Nucleic acid drugs that inhibit the production of myostatin gene mRNA

Abstract

The present invention provides a novel method for inhibiting myostatin. An antisense oligonucleotide having a base length of 15 to 30, a salt thereof or a solvate thereof, the antisense oligonucleotide having a nucleotide sequence complementary to a target site of exon 1 of a myostatin gene, and being capable of inhibiting the production of mRNA of the myostatin gene. A drug, food, feed, a drug for promoting proliferation and / or hypertrophy of muscle cells, a drug for increasing muscle mass and / or inhibiting myopenia, a drug for inhibiting the production of mRNA of a myostatin gene, and a functional inhibitor of myostatin containing the antisense oligonucleotide, a salt thereof or a solvate thereof.

Description

Technical Field

[0001] The present invention relates to a nucleic acid drug that inhibits the production of mRNA of the myostatin gene. Background Art

[0002] Myostatin is one of the TGF-β type cell growth factors and is a protein encoded by the myostatin gene. Myostatin has the effect of inhibiting the proliferation / hypertrophy of muscle cells. Therefore, inhibiting the function of myostatin will promote the proliferation / hypertrophy of muscle cells, so it has attracted attention as a therapeutic target for muscular atrophy. In fact, anti-myostatin antibodies or antisense oligonucleotides (AO) have been developed, and the antisense oligonucleotides control the splicing of the myostatin gene to produce a loss-of-function mRNA (Non-Patent Documents 1, 2). However, clinically effective myostatin inhibition methods have not yet been established.

[0003] Prior art literature

[0004] Non-patent literature

[0005] Non-patent literature 1: St Andre et al. Skeletal Muscle 2017, 7; 25;

[0006] Non-patent document 2: Kang et al. Mol Ther 2011, 19; 159-64. Summary of the invention

[0007] Problems to be solved by the invention

[0008] The object of the present invention is to provide a novel method for inhibiting myostatin.

[0009] Means for solving problems

[0010] The myostatin (MSTN) gene has a simple structure of three exons. The inventors aimed to develop antisense oligonucleotides (AOs) targeting exon 1 of the MSTN gene. Therefore, various AOs were synthesized with ENA as monomers complementary to sequences such as splicing factor binding sites within the exon. Each synthesized AO was introduced (introduced) into rhabdomyosarcoma cells, and the MSTN mRNA expressed in the cells was semi-quantitated using the RT-PCR method. Then, the AOs that reduced the expression of mRNA were identified ( Figure 3 ). It was further found that the myostatin signaling activity of cells was reduced by introducing the AO ( FIG. 8 ). These results show that the identified AO inhibits the expression of myostatin, and as a result, has the effect of causing a reduction in myostatin signaling. The present invention was completed based on these findings.

[0011] The gist of the present invention is as follows.

[0012] (1) An antisense oligonucleotide having a base length of 15 to 30, or a salt or solvate thereof, wherein the antisense oligonucleotide has a nucleotide sequence (base sequence) complementary to a target site of exon 1 of a myostatin gene and can inhibit the production of mRNA of the myostatin gene.

[0013] (2) The antisense oligonucleotide, salt or solvate thereof according to (1), wherein the nucleotide sequence of exon 1 of the myostatin gene is the nucleotide sequence of SEQ ID NO: 1, and the target site of exon 1 of the myostatin gene is present in the region of base numbers 22 to 420 of the nucleotide sequence of SEQ ID NO: 1.

[0014] (3) The antisense oligonucleotide, a salt or a solvate thereof according to (1) or (2), wherein the nucleotide sequence of the antisense oligonucleotide comprises a sequence consisting of at least 15 consecutive bases of any one of the nucleotide sequences in SEQ ID NOs: 2 to 25 (wherein t in the sequence may be u and u may be t).

[0015] (4) The antisense oligonucleotide, a salt thereof, or a solvate thereof according to any one of (1) to (3), wherein the antisense oligonucleotide has a base length of 18.

[0016] (5) The antisense oligonucleotide, salt or solvate thereof according to (4), wherein the nucleotide sequence of the antisense oligonucleotide is any one of SEQ ID NOs: 2 to 25 (wherein t in the sequence may be u and u may be t).

[0017] (6) The antisense oligonucleotide, a salt or a solvate thereof according to any one of (1) to (5), wherein at least one nucleotide is modified.

[0018] (7) The antisense oligonucleotide, salt or solvate thereof according to (6), wherein the sugar constituting the modified nucleotide is D-ribofuranose, and the hydroxyl group at the 2′ position of the D-ribofuranose is modified.

[0019] (8) The antisense oligonucleotide, salt or solvate thereof according to (7), wherein the D-ribofuranose is 2'-O-alkylated and / or 2'-O, 4'-C-alkylened.

[0020] (9) A drug comprising the antisense oligonucleotide according to any one of (1) to (8), or a pharmaceutically acceptable salt or solvate thereof.

[0021] (10) The medicament according to (9), which is used for preventing and / or treating a condition and / or disease in which myostatin is involved.

[0022] (11) The medicament according to (10), wherein the condition and / or disease in which myostatin is involved is muscular atrophy.

[0023] (12) The drug according to (11), wherein the muscular atrophy is at least one selected from the group consisting of muscular dystrophy, myopathy, spinal muscular atrophy, sarcopenia and disuse muscular atrophy.

[0024] (13) The medicament according to (10), wherein the condition and / or disease in which myostatin is involved is a condition and / or disease that produces a therapeutic effect by restoring muscle mass.

[0025] (14) The drug according to (13), wherein the condition and / or disease that produces a therapeutic effect by restoring muscle mass is at least one selected from cancer cachexia (cancer cachexia), diabetes, circulatory system disease, kidney disease and bone disease.

[0026] (15) The medicament according to (14), wherein the circulatory system disease is heart failure and / or arteriosclerosis, the kidney disease is chronic renal failure, and the bone disease is inflammatory arthritis.

[0027] (16) A food comprising the antisense oligonucleotide according to any one of (1) to (8), or a food-acceptable salt or solvate thereof.

[0028] (17) A feed comprising the antisense oligonucleotide according to any one of (1) to (8), or a feed-acceptable salt or solvate thereof.

[0029] (18) An agent for promoting proliferation and / or hypertrophy of muscle cells, comprising the antisense oligonucleotide according to any one of (1) to (8), or a salt or solvate thereof.

[0030] (19) A drug for increasing muscle mass and / or inhibiting muscle loss, comprising the antisense oligonucleotide according to any one of (1) to (8), or a salt or solvate thereof.

[0031] (20) A drug for inhibiting the production of mRNA of a myostatin gene, comprising the antisense oligonucleotide according to any one of (1) to (8), or a salt or solvate thereof.

[0032] (21) A myostatin function inhibitor comprising the antisense oligonucleotide according to any one of (1) to (8), or a salt or solvate thereof.

[0033] (22) A method for preventing and / or treating a disease in which myostatin is involved, comprising administering to a subject an effective amount of the antisense oligonucleotide, a salt or a solvate thereof according to any one of (1) to (8).

[0034] (23) A method for promoting proliferation and / or hypertrophy of muscle cells, comprising administering to a subject an effective amount of the antisense oligonucleotide, a salt or a solvate thereof according to any one of (1) to (8).

[0035] (24) A method for increasing muscle mass and / or inhibiting sarcopenia, comprising administering to a subject an effective amount of the antisense oligonucleotide, a salt or a solvate thereof according to any one of (1) to (8).

[0036] (25) A method for inhibiting the production of mRNA of a myostatin gene, the method comprising administering to a subject an effective amount of the antisense oligonucleotide, a salt or a solvate thereof according to any one of (1) to (8).

[0037] (26) A method for inhibiting the function of myostatin, comprising administering to a subject an effective amount of the antisense oligonucleotide, a salt or a solvate thereof according to any one of (1) to (8).

[0038] (27) The antisense oligonucleotide, salt or solvate thereof according to any one of (1) to (8), for use in a method for preventing and / or treating a disease in which myostatin is involved.

[0039] (28) The antisense oligonucleotide, salt or solvate thereof according to any one of (1) to (8), for use in a method for promoting proliferation and / or hypertrophy of muscle cells.

[0040] (29) The antisense oligonucleotide, salt or solvate thereof according to any one of (1) to (8), for use in a method for increasing muscle mass and / or inhibiting sarcopenia.

[0041] (30) The antisense oligonucleotide, salt or solvate thereof according to any one of (1) to (8), which is used for use in a method for inhibiting the production of mRNA of a myostatin gene.

[0042] (31) The antisense oligonucleotide, salt or solvate thereof according to any one of (1) to (8), for use in a method for inhibiting the function of myostatin.

[0043] Effects of the Invention

[0044] By using the AO of the present invention, the expression of myostatin can be inhibited and the myostatin signal transduction can be reduced.

