WO2019168331A1 - Nanovésicules issues de bactéries pseudomonas, et leur utilisation - Google Patents

Nanovésicules issues de bactéries pseudomonas, et leur utilisation Download PDF

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WO2019168331A1
WO2019168331A1 PCT/KR2019/002345 KR2019002345W WO2019168331A1 WO 2019168331 A1 WO2019168331 A1 WO 2019168331A1 KR 2019002345 W KR2019002345 W KR 2019002345W WO 2019168331 A1 WO2019168331 A1 WO 2019168331A1
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vesicles
cancer
pseudomonas
bacteria
derived
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PCT/KR2019/002345
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English (en)
Korean (ko)
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김윤근
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주식회사 엠디헬스케어
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Priority claimed from KR1020190022182A external-priority patent/KR102185982B1/ko
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Publication of WO2019168331A1 publication Critical patent/WO2019168331A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • 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
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to nanovesicles derived from Pseudomonas bacteria and their use, and more specifically, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, cardiomyopathy, dysplastic angina, diabetes mellitus, cirrhosis, using nanovesicles derived from Pseudomonas bacteria. And it relates to a diagnostic method such as atopic dermatitis, and a composition for preventing or treating the disease or inflammatory disease.
  • microbiota is a microbial community including microbes, archaea and eukarya that exist in a given settlement.
  • vesicles derived from pathogenic gram-negative bacteria such as Eshcherichia coli
  • pathogenic gram-negative bacteria such as Eshcherichia coli
  • systemic inflammatory and blood coagulation through vascular endothelial inflammatory responses when absorbed into blood vessels. It is also promoted, and insulin is absorbed in muscle cells, etc. cause insulin resistance and diabetes.
  • vesicles derived from beneficial bacteria can control the disease by controlling immune and metabolic abnormalities caused by pathogenic vesicles.
  • Th17 immune response characterized by the secretion of IL-17 cytokines, which secrete IL-6 upon exposure to bacterial derived vesicles, which induce a Th17 immune response.
  • Inflammation by the Th17 immune response is characterized by neutrophil infiltration, and TNF-alpha secreted from inflammatory cells such as macrophages plays an important role in the process of inflammation.
  • Inflammation is a local or systemic defense against damage or infection of cells and tissues, primarily by the direct response of humoral mediators that make up the immune system, or by stimulating local or systemic effector systems. It is caused by a cascade of biological reactions that take place.
  • Major inflammatory diseases include gastroenteritis, digestive diseases such as inflammatory bowelitis, oral diseases such as periodontitis, asthma, chronic obstructive pulmonary disease (COPD), respiratory diseases such as rhinitis, atopic dermatitis, hair loss, skin diseases such as psoriasis, degenerative arthritis, Arthritis, such as rheumatoid arthritis; And metabolic diseases such as obesity, diabetes, cirrhosis of the liver.
  • Pseudomonas genus bacteria are Gram-negative bacteria that inhabit widely in nature such as water, plants, and intestines, and most Pseudomonas bacteria are resistant to antibiotics.
  • Pseudomonas aeruginosa belonging to the genus (genus) is known as an antibiotic resistance bacteria, is known as a major causative agent of opportunistic infection.
  • vesicles derived from Pseudomonas spp To the diagnosis and treatment of intractable diseases such as cancer, cardiovascular disease, metabolic disease and atopic dermatitis.
  • the present inventors earnestly researched to solve the above-mentioned conventional problems, and compared with the normal persons through pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, cardiomyopathy, dysplastic angina, diabetes, cirrhosis, and atopic dermatitis, It was confirmed that the content of bacterial vesicles of Pseudomonas genus in the patient-derived sample was significantly reduced.
  • Pseudomonas aeruginosa bacteria belonging to the Pseudomonas genus bacteria were isolated in the body and cultured, and then treated with macrophages by separating the vesicles, it was confirmed that significantly inhibits the secretion of IL-6 and TNF-alpha by pathogenic vesicles To this end, the present invention has been completed.
  • an object of the present invention is to provide a method for providing information for the diagnosis of pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, cardiomyopathy, dysplastic angina, diabetes, cirrhosis, or atopic dermatitis.
  • the present invention is one or more selected from the group consisting of pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, cardiomyopathy, heteroangular angina, diabetes mellitus, cirrhosis, atopic dermatitis, and inflammatory disease It is another object to provide a composition for the prevention, treatment or amelioration of a disease.
