WO2023013719A1 - Pharmaceutical composition for treating and/or preventing cancer - Google Patents

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

The present invention relates to a pharmaceutical composition for treating and/or preventing cancer, which contains microRNA-derived polynucleotides as active ingredients.

MicroRNAs (miRNAs) are protein-untranslated RNAs of 16 to 28 bases, 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 an RNA precursor with a hairpin structure by RNA polymerase II. Cleaved to form mature miRNA. It is known that this mature miRNA is incorporated into a protein complex called RISC, interacts with multiple target gene mRNAs having complementary sequences, and suppresses gene expression.

Certain miRNAs have been suggested to be associated with human diseases including cancer, and in cancer in particular, many miRNAs such as hsa-miR-6778-5p can serve as pancreatic cancer-specific markers in the blood. It is known (Patent Document 1).

In addition, the existence of not only miRNAs involved in the proliferation of cancer cells, but also miRNAs that act to suppress cancer cells have been reported, suggesting cancer treatment methods that utilize miRNA expression patterns. As a specific example, 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), hsa-miR-513c- A method of overexpressing multiple miRNAs such as 5p in prostate cancer cell lines to suppress proliferation (Non-Patent Document 1), administering antisense oligonucleotides of many miRNAs such as hsa-miR-1321 contained in circulating exosomes in the body A method of treating hematologic cancer by using a

WO2015/182781 Japanese translation of PCT publication No. 2013-504542 WO2014/071205

BD Wang CLINICAL CANCER RESEARCH Vol. 21, 4970-4984 (2015) "Identification and Functional Validation of Reciprocal microRNA-mRNA Pairings in African American Prostate Cancer Disparities"

An object of the present invention is to identify, from various miRNAs associated with cancer, miRNAs that exert therapeutic and/or preventive effects in common on various types of cancer, and to create new novel therapeutic agents containing polynucleotides derived from the miRNAs as active ingredients. It is to provide a pharmaceutical composition for treating and/or preventing cancer.

As a result of intensive studies to solve the above problems, the present inventors have found novel polynucleotides that suppress the growth of cancer cells from miRNAs whose expression is increased or decreased in body fluids or tissues of cancer patients. was completed.

That is, the present invention has the following features (1) to (14).
(1) A pharmaceutical composition for treating and/or preventing cancer, comprising a polynucleotide comprising the base sequence represented by SEQ ID NO: 1 as an active ingredient.
(2) The pharmaceutical composition according to (1), wherein the polynucleotide has a base length of 8 to 60 bases.
(3) The medicament according to (1) or (2), wherein the polynucleotide comprises the following nucleotide sequence (a) or (b) on the 3′ end side of the nucleotide sequence represented by SEQ ID NO: 1 Composition.
(a) the nucleotide sequence represented by either SEQ ID NO: 2 or 3 (b) the nucleotide sequence represented by either SEQ ID NO: 2 or 3, in which 1 to 5 bases are deleted, substituted, inserted and /or added nucleotide sequence (4) The pharmaceutical composition according to any one of (1) to (3), wherein the polynucleotide comprises a nucleotide sequence represented by either SEQ ID NO:4 or 5.
(5) The pharmaceutical composition according to any one of (1) to (4), wherein the polynucleotide is single-stranded or double-stranded.
(6) The pharmaceutical composition according to any one 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 solid cancer.
(8) the solid cancer is breast cancer, kidney cancer, pancreatic cancer, colon cancer, lung cancer, brain tumor, gastric cancer, cervical cancer, ovarian cancer, prostate cancer, bladder cancer, esophageal cancer, liver cancer, fibrosarcoma, mast cell tumor, The pharmaceutical composition according to (7), which is selected from the group consisting of melanoma.
(9) The pharmaceutical composition according to any one of (1) to (6), wherein the cancer is 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 into a vector in the form of DNA so as to be expressible.
(12) A carrier in which the polynucleotide is selected from the group consisting of non-cationic polymer carriers, liposome carriers, dendritic carriers, nanomaterial carriers, microparticle carriers, biological structure carriers, micellar carriers, polymeric microparticles and magnetic microparticles. The pharmaceutical composition according to any one of (1) to (11), which is encapsulated therein or bound to the carrier.
(13) A pharmaceutical combination for treating and/or preventing cancer, comprising the pharmaceutical composition according to any one of (1) to (12) and an antitumor agent as active ingredients.
(14) administering the pharmaceutical composition according to any one of (1) to (12) or the pharmaceutical combination according to (13) to a subject who has or has had cancer A method of treating or preventing cancer in said subject, comprising:

The pharmaceutical composition for treating and/or preventing cancer of the present invention dramatically suppresses the growth of cancer cells of various cancer types, and is therefore useful for treating and preventing cancer.

The base sequences represented by SEQ ID NOS: 1-6 are as shown in Table 1.

