuring the expression level of the biomarker, and diagnosing degenerative arthritis using the biomarker It is intended to provide a method and a method for screening a therapeutic agent for degenerative arthritis.
또한, SNCG 발현 또는 활성 억제제를 유효성분으로 함유하는 퇴행성 질환 예방 또는 치료용 약학조성물을 제공하고자 한다.In addition, it is intended to provide a pharmaceutical composition for preventing or treating degenerative diseases containing an SNCG expression or activity inhibitor as an active ingredient.
본 발명은 SNCG (Synuclein Gamma) 유전자 또는 상기 유전자가 코딩하는 단백질을 포함하는 퇴행성 관절염 진단용 바이오마커 조성물을 제공한다.The present invention provides a biomarker composition for diagnosing degenerative arthritis comprising a synuclein gamma (SNCG) gene or a protein encoded by the gene.
본 발명은 SNCG 유전자 또는 상기 유전자가 코딩하는 단백질의 발현 또는 활성 수준을 검출하는 제제를 유효성분으로 함유하는 퇴행성 관절염 진단용 키트를 제공한다.The present invention provides a kit for diagnosing degenerative arthritis containing, as an active ingredient, an agent for detecting the expression or activity level of a SNCG gene or a protein encoded by the gene.
본 발명은 검체로부터 분리된 시료로부터 SNCG 유전자 또는 상기 유전자가 코딩하는 단백질의 발현 또는 활성 수준을 확인하는 단계를 포함하는 퇴행성 관절염 진단을 위한 정보를 제공하는 방법을 제공한다. The present invention provides a method for providing information for diagnosing degenerative arthritis comprising the step of confirming the expression or activity level of a SNCG gene or a protein encoded by the gene from a sample isolated from a specimen.
본 발명은 퇴행성 관절염 환자로부터 분리된 연골세포에 후보물질을 처리하는 단계; 상기 후보물질이 처리된 연골세포로부터 SNCG 발현 수준을 확인하는 단계; 및 상기 SNCG 발현 수준이 후보물질 비처리군과 비교하여 감소된 후보물질을 퇴행성 관절염 치료제로 제공하는 퇴행성 관절염 치료제 스크리닝 방법을 제공한다.The present invention comprises the steps of processing a candidate substance to chondrocytes isolated from a patient with degenerative arthritis; Checking the expression level of SNCG from the chondrocytes treated with the candidate substance; and a method for screening a treatment for degenerative arthritis, wherein the candidate substance having a reduced expression level of the SNCG compared to a group not treated with the candidate substance is provided as a treatment for degenerative arthritis.
본 발명은 SNCG 발현 또는 활성 억제제를 유효성분으로 함유하는 퇴행성 관절염 예방 또는 치료용 약학조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating degenerative arthritis containing an SNCG expression or activity inhibitor as an active ingredient.
본 발명은 SNCG 발현 또는 활성 억제제를 유효성분으로 함유하는 퇴행성 관절염 예방 또는 개선용 건강식품을 제공한다.The present invention provides a health food for preventing or improving degenerative arthritis, which contains a SNCG expression or activity inhibitor as an active ingredient.
본 발명은 SNCG 발현 또는 활성 조절제를 유효성분으로 함유하는 SNCG 관련 질환 예방 또는 치료용 약학조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating SNCG-related diseases, containing a SNCG expression or activity regulator as an active ingredient.
본 발명은 AZ-628 또는 PD-184352에서 선택된 화합물 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 SNCG(Synuclein Gamma) 과발현 질환의 예방 또는 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating Synuclein Gamma (SNCG) overexpression diseases, comprising a compound selected from AZ-628 or PD-184352 or a pharmaceutically acceptable salt thereof as an active ingredient.
또한, 본 발명은 AZ-628 또는 PD-184352를 유효성분으로 포함하는 SNCG(Synuclein Gamma) 과발현 질환의 예방 또는 개선용 건강기능 식품 조성물을 제공한다.In addition, the present invention provides a health functional food composition for preventing or improving Synuclein Gamma (SNCG) overexpression diseases, comprising AZ-628 or PD-184352 as an active ingredient.
본 발명에 따르면 퇴행성 관절염 발병시 SNCG 발현 및 활성이 증가하고, 이에 따라 연골 파괴를 유도하는 Mmp3, Mmp13 및 Cox2의 발현 증가를 확인하였으며, SNCG 발현 억제에 의해 상기 유전자들의 발현이 억제되는 것을 확인함에 따라, SNCG를 퇴행성 관절염 진단용 바이오마커로 제공하며, SNCG를 결핍시킨 결과, 퇴행성 관절염이 완화되는 효과를 확인함에 따라, 상기 SNCG 발현 또는 활성 억제제를 퇴행성 관절염 치료제로 제공하고자 한다.According to the present invention, SNCG expression and activity increase during the onset of degenerative arthritis, and accordingly, it was confirmed that the expression of Mmp3, Mmp13, and Cox2, which induce cartilage destruction, was increased, and it was confirmed that the expression of the above genes was suppressed by suppressing SNCG expression. Accordingly, SNCG is provided as a biomarker for diagnosing degenerative arthritis, and as a result of depletion of SNCG, the effect of degenerative arthritis is alleviated, the SNCG expression or activity inhibitor is intended to be provided as a therapeutic agent for degenerative arthritis.
또한 본 발명에 따르면, SNCG(Synuclein Gamma) 억제제인 AZ-628 또는 PD-184352는 SNCG(Synuclein Gamma) 단백질의 C-말단에 결합하여 SNCG에 의해 조절되는 퇴행성 질환과 관련 인자들인 MMP3, MMP13 및 COX2의 발현을 억제하는 것을 확인함으로써, 상기 화합물은 SNCG(Synuclein Gamma)에 의해 발병되는 퇴행성 관절염 및 파킨슨병의 치료제로 제공될 수 있다.In addition, according to the present invention, AZ-628 or PD-184352, a synuclein gamma (SNCG) inhibitor, binds to the C-terminus of the synuclein gamma (SNCG) protein and binds to the degenerative disease-related factors regulated by SNCG, MMP3, MMP13, and COX2. By confirming that the expression of is suppressed, the compound can be provided as a therapeutic agent for degenerative arthritis and Parkinson's disease caused by Synuclein Gamma (SNCG).
도 1은 SNCG 발현이 퇴행성관절염 환자 및 퇴행성관절염 유발 마우스 관절 조직에서 SNCG 발현을 확인한 결과로, 도 1A는 사람 관절에서 확인한 결과이며, 도 1B는 마우스 관절에서 확인한 결과이다.1 is a result of confirming SNCG expression in degenerative arthritis patients and degenerative arthritis-induced mouse joint tissues, FIG. 1A is the result confirmed in human joints, and FIG. 1B is the result confirmed in mouse joints.
도 2는 SNCG 발현의 변화(modulation)와 퇴행성 관절염과의 상관관계를 확인한 결과로, 도 2A는 재조합 SNCG를 농도별로 연골세포에 처리하였을 때 Mmp3, Mmp13 및 Cox2와 같은 catabolic factor가 증가됨을 확인 할 수 있었으며, 동시에 Col2 및 Sox9과 같은 anabolic factor의 발현이 감소되는 것을 확인한 웨스턴 블롯 분석 결과이며, 도 2B는 연골세포에 Ad-C 또는 Ad-SNCG로 감염된 연골 세포에서 SNCG, MMP3, MMP13 및 COX2 발현이 증가하고 동시에 Col2 및 Sox9의 발현이 감소되는 것을 확인한 웨스턴 블롯 분석 결과이며, 도 2C 및 도 2D는 사프라닌-O 염색, OARSI 등급, 골극 형성 및 연골하 골판 두께로 평가된 Ad-C 및 Ad-SNCG가 관절 내로 주입된 마우스 무릎 관절의 연골 파괴 및 퇴행성 관절염 발달을 확인한 결과로, SNCG가 과발현 되었을 때 연골파괴가 촉진되는 것을 확인한 결과이다.2 is a result of confirming the correlation between modulation of SNCG expression and degenerative arthritis, and FIG. 2A confirms that catabolic factors such as Mmp3, Mmp13 and Cox2 are increased when chondrocytes are treated with recombinant SNCG at different concentrations. 2B shows the expression of SNCG, MMP3, MMP13 and COX2 in chondrocytes infected with Ad-C or Ad-SNCG. This is the result of western blot analysis confirming that the expression of Col2 and Sox9 is increased and the expression of Sox9 is decreased at the same time, and FIG. 2C and FIG. 2D are Safranin-O staining, OARSI grade, osteophyte formation, and Ad-C and As a result of confirming cartilage destruction and the development of degenerative arthritis in the mouse knee joint injected with Ad-SNCG into the joint, it was confirmed that cartilage destruction was promoted when SNCG was overexpressed.
도 3은 SNCG 녹아웃 마우스에서 퇴행성 관절염 병인이 감소되는 것을 확인한 결과로, 도 3A는 WT 및 SNCG 녹아웃 마우스에 수술 DMM을 적용하여 퇴행성 관절염을 유도한 후 연골 파괴를 확인한 사프라닌-O 염색결과이며, 도 3B는 OARSI 등급, 골극 형성 및 연골하 골판 두께로 연골 파괴를 확인한 결과로, 값은 표시된 수의 독립된 실험의 표준 ± 표준편차로 표시되었으며, 통계 분석은 one-way ANOVA with Bonferroni's test, a non-parametric Mann-Whitney U test (A, OARSI 등급 지정, 골극 형성), 또는 two-tailed t-test (A, 연골하 골판 두께)로 수행되었다 (스케일 바 : 100 μm).Figure 3 is a result of confirming that the pathogenesis of degenerative arthritis is reduced in SNCG knockout mice. Figure 3A is a safranin-O staining result confirming cartilage destruction after inducing degenerative arthritis by applying surgical DMM to WT and SNCG knockout mice. , Figure 3B is the result of confirming cartilage destruction by OARSI grade, osteophyte formation, and subchondral bone plate thickness. Values are expressed as the standard ± standard deviation of the indicated number of independent experiments, and statistical analysis is one-way ANOVA with Bonferroni's test, a Non-parametric Mann-Whitney U test (A, OARSI grading, osteophyte formation), or two-tailed t-test (A, subchondral bone plate thickness) were performed (scale bar: 100 μm).
