WO2023068857A1 - Nouvelle souche bactérienne possédant une activité anticancéreuse et composition permettant de soulager, de prévenir ou de traiter le cancer l'utilisant - Google Patents

Nouvelle souche bactérienne possédant une activité anticancéreuse et composition permettant de soulager, de prévenir ou de traiter le cancer l'utilisant Download PDF

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WO2023068857A1
WO2023068857A1 PCT/KR2022/016092 KR2022016092W WO2023068857A1 WO 2023068857 A1 WO2023068857 A1 WO 2023068857A1 KR 2022016092 W KR2022016092 W KR 2022016092W WO 2023068857 A1 WO2023068857 A1 WO 2023068857A1
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strain
cancer
clostridium
kbl1038
tumor
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Korean (ko)
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고광표
송림
유현주
김준형
신승연
장성재
조보람
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주식회사 고바이오랩
서울대학교산학협력단
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    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • 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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/308—Foods, ingredients or supplements having a functional effect on health having an effect on cancer prevention
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • 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 Erysipelatoclostridium ramosum strains and Clostridium scindens strains having anticancer effects, and their cancer prevention, treatment, or improvement uses.
  • Probiotics refer to microorganisms and products produced by the microorganisms having antibacterial activity and enzymatic activity that help the intestinal microbial balance.
  • probiotics are defined as live bacteria in the form of single or complex strains that are supplied to humans or animals in the form of dry cells or fermentation products to improve the intestinal flora.
  • Probiotics have the characteristics of non-pathogenicity and non-toxicity, inhabit the human intestine, and must survive while going to the intestine, so they must have resistance to acids, enzymes, and bile in the intestinal environment.
  • probiotics must retain viability and activity prior to consumption in delivery foods, must be sensitive to antibiotics used to prevent infection, and must not contain antibiotic-resistant plasmids.
  • probiotics include Bacillus species ( Bac probiotics), which have an excellent ability to produce digestive enzymes (amylase, protease, lipase, cellulase, phosphatase), are microorganisms with antibacterial activity and enzyme activity that help balance intestinal microorganisms, and the microorganisms are produced
  • probiotics are defined as live bacteria in the form of single or complex strains that are supplied to humans or animals in the form of dry cells or fermentation products to improve the intestinal flora.
  • probiotics maintain their viability and activity before being consumed in delivered food, and as a preventive measure against infection. It should be sensitive to the antibiotics used and should not carry antibiotic-resistant plasmids.
  • probiotics include Bacillus sp. , which has an excellent ability to produce digestive enzymes (amylase, protease, lipase, cellulase, and phosphatase), Lactobacillus sp. Examples include photosynthetic bacteria that use substances (ammonia, hydrogen sulfide, amines, etc.) in metabolic processes to prevent odors.
  • Erysipelatoclostridium ramosum Erysipelatoclostridium ramosum
  • Clostridium Sindens Clostridium scindens
  • cancer refers to a group of abnormal cells generated by continuous division and proliferation when the balance between cell division and death is destroyed for various reasons, and is also referred to as a tumor or a neoplasm. In general, it develops in more than 100 different body parts, including organs, white blood cells, bones, and lymph nodes, and develops into serious symptoms through infiltration into surrounding tissues and metastasis to other organs (WHO, 2006).
  • causes of cancer include environmental or external factors such as chemicals, viruses, bacteria, and ionizing radiation, and internal factors such as congenital genetic mutations (Klauunig & Kamendulis, Annu Rev Pharmacol Toxicol 2004, 44:239-267 ).
  • An object of the present invention is to provide a probiotic strain showing excellent effects in the alleviation, prevention or treatment of cancer, a composition containing the same, and a composition for co-administration thereof with an anti-cancer therapeutic agent.
  • the present invention provides an Erysiphellatoclostridium ramosum strain and a Clostridium syndens strain having anticancer activity.
  • the present invention is an Erysipella toclostridium lamosum KBL1038 strain deposited with accession number KCTC14615BP, an Erysipella toclostridium lamosum KBL1039 strain deposited with accession number KCTC14609BP, and a Clostridium deposited with accession number KCTC13277BP
  • a Clostridium Sindense KBL987 strain and a Clostridium Sindense KBL1037 strain deposited under accession number KCTC14608BP are provided.
  • the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising at least one strain selected from the group consisting of at least one strain, a culture of the strain, a lysate of the strain, and an extract of the strain. .
  • the present invention provides a food composition for preventing or improving cancer, including at least one selected from the group consisting of one or more strains of the strains, cultures of the strains, lysates of the strains, and extracts of the strains.
  • the present invention provides a feed composition comprising at least one selected from the group consisting of one or more strains of the strain, a culture of the strain, a lysate of the strain, and an extract of the strain.
  • Erysipella toclostridium lamosum KBL1038 strain and KBL1039 strain and Clostridium syndens KBL987 strain and KBL1037 strain, and extracts thereof according to the present invention reduce the size of tumors when administered to animals having cancer The effect appears, and it increases immune cells related to improvement or treatment of cancer.
  • the composition comprising at least one selected from the group consisting of the strain of the present invention, its culture, lysate and extract exhibits cancer alleviation, prevention or treatment effects As a result, it can be used industrially very usefully.
  • Erysipelato Clostridium lamosum strain and Clostridium syndens strain, and their cultures, lysates and extracts according to the present invention are administered to animals with cancer It has the effect of reducing the size of the tumor when it becomes, and increases the immune cells related to the improvement or treatment of cancer.
  • the composition comprising at least one selected from the group consisting of the strain of the present invention, its culture, lysate and extract exhibits cancer alleviation, prevention or treatment effects As a result, it can be used industrially very usefully.
  • 1 is a view showing the experimental process of animal experiments to confirm the tumor size after administering the extracts of KBL1038, KBL1039, KBL987 and KBL1037 of the present invention to melanoma-induced mice.
  • Figure 2 is a graph showing the tumor volume increase inhibitory effect when Clostridium syndens KBL987 strain extract and Erysipelato Clostridium lamosum KBL1039 strain extract were administered to melanoma mice.
  • Figure 3 is a graph showing the tumor volume increase inhibitory effect when Clostridium syndens KBL1037 strain extract and Erysipelato Clostridium lamosum KBL1038 strain extract were administered to melanoma mice.
  • Figure 4 shows Clostridium syndens KBL1037 strain extract, Clostridium syndens KBL987 strain extract, Erysipella toclostridium lamosum KBL1038 strain extract and Erysipella toclostridium lamosum KBL 1039 strain extract melanoma It is a graph showing the tumor volume increase inhibitory effect when administered to mice.
  • Figure 5 is a view showing the experimental process of animal experiments to determine the tumor size after administering Erysipelato Clostridium lamosum KBL1038 strain to melanoma-induced mice.
