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WO2019168327A1 - Nanovésicules issues de bactéries micrococcus et leur utilisation - Google Patents

Nanovésicules issues de bactéries micrococcus et leur utilisation Download PDF

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Publication number
WO2019168327A1
WO2019168327A1 PCT/KR2019/002341 KR2019002341W WO2019168327A1 WO 2019168327 A1 WO2019168327 A1 WO 2019168327A1 KR 2019002341 W KR2019002341 W KR 2019002341W WO 2019168327 A1 WO2019168327 A1 WO 2019168327A1
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Prior art keywords
cancer
vesicles
micrococcus
derived
bacteria
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Ceased
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PCT/KR2019/002341
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English (en)
Korean (ko)
Inventor
김윤근
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MD Healthcare Inc
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MD Healthcare Inc
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Publication date
Priority claimed from KR1020190022167A external-priority patent/KR102118197B1/ko
Application filed by MD Healthcare Inc filed Critical MD Healthcare Inc
Priority to EP19761634.5A priority Critical patent/EP3760742A4/fr
Priority to CN201980014447.5A priority patent/CN111757943A/zh
Priority to JP2020545158A priority patent/JP2021514989A/ja
Priority to US16/563,720 priority patent/US10888591B2/en
Publication of WO2019168327A1 publication Critical patent/WO2019168327A1/fr
Anticipated expiration legal-status Critical
Priority to JP2022006126A priority patent/JP7433665B2/ja
Ceased legal-status Critical Current

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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
    • 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
    • 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 a microvesicle-derived nano-vesicles and uses thereof, more specifically, gastric cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy using nano-vesicles derived from the micrococcus bacteria , Atrial fibrillation, heteroangular angina, chronic obstructive pulmonary disease (COPD), a method for diagnosing dementia or diabetes, and the like, and a composition for preventing, ameliorating or treating the disease, including the vesicles.
  • gastric cancer pancreatic cancer
  • cholangiocarcinoma breast cancer
  • ovarian cancer bladder cancer
  • myocardial infarction cardiomyopathy using nano-vesicles derived from the micrococcus bacteria
  • Atrial fibrillation heteroangular angina
  • COPD chronic obstructive pulmonary disease
  • COPD chronic obstructive pulmonary disease
  • microbiota refers to a microbial community including microbes, archae 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 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.
  • Micrococcus bacteria are Gram-positive bacteria belonging to the micrococcus family, and are widely distributed in nature such as water, dust, and soil. Among them, Micrococc us luteus is known to produce riboflavin when grown in toxic organic pollutants such as pyridine, and absorb ultraviolet light through the lutein pigment. Micrococcus bacteria are isolated from dairy products and beer, grow in dry or high salt environments, do not form spores, but are known to survive long-term at refrigerated temperatures such as refrigerators. However, it has not been reported that micrococcus bacteria secrete extracellular vesicles, and there have been no reports on the application and diagnosis of cancer, cardiovascular disease, allergic-chronic lung disease, dementia, or metabolic disease. .
  • the present inventors earnestly researched to solve the above-mentioned conventional problems, gastric cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, atrial fibrillation, heteroangular angina,
  • COPD chronic obstructive pulmonary disease
  • dementia dementia
  • diabetic patients it was confirmed that the contents of microcacus-derived vesicles were significantly reduced.
  • the present invention provides a method for providing information for the diagnosis of gastric cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, atrial fibrillation, angina, chronic obstructive pulmonary disease (COPD), dementia or diabetes It aims to do it.
  • the present invention is a gastric cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, atrial fibrillation, angina pectoris, atopic dermatitis, psoriasis, acne, hair loss
  • Another object is to provide a composition for preventing, improving or treating macular degeneration, chronic obstructive pulmonary disease (COPD), dementia, or diabetes.
  • COPD chronic obstructive pulmonary disease
  • the present invention comprises the following steps, gastric cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, atrial fibrillation, dysplastic angina, chronic obstructive pulmonary Provides informational methods for diagnosing disease (COPD), dementia, or diabetes:
  • the present invention includes the following steps, gastric cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, atrial fibrillation, dysplastic angina, chronic obstructive pulmonary disease (COPD), dementia, or diagnosing method of diabetes Provides:
  • COPD chronic obstructive pulmonary disease
  • the sample in step (a) may be blood.
  • the primer pair in step (b) may be a primer of SEQ ID NO: 1 and SEQ ID NO: 2.