도 4는 CMap 접근법을 이용한 in silico 화합물 스크리닝으로 SNCG(Synuclein Gamma) 억제제로서 AZ-628을 선별한 결과이다.4 shows the results of selecting AZ-628 as a Synuclein Gamma (SNCG) inhibitor by in silico compound screening using the CMap approach.
도 5는 CMap 접근법을 이용한 in silico 화합물 스크리닝으로 SNCG(Synuclein Gamma) 억제제로서 PD-184352를 선별한 결과이다.5 is a result of selecting PD-184352 as a Synuclein Gamma (SNCG) inhibitor by in silico compound screening using the CMap approach.
도 6은 선별된 SNCG(Synuclein Gamma) 억제제인 AZ-628이 SNCG(Synuclein Gamma) 과발현으로 발병되는 퇴행성 관절염의 치료 효과를 평가한 결과이다.6 is a result of evaluating the therapeutic effect of AZ-628, a selected Synuclein Gamma (SNCG) inhibitor, on degenerative arthritis caused by overexpression of SNCG (Synuclein Gamma).
도 7은 선별된 SNCG(Synuclein Gamma) 억제제인 PD-184352가 SNCG(Synuclein Gamma) 과발현으로 발병되는 퇴행성 관절염의 치료 효과를 평가한 결과이다.7 is a result of evaluating the treatment effect of PD-184352, a selected Synuclein Gamma (SNCG) inhibitor, on degenerative arthritis caused by overexpression of SNCG (Synuclein Gamma).
도 8은 선별된 SNCG(Synuclein Gamma) 억제제인 AZ-628이 SNCG(Synuclein Gamma) 활성에 영향을 미치는지 Discovery studio를 활용하여 in silico binding affinity을 측정한 결과이다. AZ-628 아미노사 서열의 C-terminal에 글루타민 (Q), 글루탐산 (E), 글라이신 (G), 알라닌 (A), 라이신 (K) (노랑색 표기)로 처리된 부분에 AZ-628이 결합됨을 나타낸다.8 is a result of measuring in silico binding affinity using Discovery studio to see whether AZ-628, a selected Synuclein Gamma (SNCG) inhibitor, affects Synuclein Gamma (SNCG) activity. AZ-628 binds to the C-terminal of the AZ-628 amino acid sequence treated with glutamine (Q), glutamic acid (E), glycine (G), alanine (A), and lysine (K) (yellow color) indicate
도 9는 선별된 SNCG(Synuclein Gamma) 억제제인 PD-184352가 SNCG(Synuclein Gamma) 활성에 영향을 미치는지 Discovery studio를 활용하여 in silico binding affinity을 측정한 결과이다. AZ-628 아미노사 서열의 C-terminal에 글루타민 (Q), 글루탐산 (E), 글라이신 (G), 알라닌 (A), 라이신 (K) (노랑색 표기)로 처리된 부분에 AZ-628이 결합됨을 나타낸다.9 is a result of measuring in silico binding affinity using Discovery studio to see whether PD-184352, a selected Synuclein Gamma (SNCG) inhibitor, affects SNCG (Synuclein Gamma) activity. AZ-628 binds to the C-terminal of the AZ-628 amino acid sequence treated with glutamine (Q), glutamic acid (E), glycine (G), alanine (A), and lysine (K) (yellow color) indicate
이하, 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명의 발명자들은 퇴행성 관절염 발병 초기에 SNCG 발현 및 활성이 증가하고, 이에 따라 연골 파괴를 유도하는 Mmp3, Mmp13 및 Cox2의 발현 증가를 확인하였으며, SNCG 발현 억제에 의해 상기 유전자들의 발현이 억제되는 것을 확인함에 따라, SNCG를 퇴행성 관절염 바이오마커로 제공하여 퇴행성 관절염을 조기에 진단하며, SNCG의 발현 또는 활성을 조절하여 퇴행성 관절염 치료효과를 향상시키고자 본 발명을 완성하였다.The inventors of the present invention confirmed that SNCG expression and activity increased in the early stage of degenerative arthritis, and thus increased expression of Mmp3, Mmp13, and Cox2, which induce cartilage destruction, and found that the expression of the above genes was suppressed by suppressing SNCG expression. As confirmed, the present invention was completed in order to diagnose degenerative arthritis early by providing SNCG as a biomarker for degenerative arthritis and to improve the therapeutic effect of degenerative arthritis by regulating the expression or activity of SNCG.
본 발명은 SNCG (Synuclein Gamma) 유전자 또는 상기 유전자가 코딩하는 단백질을 포함하는 퇴행성 관절염 진단용 바이오마커 조성물을 제공할 수 있다.The present invention may provide a biomarker composition for diagnosing degenerative arthritis comprising a synuclein gamma (SNCG) gene or a protein encoded by the gene.
상기 SNCG (Synuclein Gamma)는 UniGene ID Mm.41063일 수 있다.The Synuclein Gamma (SNCG) may be UniGene ID Mm.41063.
본 발명은 SNCG 유전자 또는 상기 유전자가 코딩하는 단백질의 발현 또는 활성 수준을 검출하는 제제를 유효성분으로 함유하는 퇴행성 관절염 진단용 키트를 제공할 수 있다.The present invention can provide a kit for diagnosing degenerative arthritis containing, as an active ingredient, an agent for detecting the expression or activity level of a SNCG gene or a protein encoded by the gene.
상기 제제는 SNCG 유전자에 특이적으로 결합하는 프라이머 또는 프로브, SNCG 단백질에 특이적으로 결합하는 항체, 펩타이드, 앱타머또는 화합물로 이루어진 군에서 선택되는 것일 수 있다.The agent may be selected from the group consisting of a primer or probe that specifically binds to the SNCG gene, an antibody, a peptide, an aptamer, or a compound that specifically binds to the SNCG protein.
본 발명은 검체로부터 분리된 시료로부터 SNCG 유전자 또는 상기 유전자가 코딩하는 단백질의 발현 또는 활성 수준을 확인하는 단계를 포함하는 퇴행성 관절염 진단을 위한 정보를 제공하는 방법을 제고할 수 있다.The present invention can provide a method for providing information for diagnosing degenerative arthritis, which includes the step of confirming the expression or activity level of a SNCG gene or a protein encoded by the gene from a sample isolated from a specimen.
상기 시료는 세포, 조직, 혈액, 혈청, 뇨 및 타액으로 이루어진 군에서 선택되는 것일 수 있다.The sample may be selected from the group consisting of cells, tissues, blood, serum, urine and saliva.
상기 퇴행성 관절염 진단을 위한 정보를 제공하는 방법은 SNCG 유전자 또는 상기 유전자가 코딩하는 단백질의 발현 또는 활성 수준이 정상대조군보다 증가한 경우 퇴행성 관절염으로 진단하는 것일 수 있다.A method of providing information for diagnosing degenerative arthritis may include diagnosing degenerative arthritis when the expression or activity level of the SNCG gene or a protein encoded by the gene is higher than that of a normal control group.
본 발명은 퇴행성 관절염 환자로부터 분리된 연골세포에 후보물질을 처리하는 단계; 상기 후보물질이 처리된 연골세포로부터 SNCG 발현 수준을 확인하는 단계; 및 상기 SNCG 발현 수준이 후보물질 비처리군과 비교하여 감소된 후보물질을 퇴행성 관절염 치료제로 제공하는 퇴행성 관절염 치료제 스크리닝 방법을 제공할 수 있다.The present invention comprises the steps of processing a candidate substance to chondrocytes isolated from a patient with degenerative arthritis; Checking the expression level of SNCG from the chondrocytes treated with the candidate substance; and a method of screening for a treatment for degenerative arthritis, wherein the candidate substance, in which the expression level of the SNCG is reduced compared to a group not treated with the candidate substance, is provided as a treatment for degenerative arthritis.
본 발명의 "바이오마커"는 퇴행성 관절염이 의심되는 대상체의 조직 또는 세포를 정상 대조군의 조직 또는 세포와 구분하여 진단할 수 있는 물질로, 정상 대조군에 비하여 대상체의 조직 또는 세포에서 증가 또는 감소 양상을 보이는 단백질 또는 핵산, 지질, 당지질, 당단백질 등과 같은 유기 생체 분자 등을 포함한다.The "biomarker" of the present invention is a substance that can be diagnosed by distinguishing the tissue or cell of a subject suspected of having degenerative arthritis from the tissue or cell of a normal control group, and measures the increase or decrease in the tissue or cell of the subject compared to the normal control group. organic biomolecules such as visible proteins or nucleic acids, lipids, glycolipids, glycoproteins, and the like;
본 발명의 "진단 (diagnosis)"은 넓은 의미는 환자의 병의 실태를 모든면에 걸쳐서 판단하는 것을 의미한다. 판단의 내용은 병명, 병인, 병형, 경중, 병상의 상세한 양태 및 합병증의 유무 등이다.In the present invention, "diagnosis" in a broad sense means to judge the actual condition of a patient's disease in all aspects. The content of judgment is the name of the disease, etiology, type of disease, severity, detailed condition of the disease, and presence or absence of complications.
본 발명은 SNCG (Synuclein Gamma) 발현 또는 활성 억제제를 유효성분으로 함유하는 퇴행성 관절염 예방 또는 치료용 약학조성물을 제공할 수 있다.The present invention can provide a pharmaceutical composition for preventing or treating degenerative arthritis containing a synuclein gamma (SNCG) expression or activity inhibitor as an active ingredient.
