WO2024063545A1 - Composition comprenant une souche de lactobacillus plantarum pour améliorer la composition de métabolite intestinal - Google Patents

Composition comprenant une souche de lactobacillus plantarum pour améliorer la composition de métabolite intestinal Download PDF

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WO2024063545A1
WO2024063545A1 PCT/KR2023/014313 KR2023014313W WO2024063545A1 WO 2024063545 A1 WO2024063545 A1 WO 2024063545A1 KR 2023014313 W KR2023014313 W KR 2023014313W WO 2024063545 A1 WO2024063545 A1 WO 2024063545A1
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strain
cancer
composition
culture
lysate
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Korean (ko)
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장명호
양보기
김아람
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주식회사 지아이바이옴
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Priority claimed from KR1020230078227A external-priority patent/KR20240040601A/ko
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Priority claimed from KR1020230125612A external-priority patent/KR20240040656A/ko
Publication of WO2024063545A1 publication Critical patent/WO2024063545A1/fr

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    • 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
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/132—Amines having two or more amino groups, e.g. spermidine, putrescine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/194—Carboxylic acids, e.g. valproic acid having two or more carboxyl groups, e.g. succinic, maleic or phthalic acid
    • 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
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • It relates to a composition for improving the composition of intestinal metabolites containing Lactobacillus plantarum strains.
  • Microbiome refers to microorganisms and their entire genetic information existing in a specific environment. In addition to the human body, microbiome information is being used in various fields such as animals, agriculture, the ocean, and the environment. In particular, with the development of human microbiome research based on the development of genetic information analysis and data analysis technology, based on this, microbiome information is being used. The growth of the diagnostic and healthcare industries is expected.
  • Human intestinal microorganisms not only decompose various substances that human enzymes cannot decompose and convert them into nutrients that human cells can absorb, but also play a role in preventing pathogen infection by inhibiting the growth of harmful bacteria originating from outside. These intestinal bacteria themselves or various metabolites secreted by the bacteria play a role in activating or regulating the body's immune response by stimulating numerous immune cells present in intestinal cells.
  • the human microbiome is considered the second genome of humans, and its importance is recognized.
  • polyamines are known to play a role in the proliferation, differentiation, and development of eukaryotes.
  • Polyamines include spermine, spermidine, and the diamine precursor putrescine and are low molecular weight organic polycations with two or more amino groups.
  • the intracellular concentration of polyamines can be maintained within a certain physiological range through several regulatory mechanisms in normal cells.
  • polyamine metabolism is known to be dysregulated in many neoplastic conditions, including cancer. Polyamine levels are elevated in various types of cancer and a link between polyamine metabolism and oncogenic pathways such as mTOR and RAS pathways is known. Therefore, polyamines may have potential as therapeutic targets in the prevention and treatment of cancer.
  • One aspect is to provide a composition for improving the intestinal metabolite composition of an individual comprising a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof as an active ingredient.
  • Another aspect is to provide a health functional food for improving the intestinal metabolite composition of an individual containing a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof as an active ingredient. will be.
  • Another aspect is to provide a probiotic composition for improving the intestinal metabolite composition of an individual comprising a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof.
  • Another aspect is to provide a food composition for improving the intestinal metabolite composition of an individual comprising a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof.
  • Another aspect provides a feed composition for improving the intestinal metabolite composition of an individual comprising a Lactobacillus plantarum strain, a culture of the strain, lysate of the strain, or a mixture thereof as an active ingredient. will be.
  • Another aspect is a pharmaceutical composition for the prevention or treatment of proliferative diseases, specifically cancer, comprising a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof as an active ingredient. is to provide.
  • Another aspect is to provide an anti-cancer adjuvant comprising a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof as an active ingredient.
  • Another aspect is a method of improving the intestinal metabolite composition of a subject comprising administering to the subject an effective amount of a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof. It is provided.
  • Another aspect is a method for preventing or treating cancer comprising administering to a subject an effective amount of a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof. It is provided.
  • Another aspect provides the use of an effective amount of a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof for the manufacture of a formulation for improving the intestinal metabolite composition of an individual. will be.
  • Another aspect is an effective amount of a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof for the production of a pharmaceutical agent or health functional food for preventing or treating cancer. It provides a purpose.
  • One aspect is a composition for improving the intestinal metabolite composition of an individual comprising a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof as an active ingredient (e.g., pharmaceutical composition) is provided.
  • Another aspect provides a method of improving the intestinal metabolite composition of a subject, comprising administering to the subject an effective amount of a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof. do.
  • Another aspect provides the use of an effective amount of a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof for the manufacture of a formulation for improving the intestinal metabolite composition of an individual. .
  • Lactobacillus is a genus of aerobic or facultative anaerobic Gram-positive bacilli that is widely distributed in nature. Microorganisms belonging to the Lactobacillus genus include Lactobacillus plantarum and Sakei. As a result of research to develop a new strain with excellent anticancer effect, the present inventors selected Lactobacillus plantarum GB104 as an anticancer candidate strain. The strain was deposited at the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center under the deposit number KCTC14107BP on January 14, 2020. The strain corresponds to a probiotic strain, is harmless to the human body, and can be used without side effects.
  • Lactobacillus ( Lactobacillus ) has been renamed to Limosilactobacillus or Lactiplantibacillus , and the changed strain names in this specification can be used interchangeably.
  • Lactobacillus plantrum was changed to Lactiplantibacillus plantrum .
  • Lactobacillus plantarum GB104 may be used together with L. Plantarum GB104 strain or Lactobacillus plantarum GB104 strain (Accession Number: KCTC14107BP).
  • the strain may be a strain deposited under deposit number KCTC14107BP.
  • the strain may be a strain containing a 16S rRNA gene consisting of the nucleotide sequence of SEQ ID NO: 1.
  • the strain may be a strain having 16S rRNA comprising the nucleotide sequence of SEQ ID NO: 1 or a 16s rRNA comprising a nucleotide sequence having 97% or more nucleotide sequence identity thereto. Specifically, it has at least 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9% or 100% homology with the nucleotide sequence consisting of SEQ ID NO: 1 of the present specification.
  • the strain may be live cells, dead cells, or a cytoplasmic fraction obtained by disrupting the strain, and preferably may be live cells.
  • the term “culture” may be used interchangeably with “culture supernatant,” “culture supernatant,” “conditioned culture,” or “conditioned medium,” and can be used interchangeably with Lactobacillus strains to grow and survive in vitro. It may refer to the entire medium containing the strain, its metabolites, extra nutrients, etc. obtained by culturing the strain in a medium capable of supplying nutrients for a certain period of time.
  • the culture refers to a product obtained by culturing a probiotic strain in a known medium, and the product may or may not include the strain itself.
