Pharmaceutical composition for treating and / or preventing cancer

Abstract

There has been demand to identify miRNA that could be made to be involved in treatment and / or prevention in common across various cancer types from a variety of miRNA relating to cancer, and to provide a novel pharmaceutical composition for treating and / or preventing cancer in which a polynucleotide derived from said miRNA is employed as an active ingredient. The present invention provides an agent for treating and / or preventing cancer, said agent containing, as an active ingredient, a polynucleotide including a base sequence represented by SEQ ID NO: 1.

Description

Pharmaceutical composition for treating and / or preventing cancer

[0001] The present invention relates to a pharmaceutical composition for treating and / or preventing cancer, which comprises a polynucleotide derived from microRNA as an active ingredient.

[0002] MicroRNAs (miRNAs) are non-coding RNAs with 16-28 base pairs, and according to miRBase release 22 (http: / / www.mirbase.org / ), there are currently 2,654 types known to exist in humans. In recent years, miRNAs have attracted attention as molecules that suppress the expression of various genes in vivo. Each miRNA gene region exists on the genome, and is transcribed as a hairpin-structured RNA precursor by RNA polymerase II. This is then cleaved by two types of dsRNA cleaving enzymes with RNase III cleavage activity, Drosha in the nucleus and Dicer in the cytoplasm, to form mature miRNAs. This mature miRNA is incorporated into a protein complex called RISC, where it interacts with mRNAs of multiple target genes with complementary sequences, suppressing gene expression.

[0003] Certain miRNAs have been suggested to be associated with human diseases, including cancer. In particular, in the case of cancer, many miRNAs, such as hsa-miR-6778-5p, are known to serve as pancreatic cancer-specific markers in the blood (Patent Document 1).

[0004] Furthermore, not only miRNAs involved in the proliferation of cancer cells but also miRNAs that act to suppress cancer cells have been reported, suggesting cancer therapies that utilize miRNA expression patterns. Specific examples include a method of treating diseases such as cancer by administering activated serum containing 153 miRNAs, such as hsa-Let-7a, to upregulate miRNAs (Patent Document 2), a method of suppressing proliferation by overexpressing multiple miRNAs, such as hsa-miR-513c-5p, in prostate cancer cell lines (Non-Patent Document 1), and a method of treating blood cancer by administering antisense oligonucleotides of many miRNAs, such as hsa-miR-1321, contained in circulating exosomes in the body (Patent Document 3).

[0005] International Publication No. 2015 / 182781, Special Publication No. 2013-504542, International Publication No. 2014 / 071205

[0006] BD Wang CLINICAL CANCER RESEARCH Vol. 21, 4970-4984 (2015) “Identification and Functional Validation of Reciprocal microRNA-mRNA Pairings in Africa American Prostate Cancer Disparities”

[0007] An object of the present invention is to identify, from various cancer-related miRNAs, miRNAs that have therapeutic and / or preventive effects common to various types of cancer, and to provide a new pharmaceutical composition for treating and / or preventing cancer, which contains a polynucleotide derived from the miRNA as an active ingredient.

[0008] As a result of intensive research to solve the above-mentioned problems, the present inventors discovered a novel polynucleotide that suppresses the proliferation of cancer cells from miRNAs whose expression is increased or decreased in the body fluids or tissues of cancer patients, and thus completed the present invention.

[0009] That is, the present invention has the following features (1) to (14): (1) A pharmaceutical composition for treating and / or preventing cancer, comprising, as an active ingredient, a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1. (2) The pharmaceutical composition according to (1), wherein the polynucleotide has a nucleotide length of 8 to 60 nucleotides. (3) The pharmaceutical composition according to (1) or (2), wherein the polynucleotide comprises the nucleotide sequence (a) or (b) below at the 3'-terminal side of the nucleotide sequence represented by SEQ ID NO: 1: (a) a nucleotide sequence represented by either SEQ ID NO: 2 or 3; (b) a nucleotide sequence in which 1 to 5 nucleotides have been deleted, substituted, inserted, and / or added in the nucleotide sequence represented by either SEQ ID NO: 2 or 3. (4) The pharmaceutical composition according to any of (1) to (3), wherein the polynucleotide comprises the nucleotide sequence represented by either SEQ ID NO: 4 or 5. (5) The pharmaceutical composition according to any of (1) to (4), wherein the polynucleotide is single-stranded or double-stranded. (6) The pharmaceutical composition according to any of (1) to (5), wherein the polynucleotide is RNA. (7) The pharmaceutical composition according to any one of (1) to (6), wherein the cancer is a solid cancer. (8) The pharmaceutical composition according to (7), wherein the solid cancer is selected from the group consisting of breast cancer, kidney cancer, pancreatic cancer, colorectal cancer, lung cancer, brain tumor, gastric cancer, cervical cancer, ovarian cancer, prostate cancer, bladder cancer, esophageal cancer, liver cancer, fibrosarcoma, mast cell tumor, and melanoma. (9) The pharmaceutical composition according to any one of (1) to (6), wherein the cancer is a blood cancer. (10) The pharmaceutical composition according to (9), wherein the blood cancer is leukemia. (11) The pharmaceutical composition according to any one of (1) to (10), wherein the polynucleotide is inserted in the form of DNA into a vector in a manner such that it can be expressed. (12) The pharmaceutical composition according to any one of (1) to (11), wherein the polynucleotide is encapsulated in or bound to a carrier selected from the group consisting of non-cationic polymer carriers, liposome carriers, dendritic carriers, nanomaterial carriers, microparticle carriers, biostructure carriers, micelle carriers, polymeric microparticles, and magnetic microparticles. (13) A combination drug for the treatment and / or prevention of cancer, comprising the pharmaceutical composition according to any one of (1) to (12) and an antitumor agent as active ingredients.(14) A method for treating or preventing cancer in a subject, comprising administering to the subject the pharmaceutical composition according to any one of (1) to (12) or the combination drug according to (13).