  • the present invention including the micro-caustic bacteria-derived vesicles as an active ingredient, gastric cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, atrial fibrillation, angina, atopic dermatitis, psoriasis, acne,
  • a pharmaceutical composition for preventing or treating hair loss, macular degeneration, chronic obstructive pulmonary disease (COPD), dementia, or diabetes is provided.
  • the pharmaceutical composition may comprise an ophthalmic composition.
  • the present invention including the micro-caustic bacteria-derived vesicles as an active ingredient, gastric cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, atrial fibrillation, angina, atopic dermatitis, psoriasis, acne,
  • a food composition for preventing or improving hair loss, macular degeneration, chronic obstructive pulmonary disease (COPD), dementia, or diabetes.
  • COPD chronic obstructive pulmonary disease
  • the present invention including the micro-caustic bacteria-derived vesicles as an active ingredient, gastric cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, atrial fibrillation, angina, atopic dermatitis, psoriasis, acne, Prevention of hair loss, macular degeneration, chronic obstructive pulmonary disease (COPD), dementia, or diabetes or Provided is a therapeutic inhalant composition.
  • COPD chronic obstructive pulmonary disease
  • the present invention also provides a cosmetic composition for the prevention or improvement of atopic dermatitis, psoriasis, acne or hair loss, including a micro-caustic bacteria-derived vesicles as an active ingredient.
  • the present invention comprises the step of administering to the subject a pharmaceutical composition
  • a pharmaceutical composition comprising a microcacus-derived bacterial vesicles as an active ingredient, gastric cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, atrial
  • a pharmaceutical composition comprising a microcacus-derived bacterial vesicles as an active ingredient, gastric cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, atrial
  • methods of preventing or treating fibrillation angina, atopic dermatitis, psoriasis, acne, hair loss, macular degeneration, chronic obstructive pulmonary disease (COPD), dementia, or diabetes.
  • COPD chronic obstructive pulmonary disease
  • the present invention is microbial vesicle-derived vesicles, gastric cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, atrial fibrillation, angina pectoris, atopic dermatitis, psoriasis, acne, hair loss, macular degeneration, Provided for the prevention or treatment of chronic obstructive pulmonary disease (COPD), dementia, or diabetes.
  • COPD chronic obstructive pulmonary disease
  • the vesicles may have an average diameter of 10 to 200 nm.
  • the vesicles may be secreted naturally or artificially from bacteria of the micrococcus.
  • the micro-caucus bacteria-derived vesicles may be micrococcus luteus-derived vesicles.
  • 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.
  • Micro-caustic bacteria-derived vesicles is gastric cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, atrial fibrillation, dysplastic angina, chronic obstructive pulmonary disease (COPD), diagnostic method for diabetes or diabetes And it is expected that it can be usefully used in the composition for the prevention, improvement or treatment for the above 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 micrococcus after performing a bacterial-derived vesicle metagenome analysis present in gastric cancer patients and normal blood.
  • Figure 3 is a result of comparing the distribution of bacteria-derived vesicles of the micrococcus after performing a bacterial-derived vesicle metagenome analysis present in pancreatic cancer patients and normal blood.
  • Figure 4 is a result of comparing the distribution of bacteria-derived vesicles of the genus Micrococcus after analyzing the bacteria-derived vesicles metagenome present in patients with bile duct cancer.
  • Figure 5 is a result of comparing the distribution of bacteria-derived vesicles of the micrococcus after performing a bacterial-derived vesicle metagenome analysis present in breast cancer patients and normal blood.
  • Figure 6 is a result of comparing the distribution of bacteria-derived vesicles of the genus Micrococcus after analyzing the bacteria-derived vesicles metagenome present in ovarian cancer patients and normal blood.
  • 9 is a result of comparing the distribution of microbial bacteria-derived vesicles after performing a microorganism-derived vesicle metagenome analysis present in cardiomyopathy patients and normal blood.
  • 10 is a result of comparing the distribution of bacteria-derived vesicles of the micrococcus after analyzing the bacteria-derived vesicles metagenome present in atrial fibrillation patients and normal blood.
  • 11 is a result of comparing the distribution of bacteria-derived vesicles of the micrococcus after analyzing the bacteria-derived vesicles metagenome present in patients with heteroangular angina and normal blood.
  • COPD chronic obstructive pulmonary disease
  • Fig. 13 shows the results of comparing the distribution of bacterial-derived vesicles of the micrococcus after analyzing the bacterial-derived vesicles metagenome present in dementia patients and normal blood.
