WO2021002664A1 - Composition pour la prévention, le soulagement ou le traitement d'un cancer - Google Patents

Composition pour la prévention, le soulagement ou le traitement d'un cancer Download PDF

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WO2021002664A1
WO2021002664A1 PCT/KR2020/008569 KR2020008569W WO2021002664A1 WO 2021002664 A1 WO2021002664 A1 WO 2021002664A1 KR 2020008569 W KR2020008569 W KR 2020008569W WO 2021002664 A1 WO2021002664 A1 WO 2021002664A1
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cancer
mir
present
cell line
mimic
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PCT/KR2020/008569
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English (en)
Korean (ko)
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김백길
김지은
조남훈
장연수
강숙희
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연세대학교 산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • A23L33/13—Nucleic acids or derivatives thereof
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A61K8/606—Nucleosides; Nucleotides; Nucleic acids
    • 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
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q90/00—Cosmetics or similar toiletry preparations for specific uses not provided for in other groups of this subclass
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/308—Foods, ingredients or supplements having a functional effect on health having an effect on cancer prevention

Definitions

  • the present invention relates to a composition for preventing, improving or treating cancer.
  • Cancer is one of the most fatal diseases in the world at present, and the incidence of cancer continues to increase due to the prolongation of life expectancy and the decrease in the age of cancer. According to 2013 statistics provided by the National Cancer Center in Korea, the number of cancer-causing patients in Korea registered in the Department of Cancer Registration and Statistics in 2010 was 202,053, and the trend is increasing continuously.
  • siRNA small interfering RNA
  • the siRNA forms a ribonucleic acid protein and an RNA induced silencing complex (RISC) to cleave a messenger RNA (mRNA) having a sequence complementary to the siRNA, thereby inhibiting the protein from being produced from the mRNA.
  • RISC RNA induced silencing complex
  • mRNA messenger RNA
  • the siRNA-based anticancer agent is evaluated as a more advanced technology than the monomolecular anticancer agent in that it blocks the mRNA before the protein production stage, and that it utilizes the RNA and cell-intrinsic RISC system.
  • siRNA-based anticancer drugs have side effects caused by phenomena such as off-target effects.
  • miRNAs that control many biological processes such as cell differentiation, proliferation, apoptosis, development, immunity, metabolism, and stem cell maintenance are actively being conducted.
  • miRNAs unlike peptide or antibody therapeutics, miRNAs have a short development period, and theoretically all therapeutics can be developed if only certain disease target genes are identified.
  • miRNAs generally have very high specificity and efficacy by simultaneously regulating the expression of multiple target genes rather than one. Therefore, there is an urgent need for research on anticancer drugs made of novel miRNAs to overcome the aforementioned side effects of anticancer drugs used in anticancer chemotherapy.
  • One object of the present invention is to provide a composition for preventing, improving or treating cancer.
  • Another object of the present invention is to provide a method for screening a cancer therapeutic agent.
  • One embodiment of the present invention provides a composition for preventing, improving or treating cancer.
  • composition of the present invention may be provided as a pharmaceutical composition, a food composition, and a cosmetic composition.
  • composition of the present invention contains miR-4516 or a mimic thereof as an active ingredient.
  • the miR-4516 of the present invention is a miRNA, and may be derived from animals including humans, for example, monkeys, chimpanzees, pigs, horses, cows, sheep, dogs, cats, mice, rabbits, etc., preferably It may be of human origin.
  • the miR-4516 of the present invention may exist in a single-stranded or double-stranded form.
  • Mature miRNA molecules exist primarily as single strands, but precursor miRNA molecules may contain partial self-complementary structures (eg stem-loop structures) capable of forming double strands.
  • the miRNA of the present invention may be composed of RNA, peptide nucleic acids (PNA), or locked nucleic acid (LNA).
  • the mimic of miR-4516 of the present invention is a precursor miRNA, and may be composed of double strands, but is not limited thereto.
  • the miR-4516 of the present invention when the miR-4516 of the present invention is single-stranded, it may consist of a nucleotide sequence represented by SEQ ID NO: 1, and when the miR-4516 is double-stranded, it is a base that is complementary to SEQ ID NO: 1 and SEQ ID NO: 1
  • the sequence may be formed in a form in which SEQ ID NO: 2 is complementarily linked, but is not limited thereto.
  • RNA refers to an RNA sequence that controls expression of various genes and does not encode a protein, and regulates the level of gene expression after transcription by inhibiting the step of translating mRNA into a protein or inducing degradation of the mRNA itself.
  • the cancer of the present invention may be at least one selected from the group consisting of breast cancer, bladder cancer, cervical cancer, colon cancer, lung cancer, pancreatic cancer, stomach cancer, ovarian cancer, hematologic cancer, liver cancer, prostate cancer, and head and neck cancer, and preferably May be breast cancer, more preferably triple-negative breast cancer (TNBC), but is not limited thereto.