[0010] The pharmaceutical composition of the present invention for treating and / or preventing cancer dramatically inhibits the proliferation of cancer cells of various types of cancer, and is therefore useful for treating and preventing cancer.

[0011] The base sequences represented by SEQ ID NOs: 1 to 6 are as shown in Table 1.

[0012]

[0013] This figure shows the percentage of surviving cells of the pancreatic cancer cell line Panc-1 after introduction of synthetic RNA having the same base sequence as hsa-miR-6778-5p represented by SEQ ID NO: 4 and synthetic RNA having the same base sequence as hsa-miR-1233-5p represented by SEQ ID NO: 5, relative to the percentage of surviving cells (100%) after introduction of synthetic RNA of a negative control oligo. This figure shows the percentage of surviving cells of the cholangiocarcinoma cell line TFK-1 after introduction of synthetic RNA having the same base sequence as hsa-miR-6778-5p represented by SEQ ID NO: 4 and synthetic RNA having the same base sequence as hsa-miR-1233-5p represented by SEQ ID NO: 5, relative to the percentage of surviving cells (100%) after introduction of synthetic RNA of a negative control oligo. This figure shows the percentage of surviving cells of the colon cancer cell line HCT116 after introduction of synthetic RNA having the same base sequence as hsa-miR-6778-5p represented by SEQ ID NO: 4 and synthetic RNA having the same base sequence as hsa-miR-1233-5p represented by SEQ ID NO: 5, relative to the percentage of surviving cells (100%) after introduction of synthetic RNA of a negative control oligo. This figure shows the percentage of surviving cells of the pancreatic cancer cell line Panc-1 after introduction of synthetic RNA having the same base sequence as hsa-miR-6778-5p represented by SEQ ID NO: 4, synthetic RNA having the same base sequence as hsa-miR-1233-5p represented by SEQ ID NO: 5, and synthetic RNA having the same base sequence as hsa-miR-4488 represented by SEQ ID NO: 6, relative to the percentage of surviving cells (100%) after introduction of synthetic RNA of a negative control oligo.

[0014] The present invention will now be described in further detail.

[0015] <Polynucleotide as active ingredient> The pharmaceutical composition for treating and / or preventing cancer of the present invention is characterized in that it contains as an active ingredient a polynucleotide comprising the base sequence represented by AGUGGGAG (SEQ ID NO: 1). The polynucleotide as an active ingredient will be described below.

[0016] The nucleotide sequence represented by SEQ ID NO: 1 is a nucleotide sequence identified as a partial sequence on the 5' end of the human miRNA hsa-miR-6778-5p (miRBase Accession No. MIMAT0027456). This miRNA is known to be a part of miRNAs that serve as specific markers for pancreatic cancer (WO 2015 / 182781), but the fact that it suppresses the proliferation of pancreatic cancer and other cancer cells and that the polynucleotide of the nucleotide sequence represented by SEQ ID NO: 1, which is a partial sequence of these miRNAs, plays an important role in suppressing cancer cell proliferation are new findings made by the present inventors.

[0017] Therefore, the polynucleotide is not particularly limited as long as it contains the base sequence represented by SEQ ID NO: 1. That is, the polynucleotide may be the polynucleotide of the base sequence represented by SEQ ID NO: 1 itself, or may have another base sequence added to the 5'-end or 3'-end of the base sequence represented by SEQ ID NO: 1, but is preferably a polynucleotide in which another base sequence is added to the 3'-end of the base sequence represented by SEQ ID NO: 1. The base length of the base sequence of the polynucleotide is preferably 8 to 60 bases, more preferably 16 to 28 bases.

[0018] The base sequence added to the 3'-terminal side of the base sequence represented by SEQ ID NO: 1 is preferably a base sequence containing the following (a) or (b) as a partial sequence, more preferably a base sequence containing the following (a) or (b) at the 5'-terminal side, and even more preferably a base sequence consisting of the following (a) or (b): (a) A base sequence represented by SEQ ID NO: 2 or 3. (b) A base sequence in which 1 to 5, preferably 1 to 4, more preferably 1 to 3, even more preferably 1 to 2, and particularly preferably 1 base has been deleted, substituted, inserted and / or added in the base sequence represented by SEQ ID NO: 2 or 3.

[0019] A preferred example of the polynucleotide having another nucleotide sequence added to the 3'-end of the nucleotide sequence represented by SEQ ID NO: 1 is a polynucleotide consisting of the nucleotide sequence represented by either SEQ ID NO: 4 or 5. These two types of polynucleotides are known as miRNAs that have already been identified in humans. The names and miRBase Accession Nos. (registration numbers) of these miRNAs are as shown in Table 2.

