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WO2024085698A1 - Pharmaceutical composition for prevention or treatment of inflammatory bowel disease comprising glycogen phosphorylase (pygl) inhibitor as active ingredient - Google Patents

Pharmaceutical composition for prevention or treatment of inflammatory bowel disease comprising glycogen phosphorylase (pygl) inhibitor as active ingredient Download PDF

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WO2024085698A1
WO2024085698A1 PCT/KR2023/016299 KR2023016299W WO2024085698A1 WO 2024085698 A1 WO2024085698 A1 WO 2024085698A1 KR 2023016299 W KR2023016299 W KR 2023016299W WO 2024085698 A1 WO2024085698 A1 WO 2024085698A1
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inflammatory bowel
bowel disease
pygl
disease
formula
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Korean (ko)
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임동철
박희동
김태훈
류재윤
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Innovo Therapeutics Inc Korea
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Innovo Therapeutics Inc Korea
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4418—Non condensed pyridines; Hydrogenated derivatives thereof having a carbocyclic group directly attached to the heterocyclic ring, e.g. cyproheptadine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/91091—Glycosyltransferases (2.4)
    • G01N2333/91097—Hexosyltransferases (general) (2.4.1)
    • G01N2333/91102—Hexosyltransferases (general) (2.4.1) with definite EC number (2.4.1.-)

Definitions

  • the present invention relates to a pharmaceutical composition for the prevention or treatment of inflammatory bowel disease containing a glycogen phosphorylase (PYGL) inhibitor as an active ingredient, and a screening method for a substance for the prevention or treatment of inflammatory bowel disease.
  • PYGL glycogen phosphorylase
  • IBD Inflammatory bowel disease
  • UC ulcerative colitis
  • Crohn's disease Crohn's disease
  • UC ulcerative colitis
  • Crohn's disease Crohn's disease
  • TNF- ⁇ tumor necrosis factor- ⁇
  • 5-ASA mealamine
  • steroids are prescribed depending on symptoms
  • biological agents including immunosuppressants are also used.
  • 20-40% of ulcerative colitis patients fail conventional drug treatment, and 65-80% of patients fail to induce complete remission even with biological agents such as anti-TNF- ⁇ agents.
  • biological agents such as anti-TNF- ⁇ agents.
  • high molecular weight proteins such as biological agents cause the formation of autoantibodies and loss of response during long-term treatment, resulting in a high recurrence rate and ultimately requiring colectomy.
  • biological agents are not only expensive, but are also injectable, making them less convenient to take. Therefore, the need to develop oral small molecule new drugs with new mechanisms that improve these shortcomings continues to be raised.
  • the problem of the present invention is to provide a method for screening a substance for the prevention or treatment of inflammatory bowel disease, and to provide a method for screening a substance for the prevention or treatment of inflammatory bowel disease containing the selected glycogen phosphatase (PYGL) inhibitor as an active ingredient.
  • PYGL glycogen phosphatase
  • the problem of the present invention is to provide a method for preventing or treating inflammatory bowel disease, including the step of administering the glycogen phosphorylase (PYGL) inhibitor to a subject in need thereof.
  • PYGL glycogen phosphorylase
  • the problem of the present invention is to provide a use of the glycogen phosphorylase (PYGL) inhibitor for producing a medicament for use in the prevention or treatment of inflammatory bowel disease.
  • PYGL glycogen phosphorylase
  • a pharmaceutical composition for preventing or treating inflammatory bowel disease containing a glycogen phosphorylase (PYGL) inhibitor as an active ingredient is provided.
  • PYGL glycogen phosphorylase
  • a screening method for a substance for the prevention or treatment of inflammatory bowel disease comprising the step of determining a test substance that reduces the activity of glycogen phosphorylase (PYGL) as a candidate substance for the treatment of inflammatory bowel disease.
  • PYGL glycogen phosphorylase
  • a method for preventing or treating inflammatory bowel disease comprising administering the glycogen phosphorylase (PYGL) inhibitor to a subject in need thereof.
  • PYGL glycogen phosphorylase
  • glycogen phosphorylase (PYGL) inhibitor for preparing a medicament for use in the prevention or treatment of inflammatory bowel disease.
  • Inflammatory bowel diseases such as ulcerative colitis can be prevented or treated through a pharmaceutical composition for preventing or treating inflammatory bowel disease containing the glycogen phosphatase (PYGL) inhibitor of the present invention as an active ingredient.
  • PYGL glycogen phosphatase
  • a substance that reduces phosphorylase (PYGL) activity can be determined as a candidate for a substance for the prevention or treatment of inflammatory bowel disease.
  • Figure 1 is a graph showing the in vitro PYGL inhibition ability of test substances (INV-101, CP-91149, and UC-205686).
  • Figure 2a is a graph showing body weight changes in disease model rats induced by TNBS.
  • Figure 2b is a graph showing the body weight of rats on day 8 after the disease was induced by TNBS.
  • Figure 3 is a graph showing the degree of disease progression in disease model rats induced by TNBS.
  • Figure 4 is a photograph showing morphological changes in the large intestine of a disease model rat induced by TNBS.
  • Figure 5 is a graph showing the weight per unit length of the large intestine of a disease model rat induced by TNBS.
  • Figure 6 is a photograph of H&E staining of colon tissue of a disease model rat induced by TNBS.
  • Figure 7 is a graph showing the damage score of colonic tissue in a disease model rat induced by TNBS.
  • Figure 8 shows the expression of inflammatory cytokines in the colonic tissue of a disease model rat induced by TNBS.
  • Figure 9a shows the degree of inflammatory signal activation (RIP1, RIP3, JAK1, JAK2, JAK3) in colon tissue of a disease model rat induced by TNBS.
  • Figure 9b shows the degree of inflammatory signal activation (STAT1, STAT3, NF- ⁇ B) in colon tissue of a disease model rat induced by TNBS.
  • Figure 10 is a graph showing changes in MPO in colon tissue of a disease model rat induced by TNBS.
  • Figure 11a is a graph showing the concentration of TNF ⁇ in the blood of a disease model rat induced by TNBS.
  • Figure 11b is a graph showing the concentration of IL-6 in the blood of a disease model rat induced by TNBS.
  • Figure 11c is a graph showing the concentration of IL-1 ⁇ in the blood of TNBS-induced disease model rats.
  • Figure 11d is a graph showing the blood CRP concentration of a disease model rat induced by TNBS.
  • Figure 12a is a graph showing the concentration of glycogen phosphatase (PYGL) in the blood of a disease model rat induced by TNBS.
  • PYGL glycogen phosphatase
  • Figure 12b is a graph showing the serum glycogen phosphatase (PYGL) activity of a disease model rat induced by TNBS.
  • Figure 13 is a graph showing glycogen phosphorylase (PYGL) activity in colon tissue of a disease model rat induced by TNBS.
  • PYGL glycogen phosphorylase
  • the present invention provides a pharmaceutical composition for preventing or treating inflammatory bowel disease comprising a glycogen phosphorylase (PYGL) inhibitor as an active ingredient.
  • PYGL glycogen phosphorylase
  • the present invention also provides a method for preventing or treating inflammatory bowel disease, comprising administering the glycogen phosphorylase (PYGL) inhibitor to a subject in need thereof.
  • PYGL glycogen phosphorylase
  • the present invention also provides the use of the glycogen phosphorylase (PYGL) inhibitor for preparing a medicament for use in the prevention or treatment of inflammatory bowel disease.
  • PYGL glycogen phosphorylase
  • Glycogen phosphorylase or glycogen phosphorylase [PYGL] is an enzyme that catalyzes the breakdown of glycogen in animals by releasing glucose-1,4-phosphate from the terminal alpha-1-glycosidic bond. This enzyme exists in two forms, the active phosphorylated form, phosphorylase A, and the inactive phosphorylated form, phosphorylase B, and both forms exist as homodimers. Much research has been conducted on diabetes treatments using the glycogen breakdown inhibition effect of glycogen phosphatase inhibitors. In the present invention, glycogen phosphatase inhibitors have a therapeutic effect on inflammatory bowel disease by controlling immune cell-mediated inflammatory responses by regulating intracellular signaling and ROS generation related to immune responses.
  • the glycogen phosphorylase (PYGL) inhibitor may be any one selected from the group consisting of compounds represented by the following formulas 1 to 8, but is not limited thereto.
  • the compound represented by the following formula (1) is N-(5-hydroxy-3,4,6-trimethylpyridin-2-yl)-5-methyl-1H-indole-2-carboxamide (INV-101). ,
  • the compound represented by the following formula (3) is UC-205686,
  • the compound represented by Formula 8 below is AVE-5688.
  • the inflammatory bowel disease includes enteritis, colitis, ulcerative colitis, Crohn's disease, Crohn's apoptotic tumor, irritable bowel syndrome, bleeding rectal ulcer, ileal pouchitis, peptic ulcer, intestinal Behcet's disease, and gastritis. It may be any one selected from the group consisting of:
  • the inflammatory bowel disease may be ulcerative colitis.
  • Ulcerative colitis is a disease in which inflammation or ulcers occur in the mucosa or submucosa of the large intestine. Unlike Crohn's disease, which is an inflammatory bowel disease that occurs sporadically throughout the small and large intestine, it is characterized by lesions that start in the rectum and connect to the inside of the large intestine. Most patients with ulcerative colitis have loose stools or diarrhea mixed with blood and mucus several times a day, and in severe cases, they complain of symptoms such as abdominal pain, dehydration, fever, vomiting, and weight loss. Most ulcerative colitis has a chronic recurrent course, and the condition worsens as it relapses.
  • the present invention includes the steps of 1) treating the test substance with glycogen phosphorylase (PYGL); 2) measuring the activity of glycogen phosphorylase (PYGL); and 3) determining a test substance that reduces the activity of glycogen phosphatase (PYGL) as a candidate for the treatment of inflammatory bowel disease. It provides a screening method for a substance for the prevention or treatment of inflammatory bowel disease. .
  • the activity of glycogen phosphorylase may be measured using glucose-1-phosphate (G-1-P) detection.
  • the glucose-1-phosphate (G-1-P) is a substance produced by decomposition of glycogen by glycogen phosphatase.
  • the activity of glycogen phosphatase is proportional to the intensity of the detection signal of glucose-1-phosphate.
  • the inflammatory bowel disease includes enteritis, colitis, ulcerative colitis, Crohn's disease, Crohn's sarcoma, irritable bowel syndrome, bleeding rectal ulcer, ileal pouchitis, peptic ulcer, intestinal Behcet's disease, and gastritis. It may be any one selected from the group consisting of:
  • the pharmaceutical composition according to the present invention may be administered in various oral and parenteral dosage forms during clinical administration. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain one or more compounds and at least one excipient, such as starch, calcium carbonate, sucrose or lactose ( It is prepared by mixing lactose, gelatin, etc.
  • lubricants such as magnesium stearate and talc are also used.
  • Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups.
  • various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included.
  • Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, and emulsions.
  • Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.
  • the pharmaceutical composition according to the present invention can be administered parenterally, and parenteral administration is by subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection.
  • parenteral administration is by subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection.
  • the pharmaceutical composition is mixed with water along with a stabilizer or buffer to prepare a solution or suspension, which can be prepared in an ampoule or vial unit dosage form.
  • the composition may be sterilized and/or contain preservatives, stabilizers, wetting agents or emulsification accelerators, auxiliaries such as salts and/or buffers for adjusting osmotic pressure, and other therapeutically useful substances, and may be mixed, granulated, etc. using conventional methods. It can be formulated according to the coating or coating method.
  • Formulations for oral administration of the pharmaceutical composition according to the present invention include, for example, tablets, pills, hard/soft capsules, solutions, suspensions, emulsifiers, syrups, granules, elixirs, troches, etc., and these formulations are effective
  • diluents e.g. lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine
  • lubricants e.g. silica, talc, stearic acid and its magnesium or calcium salts and/or polyethylene glycol
  • the tablet may contain a binder such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and optionally a binder such as starch, agar, alginic acid or its sodium salt, etc.
  • a binder such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and optionally a binder such as starch, agar, alginic acid or its sodium salt, etc.
  • the releasing or boiling mixture may contain absorbents, colorants, flavoring and sweetening agents.
  • the dosage of the active ingredient contained in the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, degree of disease, form of the active ingredient, route and period of administration, and can be appropriately adjusted depending on the patient.
  • the active ingredient can be administered at a dose of 0.0001 to 1000 mg/kg per day, preferably 0.001 to 100 mg/kg, and the administration may be administered once a day or in divided doses.
  • the pharmaceutical composition of the present invention may contain the active ingredient at a weight percentage of 0.001 to 90% based on the total weight of the composition.
  • Glycogen phosphatase catalyzes the reversible reaction of (glucose)n + Pi to (glucose)n-1 + G-1-P. The reaction begins when conditions allow glycogen and Pi to react in a forward reaction. Phosphatase kinase catalyzes the conversion of inactive glycogen phosphatase B to fully active glycogen phosphatase A.
  • overall regulation depends on the interaction of a variety of factors, ranging from the action of phosphatases to feedback control by AMPs.
  • the PYGL inhibition ability of compound INV-101 was confirmed using the Glycogen Phosphorylase Colorimetric Assay Kit and rabbit muscle glycogen phosphatase (RMGPa).
  • Test substances or vehicle in 1.0% DMSO were pre-incubated in modified imidazole buffer (pH 6.8) with 5 ⁇ g/ml enzyme for 15 minutes at 37°C. UC-205686 (0.03 ⁇ M) and CP-91149 (3 ⁇ M) were used as positive controls.
  • the reaction was initiated with the addition of 4 mg/ml glycogen for another 30 min incubation period.
  • the amount of NADPH produced was analyzed spectrophotometrically at 340 nm. Substances were measured at at least 10 inhibitory concentrations (100, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0 ⁇ M) for accurate IC 50 analysis.
  • the activity of RMGPa was measured by detecting glucose-1-phosphate.
  • Colitis induced by intrarectal injection of trinitrobenzene sulfonic acid (TNBS) in mice or rats is one of the animal models in which the basic pathological and immunological mechanisms of Crohn's disease, a form of inflammatory bowel disease, can be studied.
  • TNBS trinitrobenzene sulfonic acid
  • the efficacy of oral administration of INV-101 was compared using the oral administration group of Filgotinib (30 mg/kg) and UC-205686 (10 mg/kg) as a positive control group. .
  • the animals were 7-8 week old rats (Sprague Dawley Rats) purchased from Orient Bio (Korea) and stabilized with regular solid feed for 7 days before being used in the experiment. Feed and water were supplied ad libitum during the experiment, and the temperature in the breeding room was maintained at 25 ⁇ 1°C and relative humidity at 50 ⁇ 10%. Lighting management was controlled with a 12-hour light-dark cycle using an automatic lighting controller.
  • the experimental group was divided into 7 groups (normal group, TNBS-Vehicle administered group, TNBS+Filgotinib 30 mg/kg administered group, TNBS+UC) by randomized block design with 8 animals in each group, with an average weight of 250 ⁇ 20 g. -205686 10 mg/kg administration group, TNBS+INV-101 1 mg/kg administration group, TNBS+INV-101 3 mg/kg administration group, TNBS+INV-101 10 mg/kg administration group).
  • the first oral administration of the drug on the day of NBS administration was 2 hours after TNBS administration, and the drug was administered orally at a constant time every day for 6 days from the next day.
  • each cytokine was thawed on the day of measurement and quantified using each ELISA kit (CRP, TNF ⁇ , IL-6, IL-1 ⁇ PYGL).
  • Blood PYGL activity was measured using a glycogen phosphorylase activity kit (BioVision).
  • the large intestine of the rat was removed, the tissue between 5 and 6 cm from the anus was cut into 1 cm long pieces, and the weight of the tissue was measured.
  • MPO Myeloperoxidase
  • the amount of the enzyme MPO which is an indicator for quantifying neutrophils.
  • a 1-cm section of the large intestine was washed with cold PBS, weighed, and added with lysis buffer (pH7.4, 200mM NaCl, 5mM EDTA, 10mM tris, 10% glycerin, 1mM PMSF) per 10mg of tissue weight.
  • 500 ⁇ L (1 ⁇ g/mL leupeptin and 28 ⁇ g/mL aprotinin) was added and homogenized using a Bead Blaster®D2400 homogenizer (Benchmark Scientific, NJ, USA).
  • the homogenized sample was centrifuged twice at 1500xg for 5 minutes to obtain a supernatant, and then 100 ⁇ L of this supernatant was measured using an MPO ELISA kit (HK210, Hycult Biotechnology, Netherlands).
  • MPO ELISA kit HK210, Hycult Biotechnology, Netherlands.
  • MPO activity refers to the reduction level of 1 ⁇ M hydrogen peroxide in water for 1 minute at 25°C, and was calculated as the amount of MPO contained in 1 mL of colon tissue homogenate.
  • Colon tissue was ground and proteins were extracted by ELISA and WB (p-STAT1/3, STAT1/3, NF- ⁇ p-I- ⁇ I- ⁇ RIP1/3, p-JAK1/2/3, TNF ⁇ , IL-6, IL -1 ⁇ was carried out.
  • the degree of colon tissue inflammatory signal activation was confirmed by WB, and signaling of p-STAT1/3 and NF-kB was confirmed in the cytosol and nuclear fractions, respectively.
  • PYGL activity in colon tissue was measured using a glycogen phosphorylase activity kit (Bio Visoln).
  • the large intestine of the rat was removed and the weight of the entire length from the cecum to the rectum was measured. As a result, there was edema and the weight of the intestine was significantly increased in the TNBS-only treated group compared to the vehicle-treated control group, and the test substance was administered. In one group, intestinal weight was significantly reduced compared to the TNBS treated group.
  • the INV-101 10 mg/kg administration group had the best effect ( Figure 5). In the case of the INV-101 10 mg/kg administration group, the recovery rate was 73.1%, which was significantly better than the recovery rate of 55.1% for the control drug filgotinib 30 mg/kg or 49.6% for UC-205689 10 mg/kg (Table 4).
  • H&E staining was performed to confirm the degree of damage to intestinal tissue and the degree of recovery by drug administration. Significant damage to the intestinal mucosa was confirmed in the group administered only TNBS compared to the normal control group. Recovery of the intestinal mucosa was confirmed by administration of the test compound. The improvement effect of INV-101 10mg/kg was the best ( Figure 6).
  • the TNBS-only treatment group showed ulcers in the mucosal tissue and infiltration of inflammatory cells into the lamina intestinal and submucosal layer. , vascular distribution, fibrosis (collagen fiber accumulation), and edematous changes were observed, and the degree of intestinal tissue damage was alleviated by compound administration.
  • INV-101 showed a dose-dependent effect, and the effect of the INV-101 10 mg/kg group was significantly better than the control drug filgotinib 30 mg/kg or UC-205689 10 mg/kg (Figure 7).
  • INV-101 showed a dose-dependent effect, and the effect of the INV-101 10 mg/kg group was significantly better than the control drug filgotinib 30 mg/kg or UC-205689 10 mg/kg (Figure 8).
  • INV-101 showed a dose-dependent effect, and the effect of the INV-101 10 mg/kg group was significantly better than the control drug filgotinib 30 mg/kg or UC-205689 10 mg/kg (Figure 9a).
  • the movement of STAT3 and NF- ⁇ induced by TNBS to the nucleus was inhibited to the level of the normal control by administration of 10 mg/kg of INV-101 (FIG. 9b).
  • MPO is an enzyme mainly found in neutrophils, and the presence of MPO in tissues is an indicator of neutrophil infiltration, which in turn is an indicator of inflammatory response and correlates with the level of intestinal damage caused by inflammatory colitis.
  • the amount of PYGL in the blood of TNBS-induced enteritis rats increased by about 4 times compared to normal, and was significantly suppressed in the compound administration group.
  • the inhibitory effect of INV-101 10 mg/kg was the strongest, and the remaining groups had similar levels. was shown ( Figure 12a).
  • the effect was excellent in the order of 205689 10 mg/kg > filgotinib 30 mg/kg (FIG. 12b).
  • INV-101 10 mg/kg which showed the strongest inhibitory activity, was confirmed to restore the level to the normal control level.
  • the amount of PYGL in the colonic tissue of TNBS-induced enteritis rats increased to about 3-fold compared to normal, and was significantly suppressed in the compound administration group.
  • the inhibitory effect of INV-101 10 mg/kg was the strongest, and the remaining groups showed similar levels.
  • INV-101 10 mg/kg which showed the strongest inhibitory activity, was confirmed to restore the level to the normal control level.
  • compound INV-101 restored colonic inflammation and damage induced by TNBS in a dose-dependent manner, suppressed the production of inflammatory cytokines in the blood and colonic tissue, Not only did it suppress the increase in blood PYGL, but it also significantly suppressed the increased PYGL activity. From these results, it was confirmed that compound INV-101, a PYGL inhibitor, is a therapeutic candidate with excellent efficacy in inhibiting PYGL activity and enteritis.

