WO2023204640A1 - Pharmaceutical composition for prevention or treatment of gastric mucosal damage-related diseases caused by helicobacter pylori and platform for screening for active ingredient for prevention or treatment of helicobacter pylori infection - Google Patents

Pharmaceutical composition for prevention or treatment of gastric mucosal damage-related diseases caused by helicobacter pylori and platform for screening for active ingredient for prevention or treatment of helicobacter pylori infection Download PDF

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WO2023204640A1
WO2023204640A1 PCT/KR2023/005398 KR2023005398W WO2023204640A1 WO 2023204640 A1 WO2023204640 A1 WO 2023204640A1 KR 2023005398 W KR2023005398 W KR 2023005398W WO 2023204640 A1 WO2023204640 A1 WO 2023204640A1
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helicobacter pylori
gastric
hagos
mucus
cells
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PCT/KR2023/005398
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French (fr)
Korean (ko)
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손미영
손예슬
박두상
이무승
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한국생명공학연구원
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    • 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
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K31/4025—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 not condensed and containing further heterocyclic rings, e.g. cromakalim
    • 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
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene
    • 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
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • G01N33/5005—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
    • 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
    • G01N33/5076—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 involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • G01N33/5079—Mitochondria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to a novel pharmaceutical composition for the prevention or treatment of diseases related to gastric mucosal damage, particularly diseases related to gastric mucosal damage caused by Helicobacter pylori.
  • it relates to a platform for screening active ingredients for the prevention or treatment of Helicobacter pylori infection or diseases related to gastric mucosal damage caused by the infection and a screening method using the same.
  • Helicobacter pylori infection is widely distributed around the world and is one of the most common chronic infections in humans.
  • Helicobacter pylori which infects 50% of the world's population, has been an important factor in the development of gastritis, gastric ulcer, duodenal ulcer, and gastric cancer since it was first isolated and identified from human gastric mucosa by Warren and Marshall in 1983. It is known.
  • the World Health Organization defines Helicobacter pylori as a group 1 carcinogen for stomach cancer, and if it is not artificially eradicated, Helicobacter pylori lasts a lifetime in most cases and causes various gastrointestinal diseases.
  • Helicobacter pylori infection appears in various ways depending on age, race, and education level, and is also related to socioeconomic conditions during growth. In particular, in developing countries, Helicobacter pylori infection mainly occurs in childhood, but in developed countries, while the number of infections in childhood is low, infection occurs steadily even in adulthood, with infections occurring at a rate of 0.3% to 0.5% per year.
  • Helicobacter pylori infection is a chronic infection that lasts a lifetime in most cases unless artificial eradication is attempted, but the infection route and prevention have not yet been clearly identified.
  • the age-specific characteristics of Helicobacter pylori infection in Korea are low, at an average of 17.2% in children under 15 years of age, and at an average of 66.9% in adults over 16 years of age, indicating a transition from the form of infection in developing countries to the form of infection in developed countries.
  • the infection route of Helicobacter pylori has not been completely elucidated, but research results to date have shown that first, it is transmitted from person to person, and second, infection occurs mainly within the family during childhood.
  • Factors that may affect infection with Helicobacter pylori are: Gender, body mass index, blood type, education level, economic level, presence of diabetes, presence of fatty liver, peptic ulcer, smoking and drinking history were analyzed.
  • Helicobacter pylori produces urease to decompose urea into carbon dioxide and ammonia, and the ammonia generated at this time neutralizes gastric acid and is known to be able to survive in the stomach where strong acids act, causing infection in the stomach.
  • VacA is a protein with a molecular weight of 87 kDa and is a cytotoxin that causes vacuolation of host cells. It has been reported that it suppresses the host's T cell response to Helicobacter pylori, thereby allowing infection to persist.
  • the VacA destroys the barrier effect and regulates the inflammatory response through morphological modification of gastric epithelial cells. In addition, it destroys the endosome compartment within the cell and the outer membrane of the mitochondria, causing vacuolation of the cell and activating caspase-8 and caspase-9, thereby inducing cell death. It is known to cause peptic ulcers.
  • One object of the present invention is to provide a novel pharmaceutical composition for preventing or treating diseases related to gastric mucosal damage.
  • Another object of the present invention is to provide a new food or health functional food composition for preventing or improving gastric mucosal damage.
  • Another object of the present invention is to provide a platform for screening a therapeutic agent for diseases related to gastric mucosal damage caused by Helicobacter pylori and a method for screening a therapeutic agent using the platform.
  • Another object of the present invention is to provide a platform for screening components that inhibit gastric adhesion of Helicobacter pylori and a method for screening gastric adhesion inhibitors using the same.
  • one aspect of the present invention relates to gastric mucosal damage comprising an Aurora kinase A (AURKA) inhibitor or a ubiquitin specific peptidase 14 (USP14) inhibitor as an active ingredient.
  • a composition for preventing or treating diseases is provided.
  • another aspect of the present invention provides a food or health functional food composition for preventing or improving gastric mucosal damage containing an Aurora Kinase A inhibitor or a Ubiquitin-specific Peptidase 14 inhibitor as an active ingredient. .
  • another aspect of the present invention includes treating VacA-treated human antrum gastric organoids (hAGOs) with a candidate material; And depending on whether the candidate material is treated, measuring changes in any one of the number of mucus-producing cells and the expression of mucus-producing genes and the change in mitochondrial activity level in the hAGOs.
  • hAGOs human antrum gastric organoids
  • another aspect of the present invention includes treating human antrum gastric organoids (hAGOs) treated with a Helicobacter pylori strain with a candidate material; and measuring changes in the level of Helicobacter pylori strains attached to the hAGOs, depending on whether the candidate material is treated.
  • hAGOs human antrum gastric organoids
  • Aurora Kinase A inhibitors or Ubiquitin-specific Peptidase 14 inhibitors are effective in preventing or recovering mitochondrial damage in mucus-producing cells, thereby preventing gastric ulcers caused by Helicobacter pylori. It has the effect of providing a new treatment for diseases related to mucosal damage.
  • Figure 1 is a schematic diagram schematically showing the process of step-by-step differentiation of hPSC (human pluripotent stem cells) formed from hESC (human embryonic stem cell) line H9 and finally establishment of hAGOs (human antrum gastric organoids) and microphotographs at each step. am.
  • hPSC human pluripotent stem cells
  • hESC human embryonic stem cell
  • hAGOs human antrum gastric organoids
  • Figure 2a is the result of confirming the expression of specific marker proteins of cells present in the definitive endoderm and posterior-foregut that appear during the hAGOs differentiation process
  • Figure 2b shows the expression of cell-specific marker genes shown during the differentiation process. This is the confirmed result
  • Figure 2c shows the result of confirming the expression of marker proteins specific to each cell type to confirm the various cell types present in hAGO.
  • Figure 3 shows the results of exploring the VacA treatment concentration range based on the inhibition of proliferation and increase in apoptosis in hAGOs caused by VacA treatment.
  • Figure 4 shows the results of confirming physiological and molecular biological changes such as surface area changes, epithelial thickness changes, and tight junction changes after VacA treatment based on the structural characteristics of 3D hGO.
  • Figure 5 shows the results of confirming and verifying global genome changes in hAGO after VacA treatment.
  • Figure 6 shows the results of confirming the effect of VacA treatment on mitochondrial activity in hAGOs through changes in mitochondrial morphology, gene expression, and respiration.
  • Figures 7a to 7e show the results of analyzing cells affected by VacA treatment in hAGOs through single cell genome changes, confirming the confirmation of organoid constituent cells and the expression of genes associated with mucus-producing cells.
  • Figures 8A to 8E show the results of confirming the types of cells affected by VacA treatment in hAGOs, specific physiological effects, and changes in mitochondrial activity for each cell.
  • Figure 9 shows the results of confirming changes in cell type and mitochondrial activity affected by VacA in hAGOs derived from hiPSC (human induced pluripotent stem cell).
  • Figures 10A to 10E show the results of verifying changes in cell type and mitochondrial activity due to VacA found in hAGOs in human stomach tissue infected with Helicobacter pylori.
  • Figures 11A to 11C show the results of confirming the restoration of mitochondrial activity and barrier function to confirm the effectiveness of VacA-treated hAGOs as a screening platform for treating gastric mucosal damage caused by Helicobacter pylori infection using mitochondrial regulators.
  • Figure 12 shows the results of screening therapeutic agents using the platform.
  • Figures 13 and 14a to 14d show the phenotypic analysis and cellular respiration of MLN8054, an Aurora Kinase A inhibitor selected as the most effective, and IU1, a ubiquitin-specific peptidase 14 inhibitor, using hAGOs to further treat gastric mucosal damage. This shows the results verified through analysis, ATP production ability, mucus-producing function, and tight junction expression analysis.
  • Figures 15A to 15E show the results confirming that MLN8054 and IU1 regulate mitochondrial activity through mitochondrial fusion or mitophagy.
  • Figures 16a to 16f show the gastric barrier damage recovery effect caused by VacA of MLN8054 confirmed through hAGOs by measuring mitochondrial activity-related gene expression, E-cadherin expression, tight junction expression, mucus-producing function, and mitochondrial elements. This shows the results confirmed once again in vivo .
  • Figures 17a to 17i confirm the growth of Helicobacter pylori and the ability to express VacA, and whether MLN8054 has a therapeutic effect not only when induced by VacA but also against damage to the gastric barrier caused by actual Helicobacter pylori infection. This shows the results confirmed through changes in mitochondrial activity, mucus-producing function, and tight junction expression by treating hAGOs infected with Helicobacter pylori with MLN8054.
  • Figure 18 shows an in vivo study using mice infected with Helicobacter pylori to determine whether MLN8054 has a therapeutic effect not only in the case induced by VacA, but also in the damage to the gastric barrier caused by actual Helicobacter pylori infection. ) shows the results of confirming mitochondrial elements, E-cadherin expression, and mucus-producing functions.
  • Figures 19a to 19d show that in addition to MLN8054 and IU1 among Aurora Kinase A inhibitors and Ubiquitin-specific Peptidase 14 inhibitors, hAGOs are also used for MLN8237, MK8745, MK5108, which are Aurora Kinase A inhibitors, and WP1130, which is another Ubiquitin-specific Peptidase 14 inhibitor. This shows the results confirming the therapeutic effect of gastric mucosal damage induced by VacA.
  • Figure 20 is a schematic diagram explaining the configuration and operating principle of a platform for screening inhibitors of gastric adhesion of Helicobacter pylori.
  • Figure 21 shows the results of screening a new strain with the activity of inhibiting the attachment of Helicobacter pylori to the stomach using the above platform.
  • composition for preventing or treating diseases related to gastric mucosal damage One.
  • Pharmaceutical composition for preventing or treating diseases related to gastric mucosal damage One.
  • One aspect of the present invention provides a pharmaceutical composition for preventing or treating diseases related to gastric mucosal damage.
  • the pharmaceutical composition of the present invention contains an Aurora kinase A (AURKA) inhibitor or a ubiquitin specific peptidase 14 (USP14) inhibitor as an active ingredient.
  • AURKA Aurora kinase A
  • USP14 ubiquitin specific peptidase 14
  • the Aurora Kinase A inhibitor is MLN8054, MLN8237, MK8745, MK5108, CCT-137690, lestaurtinib, NU6140, PF-03814735, SNS-314, TC-A 2317 hydrochloride, TC-S 7010, etc. or It may be a derivative thereof, such as MLN8054, MLN8237, MK8745, MK5108, etc., or a derivative thereof, for example, MLN8054 or a derivative thereof.
  • the ubiquitin-specific peptidase 14 inhibitor may be IU1, WP1130, etc., or a derivative thereof, for example, IU1 or a derivative thereof.
  • the disease related to damage to the gastric mucosa may be caused by damage to the mitochondria of gastric epithelial cells, and in particular, damage to the mitochondria of the gastric epithelial cells may be caused by Helicobacter pylori .
  • the gastric epithelial cells may be mucous-producing cells such as MUC5AC + cells, MUC6 + cells, SOX9 + cells, etc., and the mucus-producing cells may have damaged mitochondria.
  • the damage may be due to fragmentation of the mitochondria, shortening of the length, or a decrease in the respiratory capacity or efficiency of the mitochondria, and the mucus secretion of the mucus-producing cells is reduced due to the damage to the mitochondria. It may have happened.
  • the disease related to damage to the gastric mucosa may be gastritis, gastric ulcer, stomach cancer, etc.
  • the above prevention refers to all actions that prevent, suppress, or delay the onset of diseases related to gastric mucosal damage
  • the above treatment refers to all actions that improve or beneficially change diseases related to gastric mucosal damage.
  • human antrum gastric organoids hAGOs
  • hESC human embryonic stem cells
  • hiPSC human induced pluripotent stem cells
  • the pharmaceutical composition may further include, in addition to the Aurora Kinase A inhibitor or the Ubiquitin-specific Peptidase 14 inhibitor, one or more active ingredients exhibiting the same or similar functions, or may be used in combination therewith.
  • the pharmaceutical composition may further include one or more acceptable carriers.
  • 'acceptable' means that it does not inhibit the activity of the active ingredient, the Aurora Kinase A inhibitor or the Ubiquitin-specific peptidase 14 inhibitor, and does not have toxicity beyond what the subject of application can adapt to.
  • the 'carrier' is defined as a compound that facilitates the addition of a compound into cells or tissues.
  • the composition may be prepared in unit dosage form by formulating using a carrier and/or excipient, or may be manufactured by placing it in a multi-dose container, and may additionally include a dispersant or stabilizer.
  • the active ingredient contained in the composition may be transported in a carrier such as colloidal suspension, powder, saline solution, lipid, liposome, microsphere, or nano spherical particle. They may form complexes or associate with the delivery vehicle and may be used in combination with the present invention, such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation agents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancers or fatty acids. It can be delivered in vivo using delivery systems known in the art.
  • a carrier such as colloidal suspension, powder, saline solution, lipid, liposome, microsphere, or nano spherical particle. They may form complexes or associate with the delivery vehicle and may be used in combination with the present invention, such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation agents, polysaccharides, polyamino acids, dendrim
  • the carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, and polyvinyl pyrolidone. , cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc., but is not limited thereto.
  • lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. may be additionally included.
  • the carrier can be used by mixing saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these ingredients, and if necessary, other common agents such as antioxidants, buffers, and bacteriostatic agents. Additives can be added.
  • the pharmaceutical composition can be administered in various oral and parenteral formulations during actual clinical administration.
  • diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants are used. It is prepared in this way.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain herbal extracts or herbal ferments with at least one excipient, such as starch, calcium carbonate, sucrose or It is prepared by mixing lactose and gelatin. Additionally, in addition to simple excipients, lubricants such as magnesium styrate talc are also used.
  • the powder can be prepared by simply mixing the active ingredient of the present invention with a suitable pharmaceutically acceptable carrier such as lactose, starch, or microcrystalline cellulose.
  • Granules are prepared by mixing the active ingredient of the present invention, a suitable pharmaceutically acceptable carrier, and a suitable pharmaceutically acceptable binder such as polyvinylpyrrolidone and hydroxypropyl cellulose, and then mixing them with a solvent such as water, ethanol, or isopropanol. It can be manufactured using a wet granulation method or a dry granulation method using compression force. Additionally, tablets can be manufactured by mixing the granules with a suitable pharmaceutically acceptable lubricant such as magnesium stearate and then compressing the mixture into tablets using a tablet press.
  • a suitable pharmaceutically acceptable carrier such as lactose, starch, or microcrystalline cellulose.
  • 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 solutions, suspensions, emulsions, freeze-dried preparations, and suppositories.
  • Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.
  • As a base for suppositories wethepsol, macrogol, Tween 61, cacao, laurel, glycerol, gelatin, etc. can be used.
  • the active ingredient, the Aurora Kinase A inhibitor or the Ubiquitin-specific peptidase 14 inhibitor is administered as an oral agent, an injection (e.g., an intramuscular injection, an intraperitoneal injection) depending on the condition of the individual and the disease to be prevented, improved, or treated. It can be administered by injection, intravenous injection, infusion, subcutaneous injection, implant), inhalation, intranasal administration, vaginal administration, rectal administration, sublingual formulation, transdermal formulation, topical formulation, etc., but is not limited thereto. Depending on the route of administration, it may be formulated in an appropriate dosage unit form containing commonly used, non-toxic, pharmaceutically acceptable carriers, excipients, and vehicles.
  • the amount of recombinant microorganisms as an active ingredient during the above administration varies depending on the patient's weight, age, gender, health status, diet, administration time, administration method, excretion rate, target site, and severity of disease.
  • the active ingredient in the composition may be included at a concentration of 1 ⁇ M or more, such as 5 ⁇ M or more, 10 ⁇ M or more, 20 ⁇ M or more, or 50 ⁇ M or more.
  • the active ingredient has a lower limit selected from 0.05mg, 0.1mg, 0.15mg, 0.2mg, 0.3mg, 0.5mg, 1mg, 2mg, 3mg, 5mg, 10mg, 20mg, 30mg, 50mg and 100mg and/or 500mg. , 450mg, 400mg, 350mg, 320mg, 300mg, 280mg, 250mg, 200mg, 150mg and 100mg, for example, 0.05 to 500mg, 0.05 to 450mg, 0.05 to 400mg.
  • the composition when used pharmaceutically, the composition may be administered in a pharmaceutically effective amount.
  • 'pharmaceutically effective amount means an amount sufficient to treat the disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, activity of the drug, and the type and severity of the patient's disease. It can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the medical field.
  • the effective dose is generally 0.01 mg to 5000 mg per day per 1 kg of body weight of the administered subject, and may be administered in divided doses once or several times a day at certain time intervals, depending on the judgment of a doctor or pharmacist, but is not limited thereto.
  • the composition may be administered as an individual therapeutic agent, or in combination with other therapeutic agents, and may be administered simultaneously, separately, or sequentially with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.
  • the effective amount of the composition may vary depending on the patient's age, gender, condition, weight, absorption of the active ingredient in the body, inactivation rate, excretion rate, type of disease, and concomitant drug, route of administration, severity of obesity, It may increase or decrease depending on gender, weight, age, etc., and may vary depending on the severity of the condition being treated.
  • the total daily dose may be administered in divided doses several times throughout the day.
  • the daily dosage may be about 0.0001 mg/kg to about 10 g/kg, for example, about 0.001 mg/kg to about 1 g/kg once a day.
  • the administration period may be 1 day to 2 months, but may be administered without limitation until the disease prevention or treatment effect appears. Additionally, depending on the judgment of a doctor or pharmacist, it may be administered in divided doses several times a day at regular time intervals, for example, 2 to 3 times a day.
  • Another aspect of the present invention provides a food or health functional food composition for preventing or improving gastric mucosal damage.
  • the food or health functional food composition of the present invention contains an Aurora kinase A (AURKA) inhibitor or a ubiquitin specific peptidase 14 (USP14) inhibitor as an active ingredient, and the specific details thereof are as follows. As described above in ‘ 1. Pharmaceutical composition for preventing or treating diseases related to damage to the gastric mucosa ’.
  • AURKA Aurora kinase A
  • USP14 ubiquitin specific peptidase 14
  • the gastric mucosa damage may be caused by damage to the mitochondria of the gastric epithelial cells, and in particular, the damage to the mitochondria of the gastric epithelial cells may be caused by Helicobacter pylori .
  • the gastric epithelial cells may be mucous-producing cells such as MUC5AC + cells, MUC6 + cells, SOX9 + cells, etc., and the mucus-producing cells may have damaged mitochondria.
  • the damage may be due to fragmentation of the mitochondria, shortening of the length, or a decrease in the respiratory capacity or efficiency of the mitochondria, and the mucus secretion of the mucus-producing cells is reduced due to the damage to the mitochondria. It may have happened.
  • the above prevention refers to all actions that prevent, suppress, or delay damage to the gastric mucosa
  • the above improvement refers to all actions that improve or beneficially change the damage to the gastric mucosa.
  • the food includes both edible raw materials and processed substances, meaning that it contains various digestible nutrients.
  • the food composition when using the food composition as a food additive, the food composition can be added as is or used together with other foods or food ingredients, and can be used appropriately according to conventional methods.
  • Foods to which the food composition can be added may be probiotic preparations, such as meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, It includes beverages, tea drinks, alcoholic beverages, vitamin complexes, and fermented foods, and includes all health foods in the conventional sense.
  • the fermented food may be fermented milk, cheese or butter such as yogurt (hard type, soft type, drink type), lactic acid bacteria beverage, but is not limited thereto, and is not limited to any food manufactured by fermentation performed by fermentation microorganisms or lactic acid bacteria. Food and products may also be included.
  • the food composition itself may be a food.
  • the food includes ingredients commonly added during food production, such as proteins, carbohydrates, fats, nutrients, and seasonings.
  • natural carbohydrates or flavoring agents may be included as additional ingredients in addition to the active ingredient.
  • the natural carbohydrates include monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g., dextrins, cyclodextrins, etc.), or sugar alcohols (e.g., , xylitol, sorbitol, erythritol, etc.) is preferable.
  • the flavoring agent may be a natural flavoring agent (e.g., thaumatin, stevia extract, etc.) or a synthetic flavoring agent (e.g., saccharin, aspartame, etc.).
  • the health functional food composition refers to a food using raw materials or ingredients with functionality useful to the human body, and has the functionality to maintain and improve health through maintaining the normal function of the human body or activating physiological functions.
  • the health functional food of the present invention corresponds to a health functional food under the Health Functional Food Act.
  • the Aurora kinase A (AURKA) inhibitor or the ubiquitin specific peptidase 14 (USP14) inhibitor prevents or restores mitochondrial damage caused by mucus-producing cells present in the epithelium of the gastric vestibule. Therefore, the Aurora Kinase A inhibitor or Ubiquitin-specific peptidase 14 inhibitor is a functional ingredient with the function of preventing or improving gastric mucosal damage and can be included as an active ingredient in health functional foods.
  • the health functional food composition of the present invention may appropriately contain the above functional raw materials as active ingredients depending on the purpose of use (prevention or improvement). For example, it may be included at a concentration of 1 ⁇ M or more, such as 5 ⁇ M or more, 10 ⁇ M or more, 20 ⁇ M or more, or 50 ⁇ M or more.
  • the active ingredient has a lower limit selected from 0.05mg, 0.1mg, 0.15mg, 0.2mg, 0.3mg, 0.5mg, 1mg, 2mg, 3mg, 5mg, 10mg, 20mg, 30mg, 50mg and 100mg and/or 500mg.
  • 450mg, 400mg 350mg, 320mg, 300mg, 280mg, 250mg, 200mg, 150mg and 100mg, for example, 0.05 to 500mg, 0.05 to 450mg, 0.05 to 400mg. , 0.05 to 350 mg, 0.05 to 300 mg, 0.05 to 250 mg, 0.1 to 500 mg, 0.1 to 450 mg, 0.1 to 400 mg, 0.1 to 350 mg, 0.1 to 300 mg, 0.1 to 250 mg, 0.1 to 200 mg, 0.2 to 5 00mg, 0.2 to 400mg, 0.2 It may be included in an amount of 300 mg to 300 mg, 0.5 to 300 mg, 1 to 300 mg, 5 to 300 mg, or 10 to 300 mg. However, in case of long-term intake for health purposes, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.
  • the health functional food composition can be manufactured in dosage forms such as tablets, granules, soft capsules, hard capsules, syrups, beverages, pills, soft extracts, and water baths. More specifically, the health food composition of the present invention can be manufactured in dosage forms such as tablets, granules, soft capsules, hard capsules, syrups, beverages, pills, soft extracts, dried extracts, and extraction baths. In addition, the health food composition of the present invention contains ingredients commonly added in the production of health functional foods, such as excipients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, and alginic acid, in addition to the ingredients described above in each formulation.
  • ingredients commonly added in the production of health functional foods such as excipients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, and alginic acid, in addition to the ingredients described above in each formulation.
  • It may further include salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, flavorings, pigments, preservatives, etc.
  • the ratio of the added ingredients is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food composition.
  • Helicobacter pylori Helicobacter pylori Screening method for treatments for diseases related to gastric mucosal damage
  • One aspect of the present invention provides a screening method for a treatment for diseases related to gastric mucosal damage caused by Helicobacter pylori .
  • the therapeutic screening method of the present invention includes the steps of treating a candidate substance in human antrum gastric organoids (hAGOs) treated with VacA, and depending on whether the candidate substance is treated, in the hAGOs, mucus- It includes measuring a change in any one of the number of producing cells and the expression of mucus-production related genes and a change in the activity level of mitochondria.
  • hAGOs human antrum gastric organoids
  • the therapeutic screening method of the present invention first includes the step of treating VacA-treated hAGOs with a candidate substance.
  • the VacA (vacuolating cytotoxin A) is a channel-forming toxin expressed by Helicobacter pylori.
  • the VacA is treated with hAGOs, the number of mucus-producing cells present in the hAGOs is reduced or the mitochondria of the mucus-producing cells are damaged. Mitochondrial damage as described above may be due to mitochondrial fragmentation and shortening, a decrease in mitochondrial respiratory capacity and respiratory efficiency, or a decrease in the expression of genes related to mucus production.