This figure shows a synthetic RNA having the same nucleotide sequence as hsa-miR-6778-5p represented by SEQ ID NO: 4 and a synthetic RNA having the same nucleotide sequence as hsa-miR-1233-5p represented by SEQ ID NO: 5. The ratio of the cell survival number of the post-pancreatic cancer cell line Panc-1 to the cell survival number (100%) after the synthetic RNA introduction of the negative control oligo is shown. This figure shows a synthetic RNA having the same nucleotide sequence as hsa-miR-6778-5p represented by SEQ ID NO: 4 and a synthetic RNA having the same nucleotide sequence as hsa-miR-1233-5p represented by SEQ ID NO: 5. The ratio of the cell survival number of the post-cholangiocarcinoma cell line TFK-1 to the cell survival number (100%) after the synthetic RNA introduction of the negative control oligo is shown. This figure shows a synthetic RNA having the same nucleotide sequence as hsa-miR-6778-5p represented by SEQ ID NO: 4 and a synthetic RNA having the same nucleotide sequence as hsa-miR-1233-5p represented by SEQ ID NO: 5. The ratio of the cell survival number of colon cancer cell line HCT116 after colon cancer to the cell survival number (100%) after synthetic RNA introduction of the negative control oligo is shown. This figure shows a synthetic RNA having the same nucleotide sequence as hsa-miR-6778-5p represented by SEQ ID NO: 4, a synthetic RNA having the same nucleotide sequence as hsa-miR-1233-5p represented by SEQ ID NO: 5, and Cell viability after introduction of synthetic RNA of negative control oligo in pancreatic cancer cell line Panc-1 after introduction of synthetic RNA having the same base sequence as hsa-miR-4488 represented by SEQ ID NO: 6 (100 %).

The present invention will be explained in further detail.

<Polynucleotide as an active ingredient>
The pharmaceutical composition for treating and/or preventing cancer of the present invention is characterized by comprising a polynucleotide comprising a base sequence represented by AGUGGGAG (SEQ ID NO: 1) as an active ingredient. Polynucleotides serving as active ingredients are described below.

The nucleotide sequence represented by SEQ ID NO: 1 is a nucleotide sequence identified as a partial sequence on the 5' end side of human miRNA hsa-miR-6778-5p (miRBase Accession No. MIMAT0027456). This miRNA is known to be a part of miRNA that serves as a specific marker for pancreatic cancer (International Publication No. 2015/182781), but suppresses the proliferation of pancreatic cancer and other cancer cells. The present inventors have newly discovered that the polynucleotide having the base sequence represented by SEQ ID NO: 1, which is a partial sequence of miRNA, plays an important role in suppressing the proliferation of cancer cells.

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

The nucleotide sequence added to the 3′ end of the nucleotide sequence represented by SEQ ID NO: 1 is preferably a nucleotide sequence containing the following (a) or (b) as a partial sequence, more preferably the following ( It is a nucleotide sequence containing a) or (b) at the 5′ end, and more preferably a nucleotide sequence consisting of the following (a) or (b).
(a) a base sequence represented by SEQ ID NO: 2 or 3;
(b) in the nucleotide sequence represented by SEQ ID NO: 2 or 3, 1 to 5, preferably 1 to 4, more preferably 1 to 3, still more preferably 1 to 2, particularly preferably 1 A base sequence in which bases have been deleted, substituted, inserted and/or added.

A preferred specific example of the polynucleotide in which another nucleotide sequence is added to the 3′ end of the nucleotide sequence represented by SEQ ID NO: 1 consists of the nucleotide sequence represented by either SEQ ID NO: 4 or 5. Polynucleotides. These two types of polynucleotides are known as miRNAs that have already been identified in humans. Names of these miRNAs and miRBase Accession No. (Registration number) is as shown in Table 2.

hsa-miR-6778-5p, which is a miRNA having the nucleotide sequence represented by SEQ ID NO: 4, has a nucleotide sequence represented by SEQ ID NO: 1 from the 5' end to the 8th, and SEQ ID NO: 2 from the 9th. Consists of the represented base sequence. As described 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 a gene or its transcription product can suppress tumor cells.

hsa-miR-1233-5p, which is a miRNA having the nucleotide sequence represented by SEQ ID NO: 5, has the nucleotide sequence represented by SEQ ID NO: 1 from the 5' end to the 8th nucleotide sequence, and the 9th and subsequent nucleotide sequences. It is composed of the nucleotide sequence represented by number 3. Berezikov E et al., 2007, Mol Cell 28:328-336. In addition, hsa-mir-1233-1 (miRBase Accession No. MI0006323), which has a hairpin-like structure as its precursor, is known for hsa-miR-1233-5p. There are no known reports that sequence-based compounds can suppress tumor cells.

