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WO2018139703A1 - Pharmaceutical composition for treating colorectal disease containing cystatin and a protein derived from lactic acid bacteria - Google Patents

Pharmaceutical composition for treating colorectal disease containing cystatin and a protein derived from lactic acid bacteria Download PDF

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Publication number
WO2018139703A1
WO2018139703A1 PCT/KR2017/002512 KR2017002512W WO2018139703A1 WO 2018139703 A1 WO2018139703 A1 WO 2018139703A1 KR 2017002512 W KR2017002512 W KR 2017002512W WO 2018139703 A1 WO2018139703 A1 WO 2018139703A1
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Prior art keywords
cystatin
lactic acid
acid bacteria
disease
colorectal
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PCT/KR2017/002512
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French (fr)
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Myung Jun Chung
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Cell Biotech Co., Ltd.
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Publication of WO2018139703A1 publication Critical patent/WO2018139703A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • 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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/005—Enzyme inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a pharmaceutical composition for treating colorectal disease, which contains cystatin and a protein derived from lactic acid bacteria.
  • lactic acid bacteria refer to a group of bacteria that ferment sugars to obtain energy and produce a large amount of lactic acid. Lactic acid bacteria function to inhibit the propagation of harmful bacteria in the intestines and induce smooth bowel movement, thereby promoting a healthy intestinal environment. Thus, lactic acid bacteria are considered essential for modern people in whom the risk of colorectal diseases increases due to frequent stress, westernized eating habits, drinking and the like.
  • cystatin is a substance that inhibits the activity of intracellular proteases, and is currently classified into family 1 (cystatin-alpha, and cystatin-beta) that is present in cells, secretory family 2 (cystatin C, cystatin S, egg white cystatin, and milk cystatin), family 3 (kininogen), and the like.
  • Cystatin-alpha exists in leukocyte, epiehelial tissue of epidermis, digestive tracts, vagina, and etc., and the like, and functions to protect the human body from the invasion of pathogenic microbes. Cystatin-beta is present in most cytosols, and functions to inhibit the activity of proteases to thereby prevent viral infections.
  • Egg white cystatin also helps prevent viral infections, and a substance having the same function as that of egg white cystatin is oryzacystatin that is found in rice seeds.
  • Cystatin C is a functional protein that is produced in certain amounts every moment by all the cells of the human body and functions to prevent the onset of diseases and maintain normal physiology, and particularly, it is known to play an important role in the defense function of the immune system.
  • the present invention is directed to a pharmaceutical composition for treating colorectal disease, which contains cystatin and a protein derived from lactic acid bacteria.
  • the composition of the present invention has been demonstrated to have significant effects on the inhibition of colorectal cancer growth, and thus will be effectively used as a therapeutic agent against colorectal cancer in the medical field.
  • the present invention has been made in order to solve the above-described problems occurring in the prior art, and is directed to a pharmaceutical composition for treating colorectal disease, which contains cystatin and a protein derived from lactic acid bacteria.
  • probiotics refers to strains that show beneficial effects on the intestinal environment when reaching intestines after intake.
  • Most probiotics known to date are lactic acid bacteria and include some Bacillus species and the like. Since Russian scientist Elie Mechinikoff won a Nobel prize for his discovery that the reason why Bulgarians enjoy longevity is because of the intake of milk fermented with Lactobacillus , the functionalities of lactic acid bacteria as probiotics have been studied for a long period of time.
  • bacteria including lactic acid bacteria
  • these bacteria should resist gastric acid and bile acid, reach the small intestines, proliferate and colonize in the intestines, exhibit useful effects in the intestinal tracts, and should be nontoxic and nonpathogenic.
  • lactic acid bacteria refers to a group of bacteria that ferment sugars to obtain energy and produce a large amount of lactic acid.
  • the term “lactic acid bacteria” is a common name and does not indicate a taxonomic position. Those falling within the definition of lactic acid bacteria include genera such as Lactobacillus , Lactococcus , Leuconostoc , Pediococcus , Bifidobacterium and the like. Lactic acid bacteria are morphologically divided into cocci ( Lactococcus , Pediococcus , and Leuconostoc ) and bacilli ( Lactobacillus , and Bifidobacterium ), and are gram-positive.
  • lactic acid bacteria grow well in a hypoxic environment and produce lactic acid from various sugars. These lactic acid bacteria mostly show acid resistance, and have very complex auxotrophy to require many kinds of amino acids or vitamins in addition to sugars, and some of these lactic acid bacteria cannot grow if micronutrients are not added thereto. Lactic acid bacteria are widely distributed in nature, including agricultural products, foods, and human or animal bodies, and the exact place of growth of any of these lactic acid bacteria cannot be seen. Lactococcus grows at 10°C, but does not grow at 45°C, has an optimal growth temperature of about 30°C, and shows normal fermentation. Many Lactococcus strains are used as starters for milk products in food processing.
  • Pediococcus shows normal fermentation and is arranged in packets of four cells. It is classified, according to growth temperature, the optical rotation of lactic acid produced, etc., into 8 species.
  • Pediococcus together with Leuconostoc is a major genus related to fermentation, and is less connected with living animal bodies.
  • Leuconostoc shows abnormal fermentation, and is classified, according to sugar decomposition, growth, growth pH, etc., into 4 species. Lactobacillus is largely divided into two, one that shows normal fermentation, and the other that shows abnormal fermentation. It is classified, according to growth temperature, sugar decomposition, the optical rotation of lactic acid produced, etc., into 55 species and 11 subspecies.
  • Lactobacillus is a typical genus of lactic acid bacteria, and is used in various fermented foods. It is a flora present in intestinal tracts, and has an importance relationship with human or animal health. Bifidobacterium is an obligately anaerobic, gram-positive bacillus showing abnormal fermentation, and mainly produces lactic acid and acetic acid as final products.
  • the expression “protein derived from lactic acid bacteria” means a protein isolated and purified from lactic acid bacteria.
  • the lactic acid bacteria may be any lactic acid bacteria that ferment sugars to produce lactic acid, the lactic acid bacteria are preferably Lactobacillus , more preferably Lactobacillus rhamnosus .
  • protein derived from lactic acid bacteria means “P8 protein” or “P14 protein”, but is not limited thereto.
  • the term “P8 protein (protein No. 8)” means an 8-KDa protein fragment extracted from lactic acid bacteria ( Lactobacillus rhamnosus ) according to a preparation method described in an example of the present invention.
  • the P8 protein in the present invention may be defined by the nucleotide sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2.
  • the term “P14 protein (protein No. 14)” means a 14-KDa protein fragment extracted from lactic acid bacteria ( Lactobacillus rhamnosus ) according to a preparation method described in an example of the present invention.
  • the P14 protein in the present invention may be defined by the nucleotide sequence of SEQ ID NO: 3 or the amino acid sequence of SEQ ID NO: 4.
  • cystatin refers to a substance that inhibits the activity of intracellular proteases. Cystatin is currently classified into family 1 (cystatin-alpha, and cystatin-beta) that is present in cells, secretory family 2 (cystatin C, cystatin D, cystatin S, egg white cystatin, and milk cystatin), family 3 (kininogen), and the like. Cystatin-alpha exists in leukocytes, the epithelial tissue of epidermis, digestive tracts, vagina, and etc., and the like, and functions to protect the human body from the invasion of pathogenic microbes.
  • Cystatin-beta is present in most cytosols, and functions to inhibit the activity of proteases to thereby prevent viral infections.
  • Egg white cystatin also helps prevent viral infections, and a substance having the same function as that of egg white cystatin is oryzacystatin that is found in rice seeds.