[0020]

[0021] hsa-miR-6778-5p, a miRNA having the nucleotide sequence represented by SEQ ID NO: 4, is composed of the nucleotide sequence represented by SEQ ID NO: 1 from the 5' end to the 8th position, and the nucleotide sequence represented by SEQ ID NO: 2 from the 9th position onwards. As mentioned above, it is known to be a specific marker for pancreatic cancer, but there have been no reports to date that compounds using the sequence of the gene or its transcription product can suppress tumor cells.

[0022] hsa-miR-1233-5p, a miRNA having the nucleotide sequence represented by SEQ ID NO: 5, is composed of the nucleotide sequence from the 5' end to the 8th base represented by SEQ ID NO: 1, and the nucleotide sequence from the 9th base onwards represented by SEQ ID NO: 3. It was identified by the method described in Berezikov E et al., 2007, Mol Cell, Vol. 28, pp. 328-336. Furthermore, hsa-miR-1233-1 (miRBase Accession No. MI0006323), which has a hairpin-like structure, is known as the precursor of hsa-miR-1233-5p, but there have been no reports to date that compounds using the sequence of the gene or its transcription product can suppress tumor cells.

[0023] The polynucleotide may have any structure as long as it can exert an effect in treating and / or preventing cancer, for example, it may have a single-stranded, double-stranded, or triple- or more-stranded structure, but preferably has a single-stranded or double-stranded structure, and more preferably has a single-stranded structure.

[0024] The polynucleotide may be RNA, DNA, or RNA / DNA (chimera) as long as it is effective in treating and / or preventing cancer (note that, with regard to the polynucleotide, when all or part of a base sequence corresponding to a base sequence set forth in the Sequence Listing corresponds to DNA, U (uracil) in the Sequence Listing shall be read as T (thymine)), but is preferably RNA. In the case of RNA, from the viewpoint of gene regulation involved in tumor cell suppression, examples of the form include the aforementioned miRNA, as well as mRNA, rRNA, non-coding RNA, siRNA, shRNA, snoRNA, snRNA, nkRNA (registered trademark), PnkRNA (trademark), and the like, with miRNA being preferred. Note that miRNA includes naturally occurring miRNA as well as synthetic miRNA, so-called mimics.

[0025] Polynucleotides usable in the present invention may contain at least one modified nucleotide analog. The nucleotide analog may be located, for example, at the 5'-end, 3'-end, and / or internally of the RNA molecule. In particular, the incorporation of a modified nucleotide analog can provide stabilization.

[0026] Preferred nucleotide analogs are sugar- or backbone-modified ribonucleotides, more preferably ribonucleotides with modified nucleobases, i.e., ribonucleotides containing non-naturally occurring nucleobases, such as 5-position-modified uridine or cytidine, e.g., 5-methyluridine, 5-(2-amino)propyluridine, 5-methyl-2-thiouridine, 5-bromouridine, 6-azolidine, 8-position-modified adenosine and guanosine, e.g., 8-bromoguanosine, deazanucleotides, 7-deazaadenosine, O- and N-alkylated nucleotides, N6-methyladenosine, and universal bases.

[0027] Preferred sugar-modified ribonucleotides include H, OR, halo, SH, SR, and NH ２ , N.H.R., N.R. ２ The 2'-OH group may be replaced with a group selected from the group consisting of , , CN, or may contain a 2'-O, 4'-C methylene or ethylene bridge (e.g., LNA, ENA, etc.), where R is C1-C6 alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I. The sugar moiety may also be mannose, arabinose, glucopyranose, galactopyranose, 4'-thioribose, and other sugars, heterocycles, or carbocycles.

[0028] Preferred backbone-modified ribonucleotides are those in which the phosphoester group linking adjacent ribonucleotides is replaced with, for example, a phosphothioate modification, boranophosphate, 3'-(or 5'-)deoxy-3'-(or 5'-)aminophosphoramidate, hydrogen phosphonate, boranophosphate, phosphoramidate, alkyl or aryl phosphonate, and phosphotriester or phosphorus linkage. The aforementioned modifications may also be combined.

[0029] <Carrier Added to the Polynucleotide as an Active Ingredient> The pharmaceutical composition for treating and / or preventing cancer of the present invention may contain a pharmaceutically acceptable carrier in addition to the polynucleotide. A pharmaceutically acceptable carrier is a substance that facilitates the selective delivery of the polynucleotide to target cancer cells or cancer tissues, and preferably does not stimulate the organism or inhibit the activity and properties of the polynucleotide. It is also preferable that the carrier itself does not induce the production of antibodies harmful to the individual to whom the composition is administered. The size of the carrier is preferably such that it does not penetrate normal vascular walls but can penetrate neovascular walls in cancer tissues. When the carrier is approximately spherical, the diameter of the carrier can preferably be nanosized, for example, between about 1 nm and 1000 nm.