  • Figure 6 is a graph showing the tumor volume increase inhibitory effect when Erysipella toclostridium lamosum KBL1038 strain was administered to melanoma mice compared to other strains.
  • Figure 7 is a graph showing the change in tumor volume increase over three times after administration of Erysipelato Clostridium lamosum KBL1038 strain to melanoma mice (upper figure), and immune cells in tumor tissue using flow cytometry It is a diagram showing the analysis result (bottom figure).
  • FIG. 8 is a diagram illustrating an experimental process of an animal experiment for confirming the size of a tumor after administering an Erysiphelatoclostridium lamosum KBL1038 strain to a mouse in which colon cancer was induced.
  • FIG. 9 is a graph showing the change in tumor volume increase after administration of Erysipelatoclostridium lamosum KBL1038 strain to colorectal cancer mice (left figure), and the results of analyzing immune cells in tumor tissue using flow cytometry ( It is a drawing showing the figure on the right).
  • Figure 10 shows the experimental process of an animal experiment to confirm the tumor size by orally administering Erysipelato Clostridium ramosum KBL1038 strain after administering antibiotics to melanoma-induced mice for 0, 7, or 14 days. It is a drawing
  • 11 is a graph showing changes in tumor volume increase after oral administration of Erysiphelatoclostridium lamosum KBL1038 strain to melanoma mice in an antibiotic and oral model.
  • Figure 12 shows the experimental process of animal experiments to determine the size of tumors after administering the anti-PD-1 antibody, an anti-cancer treatment, alone or in combination with the Erysipelatoclostridium lamosum KBL1038 strain to melanoma-induced mice. it is a drawing
  • FIG. 13 is a graph showing changes in tumor volume increase when Erysiphelatoclostridium lamosum KBL1038 strain and anti-PD-1 antibody were administered alone or in combination to melanoma mice.
  • FIG 14 is a graph (left figure) showing changes in tumor volume increase when Erysiphelatoclostridium lamosum KBL1038 strain and anti-PD-1 antibody are administered alone or in combination to colorectal cancer mice (left figure) and tumor It is a diagram showing the result of analyzing immune cells in tissues (right figure).
  • Figure 15 shows the experimental process of animal experiments to confirm the size of tumors by administering Erysipella toclostridium ramosum KBL1038 strain to melanoma and colorectal cancer induced mice in a T cell deficient mouse model (athymic nude mouse). It is an illustrated drawing.
  • 16 is a graph showing changes in tumor volume increase of Erysiphelatoclostridium ramosum KBL1038 strain for T cell deficient mice.
  • 17 is a graph illustrating the survival rate of cancer cells after treatment with Erysipelato Clostridium lamosum KBL1038 strain in colorectal cancer cell lines HCT116 and DLD-1.
  • FIG. 18 is a diagram showing the non-settled formation ability of cancer cells after treatment with Erysipelato Clostridium lamosum KBL1038 strain in HCT116, a colorectal cancer cell line.
  • 19 is a diagram illustrating the non-settled formation ability of cancer cells after treatment with Erysipelato Clostridium lamosum KBL1038 strain in colorectal cancer cell line DLD-1.
  • FIG. 20 is a view showing analysis of non-settled formation ability of cancer cells after treatment with Erysipelato Clostridium lamosum KBL1038 strain in lung cancer cell line A549.
  • FIG. 21 is a diagram illustrating the non-settled formation ability of cancer cells after treatment with Erysipelato Clostridium lamosum KBL1038 strain in pancreatic cancer cell line PANC1.
  • the present invention is an Erysipelatoclostridium ramosum KBL1038 strain deposited under accession number KCTC14615BP, an Erysipelatoclostridium ramosum deposited under accession number KCTC14609BP, Erysipelatoclostridium ramosum KBL1039 strain, accession number KCTC13277BP Provides Clostridium scindens KBL987 strain and/or Clostridium scindens KBL1037 strain deposited with accession number KCTC14608BP.
  • the KBL1038 strain may have a 16s rDNA sequence of SEQ ID NO: 1 below.
  • the present invention is an Erysipelatoclostridium ramosum KBL1038 strain deposited under accession number KCTC14615BP, an Erysipelatoclostridium ramosum deposited under accession number KCTC14609BP, Erysipelatoclostridium ramosum KBL1039 strain, accession number KCTC13277BP Clostridium Sindens deposited as ( Clostridium scindens ) KBL987 strain and / or the basic invention with accession number KCTC14608BP Erysipelato Clostridium ramosum having anticancer activity ( Erysipelatoclostridium ramosum ) and Clostridium Sindens ( Clostridium scindens ) strains provides
  • the present invention is an Erysipelatoclostridium ramosum KBL1038 strain deposited under accession number KCTC14615BP, an Erysipelatoclostridium ramosum deposited under accession number KCTC14609BP, Erysipelatoclostridium ramosum KBL1039 strain, accession number KCTC13277BP Provides Clostridium scindens KBL987 strain and/or Clostridium scindens KBL1037 strain deposited with accession number KCTC14608BP.
  • the KBL1038 strain may have a 16s rDNA sequence of SEQ ID NO: 1 below.
  • the KBL1038 strain has a 16s rDNA sequence having 70% or more, 80% or more, 90% or more, 95% or more, 98% or more or 99% or more sequence homology with the 16s rDNA represented by SEQ ID NO: 1 It may be an Erysipella toclostridium ramosum strain.
  • the KBL1039 strain may have a 16s rDNA sequence of SEQ ID NO: 2 below.
  • the KBL1039 strain has a 16s rDNA sequence having 70% or more, 80% or more, 90% or more, 95% or more, 98% or more or 99% or more sequence homology with the 16s rDNA represented by SEQ ID NO: 2 It may be an Erysipella toclostridium ramosum strain.
  • the KBL987 strain may have a 16s rDNA sequence of SEQ ID NO: 3 below.
  • Clostridium syndense is a 16s rDNA sequence having 70% or more, 80% or more, 90% or more, 95% or more, 98% or more or 99% or more sequence homology with the 16s rDNA represented by SEQ ID NO: 3 It may be a Clostridium syndens strain having.
  • the KBL1037 strain may have a 16s rDNA sequence of SEQ ID NO: 4 below.
  • Clostridium syndense is a 16s rDNA sequence having 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more sequence homology with the 16s rDNA represented by SEQ ID NO: 4. It may be a Clostridium syndens strain having.
  • the strains may have an activity of increasing the number and activity of immune cells in cancer cells, and may suppress tumors through T cells.