  • Cystatin C is a functional protein that is produced in certain amounts every moment by all the cells of the human body and functions to prevent the onset of diseases and maintain normal physiology, and particularly, it plays an important role in the defense function of the immune system.
  • blood cystatin C levels increase in various pathologies. If blood cystatin C levels are 1 mg/L or higher, the incidence rate of heart attack, stroke, sudden cardiac death or chronic renal failure increases as the value increases. Elucidation of the secretory mechanism and function of cystatin D is still insufficient.
  • the term “colorectal disease” refers to a group of various diseases that occur in the colon.
  • the colorectal disease is preferably colorectal cancer, colorectal polyp, colitis, ischemic bowel disease, dysentery, intestinal vascular dysplasia, diverticulosis, irritable bowel syndrome, or Crohn's disease, but is not limited thereto. More preferably, the colorectal disease is colorectal cancer.
  • “pharmaceutical composition” refers to a composition to be administered for a specific purpose.
  • the pharmaceutical composition of the present invention contains, as active ingredients, cystatin and a protein derived from lactic acid bacteria.
  • the protein derived from lactic acid bacteria may be “P8 protein” or “P14 protein”
  • the cystatin may be “cystatin A” or “cystatin D”.
  • the pharmaceutical composition of the present invention may contain protein and a pharmaceutically acceptable carrier, excipient or diluent, which are involved therein.
  • the "pharmaceutically acceptable" carrier or excipient means one approved by a regulatory agency of the Federal or a state government, or one listed in the pharmacopoeia or other generally recognized pharmacopoeia for use in vertebral animals, and more particularly in humans.
  • the pharmaceutical composition of the present invention can be in the form of suspensions, solutions, or emulsions, in oily or aqueous vehicles, and can be in the form of solid or semi-solid, preferably liquid.
  • the pharmaceutical composition of the present invention can contain formulatory agents such as suspending, stabilizing, solubilizing, and/or dispersing agents, and can be sterilized.
  • the pharmaceutical composition can be stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the pharmaceutical composition of the present invention can be in sterile powder form for reconstitution with a suitable vehicle before use.
  • the pharmaceutical composition can be presented in unit dose form, in microneedle patch, in ampoules, or other unit-dose containers, or in multi-dose containers.
  • the pharmaceutical composition can be stored in a freeze-dried (lyophilized) condition requiring only the addition of sterile liquid carrier, for example, water for injection immediately prior to use.
  • sterile liquid carrier for example, water for injection immediately prior to use.
  • Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules or tablets.
  • the pharmaceutical composition of the present invention may be formulated as liquid or contained as microspheres in liquids.
  • excipients that are suitable for the pharmaceutical composition of the present invention may include preservatives, suspending agents, stabilizers, dyes, buffers, antibacterial agents, antifungal agents, and isotonic agents, for example, sugars or sodium chloride.
  • stabilizer refers to a compound optionally used in the pharmaceutical composition of the present invention in order to increase storage life.
  • additional stabilizers may be sugars, amino acids or polymers.
  • the pharmaceutical composition of the present invention can comprise one or more pharmaceutically acceptable carriers.
  • the carrier can be a solvent or dispersion medium.
  • Non-limiting examples of pharmaceutically acceptable carriers include water, saline, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), oils, and suitable mixtures thereof.
  • Non-limiting examples of sterilization techniques that are applied to the pharmaceutical composition of the present invention include filtration through a bacterial-restraining filter, terminal sterilization, incorporation of sterilizing agents, irradiation, sterilization gas irradiation, heating, vacuum drying, and freeze drying.
  • “food composition” can be used in various foods, for example, beverages, gums, teas, vitamin complexes, health supplement foods or the like, and may be used as pills, powders, granules, infusions, tablets, capsules or beverages, and the content (wt%) of the food composition in foods is not limited.
  • the food composition of the present invention may contain conventional food additives known in the art, for example, flavorings, colorants, fillers, stabilizers and the like.
  • the food composition according to the present invention is not particularly limited, as long as it is a composition containing, as essential ingredients, cystatin and the protein derived from lactic acid bacteria.
  • the food composition of the present invention may additionally contain various flavorings or natural carbohydrates as in conventional beverages.
  • the natural carbohydrates include conventional sugars, such as monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), polysaccharides (e.g., dextrin, cyclodextrin, etc.), and sugar alcohols such as xylitol, sorbitol, erythritol or the like.
  • sugar alcohols such as xylitol, sorbitol, erythritol or the like.
  • flavorings that may be used in the present invention include natural flavorings (thaumatin, stevia extracts, such as rebaudioside A, glycyrrhizin, etc.) and synthetic flavorings (saccharin, aspartame, etc.).
  • the natural carbohydrate is used in an amount of about 1-20 g, preferably about 5-12 g, based on 100 mL of the composition of the present invention.
  • the food composition of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavorings such as synthetic flavorings and natural flavorings, colorants, extenders (cheese, chocolate, etc.), pectic acid and its salt, alginic acid and its salt, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonizing agents as used in carbonated beverages, etc.
  • Such components may be used individually or in combination. Although the percentage of such additives is not of great importance, it is generally selected in a range of 0 to about 20 parts by weight based on 100 parts by weight of the composition of the present invention.
  • “administration” means introducing the composition of the present invention into a patient by any suitable method.
  • the composition of the present invention may be administered by any general route, as long as it can reach a target tissue.
  • the composition of the present invention may be administered orally, intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, intranasally, intrapulmonarily, intrarectally, intracavitally or intrathecally.
  • the pharmaceutical composition of the present invention which contains, as active ingredients, cystatin and a protein derived from lactic acid bacteria, is administered intravenously as a liquid formulation or administered as orally as a powder, tablet, capsule or liquid formulation, but is not limited thereto.
  • a method for treating colorectal disease according to the present invention may comprise administering a pharmaceutically effective amount of the pharmaceutical composition.
  • the effective amount can be determined depending on various factors, including the kind of disease, the severity of the disease, the kinds and contents of active ingredient and other ingredients in the composition, the kind of formulation, the patient’s age, body weight, general health condition, sex and diet, the time of administration, the route of administration, the secretion rate of the composition, the period of treatment, and other drugs that are concurrently used.
  • the present invention provides a pharmaceutical composition for treating colorectal disease, which contains, as active ingredients, cystatin and a protein derived from lactic acid bacteria.
  • the protein derived from lactic acid bacteria comprises an amino acid sequence represented by SEQ ID NO: 2 or 4, and the cystatin is cystatin A or cystatin D.
  • the lactic acid bacteria are any one or more selected from the group consisting of Lactobacillus rhamnosus , Lactobacillus acidophilus , Lactobacillus paracasei , Lactobacillus plantarum , Pediococcus pentosaceus , and Lactobacillus brevis .
  • the lactic acid bacteria are Lactobacillus rhamnosus .
  • the colorectal disease is any one or more selected from the group consisting of colorectal cancer, colorectal polyp, colitis, ischemic bowel disease, dysentery, intestinal vascular dysplasia, diverticulosis, irritable bowel syndrome, or Crohn's disease.
  • the colorectal disease is colorectal cancer.
  • the present invention provides a food composition for alleviating colorectal disease, which contains, as active ingredients, cystatin and a protein derived from lactic acid bacteria.
  • the protein derived from lactic acid bacteria comprises an amino acid sequence represented by SEQ ID NO: 2 or 4, and the cystatin is cystatin A or cystatin D.
  • the lactic acid bacteria are any one or more selected from the group consisting of Lactobacillus rhamnosus , Lactobacillus acidophilus , Lactobacillus paracasei , Lactobacillus plantarum , Pediococcus pentosaceus , and Lactobacillus brevis .
  • the lactic acid bacteria are Lactobacillus rhamnosus .