상기 SNCG 발현 억제제는 SNCG 유전자 또는 SNCG의 발현을 촉진하는 유전자의 mRNA에 특이적으로 결합하는 안티센스 뉴클레오티드, siRNA (small interfering RNA), shRNA (short hairpin RNA), microRNA 및 리보자임으로 이루어진 군에서 하나 이상 선택되는 것일 수 있다.The SNCG expression inhibitor is at least one from the group consisting of antisense nucleotides, small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA, and ribozymes that specifically bind to mRNA of a gene or gene promoting SNCG expression may be selected.
상기 SNCG 활성 억제제는 SNCG 활성을 촉진하는 단백질 또는 SNCG 단백질에 특이적으로 결합하는 화합물, 펩티드, 펩티드모방체, 단백질, 앱타머, 및 항체로 이루어진 군에서 하나 이상 선택되는 것일 수 있다.The SNCG activity inhibitor may be one or more selected from the group consisting of proteins that promote SNCG activity or compounds that specifically bind to SNCG proteins, peptides, peptidomimetics, proteins, aptamers, and antibodies.
본 발명의 한 구체예에서, 상기 약학조성물은 통상적인 방법에 따라 주사제, 과립제, 산제, 정제, 환제, 캡슐제, 좌제, 겔, 현탁제, 유제, 점적제 또는 액제로 이루어진 군에서 선택된 어느 하나의 제형을 사용할 수 있다.In one embodiment of the present invention, the pharmaceutical composition is any one selected from the group consisting of injections, granules, powders, tablets, pills, capsules, suppositories, gels, suspensions, emulsions, drops or liquids according to conventional methods. formulations can be used.
본 발명의 다른 구체예에서, SNCG 발현 또는 활성 억제제를 유효성분으로 함유하는 퇴행성 관절염 예방 또는 치료용 약학조성물은 약학조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제, 붕해제, 감미제, 피복제, 팽창제, 활택제, 향미제, 항산화제, 완충액, 정균제, 희석제, 분산제, 계면활성제, 결합제 및 윤활제로 이루어진 군에서 선택되는 하나 이상의 첨가제를 추가로 포함할 수 있다.In another embodiment of the present invention, a pharmaceutical composition for preventing or treating degenerative arthritis containing a SNCG expression or activity inhibitor as an active ingredient is suitable carriers, excipients, disintegrants, sweeteners, coating agents, It may further include one or more additives selected from the group consisting of an expanding agent, a lubricant, a flavoring agent, an antioxidant, a buffer, a bacteriostatic agent, a diluent, a dispersing agent, a surfactant, a binder, and a lubricant.
구체적으로 담체, 부형제 및 희석제는 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 사용할 수 있으며, 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 조성물에 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘카보네이트, 수크로스 또는 락토오스, 젤라틴 등을 섞어 조제할 수 있다. 또한 단순한 부형제 이외에 마그네슘 스티레이트, 탈크 같은 윤활제들도 사용할 수 있다. 경구를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 있으며 흔히 사용되는 단순 희석제인 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, 동결건조제제, 좌제 등이 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기재로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.Specifically, carriers, excipients and diluents are 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 oil may be used, and solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules. These solid preparations may be prepared by mixing at least one or more excipients, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions for oral use, emulsions, syrups, and the like, and various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base material of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.
본 발명의 일실시예에 따르면 상기 약학 조성물은 정맥내, 동맥내, 복강내, 근육내, 흉골내, 경피, 비측내, 흡입, 국소, 직장, 경구, 안구내 또는 피내 경로를 통해 통상적인 방식으로 대상체로 투여할 수 있다.According to one embodiment of the present invention, the pharmaceutical composition is administered in a conventional manner through intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or intradermal routes. can be administered to the subject as
상기 SNCG 발현 또는 활성 억제제의 바람직한 투여량은 대상체의 상태 및 체중, 질환의 종류 및 정도, 약물 형태, 투여경로 및 기간에 따라 달라질 수 있으며 당업자에 의해 적절하게 선택될 수 있다. 본 발명의 일실시예에 따르면 이에 제한되는 것은 아니지만 1일 투여량이 0.01 내지 200 mg/kg, 구체적으로는 0.1 내지 200 mg/kg, 보다 구체적으로는 0.1 내지 100 mg/kg 일 수 있다. 투여는 하루에 한 번 투여할 수도 있고 수회로 나누어 투여할 수도 있으며, 이에 의해 본 발명의 범위가 제한되는 것은 아니다.A preferable dosage of the SNCG expression or activity inhibitor may vary depending on the condition and weight of the subject, the type and severity of the disease, the type of drug, the route and duration of administration, and may be appropriately selected by a person skilled in the art. According to one embodiment of the present invention, but not limited thereto, the daily dosage may be 0.01 to 200 mg/kg, specifically 0.1 to 200 mg/kg, and more specifically 0.1 to 100 mg/kg. Administration may be administered once a day or divided into several administrations, and the scope of the present invention is not limited thereby.
본 발명에 있어서, 상기 '대상체'는 인간을 포함하는 포유동물일 수 있으나, 이들 예에 한정되는 것은 아니다.In the present invention, the 'subject' may be a mammal including a human, but is not limited to these examples.
본 발명은 SNCG 발현 또는 활성 억제제를 유효성분으로 함유하는 퇴행성 관절염 예방 또는 개선용 건강식품을 제공할 수 있다.The present invention can provide a health food for preventing or improving degenerative arthritis containing an SNCG expression or activity inhibitor as an active ingredient.
상기 건강식품은 상기 SNCG 발현 또는 활성 억제제 이외에 다른 식품 또는 식품 첨가물과 함께 사용되고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분의 혼합양은 그의 사용 목적 예를 들어 예방, 건강 또는 치료적 처치에 따라 적합하게 결정될 수 있다.The health food may be used together with other foods or food additives in addition to the SNCG expression or activity inhibitors, and may be appropriately used according to conventional methods. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use thereof, for example, prevention, health or therapeutic treatment.
상기 건강식품에 함유된 화합물의 유효용량은 상기 치료제의 유효용량에 준해서 사용할 수 있으나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 범위 이하일 수 있으며, 유효성분은 안전성 면에서 아무런 문제가 없기 때문에 상기 범위 이상의 양으로도 사용될 수 있음은 확실하다.The effective dose of the compound contained in the health food may be used according to the effective dose of the therapeutic agent, but may be less than the above range in the case of long-term intake for the purpose of health and hygiene or health control. Since there is no problem in terms of safety, it is certain that the components can be used in an amount greater than the above range.
상기 건강식품의 종류에는 특별한 제한이 없고, 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제등을 들 수 있다.There is no particular limitation on the type of health food, and examples include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes; and the like.
또한, 본 발명은 SNCG 발현 또는 활성 조절제를 유효성분으로 함유하는 SNCG 관련 질환 예방 또는 치료용 약학조성물을 제공할 수 있다.In addition, the present invention can provide a pharmaceutical composition for preventing or treating SNCG-related diseases containing a SNCG expression or activity regulator as an active ingredient.
또한, 본 발명은 하기 화학식 1 또는 화학식 2로 표시되는 화합물 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 SNCG(Synuclein Gamma) 과발현 질환의 예방 또는 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating Synuclein Gamma (SNCG) overexpression diseases, comprising a compound represented by Formula 1 or Formula 2 below or a pharmaceutically acceptable salt thereof as an active ingredient.
[화학식 1][Formula 1]
Figure PCTKR2022011297-appb-img-000001
Figure PCTKR2022011297-appb-img-000001
[화학식 2][Formula 2]
Figure PCTKR2022011297-appb-img-000002
Figure PCTKR2022011297-appb-img-000002
상기 약학적으로 허용 가능한 염은 약학적으로 허용 가능한 유리산(free acid)에 의해 형성된 산 부가염을 의미하고, 약학적으로 허용 가능한 염은 의약업계에서 통상적으로 사용되는 염을 의미하며, 예를 들어 칼슘, 포타슘, 소듐 또는 마그네슘 등으로 제조된 무기이온염, 염산, 질산, 인산, 브롬산, 요오드산, 과염소산 또는 황산 등으로 제조된 무기산염; 아세트산, 트라이플루오로아세트산, 시트르산, 말레산, 숙신산, 옥살산, 벤조산, 타르타르산, 푸마르산, 만델산, 프로피온산, 젖산, 글리콜산, 글루콘산, 갈락투론산, 글루탐산, 글루타르산, 글루쿠론산, 아스파르트산, 아스코르브산, 카본산 또는, 바닐릭산 등으로 제조된 유기산염; 메탄설폰산, 에탄설폰산, 벤젠설폰산, p-톨루엔설폰산 또는 나프탈렌설폰산 등으로 제조된 설폰산염; 글리신, 아르기닌, 라이신 등으로 제조된 아미노산염; 또는 트라이메틸아민, 트라이에틸아민, 암모니아, 피리딘, 피콜린 등으로 제조된 아민염 등이 있으나, 열거된 이들 염에 의해 본 발명에서 의미하는 염의 종류가 한정되는 것은 아니다.The pharmaceutically acceptable salt refers to an acid addition salt formed by a pharmaceutically acceptable free acid, and the pharmaceutically acceptable salt refers to a salt commonly used in the pharmaceutical industry, for example For example, an inorganic ion salt made of calcium, potassium, sodium or magnesium, an inorganic acid salt made of hydrochloric acid, nitric acid, phosphoric acid, hydrobromic acid, iodic acid, perchloric acid or sulfuric acid; Acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid organic acid salts made of acid, ascorbic acid, carbonic acid or vanillic acid; sulfonic acid salts prepared from methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or naphthalenesulfonic acid; amino acid salts made of glycine, arginine, lysine, and the like; or amine salts prepared with trimethylamine, triethylamine, ammonia, pyridine, picoline, etc., but the types of salts meant in the present invention are not limited by these listed salts.