  • the present invention is an Erysipelatoclostridium ramosum KBL1038 strain deposited with the accession number KCTC14615BP, an Erysipelatoclostridium ramosum deposited with accession number KCTC14609BP, Erysipelatoclostridium ramosum KBL1039 strain, accession number Clostridium Sindens deposited as KCTC13277BP ( Clostridium scindens ) KBL987 strain and / or deposited with accession number KCTC14608BP Clostridium scindens ( Clostridium scindens ) At least one or more strains of the KBL1037 strain, a culture of the strain, a lysate of the strain, and It provides a pharmaceutical composition for preventing or treating cancer comprising at least one selected from the group consisting of extracts of the strain.
  • composition of the present invention may further include other strains in addition to the above strains, and may include both other strains of Erysipelato Clostridium ramosum and Clostridium syndens, of course.
  • the strains are in the form of live cell cells, dead cell cells and dried strains
  • the present invention is Erysipelatoclostridium ramosum ( Erysipelatoclostridium ramosum )
  • At least one strain and Clostridium syndens ( Clostridium scindens ) strain It provides a pharmaceutical composition for preventing or treating cancer, comprising at least one selected from the group consisting of the above strains, cultures of the strains of the strains, lysates of the strains, and extracts of the strains.
  • the strain is Erysipelatoclostridium ramosum KBL1038 strain, deposited with accession number KCTC14615BP, Erysipelatoclostridium ramosum KBL1039 strain, accession number KCTC13277BP Clostridium Sindens deposited as ( Clostridium scindens ) KBL987 strain and Clostridium Sindens deposited as accession number KCTC14608BP ( Clostridium scindens ) It may be at least one strain of KBL1037 strain.
  • composition of the present invention may further include other strains in addition to the above strains, and may include both other strains of Erysipelato Clostridium ramosum and Clostridium syndens, of course.
  • strains may be included in the form of live cell cells, dead cell cells and dried strains, cultures thereof, lysates thereof, or extracts thereof.
  • the term "culture” refers to a product obtained by culturing a strain in a known medium, and the product may include the strain itself.
  • the medium may be selected from known liquid medium or solid medium, and may be, for example, MRS liquid medium, GAM liquid medium, MRS agar medium, GAM agar medium, and BL agar medium, but is not limited thereto.
  • lysate refers to any product obtained by disrupting a strain by enzyme treatment, homogenization, or ultrasonic treatment.
  • extract refers to a product obtained by extracting a strain with a known extraction solvent.
  • extract includes a water extract and/or an organic solvent extract of the strains according to the present invention.
  • the organic solvent extract of the strain according to the present invention may be an organic solvent extract having 1 or more and 10 or less carbon atoms.
  • a substituted or unsubstituted alcohol extract having 1 to 10 carbon atoms, 1 to 5 carbon atoms, or 1 to 3 carbon atoms may be used as the extraction solvent.
  • alcohol extracts such as, for example, methanol extracts, ethanol extracts, iso-propanol extracts, n-propanol extracts, n-butanol extracts, iso-butanol extracts, tert-butanol extracts and/or phenol extracts; ether extracts such as dialkyl ethers such as dimethyl ether, diethyl ether, and methyl ethyl ether; n-hexane, ethyl acetate, dichloromethane, chloroform and/or acetone extracts.
  • ether extracts such as dialkyl ethers such as dimethyl ether, diethyl ether, and methyl ethyl ether
  • n-hexane ethyl acetate, dichloromethane, chloroform and/or acetone extracts.
  • live cell refers to the strain itself of the present invention
  • dead cell refers to a strain that has been sterilized by heating, pressurization, or drug treatment.
  • composition of the present invention may be provided as a composition capable of being further combined with pharmaceutically acceptable additives such as carriers or media.
  • the additives used in the present invention are solvents, dispersants, coatings, absorption accelerators, controlled release agents (i.e. sustained release agents), and one or more inactive excipients (starch, polyols, granules, microfine cellulose, microcrystalline cellulose). (including, for example, cellphere, cellphere beads, diluents, lubricants, binders, disintegrants, etc.), etc.
  • tablet formulations of the disclosed compositions may be standard aqueous or Non-limiting examples of excipients for use as pharmaceutically acceptable carriers and pharmaceutically acceptable inert carriers and the additional ingredients include binders, fillers, disintegrants, lubricants. , antimicrobial agents and coating agents.
  • the content of the additives included in the pharmaceutical composition is not particularly limited and may be appropriately adjusted within the range of content used in conventional formulations.
  • cancer medically refers to a problem in the normal division, differentiation, or death control function of cells, resulting in abnormal overproliferation, infiltration of surrounding tissues or organs, formation of lumps, and destruction or transformation of existing structures. means any condition. Due to such uncontrolled and abnormal cell growth, a cell mass called a tumor is formed, infiltrating surrounding normal tissues or organs, destroying the normal tissues or organs, and taking the life of the subject.
  • prevention means delaying the onset of a disease, disorder or condition.
  • treatment means, unless otherwise stated, to reverse, alleviate, inhibit the progress of, or reverse the disease or condition to which the term applies, or one or more symptoms of the disease or condition, or It means to prevent, and the term “treatment” used in the present invention refers to the act of treating when "treating" is defined as above.
  • treatment or therapy for a disease in a mammal may include one or more of the following:
  • composition of the present invention according to the conventional method according to each purpose of use, oral formulations such as solutions, suspensions, powders, granules, tablets, capsules, pills, extracts, emulsions, syrups, aerosols, injections of sterile injection solutions It can be formulated and used in various forms such as oral administration or parenteral administration through various routes including intravenous, intraperitoneal, subcutaneous, intradermal, intramuscular, spinal, intrathecal, or rectal topical administration or injection.
  • the dosage may vary depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, administration period or interval, excretion rate, constitutional specificity, nature of the preparation, severity of the disease, etc. there is.
  • liquid formulation refers to a medicine to be taken in the form of a liquid medicine dissolved in water or an organic solvent. Compared to suspensions or solid formulations, liquid formulations have the advantage of more effective drug absorption from the intestinal tract to the systemic circulation, and the liquid formulations may contain additional solutes in addition to pharmaceuticals, and additives providing color, odor, sweetness, or stability. may also be included.
  • the term “suspending agent” refers to any agent capable of providing the desired solubility and/or dispersibility of an alginate-containing composition, i.e., an aqueous formulation that is substantially clear and free of sedimentation and lumps.
  • powder refers to finely divided drugs, chemicals, or a dry mixture of both. “Powder” may include a mixture in a lyophilized state.
  • powders may contain conventional additives used in lyophilized formulations such as lyophilized strains and lyophilized preservatives.
  • the term "granule” refers to a drug or a mixture of drugs in the form of granules, which usually passes through a sieve of 4.76 to 20 mm.
  • Granules are generally produced by wetting the powder or mixture of powders and passing the mass through a sieve or granulator of appropriate mesh size depending on the size of the granules required. Since granules, like powders, are granular, the degree of contact of the drug to the tongue is high, so when a drug having a bitter taste is used in the form of granules, it may cause discomfort to patients, especially children or the elderly.