  • the colorectal disease is any one or more selected from the group consisting of colorectal cancer, colorectal polyp, colitis, ischemic bowel disease, dysentery, intestinal vascular dysplasia, diverticulosis, irritable bowel syndrome, or Crohn's disease.
  • the colorectal disease is colorectal cancer.
  • the present invention provides a method of treating colorectal disease, the method comprising administering to a patient in need thereof a pharmaceutical composition containing an effective amount of cystatin and a protein derived from lactic acid bacteria.
  • the protein derived from lactic acid bacteria comprises an amino acid sequence represented by SEQ ID NO: 2 or 4, and the cystatin is cystatin A or cystatin D.
  • the lactic acid bacteria are any one or more selected from the group consisting of Lactobacillus rhamnosus , Lactobacillus acidophilus , Lactobacillus paracasei , Lactobacillus plantarum , Pediococcus pentosaceus , and Lactobacillus brevis .
  • the lactic acid bacteria are Lactobacillus rhamnosus .
  • the colorectal disease is any one or more selected from the group consisting of colorectal cancer, colorectal polyp, colitis, ischemic bowel disease, dysentery, intestinal vascular dysplasia, diverticulosis, irritable bowel syndrome, or Crohn's disease.
  • the colorectal disease is colorectal cancer.
  • the present invention provides a use of cystatin and a protein derived from lactic acid bacteria thereof, for preparation of a medicament for treatment of colorectal disease.
  • the protein derived from lactic acid bacteria comprises an amino acid sequence represented by SEQ ID NO: 2 or 4, and the cystatin is cystatin A or cystatin D.
  • the lactic acid bacteria are any one or more selected from the group consisting of Lactobacillus rhamnosus , Lactobacillus acidophilus , Lactobacillus paracasei , Lactobacillus plantarum , Pediococcus pentosaceus , and Lactobacillus brevis .
  • the lactic acid bacteria are Lactobacillus rhamnosus .
  • the colorectal disease is any one or more selected from the group consisting of colorectal cancer, colorectal polyp, colitis, ischemic bowel disease, dysentery, intestinal vascular dysplasia, diverticulosis, irritable bowel syndrome, or Crohn's disease.
  • the colorectal disease is colorectal cancer.
  • lactic acid bacteria are effective for the prevention, alleviation and treatment of colorectal diseases, including colorectal cancer, colitis, irritable bowel syndrome, Crohn's disease and the like, the development of therapeutic agents that are used together with lactic acid bacterial components to exhibit synergistic effects is still insufficient.
  • the pharmaceutical composition of the present invention which contains cystatin and the protein derived from lactic acid bacteria, exhibits a significantly increased inhibitory effect against colorectal cancer compared to when cystatin and the protein derived from lactic acid bacteria are used alone.
  • the pharmaceutical composition of the present invention will be effectively used as a therapeutic agent against colorectal cancer in the medical field.
  • FIG. 1 is a schematic view showing protein treatment processes in Experimental Examples 1-1 to 1-9 of the present invention.
  • FIG. 2 is a schematic view showing protein treatment processes in Experimental Examples 2-1 to 2-9 of the present invention.
  • FIG. 3 is a graph showing that the growth of colorectal cancer cells was inhibited by protein treatment in Experimental Examples 1-1 to 1-9 of the present invention.
  • FIG. 4 is a graph showing that the growth of colorectal cancer cells was inhibited by protein treatment in Experimental Examples 2-1 to 2-9 of the present invention.
  • Example 1-1 Purification of Protein from Lactic Acid Bacteria
  • the colorectal cancer cell line DLD-1 was treated with culture supernatants or cell lysates of various lactic acid bacteria to examine the anticancer activities of the supernatants or lysates. Among them, a cell lysate of Lactobacillus rhamnosus (KCTC 12202BP) having the highest anticancer activity was selected.
  • KCTC 12202BP Lactobacillus rhamnosus
  • size exclusion chromatography Sephadex G-25, desalting column, GE Healthcare
  • FPLC fast protein liquid chromatography
  • the fraction containing the isolated protein was dialyzed with 20 mM Tris buffer (pH8.0), and the protein not adsorbed onto HiTrap DEAE FF (GE Healthcare) was collected, and concentrated using a 3-KDa membrane, after which it was dialyzed again with 0.05 M phosphate (pH 6.0) buffer, adsorbed onto HiTrap DEAE SP (GE Healthcare), and sequentially separated according to the concentration gradient of 0.5M sodium chloride.
  • Colorectal cancers were treated with each of the isolated fractions to examine the anticancer activity of each fraction, and the fraction having the highest anticancer activity was concentrated and analyzed by SDS-PAGE, thereby isolating a 8-KDa protein and a 14-KDa protein, which were named “P8 protein” and “P14 protein”, respectively.
  • P8 protein or P14 protein, purified according to the method of Example 1-1 was electrophoresed, and then a band was excised from the Coomassie gel and stirred in 100 ⁇ l of a destaining solution (50% MeOH/D.W) for 5 minutes, followed by complete removal of the destaining solution. Then, the resulting material was stored in 100 ⁇ l of 200 mM ABC (ammonium bicarbonate, pH 7.8) solution for 20 minutes, followed by removal of the ABC solution.
  • a destaining solution 50% MeOH/D.W
  • the resulting material was stirred in 100 ⁇ l of acetonitrile for 2 minutes, followed by removal of the acetonitrile solution, and was stirred in 100 ⁇ l of ABC solution for 2 minutes, followed by removal of the ABC solution, and was then stirred in 100 ⁇ l of acetonitrile for 2 minutes. This process was repeated a total of three times. Next, the acetonitrile was completely removed, and the remaining gel was dried. 20 ⁇ l of trypsin solution (0.2 ⁇ g) was added to the dried gel which was then kept on ice for 45 minutes.
  • a micro-scale column was prepared using Geload tip and Poros R2 resin.
  • a 10 ml syringe was prepared such that the front of the syringe was consistent with the column so as to be able to apply pressure to the micro-column.
  • SEQ ID NO: 1 Nucleotide sequence of P8 protein gcaacagtagatcctgaaaagacattgtttctcgatgaaccaatgaacaaggtatttgactggagcaacagcgaagcacctgtacgtgatgcgctgtgggattattacatggaaaagaacagccgtgataccatcaagactgaagaagaagaaatgaaaccagtcctagacatgtccgacgatgaggtcaaagccctagcagaaaaggttctcaagaagtaa SEQ ID NO: 2 Amino acid sequence of P8 protein ATVDPEKTLFLDEPMNKVFDWSNSEAPVRDALWDYYMEKNSRDTIKTEEEMKPVLDMSDDEVKALAEKVLKK SEQ ID NO: 3 Nucleotide sequence of P14 protein gctaaat
  • colorectal cancer cell lines (HCT116 and DLD-1) was cultured in a 12-well plate at a density of 5x10 4 cells/well, and experimental examples were classified as shown in Table 2 below according to the order of treatment with P8 protein and cystatin A.
  • Experimental Example 1-1 Not treated Experimental Example 1-2 Treated once with P8 protein (20 ⁇ g) Experimental Example 1-3 Treated once with P8 protein (50 ⁇ g) Experimental Example 1-4 Treated with P8 protein (20 ⁇ g) for 3 days (once a day), and then treated with cystatin A (10 ⁇ g) for 3 days (once a day) Experimental Example 1-5 Treated with P8 protein (20 ⁇ g) for 3 days (once a day), and then treated with cystatin A (20 ⁇ g) for 3 days (once a day) Experimental Example 1-6 Treated with P8 protein (50 ⁇ g) for 3 days (once a day), and then treated with cystatin A (10 ⁇ g) for 3 days (once a day) Experimental Example 1-7 Treated with P8 protein (50 ⁇ g) for 3 days (once a day), and then treated with cystatin A (20 ⁇ g) for 3 days (once a day) Experimental Example 1-8 Treated with P8 protein (20 ⁇ g) for 6 days (once a day) Experimental Example 1
  • FIGS. 1 and 2 are schematic views showing the protein treatment processes.