  • the candidate material is treated with hAGOs where the number of mucus-producing cells is reduced or the mitochondria of the mucus-producing cells are damaged, and the candidate material may have the effect of preventing or treating diseases related to gastric mucosal damage caused by Helicobacter pylori.
  • It may be a single compound of any type expected to exist, such as nucleic acids, amino acids, monosaccharides, etc., or a polymer such as polynucleotides, peptides, proteins, polysaccharides, etc., or a molecule such as natural or synthetic organic matter, inorganic matter, or a complex thereof. , or a mixture of all of them, or microorganisms that produce them.
  • the candidate substances those that increase the number of mucus-producing cells or the expression of genes related to mucus-producing cells, which are reduced by VacA in hAGOs, or restore mitochondrial damage in mucus-producing cells, are called 'therapeutics'. do.
  • recovery from mitochondrial damage may be achieved by fusion of mitochondria morphologically, increasing their length or increasing their activity such as ATP production.
  • the therapeutic agent may restore the number and function of mucus-producing cells present in hAGOs to the state before VacA treatment.
  • the therapeutic screening method of the present invention includes measuring either the number of mucus-producing cells or the expression of mucus-producing genes and mitochondrial activity in the hAGOs, depending on whether or not the candidate substance is processed. Includes. This is based on the fact that it is possible to know whether diseases related to gastric mucosal damage caused by Helicobacter pylori can be treated by measuring changes in the activity of mucus-producing cells in hAGOs as described above. It has technical significance in that it suggests changes in the expression of genes related to production and changes in the activity level of mitochondria as new parameters that can be used to determine whether treatment is possible for diseases related to gastric mucosal damage caused by Helicobacter pylori.
  • the mucus-producing cells may be MUC5AC + cells, MUC6 + cells, SOX9 + cells, etc., and may be surface mucus cells, vestibular neck cells, epithelial precursor cells, etc. Additionally, the mucus-production-related gene may be MUC5AC, MUC6, SOX9, etc., and the expression of the gene may be at the mRNA level or at the protein level.
  • the activity level of the mitochondria can be confirmed through changes in the shape of the mitochondria or the level of ATP production. Specifically, if the mitochondria are fused and become longer in length or the amount of ATP production increases, the activity increases. Conversely, the activity of the mitochondria increases. If the length becomes shorter due to fragmentation or the amount of ATP production decreases, the activity may be reduced.
  • the therapeutic screening method of the present invention is directed to the degree of the above changes, that is, the change in the number of mucus-producing cells and/or the expression of mucus-producing genes in hAGOs, and the degree of change in the ATP production level of mitochondria. If is significant, a step of determining the candidate substance as a therapeutic agent may be further included.
  • the changes in the number of mucus-producing cells and/or the expression of mucus-producing related genes in the hAGOs, and the changes in mitochondrial ATP production levels are 'significant', the number of mucus-producing cells after treatment with the candidate substance and/or expression of mucus-production related genes, and mitochondrial ATP production levels of 3% or more, 5% or more, 7% or more, 10% or more, 15% or more, 20% or more, 30% or more, or 40% or more. This means an increase of more than 50%.
  • the therapeutic agent restores the pathological phenomenon reduced by VacA, it may be a therapeutic agent for diseases related to gastric mucosal damage caused by VacA, that is, caused by Helicobacter pylori, and specifically, caused by Helicobacter pylori. It may be a treatment for gastritis, stomach ulcers, or stomach cancer.
  • human antrum gastric organoids hAGOs
  • hESC human embryonic stem cell
  • hiPSC human induced pluripotent stem cell
  • VacA human induced pluripotent stem cell
  • Another aspect of the present invention provides a screening method for a gastric adhesion inhibitor that inhibits gastric adhesion of Helicobacter pylori .
  • the screening method for a gastric adhesion inhibitor of the present invention includes the steps of treating a candidate material in human antrum gastric organoids (hAGOs) treated with a Helicobacter pylori strain, and depending on whether or not the candidate material is treated, the hAGOs It includes measuring changes in the level of Helicobacter pylori strains remaining in the.
  • hAGOs human antrum gastric organoids
  • the screening method for a gastric adhesion inhibitor of the present invention first includes the step of treating hAGOs treated with Helicobacter pylori strain with a candidate substance.
  • the hAGOs may be two-dimensional hAGOs obtained by dissociating three-dimensional hAGOs into single cells and culturing them on a plate.
  • Helicobacter pylori strains can be inoculated into the two-dimensional hAGOs and cultured again for a certain period of time.
  • the hAGOs attach to epithelial cells through gastric adhesion-related proteins such as BabA or SabA present on the surface of the Helicobacter pylori strains.
  • a candidate material is treated with hAGOs to which Helicobacter pylori strains are attached.
  • the candidate material has the effect of preventing Helicobacter pylori from attaching to gastric epithelial cells or removing Helicobacter pylori attached to gastric epithelial cells.
  • It may be a single compound such as any type expected to have, such as nucleic acids, amino acids, monosaccharides, etc., or a polymer such as polynucleotides, peptides, proteins, polysaccharides, etc., and may be a natural or synthetic organic substance, an inorganic substance, or a complex thereof.
  • 'gastric adhesion inhibitors' refers to molecules such as, or a mixture of all of them, or microorganisms that produce them.
  • candidate substances those that reduce the level of Helicobacter pylori attached to hAGOs are called 'gastric adhesion inhibitors'.
  • the screening method for a gastric adhesion inhibitor of the present invention includes the step of measuring changes in the level of Helicobacter pylori strains remaining in the hAGOs, depending on whether or not the candidate material is treated.
  • the level of the Helicobacter pylori strain attached to the hAGOs is changed by measuring the expression level of the unique material derived from the Helicobacter pylori strain remaining in the hAGOs, such as the unique gene derived from the Helicobacter pylori strain, specifically the 16S rRNA of the Helicobacter pylori strain. You can check.
  • the gastric adhesion inhibitor screening method of the present invention determines the candidate substance as a gastric adhesion inhibitor when the degree of change as described above, that is, the degree of change in the intrinsic material derived from the Helicobacter pylori strain remaining in hAGOs is significant. Additional steps may be included. When the change in the level of the unique material derived from the Helicobacter pylori strain remaining in the hAGOs is 'significant', the unique material derived from the Helicobacter pylori strain remaining in the hAGOs after treatment of the candidate material is compared to the case where the candidate material is not treated. This means that the level of a substance has decreased by more than 3%, more than 5%, more than 7%, more than 10%, more than 15%, more than 20%, more than 30%, more than 40%, or more than 50%.
  • the gastric adhesion inhibitor inhibits Helicobacter pylori from attaching to the stomach, so it may be a preventive agent that fundamentally blocks infection by Helicobacter pylori itself, or it may be a therapeutic agent that removes Helicobacter pylori that has already attached to the stomach.
  • human antrum gastric organoids hAGOs
  • hESC human embryonic stem cell
  • hiPSC human induced pluripotent stem cell
  • hAGOs established from human pluripotent stem cells were dissociated into single cells, cultured on plates, and then inoculated with Helicobacter pylori P1 strain and co-cultured, thereby establishing the 'screening platform for inhibitors of attachment of Helicobacter pylori to the stomach' of the present invention.
  • hPSC human pluripotent stem cell
  • hESC human embryonic stem cell line H9 purchased from WiCell Research Institute (USA), and this was previously described in McCrachen et al . (2014) (McCracken et al. , Nature, 516).
  • differentiation is carried out step by step through the process of endoderm (definitive endoderm) and posterior-foregut, and finally hAGOs (human antrum gastric) organoids) were established.
  • progenitor cells SOX9
  • surface mucus cells MUC5AC and GKN1
  • antral neck cells MUC6 and TFF2
  • endocrine cells SST and GAST
  • gastric basal-like cells such as gastric parietal cells (ATP4A and ATP4B) or chief cells (MIST1, PGA and PGC) It was confirmed that there were no fundus-like cells or intestinal-like cells (intestine-like cells; MUC2 and VIL).
  • Example [1-1] evenly retain various types of cells that exist only in the antrum of the stomach.
  • Example [1-1] The hAGOs established in Example [1-1] were treated with VacA (vacuolating cytotoxin A), a channel-forming toxin of Helicobacter pylori, at various concentrations of 2ug/mL to 40ug/mL in the culture medium or in the organoid lumen. were microinjected and cultured for 48 hours, and the physiological and molecular biological changes occurring in hAGOs were confirmed.
  • Whole genome sequencing (WGS) was performed.
  • VacA treatment induced changes at the transcriptome level in hAGOs Figure 5A
  • differentially expressed genes between control and VacA-treated hAGOs genes, DEGs were most closely related to signaling pathways induced by Helicobacter pylori infection, especially inflammation- and immune-related signaling pathways (Figure 5B).
  • VacA was reported to be a toxin targeting mitochondria. Accordingly, the hAGOs established in Example [1-1] were used to determine how mitochondrial activity varies by VacA.
  • Example [1-1] From the results in Examples [1-2] to [1-4], it can be seen that the hAGOs established in Example [1-1] well reproduce the above pruning in terms of various cellular complexity. In addition, it can be seen that the above vestibule is well replicated in terms of the three-dimensional structure, barrier function of the epithelial layer by tight junctions, and mitochondrial function. Therefore, the three-dimensional hAGOs established in Example [1-1] can be said to be a suitable model for studying infection by Helicobacter pylori.
  • scRNA-seq single-cell RNA-sequencing
  • VacA affected the gene expression profile in the gastric epithelial layer of hAGOs on a global scale, based on well-known human gastric mucosa-specific markers. A total of seven major clusters were identified ( Figures 7a to 7c). In addition, by treatment with VacA, the ratio of cell types in hAGOs was changed ( Figure 7d), and the expression of genes related to gastric mucus secretion, MUC5AC and MUC6, and SOX9, a progenitor cell marker that can differentiate into gastric mucus secreting cells, was observed. It was confirmed that this decreased (Figure 7d).
  • hAGOs were formed in the same manner as in Example [1-1], and the hAGOs thus formed were treated with VacA to confirm the resulting changes.
  • Figure 9 the same changes as in Figures 8a to 8e were confirmed in hAGOs formed by differentiation from hiPSCs ( Figure 9 ).
  • VacA damages the mitochondrial structure of mainly mucus-producing cells (MUC5AC + cells and MUC6 + cells) in hAGOs, regardless of the type of human pluripotent cells used to form hAGOs, It can be seen that it not only reduces the respiratory capacity and respiratory efficiency of mitochondria, which play a very important role in ATP production and subsequent mucus secretion, but also makes the mucus layer thinner.
  • Example [2-1] confirmed in hAGOs were the same in actual clinical practice.
  • gastric tissue samples from 11 normal people and 27 patients confirmed to be infected with Helicobacter pylori were collected at Kyungpook National University Hospital, and with the consent of the 38 people, age, gender, history of Helicobacter pylori infection and treatment, history of antibiotic treatment, and medical history were collected. , demographic and clinical data such as endoscopic findings were obtained.
  • Example 2 From the results in Example 2, we hypothesized that the pathological phenomenon caused by Helicobacter pylori infection could be recovered by restoring the function of mitochondria in mucus-producing cells, and mdivi-, a mitochondrial fission inhibitor, was used. 1 was used to test the above hypothesis.
  • hAGOs treated with 20ug/mL VacA were treated with 50uM of mdivi-1 for 24 hours, and the resulting morphological and physiochemical changes were observed.
  • FIGS. 11A to 11C the mitochondria that had been fragmented by treatment with mdivi-1 were changed to a filament type (FIG. 11A), and the amount of ATP production was confirmed to be increased (FIG. 11B).
  • the fluorescent dye (FD4) microinjected into the lumen area was also confirmed to remain in the lumen area without diffusing to the surroundings ( Figure 11c), indicating that the tight junctions damaged by VacA were also fully restored. .
  • Example [3-1] 150 types of autophagy-regulators included in the ACL (autophagy compound library) (Selleckchem) were added to hAGOs treated with 20ug/mL of VacA for 48 hours. sutophagy-modulators) were treated at a concentration of 10uM each, or mdivi-1 was treated at a concentration of 50uM for 24 hours, and then the concentration of ATP was measured using CellTiter-Glo ® 3D Cell Viability Assay (Promega) according to standard protocols. was measured.
  • MLN8054 and IU1 which were confirmed to be the most effective among the AURKA inhibitors and USP14 inhibitors selected in Example [3-2], were additionally verified for their effectiveness in treating gastric mucosal damage using hAGOs.
  • hAGOs treated with 20ug/mL of VacA for 48 hours were treated with 10uM of MLN8054 or IU1 and cultured for 24 hours, and then morphological or molecular biological changes were observed.
  • VacA was activated by reacting with 10% (v/v) 0.3M HCl at 37 degrees Celsius for 30 minutes, and the mixture was neutralized by adding an equal amount of NaOH of the same concentration to 5-week-old mice that were allowed only water for 24 hours.
  • C57BL/6 mice were orally administered with PBS every 12 hours for 10 days to induce damage to the stomach tissue, and then MLN8054 was administered every 24 hours starting 3 days after starting the oral administration of VacA. It was administered orally at a dose of 1, 5, or 10 mg/kg, and 10 days after starting the oral administration of VacA, the mice were sacrificed and the entire stomach was removed from the abdominal cavity to compare the effect of MLN8054 administration. (Figure 16a).
  • Helicobacter pylori P1 strain (Chonnam National University, Professor Jong-Hwan Park's laboratory) isolated and established from a gastric ulcer patient was inoculated and cultured overnight at 37 degrees Celsius under microaerobic conditions. It was then washed with PBS, resuspended, and used to infect hAGOs and mice with Helicobacter pylori.
  • the Helicobacter pylori strain prepared as above was cultured for 48 hours and OD 600 was measured using a spectrophotometer. As a result, it was confirmed that the Helicobacter pylori strain prepared as above was growing well (FIG. 17a).
  • real-time PCR and Western blot were performed on the Helicobacter pylori strain cultured as above to confirm the transcript and protein levels of VacA, and as a result, it was confirmed that the Helicobacter pylori strain well expressed VacA. ( Figures 17b and 17c).
  • hAGOs infected with Helicobacter pylori were cultured in hAGOs (2mM L-glutamine (Invitrogen), 10mM HEPES (Invitrogen), penicillin/streptomycin, 1x N2 (Invitrogen), 1x B27 (Invitrogen), and 100ng/mL EGF (The cells were cultured in Advanced DMEM/F12 (Invitrogen) containing R&D Systems) for 24 hours, then treated with 10uM of MLN8054 and cultured again for 24 hours to observe morphological or molecular biological changes (FIG. 17d).
  • Example [3-2] In addition to MLN8054 and IU1, the effects of which were confirmed in Examples 4 and 5, the remaining AURKA inhibitors, MLN8237, MK8745, and MK5108, and the remaining USP14 inhibitor, WP1130 In addition, hAGOs were used to confirm the therapeutic effect of gastric mucosal damage induced by VacA.
  • hAGOs treated with 20ug/mL VacA for 48 hours were treated with 5uM, 10uM, and 20uM of MLN8237, or 1uM, 10uM, and 20uM of MK8745.
  • cells were treated with MK5108 at 1uM, 2uM, and 5uM, or WP1130 at 0.1uM, 0.5uM, and 1uM and cultured for 24 hours, and then morphological or molecular biological changes were observed.
  • AURKA inhibitors including MLN8054, and several USP14 inhibitors, including IU1, are effective substances in preventing or treating gastric mucosal damage caused by Helicobacter pylori infection.
  • the hAGOs produced in Example [1-1] were separated from Matrigel, washed with cold PBS, treated with 0.25% trypsin-EDTA (Gibco), and incubated at 37 degrees Celsius for 5 minutes.
  • the hAGOs dissociated into single cells were washed by centrifugation at 1000 rpm, and then inoculated 1x106 into 12-well dishes coated with 1% Matrigel and incubated in gastric vestibular organoid differentiation medium [advanced DMRM/F12, 2 mM L-glutamine, 1 % Penicillin-Streptomycin, and 15 mM HEPES buffer, 1
  • the Helicobacter pylori P1 strain cultured as above was treated and cultured with 10 MOI per well containing the two-dimensional hAGOs prepared as above, thereby reducing the level of Helicobacter pylori P1 strain remaining in the hAGOs.
  • a so-called 'Helicobacter pylori adhesion inhibitor screening platform' was established to discover substances as inhibitors of Helicobacter pylori adhesion to the stomach (FIG. 20).
  • MN NucleoSpin RNA
  • cDNA was synthesized from the extracted RNA using superscript IV (invitrogen), as shown in Table 5 below.

Abstract

The present invention provides: a method for screening for a therapeutic agent for gastric mucosal damage-related diseases caused by Helicobacter pylori; and a pharmaceutical composition for the prevention or treatment of gastric mucosal damage-related diseases, comprising a novel active ingredient discovered through this method. The present invention also provides a platform for screening for a therapeutic agent for gastric mucosal damage-related diseases caused by Helicobacter pylori, and a platform for screening for an ingredient that inhibits the gastric adherence of Helicobacter pylori.

Description

헬리코박터 파일로리에 의한 위 점막 손상 관련 질환의 예방 또는 치료용 약학적 조성물 및 헬리코박터 파일로리 감염의 예방 또는 치료용 유효성분의 스크리닝 플랫폼Pharmaceutical composition for preventing or treating diseases related to gastric mucosa damage caused by Helicobacter pylori and screening platform for active ingredients for preventing or treating Helicobacter pylori infection
관련 출원과의 상호인용Cross-citation with related applications
본 출원은 2022년 04월 22일자 한국 특허출원 제2022-0050209호에 기초한 우선권의 이익을 주장하며, 해당 한국 특허출원에 기재된 모든 내용은 본 명세서의 일부로서 포함된다.This application claims the benefit of priority based on Korean Patent Application No. 2022-0050209, dated April 22, 2022, and all contents described in the Korean Patent Application are included as part of this specification.
기술분야Technology field
본 발명은 위 점막 손상 관련 질환, 특히 헬리코박터 파일로리에 의한 위 점막 손상 관련 질환의 예방 또는 치료를 위한 신규의 약학적 조성물에 관한 것이다. 또한, 헬리코박터 파일로리 감염 또는 상기 감염에 의한 위 점막 손상 관련 질환의 예방 또는 치료를 위한 유효성분을 스크리닝하기 위한 플랫폼과 이를 이용한 스크리닝 방법에 관한 것이다.The present invention relates to a novel pharmaceutical composition for the prevention or treatment of diseases related to gastric mucosal damage, particularly diseases related to gastric mucosal damage caused by Helicobacter pylori. In addition, it relates to a platform for screening active ingredients for the prevention or treatment of Helicobacter pylori infection or diseases related to gastric mucosal damage caused by the infection and a screening method using the same.
헬리코박터 파일로리(Helicobacter pylori) 감염은 전 세계에 널리 분포되어 있으며, 인간에게 가장 흔한 만성 감염중의 하나이다. 세계 인구의 50%가 감염되어 있는 헬리코박터 파일로리는 1983년 워렌(Warren)과 마샬(Marshall)에 의해 처음으로 인체 위 점막에서 분리 동정된 이후, 위염, 위궤양, 십이지장궤양, 그리고 위암 발생에 중요한 요인으로 알려져 있다. Helicobacter pylori infection is widely distributed around the world and is one of the most common chronic infections in humans. Helicobacter pylori, which infects 50% of the world's population, has been an important factor in the development of gastritis, gastric ulcer, duodenal ulcer, and gastric cancer since it was first isolated and identified from human gastric mucosa by Warren and Marshall in 1983. It is known.
세계보건기구(WHO)에서 헬리코박터 파일로리를 위암의 제1군 발암인자로 규정하고 있으며, 헬리코박터 파일로리는 인위적으로 제균하지 않으면 대부분에서 평생 동안 지속하면서 다양한 위장질환을 유발하는 원인이 된다. 헬리코박터 파일로리 감염은 연령, 인종, 교육수준에 따라 다양하게 나타나며, 성장기의 사회경제 여건과도 관계가 있다. 특히, 개발도상국에서는 헬리코박터 파일로리 감염이 주로 유년기에 이루어지지만, 선진국에서는 유년기의 감염이 적은 반면 성년기에도 감염이 꾸준히 이루어져서 연간 0.3% 내지 0.5%의 비율로 감염이 발생하고 있다. 헬리코박터 파일로리 감염은 인위적으로 제균을 시도하지 않는 한, 대부분에서 평생 감염이 지속되는 만성 감염이나 감염 경로 및 예방 측면에서 아직 분명히 밝혀진 바가 없다. 우리나라의 헬리코박터 파일로리 감염의 연령별 특징은 15세 이하의 소아에서 평균 17.2%로 낮고, 16세 이상의 성인에서 평균 66.9%로 개발도상국의 감염 형태에서 선진국의 감염 형태로 이행되는 단계로 밝혀졌다.The World Health Organization (WHO) defines Helicobacter pylori as a group 1 carcinogen for stomach cancer, and if it is not artificially eradicated, Helicobacter pylori lasts a lifetime in most cases and causes various gastrointestinal diseases. Helicobacter pylori infection appears in various ways depending on age, race, and education level, and is also related to socioeconomic conditions during growth. In particular, in developing countries, Helicobacter pylori infection mainly occurs in childhood, but in developed countries, while the number of infections in childhood is low, infection occurs steadily even in adulthood, with infections occurring at a rate of 0.3% to 0.5% per year. Helicobacter pylori infection is a chronic infection that lasts a lifetime in most cases unless artificial eradication is attempted, but the infection route and prevention have not yet been clearly identified. The age-specific characteristics of Helicobacter pylori infection in Korea are low, at an average of 17.2% in children under 15 years of age, and at an average of 66.9% in adults over 16 years of age, indicating a transition from the form of infection in developing countries to the form of infection in developed countries.
헬리코박터 파일로리의 감염 경로는 완전히 밝혀지지 않았으나, 현재까지의 연구결과 첫째 사람에서 사람으로 전염되며, 둘째 아동기에 주로 가족 내에서 감염이 이루어지는 것으로 밝혀졌고, 헬리코박터 파일로리의 감염에 영향을 미칠 수 있는 인자는 성별, 체질량지수, 혈액형, 교육수준, 경제수준, 당뇨병 유무, 지방간 유무, 소화성 궤양, 흡연 및 음주력 등으로 분석되었다. 헬리코박터 파일로리는 요소분해효소(urease)를 생성하여 요소(urea)를 이산화탄소와 암모니아로 분해하고, 이때 발생한 암모니아가 위산을 중화시켜 강산이 작용하는 위 속에서 살아남을 수 있는 것으로 알려져 있으며, 위 내에 감염되면 점액층 혹은 점막에 부착되어 집락을 형성하고, 직접 위에 염증을 일으킴으로써 여러 소화기 질환을 일으킨다고 알려져 있다. 특히, 헬리코박터 파일로리에서 생성되는 병독인자인 VacA(vacuolating cytotoxin)과 CagA(cytotoxin-associated protein)이 검출되는 종이 다른 종보다 병원성이 강한 것으로 알려져 있다.The infection route of Helicobacter pylori has not been completely elucidated, but research results to date have shown that first, it is transmitted from person to person, and second, infection occurs mainly within the family during childhood. Factors that may affect infection with Helicobacter pylori are: Gender, body mass index, blood type, education level, economic level, presence of diabetes, presence of fatty liver, peptic ulcer, smoking and drinking history were analyzed. Helicobacter pylori produces urease to decompose urea into carbon dioxide and ammonia, and the ammonia generated at this time neutralizes gastric acid and is known to be able to survive in the stomach where strong acids act, causing infection in the stomach. It is known to attach to the mucus layer or mucous membrane, form colonies, and directly cause inflammation in the stomach, causing various digestive diseases. In particular, species in which the virulence factors VacA (vacuolating cytotoxin) and CagA (cytotoxin-associated protein) produced by Helicobacter pylori are detected are known to be more pathogenic than other species.
특히, 상기 VacA는 87kDa 분자량의 단백질이며, 숙주세포의 공포화(vacuolation)를 유발하는 세포독소(cytotoxin)로, 헬리코박터 파일로리에 대한 숙주의 T세포 반응을 억제함으로써 감염이 지속되게 한다고 보고되고 있다. 상기 VacA는 위 상피세포의 형태 변형을 통해 방벽 효과를 파괴하고 염증 반응을 조절한다. 또한 세포 내 엔도좀(endosome) 구획과 미토콘드리아의 외막을 파괴하여 세포의 공포화(vacuolation)를 유발하고, 카스파아제(caspase)-8, 카스파아제-9를 활성화시킴으로써 세포 사멸을 유도할 수 있고, 소화성 궤양을 일으키는 것으로 알려져 있다.In particular, VacA is a protein with a molecular weight of 87 kDa and is a cytotoxin that causes vacuolation of host cells. It has been reported that it suppresses the host's T cell response to Helicobacter pylori, thereby allowing infection to persist. The VacA destroys the barrier effect and regulates the inflammatory response through morphological modification of gastric epithelial cells. In addition, it destroys the endosome compartment within the cell and the outer membrane of the mitochondria, causing vacuolation of the cell and activating caspase-8 and caspase-9, thereby inducing cell death. It is known to cause peptic ulcers.
