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WO2019139360A1 - Nanovésicules issues de faecalibacterium prausnitzii et utilisations associées - Google Patents

Nanovésicules issues de faecalibacterium prausnitzii et utilisations associées Download PDF

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WO2019139360A1
WO2019139360A1 PCT/KR2019/000368 KR2019000368W WO2019139360A1 WO 2019139360 A1 WO2019139360 A1 WO 2019139360A1 KR 2019000368 W KR2019000368 W KR 2019000368W WO 2019139360 A1 WO2019139360 A1 WO 2019139360A1
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cancer
vesicles
disease
parkinson
myocardial infarction
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PCT/KR2019/000368
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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 KR1020190002607A external-priority patent/KR102282490B1/ko
Application filed by 주식회사 엠디헬스케어 filed Critical 주식회사 엠디헬스케어
Priority to CN201980007976.2A priority Critical patent/CN111587295A/zh
Priority to JP2020538572A priority patent/JP2021510300A/ja
Priority to EP19738195.7A priority patent/EP3739068A4/fr
Priority to US15/733,371 priority patent/US11666607B2/en
Publication of WO2019139360A1 publication Critical patent/WO2019139360A1/fr
Priority to JP2021164124A priority patent/JP7191411B2/ja

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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 OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • 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
    • 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
    • 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