  • each well was treated with 200 ⁇ l of a reagent (MTT formazan, 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-tetrazolium bromide) for cell survival rate measurement and incubated for 2 hours.
  • the absorbance of each well at 570 nm was measured using a microplate reader (Amersham, Biorad, USA, Japan), and based on the measured value, the cell survival rate was calculated.
  • the present invention is directed to a pharmaceutical composition for treating colorectal disease, which contains cystatin and a protein derived from lactic acid bacteria.
  • the composition of the present invention has been demonstrated to have significant effects on the inhibition of colorectal cancer growth, and thus will be effectively used as a therapeutic agent against colorectal cancer in the medical field.
  • gcaacagtag atcctgaaa gacattgttt ctcgatgaac caatgaacaa ggtatttgac tggagcaaca gcgaagcacc tgtacgtgat gcgctgtggg attattacat ggaaaagaac agccgtgata ccatcaagac tgaagaagaa atgaaaccag tcctagacat gtccgacgat gaggtcaaag ccctagcaga aaaggttctc aagaagtaaa

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Abstract

The present invention relates to a pharmaceutical composition for treating colorectal disease, which contains cystatin and a protein derived from lactic acid bacteria. The pharmaceutical composition of the present invention, which contains cystatin and the protein derived from lactic acid bacteria, exhibits a significantly increased inhibitory effect against colorectal cancer compared to when cystatin and the protein derived from lactic acid bacteria are used alone. Thus, the pharmaceutical composition of the present invention will be effectively used as a therapeutic agent against colorectal cancer in the medical field.

Description

PHARMACEUTICAL COMPOSITION FOR TREATING COLORECTAL DISEASE CONTAINING CYSTATIN AND A PROTEIN DERIVED FROM LACTIC ACID BACTERIA
The present invention relates to a pharmaceutical composition for treating colorectal disease, which contains cystatin and a protein derived from lactic acid bacteria.
Among microorganisms, lactic acid bacteria refer to a group of bacteria that ferment sugars to obtain energy and produce a large amount of lactic acid. Lactic acid bacteria function to inhibit the propagation of harmful bacteria in the intestines and induce smooth bowel movement, thereby promoting a healthy intestinal environment. Thus, lactic acid bacteria are considered essential for modern people in whom the risk of colorectal diseases increases due to frequent stress, westernized eating habits, drinking and the like.
Accordingly, many studies have been conducted on the functions of probiotics, including lactic acid bacteria (KR10-2015-0066772A, KR0232639B, and EP1082964A1, etc.), and the use of probiotics for treatment of colorectal diseases (US7887794B1, and US 7887794 B2, etc.). However, such studies are merely related to the effects of lactic acid bacteria themselves on the prevention or alleviation of colorectal diseases, and a technology that extracts an active ingredient from lactic acid bacteria and applies the active ingredient directly as a therapeutic agent against colorectal cancer or the like is still insignificant.
Meanwhile, cystatin is a substance that inhibits the activity of intracellular proteases, and is currently classified into family 1 (cystatin-alpha, and cystatin-beta) that is present in cells, secretory family 2 (cystatin C, cystatin S, egg white cystatin, and milk cystatin), family 3 (kininogen), and the like. Cystatin-alpha exists in leukocyte, epiehelial tissue of epidermis, digestive tracts, vagina, and etc., and the like, and functions to protect the human body from the invasion of pathogenic microbes. Cystatin-beta is present in most cytosols, and functions to inhibit the activity of proteases to thereby prevent viral infections. Egg white cystatin also helps prevent viral infections, and a substance having the same function as that of egg white cystatin is oryzacystatin that is found in rice seeds. Cystatin C is a functional protein that is produced in certain amounts every moment by all the cells of the human body and functions to prevent the onset of diseases and maintain normal physiology, and particularly, it is known to play an important role in the defense function of the immune system.
Therefore, the present invention is directed to a pharmaceutical composition for treating colorectal disease, which contains cystatin and a protein derived from lactic acid bacteria. The composition of the present invention has been demonstrated to have significant effects on the inhibition of colorectal cancer growth, and thus will be effectively used as a therapeutic agent against colorectal cancer in the medical field.
The present invention has been made in order to solve the above-described problems occurring in the prior art, and is directed to a pharmaceutical composition for treating colorectal disease, which contains cystatin and a protein derived from lactic acid bacteria.
However, the technical object to be achieved by the present invention is not limited to the above technical object, and other objects that are not mentioned above can be clearly understood by those skilled in the art from the following description.
Hereinafter, various embodiments described herein will be described with reference to figures. In the following description, numerous specific details are set forth, such as specific configurations, compositions, and processes, etc., in order to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in combination with other known methods and configurations. In other instances, known processes and preparation techniques have not been described in particular detail in order to not unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrase "in one embodiment" or "an embodiment" in various places throughout this specification are not necessarily referring to the same embodiment of the present invention. Additionally, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.
Unless otherwise specified in the specification, all the scientific and technical terms used in the specification have the same meanings as commonly understood by those skilled in the technical field to which the present invention pertains.
In one embodiment, the term “probiotics” refers to strains that show beneficial effects on the intestinal environment when reaching intestines after intake. Most probiotics known to date are lactic acid bacteria and include some Bacillus species and the like. Since Russian scientist Elie Mechinikoff won a Nobel prize for his discovery that the reason why Bulgarians enjoy longevity is because of the intake of milk fermented with Lactobacillus, the functionalities of lactic acid bacteria as probiotics have been studied for a long period of time. In order for bacteria (including lactic acid bacteria) to be recognized as probiotics, these bacteria should resist gastric acid and bile acid, reach the small intestines, proliferate and colonize in the intestines, exhibit useful effects in the intestinal tracts, and should be nontoxic and nonpathogenic.
About 1 kg of bacteria live in the human intestines, and the amount of bacteria present in the human intestines is substantially equal to the amount of food present in the human intestines, and bacteria account for about 40% of feces contents (excluding water) that are excreted every day. When human feces are observed with a microscope, it can be seen that the feces consist mostly of bacterial clusters, and about 99% of these bacteria are anaerobic bacteria. In the case of healthy babies who eat mother’s milk, 90% or more of feces bacteria are Bifidobacterium, and with age, Bifidobacterium gradually decrease, and intestinal harmful bacteria increases (Bifido Microfl 7:35-43, 1998). In this normal aging process, probiotics function to help maintain the distribution of intestinal flora at a healthy state.