[0030] The carrier may encapsulate or be movably linked to the polynucleotide. "Modibly linked" refers to an electronic interaction between the carrier and one or more agents. Such interactions may take the form of chemical bonds, including, but not limited to, covalent bonds, polar covalent bonds, ionic bonds, electrostatic bonds, coordinate covalent bonds, aromatic bonds, hydrogen bonds, dipole-dipole or van der Waals interactions.

[0031] The binding site between the polynucleotide and the carrier is preferably the 5'-end or 3'-end, more preferably the 5'-end.

[0032] Specific examples of the carrier include non-cationic polymer carriers, liposome carriers, dendritic carriers, nanomaterial carriers, microparticle carriers, biostructural carriers, micelle carriers, polymeric microparticles, and magnetic microparticles.

[0033] Non-cationic polymeric carriers can be, for example, anionic (i.e., negatively charged) polymers or electronically neutral, flocculent, or branched polymers that can encapsulate and / or movably bind one or more agents. They can be in the form of microparticles or nanoparticles, water-soluble or water-insoluble, and biodegradable or non-biodegradable. Suitable non-cationic polymeric carriers are known to those skilled in the art. For example, they can include poly-L-glutamic acid (PGA), poly-(γ-L-glutamylglutamine) (PGGA), poly-(γ-L-aspartylglutamine) (PGAA), poly-(lactic-co-glycolic acid) (PLGA), or a mixture of at least two polymers.

[0034] A liposome carrier refers to a lipid bilayer structure comprising lipids attached to polar hydrophilic groups that, in an aqueous medium, form a substantially closed structure capable of encapsulating and / or movably associating one or more agents. The liposome carrier may comprise a single lipid bilayer (i.e., unilamellar) or two, three, or more concentric lipid bilayers (i.e., multilamellar). The liposome carrier may be approximately spherical or ellipsoidal in shape. Suitable liposome carriers are known to those skilled in the art and can be selected based on various characteristics, such as the rigidity of the lipid bilayer, the electronic charge of the lipid bilayer, and / or the compatibility of the liposome carrier with one or both of the agents. For example, natural phospholipids such as egg phosphatidylcholine, egg phosphatidylethanolamine, soybean phosphatidylcholine, lecithin and sphingomyelin, synthetic phosphatidylcholine, lysophosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, dioctadecylamidoglycylspermine, dioleoylphosphatidylethanolamine, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride, 2,3-dioleoxyoloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propaneammonium trifluoroacetamide, phosphatidylserine and derivatives thereof, PEGylated phospholipids, etc.

[0035] The dendritic carrier may be, for example, a dendrimer, a dendron, or a derivative thereof, capable of encapsulating and / or movably attaching one or more agents. Dendrimers are macromolecules having a core and multiple shells with branching structures extending from the core. Dendrons are a type of dendrimer with branches extending from a focal point. Dendritic carriers are commercially available or can be synthesized by methods known to those skilled in the art. At least a portion of the dendritic carrier may be hydrophobic or hydrophilic. Dendritic carriers may be cationic, electronically neutral, or anionic. The core molecule may include, for example, alkyldiamines such as ethylenediamine, 1,4-diaminobutane, 1,6-diaminohexane, and 1,12-diaminodecane; amines such as ammonia; alkylimines such as cystamine, polyethyleneimine (PEI); and chlorinated phosphorus molecules such as cyclotriphosphazene and thiophosphoryl. They may also include polyalkylimines such as polypropyleneimine (PPI), DAB-Am-16, tertiary amines such as polyamidoamine (PAMAM), polyamino acids such as polylysine, and phenoxymethyl(methylhydrazono) (PMMH).

[0036] Nanomaterial carriers can be any material having a longest dimension ranging from about 1 nm to about 100 nm and capable of encapsulating and / or movably associating one or more agents. Suitable nanomaterial carriers are known to those skilled in the art and may include nanoparticles, nanopowders, nanoclusters, nanocrystals, nanospheres, nanofibers, nanotubes, nanoclusters, nanocrystals, nanospheres, nanofibers, nanotubes, nanogels, and nanorods. Examples of nanomaterial carrier materials include poly(lactic-co-glycolic acid) (PLGA), polyalkylcyanoacrylate (PACA), polyepsilon-caprolactone (PCL), polylactic acid (PLA), polyethylene glycol (PEG), poly-N-vinylcaprolactam sodium acrylate, poly-N-isopropylacrylamide, and polyvinyl acetate. In some embodiments, the nanomaterial carrier can be a fullerene, including spherical fullerenes (e.g., C60), carbon nanotubes, and fullerene derivatives.