  • tablette means a powdered medicine made easy to take by compressing it into a small disc shape. Tablets may include uncoated tablets, film-coated tablets, coated tablets, multilayer tablets, press-coated tablets, inner core tablets, orally disintegrating tablets, chewable tablets, effervescent tablets, dispersible tablets, and dissolving tablets.
  • a pharmaceutical composition refers to a product made by filling a drug into a capsule in the form of a liquid, suspension, water, powder, granule, mini-tablet, or pellet, or encapsulated with a capsule base.
  • a pharmaceutical composition according to one embodiment may include a lyophilized strain.
  • the pharmaceutical composition of one embodiment may be formulated in the form of a capsule formulation of the lyophilized strain.
  • the storage period of the strain can be improved by minimizing the loss of the strain that may occur due to heat.
  • the biological activity of the strain can be inactivated to improve formulation stability, and the strain can be more easily induced to rehydrate than other formulation methods, so that the original activity and growth ability can be quickly restored after supplying water to the dry strain.
  • the freeze-dried preparation is easy to formulate, such as tableting and encapsulation, and can be prepared in various dosage forms.
  • regeneration which is the most important feature of LBP (Live Biotherapeutic Products) such as strain preparations, can proceed very stably in freeze-dried strains.
  • the strain when the strain is formulated by lyophilization, skim milk, sugars (eg, trehalose, sucrose, maltose or glucose, etc.), sugar alcohols (eg, to prevent a rapid decrease in activity during freeze-drying of the strain)
  • sugars eg, trehalose, sucrose, maltose or glucose, etc.
  • sugar alcohols eg, to prevent a rapid decrease in activity during freeze-drying of the strain
  • mannitol, inositol, or sorbitol, etc. may be mixed with conventional freeze-drying preservatives (or protecting agents) used in the art and then freeze-dried.
  • any lyophilization method commonly used in the art can be used without particular limitation.
  • pill is meant to encompass small, round solid dosage forms containing multiparticulates mixed with binders and other excipients.
  • the term "syrup" means a thick homemade sugar or sugar substitute.
  • the syrup is an easy-to-take medicine with an unpleasant taste, such as a bitter taste, in a liquid form, and is particularly suitable for use by children.
  • the syrup may include, in addition to purified water and extract, sugar or a substitute for sugar used to give sweetness and viscosity, an antibacterial preservative, a flavoring agent, or a coloring agent, but is not limited thereto.
  • sweeteners examples include, but are not limited to, white sugar, mannitol, sorbitol, xylitol, aspartame, stevioside, fructose, lactose, sucralose, saccharin, or menthol.
  • injection refers to an aseptic preparation applied to the body through the skin or mucous membrane, and in particular, any form of administration such as subcutaneous injection, intramuscular injection, intradermal injection, and intraperitoneal injection may be used as an injection route. It is possible, and the dosage form is selected according to the characteristics of each pharmacologically active substance.
  • the composition of the present invention may be a composition for oral administration.
  • oral administration means that the active substance is administered to the gastrointestinal tract through the oral route for absorption.
  • Non-limiting examples of the formulation for oral administration include tablets, troches, lozenges, aqueous suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups or elixirs, and the like.
  • a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin; excipients such as dicalcium phosphate and the like; disintegrants such as corn starch or sweet potato starch; Lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate, or polyethylene glycol may be used, and sweeteners, aromatics, syrups, and the like may also be used.
  • a liquid carrier such as fatty oil may be additionally used in addition to the above-mentioned materials.
  • the pharmaceutical composition of the present invention may be provided as an enteric-coated enteric preparation, particularly as a unit dosage form for oral use.
  • Enteric coating in the present specification includes all types of pharmaceutically acceptable known coatings that are not decomposed by gastric acid and the coating is maintained, but sufficiently decomposed in the small intestine so that the active ingredient can be released into the small intestine. do.
  • the "enteric coating” of the present invention is maintained for at least 2 hours when artificial gastric juice, such as a pH 1 HCl solution, is brought into contact at 36°C to 38°C, and is preferably followed by a pH 6.8 KH 2 PO 4 buffer solution. Refers to coatings that disintegrate within 30 minutes in artificial intestinal juices such as
  • the enteric coating of the present invention is coated in an amount of about 16 to 30, preferably 16 to 20 or 25 mg or less per core.
  • the thickness of the enteric coating of the present invention is 5 to 100 ⁇ m, preferably 20 to 80 ⁇ m, satisfactory results are obtained as an enteric coating.
  • the material of the enteric coating is appropriately selected from known high molecular materials. Suitable polymeric materials are described in a number of well-known literature (L. Lachman et al., The Theory and Practice of Industrial Pharmacy, 3rd edition, 1986, pp. 365-373 H. Sucker et al., Pharmazeutician Technologie, Thieme, 1991, pp. 355-359; Hagers Handbuchder pharmazeutician fürtechnik, 4th edition, Vol. 7, pp.
  • the enteric coating of the present invention can be prepared using a conventional enteric coating method in which an enteric coating solution is sprayed onto a core.
  • Suitable solvents used in the enteric coating process include alcohols such as ethanol, ketones such as acetone, and halogenated hydrocarbon solvents such as dichloromethane (CH 2 Cl 2 ), and a mixed solvent of these solvents may be used.
  • An emollient such as di(di)-n-butylphthalate or triacetin is added to the coating solution in a ratio of 1 to about 0.05 to about 0.3 (coating material to softener). It is appropriate to carry out the spraying process continuously and it is possible to adjust the amount of spraying taking into account the conditions of the coating.
  • the spray pressure can be varied, and satisfactory results are generally obtained with spray pressures of about 1 to about 1.5 bar.
  • the composition of the present invention may be a composition for parenteral administration.
  • parenteral administration means intravenous, intraperitoneal, subcutaneous, intradermal, intramuscular, spinal, intrathecal or rectal topical administration or injection.
  • Parenteral administration is by injecting a suppository preparation, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection.
  • the composition may be mixed in water with a stabilizer or a buffer to prepare a solution or suspension, which may be prepared in a unit dosage form in an ampoule or vial.
  • the preferred dosage of the composition of the present invention depends on the condition and weight of the patient, age, sex, health condition, dietary constitution specificity, the nature of the preparation, the severity of the disease, the administration time of the composition, the administration method, the administration period or interval, the excretion rate, And the range may vary depending on the type of drug, and may be appropriately selected by a person skilled in the art. For example, it may be in the range of about 0.1 to 10,000 mg/kg, but is not limited thereto, and may be divided and administered once or several times a day.