상기 화학식 1로 표시되는 화합물은 AZ-628이며, 구체적으로 3-(2-시아노프로판-2-일)-N-(4-메틸-3-((3-메틸-4-옥소-3,4-디하이드로퀴나졸린-6-일)아미노)페놀)벤자마이드(3-(2-cyanopropan-2-yl)-N-(4-methyl-3-((3-methyl-4-oxo-3,4-dihydroquinazolin-6-yl)amino)phenyl)benzamide)이라 명명할 수 있다. 상기 화합물은 SNCG(Synuclein Gamma) 단백질의 C-말단에 결합하여 MMP3, MMP13 및 COX2의 발현을 억제한다.The compound represented by Formula 1 is AZ-628, specifically 3-(2-cyanopropan-2-yl)-N-(4-methyl-3-((3-methyl-4-oxo-3, 4-dihydroquinazolin-6-yl)amino)phenol)benzamide (3-(2-cyanopropan-2-yl)-N-(4-methyl-3-((3-methyl-4-oxo-3 ,4-dihydroquinazolin-6-yl)amino)phenyl)benzamide). The compound inhibits the expression of MMP3, MMP13 and COX2 by binding to the C-terminus of Synuclein Gamma (SNCG) protein.
상기 화학식 2로 표시되는 화합물은 PD-184352이며 2-((2-클로로-4-아이오도페닐)아미노)-N-(사이클로프로필메톡시)-3,4-디플루오로벤자마이드(2-((2-chloro-4-iodophenyl)amino)-N-(cyclopropylmethoxy)-3,4-difluorobenzamide)라 명명될 수 있다. 상기 화합물은 SNCG(Synuclein Gamma) 단백질의 C-말단에 결합하여 MMP3, MMP13 및 COX2의 발현을 억제한다.The compound represented by Formula 2 is PD-184352 and is 2-((2-chloro-4-iodophenyl)amino)-N-(cyclopropylmethoxy)-3,4-difluorobenzamide (2- ((2-chloro-4-iodophenyl)amino)-N-(cyclopropylmethoxy)-3,4-difluorobenzamide). The compound inhibits the expression of MMP3, MMP13 and COX2 by binding to the C-terminus of Synuclein Gamma (SNCG) protein.
상기 약학적 조성물은 SNCG(Synuclein Gamma)의 과발현으로 발병되는 질환의 환자에게 투여하기 위한 것이다. 상기 SNCG(Synuclein Gamma) 과발현 질환은 퇴행성 관절염 또는 파킨슨병이다.The pharmaceutical composition is to be administered to a patient with a disease caused by overexpression of Synuclein Gamma (SNCG). The synuclein gamma (SNCG) overexpression disease is degenerative arthritis or Parkinson's disease.
본 발명의 약학적 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다.The pharmaceutical composition of the present invention is prepared in unit dosage form or multi-dose by formulating using a pharmaceutically acceptable carrier according to a method that can be easily performed by those skilled in the art. It can be prepared by incorporating into a container.
상기 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸 히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 약학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다.The pharmaceutically acceptable carrier is one commonly used in formulation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components.
본 발명에 있어서, 상기 약학적 조성물에 포함되는 첨가제의 함량은 특별히 한정되는 것은 아니며 통상의 제제화에 사용되는 함량 범위 내에서 적절하게 조절될 수 있다.In the present invention, the content of the additives included in the pharmaceutical composition is not particularly limited and may be appropriately adjusted within the range of content used in conventional formulations.
상기 약학적 조성물은 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립, 정제, 크림, 젤, 패취, 분무제, 연고제, 경고제, 로션제, 리니멘트제, 파스타제 및 카타플라스마제로 이루어진 군으로부터 선택되는 하나 이상의 외용제 형태로 제형화될 수 있다.The pharmaceutical composition may be formulated as an aqueous solution, suspension, emulsion, etc. for injection, pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, warning agents, lotions, liniment agents, pasta agents, and cataplasma agents. It may be formulated in the form of one or more external preparations selected from the group consisting of agents.
본 발명의 약학적 조성물은 제형화를 위해 추가로 있는 약학적으로 허용가능한 담체 및 희석제를 포함할 수 있다. 상기 약학적으로 허용가능한 담체 및 희석제는 전분, 당, 및 만니톨과 같은 부형제, 칼슘 포스페이트 등과 같은 충전제 및 증량제, 카르복시메틸셀룰로오스, 히드록시프로필셀룰로오스 등과 같은 셀룰로오스 유도체, 젤라틴, 알긴산염, 및 폴리비닐 피롤리돈 등과 같은 결합제, 활석, 스테아린산 칼슘, 수소화 피마자유 및 폴리에틸렌 글리콜과 같은 윤활제, 포비돈, 크로스포비돈과 같은 붕해제, 폴리소르베이트, 세틸알코올, 및 글리세롤 등과 같은 계면활성제를 포함하나, 이에 한정되지 않는다. 상기 약학적으로 허용가능한 담체 및 희석제는 대상체에게 생물학적 및 생리학적으로 친화적인 것일 수 있다. 희석제의 예로는 염수, 수용성 완충액, 용매 및/또는 분산제(dispersion media)를 들 수 있으나, 이에 제한되는 것은 아니다.The pharmaceutical composition of the present invention may further contain pharmaceutically acceptable carriers and diluents for formulation. The pharmaceutically acceptable carriers and diluents include excipients such as starch, sugar and mannitol, fillers and extenders such as calcium phosphate, cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose, gelatin, alginates, and polyvinyl fibres. binders such as rolidone, etc., lubricants such as talc, calcium stearate, hydrogenated castor oil and polyethylene glycol, disintegrants such as povidone, crospovidone, surfactants such as polysorbates, cetyl alcohol, and glycerol; don't The pharmaceutically acceptable carrier and diluent may be biologically and physiologically compatible with the subject. Examples of diluents include, but are not limited to, saline, aqueous buffers, solvents and/or dispersion media.
본 발명의 약학적 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구 투여(예를 들어, 정맥 내, 피하, 복강 내 또는 국소에 적용)할 수 있다. 경구 투여일 경우, 정제, 트로키제 (troches), 로젠지 (lozenge), 수용성 현탁액, 유성 현탁액, 조제 분말, 과립, 에멀젼, 하드 캡슐, 소프트 캡슐, 시럽 또는 엘릭시르제 등으로 제형화될 수 있다. 비경구 투여일 경우, 주사액, 좌제, 호흡기 흡입용 분말, 스프레이용 에어로졸제, 연고, 도포용 파우더, 오일, 크림 등으로 제형화 될 수 있다.The pharmaceutical composition of the present invention may be administered orally or parenterally (eg, intravenous, subcutaneous, intraperitoneal or topical application) depending on the desired method. For oral administration, it may be formulated into tablets, troches, lozenges, aqueous suspensions, oily suspensions, powders, granules, emulsions, hard capsules, soft capsules, syrups or elixirs. In the case of parenteral administration, it may be formulated as an injection solution, suppository, powder for respiratory inhalation, aerosol for spray, ointment, powder for application, oil, cream, etc.
본 발명의 약학적 조성물의 투여량은 환자의 상태 및 체중, 연령, 성별, 건강상태, 식이 체질 특이성, 제제의 성질, 질병의 정도, 조성물의 투여시간, 투여방법, 투여기간 또는 간격, 배설율, 및 약물 형태에 따라 그 범위가 다양할 수 있으며, 이 분야 통상의 기술자에 의해 적절하게 선택될 수 있다. 예컨대, 약 0.1 내지 10,000 mg/kg의 범위일 수 있으나 이제 제한되지 않으며, 하루 일회 내지 수회에 나누어 투여될 수 있다.The dosage of the pharmaceutical composition of the present invention depends on the condition and weight of the patient, age, sex, health condition, dietary constitution specificity, the nature of the preparation, the severity of the disease, the administration time of the composition, the administration method, the administration period or interval, and the excretion rate. , And the range may vary depending on the type of drug, and may be appropriately selected by a person skilled in the art. For example, it may be in the range of about 0.1 to 10,000 mg/kg, but is not limited thereto, and may be divided and administered once or several times a day.
상기 약학적 조성물은 목적하는 방법에 따라 경구 투여되거나 비경구 투여(예를 들면, 정맥 내, 피하 내, 복강 내 또는 국소에 적용)될 수 있다. 본 발명의 약학적 조성물의 약학적 유효량, 유효 투여량은 약학적 조성물의 제제화 방법, 투여 방식, 투여 시간 및/또는 투여 경로 등에 의해 다양해질 수 있으며, 당해 기술 분야에서 통상의 지식을 가진 자는 목적하는 치료에 효과적인 투여량을 용이하게 결정하고 처방할 수 있다. 본 발명의 약학적 조성물의 투여는 하루에 1회 투여될 수 있고, 수회에 나누어 투여될 수도 있다.The pharmaceutical composition may be administered orally or parenterally (eg, intravenous, subcutaneous, intraperitoneal or topical application) depending on the desired method. The pharmaceutically effective amount and effective dose of the pharmaceutical composition of the present invention may vary depending on the formulation method, administration method, administration time and/or administration route of the pharmaceutical composition, and those skilled in the art can use the purpose Dosages effective for treatment can be easily determined and prescribed. Administration of the pharmaceutical composition of the present invention may be administered once a day, or may be divided into several administrations.
또한, 본 발명은 하기 화학식 1 또는 화학식 2로 표시되는 화합물 및 식품학적으로 허용 가능한 식품보조 첨가제를 포함하는 SNCG(Synuclein Gamma) 과발현 질환의 예방 또는 개선용 건강기능 식품 조성물In addition, the present invention is a health functional food composition for preventing or improving SNCG (Synuclein Gamma) overexpression disease comprising a compound represented by Formula 1 or Formula 2 and a food additive acceptable in food science
[화학식 1][Formula 1]
Figure PCTKR2022011297-appb-img-000003
Figure PCTKR2022011297-appb-img-000003
[화학식 2][Formula 2]
Figure PCTKR2022011297-appb-img-000004
Figure PCTKR2022011297-appb-img-000004
본 발명은 통상적으로 이용되는 식품으로써 일반적으로 사용될 수 있다.The present invention can be generally used as a commonly used food.
상기 식품보조 첨가제는 당업계에 통상적인 식품첨가제, 예를 들어 향미제, 풍미제, 착색제, 충진제, 안정화제 등을 포함하며 하기에 예시한다.The food supplement additives include conventional food additives in the art, for example, flavoring agents, flavoring agents, coloring agents, fillers, stabilizers, and the like, and are exemplified below.