[0045] This specification includes the contents described in the specification and / or drawings of the Japanese Patent Application and Japanese Patent Application No. 2019-118446 which are the basis of priority of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] [ Figure 1 ] MSTN pre-mRNA and target sites of AO. MSTN pre-mRNA produced by transcription of MSTN gene consists of exon 1, exon 2, and exon 3. AOs (AO1, AO2, AO3) complementary to the sequence in exon 1 of MSTN pre-mRNA were prepared.

[0047] [ Figure 2 ] Comparison of the effectiveness of AO1, AO2, and AO3. AO was introduced into human rhabdomyosarcoma cells, and the results of RT-PCR of MSTN mRNA are shown. a) The complementary sequences of AO1, AO2, and AO3 are shown in the □. The numbers of the nucleotide sequence are the base numbers when the base at the 5' end of exon 1 of MSTN is 1. b) The results of RT-PCR of MSTN mRNA and GAPDH mRNA in human rhabdomyosarcoma cells are shown. w / o means no AO treatment. c) The relative value is shown when MSTN / GAPDH without AO treatment is 1. * P<0.05, *** P<0.001.

[0048] [ Figure 3 ] Comparison of the effectiveness of AO designed around AO2. AO was introduced into human rhabdomyosarcoma cells, and the results of RT-PCR of MSTN mRNA are shown. a) AO is represented by -, and the complementary sequence of AO2 is shown in □. In the upper part of the nucleotide sequence, AO is shown with 3 bases offset to the 5' side and 3' side respectively relative to the sequence of AO2. In the lower part of the nucleotide sequence, AO is shown with 1 base offset to the 3' side respectively relative to the sequence of AO2. The numbers in the nucleotide sequence are base numbers when the base at the 5' end of exon 1 of MSTN is 1. b, d) The results of RT-PCR of MSTN mRNA and GAPDH mRNA in human rhabdomyosarcoma cells are shown. w / o means AO untreated. c, e) Relative values ​​are shown when MSTN / GAPDH untreated with AO is 1. * P<0.05, *** P<0.001.

[0049] [ Figure 4a] Comparison of the effectiveness of AO2+1 with AO designed in other regions. AO was introduced into human rhabdomyosarcoma cells, and the results of RT-PCR of MSTN mRNA are shown. The complementary sequence of AO in exon 1 is shown. AO is represented by - and is arranged below the complementary sequence. The numbers of the nucleotide sequence are the base numbers when the base at the 5' end of exon 1 of MSTN is 1.

[0050] [ Figure 4bc ] b) Shows the results of RT-PCR of MSTN mRNA and GAPDH mRNA in human rhabdomyosarcoma cells. w / o means untreated with AO. c) Shows the relative values ​​when MSTN / GAPDH untreated with AO is 1. * P<0.05, ** P<0.01, *** P<0.001.

[0051] [ Figure 5ab ] a) Test of the effectiveness of AO2+1 in human myoblasts. AO was introduced into human myoblasts, and the results of RT-PCR of MSTN mRNA are shown. b) The results of RT-PCR of MSTN mRNA and GAPDH mRNA in human myoblasts are shown. The relative value when MSTN / GAPDH treated with 0nM AO is 1 is shown. ** P<0.01, *** P<0.001.

[0052] [ Figure 6ab ] a) Test of the effect of AO2+1 on GDF11 expression. AO was introduced into human rhabdomyosarcoma cells, and the results of RT-PCR of MSTN mRNA and GDF11 mRNA are shown. b) The results of RT-PCR of MSTN mRNA, GDF11 mRNA, and GAPDH mRNA in human rhabdomyosarcoma cells are shown. The relative values ​​are shown when MSTN / GAPDH and GDF11 / GAPDH in cells without AO treatment (w / o) are taken as 1. *** P<0.001.

[0053] [ Figure 7 ] Myostatin signal. Myostatin activates downstream signal transduction by binding to receptors on the cell surface. If mature myostatin binds to the receptor, Smad2 / 3 is phosphorylated and phosphorylated Smad2 / 3 is transferred to the nucleus. Phosphorylated Smad2 / 3 binds to the Smad binding region in the promoter of the target gene, thereby inducing the expression of the target gene.

[0054] [ Figure 8ab] Reduction of myostatin signaling by AO2+1. Shown are the results of an examination of myostatin signaling upon AO treatment in human rhabdomyosarcoma cells.

[0055] a) Schematic diagram showing the effect of AO2+1 treatment on the in vitro myostatin transcriptional activity assay system. Myostatin signaling analysis was evaluated by the activity of luciferase induced by AO2+1 treatment and AO-free.

[0056] b) The results without AO treatment (w / o) were taken as 1 and expressed as relative values. *** P<0.001.

[0057] [ Fig. 9 ] Reduction of myostatin signaling by AO2+1. The results of a study on myostatin signaling in human myoblasts treated with AO are shown. The results of AO 0 nM treatment are expressed as 1 and are expressed as relative values. * P<0.05, ** P<0.01, *** P<0.001.

[0058] [ Fig.10 ] Proliferation promotion of human myoblasts by AO2+1. Results of studies showing cell proliferation in human myoblasts treated with and without AO (w / o). * P<0.05, *** P<0.001.

[0059] [ Fig.11 ] Target sequence of AO2+1 conserved across species. Shown is the alignment of the nucleic acid sequences of the amino acid coding region of MSTN exon 1 of human (Homo sapiens), cattle (Bos Taurus), pig (Sus scrofa), and dog (Canis Lupus familiaris) (excluding the 5' untranslated region). The target sequence of AO2+1 is shown in the □. DETAILED DESCRIPTION

[0060] Hereinafter, embodiments of the present invention will be described in more detail.

[0061] The present invention provides an antisense oligonucleotide having a base length of 15 to 30, and a salt or solvate thereof, wherein the antisense oligonucleotide has a nucleotide sequence complementary to a target site of exon 1 of a myostatin gene and can inhibit the production of mRNA of the myostatin gene.

[0062] The nucleotide sequence of exon 1 of the human myostatin gene is shown in SEQ ID NO: 1. In the present invention, when the nucleotide sequence of exon 1 of the myostatin gene is the nucleotide sequence of SEQ ID NO: 1, the target site of exon 1 of the myostatin gene may be present in the region of base numbers 22 to 420 of the nucleotide sequence of SEQ ID NO: 1.

[0063] Furthermore, in the present invention, the nucleotide sequence of the antisense oligonucleotide may comprise a sequence consisting of at least 15 consecutive bases in any one of the nucleotide sequences in SEQ ID NOs: 2 to 25 (wherein t in the sequence may be u, and u may be t).

[0064] The base length of the antisense oligonucleotide may be 18, and the nucleotide sequence of the antisense oligonucleotide may be any one of SEQ ID NOs: 2 to 25 (wherein t in the sequence may be u, and u may be t).

[0065] The nucleotides constituting the antisense oligonucleotide may be any of natural DNA, natural RNA, or modifications thereof, and preferably at least one of them is a modified nucleotide.

[0066] As modified nucleotides, there can be exemplified: sugar-modified nucleotides (e.g., nucleotides in which the hydroxyl group of D-ribofuranose is modified, such as nucleotides in which D-ribofuranose is 2'-O-alkylated, nucleotides in which D-ribofuranose is 2'-O,4'-C-alkylated, etc.), nucleotides in which the phosphodiester bond is modified (e.g., thiolated), nucleotides in which the base is modified, nucleotides in which these are combined, etc. At least one nucleotide in which D-ribofuranose is 2'-O-alkylated or 2'-O,4'-C-alkylated constituting the antisense oligonucleotide has a high binding affinity to RNA and high resistance to nucleases, so it is expected that the therapeutic effect is higher than that of natural nucleotides (i.e., oligoDNA, oligoRNA). In addition, at least one nucleotide in which the phosphodiester bond is thiolated constituting the oligonucleotide also has a high resistance to nucleases, so it is expected that the therapeutic effect is higher than that of natural nucleotides (i.e., oligoDNA, oligoRNA). Oligonucleotides containing both modified sugars and modified phosphates as described above have higher resistance to nucleases and can be expected to have higher therapeutic effects.

[0067] Regarding antisense oligonucleotides, examples of sugar modifications include: 2'-O-alkylation of D-ribofuranose (e.g., 2'-O-methylation, 2'-O-aminoethylation, 2'-O-propylation, 2'-O-allylation, 2'-O-methoxyethylation, 2'-O-butylation, 2'-O-pentylation, 2'-O-propargylation, etc.), 2'-O,4'-C-alkylation of D-ribofuranose, Alkylation (e.g., 2'-O,4'-C-ethylidene, 2'-O,4'-C-methylidene, 2'-O,4'-C-propylidene, 2'-O,4'-C-tetramethylidene, 2'-O,4'-C-pentamethylene, etc.), 3'-deoxy-3'-amino-2'-deoxy-D-ribofuranose, 3'-deoxy-3'-amino-2'-deoxy-2'-fluoro-D-ribofuranose, etc.