  • the present invention comprises the following steps, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, cardiomyopathy, dysplastic angina, diabetes, cirrhosis, or atopic dermatitis for the diagnosis of Provide information methods:
  • the present invention also provides a method for diagnosing pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, cardiomyopathy, dysplastic angina, diabetes, cirrhosis, or atopic dermatitis, comprising the following steps:
  • the sample in step (a) may be blood.
  • the primer pair in step (b) may be a primer of SEQ ID NO: 1 and SEQ ID NO: 2.
  • the present invention is one selected from the group consisting of pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, cardiomyopathy, heteroangular angina, diabetes mellitus, cirrhosis, atopic dermatitis, and inflammatory diseases, including vesicles derived from Pseudomonas genus as an active ingredient
  • a pharmaceutical composition for preventing or treating the above diseases.
  • the present invention is one selected from the group consisting of pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, cardiomyopathy, heteroangular angina, diabetes mellitus, cirrhosis, atopic dermatitis, and inflammatory diseases, including vesicles derived from Pseudomonas genus as an active ingredient
  • a food composition for preventing or improving the above diseases.
  • the present invention is one selected from the group consisting of pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, cardiomyopathy, heteroangular angina, diabetes mellitus, cirrhosis, atopic dermatitis, and inflammatory diseases, including vesicles derived from Pseudomonas genus as an active ingredient
  • an inhalant composition for preventing or treating the above diseases.
  • the inflammatory disease is acne, psoriasis, sinusitis, rhinitis, conjunctivitis, asthma, dermatitis, inflammatory collagen vascular disease, glomerulonephritis, encephalitis, inflammatory enteritis, chronic obstructive pulmonary disease, sepsis, septic shock , Pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthrosis, arthritis, inflammatory osteolysis, chronic inflammatory disease caused by viral or bacterial infection, colitis, ulcerative colitis, inflammatory bowel disease, arthritis, rheumatoid arthritis, reactive arthritis, osteoarthritis, scleroderma , Osteoporosis, atherosclerosis, myocarditis, endocarditis, pericarditis, cystic fibrosis, Hashimoto's thyroiditis, Graves' disease, leprosy, syphilis, Lyme disease,
  • the inflammatory disease may be a disease mediated by Interleukin-6 (IL-6) or Tumor necrosis factor alpha (TNF- ⁇ ).
  • IL-6 Interleukin-6
  • TNF- ⁇ Tumor necrosis factor alpha
  • the present invention also provides a cosmetic composition for the prevention or improvement of atopic dermatitis, comprising a vesicle derived from Pseudomonas genus as an active ingredient.
  • the present invention comprises administering a pharmaceutical composition comprising Pseudomonas genus bacteria-derived vesicles as an active ingredient to an individual, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, cardiomyopathy, heteroangular angina, diabetes, cirrhosis, A method for preventing or treating atopic dermatitis, and one or more diseases selected from the group consisting of inflammatory diseases.
  • the present invention also provides for the prevention or prevention of one or more diseases selected from the group consisting of Pseudomonas genus vesicles, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, cardiomyopathy, heteroangular angina, diabetes, cirrhosis, atopic dermatitis, and inflammatory diseases. Provide therapeutic use.
  • the vesicles may have an average diameter of 10 to 200 nm.
  • the vesicles may be secreted naturally or artificially from Pseudomonas genus bacteria.
  • the bacteria derived from Pseudomonas genus may be a vesicle derived from Pseudomonas aeruginosa.
  • the present inventors confirmed that the intestinal bacteria are not absorbed into the body, but the bacteria-derived vesicles are absorbed into the body through epithelial cells, distributed systemically, and excreted in vitro through the kidneys, liver, and lungs.
  • Bacterial-derived vesicles in the blood metagenomic analysis revealed that Pseudomonas bacteria-derived vesicles present in the blood of patients with pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, cardiomyopathy, dysplastic angina pectoris, diabetes mellitus, cirrhosis, and atopic dermatitis. It was confirmed that it is significantly reduced compared to.
  • Pseudomonas aeruginosa a species of Pseudomonas spp.