본 발명의 건강 음료 조성물은 지시된 비율로 필수 성분으로서 상기 추출물을 함유하는 외에는 액체성분에는 특별한 제한점은 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예로는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린; 등과 같은 통상적인 당 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제(타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진 등)) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다.The health drink composition of the present invention has no particular limitations on the liquid component except for containing the extract as an essential component in the indicated ratio, and may contain various flavors or natural carbohydrates as additional components like conventional beverages. Examples of the aforementioned natural carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; polysaccharides such as dextrins, cyclodextrins; and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (eg rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can advantageously be used. there is.
본 발명의 조성물은 여러가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 본 발명의 조성물들은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용 할 수 있다.The composition of the present invention contains various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids , a protective colloidal thickener, a pH adjusting agent, a stabilizer, a preservative, glycerin, alcohol, a carbonating agent used in carbonated beverages, and the like. In addition, the compositions of the present invention may contain fruit flesh for preparing natural fruit juice, fruit juice beverages, and vegetable beverages. These components may be used independently or in combination.
본 발명의 식품 조성물은 건강기능식품으로서 사용될 수 있다. 상기 "건강기능 식품"이라 함은 건강기능 식품에 관한 법률에 따른 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 의미하며, "기능성"이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻을 목적으로 섭취하는 것을 의미한다.The food composition of the present invention can be used as a health functional food. The term "health functional food" refers to food manufactured and processed using raw materials or ingredients having useful functionalities for the human body in accordance with the Health Functional Food Act, and "functional" refers to food that is not related to the structure and function of the human body. It refers to intake for the purpose of obtaining useful effects for health purposes such as regulating nutrients or physiological functions.
본 발명의 식품 조성물은 통상의 식품 첨가물을 포함할 수 있으며, 상기 "식품 첨가물"로서의 적합 여부는 다른 규정이 없는 한, 식품의약품안전처에 승인된 식품 첨가물 공전의 총칙 및 일반시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 판정한다.The food composition of the present invention may include conventional food additives, and the suitability as the "food additive" is determined according to the general rules of the food additive code approved by the Ministry of Food and Drug Safety and general test methods, etc., unless otherwise specified. It is judged according to the specifications and standards for the item.
상기 "식품 첨가물 공전"에 수재된 품목으로는 예를 들어, 케톤류, 글리신, 구연산칼륨, 니코틴산, 계피산 등의 화학적 합성물, 감색소, 감초추출물, 결정셀룰로오스, 고량색소, 구아검 등의 천연첨가물, L-글루타민산나트륨 제제, 면류첨가알칼리제, 보존료제제, 타르색소제제 등의 혼합제제류들을 들 수 있다.Items listed in the "Food Additive Code" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid, natural additives such as dark pigment, licorice extract, crystalline cellulose, goyang pigment, guar gum, and mixed preparations such as sodium L-glutamate preparations, noodle-added alkali preparations, preservative preparations, and tar color preparations.
본 발명의 식품 조성물은 정제, 캡슐, 분말, 과립, 액상, 환 등의 형태로 제조 및 가공할 수 있다.The food composition of the present invention can be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills and the like.
예를 들어, 캡슐 형태의 건강기능 식품 중 경질캡슐제는 통상의 경질캡슐에 본 발명에 따른 조성물을 부형제 등의 첨가제와 혼합 및 충진하여 제조할 수 있으며, 연질캡슐제는 본 발명에 따른 조성물의 부형제 등의 첨가제와 혼합하고 젤라틴 등 캡슐기제에 충진하여 제조할 수 있다. 상기 연질캡슐제는 필요에 따라 글리세린 또는 소르비톨 등의 가소제, 착색제, 보존제 등을 함유할 수 있다.For example, among the health functional foods in the form of capsules, hard capsules can be prepared by mixing and filling the composition according to the present invention with additives such as excipients in conventional hard capsules, and soft capsules can be prepared by filling the composition according to the present invention. It can be prepared by mixing with additives such as excipients and filling in a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary.
상기 부형제, 결합제, 붕해제, 활택제, 교미제, 착향제 등에 대한 용어 정의는 당업계에 공지된 문헌에 기재된 것으로 그 기능 등이 동일 내지 유사한 것들을 포함한다. 상기 식품의 종류에는 특별한 제한이 없으며, 통상적인 의미에서의 건강기능식품을 모두 포함한다.Definitions of terms for the excipients, binders, disintegrants, lubricants, corrigents, flavoring agents, etc. are described in literature known in the art, and include those having the same or similar functions. There is no particular limitation on the type of food, and it includes all health functional foods in the usual sense.
본 발명에서 용어 “예방”이란 본 발명에 따른 조성물의 투여로 질환의 억제 또는 지연시키는 모든 행위를 말한다. 본 발명에서 용어 “치료”는 본 발명에 따른 조성물의 투여로 질환의 증세가 호전되거나 이롭게 변경하는 모든 행위를 말한다. 본 발명에서 "개선"이란 본 발명의 조성물을 개체에 투여하거나 섭취시켜 질환의 나쁜 상태를 좋게 하는 모든 행위를 의미한다.In the present invention, the term "prevention" refers to all activities that inhibit or delay a disease by administering the composition according to the present invention. In the present invention, the term "treatment" refers to all activities that improve or beneficially change the symptoms of a disease by administering the composition according to the present invention. In the present invention, "improvement" means any action that improves the bad condition of a disease by administering or ingesting the composition of the present invention to a subject.
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, but the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.
<실험예><Experimental example>
하기의 실험예들은 본 발명에 따른 각각의 실시예에 공통적으로 적용되는 실험예를 제공하기 위한 것이다.The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.
1. 인간 퇴행성 관절염 샘플 및 실험 퇴행성 관절염 마우스 모델의 제작1. Preparation of human osteoarthritis samples and experimental osteoarthritis mouse models
인간 연골 샘플은 무릎 관절 전체 치환술(total knee arthroplasty)을 받은 63 ~ 80 세의 개인에게서 얻었다 (표 1). 모든 환자는 서면 동의서를 제출하였고, 샘플 수집은 가톨릭대학교의 IRB에 의해 승인되었다(UC14CNSI0150). Human cartilage samples were obtained from individuals aged 63 to 80 years who underwent total knee arthroplasty (Table 1). All patients gave written informed consent, and sample collection was approved by the IRB of The Catholic University of Korea (UC14CNSI0150).
수컷 C57BL/6 및 SNCG-/-마우스 (008843-B6.129P2-SNCG tm1vlb/J, Jackson laboratory, USA)는 아주 대학교 실험실 동물 연구 센터에서 모든 동물 절차를 승인한 Institutional Animal Care and Use Committee의 가이드라인에 따라 유지하였다. Male C57BL/6 and SNCG−/− mice (008843-B6.129P2- SNCG tm1vlb /J, Jackson laboratory, USA) were prepared according to the guidelines of the Institutional Animal Care and Use Committee, which approved all animal procedures at the Laboratory Animal Research Center of Ajou University. maintained according to
Figure PCTKR2022011297-appb-img-000005
Figure PCTKR2022011297-appb-img-000005
퇴행성 관절염 동물 모델은 수컷 C57BL/6 및 SNCG-/-마우스(008843-B6.129P2-SNCG tm1vlb/J, Jackson laboratory, USA)로 수행되었으며, 모든 동물 실험은 아주 대학교 실험실 동물 연구 센터에서 승인된 Institutional Animal Care and Use Committee의 가이드라인에 따라 수행되었다. 퇴행성 관절염 동물 모델을 제조하기 위해, 12 주령의 수컷 마우스를 DMM(Destabilization of Medial Meniscus) 수술을 적용하고, 수술 후 10주에 희생시켰다. 암컷 마우스의 경우, 여성 호르몬에 의해 나타나는 퇴행성 관절염은 실험에서 제외하였다. 관절 내 주사를 위한 아데노바이러스는 Vector Biolabs (Malvern, USA)에서 구입하였다: Ad-C (1060), Ad-SNCG(Synuclein Gamma) (ADV-223826). 야생형 마우스에 아데노바이러스(1 x 109 PFUs / 10 μL)를 매주 2회 무릎 관절에 주사하고, 첫 번째 주사 후 3 주 후에 마우스를 희생시켰다.The degenerative arthritis animal model was performed with male C57BL/6 and SNCG-/- mice (008843-B6.129P2- SNCG tm1vlb /J, Jackson laboratory, USA), and all animal experiments were performed using Institutional approved by Ajou University Laboratory Animal Research Center. It was performed according to the guidelines of the Animal Care and Use Committee. To prepare an animal model for degenerative arthritis, 12-week-old male mice were subjected to Destabilization of Medial Meniscus (DMM) surgery and sacrificed 10 weeks after surgery. In the case of female mice, degenerative arthritis caused by female hormones was excluded from the experiment. Adenoviruses for intra-articular injection were purchased from Vector Biolabs (Malvern, USA): Ad-C (1060), Ad-Synuclein Gamma (SNCG) (ADV-223826). Wild-type mice were injected twice weekly with adenovirus (1 x 10 9 PFUs/10 μL) into the knee joint, and the mice were sacrificed 3 weeks after the first injection.
2. 일차 마우스 관절 연골 세포의 분리 및 세포 배양2. Isolation and cell culture of primary mouse articular chondrocytes
마우스 관절 연골세포는 출생 후 5일 째 ICR 마우스의 연골에서 분리하고, 단백질 분해 효소와 콜라게나제로 효소적으로 분해한 후, 10 % FBS, 100 units/mL의 페니실린 및 100ug/mL의 스트렙토마이신이 보충된 DMEM (Capricorn science GmbH; Hessen, Germany)에서 유지시켰다. 3일째, 세포 (4.25 × 105 세포/웰)를 아데노 바이러스로 감염시키거나 재조합 단백질로 처리하였다. Mlkl+/+ 및 Mlkl-/- MEF는 10 % FBS 및 페니실린-스트렙토마이신이 보충된 DMEM에서 유지시켰다. Mouse articular chondrocytes were isolated from the cartilage of ICR mice on day 5 after birth, enzymatically digested with proteolytic enzyme and collagenase, and then mixed with 10% FBS, 100 units/mL of penicillin, and 100ug/mL of streptomycin. It was maintained in supplemented DMEM (Capricorn science GmbH; Hessen, Germany). On day 3, cells (4.25 × 10 5 cells/well) were infected with adenovirus or treated with recombinant protein. Mlkl+/+ and Mlkl-/- MEFs were maintained in DMEM supplemented with 10% FBS and penicillin-streptomycin.