  • the medium may be selected from known liquid media or solid media, for example, MRS liquid medium, GAM liquid medium, MRS agar medium, GAM agar medium, and BL agar medium, but is not limited thereto.
  • lysate may be used interchangeably with “lysate”, meaning a solution or suspension of cells of a microorganism such as Lactobacillus plantarum in an aqueous medium o broken down.
  • Cell lysates include, for example, macromolecules such as DNA, RNA, proteins, peptides, carbohydrates, lipids, etc. and/or micromolecules such as amino acids, sugars, fatty acids, etc., or fractions thereof.
  • the lysate also contains cell debris, which may be smooth or granular in structure.
  • the culture medium may include the culture medium itself, its concentrate, or freeze-dried product obtained by cultivating the strain, or the culture supernatant obtained by removing the strain from the culture medium, its concentrate, or freeze-dried product.
  • the culture medium may be obtained by culturing Lactobacillus plantarum in an appropriate medium (e.g., MRS plate medium) at a temperature of more than 10°C or less than 40°C for a certain period of time, for example, 4 to 50 hours. .
  • an appropriate medium e.g., MRS plate medium
  • the strain, the culture of the strain, or the lysate of the strain may contain acetylated spermidine.
  • the strain, a culture of the strain, or lysate of the strain may contain a polyamine acetylation enzyme. More specifically, it may include an enzyme involved in acetylation of polyamines including spermidine.
  • the acetylated spermidine may be N 1 -acetylspermidine, N 8 -acetylspermidine, or N 1 ,N 8 -diacetylspermidine.
  • the strain, the culture of the strain, or the lysate of the strain may contain glutaric acid or glutaconic acid.
  • the glutaric acid may be 2-oxoglutarate or 2-hydroxyglutarate. Additionally, in one embodiment, the glutaconic acid may be trans-glutarconic acid.
  • the improvement of the intestinal metabolite composition may include the following.
  • the polyamine synthase may be ornithine decarboxylase (ODC), an enzyme that synthesizes spermidine from ornithine in cells, and the polyamine decomposition enzyme may be an enzyme that synthesizes spermidine from ornithine. It may be spermidine/spermine N 1 -acetyltransferase (SSAT), an enzyme that acetylates, but this is an example and is not limited thereto.
  • ODC ornithine decarboxylase
  • SSAT spermidine/spermine N 1 -acetyltransferase
  • the activity of polyamine synthetase e.g., ornithine decarboxylase
  • polyamine decomposition occurs.
  • the activity of enzymes e.g. spermidine/spermine N 1 -acetyltransferase
  • the levels of ornithine and polyamines e.g. spermidine
  • acetylated Levels of polyamines may increase.
  • the intestinal microbial composition also called the intestinal microbiota, may refer to the complex ecosystem within the gastrointestinal tract. Specifically, it consists of the entire microbial community in the gut, including bacteria, yeast, fungi, archaea, and viruses.
  • the gut microbiota is responsible for colonic fermentation of dietary fiber, nutrient extraction, synthesis of certain vitamins, prevention of colonization by pathogens, maturation of the intestinal epithelium and immune system, release of metabolites into systemic tissues, and regulation of gastro-hormonal secretion and neurological functions. Maintains several functions. It is generally recognized that disturbance of the normal balance in the intestinal microbial composition can compromise intestinal barrier integrity, which is often observed in many different diseases.
  • the Lactobacillus plantarum GB104 strain of the present invention can restore and/or maintain a health-beneficial intestinal microbial composition through regulation of the intestinal metabolite profile (specifically, polyamine metabolism) in an individual. Accordingly, the Lactobacillus plantarum strains herein may also further provide compositions for restoring and/or maintaining a health-beneficial intestinal microbial composition (or for preventing or treating disorders associated with impaired intestinal integrity). there is.
  • Polyamine metabolism may include polyamine biosynthesis, catabolism, and transport.
  • Natural polyamines can be synthesized in the cytoplasm of all cells. This biosynthesis begins with L-methionine and L-ornithine, which are amino acids of the urea cycle, and putsrescine is formed through ornithine decarboxylation by ornithine decarboxylase (ODC).
  • Putrescine is a polyamine precursor in mammalian cells that produces spermine and spermidine when the aminopropyl group is added by decarboxylated S-adenosylmethionine (dcSAM). This dcSAM is known to be produced by S-adenosylmethionine decarboxylase 1 (SAMDC or adenosylmethionine decarboxylase 1, AMD1).
  • polyamine transport in metabolism has led to the concept that polyamines are transported into cells through specific polyamine transport systems (PTS).
  • PTS polyamine transport systems
  • Polyamine uptake through PTS is upregulated in proliferating cells, including tumor cells, suggesting that PTS plays a role in regulating intracellular polyamine concentrations.
  • polyamine levels are upregulated in various cancer cell types, polyamines may be common therapeutic targets for cancer treatment.
  • Fast-growing cells, including tumor cells exhibit higher activity of several enzymes involved in polyamine biosynthesis.
  • Polyamine levels are elevated in cancer patients and may be correlated with cancer development. High polyamine levels are known to be associated with the progression of neuroblastoma, hepatocellular carcinoma (HCC), prostate cancer, lung cancer, breast cancer, stomach cancer, and colorectal cancer (CRC).
  • HCC hepatocellular carcinoma
  • CRC colorectal cancer
  • polyamines may play a role in establishing tumor immunity, promoting the growth of cancer cells through the excretion of sperm and their metabolites.
  • Polyamines are also known to induce acquired chemical resistance to 5-fluorouracil and paclitaxel in colorectal and breast cancer. Reduction of polyamines may prevent tumor-induced immunosuppression by enhancing spontaneous IL-2 production, NK-cell activation, and recovery of T-lymphocyte populations without affecting tumor polyamines.
  • the strain, the culture of the strain, or the lysate of the strain may promote the activity of intestinal immune cells or the expression of tight junction proteins between intestinal cells.
  • the activity of the intestinal immune cells is an increase in the number of activated CD8 + T cells in immune cells, an increase in the ratio of activated CD8 + T cells in immune cells, or an increase in INF- ⁇ secretion, more specifically activation. It may include increased secretion of cytokines or interferons from immune cells.
  • the intestinal immune cells are within the small intestine or large intestine (e.g., small intestinal intraepithelial lymphocytes (IEL), small intestinal lamina basement (siLP), colonic lamina). may include immune cells within the basement (cLP)).
  • small intestinal intraepithelial lymphocytes IEL
  • small intestinal laminalitis siLP
  • colonic lamina may include immune cells within the intestinal (cLP)).
  • the tight junction proteins include claudin-1, claudin-2, claudin-3, claudin-4, and claudin-5. It may include one or more selected from the group consisting of (claudin-5), Zonula Occludens (ZO)-1, ZO-2, ZO-3, and occludin.