[0068] Regarding the antisense oligonucleotide, examples of modifications of the phosphodiester bond include a phosphorothioate bond, a methylphosphonate bond, a methylphosphonothioate bond, a phosphorodithioate bond, a phosphoramidate bond, and the like.

[0069] Regarding antisense oligonucleotides, examples of base modifications include 5-methylation, 5-fluorination, 5-bromination, 5-iodination, and N4-methylation of cytosine, 5-demethylation (uracil), 5-fluorination, 5-bromination, and 5-iodination of thymidine, N6-methylation and 8-bromination of adenine, and N2-methylation and 8-bromination of guanine.

[0070] The antisense oligonucleotide of the present invention may be in the form of a salt. When the antisense oligonucleotide of the present invention is used as a drug, the salt may be a pharmaceutically acceptable salt, and examples of such salts include: alkali metal salts such as sodium salts, potassium salts, lithium salts, alkaline earth metal salts such as calcium salts, magnesium salts, aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts and other metal salts; inorganic salts such as ammonium salts, tert-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, N-benzyl-phenethylamine Amine salts such as organic salts such as iodine, piperazine, tetramethylammonium, tris(hydroxymethyl)aminomethane salts; inorganic acid salts such as hydrohalides such as hydrofluorides, hydrochlorides, hydrobromides, and hydroiodides, nitrates, perchlorates, sulfates, and phosphates; organic acid salts such as lower alkane sulfonates such as methanesulfonates, trifluoromethanesulfonates, and ethanesulfonates, aryl sulfonates such as benzenesulfonates and p-toluenesulfonates, acetates, malates, fumarates, succinates, citrates, tartrates, oxalates, and maleates; amino acid salts such as glycine, lysine, arginine, ornithine, glutamate, and aspartate, etc. These salts can be produced by known methods.

[0071] In addition, the antisense oligonucleotide may exist in the form of a solvate (eg, a hydrate) in some cases, and may be such a solvate.

[0072] Furthermore, the antisense oligonucleotide can be administered in the form of a prodrug, and examples of the prodrug include amides, esters, carbamates, carbonates, ureides, and phosphates. These prodrugs can be produced by known methods.

[0073] There is no particular limitation on the method for synthesizing antisense oligonucleotides, and a known method can be used. Examples of the above-mentioned synthesis method include: a synthesis method based on a genetic engineering method, a chemical synthesis method, and the like. Examples of the genetic engineering method include: an in vitro transcription synthesis method, a method using a vector, and a method based on a PCR cassette. There is no particular limitation on the above-mentioned vector, and examples include: non-viral vectors such as plasmids, viral vectors, and the like. There is no particular limitation on the above-mentioned chemical synthesis method, and examples include: a phosphoramidite method and an H-phosphonate method, and the like. The above-mentioned chemical synthesis method can use, for example, a commercially available automatic nucleic acid synthesizer. The above-mentioned chemical synthesis method generally uses amidite (phosphoramidite). There is no particular limitation on the above-mentioned amidite. In the examples described below, the antisense oligonucleotide is synthesized by the phosphoramidite method using ENA-2CE phosphoramidite and 2'OMe-2CE phosphoramidite.

[0074] As for natural nucleosides and 2'-O-methyl nucleosides (i.e., 2'-O-methylguanosine, 2'-O-methyladenosine, 2'-O-methylcytosine, 2'-O-methyluridine), commercially available reagents can be used as phosphoramidite reagents. 2'-O-alkylguanosine, adenosine, cytosine, and uridine in which the alkyl group has 2 to 6 carbon atoms are described below.

[0075] 2'-O-aminoethylguanosine, adenosine, cytosine, and uridine can be synthesized according to the literature (Blommers et al. Biochemistry (1998), 37, 17714-17725).

[0076] 2'-O-propylguanosine, adenosine, cytosine, and uridine can be synthesized according to the literature (Lesnik, EA et al. Biochemistry (1993), 32, 7832-7838).

[0077] Commercially available reagents can be used for 2'-O-allylguanosine, adenosine, cytosine, and uridine.

[0078] 2'-O-methoxyethylguanosine, adenosine, cytosine, and uridine can be synthesized according to patent (US6261840) or literature (Martin, P. Helv. Chim. Acta. (1995) 78, 486-504.

[0079] 2'-O-butylguanosine, adenosine, cytosine, and uridine can be synthesized according to the literature (Lesnik, EA et al. Biochemistry (1993), 32, 7832-7838).

[0080] 2'-O-pentylguanosine, adenosine, cytosine, and uridine can be synthesized according to the literature (Lesnik, EA et al. Biochemistry (1993), 32, 7832-7838).

[0081] Commercially available reagents can be used for 2'-O-propargylguanosine, adenosine, cytosine, and uridine.

[0082] 2'-O, 4'-C-methyleneguanosine, adenosine, 5-methylcytosine and thymidine can be produced according to the method described in WO99 / 14226, and 2'-O, 4'-C-alkyleneguanosine, adenosine, 5-methylcytosine and thymidine having an alkylene group with 2 to 5 carbon atoms can be produced according to the method described in WO00 / 47599.

[0083] After coupling with a phosphoramidite reagent, the reaction is then reacted with sulfur, tetraethylthiuram disulfide (TETD, Applied Biosystems), Beaucage reagent (Glen Research), or hydrogenated xanthin to synthesize an antisense oligonucleotide having a thiophosphate bond (Tetrahedron Letters, 32, 3005 (1991), J. Am. Chem. Soc. 112, 1253 (1990), PCT / WO98 / 54198).

[0084] As the controlled pore glass (CPG) used in the synthesizer, a commercially available CPG bonded with 2'-O-methyl nucleoside can be used. In addition, with respect to 2'-O, 4'-C-methyleneguanosine, adenosine, 5-methylcytosine and thymidine, nucleosides produced according to the method described in WO99 / 14226 can be bonded to CPG according to the document (Oligonucleotide Synthesis, Edited by MJ Gait, Oxford University Press, 1984), and with respect to 2'-O, 4'-C-alkyleneguanosine, adenosine, 5-methylcytosine and thymidine having 2 to 5 carbon atoms in the alkylene group, nucleosides produced according to the method described in WO00 / 47599 can be bonded to CPG according to the same document. By using a modified CPG (described in Example 12b of Japanese Patent Application Laid-Open No. 7-87982), an oligonucleotide bonded with a 2-hydroxyethyl phosphate group at the 3' end can be synthesized. In addition, by using 3'-amino-modifier C3 CPG, 3'-amino-modifier C7 CPG, glyceryl CPG (Glen Research), 3'-specer C3 SynBase CPG 1000, 3'-specer C9 SynBase CPG 1000 (link technologies), oligonucleotides having a hydroxyalkylphosphate group or an aminoalkylphosphate group bound to the 3' terminus can be synthesized.

[0085] The antisense oligonucleotides of the present invention can be used for medicine. When used as medicine, the antisense oligonucleotide can be in the form of a pharmaceutically acceptable salt, solvate or prodrug thereof. Therefore, the present invention provides a medicine comprising the above-mentioned antisense oligonucleotide, a pharmaceutically acceptable salt or solvate thereof. The medicine can be a medicine for preventing and / or treating a condition and / or disease in which myostatin is involved. As the condition and / or disease in which myostatin is involved, there can be listed: muscular atrophy (for example, muscular atrophy is muscular dystrophy, myopathy, spinal muscular atrophy, sarcopenia, disuse muscular atrophy, etc.), a condition and / or disease that produces a therapeutic effect by restoring muscle mass (for example, cancer cachexia, diabetes, circulatory system diseases (heart failure, arteriosclerosis, etc.), kidney disease (chronic renal insufficiency, etc.), bone disease (inflammatory arthritis, etc.), etc.), but it is not limited to these. From the perspective that the inhibition of myostatin can increase the amount of skeletal muscle, it is believed that regardless of the cause of muscular atrophy, it can be used to treat all diseases presenting muscular atrophy. The increase in skeletal muscle mass can help increase the amount of exercise and improve systemic metabolism. In addition, it is expected to act on the myocardium to restore its function. On the other hand, the inhibition of myostatin is also expected to act on osteoclasts to inhibit bone destruction; activate the homeostasis-maintaining ability of vascular endothelial cells; induce cell apoptosis; and improve insulin sensitivity.

[0086] The antisense oligonucleotides of the present invention, their pharmaceutically acceptable salts, solvates or prodrugs (hereinafter referred to as "active ingredients") can be administered orally or parenterally to mammals (e.g., humans, rabbits, dogs, cats, rats, mice) alone or together with pharmacologically acceptable carriers, diluents or excipients in the form of preparations of appropriate dosage forms. The dosage also varies according to the subject of administration, the subject disease, symptoms, the route of administration, etc. For example, in the case of prevention / treatment of muscular atrophy diseases (e.g., muscular dystrophy), as a single dose of the active ingredient, about 0.1 to 50 mg / kg body weight, preferably about 0.5 mg / kg body weight, can usually be administered orally / intramuscularly / subcutaneously / intravenously at a frequency of about once a week to about once a month, preferably about once a year (preferably continuously or every other day). This amount can also be administered in other cases of parenteral administration and oral administration. In the case of particularly severe symptoms, the amount can be increased according to the symptoms. For other diseases, the above-mentioned dosage can also be referred to and the dosage can be appropriately increased or decreased.

