WO2019172600A1 - Nanovésicules issues de bactéries enhydrobacter, et leur utilisation - Google Patents

Nanovésicules issues de bactéries enhydrobacter, et leur utilisation Download PDF

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WO2019172600A1
WO2019172600A1 PCT/KR2019/002503 KR2019002503W WO2019172600A1 WO 2019172600 A1 WO2019172600 A1 WO 2019172600A1 KR 2019002503 W KR2019002503 W KR 2019002503W WO 2019172600 A1 WO2019172600 A1 WO 2019172600A1
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vesicles
enhydrobacter
cancer
bacteria
derived
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PCT/KR2019/002503
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Korean (ko)
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김윤근
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주식회사 엠디헬스케어
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Priority claimed from KR1020190024890A external-priority patent/KR102118989B1/ko
Application filed by 주식회사 엠디헬스케어 filed Critical 주식회사 엠디헬스케어
Priority to US16/978,562 priority Critical patent/US11554144B2/en
Priority to JP2020546473A priority patent/JP7013052B2/ja
Priority to EP19763721.8A priority patent/EP3763829A4/fr
Priority to CN201980017161.2A priority patent/CN111819294A/zh
Publication of WO2019172600A1 publication Critical patent/WO2019172600A1/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 enhydrobacter bacteria and their use, and more specifically, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy and atrial fibrillation using nanovesicles derived from enhydrobacter bacteria. It relates to a diagnostic method such as angina, liver cirrhosis, or diabetes mellitus, and a composition for preventing, ameliorating or treating such diseases including the vesicles.
  • 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 the immune and metabolic abnormalities caused by pathogenic vesicles.
  • Th17 immune response characterized by the secretion of Interleukin (IL) -17 cytokines, which secrete IL-6 upon exposure to bacterial vesicles, This induces a Th17 immune response.
  • IL Interleukin
  • Th17 immune response Inflammation by the Th17 immune response is characterized by neutrophil infiltration, and tumor necrosis factor-alpha (TNF- ⁇ ), which is secreted from inflammatory cells such as macrophages in the process of inflammation, plays an important role. In charge.
  • Enhydrobacter bacteria are Gram-negative bacteria belonging to gamma proteobacteria.
  • Enhydrobacter aerosaccus is the only species belonging to the genus Enhydrobacter genus, and is known as a catalase and oxidase positive bacterium.
  • Enhydrobacter vesicles there has been no report on the secretion of extracellular vesicles from Enhydrobacter bacteria, and the application of Enhydrobacter vesicles for the diagnosis and treatment of intractable diseases such as cancer, cardiovascular disease and diabetes has not been reported. There is no bar.
  • the present inventors earnestly researched to solve the above-mentioned conventional problems, and compared with the normal person through pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, dysplastic angina pectoris, cirrhosis It was confirmed that the contents of enhydrobacter bacteria-derived vesicles were significantly reduced in the samples derived from, and diabetic patients. In addition, when vesicles were isolated from enhydrobacter aerosacus bacteria belonging to the enhydrobacter bacterium and treated with macrophages, it was confirmed that significantly suppressed the secretion of IL-6 and TNF- ⁇ by pathogenic vesicles. 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, cholangiocarcinoma, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, angina, liver cirrhosis, or diabetes mellitus.
  • the present invention is a pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, angina pectoris, liver cirrhosis, or diabetes prevention, improvement or treatment containing vesicles derived from enhydrobacter bacteria as an active ingredient It is another object to provide a composition for use.
  • the present invention comprises the following steps, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, angina, liver cirrhosis, or diabetes mellitus Provide informational methods for diagnosis:
  • pancreatic cancer pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, if the content of extracellular vesicles derived from Enhydrobacter bacteria is lower than that of normal humans by quantitative analysis of the PCR products. Classify as dysplastic angina, cirrhosis, or diabetes.
  • the present invention also provides a method for diagnosing pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, heteroangular angina, liver cirrhosis, or diabetes, comprising the following steps:
