WO2020171255A1 - Souche de clostridium scindens ayant un effet inhibiteur contre la croissance de clostridium difficile - Google Patents

Souche de clostridium scindens ayant un effet inhibiteur contre la croissance de clostridium difficile Download PDF

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WO2020171255A1
WO2020171255A1 PCT/KR2019/002114 KR2019002114W WO2020171255A1 WO 2020171255 A1 WO2020171255 A1 WO 2020171255A1 KR 2019002114 W KR2019002114 W KR 2019002114W WO 2020171255 A1 WO2020171255 A1 WO 2020171255A1
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clostridium
cdi
strain
scindens
snug
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PCT/KR2019/002114
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Korean (ko)
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고광표
유현주
유준선
윤수빈
김성호
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서울대학교산학협력단
주식회사 고바이오랩
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Priority to PCT/KR2019/002114 priority Critical patent/WO2020171255A1/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
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/145—Clostridium

Definitions

  • the present invention relates to a Clostridium difficile infection (CDI) treatment pharmaceutical composition
  • CDI Clostridium difficile infection
  • CDI Clostridium difficile infection
  • AAD antibiotics-associated diarrhea
  • C. difficile shows relatively high resistance to disinfectant treatment such as alcohol, and it is difficult to properly manage and remove because it can build spores that can survive in extreme environments for many years or more.
  • disinfectant treatment such as alcohol
  • the number of CDI patients per year is around 500,000, and the death toll is 14,000 a year, a serious disease.
  • recurrence is frequent even after treatment.
  • recurrence patients are classified as a major disease because it is difficult to see great efficacy even with general antibiotic treatment.
  • CDI treatment is antibiotic prescription, which is known to show relatively low treatment success rate and high recurrence rate.
  • Metronidazole and vancomycin both have a limited treatment success rate at the time of primary treatment and show a recurrence rate of 20 to 30%, the treatment success rate at the first recurrence is 70%, and at more recurrences it decreases to 35%, but due to highly toxic strains. Inadequate for use in refractory severe CDIs and recurrent CDIs.
  • Fidaxomicin inhibits the synthesis of C.
  • FMT fecal microbiota transplantation
  • Useful microorganisms isolated in the intestine can cause changes in the intestinal ecosystem and interaction with the immune system, and as a result, maintain space and resource competition with C. difficile.
  • these When these are treated, it has been reported that they actually show the same level of therapeutic effect as metronidazole or vancomycin, and that the recurrence rate is also significantly lower (Ollech et al., 2016. Best Practice & Research Clinical Gastroenterology). Therefore, the process of securing and mass-producing functional beneficial strains that can make a big contribution to the prevention/treatment of CDI can be seen as having very important significance as a method that can supplement/replace chemical antibiotic therapy in the future.
  • Intestinal microbes play a role of protecting the intestinal epithelium from pathogens such as C. difficile, and when an imbalance of intestinal microbes is caused by certain causes such as antibiotics (dysbiosis), the pathogen penetrates the intestinal wall and prevents disease. cause.
  • IL-25 produced by intestinal microbes increased the tight junction of the intestinal wall to prevent CDI (Buonomo et al., 2016. Cell Reports). It has also been reported that there is a high correlation with the degree of diversity of (Milani et al., 2016. Scientific Reports).
  • the diversity of intestinal microflora was significantly lower than that of normal people.
  • C. difficile spores Secondary bile acids produced by beneficial strains in the intestinal microbiota were C. difficile spores. It has been reported that the growth of C. difficile is inhibited by inhibiting TCA and DCA, which are essential for anger (Winston et al., 2016. Anaerobe.).
  • An object of the present invention is to provide a Clostridium scindens SNUG 40402 (KCTC 13277 BP) strain, characterized in that it has a Clostridium difficile growth inhibitory effect.
  • Another object of the present invention is to provide a pharmaceutical composition for the treatment of Clostridium difficile infection (CDI), characterized in that it comprises a Clostridium scindens SNUG 40402 (KCTC 13277 BP) strain or a culture therefor. will be.
  • CDI Clostridium difficile infection
  • Another object of the present invention is to provide a health functional food for alleviating or improving Clostridium difficile infection (CDI), comprising a strain of Clostridium scindens SNUG 40402 (KCTC 13277 BP) or a culture therefor.
  • CDI Clostridium difficile infection
  • KCTC 13277 BP Clostridium scindens SNUG 40402