In one embodiment of the present invention, the term “lactic acid bacteria” refers to a group of bacteria that ferment sugars to obtain energy and produce a large amount of lactic acid. The term “lactic acid bacteria” is a common name and does not indicate a taxonomic position. Those falling within the definition of lactic acid bacteria include genera such as Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Bifidobacterium and the like. Lactic acid bacteria are morphologically divided into cocci (Lactococcus, Pediococcus, and Leuconostoc) and bacilli (Lactobacillus, and Bifidobacterium), and are gram-positive. These lactic acid bacteria grow well in a hypoxic environment and produce lactic acid from various sugars. These lactic acid bacteria mostly show acid resistance, and have very complex auxotrophy to require many kinds of amino acids or vitamins in addition to sugars, and some of these lactic acid bacteria cannot grow if micronutrients are not added thereto. Lactic acid bacteria are widely distributed in nature, including agricultural products, foods, and human or animal bodies, and the exact place of growth of any of these lactic acid bacteria cannot be seen. Lactococcus grows at 10°C, but does not grow at 45°C, has an optimal growth temperature of about 30°C, and shows normal fermentation. Many Lactococcus strains are used as starters for milk products in food processing. Pediococcus shows normal fermentation and is arranged in packets of four cells. It is classified, according to growth temperature, the optical rotation of lactic acid produced, etc., into 8 species. Pediococcus together with Leuconostoc is a major genus related to fermentation, and is less connected with living animal bodies. Leuconostoc shows abnormal fermentation, and is classified, according to sugar decomposition, growth, growth pH, etc., into 4 species. Lactobacillus is largely divided into two, one that shows normal fermentation, and the other that shows abnormal fermentation. It is classified, according to growth temperature, sugar decomposition, the optical rotation of lactic acid produced, etc., into 55 species and 11 subspecies. Lactobacillus is a typical genus of lactic acid bacteria, and is used in various fermented foods. It is a flora present in intestinal tracts, and has an importance relationship with human or animal health. Bifidobacterium is an obligately anaerobic, gram-positive bacillus showing abnormal fermentation, and mainly produces lactic acid and acetic acid as final products.
In one embodiment of the present invention, the expression “protein derived from lactic acid bacteria” means a protein isolated and purified from lactic acid bacteria. Herein, although the lactic acid bacteria may be any lactic acid bacteria that ferment sugars to produce lactic acid, the lactic acid bacteria are preferably Lactobacillus, more preferably Lactobacillus rhamnosus.
In the present invention, the expression “protein derived from lactic acid bacteria” means “P8 protein” or “P14 protein”, but is not limited thereto.
In one embodiment of the present invention, the term “P8 protein (protein No. 8)” means an 8-KDa protein fragment extracted from lactic acid bacteria (Lactobacillus rhamnosus) according to a preparation method described in an example of the present invention. The P8 protein in the present invention may be defined by the nucleotide sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2.
In one embodiment of the present invention, the term “P14 protein (protein No. 14)” means a 14-KDa protein fragment extracted from lactic acid bacteria (Lactobacillus rhamnosus) according to a preparation method described in an example of the present invention. The P14 protein in the present invention may be defined by the nucleotide sequence of SEQ ID NO: 3 or the amino acid sequence of SEQ ID NO: 4.
In one embodiment of the present invention, the term “cystatin” refers to a substance that inhibits the activity of intracellular proteases. Cystatin is currently classified into family 1 (cystatin-alpha, and cystatin-beta) that is present in cells, secretory family 2 (cystatin C, cystatin D, cystatin S, egg white cystatin, and milk cystatin), family 3 (kininogen), and the like. Cystatin-alpha exists in leukocytes, the epithelial tissue of epidermis, digestive tracts, vagina, and etc., and the like, and functions to protect the human body from the invasion of pathogenic microbes. Cystatin-beta is present in most cytosols, and functions to inhibit the activity of proteases to thereby prevent viral infections. Egg white cystatin also helps prevent viral infections, and a substance having the same function as that of egg white cystatin is oryzacystatin that is found in rice seeds. Cystatin C is a functional protein that is produced in certain amounts every moment by all the cells of the human body and functions to prevent the onset of diseases and maintain normal physiology, and particularly, it plays an important role in the defense function of the immune system. However, it was reported that blood cystatin C levels increase in various pathologies. If blood cystatin C levels are 1 mg/L or higher, the incidence rate of heart attack, stroke, sudden cardiac death or chronic renal failure increases as the value increases. Elucidation of the secretory mechanism and function of cystatin D is still insufficient.
In one embodiment of the present invention, the term “colorectal disease” refers to a group of various diseases that occur in the colon. The colorectal disease is preferably colorectal cancer, colorectal polyp, colitis, ischemic bowel disease, dysentery, intestinal vascular dysplasia, diverticulosis, irritable bowel syndrome, or Crohn's disease, but is not limited thereto. More preferably, the colorectal disease is colorectal cancer.
In one embodiment of the present invention, “pharmaceutical composition” refers to a composition to be administered for a specific purpose. For the purpose of the present invention, the pharmaceutical composition of the present invention contains, as active ingredients, cystatin and a protein derived from lactic acid bacteria. Preferably, the protein derived from lactic acid bacteria may be “P8 protein” or “P14 protein”, and the cystatin may be “cystatin A” or “cystatin D”. The pharmaceutical composition of the present invention may contain protein and a pharmaceutically acceptable carrier, excipient or diluent, which are involved therein. The "pharmaceutically acceptable" carrier or excipient means one approved by a regulatory agency of the Federal or a state government, or one listed in the pharmacopoeia or other generally recognized pharmacopoeia for use in vertebral animals, and more particularly in humans.
For parenteral administration, the pharmaceutical composition of the present invention can be in the form of suspensions, solutions, or emulsions, in oily or aqueous vehicles, and can be in the form of solid or semi-solid, preferably liquid. Furthermore, the pharmaceutical composition of the present invention can contain formulatory agents such as suspending, stabilizing, solubilizing, and/or dispersing agents, and can be sterilized. The pharmaceutical composition can be stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. Alternatively, the pharmaceutical composition of the present invention can be in sterile powder form for reconstitution with a suitable vehicle before use. The pharmaceutical composition can be presented in unit dose form, in microneedle patch, in ampoules, or other unit-dose containers, or in multi-dose containers. Alternatively, the pharmaceutical composition can be stored in a freeze-dried (lyophilized) condition requiring only the addition of sterile liquid carrier, for example, water for injection immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules or tablets.
In some non-limiting embodiments, the pharmaceutical composition of the present invention may be formulated as liquid or contained as microspheres in liquids. In some non-limiting embodiments, excipients that are suitable for the pharmaceutical composition of the present invention may include preservatives, suspending agents, stabilizers, dyes, buffers, antibacterial agents, antifungal agents, and isotonic agents, for example, sugars or sodium chloride. As used herein, the term "stabilizer" refers to a compound optionally used in the pharmaceutical composition of the present invention in order to increase storage life. In non-limiting embodiments, additional stabilizers may be sugars, amino acids or polymers. In addition, the pharmaceutical composition of the present invention can comprise one or more pharmaceutically acceptable carriers. The carrier can be a solvent or dispersion medium. Non-limiting examples of pharmaceutically acceptable carriers include water, saline, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), oils, and suitable mixtures thereof. Non-limiting examples of sterilization techniques that are applied to the pharmaceutical composition of the present invention include filtration through a bacterial-restraining filter, terminal sterilization, incorporation of sterilizing agents, irradiation, sterilization gas irradiation, heating, vacuum drying, and freeze drying.
In one embodiment of the present invention, “food composition” can be used in various foods, for example, beverages, gums, teas, vitamin complexes, health supplement foods or the like, and may be used as pills, powders, granules, infusions, tablets, capsules or beverages, and the content (wt%) of the food composition in foods is not limited.
The food composition of the present invention may contain conventional food additives known in the art, for example, flavorings, colorants, fillers, stabilizers and the like. The food composition according to the present invention is not particularly limited, as long as it is a composition containing, as essential ingredients, cystatin and the protein derived from lactic acid bacteria. The food composition of the present invention may additionally contain various flavorings or natural carbohydrates as in conventional beverages. Examples of the natural carbohydrates include conventional sugars, such as monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), polysaccharides (e.g., dextrin, cyclodextrin, etc.), and sugar alcohols such as xylitol, sorbitol, erythritol or the like. Examples of flavorings that may be used in the present invention include natural flavorings (thaumatin, stevia extracts, such as rebaudioside A, glycyrrhizin, etc.) and synthetic flavorings (saccharin, aspartame, etc.). The natural carbohydrate is used in an amount of about 1-20 g, preferably about 5-12 g, based on 100 mL of the composition of the present invention.
