WO2021020412A1 - Ｍｕｒｆ１を標的とする核酸医薬 - Google Patents

Ｍｕｒｆ１を標的とする核酸医薬 Download PDF

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WO2021020412A1
WO2021020412A1 PCT/JP2020/028960 JP2020028960W WO2021020412A1 WO 2021020412 A1 WO2021020412 A1 WO 2021020412A1 JP 2020028960 W JP2020028960 W JP 2020028960W WO 2021020412 A1 WO2021020412 A1 WO 2021020412A1
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seq
nucleotide sequence
oligonucleotides consisting
oligonucleotides
nucleic acid
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PCT/JP2020/028960
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English (en)
French (fr)
Japanese (ja)
Inventor
孝博 藤原
和義 冨田
邦剛 梨木
愛友美 長澤
亮 善本
尊仁 伊藤
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塩野義製薬株式会社
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Priority to US17/630,671 priority Critical patent/US20220267778A1/en
Priority to EP20847104.5A priority patent/EP4005602A1/en
Priority to JP2021535370A priority patent/JPWO2021020412A1/ja
Publication of WO2021020412A1 publication Critical patent/WO2021020412A1/ja

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    • 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
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/02—Aminoacyltransferases (2.3.2)

Definitions

  • the present invention relates to a nucleic acid drug that targets MURF1 (muscle RING finger 1). More specifically, it relates to a nucleic acid for MURF1 useful as a prophylactic or therapeutic agent for a disease with one or more symptoms selected from the group consisting of muscle loss, muscle weakness and muscle function loss.
  • a decrease in muscle mass, a decrease in muscle strength, or a decrease in muscle function is a symptom that appears due to aging, disease, or the like.
  • Diseases with one or more symptoms selected from the group consisting of muscle loss, muscle weakness, and muscle function include, for example, myogenic muscular atrophy caused by the disease of the muscle itself, command and nutrition to the muscle.
  • Diseases such as neurogenic muscular atrophy caused by damage to the supplying motor nerves, immobility or low movement due to some cause, disused muscular atrophy caused by physical inactivity such as lying down, COPD, heart failure, tuberculosis, etc. Examples include sarcopenia, which causes a decrease in muscle mass due to illness and aging.
  • no therapeutic agent for muscular atrophy has been put on the market so far, and suppression of muscle mass loss, muscle weakness, or muscle function decline is an important preventive or clinical task.
  • MURF1 Muscle RING-Finger Protein-1
  • MURF1 is one of the ubiquitin ligases that are highly expressed in skeletal muscle and myocardium, and is an enzyme involved in the decomposition of muscle proteins.
  • MURF1 is upregulated during muscular atrophy, and it is known that gene-deficient mice show resistance to various muscular atrophy, and upregulation of MURF1 has been confirmed in various muscular atrophy patients in humans (non-muscle atrophy patients).
  • Patent Documents 1, 2 etc. From these facts, it is suggested that the pharmaceutical composition having MURF1 expression inhibitory activity can be used for the treatment or prevention of diseases associated with muscular atrophy.
  • Patent Document 1 exemplifies miRNA and siRNA as antimuscular atrophy agents containing an inhibitor against the expression of the MAFbx / atlogin-1 gene and / or the Trim63 / MuRF1 gene.
  • miRNA miR-23a is specified in the examples, and while there is a specific description in the specification, no specific example of siRNA is described, and no structure or activity is suggested.
  • SiRNA for MURF1 is sold as a research reagent and is known in papers and the like.
  • Non-Patent Document 3 describes siRNA for rat MURF1.
  • Table 13 of Patent Document 2 describes siRNAs for a large number of targets, and describes siRNAs for 99 human MURF1s under IDs: 4862450 to 4862549, but biologically including suppression of MURF1 expression. No data is given.
  • An object of the present invention is to provide a nucleic acid having excellent MURF1 expression inhibitory activity.
  • the present inventors have succeeded in synthesizing a novel nucleic acid (siRNA) having an excellent MURF1 expression inhibitory activity (knockdown activity). Furthermore, we have found a target region in the mRNA of MURF1 that is particularly associated with the knockdown activity of the nucleic acid.
  • the nucleic acid of the present invention is sufficiently safe for use as a pharmaceutical.
  • the present invention relates to the following.
