WO2019168328A1 - Nanovésicules dérivées de bactéries rhizobium sp., et leur utilisation - Google Patents

Nanovésicules dérivées de bactéries rhizobium sp., et leur utilisation Download PDF

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WO2019168328A1
WO2019168328A1 PCT/KR2019/002342 KR2019002342W WO2019168328A1 WO 2019168328 A1 WO2019168328 A1 WO 2019168328A1 KR 2019002342 W KR2019002342 W KR 2019002342W WO 2019168328 A1 WO2019168328 A1 WO 2019168328A1
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cancer
vesicles
bacteria
derived
disease
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PCT/KR2019/002342
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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 KR1020190022168A external-priority patent/KR102118198B1/ko
Application filed by 주식회사 엠디헬스케어 filed Critical 주식회사 엠디헬스케어
Priority to US16/975,889 priority Critical patent/US20210002701A1/en
Priority to CN201980016149.XA priority patent/CN111819292A/zh
Priority to JP2020545153A priority patent/JP7054956B2/ja
Priority to EP19761635.2A priority patent/EP3760743A4/fr
Publication of WO2019168328A1 publication Critical patent/WO2019168328A1/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 bacteria of the genus Rizobium, and their use, and more particularly, to stomach cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma and diabetes using nanovesicles derived from bacteria of the genus Rizobium. And a method for diagnosing Parkinson's disease or dementia, and a composition for preventing, ameliorating or treating the disease, including the vesicle.
  • 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
  • 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.
  • a bacterium in rizobium is a Gram-negative bacterium that absorbs nitrogen from the soil, symbiotic with roots such as nuts, converts atmospheric nitrogen into ammonia, and provides nitrogen compounds such as glutamine to plants.
  • bacteria in rizobium secrete extracellular vesicles, and in particular, there have been no cases of application to diagnosis and treatment of intractable diseases such as cancer, neurological diseases, or metabolic diseases.
  • the present inventors earnestly researched to solve the above-mentioned problems, and as a result of metagenome analysis, the vesicle-derived vesicles of the genus Rizovium were found to have gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, Significant reductions in clinical samples of patients with diabetes, Parkinson's disease, and dementia confirmed that the disease could be diagnosed. Also, Rhizobium leguminosarum ) when the vesicles were isolated and treated with macrophages, it was confirmed that significantly inhibit the IL-6 and TNF- ⁇ secretion by pathogenic vesicles, the present invention was completed based on this.
  • an object of the present invention is to provide an information providing method for the diagnosis of gastric cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease or dementia.
  • the present invention comprises a bacterial vesicle of the genus Rizobium as an active ingredient, gastric cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, and composition for the prevention, improvement or treatment of dementia To provide another purpose.
  • the present invention comprises the following steps, for the diagnosis of gastric cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, or dementia Provide information methods:
  • the present invention provides a method for diagnosing gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease or dementia, comprising the following steps:
  • the sample in step (a) may be blood or urine.
  • 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 pharmaceutical composition for the prevention or treatment of gastric cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, or dementia, including the bacteria-derived vesicles of the genus Rizobium To provide.
  • the present invention comprises a bacteria-derived vesicles of the genus Rizobium as an active ingredient. It provides a food composition for the prevention or improvement of stomach cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, or dementia.
  • the present invention comprises the step of administering to the subject a pharmaceutical composition comprising a bacteria-derived vesicles of the genus Rizobium as an active ingredient, gastric cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson Provided are methods for preventing or treating a disease or dementia.
  • the present invention also provides a prophylactic or therapeutic use of vesicles derived from bacteria of the genus Rizobium, gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, or dementia.
  • the vesicles may have an average diameter of 10 to 200 nm.
  • the vesicles may be secreted naturally or artificially in bacteria of the genus Rizobium.
  • the bacteria-derived vesicles of the genus Rizobium may be vesicles derived from Rizium regyuminosarum.
  • the present inventors confirmed that the intestinal bacteria are not absorbed into the body, but when the bacteria-derived vesicles are absorbed into the body through epithelial cells, they are distributed systemically and excreted in vitro through the kidneys, liver, and lungs.