  • E. coli EV Escherichia coli vesicles
  • the present invention relates to vesicles derived from bacteria of the micrococcus and their use.
  • microcacus-derived vesicles from the genome of the micrococcus have gastric cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, atrial fibrillation, dysplastic angina, and chronic obstructive pulmonary disease (COPD).
  • Significant reductions were found in clinical samples of patients with diabetes, dementia, and diabetes, confirming that the disease could be diagnosed.
  • the vesicles were isolated and characterized from micrococcus luteus, which showed gastric cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, atrial fibrillation, dysplastic angina, atopic dermatitis, macular degeneration, and chronic It was confirmed that it can be used as a composition for preventing, improving or treating diseases such as obstructive pulmonary disease (COPD), dementia, and diabetes.
  • COPD obstructive pulmonary disease
  • the present invention includes the following steps, gastric cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, atrial fibrillation, dysplastic angina pectoris, chronic obstructive pulmonary disease (COPD), dementia, or diabetes mellitus Provide informational methods for diagnosis:
  • 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 the presence and disease of gastric cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, atrial fibrillation, dysplastic angina, chronic obstructive pulmonary disease (COPD), dementia, and / or diabetes To judge the level and so on.
  • COPD chronic obstructive pulmonary disease
  • 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 it also contains peptidoglycan and lipoteichoic acid, which are components of bacterial cell walls.
  • nanovesicles or vesicles are naturally secreted or artificially produced by micrococcus bacteria, and have a spherical shape and have an average diameter of 10 to 200 nm.
  • the term "metagenome” used in the present invention also referred to as "gunoelectric”, refers to the sum total of the genome including all viruses, bacteria, fungi, etc. in an isolated area such as soil, animal intestine, mainly culture It is used as a concept of genome explaining the identification of many microorganisms at once using sequencer to analyze microorganisms that are not.
  • the metagenome does not refer to one genome or genome, but to a kind of mixed dielectric as the genome of all species of one environmental unit. This is a term from the point of view of defining a species in the course of the evolution of biology in terms of functional species as well as various species that interact with each other to create a complete species.
  • rapid sequencing is used to analyze all DNA and RNA, regardless of species, to identify all species in one environment, and to identify interactions and metabolism.
  • the sample may be blood, but is not limited thereto.
  • the present invention comprises a micro-caustic bacteria-derived vesicles as an active ingredient, gastric cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, atrial fibrillation, dysplastic angina, atopic dermatitis It provides a composition for preventing, treating or improving psoriasis, acne, hair loss, macular degeneration, chronic obstructive pulmonary disease (COPD), dementia, or diabetes.
  • the composition includes food, inhalants, cosmetics and pharmaceutical compositions.
  • the food composition in the present invention includes a health functional food composition
  • the pharmaceutical composition may include an ophthalmic composition, but is not limited thereto.
  • prevention refers to gastric cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, atrial fibrillation, angina pectoris, atopic dermatitis, psoriasis, by administration of the composition according to the invention Means any action that inhibits or delays the development of acne, hair loss, macular degeneration, chronic obstructive pulmonary disease (COPD), dementia, and / or diabetes.
  • COPD chronic obstructive pulmonary disease
  • treatment refers to gastric cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, atrial fibrillation, angina pectoris, atopic dermatitis, psoriasis, by administration of the composition according to the invention It means any behavior that improves or beneficially changes the condition for acne, hair loss, macular degeneration, chronic obstructive pulmonary disease (COPD), dementia, or diabetes.
  • COPD chronic obstructive pulmonary disease
  • 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, high pressure treatment, extrusion, sonication, cell lysis, homogenization, freeze-thaw, electroporation, mechanical decomposition, chemical treatment, filtration by a filter containing micrococcus bacteria. It can be separated using one or more methods selected from the group consisting of, gel filtration chromatography, pre-flow electrophoresis, and capillary electrophoresis. In addition, it may further include a process for washing to remove impurities, concentration of the obtained vesicles and the like.
  • the pharmaceutical composition according to the invention may comprise a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carriers are conventionally used in the preparation, and include, but are not limited to, saline solution, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and the like. If necessary, other conventional additives such as antioxidants and buffers may be further included.
  • diluents, dispersants, surfactants, binders, lubricants and the like may be additionally added to formulate injectable formulations, pills, capsules, granules, or tablets such as aqueous solutions, suspensions, emulsions and the like.
  • Suitable pharmaceutically acceptable carriers and formulations can be preferably formulated according to the individual components using methods disclosed in Remington's literature.