[0037] Microparticle carriers can be particles having a longest dimension ranging from about 100 to about 1000 nm, for example. Microparticles can have any shape and any morphology. Examples of materials that can constitute microparticle carriers include poly(lactic-co-glycolic acid) (PLGA), polyalkylcyanoacrylate (PACA), polyepsilon-caprolactone (PCL), polylactic acid (PLA), PLGA, polyethylene glycol (PEG), and the like. Biostructure carriers refer to polymers or compounds in which the majority of the units of the biostructure carrier are amino acids and / or saccharides, and which can encapsulate and / or movably bind one or more agents. Suitable biostructure carriers are known to those skilled in the art and may include sugars, monosaccharides, oligosaccharides, polysaccharides, cyclic polysaccharides, acyclic polysaccharides, linear polysaccharides, branched polysaccharides, amino acids, proteins and peptides, as well as semi-synthetic derivatives thereof, such as α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, methyl β-cyclodextrin, dimethyl-β-cyclodextrin, carboxymethyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, sulfobutylether-β-cyclodextrin, tri-O-methyl-β-cyclodextrin, The sugars may include cyclodextrin, glucosyl-β-cyclodextrin, β1,3D glucan, β1,6 glucan, C-reactive protein, conalbumin, lactalbumin, ovalbumin, parvalbumin, serum albumin, technetium TC99m aggregated albumin, human serum albumin (HSA), bovine serum albumin (BSA), recombinant human serum albumin (rHSA), glucose (dextrose), fructose, galactose, xylose, ribose, sucrose, cellulose, cyclodextrin, starch, and the like.

[0038] Micellar carriers are micellar structures of lipids, any fat-soluble (i.e., lipophilic) molecules, oils, waxes, sterols, monoglycerides, diglycerides, triglycerides, phospholipids, etc. They may also include polyalkylene glycols such as polyethylene glycol (PEG), polyamino acids such as polyaspartic acid and polyglutamic acid (PGA), poly-(γ-L-glutamylglutamine) (PGGA), polyphenylene oxide (PPO), poly(ε-caprolactone) (PCL), poly(lactic-co-glycolic acid) (PLGA), diblock copolymers, etc.

[0039] The carrier may also be a conjugate, and may include a nucleotide linker, a non-nucleotide linker, or a nucleotide / non-nucleotide composite linker, polyethylene glycol, human serum albumin, or a ligand for a cell receptor capable of inducing cellular uptake, which links the sense region and the antisense region of the nucleic acid. The nucleotide linker may also be a linker having a length of two or more nucleotides, or may be a nucleic acid aptamer.

[0040] The polynucleotide may further comprise at least one selected from a pharmaceutically acceptable excipient, pharmaceutical carrier, and diluent, and may be formulated into a dosage form including, by additional addition of a diluent, dispersing agent, surfactant, binder, lubricant, or mixtures thereof, a parenteral dosage form such as an injectable dosage form, a form suitable for oral, rectal, nasal, topical, subcutaneous, vaginal, or parenteral administration, or an oral dosage form such as a pill, capsule, granule, or tablet, or a form suitable for administration by inhalation or injection.

[0041] When the polynucleotide is used as a liquid formulation, the carrier is preferably sterile and biocompatible, and other common additives such as antioxidants, buffers, bacteriostatic agents, etc. are optionally added. Preferred are large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactose, polyglycolic acid, polymeric amino acids, amino acid copolymers, lipid aggregates, hydrogels, inactive virus particles, and collagens. Liquids such as water, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, and ethanol may also be included, as well as auxiliary substances such as wetting agents or emulsifiers, pH buffering substances, etc.

[0042] Administration means introducing a pharmaceutical composition for cancer treatment containing the polynucleotide as an active ingredient into a patient by any appropriate method, including delivery of the polynucleotide by viral or non-viral techniques, or transplantation of cells expressing the polynucleotide.

[0043] The administration route can be various oral or parenteral routes as long as they can reach the target tissue, for example, oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, intranasal, inhalation, intraocular or intradermal routes.

[0044] The dosage varies depending on the purpose of administration, the method of administration, the type and size of the tumor, and the condition of the recipient (i.e., the subject) (e.g., gender, age, body weight, etc.). Typically, the dosage is administered at a lower level and increased until the desired effect is achieved. Suitable dosages of the polynucleotide may be, for example, but are not limited to, in the range of 1 pmol to 100 nmol per kilogram of body weight, 0.001 to 0.25 mg per kilogram of body weight, 0.01 to 20 μg per kg of body weight, or 0.10 to 5 μg per kg of body weight. It is preferred that such dosages be administered 1 to 10 times, more preferably 5 to 10 times.

[0045] <Cancer Suppression by Polynucleotides> The polynucleotide may be provided in a form introduced into cells. "Introducing into cells" refers to introducing an exogenous polynucleotide into cells by transfection or transduction. Transfection refers to, for example, calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofectamine transfection, and protoplast fusion. Transduction refers to transferring a gene into other cells by infection using a virus or viral vector particle (e.g., vectors such as adenovirus, adeno-associated virus, Sendai virus, and retrovirus (lentivirus, etc.)) or a plasmid vector. The vector may contain elements (e.g., a promoter) necessary for expressing the polynucleotide of the present invention, and may be prepared by known techniques (e.g., Sambrook and Russell, Molecular Cloning A Laboratory Manual (4) ｔｈ Ed., 2001), Cold Spring Harbor Laboratory Press, JP 2016-153403 A, JP 2016-025853 A, etc.) Cells into which the polynucleotide has been introduced by such a method are able to express the base sequence at a high level, and thus can be used as a cell therapy agent that suppresses cancer growth by transplanting such cells into cancer tissue.