  • the strain, the culture of the strain, or the lysate of the strain may be used to determine the activity of intestinal immune cells, specifically within the small intestine or large intestine (e.g., small intestine intraepithelial lymphocytes (IEL), small intestinal mucosa lamina propria).
  • IEL small intestinal epiepithelial lymphocytes
  • the Lactobacillus plantarum strain prevents, improves, treats, or improves the prognosis of cancer by reducing intestinal polyamines.
  • it is provided as a pharmaceutical composition for anti-cancer support, food (health functional food), feed composition, or anti-cancer supplement.
  • improving the intestinal metabolite composition of the subject may be for preventing or treating cancer.
  • the above cancers include stomach cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, gallbladder cancer, biliary tract cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, salivary gland cancer, and melanoma. It may be any one selected from the group consisting of cancer, bladder cancer, esophageal cancer, head and neck cancer, skin cancer, small intestine cancer, anal cancer, colon cancer, rectal cancer, kidney cancer, blood cancer, and lymphoma.
  • the colon cancer may be a malignant tumor that occurs in any area selected from the group consisting of the ascending colon, transverse colon, descending colon, sigmoid colon, and rectal mucosa.
  • cancer refers to a physiological condition in animals, typically characterized by abnormal or uncontrolled cell growth. Cancer and cancer pathology include, for example, metastasis, interference with normally functioning surrounding cells, release of cytokines or other secreted products at abnormal levels, inhibition or enhancement of inflammatory or immunological responses, neoplasia, and premalignancy. ), malignancy, or involvement of surrounding or distant tissues or organs, such as lymph node invasion.
  • the cancer may be gastrointestinal cancer or non-gastrointestinal cancer.
  • the gastrointestinal cancer is a malignant tumor that occurs in the gastrointestinal tract, such as the esophagus, stomach, small intestine, or large intestine.
  • the gastrointestinal cancer includes, for example, esophageal cancer, gallbladder cancer, liver cancer, biliary tract cancer, pancreatic cancer, stomach cancer, small intestine cancer, colon cancer, colon cancer, and anal cancer. It may be one or more cancers selected from the group consisting of rectal cancer, but is not limited thereto, and in one example, it may be colon cancer.
  • the non-gastrointestinal cancer includes, without limitation, malignant tumors occurring in organs other than the gastrointestinal tract or digestive system, for example, hematological cancer, leukemia, acute myeloid leukemia, neuroblastoma, retinoblastoma, lung cancer, head and neck cancer, salivary gland cancer, melanoma, It may be, but is not limited to, laryngeal cancer, prostate cancer, breast cancer, bladder cancer, kidney cancer, multiple myeloma, cervical cancer, thyroid cancer, ovarian cancer, urethral cancer, skin cancer, osteosarcoma, glioblastoma, brain tumor, or lymphoma.
  • malignant tumors occurring in organs other than the gastrointestinal tract or digestive system for example, hematological cancer, leukemia, acute myeloid leukemia, neuroblastoma, retinoblastoma, lung cancer, head and neck cancer, salivary gland cancer, melanoma
  • It may be, but is not limited to, laryngeal cancer
  • the cancer may be colon cancer, and the colon cancer includes malignant tumors that occur in one or more regions selected from the group consisting of ascending colon, transverse colon, descending colon, sigmoid colon, and rectal mucosa. do.
  • the colon cancer may be one or more types selected from the group consisting of adenocarcinoma, lymphoma, malignant carcinoid, leiomyosarcoma, Kaposi's sarcoma, and squamous cell carcinoma, but is not limited thereto.
  • the composition according to one embodiment may include 0.001% by weight to 80% by weight of the Lactobacillus plantarum strain based on the total weight of the composition. Additionally, the administered dose of the Lactobacillus plantarum strain may be 0.01 mg to 10,000 mg, 0.1 mg to 1000 mg, 1 mg to 100 mg, 0.01 mg to 1000 mg, 0.01 mg to 100 mg, 0.01 mg to 10 mg, or 0.01 mg to 1 mg. .
  • the strain is included in the composition in a therapeutically effective amount or nutritionally effective concentration, for example, the strain is 10 3 to 10 16 CFU/g, 10 3 to 10 15 CFU/g, 10 3 to 10 14 CFU/g.
  • 1X10 3 to 1X10 16 CFU/g of live or dead cells may be administered once or in divided doses.
  • the dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, gender, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity, and those skilled in the art will Taking these factors into consideration, the dosage can be adjusted appropriately.
  • the number of administrations can be one time or two or more times within the range of clinically acceptable side effects, and the administration site can be administered at one or two or more places.
  • the dosage per kg is the same as for humans, or, for example, the above-mentioned administration is based on the volume ratio (e.g., average value) of organs (e.g., heart, etc.) between the target animal and human.
  • the converted dose can be administered.
  • Possible routes of administration include oral, sublingual, parenteral (e.g., subcutaneous, intramuscular, intraarterial, intraperitoneal, intrathecal, or intravenous), rectal, topical (including transdermal), inhalation, and injection, or implantable device. Alternatively, it may include insertion of a substance.
  • the composition includes killed dried strains, and can be administered in an amount of 1g to 10g, 0.5g to 1.5g, 2.5g to 3.5g, or 4.5g to 5.5g, once a day to 3 times. It may be administered once.
  • the term "therapeutically effective amount” refers to an anticancer agent or method of the present invention for the method and use of the present invention that elicits a biological or medical response or desired therapeutic effect in the patient that researchers, doctors, or other clinicians wish to obtain. and the amount of a pharmaceutical composition containing an anticancer agent for use.
  • the therapeutically effective amount of an anticancer agent may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the anticancer agent to elicit a desired response in the individual.
  • a therapeutically effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or harmful effects.
  • the pharmaceutical composition according to one embodiment may include a pharmaceutically acceptable carrier and/or additive.
  • a pharmaceutically acceptable carrier and/or additive for example, sterilized water, physiological saline, common buffers (phosphoric acid, citric acid, other organic acids, etc.), stabilizers, salts, antioxidants (ascorbic acid, etc.), surfactants, suspending agents, isotonic agents, or preservatives.
  • it may also include combinations with organic materials such as biopolymers and inorganic materials such as hydroxyapatite, specifically collagen matrices, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers, and chemical derivatives thereof. You can.
  • the pharmaceutical composition may be an oral formulation.
  • the oral preparation comes in the form of tablets, pills, capsules, lozenges, granules, powders, suspensions, and sachets. , or it may be in the form of syrups.
  • the Lactobacillus bacteria may be dissolved or dispersed in a pharmaceutically acceptable carrier, or may be frozen in a dissolved or dispersed solution state. .
  • the pharmaceutical composition may be used as a suspending agent, solubilizing agent, stabilizer, isotonic agent, preservative, anti-adsorption agent, surfactant, diluent, excipient, pH adjuster, analgesic agent, etc., if necessary depending on the administration method or formulation. Buffers, reducing agents, antioxidants, etc. may be appropriately included.