상기와 같은 헬리코박터 파일로리의 감염에 의한 질환을 치료하기 위한 하나의 전략으로 헬리코박터 파일로리를 없애는 제균 치료법이 있는데, '프로톤펌프억제제(proton pump inhibitor, PPI)-아목시실린(amoxicillin)-클라리스로마이신(clarithromycin)'을 사용하는 3제요법을 1 내지 2주간 시행하는 방법으로서, 현재까지 가장 많이 활용되는 치료법이다. 그러나 상기와 같은 삼제요법 역시도 제균율이 80%를 넘지 못하고 있으며, 순차치료, 동시치료, 변형 순차치료법이 활용되고 있으나, 근본적인 항생제 내성 발생을 배제할 수 없고, 위 상피의 점막층(mucosal layer)에 존재하는 헬리코박터 파일로리까지 완전히 제거하는 것에는 어려움이 있다. 이에, 헬리코박터 파일로리 감염에 의한 질환의 치료시에 제균 요법과 더불어, 위 점막의 손상과 점액-생성 기능까지도 회복시킬 수 있는, 나아가 헬리코박터 파일로리의 위 부착 자체를 억제하여 원천적으로 헬리코박터 파일로리의 감염을 차단할 수 있는 새로운 예방 또는 치료 성분의 발굴이 필요한 실정이다. 아울러, 이를 위해 상기와 같은 새로운 예방 또는 치료 성분을 빠르고 정확하게 발굴해 낼 수 있는 플랫폼의 연구·개발 또한 절실하다.As a strategy to treat diseases caused by Helicobacter pylori infection as described above, there is an eradication treatment to eliminate Helicobacter pylori, which includes 'proton pump inhibitor (PPI) - amoxicillin - clarithromycin. 'This is a method of administering a three-drug therapy for 1 to 2 weeks, and is the most commonly used treatment method to date. However, even in the triple therapy described above, the eradication rate does not exceed 80%, and although sequential treatment, simultaneous treatment, and modified sequential treatment methods are used, the occurrence of fundamental antibiotic resistance cannot be ruled out, and it exists in the mucosal layer of the gastric epithelium. It is difficult to completely eliminate Helicobacter pylori. Accordingly, in the treatment of diseases caused by Helicobacter pylori infection, in addition to eradication therapy, it is possible to restore the damage to the gastric mucosa and even the mucus-producing function, and furthermore, to inhibit the attachment of Helicobacter pylori to the stomach itself to fundamentally block Helicobacter pylori infection. There is a need to discover new preventative or therapeutic ingredients. In addition, for this purpose, research and development of a platform that can quickly and accurately discover new preventive or therapeutic ingredients as mentioned above is also urgently needed.
본 발명의 일 목적은 위 점막 손상 관련 질환의 예방 또는 치료를 위한 신규의 약학적 조성물을 제공하는 것이다.One object of the present invention is to provide a novel pharmaceutical composition for preventing or treating diseases related to gastric mucosal damage.
본 발명의 다른 목적은 위 점막 손상의 예방 또는 개선을 위한 신규의 식품 또는 건강기능식품 조성물을 제공하는 것이다.Another object of the present invention is to provide a new food or health functional food composition for preventing or improving gastric mucosal damage.
본 발명의 또 다른 목적은 헬리코박터 파일로리(Helicobacter pylori)에 의한 위 점막 손상 관련 질환의 치료제를 스크리닝하기 위한 플랫폼과 이를 이용하여 치료제를 스크리닝하는 방법을 제공하는 것이다.Another object of the present invention is to provide a platform for screening a therapeutic agent for diseases related to gastric mucosal damage caused by Helicobacter pylori and a method for screening a therapeutic agent using the platform.
본 발명의 다른 목적은 헬리코박터 파일로리의 위 부착을 억제하는 성분을 스크리닝하기 위한 플랫폼과 이를 이용하여 위 부착 억제제를 스크리닝하는 방법을 제공하는 것이다.Another object of the present invention is to provide a platform for screening components that inhibit gastric adhesion of Helicobacter pylori and a method for screening gastric adhesion inhibitors using the same.
상기의 목적을 달성하기 위하여, 본 발명의 일 측면은 오로라 키나아제 A(Aurora kinase A, AURKA) 억제제 또는 유비퀴틴 특이적 펩티다아제 14(ubiquitin specific peptidase 14, USP14) 억제제를 유효성분으로 포함하는 위 점막 손상 관련 질환의 예방 또는 치료용 조성물을 제공한다.In order to achieve the above object, one aspect of the present invention relates to gastric mucosal damage comprising an Aurora kinase A (AURKA) inhibitor or a ubiquitin specific peptidase 14 (USP14) inhibitor as an active ingredient. A composition for preventing or treating diseases is provided.
또한, 상기의 목적을 달성하기 위하여, 본 발명의 다른 측면은 오로라 키나아제 A 억제제 또는 유비퀴틴 특이적 펩티다아제 14 억제제를 유효성분으로 포함하는 위 점막 손상의 예방 또는 개선용 식품 또는 건강기능식품 조성물을 제공한다.In addition, in order to achieve the above object, another aspect of the present invention provides a food or health functional food composition for preventing or improving gastric mucosal damage containing an Aurora Kinase A inhibitor or a Ubiquitin-specific Peptidase 14 inhibitor as an active ingredient. .
또한, 상기의 목적을 달성하기 위하여, 본 발명의 또 다른 측면은 VacA가 처리된 인간 전정 위 오가노이드(human antrum gastric organoids, hAGOs)에 후보 물질을 처리하는 단계; 및 상기 후보 물질의 처리 여부에 따라, 상기 hAGOs에서, 점액-생성 세포의 수 및 점액-생성 관련 유전자의 발현 중 어느 하나의 변화와 미토콘드리아의 활성 수준의 변화를 측정하는 단계;를 포함하는, 헬리코박터 파일로리(Helicobacter pylori)에 의한 위 점막 손상 관련 질환의 치료제 스크리닝 방법을 제공한다.In addition, in order to achieve the above object, another aspect of the present invention includes treating VacA-treated human antrum gastric organoids (hAGOs) with a candidate material; And depending on whether the candidate material is treated, measuring changes in any one of the number of mucus-producing cells and the expression of mucus-producing genes and the change in mitochondrial activity level in the hAGOs. Provides a screening method for treatments for diseases related to gastric mucosal damage caused by Helicobacter pylori .
또한, 상기의 목적을 달성하기 위하여, 본 발명의 다른 측면은 헬리코박터 파일로리(Helicobacter pylori) 균주가 처리된 인간 전정 위 오가노이드(human antrum gastric organoids, hAGOs)에 후보 물질을 처리하는 단계; 및 상기 후보 물질의 처리 여부에 따라, 상기 hAGOs에 부착된 헬리코박터 파일로리 균주의 수준의 변화를 측정하는 단계;를 포함하는, 헬리코박터 파일로리의 위 부착 억제제 스크리닝 방법을 제공한다.In addition, in order to achieve the above object, another aspect of the present invention includes treating human antrum gastric organoids (hAGOs) treated with a Helicobacter pylori strain with a candidate material; and measuring changes in the level of Helicobacter pylori strains attached to the hAGOs, depending on whether the candidate material is treated.
본 발명에서는 헬리코박터 파일로리의 감염에 의해 발생되는 위 전정의 점막 손상이, 위 전정의 상피에 존재하는 점액-생성 세포의 미토콘드리아가 헬리코박터 파일로리의 독소 VacA에 의해 손상됨으로써 유발되는 것임을 확인하여 헬리코박터 파일로리에 의한 위 점막 손상 관련 질환의 발병 메커니즘을 새롭게 규명하였다.In the present invention, it was confirmed that damage to the gastric vestibule mucosa caused by infection by Helicobacter pylori is caused by the mitochondria of mucus-producing cells present in the epithelium of the gastric vestibule being damaged by the toxin VacA of Helicobacter pylori. The pathogenesis mechanism of diseases related to gastric mucosal damage was newly identified.
나아가, 본 발명에서는 위와 같은 발병 메커니즘에 기초하여, 오로라 키나아제 A 억제제나 유비퀴틴 특이적 펩티다아제 14 억제제가 점액 생성 세포에서의 미토콘드리아의 손상을 방지 또는 회복하는 효과가 있음을 확인함으로써, 헬리코박터 파일로리에 의한 위 점막 손상 관련 질환에 대한 새로운 치료제를 제공한 효과가 있다.Furthermore, in the present invention, based on the above pathogenic mechanism, it was confirmed that Aurora Kinase A inhibitors or Ubiquitin-specific Peptidase 14 inhibitors are effective in preventing or recovering mitochondrial damage in mucus-producing cells, thereby preventing gastric ulcers caused by Helicobacter pylori. It has the effect of providing a new treatment for diseases related to mucosal damage.
아울러, 상기와 같이 새롭게 규명된 메커니즘에 기초하여 확립한 위 전정 오가노이드 플랫폼을 이용하여 헬리코박터 파일로리에 의한 감염을 예방 또는 치료할 수 있는 유효성분을 빠르고 정확하게 발굴해 낼 수 있는 효과도 있다.In addition, there is the effect of quickly and accurately discovering active ingredients that can prevent or treat infection by Helicobacter pylori using the gastric vestibular organoid platform established based on the newly identified mechanism as described above.
다만, 본 발명의 효과는 상기에서 언급한 효과로 제한되지 아니하며, 언급되지 않은 또 다른 효과들은 하기의 기재로부터 당업자에게 명확히 이해될 수 있을 것이다.However, the effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the following description.
도 1은 hESC(human embryonic stem cell) 라인 H9으로부터 형성한 hPSC(human pluripotent stem cell)를 단계적으로 분화시켜 최종적으로 hAGOs(human antrum gastric organoids)를 확립하는 과정을 개략적으로 나타낸 모식도 및 각 단계별 현미경 사진이다.Figure 1 is a schematic diagram schematically showing the process of step-by-step differentiation of hPSC (human pluripotent stem cells) formed from hESC (human embryonic stem cell) line H9 and finally establishment of hAGOs (human antrum gastric organoids) and microphotographs at each step. am.
도 2a hAGOs 분화과정에 나타나는 내배엽(definitive endoderm), 후측-전장(posterior-foregut)에 존재하는 세포의 특이적 마커 단백질 발현을 확인한 결과이며, 도 2b는 분화과정 동안 나타나는 세포 특이적 마커 유전자 발현을 확인한 결과이며, 도 2c는 hAGO에 존재하는 다양한 세포 유형을 확인하기 위해 각 세포 타입 특이적인 마커 단백질 발현을 확인한 결과이다.Figure 2a is the result of confirming the expression of specific marker proteins of cells present in the definitive endoderm and posterior-foregut that appear during the hAGOs differentiation process, and Figure 2b shows the expression of cell-specific marker genes shown during the differentiation process. This is the confirmed result, and Figure 2c shows the result of confirming the expression of marker proteins specific to each cell type to confirm the various cell types present in hAGO.
도 3은 VacA의 처리에 의해 hAGOs에서 나타나는 증식 저해 및 세포사멸 증가를 기반으로 VacA 처리 농도 구간 탐색을 수행한 결과이다.Figure 3 shows the results of exploring the VacA treatment concentration range based on the inhibition of proliferation and increase in apoptosis in hAGOs caused by VacA treatment.
도 4는 3차원 hGO의 구조적 특징을 기반으로 VacA 처리 후 표면적 변화, 상피 두께 변화, 밀착 연접(tight junction) 변화 등 생리학적, 분자생물학적 변화를 확인한 결과를 나타낸 것이다.Figure 4 shows the results of confirming physiological and molecular biological changes such as surface area changes, epithelial thickness changes, and tight junction changes after VacA treatment based on the structural characteristics of 3D hGO.
도 5는 VacA 처리 후 hAGO의 글로벌 유전체 변화 확인 및 검증한 결과이다. Figure 5 shows the results of confirming and verifying global genome changes in hAGO after VacA treatment.
도 6은 VacA의 처리가 hAGOs에서 미토콘드리아의 활성에 미치는 영향을 미토콘드리아 형태, 유전자 발현 변화, 호흡 변화를 통해 확인한 결과를 나타낸 것이다.Figure 6 shows the results of confirming the effect of VacA treatment on mitochondrial activity in hAGOs through changes in mitochondrial morphology, gene expression, and respiration.
도 7a 내지 도 7e는 단일 세포 유전체 변화를 통해 hAGOs에서 VacA 처리에 의해 영향을 받는 세포를 분석한 결과이며, 오가노이드 구성 세포 확인 및 점액-생성 세포 연관 유전자 발현을 확인한 것이다.Figures 7a to 7e show the results of analyzing cells affected by VacA treatment in hAGOs through single cell genome changes, confirming the confirmation of organoid constituent cells and the expression of genes associated with mucus-producing cells.
도 8a 내지 도 8e는 hAGOs에서 VacA의 처리에 의해 영향을 받는 세포의 유형과 구체적인 생리학적 영향, 세포 별 미토콘드리아 활성 변화를 확인한 결과를 나타낸 것이다.Figures 8A to 8E show the results of confirming the types of cells affected by VacA treatment in hAGOs, specific physiological effects, and changes in mitochondrial activity for each cell.
도 9는 hiPSC(human induced pluripotent stem cell) 유래 hAGOs에서 VacA에 의해 영향을 받는 세포의 유형 및 미토콘드리아 활성 변화를 확인한 결과를 나타낸 것이다. Figure 9 shows the results of confirming changes in cell type and mitochondrial activity affected by VacA in hAGOs derived from hiPSC (human induced pluripotent stem cell).
도 10a 내지 도 10e는 hAGOs에서 발견된 VacA에 의한 세포 유형 변화와 미토콘드리아 활성 변화를 실제 헬리코박터 파일로리에 감염된 인간의 위 조직에서 검증한 결과를 나타낸 것이다.Figures 10A to 10E show the results of verifying changes in cell type and mitochondrial activity due to VacA found in hAGOs in human stomach tissue infected with Helicobacter pylori.
도 11a 내지 도 11c는 미토콘드리아 조절자를 활용하여 헬리코박터 파일로리 감염에 의한 위 점막 손상 치료제 스크리닝 플랫폼으로서 VacA가 처리된 hAGOs의 유효성 확인을 위해 미토콘드리아 활성 및 장벽 기능 회복을 확인한 결과이다.Figures 11A to 11C show the results of confirming the restoration of mitochondrial activity and barrier function to confirm the effectiveness of VacA-treated hAGOs as a screening platform for treating gastric mucosal damage caused by Helicobacter pylori infection using mitochondrial regulators.
도 12은 상기 플랫폼을 이용하여 치료제를 스크리닝한 결과를 나타낸 것이다.Figure 12 shows the results of screening therapeutic agents using the platform.
도 13 및 도 14a 내지 도 14d는 가장 효과가 우수한 것으로 선별된 오로라 키나아제 A 억제제인 MLN8054와 유비퀴틴 특이적 펩티다아제 14 억제제인 IU1에 대하여, hAGOs를 이용하여 추가적으로 위 점막 손상 치료 효과를 표현형 분석, 세포 호흡 분석, ATP 생성능, 점액-생성 기능, 밀착 연접 발현 분석을 통해 검증한 결과를 나타낸 것이다.Figures 13 and 14a to 14d show the phenotypic analysis and cellular respiration of MLN8054, an Aurora Kinase A inhibitor selected as the most effective, and IU1, a ubiquitin-specific peptidase 14 inhibitor, using hAGOs to further treat gastric mucosal damage. This shows the results verified through analysis, ATP production ability, mucus-producing function, and tight junction expression analysis.
도 15a 내지 도 15e는 MLN8054와 IU1이 미토콘드리아 융합(fusion) 또는 미토파지(mitophagy)를 통해 미토콘드리아 활성을 조절함을 확인한 결과를 나타낸 것이다. Figures 15A to 15E show the results confirming that MLN8054 and IU1 regulate mitochondrial activity through mitochondrial fusion or mitophagy.
도 16a 내지 도 16f는 hAGOs를 통해 확인된 MLN8054의 VacA에 의한 위 장벽 손상 회복 효과를 미토콘드리아 활성 연관 유전자 발현, E-카드헤린(E-cadherin) 발현, 밀착 연접 발현, 점액-생성 기능 및 미토콘드리아 요소를 통해 인 비보(in vivo)에서 다시 한번 확인한 결과를 나타낸 것이다.Figures 16a to 16f show the gastric barrier damage recovery effect caused by VacA of MLN8054 confirmed through hAGOs by measuring mitochondrial activity-related gene expression, E-cadherin expression, tight junction expression, mucus-producing function, and mitochondrial elements. This shows the results confirmed once again in vivo .
도 17a 내지 도 17i는 헬리코박터 파일로리의 성장 및 VacA 발현능을 확인하고, MLN8054에 대하여, VacA에 의해 유도된 경우뿐만 아니라, 실제 헬리코박터 파일로리 감염에 의한 위 장벽의 손상에 대해서도 치료 효과가 있는지 여부를, 헬리코박터 파일로리로 감염된 hAGOs에 MLN8054를 처리하여 미토콘드리아 활성 변화, 점액-생성 기능, 밀착 연접 발현을 통해 확인한 결과를 나타낸 것이다.Figures 17a to 17i confirm the growth of Helicobacter pylori and the ability to express VacA, and whether MLN8054 has a therapeutic effect not only when induced by VacA but also against damage to the gastric barrier caused by actual Helicobacter pylori infection. This shows the results confirmed through changes in mitochondrial activity, mucus-producing function, and tight junction expression by treating hAGOs infected with Helicobacter pylori with MLN8054.
도 18은 MLN8054에 대하여, VacA에 의해 유도된 경우뿐만 아니라, 실제 헬리코박터 파일로리의 감염에 의해 유발된 위 장벽의 손상에 대해서도 치료 효과가 있는지 여부를, 헬리코박터 파일로리로 감염된 마우스를 통해 인 비보(in vivo)에서 미토콘드리아 요소, E-카드헤린(E-cadherin)발현, 점액-생성 기능을 확인한 결과를 나타낸 것이다.Figure 18 shows an in vivo study using mice infected with Helicobacter pylori to determine whether MLN8054 has a therapeutic effect not only in the case induced by VacA, but also in the damage to the gastric barrier caused by actual Helicobacter pylori infection. ) shows the results of confirming mitochondrial elements, E-cadherin expression, and mucus-producing functions.
도 19a 내지 도 19d는 오로라 키나아제 A 억제제와 유비퀴틴 특이적 펩티다아제 14 억제제 중 MLN8054와 IU1 외에, 다른 오로라 키나아제 A 억제제인 MLN8237, MK8745, MK5108과 다른 유비퀴틴 특이적 펩티다아제 14 억제제인 WP1130에 대해서도, hAGOs를 이용하여 VacA에 의해 유도된 위 점막 손상의 치료 효과를 확인한 결과를 나타낸 것이다.Figures 19a to 19d show that in addition to MLN8054 and IU1 among Aurora Kinase A inhibitors and Ubiquitin-specific Peptidase 14 inhibitors, hAGOs are also used for MLN8237, MK8745, MK5108, which are Aurora Kinase A inhibitors, and WP1130, which is another Ubiquitin-specific Peptidase 14 inhibitor. This shows the results confirming the therapeutic effect of gastric mucosal damage induced by VacA.
도 20은 헬리코박터 파일로리의 위 부착 억제제를 스크리닝하기 위한 플랫폼의 구성 및 작동 원리를 설명한 개략도이다.Figure 20 is a schematic diagram explaining the configuration and operating principle of a platform for screening inhibitors of gastric adhesion of Helicobacter pylori.
도 21은 상기 플랫폼을 이용하여 헬리코박터 파일로리의 위 부착을 억제하는 활성을 갖는 신규 균주를 스크리닝한 결과를 나타낸 것이다.Figure 21 shows the results of screening a new strain with the activity of inhibiting the attachment of Helicobacter pylori to the stomach using the above platform.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
1. One. 위 점막 손상 관련 질환의 예방 또는 치료용 약학적 조성물Pharmaceutical composition for preventing or treating diseases related to gastric mucosal damage
본 발명의 일 측면은 위 점막 손상 관련 질환의 예방 또는 치료용 약학적 조성물을 제공한다.One aspect of the present invention provides a pharmaceutical composition for preventing or treating diseases related to gastric mucosal damage.
상기 본 발명의 약학적 조성물은 오로라 키나아제 A(Aurora kinase A, AURKA) 억제제 또는 유비퀴틴 특이적 펩티다아제 14(ubiquitin specific peptidase 14, USP14) 억제제를 유효성분으로 포함한다.The pharmaceutical composition of the present invention contains an Aurora kinase A (AURKA) inhibitor or a ubiquitin specific peptidase 14 (USP14) inhibitor as an active ingredient.
상기 오로라 키나아제 A 억제제는 MLN8054, MLN8237, MK8745, MK5108, CCT-137690, 레스타르티닙(lestaurtinib), NU6140, PF-03814735, SNS-314, TC-A 2317 염산염(hydrochloride), TC-S 7010 등 또는 이들의 유도체일 수 있고, MLN8054, MLN8237, MK8745, MK5108 등 또는 이들의 유도체일 수 있으며, 예컨대 MLN8054 또는 이의 유도체일 수 있다.The Aurora Kinase A inhibitor is MLN8054, MLN8237, MK8745, MK5108, CCT-137690, lestaurtinib, NU6140, PF-03814735, SNS-314, TC-A 2317 hydrochloride, TC-S 7010, etc. or It may be a derivative thereof, such as MLN8054, MLN8237, MK8745, MK5108, etc., or a derivative thereof, for example, MLN8054 or a derivative thereof.
또한, 상기 유비퀴틴 특이적 펩티다아제 14 억제제는 IU1, WP1130 등 또는 이들의 유도체일 수 있고, 예컨대 IU1 또는 이의 유도체일 수 있다.Additionally, the ubiquitin-specific peptidase 14 inhibitor may be IU1, WP1130, etc., or a derivative thereof, for example, IU1 or a derivative thereof.
한편, 상기 위 점막 손상 관련 질환은 위 상피세포의 미토콘드리아가 손상됨으로써 유발되는 것일 수 있고, 특히 상기 위 상피세포의 미토콘드리아의 손상은 헬리코박터 파일로리(Helicobacter pylori)에 의한 것일 수 있다. 상기 위 상피세포는 MUC5AC+ 세포, MUC6+ 세포, SOX9+ 세포 등과 같은 점액-생성 세포들(mucous-producing cells)일 수 있고, 상기 점액-생성 세포는 미토콘드리아의 손상된 것일 수 있다. 또한, 상기 손상은 미토콘드리아가 단편화(fragmentation)되거나, 길이가 짧아지거나, 미토콘드리아의 호흡 용량이나 호흡 효율이 감소된 것일 수 있고, 상기와 같은 미토콘드리아의 손상으로 인해 상기 점액-생성 세포의 점액 분비가 감소된 것일 수 있다. 예컨대, 상기 위 점막 손상 관련 질환은 위염, 위궤양, 위암 등일 수 있다.Meanwhile, the disease related to damage to the gastric mucosa may be caused by damage to the mitochondria of gastric epithelial cells, and in particular, damage to the mitochondria of the gastric epithelial cells may be caused by Helicobacter pylori . The gastric epithelial cells may be mucous-producing cells such as MUC5AC + cells, MUC6 + cells, SOX9 + cells, etc., and the mucus-producing cells may have damaged mitochondria. In addition, the damage may be due to fragmentation of the mitochondria, shortening of the length, or a decrease in the respiratory capacity or efficiency of the mitochondria, and the mucus secretion of the mucus-producing cells is reduced due to the damage to the mitochondria. It may have happened. For example, the disease related to damage to the gastric mucosa may be gastritis, gastric ulcer, stomach cancer, etc.
상기 예방은 위 점막 손상 관련 질환의 유발을 방지하거나 억제 또는 지연시키는 모든 행위를 말하고, 상기 치료는 위 점막 손상 관련 질환을 호전시키거나 이롭게 변경시키는 모든 행위를 말한다.The above prevention refers to all actions that prevent, suppress, or delay the onset of diseases related to gastric mucosal damage, and the above treatment refers to all actions that improve or beneficially change diseases related to gastric mucosal damage.