  • TNBC triple-negative breast cancer
  • the "triple-negative breast cancer” of the present invention is a type of breast cancer in which the expression of estrogen receptor, progesterone receptor, and HER2 corresponding to the therapeutic target gene is all negative, and triple-negative breast cancer is histological differentiation compared to non-triple-negative breast cancer patients
  • the grade 3 rate of is high, the recurrence period is fast, and the prognosis is very poor.
  • the triple-negative breast cancer has a limitation in that drugs such as Herceptin, which is a HER2 target antibody drug used as a treatment for breast cancer, cannot be used. Therefore, in the case of using the miR-4516 of the present invention, there is an advantage of being able to very effectively prevent, improve or treat triple negative breast cancer, which is difficult to treat with conventional breast cancer treatments.
  • the miR-4516 of the present invention may target the 3'-UTR of mRNA transcribed from the FOSL1 (FOS-like antigen 1) gene.
  • the miR-4516 of the present invention may degrade FOSL1 mRNA by binding to 3'-UTR of mRNA transcribed from FOSL1 gene or inhibit translation.
  • the "FOSL1 gene” of the present invention is that the expression level of the gene and the protein encoded by it is increased specifically in TNBC compared to breast cancer, particularly non-TNBC, and the miR-4516 of the present invention is the mRNA of FOSL1.
  • the expression of FOSL1 mRNA and protein can be very effectively inhibited to exert a preventive or therapeutic effect on cancer.
  • the "miRNA” of the present invention may all be included even if some of the nucleotide sequences constituting the miRNA are modified by deletion, substitution, or insertion as long as it can function functionally equivalent to the miR-4516. For example, it may include those having 80%, 90%, 95%, or 99% homology with the base sequence constituting the miR-4516.
  • the "homology" of the present invention is intended to indicate the degree of similarity with the nucleotide sequence of a wild type gene, and includes a sequence having the same percentage or more of the same sequence as the nucleotide sequence of the present invention. Such homology can also be determined by visually comparing two sequences, but can be determined using a bioinformatic algorithm that analyzes the degree of homology by arranging the sequences to be compared side by side. The homology between the two base sequences can be expressed as a percentage. Useful automated algorithms are available in the GAP, BESTFIT, FASTA and TFASTA computer software modules of the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, W, USA).
  • Alignment algorithms automated in the module include Needleman & Wunsch, Pearson & Lipman, and Smith & Waterman sequence alignment algorithms. Algorithms and homology determinations for other useful arrangements can be automated in software including FASTP, BLAST, BLAST2, PSIBLAST and CLUSTAL W.
  • the miR-4516 of the present invention may be included in an expression vector and provided for intracellular delivery.
  • miRNA nucleic acid molecules can be introduced into cells using various transformation techniques such as a complex of nucleic acids and DEAE-dextran, complex of nucleic acids and nuclear proteins, complex of nucleic acids and lipids, etc. It may be provided in a form contained in a delivery system that enables efficient introduction of.
  • the carrier is a vector, and both viral vectors and non-viral vectors can be used.
  • a viral vector for example, lentivirus, retrovirus, adenovirus, herpes virus, and abipox virus vector, etc. can be used, It is not limited thereto.
  • the expression vector of the present invention may further include a selection marker to facilitate selection of transformed cells.
  • Markers that can exhibit a selectable phenotype for example drug resistance, auxotrophic, resistance to cytotoxic agents, or expression of surface proteins, i.e. green fluorescent protein, puromycin, neomycin, hygromycin, histi Dinol dehydrogenase (hisD) and guanine phosphoribosyltransferase (Gpt) may further be included, but the present invention is not limited thereto.
  • the miR-4516 of the present invention may be provided in a form introduced into a cell.
  • the method of introducing miR-4516 into cells is a conventional method, for example, G-fectin, Mirus TrasIT-TKO lipophilic reagent, lipofectin, lipofectamine, cellfectin, cationic phospholipid nanoparticles, A method of introducing into a cell together with a delivery reagent including a cationic polymer, a cationic micelle, a cationic emulsion, or a liposome, and the like, but is not limited thereto.
  • the "cell” of the present invention may include any cells constituting the tumor microenvironment around cancer cells, and may be, for example, fibroblasts, but is not limited thereto.
  • the "prevention" of the present invention may include, without limitation, any act of blocking or suppressing or delaying symptoms caused by cancer using the composition of the present invention.
  • the “improvement” or “treatment” of the present invention may include, without limitation, any action that improves or benefits the symptoms caused by cancer using the composition of the present invention.
  • the pharmaceutical composition of the present invention may be characterized in that it is in the form of capsules, tablets, granules, injections, ointments, powders, or beverages, and the pharmaceutical composition may be characterized for human.
  • the pharmaceutical composition of the present invention is not limited thereto, but is formulated and used in the form of oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories, and sterile injection solutions according to conventional methods.
  • the pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers can be used as binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, flavoring agents, etc. for oral administration, and buffers, preservatives, painlessness, etc. for injections.
  • An agent, a solubilizing agent, an isotonic agent, a stabilizer, and the like may be mixed and used, and in the case of topical administration, a base agent, an excipient, a lubricant, a preservative, and the like may be used.