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Abstract

The present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory bowel disease, comprising a glycogen phosphorylase (PYGL) inhibitor as an active ingredient, and a method for screening for a material for the prevention or treatment of inflammatory bowel disease. By using the pharmaceutical composition for the prevention or treatment of inflammatory bowel disease, comprising a PYGL inhibitor as an active ingredient, according to the present invention, inflammatory bowel disease such as ulcerative colitis can be prevented or treated. Also, through the method for screening for a material for the prevention or treatment of inflammatory bowel disease, according to the present invention, a material that reduces PYGL activity can be determined as a candidate material for the prevention or treatment of inflammatory bowel disease.

Description

글리코겐 가인산분해효소(PYGL) 저해제를 유효성분으로 포함하는 염증성 장 질환의 예방 또는 치료용 약학 조성물Pharmaceutical composition for preventing or treating inflammatory bowel disease containing a glycogen phosphorylase (PYGL) inhibitor as an active ingredient

본 발명은 글리코겐 가인산분해효소(PYGL) 저해제를 유효성분으로 포함하는 염증성 장 질환의 예방 또는 치료용 약학 조성물, 및 염증성 장 질환의 예방 또는 치료용 물질의 스크리닝 방법에 관한 것이다.The present invention relates to a pharmaceutical composition for the prevention or treatment of inflammatory bowel disease containing a glycogen phosphorylase (PYGL) inhibitor as an active ingredient, and a screening method for a substance for the prevention or treatment of inflammatory bowel disease.

염증성 장 질환(inflammatory bowel disease, IBD)은 자가면역질환의 일종으로, 장에 만성적인 원인불명의 염증을 일으키는 질환이다. 상기 염증성 장 질환은 궤양성 대장염(ulcerative colitis, UC) 및 크론병(Crohn’s disease)을 포함하고, 정확한 병인은 아직 밝혀지지 않았으나, 유전학적 소인이 있는 사람에서 장관 내 세균에 대해 부적절한 면역반응에 의해 유발된다. 선천면역세포인 호중구와 대식세포는 물론 후천면역세포인 림프구 등 장면역계의 지속적이거나 부적절한 활성화는 결국 점막 파괴 및 궤양을 초래한다. 염증 상태의 장관의 점막에서 다양한 염증성 사이토카인들이 생성·분비되며 이 중 TNF-α(종양괴사인자-α)는 궤양성 대장염 환자의 대장 루멘과 대장 상피세포에서 높게 나타나며, 최근 연구에 의하면 TNF-α는 궤양성 대장염의 병인으로 중요한 역할을 한다고 알려져 있다.Inflammatory bowel disease (IBD) is a type of autoimmune disease that causes chronic, unexplained inflammation in the intestines. The inflammatory bowel disease includes ulcerative colitis (UC) and Crohn's disease, and although the exact etiology is not yet known, it is caused by an inappropriate immune response to intestinal bacteria in people with a genetic predisposition. It is triggered. Continuous or inappropriate activation of the intestinal immune system, including neutrophils and macrophages, which are innate immune cells, as well as lymphocytes, which are adaptive immune cells, ultimately results in mucosal destruction and ulceration. Various inflammatory cytokines are produced and secreted in the intestinal mucosa in an inflamed state, and among these, TNF-α (tumor necrosis factor-α) is highly expressed in the colonic lumen and colonic epithelial cells of patients with ulcerative colitis. Recent studies have shown that TNF-α α is known to play an important role in the pathogenesis of ulcerative colitis.