[0046] <Types of Cancer> In the present invention, tumor and cancer refer to malignant neoplasms and are used interchangeably. The target cancer is not particularly limited, but specific examples of solid cancers include cancers and cancer cells in the bladder, bone, bone marrow, brain, breast, colorectum, esophagus, digestive tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testicle, tongue, blood, or uterus. Preferred examples include breast cancer, kidney cancer, pancreatic cancer, colon cancer, lung cancer, brain tumor, stomach cancer, cervical cancer, uterine cancer, ovarian cancer, prostate cancer, bladder cancer, esophageal cancer, liver cancer, fibrosarcoma, mast cell tumor, and melanoma. These specific cancers include, for example, breast adenocarcinoma, hybrid breast adenocarcinoma, malignant mixed breast tumor, intraductal papillary adenocarcinoma, lung adenocarcinoma, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, glioma, which is a neuroepithelial tissue tumor, ependymoma, neuronal cell tumor, embryonal neuroectodermal tumor, schwannoma, neurofibroma, meningioma, chronic lymphocytic leukemia, lymphoma, gastrointestinal lymphoma, digestive lymphoma, small to medium cell lymphoma, cecal cancer, ascending colon cancer, descending colon cancer, These include, but are not limited to, transverse colon cancer, sigmoid colon cancer, rectal cancer, ovarian epithelial cancer, germ cell tumor, stromal cell tumor, pancreatic ductal carcinoma, invasive pancreatic ductal carcinoma, pancreatic adenocarcinoma, acinar cell carcinoma, adenosquamous carcinoma, giant cell tumor, intraductal papillary mucinous neoplasm, mucinous cystadenocarcinoma, pancreatoblastoma, serous cystadenocarcinoma, solid papillary carcinoma, gastrinoma, glucagonoma, insulinoma, multiple endocrine neoplasia, nonfunctioning insulinoma, somatostatinoma, and VIP-secreting tumor. Specific examples of blood cancers include leukemia.

[0047] Furthermore, preferred subjects of the present invention are mammals, including, for example, primates such as humans, livestock such as cows, pigs, sheep, and horses, pet animals such as dogs and cats, and mammals kept in zoos, with humans being preferred.

[0048] <Types of Antitumor Agents> In the present invention, a pharmaceutical composition for treating and / or preventing cancer containing the polynucleotide as an active ingredient can be administered in combination with another antitumor agent (i.e., a pharmaceutical composition containing another antitumor agent) (referred to as a "combination pharmaceutical") to a subject to enhance the antitumor effect. The pharmaceutical composition for treating and / or preventing cancer of the present invention and the other antitumor agent (i.e., a pharmaceutical composition containing the other antitumor agent) can be administered to a subject simultaneously or separately. When administered separately, either pharmaceutical composition can be administered first or second, and the administration interval, dosage, administration route, and number of administrations can be appropriately selected by a specialist. The separate pharmaceutical dosage forms to be administered simultaneously also include, for example, a pharmaceutical composition (also referred to as a "combination pharmaceutical") obtained by mixing the pharmaceutical composition for treating and / or preventing cancer and the other antitumor agent in a pharmaceutically acceptable carrier (or medium) and formulating the mixture.

[0049] Examples of the antitumor agent include the following antitumor agents known in the literature.

[0050] Alkylating agents such as thiotepa and cyclosulfamide; alkyl sulfonates such as (i.e., "such as") busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines such as altretamine, triethyleneamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolamine; acetogenins such as bullatacin and bullatasinone; camptothecins; bryostatin; kallistatin; cryptophycin 1, cryptophycin 8; dolastatin; dolastatin; Examples of suitable antihistamines include ocarmycin; erosirobin; pancratistatin; sarcodictine; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, colofosfamide, and estramustine; ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, temozolomide, nobembicine; phenesterine, prednimustine, trofosfamide, and uracil mustard; and nitrosoureas such as bendamustine, carmustine, chlorozotocin, streptozocin, fotemustine, lomustine, nimustine, and laninustine.

[0051] Examples of anticancer antibiotics include calicheamicin, dynemicin, clodronate, esperamicin, aclacinomycin, actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, adriamycin (doxorubicin), bleomycin, aclarubicin, amrubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin.

[0052] Examples of antimetabolites include folic acid analogs such as denopterin, pteropterin, methotrexate, trimetrexate, and pemetrexed; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, cladribine, and clofarabine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, trifluridine, capecitabine, 5-FU, gemcitabine, S-1, and tegafur; and hydroxycarbamide, nelarabine, and azacitidine.

[0053] Examples of hormone preparations include anastrozole, bicalutamide, degarelix, estramustine, exemestane, flutamide, fulvestrant, goserelin, letrozole, leuprorelin, medroxyprogesterone, mepitiostane, octreotide, tamoxifen, and toremifene; androgen preparations such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone, and enzalutamide; antiadrenal preparations such as aminoglutethimide, mitotane, and trilostane; furoic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestravcil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, and elformitine. , elliptinium acetate, epothilone, etoglucide, lentinan, lonidamine, maytansine, ansamitocin, abiraterone, mitoguazone, mitoxantrone, mopidamol, nitraelin, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide, procarbazine, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid, triazicon, roridin A, anguidine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside, BCG, krestin, picibanil and the like.