  • (1-1) At least 15 complementary to the base sequence consisting of positions 188 to 229, 1039 to 1060, 1427 to 1447, 1510 to 1530, or 1715 to 1737 of SEQ ID NO: 609.
  • the nucleic acid according to (1-1) which contains a base sequence complementary to the base sequence.
  • Oligonucleotides consisting of the nucleotide sequence of SEQ ID NO: 33 and oligonucleotides consisting of the nucleotide sequence of SEQ ID NO: 34 Oligonucleotides consisting of the nucleotide sequence of SEQ ID NO: 37 or 625 and oligonucleotides consisting of the nucleotide sequence of SEQ ID NO: 38, Oligonucleotides consisting of the nucleotide sequence of SEQ ID NO: 626 and oligonucleotides consisting of the nucleotide sequence of SEQ ID NO: 42, Oligonucleotides consisting of the nucleotide sequence of SEQ ID NO: 49 and oligonucleotides consisting of the nucleotide sequence of SEQ ID NO: 50, Oligonucleotides consisting of the nucleotide sequence of SEQ ID NO: 55 and oligonucleotides consisting of the nucleotide sequence of SEQ ID
  • the nucleic acid according to (5) which suppresses the expression of MURF1.
  • the nucleic acid according to (7) which is a siRNA containing an overhang at the 3'end of the sense strand and / or the antisense strand.
  • (11) The pharmaceutical composition according to (10), wherein the disease is a disease with one or more symptoms selected from the group consisting of a decrease in muscle mass, a decrease in muscle strength, and a decrease in muscle function.
  • the nucleic acids of the present invention exhibit excellent MURF1 expression-suppressing activity and are associated with one or more symptoms selected from the group consisting of pharmaceuticals, especially diseases associated with MURF1, such as muscle loss, muscle weakness and muscle function decline. It is very useful as a medicine for the prevention or treatment of diseases.
  • nucleic acid of the present invention can be any nucleic acid known in the art as a nucleic acid that can be used as a pharmaceutical product.
  • siRNA siRNA
  • antisense oligonucleotide shRNA
  • miRNA miRNA
  • the siRNA also includes a single-stranded oligonucleotide siRNA (see WO2015 / 168661 etc.).
  • an antisense oligonucleotide a double-stranded oligonucleotide may be formed together with a hybridizable sequence (see WO2013 / 089283, etc.).
  • MURF1 is mentioned as a target gene of the nucleic acid of the present invention.
  • human MURF1, mouse Murf1, and the like can be mentioned, but the present invention is not limited thereto.
  • MURF1 is a known protein.
  • the human MURF1 mRNA sequence (GenBank: NM_032588.3) is set forth in SEQ ID NO: 609 of the Sequence Listing, and the amino acid sequence (GenPept: NP_115977.2) is set forth in SEQ ID NO: 610.
  • the mRNA sequence of mouse Murf1 (GenBank: NM_001039048.2) is shown in SEQ ID NO: 611 in the Sequence Listing, and the amino acid sequence (GenPept: NP_115977.2) is shown in SEQ ID NO: 612.
  • MURF1 in the present invention is not limited to these sequences, and the number of mutations and mutation sites of amino acids and mRNAs are limited as long as the function of the protein consisting of the amino acid sequence of SEQ ID NO: 610 or 612 is maintained. Make it not exist.
  • nucleic acid of the present invention includes For the base sequence consisting of positions 188 to 229 (more preferably, positions 193 to 229), positions 1039 to 1060, positions 1427 to 1447, positions 1510 to 1530, or positions 1715 to 1737 of SEQ ID NO: 609.
  • nucleic acids that suppress the expression of MURF1 including oligonucleotides consisting of 15 to 30 nucleotides having a complementary base sequence of at least 15 bases or more.
  • the nucleic acid may further contain a base sequence complementary to the base sequence.
  • Each of the target regions is a region of human MURF1 mRNA that is particularly related to nucleic acid knockdown activity.
  • an oligonucleotide that is "complementary" to the target region is included in the nucleic acid of the invention, as long as it is a substantially complementary sequence, regardless of its length, nucleotide modification or mutation.
  • the "substantially complementary sequence” is an oligo having at least 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more homology with the completely complementary sequence of the above base sequence. Nucleotides can be mentioned.
  • the homology is shown by the score by using, for example, the search program BLAST using the algorithm developed by Altschul et al. (The Journal of Molecular Biology, 215, 403-410 (1990)). ..