궤양성 대장염 치료를 위하여 1차 약제로 5-ASA (메살라민)을 처방하거나, 증상에 따라 스테로이드를 처방하기도 하며, 면역억제제를 포함한 생물학적 제제가 사용되기도 한다. 현재 궤양성 대장염 환자의 20-40%는 통상적인 약물치료에 실패하거나, 65-80%의 환자는 항 TNF- α 제제로 대표되는 생물학적 제제로도 완전한 관해를 유도하는데 실패하고 있다. 관해가 유도되더라도 생물학적 제제와 같은 고분자 단백질은 자가 항체가 형성되어 장기 치료시 반응이 소실되어 재발률이 높고, 결국 대장절제술을 받아야 하는 비율이 높다. 또한, 생물학적 제제는 고가의 비용은 물론이고, 주사제이므로 복용편의성이 낮다. 따라서, 이러한 단점을 개선한 새로운 기전의 경구용 저분자 신약의 개발의 필요성이 지속적으로 제기되어 있다.To treat ulcerative colitis, 5-ASA (mesalamine) is prescribed as a first-line drug, steroids are prescribed depending on symptoms, and biological agents including immunosuppressants are also used. Currently, 20-40% of ulcerative colitis patients fail conventional drug treatment, and 65-80% of patients fail to induce complete remission even with biological agents such as anti-TNF-α agents. Even if remission is induced, high molecular weight proteins such as biological agents cause the formation of autoantibodies and loss of response during long-term treatment, resulting in a high recurrence rate and ultimately requiring colectomy. In addition, biological agents are not only expensive, but are also injectable, making them less convenient to take. Therefore, the need to develop oral small molecule new drugs with new mechanisms that improve these shortcomings continues to be raised.

본 발명의 해결 과제는 염증성 장 질환의 예방 또는 치료용 물질을 스크리닝 하는 방법을 제공하고, 이에 따라 선별된 글리코겐 가인산분해효소(PYGL) 저해제를 유효성분으로 포함하는 염증성 장 질환의 예방 또는 치료용 약학 조성물을 제공하는 것이다. The problem of the present invention is to provide a method for screening a substance for the prevention or treatment of inflammatory bowel disease, and to provide a method for screening a substance for the prevention or treatment of inflammatory bowel disease containing the selected glycogen phosphatase (PYGL) inhibitor as an active ingredient. To provide a pharmaceutical composition.

또한, 본 발명의 해결 과제는 상기 글리코겐 가인산분해효소(PYGL) 저해제를, 이를 필요로 하는 대상(subject)에게 투여하는 단계를 포함하는 염증성 장 질환의 예방 또는 치료 방법을 제공하는 것이다.In addition, the problem of the present invention is to provide a method for preventing or treating inflammatory bowel disease, including the step of administering the glycogen phosphorylase (PYGL) inhibitor to a subject in need thereof.

또한, 본 발명의 해결 과제는 염증성 장 질환의 예방 또는 치료에 사용하기 위한 약제(medicament)를 제조하기 위한, 상기 글리코겐 가인산분해효소(PYGL) 저해제의 용도(use)를 제공하는 것이다.In addition, the problem of the present invention is to provide a use of the glycogen phosphorylase (PYGL) inhibitor for producing a medicament for use in the prevention or treatment of inflammatory bowel disease.

본 발명이 해결하고자 하는 과제는 이상에서 언급한 해결 과제로 제한되지 않으며, 언급되지 않은 또 다른 기술적 과제들은 아래의 기재로부터 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.The problem to be solved by the present invention is not limited to the problems mentioned above, and other technical problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

상기 기술적 과제를 달성하기 위하여, 본 발명의 일 측면에 따라, 글리코겐 가인산분해효소(PYGL) 저해제를 유효성분으로 포함하는 염증성 장 질환의 예방 또는 치료용 약학 조성물이 제공된다. In order to achieve the above technical problem, according to one aspect of the present invention, a pharmaceutical composition for preventing or treating inflammatory bowel disease containing a glycogen phosphorylase (PYGL) inhibitor as an active ingredient is provided.

본 발명의 다른 측면에 따라, 1) 글리코겐 가인산분해효소(PYGL)에 시험 물질을 처리하는 단계;According to another aspect of the present invention, 1) treating the test substance with glycogen phosphatase (PYGL);

2) 글리코겐 가인산분해효소(PYGL)의 활성을 측정하는 단계; 및2) measuring the activity of glycogen phosphorylase (PYGL); and

3) 상기 글리코겐 가인산분해효소(PYGL)의 활성을 감소시키는 시험 물질을 염증성 장 질환 치료제의 후보 물질로 결정하는 단계를 포함하는, 염증성 장 질환의 예방 또는 치료용 물질의 스크리닝 방법이 제공된다.3) A screening method for a substance for the prevention or treatment of inflammatory bowel disease is provided, comprising the step of determining a test substance that reduces the activity of glycogen phosphorylase (PYGL) as a candidate substance for the treatment of inflammatory bowel disease.

본 발명의 또 다른 측면에 따라, 상기 글리코겐 가인산분해효소(PYGL) 저해제를, 이를 필요로 하는 대상(subject)에게 투여하는 단계를 포함하는 염증성 장 질환의 예방 또는 치료 방법이 제공된다.According to another aspect of the present invention, a method for preventing or treating inflammatory bowel disease is provided, comprising administering the glycogen phosphorylase (PYGL) inhibitor to a subject in need thereof.

본 발명의 또 다른 측면에 따라, 염증성 장 질환의 예방 또는 치료에 사용하기 위한 약제(medicament)를 제조하기 위한, 상기 글리코겐 가인산분해효소(PYGL) 저해제의 용도(use)가 제공된다.According to another aspect of the present invention, there is provided the use of the glycogen phosphorylase (PYGL) inhibitor for preparing a medicament for use in the prevention or treatment of inflammatory bowel disease.

본 발명의 글리코겐 가인산분해효소(PYGL) 저해제를 유효성분으로 포함하는 염증성 장 질환의 예방 또는 치료용 약학 조성물을 통해 궤양성 대장염 등의 염증성 장 질환을 예방 또는 치료할 수 있다. Inflammatory bowel diseases such as ulcerative colitis can be prevented or treated through a pharmaceutical composition for preventing or treating inflammatory bowel disease containing the glycogen phosphatase (PYGL) inhibitor of the present invention as an active ingredient.

본 발명의 염증성 장 질환의 예방 또는 치료용 물질의 스크리닝 방법을 통해 가인산분해효소(PYGL) 활성을 감소시키는 물질을 염증성 장 질환의 예방 또는 치료용 물질의 후보물질로 결정할 수 있다.Through the screening method for a substance for the prevention or treatment of inflammatory bowel disease of the present invention, a substance that reduces phosphorylase (PYGL) activity can be determined as a candidate for a substance for the prevention or treatment of inflammatory bowel disease.

본 발명의 효과는 상기한 효과로 한정되는 것은 아니며, 본 발명의 설명 또는 청구범위에 기재된 발명의 구성으로부터 추론 가능한 모든 효과를 포함하는 것으로 이해되어야 한다.The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the description or claims of the present invention.

첨부된 도면은 해당 기술 분야의 통상의 기술자에게 본 발명의 내용을 보다 상세하게 설명하기 위한 것으로 본 발명의 기술적 사상이 이에 한정되는 것은 아니다. The attached drawings are intended to explain the present invention in more detail to those skilled in the art, and the technical idea of the present invention is not limited thereto.

도 1은 시험물질(INV-101, CP-91149 및 UC-205686)의 in vitro PYGL 저해능을 나타내는 그래프이다. Figure 1 is a graph showing the in vitro PYGL inhibition ability of test substances (INV-101, CP-91149, and UC-205686).

도 2a는 TNBS에 의해 유도된 질환 모델 랫트의 체중 변화를 나타낸 그래프이다. Figure 2a is a graph showing body weight changes in disease model rats induced by TNBS.

도 2b는 TNBS에 의해 질병 유도된 후 8일차의 랫트의 체중을 나타낸 그래프이다. Figure 2b is a graph showing the body weight of rats on day 8 after the disease was induced by TNBS.

도 3은 TNBS에 의해 유도된 질환 모델 랫트의 질병 진행 정도를 나타낸 그래프이다. Figure 3 is a graph showing the degree of disease progression in disease model rats induced by TNBS.

도 4는TNBS에 의해 유도된 질환 모델 랫트의 대장의 형태학적 변화를 나타낸 사진이다. Figure 4 is a photograph showing morphological changes in the large intestine of a disease model rat induced by TNBS.

도 5는 TNBS에 의해 유도된 질환 모델 랫트의 대장의 단위 길이당 무게를 나타낸 그래프이다. Figure 5 is a graph showing the weight per unit length of the large intestine of a disease model rat induced by TNBS.

도 6은 TNBS에 의해 유도된 질환 모델 랫트의 대장 조직의 H&E 염색 사진이다. Figure 6 is a photograph of H&E staining of colon tissue of a disease model rat induced by TNBS.

도 7은 TNBS에 의해 유도된 질환 모델 랫트의 대장 조직의 손상 정도 점수를 나타낸 그래프이다. Figure 7 is a graph showing the damage score of colonic tissue in a disease model rat induced by TNBS.

도 8은 TNBS에 의해 유도된 질환 모델 랫트의 대장 조직에서 염증 사이토카인 발현을 나타낸 것이다. Figure 8 shows the expression of inflammatory cytokines in the colonic tissue of a disease model rat induced by TNBS.

도 9a는 TNBS에 의해 유도된 질환 모델 랫트의 대장 조직의 염증 신호 활성화 정도를 나타낸 것이다(RIP1, RIP3, JAK1, JAK2, JAK3). Figure 9a shows the degree of inflammatory signal activation (RIP1, RIP3, JAK1, JAK2, JAK3) in colon tissue of a disease model rat induced by TNBS.

도 9b는 TNBS에 의해 유도된 질환 모델 랫트의 대장 조직의 염증 신호 활성화 정도를 나타낸 것이다(STAT1, STAT3, NF-κB). Figure 9b shows the degree of inflammatory signal activation (STAT1, STAT3, NF-κB) in colon tissue of a disease model rat induced by TNBS.

도 10은 TNBS에 의해 유도된 질환 모델 랫트의 대장 조직의 MPO 변화를 나타낸 그래프이다. Figure 10 is a graph showing changes in MPO in colon tissue of a disease model rat induced by TNBS.

도 11a는 TNBS에 의해 유도된 질환 모델 랫트의 혈중 TNFα 농도를 나타낸 그래프이다. Figure 11a is a graph showing the concentration of TNFα in the blood of a disease model rat induced by TNBS.

도 11b는 TNBS에 의해 유도된 질환 모델 랫트의 혈중 IL-6 농도를 나타낸 그래프이다. Figure 11b is a graph showing the concentration of IL-6 in the blood of a disease model rat induced by TNBS.

도 11c는 TNBS에 의해 유도된 질환 모델 랫트의 혈중 IL-1β 농도를 나타낸 그래프이다. Figure 11c is a graph showing the concentration of IL-1β in the blood of TNBS-induced disease model rats.

도 11d는 TNBS에 의해 유도된 질환 모델 랫트의 혈중 CRP 농도를 나타낸 그래프이다. Figure 11d is a graph showing the blood CRP concentration of a disease model rat induced by TNBS.

도 12a는 TNBS에 의해 유도된 질환 모델 랫트의 혈중 글리코겐 가인산분해효소(PYGL) 농도를 나타낸 그래프이다. Figure 12a is a graph showing the concentration of glycogen phosphatase (PYGL) in the blood of a disease model rat induced by TNBS.

도 12b는 TNBS에 의해 유도된 질환 모델 랫트의 혈청중(serum) 글리코겐 가인산분해효소(PYGL) 활성을 나타낸 그래프이다. Figure 12b is a graph showing the serum glycogen phosphatase (PYGL) activity of a disease model rat induced by TNBS.

도 13은 TNBS에 의해 유도된 질환 모델 랫트의 대장 조직의 글리코겐 가인산분해효소(PYGL) 활성을 나타낸 그래프이다. Figure 13 is a graph showing glycogen phosphorylase (PYGL) activity in colon tissue of a disease model rat induced by TNBS.

이하, 본 발명을 더욱 상세하게 설명한다. 다만, 이는 예시로서 제시되는 것으로, 이에 의해 본 발명이 제한되지는 않으며, 본 발명은 후술할 청구범위의 범주에 의해 정의될 뿐이다. Hereinafter, the present invention will be described in more detail. However, this is presented as an example, and the present invention is not limited thereby, and the present invention is only defined by the scope of the claims to be described later.