In addition, the food composition of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavorings such as synthetic flavorings and natural flavorings, colorants, extenders (cheese, chocolate, etc.), pectic acid and its salt, alginic acid and its salt, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonizing agents as used in carbonated beverages, etc. Such components may be used individually or in combination. Although the percentage of such additives is not of great importance, it is generally selected in a range of 0 to about 20 parts by weight based on 100 parts by weight of the composition of the present invention.
In one embodiment of the present invention, “administration” means introducing the composition of the present invention into a patient by any suitable method. The composition of the present invention may be administered by any general route, as long as it can reach a target tissue. Specifically, the composition of the present invention may be administered orally, intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, intranasally, intrapulmonarily, intrarectally, intracavitally or intrathecally. However, the pharmaceutical composition of the present invention, which contains, as active ingredients, cystatin and a protein derived from lactic acid bacteria, is administered intravenously as a liquid formulation or administered as orally as a powder, tablet, capsule or liquid formulation, but is not limited thereto.
A method for treating colorectal disease according to the present invention may comprise administering a pharmaceutically effective amount of the pharmaceutical composition. In the present invention, the effective amount can be determined depending on various factors, including the kind of disease, the severity of the disease, the kinds and contents of active ingredient and other ingredients in the composition, the kind of formulation, the patient’s age, body weight, general health condition, sex and diet, the time of administration, the route of administration, the secretion rate of the composition, the period of treatment, and other drugs that are concurrently used.
In one embodiment, the present invention provides a pharmaceutical composition for treating colorectal disease, which contains, as active ingredients, cystatin and a protein derived from lactic acid bacteria. In the pharmaceutical composition, the protein derived from lactic acid bacteria comprises an amino acid sequence represented by SEQ ID NO: 2 or 4, and the cystatin is cystatin A or cystatin D. Furthermore, the lactic acid bacteria are any one or more selected from the group consisting of Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus plantarum, Pediococcus pentosaceus, and Lactobacillus brevis. Preferably, the lactic acid bacteria are Lactobacillus rhamnosus. In addition, the colorectal disease is any one or more selected from the group consisting of colorectal cancer, colorectal polyp, colitis, ischemic bowel disease, dysentery, intestinal vascular dysplasia, diverticulosis, irritable bowel syndrome, or Crohn's disease. Preferably, the colorectal disease is colorectal cancer.
In another embodiment of the present invention, the present invention provides a food composition for alleviating colorectal disease, which contains, as active ingredients, cystatin and a protein derived from lactic acid bacteria. In the food composition, the protein derived from lactic acid bacteria comprises an amino acid sequence represented by SEQ ID NO: 2 or 4, and the cystatin is cystatin A or cystatin D. Furthermore, the lactic acid bacteria are any one or more selected from the group consisting of Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus plantarum, Pediococcus pentosaceus, and Lactobacillus brevis. Preferably, the lactic acid bacteria are Lactobacillus rhamnosus. In addition, the colorectal disease is any one or more selected from the group consisting of colorectal cancer, colorectal polyp, colitis, ischemic bowel disease, dysentery, intestinal vascular dysplasia, diverticulosis, irritable bowel syndrome, or Crohn's disease. Preferably, the colorectal disease is colorectal cancer.
In another embodiment of the present invention, the present invention provides a method of treating colorectal disease, the method comprising administering to a patient in need thereof a pharmaceutical composition containing an effective amount of cystatin and a protein derived from lactic acid bacteria. In the method, the protein derived from lactic acid bacteria comprises an amino acid sequence represented by SEQ ID NO: 2 or 4, and the cystatin is cystatin A or cystatin D. Furthermore, the lactic acid bacteria are any one or more selected from the group consisting of Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus plantarum, Pediococcus pentosaceus, and Lactobacillus brevis. Preferably, the lactic acid bacteria are Lactobacillus rhamnosus. In addition, the colorectal disease is any one or more selected from the group consisting of colorectal cancer, colorectal polyp, colitis, ischemic bowel disease, dysentery, intestinal vascular dysplasia, diverticulosis, irritable bowel syndrome, or Crohn's disease. Preferably, the colorectal disease is colorectal cancer.
In still another embodiment of the present invention, the present invention provides a use of cystatin and a protein derived from lactic acid bacteria thereof, for preparation of a medicament for treatment of colorectal disease. In the use, the protein derived from lactic acid bacteria comprises an amino acid sequence represented by SEQ ID NO: 2 or 4, and the cystatin is cystatin A or cystatin D. Furthermore, the lactic acid bacteria are any one or more selected from the group consisting of Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus plantarum, Pediococcus pentosaceus, and Lactobacillus brevis. Preferably, the lactic acid bacteria are Lactobacillus rhamnosus. In addition, the colorectal disease is any one or more selected from the group consisting of colorectal cancer, colorectal polyp, colitis, ischemic bowel disease, dysentery, intestinal vascular dysplasia, diverticulosis, irritable bowel syndrome, or Crohn's disease. Preferably, the colorectal disease is colorectal cancer.
Hereinafter, each step of the present invention will be described in detail.
Although it is known that lactic acid bacteria are effective for the prevention, alleviation and treatment of colorectal diseases, including colorectal cancer, colitis, irritable bowel syndrome, Crohn's disease and the like, the development of therapeutic agents that are used together with lactic acid bacterial components to exhibit synergistic effects is still insufficient.
The pharmaceutical composition of the present invention, which contains cystatin and the protein derived from lactic acid bacteria, exhibits a significantly increased inhibitory effect against colorectal cancer compared to when cystatin and the protein derived from lactic acid bacteria are used alone. Thus, the pharmaceutical composition of the present invention will be effectively used as a therapeutic agent against colorectal cancer in the medical field.
FIG. 1 is a schematic view showing protein treatment processes in Experimental Examples 1-1 to 1-9 of the present invention.
FIG. 2 is a schematic view showing protein treatment processes in Experimental Examples 2-1 to 2-9 of the present invention.
FIG. 3 is a graph showing that the growth of colorectal cancer cells was inhibited by protein treatment in Experimental Examples 1-1 to 1-9 of the present invention.
FIG. 4 is a graph showing that the growth of colorectal cancer cells was inhibited by protein treatment in Experimental Examples 2-1 to 2-9 of the present invention.
When the P8 protein and cystatin proteins were administered in combination, the growth of the cancer cells (HCT116 and DLD-1 cells) was significantly inhibited compared to when the P8 protein and cystatin proteins were administered alone.
Hereinafter, the present invention will be described in further detail. It will be obvious to those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1: Isolation and Purification of Protein from Lactic Acid Bacteria
Example 1-1: Purification of Protein from Lactic Acid Bacteria
The colorectal cancer cell line DLD-1 was treated with culture supernatants or cell lysates of various lactic acid bacteria to examine the anticancer activities of the supernatants or lysates. Among them, a cell lysate of Lactobacillus rhamnosus (KCTC 12202BP) having the highest anticancer activity was selected.
To purify an anticancer protein as an active ingredient from the cell lysate of Lactobacillus rhamnosus, size exclusion chromatography (Sephadex G-25, desalting column, GE Healthcare) was performed using a fast protein liquid chromatography (FPLC) system (GE Healthcare) to isolate only a protein.