  • pancreatic cancer pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, if the content of extracellular vesicles derived from Enhydrobacter bacteria is lower than that of normal humans by quantitative analysis of the PCR products. Determining dysplastic angina, cirrhosis, or diabetes.
  • 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 a pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, dysplastic angina, It provides a pharmaceutical composition for the prevention or treatment of chronic liver disease or diabetes.
  • the present invention is a pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, dysplastic angina, Provided is a food composition for preventing or improving chronic liver disease or diabetes.
  • the present invention is an enhydrobacter bacteria-derived vesicles, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, heteroangular angina, Provided for the prevention or treatment of chronic liver disease or diabetes.
  • the present invention comprises administering to a subject a pharmaceutical composition comprising an enhydrobacter bacteria-derived vesicle as an active ingredient, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation,
  • a pharmaceutical composition comprising an enhydrobacter bacteria-derived vesicle as an active ingredient, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation.
  • the vesicles may have an average diameter of 10 to 200 nm.
  • the vesicles may be secreted naturally or artificially in the enhydrobacterium bacteria.
  • the enhydrobacter bacteria-derived vesicles may be vesicles derived from enhydrobacter aeroscus.
  • the present inventors confirmed that the 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.
  • Analysis of existing bacterial-derived vesicles metagenome revealed that vesicles derived from pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, heterozygous angina, cirrhosis, and blood of diabetic patients It was confirmed that the significantly reduced compared to the normal person.
  • the vesicles derived from the enhydrobacter bacterium according to the present invention are pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, dysplastic angina, liver cirrhosis, or diagnostic method for diabetes, and prevention of the disease. It is expected that the present invention can be usefully used in improving or treating compositions.
  • Figure 1a is a picture of the distribution of bacteria and vesicles by time after oral administration of bacteria and bacteria-derived vesicles (EV) to the mouse
  • Figure 1b is 12 hours after oral administration
  • blood blood
  • kidney 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 enhydrobacter bacteria-derived vesicles after performing a bacterial-derived vesicle metagenome analysis present in patients with pancreatic cancer and normal blood.
  • Figure 3 is a result of comparing the distribution of enhydrobacter bacteria-derived vesicles after performing a bacterial-derived vesicle metagenome analysis present in patients with bile duct cancer.
  • Figure 4 is a result of comparing the distribution of enhydrobacter bacteria-derived vesicles after performing the bacteria-derived vesicles metagenome analysis present in breast cancer patients and normal blood.
  • 5 is a result of comparing the distribution of enhydrobacter bacterium-derived vesicles after performing a bacterial-derived vesicle metagenome analysis present in ovarian cancer patients and normal blood.
  • Figure 6 is a result of comparing the distribution of enhydrobacter bacteria-derived vesicles after performing a bacterial-derived vesicle metagenome analysis present in lymphoma patients and normal blood.
  • 9 is a result of comparing the distribution of enhydrobacter bacteria-derived vesicles after performing a bacterial-derived vesicle metagenome analysis present in patients with atrial fibrillation and normal blood.
  • 10 is a result of comparing the distribution of enhydrobacter bacterium-derived vesicles after performing a bacterial-derived vesicle metagenome analysis present in patients with heterozygous angina and normal blood.
  • 11 is a result of comparing the distribution of enhydrobacter bacteria-derived vesicles after performing the bacterial-derived vesicle metagenome analysis present in the liver cirrhosis transducer and normal blood.
  • E. coli EV E. coli vesicles
  • E. coli EV Escherichia coli vesicles
  • E. hydrophilis The results of the evaluation of the inflammatory mediators IL-6 and TNF- ⁇ secretion (PC: positive control; LP: Lactobacillus plantarum EVs; EA: Enhydrobacter aerosaccus EVs).
  • the present invention relates to vesicles derived from enhydrobacter bacteria and uses thereof.