  • Bacterial-derived vesicle metagenome analysis in blood or urine results in clinical samples of gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, and blood or urine in patients with dementia. It was confirmed that the vesicle-derived bacteria in rizobium were significantly reduced compared to normal people.
  • Bacterial-derived vesicles of the genus Rizobium according to the present invention is a method for diagnosing gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, or dementia, and preventing, improving or It is expected that the present invention may be usefully used in therapeutic 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, urine
  • 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 bacteria-derived vesicles of the genus Rizobium after the analysis of bacteria-derived vesicles metagenome present in gastric cancer patients and normal urine.
  • Figure 3 is a result of comparing the distribution of bacteria-derived vesicles of the genus Rizobium after analyzing the bacteria-derived vesicles metagenome present in colon cancer patients and normal urine.
  • Figure 4 is a result of comparing the distribution of bacteria-derived vesicles of the genus Rizobium after analyzing the bacteria-derived vesicles metagenome present in breast cancer patients and normal urine.
  • Figure 5 is a result of comparing the distribution of bacteria-derived vesicles of the genus Rizobium after analyzing the bacteria-derived vesicles metagenome present in ovarian cancer patients and normal urine.
  • Figure 6 is a result of comparing the distribution of bacteria-derived vesicles of the genus Rizobium after analyzing the bacteria-derived vesicles metagenome present in bladder cancer patients and normal urine.
  • Figure 7 is a result of comparing the distribution of bacteria-derived vesicles of the genus Rizobium after analysis of bacteria-derived vesicles metagenome present in the prostate cancer patients and normal urine.
  • Figure 9 is a result of comparing the distribution of bacteria-derived vesicles of the genus Rizobium after the analysis of bacteria-derived vesicles metagenome present in diabetic patients and normal urine.
  • Figure 10 is a result of comparing the distribution of bacteria-derived vesicles of the genus Rizobium after analyzing the bacterial-derived vesicles metagenome present in Parkinson's disease patients and normal urine.
  • 11 is a result of comparing the distribution of bacteria-derived vesicles of Rizobium after analysis of bacteria-derived vesicles metagenome present in dementia patients and normal blood.
  • FIG. 12 is a pre-treatment of lysobium-derived vesicles prior to treatment with Escherichia coli vesicles ( E. coli EV) to evaluate the anti-inflammatory and immunomodulatory effects of lysodium regyuminosarum-derived vesicles.
  • E. coli EV Escherichia coli vesicles
  • the present invention relates to vesicles derived from bacteria of the genus Rizobium and uses thereof.
  • metagenome analysis significantly reduced vesicle-derived vesicles of Rizovium in clinical samples of patients with gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, and dementia. It was confirmed that the disease can be diagnosed.
  • the vesicles were isolated and analyzed from Rhizobium leguminosarum, and gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, or dementia. It was confirmed that it can be used as a composition for preventing, improving or treating diseases.
  • the present invention provides a method for providing information for the diagnosis of gastric cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, or dementia, comprising the following steps:
  • 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 the onset and level of disease, such as gastric cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, and / or dementia.
  • nanovesicle refers to a structure of nanoscale membranes secreted by various bacteria.
  • Gram-negative bacteria-derived vesicles or outer membrane vesicles contain toxic proteins, bacterial DNA and RNA as well as lipopolysaccharides, and gram-positive bacteria-derived vesicles.
  • OMVs outer membrane vesicles
  • proteins and nucleic acids 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 from bacteria of the genus Rizobium, and have a spherical shape and have an average diameter of 10 to 100 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 or urine, but is not limited thereto.
  • the present invention comprises a bacterial vesicles of the genus Rizobium as an active ingredient, gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, or prevention of dementia, It provides a composition for treatment or improvement.
  • 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 means the inhibition of gastric cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, and / or dementia by administration of a composition according to the present invention. Or any action that delays onset.
  • treatment refers to symptoms of gastric cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, and / or dementia by administration of the composition according to the present invention. Means any action that improves or changes beneficial;
  • 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 are centrifuged, ultra-fast centrifugation, autoclaving, extrusion, sonication, cell lysis, homogenization, freeze-thaw, electroporation, mechanical degradation, chemical treatment, filtration by a culture medium containing bacteria in the riboseum 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 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).