본 발명은, 글리코겐 가인산분해효소(PYGL) 저해제를 유효성분으로 포함하는 염증성 장 질환의 예방 또는 치료용 약학 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating inflammatory bowel disease comprising a glycogen phosphorylase (PYGL) inhibitor as an active ingredient.

본 발명은 또한, 상기 글리코겐 가인산분해효소(PYGL) 저해제를, 이를 필요로 하는 대상(subject)에게 투여하는 단계를 포함하는 염증성 장 질환의 예방 또는 치료 방법을 제공한다.The present invention also provides a method for preventing or treating inflammatory bowel disease, comprising administering the glycogen phosphorylase (PYGL) inhibitor to a subject in need thereof.

본 발명은 또한, 염증성 장 질환의 예방 또는 치료에 사용하기 위한 약제 (medicament)를 제조하기 위한, 상기 글리코겐 가인산분해효소(PYGL) 저해제의 용도(use)를 제공한다.The present invention also provides the use of the glycogen phosphorylase (PYGL) inhibitor for preparing a medicament for use in the prevention or treatment of inflammatory bowel disease.

글리코겐 가인산분해효소 또는 글리코겐 인산화효소(glycogen phosphorylase[PYGL])는 말단 알파-1-글리코시드 결합에서 글루코오스-1,4-포스페이트를 방출함으로써 동물에서 글리코겐의 분해를 촉매하는 효소이다. 이 효소는 활성 인산화 형태인 phosphorylase A와 비활성 인산화 형태인 phosphorylase B의 두 가지 형태로 존재하고, 두 가지 형태는 모두 동종이량체로 존재한다. 글리코겐 가인산분해효소 저해제의 글리코겐 분해 억제 효과를 이용하여 당뇨병 치료제 연구가 많이 진행되었다. 본 발명에서, 글리코겐 가인산분해효소 저해제는 면역반응과 관련된 세포내 신호전달 및 ROS 발생을 조절하여 면역세포 매개 염증반응을 조절함으로써 염증성 장 질환 치료효과를 가진다. 기존에 알려진 염증성 장 질환 치료제 약물들은 사이토카인을 저해하는 메커니즘을 가지고 있어 예기치 못한 부작용이 발생할 수 있으나, PYGL 저해제는 면역세포 대사 조절을 통해 염증반응을 조절하여 염증성 장 질환을 치료하는 것이므로 위와 같은 부작용이 없다는 장점이 있다.Glycogen phosphorylase, or glycogen phosphorylase [PYGL], is an enzyme that catalyzes the breakdown of glycogen in animals by releasing glucose-1,4-phosphate from the terminal alpha-1-glycosidic bond. This enzyme exists in two forms, the active phosphorylated form, phosphorylase A, and the inactive phosphorylated form, phosphorylase B, and both forms exist as homodimers. Much research has been conducted on diabetes treatments using the glycogen breakdown inhibition effect of glycogen phosphatase inhibitors. In the present invention, glycogen phosphatase inhibitors have a therapeutic effect on inflammatory bowel disease by controlling immune cell-mediated inflammatory responses by regulating intracellular signaling and ROS generation related to immune responses. Existing drugs for the treatment of inflammatory bowel disease have mechanisms that inhibit cytokines, which may cause unexpected side effects. However, PYGL inhibitors treat inflammatory bowel disease by controlling the inflammatory response through regulation of immune cell metabolism, so the above side effects are unlikely. There is an advantage to not having this.

본 발명에서, 상기 글리코겐 가인산분해효소(PYGL) 저해제는 하기 화학식 1 내지 8로 표시되는 화합물로 이루어지는 군으로부터 선택되는 어느 하나일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the glycogen phosphorylase (PYGL) inhibitor may be any one selected from the group consisting of compounds represented by the following formulas 1 to 8, but is not limited thereto.

하기 화학식 1로 표시되는 화합물은 N-(5-하이드록시-3,4,6-트라이메틸피리딘-2-일)-5-메틸-1H-인돌-2-카복사마이드(INV-101)이고, The compound represented by the following formula (1) is N-(5-hydroxy-3,4,6-trimethylpyridin-2-yl)-5-methyl-1H-indole-2-carboxamide (INV-101). ,

하기 화학식 2로 표시되는 화합물은 CP-91149이고,The compound represented by the following formula (2) is CP-91149,

하기 화학식 3으로 표시되는 화합물은UC-205686이고,The compound represented by the following formula (3) is UC-205686,

하기 화학식 4로 표시되는 화합물은 PSN-357이고,The compound represented by the following formula (4) is PSN-357,

하기 화학식 5로 표시되는 화합물은GSK-1362885이고,The compound represented by Formula 5 below is GSK-1362885,

하기 화학식 6으로 표시되는 화합물은 CP-368296 (Ingliforib)이고,The compound represented by Formula 6 below is CP-368296 (Ingliforib),

하기 화학식 7로 표시되는 화합물은 Z13865200141이고,The compound represented by the following formula (7) is Z13865200141,

하기 화학식 8로 표시되는 화합물은 AVE-5688이다.The compound represented by Formula 8 below is AVE-5688.

<화학식 1><Formula 1>

Figure PCTKR2023016299-appb-img-000001
Figure PCTKR2023016299-appb-img-000001

<화학식 2><Formula 2>

Figure PCTKR2023016299-appb-img-000002
Figure PCTKR2023016299-appb-img-000002

<화학식 3><Formula 3>

Figure PCTKR2023016299-appb-img-000003
Figure PCTKR2023016299-appb-img-000003

<화학식 4><Formula 4>

Figure PCTKR2023016299-appb-img-000004
Figure PCTKR2023016299-appb-img-000004

<화학식 5><Formula 5>

Figure PCTKR2023016299-appb-img-000005
Figure PCTKR2023016299-appb-img-000005

<화학식 6><Formula 6>

Figure PCTKR2023016299-appb-img-000006
Figure PCTKR2023016299-appb-img-000006

<화학식 7><Formula 7>

Figure PCTKR2023016299-appb-img-000007
Figure PCTKR2023016299-appb-img-000007

<화학식 8><Formula 8>

Figure PCTKR2023016299-appb-img-000008
Figure PCTKR2023016299-appb-img-000008

본 발명에서, 상기 염증성 장 질환은 장염, 대장염, 궤양성 대장염, 크론병, 크론친화성 세포종, 과민성 대장 증후군, 출혈성 직장 궤양, 회장낭 염(pouchitis), 소화성 궤양, 장관형 베체트병, 및 위염으로 이루어진 군으로부터 선택되는 어느 하나일 수 있다. In the present invention, the inflammatory bowel disease includes enteritis, colitis, ulcerative colitis, Crohn's disease, Crohn's apoptotic tumor, irritable bowel syndrome, bleeding rectal ulcer, ileal pouchitis, peptic ulcer, intestinal Behcet's disease, and gastritis. It may be any one selected from the group consisting of:

본 발명에서, 상기 염증성 장 질환은 궤양성 대장염일 수 있다. 궤양성 대장염은 대장의 점막 또는 점막하층에 염증 또는 궤양이 생기는 질환이며, 소장 및 대장 전체에 산발적으로 발생하는 염증성 장 질환인 크론병과는 다르게 직장에서 시작되어 대장 안쪽으로 연결된 병변을 특징으로 한다. 궤양성 대장염 환자의 대부분은 하루 수회의 혈액과 점액이 섞인 묽은 변 또는 설사를 하며, 심한 경우 복통, 탈수, 발열, 구토, 체중 감소 등의 증상을 호소한다. 대부분의 궤양성 대장염은 만성 재발성 경과를 보이며, 재발할수록 상태가 악화된다.In the present invention, the inflammatory bowel disease may be ulcerative colitis. Ulcerative colitis is a disease in which inflammation or ulcers occur in the mucosa or submucosa of the large intestine. Unlike Crohn's disease, which is an inflammatory bowel disease that occurs sporadically throughout the small and large intestine, it is characterized by lesions that start in the rectum and connect to the inside of the large intestine. Most patients with ulcerative colitis have loose stools or diarrhea mixed with blood and mucus several times a day, and in severe cases, they complain of symptoms such as abdominal pain, dehydration, fever, vomiting, and weight loss. Most ulcerative colitis has a chronic recurrent course, and the condition worsens as it relapses.

또한, 본 발명은 1) 글리코겐 가인산분해효소(PYGL)에 시험 물질을 처리하는 단계; 2) 글리코겐 가인산분해효소(PYGL)의 활성을 측정하는 단계; 및 3) 상기 글리코겐 가인산분해효소(PYGL)의 활성을 감소시키는 시험 물질을 염증성 장 질환 치료제의 후보 물질로 결정하는 단계를 포함하는, 염증성 장 질환의 예방 또는 치료용 물질의 스크리닝 방법을 제공한다.In addition, the present invention includes the steps of 1) treating the test substance with glycogen phosphorylase (PYGL); 2) measuring the activity of glycogen phosphorylase (PYGL); and 3) determining a test substance that reduces the activity of glycogen phosphatase (PYGL) as a candidate for the treatment of inflammatory bowel disease. It provides a screening method for a substance for the prevention or treatment of inflammatory bowel disease. .

본 발명에서, 상기 글리코겐 가인산분해효소(PYGL)의 활성의 측정은 포도당-1-인산(G-1-P) 검출을 이용하여 측정하는 것일 수 있다. In the present invention, the activity of glycogen phosphorylase (PYGL) may be measured using glucose-1-phosphate (G-1-P) detection.

상기 포도당-1-인산(G-1-P)은 글리코겐이 글리코겐 가인산분해효소에 의해 분해되어 생성되는 물질이다. 글리코겐 가인산분해효소의 활성은 포도당-1-인산의 검출 신호의 세기에 비례한다.The glucose-1-phosphate (G-1-P) is a substance produced by decomposition of glycogen by glycogen phosphatase. The activity of glycogen phosphatase is proportional to the intensity of the detection signal of glucose-1-phosphate.

본 발명에서, 상기 염증성 장 질환은 장염, 대장염, 궤양성 대장염, 크론병, 크론친화성 세포종, 과민성 대장 증후군, 출혈성 직장 궤양, 회장낭 염(pouchitis), 소화성 궤양, 장관형 베체트병, 및 위염으로 이루어진 군으로부터 선택되는 어느 하나일 수 있다.In the present invention, the inflammatory bowel disease includes enteritis, colitis, ulcerative colitis, Crohn's disease, Crohn's sarcoma, irritable bowel syndrome, bleeding rectal ulcer, ileal pouchitis, peptic ulcer, intestinal Behcet's disease, and gastritis. It may be any one selected from the group consisting of:

본 발명에 따른 상기 약학 조성물은 임상 투여시에 경구 및 비경구의 여러 가지 제형으로 투여될 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등 이 포함되며, 이러한 고형제제는 하나 이상의 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 탄산칼슘, 수크로오스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 스테아린산 마그네슘, 탈크 등과 같은 윤활제들도 사용된다. 경구투여를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제가 포함된다. 비수성용제, 현탁용제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다.The pharmaceutical composition according to the present invention may be administered in various oral and parenteral dosage forms during clinical administration. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain one or more compounds and at least one excipient, such as starch, calcium carbonate, sucrose or lactose ( It is prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, and emulsions. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.

본 발명에 따른 상기 약학 조성물은 비경구 투여할 수 있으며, 비경구 투여는 피하주사, 정맥주사, 근육 내 주사 또는 흉부 내 주사를 주입하는 방법에 의한다. 이때, 비경구 투여용 제형으로 제제화하기 위하여 상기 약학 조성물을 안정제 또는 완충제와 함께 물에 혼합하여 용액 또는 현탁액으로 제조하고, 이를 앰플 또는 바이알 단위 투여형으로 제조할 수 있다. 상기 조성물은 멸균되고/되거나 방부제, 안정화제, 수화제 또는 유화 촉진제, 삼투압 조절을 위한 염 및/또는 완충제 등의 보조제, 및 기타 치료적으로 유용한 물질을 포함할 수 있으며, 통상적인 방법인 혼합, 과립화 또는 코팅 방법에 따라 제제화할 수 있다.The pharmaceutical composition according to the present invention can be administered parenterally, and parenteral administration is by subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection. At this time, in order to formulate a formulation for parenteral administration, the pharmaceutical composition is mixed with water along with a stabilizer or buffer to prepare a solution or suspension, which can be prepared in an ampoule or vial unit dosage form. The composition may be sterilized and/or contain preservatives, stabilizers, wetting agents or emulsification accelerators, auxiliaries such as salts and/or buffers for adjusting osmotic pressure, and other therapeutically useful substances, and may be mixed, granulated, etc. using conventional methods. It can be formulated according to the coating or coating method.