  • nucleic acid that suppresses the expression of MURF1 for example, as a nucleic acid containing an oligonucleotide consisting of 15 to 30 nucleotides having a base sequence of at least 15 bases or more complementary to the base sequence consisting of positions 188 to 229 of SEQ ID NO: 609. Examples thereof include SNG-1 to SNG-19, SNG-305 and SNG-306.
  • a nucleic acid that suppresses the expression of MURF1 for example, as a nucleic acid containing an oligonucleotide consisting of 15 to 30 nucleotides having a base sequence of at least 15 bases or more complementary to the base sequence consisting of positions 1039 to 1060 of SEQ ID NO: 609.
  • Examples thereof include SNG-191 to SNG-195, SNG-314 and SNG-315.
  • a nucleic acid that suppresses the expression of MURF1 for example, as a nucleic acid containing an oligonucleotide consisting of 15 to 30 nucleotides having a base sequence of at least 15 bases or more complementary to the base sequence consisting of positions 1427 to 1447 of SEQ ID NO: 609. Examples thereof include SNG-239 to SNG-242, SNG-317 and SNG-318.
  • nucleic acid that suppresses the expression of MURF1 for example, as a nucleic acid containing an oligonucleotide consisting of 15 to 30 nucleotides having a base sequence of at least 15 bases or more complementary to the base sequence consisting of positions 1510 to 1530 of SEQ ID NO: 609. Examples thereof include SNG-285 to SNG-298.
  • a nucleic acid that suppresses the expression of MURF1 for example, as a nucleic acid containing an oligonucleotide consisting of 15 to 30 nucleotides having a base sequence of at least 15 bases or more complementary to the base sequence consisting of positions 1715 to 1737 of SEQ ID NO: 609. Examples thereof include SNG-300 to SNG-304.
  • the MURF1 expression inhibitory activity can be measured by a known method. For example, it can be measured by the method described in Examples described later.
  • nucleic acid of the present invention includes a nucleic acid containing the following base sequence (a) or (b).
  • the nucleic acid may further contain a base sequence complementary to the base sequence.
  • nucleic acid containing the nucleotide sequence of SEQ ID NO: 619 examples include SNG-13 to SNG-19, SNG-305 and SNG-306.
  • nucleic acid containing the nucleotide sequence of SEQ ID NO: 622 examples include SNG-291 to SNG-298.
  • nucleic acid containing the nucleotide sequence of SEQ ID NO: 623 examples include SNG-300 to SNG-304.
  • nucleic acids containing a contiguous base sequence of at least 15 bases in the base sequence of SEQ ID NO: 620 include SNG-191 to SNG-195, SNG-314 and SNG-315. ..
  • nucleic acids containing a contiguous base sequence of at least 15 bases in the base sequence of SEQ ID NO: 621 include SNG-239 to SNG-242, SNG-317 and SNG-318. ..
  • E SEQ ID NO: 22, 34, 38, 50, 76, 80, 158, 176, 188, 194, 210, 216, 222, 226, 232, 236, 244, 254, 266, 278, 282, 302, 336 Nucleic acid containing the base sequence of 364, 386, 394, 464, 472, 476, 482, 510, 564, 568, 574 or 578 and suppressing the expression of MURF1; or
  • F SEQ ID NOs: 22, 34, 38, 50, 76, 80, 158, 176, 188, 194, 210, 216, 222, 226, 232, 236, 244, 254, 266, 278, 282, 302, 336.
  • the nucleic acid may further contain a base sequence complementary to the base sequence.
  • the "nucleic acid" of the present invention is included in the nucleic acid of the present invention as long as it contains the base sequence and has human MURF1 expression inhibitory activity, regardless of the length or the presence or absence of nucleotide modification.
  • 1 to 3 bases preferably means 1 or 2 bases.
  • the type of mutation may be the same or different, and is 1 or 2 or more selected from deletions, substitutions and insertions. Deletion, substitution or insertion is also included in the nucleic acid of the present invention as long as it has an effect of suppressing the expression of the target gene (MURF1).
  • nucleic acid examples include siRNA (including single-stranded oligonucleotide siRNA), antisense oligonucleotide, shRNA or miRNA. More preferably, it is siRNA or antisense oligonucleotide. Particularly preferably, a siRNA or antisense oligonucleotide having an oligonucleotide having the nucleotide sequence according to the above (3-1), (3-2), (4-1) or (4-2), the above (5). It is a siRNA having the base sequence described in 1.