  • the present invention Erysipelato Clostridium Ramosum ( Erysipelatoclostridium ramosum ) Strain and Clostridium Sindens ( Clostridium scindens ) At least one strain, a culture of the strain of the strain, a lysate of the strain, and the strain It provides a food composition or food additive composition for preventing or alleviating cancer, including at least one selected from the group consisting of an extract of.
  • the strain may be at least one strain of Erysipelato Clostridium lamosum KBL1038 strain, KBL1039 strain, Clostridium syndens KBL987 strain and KBL1037 strain.
  • the food may be a health functional food.
  • the food composition or composition for food additives may be included in foods effective in preventing or alleviating cancer.
  • the food composition of the present invention can be easily utilized as a main ingredient, supplementary ingredient, food additive, health functional food or functional beverage of food.
  • the food composition means a natural product or processed product containing one or more nutrients, and preferably means a product that can be directly eaten through a certain degree of processing, and in a conventional sense, food, It refers to food additives, health functional foods, and functional beverages.
  • Foods to which the food or food additive composition according to the present invention can be added include, for example, various foods, beverages, chewing gum, tea, vitamin complexes, and functional foods.
  • food includes special nutritional food (eg, formula milk, infant food, baby food, etc.), processed meat product, fish meat product, tofu, jelly, noodles (eg, ramen, noodles, etc.), bread, health supplement food, seasoning Foods (eg, soy sauce, soybean paste, gochujang, mixed paste, etc.), sauces, confectionery (eg, snacks), candies, chocolates, chewing gum, ice cream, dairy products (eg, fermented milk, cheese, etc.), other processed foods, kimchi, It includes, but is not limited to, pickled foods (various types of kimchi, pickled vegetables, etc.), beverages (eg, fruit drinks, vegetable drinks, soy milk, fermented beverages, etc.), and natural seasonings (eg, ramen soup, etc.).
  • the food, beverage or food includes, but
  • the term “health functional food” refers to a food group or food composition that has added value so that the function of the food acts for a specific purpose by using physical, biochemical, or bioengineering methods, etc. It refers to food designed and processed to fully express the body's regulatory functions related to disease prevention and recovery.
  • the functional food may include food additives that are acceptable in food science, and may further include appropriate carriers, excipients, and diluents commonly used in the manufacture of functional foods.
  • the food containing the food composition of the present invention is various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and fillers (cheese, chocolate, etc.), pectins acids and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. and can be used.
  • the amount of the composition according to the present invention may be 0.001% to 100% by weight of the total weight of the food, preferably 1% to 99% by weight. %, and in the case of beverages, it may be included in a ratio of 0.001 g to 10 g, preferably 0.01 g to 1 g based on 100 mL, but for health and hygiene purposes or health control purposes In the case of long-term ingestion, it may be less than the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.
  • the composition for food or food additives of the present invention is at least one of Erysipella toclostridium lamosum strains (eg KBL1038 strain and / or KBL1039 strain) and Clostridium syndens strain (eg KBL987 strain and / or KBL1037 strain)
  • the above strains may be added independently or to an acceptable carrier, or prepared in the form of a composition suitable for consumption by humans or animals. That is, it can be added to and used in foods that do not contain other probiotic bacteria and foods that already contain some probiotic bacteria.
  • the present invention Erysipelato Clostridium Ramosum ( Erysipelatoclostridium ramosum ) Strain and Clostridium Sindens ( Clostridium scindens ) At least one strain, a culture of the strain of the strain, a lysate of the strain, and the strain It provides a composition for adding animal feed or animal feed, including at least one selected from the group consisting of extracts of.
  • the strain may be at least one strain of Erysipelato Clostridium lamosum KBL1038 strain, KBL1039 strain, Clostridium syndens KBL987 strain and KBL1037 strain.
  • the additive for animal feed of the present invention may be in the form of a dry or liquid formulation, and is an Erysipherato Clostridium ramosum strain (eg, KBL1038 strain and / or KBL1039 strain) and a Clostridium syndens strain (eg, KBL987 strain and / or KBL1037 strain) may further include other non-pathogenic microorganisms in addition to at least one or more strains.
  • an Erysipherato Clostridium ramosum strain eg, KBL1038 strain and / or KBL1039 strain
  • a Clostridium syndens strain eg, KBL987 strain and / or KBL1037 strain
  • At least one strain of the Erysipelato Clostridium ramosum strain eg KBL1038 strain and / or KBL1039 strain
  • Clostridium syndens strain eg KBL987 strain and / or KBL1037 strain
  • various grains and soybean proteins including peanuts, peas, sugar beets, pulp, grain by-products, animal intestine powder and fish meal powder, etc. may be used as raw materials for feed, and these may be used unprocessed or processed without limitation.
  • the present invention Erysipelato Clostridium Ramosum ( Erysipelatoclostridium ramosum ) Strain and Clostridium Sindens ( Clostridium scindens ) At least one strain, a culture of the strain of the strain, a lysate of the strain, and the strain It provides a cancer prevention or treatment method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising at least one selected from the group consisting of an extract of to a subject in need of treatment.
  • the strain may be at least one strain of Erysipelato Clostridium lamosum KBL1038 strain, KBL1039 strain, Clostridium syndens KBL987 strain and KBL1037 strain.
  • the present invention in the manufacture of a drug for preventing or treating cancer, Erysipelato Clostridium ramosum ( Erysipelatoclostridium ramosum ) At least one strain of the strain and Clostridium Sindens ( Clostridium scindens ) strain, of the strain of the strain It provides the use of a composition comprising at least one selected from the group consisting of a culture, a lysate of the strain, and an extract of the strain.
  • the strain may be at least one strain of Erysipelato Clostridium lamosum KBL1038 strain, KBL1039 strain, Clostridium syndens KBL987 strain and KBL1037 strain.
  • the present invention Erysipelato Clostridium Ramosum ( Erysipelatoclostridium ramosum ) Strain and Clostridium Sindens ( Clostridium scindens ) At least one strain, a culture of the strain of the strain, a lysate of the strain, and the strain It provides a use for preventing or treating cancer of a pharmaceutical composition comprising at least one selected from the group consisting of extracts of.
  • the strain may be at least one strain of Erysipelato Clostridium lamosum KBL1038 strain, KBL1039 strain, Clostridium syndens KBL987 strain and KBL1037 strain.
  • the pharmaceutical composition of the present invention may be used alone or in other therapies, such as surgery, radiation therapy, gene therapy, immunotherapy, bone marrow transplantation, stem cell transplantation, hormone therapy, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, chemotherapy. etc. can be performed.
  • therapies such as surgery, radiation therapy, gene therapy, immunotherapy, bone marrow transplantation, stem cell transplantation, hormone therapy, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, chemotherapy. etc. can be performed.