  • the present invention provides a Clostridium scindens SNUG 40402 (KCTC 13277 BP) strain, characterized in that it has a Clostridium difficile growth inhibitory effect.
  • Clostridium difficile may be dependent on bile acids.
  • the concentration of the bile acid may be 0.2 to 2.0% (wt/vol).
  • the present invention provides a pharmaceutical composition for the treatment or prevention of Clostridium difficile infection (CDI), characterized in that it comprises a Clostridium scindens SNUG 40402 (KCTC 13277 BP) strain or a culture therefor. .
  • CDI Clostridium difficile infection
  • the present invention provides a functional food for alleviating or improving Clostridium difficile infection (CDI), comprising a strain of Clostridium scindens SNUG 40402 (KCTC 13277 BP) or a culture therefor. .
  • CDI Clostridium difficile infection
  • the Clostridium difficile infection may be a recurrent CDI.
  • Clostridium difficile infection may be caused by an intestinal microbial imbalance.
  • the present invention provides a method for preventing, improving or treating CDI by administering an effective amount of Clostridium scindens SNUG 40402 (KCTC 13277 BP) strain to an individual in need thereof.
  • the present invention provides the use of a Clostridium scindens SNUG 40402 (KCTC 13277 BP) strain for inhibiting the growth of Clostridium difficile in an individual.
  • the present invention provides the use of a strain of Clostridium scindens SNUG 40402 (KCTC 13277 BP) for the manufacture of a pharmaceutical formulation for preventing, improving or treating CDI.
  • the present invention relates to a strain of Clostridium scindens SNUG 40402 (KCTC 13277 BP), characterized in that it has a growth inhibitory effect of Clostridium difficile, is effective in CDI treatment and symptom improvement, and , In particular, it is very effective in treating recurrent CDI and improving symptoms.
  • FIG. 2 is a schematic diagram of the establishment of a C.difficile infection prevention model.
  • Figure 3 is a result of experimenting the body weight change after treatment with Clostridium scindens SNUG 40402 in the C. difficile infection prevention model.
  • Figure 3a is the result of a comparative experiment with the control strain
  • Figure 3b is the result of measuring the weight change after treatment with Clostridium scindens SNUG 40402
  • Figure 3c is the result of measuring the change in weight after treatment with Clostridium scindens SNUG 40402 over time.
  • Figure 4 is a result of testing the C.difficile proliferation inhibitory effect of Clostridium scindens SNUG 40402 in a C.difficile infection prevention model.
  • Fig. 5 is a result of an experiment on the sensitizing effect of Clostridium scindens SNUG 40402 on C.difficile Toxin in a C.
  • the present invention provides a Clostridium scindens (Clostridium scindens) SNUG 40402 (KCTC 13277 BP) strain, characterized in that it has a Clostridium difficile (C.difficile) growth inhibitory effect.
  • Clostridium scindens SNUG 40402 (KCTC 13277 BP) strain was isolated from feces provided by healthy adult volunteers. Since the strain is an absolute anaerobic strain, the separation process was performed under an anaerobic system.
  • Clostridium difficile (C.difficile) is a gram-positive bacterium, an anaerobic bacterium, is a relatively large bacterium with a length of 2-17 ⁇ m, has a structure such as flagella or projections, and is a bacterium living in human intestine. Clostridium difficile is likely to occur even after administration of antibiotics such as clindamycin, lincomycin, amoxicillin, cefalothin, cefazoline, etc. After the bacterial flora is destroyed, colonization can occur due to toxic C. difficile.
  • Clostridium difficile produces toxin A (enterotoxin) with a molecular weight of about 310,000 and toxin B (cytotoxin) with a molecular weight of about 270,000.
  • Toxin A first injures the intestinal epithelial cells and then invades the intestinal membrane at the site of toxin B stage and exerts a poison effect.
  • Clostridium difficile strains The growth inhibitory effect on Clostridium difficile strains can be confirmed through an inhibition assay.
  • the growth inhibition effect may be due to the baiCD gene of a Clostridium scindens strain.
  • the baiCD gene can encode 7 alpha-dehydrogenase. 7
  • the alpha-dehydrogenase gene can convert cholic acid, a primary bile acid (bile acid), into deoxycholic acid (DCA), a secondary bile acid, and this DAC can inhibit the growth of C.difficile.
  • inhibition of growth of C.difficile may be due to inhibition of spore germination.
  • Clostridium scindens SNUG 40402 strain is a deposited strain and was deposited with the Biological Resource Center on May 29, 2017, and the accession number is KCTC 13277 BP.
  • Clostridium difficile may be dependent on bile acids.
  • the concentration of a bile acid capable of exhibiting a growth inhibitory effect of Clostridium difficile may be 0.2 to 2.0% (wt/vol).
  • the present invention provides a pharmaceutical composition for the treatment or prevention of Clostridium difficile infection (CDI), characterized in that it comprises a Clostridium scindens SNUG 40402 (KCTC 13277 BP) strain or a culture therefor.