The polynucleotide may have any structure as long as it can exhibit cancer treatment and/or prevention effects, for example, it may have a single-stranded, double-stranded, or three or more multiplex structure, but is preferably is a single-stranded or double-stranded structure, more preferably a single-stranded structure.

The polynucleotide may be RNA, DNA, or RNA/DNA (chimera) as long as it can exert an effect of cancer treatment and/or prevention (the polynucleotide described in the sequence list). When all or part of the nucleotide sequence corresponding to the nucleotide sequence of corresponds to DNA, U (uracil) in the sequence listing shall be replaced with T (thymine)), preferably RNA. In the case of RNA, from the viewpoint of gene regulation involved in suppressing tumor cells, in addition to the above-mentioned miRNA, mRNA, rRNA, non-coding RNA, siRNA, shRNA, snoRNA, snRNA, nkRNA (registered trademark) , PnkRNA (trademark) and the like, preferably miRNA. Note that miRNAs include not only naturally occurring miRNAs but also synthetic miRNAs called so-called mimics.

A polynucleotide that can be used in the present invention can contain at least one modified nucleotide analogue. Nucleotide analogues can be placed, for example, at the 5' end, 3' end and/or internally of the RNA molecule. In particular, it can be stabilized by incorporating modified nucleotide analogues.

Preferred nucleotide analogues are sugar- or backbone-modified ribonucleotides, more preferably nucleobase-modified ribonucleotides, that is, ribonucleotides containing non-naturally occurring nucleobases. That is, non-naturally occurring nucleobases include uridine or cytidine modified at the 5-position, such as 5-methyluridine, 5-(2-amino)propyluridine, 5-methyl-2-thiouridine, or 5- Bromouridine, 6-azouridine, adenosines and guanosines modified at position 8, such as 8-bromoguanosine, deazanucleotides, 7-deazaadenosine; O- and N-alkylated nucleotides, N6-methyladenosine, universal bases and so on.

Preferred sugar-modified ribonucleotides include replacing the 2'OH group with a group selected from the group consisting of H, OR, halo, SH, SR, _NH2 , NHR, _NR2 , CN, or 2'-O, 4 It may contain '-C methylene bridges and ethylene bridges (eg, LNA, ENA, etc.). where R is C1-C6 alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I; Sugar moieties may also be mannose, arabinose, glucopyranose, galactopyranose, 4'-thioribose and other sugars, heterocycles or carbocycles.

Preferred backbone-modified ribonucleotides include phosphoester groups that link adjacent ribonucleotides, such as modified groups of phosphothioate groups, boranophosphate, 3′-(or 5′-)deoxy-3′-(or 5′-) '-) substituted with aminophosphoramidates, hydrogen phosphonates, boranophosphate esters, phosphoramidates, alkyl or aryl phosphonates and phosphotriesters or phosphorus linkages. Combinations of the aforementioned modifications may also be used.

<Carrier added to polynucleotide as 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 selective delivery of said polynucleotide to a target cancer cell or tissue, is non-irritating to the organism, and does not affect the activity and properties of said polynucleotide. and preferably does not itself induce the production of antibodies harmful to the individual to whom the composition is administered. Regarding the size of the carrier, a size that does not permeate normal vascular walls but allows permeation through neovascular walls in cancer tissue is preferred. When the carrier is a substantially spherical body, the diameter of the carrier can preferably be nano-sized, for example, about 1 nm or more and less than 1000 nm.

The carrier may enclose the polynucleotide, or may be movably bound thereto. "Mobably associated" refers to electronic interactions between a carrier and one or more agents. Such interactions may be in any form of chemical bonding including, but not limited to, covalent, polar covalent, ionic, electrostatic, coordinative, aromatic, hydrogen bonding, dipole or van der Waals interactions. can be taken.

The binding portion between the polynucleotide and the carrier is preferably the 5'-terminal side or the 3'-terminal side, more preferably the 5'-terminal side.

Specific examples of the carrier include non-cationic polymer carriers, liposome carriers, dendritic carriers, nanomaterial carriers, microparticle carriers, biostructural carriers, micellar carriers, polymer microparticles and magnetic fine particles.

Non-cationic polymeric carriers may entrap and/or movably associate one or more agents, such as anionic (i.e., negatively charged) polymers or electronically neutral flocculents. Alternatively, it can be a branched polymer. The form may be microparticulate or nanoparticulate, water soluble or water insoluble, biodegradable or non-biodegradable. Suitable non-cationic polymeric carriers are known to those skilled in the art. For example, poly-L-glutamic acid (PGA), poly-(γ-L-glutamylglutamine) (PGGA), poly-(γ-L-aspartylglutamine) (PGAA), poly-(lactic-co-glycolic acid) (PLGA) and may include a mixture of at least two polymers.