  • composition of the present invention may be administered in combination with an anti-cancer therapeutic agent. That is, the composition of the present invention may be a composition for concomitant administration of anticancer drugs.
  • composition of the present invention may further contain an anti-cancer agent.
  • the composition of the present invention may include the strain and the anticancer agent as a single agent, or may be included as separate agents.
  • the composition of the present invention may include a first agent containing a strain and a second agent including an anti-cancer agent.
  • the composition of the present invention may include the first agent and the second agent as separate and separate agents.
  • the first agent and the second agent may be administered through the same route of administration or different routes of administration.
  • the first agent and the second agent may be administered simultaneously or sequentially via the same route of administration or separate routes of administration.
  • examples of anticancer therapeutics include chemotherapeutic agents, targeted anticancer therapeutics, and immunotherapeutic agents.
  • the immunotherapeutic agent is an immune checkpoint inhibitor, an immune checkpoint protein activity inhibitor, an immune checkpoint protein expression inhibitor, an immune cell therapy (eg, a tumor infiltrating lymphocyte, a T cell receptor or a chimeric antigen receptor cell therapy, etc.) and an anticancer agent. It may be at least one selected from vaccines.
  • an immunotherapeutic agent may be an immune checkpoint inhibitor.
  • the term “immune checkpoint inhibitor” refers to MHC class presentation, T cell presentation and/or differentiation, B cell presentation and/or differentiation, and cytokine, chemokine or immune cell proliferation and/or differentiation. It refers to any substance that prevents suppression of any mechanism in the immune system, such as signal transduction for cancer, and can treat cancer by suppressing immune evasion of cancer by blocking immune checkpoints that prevent the progression of immune responses in cancer with high immunosuppression. .
  • non-limiting examples of immune checkpoint inhibitors include anti-CTLA4 antibodies or antigen-binding fragments thereof; anti-PD-L1 antibody or antigen-binding fragment thereof; and one or more immune checkpoint inhibitors selected from the group consisting of anti-PD-1 antibodies or antigen-binding fragments thereof.
  • non-limiting examples of immune checkpoint inhibitors in the present invention include anti-PD-1 antibodies or antigen-binding fragments thereof.
  • Non-limiting examples of immune checkpoint inhibitors in the present invention include pembrolizumab, nivolumab, semiflamab, atezolizumab, avelumab, durvalumab, and/or ipilimumab and antigen-binding fragments thereof. there is.
  • composition of the present invention may exhibit a synergistic or additive effect on cancer alleviation, prevention, or treatment by being administered simultaneously with an immune checkpoint inhibitor or sequentially administered in combination with an immune checkpoint inhibitor.
  • synergistic or additive effects on tumor size reduction can be achieved by administering the composition of the present invention in combination with the aforementioned immune checkpoint inhibitor.
  • the cancer is lung cancer, non-small cell lung cancer, gastric cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin cancer (eg, melanoma), uterine cancer, ovarian cancer, colon cancer, colorectal cancer, breast cancer, uterine sarcoma , fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, esophageal cancer, laryngeal cancer, small intestine cancer, thyroid cancer, parathyroid cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, multiple myeloma, chronic or acute leukemia , may include at least one selected from the group consisting of childhood solid tumor, lymphoma, bladder cancer, renal cancer, renal cell carcinoma, renal pelvic carcinoma, axial contraction tumor, brain stem glioma, Merkel cell tumor, urinary tract tumor, and pituitary adenoma. .
  • cancer may include at least one selected from the group consisting of non-small cell lung cancer, melanoma, colorectal cancer, colorectal cancer, renal cancer, and liver cancer.
  • the cancer alleviating, preventive and therapeutic effects of the extracts of the strains were confirmed. Specifically, after culturing Erysipella toclostridium lamosum KBL1038 strain, Erysipella toclostridium lamosum KBL1039 strain, Clostridium syndens KBL987 strain and Clostridium syndens KBL1037 strain, their extracts were black By treating species-induced mice, the tumor volume increase inhibitory effect was confirmed.
  • the cancer alleviating, preventive and therapeutic effects of the extracts of the strains were confirmed. Specifically, after culturing Erysipella toclostridium lamosum KBL1038 strain, Erysipella toclostridium lamosum KBL1039 strain, Clostridium syndens KBL987 strain and Clostridium syndens KBL1037 strain, their extracts were black By treating species-induced mice, the tumor volume increase inhibitory effect was confirmed.
  • the strains were first cultured. Each of the strains was cultured in YBHI medium supplemented with 0.5% cysteine, activated through a total of two subcultures at 9 and 12 hour intervals, and then used in the experiment.
  • the above strains cultured in 1.8 liter YBHI medium were centrifuged at 6000 g, 20 minutes, 4 ° C, the culture medium was removed, and the pellet was reproduced with 40 mL of phosphate-buffered saline (PBS). Resuspension was made. After washing with PBS to completely remove the culture medium, it was centrifuged under the same conditions. After centrifugation, the supernatant was discarded, and the remaining pellet was again dissolved in 20 mL of PBS, and then ultrasonicated using a sonicator for 60 minutes or treated three times for 30 seconds using an ultra-high speed blender.
  • PBS phosphate-buffered saline
  • mice For tumor induction, 5-week-old C57BL/6 male mice were used. Mice introduced into the animal testing facility were subjected to a one-week acclimatization and stabilization period, and their weights were measured to match the mean and standard deviation of the weights among the animals.
  • each mouse was subcutaneously injected with a B16F10 suspension of melanoma cells at 1x10 6 cells/100 ⁇ l to induce cancer.
  • 6 animals per group were randomized, 3 animals per cage, and the extracts prepared from the 7th day after induction (Erysipherato Clostridium lamosum KBL1038, KBL1039 strains and Clostridium syndens KBL987 , KBL1037 strain extract) was intraperitoneally administered daily at 5 ⁇ g/100 ⁇ l/mouse.
  • mice administered with the Clostridium syndens KBL987 strain extract and the Erysiphelatoclostridium ramosum KBL1038 strain extract of the present invention were mice administered with PBS or Roseburia intranasal strain extract and S.
  • the size of the tumor was significantly reduced compared to the mice administered with Kerichia coli strain extract (FIG. 2).
  • mice administered with the Clostridium syndens KBL1037 strain extract and the Erysiphelatoclostridium ramosum KBL1038 strain extract of the present invention mice administered with PBS, Roseburia internalis, Escherichia coli And the size of the tumor was reduced compared to the mice administered with Clostridium clostrdioform extract (FIG. 3).
  • Clostridium Sindens KBL987 strain extract, Clostridium Sindens KBL1037 strain extract, Erysipelato Clostridium ramosum KBL1038 strain extract and Erysipelato Clostridium ramosum KBL1039 strain extract of the present invention were administered , The size of tumors was significantly reduced compared to mice administered with PBS, and the size of tumors was reduced compared to mice administered with extracts of Roseburia intranas and Lactobacillus luminis strains (FIG. 4).