  • CDI Clostridium difficile infection
  • CDI may be caused by spore-forming bacteria, specifically Clostridium difficile strain, and may also be accompanied by diarrhea, high temperature, and abnormal pain.
  • the CDI may be a type of antibiotic-associated diarrhea.
  • CDI may have occurred in patients receiving broad spectrum antibiotics (especially clindamycin) that alter the ecological balance of the intestinal flora.
  • CDI may occur in patients with normal bile secretion in the liver.
  • the CDI may be mild CDI, severe CDI, complex CDI, or recurrent CDI, and more preferably, may be recurrent CDI.
  • the severity of the CDI can be determined based on age, whether systemic antibiotics are used, the number of white blood cells, albumin, and serum creatine.
  • the number of leukocytes may be less than 15,000 cells/ml, and the serum creatine may be less than 1.5 times premorbid level
  • the number of white blood cells is 15,000 cells/ml or more, and serum creatine is 1.5 times premorbid level. It may be abnormal, and complex (complicated CDI) may involve hypotension, shock, rainy season, megacolon, and recurrent CDI may mean recurrence within 8 weeks after successful treatment for CDI.
  • the pharmaceutical composition according to the present invention can be administered to mammals including humans by various routes.
  • the mode of administration may be any method commonly used, for example, may be administered by oral, dermal, intravenous, intramuscular, subcutaneous, and the like, preferably orally.
  • the pharmaceutical compositions of the present invention are each parenterally in the form of oral dosage forms such as powders, granules, tablets, capsules, ointments, suspensions, emulsions, syrups, aerosols, or transdermal preparations, suppositories, and sterile injectable solutions according to conventional methods. It can be formulated and used as an old formulation.
  • the pharmaceutical composition of the present invention may further contain adjuvants such as a pharmaceutically suitable and physiologically acceptable carrier, excipient, and diluent.
  • Carriers, excipients and diluents that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium. Silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils.
  • diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants can be used.
  • the pharmaceutical composition of the present invention may be administered alone, but generally, a pharmaceutical composition selected in consideration of the mode of administration and standard pharmaceutical practice.
  • the composition containing the strain of Clostridium syndense of the present invention may be administered in the form of a tablet containing starch or lactose, or in the form of a capsule containing an excipient or alone. It can be administered orally, orally, or under the tongue, in the form of elixirs or suspensions containing chemicals to give off or color.
  • the dosage of the pharmaceutical composition containing the Clostridium syndense strain of the present invention may vary depending on the patient's age, weight, sex, dosage form, health condition and degree of disease, and at certain time intervals according to the judgment of a doctor or pharmacist. It may be administered in divided doses from once a day to several times.
  • the daily dosage may be 0.1 to 500 mg/kg, preferably 0.5 to 300 mg/kg, based on the content of the active ingredient.
  • the above dosage is an example of an average case, and the dosage may be high or low depending on individual differences.
  • the daily dosage of the composition containing the mixed extract of the present invention is less than the above dosage, a significant effect cannot be obtained, and if it exceeds that, it is not only uneconomical, but also outside the range of the normal dosage, there is a concern that undesirable side effects may occur. It may occur, so it is better to set it within the above range.
  • Clostridium scindens (Clostridium scindens) SNUG 40402 (KCTC 13277 BP) strain or CDI (Clostridium difficile infection) alleviation or improvement health functional food comprising a strain or a culture thereof Provides.
  • the health functional food may be various beverages, fermented milk, food additives, and the like.
  • the content of the Clostridium syndense strain as an active ingredient contained in the health functional food is not particularly limited appropriately depending on the form of the food, the desired use, etc., and may be added in an amount of 0.01 to 15% by weight of the total food ,
  • the health beverage composition may be added in an amount of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml.
  • natural carbohydrates examples include monosaccharides such as disaccharides such as glucose and fructose, such as maltose, sucrose, and the like, and polysaccharides such as dextrin, cyclodextrin, and the like. Sugar and sugar alcohols such as xylitol, sorbitol, and erythritol.
  • sweetitol a sugar alcohol
  • erythritol erythritol.
