WO2023204640A1 - Composition pharmaceutique pour la prévention ou le traitement de maladies liées à des lésions de la muqueuse gastrique provoquées par helicobacter pylori et plateforme pour le criblage d'un principe actif pour la prévention ou le traitement d'une infection par helicobacter pylori - Google Patents

Composition pharmaceutique pour la prévention ou le traitement de maladies liées à des lésions de la muqueuse gastrique provoquées par helicobacter pylori et plateforme pour le criblage d'un principe actif pour la prévention ou le traitement d'une infection par helicobacter pylori 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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English (en)
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 or sparfloxacin
    • 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.

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Abstract

La présente invention concerne : un procédé de criblage d'un agent thérapeutique pour des maladies liées à des lésions de la muqueuse gastrique provoquées par Helicobacter pylori ; et une composition pharmaceutique pour la prévention ou le traitement de maladies liées à des lésions de la muqueuse gastrique, comprenant un nouveau principe actif découvert par ce procédé. La présente invention concerne également une plateforme de criblage d'un agent thérapeutique pour des maladies liées à des lésions de la muqueuse gastrique provoquées par Helicobacter pylori, et une plateforme de criblage d'un ingrédient qui inhibe l'adhérence gastrique d'Helicobacter pylori.
PCT/KR2023/005398 2022-04-22 2023-04-20 Composition pharmaceutique pour la prévention ou le traitement de maladies liées à des lésions de la muqueuse gastrique provoquées par helicobacter pylori et plateforme pour le criblage d'un principe actif pour la prévention ou le traitement d'une infection par helicobacter pylori WO2023204640A1 (fr)

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US20200016146A1 (en) * 2016-04-06 2020-01-16 The Hospital For Sick Children Compositions and methods for treating helicobacter pylori infection

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