Definitions

  • the present invention relates to a nano-vesicle derived from a pathogenic bacterium, such as a gastric cancer, a colon cancer, a liver cancer, a pancreatic cancer, a cholangiocarcinoma, an ovarian cancer, , A bladder cancer, a lymphoma, myocardial infarction, atrial fibrillation, and Parkinson's disease, and a composition for preventing, improving or treating the disease including the vesicle.
  • a pathogenic bacterium such as a gastric cancer, a colon cancer, a liver cancer, a pancreatic cancer, a cholangiocarcinoma, an ovarian cancer, , A bladder cancer, a lymphoma, myocardial infarction, atrial fibrillation, and Parkinson's disease, and a composition for preventing, improving or treating the disease including the vesicle.
  • microbiota or microbiome refers to a microbial community, including true bacteria, archaea, and eukarya in a given settlement.
  • the mucous membrane forms a physical barrier that can not pass through particles of 200 nanometers (nm) or larger, and can not pass through the mucous membrane when the bacteria are symbiotic to the mucous membrane.
  • the bacterial-derived vesicles are less than 100 nanometers in size, It passes through epithelial cells through the mucosa and is absorbed by our body.
  • the locally secreted bacterial-derived vesicles are absorbed through the epithelial cells of the mucosa to induce a local inflammatory reaction.
  • the vesicles passing through the epithelium are systemically absorbed through the lymphatic vessels and distributed to each organ, It regulates immune and inflammatory responses.
  • vesicles originating from pathogenic Gram-negative bacteria such as E. coli ( Eshcherichia coli ) cause colitis locally, and when they are absorbed into the blood vessels, the vascular endothelial cell inflammatory response causes systemic inflammation, stool, And is also absorbed by insulin-acting muscle cells and the like, leading to insulin resistance and diabetes.
  • beneficial bacteria-derived vesicles can control disease by controlling immune function and metabolic dysfunction caused by pathogenic vesicles.
  • the immune response to bacterial-derived factors such as vesicles is a Th17 immune response characterized by interleukin (IL) -17 cytokine secretion, which upon exposure to bacterial-derived vesicles secretes IL-6, Induce an immune response.
  • IL interleukin
  • Tumor necrosis factor-alpha (TNF- ⁇ ) which is secreted by inflammatory cells such as neutrophils and macrophages during the inflammatory process, is characterized by neutrophil infiltration by Th17 immune response It plays an important role in inflammation and cancer development.
  • the present invention provides a method of treating a clinical sample of gastric cancer, colon cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, and Parkinson's disease in comparison with a normal human parvovirus- And it is confirmed that the disease can be diagnosed.
  • the vesicles were isolated from the pathogenic bacterium, and the characteristics thereof were analyzed.
  • the present inventors have intensively studied to solve the above-mentioned conventional problems.
  • the present inventors have found that gastric cancer, colon cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, And that the content of paracetamycinum purosunicus-derived vesicles in the samples derived from patients with Parkinson's disease was significantly reduced.
  • the vesicles were isolated from the cells of the pathogenic bacterium, the inhibition of IL-6 and TNF-alpha secretion by the pathogenic vesicles was remarkably suppressed. Based on this, the present invention was completed .
  • the present invention also relates to a pharmaceutical composition for treating gastric cancer, colon cancer, hepatic cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, and Parkinson's disease comprising a parasite derived from a pathogenic bacterium, , Or a composition for preventing, ameliorating, or treating one or more diseases selected from the group consisting of:
  • the present invention provides a method of treating gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, Provides a method of providing information for diagnosis:
  • the present invention also provides a diagnostic method for gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, biliary cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, or Parkinson's disease comprising the steps of:
  • the sample in step (a) may be feces, blood, or urine.
  • the primer pair in step (b) may be a primer of SEQ ID NO: 1 or SEQ ID NO: 2.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising gastric cancer, colon cancer, hepatic cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, and Parkinson's disease, which comprises a vesicle derived from a pathogenic bacterium
  • the present invention provides a pharmaceutical composition for preventing or treating at least one disease selected from the group consisting of:
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising gastric cancer, colon cancer, hepatic cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, and Parkinson's disease, which comprises a vesicle derived from a pathogenic bacterium, At least one disease prevention or improvement food composition selected from the group consisting of:
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutical composition comprising an effective amount of a bacterium belonging to the genus Acanthopanax senticosus as an active ingredient, , Myocardial infarction, atrial fibrillation, and Parkinson ' s disease.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising at least one compound selected from the group consisting of gastric cancer, colon cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation and Parkinson's disease It provides a preventive or therapeutic use of the disease.
  • the vesicles may have an average diameter of 10 to 200 nm.
  • the vesicle may be naturally or artificially secreted from the pathogenic bacterium.
  • the present inventors confirmed that intestinal bacteria are not absorbed into the body but they are absorbed into the body through epithelial cells in the case of bacterial-derived vesicles, and are excreted through the kidneys, liver, and lungs by systemic distribution.
  • Stomach cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, and Parkinson's disease by analysis of a bacterial-derived vesicle metagenome present in blood, urine or blood It was confirmed that the vesicles derived from the pathogenic bacterium Pausus nichia in the urine were significantly decreased compared to the normal subjects.