[0054] Other anticancer agents, such as those derived from plants, include, for example, docetaxel, etoposide, teniposide, irinotecan, nogitecan, paclitaxel, cabazitaxel, vinblastine, vincristine, vindesine, vinorelbine, carboplatin, cisplatin, dacarbazine, eribulin, L-asparagine kinase, miriplatin, mitoxantrone, nedaplatin, oxaliplatin, pentostatin, procarbazine, arsenic trioxide, sobuzoxane, tamibarotene, mitoxantrone, novantrone, edatrexate, ibandronate, topoisomerase inhibitors, difluoromethylolnithine (DMFO), and retinoic acid.

[0055] Examples of molecular targeted drugs include afatinib, axitinib, alectinib, bevacizumab, cetuximab, crizotinib, erlotinib, everolimus, gefitinib, lapatinib, ramucirumab, panitumumab, pazopanib, pertuzumab, nivolumab, regorafenib, lenvatinib, sorafenib, sunitinib, temisirolimus, trastuzumab, bosutinib, pembrolizumab, venetoclax, and the like, as well as pharmaceutically acceptable salts or derivatives thereof. ２１１ At, １３１ I, １２５ I, ９０ Y. １８６ Re, １８８ Re, １５３ Sm, ２１２ Bi, ３２ P. １７５ Lu, １７６ Lu, ８９ Sr, ２２３ Ra, １６１The radioactive isotope may be a radioactive isotope such as Tb. Preferably, the radioactive isotope is one that is effective for the treatment or diagnosis of tumors, and such a radioactive isotope may also be included in the pharmaceutical composition for treating and / or preventing cancer of the present invention. <Treatment and Prevention Methods> The present invention also provides a method for treating and / or preventing cancer in a subject suffering from (or having previously suffered from) cancer, comprising administering to the subject the pharmaceutical composition for treating and / or preventing cancer of the present invention or a combination drug comprising the pharmaceutical composition and another antitumor agent.

[0056] The term "prevention" as used herein includes the prevention of cancer recurrence to reduce the risk of recurrence after cancer treatment in cancer therapies such as surgery, chemotherapy, radiotherapy, immunotherapy, and the like.

[0057] The same applies to the pharmaceutical compositions, combination drugs, polynucleotides as active ingredients, dosages, administration methods, formulations, types of target cancers, etc., as explained above.

[0058] The present invention will be explained in more detail by the following examples, but the scope of the present invention is not limited to these examples.

[0059] Example 1 Efficacy of Synthetic RNAs against Pancreatic Cancer Cells The efficacy of synthetic RNAs having the same base sequence as hsa-miR-6778-5p represented by SEQ ID NO: 4 and synthetic RNAs having the same base sequence as hsa-miR-1233-5p represented by SEQ ID NO: 5 against pancreatic cancer cells was evaluated.

[0060] Pancreatic cancer cells from the Panc-1 cell line (ATCC) were plated in DMEM medium (Nacalai Tesque, Japan) containing 10% FBS and incubated at 37°C and 5% CO ２ The cells were cultured under the following conditions: 6 × 10 cells per well in a 96-well plate. ３ The cells were plated and transfected with synthetic RNA products (mirVana, Thermo Fisher Scientific) having the base sequences shown in SEQ ID NOs: 4 and 5 at a concentration of 30 nM. ＴＭmiRNA Mimics) or negative control oligo (Thermo Fisher Scientific, mirVana ＴＭ miRNA mimic, negative control) was transfected using Lipofectamine RNAiMAX (Thermo Fisher Scientific). After 24 hours, the culture medium was replaced, and the cells were counted for 4 days. Cell counts were determined by measuring ATP activity using Celtiter-glo (Promega) reagent, and the number of viable cells was calculated. n = 3, and the graph shows the mean ± standard deviation.

[0061] As a result, the cell survival rates of pancreatic cancer cells transfected with synthetic RNAs having the base sequences shown in SEQ ID NOs: 4 and 5 were 57% and 47%, respectively, compared to pancreatic cancer cells transfected with a negative control oligo. The results are shown in Figure 1.

[0062] Example 2: Efficacy of synthetic RNA against cholangiocarcinoma cells The efficacy of synthetic RNA having the same base sequence as hsa-miR-6778-5p represented by SEQ ID NO: 4 and synthetic RNA having the same base sequence as hsa-miR-1233-5p represented by SEQ ID NO: 5 against cholangiocarcinoma cells was evaluated. TFK-1 cell line (ATCC) was plated as cholangiocarcinoma cells in RPMI medium (Nacalai Tesque) containing 10% FBS, and incubated at 37°C and 5% CO ２ The cells were cultured under the following conditions: 3 × 10 cells per well in a 96-well plate. ３ The cells were plated and transfected with synthetic RNA products (mirVana, Thermo Fisher Scientific) having the base sequences shown in SEQ ID NOs: 4 and 5 at a concentration of 30 nM. ＴＭ miRNA Mimics) or negative control oligo (Thermo Fisher Scientific, mirVana ＴＭmiRNA mimic, negative control) was transfected using Lipofectamine RNAiMAX (Thermo Fisher Scientific). After 24 hours, the culture medium was replaced, and the cells were counted for 4 days. Cell counts were determined by measuring ATP activity using Celtiter-glo (Promega) reagent, and the number of viable cells was calculated. n = 3, and the graph shows the mean ± standard deviation.