  • vesicles were isolated and characterized from enhydrobacter aerosacus, the only species belonging to the enhydrobacter bacterium, and found to be pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, heteroangular angina, It was confirmed that it can be used as a composition for preventing, improving or treating diseases such as chronic liver disease and diabetes.
  • the present invention provides a method for providing information for diagnosing pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, heteroangular angina, liver cirrhosis, or diabetes, comprising the following steps:
  • pancreatic cancer pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, if the content of extracellular vesicles derived from Enhydrobacter bacteria is lower than that of normal humans by quantitative analysis of the PCR products. Classify as dysplastic angina, cirrhosis, or diabetes.
  • Diagnosis in the broad sense means to determine the actual condition of the patient's disease 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, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, dysplastic angina, and / or diabetes, and the level of the disease.
  • nanovesicle refers to a structure of nanoscale membranes 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.
  • nanovesicles or vesicles are naturally secreted or artificially produced by enhydrobacter bacteria, and have a spherical shape and have an average diameter of 10 to 200 nm.
  • the term "metagenome” used in the present invention also referred to as "gunoelectric”, refers to the sum total of the genome including all viruses, bacteria, fungi, etc. in an isolated area such as soil, animal intestine, mainly culture It is used as a concept of genome explaining the identification of many microorganisms at once using sequencer to analyze microorganisms that are not.
  • 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 present invention comprises an enhydrobacter bacteria-derived vesicles as an active ingredient, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, dysplastic angina, chronic liver disease, Or it provides a composition for preventing, treating or improving diabetes.
  • the composition comprises a food composition and a pharmaceutical composition, in the present invention the food composition comprises a nutraceutical composition.
  • the composition of the present invention may be a formulation of an oral nebulizer or inhalant.
  • prevention refers to pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, angina, chronic liver disease, and / or by administration of a composition according to the invention. It means any action that suppresses or delays the onset of diabetes.
  • treatment means pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, dysplastic angina, chronic liver disease, and / or by administration of a composition according to the invention. Means any behavior that improves or beneficially changes the symptoms of diabetes.
  • the term “improvement” means any action that at least reduces the parameters associated with the condition being treated, for example, the extent of symptoms.
  • the vesicles were centrifuged, ultra-fast centrifugation, autoclaving, extrusion, sonication, cell lysis, homogenization, freeze-thaw, electroporation, mechanical degradation, chemical treatment, filtration by a culture medium containing enhydrobacter bacteria. It can be separated 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 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 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).
  • the vesicles isolated from the blood of normal people matched age and sex to patients with pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, angina with angina, liver cirrhosis, and diabetes Bacterial metagenome analysis was performed using.
  • enhydrobacter bacterial-derived vesicles were significantly reduced in clinical samples of pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, angina, liver cirrhosis, and diabetic patients compared to normal samples. (See Examples 3 to 13).
  • Example 1 Analysis of absorption, distribution, and excretion of intestinal bacteria and bacterial-derived vesicles
  • the experiment was performed as follows. Fluorescently labeled bacteria and vesicle-derived vesicles were administered to the gastrointestinal tract at a dose of 50 ⁇ g, respectively, and the fluorescence was measured after 0, 5, 3, 6 and 12 hours. As a result of observing the whole image of the mouse, as shown in FIG. 1A, the bacteria were not absorbed systemically, but in the case of bacterial-derived vesicles, they were absorbed systemically 5 minutes after administration, and the bladder fluorescence after 3 hours This was observed strongly, indicating that the vesicles were excreted by the urinary system. In addition, the vesicles were found to exist in the body until 12 hours of administration.