[0087] As preparations for oral administration, there can be listed: solid dosage forms or liquid dosage forms, specifically, tablets (including sugar-coated tablets, film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups, emulsions, suspensions, etc. The preparations can be manufactured using conventional methods and can contain carriers, diluents or excipients commonly used in the field of preparations. For example, as carriers and excipients for tablets, there can be listed: lactose, starch, sucrose, magnesium stearate, etc.

[0088] As preparations for parenteral administration, for example, injections and suppositories can be listed. Injections can be in the form of intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, and the like. Such injections are prepared by conventional methods, i.e., by dissolving, suspending, or emulsifying the active ingredient in a sterile aqueous or oily liquid commonly used for injections. As aqueous liquids for injection, physiological saline, isotonic liquids containing glucose or other adjuvants, etc., can be used in combination with appropriate cosolvents, such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)), etc. As oily liquids, sesame oil, soybean oil, etc. can be listed, and benzyl benzoate, benzyl alcohol, etc. can be used in combination as cosolvents. The prepared injection is usually filled in an appropriate ampoule. Suppositories for rectal administration can be prepared by mixing the active ingredient in a conventional suppository base.

[0089] The above-mentioned oral or parenteral pharmaceutical preparations can be prepared into dosage forms of dosage units suitable for the dosage of the active ingredient. Such dosage forms of dosage units include tablets, pills, capsules, injections (ampoules), suppositories, etc., and each dosage unit dosage form preferably contains 0.1 to 1.000 mg of the active ingredient.

[0090] The antisense oligonucleotides of the present invention can also be used in food and feed. For example, the antisense oligonucleotides of the present invention are used as additives for food or feed, or as supplements for humans or animals. The antisense oligonucleotides of the present invention can be in the form of salts, solvates or prodrugs acceptable to food or feed. Therefore, the present invention provides food, which contains the above-mentioned antisense oligonucleotides, salts or solvates acceptable to food thereof. In addition, the present invention also provides feed, which contains the above-mentioned antisense oligonucleotides, salts or solvates acceptable to feed thereof. As an example of salts, solvates or prodrugs acceptable to food or feed, pharmaceutically acceptable salts, solvates or prodrugs can be listed, and see above for them.

[0091] The antisense oligonucleotides of the present invention can be used to promote the proliferation and / or hypertrophy of muscle cells. Myocytes are contractile cells that form muscle tissues of humans or animals, including skeletal muscle cells, smooth muscle cells, cardiomyocytes, etc. The myostatin inhibition of the antisense oligonucleotides of the present invention has an effect in myoblasts, and by inducing the proliferation promotion / differentiation promotion of myoblasts, the proliferation / hypertrophy of muscle cells can be caused as a result. Myocytes also include precursor cells such as myoblasts. The antisense oligonucleotides of the present invention can be in the form of salts, solvates or prodrugs. As an example of a salt, solvate or prodrug, pharmaceutically acceptable salts, solvates or prodrugs can be listed, and these are described above. As long as the animal is an animal that expresses myostatin, examples can be given: mammals such as cats, dogs, sheep, pigs, cattle, chickens, turkeys, and fish such as salmon, trout, cod, tuna, and yellowtail fish, and other farmed animals for use and consumption. Therefore, the present invention provides: an agent for promoting the proliferation and / or hypertrophy of muscle cells, the agent comprising the above-mentioned antisense oligonucleotides, their salts or solvates. In addition, the present invention also provides: a method for promoting proliferation and / or hypertrophy of muscle cells, the method comprising: administering an effective amount of the above-mentioned antisense oligonucleotide, its salt or solvate to a subject. The subject can be a human or an animal. For animals, see above. The dosage form, dosage, route of administration, frequency of administration, etc. of the medicament can be based on the above-mentioned drugs and can be appropriately changed to obtain the desired effect.

[0092] In addition, the antisense oligonucleotides of the present invention can be used to promote muscle formation or inhibit muscle loss in humans or animals. The antisense oligonucleotides of the present invention may be in the form of salts, solvates or prodrugs. As an example of a salt, solvate or prodrug, pharmaceutically acceptable salts, solvates or prodrugs may be cited, as described above. As long as the animal expresses myostatin, examples may include mammals such as cats, dogs, sheep, pigs, cattle, chickens, turkeys, and fish such as salmon, trout, cod, tuna, and yellowtail fish, and other farmed animals used for work and food. Therefore, the present invention provides a medicament for promoting muscle formation and / or inhibiting muscle loss, which comprises the antisense oligonucleotides, salts or solvates thereof described above. In addition, the present invention also provides a method for promoting muscle formation and / or inhibiting muscle loss, which comprises administering an effective amount of the antisense oligonucleotides, salts or solvates thereof to a subject. The subject may be a human or an animal. See above for animals. The dosage form, dosage, route of administration, frequency of administration, etc. of the medicament may be based on the above-mentioned drugs, and may be appropriately changed to obtain the desired effect.

[0093] The food to which the antisense oligonucleotide of the present invention, its food-acceptable salt, solvate or prodrug is added may be any food, such as plant food, animal food, fungal food, fresh food, processed food, targeted food, cooking / seasoning material, beverage, health food, space food, pet food, etc.

[0094] In the food of the present invention, it is possible to add: common ingredients such as protein, lipid, carbohydrate, sodium; minerals such as potassium, calcium, magnesium, phosphorus; trace elements such as iron, zinc, copper, selenium, chromium; vitamins such as vitamin A, β-carotene, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, niacin, folic acid, vitamin D3, vitamin E, biotin, pantothenic acid; coenzyme Q10, α-lipoic acid, galacto-oligosaccharide, dietary fiber, excipients (water, carboxymethyl cellulose, lactose, etc.), sweeteners, flavoring agents (malic acid, citric acid, amino acids, etc.), spices, etc. In the case of making the food of the present invention into a liquid preparation, water, saline, soup, milk, juice, etc. can be used as a liquid for dispersing or dissolving food ingredients. The food of the present invention can be made into shapes such as powder, granules, tablets, liquid preparations, etc. In order to make it easy for patients or the elderly to ingest, it is preferably made into a gel-like product such as jelly.

[0095] The feed to which the antisense oligonucleotide of the present invention, its feed-acceptable salt, solvate or prodrug may be added may be any feed, such as single feeds such as cereals, vegetable oils and cakes, brans, manufactured cakes, animal feeds, or mixed feeds containing a variety of feed ingredients or feed additives.

[0096] The feed of the present invention may be added with: antioxidants (ethoxyquin, butylated hydroxytoluene, butylated hydroxyanisole, etc.), mildew inhibitors (propionic acid, calcium propionate, sodium propionate, etc.), binders (sodium alginate, sodium caseinate, sodium carboxymethyl cellulose, propylene glycol, sodium polyacrylate, etc.), emulsifiers (glycerol fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene glycerol fatty acid esters, etc.), regulators (formic acid, etc.), amino acids, etc. (aminoacetic acid, DL-alanine, L-arginine, L-lysine hydrochloride, L-carnitine, guanidine acetic acid, L- Sodium glutamate, taurine, 2-deamino-2-hydroxymethionine, DL-tryptophan, L-tryptophan, L-threonine, L-valine, DL-methionine, L-lysine sulfate, etc.), vitamins (L-ascorbic acid, calcium L-ascorbate, sodium L-ascorbate, sodium calcium L-ascorbate-2-phosphate, magnesium L-ascorbate-2-phosphate, acetyl menadione, inositol, dibenzoylthiamine hydrochloride, ergocalciferol, choline chloride, thiamine hydrochloride, pyridoxine hydrochloride, beta-carotene, cholecalciferol, dl-alpha-tocopherol acetate, cyanocobalamin, thiamine nitrate, niacin, nicotinic acid Acid amide, para-aminobenzoic acid, D-calcium pantothenate, DL-calcium pantothenate, d-biotin, vitamin A powder, vitamin A oil, vitamin D powder, vitamin D3 oil, vitamin E powder, 25-hydroxycholecalciferol, dimethylpyrimidine alcohol menadione bisulfite, sodium menadione bisulfite, folic acid, riboflavin, riboflavin butyrate, etc.), minerals (potassium chloride, ferric citrate, calcium gluconate, sodium ferric citrate succinate, magnesium oxide, aluminum hydroxide, zinc carbonate, cobalt carbonate, sodium bicarbonate, magnesium carbonate, manganese carbonate, 2-deamino-2-hydroxymethionine zinc, DL-threonine iron, calcium lactate, fumaric acid Ferrous iron, peptide zinc, peptide iron, peptide copper, peptide manganese, potassium iodide, potassium iodate, calcium iodate, zinc sulfate (dry), zinc sulfate (crystal), zinc methionine sulfate, sodium sulfate (dry), magnesium sulfate (dry), magnesium sulfate (crystal), cobalt sulfate (dry), cobalt sulfate (crystal), ferrous sulfate (dry), copper sulfate (dry), copper sulfate (crystal), manganese sulfate, potassium monohydrogen phosphate (dry), sodium monohydrogen phosphate (dry), potassium dihydrogen phosphate (dry), sodium dihydrogen phosphate (dry), sodium dihydrogen phosphate (crystal), etc.), pigments, synthetic antibacterial agents, antibiotics, flavorings, flavoring agents, enzymes, probiotics, organic acids, etc.