본 발명에 따른 상기 약학 조성물의 경구 투여용 제형으로는 예를 들면 정제, 환제, 경/연질 캅셀제, 액제, 현탁제, 유화제, 시럽제, 과립제, 엘릭시르제, 트로키제 등이 있는데, 이들 제형은 유효성분 이외에 희석제(예: 락토즈, 덱스트로즈, 수크로즈, 만니톨, 솔비톨, 셀룰로즈 및/또는 글리신), 활택제(예: 실리카, 탈크, 스테아르산 및 그의 마그네슘 또는 칼슘염 및/또는 폴리에틸렌 글리콜)를 포함할 수 있다. 정제는 마그네슘 알루미늄 실리케이트, 전분 페이스트, 젤라틴, 메틸셀룰로즈, 나트륨 카복시메틸셀룰로즈 및/또는 폴리비닐피롤리딘 등과 같은 결합제를 포함할 수 있으며, 경우에 따라 전분, 한천, 알긴산 또는 그의 나트륨 염 등과 같은 붕해제 또는 비등 혼합물 및/또는 흡수제, 착색제, 향미제 및 감미제를 포함할 수 있다.Formulations for oral administration of the pharmaceutical composition according to the present invention include, for example, tablets, pills, hard/soft capsules, solutions, suspensions, emulsifiers, syrups, granules, elixirs, troches, etc., and these formulations are effective In addition to the ingredients, diluents (e.g. lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine), lubricants (e.g. silica, talc, stearic acid and its magnesium or calcium salts and/or polyethylene glycol) may include. The tablet may contain a binder such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and optionally a binder such as starch, agar, alginic acid or its sodium salt, etc. The releasing or boiling mixture may contain absorbents, colorants, flavoring and sweetening agents.

본 발명의 상기 부형제, 결합제, 붕해제, 활택제, 착색제, 향미제 등에 대한 용어 정의는 당업계에 공지된 문헌에 기재된 것으로 그 기능 등이 동일 내지 유사한 것들을 포함한다.Definitions of the excipients, binders, disintegrants, lubricants, colorants, flavoring agents, etc. of the present invention are those described in literature known in the art and include those with the same or similar functions.

본 발명의 약학 조성물에 포함되는 유효성분의 투여량은 환자의 상태 및 체중, 질병의 정도, 유효성분 형태, 투여 경로 및 기간에 따라 다르며, 환자에 따라 적절하게 조절될 수 있다. 예를 들면, 상기 유효성분은 1일 0.0001 내지 1000 mg/kg으로, 바람직하게는 0.001 내지 100 mg/kg의 용량으로 투여할 수 있으며, 상기 투여는 하루에 한번 또는 수회 나누어 투여할 수도 있다. 또한, 본 발명의 약학 조성물은 조성물 총 중량에 대하여 상기 유효성분을 0.001 내지 90 % 중량백분율로 포함할 수 있다.The dosage of the active ingredient contained in the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, degree of disease, form of the active ingredient, route and period of administration, and can be appropriately adjusted depending on the patient. For example, the active ingredient can be administered at a dose of 0.0001 to 1000 mg/kg per day, preferably 0.001 to 100 mg/kg, and the administration may be administered once a day or in divided doses. Additionally, the pharmaceutical composition of the present invention may contain the active ingredient at a weight percentage of 0.001 to 90% based on the total weight of the composition.

이하, 본 발명을 실시예 및 실험예를 통하여 더욱 상세히 설명한다. 그러나, 하기 실시예 및 실험예는 본 발명을 예시하기 위한 것으로, 본 발명의 범위가 이에 제한되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples and experimental examples. However, the following examples and experimental examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto.

실시예. N-(5-하이드록시-3,4,6-트라이메틸피리딘-2-일)-5-메틸-1H-인돌-2-카복사마이드 (화합물 INV-101)Example. N-(5-hydroxy-3,4,6-trimethylpyridin-2-yl)-5-methyl-1H-indole-2-carboxamide (Compound INV-101)

본 발명의 실시예로서 사용한 화합물 N-(5-하이드록시-3,4,6-트라이메틸피리딘-2-일)-5-메틸-1H-인돌-2-카복사마이드 [N-(5-hydroxy-3,4,6-trimethylpyridin-2-yl)-5-methyl-1H-indole-2-carboxamide, INV-101] 의 화학식은 하기 화학식 1로 표시된다. Compound N-(5-hydroxy-3,4,6-trimethylpyridin-2-yl)-5-methyl-1H-indole-2-carboxamide [N-(5-) used as an example of the present invention The chemical formula of [hydroxy-3,4,6-trimethylpyridin-2-yl)-5-methyl-1H-indole-2-carboxamide, INV-101] is represented by Formula 1 below.

<화학식 1><Formula 1>

Figure PCTKR2023016299-appb-img-000009
Figure PCTKR2023016299-appb-img-000009

1H-NMR ((CD3)2SO) δ 11.54 (d, J= 1.1 Hz, 1H), 10.22 (s, 1H), 8.63 (s, 1H), 7.41 (s, 1H), 7.30 (dd, J = 12.6, 4.9 Hz, 2H), 7.03 (dd, J = 8.4, 1.4 Hz, 1H), 2.36 (d, J = 8.1 Hz, 6H), 2.17 (s, 3H), 2.04 (s, 3H). 1 H-NMR ((CD 3 ) 2 SO) δ 11.54 (d, J= 1.1 Hz, 1H), 10.22 (s, 1H), 8.63 (s, 1H), 7.41 (s, 1H), 7.30 (dd, J = 12.6, 4.9 Hz, 2H), 7.03 (dd, J = 8.4, 1.4 Hz, 1H), 2.36 (d, J = 8.1 Hz, 6H), 2.17 (s, 3H), 2.04 (s, 3H).

실험예 1. 화합물 INV-101 (실시예)의 PYGL 저해능 확인 시험Experimental Example 1. Test to confirm the PYGL inhibition ability of compound INV-101 (Example)

글리코겐 가인산분해효소(GPase)는 가역 반응인 (글루코스)n + Pi에서 (글루코스)n-1 + G-1-P로의 반응을 촉매한다. 반응은 글리코겐과 Pi가 순방향 반응으로 반응할 수 있는 조건이 되면 시작된다. 가인산분해효소 키나아제는 비활성 글리코겐 가인산분해효소 B를 완전 활성 글리코겐 가인산분해효소 A로 전환하는 것을 촉매한다. 그러나 전반적인 조절은 포스파타제(phosphatase)의 작용에서 AMP에 의한 피드백 제어에 이르기까지 다양한 요인의 상호 작용에 따라 달라진다.Glycogen phosphatase (GPase) catalyzes the reversible reaction of (glucose)n + Pi to (glucose)n-1 + G-1-P. The reaction begins when conditions allow glycogen and Pi to react in a forward reaction. Phosphatase kinase catalyzes the conversion of inactive glycogen phosphatase B to fully active glycogen phosphatase A. However, overall regulation depends on the interaction of a variety of factors, ranging from the action of phosphatases to feedback control by AMPs.

글리코겐 가인산분해효소 비색 분석 키트(Glycogen Phosphorylase Colorimetric Assay Kit)를 이용하고, 토끼 근육 글리코겐 가인산분해효소(RMGPa)를 사용하여, 화합물 INV-101의 PYGL 저해능을 확인하였다. 시험 물질 또는 비이클(vehicle, 1.0% DMSO 내)을 37 ℃에서 15분 동안 5μg/ml의 효소와 함께 변형된(modified) 이미다졸 완충액(pH 6.8)에서 사전 인큐베이션시켰다. 양성 대조군으로 UC-205686(0.03 μM) 및 CP-91149(3 μM)을 사용하였다. 반응은 다른 30분 인큐베이션 기간 동안 4mg/ml 글리코겐의 첨가로 개시되었다. 생성된 NADPH의 양은 340 nm에서 분광광도계로 분석하였다. 물질들은 정확한 IC50 분석을 위하여 최소 10개의 저해 농도(100, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0 μM)에서 측정하였다. RMGPa의 활성은 글루코오스-1-포스페이트(glucose-1-phosphate)를 검출하여 측정하였다.The PYGL inhibition ability of compound INV-101 was confirmed using the Glycogen Phosphorylase Colorimetric Assay Kit and rabbit muscle glycogen phosphatase (RMGPa). Test substances or vehicle (in 1.0% DMSO) were pre-incubated in modified imidazole buffer (pH 6.8) with 5 μg/ml enzyme for 15 minutes at 37°C. UC-205686 (0.03 μM) and CP-91149 (3 μM) were used as positive controls. The reaction was initiated with the addition of 4 mg/ml glycogen for another 30 min incubation period. The amount of NADPH produced was analyzed spectrophotometrically at 340 nm. Substances were measured at at least 10 inhibitory concentrations (100, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0 μM) for accurate IC 50 analysis. The activity of RMGPa was measured by detecting glucose-1-phosphate.

상기 물질들의 RMGPa 저해능 결과는 도 1 및 하기 표 1과 같다. PYGL 저해제로 알려져 있는 CP-91149 및 UC-205686의 IC50는 각각 2.53 μM 및 0.016 μM이고, INV-101의 IC50는 0.085 μM 로 측정되었다. 상기 실험예 1 의 결과로부터, 본 발명의 화합물 INV-101은 강력한 PYGL 저해제임을 알 수 있다. The results of the RMGPa inhibition ability of the above substances are shown in Figure 1 and Table 1 below. The IC 50 of CP-91149 and UC-205686, which are known PYGL inhibitors, were 2.53 μM and 0.016 μM, respectively, and the IC 50 of INV-101 was measured at 0.085 μM. From the results of Experimental Example 1, it can be seen that the compound INV-101 of the present invention is a strong PYGL inhibitor.

화합물compound 종bell 농도density % Inh.%Inh. IC50 IC 50 CP-91149CP-91149 토끼rabbit 3μM3μM 6464 2.53 μM2.53 μM INV-101
(BJ-80F)INV-101
(BJ-80F) 토끼rabbit 0.1μM0.1μM 5757 0.085 μM0.085 μM UC-205686UC-205686 토끼rabbit 0.03μM0.03μM 7474 0.016 μM0.016 μM

실험예 2. TNBS로 유도한 염증성 장 질환 랫트 모델에서 PYGL 저해제인 화합물 INV-101의 경구 투여 Experimental Example 2. Oral administration of compound INV-101, a PYGL inhibitor, in a TNBS-induced inflammatory bowel disease rat model in vivoin vivo 효능 시험 efficacy test

염증성 장 질환의 동물 모델은 여러 유형이 있으나 화학물질로 병변을 야기하는 모델이 자주 이용되고 있다. 마우스 또는 랫트에서 트리니트로벤젠 설폰산(TNBS)을 직장내에 주입하여 유도된 대장염은 염증성 장 질환의 한 형태인 크론병에 대한 기본 병리학적 및 면역학적 메커니즘을 연구할 수 있는 동물 모델 중 하나이다. TNBS로 유도한 염증성 장 질환 동물모델에서 필고티닙(Filgotinib)(30 mg/kg), UC-205686(10 mg/kg) 경구투여 그룹을 양성대조군으로 하여 INV-101의 경구투여 효능을 비교하였다.There are several types of animal models of inflammatory bowel disease, but models that cause lesions with chemicals are frequently used. Colitis induced by intrarectal injection of trinitrobenzene sulfonic acid (TNBS) in mice or rats is one of the animal models in which the basic pathological and immunological mechanisms of Crohn's disease, a form of inflammatory bowel disease, can be studied. In an inflammatory bowel disease animal model induced by TNBS, the efficacy of oral administration of INV-101 was compared using the oral administration group of Filgotinib (30 mg/kg) and UC-205686 (10 mg/kg) as a positive control group. .