  • 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 blood of pancreatic cancer patients and 271 blood of normal people who matched age and sex were extracted from vesicles present in the blood and subjected to metagenomic analysis, followed by enhydrobacter bacteria. The distribution of the derived vesicles was evaluated. As a result, it was confirmed that the vesicles derived from the enhydrobacter bacterium were significantly reduced in the blood of pancreatic cancer patients compared to the normal blood (see Table 2 and FIG. 2).
  • Example 4 Bacterial-derived vesicles from bile duct cancer patients Metagenome analysis
  • Example 2 blood was extracted from vesicles present in the blood of 96 breast cancer patients and 192 normal blood patients whose age and sex were matched. The distribution of the derived vesicles was evaluated. As a result, it was confirmed that enhydrobacter bacteria-derived vesicles 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 blood of ovarian cancer patients and 139 blood of normal and matched age and sex were extracted from the vesicles present in the blood to perform a metagenome analysis, followed by enhydrobacter The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicles derived from the enhydrobacter bacterium were significantly reduced in the blood of ovarian cancer patients compared to the normal blood (see Table 5 and FIG. 5).
  • Example 2 In the method of Example 2, the blood of 63 lymphatic patients and 53 blood of normal people who matched age and sex were extracted from vesicles in the blood and subjected to metagenomic analysis, followed by enhydrobacter bacteria. The distribution of the derived vesicles was evaluated. As a result, it was confirmed that the vesicles derived from the enhydrobacter bacterium were significantly reduced in the blood of lymphoma patients compared to normal blood (see Table 6 and FIG. 6).
  • Example 2 the blood of 80 patients with heteroangular angina and 80 normal blood whose age and sex were matched were extracted from the vesicles present in the blood and subjected to a metagenomic analysis, followed by enhydrobacter The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived vesicles derived from the bacterium from the angina pectoris significantly reduced compared to normal blood (see Table 10 and FIG. 10).
  • Example 2 the blood was extracted from vesicles of 130 livers and 145 normal blood persons whose ages and genders were matched. The distribution of the derived vesicles was evaluated. As a result, it was confirmed that enhydrobacter bacteria-derived vesicles were significantly reduced in the blood of liver cirrhosis compared to normal blood (see Table 11 and FIG. 11).
  • Example 2 the blood of 61 diabetic patients and 122 normal blood of age and sex matched were extracted from the vesicles present in the blood and subjected to a metagenomic analysis, followed by enhydrobacter bacteria. The distribution of the derived vesicles was evaluated. As a result, it was confirmed that enhydrobacter bacteria-derived vesicles were significantly reduced in blood of diabetic patients compared to normal blood (see Table 12 and FIG. 12).
  • the enhydrobacter aeroscus strain belonging to the enhydrobacter bacterium was isolated from the environmental sample, and then cultured to separate the vesicles thereof.
  • Enhydrobacter aerosacus strains were incubated in BHI (brain heart infusion) medium until absorbance (OD 600 ) was 1.0 to 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. for 15 minutes, filtered through a 0.45 ⁇ m filter, and the filtered supernatant was used as a 100 kDa hollow filter membrane using a QuixStand benchtop system (GE Healthcare, UK).
  • 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.
  • endobacterial-derived vesicles from mouse macrophage Raw 264.7 cells were treated with various concentrations (0.1, 1, 10 ⁇ g / Ml) and then E. coli- derived vesicles, E. coli , an inflammatory pathogenic vesicle. EV) was treated to measure the amount of secretion of inflammatory mediators (IL-6, TNF- ⁇ , etc.).
  • Lactobacillus plantarum derived vesicles (1 ⁇ g / ml) were used as beneficial control vesicles.
  • the vesicles derived from the enhydrobacter bacterium according to the present invention include pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, heterogeneous angina pectoris, liver cirrhosis, or a diagnosis method for diabetes, as well as prevention of the above diseases, As it can be used as a composition for improvement or treatment, it is expected to be usefully used in the related medical and food industries.