본 발명의 구체적인 실시예들에서는, 인간 전분화능줄기세포(human pluripotent stem cell)인 hESC(human embryonic stem cell)과 hiPSC(human induced pluripotent stem cell)로부터 각각 hAGOs(human antrum gastric organoids)를 확립하였고, 이와 같이 인간 전분화능줄기세포로부터 확립된 hAGOs가 기관(organ) 수준의 위(stomach)의 '전정(antrum)'를 잘 모사(mimic)하는 3D-오가노이드임을 확인하고, 이러한 hAGOs와 마우스 각각에 헬리코박터 파일로리 균주를 감염시켰을 때 두 모델 모두에서 공통적으로 VacA에 의해 위 전정의 상피에 존재하는 점액 생성 세포의 미토콘드리아가 손상되고 그에 따른 미토콘드리아의 활성 저하로 인해 점액-생성 세포의 밀착 연접이나 점액 생성이 저하됨으로써 위 점막의 손상이 동일하게 유발됨을 확인하는 한편, 상기 hAGOs 및 마우스 모델을 통해 오로라 키나아제 A 억제제나 유비퀴틴 특이적 펩티다아제 14 억제제에 의해 상기와 같은 점액-생성 세포에서의 미토콘드리아 손상이 방지 또는 회복됨을 확인하였다(도 17 내지 도 19 참고).In specific embodiments of the present invention, human antrum gastric organoids (hAGOs) were established from human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC), which are human pluripotent stem cells, respectively. In this way, it was confirmed that hAGOs established from human pluripotent stem cells are 3D-organoids that well mimic the 'antrum' of the stomach at the organ level, and each of these hAGOs and mice was confirmed. When infected with a Helicobacter pylori strain, the mitochondria of mucus-producing cells present in the epithelium of the gastric vestibule are commonly damaged by VacA in both models, and the resulting decrease in mitochondrial activity causes tight junctions of mucus-producing cells and mucus production. While it was confirmed that damage to the gastric mucosa was equally caused by degradation, mitochondrial damage in the above mucus-producing cells was prevented or restored by Aurora Kinase A inhibitors or ubiquitin-specific peptidase 14 inhibitors through the hAGOs and mouse models. was confirmed (see Figures 17 to 19).
한편, 상기 약학적 조성물은 오로라 키나아제 A 억제제 또는 유비퀴틴 특이적 펩티다아제 14 억제제 외에, 이와 동일 또는 유사한 기능을 나타내는 유효성분 1종 이상을 더 포함하거나, 또는 이와 함께 병용될 수 있다. Meanwhile, the pharmaceutical composition may further include, in addition to the Aurora Kinase A inhibitor or the Ubiquitin-specific Peptidase 14 inhibitor, one or more active ingredients exhibiting the same or similar functions, or may be used in combination therewith.
또한, 상기 약학적 조성물은 추가로 허용 가능한 담체를 1종 이상 포함할 수 있다. 상기 '허용 가능한'의 의미는 유효성분인 상기 오로라 키나아제 A 억제제 또는 유비퀴틴 특이적 펩티다아제 14 억제제의 활성을 억제하지 않으면서 적용 대상이 적응 가능한 이상의 독성을 지니지 않는다는 의미이다. 상기 '담체'는 세포 또는 조직 내로의 화합물의 부가를 용이하게 하는 화합물로 정의된다. 상기 조성물은 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기내에 내입시켜 제조될 수 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다. 또한, 상기 조성물이 포함하는 상기 유효성분은 콜로이드 현탁액, 분말, 식염수, 지질, 리포좀, 미소구체(microspheres), 또는 나노 구형입자와 같은 담체에 운반될 수 있다. 이들은 운반 수단과 복합체를 형성하거나 관련될 수 있고, 지질, 리포좀, 미세입자, 금, 나노입자, 폴리머, 축합 반응제, 다당류, 폴리아미노산, 덴드리머, 사포닌, 흡착 증진 물질 또는 지방산과 같은 본 발명이 속하는 기술분야에 공지된 운반 시스템을 사용하여 생체 내 운반될 수 있다. 이 외에도, 상기 담체는 제제시 통상적으로 이용되는 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아, 고무, 인산칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세 결정성 셀룰로스, 폴리비닐 피로리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필 히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함할 수 있으나, 이에 제한되는 것은 아니다. 또한, 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 상기 담체는 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로스 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 이용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다.Additionally, the pharmaceutical composition may further include one or more acceptable carriers. The meaning of 'acceptable' means that it does not inhibit the activity of the active ingredient, the Aurora Kinase A inhibitor or the Ubiquitin-specific peptidase 14 inhibitor, and does not have toxicity beyond what the subject of application can adapt to. The 'carrier' is defined as a compound that facilitates the addition of a compound into cells or tissues. The composition may be prepared in unit dosage form by formulating using a carrier and/or excipient, or may be manufactured by placing it in a multi-dose container, and may additionally include a dispersant or stabilizer. Additionally, the active ingredient contained in the composition may be transported in a carrier such as colloidal suspension, powder, saline solution, lipid, liposome, microsphere, or nano spherical particle. They may form complexes or associate with the delivery vehicle and may be used in combination with the present invention, such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation agents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancers or fatty acids. It can be delivered in vivo using delivery systems known in the art. In addition, the carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, and polyvinyl pyrolidone. , cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc., but is not limited thereto. In addition, in addition to the above ingredients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. may be additionally included. The carrier can be used by mixing saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these ingredients, and if necessary, other common agents such as antioxidants, buffers, and bacteriostatic agents. Additives can be added.
상기 약학적 조성물은 실제 임상 투여 시에 경구 및 비경구의 여러 가지 제형으로 투여될 수 있는데, 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구 투여를 위한 고형 제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형 제제는 생약 추출물 또는 생약 발효물에 적어도 하나 이상의 부형제, 예를 들면, 전분, 탄산칼슘, 수크로스 또는 락토오스, 젤라틴 등을 섞어 조제된다. 또한, 단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용된다. 상기 산제는 본 발명의 유효성분과 유당, 전분, 미결정셀룰로오스 등 약학적으로 허용가능한 적당한 담체를 단순 혼합함으로써 제조될 수 있다. 과립제는 본 발명의 상기 유효성분, 약학적으로 허용 가능한 적당한 담체 및 폴리비닐피롤리돈, 히드록시프로필셀룰로오스 등의 약학적으로 허용 가능한 적당한 결합제를 혼합한 후, 물, 에탄올, 이소프로판올 등의 용매를 이용한 습식과립법 또는 압축력을 이용한 건식과립법을 이용하여 제조될 수 있다. 또한 정제는 상기 과립제를 마그네슘스테아레이트 등의 약학적으로 허용 가능한 적당한 활택제와 혼합한 후, 타정기를 이용하여 타정함으로써 제조될 수 있다. 경구 투여를 위한 액상 제제로는 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. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁용제로는 프로필렌글리콜, 폴리에틸렌글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔, 마크로골, 트윈 61, 카카오지, 라우린지, 글리세롤, 젤라틴 등이 사용될 수 있다.The pharmaceutical composition can be administered in various oral and parenteral formulations during actual clinical administration. When formulated, diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants are used. It is prepared in this way. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain herbal extracts or herbal ferments with at least one excipient, such as starch, calcium carbonate, sucrose or It is prepared by mixing lactose and gelatin. Additionally, in addition to simple excipients, lubricants such as magnesium styrate talc are also used. The powder can be prepared by simply mixing the active ingredient of the present invention with a suitable pharmaceutically acceptable carrier such as lactose, starch, or microcrystalline cellulose. Granules are prepared by mixing the active ingredient of the present invention, a suitable pharmaceutically acceptable carrier, and a suitable pharmaceutically acceptable binder such as polyvinylpyrrolidone and hydroxypropyl cellulose, and then mixing them with a solvent such as water, ethanol, or isopropanol. It can be manufactured using a wet granulation method or a dry granulation method using compression force. Additionally, tablets can be manufactured by mixing the granules with a suitable pharmaceutically acceptable lubricant such as magnesium stearate and then compressing the mixture into tablets using a tablet press. 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 solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, wethepsol, macrogol, Tween 61, cacao, laurel, glycerol, gelatin, etc. can be used.
상기 약학적 조성물의 경우, 유효성분인 상기 오로라 키나아제 A 억제제 또는 유비퀴틴 특이적 펩티다아제 14 억제제는 예방, 개선 또는 치료해야 할 질환 및 개체의 상태에 따라 경구제, 주사제(예를 들어, 근육주사, 복강주사, 정맥주사, 주입(infusion), 피하주사, 임플란트), 흡입제, 비강투여제, 질제, 직장투여제, 설하제, 트랜스더말제, 토피칼제 등으로 투여될 수 있으나, 이에 제한되는 것은 아니다. 투여 경로에 따라 통상적으로 사용되고 비독성인, 약학적으로 허용 가능한 운반체, 첨가제, 비히클을 포함하는 적당한 투여 유닛 제형으로 제제화될 수 있다. 상기 투여시 유효성분인 재조합 미생물의 양은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여 방법, 배설율, 목적 부위 및 질환의 중증도 등에 따라 그 범위가 다양하다. 상기 조성물 내의 상기 유효성분은 1μM 이상, 예컨대 5μM 이상, 10μM 이상, 20μM 이상 또는 50μM 이상의 농도로 포함될 수 있다. 또한, 상기 유효성분은 0.05mg, 0.1mg, 0.15mg, 0.2mg, 0.3mg, 0.5mg, 1mg, 2mg, 3mg, 5mg, 10mg, 20mg, 30mg, 50mg 및 100mg로부터 선택된 하나의 하한선 및/또는 500mg, 450mg, 400mg, 350mg, 320mg, 300mg, 280mg, 250mg, 200mg, 150mg 및 100mg로부터 선택된 하나의 상한선으로 구성된 범위의 양으로 함유될 수 있으며, 일례로, 0.05 내지 500mg, 0.05 내지 450mg, 0.05 내지 400mg, 0.05 내지 350mg, 0.05 내지 300mg, 0.05 내지 250mg, 0.1 내지 500mg, 0.1 내지 450mg, 0.1 내지 400mg, 0.1 내지 350mg, 0.1 내지 300mg, 0.1 내지 250mg, 0.1 내지 200mg, 0.2 내지 500mg, 0.2 내지 400mg, 0.2 내지 300mg, 0.5 내지 300mg, 1 내지 300mg, 5 내지 300mg, 또는 10 내지 300mg의 양으로 함유될 수 있다.In the case of the pharmaceutical composition, the active ingredient, the Aurora Kinase A inhibitor or the Ubiquitin-specific peptidase 14 inhibitor, is administered as an oral agent, an injection (e.g., an intramuscular injection, an intraperitoneal injection) depending on the condition of the individual and the disease to be prevented, improved, or treated. It can be administered by injection, intravenous injection, infusion, subcutaneous injection, implant), inhalation, intranasal administration, vaginal administration, rectal administration, sublingual formulation, transdermal formulation, topical formulation, etc., but is not limited thereto. Depending on the route of administration, it may be formulated in an appropriate dosage unit form containing commonly used, non-toxic, pharmaceutically acceptable carriers, excipients, and vehicles. The amount of recombinant microorganisms as an active ingredient during the above administration varies depending on the patient's weight, age, gender, health status, diet, administration time, administration method, excretion rate, target site, and severity of disease. The active ingredient in the composition may be included at a concentration of 1 μM or more, such as 5 μM or more, 10 μM or more, 20 μM or more, or 50 μM or more. In addition, the active ingredient has a lower limit selected from 0.05mg, 0.1mg, 0.15mg, 0.2mg, 0.3mg, 0.5mg, 1mg, 2mg, 3mg, 5mg, 10mg, 20mg, 30mg, 50mg and 100mg and/or 500mg. , 450mg, 400mg, 350mg, 320mg, 300mg, 280mg, 250mg, 200mg, 150mg and 100mg, for example, 0.05 to 500mg, 0.05 to 450mg, 0.05 to 400mg. , 0.05 to 350 mg, 0.05 to 300 mg, 0.05 to 250 mg, 0.1 to 500 mg, 0.1 to 450 mg, 0.1 to 400 mg, 0.1 to 350 mg, 0.1 to 300 mg, 0.1 to 250 mg, 0.1 to 200 mg, 0.2 to 5 00mg, 0.2 to 400mg, 0.2 It may be contained in an amount of from 300 mg to 300 mg, 0.5 to 300 mg, 1 to 300 mg, 5 to 300 mg, or 10 to 300 mg.
또한, 상기 조성물을 약학적으로 사용하는 경우, 상기 조성물은 약학적으로 유효한 양으로 투여될 수 있다. 본 발명에 있어서 '약학적으로 유효한 양'은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 상기 유효 용량은 투여 개체의 몸무게 1kg 당 일반적으로 1일 0.01mg 내지 5000mg이며, 의사 또는 약사의 판단에 따라 일정 시간간격으로 1일 1회 내지 수회로 분할 투여할 수도 있으나, 이에 제한되지 않는다. 상기 조성물은 개별 치료제로 투여하거나, 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 동시에, 별도로, 또는 순차적으로 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기 요소들을 모두 고려하여 부작용없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 통상의 기술자에 의해 용이하게 결정될 수 있다. 구체적으로 상기 조성물의 유효량은 환자의 연령, 성별, 상태, 체중, 체내에 활성 성분의 흡수도, 불활성율, 배설 속도, 질병 종류, 병용되는 약물에 따라 달라질 수 있으며, 투여 경로, 비만의 중증도, 성별, 체중, 연령 등에 따라 증감될 수 있으며, 치료하고 있는 상태의 심각성 등에 따라 다양할 수 있다. 필요에 따라 편리성을 위하여 1일 총 투여량을 하루 동안 여러 번 나누어 투여될 수 있다. 일례로 일일 투여량은 매일 약 0.0001 mg/kg 내지 약 10 g/kg으로, 예컨대 약 0.001 mg/kg 내지 약 1 g/kg의 양을 1일 1회 투여할 수 있다. 아울러, 투여 기간은 1일 내지 2개월일 수 있으나, 질환의 예방 또는 치료 효과가 나타날 때까지 제한 없이 투여될 수 있다. 또한, 의사 또는 약사의 판단에 따라 일정 시간 간격으로 1일 수회, 예컨대 하루 2회 내지 3회 분할 투여될 수 있다.Additionally, when the composition is used pharmaceutically, the composition may be administered in a pharmaceutically effective amount. In the present invention, 'pharmaceutically effective amount' means an amount sufficient to treat the disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, activity of the drug, and the type and severity of the patient's disease. It can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the medical field. The effective dose is generally 0.01 mg to 5000 mg per day per 1 kg of body weight of the administered subject, and may be administered in divided doses once or several times a day at certain time intervals, depending on the judgment of a doctor or pharmacist, but is not limited thereto. The composition may be administered as an individual therapeutic agent, or in combination with other therapeutic agents, and may be administered simultaneously, separately, or sequentially with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art. Specifically, the effective amount of the composition may vary depending on the patient's age, gender, condition, weight, absorption of the active ingredient in the body, inactivation rate, excretion rate, type of disease, and concomitant drug, route of administration, severity of obesity, It may increase or decrease depending on gender, weight, age, etc., and may vary depending on the severity of the condition being treated. If necessary, for convenience, the total daily dose may be administered in divided doses several times throughout the day. For example, the daily dosage may be about 0.0001 mg/kg to about 10 g/kg, for example, about 0.001 mg/kg to about 1 g/kg once a day. In addition, the administration period may be 1 day to 2 months, but may be administered without limitation until the disease prevention or treatment effect appears. Additionally, depending on the judgment of a doctor or pharmacist, it may be administered in divided doses several times a day at regular time intervals, for example, 2 to 3 times a day.
2. 2. 위 점막 손상의 예방 또는 개선용 식품 또는 건강기능식품 조성물Food or health functional food composition for preventing or improving gastric mucosal damage
본 발명의 다른 측면은 위 점막 손상의 예방 또는 개선용 식품 또는 건강기능식품 조성물을 제공한다.Another aspect of the present invention provides a food or health functional food composition for preventing or improving gastric mucosal damage.
상기 본 발명의 식품 또는 건강기능식품 조성물은 오로라 키나아제 A(Aurora kinase A, AURKA) 억제제 또는 유비퀴틴 특이적 펩티다아제 14(ubiquitin specific peptidase 14, USP14) 억제제를 유효성분으로 포함하며, 이들에 대한 구체적인 사항은 위 '1. 위 점막 손상 관련 질환의 예방 또는 치료용 약학적 조성물 ' 항목에서 설명한 바와 같다.The food or health functional food composition of the present invention contains an Aurora kinase A (AURKA) inhibitor or a ubiquitin specific peptidase 14 (USP14) inhibitor as an active ingredient, and the specific details thereof are as follows. As described above in ‘ 1. Pharmaceutical composition for preventing or treating diseases related to damage to the gastric mucosa ’.
특히, 상기 위 점막 손상은 위 상피세포의 미토콘드리아가 손상됨으로써 유발되는 것일 수 있고, 특히 상기 위 상피세포의 미토콘드리아의 손상은 헬리코박터 파일로리(Helicobacter pylori)에 의한 것일 수 있다. 상기 위 상피세포는 MUC5AC+ 세포, MUC6+ 세포, SOX9+ 세포 등과 같은 점액-생성 세포들(mucous-producing cells)일 수 있고, 상기 점액-생성 세포는 미토콘드리아의 손상된 것일 수 있다. 또한, 상기 손상은 미토콘드리아가 단편화(fragmentation)되거나, 길이가 짧아지거나, 미토콘드리아의 호흡 용량이나 호흡 효율이 감소된 것일 수 있고, 상기와 같은 미토콘드리아의 손상으로 인해 상기 점액-생성 세포의 점액 분비가 감소된 것일 수 있다.In particular, the gastric mucosa damage may be caused by damage to the mitochondria of the gastric epithelial cells, and in particular, the damage to the mitochondria of the gastric epithelial cells may be caused by Helicobacter pylori . The gastric epithelial cells may be mucous-producing cells such as MUC5AC + cells, MUC6 + cells, SOX9 + cells, etc., and the mucus-producing cells may have damaged mitochondria. In addition, the damage may be due to fragmentation of the mitochondria, shortening of the length, or a decrease in the respiratory capacity or efficiency of the mitochondria, and the mucus secretion of the mucus-producing cells is reduced due to the damage to the mitochondria. It may have happened.
상기 예방은 위 점막의 손상을 방지하거나 억제 또는 지연시키는 모든 행위를 말하고, 상기 개선은 위 점막 손상을 호전시키거나 이롭게 변경시키는 모든 행위를 말한다.The above prevention refers to all actions that prevent, suppress, or delay damage to the gastric mucosa, and the above improvement refers to all actions that improve or beneficially change the damage to the gastric mucosa.
한편, 상기 식품은 먹을 수 있는 원재료 및 가공물질을 모두 포함하는 것으로서, 여러 가지 소화될 수 있는 영양소를 함유하는 것을 의미한다. Meanwhile, the food includes both edible raw materials and processed substances, meaning that it contains various digestible nutrients.
상기 식품 조성물을 식품첨가물로 사용하는 경우, 상기 식품 조성물을 그대로 첨가하거나 다른 식품 또는 식품성분과 함께 사용할 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. When using the food composition as a food additive, the food composition can be added as is or used together with other foods or food ingredients, and can be used appropriately according to conventional methods.
상기 식품의 종류에 특별한 제한은 없다. 상기 식품 조성물을 첨가할 수 있는 식품은 프로바이오틱스 제제일 수 있으며, 예컨대 육류, 소시지, 빵, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차 드링크제, 알콜 음료, 비타민 복합제 및 발효 식품등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함한다. 특히, 상기 발효 식품은 요구르트(하드 타입, 소프트 타입, 드링크 타입), 유산균 음료 등의 발효유, 치즈 또는 버터일 수 있으나, 이에 제한되는 것은 아니며, 발효 미생물 또는 유산균이 수행하는 발효에 의해 제조되는 어떠한 식품, 제품이라도 포함될 수 있다. There are no particular restrictions on the type of food. Foods to which the food composition can be added may be probiotic preparations, such as meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, It includes beverages, tea drinks, alcoholic beverages, vitamin complexes, and fermented foods, and includes all health foods in the conventional sense. In particular, the fermented food may be fermented milk, cheese or butter such as yogurt (hard type, soft type, drink type), lactic acid bacteria beverage, but is not limited thereto, and is not limited to any food manufactured by fermentation performed by fermentation microorganisms or lactic acid bacteria. Food and products may also be included.
또한, 상기 식품 조성물은 그 자체가 식품일 수도 있다. 상기 식품은 식품 제조 시에 통상적으로 첨가되는 성분을 포함하며, 예를 들어, 단백질, 탄수화물, 지방, 영양소 및 조미제를 포함한다. 예컨대, 드링크제로 제조되는 경우에는 유효성분 이외에 천연 탄수화물 또는 향미제를 추가 성분으로서 포함할 수 있다. 상기 천연 탄수화물은 모노사카라이드(예컨대, 글루코오스, 프럭토오스 등), 디사카라이드(예컨대, 말토스, 수크로오스 등), 올리고당, 폴리사카라이드(예컨대, 덱스트린, 시클로덱스트린 등) 또는 당알코올(예컨대, 자일리톨, 소르비톨, 에리쓰리톨 등)인 것이 바람직하다. 상기 향미제는 천연 향미제(예컨대, 타우마틴, 스테비아 추출물 등)와 합성 향미제(예컨대, 사카린, 아스파르탐 등)를 이용할 수 있다.Additionally, the food composition itself may be a food. The food includes ingredients commonly added during food production, such as proteins, carbohydrates, fats, nutrients, and seasonings. For example, when manufactured as a drink, natural carbohydrates or flavoring agents may be included as additional ingredients in addition to the active ingredient. The natural carbohydrates include monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g., dextrins, cyclodextrins, etc.), or sugar alcohols (e.g., , xylitol, sorbitol, erythritol, etc.) is preferable. The flavoring agent may be a natural flavoring agent (e.g., thaumatin, stevia extract, etc.) or a synthetic flavoring agent (e.g., saccharin, aspartame, etc.).
한편, 상기 건강기능식품 조성물은 인체에 유용한 기능성을 가진 원료나 성분을 사용한 식품으로서, 인체의 정상적인 기능을 유지하거나 생리기능 활성화를 통하여 건강을 유지하고 개선할 수 있는 기능성을 가진 식품을 의미한다. 본 발명의 건강기능식품은 건강기능식품에 관한 법률상의 건강기능식품에 해당한다. 상기 오로라 키나아제 A(Aurora kinase A, AURKA) 억제제 또는 유비퀴틴 특이적 펩티다아제 14(ubiquitin specific peptidase 14, USP14) 억제제는 위 전정의 상피에 존재하는 점액-생성 세포에서 유발되는 미토콘드리아의 손상을 방지 또는 회복할 수 있으므로, 상기 오로라 키나아제 A 억제제 또는 유비퀴틴 특이적 펩티다아제 14 억제제는 위 점막 손상을 예방 또는 개선하는 기능을 가진 기능성 원료로서 건강기능식품에 유효성분으로 포함될 수 있다.Meanwhile, the health functional food composition refers to a food using raw materials or ingredients with functionality useful to the human body, and has the functionality to maintain and improve health through maintaining the normal function of the human body or activating physiological functions. The health functional food of the present invention corresponds to a health functional food under the Health Functional Food Act. The Aurora kinase A (AURKA) inhibitor or the ubiquitin specific peptidase 14 (USP14) inhibitor prevents or restores mitochondrial damage caused by mucus-producing cells present in the epithelium of the gastric vestibule. Therefore, the Aurora Kinase A inhibitor or Ubiquitin-specific peptidase 14 inhibitor is a functional ingredient with the function of preventing or improving gastric mucosal damage and can be included as an active ingredient in health functional foods.
일반적으로, 본 발명의 건강기능식품 조성물은 상기 기능성 원료를 유효성분으로 그의 사용 목적(예방 또는 개선)에 따라 적절하게 포함할 수 있다. 예컨대 1μM 이상, 예컨대 5μM 이상, 10μM 이상, 20μM 이상 또는 50μM 이상의 농도로 포함될 수 있다. 또한, 상기 유효성분은 0.05mg, 0.1mg, 0.15mg, 0.2mg, 0.3mg, 0.5mg, 1mg, 2mg, 3mg, 5mg, 10mg, 20mg, 30mg, 50mg 및 100mg로부터 선택된 하나의 하한선 및/또는 500mg, 450mg, 400mg, 350mg, 320mg, 300mg, 280mg, 250mg, 200mg, 150mg 및 100mg로부터 선택된 하나의 상한선으로 구성된 범위의 양으로 함유될 수 있으며, 일례로, 0.05 내지 500mg, 0.05 내지 450mg, 0.05 내지 400mg, 0.05 내지 350mg, 0.05 내지 300mg, 0.05 내지 250mg, 0.1 내지 500mg, 0.1 내지 450mg, 0.1 내지 400mg, 0.1 내지 350mg, 0.1 내지 300mg, 0.1 내지 250mg, 0.1 내지 200mg, 0.2 내지 500mg, 0.2 내지 400mg, 0.2 내지 300mg, 0.5 내지 300mg, 1 내지 300mg, 5 내지 300mg, 또는 10 내지 300mg의 양으로 포함될 수 있다. 그러나 건강을 목적으로 장기간 섭취하는 경우에는 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로 사용될 수 있다.In general, the health functional food composition of the present invention may appropriately contain the above functional raw materials as active ingredients depending on the purpose of use (prevention or improvement). For example, it may be included at a concentration of 1 μM or more, such as 5 μM or more, 10 μM or more, 20 μM or more, or 50 μM or more. In addition, the active ingredient has a lower limit selected from 0.05mg, 0.1mg, 0.15mg, 0.2mg, 0.3mg, 0.5mg, 1mg, 2mg, 3mg, 5mg, 10mg, 20mg, 30mg, 50mg and 100mg and/or 500mg. , 450mg, 400mg, 350mg, 320mg, 300mg, 280mg, 250mg, 200mg, 150mg and 100mg, for example, 0.05 to 500mg, 0.05 to 450mg, 0.05 to 400mg. , 0.05 to 350 mg, 0.05 to 300 mg, 0.05 to 250 mg, 0.1 to 500 mg, 0.1 to 450 mg, 0.1 to 400 mg, 0.1 to 350 mg, 0.1 to 300 mg, 0.1 to 250 mg, 0.1 to 200 mg, 0.2 to 5 00mg, 0.2 to 400mg, 0.2 It may be included in an amount of 300 mg to 300 mg, 0.5 to 300 mg, 1 to 300 mg, 5 to 300 mg, or 10 to 300 mg. However, in case of long-term intake for health purposes, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.
