WO2023008978A1 - Composition ciblant le sncg pour le traitement ou le diagnostic d'une maladie dégénérative - Google Patents

Composition ciblant le sncg pour le traitement ou le diagnostic d'une maladie dégénérative Download PDF

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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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English (en)
Korean (ko)
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양시영
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아주대학교산학협력단
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Priority claimed from KR1020210100767A external-priority patent/KR20230018813A/ko
Priority claimed from KR1020210100766A external-priority patent/KR20230018812A/ko
Priority claimed from KR1020210101683A external-priority patent/KR20230020593A/ko
Application filed by 아주대학교산학협력단 filed Critical 아주대학교산학협력단
Publication of WO2023008978A1 publication Critical patent/WO2023008978A1/fr

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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

La présente invention concerne une composition de biomarqueur pour diagnostiquer l'arthrose, comprenant un gène de synucléine gamma (SNCG) ou une protéine codée par le gène de SNCG, et un procédé de diagnostic l'utilisant. Il a été confirmé que lors de la pathogenèse de l'arthrose, l'expression et l'activité de SNCG augmentaient, et, par conséquent, l'expression de Mmp 3, Mmp 13 et Cox2, qui induisent la destruction du cartilage, augmentaient et que l'expression des gènes mentionnés ci-dessus était inhibée par l'inhibition de l'expression de SNCG. En conséquence, la présente invention concerne la fourniture de SNCG en tant que biomarqueur pour diagnostiquer l'arthrose et pour fournir, avec la confirmation d'un effet d'amélioration de l'arthrose suite à la déplétion de l'expression de SNCG, un inhibiteur d'expression ou d'activité de SNCG en tant qu'agent thérapeutique pour l'arthrose. La présente invention, en confirmant que AZ-628 ou PD-184352 en tant qu'inhibiteur de SNCG se lie à l'extrémité C-terminale de la protéine SNCG et inhibe l'expression de MMP3, MMP13, et COX2, qui sont des facteurs associés à des maladies dégénératives et contrôlés par la SNCG, concerne le composé en tant qu'agent thérapeutique pour l'arthrose et la maladie de Parkinson qui sont provoquées par la SNCG.
PCT/KR2022/011297 2021-07-30 2022-08-01 Composition ciblant le sncg pour le traitement ou le diagnostic d'une maladie dégénérative WO2023008978A1 (fr)

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KR1020210100767A KR20230018813A (ko) 2021-07-30 2021-07-30 Pd-184352를 유효성분으로 포함하는 sncg 과발현 질환의 예방 또는 치료용 약학적 조성물
KR1020210100766A KR20230018812A (ko) 2021-07-30 2021-07-30 Az-628을 유효성분으로 포함하는 sncg 과발현 질환의 예방 또는 치료용 약학적 조성물
KR10-2021-0100767 2021-07-30
KR10-2021-0100766 2021-07-30
KR10-2021-0101683 2021-08-03
KR1020210101683A KR20230020593A (ko) 2021-08-03 2021-08-03 Sncg를 표적으로 하는 퇴행성 관절염 치료 또는 진단용 조성물

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