  • the length of each strand constituting the nucleic acid without the following overhang or terminal modification is preferably 15 to 30 nucleotides. For example, the lengths are 15-25, 17-25, 17-23, 17-21, 19-21 nucleotides.
  • an overhang may be contained at the 3'end of the sense strand and / or the antisense strand.
  • An "overhang” is a nucleotide protruding from a double-stranded structure when the 3'end of a single strand of an siRNA extends beyond the 5'end of another strand (or vice versa). means. Any nucleotide used as an overhang known in the art can be used. For example, 1 to 6 nucleotides, 1 to 5 nucleotides, 1 to 3 nucleotides, 2 or 3 nucleotides (dTdT, U (2'-OMe) U (2'-OMe), U (2'-OMe) A (2').
  • the nucleic acid of the present invention has not only MURF1 expression-suppressing activity but also medicinal usefulness, and has any or all of the following excellent characteristics.
  • Nucleotides may be modified in the nucleic acids of the invention. Appropriately modified nucleic acids have any or all of the following characteristics as compared to unmodified nucleic acids. a) High affinity with the target gene. b) High resistance to nucleases. c) Pharmacokinetics improve. d) Tissue migration is high. e) Poor immune response and cytotoxicity. Therefore, the modified nucleic acid is less likely to be degraded in vivo as compared with the unmodified nucleic acid, and can more stably inhibit the expression of the target gene.
  • nucleic acid of the present invention Any nucleotide modification known in the art can be used in the nucleic acid of the present invention.
  • Known nucleotide modifications include phosphoric acid modification, nucleobase modification, and sugar modification.
  • Examples of the phosphoric acid modification include phosphoric acid diester bond, S-oligo (phospholothioate), D-oligo (phosphodiester), M-oligo (methylphosphonate), borane phosphate and the like possessed by natural nucleic acids. ..
  • nucleobase modification include 5-methylcytosine, 5-hydroxymethylcytosine, 5-propynylcytosine and the like.
  • sugar modifications e.g., 2'-O-CH 2 -CH 2 -O-CH 3 (2'MOE), LNA (Locked nucleic acid), 2'-OMe, 2'-Fluoro, BNA (Bridged Nucleic Acid ), AmNA (see WO2011 / 052436), TrNA (see WO2014 / 126229), 2'-Deoxy and the like.
  • Nucleotide modification and modification methods known in the art are also disclosed in, for example, the following patent documents.
  • the 3'end and / or 5'end of the nucleic acid of the present invention may have a modifying group.
  • a debased (Abasic) nucleotide may be inserted into the nucleic acid.
  • a known ligand may be added to the 3'end and / or 5'end of the nucleic acid of the present invention (including nucleic acids containing overhangs and terminal modifications).
  • Cells of the Nucleic Acids of the Invention To Enable Tracing of the Nucleic Acids of the Invention, To Improve the Pharmacodynamics or Pharmacodynamics of the Nucleic Acids of the Present Invention, To Improve the Stability or Binding Affinities of the Nucleic Acids of the Present Invention
  • Transport carriers consisting of a single molecule or multiple molecules (liposomes, lipid nanoparticles (LNPs), polymers, micelles) to improve intracellular kinetics, including uptake, or to achieve all or any of them.
  • reporter molecules for example, reporter molecules, lipids (fatty acids, fatty chains, cholesterol, phospholipids, etc.), sugars (N-acetyl-galactosamine, etc.), vitamins, peptides (membrane-penetrating peptides, cell-targeting peptides, receptor-binding peptides, endosome escape-promoting peptides, etc.) , RGD peptide, peptide having high affinity with blood components, tissue target peptide, etc.), PEG (polyethylene glycol), dye, fluorescent molecule and the like.
  • reporter molecules for example, reporter molecules, lipids (fatty acids, fatty chains, cholesterol, phospholipids, etc.), sugars (N-acetyl-galactosamine, etc.), vitamins, peptides (membrane-penetrating peptides, cell-targeting peptides, receptor-binding peptides, endosome escape-promoting peptides, etc.) , RGD
  • linker any linker used in the art can be used.
  • polar linkers for example, oligonucleotide linkers
  • alkylene linkers for example, ethylene glycol linkers, ethylenediamine linkers and the like can be mentioned.
  • the linker can be synthesized with reference to a method known in the art.