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Abstract

La présente invention concerne des vésicules issues de bactéries Enhydrobacter, et leur utilisation. Les présents inventeurs ont expérimentalement confirmé que : comparées à celles d'une personne normale, les vésicules dans des échantillons cliniques provenant de patients souffrant d'un cancer du pancréas, d'un cholangiocarcinome, d'un cancer du sein, d'un cancer de l'ovaire, d'un lymphome, d'un infarctus du myocarde, d'une cardiomyopathie, d'une fibrillation auriculaire, d'une angine variante, d'une cirrhose ou d'un diabète, sont considérablement réduites ; et lorsque les vésicules isolées d'une souche sont administrées, la sécrétion des médiateurs inflammatoires par des vésicules pathogènes, telles que les vésicules issues d'Escherichia coli, est inhibée de façon notable. Ainsi, les vésicules issues de bactéries Enhydrobacter, selon la présente invention, peuvent être utilement utilisées aux fins de développer : une méthode de diagnostic du cancer du pancréas, du cholangiocarcinome, du cancer du sein, du cancer de l'ovaire, du lymphome, de l'infarctus du myocarde, d'une cardiomyopathie, d'une fibrillation auriculaire, d'une angine variante, d'une cirrhose ou d'un diabète ; et une composition pour prévenir, faire régresser ou traiter lesdites maladies.
PCT/KR2019/002503 2018-03-05 2019-03-05 Nanovésicules issues de bactéries enhydrobacter, et leur utilisation WO2019172600A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
US16/978,562 US11554144B2 (en) 2018-03-05 2019-03-05 Nanovesicles derived from enhydrobacter bacteria, and use thereof
JP2020546473A JP7013052B2 (ja) 2018-03-05 2019-03-05 エンヒドロバクター細菌由来のナノ小胞及びその用途
EP19763721.8A EP3763829A4 (fr) 2018-03-05 2019-03-05 Nanovésicules issues de bactéries enhydrobacter, et leur utilisation
CN201980017161.2A CN111819294A (zh) 2018-03-05 2019-03-05 来源于栖水菌种细菌的纳米囊泡及其用途

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KR20180026037 2018-03-05
KR10-2018-0026037 2018-03-05
KR1020190024890A KR102118989B1 (ko) 2018-03-05 2019-03-04 엔히드로박터 세균 유래 나노소포 및 이의 용도
KR10-2019-0024890 2019-03-04

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20110025068A (ko) * 2009-09-01 2011-03-09 주식회사이언메딕스 장내 공생 세균유래 세포밖 소포체, 및 이를 이용한 질병모델, 백신, 후보 약물 탐색 방법, 및 진단 방법
KR20160073157A (ko) * 2014-12-16 2016-06-24 이화여자대학교 산학협력단 세균 유래의 나노소포체를 이용한 세균성 감염질환 원인균 동정방법
KR20180006303A (ko) * 2016-07-08 2018-01-17 주식회사 엠디헬스케어 프로피오니박테리움 속 세균 유래 나노소포 및 이의 용도
KR20180018354A (ko) * 2016-08-12 2018-02-21 주식회사 엠디헬스케어 바실러스 속 세균 유래 나노소포 및 이의 용도
KR101833348B1 (ko) * 2016-12-26 2018-03-02 주식회사 엠디헬스케어 세균 메타게놈 분석을 통한 유방암 진단방법

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20110025068A (ko) * 2009-09-01 2011-03-09 주식회사이언메딕스 장내 공생 세균유래 세포밖 소포체, 및 이를 이용한 질병모델, 백신, 후보 약물 탐색 방법, 및 진단 방법
KR20160073157A (ko) * 2014-12-16 2016-06-24 이화여자대학교 산학협력단 세균 유래의 나노소포체를 이용한 세균성 감염질환 원인균 동정방법
KR20180006303A (ko) * 2016-07-08 2018-01-17 주식회사 엠디헬스케어 프로피오니박테리움 속 세균 유래 나노소포 및 이의 용도
KR20180018354A (ko) * 2016-08-12 2018-02-21 주식회사 엠디헬스케어 바실러스 속 세균 유래 나노소포 및 이의 용도
KR101833348B1 (ko) * 2016-12-26 2018-03-02 주식회사 엠디헬스케어 세균 메타게놈 분석을 통한 유방암 진단방법

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3763829A4 *

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