  • the bacterial vesicles of the genus Pseudomonas is pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, cardiomyopathy, dysplastic angina, diabetes, liver cirrhosis, and diagnostic method for atopic dermatitis, and the disease or It is expected that the present invention may be usefully used in a prophylactic or therapeutic composition for inflammatory diseases.
  • Figure 1a is a photograph of the distribution of bacteria and vesicles by time after administration of bacteria and bacteria-derived vesicles (EV) to the mouth
  • Figure 1b is 12 hours after oral administration
  • blood blood
  • kidneys Figure shows the distribution of bacteria, vesicles and vesicles in the body, liver and various organs.
  • Figure 2 is a result of comparing the distribution of bacteria-derived vesicles of the genus Pseudomonas after analyzing the bacteria-derived vesicles metagenome present in the pancreatic cancer patients and normal blood.
  • Figure 3 is a result of comparing the distribution of bacteria-derived vesicles of the genus Pseudomonas after the analysis of bacteria-derived vesicles metagenome present in patients with bile duct cancer.
  • 5 is a result of comparing the distribution of bacteria-derived vesicles of Pseudomonas genus after analyzing the bacteria-derived vesicles metagenome present in ovarian cancer patients and normal blood.
  • Figure 6 is a result of comparing the distribution of bacteria-derived vesicles of the genus Pseudomonas after analyzing the bacteria-derived vesicles metagenome present in the bladder cancer patients and normal blood.
  • 11 is a result of comparing the distribution of vesicles derived from Pseudomonas genus after analyzing the bacterial-derived vesicles metagenome present in atopic dermatitis patients and normal blood.
  • E. coli EV Escherichia coli vesicles
  • PC positive control
  • LP Lactobacillus plantarum EVs
  • PA Pseudomonas aerouginosa EVs
  • the present invention relates to a vesicle derived from Pseudomonas genus and its use.
  • the present inventors have found that the vesicle-derived vesicles of Pseudomonas genus are significantly reduced in clinical samples of patients with pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, cardiomyopathy, dysplastic angina pectoris, diabetes mellitus, cirrhosis, and atopic dermatitis compared to normal individuals. Confirmed that the disease can be diagnosed.
  • the vesicles were isolated and characterized from Pseudomonas aeruginosa, which belongs to the Pseudomonas spp.
  • Bacteria, and diseases such as pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, cardiomyopathy, dysplastic angina, diabetes, cirrhosis, and atopic dermatitis. It was confirmed that it can be used as a composition for preventing or treating.
  • the present invention provides a method for providing information for diagnosing pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, cardiomyopathy, heteroangular angina, diabetes, cirrhosis, or atopic dermatitis, comprising the following steps:
  • Diagnosis means, in a broad sense, to determine the actual condition of a patient in all aspects. The content of the judgment is the name of the disease, the etiology, the type of disease, the seriousness, the detailed mode of the condition, the presence or absence of complications, and the prognosis. Diagnosis in the present invention is to determine whether the pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, cardiomyopathy, dysplastic angina, diabetes, cirrhosis, and atopic dermatitis and the level of disease.
  • nanovesicles Nanovesicle or vesicles (Vesicles) refers to the structure of the nano-size membrane secreted by various bacteria. Vesicles derived from gram-negative bacteria, or outer membrane vesicles (OMVs), contain toxic proteins, bacterial DNA and RNA as well as endotoxins, and gram-positive bacteria-derived vesicles. In addition to proteins and nucleic acids, it also contains peptidoglycan and lipoteichoic acid, which are components of bacterial cell walls. In the present invention, nanovesicles or vesicles are naturally secreted or artificially produced by Pseudomonas spp., And have a spherical shape and have an average diameter of 10 to 100 nm.
  • metagenome also referred to as a military genome, refers to the sum total of the genome including all viruses, bacteria, fungi, etc. in an isolated area such as soil and animal intestine. It is used as a concept of genome to explain the identification of many microorganisms at once using sequencer for analysis.
  • the metagenome does not refer to one genome or genome, but to a kind of mixed dielectric as the genome of all species of one environmental unit. This is a term from the point of view of defining a species in the course of the evolution of biology in terms of functional species as well as various species that interact with each other to create a complete species.
  • rapid sequencing is used to analyze all DNA and RNA, regardless of species, to identify all species in one environment, and to identify interactions and metabolism.
  • the sample may be blood, but is not limited thereto.
  • the primer pair in step (b) may be a primer of SEQ ID NO: 1 and SEQ ID NO: 2.