  • the present invention provides a method for diagnosing gastric cancer, colon cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, and Parkinson's disease, and It is anticipated that it may be usefully used in a composition for preventing, ameliorating or treating a food or a drug for the above diseases.
  • FIG. 1A is a photograph of the distribution pattern of bacteria and vesicles by time after oral administration of bacteria and bacterial-derived vesicles (EV) to a mouse.
  • FIG. 1B is a photograph of blood, kidney , Liver and various organs were extracted to evaluate the distribution patterns of bacteria and vesicles in the body.
  • FIG. 2 shows the results of a comparison of the distribution of vesicles derived from Pataribacterium fruticus Niichus after the analysis of the bacterial-derived vesicle metagenomes present in gastric cancer patients and normal human blood.
  • FIG. 3 shows the results of a comparison of the distribution of vesicles derived from Pataribacterium furosinii after the analysis of bacterial-derived vesicle metagenomes existing in colon cancer patients and normal feces.
  • FIG. 4 shows the results of a comparison of the distribution of vesicles derived from Pataribacterium fruticus Niichus after the analysis of bacterial-derived vesicle metagenomes existing in patients with liver cancer and normal human blood.
  • FIG. 5 shows the results of a comparison of the distribution of vesicles derived from Pataribacterium furosinii after the analysis of bacterial-derived vesicle metagenomes existing in pancreatic cancer patients and normal human blood.
  • Fig. 6 shows the results of a comparison of the distribution of vesicles derived from Pataribacterium furosinii after the analysis of bacterial-derived vesicle metagenomes existing in patients with biliary cancer and normal human blood.
  • Fig. 7 shows the results of a comparison of the distribution of vesicles derived from Pataribacterium furosinii after the analysis of bacterial-derived vesicle metagenomes existing in ovarian cancer patients and normal human blood.
  • FIG. 8 shows the results of a comparison of the distribution of vesicles derived from Pataribacterium frutosa Niichi after the analysis of the vesicle-derived vesicle metagenomes in bladder cancer patients and normal blood.
  • FIG. 9 shows the results of a comparison of the distribution of vesicles derived from Pataribacterium furosinii after the analysis of bacterial-derived vesicle metagenomes existing in lymphoma patients and normal blood.
  • Fig. 10 shows the results of a comparison of the distribution of vesicles derived from the pathogenic bacterium pneumoniae after the analysis of the bacterial-derived vesicle metagenomes present in the myocardial infarction patients and the normal blood.
  • Fig. 11 shows the results of a comparison of the distribution of vesicles derived from Pataribacterium furosinii after the analysis of the bacterial-derived vesicle metagenomes present in the atrial fibrillation and normal human blood.
  • Fig. 12 shows the results of a comparison of the distribution of vesicles derived from the pathogenic bacterium pneumoniae after the analysis of the bacterial-derived vesicle metagenomes present in Parkinson's disease patients and normal human urine.
  • FIGS. 13A and 13B are graphs showing the effect of various concentrations of the paracetamol-derived microbial cells from Macallium bacterium on the macrophage to evaluate the inflammatory mediator effect of the paracase derived from the pathogenic bacterium Escherichia coli As a result of comparison with the case of treatment with E. coli EV, Fig. 13A compares IL-6 secretion and Fig. 13B compares the secretion of TNF-a.
  • FIG. 14A and 14B are graphs showing the results of pretreatment of the paracase derived from the pathogenic bacterium E. coli EV prior to the treatment with the E. coli EV to evaluate the antiinflammatory and immunomodulatory effects of the paracase from the pathogenic bacterium, As a result of evaluating the effect on the inflammatory mediator secretion by E. coli vesicle, FIG. 14A compares IL-6 secretion and FIG. 14B compares the secretion of TNF- ⁇ .
  • the present invention relates to vesicles derived from Lactobacillus brevis and their uses.
  • the present inventors have conducted metagenomic analysis to determine whether a sample derived from Pataribacterium frutosa Niichia can be obtained from samples derived from gastric cancer, colon cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, It was confirmed that the content of vesicles was significantly reduced, and thus the present invention was completed based on this finding.
  • the present invention provides a method for providing information for diagnosis of gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, or Parkinson's disease comprising the following steps:
  • diagnosis means, in a broad sense, judging the actual condition of a patient in all aspects. The contents of the judgment are the pathology, etiology, pathology, severity, details of the disease, presence of complications, and prognosis.
  • the diagnosis in the present invention is to judge whether or not the disease such as gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, and / or Parkinson's disease,
  • Nanovesicle or "Vesicle” as used in the present invention means a structure composed of nano-sized membranes secreted from various bacteria.
  • Gram-negative bacteria-derived vesicles or outer membrane vesicles have not only lipopolysaccharides but also toxic proteins and bacterial DNA and RNA, and gram-positive bacteria-derived vesicles Has peptidoglycan and lipoteichoic acid which are cell wall components of bacteria as well as protein and nucleic acid.
  • nano-vesicles or vesicles are naturally secreted or artificially produced in the pathogenic bacterium, and have a spherical shape and an average diameter of 10 to 200 nm.
  • &quot metagenome &quot
  • metagenome &quot is also referred to as 'whole milk', and refers to the total of genomes including all viruses, bacteria, fungi, etc. in isolated regions such as soil and animal fields. It is used as a concept of a genome to explain the identification of many microorganisms at once by using a sequencer to analyze microorganisms.
  • a metagenome is not a genome or a genome, but a kind of mixed genome as a genome of all species of an environmental unit. This is a term derived from the viewpoint that when defining a species in the course of omics biology development, it functions not only as an existing species but also as a species that interacts with various species to form a complete species.
  • it is the subject of techniques that analyze all DNA and RNA regardless of species, identify all species in an environment, identify interactions, and metabolism using rapid sequencing.