Liposomal carriers are lipids attached to polar hydrophilic groups that form a substantially closed structure capable of encapsulating and/or movably associated with one or more agents in an aqueous medium. represents a lipid bilayer structure, which may comprise a single lipid bilayer (ie, unilamellar), or may comprise two or more concentric lipid bilayers (ie, multilamellar). A liposome carrier may be generally spherical or generally elliptical in shape. Suitable liposome carriers are known to those of skill in the art and are selected based on a variety of properties, such as the rigidity of the lipid bilayer, the electronic charge of the lipid bilayer and/or the compatibility of one or both of the agents with the liposome carrier. can do. For example, natural phospholipids such as egg phosphatidylcholine, egg phosphatidylethanolamine, soybean phosphatidylcholine, lecithin and sphingomyelin, synthetic phosphatidylcholine, lysophosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, dioctadecylamidoglycylspermine, dioleoylphosphatidyl. Ethanolamine, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride, 2,3-diolexioroxy-N-[2-(sperminecarboxamido)ethyl ]-N,N-dimethyl-1-propaneammonium trifluoroacetamide, phosphatidylserine and derivatives thereof, PEGylated phospholipids, and the like.

A dendritic carrier may enclose and/or movably bind one or more agents, and may be, for example, a dendrimer, dendron, or derivative thereof. Dendrimers are macromolecules that have a core and multiple shells of branched structures emanating from the core. Dendrons are a type of dendrimer with branches radiating out from a focal point. Dendritic supports are commercially available or can be synthesized by methods known to those skilled in the art. At least a portion of the dendritic support may be hydrophobic or hydrophilic. Dendritic carriers can be cationic and can be electronically neutral or anionic. Core molecules may also include alkyldiamines such as ethylenediamine, 1,4-diaminobutane, 1,6-diaminohexane and 1,12-diaminodecane, amines such as ammonia, cystamine, polyethylenimine (PEI) and the like. Chlorinated phosphorus molecules such as alkylimine, cyclotriphosphazene and thiophosphoryl may also be included. Also included are polypropyleneimine (PPI), polyalkylimines such as DAB-Am-16, tertiary amines such as polyamidoamine (PAMAM), polyamino acids such as polylysine, and phenoxymethyl(methylhydrazono) (PMMH). It's okay.

A nanomaterial carrier can be, for example, a material having a longest dimension ranging from about 1 nm to about 100 nm and capable of encapsulating and/or movably binding one or more agents. Suitable nanomaterial carriers are known to those skilled in the art and include nanoparticles, nanopowders, nanoclusters, nanocrystals, nanospheres, nanofibers, nanotubes, nanoclusters, nanocrystals, nanospheres, nanofibers, nanotubes, nanogels and nanorods. may include Substances constituting the nanomaterial carrier include poly-(lactic-co-glycolic acid) (PLGA), polyalkylcyanoacrylate (PACA), polyepsilon-caprolactone (PCL), polylactic acid (PLA), and polyethylene glycol. (PEG), poly-N-vinylcaprolactam sodium acrylate, poly-N-isopropylacrylamide, polyvinyl acetate and the like. Also, in some embodiments, the nanomaterial carrier may be a fullerene, and may include spherical fullerenes (eg, C60), carbon nanotubes, fullerene derivatives.

A microparticle carrier can be, for example, a particle having a longest dimension in the range of about 100 to about 1000 nm. Microparticles may have any shape and any morphology. Substances constituting microparticle carriers include poly-(lactic-co-glycolic acid) (PLGA), polyalkylcyanoacrylate (PACA), polyepsilon-caprolactone (PCL), polylactic acid (PLA), PLGA, and polyethylene glycol. (PEG) and the like are examples of biostructure carriers, in which many units of the biostructure carrier are amino acids and/or saccharides, and one or more agents can be encapsulated and/or movably bound. A polymer or compound that contains Suitable biostructure carriers are known to those skilled in the art and include sugars, monosaccharides, oligosaccharides, polysaccharides, cyclic polysaccharides, acyclic polysaccharides, linear polysaccharides, branched polysaccharides, amino acids, proteins and peptides, and semisyntheses thereof. Derivatives may also be included, such as α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, methyl β-cyclodextrin, dimethyl-β-cyclodextrin, carboxymethyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin. Dextrin, sulfobutyl ether-β-cyclodextrin, tri-O-methyl-β-cyclodextrin, glucosyl-β-cyclodextrin, β1,3D-glucan, β1,6-glucan, C-reactive protein, conalbumin, lactalbumin, ovo Albumin, 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.

A micelle carrier is a micelle structure of lipids, any fat-soluble (that is, lipophilic) molecules, oils, waxes, sterols, monoglycerides, diglycerides, triglycerides, phospholipids, and the like. 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, and the like.