  • natural flavoring agents tacmatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.)
  • synthetic flavoring agents sacharin, aspartame, etc.
  • the ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.
  • the health functional food of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring agents and enhancers (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid, and It may contain salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like.
  • the health functional food of the present invention may contain flesh for the manufacture of natural fruit juice and fruit juice beverage and vegetable beverage. These components may be used independently or in combination. The proportion of these additives is not so important, but is generally selected from 0 to about 20 parts by weight per 100 parts by weight of the health functional food of the present invention.
  • Clostridium scindens SNUG 40402 (KCTC 13277 BP) strain is administered to an individual in need thereof using the Clostridium difficile growth inhibitory effect of the Clostridium scindens SNUG 40402 (KCTC 13277 BP) strain. It provides a method of inhibiting or enhancing the curative ability of an individual including the step of CDI (Clostridium difficile infection), and a method of preventing, improving, or treating CDI.
  • CDI Clostridium difficile infection
  • Clostridium scindens SNUG 40402 (KCTC 13277 BP) strain for inhibiting the growth of Clostridium difficile. It provides the use of the Clostridium scindens SNUG 40402 (KCTC 13277 BP) strain for enhancing the CDI inhibitory or healing ability of an individual, and the use of the Clostridium scindens SNUG 40402 (KCTC 13277 BP) strain for the prevention, improvement or treatment of CDI.
  • The'individual' refers to an animal that is expected to exert an effect according to the Clostridium scindens SNUG 40402 (KCTC 13277 BP) strain by administering a composition containing the strain or strain according to the present invention as an active ingredient, and the animal includes, for example, dogs, Monkeys, goats, pigs, rats and the like are included, and are preferably humans.
  • The'prevention' refers to any action of inhibiting or delaying the progress of CDI by administration of a composition comprising the strain or strain according to the present invention as an active ingredient.
  • The'treatment' or'improvement' refers to any action in which symptoms of CDI are improved or beneficially changed by administration of a composition comprising the strain or strain according to the present invention as an active ingredient.
  • The'administration' means providing a pharmaceutical composition comprising a predetermined strain or strain according to the present invention as an active ingredient to an individual by any suitable method.
  • the research team intensively isolated candidate strains from feces provided by more than 40 healthy adult volunteers. Considering that most of the candidate strains are obligate anaerobes, all processes were performed under the anaerobic system of Go Bio Lab.
  • the inhibitory ability of C. difficile was confirmed using the supernatant obtained after culturing the isolated strain for 24 hours.
  • the strains used for the C. difficile inhibitory effect comparison experiment were C. difficile KCTC 5009 strain and C. difficile ATCC43255 strain, and the growth inhibitory effect on the strain was compared.
  • the CD culture solution and the prepared test strain supernatant were mixed at a ratio of 1:1 at 37°C. Incubated for 24 hours, OD was measured to evaluate the CD growth rate.
  • the test strain was inoculated into a liquid medium added with bile acids at 0, 0.5, 1% concentration and cultured at 37° C. for 24 hours, and then the supernatant was used. After diluting the OD of the C. difficile strain cultured in the liquid medium for 4 hours to 0.3 and re-inoculating the liquid medium at a concentration of 2%, the CD culture solution and the prepared test strain supernatant were mixed at a ratio of 1:1 at 37°C. Incubated for 24 hours, OD was measured to evaluate the CD growth rate.
  • CDI prophylaxis animal model C57BL/6 mice that were negatively administered antibiotics for 3 days to disturb intestinal microbial strains were orally administered the candidate strain for 2 days, followed by intraperitoneal administration of clindamycin and infection with C. difficile, resulting in CDI symptoms ( Body weight change) and the efficacy on survival were measured every day for 3 weeks (see Fig. 2).
  • RNA-later was used to prevent changes in RNA, and stored in a deep freezer at -80 degrees before analysis.
  • RNA was extracted from the obtained colon tissue using a Total RNA extraction kit, the expression level of TNF- ⁇ , an inflammation inducing factor, among the libraries converted to cDNA using the cDNA synthesis kit was quantitatively analyzed using real-time quantitative PCR.
  • Clostridium scindens SNUG 40402 significantly reduced the expression of TNF- ⁇ , a major inflammatory indicator, thereby inhibiting the reduction of the length of the bowel and reducing inflammation in the intestine. (See Fig. 7)