1. 시험 방법1. Test method

(1) 실험동물 및 그룹화(1) Experimental animals and grouping

동물은 7-8주령된 랫트(Sprague Dawley Rat)를 오리엔트바이오(Korea)로부터 구입하여 7일간 일반 고형사료로 안정화시킨 후 실험에 이용하였다. 실험 기간 중 사료와 물을 자유로이 공급하였고, 사육실의 온도는 25±1 ℃상대습도는 50±10%로 유지시켰다. 점등 관리는 자동조명조절기에 의해 12시간 명암주기(light-dark cycle)로 조절하였다. 실험군은 각 군당 8마리로 하여 평균 체중이 250±20 g이 되도록 무작위 블록 설계(randomized block design)에 의하여 7군(정상군, TNBS-Vehicle 투여군, TNBS+Filgotinib 30 mg/kg 투여군, TNBS+UC-205686 10 mg/kg 투여군, TNBS+INV-101 1 mg/kg투여군, TNBS+INV-101 3 mg/kg투여군, TNBS+INV-101 10 mg/kg투여군)으로 나누어 실험하였다.The animals were 7-8 week old rats (Sprague Dawley Rats) purchased from Orient Bio (Korea) and stabilized with regular solid feed for 7 days before being used in the experiment. Feed and water were supplied ad libitum during the experiment, and the temperature in the breeding room was maintained at 25±1°C and relative humidity at 50±10%. Lighting management was controlled with a 12-hour light-dark cycle using an automatic lighting controller. The experimental group was divided into 7 groups (normal group, TNBS-Vehicle administered group, TNBS+Filgotinib 30 mg/kg administered group, TNBS+UC) by randomized block design with 8 animals in each group, with an average weight of 250±20 g. -205686 10 mg/kg administration group, TNBS+INV-101 1 mg/kg administration group, TNBS+INV-101 3 mg/kg administration group, TNBS+INV-101 10 mg/kg administration group).

(2) TNBS 직장 투여 장염 유발(2) TNBS rectal administration causes enteritis

24시간 절식한 랫트를 디에틸 에테르로 마취한 후, 폴리에틸렌 카테터를 연결한 1 mL 주사기를 이용하여 결장의 내강에 50 v/v% 에탄올로 희석한 5% TNBS를 0.8 mL을 천천히 주입한 후, 항문으로 5% TNBS가 새어 나오는 것을 방지하기 위하여 랫트를 거꾸로 세운 상태에서 60초 동안 정치시켰다. 대조군은 비이클(vehicle) [50v/v% ethanol]만을 다른 군과 마찬가지 방법으로 주입하였다.After anesthetizing a rat that had fasted for 24 hours with diethyl ether, 0.8 mL of 5% TNBS diluted with 50 v/v% ethanol was slowly injected into the lumen of the colon using a 1 mL syringe connected to a polyethylene catheter. To prevent 5% TNBS from leaking through the anus, the rat was left upside down for 60 seconds. The control group was injected with only vehicle [50v/v% ethanol] in the same manner as the other groups.

(3) 약물 투여(3) Drug administration

NBS를 투여한 당일 약물 투여는 TNBS 투여 2시간 후 약물의 최초 경구 투여를 실시하고 다음 날부터 6일 동안 매일 일정한 시간에 경구 투여하였다.The first oral administration of the drug on the day of NBS administration was 2 hours after TNBS administration, and the drug was administered orally at a constant time every day for 6 days from the next day.

(4) 체중 관찰(4) Weight observation

디지털 체중계를 이용하여 절식 단계부터 TNBS 투여 및　약물　투여과정 동안 각 랫트의 체중 변화를 관찰하였다.Using a digital scale, we observed the change in body weight of each rat from the fasting phase to the TNBS administration and drug administration process.

(5) 심장채혈 및 혈액 분석(5) Heart blood collection and blood analysis

최후 약물 투여 전 24시간 절식시킨 후 최후 약물 투여 1시간 내에 랫트를 마취시킨 후 심장채혈을 실시하였다.Rats were fasted for 24 hours before the last drug administration, then anesthetized within 1 hour of the last drug administration, and heart blood was collected.

(가) 혈청 사이토카인 측정(A) Serum cytokine measurement

혈청을 분리한 후 -80 ℃냉동고에 분주하여 보관하였고, 각 사이토카인 측정 당일에 해동하여 각 ELISA 키트(CRP, TNFα, IL-6, IL-1βPYGL)를 이용하여 정량하였다.After separation of the serum, it was aliquoted and stored in a -80°C freezer, and each cytokine was thawed on the day of measurement and quantified using each ELISA kit (CRP, TNFα, IL-6, IL-1βPYGL).

(나) 혈액 PYGL 활성 측정(B) Measurement of blood PYGL activity

글리코겐 가인산분해효소 활성 키트(Glycogen phosphorylase activity kit, BioVision)를 이용하여 혈액 PYGL 활성을 측정하였다.Blood PYGL activity was measured using a glycogen phosphorylase activity kit (BioVision).

(6) 대장 무게 측정(6) Measurement of colon weight

랫트의 대장을 적출하여 항문으로부터 5-6 cm 사이의 조직을 1 cm 길이로 잘라서 조직의 무게를 측정하였다.The large intestine of the rat was removed, the tissue between 5 and 6 cm from the anus was cut into 1 cm long pieces, and the weight of the tissue was measured.

(7) Myeloperoxidase (MPO) 측정(7) Myeloperoxidase (MPO) measurement

조직 내 호중구의 침윤한 정도를 확인하기 위해 호중구 정량하는 지표인 효소 MPO 양을 측정하였다. 대장 1 cm 크기를 차가운 PBS로 세척하고 무게를 측정하여 조직의 무게 10 mg당 용해 완충액(lysis buffer, pH7.4, 200 mM NaCl, 5 mM EDTA, 10 mM tris, 10% glycerin, 1 mM PMSF, 1 μg/mL leupeptin and 28 μg/mL aprotinin) 500 μL를 첨가한 후 Bead Blaster®D2400 homogenizer (Benchmark Scientific, NJ, USA)를 이용하여 균질화하였다. 균질화한 시료를 1500ｘg, 5분으로 2회 원심분리하여 상등액을 얻은 다음, 이 상등액 100 μL를 MPO ELISA 키트(HK210, Hycult Biotechnology, Netherlands)를 이용하여 측정하였다. 항-랫트 MPO 항체(Anti-rat MPO antibody)가 코팅된 96 well에 앞의 상등액을 100 μL 첨가하여 실온에서 1시간 반응시킨 후 세척 완충액으로 3회 세척하고 여기에 재구성 추적자 (reconstituted tracer)을 100 μl 첨가하여 실온에서 1시간 반응하고 3회 반복 세척한 다음, 스트렙타비딘-과산화효소 접합체(streptavidin-peroxidase conjugate)를 100 μL 첨가하고 실온에서 1시간 반응한 후, 세척하고 TMB 기질 용액(substrate solution)을 100 μL 가하여 반응 30분 후에 반응 종결액(stop solution) 100 μl로 정지시킨 다음 450 nm에서 흡광도를 측정하였다. MPO 활성도는 25 ℃에서 1분 동안 물에서 과산화수소 1 μM이 환원되는 수치를 의미하여 이것을 대장 조직 균질액 1 mL에 포함된 MPO 양으로 계산하였다.To confirm the degree of neutrophil infiltration within the tissue, the amount of the enzyme MPO, which is an indicator for quantifying neutrophils, was measured. A 1-cm section of the large intestine was washed with cold PBS, weighed, and added with lysis buffer (pH7.4, 200mM NaCl, 5mM EDTA, 10mM tris, 10% glycerin, 1mM PMSF) per 10mg of tissue weight. 500 μL (1 μg/mL leupeptin and 28 μg/mL aprotinin) was added and homogenized using a Bead Blaster®D2400 homogenizer (Benchmark Scientific, NJ, USA). The homogenized sample was centrifuged twice at 1500xg for 5 minutes to obtain a supernatant, and then 100 μL of this supernatant was measured using an MPO ELISA kit (HK210, Hycult Biotechnology, Netherlands). Add 100 μL of the above supernatant to 96 wells coated with anti-rat MPO antibody, react at room temperature for 1 hour, wash three times with washing buffer, and add 100 μL of reconstituted tracer to it. Add μl, react at room temperature for 1 hour, wash three times, add 100 μl of streptavidin-peroxidase conjugate, react for 1 hour at room temperature, wash, and wash with TMB substrate solution. ) was added and the reaction was stopped 30 minutes later with 100 μl of stop solution, and then the absorbance was measured at 450 nm. MPO activity refers to the reduction level of 1 μM hydrogen peroxide in water for 1 minute at 25°C, and was calculated as the amount of MPO contained in 1 mL of colon tissue homogenate.

(8) 대장 조직 병리 관찰(8) Observation of colon tissue pathology

10% 중성 완충 포르말린(neutral buffered formalin)에 고정시킨 대장 조직의 H&E 염색 및 면역염색을 진행하였다. H&E staining and immunostaining of colon tissue fixed in 10% neutral buffered formalin were performed.

(가) H&E 염색 및 조직 손상 측정(A) H&E staining and tissue damage measurement

H&E 염색 후 현미경 하에서 관찰하여 점막 손상 및 회복 정도를 하기 표 2와 같이 점수화하였다.After H&E staining, it was observed under a microscope and the degree of mucosal damage and recovery was scored as shown in Table 2 below.

점막상피
1. 궤양: 없음 (0); 경증(mild) 표면 (1); 보통 (2); 광범위한 전체 두께 (extensive-full thickness) (3)mucosal epithelium
1. Ulcer: None (0); Mild surface (1); Normal (2); extensive-full thickness (3) 고유층 (Lamina propria)2. 염증 세포 침윤물: 없음 (0); 경증(mild) (1); 보통 (2); 심함 (3)
3. 혈관성: 없음 (0); 경증(mild) (1); 보통 (2); 심함 (3)
4. 콜라겐 침착: 없음 (0); 경증(mild) (1); 보통 (2); 심함 (3)Lamina propria2. Inflammatory cell infiltrate: none (0); mild (1); Normal (2); Severe (3)
3. Vascular: None (0); mild (1); Normal (2); Severe (3)
4. Collagen deposition: none (0); mild (1); Normal (2); Severe (3) 점막하층5. 염증세포 침윤물: 없음 (0); 경증(mild) (1); 보통 (2); 심함 (3)
6. 콜라겐 침착: 없음 (0); 경증(mild) (1); 보통 (2); 심함 (3)
7. 혈관성: 없음 (0); 경증(mild) (1); 보통 (2); 심함 (3)
8. 부종: 없음 (0); 경증(mild) (1); 보통 (2); 심함 (3)Submucosa 5. Inflammatory cell infiltrate: none (0); mild (1); Normal (2); Severe (3)
6. Collagen deposition: none (0); mild (1); Normal (2); Severe (3)
7. Vascular: none (0); mild (1); Normal (2); Severe (3)
8. Edema: None (0); mild (1); Normal (2); Severe (3) 점막상피1. 궤양: 없음 (0); 경증(mild) 표면 (1); 보통 (2); 광범위한 전체 두께 (extensive-full thickness) (3)Mucosal epithelium 1. Ulcer: None (0); Mild surface (1); Normal (2); extensive-full thickness (3) 고유층 (Lamina propria)Lamina propria 최대 점수: 30Maximum score: 30

(나) 대장 조직 사이토카인 발현 변화 측정 (ELISA 및 WB)(B) Measurement of changes in colonic tissue cytokine expression (ELISA and WB)

대장 조직을 갈아서 단백질을 추출하여 ELISA 및 WB (p-STAT1/3, STAT1/3, NF-κp-I-κI-κ RIP1/3, p-JAK1/2/3, TNFαα, IL-6, IL-1β을 실시하였다.Colon tissue was ground and proteins were extracted by ELISA and WB (p-STAT1/3, STAT1/3, NF-κp-I-κI-κ RIP1/3, p-JAK1/2/3, TNFαα, IL-6, IL -1β was carried out.

(다) 대장 조직 염증신호 활성화(phosphorylation) 확인(C) Confirmation of phosphorylation of inflammatory signals in colon tissue

대장 조직 염증신호 활성화 정도는 WB으로 확인하였으며, p-STAT1/3 과 NF-kB의 시그널링(signaling)은 세포질(cytosol)과 핵(Nucleus) 분획 각각에 대해 확인하였다.The degree of colon tissue inflammatory signal activation was confirmed by WB, and signaling of p-STAT1/3 and NF-kB was confirmed in the cytosol and nuclear fractions, respectively.

(9) 대장 조직의 PYGL 활성 측정(9) Measurement of PYGL activity in colon tissue

글리코겐 가인산분해효소 활성 키트(Glycogen phosphorylase activity kit, Bio Visoln)를 이용하여 대장 조직의 PYGL 활성을 측정하였다. PYGL activity in colon tissue was measured using a glycogen phosphorylase activity kit (Bio Visoln).