  • Example 1 Analysis of absorption, distribution, and excretion of intestinal bacteria and bacterial-derived vesicles
  • the intestinal bacteria and enteric bacteria-derived vesicles are absorbed systemically, 50 ⁇ g of fluorescently labeled bacteria and bacteria-derived vesicles are administered in the same manner as described above in order to evaluate the infiltration into various organs.
  • 50 ⁇ g of fluorescently labeled bacteria and bacteria-derived vesicles are administered in the same manner as described above in order to evaluate the infiltration into various organs.
  • urine, heart, lung, liver, kidney, spleen, fat and muscle were collected.
  • the bacteria-derived vesicles were distributed in the urine, heart, lung, liver, spleen, fat, muscle, kidney, it was found that the bacteria are not absorbed.
  • 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). After converting the SFF file into a sequence file (.fasta) and a nucleotide qualityscore file, the credit rating of the lead was confirmed, and the window (20 bps) average base call accuracy was less than 99% (Phred score ⁇ 20). . For operational taxonomy unit (OTU) analysis, clustering is performed according to sequence similarity using UCLUST and USEARCH.
  • OFUTU operational taxonomy unit
  • Genus is 94%, family is 90%, order is 85%, and steel ( class is 80% and phylum is clustered based on 75% sequence similarity and the phylum, class, order, family and genus levels of each OTU Sorting was performed, and bacteria with greater than 97% sequence similarity at the genus level were profiled (QIIME) using BLASTN and GreenGenes' 16S RNA sequence database (108,453 sequences).
  • Example 2 In the method of Example 2, the urine of 61 patients with gastric cancer and 120 urine of normal age matched with age and sex were extracted from vesicles present in urine and subjected to metagenomic analysis. The distribution of vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Rizobium were significantly reduced in the urine of gastric cancer patients compared to normal urine (see Table 2 and FIG. 2).
  • Example 2 the genes were extracted from the vesicles in the urine of 38 colon cancers and the 38 urine of normal people, and the metagenome analysis was performed, and then the distribution of the bacterial vesicles of the genus Rizobium was evaluated. It was. As a result, it was confirmed that the vesicle-derived bacteria of Rizobium were significantly reduced in urine of colorectal cancer patients compared to normal urine (see Table 3 and FIG. 3).
  • Example 2 the genes were extracted from vesicles of 127 breast cancer patients and 220 urine people of normal age who matched their age and sex. The distribution of vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Rizobium were significantly reduced in the urine of breast cancer patients compared to normal urine (see Table 4 and FIG. 4).
  • the urine of 135 ovarian cancer patients and 136 normal urine of age and sex matched by the method of Example 2 were extracted from vesicles present in the urine, and then subjected to metagenomic analysis. The distribution of the derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of the lyzobiium significantly decreased in the urine of ovarian cancer patients compared to normal urine (see Table 5 and FIG. 5).
  • Example 7 Bladder-derived vesicles from bladder cancer patients Metagenome analysis
  • Example 2 In the method of Example 2, the urine of bladder cancer patients and urine of 107 normal people whose age and sex were matched were extracted from vesicles present in the urine, followed by metagenome analysis. The distribution of vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Rizobium were significantly reduced in the urine of bladder cancer patients compared to normal urine (see Table 6 and FIG. 6).
  • the urine of 55 prostate cancer patients and the urine of 159 normal urine were extracted by the method of Example 2, and the genes were extracted from the vesicles present in the urine and subjected to metagenomic analysis. The distribution of was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Rizobium were significantly reduced in the urine of prostate cancer patients compared to normal urine (see Table 7 and FIG. 7).
  • Example 2 blood was collected from 160 bloods of asthma patients and 114 normal bloods of age and sex, and genes were extracted from vesicles present in the blood, followed by metagenome analysis. The distribution of vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Rizobium were significantly reduced in the blood of asthma patients compared to normal blood (see Table 8 and FIG. 8).
  • Example 2 In the method of Example 2, the urine of 60 diabetic patients and the urine of 134 normal persons whose age and sex were matched were extracted from the vesicles present in the urine and subjected to metagenomic analysis. The distribution of vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Rizobium were significantly reduced in urine of diabetic patients compared to normal urine (see Table 9 and FIG. 9).