The carrier may also be a conjugate, a nucleotide linker, a non-nucleotide linker, or a nucleotide/non-nucleotide composite linker, polyethylene glycol, human serum albumin, or cellular uptake linking the sense and antisense regions of the nucleic acid. may also include ligands for cellular receptors capable of inducing . Also, the nucleotide linker may be a linker with a length of 2 or more nucleotides, or may be a nucleic acid aptamer.

The polynucleotide may further comprise at least one selected from pharmaceutically acceptable excipients, pharmaceutical carriers and diluents, diluents, dispersants, surfactants, binders, lubricants. or a parenteral dosage form such as an injectable dosage form, or a form suitable for buccal, rectal, nasal, topical, subcutaneous, vaginal or parenteral administration, or a pill, capsule, with the additional addition of a mixture thereof. , in oral dosage forms such as granules or tablets, or in dosage forms including forms suitable for administration by inhalation or injection.

When the polynucleotide is used as a liquid formulation, the carrier is preferably sterile and biocompatible, and other common additives such as antioxidants, buffers, and bacteriostats may be added. Preferred are large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactose, polyglycolic acid, polymeric amino acids, amino acid copolymers, lipid aggregates, hydrogels and inert virus particles, collagens. It may also contain liquids such as water, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol and ethanol, wetting or emulsifying agents, pH buffering. Auxiliary substances such as substances may also be included.

Administering means introducing into a patient a pharmaceutical composition for treating cancer comprising said polynucleotide as an active ingredient by any suitable method, including delivery of said polynucleotide by viral or non-viral techniques, or Including transplantation of cells expressing the polynucleotide.

The administration route can be administered via various oral or parenteral routes as long as the target tissue can be reached. For example, administration can be by oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, intranasal, inhalation, intraocular or intradermal routes.

The dosage varies depending on the purpose of administration, method of administration, tumor type, size, and subject (i.e., subject) circumstances (sex, age, weight, etc.). Typically, dosage is administered at lower levels and increased until the desired effect is achieved. Suitable doses of said polynucleotides may, for example, range from 1 pmol to 100 nmol per kilogram of body weight, range from 0.001 to 0.25 mg per kilogram of body weight, The range may be, but is not limited to, 0.10 to 5 μg/kg body weight. Such doses are preferably administered 1 to 10 times, more preferably 5 to 10 times.

<Cancer Suppression by Polynucleotide>
The polynucleotide may be provided in the form introduced into the cell. By "introduced into a cell" is meant the entry of an exogenous polynucleotide into a cell by transfection or transduction. Transfection may be performed by, for example, calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofectamine transfection, and plasma transfection. Transduction refers to methods such as plasma fusion, and transduction refers to viruses or viral vector particles (e.g. vectors such as adenoviruses, adeno-associated viruses, Sendai viruses, retroviruses (such as lentiviruses)) by means of infection. or using a plasmid vector to transfer genes into other cells. The vector can contain elements (e.g., promoters, etc.) necessary for expressing the polynucleotide in the present invention, and can be produced by known techniques (e.g., Sambrook and Russell, Molecular Cloning A Laboratory Manual ( ^4th Ed., 2001), Cold Spring Harbor Laboratory Press, JP-A-2016-153403, JP-A-2016-025853, etc.). Since cells into which the polynucleotide has been introduced by such a method can express the base sequence at a high level, a cell therapeutic agent that suppresses the growth of cancer by transplanting such cells into cancer tissue. can be used as

<type of cancer>
In the present invention tumor and cancer refer to malignant neoplasms and are used interchangeably. There are no particular restrictions on the target cancer, but specific examples of solid cancer include bladder, bone, bone marrow, brain, breast, colon/rectum, esophagus, gastrointestinal tract, gingiva, head, kidney, liver, lung, and nasopharynx. , cervix, ovary, prostate, skin, stomach, testis, tongue, blood, or cancer and cancer cells in the uterus. Preferably, breast cancer, renal cancer, pancreatic cancer, colon cancer, lung cancer, brain cancer, stomach cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, bladder cancer, esophageal cancer, liver cancer, fibrosarcoma, mast cell tumor and melanoma is mentioned. These specific cancers include, for example, mammary adenocarcinoma, combined mammary adenocarcinoma, malignant mixed malignant mammary tumor, intraductal papillary adenocarcinoma, lung adenocarcinoma, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, neuroepithelial carcinoma Glioma, ependymoma, neurocellular tumor, fetal neuroectodermal tumor, schwannoma, neurofibroma, meningioma, chronic lymphocytic leukemia, lymphoma, gastrointestinal tract type lymphoma, gastrointestinal lymphoma, small to medium cell lymphoma, cecal cancer, ascending colon cancer, descending colon cancer, transverse colon cancer, sigmoid colon cancer, rectal cancer, ovarian epithelial cancer, germ cell tumor, stromal cell tumor , pancreatic ductal carcinoma, invasive pancreatic ductal carcinoma, adenocarcinoma of pancreatic cancer, acinic cell carcinoma, adenosquamous cell carcinoma, giant cell tumor, intraductal papillary mucinous tumor, mucinous cystadenocarcinoma, pancreatoblastoma, serous cyst gland Carcinoma, solid papillary carcinoma, gastrinoma, glucagonoma, insulinoma, multiple endocrine neoplasia, nonfunctioning islet cell tumor, somatostatinoma, VIP-producing tumors include, but are not limited to. A specific example of blood cancer is leukemia.