  • Clostridium Sindens KBL987 strain extract Clostridium Sindens KBL1037 strain extract, Erysiphera toclostridium ramosum KBL1038 strain extract and Erysipellato of the present invention
  • the Clostridium ramosum KBL1039 strain extract has the effect of significantly suppressing the increase in tumor size compared to PBS or other strain extracts.
  • tumors derived from B16F10 cells are less sensitive to anticancer drugs than tumors derived from other tumor cells, the tumor size inhibitory effect observed after administration of the strain extract of the present invention was more unpredictable. From this, it was confirmed that the significantly superior cancer alleviation, prevention or treatment effect of the present invention was obtained.
  • each C57BL/6 mouse was subcutaneously injected with a B16F10 suspension of melanoma cells at 5x10 5 cells/100 ⁇ l to induce cancer.
  • 7 animals per group were randomly assigned to 3 or 4 animals per cage, and from the 7th day after induction, the pasteurized bacteria of the Erysiphelatoclostridium ramosum KBL1038 strain were 1x10 9 cells/200 ⁇ l/mouse was intraperitoneally administered once every 3 days for a total of 5 times.
  • mice administered with the Erysipelato Clostridium lamosum KBL1038 strain of the present invention were mice administered with PBS, or Enterococcus faecalis SNUV414 strain, Clostridium syndens SNUG40297 and Baylonella Parbula strain administration. Compared to mice, the tumor size was significantly reduced (FIG. 6).
  • the KBL1038 strain was intraperitoneally administered once every 3 days for a total of 4 times in the same way, and then calipers twice or 3 times a week from the 6th day to 20-21 after induction
  • the volume change of the tumor was measured using and shown in FIG. 7 , and then immune cells were analyzed in the tumor tissue using a flow cytometer, and the results were shown in FIG. 7 .
  • mice administered with the strain of Erysiphelatoclostridium lamosum KBL1038 of the present invention had a significantly reduced tumor size compared to mice administered with PBS (upper part of FIG. 7).
  • CD8 T cells secreting the inflammatory factor IFN ⁇ and the cell killing factor Granzyme B increased in the tumor tissue of mice administered with the KBL1038 strain (bottom of FIG. 7).
  • the KBL1038 strain of the present invention had an effect of inhibiting tumor growth in melanoma-induced mice, and immune cells that play an important role in anti-cancer mechanisms It was confirmed that there is an effect of increasing the distribution of .
  • colon cancer cells MC38
  • MC38 colon cancer cells
  • 6 animals per group were randomized, 3 animals per cage, and from the 7th day after the induction, the prepared bacteria of the Erysiphelatoclostridium lamosum KBL1038 strain were 1x10 9 cells/ 200 ⁇ l/mouse was intraperitoneally administered once every 3 days for a total of 4 times.
  • 200 ⁇ l of PBS was administered.
  • mice administered with the strain of Erysiphelatoclostridium lamosum KBL1038 of the present invention had a significantly reduced tumor size compared to mice administered with PBS (left side of FIG. 9).
  • CD8 and CD4 T cells secreting the inflammatory factor IFN ⁇ and the cell killing factor Granzyme B were increased in the tumor tissues of mice administered with the KBL1038 strain (right side of FIG. 9).
  • cancer was induced by subcutaneous injection of 5x10 5 cells/100 ⁇ l of B16F10 suspension, which is a melanoma cell, into each C57BL/6 mouse, and antibiotics (5 g/L Streptomycin, 1 g/L Colistin sulfate, 1 g/L Ampicillin ) was supplied for 0, 7 or 14 days, after which the KBL1038 strain was supplied at various doses (5x10 8 cells/200 ⁇ l/mouse, 1x10 9 cells/200 ⁇ l/mouse, 5x10 9 cells/200 ⁇ l/mouse). It was orally administered for 7 days. For the negative control group, 200 ⁇ l of PBS was administered.
  • the volume change of the tumor was measured using a caliper 2 or 3 times and shown in FIG. 11 .
  • the tumor size was reduced compared to the PBS-administered mice, especially at the doses of 1x10 9 cells/200 ⁇ l/mouse and 5x10 9 cells/200 ⁇ l/mouse. showed a significant reduction in In addition, when the KBL1038 strain was administered at a dose of 5x10 9 cells/200 ⁇ l/mouse in mice after administration of antibiotics, the tumor size was significantly reduced (FIG. 11).
  • mice orally administered with the KBL1038 strain at a concentration of 5x10 9 cells / 200 ⁇ l / mouse the size of the tumor is significantly reduced compared to mice administered with PBS, which is a negative control, regardless of the antibiotic supply period.
  • the KBL1038 strain of the present invention shows excellent tumor suppression effect even when administered by oral administration as well as intraperitoneal administration, and tumor suppression of the orally administered KBL1038 strain even when antibiotics are administered It was confirmed that the effect appeared.
  • each C57BL/6 mouse was subcutaneously injected with a B16F10 suspension of melanoma cells at 5x10 5 cells/100 ⁇ l to induce cancer.
  • 6 animals per group were randomized, 3 animals per cage, and the dead cells of the Erysipella toclostridium lamosum KBL1038 strain prepared from the 10th day after the induction (1x10 9 cells / 200 ⁇ l / mouse) ) and immune checkpoint inhibitor anti-PD-1 antibody (BioxCell, Cat# BE0146, 150 ⁇ g/mouse/time) were intraperitoneally administered once every 3 days for a total of 3 times.
  • 200 ⁇ l of PBS was administered (FIG. 12).
  • mice administered with the Erysiphelatoclostridium lamosum KBL1038 strain alone and in combination with the anti-PD-1 antibody of the present invention mice administered with PBS or anti-PD-1 antibody The size of the tumor was significantly reduced compared to the mice administered with only the mouse alone (FIG. 13).
  • the KBL1038 strain of the present invention not only has a superior tumor suppression effect compared to the anti-PD-1 antibody, which is an existing anti-cancer drug, but also uses the KBL1038 strain in combination with the anti-PD-1 antibody. Even when administered, it was confirmed that the tumor inhibitory effect was very excellent.
  • colon cancer cell MC38 was subcutaneously injected into each C57BL/6 mouse at 1x10 5 cells/100 ⁇ l to induce cancer.
  • 6 animals per group were randomized, 3 animals per cage, and the dead cells of the Erysiphelatoclostridium lamosum KBL1038 strain prepared from the 10th day after the induction (1x10 9 cells / 200 ⁇ l / mouse) ) and immune checkpoint inhibitor anti-PD-1 antibody (BioxCell, Cat# BE0146, 150 ⁇ g/mouse/time) were intraperitoneally administered once every 3 days for a total of 3 times.