상기 건강기능식품 조성물은 정제, 과립제, 연질캅셀제, 경질캅셀제, 시럽, 음료수, 환(丸), 연조엑스제, 중탕액 등의 제형으로 제조될 수 있다. 보다 구체적으로, 본 발명의 건강식품 조성물은 정제, 과립제, 연질캅셀제, 경질캅셀제, 시럽, 음료수, 환(丸), 연조엑스제, 건조엑스제, 추출중탕액 등의 제형으로 제조될 수 있다. 또한, 본 발명의 건강식품 조성물은 각각의 제형에 상기 기재한 성분들 이외에 건강기능식품의 제조에 통상적으로 첨가되는 성분, 예컨대 부형제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제, 향료, 색소, 방부제 등을 더 포함할 수 있다. 이러한 상기 첨가되는 성분의 비율은 크게 중요하진 않지만 상기 건강기능식품 조성물 100 중량부에 대하여, 0.01 내지 0.1 중량부의 범위에서 선택되는 것이 일반적이다.The health functional food composition can be manufactured in dosage forms such as tablets, granules, soft capsules, hard capsules, syrups, beverages, pills, soft extracts, and water baths. More specifically, the health food composition of the present invention can be manufactured in dosage forms such as tablets, granules, soft capsules, hard capsules, syrups, beverages, pills, soft extracts, dried extracts, and extraction baths. In addition, the health food composition of the present invention contains ingredients commonly added in the production of health functional foods, such as excipients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, and alginic acid, in addition to the ingredients described above in each formulation. It may further include salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, flavorings, pigments, preservatives, etc. The ratio of the added ingredients is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food composition.
3. 3. 헬리코박터 파일로리(Helicobacter pylori ( Helicobacter pyloriHelicobacter pylori )에 의한 위 점막 손상 관련 질환의 치료제 스크리닝 방법) Screening method for treatments for diseases related to gastric mucosal damage
본 발명의 일 측면은 헬리코박터 파일로리(Helicobacter pylori)에 의한 위 점막 손상 관련 질환의 치료제 스크리닝 방법을 제공한다.One aspect of the present invention provides a screening method for a treatment for diseases related to gastric mucosal damage caused by Helicobacter pylori .
본 발명의 상기 치료제 스크리닝 방법은, VacA가 처리된 인간 전정 위 오가노이드(human antrum gastric organoids, hAGOs)에 후보 물질을 처리하는 단계와, 상기 후보 물질의 처리 여부에 따라, 상기 hAGOs에서, 점액-생성 세포의 수 및 점액-생성 관련 유전자의 발현 중 어느 하나의 변화와 미토콘드리아의 활성 수준의 변화를 측정하는 단계를 포함한다.The therapeutic screening method of the present invention includes the steps of treating a candidate substance in human antrum gastric organoids (hAGOs) treated with VacA, and depending on whether the candidate substance is treated, in the hAGOs, mucus- It includes measuring a change in any one of the number of producing cells and the expression of mucus-production related genes and a change in the activity level of mitochondria.
본 발명의 상기 치료제 스크리닝 방법은, 먼저, VacA가 처리된 hAGOs에 후보 물질을 처리하는 단계를 포함한다.The therapeutic screening method of the present invention first includes the step of treating VacA-treated hAGOs with a candidate substance.
상기 VacA(vacuolating cytotoxin A)는 헬리코박터 파일로리가 발현하는 채널-형성 독소이다. 상기 VacA가 hAGOs에 처리되면, hAGOs에 존재하는 점액-생성 세포의 수가 감소되거나, 상기 점액-생성 세포의 미토콘드리아가 손상된다. 상기와 같은 미토콘드리아의 손상은 미토콘드리아가 단편화(fragmentation)되어 짧아지거나, 미토콘드리아의 호흡 용량과 호흡 효율이 저하된 것일 수 있고, 점액-생성 관련 유전자들의 발현이 감소된 것일 수도 있다.The VacA (vacuolating cytotoxin A) is a channel-forming toxin expressed by Helicobacter pylori. When the VacA is treated with hAGOs, the number of mucus-producing cells present in the hAGOs is reduced or the mitochondria of the mucus-producing cells are damaged. Mitochondrial damage as described above may be due to mitochondrial fragmentation and shortening, a decrease in mitochondrial respiratory capacity and respiratory efficiency, or a decrease in the expression of genes related to mucus production.
상기와 같이 점액-생성 세포의 수가 감소되거나 점액-생성 세포의 미토콘드리아가 손상된 hAGOs에 후보 물질이 처리되는데, 상기 후보 물질은 헬리코박터 파일로리에 의한 위 점막 손상 관련 질환을 예방 또는 치료할 수 있는 효과를 가질 수 있을 것으로 예상되는 모든 종류, 예컨대 핵산, 아미노산, 단당류 등과 같은 단일 화합물일 수도 있고, 폴리뉴클레오티드, 펩티드, 단백질, 다당류 등과 같은 중합체일 수도 있으며, 천연 또는 합성의 유기물, 무기물 또는 이들의 복합체 등의 분자, 또는 이들 모두의 혼합물 또는 이들을 생성하는 미생물 등을 의미한다. 상기 후보 물질들 중, hAGOs에서 상기와 같은 VacA에 의하여 감소된 점액-생성 세포의 수나 점액-생성 관련 유전자의 발현을 증가시키거나, 점액-생성 세포의 미토콘드리아의 손상을 회복시키는 것을 '치료제'라고 한다. 특히, 상기 미토콘드리아의 손상 회복은 형태학적으로 미토콘드리아가 융합(fusion)되어 길이가 길어지거나 ATP 생성 등의 활성이 증가되는 것일 수 있다. 다시 말해서, 상기 치료제는 hAGOs에 존재하는 점액-생성 세포의 수와 기능을 VacA가 처리되기 전의 상태로 회복시키는 것일 수 있다.As described above, the candidate material is treated with hAGOs where the number of mucus-producing cells is reduced or the mitochondria of the mucus-producing cells are damaged, and the candidate material may have the effect of preventing or treating diseases related to gastric mucosal damage caused by Helicobacter pylori. It may be a single compound of any type expected to exist, such as nucleic acids, amino acids, monosaccharides, etc., or a polymer such as polynucleotides, peptides, proteins, polysaccharides, etc., or a molecule such as natural or synthetic organic matter, inorganic matter, or a complex thereof. , or a mixture of all of them, or microorganisms that produce them. Among the candidate substances, those that increase the number of mucus-producing cells or the expression of genes related to mucus-producing cells, which are reduced by VacA in hAGOs, or restore mitochondrial damage in mucus-producing cells, are called 'therapeutics'. do. In particular, recovery from mitochondrial damage may be achieved by fusion of mitochondria morphologically, increasing their length or increasing their activity such as ATP production. In other words, the therapeutic agent may restore the number and function of mucus-producing cells present in hAGOs to the state before VacA treatment.
따라서, 본 발명의 상기 치료제 스크리닝 방법은, 상기와 같은 후보 물질의 처리 여부에 따라, 상기 hAGOs에서, 점액-생성 세포의 수 및 점액-생성 관련 유전자의 발현 중 어느 하나와 미토콘드리아 활성을 측정하는 단계를 포함한다. 이는 상기와 같이 hAGOs에서 점액-생성 세포의 활성의 변화를 측정함으로써 헬리코박터 파일로리에 의한 위 점막 손상 관련 질환을 치료할 수 있는지 여부를 알 수 있다는 점에 기반한 것으로서, hAGOs에서 점액-생성 세포의 수나 점액-생성 관련 유전자의 발현의 변화, 그리고 미토콘드리아의 활성 수준의 변화를 헬리코박터 파일로리에 의한 위 점막 손상 관련 질환의 치료 가부 판단에 이용할 수 있는 새로운 파라미터로 제시한 것에 기술적 의의가 있다.Accordingly, the therapeutic screening method of the present invention includes measuring either the number of mucus-producing cells or the expression of mucus-producing genes and mitochondrial activity in the hAGOs, depending on whether or not the candidate substance is processed. Includes. This is based on the fact that it is possible to know whether diseases related to gastric mucosal damage caused by Helicobacter pylori can be treated by measuring changes in the activity of mucus-producing cells in hAGOs as described above. It has technical significance in that it suggests changes in the expression of genes related to production and changes in the activity level of mitochondria as new parameters that can be used to determine whether treatment is possible for diseases related to gastric mucosal damage caused by Helicobacter pylori.
상기 점액-생성 세포는 MUC5AC+ 세포, MUC6+ 세포, SOX9+ 세포 등일 수 있고, 표면 점액 세포, 전정 경부 세포, 상피 전구체 세포 등일 수 있다. 또한, 상기 점액-생성 관련 유전자는 MUC5AC, MUC6, SOX9 등일 수 있으며, 상기 유전자의 발현은 mRNA 수준에서의 발현 또는 단백질 수준일 수 있다.The mucus-producing cells may be MUC5AC + cells, MUC6 + cells, SOX9 + cells, etc., and may be surface mucus cells, vestibular neck cells, epithelial precursor cells, etc. Additionally, the mucus-production-related gene may be MUC5AC, MUC6, SOX9, etc., and the expression of the gene may be at the mRNA level or at the protein level.
또한, 상기 미토콘드리아의 활성 수준은 미토콘드리아의 형태 변화 또는 ATP의 생성 수준을 통해 확인할 수 있고, 구체적으로 미토콘드리아가 융합(fusion)되어 길이가 길어지거나 ATP 생성량이 증가되면 활성이 증가된 것이고, 반대로 미토콘드리아가 단편화(fragmentation)되어 길이가 짧아지거나 ATP 생성량이 감소되면 활성이 감소된 것일 수 있다.In addition, the activity level of the mitochondria can be confirmed through changes in the shape of the mitochondria or the level of ATP production. Specifically, if the mitochondria are fused and become longer in length or the amount of ATP production increases, the activity increases. Conversely, the activity of the mitochondria increases. If the length becomes shorter due to fragmentation or the amount of ATP production decreases, the activity may be reduced.
또한, 본 발명의 상기 치료제 스크리닝 방법은, 상기와 같은 변화의 정도, 즉 hAGOs에서 점액-생성 세포의 수 및/또는 점액-생성 관련 유전자의 발현의 변화, 그리고 미토콘드리아의 ATP 생성 수준의 변화의 정도가 유의미한 경우에, 상기 후보 물질을 치료제로 판단하는 단계를 더 포함할 수 있다. 상기 hAGOs에서 점액-생성 세포의 수 및/또는 점액-생성 관련 유전자의 발현의 변화, 그리고 미토콘드리아의 ATP 생성 수준의 변화가 '유의미한 경우'는, 후보 물질의 처리 이후에 상기 점액-생성 세포의 수 및/또는 점액-생성 관련 유전자의 발현, 그리고 미토콘드리아의 ATP 생성 수준이 3% 이상, 5% 이상, 7% 이상, 10% 이상, 15% 이상, 20% 이상, 30% 이상, 40% 이상 또는 50% 이상 증가한 경우를 의미한다.In addition, the therapeutic screening method of the present invention is directed to the degree of the above changes, that is, the change in the number of mucus-producing cells and/or the expression of mucus-producing genes in hAGOs, and the degree of change in the ATP production level of mitochondria. If is significant, a step of determining the candidate substance as a therapeutic agent may be further included. If the changes in the number of mucus-producing cells and/or the expression of mucus-producing related genes in the hAGOs, and the changes in mitochondrial ATP production levels are 'significant', the number of mucus-producing cells after treatment with the candidate substance and/or expression of mucus-production related genes, and mitochondrial ATP production levels of 3% or more, 5% or more, 7% or more, 10% or more, 15% or more, 20% or more, 30% or more, or 40% or more. This means an increase of more than 50%.
한편, 상기 치료제는 VacA에 의해 감소된 병리학적 현상을 회복하는 것이므로, VacA에 의해 유발된, 즉 헬리코박터 파일로리에 의해 유발된 위 점막 손상 관련 질환의 치료제일 수 있고, 구체적으로 헬리코박터 파일로리에 의해 유발된 위염, 위궤양 또는 위암의 치료제일 수 있다.Meanwhile, since the therapeutic agent restores the pathological phenomenon reduced by VacA, it may be a therapeutic agent for diseases related to gastric mucosal damage caused by VacA, that is, caused by Helicobacter pylori, and specifically, caused by Helicobacter pylori. It may be a treatment for gastritis, stomach ulcers, or stomach cancer.
본 발명의 구체적인 실시예에서는 인간 전분화능줄기세포(human pluripotent stem cell)인 hESC(human embryonic stem cell)과 hiPSC(human induced pluripotent stem cell)로부터 각각 hAGOs(human antrum gastric organoids)를 확립하였고, 이와 같이 인간 전분화능줄기세포로부터 확립된 hAGOs에 VacA를 처리한 본 발명의 '헬리코박터 파일로리 감염에 의한 위 점막 손상 치료제 스크리닝 플랫폼'에 여러 후보물질을 처리하여, 상기 플랫폼에서 VacA의 처리에 의해 감소되었던 ATP 생성 수준을 유의하게 회복시키는 것으로 확인된 9종의 물질 중 오로라 키나아제 A 억제제나 유비퀴틴 특이적 펩티다아제 14 억제제를 선별하였다(도 12 참고). 그리고, 상기와 같이 선별된 오로라 키나아제 A 억제제나 유비퀴틴 특이적 펩티다아제 14 억제제들이 헬리코박터 파일로리의 감염에 의해 유발된 손상을 치료하는 효과가 있음을 인 비트로 또는 인 비보에서 다시금 확인하였다(도 13 내지 도 19 참고). 상기와 같은 결과로부터, 본 발명의 '헬리코박터 파일로리 감염에 의한 위 점막 손상 치료제 스크리닝 플랫폼'에 의해 빠르고 정확하게 헬리코박터 파일로리 감염에 의한 위 손상 치료제를 스크리닝해 낼 수 있음을 알 수 있다.In a specific embodiment of the present invention, human antrum gastric organoids (hAGOs) were established from hESC (human embryonic stem cell) and hiPSC (human induced pluripotent stem cell), which are human pluripotent stem cells, respectively. By treating hAGOs established from human pluripotent stem cells with VacA, several candidate substances were processed into the 'screening platform for the treatment of gastric mucosal damage caused by Helicobacter pylori infection' of the present invention, thereby generating ATP, which was reduced by treatment with VacA in the platform. Among the nine substances confirmed to significantly restore the level, Aurora Kinase A inhibitor and Ubiquitin-specific peptidase 14 inhibitor were selected (see Figure 12). In addition, it was confirmed again in vitro or in vivo that the Aurora Kinase A inhibitors or Ubiquitin-specific peptidase 14 inhibitors selected as above were effective in treating damage caused by Helicobacter pylori infection ( FIGS. 13 to 19 reference). From the above results, it can be seen that the 'screening platform for the treatment of gastric mucosal damage caused by Helicobacter pylori infection' of the present invention can quickly and accurately screen for the treatment of gastric damage caused by Helicobacter pylori infection.
4. 4. 헬리코박터 파일로리(Helicobacter pylori ( Helicobacter pyloriHelicobacter pylori )의 위 부착 억제제 스크리닝 방법) Gastric adhesion inhibitor screening method
본 발명의 다른 측면은 헬리코박터 파일로리(Helicobacter pylori)의 위 부착을 억제하는 위 부착 억제제의 스크리닝 방법을 제공한다.Another aspect of the present invention provides a screening method for a gastric adhesion inhibitor that inhibits gastric adhesion of Helicobacter pylori .
본 발명의 상기 위 부착 억제제 스크리닝 방법은, 헬리코박터 파일로리 균주가 처리된 인간 전정 위 오가노이드(human antrum gastric organoids, hAGOs)에 후보 물질을 처리하는 단계와, 상기 후보 물질의 처리 여부에 따라, 상기 hAGOs에 잔존하는 헬리코박터 파일로리 균주의 수준의 변화를 측정하는 단계를 포함한다.The screening method for a gastric adhesion inhibitor of the present invention includes the steps of treating a candidate material in human antrum gastric organoids (hAGOs) treated with a Helicobacter pylori strain, and depending on whether or not the candidate material is treated, the hAGOs It includes measuring changes in the level of Helicobacter pylori strains remaining in the.
본 발명의 상기 위 부착 억제제 스크리닝 방법은, 먼저, 헬리코박터 파일로리 균주가 처리된 hAGOs에 후보 물질을 처리하는 단계를 포함한다.The screening method for a gastric adhesion inhibitor of the present invention first includes the step of treating hAGOs treated with Helicobacter pylori strain with a candidate substance.
상기 hAGOs는 3차원 hAGOs를 단일세포로 해리하여 평판 배양한 2차원 hAGOs일 수 있다. 상기 2차원 hAGOs에 헬리코박터 파일로리 균주를 접종하고, 다시 일정 기간 동안 배양할 수 있다. 상기와 같이 hAGOs에 헬리코박터 파일로리 균주를 접종하여 공배양하면, 헬리코박터 파일로리 균주 표면에 존재하는 BabA 또는 SabA 등과 같은 위 부착 관련 단백질을 통해 상기 hAGOs의 상피세포에 부착된다.The hAGOs may be two-dimensional hAGOs obtained by dissociating three-dimensional hAGOs into single cells and culturing them on a plate. Helicobacter pylori strains can be inoculated into the two-dimensional hAGOs and cultured again for a certain period of time. When hAGOs are inoculated with Helicobacter pylori strains and co-cultured as described above, the hAGOs attach to epithelial cells through gastric adhesion-related proteins such as BabA or SabA present on the surface of the Helicobacter pylori strains.
상기와 같이 헬리코박터 파일로리 균주들이 부착된 hAGOs에 후보 물질이 처리되는데, 상기 후보 물질은 헬리코박터 파일로리가 위의 상피세포에 부착되는 것을 방지하거나, 위의 상피세포에 부착된 헬리코박터 파일로리를 떼어낼 수 있는 효과를 가질 수 있을 것으로 예상되는 모든 종류, 예컨대 핵산, 아미노산, 단당류 등과 같은 단일 화합물일 수도 있고, 폴리뉴클레오티드, 펩티드, 단백질, 다당류 등과 같은 중합체일 수도 있으며, 천연 또는 합성의 유기물, 무기물 또는 이들의 복합체 등의 분자, 또는 이들 모두의 혼합물 또는 이들을 생성하는 미생물 등을 의미한다. 상기 후보 물질들 중, hAGOs에 부착된 헬리코박터 파일로리의 수준을 감소시키는 것을 '위 부착 억제제'라고 한다.As described above, a candidate material is treated with hAGOs to which Helicobacter pylori strains are attached. The candidate material has the effect of preventing Helicobacter pylori from attaching to gastric epithelial cells or removing Helicobacter pylori attached to gastric epithelial cells. It may be a single compound such as any type expected to have, such as nucleic acids, amino acids, monosaccharides, etc., or a polymer such as polynucleotides, peptides, proteins, polysaccharides, etc., and may be a natural or synthetic organic substance, an inorganic substance, or a complex thereof. refers to molecules such as, or a mixture of all of them, or microorganisms that produce them. Among the candidate substances, those that reduce the level of Helicobacter pylori attached to hAGOs are called 'gastric adhesion inhibitors'.
따라서, 본 발명의 상기 위 부착 억제제 스크리닝 방법은, 상기와 같은 후보 물질의 처리 여부에 따라, 상기 hAGOs에 잔존하는 헬리코박터 파일로리 균주의 수준의 변화를 측정하는 단계를 포함한다. 상기 hAGOs에 부착된 헬리코박터 파일로리 균주의 수준은, 상기 hAGOs에 잔존하는 헬리코박터 파일로리 균주 유래의 고유 물질, 예컨대 헬리코박터 파일로리 균주 유래의 고유 유전자, 구체적으로 헬리코박터 파일로리 균주의 16S rRNA의 발현 수준을 측정함으로써 그 변화를 확인할 수 있다.Accordingly, the screening method for a gastric adhesion inhibitor of the present invention includes the step of measuring changes in the level of Helicobacter pylori strains remaining in the hAGOs, depending on whether or not the candidate material is treated. The level of the Helicobacter pylori strain attached to the hAGOs is changed by measuring the expression level of the unique material derived from the Helicobacter pylori strain remaining in the hAGOs, such as the unique gene derived from the Helicobacter pylori strain, specifically the 16S rRNA of the Helicobacter pylori strain. You can check.
또한, 본 발명의 상기 위 부착 억제제 스크리닝 방법은, 상기와 같은 변화의 정도, 즉 hAGOs에 잔존하는 헬리코박터 파일로리 균주 유래의 고유 물질의 변화의 정도가 유의미한 경우에, 상기 후보 물질을 위 부착 억제제로 판단하는 단계를 더 포함할 수 있다. 상기 hAGOs에 잔존하는 헬리코박터 파일로리 균주 유래의 고유 물질의 수준의 변화가 '유의미한 경우'는, 후보 물질을 처리하지 않은 경우에 비해, 후보 물질의 처리 이후에 상기 hAGOs에 잔존하는 헬리코박터 파일로리 균주 유래의 고유 물질의 수준이 3% 이상, 5% 이상, 7% 이상, 10% 이상, 15% 이상, 20% 이상, 30% 이상, 40% 이상 또는 50% 이상 감소한 경우를 의미한다.In addition, the gastric adhesion inhibitor screening method of the present invention determines the candidate substance as a gastric adhesion inhibitor when the degree of change as described above, that is, the degree of change in the intrinsic material derived from the Helicobacter pylori strain remaining in hAGOs is significant. Additional steps may be included. When the change in the level of the unique material derived from the Helicobacter pylori strain remaining in the hAGOs is 'significant', the unique material derived from the Helicobacter pylori strain remaining in the hAGOs after treatment of the candidate material is compared to the case where the candidate material is not treated. This means that the level of a substance has decreased by more than 3%, more than 5%, more than 7%, more than 10%, more than 15%, more than 20%, more than 30%, more than 40%, or more than 50%.
한편, 상기 위 부착 억제제는 헬리코박터 파일로리가 위에 부착되는 것을 억제하는 것이므로, 헬리코박터 파일로리에 의한 감염 자체를 원천적으로 차단하는 예방제일 수도 있고, 이미 위에 부착된 헬리코박터 파일로리를 떼어내는 치료제일 수도 있다.Meanwhile, the gastric adhesion inhibitor inhibits Helicobacter pylori from attaching to the stomach, so it may be a preventive agent that fundamentally blocks infection by Helicobacter pylori itself, or it may be a therapeutic agent that removes Helicobacter pylori that has already attached to the stomach.
본 발명의 구체적인 실시예에서는 인간 전분화능줄기세포(human pluripotent stem cell)인 hESC(human embryonic stem cell)과 hiPSC(human induced pluripotent stem cell)로부터 각각 hAGOs(human antrum gastric organoids)를 확립하였고, 이와 같이 인간 전분화능줄기세포로부터 확립된 hAGOs를 단일세포로 해리하여 평판 배양한 다음 헬리코박터 파일로리 P1 균주를 접종하여 공배양함으로써 본 발명의 '헬리코박터 파일로리의 위에 대한 부착 억제제 스크리닝 플랫폼'을 확립하였다. 상기와 같이 확립한 '헬리코박터 파일로리의 위에 대한 부착 억제제 스크리닝 플랫폼'에 여러 후보물질(신규 균주들의 배양물)을 처리한 다음, 상기 플랫폼에 잔존하는 헬리코박터 파일로리 P1 균주의 16S rRNA의 양을 측정하여, 상기 헬리코박터 파일로리 P1 균주의 16S rRNA의 양을 유의하게 감소시키는 것으로 확인된 5종의 신규 균주를 선별할 수 있었다(도 21 참고).In a specific embodiment of the present invention, human antrum gastric organoids (hAGOs) were established from hESC (human embryonic stem cell) and hiPSC (human induced pluripotent stem cell), which are human pluripotent stem cells, respectively. hAGOs established from human pluripotent stem cells were dissociated into single cells, cultured on plates, and then inoculated with Helicobacter pylori P1 strain and co-cultured, thereby establishing the 'screening platform for inhibitors of attachment of Helicobacter pylori to the stomach' of the present invention. Several candidate substances (cultures of new strains) were processed on the 'Helicobacter pylori adhesion inhibitor screening platform to the stomach' established as above, and then the amount of 16S rRNA of the Helicobacter pylori P1 strain remaining on the platform was measured, Five new strains that were confirmed to significantly reduce the amount of 16S rRNA of the Helicobacter pylori P1 strain were selected (see Figure 21).