  • Pharmaceutically acceptable carriers and agents suitable for the present invention including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, 19th ed., 1995.
  • the pharmaceutical composition according to one embodiment is formulated in unit dosage form using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person skilled in the art to which the invention pertains. It can be manufactured by or by placing it in a multi-capacity container.
  • the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of powder, granules, tablets or capsules.
  • the pharmaceutical composition is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat the disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, activity of the drug, and the type and severity of the patient's disease. It can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the medical field.
  • the composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.
  • Another aspect is to provide a health functional food for improving the intestinal metabolite composition of an individual containing a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof as an active ingredient. will be.
  • improving the intestinal metabolite composition of the subject may include proliferation of beneficial intestinal bacteria, suppression of harmful bacteria, improvement of intestinal health by regulating immunity, or improvement of bowel activity.
  • the strain, the culture of the strain, or the lysate of the strain may contain acetylated spermidine or polyamine acetylation enzyme.
  • the acetylated spermidine may be N 1 -acetylspermidine, N 8 -acetylspermidine, or N 1 ,N 8 -diacetylspermidine.
  • the strain, the culture of the strain, or the lysate of the strain may contain glutaric acid or glutaconic acid.
  • the glutaric acid may be 2-oxoglutarate or 2-hydroxyglutarate. Additionally, in one embodiment, the glutaconic acid may be trans-glutarconic acid.
  • improving intestinal metabolite composition may include:
  • the strain, the culture of the strain, or the lysate of the strain may promote or up-regulate the activity of intestinal immune cells or the expression of tight junction proteins between intestinal cells.
  • the activity of the intestinal immune cells is an increase in the number of activated CD8 + T cells in immune cells, an increase in the ratio of activated CD8 + T cells in immune cells, or an increase in INF- ⁇ secretion, more specifically activation. It may include increased secretion of cytokines or interferons from immune cells.
  • the intestinal immune cells are within the small intestine or large intestine (e.g., small intestinal intraepithelial lymphocytes (IEL), small intestinal lamina basement (siLP), colonic lamina). may include immune cells within the basement (cLP)).
  • small intestinal intraepithelial lymphocytes IEL
  • small intestinal laminalitis siLP
  • colonic lamina may include immune cells within the intestinal (cLP)).
  • the tight junction proteins include claudin-1, claudin-2, claudin-3, claudin-4, and claudin-5. It may include one or more selected from the group consisting of (claudin-5), Zonula Occludens (ZO)-1, ZO-2, ZO-3, and occludin.
  • the health functional food may be an oral preparation.
  • the strain, culture of the strain, lysate of the strain or a mixture thereof, route of administration, administration method, and administration dose are as described above.
  • the health functional food may further include a foodologically acceptable carrier.
  • the term “foodologically acceptable” means that the compound exhibits non-toxic properties to cells or humans exposed to the compound.
  • the term “improvement” may refer to any action that at least reduces the severity of a parameter related to the condition being treated, for example, a symptom.
  • the health functional food can be used simultaneously or separately with a drug for treatment before or after the onset of the disease in order to prevent or improve cancer.
  • the active ingredient can be added directly to the food or used together with other foods or food ingredients, and can be used appropriately according to conventional methods.
  • the mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention or improvement).
  • the health functional food may be added in an amount of about 15% by weight or less, more specifically about 10% by weight or less, based on the raw materials.
  • the amount may be below the above range.
  • the health functional food may be formulated with one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid formulations, further including one or more of carriers, diluents, excipients, and additives.
  • Foods to which compounds according to one aspect can be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gum, tea, vitamin complexes, health functional foods, etc.
  • the carriers, excipients, diluents and additives include lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose. , polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. It may be at least one selected from.
  • the health functional food may contain other ingredients as essential ingredients without any particular restrictions.
  • the health functional food may contain various flavoring agents or natural carbohydrates as additional ingredients.
  • natural carbohydrates include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol.
  • natural flavoring agents thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)
  • synthetic flavoring agents sacharin, aspartame, etc.
  • the ratio of the natural carbohydrates can be appropriately determined by the selection of a person skilled in the art.
  • health functional foods include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and salts thereof. , alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. These components can be used independently or in combination, and the proportions of these additives can also be appropriately selected by those skilled in the art.
  • the health functional food may be provided by mixing with conventionally known health functional food for preventing or improving cancer or other existing health functional food, and the health functional food for preventing or improving cancer is known to be a metabolic disease. It may be a health functional food for the prevention or improvement of, an existing health functional food, or a newly developed health functional food.
  • the health functional food contains other health functional foods that have the effect of preventing or improving cancer, it is important to mix the amount to obtain the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art. there is.
  • the food composition for preventing or improving cancer includes all forms such as functional food, nutritional supplement, health food, and food additives, and the above type of food composition It can be manufactured in various forms according to conventional methods known in the art.
  • compositions herein may be considered food supplements.
  • Food supplements also known as dietary supplements or nutritional supplements, can be considered another pharmaceutical product. It is intended to supplement the diet and provide nutrients or beneficial ingredients that may not be available or consumed in sufficient amounts in the normal diet.
  • Most food supplements are considered foods, but sometimes they are considered drugs, natural health products, or nutraceutical products.
  • food supplements include health functional foods. Food supplements are usually sold over the counter without a prescription. When food supplements take the form of pills or capsules, they contain the same excipients used in pharmaceuticals. However, food supplements may take the form of food fortified with some nutrients (e.g. infant formula). Accordingly, in certain embodiments, the compositions of the present invention are food supplements.
  • composition according to the present invention can be administered as is or mixed with a suitable edible liquid or solid or in the form of tablets, pills, capsules, lozenges, granules, powders. ), suspensions, sachets, syrups, or may be freeze-dried in the form of unit doses. It may also be in the form of monodoses of a lyophilized composition that are mixed in a separate liquid container provided prior to administration.
  • composition of the present invention may be included in various edible foods and foods such as milk products for infants.
  • the term “edible product” is broadly defined to include any product that can be ingested by an animal, in any form (e.g., a product that is absorbed by the sense organs). products that can be imported).
  • the term "food product” is understood as an edible product that provides nutritional support to the body.
  • Foods of particular interest are food supplements and infant formulas.
  • Foods preferably include oatmeal porridge, lactic acid fermented foods, resistant starch, dietary fibers, carbohydrates, proteins and glycated proteins. It includes carrier materials such as glycosylated proteins.
  • bacterial cells of the invention are homogenized with other ingredients, such as cereals or powdered milk, to form infant formula.
  • Another aspect provides a feed composition for improving the intestinal metabolite composition of an individual comprising a Lactobacillus plantarum strain, a culture of the strain, lysate of the strain, or a mixture thereof as an active ingredient. will be.