  • the linker is preferably an oligonucleotide linker.
  • the length of the oligonucleotide linker is 2 to 10 bases, 2 to 5 bases, 2 bases, 3 bases, 4 bases, and 5 bases.
  • dG, dGdG, dGdGdGdG, dGdGdGdG, dT, dTdT, dTdTdTdT, dTdTdTdTdT and the like can be mentioned.
  • Ligs and linkers known in the art and methods for synthesizing them are also disclosed, for example, in the following patent documents. WO2009 / 126933, WO2012 / 037554, WO2009 / 0693313, WO2009 / 123185, WO2013 / 089283, WO2015 / 105083, WO2018 / 181428, etc.
  • the nucleic acid of the present invention (or a modified product thereof) can be synthesized by a conventional method, and can be easily synthesized by, for example, a commercially available nucleic acid automatic synthesizer (for example, Applied Biosystems, Dainippon Seiki Co., Ltd., etc.). it can.
  • the synthesis method includes a solid-phase synthesis method using phosphoramidite, a solid-phase synthesis method using hydrogen phosphonate, and the like. For example, it is disclosed in Tetrahedron Letters 22, 1859-1862 (1981), WO2011 / 052436 and the like.
  • the nucleic acids of the invention are any pharmaceutically acceptable that may provide (directly or indirectly) a biologically active metabolite or residue thereof when administered to animals, including humans. Includes salts, esters, or salts of such esters, or any other equivalent. That is, it includes prodrugs and pharmaceutically acceptable salts of the nucleic acids of the invention, pharmaceutically acceptable salts of the prodrugs, and other bioequivalents.
  • a “prodrug” is an inactive or less active form that is converted into an active form (ie, a drug) in vivo or cells by the action and / or state of an endogenous enzyme or other chemical. It is a derivative.
  • Prodrugs of the nucleic acids of the invention can be prepared according to the methods described in WO93 / 24510, WO94 / 26764, WO2004 / 063331 and the like.
  • a “pharmaceutically acceptable salt” is a physiologically and pharmaceutically acceptable salt of the nucleic acid of the invention, i.e., a toxicology that retains the desired biological activity of the nucleic acid and is not desired therein. A salt that does not have a positive effect.
  • Pharmaceutically acceptable salts include, for example, alkali metals (eg, lithium, sodium, potassium, etc.), alkaline earth metals (eg, calcium, barium, etc.), magnesium, transition metals (eg, zinc, iron, etc.), Salts with ammonia, organic bases (eg, trimethylamine, triethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, meglumin, diethanolamine, ethylenediamine, pyridine, picolin, quinoline, etc.) and amino acids, or inorganic acids (eg, hydrochloric acid, Sulfuric acid, nitrate, carbonic acid, hydrobromic acid, phosphoric acid, hydroiodic acid, etc.), and organic acids (eg, formic acid, acetic acid, propionic acid, trifluoroacetic acid, citric acid, lactic acid, tartaric acid, oxalic acid, maleic acid) , Fumaric acid, mandelic acid, glut
  • the present invention also includes pharmaceutical compositions containing the nucleic acids of the present invention.
  • any administration method and preparation known in the art can be used in addition to the above-mentioned modification method and method of adding a ligand.
  • Nucleic acid administration methods and formulations are also disclosed, for example, in the following literature. WO2008 / 042973, WO2009 / 127060, WO2011 / 064130, WO2011 / 123468, WO2011 / 153542, WO2013 / 074974, WO2013 / 075035, WO2013 / 163258, WO2013 / 192486, etc.
  • the pharmaceutical composition of the present invention can be administered by various methods depending on whether topical or systemic treatment is desired or the area to be treated.
  • the administration method may be, for example, topical (including eye drops, vagina, rectum, nasal cavity, and transdermal), oral, or parenteral.
  • Parenteral administration includes intravenous injection or infusion, subcutaneous, intraperitoneal or intramuscular injection, pulmonary administration by inhalation or inhalation, intradural administration, intraventricular administration and the like.
  • compositions such as transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders can be used.
  • preparations such as transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders can be used.
  • the composition for oral administration include powders, granules, suspensions or solutions dissolved in water or a non-aqueous medium, capsules, powders, tablets and the like.
  • Compositions for parenteral, subdural, or intracerebroventricular administration include sterile aqueous solutions containing buffers, diluents and other suitable additives.