The fraction containing the isolated protein was dialyzed with 20 mM Tris buffer (pH8.0), and the protein not adsorbed onto HiTrap DEAE FF (GE Healthcare) was collected, and concentrated using a 3-KDa membrane, after which it was dialyzed again with 0.05 M phosphate (pH 6.0) buffer, adsorbed onto HiTrap DEAE SP (GE Healthcare), and sequentially separated according to the concentration gradient of 0.5M sodium chloride. Colorectal cancers were treated with each of the isolated fractions to examine the anticancer activity of each fraction, and the fraction having the highest anticancer activity was concentrated and analyzed by SDS-PAGE, thereby isolating a 8-KDa protein and a 14-KDa protein, which were named “P8 protein” and “P14 protein”, respectively.
Example 1-2: Identification of Protein Derived from Lactic Acid Bacteria
P8 protein or P14 protein, purified according to the method of Example 1-1, was electrophoresed, and then a band was excised from the Coomassie gel and stirred in 100 μl of a destaining solution (50% MeOH/D.W) for 5 minutes, followed by complete removal of the destaining solution. Then, the resulting material was stored in 100 μl of 200 mM ABC (ammonium bicarbonate, pH 7.8) solution for 20 minutes, followed by removal of the ABC solution. Then, the resulting material was stirred in 100 μl of acetonitrile for 2 minutes, followed by removal of the acetonitrile solution, and was stirred in 100 μl of ABC solution for 2 minutes, followed by removal of the ABC solution, and was then stirred in 100 μl of acetonitrile for 2 minutes. This process was repeated a total of three times. Next, the acetonitrile was completely removed, and the remaining gel was dried. 20 μl of trypsin solution (0.2 μg) was added to the dried gel which was then kept on ice for 45 minutes. Next, the solution other than the trypsin solution absorbed into the gel was removed, and 70 μl of 50 mM ABC solution was added to the gel which was then stored at 37°C for 12 hours or more. A micro-scale column was prepared using Geload tip and Poros R2 resin. A 10 ml syringe was prepared such that the front of the syringe was consistent with the column so as to be able to apply pressure to the micro-column. 20 μl of formic acid solution was added to the prepared column and passed through the column by means of the syringe (equilibration), and 30 μl of a peptide solution made in in-gel digestion was added to the micro-column and passed through the column by means of the syringe (loading). 20 μl of 5% formic acid solution was added to the column and passed through the column by means of the syringe, and 1.5 μl of 50% methanol/49% H2O/1% formic acid solution was added to the column, and the peptide was eluted from the column by the syringe (rinse). 1.5 μl of the eluate was placed in a nanoelectrospray needle (EconoTipTM, New Objective, USA) which was then placed into a Q-TOF source (elution), and the amino acid sequence of the isolated protein was analyzed using the Hybrid Quadrupole-TOF LC/MS/MS mass spectrometer (AB Sciex Instruments, CA 94404 USA). The amino acid sequence of the analyzed P8 protein or P14 protein and a nucleotide sequence corresponding thereto is shown in Table 1 below.
SEQ ID NO Definition Sequence
SEQ ID NO: 1 Nucleotide sequence of P8 protein gcaacagtagatcctgaaaagacattgtttctcgatgaaccaatgaacaaggtatttgactggagcaacagcgaagcacctgtacgtgatgcgctgtgggattattacatggaaaagaacagccgtgataccatcaagactgaagaagaaatgaaaccagtcctagacatgtccgacgatgaggtcaaagccctagcagaaaaggttctcaagaagtaa
SEQ ID NO: 2 Amino acid sequence of P8 protein ATVDPEKTLFLDEPMNKVFDWSNSEAPVRDALWDYYMEKNSRDTIKTEEEMKPVLDMSDDEVKALAEKVLKK
SEQ ID NO: 3 Nucleotide sequence of P14 protein gctaaatcacaagatcaatttaacgaaaaagctggtaaaaaaattacagtttcagatgaagctgttgataaagctgctaaaaaaattgaacaagttggttacgttacagaaaaagatgttccagaaatgattgatcgtgattacacacgtgctctttcaaaaaaagtttcagctaaacttcatcaagataaagatgatgattacttttacgaagaaccatttgattacgaaaacggtcgtattgctaacattatttgggatatggataaaattaaaacacgtgaagaagctatgaaaacacttgctaacgaacttggtcttacagttccaaaaattgttatgcgtaaagttgatgaacaagttttt
SEQ ID NO: 4 Amino acid sequence of P14 protein AKSQDQFNEKAGKKITVSDEAVDKAAKKIEQVGYVTEKDVPEMIDRDYTRALSKKVSAKLHQDKDDDYFYEEPFDYENGRIANIIWDMDKIKTREEAMKTLANELGLTVPKIVMRKVDEQVF
Example 2: Analysis of the Effects of Cystatin and Protein Derived from Lactic Acid Bacteria against Colorectal Cancer Cells
Each of colorectal cancer cell lines (HCT116 and DLD-1) was cultured in a 12-well plate at a density of 5x104 cells/well, and experimental examples were classified as shown in Table 2 below according to the order of treatment with P8 protein and cystatin A.
Experimental Example Definition
Experimental Example 1-1 Not treated
Experimental Example 1-2 Treated once with P8 protein (20 μg)
Experimental Example 1-3 Treated once with P8 protein (50 μg)
Experimental Example 1-4 Treated with P8 protein (20 μg) for 3 days (once a day), and then treated with cystatin A (10 μg) for 3 days (once a day)
Experimental Example 1-5 Treated with P8 protein (20 μg) for 3 days (once a day), and then treated with cystatin A (20 μg) for 3 days (once a day)
Experimental Example 1-6 Treated with P8 protein (50 μg) for 3 days (once a day), and then treated with cystatin A (10 μg) for 3 days (once a day)
Experimental Example 1-7 Treated with P8 protein (50 μg) for 3 days (once a day), and then treated with cystatin A (20 μg) for 3 days (once a day)
Experimental Example 1-8 Treated with P8 protein (20 μg) for 6 days (once a day)
Experimental Example 1-9 Treated with P8 protein (50 μg) for 6 days (once a day)
Experimental Example 2-1 Not treated
Experimental Example 2-2 Treated once with cystatin A (10 μg)
Experimental Example 2-3 Treated once with cystatin A (20 μg)
Experimental Example 2-4 Treated with cystatin A (10 μg) for 3 days (once a day), and then treated with P8 protein (20 μg) for 3 days (once a day)
Experimental Example 2-5 Treated with cystatin A (10 μg) for 3 days (once a day), and then treated with P8 protein (50 μg) for 3 days (once a day)
Experimental Example 2-6 Treated with cystatin A (20 μg) for 3 days (once a day), and then treated with P8 protein (20 μg) for 3 days (once a day)
Experimental Example 2-7 Treated with cystatin A (20 μg) for 3 days (once a day), and then treated with P8 protein (50 μg) for 3 days (once a day)
Experimental Example 2-8 Treated with cystatin A (10 μg) for 6 days (once a day)
Experimental Example 2-9 Treated with cystatin A (20 μg) for 6 days (once a day)
As shown in Table 2 above, the cell line HCT116 or DLD-1 was treated with the protein in each of Experimental Examples 1-1 to 1-9 and Experimental Examples 2-1 and 2-9. As a negative control, 0.1% PBS, a buffer solution used for purification of the protein was used. More specifically, FIGS. 1 and 2 are schematic views showing the protein treatment processes.
After completion of the protein treatment, each well was treated with 200 μl of a reagent (MTT formazan, 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-tetrazolium bromide) for cell survival rate measurement and incubated for 2 hours. The absorbance of each well at 570 nm was measured using a microplate reader (Amersham, Biorad, USA, Japan), and based on the measured value, the cell survival rate was calculated. As a result, it was shown that, when the P8 protein and cystatin proteins were administered in combination, the growth of the cancer cells (HCT116 and DLD-1 cells) was significantly inhibited compared to when the P8 protein and cystatin proteins were administered alone. In addition, it was shown that the order of treatment with P8 protein and cystatin had no great effect on the inhibition of the cancer cells.