  • the strain, culture of the strain, lysate of the strain or a mixture thereof, route of administration, administration method, and administration dose are as described above.
  • the feed composition can be manufactured by adding the mixed strain composition in an appropriate effective concentration range according to various feed production methods known in the art, and can be used as a feed additive composition for the purpose of preventing or improving aging-related diseases.
  • the “feed” may mean any natural or artificial diet, meal, etc., or an ingredient of the meal, for or suitable for eating, ingestion, and digestion by an animal.
  • the type of feed is not particularly limited, and feed commonly used in the technical field can be used.
  • Non-limiting examples of the feed include plant feeds such as grains, root fruits, food processing by-products, algae, fiber, pharmaceutical by-products, oils, starches, cucurbits or grain by-products: proteins, non-lipids, Examples include animal feeds such as fats and oils, minerals, fats and oils, single-cell proteins, zooplanktons, or food.
  • an anti-cancer adjuvant comprising a Lactobacillus plantarum strain, a culture of the strain, lysate of the strain, or a mixture thereof as an active ingredient.
  • the strain, culture of the strain, lysate of the strain or a mixture thereof, route of administration, administration method, and administration dose are as described above.
  • “adjuvant” refers to an agent that assists the efficacy of the main drug, that is, an anticancer agent, to improve and/or enhance the therapeutic effect, or to prevent or alleviate the harmful effects of the main drug. .
  • an anticancer agent By improving the composition of intestinal metabolites, the Lactobacillus plantarum strain of the present invention can improve the anticancer effect of other anticancer agents without itself being burdensome to the human body.
  • Conventional treatments that can be used in combination with other anticancer agents may be selected from the group consisting of chemical anticancer agents for chemotherapy, targeted anticancer agents, antibody therapy, immunotherapy agents, and combinations thereof.
  • the term “chemical anti-cancer agent” is also referred to as an anti-tumor drug (Antineoplastic agent) or a cytotoxic agent. It is a general term for drugs that exhibit anticancer activity mainly by acting directly on DNA to block DNA replication, transcription, and translation processes, or by interfering with the synthesis of nucleic acid precursors in metabolic pathways and inhibiting cell division.
  • the chemical anticancer agent may be any one selected from the group consisting of an alkylating agent, microtubule inhibitor, antimetabolite, and topoisomerase inhibitor.
  • the anti-tumor drug acts not only on tumor cells but also on normal cells and exhibits cytotoxicity. Chemotherapy agents can be used for maintenance therapy.
  • the term “maintenance therapy” in this specification refers to treating cancer with drugs after initial anti-cancer treatment, and refers to a treatment method performed to prevent or delay the recurrence of cancer.
  • targeted anticancer agent refers to a treatment that kills cancer cells specifically by blocking signals involved in the growth and development of cancer by targeting specific proteins or specific genetic changes that occur frequently only in cancer cells. It is classified into monoclonal antibodies that react outside the cell and small molecule substances that act inside the cell. Monoclonal antibodies are anticancer drugs that block cancer cell-inducing signals transmitted outside the cell and act on initiation signals related to proliferation and death, while small molecule substances act on complex signaling that occurs inside cells.
  • the targeted proteins are epidermal growth factor receptor (EGFR), vascular growth factor receptor (VEGFR), CD20, CD38, RNAK-L, BTK, Bcr-abl, PDGFR/FGFR family, MEK/RAF, and HER2/Neu. , Ubiquitin, JAK, MAP2K, ALK, PARP, tumor growth factor ⁇ receptor (TGF ⁇ R), Proteasome, Bcl-2, C-Met, VR1, VR2, VR3, c-kit, AXL, RET, Braf, DNA It may be methyltransferase (DNMT), CDK4/6, STING, etc.
  • EGFR epidermal growth factor receptor
  • VEGFR vascular growth factor receptor
  • CD20 CD38
  • RNAK-L RNAK-L
  • BTK vascular growth factor receptor
  • Bcr-abl PDGFR/FGFR family
  • MEK/RAF MEK/RAF
  • HER2/Neu HER2/Neu.
  • Ubiquitin JAK
  • antibody therapeutic agent refers to a therapeutic agent that exhibits an anti-cancer effect using an antibody that recognizes a specific protein of cancer cells as an antigen.
  • Antibody treatments may include Cetuximab, Trastuzumab, Emtansine, Emtansine, Rituximab, Ibritumomab, Tositumomab, Brentuximab, Ofatumumab, Obinutuzumab, Necitumumab, Bevacizumab, Ramucirumab, Nivolumab, Pembrolizumab, Atezolizumab, Durvalumab, Ipilimumab, etc.
  • immune anti-cancer agent refers to a substance that inhibits the activity of immune checkpoint proteins that inhibit the differentiation, proliferation, and activity of immune cells, preventing cancer cells from exercising the function of evading the immune system. It is known to eliminate cancer cells.
  • the immuno-anticancer agents include 2B4, 4-1BB (CD137), AaR, B7-H3, B7-H4, BAFFR, BTLA, CD2, CD7, CD27, CD28, CD30, CD40, CD80, CD83 ligand, CD86, CD160, CD200, CDS, CEACAM, CTLA-4, GITR, HVEM, ICAM-1, KIR, LAG-3, LAIR1, LFA-1 (CD 11 a/CD 18), LIGHT, NKG2C, NKp80, OX40, PD-1, PD- It may be an antibody against any one selected from the group consisting of L1, PD-L2, SLAMF7, TGFRp, TIGIT, Tim3, and VISTA.
  • anti-CTLA-4 antibody anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-B7-H4 antibody, anti-HVEM antibody, anti-TIM3 antibody, anti-GAL9 antibody, anti-LAG3 antibody, It may be any one selected from the group consisting of anti-VISTA antibody, anti-KIR antibody, anti-BTLA antibody, and anti-TIGIT antibody, but is not limited thereto.
  • strain, its culture, or its lysate according to one embodiment may be administered in combination with the other anticancer drugs.
  • the term “combination therapy” or “combination administration” or “in combination” refers to any form of simultaneous or concurrent treatment using at least two separate therapeutic agents.
  • the components of the combination therapy may be administered simultaneously, sequentially, or in any order.
  • the components may be administered in any suitable manner, in different doses or at different frequencies of administration or via different routes.
  • the combined administration includes Lactobacillus Plantarum strains; And the anticancer agent or antibiotic may be administered simultaneously, or the anticancer agent or antibiotic may be administered after administering the Lactobacillus plantarum strain.
  • the combination therapy according to the present invention is an efficacy that can be obtained by administering one or the rest of the components of the combination therapy at a conventional dose, for example, the efficacy measured through the degree of response, response rate, time to disease progression, or survival time. More therapeutically superior can be defined as being able to provide synergistic effects. For example, if the therapeutic efficacy is superior to the efficacy obtained by using each of the above alone, the efficacy of the combination treatment is synergistic.