  • the patient-derived sample may be feces, blood, or urine, but is not limited thereto.
  • the present invention provides a pharmaceutical composition for preventing or treating gastric cancer, colon cancer, hepatic cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, And Parkinson's disease.
  • the present invention also provides a pharmaceutical composition for preventing or treating at least one disease selected from the group consisting of Parkinson's disease and Parkinson's disease.
  • the present invention provides a pharmaceutical composition for treating gastric cancer, colon cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation , And Parkinson ' s disease.
  • prevention used in the present invention refers to the administration of a food or drug composition according to the present invention to treat gastric cancer, colon cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, and / Parkinson 's disease, etc.
  • treatment refers to the treatment of gastric cancer, colon cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, and / Parkinson 's disease, etc.
  • improvement means all actions that at least reduce the degree of symptom associated with the condition being treated.
  • the vesicles can be obtained by culturing a culture solution containing a pathogenic bacterium belonging to the genus Saccharomyces cerevisiae by centrifugation, ultrafast centrifugation, high pressure treatment, extrusion, ultrasonic degradation, cell lysis, homogenization, freezing-thawing, electroporation, Separation by using one or more methods selected from the group consisting of filtration, gel filtration chromatography, pre-flow electrophoresis, and capillary electrophoresis. Further, it may further include processes such as washing for removal of impurities and concentration of the resulting vesicles.
  • bacterial and bacterial-derived vesicles were orally administered to mice to evaluate the absorption, distribution, and excretion of bacteria and vesicles in the body.
  • the vesicles were not absorbed through the intestinal membrane, Were absorbed and distributed systemically and excreted through the kidneys and liver (see Example 1).
  • the stool, blood, or blood of a normal person who matched age and sex in gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, Bacterial metagenomic analysis was carried out using vesicles isolated from urine.
  • a clinical sample of patients with gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, and Parkinson's disease See Examples 3 to 13).
  • vesicles secreted from the cultured Paciarum bacterium were able to demonstrate immunomodulatory and anti-inflammatory effects.
  • the vesicles derived from the various strains of P. falcus nichia After treatment with phagocytes, E. coli-derived vesicles, which are the causative factors of inflammatory diseases, were treated to evaluate inflammatory mediator secretion.
  • IL-6 and TNF-a secretion by E. coli-derived vesicles was effectively suppressed by the parasites derived from Parka bacteria (See Example 15).
  • the pharmaceutical composition according to the present invention may comprise a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carriers are those conventionally used in the formulation and include, but are not limited to, saline, sterilized water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, And may further contain other conventional additives such as antioxidants and buffers as needed.
  • it may be formulated into injectable formulations, pills, capsules, granules, or tablets such as aqueous solutions, suspensions, emulsions and the like by additionally adding diluents, dispersants, surfactants, binders, lubricants and the like.
  • Suitable pharmaceutically acceptable carriers and formulations can be suitably formulated according to the respective ingredients using the methods disclosed in Remington's reference.
  • the pharmaceutical composition of the present invention is not particularly limited to a formulation, but may be formulated into an injection, an inhalant, an external preparation for skin, or an oral ingestion agent.
  • the pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenous, subcutaneous, skin, nasal, or airway) according to the desired method, The type of administration, the route of administration, and the time, but may be suitably selected by those skilled in the art.
  • a pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level is determined by the type of disease, severity, activity of the drug, The time of administration, the route of administration and the rate of excretion, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts.
  • the composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal 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 body weight of the patient. Generally, 0.001 to 150 mg, preferably 0.01 to 100 mg per 1 kg of body weight is administered daily or every other day Or one to three times a day. However, the dosage may be varied depending on the route of administration, the severity of obesity, sex, weight, age, etc. Therefore, the dosage is not limited to the scope of the present invention by any means.
  • the food composition of the present invention comprises a health functional food composition.
  • the food composition according to the present invention can be used as it is or in combination with other food or food ingredients, and can be suitably used according to conventional methods.
  • the amount of the active ingredient to be mixed can be suitably determined according to the intended use (for prevention or improvement).
  • the composition of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material, in the production of food or beverage.
  • the amount may be less than the above range.
  • the food composition of the present invention has no particular limitation on the ingredients other than those containing the active ingredient as an essential ingredient in the indicated ratios 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 polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol.
  • Natural flavors tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above .
  • the ratio of the above-mentioned natural carbohydrate can be appropriately determined by a person skilled in the art.
  • the food composition of the present invention can be used as a flavoring agent such as a variety of nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and thickening agents (cheese, chocolate etc.), pectic acid and its salts, Salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. These components may be used independently or in combination. The ratios of these additives can also be appropriately selected by those skilled in the art.