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Abstract

La présente invention concerne une souche de Clostridium scindens SNUG 40402 (KCTC 13277 BP) ayant un effet inhibiteur contre la croissance de Clostridium difficile.
PCT/KR2019/002114 2019-02-21 2019-02-21 Souche de clostridium scindens ayant un effet inhibiteur contre la croissance de clostridium difficile WO2020171255A1 (fr)

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Publication number Priority date Publication date Assignee Title
US20170087196A1 (en) * 2014-05-19 2017-03-30 Memorial Sloan-Kettering Cancer Center Methods and compositions for reducing clostridium difficile infection
KR20190033897A (ko) * 2017-09-22 2019-04-01 주식회사 고바이오랩 클로스트리디움 디피실레(Clostridium difficile) 성장 억제효과를 갖는 클로스트리디움 신덴스 균주

Patent Citations (2)

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US20170087196A1 (en) * 2014-05-19 2017-03-30 Memorial Sloan-Kettering Cancer Center Methods and compositions for reducing clostridium difficile infection
KR20190033897A (ko) * 2017-09-22 2019-04-01 주식회사 고바이오랩 클로스트리디움 디피실레(Clostridium difficile) 성장 억제효과를 갖는 클로스트리디움 신덴스 균주

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CHARLIE G BUFFIE; VANNI BUCCI; RICHARD R STEIN; PETER T MCKENNEY; LILAN LING; ASIA GOBOURNE; DANIEL NO; HUI LIU; MELISSA KINNEBREW: "Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile", NATURE, vol. 517, 7533, 22 October 2014 (2014-10-22), pages 205 - 208, XP055363406, ISSN: 0028-0836, DOI: 10.1038/nature13828 *
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