  • the pharmaceutical composition of the present invention requires various pharmaceutical additives such as excipients, binders, wetting agents, disintegrants, lubricants, diluents, etc. suitable for the dosage form in an effective amount of the nucleic acid of the present invention. It can be obtained by mixing according to the above. In the case of an injection, it may be sterilized together with an appropriate carrier to prepare a preparation.
  • pharmaceutical additives such as excipients, binders, wetting agents, disintegrants, lubricants, diluents, etc.
  • excipients include lactose, sucrose, glucose, starch, calcium carbonate, crystalline cellulose and the like.
  • binder include methyl cellulose, carboxymethyl cellulose, hydroxypropyl cellulose, gelatin, polyvinylpyrrolidone and the like.
  • disintegrant include carboxymethyl cellulose, sodium carboxymethyl cellulose, starch, sodium alginate, agar powder, sodium lauryl sulfate and the like.
  • lubricant include talc, magnesium stearate, macrogol and the like.
  • cacao butter, macrogol, methyl cellulose and the like can be used as the base of the suppository.
  • a solubilizing agent, suspending agent, emulsifier, stabilizer, preservative, isotonic agent, etc. which are usually used, are appropriately added. You may. In the case of oral administration, a flavoring agent, a fragrance agent or the like may be added.
  • Optimal doses will vary depending on the relative potency of the individual nucleic acids, but can generally be calculated based on IC50s or EC50s in in vitro and in vivo animal studies. For example, given the molecular weight of the nucleic acid (derived from the nucleic acid sequence and chemical structure) and an effective dose (experimentally derived), such as IC50, the dose expressed in mg / kg is usually used. Calculated according to.
  • 0.001 to 10 mg / kg per day can be mentioned.
  • an injectable preparation it can be administered for a certain period of time, for example, for 5 to 180 minutes. It can also be administered once to several times a day, or at intervals of one to several days (for example, every two weeks).
  • the pharmaceutical composition of the present invention has MURF1 expression inhibitory activity, it can be used for the prevention or treatment of diseases associated with MURF1.
  • Diseases associated with MURF1 include diseases with one or more symptoms selected from the group consisting of decreased muscle mass, decreased muscle strength and decreased muscle function. For example, disused muscular atrophy (due to patients who are expected to be hospitalized for a certain period of time due to femoral fracture, pneumonia, etc., or use of gypsum, etc.), motor instability, locomotive syndrome, bad fluid.
  • ICU-acquired weakness muscle weakness that occurs while in the ICU
  • COPD pathological muscular atrophy
  • COPD Choronic Obstructive Pulmonary Disease
  • heart failure tuberculosis
  • cancer diabetes
  • AIDS Acquireid Disease
  • Peripheral neuropathy muscle loss
  • drug-induced myopathy muscle atrophy due to steroid treatment, cancer chemotherapy, etc.
  • age-related loss of muscle mass loss of muscle strength, and loss of muscle function
  • siRNA homologous to human and mouse SiRNA was designed to target human and mouse MURF1 mRNA.
  • the mRNA sequence used in the design is human MURF1 (GenBank: NM_032588.3, SEQ ID NO: 609), mouse Murf1 (GenBank: NM_001039048.2, SEQ ID NO: 611).
  • the designed siRNA is a duplex, which consists of a 19-base antisense strand and a 19-base sense strand.
  • the antisense strand is the complementary sequence of the mRNA sequence
  • the sense strand is the complementary sequence of the antisense strand.
  • the siRNA was designed so that the 2nd to 17th bases at the 5'end of the antisense strand had 100% homology to human and mouse mRNA.
  • the designed sequences (SNG-1 to SNG320) are shown in Tables 1 to 16. In the table, the target site indicates the position in SEQ ID NO: 609, and the capital letter in the base sequence means RNA.
  • Example 2 In vitro model mouse cell culture Mouse melanoma cell line B16, MEM (Thermo Fisher Scientific) + 10% fetal bovine serum (FBS) (Hycron) + Penicillin (100 units / mL) (Thermo Fisher Scientific) + Streptomycin The cells were cultured at (100 ug / mL) (Thermo Fisher Scientific) and maintained at 37 ° C., 95-98% humidity and 5% CO 2 .