[0063] As a result, the cell survival rates of the cholangiocarcinoma cells transfected with the synthetic RNAs having the base sequences shown in SEQ ID NOs: 4 and 5 were 67% and 25%, respectively. The results are shown in Figure 2.

[0064] Example 3: Efficacy of synthetic RNA against colon cancer cells The efficacy of synthetic RNA having the same base sequence as hsa-miR-6778-5p represented by SEQ ID NO: 4 and synthetic RNA having the same base sequence as hsa-miR-1233-5p represented by SEQ ID NO: 5 against colon cancer cells was evaluated.

[0065] HCT116 cell line (ATCC) was used as colon cancer cells and plated in McCoy's medium (Nacalai Tesque) containing 10% FBS. The cells were incubated at 37°C and 5% CO ２ The cells were cultured under the following conditions: 6 × 10 cells per well in a 96-well plate. ３ The cells were plated and transfected with synthetic RNA products (mirVana, Thermo Fisher Scientific) having the base sequences shown in SEQ ID NOs: 4 and 5 at a concentration of 30 nM. ＴＭ miRNA Mimics) or negative control oligo (Thermo Fisher Scientific, mirVana ＴＭ miRNA mimic, negative control) was transfected using Lipofectamine RNAiMAX (Thermo Fisher Scientific). After 24 hours, the culture medium was replaced, and the cells were counted for 4 days. Cell counts were determined by measuring ATP activity using Celtiter-glo (Promega) reagent, and the number of viable cells was calculated. n = 3, and the graph shows the mean ± standard deviation.

[0066] As a result, the cell survival rates of colon cancer cells transfected with synthetic RNAs having the base sequences shown in SEQ ID NOs: 4 and 5 were 22% and 8%, respectively, compared to colon cancer cells transfected with a negative control oligo. The results are shown in Figure 3.

[0067] Comparative Example 1: Efficacy of synthetic RNA against pancreatic cancer cells A synthetic RNA (mirVana®, Thermo Fisher Scientific) having the same base sequence as hsa-miR-4488 (SEQ ID NO: 6), a known cancer marker, was synthesized. ＴＭ The efficacy of miRNA mimics against pancreatic cancer was evaluated according to the method described in Example 1.

[0068] As a result, the cell survival rate of pancreatic cancer cells transfected with synthetic RNAs having the base sequences shown in SEQ ID NOs: 4 and 5 was 60% or less, whereas the cell survival rate of pancreatic cancer cells transfected with synthetic RNAs having the base sequence shown in SEQ ID NO: 6 was 92%, indicating little effect. The results are shown in Figure 4.

[0069] The pharmaceutical composition for cancer treatment of the present invention is useful for treating and / or preventing cancer.

[0070] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A pharmaceutical composition for treating and / or preventing cancer, comprising a polynucleotide containing the nucleotide sequence represented by SEQ ID NO:1 as an active ingredient.

2. The pharmaceutical composition according to Claim 1, wherein the polynucleotide has a base length of 8 to 60 bases.

3. The pharmaceutical composition according to Claim 1, wherein the polynucleotide contains the following base sequence (a) or (b) on the 3'-terminal side of the base sequence represented by SEQ ID NO:

1. (a) A base sequence represented by any of SEQ ID NO:2 or 3 (b) A base sequence in which 1 to 5 bases are deleted, substituted, inserted and / or added in the base sequence represented by any of SEQ ID NO:2 or 3

4. The pharmaceutical composition according to any one of Claims 1 to 3, wherein the polynucleotide contains the base sequence represented by any of SEQ ID NO:4 or 5.

5. The pharmaceutical composition according to any one of Claims 1 to 3, wherein the polynucleotide is single-stranded or double-stranded.

6. The pharmaceutical composition according to any one of Claims 1 to 3, wherein the polynucleotide is RNA.

7. The pharmaceutical composition according to any one of Claims 1 to 3, wherein the cancer is solid cancer.

8. The pharmaceutical composition according to Claim 7, wherein the solid cancer is selected from the group consisting of breast cancer, renal cancer, pancreatic cancer, colorectal cancer, lung cancer, brain tumor, gastric cancer, cervical cancer, ovarian cancer, prostate cancer, bladder cancer, esophageal cancer, liver cancer, fibrosarcoma, mastocytoma and melanoma.

9. The pharmaceutical composition according to any one of Claims 1 to 3, wherein the cancer is hematological cancer.

10. The pharmaceutical composition according to Claim 9, wherein the hematological cancer is leukemia.

11. The pharmaceutical composition according to any one of Claims 1 to 3, wherein the polynucleotide is inserted into a vector in an expressible form in the form of DNA.

12. The pharmaceutical composition according to any one of Claims 1 to 3, wherein the polynucleotide is encapsulated in or bound to a carrier selected from the group consisting of a non-cationic polymer carrier, a liposome carrier, a dendritic carrier, a nanomaterial carrier, a microparticle carrier, a biological structure carrier, a micelle carrier, polymer microparticles and magnetic microparticles.

13. A combined pharmaceutical for treating and / or preventing cancer, comprising the pharmaceutical composition according to any one of Claims 1 to 3 and an antitumor agent as active ingredients.