3. 시약3. Reagents
항체는 Abcam (Cambridge, UK; SNCG, COX2, MMP3, MMP13), Col2a1 (Merck, USA), Sox9 (Novus, Littleton, USA) 및 Erk (610408, Beckton Dickinson, New Jersey, USA) 로부터 구입하였다. Recombinant SNCG는 Abcam에서 구입하였다.Antibodies were purchased from Abcam (Cambridge, UK; SNCG, COX2, MMP3, MMP13), Col2a1 (Merck, USA), Sox9 (Novus, Littleton, USA) and Erk (610408, Beckton Dickinson, New Jersey, USA). Recombinant SNCG was purchased from Abcam.
4. 웨스턴 블로팅4. Western blotting
세포를 M2 완충액에 용해하고, 마우스 조직은 50mM Tris-HCl (pH 7.5), 150mM NaCl, 50mM NaF, 1 % Tween 20, 0.2 % NP-40 및 프로테아제 저해제로 구성된 용해 완충액에 용해하였다. 동일한 양의 세포 추출물을 SDS-PAGE (6 % 스태킹 겔 및 10 % 러닝 겔)로 분리하고 면역 블롯팅으로 분석하였다.Cells were lysed in M2 buffer, and mouse tissue was lysed in lysis buffer consisting of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 50 mM NaF, 1% Tween 20, 0.2% NP-40 and protease inhibitors. Equal amounts of cell extracts were separated by SDS-PAGE (6% stacking gel and 10% running gel) and analyzed by immunoblotting.
5. 조직학 및 면역 조직 화학5. Histology and Immunohistochemistry
인간 퇴행성 관절염 연골과 마우스 무릎 관절을 4 % 파라포름알데히드에 고정하고 파라핀에 포매하였다. 마우스 무릎 관절은 0.5 M EDTA (pH 7.4)에서 2 주 동안 석회화하였다. 파라핀 포매 샘플은 사프라닌-O (Safranin-O), 알리시안 블루 (Alcian blue) 또는 면역 염색으로 염색하였다. 연골 파괴는 실험 그룹핑에 대해 알지 못하는 세 명의 관찰자에 의해 실험 퇴행성 관절염 마우스 모델에서 평가되었으며, OARSI (국제 퇴행성 관절염 연구 학회) 등급 시스템 (등급 0-6)에 따라 점수가 매겨졌다. OARSI 점수는 각 마우스에 대한 평균 최대 점수로 제시되었다. 대표적인 사프라닌-O 염색 이미지는 각 섹션의 최고 고도 병변(advanced lesions)에서 선택되었으며 이전에 설명한대로 골극 성숙도(osteophyte maturity)를 정량화하였다. 연골하 골 경화증(Subchondral bone sclerosis)은 연골하 골판 두께를 측정하여 결정하였다. Human degenerative arthritic cartilage and mouse knee joints were fixed in 4% paraformaldehyde and embedded in paraffin. Mouse knee joints were calcified in 0.5 M EDTA (pH 7.4) for 2 weeks. Paraffin-embedded samples were stained with Safranin-O, Alcian blue or immunostain. Cartilage destruction was assessed in an experimental osteoarthritis mouse model by three observers blinded to the experimental grouping, and scored according to the OARSI (International Society for the Study of Degenerative Arthritis) grading system (grade 0-6). OARSI scores were presented as the average maximum score for each mouse. Representative safranin-O staining images were selected from the advanced lesions of each section and osteophyte maturity was quantified as previously described. Subchondral bone sclerosis was determined by measuring the thickness of the subchondral bone plate.
6. CMap 데이터 베이스 분석 및 Discovery studio 분석6. CMap database analysis and Discovery studio analysis
CMap 데이터 베이스에서 약 20,000 개의 소분자(small molecules)에 대한 약물 유도 전사체 데이터로부터 연골 세포에서 SNCG(Synuclein Gamma) 과발현에 의해 상향 조절된 후 하향 조절되는 유전자의 우선순위를 정하여, SNCG(Synuclein Gamma) 과발현에 대한 발현 억제 시그니처를 갖는 화합물을 선별하였다. SNCG(Synuclein Gamma) 발현 억제 가능성을 가진 화합물 중에서 Discovery studio에서 SNCG와 CMap에 의해 분석된 화합물 중에서 가장 affinity가 높은 화합물을 선정하였다.From the drug-induced transcriptome data for about 20,000 small molecules in the CMap database, genes that are up-regulated and then down-regulated by SNCG (Synuclein Gamma) overexpression in chondrocytes are prioritized, and SNCG (Synuclein Gamma) Compounds with an expression inhibition signature for overexpression were selected. Among the compounds with the potential to inhibit the expression of Synuclein Gamma (SNCG), the compound with the highest affinity was selected among the compounds analyzed by SNCG and CMap in Discovery studio.
7. 통계분석7. Statistical analysis
모든 실험은 4 회 이상 독립적으로 수행되었다. Shapiro-Wilk normality test, Levene's homogeneity of variance test, 및 two-tailed independent t-test를 사용하여 두 개의 독립 그룹을 비교하였다. Shapiro-Wilk test, Levene's test, 및 one-way analysis of variance with Bonferroni's post-hoc test를 이용하여 다수의 비교를 수행하였다. non-parametric Mann-Whitney U tests를 이용하여 서수 등급 시스템(ordinal grading system)을 기반으로 한 데이터를 분석하였다. 0.05 미만의 P 값을 통계적으로 유의한 것으로 간주되었다.All experiments were performed independently at least 4 times. Two independent groups were compared using Shapiro-Wilk normality test, Levene's homogeneity of variance test, and two-tailed independent t-test. Multiple comparisons were performed using the Shapiro-Wilk test, Levene's test, and one-way analysis of variance with Bonferroni's post-hoc test. Data based on an ordinal grading system were analyzed using non-parametric Mann-Whitney U tests. A P value of less than 0.05 was considered statistically significant.
<실시예 1> SNCG(Synuclein Gamma) 과발현에 의한 유전자 발현 양상 변화와 퇴행성 관절염과의 연관성 확인<Example 1> Confirmation of correlation between changes in gene expression patterns due to overexpression of Synuclein Gamma (SNCG) and degenerative arthritis
골관절에서 SNCG의 가능한 역할을 확인하기 위해, 인간 및 마우스 퇴행성 관절염 비손상 및 손상된 연골 샘플에서 SNCG 발현을 조사하였다. 손상된 퇴행성 관절염 연골에서는 비손상된 샘플에 비해 Mmp3, Mmp13 및 Cox2의 발현이 증가함을 확인하였고 동시에 SNCG 발현이 현저히 높게 나타나는 것을 확인하였다 (도 1A; human, 1B; mouse).To confirm the possible role of SNCG in bone joints, SNCG expression was investigated in human and mouse degenerative arthritis intact and damaged cartilage samples. In the damaged degenerative arthritic cartilage, it was confirmed that the expression of Mmp3, Mmp13, and Cox2 increased compared to the non-injured sample, and at the same time, it was confirmed that the expression of SNCG was significantly higher (Fig. 1A; human, 1B; mouse).
<실시예 2> SNCG에 의한 퇴행성 관절염 발병의 조절 확인<Example 2> Confirmation of regulation of degenerative arthritis by SNCG
MMP3, MMP13은 퇴행성 관절염 발병에서 중요한 역할을 하는 것으로 알려져 있고, Cox2는 주로 염증에 관여하며 콜라게나제(collagenas) 및 아그레카나제(aggrecanase) 활성화에 의해 연골 기질 분해를 유발한다. SNCG(Synuclein Gamma) 과발현과 퇴행성 관절염 사이의 관련성을 분석하기 위해, 연골세포에 SNCG을 처리하거나 과발현을 유도하여 관련 인자의 변화를 확인하였다. 도 2A에 나타난 바와 같이, 재조합 SNCG(Recombinant SNCG)를 연골세포에 처리하는 경우, Mmp3, Mmp13 및 Cox2와 같은 이화작용 인자의 발현이 증가되고, Col2 및 Sox9과 같은 동화작용 인자의 발현이 감소하는 것을 웨스턴 블롯으로 확인하였다. 또한, 도 2B에 나타난 바와 같이, 연골세포에 아데노바이러스 대조군(Ad-C) 또는 아데노바이러스 SNCG(Ad-SNCG)로 감염키는 경우, 아데노바이러스 SNCG(Ad-SNCG)로 SNCG의 과발현이 유도된 연골세포에서 SNCG, MMP3, MMP13 및 COX2의 발현이 증가하고, Col2 및 Sox9의 발현이 감소되는 것을 웨스턴 블롯으로 확인하였다. 또한, 아데노바이러스가 관절 내로 주입된 마우스 무릎 관절의 연골 파괴 및 퇴행성 관절염 발달 정도를 Safranin-O 염색, OARSI 등급, 골극 형성 및 연골하 골판 두께로 확인한 결과, 도 2C, 2D에 나타난 바와 같이, SNCG가 과발현된 경우 연골 파괴가 촉진되고, 퇴행성 관절염 증상이 유발되는 것을 확인하였다.MMP3 and MMP13 are known to play important roles in the pathogenesis of degenerative arthritis, and Cox2 is mainly involved in inflammation and induces cartilage matrix degradation by activating collagenase and aggrecanase. In order to analyze the relationship between synuclein gamma (SNCG) overexpression and degenerative arthritis, chondrocytes were treated with SNCG or overexpression was induced to confirm changes in related factors. As shown in Figure 2A, when recombinant SNCG (Recombinant SNCG) is treated with chondrocytes, the expression of catabolic factors such as Mmp3, Mmp13 and Cox2 is increased, and the expression of anabolic factors such as Col2 and Sox9 is decreased. It was confirmed by Western blot. In addition, as shown in FIG. 2B, when chondrocytes were infected with adenovirus control (Ad-C) or adenovirus SNCG (Ad-SNCG), overexpression of SNCG was induced by adenovirus SNCG (Ad-SNCG). It was confirmed by Western blot that the expression of SNCG, MMP3, MMP13, and COX2 increased, and the expression of Col2 and Sox9 decreased in chondrocytes. In addition, as a result of confirming the degree of cartilage destruction and degenerative arthritis development of the mouse knee joint injected with adenovirus into the joint by Safranin-O staining, OARSI grade, osteophyte formation, and subchondral bone plate thickness, as shown in FIGS. 2C and 2D, SNCG When was overexpressed, it was confirmed that cartilage destruction was promoted and degenerative arthritis symptoms were induced.