  • the present invention is a pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, cardiomyopathy, dysplastic angina, diabetes mellitus, cirrhosis, atopic dermatitis, and inflammatory diseases, including vesicles derived from Pseudomonas genus as an active ingredient
  • the composition comprises a pharmaceutical composition, oral composition, or inhalant composition.
  • the composition of the present invention may be a formulation of an oral spray or a nasal spray.
  • the term “inflammatory disease” refers to a disease caused by an inflammatory response in a mammalian body.
  • the inflammatory disease includes acne, psoriasis, sinusitis, rhinitis, conjunctivitis, Asthma, dermatitis, inflammatory collagen vascular disease, glomerulonephritis, encephalitis, inflammatory enteritis, chronic obstructive pulmonary disease, sepsis, septic shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthrosis, arthritis, inflammatory osteolysis, viral or bacterial infection Chronic inflammatory diseases, colitis, ulcerative colitis, inflammatory bowel disease, arthritis, rheumatoid arthritis, reactive arthritis, osteoarthritis, scleroderma, osteoporosis, atherosclerosis, myocarditis, endocarditis, pericarditis, cystic fibrosis, Hashimoto's
  • prophylaxis is selected from the group consisting of pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, cardiomyopathy, angina, diabetes mellitus, cirrhosis, atopic dermatitis, and inflammatory diseases by administration of a composition according to the present invention. Any action that inhibits or delays the development of one or more diseases.
  • treatment is selected from the group consisting of pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, cardiomyopathy, angina, diabetes, cirrhosis, atopic dermatitis, and inflammatory diseases by administration of a composition according to the present invention. Any action that improves or beneficially changes the symptoms of one or more diseases.
  • improvement means any action that at least reduces the parameters associated with the condition being treated, for example, the extent of symptoms.
  • the vesicles are centrifuged, ultra-fast centrifugation, high pressure treatment, extrusion, sonication, cell lysis, homogenization, freeze-thaw, electroporation, mechanical degradation, chemical treatment, filtration by filter,
  • the separation can be carried out using one or more methods selected from the group consisting of gel filtration chromatography, pre-flow electrophoresis, and capillary electrophoresis. In addition, it may further include a process for washing to remove impurities, concentration of the obtained vesicles and the like.
  • the pharmaceutical composition according to the invention may comprise a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carriers are conventionally used in the preparation, and include, but are not limited to, saline solution, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and the like. If necessary, other conventional additives such as antioxidants and buffers may be further included.
  • diluents, dispersants, surfactants, binders, lubricants and the like may be additionally added to formulate injectable formulations, pills, capsules, granules, or tablets such as aqueous solutions, suspensions, emulsions and the like.
  • Suitable pharmaceutically acceptable carriers and formulations can be preferably formulated according to the individual components using methods disclosed in Remington's literature.
  • the pharmaceutical composition of the present invention is not particularly limited in formulation, but may be formulated as an injection, inhalant, external preparation for skin, oral ingestion, and the like.
  • the pharmaceutical composition of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, skin, nasal, airways) according to the desired method, and the dosage is determined by the condition and weight of the patient, disease Depending on the degree, drug form, route of administration, and time, it may be appropriately selected by those skilled in the art.
  • the pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount.
  • the pharmaceutically effective amount means an amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to the medical treatment, and the effective dose level refers to the type of disease, the severity, the activity of the drug and the drug. Sensitivity, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts.
  • the composition according to the present invention may be administered as a separate therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.
  • the effective amount of the pharmaceutical composition according to the present invention may vary depending on the age, sex and weight of the patient, and generally 0.001 to 150 mg, preferably 0.01 to 100 mg daily or every other day, per kg of body weight Or divided into 1 to 3 times a day.
  • the dosage may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention in any way.
  • the present invention is a pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, cardiomyopathy, dysplastic angina, diabetes mellitus, cirrhosis, atopic dermatitis, and inflammatory diseases, including vesicles derived from Pseudomonas genus as an active ingredient It provides a food composition for the prevention or amelioration of one or more diseases selected from the group consisting of.
  • the food composition of the present invention includes a nutraceutical composition.
  • the food composition according to the present invention may be used as it is, or may be used in combination with other foods or food ingredients, or may be appropriately used according to conventional methods.
