WO2019172597A1 - Nanovésicules dérivées de bactéries methylobacterium sp. et leur utilisation - Google Patents

Nanovésicules dérivées de bactéries methylobacterium sp. et leur utilisation Download PDF

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WO2019172597A1
WO2019172597A1 PCT/KR2019/002497 KR2019002497W WO2019172597A1 WO 2019172597 A1 WO2019172597 A1 WO 2019172597A1 KR 2019002497 W KR2019002497 W KR 2019002497W WO 2019172597 A1 WO2019172597 A1 WO 2019172597A1
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cancer
vesicles
derived
methylobacterium
bacteria
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PCT/KR2019/002497
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Korean (ko)
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김윤근
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주식회사 엠디헬스케어
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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 the bacteria of the genus Methylbacteria, and more specifically, to the pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction using nanovesicles derived from the bacteria of the genus Methylbacteria. It relates to a diagnostic method such as cardiomyopathy, angina pectoris, diabetes mellitus, or Parkinson's disease, 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
  • 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.
  • Methylobacterium jeotgali Bacteria in Methylobacterium are Gram-negative bacteria in soil and water, among them Methylobacterium jeotgali ) is the first aerobic bacterium identified in Korean salted fish. However, it has not been reported that the bacteria of methyllobacterium secrete extracellular vesicles, and in particular, there have been no cases of application to the diagnosis and treatment of intractable diseases such as cancer or cardiovascular-brain diseases.
  • 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, bladder cancer, lymphoma, myocardial infarction, cardiomyopathy, dysplastic angina, diabetes, In the sample derived from Parkinson's disease patients, it was confirmed that the content of bacteria-derived vesicles of Methylbacteria bacterium was significantly reduced. In addition, when the vesicles were isolated from Methylbacterium spp.
  • an object of the present invention is to provide a method for providing information for the diagnosis of pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, cardiomyopathy, dysplastic angina, diabetes, or Parkinson's disease.
  • the present invention is a pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, cardiomyopathy, dysplastic angina pectoris, diabetes mellitus, or Parkinson's disease, including the vesicles derived from the bacteria of the genus Methylbacterium as an active ingredient
  • Another object is to provide a therapeutic composition.
  • the present invention comprises the following steps, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, cardiomyopathy, dysplastic angina, diabetes, or Parkinson's disease Provide informational methods for diagnosis:
  • the present invention also provides a method for diagnosing pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, cardiomyopathy, heteroangular angina, diabetes, or Parkinson's disease, 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 pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, cardiomyopathy, angina pectoris, diabetes mellitus, or Parkinson's disease, comprising a vesicle derived from the bacteria of the genus Methylbacterium as an active ingredient
  • a therapeutic pharmaceutical composition comprising a vesicle derived from the bacteria of the genus Methylbacterium as an active ingredient.
  • the present invention is a pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, cardiomyopathy, angina pectoris, diabetes mellitus, or Parkinson's disease, comprising a vesicle derived from the bacteria of the genus Methylbacterium as an active ingredient It provides a food composition for improvement.
  • the present invention comprises administering to a subject a pharmaceutical composition
  • a pharmaceutical composition comprising a bacterium-derived vesicle of the genus Methylbacterium as an active ingredient, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, cardiomyopathy , Methods of preventing or treating dysplastic angina, diabetes, or Parkinson's disease.
  • the present invention provides a use of the vesicles derived from the bacteria of the genus Methylobacterium, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, cardiomyopathy, dysplastic angina, diabetes, or Parkinson's disease. .
  • the vesicles may have an average diameter of 10 to 200 nm.
  • the vesicles may be secreted naturally or artificially from the bacteria of the genus Methylbacteria.
  • the bacterium-derived vesicles of the genus Methylobacterium may be a vesicle-derived vesicles derived from Methylbacterium.
  • the present inventors confirmed that intestinal bacteria are not absorbed into the body, but in the case of bacterial-derived vesicles, they are absorbed into the body through epithelial cells, distributed systemically, and excreted in vitro through the kidneys, liver, and lungs.
  • Methylbacterium in the blood or urine of patients with pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, cardiomyopathy, dysplastic angina, diabetes mellitus, and Parkinson's disease It was confirmed that the vesicle-derived genus was significantly reduced compared to the normal person.
  • the bacterium-derived vesicles according to the present invention are pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, cardiomyopathy, dysplastic angina, diabetes, or Parkinson's disease, and the diagnostic method. It is expected that the present invention may be usefully used in a composition for preventing, ameliorating or treating diseases.
  • 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 bacteria-derived vesicles of the genus Merobacterium after the analysis of bacteria-derived vesicles metagenome present in the pancreatic cancer patients and normal blood.
  • Figure 3 is a result of comparing the distribution of bacteria-derived vesicles of the genus Merobacterium after the analysis of bacteria-derived vesicles metagenome present in patients with bile duct cancer.
  • Figure 4 is a result of comparing the distribution of bacteria-derived vesicles of the genus Merobacterium after the analysis of bacteria-derived vesicles metagenome present in breast cancer patients and normal blood.