[0097] The intake of food or feed can be carried out in an intake amount, frequency, and duration in which a desired effect (for example, promotion of muscle formation) is confirmed.

[0098] The antisense oligonucleotide of the present invention can inhibit the production of mRNA of the myostatin gene. Therefore, the present invention provides: a pharmaceutical agent for inhibiting the production of mRNA of the myostatin gene, the pharmaceutical agent containing the above-mentioned antisense oligonucleotide. The pharmaceutical agent of the present invention can be used as a drug for humans or animals, an additive or supplement for food or feed, a growth promoter for animals, or an experimental reagent. The antisense oligonucleotide of the present invention may be in the form of a salt, a solvate or a prodrug. As an example of a salt, a solvate or a prodrug, there can be cited: a pharmaceutically acceptable salt, a solvate or a prodrug, and these are described above.

[0099] The antisense oligonucleotide of the present invention can inhibit the function of myostatin. Therefore, a myostatin function inhibitor is provided, which comprises the above-mentioned antisense oligonucleotide of the present invention. The myostatin function inhibitor of the present invention can be used as a drug for humans or animals, an additive or supplement for food or feed, a growth promoter for animals, or an experimental reagent. The antisense oligonucleotide may be in the form of a salt, a solvate or a prodrug. As an example of a salt, a solvate or a prodrug, there can be cited: a pharmaceutically acceptable salt, a solvate or a prodrug, as described above.

[0100] When used as an experimental reagent, the expression of myostatin can be inhibited by treating cells, tissues or organs expressing myostatin with the antisense oligonucleotide of the present invention, its salt or solvate. The antisense oligonucleotide of the present invention, its salt and solvate can be used in an amount effective to inhibit the expression of myostatin. Examples of cells expressing myostatin include muscle cells, myoma cells, cancer cells of digestive organs / lungs / esophagus, etc. In addition, in addition to cells derived from natural sources, examples include recombinant cells into which myostatin genes have been introduced. Examples of tissues and organs expressing myostatin include skeletal muscle, myocardium, blood vessels, kidneys, digestive tract, uterus, liver, pancreas, lungs, etc. The expression of myostatin can be analyzed by analyzing myostatin mRNA in a sample using RT-PCR, or by detecting myostatin protein in a sample using Western blotting, or by detecting it using mass spectrometry. Example

[0101] Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.

[0102] [Examples 1 to 24] Synthesis of antisense oligonucleotide (AO)

[0103] The antisense oligonucleotides (AO) shown in Table 1 were synthesized. The sequence positions of the AO complementary to MSTN pre-mRNA are shown in Figure 1~ 4. Introducing ENA (registered trademark) (2'-O,4'-C-ethylene bridged nucleic acid) into C (cytosine) and T (thymine) in the sequence of AO improves affinity and stability.

[0104] Synthesis of CaCuggaCCaGCaaCaau Synthesis of (MSTN_Ex1_AO1) (Example 1)

[0105] A nucleic acid automatic synthesizer (DNA / RNA synthesizer NTS H-6 manufactured by Techno Service, Japan) was used to synthesize the nucleic acid. μ The reaction was carried out on a mol scale. The concentrations of solvents, reagents, and phosphoramidites in each synthesis cycle were the same as those in the case of synthesizing natural oligonucleotides. Reagents and 2'-O-methyl nucleoside phosphoramidites (adenosine product number 10-3100-10, guanosine product number 10-3121-10) were products manufactured by Glen Research. Solvents were products of Wako Pure Chemical Industries, Ltd. Non-natural phosphoramidites were compounds of Example 22 (5'-O-dimethoxytrityl-2'-O, 4'-C-ethylidene-4-N-benzoyl-5-methylcytidine-3'-O-(2-cyanoethyl N,N-diisopropyl) phosphoramidite) and Example 9 (5'-O-dimethoxytrityl-2'-O, 4'-C-ethylidene-5-methyluridine-3'-O-(2-cyanoethyl N,N-diisopropyl) phosphoramidite) of Japanese Patent Application Laid-Open No. 2000-297097. The title compound was synthesized using universal controlled pore glass (CPG) (manufactured by Glen Research, product number 25-5040) as a solid phase support. The time required for the condensation of the amidite was 15 minutes.

[0106] The protected oligonucleotide analog with the target sequence is subjected to heat treatment in concentrated ammonia (55°C, 8 hours), the oligomer is cut from the support, and the protective group cyanoethyl on the phosphorus atom and the protective group on the nucleic acid base are removed at the same time. Using a Glen-Pak DNA purification kit (Glen Research, product number 60-5100), the ammonia solution is de-DMTed in the kit according to the method recommended by GlenResearch, and the recovered solution is distilled under reduced pressure, and the residue is purified by reverse phase HPLC (LC-2a manufactured by Shimadzu Corporation, column (Triart C18 (10×150mm) manufactured by YMC), A solution: 0.1M triethylamine acetate aqueous solution (TEAA), pH7.0; B solution: acetonitrile, B%: 10%→25% (30 minutes, linear gradient); 50°C; 4.7mL / min; 280nm). After the solvent was distilled off, the residue was dissolved in a 10 mM NaOH solution and replaced with pure water by ultrafiltration using a Microsep centrifugal filter device (manufactured by Pall Corporation, Japan, product number MCP003C). The target compound was obtained after freeze-drying.

[0107] Synthesis of augCaTTaCaCagCCCCu (MSTN_Ex1_AO2) (Example 2)

[0108] In the same manner as the compound of Example 1, the compound of Example 2 having the target sequence was synthesized.

[0109] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6395.311, found value: 6392.585).

[0110] The nucleotide sequence of the compound is complementary to nucleotide numbers 240-257 of human myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank Accession No. NG_009800.1).

[0111] Synthesis of gauuuaguguuuuguCuC (MSTN_Ex1_AO3) (Example 3)

[0112] In the same manner as the compound of Example 1, the compound of Example 3 having the target sequence was synthesized.

[0113] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6226.921, found value: 6220.691).

[0114] The nucleotide sequence of the compound is complementary to nucleotide numbers 265-282 of human myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank Accession No. NG_009800.1).

[0115] Synthesis of aagTaCaTgCaTTaCaCa (MSTN_Ex1_AO2-6) (Example 4)

[0116] In the same manner as the compound of Example 1, the compound of Example 4 having the target sequence was synthesized.

[0117] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6441.351, found value: 6435.8281).

[0118] The nucleotide sequence of the compound is complementary to nucleotide numbers 246-263 of human myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank Accession No. NG_009800.1).

[0119] Synthesis of TaCaTgCaTTaCaCagCC (MSTN_Ex1_AO2-3) (Example 5)

[0120] In the same manner as the compound of Example 1, the compound of Example 5 having the target sequence was synthesized.

[0121] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6445.375, found value: 6439.7319).

[0122] The nucleotide sequence of the compound is complementary to nucleotide numbers 243-260 of human myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank Accession No. NG_009800.1).

[0123] Synthesis of CaTTaCaCagCCCCTCTT (MSTN_Ex1_AO2+3) (Example 6)

[0124] In the same manner as the compound of Example 1, the compound of Example 6 having the target sequence was synthesized.

[0125] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6436.396, found value: 6430.9077).

[0126] The nucleotide sequence of the compound is complementary to nucleotide numbers 237-254 of human myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank Accession No. NG_009800.1).

[0127] Synthesis of TaCaCagCCCCTCTTTTT (MSTN_Ex1_AO2+6) (Example 7)

[0128] In the same manner as the compound of Example 1, the compound of Example 7 having the target sequence was synthesized.

[0129] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6440.376, found value: 6435.562).

[0130] The nucleotide sequence of the compound is complementary to nucleotide numbers 234-251 of human myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank Accession No. NG_009800.1).

[0131] Synthesis of aCagCCCCTCTTTTTCCa (MSTN_Ex1_AO2+9) (Example 8)

[0132] In the same manner as the compound of Example 1, the compound of Example 8 having the target sequence was synthesized.