  • the formulation of the pharmaceutical composition of the present invention can be variously prepared by mixing with a pharmaceutically acceptable carrier as described above.
  • a pharmaceutically acceptable carrier for example, for oral administration, it can be prepared in the form of tablets, troches, capsules, elixir, suspension, syrup, wafers, etc., and in the case of injections, it can be prepared in unit dosage ampoules or multiple dosage forms. have. Others, solutions, suspensions, tablets, capsules, can be formulated as sustained-release preparations.
  • Suitable carriers, excipients and diluents for the formulation of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium Silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like may be used.
  • fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, and the like may additionally be included.
  • the route of administration of the pharmaceutical composition of the present invention is not limited thereto, but oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, Includes sublingual or rectal. Oral or parenteral administration is preferred.
  • the "parenteral" of the present invention includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
  • the pharmaceutical composition of the present invention can also be administered in the form of suppositories for rectal administration.
  • the pharmaceutical composition of the present invention depends on a number of factors including the activity of the specific compound used, age, weight, general health, sex, formulation, time of administration, route of administration, excretion rate, drug formulation and the severity of the specific disease to be prevented or treated. It may vary in various ways, and the dosage of the pharmaceutical composition varies depending on the patient's condition, weight, degree of disease, drug form, route and duration of administration, but may be appropriately selected by those skilled in the art, and 0.0001 to 50 mg/day It can be administered in kg or 0.001 to 50 mg/kg. Administration may be administered once a day, or may be divided several times. The above dosage does not in any way limit the scope of the present invention.
  • the pharmaceutical composition according to the present invention may be formulated as a pill, dragee, capsule, liquid, gel, syrup, slurry, or suspension.
  • the food composition of the present invention may be prepared in the form of various foods, for example, beverages, gums, teas, vitamin complexes, powders, granules, tablets, capsules, confectionery, rice cakes, and bread.
  • the amount may be added in a proportion of 0.1 to 50% of the total weight, but is not limited thereto.
  • the food composition of the present invention is prepared in the form of a beverage
  • various flavoring agents or natural carbohydrates, etc. as an additional component like a normal beverage.
  • natural carbohydrates monosaccharides such as glucose, disaccharides such as fructose, and common sugars such as sucrose and polysaccharides, dextrins, cyclodextrins, and sugar alcohols such as xylitol, sorbitol, and erythritol are used.
  • natural carbohydrates monosaccharides such as glucose, disaccharides such as fructose, and common sugars such as sucrose and polysaccharides, dextrins, cyclodextrins, and sugar alcohols such as xylitol, sorbitol, and erythritol are used.
  • sugar alcohols such as xylitol, sorbitol, and erythritol
  • the flavoring agent may be a natural flavoring agent (taumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and a synthetic flavoring agent (saccharin, aspartame, etc.).
  • a natural flavoring agent for example, rebaudioside A, glycyrrhizin, etc.
  • a synthetic flavoring agent sacharin, aspartame, etc.
  • the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavors and natural flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, It may further include a pH adjusting agent, a stabilizer, a preservative, glycerin, alcohol, a carbonation agent used in carbonated beverages, and the like.
  • the above components of the present invention may be used independently or in combination.
  • the ratio of the additive does not correspond to the core element of the present invention, it may be selected from 0.1 to about 50 parts by weight per 100 parts by weight of the food composition of the present invention, but is not limited thereto.
  • the cosmetic composition of the present invention is a lotion, nutritional lotion, nutritional essence, massage cream, beauty bath water additive, body lotion, body milk, bath oil, baby oil, baby powder, shower gel, shower cream, sunscreen lotion, sunscreen cream , Suntan cream, skin lotion, skin cream, sunscreen cosmetics, cleansing milk, depilatory makeup, face and body lotion, face and body cream, skin whitening cream, hand lotion, hair lotion, cosmetic cream, jasmine oil, bath Manufactured in the form of soap, water soap, beauty soap, shampoo, hand sanitizer (hand cleaner), medicinal soap, medical use, cream soap, facial wash, body cleaner, scalp cleaner, hair rinse, makeup soap, tooth whitening gel, toothpaste, etc. Can be.
  • the composition of the present invention may further include a solvent commonly used in the manufacture of cosmetic compositions, or an appropriate carrier, excipient, or diluent.
  • the type of solvent that can be further added to the cosmetic composition of the present invention is not particularly limited, but, for example, water, saline, DMSO, or a combination thereof may be used.
  • excipients or diluents purified water, oil, wax, fatty acids, fatty alcohols, fatty acid esters, surfactants, humectants, thickeners, antioxidants, viscosity stabilizers, chelating agents, buffers, lower alcohols, etc. Included, but not limited to.
  • whitening agents, moisturizing agents, vitamins, sunscreen agents, perfumes, dyes, antibiotics, antibacterial agents, and antifungal agents may be included as needed.
  • Hydrogenated vegetable oil, castor oil, cottonseed oil, olive oil, palm seed oil, jojoba oil, and avocado oil may be used as the oil of the present invention, and waxes include beeswax, spermaceti, carnauba, candelilla, montan, ceresin, liquid paraffin , Lanolin can be used.