Preferred subjects to be treated in the present invention are mammals. It includes mammals and the like, preferably humans.

<Types of antitumor agents>
In the present invention, a pharmaceutical composition comprising a pharmaceutical composition for the treatment and/or prevention of cancer containing the polynucleotide as an active ingredient and another antitumor agent (i.e., a pharmaceutical composition containing another antitumor agent) in combination ( (referred to as a "combination drug") can be co-administered to a subject to increase the anti-tumor effect. The pharmaceutical composition for treating and/or preventing cancer of the present invention and another anti-tumor agent (that is, a pharmaceutical composition containing another anti-tumor agent) can be administered to a subject simultaneously or separately. When administered separately, either pharmaceutical composition may be administered first or later, and their administration interval, dosage, administration route and administration frequency can be appropriately selected by a specialist. In another pharmaceutical dosage form for simultaneous administration, for example, the pharmaceutical composition for treating and/or preventing cancer and another antitumor agent are mixed in a pharmaceutically acceptable carrier (or medium) and formulated. A pharmaceutical composition (also referred to as a “mixed drug”) obtained by

Antitumor agents include the following antitumor agents known in literature.

alkylsulfonates such as (i.e., "such as") busulfan, improsulfan and piposulfan, as alkylating agents such as thiotepa and cyclosulfamide; aziridines such as benzodopa, carbocone, metledopa, and uredopa; amines, ethyleneimines such as triethyleneamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolamine; acetogenins such as bratacin and bratacinone; camptothecin; bryostatin; callistatin; cryptophycin 1, cryptophycin 8; dolastatin; duocarmycin; elothrobin; pancratistatin; sarcodictin; spongestatin; , novenbicin; phenesterin, prednimustine, trophosphamide, uracil mustard; nitrosoureas such as bendamustine, carmustine, chlorozotocin, streptozocin, fotemustine, lomustine, nimustine and laninnustine.

Anticancer antibiotics include calicheamicin, dynemycin, clodronate, esperamycin, aclacinomycin, actinomycin, auslamycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, cardinophylline, chromomycin, dactinomycin, daunorubicin, detrubicin, 6-diazo-5-oxo-L-norleucine, adriamycin (doxorubicin), bleomycin, aclarubicin, amrubicin, epirubicin, ethorubicin, idarubicin, merceromycin, mitomycin C, mycophenolic acid, nogaramycin, olibomycin, peplomycin, potofilomycin, puromycin, keramycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin and the like.

Antimetabolites such as folic acid analogues such as denopterin, pteropterin, methotrexate, trimetrexate, pemetrexed; purine analogues such as fludarabine, 6-mercaptopurine, thiamipurine, thioguanine, cladribine, clofarabine; ancitabine, azacytidine, 6 - pyrimidine analogues such as azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxyuridine, trifluridine, capecitabine, 5-FU, gemcitabine, S-1, tegafur; hydroxycarbamide, nerarabine, azacitidine, and the like.

Hormonal agents such as anastrozole, bicalutamide, degarelix, estramustine, exemestane, flutamide, fulvestrant, goserelin, letrozole, luprin, medroxyprogesterone, mepitiostane, octreotide, tamoxifen, toremifene, e.g. carsterone, propion androgenic agents such as dromostanolone acid, epithiostanol, mepitiostane, testolactone, enzalutamide; anti-adrenal agents such as aminoglutethimide, mitotane, trilostane; uracil, amsacrine, bestrabcil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elformitin, elliptinium acetate, epothilone, etogluside, lentinan, lonidamine, maytansine, ansamitocin, abiraterone, mitguazone, mitoxantrone, mopidanmol, nitraeline, Pentostatin, fenamet, pirarubicin, losoxantrone, podophyllic acid, 2-ethylhydrazide, procarbazine, lazoxan, rhizoxin, schizophyllan, spirogermanium, tenureazonic acid, triazicone, roridin A, anguidine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitractol, pipobroman, gacytosine, arabinoside, BCG, krestin, picibanil and the like.

Other anticancer agents derived from plants, such as docetaxel, etoposide, teniposide, irinotecan, topotecan, paclitaxel, cabazitaxel, vinblastine, vincristine, vindesine, vinorelbine, carboplatin, cisplatin, dacarbazine, eribulin, L-asparakinase, miriplatin, mitoxantrone, nedaplatin, oxaliplatin, pentostatin, procarbazine, arsenic trioxide, sobuzoxan, tamibarotene, mitoxantrone, novantrone, edatrexate, ibandronate, topoisomerase inhibitors, difluoromethylolnithine (DMFO), retinoic acid and the like.