  • the pharmaceutical composition of the present invention is not particularly limited in formulation, but may be formulated as an injection, inhalant, external skin preparation, ophthalmic, 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 inhalant composition of the present invention may be added to the inhalant as it is, or used in combination with other ingredients, and may be suitably used according to conventional methods.
  • the amount of the active ingredient to be mixed may be suitably determined depending on the purpose of use (prophylactic or therapeutic).
  • the 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 cosmetic composition of the present invention may include components commonly used in cosmetic compositions, as well as microbe decay-derived vesicles, such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances Adjuvants, and carriers.
  • composition of the present invention may be used in addition to microvesicles-derived microvesicles, organic sunscreens that have been conventionally used as long as they do not impair the skin protection effect by reacting with microvesicles-derived microvesicles.
  • organic sunscreen examples include glyceryl pava, drometrizole trisiloxane, drometrizole, digaloyltrioleate, disodium phenyldibenzimidazole tetrasulfonate, diethylhexyl butamidotriazone, diethylamino Hydroxybenzoylhexylbenzoate, die-methoxycinnamate, a mixture of lowson and dihydroxyacetone, methylenebis-benzotriazolyltetramethylbutylphenol, 4-methylbenzylidene camphor, menthyl anthranilate, benzophenone -3 (oxybenzone), benzophenone-4, benzophenone-8 (dioxyphenbenzone), butylmethoxydibenzoylmethane, bisethylhexyloxyphenol methoxyphenyltriazine, synoxate, ethyldihydroxypropylpava, Oct
  • Examples of products to which the cosmetic composition of the present invention may be added include, for example, cosmetics such as astringent cosmetics, soft cosmetics, nourishing cosmetics, various creams, essences, packs, foundations, and the like, cleansing agents, soaps, treatments, and essences.
  • Specific formulations of the cosmetic composition of the present invention include skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, essence, nutrition essence, pack, Formulations such as soaps, shampoos, cleansing foams, cleansing lotions, cleansing creams, body lotions, body cleansers, emulsions, lipsticks, makeup bases, foundations, press powders, loose powders, eye shadows and the like.
  • the bacteria and bacteria-derived vesicles orally administered to observe the body absorption, distribution, and excretion of the bacteria and vesicles in the body in the case of bacteria is not absorbed through the intestinal membrane, the vesicles are administered 5 It was confirmed that it was absorbed within minutes and distributed systemically and excreted through the kidney, liver, and the like (see Example 1).
  • age and sex in gastric cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, atrial fibrillation, angina, chronic obstructive pulmonary disease (COPD), dementia, and diabetes patients Bacterial metagenome analysis was performed using vesicles isolated from the blood of healthy individuals.
  • micrococcus luteus strains were cultured to evaluate whether vesicles secreted therefrom exhibited immunomodulatory and anti-inflammatory effects. Later, by treating E. coli-derived vesicles, the inflammatory disease-causing factors, and evaluating the secretion of inflammatory mediators, it was confirmed that the micro-cuscus luteus-derived vesicles effectively suppressed IL-6 and TNF- ⁇ secretion by E. coli-derived vesicles ( See Example 18).
  • 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). 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 the blood of 66 gastric cancer patients and 198 normal blood patients whose age and sex were matched were extracted from the vesicles in the blood and subjected to metagenomic analysis. The distribution of vesicles was evaluated. As a result, it was confirmed that microvesicle-derived microvesicles were significantly reduced in the blood of gastric cancer patients compared to normal blood (see Table 2 and FIG. 2).
  • Example 4 Bacterial-derived vesicles from pancreatic cancer patients Metagenome analysis
  • Example 2 In the method of Example 2, 176 bloods of pancreatic cancer patients and 271 bloods of normal age matched with age and sex were extracted from the vesicles in the blood and subjected to metagenomic analysis, followed by micrococcus-derived bacteria. The distribution of vesicles was evaluated. As a result, it was confirmed that microvesicle-derived microvesicles were significantly reduced in the blood of pancreatic cancer patients compared to normal blood (see Table 3 and FIG. 3).
  • Example 2 the genes were extracted from the vesicles in the blood of 96 breast cancer patients and 192 normal blood patients whose ages and genders were matched. The distribution of vesicles was evaluated. As a result, it was confirmed that microbe decay-derived vesicles significantly reduced in the blood of breast cancer patients compared to normal blood (see Table 5 and Figure 5).