  • Stearic acid, linoleic acid, linolenic acid, and oleic acid may be used as the fatty acid of the present invention, and as fatty acid alcohols, cetyl alcohol, octyl dodecanol, oleyl alcohol, panthenol, lanolin alcohol, stearyl alcohol, hexadecanol
  • fatty acid ester isopropyl myristate, isopropyl palmitate, and butyl stearate
  • surfactant cationic surfactants, anionic surfactants, and nonionic surfactants known in the art can be used, and surfactants derived from natural products are preferred as far as possible.
  • hygroscopic agents, thickeners, antioxidants, and the like which are widely known in the cosmetic field, may be included, and the types and amounts thereof are as known in the art.
  • Another embodiment of the present invention provides a method for screening a cancer therapeutic agent.
  • the screening method of the present invention comprises the steps of treating a target candidate material in a biological sample isolated from a cancer patient; And determining the level of miR-4516 present in the biological sample.
  • the step of selecting the candidate substance as a cancer treatment may be further included.
  • the information on cancer, triple negative breast cancer and miR-4516 is the same as described in the composition, and thus is omitted to avoid excessive complexity of the specification.
  • the biological sample of the present invention refers to any substance, biological body fluid, tissue or cell obtained from or derived from an individual, for example, whole blood, leukocytes, peripheral blood mononuclear cells ( peripheral blood mononuclear cells), leukocyte buffy coat, blood including plasma and serum, sputum, tears, mucus, nasal washes, nasal cavity Nasal aspirate, breath, urine, semen, saliva, peritoneal washings, pelvic fluids, cystic fluid, Meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate aspirate), synovial fluid, joint aspirate, organ secretions, cells, cell extracts, or cerebrospinal fluid, but are limited thereto. It is not.
  • the candidate substance of the present invention refers to a drug for testing whether there is an activity for preventing, ameliorating or treating cancer symptoms, that is, an activity capable of increasing the expression level of miR-4516, protein, oligo Includes any molecule such as peptides, organic molecules, polysaccharides, polynucleotides and a wide range of compounds. These candidate substances may include not only natural substances but also synthetic substances.
  • Measurement of the level of the present miR-4516 of the present invention using a formulation capable of measuring the level of the presence of the miR-4516, RT-PCR, quantitative real time PCR (quantified real time PCR), competitive RT-PCR ( Competitive RT-PCR), real time quantitative RT-PCR (RT-PCR), RNase protection assay (RPA), Northern blot assay, DNA chip analysis, etc. It may be used for, but is not limited thereto.
  • the agent capable of measuring the level of the miR-4516 present of the present invention may be a primer or probe specific for miR-4516, but is not limited thereto.
  • the "primer” of the present invention is a base sequence having a short free 3'hydroxyl group and can form a complementary template strand and a base pair, and start for template strand copying It refers to a short nucleotide sequence that functions as a point.
  • the primers can initiate DNA synthesis in the presence of a reagent for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer solution and temperature.
  • the "probe” of the present invention refers to a nucleic acid fragment such as RNA or DNA corresponding to a base capable of specifically binding to the gene, and such a probe is the presence or absence of a specific gene and mRNA transcribed therefrom, a gene It may be labeled to confirm the expression level (expression amount) of the mRNA transcribed from.
  • the probe may be manufactured in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, an RNA probe, etc., but is not limited thereto.
  • the primer or probe of the present invention can be easily prepared by a person skilled in the art by referring to a conventional method based on the nucleotide sequence represented by SEQ ID NO: 1.
  • a method for preventing or treating cancer comprising administering to an individual an effective amount of a composition comprising the miR-4516 or mimic thereof according to the present invention as an active ingredient will be.
  • the "individual” is an individual in need of prevention or treatment of cancer, and may include both mammals and non-mammals.
  • examples of the mammal include humans, non-human primates such as chimpanzees, other apes or monkey species; Livestock animals such as cattle, horses, sheep, goats, pigs; Domesticated animals such as rabbits, dogs or cats; Experimental animals, for example rodents, such as rats, mice, guinea pigs, and the like may be included, but are not limited thereto.
  • examples of the non-mammals in the present invention may include birds or fish, but are not limited thereto.
  • the term "administration" refers to a process of introducing the active ingredient of the present invention to an individual by any appropriate method, and the formulation of the compound administered as described above is not particularly limited, and is in a solid form, liquid It can be administered in a form of a formulation or a aerosol formulation for aspiration, and can be administered in a solid form formulation intended to be converted into a liquid formulation for oral or parenteral administration immediately before use, for example, powders, granules, capsules. , Tablets, oral dosage forms such as aqueous suspensions, external preparations, suppositories, and sterile injection solutions may be formulated and administered, but are not limited thereto.
  • a pharmaceutically acceptable carrier may be additionally administered together with the compound of the present invention.