  • IgG was administered.
  • the tumor size was significantly reduced compared to the mice administered with IgG (Fig. left of 14).
  • CD4 T cells expressing the immunosuppressive factors FoxP3 and PD-1 decreased, and CD8 T cells secreting the cell killing factor Granzyme B increased in the tumor tissue of mice administered with the combination. (Right in Fig. 14).
  • melanoma cells B16F10 and colon cancer cells MC38 were subcutaneously injected at 2x10 5 cells/100 ⁇ l into each athymic nude mouse to induce melanoma and colon cancer, respectively.
  • 7 animals per group were randomized, and dead cells (1x10 9 cells/200 ⁇ l/mouse) of the Erysiphelatoclostridium lamosum KBL1038 strain prepared from day 7 after induction were added for 3 days. was administered intraperitoneally once for a total of 4 times.
  • 200 ⁇ l of PBS was administered for the negative control group.
  • the anticancer effect of the KBL1038 strain of the present invention does not appear, and some of the mechanisms that allow the anticancer effect of the KBL1038 strain to appear are tumor mediated by T cells. It was found to be suppressed.
  • colorectal cancer cell lines HCT116 and DLD-1 were treated and then the survival rate of cancer cells was measured.
  • a colon cancer cell line (HCT116, DLD-1) was cultured in a 96-well culture plate (HCT116: 1x10 4 cells/well, DLD-1: 2x10 3 cells/well), and Erysipella toclostridium lamosum Killed cells of strain KBL1038 (killed by pasteurization, 70 °C, 30 minutes) are treated at a ratio of 1:10 1 , 1:10 2 , 1:10 3 , 1:10 4 to the number of cancer cells, and then treated for 24 to 72 hours reacted After 72 hours of strain treatment, cell viability was measured by dissolving formazan reduced by reductase in mitochondria in DMSO using an MTT assay kit (Promega, #G4000) and measuring absorbance at 570 nm.
  • the survival rate of the colorectal cancer cell line was significantly decreased in a concentration-dependent manner after 72 hours of treatment with the KBL1038 strain (FIG. 17).
  • HCT116 is a human-derived colorectal cancer cell line having a KRAS mutation. After treatment with KBL1038 strain, it was confirmed whether the non-establishment growth ability of HCT116 was reduced.
  • the colorectal cancer cell line HCT116 was cultured in a 6-well culture plate (HCT116: 5x10 3 cells/well, bottom agar: 0.6%, top agar: 0.3%), and the dead cells of the Erysipella toclostridium lamosum KBL1038 strain (pasteurization, 70 °C, dead cells by way of 30 minutes) was treated at a ratio of 1:10 2 , 1:10 3 to the number of cancer cells and cultured for 3 to 4 weeks. Then, in order to confirm the non-fixation formation reaction of the colorectal cancer cell line, it was observed through staining (0.005% Crystal violet in 10% NBF staining). After 3 to 4 weeks of treatment with the strain, the number of clusters, the size of the clusters, and the number of clusters of a certain size or larger were measured to analyze non-settled growth ability.
  • DLD-1 is a human p53-defective colorectal cancer cell line.
  • the colon cancer cell line DLD-1 was cultured in a 6-well culture plate (DLD-1: 1x10 4 cells/well, bottom agar: 0.6%, top agar: 0.3%), and Erysipella toclostridium lamosum Killed cells of the KBL1038 strain (killed by pasteurization, 70 ° C., 30 minutes) were treated at a ratio of 1:10 2 and 1:10 3 to the number of cancer cells and cultured for 3 to 4 weeks. Then, in order to confirm the non-fixation formation reaction of the colorectal cancer cell line, it was observed through staining (0.005% Crystal violet in 10% NBF staining). After 3 to 4 weeks of treatment with the strain, the number of clusters, the size of the clusters, and the number of clusters of a certain size or larger were measured to analyze non-settled growth ability.
  • the KBL1038 strain of the present invention showed an inhibitory effect on non-settled formation ability not only in HCT116 but also in the DLD-1 colorectal cancer cell line.
  • Example 8 Confirmation of non-establishment formation inhibitory effect of lung cancer and pancreatic cancer cell lines of Erysipelato Clostridium ramosum KBL1038 strain
  • the lung cancer cell line A549 was cultured in a 6-well culture plate (A549: 1x10 4 cells/well, bottom agar: 0.6%, top agar: 0.3%), and dead cells of the Erysipella toclostridium lamosum KBL1038 strain ( pasteurization, 70 °C, 30 minutes) were treated at a ratio of 1:10 2 , 1:10 3 to the number of cancer cells and cultured for 3 to 4 weeks. Then, in order to confirm the non-fixation formation reaction of the colorectal cancer cell line, it was observed through staining (0.005% Crystal violet in 10% NBF staining). After 3 to 4 weeks of treatment with the strain, the number of clusters, the size of the clusters, and the number of clusters of a certain size or larger were measured to analyze non-settled growth ability.
  • the KBL1038 strain of the present invention has an effect of inhibiting the ability of lung cancer cell lines to form non-settled cells.
  • pancreatic cancer cell line PANC1 pancreatic cancer cell line
  • the pancreatic cancer cell line PANC1 was cultured in a 6-well culture plate (PANC1: 5x10 3 cells/well, bottom agar: 0.6%, top agar: 0.3%), and dead cells of the Erysipella toclostridium lamosum KBL1038 strain ( pasteurization, 70 °C, 30 minutes) were treated at a ratio of 1:10 2 , 1:10 3 to the number of cancer cells and cultured for 3 to 4 weeks. Then, in order to confirm the non-fixation formation reaction of the colorectal cancer cell line, it was observed through staining (0.005% Crystal violet in 10% NBF staining). After 3 to 4 weeks of treatment with the strain, the number of clusters, the size of the clusters, and the number of clusters of a certain size or larger were measured to analyze non-settled growth ability.
  • the KBL1038 strain of the present invention exhibits an effect of inhibiting non-settled formation to some extent even in pancreatic cancer cell lines.

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Abstract

La présente invention concerne une souche d'Erysipelatoclostridium ramosum et/ou une souche de Clostridium scindens, et leur utilisation pour prévenir, traiter ou soulager le cancer.
PCT/KR2022/016092 2021-10-20 2022-10-20 Nouvelle souche bactérienne possédant une activité anticancéreuse et composition permettant de soulager, de prévenir ou de traiter le cancer l'utilisant WO2023068857A1 (fr)

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PCT/KR2022/016092 WO2023068857A1 (fr) 2021-10-20 2022-10-20 Nouvelle souche bactérienne possédant une activité anticancéreuse et composition permettant de soulager, de prévenir ou de traiter le cancer l'utilisant

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