2. 시험 결과2. Test results

(1) TNBS로 유도한 장염증 랫트 모델에서 약물 투여에 따른 체중변화(1) Body weight change following drug administration in a rat model of intestinal inflammation induced by TNBS

TNBS 처리 전의 몸무게를 기준으로 7일간 매일 일정 시간에 몸무게의 변화를 관찰한 결과, 도 2a에서 보는 바와 같이 정상 대조군의 체중은 계속해서 몸무게가 증가함을 보이고, TNBS 단독 투여군은 몸무게가 서서히 감소하여 투여 8일째에는 실험 시작 시점 대비 23%의 체중 감소가 확인되었다(도 2b). 각 약물투여군의 체중 회복률은 하기 표 3과 같다.As a result of observing changes in body weight at certain times every day for 7 days based on the body weight before TNBS treatment, as shown in Figure 2a, the body weight of the normal control group continued to increase, and the body weight of the group administered TNBS alone gradually decreased. On the 8th day of administration, a 23% weight loss compared to the start of the experiment was confirmed (Figure 2b). The weight recovery rate for each drug administration group is shown in Table 3 below.

체중 회복률 (%)Weight recovery rate (%) Filgotinib (30mg/kg)Filgotinib (30mg/kg) INV-101 (1mg/kg)INV-101 (1mg/kg) INV-101 (3mg/kg)INV-101 (3mg/kg) INV-101 (10mg/kg)INV-101 (10mg/kg) UC-205686 (10mg/kg)UC-205686 (10mg/kg) 22.59 ± 25.022.59 ± 25.0 29.99 ± 23.429.99 ± 23.4 21.15 ± 13.521.15 ± 13.5 44.69 ± 24.244.69 ± 24.2 28.84 ± 25.828.84 ± 25.8

질병 진행 정도(Disease Activity Index, DAI)로 확인한 결과, TNBS 단독 투여군의 경우 질병 진행 정도가 TNBS 투여 5일째에 최고에 달한 후 감소되는 경향을 보였다. 약물투여군의 경우 INV-101 10mg/kg 투여군의 경우 약물투여 3일째 이후부터 확연한 감소를 보였다(도 3). 반면 필고티닙 30 mg/kg, UC-205689 10 mg/kg, INV-101 1 mg/kg, INV-101 3mg/kg 의 경우는 서로간의 유의성은 없으나, TNBS 단독군에 비해 질병정도가 감소하였다.As a result of the disease progression (Disease Activity Index, DAI), in the group administered TNBS alone, the degree of disease progression peaked on the 5th day of TNBS administration and then tended to decrease. In the case of the drug administration group, the INV-101 10mg/kg administration group showed a clear decrease after the 3rd day of drug administration (Figure 3). On the other hand, there was no significance between filgotinib 30 mg/kg, UC-205689 10 mg/kg, INV-101 1 mg/kg, and INV-101 3mg/kg, but the disease severity decreased compared to the TNBS group alone. .

(2) 대장 조직 형태 및 무게 변화(2) Changes in colon tissue morphology and weight

(가) 대장의 형태학적 변화(A) Morphological changes in the large intestine

약물투여 7일 후에 대장을 적출하여 육안으로 살펴본 결과, TNBS를 단독으로 처리한 랫트의 대장은 대조군에 비하여 부종과 유착이 많이 관찰되었으며 충수돌기의 부종 또한 증가하였다. 또 일부에서는 염증이 심화되어 다른 장기 조직들과 유착현상이 나타났다(도 4). 약물을 처리한 군에서는 TNBS군에서 나타난 부종과 충혈이 억제되었으며 특히 INV-101의 경우 농도의존적 효과를 보였으며, INV-101 10 mg/kg 투여군의 경우는 정상대조군과 유사할 정도로 회복을 보였으며, 대조약물 필고티닙 30 mg/kg 또는 UC-205689 10 mg/kg보다 월등히 우수하였다.As a result of removing the large intestine 7 days after drug administration and examining it with the naked eye, the large intestine of rats treated with TNBS alone showed more edema and adhesions than the control group, and the swelling of the appendix also increased. In addition, in some cases, inflammation worsened and adhesion with other organ tissues appeared (Figure 4). In the drug-treated group, the edema and congestion that appeared in the TNBS group were suppressed, and in particular, INV-101 showed a concentration-dependent effect. The INV-101 10 mg/kg administered group showed recovery similar to the normal control group. , it was significantly superior to the control drug filgotinib 30 mg/kg or UC-205689 10 mg/kg.

(나) 대장 무게 변화(B) Change in weight of large intestine

랫트의 대장을 적출하여 맹장(cecum)부터 직장까지 전체 길이의 무게를 측정한 결과, 비이클-처리 대조군에 비해 TNBS 단독 처리군의 경우 부종이 있으며 장의 무게가 유의적으로 증가하였고, 시험 물질을 투여한 그룹에서는 장의 무게가 TNBS 처리군에 비해 유의적으로 감소하였다. INV-101 10 mg/kg 투여 그룹의 효과가 가장 우수하였다(도 5). INV-101 10mg/kg 투여군의 경우는 회복률이 73.1%였으며, 대조약물 필고티닙 30 mg/kg의 회복률 55.1% 또는 UC-205689 10 mg/kg의 회복률 49.6%보다 월등히 우수하였다(표 4).The large intestine of the rat was removed and the weight of the entire length from the cecum to the rectum was measured. As a result, there was edema and the weight of the intestine was significantly increased in the TNBS-only treated group compared to the vehicle-treated control group, and the test substance was administered. In one group, intestinal weight was significantly reduced compared to the TNBS treated group. The INV-101 10 mg/kg administration group had the best effect (Figure 5). In the case of the INV-101 10 mg/kg administration group, the recovery rate was 73.1%, which was significantly better than the recovery rate of 55.1% for the control drug filgotinib 30 mg/kg or 49.6% for UC-205689 10 mg/kg (Table 4).

대장의 단위길이당 무게 회복률 (%)Weight recovery rate per unit length of the large intestine (%) Filgotinib (30mg/kg)Filgotinib (30mg/kg) INV-101 (1mg/kg)INV-101 (1mg/kg) INV-101 (3mg/kg)INV-101 (3mg/kg) INV-101 (10mg/kg)INV-101 (10mg/kg) UC-205686 (10mg/kg)UC-205686 (10mg/kg) 55.13 ± 22.655.13 ± 22.6 58.20 ± 30.158.20 ± 30.1 25.03 ± 30.425.03 ± 30.4 73.11 ± 25.973.11 ± 25.9 49.6 ± 45.249.6 ± 45.2

(3) 적출 대장의 조직 병리(3) Histopathology of extracted colon

(가) H&E 염색 및 조직 손상 정도(A) H&E staining and degree of tissue damage

장 조직의 손상 정도 및 약물 투여에 의한 회복 정도를 확인하기 위해 H&E 염색을 실시하고, 정상대조군에 비해 TNBS 단독 투여 군에서 현저한 장점막 손상을 확인하였으며, 시험 화합물 투여에 의해 장점막 회복을 확인할 수 있었으며, INV-101 10mg/kg의 개선 효과가 가장 우수하였다(도 6).H&E staining was performed to confirm the degree of damage to intestinal tissue and the degree of recovery by drug administration. Significant damage to the intestinal mucosa was confirmed in the group administered only TNBS compared to the normal control group. Recovery of the intestinal mucosa was confirmed by administration of the test compound. The improvement effect of INV-101 10mg/kg was the best (Figure 6).

표 2의 평가법에 의해 H&E로 염색된 조직의 손상의 정도를 측정하여 점수로 변환하여 분석한 결과, TNBS 단독 처리군에서는 점막조직의 궤양, 고유층 (Lamina propria)과 점막하층으로 염증세포의 침윤, 혈관분포, 섬유화(collagen fiber 축적) 및 부종성 변화를 나타내었으며, 화합물 투여에 의해 장조직 손상 정도가 완화되었다. INV-101은 용량의존적 효과를 보였으며, INV-101 10mg/kg 투여군의 효과가 대조약물 필고티닙 30 mg/kg 또는 UC-205689 10 mg/kg보다 월등히 우수하였다(도 7).As a result of measuring the degree of damage to the tissue stained with H&E using the evaluation method in Table 2 and converting it into a score, the TNBS-only treatment group showed ulcers in the mucosal tissue and infiltration of inflammatory cells into the lamina propria and submucosal layer. , vascular distribution, fibrosis (collagen fiber accumulation), and edematous changes were observed, and the degree of intestinal tissue damage was alleviated by compound administration. INV-101 showed a dose-dependent effect, and the effect of the INV-101 10 mg/kg group was significantly better than the control drug filgotinib 30 mg/kg or UC-205689 10 mg/kg (Figure 7).

(나) 대장 조직 사이토카인 발현 변화(B) Changes in colonic tissue cytokine expression

TNBS로 유도된 장염 마우스의 대장 조직에서 염증 사이토카인 발현(TNFα, IL-6, IL-1βIL-12, IL-17, NOS2)이 현저히 증가되어 있으며, 화합물 투여에 의해 거의 감소함을 확인하였다. INV-101은 용량의존적 효과를 보였으며, INV-101 10mg/kg 투여군의 효과가 대조약물 필고티닙 30 mg/kg 또는 UC-205689 10 mg/kg보다 월등히 우수하였다(도 8).It was confirmed that the expression of inflammatory cytokines (TNFα, IL-6, IL-1βIL-12, IL-17, NOS2) was significantly increased in the colonic tissue of mice with enteritis induced by TNBS, and was almost decreased by compound administration. INV-101 showed a dose-dependent effect, and the effect of the INV-101 10 mg/kg group was significantly better than the control drug filgotinib 30 mg/kg or UC-205689 10 mg/kg (Figure 8).

(다) 대장 조직 염증신호 활성화 정도(C) Degree of activation of inflammatory signals in colon tissue

대장 조직에서 TNBS에 의해 유도된 RIP-1/RIP-3의 인산화와 JAK2/JAK3의 인산화 및 STAT1/STAT3의 인산화가 화합물 투여에 의해 유의하게 억제됨을 확인하였다. INV-101은 용량의존적 효과를 보였으며, INV-101 10 mg/kg 투여군의 효과가 대조약물 필고티닙 30 mg/kg 또는 UC-205689 10 mg/kg보다 월등히 우수하였다(도 9a). 또한, TNBS에 의해 유도된 STAT3와 NF-κ가 핵으로 이동하는 것을 INV-101 10 mg/kg 투여에 의해 정상대조군 수준으로 억제됨을 확인하였다(도 9b).It was confirmed that phosphorylation of RIP-1/RIP-3, phosphorylation of JAK2/JAK3, and STAT1/STAT3 induced by TNBS in colon tissue was significantly inhibited by compound administration. INV-101 showed a dose-dependent effect, and the effect of the INV-101 10 mg/kg group was significantly better than the control drug filgotinib 30 mg/kg or UC-205689 10 mg/kg (Figure 9a). In addition, it was confirmed that the movement of STAT3 and NF-κ induced by TNBS to the nucleus was inhibited to the level of the normal control by administration of 10 mg/kg of INV-101 (FIG. 9b).

(4) 대장 조직 중 MPO 변화(4) MPO changes in colon tissue

MPO는 호중구에서 주로 발견되는 효소로서 조직에서 MPO의 존재는 호중구 침윤의 지표가 되며, 이는 곧 염증반응의 지표로서 염증성 대장염에 의한 장 손상 수치와 상관성을 나타낸다. MPO is an enzyme mainly found in neutrophils, and the presence of MPO in tissues is an indicator of neutrophil infiltration, which in turn is an indicator of inflammatory response and correlates with the level of intestinal damage caused by inflammatory colitis.

정상군에 비해 TNBS처리군에서 MPO 수치가 현저히 높게 나타났으며 화합물 투여에 의해 MPO 활성이 유의적으로 감소하였다. INV-101 10 mg/kg 투여군의 효과가 대조약물 필고티닙 30 mg/kg 또는 UC-205689 10 mg/kg보다 월등히 우수하였다(도 10).Compared to the normal group, MPO levels were significantly higher in the TNBS-treated group, and MPO activity was significantly decreased by compound administration. The effect of the INV-101 10 mg/kg administration group was significantly better than the control drug filgotinib 30 mg/kg or UC-205689 10 mg/kg (Figure 10).