  • Example 2 In the method of Example 2, 137 bloods of ovarian cancer patients and 139 bloods of normal age and sex matched were extracted from the vesicles in the blood and subjected to metagenomic analysis, followed by bacteria of the micrococcus. The distribution of the derived vesicles was evaluated. As a result, it was confirmed that microvesicle-derived vesicles are significantly reduced in the blood of ovarian cancer patients compared to normal blood (see Table 6 and FIG. 6).
  • the blood of 91 bladder cancer patients and blood of 176 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 a metagenome analysis. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that microvesicle-derived microvesicles were significantly reduced in the blood of bladder cancer patients compared to normal blood (see Table 7 and FIG. 7).
  • Example 2 blood was collected from 57 patients with myocardial infarction and 163 normal blood patients whose ages and genders were matched. The distribution of the derived vesicles was evaluated. As a result, it was confirmed that microvesicle-derived microvesicles are significantly reduced in the blood of myocardial infarction patients compared to normal blood (see Table 8 and FIG. 8).
  • Example 2 the genes were extracted from the vesicles in the blood and 163 normal people whose age and sex were matched with the blood of 72 cardiomyopathy patients, and then subjected to metagenomic analysis. The distribution of the derived vesicles was evaluated. As a result, it was confirmed that microbe decay-derived vesicles significantly reduced in the blood of cardiomyopathy patients compared to normal blood (see Table 9 and Figure 9).
  • Example 2 the blood of 80 patients with heterocystic angina and 80 normal blood whose age and sex were matched were extracted from the vesicles in the blood and subjected to metagenomic analysis. The distribution of the derived vesicles was evaluated. As a result, it was confirmed that microvesicle-derived vesicles are significantly reduced in blood of patients with heteroangular angina compared with normal blood (see Table 11 and FIG. 11).
  • Example 2 In the method of Example 2, 205 bloods of COPD patients and 231 bloods of normal and matched age and sex were extracted from the vesicles in the blood and subjected to metagenomic analysis. The distribution of vesicles was evaluated. As a result, it was confirmed that microvesicle-derived microvesicles were significantly reduced in blood of COPD patients compared to normal blood (see Table 12 and FIG. 12).
  • Example 2 the genes were extracted from the vesicles present in the blood of 67 people with dementia and 70 blood of normal people who matched their age and sex. The distribution of vesicles was evaluated. As a result, it was confirmed that microbe decay-derived vesicles significantly reduced in the blood of dementia patients compared to normal blood (see Table 13 and Figure 13).
  • 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, followed by micrococcus bacteria. The distribution of vesicles was evaluated. As a result, it was confirmed that microvesicle-derived vesicles are significantly reduced in the blood of diabetic patients compared to normal blood (see Table 14 and FIG. 14).
  • Micrococcus luteus was subcultured after incubating in de Man-Rogosa and Sharpe (MRS) medium until the absorbance (OD 600 ) was 1.0 to 1.5 in an aerobic chamber at 37 ° C. Then, the culture medium supernatant containing the strain was recovered and centrifuged at 10,000 g, 4 ° C.
  • micrococcus luteus-derived vesicles were collected in raw 264.7 cells, a mouse macrophage. 1, 10 ⁇ g / ml), followed by ELISA.
  • DMEM Dulbeco's Modified Eagle's Medium
  • the capture antibody was diluted in phosphate buffered saline (PBS), and 50 ⁇ l was dispensed into 96 well polystyrene plates according to the working concentration, followed by reaction at 4 ° C. overnight. I was. After washing three times with 100 ⁇ l of PBST (PBS containing 0.05% tween-20), 100 ⁇ l of RD (PBS containing 1% BSA) solution was dispensed and blocked for 1 hour at room temperature. 50 ⁇ l of the sample and standard were dispensed according to the concentration and reacted at room temperature for 2 hours. Then, after washing three times with 100 ⁇ l of PBST, the detection antibody was diluted in RD and 50 ⁇ l aliquots were adjusted according to the working concentration, and reacted at room temperature for 2 hours.
  • PBS phosphate buffered saline
  • micrococcus luteus-derived vesicles were concentrated in Raw 264.7 cells. After treatment with (0.1, 1, 10 ⁇ g / ml), the secretion amount of inflammatory mediators (IL-6, TNF- ⁇ , etc.) was measured by treatment with E. coli EV ( E. coli EV), an inflammatory pathogenic vesicle.