  • the mixing amount of the active ingredient can be suitably determined according to the purpose of use (prevention or improvement).
  • the compositions of the invention are added in amounts of up to 15% by weight, preferably up to 10% by weight relative to the raw materials.
  • the amount may be below the above range.
  • the food composition of the present invention in addition to containing the active ingredient as an essential ingredient in the indicated ratio, there are no particular restrictions on other ingredients, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks.
  • natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol.
  • natural flavoring agents such as, tauumatin, stevia extract, for example, rebaudioside A, glycyrrhizin, etc.
  • synthetic flavoring agents sacharin, aspartame, etc.
  • the proportion of the natural carbohydrate can be appropriately determined by the choice of those skilled in the art.
  • the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like.
  • these components can be used independently or in combination.
  • the proportion of such additives may also be appropriately selected by those skilled in the art.
  • the active ingredient may be added to the inhalant as it is, or may be used together with other ingredients, and may be appropriately used according to conventional methods.
  • the amount of the active ingredient to be mixed may be suitably determined depending on the purpose of use (prophylactic or therapeutic).
  • the inflammatory disease may be a disease mediated by IL-6 or TNF- ⁇ , but is not limited thereto.
  • the present invention provides a cosmetic composition for preventing or improving atopic dermatitis, comprising a vesicle derived from Pseudomonas genus as an active ingredient.
  • the cosmetic composition of the present invention may include not only vesicles derived from Pseudomonas bacteria, but also components commonly used in cosmetic compositions, and include, for example, conventional auxiliaries such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and perfumes, And a carrier.
  • conventional auxiliaries such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and perfumes, And a carrier.
  • composition of the present invention may be used in addition to the vesicles derived from Pseudomonas bacteria, organic sunscreens that have been conventionally used insofar as they do not impair the skin protection effect by reacting with the vesicles derived from Pseudomonas bacteria.
  • organic sunscreen examples include glyceryl pava, drometrizole trisiloxane, drometrizole, digaloyltrioleate, disodium phenyldibenzimidazole tetrasulfonate, diethylhexyl butamidotriazone, diethylamino Hydroxybenzoylhexylbenzoate, die-methoxycinnamate, a mixture of lowson and dihydroxyacetone, methylenebis-benzotriazolyltetramethylbutylphenol, 4-methylbenzylidene camphor, menthyl anthranilate, benzophenone -3 (oxybenzone), benzophenone-4, benzophenone-8 (dioxyphenbenzone), butylmethoxydibenzoylmethane, bisethylhexyloxyphenol methoxyphenyltriazine, synoxate, ethyldihydroxypropylpava, Oct
  • Examples of products to which the cosmetic composition of the present invention may be added include, for example, cosmetics such as astringent cosmetics, soft cosmetics, nourishing cosmetics, various creams, essences, packs, foundations, and the like, cleansing agents, soaps, treatments, and essences.
  • Specific formulations of the cosmetic composition of the present invention include skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, essence, nutrition essence, pack, Formulations such as soaps, shampoos, cleansing foams, cleansing lotions, cleansing creams, body lotions, body cleansers, emulsions, lipsticks, makeup bases, foundations, press powders, loose powders, eye shadows and the like.
  • the bacteria and bacteria-derived vesicles orally administered to observe the body absorption, distribution, and excretion of the bacteria and vesicles in the body in the case of bacteria is not absorbed through the intestinal membrane, the vesicles are administered 5 It was confirmed that it was absorbed within minutes and distributed systemically and excreted through the kidney, liver, and the like (see Example 1).
  • vesicles, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, cardiomyopathy, dysplastic angina, diabetes, cirrhosis, and atopic dermatitis patients using age- and sex-matched vesicles isolated from the blood of normal people Bacterial metagenome analysis was performed. As a result, it was confirmed that the vesicles derived from Pseudomonas bacteria were significantly decreased in clinical samples of patients with pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, cardiomyopathy, dysplastic angina, diabetes, cirrhosis, and atopic dermatitis compared to normal samples. (See Examples 3 to 12).
  • the Pseudomonas aeruginosa strains were cultured to evaluate whether the secreted vesicles exhibit immunomodulatory and anti-inflammatory effects, and treated with Pseudomonas aeruginosa-derived vesicles of various concentrations in macrophages. Later, by treating E. coli-derived vesicles, the inflammatory disease-causing factors, and evaluating the secretion of inflammatory mediators, it was confirmed that Pseudomonas aeruginosa-derived vesicles effectively suppressed IL-6 and TNF- ⁇ secretion by E. coli-derived vesicles See Example 13.