  • the pharmaceutically acceptable carrier may be used as a binder, a lubricant, a disintegrant, an excipient, a solubilizing agent, a dispersant, a stabilizer, a suspending agent, a coloring agent, a flavoring agent, etc. for oral administration, and in the case of an injection, a buffer agent, Preservatives, painless agents, solubilizers, isotonic agents, stabilizers, etc. can be mixed and used, and in the case of topical administration, base agents, excipients, lubricants, preservatives, and the like can be used.
  • Formulations of the compounds of the present invention can be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above.
  • a pharmaceutically acceptable carrier for example, for oral administration, it can be prepared in the form of tablets, troches, capsules, elixir, suspension, syrup, wafers, etc., and in the case of injections, it can be prepared in unit dosage ampoules or multiple dosage forms.
  • Others, solutions, suspensions, tablets, capsules, can be formulated as sustained-release preparations.
  • suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil may be used.
  • fillers, anti-aggregating agents, lubricants, wetting agents, flavoring agents, emulsifying agents, preservatives, and the like may additionally be included.
  • the route of administration of the compound according to the present invention is not limited to these, but oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual Or work. Oral or parenteral administration is preferred.
  • parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
  • the pharmaceutical composition of the present invention can also be administered in the form of suppositories for rectal administration.
  • an “effective amount” refers to an amount sufficient of an agent to provide a desired biological result.
  • the result may be a reduction and/or alleviation of signs, symptoms or causes of the disease, or any other desirable change in the biological system.
  • an “effective amount” for therapeutic use is the amount of a compound disclosed herein that is required to provide a clinically significant reduction in disease.
  • An appropriate “effective” amount in any individual case can be determined by one of skill in the art using routine experimentation.
  • the expression “effective amount” generally refers to the amount in which the active substance has a therapeutic effect.
  • the active substance is a prevention, amelioration or treatment of cancer.
  • the compounds of the present invention vary depending on a number of factors, including the activity of the specific compound used, age, weight, general health, sex, formulation, time of administration, route of administration, excretion rate, drug formulation and the severity of the particular disease to be prevented or treated. It may vary, and the dosage of the compound varies depending on the patient's condition, weight, degree of disease, drug form, route and duration of administration, but may be appropriately selected by those skilled in the art, and from 0.0001 to 100 mg/kg per day or from 0.001 to It can be administered at 100mg/kg. Administration may be administered once a day, or may be divided several times. The above dosage does not in any way limit the scope of the present invention.
  • the compound according to the present invention may be formulated as a pill, dragee, capsule, liquid, gel, syrup, slurry, or suspension.
  • the compounds of the present invention can be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy, and methods using biological response modifiers.
  • the cancer of the present invention may be at least one selected from the group consisting of breast cancer, bladder cancer, cervical cancer, colon cancer, lung cancer, pancreatic cancer, stomach cancer, ovarian cancer, hematologic cancer, liver cancer, prostate cancer, and head and neck cancer, and preferably May be breast cancer, more preferably triple-negative breast cancer (TNBC), but is not limited thereto.
  • TNBC triple-negative breast cancer
  • composition comprising miR-4516 or a mimic thereof according to the present invention as an active ingredient specifically binds to the 3'UTR of the mRNA transcribed from the FOSL1 (FOS-like antigen 1) gene, and the mRNA of the FOSL1 By inhibiting the translation of can effectively inhibit the growth of cancer cells, it can be used for prevention, improvement or treatment of cancer. Furthermore, by confirming the change in the expression level of miR-4516 by the candidate substance, it is possible to effectively screen a therapeutic agent for cancer.
  • FOSL1 FOS-like antigen 1
  • NF normal fibroblast
  • CAF cancer-associated fibroblast
  • FIG. 2 shows the results of confirming the effect of reducing the proliferation of breast cancer cell lines by overexpression of miR-4516 according to an embodiment of the present invention through cell growth rate analysis using CCK-8.
  • FIG. 3 is a fluorescence staining for the effect of inhibiting cell proliferation according to co-culture of an NF cell line or CAF cell line and a breast cancer cell line (MCF7 or MDA-MB-231) transformed to overexpress miR-4516 according to an embodiment of the present invention. It shows the results confirmed through.
  • FIG. 4 shows the results of measuring luciferase activity in order to identify a target gene having binding activity to miR-4516 according to an embodiment of the present invention.
  • 5A shows changes in the mRNA expression level of FOSL1, PAX5, or TCL1A in a breast cancer cell line overexpressing miR-4516 according to an embodiment of the present invention through real-time polymerase chain reaction.
  • 5B shows the result of confirming the change in the expression level of the protein of FOSL1, PAX5 or TCL1A in the breast cancer cell line overexpressing miR-4516 according to an embodiment of the present invention through Western blot analysis.
  • Figure 6a is a triple negative breast cancer (TNBC) cell line by translational inhibition of FOSL1 suppressed by overexpression of miR-4516 according to an embodiment of the present invention, confirming the expression level of the gene through real-time polymerase chain reaction It shows the results.
  • TNBC triple negative breast cancer
  • 6B is a result of confirming the expression level of the protein through Western blot analysis in a triple negative breast cancer (TNBC) cell line by translational inhibition of FOSL1 inhibited by overexpression of miR-4516 according to an embodiment of the present invention. Is shown.