  • Example 1 Analysis of intestinal absorption, distribution, and excretion of intestinal bacteria and bacterial-derived vesicles
  • Example 2 Analysis of bacterial-derived vesicle metagenomes in clinical samples
  • the DNA extracted by the above method was amplified using the above 16S rDNA primer, followed by sequencing (Illumina MiSeq sequencer), outputting the result as a Standard Flowgram Format (SFF) file, and using GS FLX software (v2.9)
  • SFF Standard Flowgram Format
  • GS FLX software v2.9
  • the SFF file was converted into a sequence file (.fasta) and a nucleotide quality score file to confirm the creditworthiness of the lead and to remove the portion with a window (20 bps) average base call accuracy less than 99% (Phred score ⁇ 20) .
  • OTU Operational Taxonomy Unit
  • clustering is performed according to sequence similarity using UCLUST and USEARCH.
  • the genus is 94%
  • the family is 90%
  • the order is 85% class, 80%
  • phylum are clustering based on 75% sequence similarity, and the phylum, class, order, family, genus level
  • the bacteria were profiled (QIIME) with a sequence similarity greater than 97% at the genus level using BLASTN and GreenGenes 16S RNA sequence database (108,453 sequence).
  • Example 4 Analysis of vesicle meta-genomes from feces bacteria of colon cancer patients
  • Example 5 Analysis of vesicle meta-genome derived from blood germ in patients with liver cancer
  • Genomic DNA was extracted from the vesicles present in the blood of 331 normal persons whose age and gender matched 86 liver cancer patients by the method of Example 2, and the results were analyzed by metagenomic analysis. The distribution of vesicles was evaluated. As a result, it was confirmed that the vesicles derived from Pataribacterium frutosa Nichia were significantly reduced in the blood of the liver cancer patients compared to the normal blood (see Table 4 and Fig. 4).
  • Example 6 Analysis of vesicle meta-genome from blood microbes in pancreatic cancer patients
  • Example 8 Analysis of vesicle meta-genome derived from blood bacterium of ovarian cancer patient
  • Example 9 Analysis of vesicle meta-genome from blood microbes of bladder cancer patients
  • a total of 176 blastomeric blood samples were collected from 91 bladder cancer patients and normal 176 blood samples from patients with bladder cancer.
  • the genes were extracted from the vesicles present in the blood and analyzed by metagenomic analysis. The distribution of vesicles was evaluated. As a result, it was confirmed that the vesicles derived from Pataribacterium flaus Nichia were significantly reduced in the blood of the bladder cancer patients compared to the normal blood (see Table 8 and Fig. 8).
  • Example 10 Analysis of vesicle metagenomes derived from urine bacteria from patients with lymphoma
  • the gene was extracted from the vesicles present in the blood of 53 normal persons who matched the age and sex of 63 lymphoma patients according to the method of Example 2, and the distribution of the vesicles derived from the pathogenic bacterium Pausus nichia was evaluated Respectively. As a result, it was confirmed that vesicles derived from L. pneumoniae were significantly reduced in the blood of lymphoma patients compared with normal blood (see Table 9 and Fig. 9).
  • Example 11 Analysis of vesicle-derived vesicle meta-genome from myocardial infarction patients
  • Example 12 Analysis of vesicle meta-genomes from blood microbes in patients with atrial fibrillation
  • Example 13 Analysis of vesicle metagenomes from urine bacteria of Parkinson's disease patients
  • Example 14 Separation of follicles derived from Lactobacillus brevis and induction of inflammation
  • Saccharomyces cerevisiae strains were subcultured in a BHI (brain heart infusion) medium until the absorbance (OD600) became 1.0 to 1.5 in an anaerobic chamber at 37 ° C.
  • the culture supernatant which did not contain the strain, was recovered and centrifuged at 10,000 g for 15 minutes at 4 ° C, filtered through a 0.45 ⁇ m filter, and the supernatant was applied to a 100 kDa hollow filter membrane using a QuixStand benchtop system (GE Healthcare, UK) And concentrated to a volume of 200 ml through ultrafiltration.
  • the resulting supernatant was filtered once again with a 0.22 ⁇ m filter, and the filtered supernatant was centrifuged at 150,000 g at 4 ° C. for 3 hours.
  • the pellet was then suspended in Dulbecco's Phosphate Buffered Saline (DPBS). Density gradient centrifugation was then performed with 10%, 40%, and 50% OptiPrep solutions (Axis-Shield PoC AS, Norway) and the OptiPrep solution was diluted with HEPES-buffered saline (20 mM HEPES , 150 mM NaCl, pH 7.4).
  • Raw 264.7 cells a mouse macrophage cell line, were infected with various concentrations (0.1, 1, 10 Mu] g / ml), and the secretion amount of the inflammatory mediator (IL-6, TNF- [alpha]) was measured. More specifically, Raw 264.7 cells were plated at 1 ⁇ 10 5 in 24-well cell culture plates and cultured in DMEM (Dulbeco's Modified Eagle's Medium) complete medium for 24 hours. The culture supernatant was collected in a 1.5 ml tube and centrifuged at 3000 g for 5 minutes. The supernatant was collected and stored at 4 ° C for ELISA analysis.
  • DMEM Dulbeco's Modified Eagle's Medium
  • the mouse macrophage cell line Raw 264.7 cells, E. coli- derived vesicles were treated with various concentrations (0.1, 1, 10 ⁇ ⁇ / ml) and E. coli EV) to measure the secretion of inflammatory mediators (IL-6, TNF- ⁇ ). More specifically, Raw 264.7 cells were plated on 24-well cell culture plates at 1 ⁇ 10 5 cells and then cultured in DMEM complete medium for 24 hours. The culture supernatant was collected in a 1.5 ml tube and centrifuged at 3000 g for 5 minutes. The supernatant was collected and stored at 4 ° C for ELISA analysis.
  • Raw 264.7 cells were plated on 24-well cell culture plates at 1 ⁇ 10 5 cells and then cultured in DMEM complete medium for 24 hours. The culture supernatant was collected in a 1.5 ml tube and centrifuged at 3000 g for 5 minutes. The supernatant was collected and stored at 4 ° C for ELISA analysis.
  • the parasite derived from the pathogenic bacterium of the present invention is useful as a diagnostic method for gastric cancer, colon cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation or Parkinson's disease, It can be used as a composition for prevention, improvement or treatment of food or medicines in Korea, so that it is expected to be useful for the related pharmaceutical industry and food industry.