  • Figure 5 is a result of comparing the distribution of bacteria-derived vesicles of the genus Methylobacterium after the analysis of bacteria-derived vesicles metagenome present in ovarian cancer patients and normal blood.
  • Figure 6 is a result of comparing the distribution of bacteria-derived vesicles of the genus Merobacterium after analyzing the bacteria-derived vesicles metagenome present in bladder cancer patients and normal blood.
  • Figure 8 is a result of comparing the distribution of bacteria-derived vesicles of the genus Merobacterium after the analysis of bacteria-derived vesicles metagenome present in myocardial infarction patients and normal blood.
  • 10 is a result of comparing the distribution of bacteria-derived vesicles of the genus Methylobacterium after analyzing the bacteria-derived vesicles metagenome present in patients with heteroangular angina and normal blood.
  • 11 is a result of comparing the distribution of bacteria-derived vesicles of the genus Merobacterium after the analysis of bacteria-derived vesicles metagenome present in diabetic patients and normal blood.
  • Figure 13 is a pre-treatment of M. tuberculosis-derived vesicles before the treatment of E. coli EV, a pathogenic vesicle ( E. coli EV), in order to evaluate the anti-inflammatory and immunomodulatory effect of Methylbacterium chopped vesicles, E. coli vesicles Is a result of evaluating the effect on the secretion of IL-6 and TNF- ⁇ .
  • the present invention relates to vesicles derived from bacteria of the genus Methylobacterium and uses thereof.
  • the present inventors conducted a metagenome analysis of vesicle-derived vesicles, bile duct cancers, breast cancers, ovarian cancers, bladder cancers, lymphomas, myocardial infarction, cardiomyopathy, heteroangular angina, diabetes, and Parkinson's disease patients compared to normal people. Confirming that the sample is significantly reduced, it was confirmed that the disease can be diagnosed. In addition, the vesicles were isolated and characterized from Methylbacterium spp.
  • the present invention provides a method for providing information for diagnosing pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, cardiomyopathy, heteroangular angina, diabetes, or Parkinson's disease, 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 whether the pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, cardiomyopathy, dysplastic angina, diabetes, and / or Parkinson's disease and the level of the disease.
  • vesicle 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 it also contains peptidoglycan and lipoteichoic acid, which are components of bacterial cell walls.
  • the nanovesicles or vesicles are naturally secreted or artificially produced from bacteria of the genus Methylobacterium, 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 or urine, but is not limited thereto.
  • the present invention is a pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, cardiomyopathy, dysplastic angina, diabetes, or Provided is a composition for preventing, treating or improving Parkinson's disease.
  • 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, bladder cancer, lymphoma, myocardial infarction, cardiomyopathy, dysplastic angina, diabetes, and / or Parkinson's disease, etc., by administration of a composition according to the present invention. Means any action that inhibits or delays onset.
  • treatment refers to pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, cardiomyopathy, dysplastic angina pectoris, diabetes mellitus, and / or Parkinson's disease by administration of the composition according to the invention. It means any activity that improves or beneficially changes the condition.
  • 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, high pressure treatment, extrusion, sonication, cell lysis, homogenization, freeze-thaw, electroporation, mechanical degradation, chemical treatment, filter Separation may be performed using one or more methods selected from the group consisting of filtration by, 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 the bacteria-derived vesicles orally administered to evaluate the absorption, distribution, and excretion of the bacteria and vesicles in the body in the case of bacteria is not absorbed through the intestinal vesicles 5 minutes administration It was confirmed that it was absorbed within the body and distributed systemically and excreted through the kidney, liver, and the like (see Example 1).
  • pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, cardiomyopathy, dysplastic angina pectoris, diabetes mellitus, and Parkinson's disease in patients with age and sex matched in the blood or urine
  • pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, cardiomyopathy, dysplastic angina pectoris, diabetes mellitus, and Parkinson's disease It was confirmed that it is reduced (see Examples 3 to 13).
  • E. coli-derived vesicles were treated to evaluate the secretion of inflammatory mediators.
  • E. coli-derived vesicle-derived IL-6 and TNF- ⁇ secretion was efficiently obtained from Methylbacterium spp. Inhibition was confirmed (see Example 14).
  • Example 1 Analysis of absorption, distribution, and excretion of intestinal bacteria and bacterial-derived vesicles
  • 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. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicles derived from the bacterium of the bacterium in the pancreatic cancer significantly reduced compared to normal blood (see Table 2 and Figure 2).
  • Example 4 Bacterial-derived vesicles from bile duct cancer patients Metagenome analysis
  • the blood of 79 patients with cholangiocarcinoma patients and blood of 259 normal people whose age and sex were matched by the method of Example 2 were extracted from the vesicles in the blood and subjected to metagenomic analysis.
  • the distribution of bacterial derived vesicles was evaluated.
  • it was confirmed that the vesicle-derived bacteria of the methylobacterium was significantly reduced in the blood of bile duct cancer patients compared to the normal blood (see Table 3 and Figure 3).