  • E. coli EV E. coli EV
  • Micro-caustic bacteria-derived vesicles is gastric cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, atrial fibrillation, dysplastic angina, chronic obstructive pulmonary disease (COPD), diagnosis of dementia or diabetes
  • gastric cancer pancreatic cancer
  • cholangiocarcinoma cholangiocarcinoma
  • breast cancer ovarian cancer
  • bladder cancer myocardial infarction
  • cardiomyopathy cardiomyopathy
  • atrial fibrillation dysplastic angina
  • chronic obstructive pulmonary disease (COPD) diagnosis of dementia or diabetes
  • COPD chronic obstructive pulmonary disease

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Abstract

La présente invention concerne des vésicules issues de bactéries Micrococcus et leur utilisation. La présente invention a permis de confirmer expérimentalement que les vésicules ont été réduites de manière significative dans des échantillons de patients atteints d'un cancer gastrique, d'un cancer du pancréas, d'un cancer du canal cholédoque, d'un cancer du sein, d'un cancer de l'ovaire, d'un cancer de la vessie, d'un infarctus du myocarde, d'une cardiomyopathie, de fibrillation auriculaire, de l'angor de Prinzmetal, d'une bronchopneumopathie chronique obstructive (BPCO), de démence et de diabète par rapport à une personne normale, et que, lors de l'injection de vésicules séparées de la souche, la sécrétion d'un médiateur inflammatoire par des vésicules pathogènes telles que des vésicules issues d'Escherichia coli ont été remarquablement inhibée. Par conséquent, les vésicules issues de bactéries Micrococcus selon la présente invention peuvent être utilisées de manière utile dans le but de développer un procédé de diagnostic du cancer gastrique, du cancer du pancréas, du cancer du canal cholédoque, du cancer du sein, du cancer de l'ovaire, du cancer de la vessie, de l'infarctus du myocarde, de la cardiomyopathie, de la fibrillation auriculaire, de l'angor de Prinzmetal, de la BPCO, de la démence ou du diabète et une composition pour prévenir, soulager ou traiter les maladies.
PCT/KR2019/002341 2018-02-28 2019-02-27 Nanovésicules issues de bactéries micrococcus et leur utilisation Ceased WO2019168327A1 (fr)

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EP19761634.5A EP3760742A4 (fr) 2018-02-28 2019-02-27 Nanovésicules issues de bactéries micrococcus et leur utilisation
CN201980014447.5A CN111757943A (zh) 2018-02-28 2019-02-27 来源于微球菌属细菌的纳米囊泡及其用途
JP2020545158A JP2021514989A (ja) 2018-02-28 2019-02-27 マイクロコッカス属細菌由来ナノ小胞及びその用途
US16/563,720 US10888591B2 (en) 2018-02-28 2019-09-06 Nano-vesicles derived from genus Micrococcus bacteria and use thereof
JP2022006126A JP7433665B2 (ja) 2018-02-28 2022-01-19 マイクロコッカス属細菌由来ナノ小胞及びその用途

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Cited By (3)

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JP2024501034A (ja) * 2020-12-28 2024-01-10 エムディー ヘルスケア インコーポレイテッド マイクロコッカス・ルテウス由来細胞外小胞を含む神経発達疾患、神経疾患、または精神疾患の予防または治療用組成物
JP2024501033A (ja) * 2020-12-28 2024-01-10 エムディー ヘルスケア インコーポレイテッド ミクロコッカス・ルテウス由来細胞外小胞を含む眼疾患の予防または治療用組成物
EP4268835A4 (fr) * 2020-12-28 2024-11-27 MD Healthcare Inc. Composition pour la prévention ou le traitement d'une maladie pulmonaire neutrophile comprenant des vésicules extracellulaires dérivées de micrococcus luteus

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JP2024501034A (ja) * 2020-12-28 2024-01-10 エムディー ヘルスケア インコーポレイテッド マイクロコッカス・ルテウス由来細胞外小胞を含む神経発達疾患、神経疾患、または精神疾患の予防または治療用組成物
JP2024501033A (ja) * 2020-12-28 2024-01-10 エムディー ヘルスケア インコーポレイテッド ミクロコッカス・ルテウス由来細胞外小胞を含む眼疾患の予防または治療用組成物
EP4268835A4 (fr) * 2020-12-28 2024-11-27 MD Healthcare Inc. Composition pour la prévention ou le traitement d'une maladie pulmonaire neutrophile comprenant des vésicules extracellulaires dérivées de micrococcus luteus

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