Molecular targeted drugs such as afatinib, axitinib, alectinib, bevacizumab, cetuximab, crizotinib, erlotinib, everolimus, gefitinib, lapatinib, ramucirumab, panitumumab, pazopanib, pertuzumab, nivolumab, regorafenib, lenvatinib, sorafenib, sunitinib, temisilolimus, , pembrolizumab, venetoclax, etc., and pharmaceutically acceptable salts or derivatives thereof. Furthermore, 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, ¹⁶¹ Tb, which are known in literature, etc. , and other radioactive isotopes. The radioisotope is preferably effective for treating or diagnosing tumors, and such a radioisotope may also be included in the pharmaceutical composition for cancer treatment and/or prevention of the present invention.
<Treatment and prophylaxis>
Furthermore, the present invention provides a pharmaceutical composition for the treatment and/or prevention of cancer of the present invention, or a combination pharmaceutical comprising the pharmaceutical composition and the other antitumor agent of the present invention. Also provided is a method of treating and/or preventing cancer in a subject, comprising administering to the subject (or who has had cancer).

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

Regarding the above pharmaceutical composition, combination drug, polynucleotide as an active ingredient, dosage, usage, dosage form, type of target cancer, etc., the contents explained above apply to this section as well.

The present invention will be explained more specifically by the following examples. However, the scope of the invention shall not be limited by this example.

<Example 1> Efficacy of synthetic RNA against pancreatic cancer cells Synthetic RNA having the same base sequence as hsa-miR-6778-5p represented by SEQ ID NO: 4, hsa-miR-1233-5p represented by SEQ ID NO: 5 The effectiveness of each synthetic RNA having the same base sequence as that for pancreatic cancer cells was evaluated.

Panc-1 cell line (ATCC) as pancreatic cancer cells was plated in DMEM medium (Nacalai Tesque, Japan) containing 10% FBS and cultured at 37° C. and 5% CO ₂ . 6×10 ³ cells were seeded per well in a 96-well plate, and synthetic RNA products having the base sequences represented by SEQ ID NOs: 4 and 5 (mirVana ^™ miRNA, Thermo Fisher Scientific, Inc.) were added at a concentration of 30 nM, respectively. Mimics) or a negative control oligo (Thermo Fisher Scientific, mirVana ^™ miRNA Mimic, Negative Control) was transfected using Lipofectamine RNAiMAX (Thermo Fisher Scientific). After 24 hours, the culture medium was changed and the number of cells was counted for 4 days. The number of cells was determined by measuring ATP activity using Celtiter-glo (Promega) reagent to determine the number of viable cells. The analysis was performed at n=3, and the graph shows the mean±standard deviation.

As a result, pancreatic cancer cells transfected with synthetic RNA products having nucleotide sequences represented by SEQ ID NOs: 4 and 5 had cell survival rates of 57% and 47%, respectively, compared to pancreatic cancer cells transfected with the negative control oligo. Met. The results are shown in FIG.

<Example 2> Efficacy of synthetic RNA against cholangiocarcinoma cells Synthetic RNA having the same base sequence as hsa-miR-6778-5p represented by SEQ ID NO: 4, hsa-miR-1233- represented by SEQ ID NO: 5 Each synthetic RNA having the same nucleotide sequence as 5p was evaluated for efficacy against cholangiocarcinoma cells. TFK-1 cell line (ATCC) as cholangiocarcinoma cells was plated in RPMI medium (Nacalai Tesque) containing 10% FBS and cultured at 37° C. and 5% CO ₂ . 3×10 ³ cells were seeded per well in a 96-well plate, and RNA synthetic products (mirVana ^™ miRNA, Thermo Fisher Scientific, Inc.) having nucleotide sequences represented by SEQ ID NOs: 4 and 5 were added at a concentration of 30 nM, respectively. Mimics) or a negative control oligo (Thermo Fisher Scientific, mirVana ^™ miRNA Mimic, Negative Control) was transfected using Lipofectamine RNAiMAX (Thermo Fisher Scientific). After 24 hours, the culture medium was changed and the number of cells was counted for 4 days. The number of cells was determined by measuring ATP activity using Celtiter-glo (Promega) reagent to determine the number of viable cells. The analysis was performed at n=3, and the graph shows the mean±standard deviation.

As a result, the cell survival rates of cholangiocarcinoma cells transfected with synthetic RNA products having nucleotide sequences represented by SEQ ID NOs: 4 and 5 were 67% and 25%, respectively. The results are shown in FIG.