<실시예 3> SNCG 발현 억제에 의한 퇴행성관절염 억제 효과 확인<Example 3> Confirmation of degenerative arthritis inhibitory effect by inhibiting SNCG expression
SNCG 결핍 마우스는 미국 Jackson Laboratory (Bar Harbor, ME USA)에서 구입하였으며, SNCG 결핍 마우스에서 인간 퇴행성 관절염 개발에 가장 적합한 방법인 DMM를 처리하여 퇴행성 관절염 모델을 유도하였다.SNCG-deficient mice were purchased from Jackson Laboratory (Bar Harbor, ME USA), and degenerative arthritis models were induced by treating SNCG-deficient mice with DMM, which is the most suitable method for developing human degenerative arthritis.
그 결과, 도 3A 및 도 3B와 같이 연골 파괴, 퇴행성 관절염 발현(manifestation) 및 이화 인자 발현은 WT 마우스에 비해 이들 마우스에서 현저하게 감소되는 것을 확인할 수 있었다.As a result, as shown in FIGS. 3A and 3B , cartilage destruction, degenerative arthritis manifestation, and catabolic factor expression were significantly reduced in these mice compared to WT mice.
상기 결과로부터 인간 퇴행성 관절염 연골에서 SNCG 상향 조절되는 것과 일치하게, SNCG 결핍은 DMM 유발 마우스 모델에서 퇴행성 관절염을 약화(attenuate)시키는 것이 확인되었으며, 상기 결과로부터 SNCG가 퇴행성 관절염 발병에서 중요한 역할을 한다는 것이 확인되었다.Consistent with the up-regulation of SNCG in human osteoarthritis cartilage from the above results, it was confirmed that SNCG deficiency attenuates degenerative arthritis in a DMM-induced mouse model, and from the above results, it was confirmed that SNCG plays an important role in the development of degenerative arthritis. Confirmed.
<실시예 4> CMap를 사용한 SNCG(Synuclein Gamma) 발현과 관련된 약물 선별<Example 4> Selection of drugs related to SNCG (Synuclein Gamma) expression using CMap
CMap 접근 방식으로 in silico 화합물 스크리닝을 통하여 SNCG의 발현을 억제하여 퇴행성 관절염의 치료 효과를 나타내는 약물을 검색하였다. CMap 데이터베이스에서 약 20,000 개의 소분자(small molecules)에 대한 약물 유도 전사체 데이터로부터 연골 세포에서 SNCG(Synuclein Gamma) 과발현에 의해 상향 조절된 후 하향 조절되는 유전자의 우선순위를 정하여, SNCG(Synuclein Gamma) 과발현에 대해 SNCG(Synuclein Gamma) 발현 억제 시그니처를 갖는 화합물을 검색하였다. 도 4 및 도 5에 나타난 바와 같이, 검색 결과 하기 화학식 1로 표시되는 화합물 또는 하기 화학식 2로 표시되는 화합물이 선정되었으며, 선정된 화학식 1로 표시되는 화합물은 이하 AZ-628이라고 명했고, 화학식 2로 표시되는 화합물은 이하 PD-184352이라고 명했다.Through in silico compound screening using the CMap approach, drugs that inhibit the expression of SNCG and exhibit therapeutic effects on degenerative arthritis were searched for. From the drug-induced transcriptome data for about 20,000 small molecules in the CMap database, genes that are up-regulated and then down-regulated by SNCG (Synuclein Gamma) overexpression in chondrocytes are prioritized, and SNCG (Synuclein Gamma) overexpression A compound having a synuclein gamma (SNCG) expression inhibitory signature was searched for. As shown in FIGS. 4 and 5, as a result of the search, a compound represented by Formula 1 or a compound represented by Formula 2 was selected, and the selected compound represented by Formula 1 was named AZ-628, and Formula 2 The compound represented by is hereinafter named PD-184352.
[화학식 1][Formula 1]
Figure PCTKR2022011297-appb-img-000006
Figure PCTKR2022011297-appb-img-000006
[화학식 2][Formula 2]
Figure PCTKR2022011297-appb-img-000007
Figure PCTKR2022011297-appb-img-000007
<실시예 5> AZ-628에 의한 SNCG(Synuclein Gamma) 매개 MMP3, MMP13 및 COX2 발현 억제 평가<Example 5> Evaluation of SNCG (Synuclein Gamma) mediated MMP3, MMP13 and COX2 expression inhibition by AZ-628
실시예 4에서 선정된 AZ-628이 퇴행성 관절염 치료제로서 유효한지 평가하기 위해, SNCG(Synuclein Gamma)에 의해 과발현되는 MMP3, MMP13 및 COX2의 발현에 미치는 영향을 평가하였다. 도 6에 나타난 바와 같이, 아데노바이러스로 SNCG(Synuclein Gamma) 과발현이 유도된 연골세포에 AZ-628를 처리한 결과, SNCG(synuclein gamma) 과발현에 의한 유도된 MMP3, MMP13 및 COX2의 발현이 AZ-628 투여 용량에 따라 감소되는 것으로 나타났다.In order to evaluate whether AZ-628 selected in Example 4 is effective as a treatment for degenerative arthritis, the effect on the expression of MMP3, MMP13 and COX2 overexpressed by Synuclein Gamma (SNCG) was evaluated. As shown in FIG. 6, as a result of treating AZ-628 to chondrocytes in which SNCG (Synuclein Gamma) overexpression was induced by adenovirus, the expression of MMP3, MMP13, and COX2 induced by SNCG (synuclein gamma) overexpression was 628 was found to decrease with the administered dose.
<실시예 6> PD-184352에 의한 SNCG(Synuclein Gamma) 매개 MMP3, MMP13 및 COX2 발현 억제 평가<Example 6> Assessment of Synuclein Gamma (SNCG) mediated MMP3, MMP13 and COX2 expression inhibition by PD-184352
실시예 4에서 선정된 PD-184352이 퇴행성 관절염 치료제로서 유효한지 평가하기 위해, SNCG(Synuclein Gamma)에 의해 과발현되는 MMP3, MMP13 및 COX2의 발현에 미치는 영향을 평가하였다. 도 7에 나타난 바와 같이, 아데노바이러스로 SNCG(Synuclein Gamma) 과발현이 유도된 연골세포에 PD-184352를 처리한 결과, SNCG(synuclein gamma) 과발현에 의한 유도된 MMP3, MMP13 및 COX2의 발현이 PD-184352 투여 용량에 따라 감소되는 것으로 나타났다.In order to evaluate whether PD-184352 selected in Example 4 is effective as a therapeutic agent for degenerative arthritis, its effect on the expression of MMP3, MMP13 and COX2 overexpressed by Synuclein Gamma (SNCG) was evaluated. As shown in FIG. 7, as a result of treating PD-184352 to chondrocytes in which SNCG (Synuclein Gamma) overexpression was induced by adenovirus, the expression of MMP3, MMP13, and COX2 induced by SNCG (synuclein gamma) overexpression was found to be PD-184352. 184352 has been shown to decrease with the administered dose.
<실시예 7> Discovery Studio를 활용한 SNCG(Synuclein Gamma) 및 AZ-628 연관성 분석<Example 7> Synuclein Gamma (SNCG) and AZ-628 correlation analysis using Discovery Studio
상기 실시예 4에서 선정된 AZ-628과 SNCG(Synuclein Gamma)와의 관련성을 분석하기 위해, Discovery studio를 활용하여 분자 도킹 분석을 실시하였다. 도 8에 나타난 바와 같이, SNCG(Synuclein Gamma) 및 AZ-628과의 in silico binding affinity를 측정한 결과, SNCG의 아미노산 서열 중 C-terminal 잔기에 AZ-628이 결합됨을 확인하였다. 상기 결과는 AZ-628이 SNCG(Synuclein Gamma) 아미노사 C-terminal의 특정 잔기에 결합하여 SNCG(Synuclein Gamma) 활성을 억제할 수 있음을 입증한다.In order to analyze the relationship between AZ-628 selected in Example 4 and Synuclein Gamma (SNCG), molecular docking analysis was performed using Discovery studio. As shown in FIG. 8, as a result of measuring the in silico binding affinity with SNCG (Synuclein Gamma) and AZ-628, it was confirmed that AZ-628 was bound to the C-terminal residue in the amino acid sequence of SNCG. The above results demonstrate that AZ-628 can bind to a specific residue of the synuclein gamma (SNCG) amino chain C-terminal to inhibit the activity of synuclein gamma (SNCG).