[0133] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6439.392, found value: 6434.6094).

[0134] The nucleotide sequence of the compound is complementary to nucleotide numbers 231-248 of human myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank Accession No. NG_009800.1).

[0135] Synthesis of TgCaTTaCaCagCCCCTC (MSTN_Ex1_AO2+1) (Example 9)

[0136] In the same manner as the compound of Example 1, the compound of Example 9 having the target sequence was synthesized.

[0137] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6449.399, found value: 6443.8228).

[0138] The nucleotide sequence of the compound is complementary to nucleotide numbers 239-256 of human myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank Accession No. NG_009800.1).

[0139] Synthesis of gCaTTaCaCagCCCCTCT (MSTN_Ex1_AO2+2) (Example 10)

[0140] In the same manner as the compound of Example 1, the compound of Example 10 having the target sequence was synthesized.

[0141] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6449.399, found value: 6443.707).

[0142] The nucleotide sequence of the compound is complementary to nucleotide numbers 238-255 of human myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank Accession No. NG_009800.1).

[0143] Synthesis of aTTaCaCagCCCCTCTTT (MSTN_Ex1_AO2+4) (Example 11)

[0144] In the same manner as the compound of Example 1, the compound of Example 11 having the target sequence was synthesized.

[0145] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6437.38, found value: 6431.9209).

[0146] The nucleotide sequence of the compound is complementary to nucleotide numbers 236-253 of human myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank Accession No. NG_009800.1).

[0147] Synthesis of TTaCaCagCCCCTCTTTT (MSTN_Ex1_AO2+5) (Example 12)

[0148] In the same manner as the compound of Example 1, the compound of Example 12 having the target sequence was synthesized.

[0149] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6440.376, found value: 6434.8232).

[0150] The nucleotide sequence of the compound is complementary to nucleotide numbers 235-252 of human myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank Accession No. NG_009800.1).

[0151] Synthesis of TgTaCagTCTgagagaCa (MSTN_Ex1_AO4) (Example 13)

[0152] In the same manner as the compound of Example 1, the compound of Example 13 having the target sequence was synthesized.

[0153] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6487.336, found value: 6481.8237).

[0154] The nucleotide sequence of the compound is complementary to nucleotide numbers 22-39 of human myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank Accession No. NG_009800.1).

[0155] Synthesis of aaTgCaTgTaCagTCTga (MSTN_Ex1_AO4-6) (Example 14)

[0156] In the same manner as the compound of Example 1, the compound of Example 14 having the target sequence was synthesized.

[0157] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6474.333, found value: 6468.584).

[0158] The nucleotide sequence of the compound is complementary to nucleotide numbers 28-45 of human myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank Accession No. NG_009800.1).

[0159] Synthesis of TTgCTTTTgagTaaTgCC (MSTN_Ex1_AO5) (Example 15)

[0160] In the same manner as the compound of Example 1, the compound of Example 15 having the target sequence was synthesized.

[0161] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6483.321, found value: 6477.7007).

[0162] The nucleotide sequence of the compound is complementary to nucleotide numbers 58-75 of human myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank Accession No. NG_009800.1).

[0163] Synthesis of aaTCaaTaTaaTCTTTTT (MSTN_Ex1_AO6) (Example 16)

[0164] In the same manner as the compound of Example 1, the compound of Example 16 having the target sequence was synthesized.

[0165] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6420.309, found value: 6414.5815).

[0166] The nucleotide sequence of the compound is complementary to nucleotide numbers 108-125 of human myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank Accession No. NG_009800.1).

[0167] Synthesis of TTgCTCaCTgTTCTCaTT (MSTN_Ex1_AO7) (Example 17)

[0168] In the same manner as the compound of Example 1, the compound of Example 17 having the target sequence was synthesized.

[0169] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6458.343, found value: 6452.9326).

[0170] The nucleotide sequence of the compound is complementary to nucleotide numbers 203-220 of human myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank Accession No. NG_009800.1).

[0171] Synthesis of CTTgaagaTTTagTgTTT (MSTN_Ex1_AO3-6) (Example 18)

[0172] In the same manner as the compound of Example 1, the compound of Example 18 having the target sequence was synthesized.

[0173] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6482.293, found value: 6476.7026).

[0174] The nucleotide sequence of the compound is complementary to nucleotide numbers 271-288 of human myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank Accession No. NG_009800.1).

[0175] Synthesis of TCTaTTCTTgaagaTTTa (MSTN_Ex1_AO3-12) (Example 19)

[0176] In the same manner as the compound of Example 1, the compound of Example 19 having the target sequence was synthesized.

[0177] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6452.307, found value: 6446.8232).

[0178] The nucleotide sequence of the compound is complementary to nucleotide numbers 277-294 of human myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank Accession No. NG_009800.1).

[0179] Synthesis of TaCTgaggaTTTgTaTCT (MSTN_Ex1_AO8) (Example 20)

[0180] In the same manner as the compound of Example 1, the compound of Example 20 having the target sequence was synthesized.

[0181] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6481.309, found value: 6475.8228).

[0182] The nucleotide sequence of the compound is complementary to nucleotide numbers 303-320 of human myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank Accession No. NG_009800.1).

[0183] Synthesis of CaTCTTTgCTgaTgTTag (MSTN_Ex1_AO9) (Example 21)

[0184] In the same manner as the compound of Example 1, the compound of Example 21 having the target sequence was synthesized.

[0185] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6483.321, found value: 6477.6953).

[0186] The nucleotide sequence of the compound is complementary to nucleotide numbers 342-359 of human myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank Accession No. NG_009800.1).

[0187] Synthesis of gTTgTCTTaTaaCaTCTT (MSTN_Ex1_AO9-12) (Example 22)

[0188] In the same manner as the compound of Example 1, the compound of Example 22 having the target sequence was synthesized.

[0189] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6454.319, found value: 6448.7021).

[0190] The nucleotide sequence of the compound is complementary to nucleotide numbers 354-371 of human myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank Accession No. NG_009800.1).

[0191] Synthesis of TgaTCaaTCagTTCCCgg (MSTN_Ex1_AO10) (Example 23)

[0192] In the same manner as the compound of Example 1, the compound of Example 23 having the target sequence was synthesized.

[0193] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6478.357, found value: 6472.9458).

[0194] The nucleotide sequence of the compound is complementary to nucleotide numbers 394-411 of human myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank Accession No. NG_009800.1).

[0195] Synthesis of aCaTCaTaCTgaTCaaTC (MSTN_Ex1_AO10-9) (Example 24)

[0196] In the same manner as the compound of Example 1, the compound of Example 24 having the target sequence was synthesized.

[0197] This compound was identified by negative ion MALDI-TOFMS (calculated value: 6430.36, found value: 6424.8286).

[0198] The nucleotide sequence of the compound is complementary to nucleotide numbers 403-420 of human myostatin (MSTN), RefSeqGene (LRG_200) on chromosome 2 (Gene Bank Accession No. NG_009800.1).

[0199] (Table 1)

[0200]

[0201] [Test example]

[0202] Experimental methods

[0203] 1. Evaluation of MSTN mRNA Amount

[0204] The expression changes of MSTN mRNA caused by AO were evaluated by RT-PCR using human rhabdomyosarcoma cells (CRL-2016, ATCC) and human myoblasts (Wada et al. Development 2002, 129; 2987-2995).

[0205] AO transfection

[0206] 1) In 100 μ l Opti-MEM medium (31985070, Thermo Fisher Scientific) was mixed with 2 μ l AO (mixed with MilliQ sterile water to 50 pmol / μ l solution). AO untreated is mixed 2 μ l MilliQ sterilized water;

[0207] 2) Use another tube at 100 μ l Opti-MEM medium (31985070, Thermo Fisher Scientific) was mixed with 4 μ Lipofectamine TM2000 transfection reagent (11668019, Thermo Fisher Scientific);

[0208] 3) Mix liquid 1) and liquid 2) and let stand at room temperature for 20 minutes;

[0209] 4) After human rhabdomyosarcoma cells cultured in a 12-well plate were washed once with PBS, 800 μL of μ lOpti-MEM medium (31985070, Thermo Fisher Scientific);

[0210] 5) Add solution 3) to solution 4) (final AO concentration is 100 nM), culture for 3 hours at 37°C and 5% CO2, then replace the culture medium with RPMI medium (22400-089, gibco) containing 10% FBS (10270-106, gibco) and continue the culture;

[0211] 6) Human myoblasts were cultured in DMEM medium (043-30085, Wako) containing 20% ​​FBS (10270-106, Gibco) and 2% Ultroser G, and AO was transfected at a final concentration of 0, 100, 200, and 400 nM.