  • the intestinal bacteria and bacterial-derived vesicles were systemically absorbed, in order to evaluate the infiltration into various organs, 50 ⁇ g of fluorescently labeled bacteria and bacterial-derived vesicles were administered as described above, followed by 12 hours of administration. Blood, heart, liver, kidneys, spleen, fat and muscle were collected later. As a result of fluorescence observation in the collected tissue, as shown in FIG. 1B, the vesicle-derived bacteria were distributed in blood, heart, lung, liver, kidney, spleen, fat, muscle and kidney, but bacteria were not absorbed. (See FIG. 1B).
  • Blood was first placed in a 10 ml tube and the suspension was allowed to settle by centrifugation (3,500 ⁇ g, 10 min, 4 ° C.) and only the supernatant was transferred to a new 10 ml tube. After removing bacteria and foreign substances using a 0.22 ⁇ m filter, it was transferred to centripreigugal filters (50 kD) and centrifuged at 1500 x g and 4 ° C for 15 minutes to discard materials smaller than 50 kD and concentrated to 10 ml.
  • centripreigugal filters 50 kD
  • DNA extracted by the above method was amplified using the above 16S rDNA primers, followed by sequencing (Illumina MiSeq sequencer), and the results were outputted in a Standard Flowgram Format (SFF) file, using GS FLX software (v2.9).
  • SFF Standard Flowgram Format
  • GS FLX Standard Flowgram Format
  • OTU operational taxonomy unit
  • clustering is performed according to sequence similarity using UCLUST and USEARCH, genus 94%, family 90%, order 85%, class 80%, phylum 75% sequence similarity
  • Clustering is based on the phylum, class, order, family, and genus levels of each OTU, and BLASTN and GreenGenes' 16S RNA sequence database (108,453 sequences) is used to identify bacteria with greater than 97% sequence similarity at the genus level.
  • Was profiled QIIME).
  • Example 2 In the method of Example 2, 176 bloods of pancreatic cancer patients and 271 bloods of normal people whose age and sex were matched were extracted from vesicles present in the blood and subjected to metagenomic analysis, followed by bacteria derived from Pseudomonas genus. The distribution of was evaluated. As a result, it was confirmed that the vesicles derived from Pseudomonas bacteria were significantly reduced in the blood of pancreatic cancer patients compared to normal blood (see Table 2 and FIG. 2).
  • Example 2 the genes were extracted from the vesicles in the breast cancer patients and 192 normal blood patients whose ages and genders were matched. The distribution of was evaluated. As a result, it was confirmed that the vesicles derived from Pseudomonas bacteria were significantly reduced in the blood of breast cancer patients compared to normal blood (see Table 4 and FIG. 4).
  • Example 2 In the method of Example 2, 137 bloods of ovarian cancer patients and 139 bloods of normal age and sex matched were extracted from vesicles in the blood and subjected to metagenomic analysis, followed by bacteria of the genus Pseudomonas. The distribution of vesicles was evaluated. As a result, it was confirmed that vesicles derived from Pseudomonas bacteria were significantly reduced in blood of ovarian cancer patients compared to normal blood (see Table 5 and FIG. 5).
  • Example 2 the genes were extracted from the vesicles in the bladder cancer patients and 176 normal blood patients whose ages and genders were matched. The distribution of was evaluated. As a result, it was confirmed that the vesicles derived from Pseudomonas bacteria were significantly reduced in the blood of bladder cancer patients compared to normal blood (see Table 6 and FIG. 6).
  • Example 2 blood was collected from 61 diabetic patients and 122 normal blood of age and sex, and the genes were extracted from the vesicles in the blood and subjected to metagenomic analysis. The distribution of was evaluated. As a result, it was confirmed that bacteria derived from Pseudomonas bacteria were significantly reduced in blood of diabetic patients compared to normal blood (see Table 9 and FIG. 9).
  • Example 2 the genes were extracted from the vesicles of 140 liver cirrhosis patients and 162 normal persons whose ages and genders were matched. The distribution of was evaluated. As a result, it was confirmed that the vesicles derived from Pseudomonas bacteria were significantly reduced in the blood of liver cirrhosis compared to normal blood (see Table 10 and FIG. 10).