<실시예 8> Discovery Studio를 활용한 SNCG(Synuclein Gamma) 및 PD-184352 연관성 분석<Example 8> Synuclein Gamma (SNCG) and PD-184352 correlation analysis using Discovery Studio
상기 실시예 4에서 선정된 PD-184352과 SNCG(Synuclein Gamma)와의 관련성을 분석하기 위해, Discovery studio를 활용하여 분자 도킹 분석을 실시하였다. 도 9에 나타난 바와 같이, SNCG(Synuclein Gamma) 및 PD-184352과의 in silico binding affinity를 측정한 결과, SNCG의 아미노산 서열 중 C-terminal 잔기에 PD-184352이 결합됨을 확인하였다. 상기 결과는 PD-184352이 SNCG(Synuclein Gamma) 아미노사 C-terminal의 특정 잔기에 결합하여 SNCG(Synuclein Gamma) 활성을 억제할 수 있음을 입증한다.In order to analyze the relationship between PD-184352 selected in Example 4 and Synuclein Gamma (SNCG), molecular docking analysis was performed using Discovery studio. As shown in FIG. 9, as a result of measuring the in silico binding affinity with Synuclein Gamma (SNCG) and PD-184352, it was confirmed that PD-184352 binds to the C-terminal residue in the amino acid sequence of SNCG. The above results demonstrate that PD-184352 can bind to a specific residue of the synuclein gamma (SNCG) amino chain C-terminal to inhibit the activity of synuclein gamma (SNCG).
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.Having described specific parts of the present invention in detail above, it is clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (19)

  1. SNCG (Synuclein Gamma) 유전자 또는 상기 유전자가 코딩하는 단백질을 포함하는 퇴행성 관절염 진단용 바이오마커 조성물.A biomarker composition for diagnosing degenerative arthritis comprising a synuclein gamma (SNCG) gene or a protein encoded by the gene.
  2. SNCG 유전자 또는 상기 유전자가 코딩하는 단백질의 발현 또는 활성 수준을 검출하는 제제를 유효성분으로 함유하는 퇴행성 관절염 진단용 키트.A kit for diagnosing degenerative arthritis containing, as an active ingredient, an agent for detecting the expression or activity level of a SNCG gene or a protein encoded by the gene.
  3. 청구항 2에 있어서, 상기 제제는 SNCG 유전자에 특이적으로 결합하는 프라이머 또는 프로브, SNCG 단백질에 특이적으로 결합하는 항체, 펩타이드, 앱타머또는 화합물로 이루어진 군에서 선택되는 것을 특징으로 하는 퇴행성 관절염 진단용 키트.The kit for diagnosis of degenerative arthritis according to claim 2, wherein the agent is selected from the group consisting of a primer or probe specifically binding to the SNCG gene, an antibody, a peptide, an aptamer, or a compound binding specifically to the SNCG protein. .
  4. 검체로부터 분리된 시료로부터 SNCG 유전자 또는 상기 유전자가 코딩하는 단백질의 발현 또는 활성 수준을 확인하는 단계를 포함하는 퇴행성 관절염 진단을 위한 정보를 제공하는 방법.A method for providing information for diagnosing degenerative arthritis comprising the step of confirming the expression or activity level of a SNCG gene or a protein encoded by the gene from a sample separated from the specimen.
  5. 청구항 4에 있어서, 상기 시료는 세포, 조직, 혈액, 혈청, 뇨 및 타액으로 이루어진 군에서 선택되는 것을 특징으로 하는 퇴행성 관절염 진단을 위한 정보를 제공하는 방법.The method of claim 4, wherein the sample is selected from the group consisting of cells, tissues, blood, serum, urine and saliva.
  6. 청구항 4에 있어서, 상기 퇴행성 관절염 진단을 위한 정보를 제공하는 방법은 SNCG 유전자 또는 상기 유전자가 코딩하는 단백질의 발현 또는 활성 수준이 정상대조군보다 증가한 경우 퇴행성 관절염으로 진단하는 것을 특징으로 하는 퇴행성 관절염 진단을 위한 정보를 제공하는 방법.The method according to claim 4, wherein the method for providing information for the diagnosis of degenerative arthritis is diagnosed as degenerative arthritis when the expression or activity level of the SNCG gene or the protein encoded by the gene is higher than that of the normal control group. How to provide information for.
  7. 퇴행성 관절염 환자로부터 분리된 연골세포에 후보물질을 처리하는 단계; processing a candidate substance into chondrocytes isolated from a patient with degenerative arthritis;
    상기 후보물질이 처리된 연골세포로부터 SNCG 발현 수준을 확인하는 단계; 및Checking the expression level of SNCG from the chondrocytes treated with the candidate substance; and
    상기 SNCG 발현 수준이 후보물질 비처리군과 비교하여 감소된 후보물질을 퇴행성 관절염 치료제로 제공하는 퇴행성 관절염 치료제 스크리닝 방법.A method for screening a treatment for degenerative arthritis, wherein the SNCG expression level is reduced compared to a group not treated with the candidate substance to provide a candidate substance as a treatment for degenerative arthritis.
  8. SNCG (Synuclein Gamma) 발현 또는 활성 억제제를 유효성분으로 함유하는 퇴행성 관절염 예방 또는 치료용 약학조성물.A pharmaceutical composition for preventing or treating degenerative arthritis containing a Synuclein Gamma (SNCG) expression or activity inhibitor as an active ingredient.
  9. 청구항 8에 있어서, 상기 SNCG 발현 억제제는 SNCG 유전자 또는 SNCG의 발현을 촉진하는 유전자의 mRNA에 특이적으로 결합하는 안티센스 뉴클레오티드, siRNA, shRNA, microRNA 및 리보자임으로 이루어진 군에서 하나 이상 선택되는 것을 특징으로 하는 퇴행성 관절염 예방 또는 치료용 약학조성물.The method according to claim 8, wherein the SNCG expression inhibitor is at least one selected from the group consisting of antisense nucleotides, siRNA, shRNA, microRNA, and ribozymes that specifically bind to mRNA of a gene or a gene promoting SNCG expression. A pharmaceutical composition for preventing or treating degenerative arthritis.
  10. 청구항 8에 있어서, 상기 SNCG 활성 억제제는 SNCG 활성을 촉진하는 단백질 또는 SNCG 단백질에 특이적으로 결합하는 화합물, 펩티드, 펩티드모방체, 단백질, 앱타머, 및 항체로 이루어진 군에서 하나 이상 선택되는 것을 특징으로 하는 퇴행성 관절염 예방 또는 치료용 약학조성물.The method according to claim 8, wherein the SNCG activity inhibitor is one or more selected from the group consisting of a protein that promotes SNCG activity or a compound that specifically binds to a SNCG protein, a peptide, a peptidomimetic, a protein, an aptamer, and an antibody. A pharmaceutical composition for preventing or treating degenerative arthritis.
  11. SNCG 발현 또는 활성 억제제를 유효성분으로 함유하는 퇴행성 관절염 예방 또는 개선용 건강식품.A health food for preventing or improving degenerative arthritis containing an SNCG expression or activity inhibitor as an active ingredient.
  12. SNCG 발현 또는 활성 조절제를 유효성분으로 함유하는 SNCG 관련 질환 예방 또는 치료용 약학조성물.A pharmaceutical composition for preventing or treating SNCG-related diseases, containing an SNCG expression or activity regulator as an active ingredient.
  13. 하기 화학식 1 또는 화학식 2로 표시되는 화합물 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 SNCG(Synuclein Gamma) 과발현 질환의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating Synuclein Gamma (SNCG) overexpression diseases, comprising a compound represented by Formula 1 or Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient.
    [화학식 1][Formula 1]
    Figure PCTKR2022011297-appb-img-000008
    Figure PCTKR2022011297-appb-img-000008
    [화학식 2][Formula 2]
    Figure PCTKR2022011297-appb-img-000009
    Figure PCTKR2022011297-appb-img-000009
  14. 제13항에 있어서, 상기 약학적 조성물은 SNCG(Synuclein Gamma)의 과발현으로 발병되는 질환의 환자에게 투여하기 위한 것을 특징으로 하는 약학적 조성물.[Claim 14] The pharmaceutical composition according to claim 13, which is characterized in that it is administered to a patient suffering from a disease caused by overexpression of Synuclein Gamma (SNCG).
  15. 제13항에 있어서, 상기 화합물은 MMP3, MMP13 및 COX2의 발현을 억제하는 것을 특징으로 하는 약학적 조성물.The pharmaceutical composition according to claim 13, wherein the compound inhibits the expression of MMP3, MMP13 and COX2.
  16. 제13항에 있어서, 상기 화합물은 SNCG(Synuclein Gamma) 단백질의 C-말단에 결합하여 SNCG(Synuclein Gamma)의 발현을 억제하는 것을 특징으로 하는 약학적 조성물.The pharmaceutical composition according to claim 13, wherein the compound inhibits the expression of Synuclein Gamma (SNCG) by binding to the C-terminus of the protein.
  17. 제13항에 있어서, 상기 SNCG(Synuclein Gamma) 과발현 질환은 퇴행성 관절염 또는 파킨슨병인 것을 특징으로 하는 약학적 조성물.The pharmaceutical composition according to claim 13, wherein the Synuclein Gamma (SNCG) overexpression disease is degenerative arthritis or Parkinson's disease.
  18. 하기 화학식 1 또는 화학식 2로 표시되는 화합물 및 식품학적으로 허용 가능한 식품보조 첨가제를 포함하는 SNCG(Synuclein Gamma) 과발현 질환의 예방 또는 개선용 건강기능 식품 조성물.A health functional food composition for preventing or improving Synuclein Gamma (SNCG) overexpression disease, comprising a compound represented by Formula 1 or Formula 2 and food additives acceptable in food science.
    [화학식 1][Formula 1]
    Figure PCTKR2022011297-appb-img-000010
    Figure PCTKR2022011297-appb-img-000010
    [화학식 2][Formula 2]
    Figure PCTKR2022011297-appb-img-000011
    Figure PCTKR2022011297-appb-img-000011
  19. 제18항에 있어서, 상기 상기 SNCG(Synuclein Gamma) 과발현 질환은 퇴행성 관절염 또는 파킨슨병인 것을 특징으로 하는 건강기능 식품 조성물.[Claim 19] The health functional food composition according to claim 18, wherein the synuclein gamma (SNCG) overexpression disease is degenerative arthritis or Parkinson's disease.
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