  • FBS fetal bovine serum
  • Penicillin 100 units / mL
  • Streptomycin Streptomycin
  • Example 3 Evaluation of siRNA for Murf1
  • the antisense strand of siRNA designed in Example 1 and the siRNA duplex having dTdT added as an overhang to the 3'end of the sense strand were purchased from SIGMA.
  • siRNA duplexes were introduced into cells using Lipofectamine® 3000 (Thermo Fisher Scientific) and added to cell culture medium to a final concentration of siRNA duplexes of 10 nmol / L. Twenty-four hours after the introduction, cells were collected by CellAmp TM Direct RNA Prep Kit for RT-PCR (Takara Bio), and real-time PCR was performed. Gapdh was used as an intrinsic control.
  • the primer sequence used to measure the expression level of mouse Murf1 was Fw primer: (SEQ ID NO: 613); TGTCTCACGTGTGTGAGGTGCCTA Rv primer: (SEQ ID NO: 614); CACCAGCATGGAGATAGCAGTTAC Using The primer sequence used to measure the expression level of mouse Gapdh is Fw primer: (SEQ ID NO: 615); TGTGTCCGTCGTGGATCTGA Rv primer: (SEQ ID NO: 616); TTGCTGTTGAGTCGCAGGAG was used. The results of knockdown activity are shown in Table 17.
  • the difference ( ⁇ Ct) between the expression level (Ct value) of mouse Murf1 and the expression level (Ct value) of mouse Gapdh in the cells into which each siRNA duplex was introduced is shown in Table 18 of SNG-305.
  • ⁇ Ct (Expression level of Gapdh-expression level of SNG-305)
  • ⁇ Ct -(Expression level of Gapdh-expression level of each siRNA duplex)
  • uppercase letters mean RNA and lowercase letters mean DNA.
  • SNG-305 is a siRNA duplex designed for mouse Murf1 mRNA and has no homology with human MURF1, but the final concentration of siRNA duplex is 10 nmol / in mouse B16 cells using Lipofectamine 3000. It was added to L and used as a positive control because it showed 89% strong knockdown activity 24 hours after introduction. As a result, it was found that the siRNA of the present application suppresses the expression of mouse Murf1 because it exhibits a strong knockdown activity almost equivalent to that of SNG-305. Furthermore, since the siRNA of the present application has homology with human MURF1, it was suggested that it suppresses the expression of human MURF1.
  • Example 4 In vitro model Human cell culture Human skeletal myoblasts are cultured in SkGM-2 BulletKit (Lonza), and then DMEM (Thermo Fisher Scientific) + 2% horse serum (Thermo Fisher Scientific) + Penicillin ( The cells were cultured at 100 units / mL (Thermo Fisher Scientific) + Streptomycin (100 ug / mL) (Thermo Fisher Scientific) and maintained at 37 ° C., 95-98% humidity and 5% CO 2 . The culture vessel was used after coating with Matrigel (Corning).
  • Example 5 Evaluation of siRNA against MURF1
  • the antisense strand of some siRNAs designed in Example 1 and the siRNA duplex having dTdT added as an overhang to the 3'end of the sense strand were purchased from SIGMA.
  • a knockdown experiment was performed on human skeletal myoblasts cultured under the conditions of Example 4.
  • SiRNA duplexes were introduced into cells using Lipofectamine® 3000 (Thermo Fisher Scientific) and added to cell culture medium to a final concentration of siRNA duplexes of 20 nmol / L. Twenty-four hours after the introduction, cells were collected by CellAmp TM Direct RNA Prep Kit for RT-PCR (Takara Bio), and real-time PCR was performed. GAPDH was used as an intrinsic control.
  • the primer sequence used to measure the expression level of human MURF1 Fw primer (SEQ ID NO: 640); CGTGTGCAGACCATCATCATC Rv primer: (SEQ ID NO: 641); CAACGTGTCAAACTTCTGGCTCA Using The primer sequence used to measure the expression level of human GAPDH is Fw primer: (SEQ ID NO: 642); GCACCGTCAAGGCTGAGAC Rv primer: (SEQ ID NO: 643); TGGTGAAGACGCCAGTGGA was used. The results of mRNA residual rate are shown in Table 19.
  • the nucleic acid of the present invention exhibits MURF1 expression inhibitory activity. Therefore, the compound of the present invention is very useful as a medicine for the prevention or treatment of a disease associated with one or more symptoms selected from the group consisting of a decrease in muscle mass, a decrease in muscle strength, and a decrease in muscle function.

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