이하, 본 발명을 실시예에 의하여 상세히 설명한다.Hereinafter, the present invention will be described in detail through examples.
단, 하기 실시예는 본 발명을 구체적으로 예시하는 것이며, 본 발명의 내용이 하기 실시예에 의해 한정되지 아니한다.However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.
[실시예 1][Example 1]
헬리코박터 파일로리에 의한 감염을 연구하기 위한 3D-오가노이드 모델의 확립 및 평가Establishment and evaluation of a 3D-organoid model to study infection by Helicobacter pylori
[1-1] 기관(organ) 수준의 위(stomach)의 '전정(antrum)'를 모사(mimic)할 수 있는 3D-오가노이드(hAGOs)의 확립[1-1] Establishment of 3D-organoids (hAGOs) that can mimic the ‘antrum’ of the stomach at the organ level
WiCell Research Institute(미국)에서 구입한 hESC(human embryonic stem cell) 라인 H9으로부터 hPSC(human pluripotent stem cell)을 형성하였고, 이를 종래 McCrachen et al.(2014)의 문헌(McCracken et al,, Nature, 516: 400-404 (2014))에 개시된 방법에 따라, 도 1에 도시된 바와 같이, 내배엽(definitive endoderm), 후측-전장(posterior-foregut) 과정을 거쳐 단계적으로 분화시켜 최종적으로 hAGOs(human antrum gastric organoids)를 확립하였다.hPSC (human pluripotent stem cell) was formed from hESC (human embryonic stem cell) line H9 purchased from WiCell Research Institute (USA), and this was previously described in McCrachen et al . (2014) (McCracken et al. , Nature, 516). According to the method disclosed in: 400-404 (2014), as shown in Figure 1, differentiation is carried out step by step through the process of endoderm (definitive endoderm) and posterior-foregut, and finally hAGOs (human antrum gastric) organoids) were established.
[1-2] hAGOs의 세포 구성 확인[1-2] Confirmation of cellular composition of hAGOs
상기 실시예 [1-1]에서 확립한 hAGOs에서 세포 유형별 특이적 마커를 확인하였다.Specific markers for each cell type were identified in the hAGOs established in Example [1-1].
구체적으로, 먼저, 상기 hAGOs 분화 단계 별 샘플을 제작한 다음, 이를 대상으로 면역형광(immunofluorescence) 분석을 수행하여 분화된 진정내배엽(definitive endoderm, DE)과 후측-전장(posterior-foregut) 스페로이드에 특이적인 마커들을 확인하였다. 그 결과, 도 2a에 도시된 바와 같이, 진정내배엽에 특이적인 마커인 FOXA2, SOX17과, 후측-전장 스페로이드에 특이적인 마커인 SOX2, PDX1, HNF6 및 HNF1β가 확인되었다.Specifically, first, samples were prepared for each stage of hAGOs differentiation, and then immunofluorescence analysis was performed on the samples to differentiate the differentiated definitive endoderm (DE) and posterior-foregut spheroids. Specific markers were identified. As a result, as shown in Figure 2a, FOXA2 and SOX17, which are specific markers for the euendoderm, and SOX2, PDX1, HNF6 and HNF1β, which are specific markers for the posterior-foregut spheroid, were identified.
또한, 상기 hAGOs 분화 단계별 세포에서 총 RNA를 분리하여 실시간-PCR을 수행하여, 그리고 추가적인 면역형광 분석을 통해, 상기 hAGOs가 어떠한 세포들로 구성되어 있는지 확인하였다. 그 결과, 도 2b 및 도 2c에 도시된 바와 같이, 전구세포(progenitor cells; SOX9), 표면 점액 세포(surface mucus cells; MUC5AC 및 GKN1), 전정 목 세포(antral neck cells; MUC6 및 TFF2) 및 내분비 세포(endocrine cells; SST 및 GAST)가 존재하는 것으로 확인된 반면, 위의 벽세포((parietal cells; ATP4A 및 ATP4B)나 주세포(chief cells; MIST1, PGA 및 PGC) 등과 같은 위 기저부-유사 세포들(fundus-like cells)이나, 장-유사 세포들(intestine-like cells; MUC2 및 VIL)은 존재하지 않는 것으로 확인되었다.In addition, total RNA was isolated from cells at each stage of hAGO differentiation, real-time-PCR was performed, and through additional immunofluorescence analysis, it was confirmed what kind of cells the hAGOs were composed of. As a result, as shown in Figures 2b and 2c, progenitor cells (SOX9), surface mucus cells (MUC5AC and GKN1), antral neck cells (MUC6 and TFF2), and endocrine cells Endocrine cells (SST and GAST) were found to be present, while gastric basal-like cells such as gastric parietal cells (ATP4A and ATP4B) or chief cells (MIST1, PGA and PGC) It was confirmed that there were no fundus-like cells or intestinal-like cells (intestine-like cells; MUC2 and VIL).
상기와 같은 결과로부터, 상기 실시예 [1-1]에서 확립한 hAGOs가 위(stomach)의 전정(antrum)에만 존재하는 다양한 유형의 세포들을 고르게 보유하고 있음을 알 수 있다.From the above results, it can be seen that the hAGOs established in Example [1-1] evenly retain various types of cells that exist only in the antrum of the stomach.
[1-3] 헬리코박터 파일로리의 감염에 따른 hAGOs의 생리학적 및 분자생물학적 현상 확인[1-3] Confirmation of physiological and molecular biological phenomena of hAGOs following infection with Helicobacter pylori
상기 실시예 [1-1]에서 확립한 hAGOs를 헬리코박터 파일로리의 채널-형성 독소인 VacA(vacuolating cytotoxin A)를 2ug/mL 내지 40ug/mL의 다양한 농도로 배양 배지에 처리하거나 오가노이드 내강(lumen)에 미세주입하여 48시간 동안 배양하였고, 그에 따라 hAGOs에서 일어나는 생리학적 변화와 분자생물학적 변화를 확인하였다.The hAGOs established in Example [1-1] were treated with VacA (vacuolating cytotoxin A), a channel-forming toxin of Helicobacter pylori, at various concentrations of 2ug/mL to 40ug/mL in the culture medium or in the organoid lumen. were microinjected and cultured for 48 hours, and the physiological and molecular biological changes occurring in hAGOs were confirmed.
그 결과, 도 3에 도시된 바와 같이, 20ug/mL의 VacA가 처리된 hAGOs에서 Ki67+ 세포의 수가 현저하게 감소하였고, 절단된 카스파아제-3+ 세포의 수가 현저히 증가하였다(도 3의 A). 그리고 세포 생존도는 대조군 대비 0.6배 감소하였고(도 3의 B), 카스파아제 3/7 활성은 대조군 대비 1.67배 증가하였다(도 3의 C). 상기와 같은 결과들은, 종래 문헌들에서 보고되어 온 위 상피세포주에서 VacA-유도성 세포사멸의 현상과 일치하는 것이다.As a result, as shown in Figure 3, the number of Ki67 + cells was significantly reduced in hAGOs treated with 20ug/mL of VacA, and the number of cleaved caspase-3 + cells was significantly increased (Figure 3A). . And cell viability decreased 0.6-fold compared to the control group (Figure 3B), and caspase 3/7 activity increased 1.67-fold compared to the control group (Figure 3C). The above results are consistent with the phenomenon of VacA-induced apoptosis in gastric epithelial cell lines that has been reported in previous literature.
또한, 도 4에 도시된 바와 같이, VacA 처리에 의해, hAGOs가 수축하였고 hAGOs의 표면적이 66.13%로 감소하였으며(도 4의 A), 상피층의 두께가 32.98±30.3um로 얇아졌고 곡률(curvature)도 감소하였다(도 4의 B). 또한, 밀착 연접(tight junction) 단백질의 위치가 변화하였고(도 4의 C), 밀착 연접(tight junction) 관련 ZO-1, OCLN, CLDN1, CLDN3, CLDN5 등의 분자들의 mRNA 발현도 감소되었다(도 4의 D). 나아가, hAGOs의 내강 영역(luminal region)으로 형광 염료(fluorescein isothiocyanate-dextra 4kDa, FD4)를 미세주입하고 그 움직임을 모니터링하는 투과성 분석(permeability assay)을 통해, 상기 형광 염료가 주변 배지로 빠르게 확산되어 버리는 것을 확인함으로써 밀착 연접이 손상되었음을 분명히 확인하였다(도 4의 E).In addition, as shown in Figure 4, by VacA treatment, hAGOs shrank and the surface area of hAGOs decreased to 66.13% (Figure 4A), and the thickness of the epithelial layer became thinner to 32.98 ± 30.3um and curvature. also decreased (B in Figure 4). In addition, the location of tight junction proteins changed (Figure 4C), and the mRNA expression of tight junction-related molecules such as ZO-1, OCLN, CLDN1, CLDN3, and CLDN5 was decreased (Figure 4C). D of 4). Furthermore, through a permeability assay that microinjects a fluorescent dye (fluorescein isothiocyanate-dextra 4kDa, FD4) into the luminal region of hAGOs and monitors its movement, the fluorescent dye rapidly diffuses into the surrounding medium. By confirming the discarding, it was clearly confirmed that the tight junctions were damaged (Figure 4E).
뿐만 아니라, VacA가 처리된 hAGOs에서 총 RNA를 분리한 다음, QuantSeq 3' mRNA-Seq Library Prep Kit(Lexogen)를 이용하여 제조사의 지시에 따라 라이브러리를 제조하였고, 상기 라이브러리를 대상으로 NetSeq500(Illumina)으로 전장 유전체 분석(whole genome sequencing, WGS)을 수행하였다. 그 결과, 도 5에 도시된 바와 같이, VacA 처리에 의해 hAGOs에서 전사체(transcriptome) 수준에서의 변화가 유발되었고(도 5의 A), 대조군과 VacA가 처리된 hAGOs 간의 차등 발현 유전자(differentially expressed genes, DEGs)들이 대부분 헬리코박터 파일로리 감염에 의해 유도되는 신호전달 경로, 특히 염증과 면역 관련 신호전달 경로와 밀접하게 연관된 것들로 확인되었다(도 5의 B). 나아가, VacA가 처리된 hAGOs의 배양 배지에서는 IL-6와 IL-8의 분비량도 증가된 것으로 관찰되어(도 5의 C), VacA에 의하여 염증도 유발됨이 분명히 확인되었다(도 5의 C).In addition, total RNA was isolated from VacA-treated hAGOs, and then a library was prepared using the QuantSeq 3' mRNA-Seq Library Prep Kit (Lexogen) according to the manufacturer's instructions, and the library was subjected to NetSeq500 (Illumina). Whole genome sequencing (WGS) was performed. As a result, as shown in Figure 5, VacA treatment induced changes at the transcriptome level in hAGOs (Figure 5A), and differentially expressed genes between control and VacA-treated hAGOs genes, DEGs) were most closely related to signaling pathways induced by Helicobacter pylori infection, especially inflammation- and immune-related signaling pathways (Figure 5B). Furthermore, increased secretion of IL-6 and IL-8 was observed in the culture medium of hAGOs treated with VacA (Figure 5C), clearly confirming that inflammation is also induced by VacA (Figure 5C). .
결국, VacA가 처리된 hAGOs에서는 유전자 발현이나 염증성 사이토카인의 분비량의 측면에서도, 종래 문헌들에서 보고된 헬리코박터 파일로리에 감염된 위에서와 동일한 현상이 나타나는 것으로 확인되었다.In the end, it was confirmed that the same phenomenon as the Helicobacter pylori-infected stomach reported in previous literature occurred in hAGOs treated with VacA in terms of gene expression and secretion of inflammatory cytokines.
[1-4] hAGOs에서 헬리코박터 파일로리의 감염이 미토콘드리아의 활성에 미치는 영향 확인[1-4] Confirmation of the effect of Helicobacter pylori infection on mitochondrial activity in hAGOs
종래 VacA는 미토콘드리아를 표적화하는 독소로 보고된 바 있다. 이에, 상기 실시예 [1-1]에서 확립한 hAGOs를 이용하여 VacA에 의해 미토콘드리아의 활성이 어떻게 달라지는지 확인하였다.Previously, VacA was reported to be a toxin targeting mitochondria. Accordingly, the hAGOs established in Example [1-1] were used to determine how mitochondrial activity varies by VacA.
구체적으로, 20ug/mL의 VacA가 48시간 동안 처리된 hAGOs에서 TOM20(translocase of the outer membrane 20)과 같은 미토콘드리아 단백질 이송(mitochondrial protein import)의 마커를 염색하여, 도 6의 A에 도시된 바와 같이, 미토콘드리아의 단편화(fragmentation)가 현저히 증가되었음을 확인하였다(도 6의 A). 또한, VacA가 처리된 hAGOs에서 총 RNA를 분리하고 실시간-PCR을 수행하여, 도 6의 B에 도시된 바와 같이, NDUFA2, NDUFS3, SDHA, SDHD, UQCRQ, COX10, MT-ATP6 등과 같은 산화적-인산화 관련 유전자들의 발현이 감소되었음을 확인하였다(도 6의 B). 나아가, Seahorse XFe96 Flux Analyzer(Agilent)를 이용하여 VacA가 처리된 hAGOs에서 OCR(oxygen consumption rate), 최대 호흡(maximal respiration), 미토콘드리아 ATP 생성률 등을 측정하여, 도 6의 C 내지 E에 도시된 바와 같이, 호흡 용량 및 호흡 효율과 같은 미토콘드리아의 활성 자체도 전반적으로 저하됨을 확인하였다(도 6의 C 내지 E).Specifically, 20ug/mL of VacA stained markers of mitochondrial protein import such as TOM20 (translocase of the outer membrane 20) in hAGOs treated for 48 hours, as shown in A in Figure 6. , it was confirmed that mitochondrial fragmentation was significantly increased (Figure 6A). In addition, total RNA was isolated from VacA-treated hAGOs and real-time-PCR was performed, and as shown in Figure 6B, oxidative- such as NDUFA2, NDUFS3, SDHA, SDHD, UQCRQ, COX10, MT-ATP6, etc. It was confirmed that the expression of phosphorylation-related genes was decreased (Figure 6B). Furthermore, OCR (oxygen consumption rate), maximal respiration, mitochondrial ATP production rate, etc. were measured in VacA-treated hAGOs using the Seahorse Likewise, it was confirmed that mitochondrial activity itself, such as respiratory capacity and respiratory efficiency, was also overall reduced (Figure 6, C to E).
결국, VacA가 처리된 hAGOs에서는 미토콘드리아의 활성의 측면에서도, 종래 문헌들에서 보고된 헬리코박터 파일로리에 감염된 위에서와 동일한 현상이 나타나는 것으로 확인되었다.In the end, it was confirmed that in terms of mitochondrial activity, the same phenomenon as the Helicobacter pylori-infected stomach reported in previous literature occurred in hAGOs treated with VacA.
[1-5] 헬리코박터 파일로리에 의한 감염을 연구하기 위한 모델로서의 hAGOs의 적합성 평가[1-5] Evaluation of the suitability of hAGOs as a model for studying infection by Helicobacter pylori
상기 실시예 [1-2] 내지 [1-4]에서의 결과로부터, 상기 실시예 [1-1]에서 확립한 hAGOs가 다양한 세포 구성(cellular complexity)의 측면에서 위의 전정을 잘 재현하고 있을 뿐만 아니라, 3차원 구조나 밀착 연접에 의한 상피층의 장벽 기능, 그리고 미토콘드리아의 기능의 측면에서도 위의 전정을 잘 모사하고 있고 있음을 알 수 있다. 따라서 상기 실시예 [1-1]에서 확립한 3차원의 hAGOs는 헬리코박터 파일로리에 의한 감염을 연구하기에 적합한 모델이라 할 수 있다.From the results in Examples [1-2] to [1-4], it can be seen that the hAGOs established in Example [1-1] well reproduce the above pruning in terms of various cellular complexity. In addition, it can be seen that the above vestibule is well replicated in terms of the three-dimensional structure, barrier function of the epithelial layer by tight junctions, and mitochondrial function. Therefore, the three-dimensional hAGOs established in Example [1-1] can be said to be a suitable model for studying infection by Helicobacter pylori.
[실시예 2][Example 2]
hAGOs를 이용한, 헬리코박터 파일로리의 감염의 영향에 대한 상세 확인Detailed confirmation of the effects of Helicobacter pylori infection using hAGOs
[2-1] hAGOs에서 VacA에 의해 영향을 받는 세포 유형의 확인[2-1] Identification of cell types affected by VacA in hAGOs
hAGOs에서 VacA에 의하여 영향을 받는 세포 유형이 어떠한 것인지 확인하기 위하여, 20ug/mL의 VacA가 처리된 hAGOs를 대상으로 단일-세포 RNA-서열분석(single-cell RNA-sequencing, scRNA-seq)을 수행하였다.To confirm which cell types are affected by VacA in hAGOs, single-cell RNA-sequencing (scRNA-seq) was performed on hAGOs treated with 20ug/mL VacA. did.
그 결과, 도 7a 내지 도 7e에 도시된 바와 같이, VacA는 hAGOs의 위 상피층에서 광범위(global scale)하게 유전자 발현 프로파일에 영향을 미쳤고, 잘 알려진 인간 위 점막(mucosa)-특이적 마커를 기준으로 총 7개의 주요 클러스터들이 확인되었다(도 7a 내지 도 7c). 또한, VacA의 처리에 의해, hAGOs 내의 세포 유형의 비율이 변하였고(도 7d), 위 점액의 분비와 관련된 유전자들인 MUC5AC, MUC6와 위 점액분비세포로 분화할 수 있는 전구세포 마커인 SOX9의 발현이 감소한 것으로 확인되었다(도 7d).As a result, as shown in Figures 7a to 7e, VacA affected the gene expression profile in the gastric epithelial layer of hAGOs on a global scale, based on well-known human gastric mucosa-specific markers. A total of seven major clusters were identified (Figures 7a to 7c). In addition, by treatment with VacA, the ratio of cell types in hAGOs was changed (Figure 7d), and the expression of genes related to gastric mucus secretion, MUC5AC and MUC6, and SOX9, a progenitor cell marker that can differentiate into gastric mucus secreting cells, was observed. It was confirmed that this decreased (Figure 7d).
아울러, 상기와 같은 scRNA-seq 결과를 다시 한번 확인하기 위하여, VacA가 처리된 hAGOs를 대상으로 면역형광 분석을 수행하여 위 전장-특이적 마커들의 수준을 확인하였다.In addition, in order to confirm the above scRNA-seq results once again, immunofluorescence analysis was performed on VacA-treated hAGOs to confirm the levels of the above full-length-specific markers.
그 결과, 도 8a 내지 도 8e에 도시된 바와 같이, 표면 점액 세포(surface mucous cell)의 마커인 MUC5AC, 전정 경부 세포(antral neck cell)의 마커인 MUC6, 상피 전구체 세포(epithelial progenitor cell)의 마커인 SOX9, 그리고 비록 적지만 내분비 세포(endocrine cell)의 마커인 SST가 모두 감소하였고(도 8a 및 도 8b), 특히 MUC5AC의 발현은 시간-의존적으로 확연히 감소하여(도 8c), VacA에 의하여 hAGOs에서 점액-생성 세포의 수가 전반적으로 감소됨을 확인하였다. 이는 VacA가 처리된 hAGOs를 PAS(Periodic acid-Schiff) 염색으로 관찰하였을 때 점액층의 두께가 매우 얇아진 것과 일치하는 결과이다(도 8d).As a result, as shown in Figures 8a to 8e, MUC5AC, a marker of surface mucous cells, MUC6, a marker of antral neck cells, and a marker of epithelial progenitor cells SOX9 and, although to a small extent, SST, a marker of endocrine cells, were all decreased (Figures 8a and 8b), and in particular, the expression of MUC5AC was significantly decreased in a time-dependent manner (Figure 8c), indicating that hAGOs were inhibited by VacA. It was confirmed that the number of mucus-producing cells was overall reduced. This result is consistent with the fact that the thickness of the mucus layer became very thin when VacA-treated hAGOs were observed with PAS (Periodic acid-Schiff) staining (Figure 8d).
나아가, VacA의 처리에 의하여, 어떠한 세포의 미토콘드리아가 손상되는지 확인하였다. 각각의 세포 군집에서 TOM20과 세포-유형-특이적 마커들을 함께 염색하여 평균적인 미토콘드리아의 길이를 확인한 결과, 도 8의 E에 도시된 바와 같이, MUC5AC+ 세포 군집에서는 4.24±0.18um에서 4.96±0.03um로, MUC6+ 세포 군집에서는 4.01±0.31um에서 1.54±0.17um로, SOX9+ 세포 군집에서는 4.04±0.25um에서 2.64±0.15um로, VacA의 처리에 의해 점액-생성 세포들에서 미토콘드리아의 평균 길이가 현저하게 짧아진 것으로 확인되었다(도 8e).Furthermore, it was confirmed which cell mitochondria were damaged by treatment with VacA. As a result of confirming the average mitochondrial length by staining with TOM20 and cell-type-specific markers in each cell population, it ranged from 4.24 ± 0.18 μm to 4.96 ± 0.03 in the MUC5AC + cell population, as shown in Figure 8E. In μm, the average length of mitochondria in mucin-producing cells by treatment with VacA increased from 4.01 ± 0.31 μm to 1.54 ± 0.17 μm in the MUC6 + cell population and from 4.04 ± 0.25 μm to 2.64 ± 0.15 μm in the SOX9 + cell population. was confirmed to be significantly shorter (Figure 8e).
아울러, hAGO를 형성하는데 이용된 인간 전분화능줄기세포의 종류에 따라 VacA의 처리에 따른 효과가 달라질 수 있는지 확인하기 위하여, hESC(human embryonic stem cell)가 아닌 hiPSC(human induced pluripotent stem cell)를 이용하여 상기 실시예 [1-1]에서와 동일한 방법으로 hAGOs를 형성하였고, 이렇게 형성된 hAGOs를 대상으로 VacA를 처리하여 그에 따른 변화를 확인하였다. 그 결과, 도 9에 도시된 바와 같이, hiPSC로부터 분화되어 형성된 hAGOs에서도 도 8a 내지 도 8e에서와 동일한 변화가 확인되었다(도 9).In addition, to determine whether the effect of VacA treatment may vary depending on the type of human pluripotent stem cell used to form hAGO, hiPSC (human induced pluripotent stem cell) rather than hESC (human embryonic stem cell) was used. Thus, hAGOs were formed in the same manner as in Example [1-1], and the hAGOs thus formed were treated with VacA to confirm the resulting changes. As a result, as shown in Figure 9, the same changes as in Figures 8a to 8e were confirmed in hAGOs formed by differentiation from hiPSCs ( Figure 9 ).
상기와 같은 결과로부터, VacA는, hAGOs를 형성하는데 이용된 인간 전분화능즐기세포의 종류에 상관 없이, hAGOs에서 주로 점액-생성 세포들(MUC5AC+ 세포 및 MUC6+ 세포)의 미토콘드리아 구조를 손상시켜, ATP 생성과 뒤이은 점액 분비에 매우 중요한 역할을 하는 미토콘드리아의 호흡 용량 및 호흡 효율을 감소시킬 뿐만 아니라, 점액층을 얇아지게 만듦을 알 수 있다.From the above results, VacA damages the mitochondrial structure of mainly mucus-producing cells (MUC5AC + cells and MUC6 + cells) in hAGOs, regardless of the type of human pluripotent cells used to form hAGOs, It can be seen that it not only reduces the respiratory capacity and respiratory efficiency of mitochondria, which play a very important role in ATP production and subsequent mucus secretion, but also makes the mucus layer thinner.
[2-2] 헬리코박터 파일로리에 감염된 인간 위 조직의 점액 생성 세포에서, 미토콘드리아 손상 여부 확인[2-2] Determination of mitochondrial damage in mucus-producing cells of human stomach tissue infected with Helicobacter pylori
헬리코박터 파일로리에 감염된 인간 환자의 위 조직을 이용하여, hAGOs에서 확인된 상기 실시예 [2-1]의 결과가 실제 임상에서도 동일하게 나타나는 것인지를 확인하였다.Using stomach tissue from a human patient infected with Helicobacter pylori, it was confirmed whether the results of Example [2-1] confirmed in hAGOs were the same in actual clinical practice.
이를 위해, 경북대학교병원에서 정상인 11명과 헬리코박터 파일로리에 감염된 것으로 확진된 환자 27명의 위 조직 샘플을, 상기 38명의 동의 하에, 이들에 대한 연령, 성별, 헬리코박터 파일로리 감염 및 치료 전력, 항생제 치료 전력, 병력, 내시경 소견 등의 인구통계학적(demographic) 및 임상 자료와 함께 입수하였다.For this purpose, gastric tissue samples from 11 normal people and 27 patients confirmed to be infected with Helicobacter pylori were collected at Kyungpook National University Hospital, and with the consent of the 38 people, age, gender, history of Helicobacter pylori infection and treatment, history of antibiotic treatment, and medical history were collected. , demographic and clinical data such as endoscopic findings were obtained.