  • the urine of 39 patients with Parkinson's disease and 76 urine of age and sex matched by the method of Example 2 were extracted from vesicles present in the urine, followed by a metagenome analysis, followed by bacteria of the genus Rizovium. The distribution of the derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Rizobium were significantly reduced in the urine of Parkinson's disease patients compared to normal urine (see Table 10 and FIG. 10).
  • 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. Then, the protein was quantified using BCA (Bicinchoninic acid) assay, and the obtained vesicles were tested.
  • BCA Boroninic acid
  • the concentration of rizobium regyuminosalum-derived vesicles in raw 264.7 cells, a mouse macrophage was measured at various concentrations (0.1, 1, 10 ⁇ g / Ml) and then E. coli- derived vesicles from E. coli , an inflammatory disease pathogenic vesicle. EV) was treated to measure the amount of secretion of inflammatory mediators (IL-6, TNF- ⁇ , etc.).
  • Raw 264.7 cells were dispensed into 24-well cell culture plates by 1 ⁇ 10 5 cells and then cultured in Dulbeco's Modified Eagle's Medium (DMEM) complete medium for 24 hours. Then, the culture supernatant was collected in a 1.5 ml tube, centrifuged at 3000 g for 5 minutes, the supernatant was collected and stored at 4 ° C., followed by ELISA analysis.
  • DMEM Dulbeco's Modified Eagle's Medium
  • IL-6 and TNF- ⁇ secretion by E. coli-derived vesicles is significantly suppressed (see Fig. 12). This means that Rizobium regyuminosarum-derived vesicles can effectively suppress the occurrence of inflammation induced by pathogenic vesicles such as E. coli-derived vesicles.
  • Bacterial-derived vesicles of the genus Rizobium according to the present invention can be used for the prevention, improvement or treatment of gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, or dementia as well as diagnostic methods for the disease. Since it can be used as a composition for the medical and food industry is expected to be usefully utilized.

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Abstract

La présente invention concerne des vésicules provenant de bactéries Rhizobium sp., et leur utilisation. Les présents inventeurs ont constaté expérimentalement que les vésicules dans des échantillons cliniques provenant de patients présentant un cancer de l'estomac, un cancer colorectal, un cancer du sein, un cancer de l'ovaire, un cancer de la vessie, un cancer de la prostate, l'asthme, le diabète, la maladie de Parkinson et la démence sont significativement réduites comparativement à celles provenant de personnes normales, et lorsque les vésicules provenant des souches sont administrées, la sécrétion de médiateurs inflammatoires par des vésicules pathogènes telles que les vésicules dérivées d'E. coli est significativement inhibée. Par conséquent, les vésicules provenant de bactéries Rhizobium sp., selon la présente invention, peuvent efficacement être utilisées aux fins de développer un procédé de diagnostic du cancer de l'estomac, du cancer colorectal, du cancer du sein, du cancer de l'ovaire, du cancer de la vessie, du cancer de la prostate, de l'asthme, du diabète, de la maladie de Parkinson ou de la démence, et une composition de prévention, de soulagement ou de traitement de maladies.
PCT/KR2019/002342 2018-02-28 2019-02-27 Nanovésicules dérivées de bactéries rhizobium sp., et leur utilisation WO2019168328A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
US16/975,889 US20210002701A1 (en) 2018-02-28 2019-02-27 Nanovesicles derived from rhizobium sp. bacteria, and use thereof
CN201980016149.XA CN111819292A (zh) 2018-02-28 2019-02-27 来源于根瘤菌种细菌的纳米囊泡及其用途
JP2020545153A JP7054956B2 (ja) 2018-02-28 2019-02-27 リゾビウム属細菌由来のナノ小胞およびその用途
EP19761635.2A EP3760743A4 (fr) 2018-02-28 2019-02-27 Nanovésicules dérivées de bactéries rhizobium sp., et leur utilisation

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KR10-2018-0024894 2018-02-28
KR20180024894 2018-02-28
KR10-2019-0022168 2019-02-26
KR1020190022168A KR102118198B1 (ko) 2018-02-28 2019-02-26 리조비움 속 세균 유래 나노소포 및 이의 용도

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