(5) 혈액 분석(5) Blood analysis

(가) 혈청 사이토카인 측정(A) Serum cytokine measurement

혈중 사이토카인의 변화 정도를 각각의 엘라이자 키트(ELISA kit)를 이용하여 측정한 결과, TNBS 단독 투여군에서 증가된 사이토카인인 TNFα, IL-6, IL-1βCRP이 화합물 처리에 의해 유의하게 억제됨을 확인하였다. 염증 사이토카인 억제 정도는 INV-101 10 mg/kg > INV-101 3 mg/kg = UC-205689 10 mg/kg > INV-101 1 mg/kg > 필고티닙 30 mg/kg 의 순으로 효과가 우수하였다(도 11a 내지 11d).As a result of measuring the degree of change in circulating cytokines using each ELISA kit, it was found that TNFα, IL-6, and IL-1βCRP, which were increased cytokines in the group administered TNBS alone, were significantly suppressed by compound treatment. Confirmed. The degree of inflammatory cytokine inhibition is in the following order: INV-101 10 mg/kg > INV-101 3 mg/kg = UC-205689 10 mg/kg > INV-101 1 mg/kg > Filgotinib 30 mg/kg. It was excellent (Figures 11a to 11d).

(나) 혈액 중 PYGL 분비 양 및 활성 변화(B) Changes in the amount and activity of PYGL secretion in the blood

TNBS로 유도된 장염 랫트의 혈액 중 PYGL의 양이 정상 대비 4배 정도 증가하였으며, 화합물 투여 그룹에서 유의하게 억제되었으며, INV-101 10 mg/kg 의 억제효과가 가장 강력하였으며, 나머지 그룹은 유사한 수준을 보였다(도 12a). 반면, TNBS 투여군에서 현저히 증가된 혈중 PYGL 효소 활성은 INV-101에 의해 용량의존적으로 억제되었으며, INV-101 10 mg/kg > INV-101 3 mg/kg > INV-101 1 mg/kg = UC-205689 10 mg/kg > 필고티닙 30 mg/kg의 순으로 효과가 우수하였다(도 12b). 특히 가장 강력한 억제활성을 보인 INV-101 10 mg/kg의 경우는 정상 대조군 수준으로 회복시킴을 확인하였다.The amount of PYGL in the blood of TNBS-induced enteritis rats increased by about 4 times compared to normal, and was significantly suppressed in the compound administration group. The inhibitory effect of INV-101 10 mg/kg was the strongest, and the remaining groups had similar levels. was shown (Figure 12a). On the other hand, the significantly increased blood PYGL enzyme activity in the TNBS administration group was suppressed by INV-101 in a dose-dependent manner, INV-101 10 mg/kg > INV-101 3 mg/kg > INV-101 1 mg/kg = UC- The effect was excellent in the order of 205689 10 mg/kg > filgotinib 30 mg/kg (FIG. 12b). In particular, INV-101 10 mg/kg, which showed the strongest inhibitory activity, was confirmed to restore the level to the normal control level.

(6) 대장 조직의 PYGL 활성 (6) PYGL activity in colon tissue

TNBS로 유도된 장염 랫트의 대장 조직의 PYGL의 양이 정상 대비 3배 정도 증가하였으며, 화합물 투여 그룹에서 유의하게 억제되었다. INV-101 10 mg/kg의 억제효과가 가장 강력하였으며, 나머지 그룹은 유사한 수준을 보였다. 반면, TNBS 투여군에서 현저히 증가된 대장 PYGL 효소 활성은 INV-101에 의해 용량의존적으로 억제되었으며, INV-101 10 mg/kg > INV-101 3 mg/kg > INV-101 1 mg/kg = UC-205689 10 mg/kg = 필고티닙 30 mg/kg의 순으로 효과가 우수하였다(도 13). 특히 가장 강력한 억제활성을 보인 INV-101 10 mg/kg의 경우는 정상 대조군 수준으로 회복시킴을 확인하였다.The amount of PYGL in the colonic tissue of TNBS-induced enteritis rats increased to about 3-fold compared to normal, and was significantly suppressed in the compound administration group. The inhibitory effect of INV-101 10 mg/kg was the strongest, and the remaining groups showed similar levels. On the other hand, the significantly increased colonic PYGL enzyme activity in the TNBS administration group was inhibited by INV-101 in a dose-dependent manner, INV-101 10 mg/kg > INV-101 3 mg/kg > INV-101 1 mg/kg = UC- The effect was excellent in the following order: 205689 10 mg/kg = filgotinib 30 mg/kg (Figure 13). In particular, INV-101 10 mg/kg, which showed the strongest inhibitory activity, was confirmed to restore the level to the normal control level.

상기 실험예 2의 결과에 의하면, 본 발명의 PYGL 저해제인 화합물 INV-101은 TNBS로 유도한 대장 염증 및 손상을 용량의존적으로 회복시켰으며, 혈중 및 대장 조직의 염증 사이토카인의 생성을 억제하고, 혈중PYGL의 증가를 억제할 뿐 아니라 증가된 PYGL 활성도 유의하게 억제하였다. 이러한 결과로부터 PYGL 저해제인 화합물 INV-101이 PYGL 활성 억제와 장염을 억제하는 효능이 매우 우수한 치료제 후보물질임이 확인되었다.According to the results of Experimental Example 2, compound INV-101, a PYGL inhibitor of the present invention, restored colonic inflammation and damage induced by TNBS in a dose-dependent manner, suppressed the production of inflammatory cytokines in the blood and colonic tissue, Not only did it suppress the increase in blood PYGL, but it also significantly suppressed the increased PYGL activity. From these results, it was confirmed that compound INV-101, a PYGL inhibitor, is a therapeutic candidate with excellent efficacy in inhibiting PYGL activity and enteritis.

또한, 상기 실험예 2의 결과에 따라, PYGL 저해능을 가지는 물질이 자가면역 질환의 예방 또는 치료에 유용하게 사용될 수 있음을 알 수 있으며, 기존약물을 대체하여 사용 가능함을 알 수 있다.In addition, according to the results of Experimental Example 2, it can be seen that substances having PYGL inhibition ability can be usefully used in the prevention or treatment of autoimmune diseases, and can be used as a replacement for existing drugs.

전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. 예를 들어, 단일형으로 설명되어 있는 각 구성 요소는 분산되어 실시될 수도 있으며, 마찬가지로 분산된 것으로 설명되어 있는 구성 요소들도 결합된 형태로 실시될 수 있다.The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive. For example, each component described as single may be implemented in a distributed manner, and similarly, components described as distributed may also be implemented in a combined form.

본 발명의 범위는 후술하는 청구범위에 의하여 나타내어지며, 청구범위의 의미 및 범위 그리고 그 균등 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.The scope of the present invention is indicated by the claims described below, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be construed as being included in the scope of the present invention.

Claims (7)

글리코겐 가인산분해효소(PYGL) 저해제를 유효성분으로 포함하는, 염증성 장 질환의 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating inflammatory bowel disease, comprising a glycogen phosphorylase (PYGL) inhibitor as an active ingredient. 제1항에 있어서, 상기 글리코겐 가인산분해효소(PYGL) 저해제는 하기 화학식 1 내지 8로 표시되는 화합물로 이루어지는 군으로부터 선택되는 1종 이상인, 염증성 장 질환의 예방 또는 치료용 약학 조성물:The pharmaceutical composition for preventing or treating inflammatory bowel disease according to claim 1, wherein the glycogen phosphatase (PYGL) inhibitor is at least one selected from the group consisting of compounds represented by the following formulas 1 to 8: <화학식 1><Formula 1>
Figure PCTKR2023016299-appb-img-000010
Figure PCTKR2023016299-appb-img-000010
 <화학식 2><Formula 2>
Figure PCTKR2023016299-appb-img-000011
Figure PCTKR2023016299-appb-img-000011
 <화학식 3><Formula 3>
Figure PCTKR2023016299-appb-img-000012
Figure PCTKR2023016299-appb-img-000012
 <화학식 4><Formula 4>
Figure PCTKR2023016299-appb-img-000013
Figure PCTKR2023016299-appb-img-000013
 <화학식 5><Formula 5>
Figure PCTKR2023016299-appb-img-000014
Figure PCTKR2023016299-appb-img-000014
 <화학식 6><Formula 6>
Figure PCTKR2023016299-appb-img-000015
Figure PCTKR2023016299-appb-img-000015
 <화학식 7><Formula 7>
Figure PCTKR2023016299-appb-img-000016
Figure PCTKR2023016299-appb-img-000016
 <화학식 8><Formula 8>
Figure PCTKR2023016299-appb-img-000017
.
Figure PCTKR2023016299-appb-img-000017
. 제1항에 있어서, 상기 염증성 장 질환은 장염, 대장염, 궤양성 대장염, 크론병, 크론친화성 세포종, 과민성 대장 증후군, 출혈성 직장 궤양, 회장낭 염(pouchitis), 소화성 궤양, 장관형 베체트병, 및 위염으로 이루어진 군으로부터 선택되는 1종 이상인, 염증성 장 질환의 예방 또는 치료용 약학 조성물.The method of claim 1, wherein the inflammatory bowel disease is enteritis, colitis, ulcerative colitis, Crohn's disease, Crohn's sarcoma, irritable bowel syndrome, bleeding rectal ulcer, ileal pouchitis, peptic ulcer, intestinal Behcet's disease, and A pharmaceutical composition for preventing or treating inflammatory bowel disease, which is at least one selected from the group consisting of gastritis. 제3항에 있어서, 상기 염증성 장 질환은 궤양성 대장염인, 염증성 장 질환의 예방 또는 치료용 약학 조성물.The pharmaceutical composition for preventing or treating inflammatory bowel disease according to claim 3, wherein the inflammatory bowel disease is ulcerative colitis. 1) 글리코겐 가인산분해효소(PYGL)에 시험 물질을 처리하는 단계;1) treating the test substance with glycogen phosphatase (PYGL); 2) 글리코겐 가인산분해효소(PYGL)의 활성을 측정하는 단계; 및2) measuring the activity of glycogen phosphorylase (PYGL); and 3) 상기 글리코겐 가인산분해효소(PYGL)의 활성을 감소시키는 시험 물질을 염증성 장 질환 치료제의 후보 물질로 결정하는 단계3) Determining the test substance that reduces the activity of glycogen phosphatase (PYGL) as a candidate substance for the treatment of inflammatory bowel disease. 를 포함하는, 염증성 장 질환의 예방 또는 치료용 물질의 스크리닝 방법.A screening method for a substance for preventing or treating inflammatory bowel disease, including. 제5항에 있어서, 상기 글리코겐 가인산분해효소(PYGL)의 활성의 측정은 포도당-1-인산(G-1-P) 검출을 이용하여 측정하는 것인, 염증성 장 질환의 예방 또는 치료용 물질의 스크리닝 방법.The substance for preventing or treating inflammatory bowel disease according to claim 5, wherein the activity of glycogen phosphatase (PYGL) is measured using glucose-1-phosphate (G-1-P) detection. Screening method. 제5항에 있어서, 상기 염증성 장 질환은 장염, 대장염, 궤양성 대장염, 크론병, 크론친화성 세포종, 과민성 대장 증후군, 출혈성 직장 궤양, 회장낭 염(pouchitis), 소화성 궤양, 장관형 베체트병, 및 위염으로 이루어진 군으로부터 선택되는 1종 이상인, 염증성 장 질환의 예방 또는 치료용 물질의 스크리닝 방법.The method of claim 5, wherein the inflammatory bowel disease is enteritis, colitis, ulcerative colitis, Crohn's disease, Crohn's apoptotic tumor, irritable bowel syndrome, bleeding rectal ulcer, ileal pouchitis, peptic ulcer, intestinal Behcet's disease, and A screening method for a substance for preventing or treating inflammatory bowel disease, which is at least one selected from the group consisting of gastritis.
PCT/KR2023/016299 2022-10-20 2023-10-19 Pharmaceutical composition for prevention or treatment of inflammatory bowel disease comprising glycogen phosphorylase (pygl) inhibitor as active ingredient Ceased WO2024085698A1 (en)

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WO2019087129A1 (en) * 2017-11-03 2019-05-09 Universite De Montreal Compounds and use thereof in the expansion of stem cells and/or progenitor cells
KR20210035454A (en) * 2019-09-24 2021-04-01 주식회사 이노보테라퓨틱스 Heteroarylamidopyridinol derivatives and pharmaceutical composition for use in preventing or treating autoimmune diseases containing the same as an active ingredient

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