  • administered simultaneously is not particularly limited and means that the components of the combination therapy are administered substantially simultaneously, for example as a mixture or in an immediately following sequence.
  • the term “sequentially administered” is not particularly limited and means that the components of the combination therapy are not administered simultaneously, but are administered one by one or in batches with a specific time interval between administrations.
  • the time interval may be the same or different between the respective administrations of the components of the combination therapy and may be selected, for example, in the range of 2 minutes to 96 hours, 1 day to 7 days or 1 week, 2 weeks or 3 weeks.
  • the time interval between administrations can range from minutes to hours, for example from 2 minutes to 72 hours, 30 minutes to 24 hours, or 1 to 12 hours. Additional examples include time intervals ranging from 24 to 96 hours, 12 to 36 hours, 8 to 24 hours, and 6 to 12 hours.
  • the Lactobacillus plantarum strain it is possible to reduce intestinal polyamines (e.g., spermidine), thereby preventing, improving, treating, or improving prognosis of cancer, and pharmaceutical compositions and foods for anti-cancer assistance. It has the effect of being useful as a (health functional food), feed composition, and anti-cancer supplement.
  • intestinal polyamines e.g., spermidine
  • Figure 1 is a graph showing the relative difference in metabolite content in GB104 culture supernatant and MRS culture supernatant (control) as metabolic pathway through CE-TOF-MS.
  • Figure 2 is a table showing the change in trans-Glutaconic content among metabolites in GB104 culture supernatant, L. Plantarum F0077 strain, and MRS culture supernatant (control) as fold change based on the total strain.
  • Figure 3 is a table showing the change in content of spermidine and three types of acetylated spermidines among metabolites in GB104 culture supernatant and MRS culture supernatant (control) as fold change based on the overall average.
  • Figure 4 is a graph showing the relative difference in content of spermidine and three types of acetylated spermidine among metabolites in GB104 culture supernatant and MRS culture supernatant (control).
  • Figure 5 is a graph showing the relative changes in spermidine and acetylspermidine content after treatment of GB104 culture supernatant and MRS culture supernatant (control group) in colon cancer cell line HCT116.
  • Figure 6 is a graph showing changes in expression of polyamine synthase ODC and polyamine decomposition enzyme SSAT after treatment of colon cancer cell lines HCT116 and HT-29 with GB104 culture supernatant and MRS culture supernatant (control group);
  • ODC Ornithine decarboxylase
  • SSAT Spermidine/spermine N 1 -acetyltransferase.
  • Figure 7 is a graph showing the relative change in spermidine content in feces after treatment of GB104 and PBS (control group) in mice transplanted with colon cancer cells MC38, respectively.
  • Figure 8 is a graph showing changes in N 1 -acetylspermidine/spermidine and ornithine content in tumors after treating mice transplanted with MC38 colon cancer cells with GB104 and PBS (control group), respectively.
  • Figure 9 is a graph showing the change in spermidine content in feces after antibiotic-treated mice were treated with GB104 and PBS (control group), respectively.
  • Figure 10 is a graph showing the change in the number of immune cells in the intestine after antibiotic-treated mice were treated with GB104 and PBS (control group), respectively.
  • Figure 11 is a graph showing changes in tight junction-related gene expression in intestinal tissue after antibiotic-treated mice were treated with GB104 and PBS (control group), respectively; ZO: Zonula Occludens.
  • Figure 12 is a graph showing the survival rate of cells after treating the colon cancer cell line HCT116 with culture supernatants of GB104 and other strains, respectively.
  • Figure 13 is a graph showing the cell cycle after treating the colon cancer cell line HCT116 with culture supernatants of GB104 and the comparison strain (WCFS1), respectively.
  • Figure 14 is a graph showing cell death after treating the colon cancer cell line HCT116 with culture supernatants of GB104 and the comparison strain (WCFS1), respectively.
  • Lactobacillus Plantarum GB104 was isolated from a vaginal sample of a healthy woman who visited the hospital for health checkup. First, vaginal samples were collected with a swab, inoculated into Rogosa SL (MRS) plate medium, and cultured in an anaerobic chamber at 37°C for 48 hours. When bacterial colonies grew, single colonies were subcultured onto new MRS plate medium for pure isolation. After pure isolation, the strain was cultured using MRS medium. Next, among the cultured strains, Lactobacillus plantarum has an inhibitory effect on fat cell accumulation and has low cytotoxicity. Strain GB104 was finally selected.
  • MRS Rogosa SL
  • the 16S rRNA gene sequence obtained through PCR using primers targeting the 16S rRNA gene was analyzed by Sanger sequencing method, and Lactobacillus Plantarum
  • the 16S rRNA sequence of GB104 is shown as SEQ ID NO: 1.
  • the present inventors named the GB104 strain as “ Lactobacillus plantarum GB104” (Accession number: KCTC 14107BP) and transferred it to the Korean collection for type cultures (KCTC) at the Korea Research Institute of Bioscience and Biotechnology in 2020. It was deposited on the 14th of February.
  • Lactobacillus plantarum was changed to Lactiplantibacillus plantarum .
  • the changed strain names of existing strains are described interchangeably.
  • Metabolome analysis of MRS culture supernatant (control) and GB104 culture supernatant was conducted by requesting Human Metabolome Technologies (HMT). Milli-Q water containing internal standards was added to the MRS culture supernatant and GB104 culture supernatant samples, and CE-TOF-MS analysis was performed. Cation and anion metabolite analysis was performed using the Agilent CE-TOF-MS system (Agilent Technologies Inc.), and the results are shown in Figure 1. Separation of metabolites was achieved through fused silica capillary. Putative metabolites analyzed in HMT were identified through the HMT internal library.
  • Figure 1 is a graph showing the relative difference in metabolite content in GB104 culture supernatant and MRS culture supernatant (control) as metabolic pathway through CE-TOF-MS.
  • Figure 2 is a table showing the change in trans-Glutaconic content among metabolites in GB104 culture supernatant, L. Plantarum F0077 strain, and MRS culture supernatant (control) as fold change based on the total strain.
  • Figure 3 is a table showing the change in content of spermidine and three types of acetylated spermidine among metabolites in GB104 culture supernatant and MRS culture supernatant (control) as fold change based on the overall average.
  • Figure 4 is a graph showing the relative difference in content of spermidine and three types of acetylated spermidine among metabolites in GB104 culture supernatant and MRS culture supernatant (control).
  • a total of 296 metabolites were analyzed, including both substances identified through the HMT internal library and unknown substances.
  • Metabolites produced and destroyed by GB104 were further analyzed using the OPLS-DA model, which is used to confirm differences between the two groups, and 87 metabolites were selected out of a total of 296 metabolites.