  • TNBC triple negative breast cancer
  • 6C is a result of confirming the proliferation inhibitory effect through analysis using CCK-8 in a triple negative breast cancer (TNBC) cell line by translational inhibition of FOSL1 suppressed by overexpression of miR-4516 according to an embodiment of the present invention Is shown.
  • TNBC triple negative breast cancer
  • FIG. 7 shows the result of confirming the proliferation inhibitory effect of the TNBC cell line by treatment with miR-4516 mimic according to an embodiment of the present invention through analysis using CCK-8.
  • FIG. 8 shows the results of confirming the proliferation inhibitory effect of the TNBC cell line by treatment with the miR-4516 mimic even when co-cultured with the CAF cell line and the breast cancer cell line according to an embodiment of the present invention through analysis using CCK-8. .
  • FIG. 9 is a cell growth rate analysis using CCK-8 that the effect of reducing the proliferation of the breast cancer cell line according to the co-culture of the overexpressed CAF cell line of miR-4516 and the breast cancer cell line according to an embodiment of the present invention is inhibited by the treatment of GW4869. It shows the results confirmed through.
  • FIG. 10 shows the results of comparing the proliferation inhibitory effect in non-TNBC cell lines and TNBC cell lines by treatment with miR-4516 mimics according to an embodiment of the present invention.
  • 11A shows the mRNA expression level of miR-4516 in the tissue of a breast cancer patient according to an embodiment of the present invention.
  • 11B shows the result of confirming the expression level of the protein of FOSL1 in the tissue of a breast cancer patient according to an embodiment of the present invention through tissue immunostaining.
  • One object of the present invention is to provide a composition for preventing, improving or treating cancer.
  • NF normal fibroblast
  • CAF cancer-associated fibroblast
  • ExoQuick-TC TM (System Bioscience, Mountain View) was added to the concentrate and incubated overnight at 4° C., followed by centrifugation at 3,500 g for 30 minutes to obtain a pellet. The pellet was washed with phosphate buffered saline (PBS), and diluted in DMEM/F12 medium containing 1% penicillin/streptomycin.
  • PBS phosphate buffered saline
  • FBS Fetal bovine serum
  • antibiotics 100 U/ml penicillin and 10 mg/ml streptomycin
  • RNA was extracted from the exosomes (NF cell line or CAF cell line) of Preparation Example 1 using Trizol, and cDNA was synthesized from the total RNA using TOPscript TM cDNA synthesis kit (ENGINEOMICS). At this time, in order to amplify miRNA, the total RNA was polyadenylate using E. coli poly(A) polymerase I before cDNA synthesis.
  • TOPscript TM cDNA synthesis kit ENGINEOMICS
  • miR-199b-5p, miR-29b-3p and miR-4516 were statistically significantly decreased in expression levels in exosomes isolated from CAF cells.
  • miR-4516 it was confirmed that the expression level was reduced by 2 times or more.
  • the expression level of miR-4516 according to the present invention is specifically reduced in CAF cells, which are tumor microenvironment cells that can contribute to cancer progression, compared to normal fibroblasts that can inhibit the progression of cancer. Can be seen.
  • miR-4516 consisting of the nucleotide sequence represented by SEQ ID NO: 1 in the MCF7 or MDA-MB-231 cell line described in Preparation Example 2 above was The inserted pENTR TM /H1/TO vector (Invitrogen) was transformed. At this time, as a control, only the pENTR TM /H1/TO vector in which the miR-4516 was not inserted into each of the cell lines was transformed.
  • Each of the transformed cell lines was diluted in the culture medium of Preparation Example 2, and then 100 ⁇ l of the cell dilution was dispensed into a 96-well plate. Then, 10 ⁇ l of CCK-8 (Dojindo Molecular Technology) was additionally added to each well of the plate and incubated for an hour at 37° C. and 5% CO 2 , and absorbance was measured at 450 nm, The results are shown in FIG. 2.
  • the overexpression of miR-4516 according to the present invention can inhibit the proliferation of cancer, and in particular, it can be more effectively inhibited the proliferation of cancer in the highly invasive TNBC cell line compared to the low invasive cell line.
  • the overexpression of miR-4516 was induced in CAF cell lines in the same manner as in the transformation method described in Example 2 above. Then, the NF cell line or CAF cell line transformed to overexpress miR-4516 was dispensed into a 6-well plate, and serum-free DMEM/F12 medium containing CellTracker green CMFDA dye (Invitrogen) was added to stain the cell line. I did. After additionally dispensing the MCF7 cell line or the MDA-MB-231 cell line stained with red dye to the plate containing the stained cell line, incubated for 3 days at 37° C. and 5% CO 2 , and under a fluorescence microscope The cell line was confirmed through and the results are shown in FIG. 3.
  • the vector containing miR-4516 was co-cultured with the transformed CAF cell line and Proliferation of the MDA-MB-231 cell line was remarkably suppressed.
  • the inhibition of proliferation by the CAF cell line transformed with the vector containing miR-4516 was higher in the MDA-MB-231 cell line (MDA/CAF) than in MCF7 (MCF7/CAF).