  • Example 2 the genes were extracted from vesicles in the blood of 96 breast cancer patients and 192 normal blood patients whose ages and genders were matched. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Methylbacterium were significantly reduced in the blood of breast cancer patients compared to normal blood (see Table 4 and FIG. 4).
  • Example 2 137 blood of ovarian cancer patients and 139 normal blood of age and sex matched were extracted from the vesicles present in the blood and subjected to metagenomic analysis, and then methyllobacterium The distribution of vesicles derived from the genus bacteria was evaluated. As a result, it was confirmed that the vesicles derived from the bacteria of the methylobacterium significantly decreased in the blood of ovarian cancer patients compared to the normal blood (see Table 5 and Figure 5).
  • Example 2 the genes were extracted from vesicles in the bladder cancer patients and 176 normal blood patients whose ages and genders were matched. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of the methylobacterium is significantly reduced in the blood of bladder cancer patients compared to normal blood (see Table 6 and Figure 6).
  • Example 2 the genes were extracted from the vesicles in the blood and the normal blood of 63 patients with lymphoma and 53 normal persons whose age and sex were matched. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicles derived from the bacterium of methylobacterium significantly decreased in the blood of lymphoma patients compared to normal blood (see Table 7 and FIG. 7).
  • Example 2 the blood of 57 patients with myocardial infarction and 163 normal blood patients whose age and sex were matched were extracted from the vesicles present in the blood and subjected to metagenomic analysis, followed by methyllobacterium. Distribution of vesicles derived from the genus bacteria was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of the methylobacterium was significantly reduced in the blood of myocardial infarction patients compared to normal blood (see Table 8 and FIG. 8).
  • Example 2 the blood of 80 patients with angina and 80 normal blood whose age and sex were matched were extracted from the vesicles present in the blood and subjected to metagenomic analysis, followed by methyllobacterium. The distribution of vesicles derived from the genus bacteria was evaluated. As a result, it was confirmed that the vesicles derived from the bacterium of methylobacterium significantly decreased in the blood of patients with heterophagia compared to normal blood (see Table 10 and FIG. 10).
  • 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 metagenomic analysis. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the bacteria-derived vesicles of the genus Methylobacterium were significantly reduced in blood of diabetic patients compared to normal blood (see Table 11 and FIG. 11).
  • 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 the vesicles present in the urine to perform a metagenomic analysis, followed by methyllobacterium
  • the distribution of vesicles derived from the genus bacteria was evaluated. As a result, it was confirmed that the vesicles derived from the bacterium of Methylbacterium were significantly reduced in the urine of Parkinson's disease patients compared to normal urine (see Table 12 and FIG. 12).
  • Methylbacterium chopped strain was cultured in a BHI (brain heart infusion) medium until the absorbance (OD 600 ) in the aerobic chamber at 37 °C 1.0 to 1.5 and then sub-culture. 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).
  • BHI brain heart infusion
  • 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.
  • Methylbacterium chopped vesicle-derived vesicles were injected into 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.). More specifically, 1 ⁇ 10 5 Raw 264.7 cells were dispensed into 24-well cell culture plates, and then cultured in DMEM complete medium for 24 hours. 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.
  • IL-6 IL-6, TNF- ⁇ , etc.
  • Bacterial-derived vesicles of the genus Methylobacterium according to the present invention may be used for pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, cardiomyopathy, dysplastic angina, diabetes, or Parkinson's disease, as well as for diagnosis of the disease. Since it can be used as a composition for preventing, improving or treating, 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 dérivées de bactéries Methylobacterium sp. et une de leurs utilisations. Les présents inventeurs ont confirmé par expérimentation que : comparativement à celles d'une personne en bonne santé, les vésicules sont significativement réduites dans les échantillons cliniques de patients souffrant de cancer du pancréas, cholangiocarcinome, cancer du sein, cancer de l'ovaire, cancer de la vessie, lymphome, infarctus du myocarde, cardiomyopathie, angine de poitrine, diabète et maladie de Parkinson; et quand des vésicules isolées d'une souche sont administrées, la sécrétion de médiateurs inflammatoires par des vésicules pathogènes, telles que des vésicules dérivées d'Escherichia coli, est inhibée de manière notable. Ainsi, les vésicules dérivées de bactéries Methylobacterium sp., selon la présente invention, peuvent être utilement employées dans le but de développer : une méthode de diagnostic du cancer du pancréas, cholangiocarcinome, cancer du sein, cancer de l'ovaire, cancer de la vessie, lymphome, infarctus du myocarde, d'une cardiomyopathie, angine de poitrine, du diabète et de la maladie de Parkinson; et une composition pour prévenir, améliorer ou traiter lesdites maladies.
PCT/KR2019/002497 2018-03-05 2019-03-05 Nanovésicules dérivées de bactéries methylobacterium sp. et leur utilisation WO2019172597A1 (fr)

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KR10-2018-0026036 2018-03-05
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KR1020190024889A KR102118201B1 (ko) 2018-03-05 2019-03-04 메틸로박테리움 속 세균 유래 나노소포 및 이의 용도

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