[0212] RNA modulation

[0213] 1) After culturing the cells transfected with each AO for 24 hours, they were washed once with PBS and 300 μL of μ l RNA extraction reagent from High Purity RNA Isolation Kit (#11828665001, Roche Life Science);

[0214] 2) After standing at room temperature for 5 minutes, the RNA extraction reagent in the well was recovered into the tube;

[0215] 3) RNA was extracted according to the protocol of the High Purity RNA Isolation Kit (#11828665001, Roche Life Science), and 50 μ l RNA dissolution solution.

[0216] Reverse transcription reaction

[0217] 1) Add random primers (#48190011, Thermo Fisher Scientific) and dNTPs (Takara) to 500 ng RNA, incubate at 65°C for 5 minutes and at 25°C for 10 minutes;

[0218] 2) Add M-MLV reverse transcriptase (#28025013, Thermo Fisher Scientific), RNaseOUT TM Recombinant ribonuclease inhibitor (#10777-019, Thermo Fisher Scientific), DTT (attached to MLVRT), and buffer (attached to MLVRT) were added, and the cells were incubated at 37°C for 55 minutes and at 70°C for 10 minutes to obtain cDNA.

[0219] PCR reaction and confirmation of reaction products

[0220] 1) Relative to the obtained 2 μ l cDNA, add 1 μ 1 Primer MSTN Ex1_F1 (5'-agattcactggtgtggcaag-3': SEQ ID NO: 26), 1 μ l MSTN R2 (5'-tgcatgacatgtctttgtgc-3': SEQ ID NO: 27), 0.1 μ l TaKaRa Ex Taq (registered trademark) DNA polymerase (#RR001A, Takara), 1.6 μ l dNTPs (attached to TaKaRa Ex Taq (registered trademark)), 2 μ l Buffer (10×), 12.3 μ l MilliQ sterilized water;

[0221] 2) Heat at 94°C for 3 minutes;

[0222] 3) 30 cycles of 94°C for 0.5 min / 60°C for 0.5 min / 72°C for 1.5 min;

[0223] 4) Heat at 72°C for 3 minutes;

[0224] 5) For the reaction products of the PCR reaction, add Midri Green Direct DNA stain (NE-MG06, Japan Genetics) and loading buffer (Takara), use 2% agarose gel electrophoresis, and visualize it using a gel photography device. In addition, use Agilent 2100 Bioanalyzer Electrophoresis System (Agilent Technologies, Inc.) to perform electrophoresis and quantification of PCR reaction products;

[0225] 6) Using primers GAPDH H_F (5'-cccttcattgacctcaac-3': SEQ ID NO: 28) and GAPDHH_R (5'-ttcacacccatgacgaac-3': SEQ ID NO: 29), perform the above steps 1) to 5) for GAPDH;

[0226] 7) Using primers GDF11Ex1F4 (5'-ctgcagcagatcctggacct-3': SEQ ID NO: 34) and GDF11Ex1R4 (5'-catgaacatgtactcgcact-3': SEQ ID NO: 35), perform the above steps 1) to 5) for GDF11;

[0227] 8) The PCR amplification products separated by agarose electrophoresis were semi-quantified using Image J, and relative comparison was performed with MSTN / GAPDH without AO treatment as 1. Similarly, in the PCR amplification products quantified using the Agilent 2100 Bioanalyzer electrophoresis system, relative comparison was also performed with MSTN / GAPDH and GDF11 / GAPDH without AO treatment as 1.

[0228] 2. Evaluation of Myostatin Signaling

[0229] Myostatin signaling was evaluated by introducing a reporter gene (SBE4-Luc plasmid, #16495, Addgene) into human rhabdomyosarcoma cells (CRL-2061, ATCC) and human myoblasts (Wada et al. Development 2002, 129; 2987-2995) and measuring the luminescence of the expressed luciferase.

[0230] AO transfection

[0231] 1) In 100 μ l Opti-MEM medium (31985070, Thermo Fisher Scientific) was mixed with 2 μ l AO2+1 (mixed with MilliQ sterile water to 50pmol / μ l solution);

[0232] 2) Use another tube at 100 μ l Opti-MEM medium (31985070, Thermo Fisher Scientific) was mixed with 3 μ g SBE4-Luc plasmid, 1 μg pSV-β-galactosidase control vector (E108A, Promega) and 4 μ Lipofectamine TM 3000 transfection reagent (11668019, Thermo Fisher Scientific), 8 μ l P3000 reagent (11668019, Thermo Fisher Scientific);

[0233] 3) Mix liquid 1) and liquid 2) and let stand at room temperature for 15 minutes;

[0234] 4) After human rhabdomyosarcoma cells cultured in a 12-well plate were washed once with PBS, 800 μL of μ lOpti-MEM medium (31985070, Thermo Fisher Scientific);

[0235] 5) Add solution 3) to solution 4) (final AO concentration is 100 nM), culture for 3 hours at 37°C and 5% CO2, then replace the culture medium with RPMI medium (22400-089, gibco) containing 10% FBS (10270-106, gibco) and continue the culture;

[0236] 6) Human myoblasts were cultured in DMEM medium (043-30085, Wako) containing 20% ​​FBS (10270-106, Gibco) and 2% Ultroser G, and transfected with AO at final concentrations of 0, 50, 100, 150, 200, 300, and 400 nM.

[0237] Preparation of cell extract

[0238] 1) After 24 hours from transfection, the cells were washed once with PBS and 250 μL of Luciferase Assay System with Reporter Lysis Buffer (E4030, Promega) was added to the wells. μ lReporter gene lysis buffer;

[0239] 2) Centrifuge the cell lysate from 1) at 15,000 rpm and 4°C for 10 minutes to obtain the supernatant;

[0240] 3) Protein quantification in cell extracts was performed using the Qubit (registered trademark) Protein Assay Kit (ProteinAssay Kit, #Q33211, Thermo Fisher Scientific) according to the protocol.

[0241] Luciferase activity assay

[0242] 1) Mix 100 wells in a 96-well plate μ l cell extract and 100 μ lLuciferase substrate (Luciferase Assay System with Reporter Lysis Buffer, #E4030, Promega);

[0243] 2) Using the multi-label plate reader ARVO TM The luciferase luminescence signal was measured by X3 (PerkinElmer).

[0244] β-Galactosidase activity assay

[0245] 1) Mix 100 wells in a 96-well plate μ l cell extract and 100 μ l β-galactosidase substrate (β-galactosidase enzyme assay system with reporter gene lysis buffer, #E2000, Promega), incubated at 37°C for 1 hour;

[0246] 2) Add 100 μ l 1M sodium carbonate was added to stop the reaction, and then the plate was read using the multi-label plate reader ARVO TM X3 (PerkinElmer) was used to measure the absorbance at 420 nm.

[0247] Activity evaluation

[0248] The extract from AO-untreated cells was measured after normalization by dividing the β-galactosidase activity value by the luciferase activity value, and the result obtained was regarded as 1, and the evaluation was performed based on the relative value.

[0249] 3. Determination of Cell Proliferation

[0250] Cell proliferation was evaluated by using the Cell Counting Kit-8 (347-07621, Dojindo Chemical Research Institute) for the proliferation of human myoblasts (Wada et al. Development 2002, 129; 2987-2995).

[0251] AO transfection

[0252] 1) In 50 μl Opti-MEM medium (31985070, Thermo Fisher Scientific) mixed with 0.4 μ l AO2+1 (mixed with MilliQ sterile water to 50pmol / μ l solution);

[0253] 2) Use another tube and μ l Opti-MEM medium (31985070, Thermo Fisher Scientific) was mixed with 0.4 μ Lipofectamine TM 3000 transfection reagent (11668019, Thermo Fisher Scientific);

[0254] 3) Mix liquid 1) and liquid 2) and let stand at room temperature for 15 minutes;

[0255] 4) After human myoblasts cultured in a 96-well plate were washed once with PBS, solution 3) was added to the wells (final AO concentration was 200 nM), and cultured for 3 hours at 37°C and 5% CO2. The culture medium was then replaced with DMEM medium (043-30085, Wako) containing 20% ​​FBS (10270-106, Gibco) and 2% Ultroser G, and the culture was continued.

[0256] Measurements of living cells

[0257] Immediately after AO transfection (day 0), 2 days later, and 3 days later, 10 μ l Cell Counting Kit-8 (347-07621, Dojin Chemical Research Institute), after incubation at 37°C and 5% CO2 for 1 hour, the absorbance at 450nm was measured to evaluate the cell number.

[0258] Alignment-based nucleic acid sequence comparison

[0259] The nucleic acid sequences of the amino acid coding regions of exon 1 of human myostatin (NM_005259.2), bovine myostatin (NC_037329.1), porcine myostatin (NC_010457.5), and dog myostatin (NM_001002959.1) were aligned. The multiple sequence alignment program Clustal Omega (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ) of EMBL-EBI was used for the alignment. The sequences used for the alignment and comparison of the nucleic acid sequences of exon 1 of human, bovine, porcine, and dog myostatin are shown in SEQ ID NOs: 30 to 33.