<Example 3> Efficacy of synthetic RNA against colon cancer cells Synthetic RNA having the same base sequence as hsa-miR-6778-5p represented by SEQ ID NO: 4, hsa-miR-1233- represented by SEQ ID NO: 5 Each synthetic RNA having the same nucleotide sequence as 5p was evaluated for efficacy against colon cancer cells.

As colon cancer cells, HCT116 cell line (ATCC) was plated in McCoy's medium (Nacalai Tesque) containing 10% FBS and cultured at 37° C. and 5% CO ₂ . 6×10 ³ cells were seeded per well in a 96-well plate, and synthetic RNA products having the base sequences represented by SEQ ID NOs: 4 and 5 (mirVana ^™ miRNA, Thermo Fisher Scientific, Inc.) were added at a concentration of 30 nM, respectively. Mimics) or a negative control oligo (Thermo Fisher Scientific, mirVana ^™ miRNA Mimic, Negative Control) was transfected using Lipofectamine RNAiMAX (Thermo Fisher Scientific). After 24 hours, the culture medium was changed and the number of cells was counted for 4 days. The number of cells was determined by measuring ATP activity using Celtiter-glo (Promega) reagent to determine the number of viable cells. The analysis was performed at n=3, and the graph shows the mean±standard deviation.

As a result, colon cancer cells transfected with each of the synthetic RNA products having nucleotide sequences represented by SEQ ID NOs: 4 and 5 had a cell survival rate of 22% compared to colon cancer cells transfected with the negative control oligo. and 8%. The results are shown in FIG.

<Comparative Example 1> Efficacy of synthetic RNA against pancreatic cancer cells Synthetic RNA having the same nucleotide sequence as hsa-miR-4488 represented by SEQ ID NO: 6, which is known as a cancer marker (Thermo Fisher Scientific, mirVana ^TM The efficacy of miRNA Mimics) against pancreatic cancer was evaluated according to the method described in Example 1.

As a result, the pancreatic cancer cells transfected with the synthetic RNA products having the base sequences represented by SEQ ID NO: 4 and 5, respectively, had a cell survival rate of 60% or less, whereas the cell survival rate was 60% or less. The cell survival rate of pancreatic cancer cells transfected with synthetic RNA products having nucleotide sequences of 92% was almost ineffective. The results are shown in FIG.

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

All publications, patents and patent applications cited herein are hereby incorporated by reference as is.

Claims (14)

A pharmaceutical composition for treating and/or preventing cancer, comprising a polynucleotide comprising the base sequence represented by SEQ ID NO: 1 as an active ingredient. The pharmaceutical composition according to claim 1, wherein the polynucleotide has a base length of 8 to 60 bases. 2. The pharmaceutical composition according to claim 1, wherein the polynucleotide comprises the following nucleotide sequence (a) or (b) at the 3' end of the nucleotide sequence represented by SEQ ID NO:1.
(a) the nucleotide sequence represented by either SEQ ID NO: 2 or 3 (b) the nucleotide sequence represented by either SEQ ID NO: 2 or 3, in which 1 to 5 bases are deleted, substituted, inserted and / or added base sequence The pharmaceutical composition according to any one of claims 1 to 3, wherein the polynucleotide comprises a base sequence represented by either SEQ ID NO: 4 or 5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the polynucleotide is single-stranded or double-stranded. The pharmaceutical composition according to any one of claims 1 to 5, wherein the polynucleotide is RNA. The pharmaceutical composition according to any one of claims 1 to 6, wherein the cancer is solid cancer. The solid cancer consists of breast cancer, kidney cancer, pancreatic cancer, colon cancer, lung cancer, brain cancer, stomach cancer, cervical cancer, ovarian cancer, prostate cancer, bladder cancer, esophageal cancer, liver cancer, fibrosarcoma, mast cell tumor and melanoma. 8. A pharmaceutical composition according to claim 7, selected from the group. The pharmaceutical composition according to any one of claims 1 to 6, wherein the cancer is blood cancer. The pharmaceutical composition according to claim 9, wherein said blood cancer is leukemia. The pharmaceutical composition according to any one of claims 1 to 10, wherein the polynucleotide is inserted into a vector in the form of DNA so that it can be expressed. The polynucleotide is encapsulated in a carrier selected from the group consisting of non-cationic polymer carriers, liposome carriers, dendritic carriers, nanomaterial carriers, microparticle carriers, biological structure carriers, micellar carriers, polymeric microparticles and magnetic microparticles. The pharmaceutical composition according to any one of claims 1 to 11, which is attached to or bound to said carrier. A combination drug for treating and/or preventing cancer, comprising the pharmaceutical composition according to any one of claims 1 to 12 and an antitumor agent as active ingredients. administering the pharmaceutical composition of any one of claims 1 to 12, or the pharmaceutical combination of claim 13, to a subject with or who has had cancer, A method of treating or preventing cancer in said subject.

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