먼저, 상기 자료들을 분석하였다. 그 결과, 아래 표 1에 기재된 바와 같이, 호중구 침투(neutrophil infiltration), 단핵세포 침투(mononuclear cell infiltration) 및 위축 점수(atrophy scores)는 헬리코박터 파일로리 감염 환자들에서 훨씬 높은 것으로 확인되었다.First, the above data were analyzed. As a result, as shown in Table 1 below, neutrophil infiltration, mononuclear cell infiltration, and atrophy scores were found to be much higher in Helicobacter pylori-infected patients.
[표 1][Table 1]
Figure PCTKR2023005398-appb-img-000001
Figure PCTKR2023005398-appb-img-000001
아울러, 상기 위 조직 샘플의 위 점막(gastric mucosa)을 대상으로 면역형광 및 면역조직화학 분석을 수행하는 한편, 실시간-PCR을 수행하였다. 그 결과, 도 10a 내지 도 10e에 도시된 바와 같이, 헬리코박터 파일로리 감염 환자 유래의 샘플에서, 위 상피세포의 세포질에 VacA가 높은 수준으로 존재하는 것으로 확인되었을 뿐만 아니라(도 10a), 위 상피 장벽이 손상되고, ZO-1, CLDN1, OCLN 등과 같은 밀착 연접 관련 유전자들의 발현 역시 전사 수준 및 단백질 수준에서 모두 감소된 것으로 확인되었다(도 10b 및 도 10c). 또한, 헬리코박터 파일로리 감염 환자 유래의 샘플에서 MUC5AC의 발현이 특별히 현저하게 감소되어(도 10d), 위 점막 장벽이 심하게 손상된 것으로 확인되었다. 한편, 헬리코박터 파일로리 감염 환자 유래의 샘플에서는 점액을 생성하는 MUC5AC+ 세포들에서 미토콘드리아가 단편화되어 있을 뿐만 아니라(도 10d), 그 길이도 매우 짧아져 있는 것으로 확인되었다(도 10e).상기와 같은 결과로부터, 상기 실시예 [2-1]을 통해 hAGOs에서 확인된 점액-생성 세포들에서의 미토콘드리아 손상 등과 같은 현상들이, 실제 헬리코박터 파일로리에 감염된 환자의 조직에서도 동일하게 나타남을 알 수 있고, 나아가 병리학적 현상의 측면에서도 VacA가 처리된 hAGO 모델이 VacA-양성 헬리코박터 파일로리를 보균하고 있는 위장계를 잘 모사하고 있음을 알 수 있다.In addition, immunofluorescence and immunohistochemical analysis were performed on the gastric mucosa of the stomach tissue sample, while real-time-PCR was performed. As a result, as shown in FIGS. 10A to 10E, not only was it confirmed that VacA was present at a high level in the cytoplasm of gastric epithelial cells in samples derived from patients infected with Helicobacter pylori (FIG. 10A), but also the gastric epithelial barrier was It was confirmed that the expression of damaged, tight junction-related genes such as ZO-1, CLDN1, OCLN, etc. was also reduced at both the transcription and protein levels (Figures 10b and 10c). In addition, the expression of MUC5AC was particularly significantly reduced in samples derived from patients infected with Helicobacter pylori (Figure 10d), confirming that the gastric mucosal barrier was severely damaged. Meanwhile, in samples from Helicobacter pylori-infected patients, it was confirmed that mitochondria in mucus-producing MUC5AC + cells were not only fragmented (Figure 10d), but their length was also greatly shortened (Figure 10e). Results similar to those described above. From this, it can be seen that the phenomena such as mitochondrial damage in mucus-producing cells identified in hAGOs through Example [2-1] appear equally in the tissues of patients actually infected with Helicobacter pylori, and furthermore, the pathological In terms of the phenomenon, it can be seen that the VacA-treated hAGO model well replicates the gastrointestinal system harboring VacA-positive Helicobacter pylori.
[실시예 3][Example 3]
헬리코박터 파일로리 감염에 의한 위 점막 손상의 치료제를 발굴하기 위한 플랫폼의 확립 및 이를 이용한 치료제의 스크리닝Establishment of a platform to discover treatments for gastric mucosal damage caused by Helicobacter pylori infection and screening of treatments using this platform
[3-1] 치료제 발굴을 위한 플랫폼 확립[3-1] Establishment of a platform for discovering treatments
상기 실시예 2에서의 결과로부터, 점액-생성 세포들에서 미토콘드리아의 기능을 회복시킴으로써 헬리코박터 파일로리 감염에 의한 병리학적 현상을 회복할 수 있을 것이라는 가설을 세우고, 미토콘드리아 분열 억제제(mitochondrial fission inhibitor)인 mdivi-1을 이용하여 상기 가설을 검증해 보았다.From the results in Example 2, we hypothesized that the pathological phenomenon caused by Helicobacter pylori infection could be recovered by restoring the function of mitochondria in mucus-producing cells, and mdivi-, a mitochondrial fission inhibitor, was used. 1 was used to test the above hypothesis.
구체적으로, 20ug/mL의 VacA가 처리된 hAGOs에 50uM의 mdivi-1을 24시간 동안 처리한 다음, 그에 따른 형태학적 및 생리화학적 변화를 관찰하였다.Specifically, hAGOs treated with 20ug/mL VacA were treated with 50uM of mdivi-1 for 24 hours, and the resulting morphological and physiochemical changes were observed.
그 결과, 도 11a 내지 도 11c에 도시된 바와 같이, mdivi-1의 처리에 의해 단편화되었던 미토콘드리아가 필라멘트형으로 변화되었고(도 11a), ATP 생성량이 증가된 것으로 확인되었다(도 11b). 아울러, 내강 영역으로 미세주입된 형광 염료(FD4) 또한 주변으로 확산되어 버리지 않고 내강 영역에 그대로 존재하고 있는 것으로 확인되어(도 11c), VacA에 의해 손상되었던 밀착 연접 또한 온전하게 회복되었음을 알 수 있다.As a result, as shown in FIGS. 11A to 11C, the mitochondria that had been fragmented by treatment with mdivi-1 were changed to a filament type (FIG. 11A), and the amount of ATP production was confirmed to be increased (FIG. 11B). In addition, the fluorescent dye (FD4) microinjected into the lumen area was also confirmed to remain in the lumen area without diffusing to the surroundings (Figure 11c), indicating that the tight junctions damaged by VacA were also fully restored. .
상기와 같은 결과로부터, 미토콘드리아의 활성 수준을 확인할 수 있는 대표적인 특징인 미토콘드리아의 형태(morphology)와 ATP 생성 수준을 회복시킴으로써, 헬리코박터 파일로리 감염에 의한 상피 장벽의 손상도 회복시킬 수 있고, 따라서 상기 가설이 유효한 것임을 분명히 알 수 있다.From the above results, by restoring mitochondrial morphology and ATP production level, which are representative characteristics that can confirm the level of mitochondrial activity, damage to the epithelial barrier caused by Helicobacter pylori infection can also be restored, and thus the above hypothesis is supported. It is clear that it is valid.
이에, 상기 가설에 따라, hAGOs를 이용하여 VacA에 의해 손상된 점액-생성 세포들의 미토콘드리아의 형태(morphology)와 ATP 생성 수준을 회복시킬 수 있는 물질을 헬리코박터 파일로리 감염에 의한 위 점막 손상의 치료제로 발굴해 내는, 이른바 '헬리코박터 파일로리 감염에 의한 위 점막 손상 치료제 스크리닝 플랫폼'을 확립하였다.Accordingly, according to the above hypothesis, a substance that can restore the mitochondrial morphology and ATP production level of mucus-producing cells damaged by VacA using hAGOs was discovered as a treatment for gastric mucosal damage caused by Helicobacter pylori infection. We have established a so-called 'screening platform for treatments for gastric mucosal damage caused by Helicobacter pylori infection'.
[3-2] 상기 플랫폼을 이용한 치료제의 발굴[3-2] Discovery of treatments using the above platform
상기 실시예 [3-1]에서 확립된 플랫폼에 따라, 20ug/mL의 VacA가 48시간 동안 처리된 hAGOs에, ACL(autophagy compound library)(Selleckchem)에 포함된 150종의 자가포식-조절제들(sutophagy-modulators)을 각각 10uM의 농도씩 처리하거나, 또는 mdivi-1을 50uM의 농도로 24시간 동안 처리한 다음, CellTiter-Glo® 3D Cell Viability Assay(Promega)를 이용하여 표준 프로토콜에 따라 ATP의 농도를 측정하였다.According to the platform established in Example [3-1], 150 types of autophagy-regulators included in the ACL (autophagy compound library) (Selleckchem) were added to hAGOs treated with 20ug/mL of VacA for 48 hours. sutophagy-modulators) were treated at a concentration of 10uM each, or mdivi-1 was treated at a concentration of 50uM for 24 hours, and then the concentration of ATP was measured using CellTiter-Glo ® 3D Cell Viability Assay (Promega) according to standard protocols. was measured.
그 결과, 도 12에 도시된 바와 같이, 총 9종의 화합물들이 VacA의 처리에 의해 감소되었던 hAGOs에서의 ATP 생성 수준을 유의하게 회복시키는 것으로 확인되었다(도 12). 그 중에서 효과가 가장 우수한 것으로 확인된 두 개의 화합물을 중심으로 분류하여, 특별히 오로라 키나아제 A(Aurora kinase A, AURKA) 억제제인 MLN8054, MLN8237, MK8745, MK5108과, 유비퀴틴 특이적 펩티다아제 14(ubiquitin specific peptidase 14, USP14) 억제제인 IU1, WP1130을 선별하였다.As a result, as shown in Figure 12, a total of 9 compounds were confirmed to significantly restore the level of ATP production in hAGOs, which had been reduced by treatment with VacA (Figure 12). Among them, we classified them based on the two compounds that were confirmed to be the most effective, especially Aurora kinase A (AURKA) inhibitors MLN8054, MLN8237, MK8745, MK5108, and ubiquitin specific peptidase 14. , USP14) inhibitors IU1 and WP1130 were selected.
[실시예 4][Example 4]
MLN8054 및 IU1의 위 점막 손상 치료 효과의 검증 - 1Verification of the effectiveness of MLN8054 and IU1 in treating gastric mucosal damage - 1
[4-1] hAGOs를 이용한, VacA에 의해 유도된 손상에 대한 치료 효과의 확인[4-1] Confirmation of treatment effect on damage induced by VacA using hAGOs
상기 실시예 [3-2]에서 선별된 AURKA 억제제와 USP14 억제제 중 가장 효과가 우수한 것으로 확인된 MLN8054와 IU1에 대하여, hAGOs를 이용하여 추가적으로 위 점막 손상 치료 효과를 검증하였다. 이를 위해, 20ug/mL의 VacA가 48시간 동안 처리된 hAGOs에 MLN8054 또는 IU1을 10uM씩 처리하여 24시간 동안 배양한 다음, 형태학적 또는 분자생물학적 변화를 관찰하였다.MLN8054 and IU1, which were confirmed to be the most effective among the AURKA inhibitors and USP14 inhibitors selected in Example [3-2], were additionally verified for their effectiveness in treating gastric mucosal damage using hAGOs. For this purpose, hAGOs treated with 20ug/mL of VacA for 48 hours were treated with 10uM of MLN8054 or IU1 and cultured for 24 hours, and then morphological or molecular biological changes were observed.
그 결과, 도 13 내지 도 15에 도시된 바와 같이, VacA에 의해 손상되었던 형태가 MLN8054 또는 IU1에 의해 다시 회복되었고(도 13의 A), VacA에 의해 감소되었던 OCR과 미토콘드리아 ATP 생성률이 mdivi-1이 처리되었을 때와 비슷한 수준으로 회복되었다(도 13의 B, C). 또한, VacA에 의해 손상되었던 점액-생성 기능과 상피층의 두께가 정상으로 회복되었고(도 14a), 밀착 연접 관련 유전자들의 전사-수준에서의 발현이나 ZO-1 및 CLDN1의 단백질 조립 역시 눈에 띄게 증가하였으며(도 14b 및 도 14c), 그 결과 상피층의 장벽 기능 역시 온전하게 회복되었음을 형광 염료 투과성 분석을 통해 다시 한번 확인하였다(도 14d).As a result, as shown in Figures 13 to 15, the form damaged by VacA was restored by MLN8054 or IU1 (A of Figure 13), and the OCR and mitochondrial ATP production rate, which had been reduced by VacA, were reduced by mdivi-1 It recovered to a similar level as when treated (Figure 13B, C). In addition, the mucus-producing function and epithelial layer thickness damaged by VacA were restored to normal (Figure 14a), and the transcription-level expression of tight junction-related genes and protein assembly of ZO-1 and CLDN1 were also noticeably increased. (Figures 14b and 14c), and as a result, it was confirmed once again through fluorescent dye permeability analysis that the barrier function of the epithelial layer was also fully restored (Figure 14d).
한편, MLN8054가 처리된 경우에는, VacA에 의해 감소되었던 미토콘드리아의 호흡에 관여하는 중요한 단백질인 ATP5A, ATP5B의 발현이나 미토콘드리아의 분열에 관여하는 중요한 단백질인 MFN1, MFN2의 발현이 다시 회복되었고(도 15a 및 도 15b), VacA의 처리에 의해 증가되었던 미토콘드리아의 단편화 역시 감소된 것으로 확인되었다(도 15c). 그리고 IU1이 처리된 경우에는, VacA에 의해 감소되었던, 미토파지(mitophagy)의 활성화, 손상된 미토콘드리아의 제거 및 미토콘드리아의 기능 회복에 중요하게 관여하는 PINK/Parkin 신호전달 경로 상의 PINK1, Parkin, p-AMPK, LC3-II 등의 단백질들의 발현이 다시 회복되었고(도 15d), 축적된 p62와 손상된 미토콘드리아가 미토파지에 의해 제거된 것으로 확인되었다(도 15d 및 도 15e).On the other hand, when treated with MLN8054, the expression of ATP5A and ATP5B, important proteins involved in mitochondrial respiration, and the expression of MFN1 and MFN2, important proteins involved in mitochondrial division, which had been reduced by VacA, were restored (Figure 15a) and Figure 15b), mitochondrial fragmentation, which had been increased by treatment with VacA, was also confirmed to be reduced ( Figure 15c). And in the case of IU1 treatment, PINK1, Parkin, and p-AMPK in the PINK/Parkin signaling pathway, which is significantly involved in the activation of mitophagy, removal of damaged mitochondria, and restoration of mitochondrial function, which was reduced by VacA. Expression of proteins such as , LC3-II was restored again (Figure 15d), and it was confirmed that accumulated p62 and damaged mitochondria were removed by mitophagy (Figures 15d and 15e).
[4-2] 인 비보([4-2] In vivo ( in vivoin vivo )에서, VacA에 의해 유도된 손상에 대한 치료 효과의 효과의 확인), confirmation of the effectiveness of the treatment effect on damage induced by VacA.
상기 실시예 [4-1]에서 hAGOs를 통해 확인된 MLN8054의 VacA에 의한 위 장벽 손상 회복 효과를 인 비보(in vivo)에서 다시 한번 확인하였다.The effect of MLN8054 on recovering gastric barrier damage caused by VacA, which was confirmed through hAGOs in Example [4-1], was confirmed again in vivo .
구체적으로, VacA를 10%(v/v)의 0.3M HCl로 섭씨 37도에서 30분 동안 반응시켜 활성화시고 동일 농도의 NaOH를 동량으로 첨가하여 중화시킨 혼합물을, 24시간 동안 물만 허용한 5주령의 C57BL/6 마우스들에게 10일 동안 매 12시간 마다 PBS와 함께 경구투여하여 위 조직의 손상을 유발한 다음, 상기 VacA의 경구투여를 시작한지 3일이 경과된 시점부터 매 24시간 마다 MLN8054를 1, 5 또는 10mg/kg의 투여량으로 경구투여하였고, 상기 VacA의 경구투여를 시작한지 10일이 경과된 시점에 상기 마우스들을 희생하고 복강으로부터 전체 위장을 적출하여 MLN8054의 투여에 따른 효과를 비교하였다(도 16a).Specifically, VacA was activated by reacting with 10% (v/v) 0.3M HCl at 37 degrees Celsius for 30 minutes, and the mixture was neutralized by adding an equal amount of NaOH of the same concentration to 5-week-old mice that were allowed only water for 24 hours. C57BL/6 mice were orally administered with PBS every 12 hours for 10 days to induce damage to the stomach tissue, and then MLN8054 was administered every 24 hours starting 3 days after starting the oral administration of VacA. It was administered orally at a dose of 1, 5, or 10 mg/kg, and 10 days after starting the oral administration of VacA, the mice were sacrificed and the entire stomach was removed from the abdominal cavity to compare the effect of MLN8054 administration. (Figure 16a).
그 결과, VacA의 투여에 의해 위 전장에서 Zo-1, Ocln, Cldn4, Cldn7, Cldn18 등의 밀착 연접 관련 유전자들의 발현이 감소되었지만, MLN8054의 투여에 의해 농도의존적으로 상기 유전자들 발현이 다시금 회복되는 것으로 확인되었다(도 16b). 또한, E-카드헤린(E-cadherin) 염색을 통해 상피 조직도 온전히 회복되었음을(도 16c), TOM20 단백질 수준 측정을 통해 Muc5ac+ 세포들에서 Muc5ac와 미토콘드리아의 요소들의 발현 수준도 회복되었음을(도 16d), 실시간-PCR을 통해 atp5, CoxIV 등과 같은 미토콘드리아의 전자전달계 복합체 요소들의 전사-수준 역시 회복되었음을(도 16e), 그리고 PAS 염색을 통해 위 전장에서 점액층의 손상도 효과적으로 회복되었음을(도 16f), 각각 확인하였다.As a result, the expression of tight junction-related genes such as Zo-1, Ocln, Cldn4, Cldn7, and Cldn18 in the gastric foregut was reduced by the administration of VacA, but the expression of these genes was restored in a concentration-dependent manner by the administration of MLN8054. It was confirmed that (Figure 16b). In addition, E-cadherin staining showed that the epithelial tissue was fully recovered (Figure 16c), and the expression levels of Muc5ac and mitochondrial elements in Muc5ac + cells were also restored through TOM20 protein level measurement (Figure 16d). , the transcription levels of mitochondrial electron transport complex elements such as atp5 and CoxIV were also restored through real-time PCR (Figure 16e), and damage to the mucus layer in the stomach forearm was effectively restored through PAS staining (Figure 16f), respectively. Confirmed.
[실시예 5][Example 5]
MLN8054 및 IU1의 위 점막 손상 치료 효과의 검증 - 2Verification of the effectiveness of MLN8054 and IU1 in treating gastric mucosal damage - 2
상기 실시예 [4-1] 및 [4-2]를 통해 VacA에 의해 유도된 위 장벽의 손상 회복 효과가 확인된 MLN8054에 대하여, VacA에 의해 유도된 경우뿐만 아니라, 실제 헬리코박터 파일로리의 감염에 의해 유발된 위 장벽의 손상에 대해서도 치료 효과가 있는지 여부를 확인하였다.With respect to MLN8054, which was confirmed to have an effect in recovering damage to the gastric barrier induced by VacA through Examples [4-1] and [4-2], not only when induced by VacA, but also when actually infected with Helicobacter pylori. It was also confirmed whether there was a treatment effect on the induced damage to the gastric barrier.
[5-1] 헬리코박터 파일로리의 배양[5-1] Culture of Helicobacter pylori
먼저, 10% FBS, 1ug/mL 니스타틴(nystatin)(Kisanbio), 5ug/mL 트리메소프림(trimethoprim)(Kisanbio) 및 10ug/mL 반코마이신(vancomycin)(Kisanbio)이 첨가된 브루셀라 브로스(Brucella broth)에 위궤양 환자에서 분리하여 확립한 헬리코박터 파일로리 P1 균주(전남대학교, 박종환 교수 연구실)를 접종하고, 미호기성(microaerobic) 조건에서 섭씨 37도로 밤새 배양하였다. 그런 다음 PBS로 세척하고 재현탁하여, hAGOs와 마우스에 헬리코박터 파일로리의 감염을 위해 이용하였다.First, Brucella broth supplemented with 10% FBS, 1 ug/mL nystatin (Kisanbio), 5 ug/mL trimethoprim (Kisanbio), and 10 ug/mL vancomycin (Kisanbio). Helicobacter pylori P1 strain (Chonnam National University, Professor Jong-Hwan Park's laboratory) isolated and established from a gastric ulcer patient was inoculated and cultured overnight at 37 degrees Celsius under microaerobic conditions. It was then washed with PBS, resuspended, and used to infect hAGOs and mice with Helicobacter pylori.
[5-2] hAGOs를 이용한, 헬리코박터 파일로리의 감염에 의해 유발된 손상에 대한 치료 효과의 확인[5-2] Confirmation of treatment effect on damage caused by Helicobacter pylori infection using hAGOs
먼저, 상기와 같이 준비된 헬리코박터 파일로리 균주를 48시간 동안 배양하면서 분광광도계(spectrophotometer)로 OD600을 측정하였다. 그 결과, 상기와 같이 준비된 헬리코박터 파일로리 균주는 잘 성장하고 있는 것으로 확인되었다(도 17a). 뿐만 아니라, 상기와 같이 배양된 헬리코박터 파일로리 균주를 대상으로 실시간-PCR과 웨스턴 블럿을 수행하여 VacA의 전사체와 단백질의 수준을 확인하였고, 그 결과 상기 헬리코박터 파일로리 균주가 VacA를 잘 발현하고 있음을 확인하였다(도 17b 및 도 17c). First, the Helicobacter pylori strain prepared as above was cultured for 48 hours and OD 600 was measured using a spectrophotometer. As a result, it was confirmed that the Helicobacter pylori strain prepared as above was growing well (FIG. 17a). In addition, real-time PCR and Western blot were performed on the Helicobacter pylori strain cultured as above to confirm the transcript and protein levels of VacA, and as a result, it was confirmed that the Helicobacter pylori strain well expressed VacA. (Figures 17b and 17c).
그런 다음, 상기 실시예 [5-1]에서 배양하여 PBS로 세척한 헬리코박터 파일로리 P1 균주 109 CFU/mL를 모세관 팁(capillary tip)에 탑재(loading)하고, CellTram 4r Oil(Eppendorf)과 미세조작 시스템(micromanipulation system)(Eppendorf)을 이용하여 hAGOs의 내강 영역으로 1x106 CFU(10MOI)의 헬리코박터 파일로리 균주를 미세주입한 후, 무-항생제 조건에서 48시간 동안 배양하여, hAGOs를 헬리코박터 파일로리 균주로 감염시켰다(도 17d). Then, 10 9 CFU/mL of the Helicobacter pylori P1 strain cultured in Example [5-1] and washed with PBS was loaded onto the capillary tip and micromanipulated with CellTram 4r Oil (Eppendorf). Using a micromanipulation system (Eppendorf), 1x10 6 CFU (10MOI) of the Helicobacter pylori strain was microinjected into the luminal region of hAGOs, and then cultured for 48 hours under antibiotic-free conditions to infect the hAGOs with the Helicobacter pylori strain. (Figure 17d).
상기와 같이 헬리코박터 파일로리로 감염된 hAGOs를, hAGOs 배양액(2mM L-글루타민(Invitrogen), 10mM HEPES(Invitrogen), 페니시린/스트렙토마이신, 1x N2(Invitrogen), 1x B27(Invitrogen) 및 100ng/mL EGF(R&D Systems)가 포함된 Advanced DMEM/F12(Invitrogen))에서 24시간 동안 배양한 다음, 10uM의 MLN8054를 처리하고 다시 24시간 동안 배양하여, 형태학적 또는 분자생물학적 변화를 관찰하였다(도 17d).As described above, hAGOs infected with Helicobacter pylori were cultured in hAGOs (2mM L-glutamine (Invitrogen), 10mM HEPES (Invitrogen), penicillin/streptomycin, 1x N2 (Invitrogen), 1x B27 (Invitrogen), and 100ng/mL EGF ( The cells were cultured in Advanced DMEM/F12 (Invitrogen) containing R&D Systems) for 24 hours, then treated with 10uM of MLN8054 and cultured again for 24 hours to observe morphological or molecular biological changes (FIG. 17d).