  • the blood of 25 atopic dermatitis patients and 113 normal blood patients whose age and sex were matched by the method of Example 2 were extracted from vesicles in the blood and subjected to a metagenome analysis, followed by bacteria of the genus Pseudomonas. The distribution of vesicles was evaluated. As a result, it was confirmed that vesicles derived from Pseudomonas bacteria were significantly reduced in blood of atopic dermatitis patients compared to normal blood (see Table 11 and FIG. 11).
  • Pseudomonas aeruginosa bacteria belonging to Pseudomonas genus bacteria were isolated in the body and cultured in vitro, and their vesicles were separated. Pseudomonas aeruginosa strains were incubated in BHI (brain heart infusion) medium until absorbance (OD600) was 1.0-1.5 in an aerobic chamber at 37 ° C. and then sub-cultured. Thereafter, the culture supernatant containing no strain was recovered, centrifuged at 10,000 g, 4 ° C.
  • the filtered supernatant was used as a 100 kDa hollow filter membrane using a QuixStand benchtop system (GE Healthcare, UK). Concentrated to 200 ml volume by ultrafiltration. Then, the concentrated supernatant was once again filtered with a 0.22 ⁇ m filter, and the filtered supernatant was ultracentrifuged at 150,000 g, 4 ° C. for 3 hours, and the pellet was suspended in DPBS.
  • Optiprep solution was prepared using HEPES-buffered saline (20 mM HEPES) to prepare low density solution. , 150 mM NaCl, pH 7.4) was used. After centrifugation at 200,000 g, 4 ° C for 2 hours, each solution fractionated with the same volume of 1 ml from the upper layer was further subjected to ultracentrifugation for 15 hours at 150,000 g, 4 ° C. Thereafter, the protein was quantified by BCA assay, and the obtained vesicles were tested.
  • Pseudomonas aeruginosa-derived vesicles were treated at various concentrations (0.1, 1, 10 ⁇ g / ml) in Raw 264.7 cells.
  • E. coli- derived vesicles EV was treated to determine the secretion amount of the inflammatory mediators (IL-6 and TNF- ⁇ ). More specifically, 1 ⁇ 10 5 Raw 264.7 cells were dispensed into 24-well cell culture plates, and then cultured in DMEM complete medium for 24 hours.
  • Pseudomonas genus-derived vesicles are pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, cardiomyopathy, dysplastic angina, diabetes, cirrhosis, or a method for diagnosing atopic dermatitis, and a food or drug for the disease or inflammatory disease. It is expected that the present invention can be usefully used for the prevention, treatment, or improvement of the composition.

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Abstract

La présente invention concerne des vésicules issues de bactéries Pseudomonas et leur utilisation. La présente invention a permis de confirmer expérimentalement que des vésicules ont été réduites de manière significative dans des échantillons cliniques de patients atteints d'un cancer du pancréas, d'un cholangiocarcinome, d'un cancer du sein, d'un cancer de l'ovaire, d'un cancer de la vessie, d'une cardiomyopathie, d'un angor de repos, de diabète, de cirrhose hépatique, et de dermatite atopique par rapport à une personne normale, et que lorsque des vésicules isolées à partir de la souche sont administrées, un médiateur inflammatoire libéré par des vésicules pathogènes telles que des vésicules issues d'Escherichia coli est supprimé de manière surprenante. Par conséquent, les vésicules issues de bactéries Pseudomonas selon la présente invention peuvent être utilisées efficacement pour mettre au point une méthode de diagnostic du cancer du pancréas, du cholangiocarcinome, du cancer du sein, du cancer des ovaires, du cancer de la vessie, d'une cardiomyopathie, d'un angor de repos, du diabète, de la cirrhose hépatique et d'une dermatite atomique et pour mettre au point une composition destinée à prévenir ou traiter les maladies susmentionnées ou des maladies inflammatoires.
PCT/KR2019/002345 2018-02-28 2019-02-27 Nanovésicules issues de bactéries pseudomonas, et leur utilisation WO2019168331A1 (fr)

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KR20110038575A (ko) * 2009-10-08 2011-04-14 주식회사이언메딕스 실내 공기유래 세포밖 소포체를 포함하는 조성물 및 이의 용도
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