[0260] Experimental Results

[0261] To inhibit the expression of myostatin (MSTN), three 18-base AOs with sequences complementary to exon 1 of MSTN pre-mRNA were prepared ( Figure 1 , Figure 2 a). Each AO was prepared based on the predicted binding of splicing factors in MSTN pre-mRNA. After human rhabdomyosarcoma cells were treated with each AO, the amount of MSTN mRNA was examined by RT-PCR. It was observed that after 24 hours of treatment, the amount of MSTN mRNA was significantly reduced by AO1 and AO2 ( Figure 2 ).

[0262] Since AO2 reduced MSTN mRNA more than AO1, the optimal AO sequence was screened with AO2 as the center. First, AOs with 3 bases shifted before and after the sequence of AO2 were prepared, and 6 AOs targeting the 231st to 263rd bases of MSTN exon 1 (SEQ ID NO: 1) were tested ( Figure 3 a). The test results in human rhabdomyosarcoma cells showed that after 24 hours of treatment with AO2-3, AO2, AO2+3, AO2+6, and AO2+9, a significant decrease in MSTN mRNA was observed ( Figure 3 b, c). Among them, AO2+3 showed the greatest effect of reducing MSTN mRNA, so AOs with 1 base shifted around AO2+3 were prepared, and 7 AOs targeting bases 234 to 257 of MSTN exon 1 (SEQ ID NO: 1) were tested ( Figure 3 a). The test results in human rhabdomyosarcoma cells showed that a significant decrease in MSTN mRNA was observed after 24 hours of treatment with AO2, AO2+1, AO2+2, AO2+3, AO2+4, AO2+5, and AO2+6 ( Figure 3d, e). Through the above screening, AO2+1 was obtained as the most effective AO for inhibiting MSTN expression.

[0263] Since AO targeting exon 1 of MSTN can inhibit MSTN expression, AOs other than the surrounding sequences of AO2+1 were prepared and compared with AO2+1 for effectiveness. 12 AOs targeting the range of bases 22 to 420 of MSTN exon 1 (SEQ ID NO: 1) were prepared, and 13 of them, including AO2+1, were treated in human rhabdomyosarcoma cells for 24 hours. As a result, it was found that all AOs tended to inhibit MSTN mRNA expression (Figure 4). In particular, AO6, AO7, AO3-6, AO3-12, AO8, AO9, AO9-12, AO10, and AO10-9 significantly reduced MSTN mRNA. When comparing the reduction rates of these MSTN mRNAs, AO2+1 was shown to be the most effective. Moreover, the effect of AO2+1 was also shown in human myoblasts (Figure 5). On the other hand, AO2+1 had no effect on the expression of GDF11 (Figure 6).

[0264] If mature myostatin interacts with receptors on the cell membrane, the transcription factor Smad2 / 3 is phosphorylated and transferred to the nucleus, inducing the expression of target genes ( Figure 7 ). The activation of the myostatin signal was evaluated using an in vitro myostatin transcriptional activity assay system (Figure 8). In the in vitro myostatin transcriptional activity assay system, a plasmid in which a luciferase gene is configured downstream of the Smad binding promoter is used to measure the activity of the luciferase expressed by the plasmid, thereby testing the myostatin signal. As a result of the myostatin signal test in human rhabdomyosarcoma cells and human myoblasts, the luciferase activity was inhibited by the treatment with AO2+1, thereby showing that AO2+1 inhibits the myostatin signal (Figures 8 and 9). Moreover, AO2+1 also showed a proliferation-promoting effect on human myoblasts ( Fig.10 ).

[0265] Investigation

[0266] Since inhibition of myostatin function promotes muscle formation, it has attracted attention as a method for treating muscular atrophy. In fact, clinical trials for muscular atrophy diseases represented by Duchenne muscular dystrophy (DMD) are underway for antibodies or peptides that inhibit the function of myostatin. On the other hand, off-target effects that target proteins other than myostatin are considered problematic in these antibodies or peptides. For example, mature myostatin that activates myostatin signaling has 90% amino acid identity with mature GDF11 of the same TGF-β family, and myostatin inhibitory peptides also inhibit GDF11 (Osawa et al., PLoS One, 2015). In contrast, AO2+1 in this study had no effect on the expression of GDF11 mRNA. In addition, loss-of-function AOs aimed at skipping exon 2 of MSTN pre-mRNA have been developed to date, but clinically effective AOs have not yet been obtained. The AO of the present invention is an expression-inhibiting type that targets exon 1 of MSTN pre-mRNA, and its actions and effects are different from those of conventional loss-of-function AOs, and therefore it is expected to be useful as a therapeutic drug.

[0267] AO2+1 of the present invention has a particularly high effectiveness, not only inhibiting the expression of MSTN mRNA, but also inhibiting myostatin signals and promoting the proliferation of myoblasts. There are many diseases for which myostatin inhibition is considered to be effective, and as a drug, it can be used for the prevention and / or treatment of muscular atrophy diseases (e.g., muscular dystrophy, spinal muscular atrophy, sarcopenia, disuse muscular atrophy), circulatory system diseases (e.g., heart failure, arteriosclerosis, etc.), kidney diseases (e.g., chronic renal failure, etc.), bone diseases (e.g., inflammatory arthritis, etc.), cancer or diabetes. Moreover, myostatin inhibition also increases skeletal muscle mass, seeks to increase the amount of exercise, and also helps to improve the metabolism of the whole body. In addition, it can be expected to act on the myocardium to restore its function. Moreover, myostatin inhibition is also expected to act on osteoclasts to inhibit bone destruction, activate the homeostasis maintenance ability of vascular endothelial cells, induce cell apoptosis, and improve sensitivity to insulin.

[0268] The target sequence of AO2+1 of the present invention is also conserved in cattle or pigs, and can also be used to promote the growth of these livestock ( Fig.11 ). Inhibition of MSTN expression by administering AO to livestock does not correspond to the production of genetically modified organisms, so it is easy to apply. In addition, it is believed that it is also effective for dogs and can be used for sarcopenia in dogs as pets ( Fig.11 ).

[0269] All publications, patents and patent applications cited in this specification are incorporated herein by reference.

[0270] Industrial Applicability

[0271] The present invention can be used as a nucleic acid drug for inhibiting the production of mRNA of the myostatin gene.

[0272] [Sequence Listing Free Text]

[0273] <SEQ ID NO: 1> shows the nucleotide sequence information of exon 1 of MSTN.

[0274] (Total 506 bases. The start codon (atg) is shown in the □)

[0275]

[0276] <SEQ ID NO: 2 to 25> show the nucleotide sequences of AO synthesized in the Examples. The nucleotides constituting the antisense oligonucleotide may be any of natural DNA, natural RNA, DNA / RNA chimeras, and modifications thereof, and at least one may be a modified nucleotide.

[0277] <SEQ ID NOs: 26 to 29> show the nucleotide sequences of primers used in the test examples.

[0278] <SEQ ID NO: 30-33> shows sequences used for comparison of nucleic acid sequences of myostatin exon 1 of human, bovine, porcine, and dog. The comparison was performed using the multiple sequence alignment of EMBL-EBI. Only the amino acid coding region of exon 1 was compared. The names in parentheses are in BLAST. The sequences in □ are the target sequences of AO2+1.

[0279] Ex1 (ORF)

[0280]

[0281]

[0282] <SEQ ID NOs: 34 and 35> show the nucleotide sequences of primers GDF11Ex1F4 and GDF11Ex1R4.

Claims

1. An antisense oligonucleotide having a base length of 18, or a salt or solvate thereof, wherein the antisense oligonucleotide has a nucleotide sequence complementary to a target site of exon 1 of a myostatin gene and can inhibit the production of mRNA of the myostatin gene. The nucleotide sequence of exon 1 of the myostatin gene is the nucleotide sequence of SEQ ID NO: 1, The nucleotide sequence of the antisense oligonucleotide is TgCaTTaCaCagCCCCTC (MSTN_Ex1_AO2+1), in, In the above nucleotide sequence, t may be u, and u may be t.

2. The antisense oligonucleotide, salt or solvate thereof according to claim 1, wherein At least 1 nucleotide is modified.

3. The antisense oligonucleotide, salt or solvate thereof according to claim 2, wherein: The sugar constituting the modified nucleotide is D-ribofuranose, and the hydroxyl group at the 2' position of D-ribofuranose is modified.

4. The antisense oligonucleotide, salt or solvate thereof according to claim 3, wherein D-ribofuranose is 2'-O-alkylated and / or 2'-O,4'-C-alkylened.

5. A medicament comprising the antisense oligonucleotide according to any one of claims 1 to 4, or a pharmaceutically acceptable salt or solvate thereof.

6. A pharmaceutical agent comprising the antisense oligonucleotide, a salt or a solvate thereof according to any one of claims 1 to 4.

7. A myostatin function inhibitor comprising the antisense oligonucleotide, a salt or a solvate thereof according to any one of claims 1 to 4.

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