  • various metabolites such as amino acids, peptides, nucleosides, nucleotides, organic acids, and saccharides were included. What was particularly noteworthy was the change in polyamines, which are known to be involved in the proliferation of cancer cells.
  • GB104 acetylated spermidine, a polyamine component, thereby reducing spermidine but increasing acetylated spermidine.
  • Human colon cancer cell line (HCT116) was distributed into each well of a 6-well plate at 2X10 5 cells and cultured for 24 hours, then treated with the culture supernatant of L. Plantarum GB104 strain at a concentration of 10% and incubated at 37°C with 5% concentration. Cultured for 24 hours under CO 2 conditions. The culture supernatant was obtained by culturing the L. Plantarum strain in MRS medium for 8, 16, and 24 hours, respectively, and then centrifuging the strain to precipitate the supernatant, which was then filtered through a 0.22 ⁇ m filter to obtain supernatant for each culture time.
  • Polyamines are synthesized from ornithine in cells by ODC (Ornithine decarboxylase) and decomposed by acetylation by SSAT (Spermidine/spermine N 1 -acetyltransferase). It has been reported that increased SSAT expression in cancer cells inhibits the proliferation of cancer cells, and high SSAT expression in cancer tissues leads to a good prognosis for anticancer treatment.
  • Figure 5 is a graph showing the relative changes in spermidine and acetylspermidine content after treatment of GB104 culture supernatant and MRS culture supernatant (control group) in colon cancer cell line HCT116.
  • Figure 6 is a graph showing changes in expression of polyamine synthase ODC and polyamine decomposition enzyme SSAT after treatment of colon cancer cell lines HCT116 and HT-29 with GB104 culture supernatant and MRS culture supernatant (control group);
  • ODC Ornithine decarboxylase
  • SSAT Spermidine/spermine N 1 -acetyltransferase.
  • Plantarum GB104 strain Changes in spermidine content in feces and tumors due to administration of L. Plantarum GB104 strain were confirmed in the colon carcinoma MC-38 allograft model. A tumor model was established by subcutaneously injecting 100 ⁇ L of 2 On the 5th day of tumor cell injection, only mice with tumor sizes within the range of 20-40 mm 3 were selected and each group was randomly set, and then L. Plantarum GB104 strain was administered to the animal model at 1x10 9 CFU per mouse. It was administered orally every day from the 6th day until just before the end of the test.
  • feces were obtained from mice in all groups (0 day samples), and on day 12, feces were again obtained from mice in all groups (7 day samples).
  • Tumor samples were obtained from mice at the end of the test, on day 20 of tumor cell injection. Extraction and derivatization of polyamines from mouse fecal and tumor samples were performed and HPLC-DAD analysis was performed. As a result, polyamines were detected in fecal and tumor samples, and it was confirmed that the change in polyamine content was caused by GB104.
  • Changes in spermidine content in feces were expressed as a fold by dividing the area of the spermidine peak in the fecal sample after oral administration of GB104 for 7 days by the area of the spermidine peak in the fecal sample collected immediately before oral administration of GB104, and this value was expressed as a fold A comparison by star is shown in Figure 7.
  • the change in acetylspermidine content in the tumor was expressed by dividing the area of the N 1 -acetylspermidine peak by the area of the spermidine peak, and the change in ornithine content was shown in Figure 8 as the ornithine peak area value.
  • Figure 7 is a graph showing the relative change in spermidine content in feces after treatment of GB104 and PBS (control group) in mice transplanted with colon cancer cells MC38, respectively.
  • Figure 8 is a graph showing changes in N 1 -acetylspermidine/spermidine and ornithine content in tumors after treating mice transplanted with MC38 colon cancer cells with GB104 and PBS (control group), respectively.
  • GB104 reduced spermidine content in a dose-dependent manner, and in particular, the change in spermidine content in the group administered GB104 (treatment dose 1 x 109 CFU/200 ⁇ L/head) compared to the control group. decreased significantly.
  • the proportion of acetylspermidine was high in the tumors of mice treated with GB104, which means that GB104 not only weakens the proliferation ability of cancer cells by acetylating polyamines, but also facilitates the decomposition of polyamines.
  • ornithine a substrate of polyamine synthetase ODC, increased in the tumors of mice treated with GB104, showing that GB104 inhibited the activity of polyamine synthetase.
  • mice were provided with sterilized regular drinking water instead of drinking water containing antibiotics until the end of the test.
  • Freeze-dried GB104 was suspended in D-PBS to adjust the dose to 1 After the antibiotic treatment was terminated, feces were obtained from mice in all groups immediately before GB104 administration (0 day samples), and feces were again obtained from mice in all groups on the 7th day of oral administration of GB104 (7 day samples). Extraction and derivatization of polyamines from mouse fecal samples were performed and HPLC-DAD analysis was performed. Spermidine was detected in the fecal sample, and it was confirmed that the change in spermidine content was caused by GB104.
  • the change in spermidine content was expressed as a fold by dividing the area of the spermidine peak of the fecal sample after oral administration of GB104 for 7 days by the area of the spermidine peak of the fecal sample collected immediately before oral administration of GB104, and this value was calculated for each group. Comparison was made and this is shown in Figure 9.
  • Figure 9 is a graph showing the change in spermidine content in feces after antibiotic-treated mice were treated with GB104 and PBS (control group), respectively.
  • Antibiotics were mixed into sterilized drinking water and kept out of the light, and the mice were allowed to freely consume this drinking water for 7 days. Additionally, the antibiotic-treated drinking water was replaced every 2 to 3 days. From day 7 onwards, mice were provided with sterilized regular drinking water instead of drinking water containing antibiotics until the end of the test. Freeze-dried GB104 was suspended in D-PBS and orally administered at 1 has been removed. The intestinal tissue was opened by making a longitudinal incision, washed with PBS, and cut into 1-2 cm long pieces.
  • IEL Intraepithelial lymphocytes
  • Pieces of intestinal tissue from which epithelial cells were removed were washed with PBS, finely chopped, and added to enzyme medium (small intestine: 400 U/ml Collagenase D, 10 ⁇ g/ml DNase I, large intestine: 800 U/ml Collagenase D, 10 ⁇ g/ml DNase I).
  • enzyme medium small intestine: 400 U/ml Collagenase D, 10 ⁇ g/ml DNase I
  • large intestine 800 U/ml Collagenase D, 10 ⁇ g/ml DNase I
  • RPMI 400 medium containing 3% FBS 20 mM HEPES, 100 U/ml Penicillin, 100 ⁇ g/ml Streptomycin, 1 mM Sodium Pyruvate, and 1 mM NEAA
  • the enzyme reaction was stopped by treatment with 10 mM EDTA, and the cells passed through the strainer were resuspended in 40% Percoll solution, and 75% Percoll solution was added thereto and centrifuged. After centrifugation, the middle layer was recovered and lamina intestinal (LP) cells were separated. The isolated immune cells were stained using a fluorescent antibody matching the marker of the cells to be identified and then analyzed using a FACSymphony device, and the results are shown in Figure 10.