  • miR-4516 according to the present invention can inhibit the proliferation of cancer by being overexpressed in the CAF cell line, and in particular, it can be more effectively inhibited in the high invasive TNBC cell line than in the low invasive cell line. have.
  • the nucleotide sequence of the 3'UTR (non-translated region) of the mRNA of FOSL1, PAX5 or TCL1A was inserted into the pGL3 control vector. Then, overlap extension PCR was performed to induce a mutation in the nucleotide sequence of 3'UTR, which is expected to bind to miR-4516 inserted in the vector. Thereafter, in the same manner as in Example 2, a pGL3 control vector (normal 3'UTR or mutant induced 3'UTR) and a miR-4516-inserted vector or pRL-TK vector were co-transformed in the HEK293T cell line, and 2 days During incubation. Luciferase activity was measured according to a conventional method using the cell lysate obtained from the co-transformed cells, and the results are shown in FIG. 4.
  • miR-4516 specifically binds to the 3'UTR region of FOSL1, PAX5 and TCL1A mRNA, thereby inducing the mRNA to be degraded, and furthermore, a gene such as FOSL1 that is involved in cancer progression. It can be seen that the progression of cancer can be very effectively inhibited by degrading the mRNA transcribed from.
  • Example 2 In the same manner as in Example 2, in order to induce overexpression of miR-4516 in the MCF7 cell line or the MDA-MB-231 cell line, the pENTR TM /H1/TO vector inserted with miR-4516 was transformed. At this time, only the pENTR TM /H1 /TO vector was transformed as a control.
  • the transformed MCF7 cell line and MDA-MB-231 cell line were dissolved in 50 ⁇ l of PRO-PREP protein extraction solution (iNtRON Biotechnology), sufficiently homogenized with a 30 gauge needle, and incubated at 4° C. for 30 minutes. . Then, it was centrifuged at 15,000 g to obtain a protein extract.
  • the protein extract quantified through the Bradford method was electrophoresed on a 10% polyacrylamide gel by 20 ⁇ g, transferred to a PVDF membrane (Millipore), and then FOSL1 (Abcam), PAX5 (Abcam), TCL1A (Abcam) And GAPDH (SantaCruz) specific antibody was used to perform Western blot analysis, and the results are shown in b of FIG. 5.
  • miR-4516 according to the present invention can suppress the growth of TNBC cell lines by remarkably inhibiting the translation of FOSL1 present at a high level in the MDA-MB-231 cell line, which is TNBC, compared to the non-TNBC MCF cell line. You can see that there is.
  • the BT-20 cell line or the Hs578T cell line was transformed with a pENTR TM /H1/TO vector into which miR-4516 was inserted in the same manner as in Example 2.
  • a pENTR TM /H1/TO vector into which miR-4516 was inserted in the same manner as in Example 2.
  • only the pENTR TM /H1 /TO vector was transformed as a control.
  • the expression levels of FOSL1 protein and mRNA were checked in the cell lines in the same manner as in Example 5, and the results are shown in FIGS. 6A and 6B.
  • the growth rate of the cell lines was measured using CCK-8 in the same manner as in Example 2, and the results are shown in FIG. 6C.
  • the protein of FOSL1 appeared at a high level in the BT-20 cell line and the Hs578T cell line, which are TNBC cell lines, and when miR-4516 was overexpressed, the protein and mRNA of the FOSL1 were present. The level was significantly reduced.
  • miR-4516 can effectively inhibit the growth of TNBC cell lines by inducing mRNA degradation of FOSL1, which is specifically expressed in TNBC cell lines, compared to non-TNBC cell lines. have.
  • TNBC TNBC
  • MDA-MB-231 cell line MDA-MB-231 cell line
  • BT-20 cell line BT-20 cell line
  • Hs578T cell line Hs578T cell line
  • miR-4516 mimics consisting of double-stranded nucleotide sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2 were treated at a concentration of 10 nM
  • CCK-8 was used in the same manner as in Example 2.
  • FIG. 7 a control miRNA of 10 nM concentration was treated in place of the miR-4516 mimic.
  • Antisense 3'- CCCGACCCUUCUCCCUU-5' (SEQ ID NO: 2)
  • the MDA-MB-231 cell line was 12.1
  • the BT-20 cell line was 9.8
  • the Hs578T cell line In the case of 8.5, the growth rate decreased.
  • the miR-4516 mimic according to the present invention exhibits an effect of inhibiting the growth of the cell line in the same manner as miR-4516 in the TNBC cell line.
  • MDA-MB-231 cell line, BT-20 cell line or Hs578T under the same conditions as in Example 7 above.
  • the cell line was treated with miR-4516 mimic, and a Transwell insert in which the CAF cell line was dispensed was inserted into the wells of each of the cell lines. Then, incubate for 1 day in DMEM or DMEM/F12 medium containing 1% penicillin/streptomycin and 10% FBS, and measure the growth rate of the cell lines using CCK-8 in the same manner as in Example 2. , The results are shown in FIG. 8.