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Abstract

La présente invention concerne des vésicules issues de Faecalibacterium prausnitzii et des utilisations associées. Il a été confirmé expérimentalement par les présents inventeurs que la proportion des vésicules a été significativement réduite dans des échantillons cliniques provenant de patients atteints d'un cancer gastrique, d'un cancer colorectal, d'un cancer du foie, d'un cancer du pancréas, d'un cholangiocarcinome, d'un cancer de l'ovaire, d'un cancer de la vessie, d'un lymphome, d'un infarctus du myocarde, d'une fibrillation auriculaire, et de la maladie de Parkinson par comparaison avec une personne normale et que les vésicules, après administration, une fois séparées de la souche, inhibent significativement la sécrétion de médiateurs inflammatoires provoquée par des vésicules pathogènes, telles que des vésicules issues de bacilles du côlon. Les vésicules issues de Faecalibacterium prausnitzii selon la présente invention sont censées être utilisées de manière avantageuse dans le but de développer une méthode de diagnostic du cancer gastrique, du cancer colorectal, du cancer du foie, du cancer du pancréas, du cholangiocarcinome, du cancer de l'ovaire, du cancer de la vessie, du lymphome, de l'infarctus du myocarde, de la fibrillation auriculaire et/ou de la maladie de Parkinson, et une composition pour prévenir, soulager ou traiter lesdites maladies.
PCT/KR2019/000368 2018-01-12 2019-01-10 Nanovésicules issues de faecalibacterium prausnitzii et utilisations associées WO2019139360A1 (fr)

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CN201980007976.2A CN111587295A (zh) 2018-01-12 2019-01-10 来源于普氏粪杆菌的纳米囊泡及其用途
JP2020538572A JP2021510300A (ja) 2018-01-12 2019-01-10 フィーカリバクテリウム・プラウスニッツィイ由来のナノ小胞およびその用途
EP19738195.7A EP3739068A4 (fr) 2018-01-12 2019-01-10 Nanovésicules issues de faecalibacterium prausnitzii et utilisations associées
US15/733,371 US11666607B2 (en) 2018-01-12 2019-01-10 Nanovesicles derived from Faecalibacterium prausnitzii and uses thereof
JP2021164124A JP7191411B2 (ja) 2018-01-12 2021-10-05 フィーカリバクテリウム・プラウスニッツィイ由来のナノ小胞およびその用途

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KR1020190002607A KR102282490B1 (ko) 2018-01-12 2019-01-09 패칼리박테리움 프라우스니찌 유래 나노소포 및 이의 용도

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115942944A (zh) * 2020-06-11 2023-04-07 伊夫罗生物科学公司 使用颤螺旋菌科微生物胞外囊泡治疗疾病和障碍的组合物和方法
WO2025011240A1 (fr) * 2023-07-13 2025-01-16 慕恩（广州）生物科技有限公司 Composition pharmaceutique et utilisation associée

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