그 결과, 도 17e 내지 도 17i에 도시된 바와 같이, 헬리코박터 파일로리 감염에 의해 감소되었던 ATP 생성 수준이 MLN8054의 처리에 의해 눈에 띄게 회복되었고(도 17e), 미토콘드리아의 초산화물(superoxide) 지표인 MitoSOX+ 신호도 MLN8054의 처리에 의해 완전히 사라졌으며(도 17f), TOM20 염색을 통해 미토콘드리아의 길이와 점액-생성 기능이 MLN8054의 처리에 의해 회복되었음은 물론(도 17g 및 도 17h), 밀착 연접 관련 단백질들의 발현과 조립 역시 MLN8054의 처리에 의해 증가된 것으로 확인되었다(도 17i).As a result, as shown in Figures 17e to 17i, the level of ATP production, which had been reduced by Helicobacter pylori infection, was noticeably restored by treatment with MLN8054 (Figure 17e), and MitoSOX, an indicator of mitochondrial superoxide, was significantly restored by treatment with MLN8054 (Figure 17e). The + signal was also completely disappeared by treatment with MLN8054 (Figure 17f), and TOM20 staining showed that mitochondrial length and mucus-producing function were restored by treatment with MLN8054 (Figures 17g and 17h), as well as tight junction-related proteins. It was confirmed that their expression and assembly were also increased by treatment with MLN8054 (Figure 17i).
[5-3] 인 비보([5-3] In vivo ( in vivoin vivo )에서, 헬리코박터 파일로리의 감염에 의해 유발된 손상에 대한 치료 효과의 확인), confirmation of the therapeutic effect on damage caused by infection with Helicobacter pylori
상기 실시예 [5-2]에서 hAGOs를 통해 확인된 MLN8054의 헬리코박터 파일로리의 감염에 의한 위 장벽 손상 회복 효과를 인 비보(in vivo)에서 다시 한번 확인하였다.The effect of MLN8054 on recovering gastric barrier damage caused by Helicobacter pylori infection, confirmed through hAGOs in Example [5-2], was confirmed again in vivo .
구체적으로, 상기 실시예 [5-1]에서 배양하여 PBS로 세척한 헬리코박터 파일로리 P1 균주 2.23x109 CFU를, 24시간 동안 물만 허용한 5주령의 C57BL/6 마우스들에게 하루에 한번씩 4일 동안 경구투여하여, 상기 마우스를 헬리코박터 파일로리 균주로 감염시켰다. 그런 다음, 헬리코박터 파일로리 균주를 경구투여하기 시작한지 19일이 경과된 시점부터 매 24시간 마다 MLN8054를 10mg/kg의 투여량으로 경구투여하였고, 상기 헬리코박터 파일로리 균주의 경구투여를 시작한지 28일이 경과된 시점에 상기 마우스들을 희생하고 복강으로부터 전체 위장을 적출하여 MLN8054의 투여에 따른 효과를 비교하였다(도 18의 A).Specifically, 2.23x10 9 CFU of the Helicobacter pylori P1 strain cultured in Example [5-1] and washed with PBS was orally administered to 5-week-old C57BL/6 mice that were allowed only water for 24 hours once a day for 4 days. By administration, the mice were infected with the Helicobacter pylori strain. Then, 19 days after the start of oral administration of the Helicobacter pylori strain, MLN8054 was orally administered at a dose of 10 mg/kg every 24 hours, and 28 days had passed since the start of oral administration of the Helicobacter pylori strain. At this point, the mice were sacrificed and the entire stomach was removed from the abdominal cavity to compare the effect of MLN8054 administration (Figure 18A).
그 결과, 도 18의 B 내지 D에 도시된 바와 같이, 헬리코박터 파일로리로 감염된 마우스의 경우 위 전정의 내강(lumen)과 상피층에서 헬리코박터 파일로리 균주와 이로부터 발현되는 VacA가 검출되었고(도 18의 B), 미토콘드리아의 요소들이 감소하여 Muc5ac의 합성에도 장애가 발생하는 것으로 확인되었다(도 18의 C 및 D). 그러나, 상기와 같은 문제점들이 MLN8054의 투여에 의해 모두 해소 및 회복되었음을 확인하였는데, 구체적으로 E-카드헤린(E-cadherin) 염색을 통해 상피 조직이 온전히 회복되었음을(도 18의 C), TOM20 단백질 수준 측정을 통해 Muc5ac+ 세포들에서 Muc5ac와 미토콘드리아의 요소들의 발현 수준도 회복되었음을(도 18의 C), 그리고 면역형광 분석과 PAS 염색을 통해 MUC5AC의 단백질 발현 수준도 향상되었음을(도 18의 C 및 D), 각각 확인하였다.As a result, as shown in B to D of Figure 18, in the case of mice infected with Helicobacter pylori, the Helicobacter pylori strain and VacA expressed therefrom were detected in the lumen and epithelial layer of the stomach vestibule (Figure 18B). , it was confirmed that mitochondrial elements were reduced and that the synthesis of Muc5ac was also impaired (Figure 18, C and D). However, it was confirmed that all of the above problems were resolved and recovered by the administration of MLN8054. Specifically, the epithelial tissue was fully recovered through E-cadherin staining (C of Figure 18), and TOM20 protein level. Measurements showed that the expression levels of Muc5ac and mitochondrial elements were recovered in Muc5ac + cells (Figure 18C), and that the protein expression level of MUC5AC was also improved through immunofluorescence analysis and PAS staining (Figures 18C and D). ), each was confirmed.
[실시예 6][Example 6]
다른 AURKA 억제제와 USP14 억제제의 위 점막 손상 치료 효과의 검증Validation of the effectiveness of different AURKA inhibitors and USP14 inhibitors in treating gastric mucosal damage
상기 실시예 [3-2]에서 선별된 AURKA 억제제와 USP14 억제제 중, 상기 실시예 4 및 5를 통해 효과가 확인된 MLN8054와 IU1 외에, 나머지 AURKA 억제제인 MLN8237, MK8745, MK5108과 나머지 USP14 억제제인 WP1130에 대해서도, hAGOs를 이용하여 VacA에 의해 유도된 위 점막 손상의 치료 효과를 확인해 보았다.Among the AURKA inhibitors and USP14 inhibitors selected in Example [3-2], in addition to MLN8054 and IU1, the effects of which were confirmed in Examples 4 and 5, the remaining AURKA inhibitors, MLN8237, MK8745, and MK5108, and the remaining USP14 inhibitor, WP1130 In addition, hAGOs were used to confirm the therapeutic effect of gastric mucosal damage induced by VacA.
구체적으로, 상기 실시예 [4-1]과 동일한 방법으로, 20ug/mL의 VacA가 48시간 동안 처리된 hAGOs에, MLN8237를 5uM, 10uM, 20uM씩 처리하거나, MK8745를 1uM, 10uM, 20uM씩 처리하거나, MK5108를 1uM, 2uM, 5uM씩 처리하거나, 또는 WP1130를 0.1uM, 0.5uM, 1uM씩 처리하여 24시간 동안 배양한 다음, 형태학적 또는 분자생물학적 변화를 관찰하였다.Specifically, in the same manner as in Example [4-1], hAGOs treated with 20ug/mL VacA for 48 hours were treated with 5uM, 10uM, and 20uM of MLN8237, or 1uM, 10uM, and 20uM of MK8745. Alternatively, cells were treated with MK5108 at 1uM, 2uM, and 5uM, or WP1130 at 0.1uM, 0.5uM, and 1uM and cultured for 24 hours, and then morphological or molecular biological changes were observed.
그 결과, 도 19a 내지 도 19d에 도시된 바와 같이, MLN8237, MK8745, MK5108 및 WP1130의 처리에 의해, VacA에 의해 감소되었던 미토콘드리아 ATP 생성률이 회복되었고(도 19a 및 도 19b), VacA에 의해 손상되었던 점액-생성 및 밀착 연접 관련 유전자들의 발현 또한 눈에 띄게 증가된 것으로 확인되었다(도 19c 및 도 19d).As a result, as shown in FIGS. 19A to 19D, the mitochondrial ATP production rate, which had been reduced by VacA, was restored by treatment with MLN8237, MK8745, MK5108, and WP1130 (FIGS. 19A and 19B), and the mitochondrial ATP production rate that had been damaged by VacA was restored. Expression of mucus-producing and tight junction-related genes was also confirmed to be noticeably increased (Figures 19c and 19d).
상기와 같은 결과로부터, MLN8054를 비롯한 여러 AURKA 억제제와 IU1을 비롯한 여러 USP14 억제제가 헬리코박터 파일로리의 감염에 의한 위 점막 손상을 예방 또는 치료하는데 효과가 있는 물질임을 분명히 알 수 있다.From the above results, it is clear that several AURKA inhibitors, including MLN8054, and several USP14 inhibitors, including IU1, are effective substances in preventing or treating gastric mucosal damage caused by Helicobacter pylori infection.
[실시예 7][Example 7]
헬리코박터 파일로리의 위에 대한 부착 억제제를 발굴하기 위한 플랫폼의 확립Establishment of a platform to discover inhibitors of Helicobacter pylori adhesion to the stomach
[7-1] 부착 억제제 발굴을 위한 플랫폼 확립[7-1] Establishment of a platform for discovery of adhesion inhibitors
상기 실시예 [1-1]에서 제작한 hAGOs를 마트리젤로부터 분리한 후, 차가운 PBS로 세척하고 0.25%의 트립신-EDTA(Gibco)를 처리하여 섭씨 37도에서 5분간 배양하였다. 단일세포로 해리된 hAGOs를 1000rpm으로 원심분리하여 세척하고 1% 마트리젤이 코팅된 12-웰 디쉬에 1x106 개씩 접종하여 위 전정 오가노이드 분화 배지[advanced DMRM/F12, 2 mM L-glutamine, 1% Penicillin-Streptomycin, and 15 mM HEPES buffer, 1X N2, 1X B27, 100ng/mL의 EGF]로 3일 동안 배양하여 2차원 hAGOs를 준비하였다. The hAGOs produced in Example [1-1] were separated from Matrigel, washed with cold PBS, treated with 0.25% trypsin-EDTA (Gibco), and incubated at 37 degrees Celsius for 5 minutes. The hAGOs dissociated into single cells were washed by centrifugation at 1000 rpm, and then inoculated 1x106 into 12-well dishes coated with 1% Matrigel and incubated in gastric vestibular organoid differentiation medium [advanced DMRM/F12, 2 mM L-glutamine, 1 % Penicillin-Streptomycin, and 15 mM HEPES buffer, 1
한편, 5-10%의 O2와 5-10%의 CO2를 포함하는 섭씨 37도의 미세호기성 배양 조건에서, 10%의 FBS(Gibco), 1ug/mL의 니스타틴(nystatin)(Kisanbio), 5ug/mL의 트리메소프림(trimethoprim)(Kisanbio) 및 10ug/mL의 반코마이신(vancomycin)(Kisanbio)을 함유하는 브루셀라(Brucella) 배지로 헬리코박터 파일로리 P1 균주를 배양하여 OD600이 0.18-0.2에 도달할 때(지수기(exponential-mid log phase) 전)까지 성장시켰다.Meanwhile, under microaerobic culture conditions at 37 degrees Celsius containing 5-10% O 2 and 5-10% CO 2 , 10% FBS (Gibco), 1 ug/mL nystatin (Kisanbio), Helicobacter pylori P1 strain was cultured with Brucella medium containing 5ug/mL trimethoprim (Kisanbio) and 10ug/mL vancomycin (Kisanbio) until the OD 600 reached 0.18-0.2. It was grown until the exponential phase (before the exponential-mid log phase).
그런 다음, 상기와 같이 배양된 헬리코박터 파일로리 P1 균주를, 상기와 같이 준비된 2차원 hAGOs가 포함된 각 웰당 10MOI의 수로 처리하고 배양하여, 상기 hAGOs에 잔존하는 헬리코박터 파일로리 P1 균주의 수준을 감소시킬 수 있는 물질을 헬리코박터 파일로리의 위에 대한 부착 억제제로 발굴해 내는, 이른바 ‘헬리코박터 파일로리의 위에 대한 부착 억제제 스크리닝 플랫폼’을 확립하였다(도 20).Then, the Helicobacter pylori P1 strain cultured as above was treated and cultured with 10 MOI per well containing the two-dimensional hAGOs prepared as above, thereby reducing the level of Helicobacter pylori P1 strain remaining in the hAGOs. A so-called 'Helicobacter pylori adhesion inhibitor screening platform' was established to discover substances as inhibitors of Helicobacter pylori adhesion to the stomach (FIG. 20).
[7-2] 상기 플랫폼을 이용한 부착 억제제의 발굴[7-2] Discovery of adhesion inhibitors using the above platform
위 실시예 7-1에서 확립된 플랫폼에 따라, 10MOI의 헬리코박터 파일로리 P1 균주가 처리된 2차원 hAGOs에, 21종의 프로바이오틱스 배양액을 1%의 농도로 처리하여 4시간 동안 공배양하였다.According to the platform established in Example 7-1 above, 21 types of probiotic cultures were treated at a concentration of 1% on two-dimensional hAGOs treated with a 10MOI Helicobacter pylori P1 strain and co-cultured for 4 hours.
그런 다음, 1% DEPC가 첨가된 1ml PBS로 5회 세척하고, NucleoSpin RNA (MN)으로 RNA를 추출하였고, 추출된 RNA로부터 superscript IV(invitrogen)를 이용하여 cDNA를 합성하였으며, 하기 표 5에 기재된 프라이머쌍을 이용하여 7500 Fast Real-time PCR 시스템(Applied Biosystems, Foster City, CA, USA)으로 qRT-PCR을 수행하여 인간 위 전정 오가노이드의 GAPDH 유전자와 헬리코박터 파일로리 균주의 16S rRNA의 양을 측정함으로써, 2차원 hAGOs에 부착된 헬리코박터 파일로리 균주의 수를 측정하였다.Then, it was washed five times with 1ml PBS containing 1% DEPC, RNA was extracted with NucleoSpin RNA (MN), and cDNA was synthesized from the extracted RNA using superscript IV (invitrogen), as shown in Table 5 below. By measuring the amount of the GAPDH gene of human gastric vestibular organoids and the 16S rRNA of Helicobacter pylori strains by performing qRT-PCR using a 7500 Fast Real-time PCR system (Applied Biosystems, Foster City, CA, USA) using primer pairs. , the number of Helicobacter pylori strains attached to two-dimensional hAGOs was measured.
[표 2][Table 2]
Figure PCTKR2023005398-appb-img-000002
Figure PCTKR2023005398-appb-img-000002
그 결과, 도 21에 도시된 바와 같이, 21종의 프로바이오틱스 배양액 중, 헬리코박터 파일로리 균주에 MRS 배지를 처리한 경우에 비해 hAGOs에 대한 헬리코박터 파일로리 균주의 부착력이 저하된 것들을 선별할 수 있었고, 대표 28, 대표 150, 대표 295 및 대표 299 균주의 배양액을 헬리코박터 파일로리 균주의 부착 억제 활성이 특히 우수한 것으로 판정할 수 있었다(도 21).As a result, as shown in Figure 21, among 21 types of probiotic cultures, it was possible to select those in which the adhesion of the Helicobacter pylori strain to hAGOs was reduced compared to the case where the Helicobacter pylori strain was treated with MRS medium, representative 28, The culture media of Representative 150, Representative 295, and Representative 299 strains were determined to have particularly excellent anti-adhesion activity of Helicobacter pylori strains (FIG. 21).
상기에서는 본 발명의 바람직한 실시예를 예시적으로 설명하였으나, 본 발명의 범위는 상기와 같은 특정 실시예에만 한정되지 아니하며, 해당 분야에서 통상의 지식을 가진 자라면 본 발명의 청구범위에 기재된 범주 내에서 적절하게 변경이 가능할 것이다.Although preferred embodiments of the present invention have been described above by way of example, the scope of the present invention is not limited to the specific embodiments described above, and those skilled in the art will understand that the scope of the present invention is within the scope stated in the claims of the present invention. It will be possible to change it appropriately.

Claims (22)

  1. 오로라 키나아제 A(Aurora kinase A, AURKA) 억제제 또는 유비퀴틴 특이적 펩티다아제 14(ubiquitin specific peptidase 14, USP14) 억제제를 유효성분으로 포함하는 위 점막 손상 관련 질환의 예방 또는 치료용 조성물.A composition for preventing or treating diseases related to gastric mucosal damage, comprising an Aurora kinase A (AURKA) inhibitor or a ubiquitin specific peptidase 14 (USP14) inhibitor as an active ingredient.
  2. 청구항 1에 있어서,In claim 1,
    상기 오로라 키나아제 A 억제제는 MLN8054, MLN8237, MK8745 및 MK5108로 구성된 군에서 선택되는 적어도 하나인 것인 약학적 조성물.A pharmaceutical composition wherein the Aurora Kinase A inhibitor is at least one selected from the group consisting of MLN8054, MLN8237, MK8745 and MK5108.
  3. 청구항 1에 있어서,In claim 1,
    상기 유비퀴틴 특이적 펩티다아제 14 억제제는 IU1 및 WP1130으로 구성된 군에서 선택되는 적어도 하나인 것인 약학적 조성물.A pharmaceutical composition wherein the ubiquitin-specific peptidase 14 inhibitor is at least one selected from the group consisting of IU1 and WP1130.
  4. 청구항 1 내지 청구항 3 중 어느 한 항에 있어서,The method according to any one of claims 1 to 3,
    상기 위 점막 손상 관련 질환은 위 상피세포의 미토콘드리아가 손상됨으로써 유발되는 것인 약학적 조성물.A pharmaceutical composition in which the disease related to gastric mucosal damage is caused by damage to mitochondria of gastric epithelial cells.
  5. 청구항 4에 있어서,In claim 4,
    상기 위 상피세포의 미토콘드리아의 손상은 헬리코박터 파일로리(Helicobacter pylori)에 의한 것인 약학적 조성물.A pharmaceutical composition wherein damage to mitochondria of the stomach epithelial cells is caused by Helicobacter pylori .
  6. 청구항 4에 있어서,In claim 4,
    상기 손상은 단편화(fragmentation)인 것인 약학적 조성물. A pharmaceutical composition wherein the damage is fragmentation.
  7. 청구항 4에 있어서,In claim 4,
    상기 위 상피세포는 점액-생성 세포(mucous-producing cell)인 것인 약학적 조성물.A pharmaceutical composition wherein the gastric epithelial cells are mucous-producing cells.
  8. 청구항 7에 있어서,In claim 7,
    상기 점액-생성 세포는 미토콘드리아의 손상으로 인해 점액의 분비가 감소된 것인 약학적 조성물.A pharmaceutical composition wherein the mucus-producing cells have reduced mucus secretion due to mitochondrial damage.
  9. 청구항 4에 있어서,In claim 4,
    상기 위 점막 손상 관련 질환은 위염, 위궤양 또는 위암인 것인 약학적 조성물.A pharmaceutical composition wherein the disease related to gastric mucosal damage is gastritis, gastric ulcer, or gastric cancer.
  10. 오로라 키나아제 A(Aurora kinase A, AURKA) 억제제 또는 유비퀴틴 특이적 펩티다아제 14(ubiquitin specific peptidase 14, USP14) 억제제를 유효성분으로 포함하는 위 점막 손상의 예방 또는 개선용 식품 또는 건강기능식품 조성물.A food or health functional food composition for preventing or improving gastric mucosal damage containing an Aurora kinase A (AURKA) inhibitor or a ubiquitin specific peptidase 14 (USP14) inhibitor as an active ingredient.
  11. VacA가 처리된 인간 전정 위 오가노이드(human antrum gastric organoids, hAGOs)에 후보 물질을 처리하는 단계; 및Processing the candidate material into VacA-treated human antrum gastric organoids (hAGOs); and
    상기 후보 물질의 처리 여부에 따라, 상기 hAGOs에서, 점액-생성 세포의 수 및 점액-생성 관련 유전자의 발현 중 어느 하나의 변화와 미토콘드리아의 활성 수준의 변화를 측정하는 단계;를 포함하는,Depending on whether or not the candidate material is treated, measuring a change in any one of the number of mucus-producing cells and the expression of mucus-producing genes and a change in the activity level of mitochondria in the hAGOs, comprising:
    헬리코박터 파일로리(Helicobacter pylori)에 의한 위 점막 손상 관련 질환의 치료제 스크리닝 방법.Method for screening treatments for diseases related to gastric mucosal damage caused by Helicobacter pylori.
  12. 청구항 11에 있어서,In claim 11,
    상기 점액-생성 세포는 표면 점액 세포 및 전정 경부 세포로 구성된 군에서 선택되는 적어도 하나인 것인 방법.A method wherein the mucus-producing cells are at least one selected from the group consisting of superficial mucus cells and vestibular neck cells.
  13. 청구항 11에 있어서,In claim 11,
    상기 점액-생성 관련 유전자는 MUC5AC, MUC 및 SOX9으로 구성된 군에서 선택되는 적어도 하나인 것인 방법.A method wherein the mucus-production related gene is at least one selected from the group consisting of MUC5AC, MUC, and SOX9.
  14. 청구항 11에 있어서,In claim 11,
    상기 발현은 mRNA 수준에서의 발현 또는 단백질 수준에서의 발현인 것인, 방법.The method wherein the expression is expression at the mRNA level or expression at the protein level.
  15. 청구항 11에 있어서,In claim 11,
    상기 미토콘드리아의 활성은 미토콘드리아의 ATP 생성 수준 또는 형태(morphology) 변화를 확인하는 방법.The activity of the mitochondria is a method of confirming changes in the ATP production level or morphology of the mitochondria.
  16. 청구항 11에 있어서,In claim 11,
    상기 후보 물질의 처리 전과 비교하여, 상기 후보 물질의 처리 후에 hAGOs에서 측정된 점액-생성 세포의 수 및 점액-생성 관련 유전자의 발현 중 어느 하나와 미토콘드리아의 ATP 생성 수준이 증가된 경우, 상기 후보 물질을 상기 치료제로 판정하는 단계;를 더 포함하는 것인 방법.If either the number of mucus-producing cells or the expression of mucus-production-related genes and the level of mitochondrial ATP production measured in hAGOs are increased after treatment of the candidate material, compared to before treatment of the candidate material, the candidate material The method further comprising determining that the therapeutic agent is the therapeutic agent.
  17. 청구항 11에 있어서,In claim 11,
    상기 후보 물질의 처리 전과 비교하여, 상기 후보 물질의 처리 후에 hAGOs에서 측정된 점액-생성 세포의 수 및 점액-생성 관련 유전자의 발현 중 어느 하나와 미토콘드리아 융합(fusion) 혹은 길이가 증가된 경우, 상기 후보 물질을 상기 치료제로 판정하는 단계;를 더 포함하는 것인 방법.Compared to before treatment of the candidate material, if either the number of mucus-producing cells or the expression of mucus-producing genes and the mitochondrial fusion or length measured in hAGOs are increased after treatment of the candidate material, The method further comprising determining a candidate substance as the therapeutic agent.
  18. 청구항 11에 있어서,In claim 11,
    상기 위 점막 손상 관련 질환은 위염, 위궤양 또는 위암인 것인 방법.The method wherein the disease related to damage to the gastric mucosa is gastritis, gastric ulcer, or gastric cancer.
  19. 헬리코박터 파일로리(Helicobacter pylori) 균주가 처리된 인간 전정 위 오가노이드(human antrum gastric organoids, hAGOs)에 후보 물질을 처리하는 단계; 및Processing the candidate material into human antrum gastric organoids (hAGOs) treated with Helicobacter pylori strain; and
    상기 후보 물질의 처리 여부에 따라, 상기 hAGOs에 잔존하는 헬리코박터 파일로리 균주의 수준의 변화를 측정하는 단계;를 포함하는,Including, measuring changes in the level of Helicobacter pylori strains remaining in the hAGOs, depending on whether or not the candidate material is treated.
    헬리코박터 파일로리의 위 부착 억제제 스크리닝 방법.Screening method for gastric adhesion inhibitors of Helicobacter pylori.
  20. 청구항 19에 있어서,In claim 19,
    상기 인간 전정 위 오가노이드는 단일세포로 해리되어 평판 배양된 것인 방법.A method wherein the human vestibular organoid is dissociated into single cells and cultured on a plate.
  21. 청구항 19 또는 청구항 20에 있어서,In claim 19 or claim 20,
    상기 hAGOs에 잔존하는 헬리코박터 파일로리 균주의 수준의 변화는, 상기 hAGOs에서 헬리코박터 파일로리 균주의 16S rRNA의 양을 측정함으로써 평가하는 것인 방법.A method in which the change in the level of Helicobacter pylori strains remaining in the hAGOs is evaluated by measuring the amount of 16S rRNA of the Helicobacter pylori strains in the hAGOs.
  22. 청구항 21에 있어서,In claim 21,
    상기 후보 물질을 처리하지 않은 경우와 비교하여, 상기 후보 물질의 처리 후에 hAGOs에서 측정된 헬리코박터 파일로리 균주의 16S rRNA의 수준이 감소된 경우, 상기 후보 물질을 상기 위 부착 억제제로 판정하는 단계;를 더 포함하는 것인 방법.If the level of 16S rRNA of Helicobacter pylori strain measured in hAGOs is reduced after treatment of the candidate material compared to the case of not treating the candidate material, determining the candidate material as the gastric adhesion inhibitor; further How to include it.
PCT/KR2023/005398 2022-04-22 2023-04-20 Pharmaceutical composition for prevention or treatment of gastric mucosal damage-related diseases caused by helicobacter pylori and platform for screening for active ingredient for prevention or treatment of helicobacter pylori infection WO2023204640A1 (en)

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Citations (3)

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