Although the present disclosure has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only of a preferred embodiment thereof, and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.
The present invention is directed to a pharmaceutical composition for treating colorectal disease, which contains cystatin and a protein derived from lactic acid bacteria. The composition of the present invention has been demonstrated to have significant effects on the inhibition of colorectal cancer growth, and thus will be effectively used as a therapeutic agent against colorectal cancer in the medical field.
SEQ ID NO: 1 -
gcaacagtag atcctgaaaa gacattgttt ctcgatgaac caatgaacaa ggtatttgac tggagcaaca gcgaagcacc tgtacgtgat gcgctgtggg attattacat ggaaaagaac agccgtgata ccatcaagac tgaagaagaa atgaaaccag tcctagacat gtccgacgat gaggtcaaag ccctagcaga aaaggttctc aagaagtaa
SEQ ID NO: 2 -
Ala Thr Val Asp Pro Glu Lys Thr Leu Phe Leu Asp Glu Pro Met Asn Lys Val Phe Asp Trp Ser Asn Ser Glu Ala Pro Val Arg Asp Ala Leu Trp Asp Tyr Tyr Met Glu Lys Asn Ser Arg Asp Thr Ile Lys Thr Glu Glu Glu Met Lys Pro Val Leu Asp Met Ser Asp Asp Glu Val Lys Ala Leu Ala Glu Lys Val Leu Lys Lys
SEQ ID NO: 3 -
gctaaatcac aagatcaatt taacgaaaaa gctggtaaaa aaattacagt ttcagatgaa gctgttgata aagctgctaa aaaaattgaa caagttggtt acgttacaga aaaagatgtt ccagaaatga ttgatcgtga ttacacacgt gctctttcaa aaaaagtttc agctaaactt catcaagata aagatgatga ttacttttac gaagaaccat ttgattacga aaacggtcgt attgctaaca ttatttggga tatggataaa attaaaacac gtgaagaagc tatgaaaaca cttgctaacg aacttggtct tacagttcca aaaattgtta tgcgtaaagt tgatgaacaa gttttt
SEQ ID NO: 4 -
Ala Lys Ser Gln Asp Gln Phe Asn Glu Lys Ala Gly Lys Lys Ile Thr Val Ser Asp Glu Ala Val Asp Lys Ala Ala Lys Lys Ile Glu Gln Val Gly Tyr Val Thr Glu Lys Asp Val Pro Glu Met Ile Asp Arg Asp Tyr Thr Arg Ala Leu Ser Lys Lys Val Ser Ala Lys Leu His Gln Asp Lys Asp Asp Asp Tyr Phe Tyr Glu Glu Pro Phe Asp Tyr Glu Asn Gly Arg Ile Ala Asn Ile Ile Trp Asp Met Asp Lys Ile Lys Thr Arg Glu Glu Ala Met Lys Thr Leu Ala Asn Glu Leu Gly Leu Thr Val Pro Lys Ile Val Met Arg Lys Val Asp Glu Gln Val Phe

Claims (28)

  1. A pharmaceutical composition for treating colorectal disease, which contains, as active ingredients, cystatin and a protein derived from lactic acid bacteria.
  2. The pharmaceutical composition of claim 1, wherein the protein derived from lactic acid bacteria is a protein comprising an amino acid sequence represented by SEQ ID NO: 2 or 4.
  3. The pharmaceutical composition of claim 1, wherein the cystatin is cystatin A or cystatin D.
  4. The pharmaceutical composition of claim 1, wherein the lactic acid bacteria are any one or more selected from the group consisting of Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus plantarum, Pediococcus pentosaceus, and Lactobacillus brevis.
  5. The pharmaceutical composition of claim 4, wherein the lactic acid bacteria are Lactobacillus rhamnosus.
  6. The pharmaceutical composition of claim 1, wherein the colorectal disease is any one or more selected from the group consisting of colorectal cancer, colorectal polyp, colitis, ischemic bowel disease, dysentery, intestinal vascular dysplasia, diverticulosis, irritable bowel syndrome, and Crohn's disease.
  7. The pharmaceutical composition of claim 6, wherein the colorectal disease is colorectal cancer.
  8. A food composition for alleviating colorectal disease, which contains, as active ingredients, cystatin and a protein derived from lactic acid bacteria.
  9. The food composition of claim 8, wherein the protein derived from lactic acid bacteria is a protein comprising an amino acid sequence represented by SEQ ID NO: 2 or 4.
  10. The food composition of claim 8, wherein the cystatin is cystatin A or cystatin D.
  11. The food composition of claim 8, wherein the lactic acid bacteria are any one or more selected from the group consisting of Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus plantarum, Pediococcus pentosaceus, and Lactobacillus brevis.
  12. The food composition of claim 11, wherein the lactic acid bacteria are Lactobacillus rhamnosus.
  13. The food composition of claim 8, wherein the colorectal disease is any one or more selected from the group consisting of colorectal cancer, colorectal polyp, colitis, ischemic bowel disease, dysentery, intestinal vascular dysplasia, diverticulosis, irritable bowel syndrome, and Crohn's disease.
  14. The food composition of claim 13, wherein the colorectal disease is colorectal cancer.
  15. A method of treating colorectal disease, which comprising administering to a subject in need thereof a pharmaceutical composition containing a therapeutically effective amount of cystatin and a protein derived from lactic acid bacteria.
  16. The method of claim 15, wherein the protein derived from lactic acid bacteria is a protein comprising an amino acid sequence represented by SEQ ID NO: 2 or 4.
  17. The method of claim 15, wherein the cystatin is cystatin A or cystatin D.
  18. The method of claim 15, wherein the lactic acid bacteria are any one or more selected from the group consisting of Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus plantarum, Pediococcus pentosaceus, and Lactobacillus brevis.
  19. The method of claim 18, wherein the lactic acid bacteria are Lactobacillus rhamnosus.
  20. The method of claim 15, wherein the colorectal disease is any one or more selected from the group consisting of colorectal cancer, colorectal polyp, colitis, ischemic bowel disease, dysentery, intestinal vascular dysplasia, diverticulosis, irritable bowel syndrome, and Crohn's disease.
  21. The method of claim 20, wherein the colorectal disease is colorectal cancer.
  22. Use of cystatin and a protein derived from lactic acid bacteria thereof, for preparation of a medicament for treatment of colorectal disease.
  23. The use of claim 22, wherein the protein derived from lactic acid bacteria is a protein comprising an amino acid sequence represented by SEQ ID NO: 2 or 4.
  24. The use of claim 22, wherein the cystatin is cystatin A or cystatin D.
  25. The use of claim 22, wherein the lactic acid bacteria are any one or more selected from the group consisting of Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus plantarum, Pediococcus pentosaceus, and Lactobacillus brevis.
  26. The use of claim 25, wherein the lactic acid bacteria are Lactobacillus rhamnosus.
  27. The use of claim 22, wherein the colorectal disease is any one or more selected from the group consisting of colorectal cancer, colorectal polyp, colitis, ischemic bowel disease, dysentery, intestinal vascular dysplasia, diverticulosis, irritable bowel syndrome, and Crohn's disease.
  28. The use of claim 27, wherein the colorectal disease is colorectal cancer.
PCT/KR2017/002512 2017-01-25 2017-03-08 Pharmaceutical composition for treating colorectal disease containing cystatin and a protein derived from lactic acid bacteria WO2018139703A1 (en)

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