  • the miR-4516 mimic according to the present invention can very effectively inhibit cell growth even in a TNBC cell line co-cultured with a CAF cell line similar to a tumor microenvironment.
  • the CAF cell line transformed with the miR-4516-inserted pENTR TM /H1/TO vector was treated with GW4869 (Cas no. 6823-69-4) in a Transwell insert.
  • the MDA-MB-231 cell line, BT-20 cell line, or Hs578T cell line was inserted onto a 6-well plate dispensed and co-cultured in the same manner as in Example 8.
  • the growth rate of the cell lines was measured using CCK-8 in the same manner as in Example 2, and the results are shown in FIG. 9.
  • the growth rates of the TNBC cell lines were 10.3 (MDA-MB-231 cell line), 7.8 (BT-20 cell line), and 9.1, respectively. (Hs578T cell line) increased as compared to the case not treated with GW4869.
  • miR-4516 according to the present invention is released through the exosomes of the CAF cell line and can very effectively inhibit the growth of the TNBC cell line.
  • non-TNBC cell line BT-474 cell line, MCF 7 cell line and SK-BR-3 cell line, and TNBC cell line MDA-MB-231 cell line, BT-20 cell line, and Hs578T cell line in the same manner as in Example 7 miR-4516
  • the mimic was treated with 0 nM, 4 nM, 10 nM, 50 nM or 100 nM
  • the growth rate of the cell lines was measured using CCK-8 in the same manner as in Example 2, and the results are shown in FIG. Indicated.
  • miR-4516 according to the present invention can very effectively inhibit the proliferation of TNBC cell lines, not non-TNBC cell lines.
  • RNA was extracted from non-TNBC or TNBC patient tissue in the same manner as in Example 1, and then primers specific for miR-4516 and FOSL1 were used. Thus, real-time polymerase chain reaction was performed. Then, the correlation of the mRNA expression levels of miR-4516 and FOSL1 was analyzed by Prism 6 for Windows (GraphPad Software, Inc., La Jolla, CA, USA).
  • the patient's tissue sectioned to 4 ⁇ m was placed on a slide coated with silane, deparaffinized, and then phosphate buffered saline containing 0.3% (v/v) hydrogen peroxide and pH 6.5, 10 mM sodium citrate. After adding the buffer, it was reacted in a 700 W microwave for 15 minutes. Thereafter, the slide was blocked for 30 minutes using a buffer containing 1% (w/v) bovine serum albumin, reacted with an antibody specific to FOSL1, and then visualized, and the results are shown in FIG. It is shown in b.
  • the mRNA expression level of FOSL1 targeted by miR-4516 according to the present invention is specifically increased in TNBC, and this phenomenon is caused by a decrease in miR-4516.
  • the present invention relates to a composition for preventing, improving or treating cancer.
  • SEQ ID NO: 1 miR-4516
  • SEQ ID NO: 2 miR-4516
  • SEQ ID NO: 3 miR-132-3p forward primer
  • SEQ ID NO: 4 miR-132-3p reverse primer
  • SEQ ID NO: 5 miR-199b-5p forward primer
  • SEQ ID NO: 6 miR-199b-5p reverse primer
  • SEQ ID NO: 7 miR-29b-3p forward primer
  • SEQ ID NO: 8 miR-29b-3p reverse primer
  • SEQ ID NO: 9 miR-1253 forward primer
  • SEQ ID NO: 10 miR-1253 reverse primer
  • SEQ ID NO: 11 miR-1915 forward primer
  • SEQ ID NO: 12 miR-1915 reverse primer
  • SEQ ID NO: 13 miR-144-3p forward primer
  • SEQ ID NO: 14 miR-144-3p reverse primer
  • SEQ ID NO: 15 miR-320e forward primer
  • SEQ ID NO: 16 miR-320e reverse primer
  • SEQ ID NO: 17 miR-4516 forward primer
  • SEQ ID NO: 18 miR-4516 reverse primer
  • SEQ ID NO: 19 FOSL1 forward primer
  • SEQ ID NO: 20 FOSL1 reverse primer
  • SEQ ID NO: 21 PAX5 forward primer
  • SEQ ID NO: 22 PAX5 reverse primer
  • SEQ ID NO: 23 TCL1A forward primer
  • SEQ ID NO: 24 TCL1A reverse primer

Abstract

La présente invention concerne une composition destinée à prévenir, soulager ou traiter un cancer. MiR-4516 ou un analogue de celui-ci selon la présente invention peut inhiber efficacement la croissance de cellules cancéreuses en se liant spécifiquement à la 3'UTR d'un ARNm transcrit à partir d'un gène d'antigène de type FOS 1 (FOSL1) et donc inhiber la traduction de l'ARNm, et peut ainsi être utilisé pour prévenir, soulager ou traiter un cancer. En outre, un agent thérapeutique pour un cancer peut être efficacement sélectionné par confirmation d'un changement du niveau d'expression de miR-4516 au moyen d'un matériau candidat.
PCT/KR2020/008569 2019-07-02 2020-07-01 Composition pour la prévention, le soulagement ou le traitement d'un cancer WO2021002664A1 (fr)

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