  • Figure 10 is a graph showing the change in the number of immune cells in the intestine after antibiotic-treated mice were treated with GB104 and PBS (control group), respectively.
  • Plantarum GB104 strain showed cytotoxic T cells (known as immune cells that directly inhibit cancer growth) compared to the control group. It was confirmed that the proportion of CD8 + T cells) was significantly increased, and the IFN- ⁇ secreted by these cells was also significantly increased. Through this, it was confirmed that the L. Plantarum GB104 strain had an effect on the increase and activation of CD8 + anti-tumor immune T cells.
  • Antibiotics were mixed into sterilized drinking water and kept out of the light, and the mice were allowed to freely consume this drinking water for 7 days. Additionally, the antibiotic-treated drinking water was replaced every 2-3 days. From day 7 onwards, mice were provided with sterilized regular drinking water instead of drinking water containing antibiotics until the end of the test. Freeze-dried GB104 was suspended in D-PBS and adjusted to a dose of 1x10 9 CFU/head, and then the test substance was orally administered at 200 ⁇ L per mouse daily for 14 days from the end of antibiotic treatment using an oral zonde.
  • Figure 11 is a graph showing changes in tight junction-related gene expression in intestinal tissue after antibiotic-treated mice were treated with GB104 and PBS (control group), respectively; ZO: Zonula Occludens.
  • GB104 increased the expression level of tight junction genes ( ZO-1, Occludin, and Claudin-4 ) in small intestine tissue. Through this, it was confirmed that GB104 can enhance gut barrier function by increasing the expression of tight junction genes.
  • L. Plantarum culture supernatants including L. Plantarum GB104 strain, were each processed, and cell viability was screened through MTT analysis.
  • Human colon cancer cell line HCT116 cells were dispensed into each well of a 96-well plate at 2X10 3 cells and cultured for 24 hours. Then, 10% culture supernatant of various L. Plantarum strains was added in a culture medium supplemented with DFMO and aminoguanidine. concentration and cultured for 72 hours under conditions of 37°C and 5% CO 2 . The culture supernatant was obtained by culturing the L. Plantarum strain in MRS medium, precipitating the strain by centrifugation, collecting only the supernatant, and filtering it through a 0.22 ⁇ m filter.
  • MTT Cell Proliferation Kit I
  • Figure 12 is a graph showing the survival rate of cells after treating the colon cancer cell line HCT116 with culture supernatants of GB104 and other strains, respectively.
  • the various L. Plantarum culture supernatants did not show the same cancer cell growth inhibitory effect, and in particular, the GB104 culture supernatant showed an effect of reducing the cancer cell survival rate by about 95%, making it the most effective in inhibiting the growth of cancer cells. was confirmed.
  • Plantarum GB104 culture supernatant was processed using the human colon cancer cell line, and changes in the cancer cell cycle were confirmed using flow cytometry.
  • Human colon cancer cell line HCT116 cells were distributed into each well of a 6-well plate at 5 ⁇ 10 4 cells and cultured for 24 hours. Then, 10% of the culture supernatant of the L. Plantarum GB104 strain was added in a culture medium containing DFMO and aminoguanidine. concentration and cultured for 48 hours under conditions of 37°C and 5% CO 2 . The culture supernatant was obtained by culturing the L. Plantarum strain in MRS medium, precipitating the strain by centrifugation, collecting only the supernatant, and filtering it through a 0.22 ⁇ m filter.
  • Figure 13 is a graph showing the cell cycle after treating the colon cancer cell line HCT116 with culture supernatants of GB104 and the comparison strain (WCFS1), respectively.
  • Plantarum GB104 culture supernatant was treated using the human colon cancer cell line, and the cancer cell killing effect was confirmed using flow cytometry.
  • Human colon cancer cell line HCT116 cells were distributed into each well of a 6-well plate at 5 ⁇ 10 4 cells and cultured for 24 hours. Then, 10% of the culture supernatant of the L. Plantarum GB104 strain was added in a culture medium containing DFMO and aminoguanidine. concentration and cultured for 48 hours under conditions of 37°C and 5% CO 2 . The culture supernatant was obtained by culturing the L. Plantarum strain in MRS medium, precipitating the strain by centrifugation, collecting only the supernatant, and filtering it through a 0.22 ⁇ m filter. After 48 hours, the cells were removed by treatment with trypsin-EDTA and then harvested by centrifugation.
  • Figure 14 is a graph showing cell death after treating the colon cancer cell line HCT116 with culture supernatants of GB104 and the comparison strain (WCFS1), respectively.

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Abstract

La présente invention concerne une composition comprenant une souche de Lactobacillus plantarum pour améliorer la composition du métabolite intestinal. La souche de Lactobacillus plantarum, selon un mode de réalisation, peut réduire les polyamines intestinales (par exemple, la spermidine) et, ainsi, la présente invention peut être efficacement utilisée en tant que composition pharmaceutique, aliment (aliment fonctionnel de santé), composition d'alimentation et adjuvant anticancéreux, tous étant destinés à prévenir, soulager et traiter le cancer ou améliorer le pronostic de celui-ci et assister un traitement anticancéreux.
PCT/KR2023/014313 2022-09-20 2023-09-20 Composition comprenant une souche de lactobacillus plantarum pour améliorer la composition de métabolite intestinal WO2024063545A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
KR10-2022-0118945 2022-09-20
KR20220118945 2022-09-20
KR10-2023-0078227 2023-06-19
KR1020230078227A KR20240040601A (ko) 2022-09-20 2023-06-19 락토바실러스 플란타룸 균주를 포함하는 장내 대사산물 조성의 개선을 위한 조성물
KR10-2023-0125612 2023-09-20
KR1020230125612A KR20240040656A (ko) 2022-09-20 2023-09-20 폴리아민 대사에 관여하는 락토바실러스 플란타룸을 포함하는 조성물

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KR20210090570A (ko) * 2020-01-10 2021-07-20 이뮤노바이옴 주식회사 신규한 락토바실러스 플란타룸(Lactobacillus plantarum) 균주, 균주 유래 다당체 및 이의 용도

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KR20180118362A (ko) * 2017-04-21 2018-10-31 한동대학교 산학협력단 항염증 및 대사성 질환 개선 효과를 갖는 락토바실러스 플란타룸 균주 및 이의 용도
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KR20210090570A (ko) * 2020-01-10 2021-07-20 이뮤노바이옴 주식회사 신규한 락토바실러스 플란타룸(Lactobacillus plantarum) 균주, 균주 유래 다당체 및 이의 용도

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