Compounds and pharmaceutical compositions for modulating myostatin expression

Abstract

A modified oligonucleotide or a pharmaceutically permissible salt thereof, comprising 12 to 30 linked nucleosides having the activity of inhibiting myotropic hormone expression, wherein the modified oligonucleotide contains a base sequence of 8 or more consecutive bases that are 100% complementary to a specific region of the base sequence of Sequence No. 1 in the sequence listing, and the full-length base sequence of the modified oligonucleotide has more than 85% complementarity to the equivalent portion of the base sequence in Sequence No. 1 in the sequence listing.

Description

Technical Field

[0001] This invention relates to a compound for reducing at least one of the pre-mRNA level, mRNA level and protein level of myoregulin (MRLN) in animals, and a pharmaceutical composition containing the compound. Background Technology

[0002] Myomodulin (MRLN), encoded by long noncoding RNA, has been discovered as a micropeptide specifically expressed in skeletal muscle that regulates calcium levels in the sarcoplasmic reticulum (Non-Patent Literature 1). It has been reported that treatment with MRLN siRNA, which inhibits MRLN mRNA expression, in cells derived from Duchenne muscular dystrophy (DMD) patients increases calcium uptake into the sarcoplasmic reticulum (Patent Literature 1). Furthermore, it has been reported that administration of MRLN antisense oligonucleotides to DMD model mice (mdx mice) and myofascial disease model mice (sarcoglycan β knockout mice), respectively, reduced MRLN gene expression, which is a clinical marker of muscular dystrophy in both mouse models, resulting in decreased creatine kinase leakage due to muscle dysfunction and myocyte death (Patent Literature 1). Myofascial disease refers to the general term for limb girdle muscular dystrophy (LGMD) types 2C-2F.

[0003] Thus, inhibiting the expression of MRLN can increase calcium uptake into the sarcoplasmic reticulum, and is therefore effective in the treatment or prevention of muscle diseases such as muscular dystrophy, which is accompanied by muscle dysfunction, myocyte death, and / or decreased muscle contractility caused by abnormal calcium influx into the cytoplasm.

[0004] In this context, there is a need for superior antisense oligonucleotides that inhibit MRLN expression in order to treat muscle diseases. However, the MRLN antisense oligonucleotides reported to date are designed based on the base sequence of the mouse MRLN gene (Patent Document 1), making them difficult to apply clinically to the treatment or prevention of muscle diseases in humans. Existing technical documents Patent documents

[0005] Patent Document 1: WO2022 / 014703 Non-patent literature

[0006] Non-patent literature 1: Cell, 160(4): 595-606 (2015) Summary of the Invention The problem that the invention aims to solve

[0007] This invention provides a compound for inhibiting MRLN expression. Methods for solving problems

[0008] The inventors conducted in-depth research and discovered modified oligonucleotides that inhibit MRLN expression, thus completing this invention.

[0009] The present invention includes the following methods. [1] A modified oligonucleotide or a pharmaceutically permissible salt thereof, comprising 12-30 linked nucleosides having the activity of inhibiting myotropic hormone expression, wherein the modified oligonucleotide comprises positions 15-41, 62-108, 448-471, 649-666, 848-863, 1104-1119, 1232-1254, 1691-1719, 1735-1751, and 2195-2196, starting from the 5' end of the sequence of sequence number 1 in the sequence listing. 211th position, 2289-2310th position, 2479-2512th position, 2616-2631st position, 2692-2716th position, 2751-2766th position, 2893-2909th position, 2979-2995th position, 3431-3447th position, 3564-3585th position, 3601-3617th position, 3721-3736th position, 4074-4089th position, 4127-4142nd position, 4156-4171st position, 4277-4292nd position, 4305-4322nd position, 4500-4515th position Positions 4613-4628, 5039-5055, 5063-5079, 5103-5119, 5522-5540, 5727-5742, 5772-5803, 5998-6013, 6272-6288, 6695-6711, 6763-6778, 7224-7247, 8013-8028, 9844-9864, 14649-14665, 14903-14920, 14951-1499 The modified oligonucleotide has a 100% complementary sequence of any eight or more consecutive bases in any of the following base sequences: position 5, positions 16113-16129, positions 16235-16254, positions 16256-16273, positions 16281-16306, positions 16316-16336, positions 16514-16536, positions 16578-16593, positions 16610-16637, or positions 16724-16739. The full-length base sequence of the modified oligonucleotide has more than 85% complementarity with the equivalent-length portion of the base sequence of sequence number 1 in the sequence listing. [2] The modified oligonucleotide or a pharmaceutically permissible salt thereof according to [1], wherein the modified oligonucleotide comprises positions 1232-1254, 1691-1719, 1735-1751, 2195-2211, 2289-2308, 3431-3446, 4127-4142, 4156-4171, 4500-4515, 5522-5537, 5727-5742, 5772-5803, ... The modified oligonucleotide has a 100% complementary sequence of any eight or more consecutive bases in any of the following base sequences: positions 5998-6013, 6272-6287, 6763-6778, 8013-8028, 9846-9864, 14963-14978, 16235-16254, 16256-16273, 16281-16306, or 16316-16331. The full-length base sequence of the modified oligonucleotide has more than 85% complementarity with the equivalent-length portion of the base sequence of sequence number 1 in the sequence listing. [3] A modified oligonucleotide or a pharmaceutically permissible salt thereof, which is a modified oligonucleotide or a pharmaceutically permissible salt thereof composed of 12 to 30 linked nucleosides having the activity of inhibiting myotropic hormone expression, wherein the modified oligonucleotide comprises a base sequence that is 100% complementary to any eight or more consecutive bases in any of the following base sequences from the 5' end of sequence number 2 of the sequence listing: positions 15 to 41, 62 to 91, 100 to 129, 160 to 225, 227 to 244, 252 to 277, 287 to 307, 485 to 507, 549 to 564, 581 to 608 or 695 to 710, and the full-length base sequence of the modified oligonucleotide is more than 85% complementary to the equivalent-length portion of the base sequence of sequence number 2 of the sequence listing. [4] The modified oligonucleotide or a pharmaceutically permissible salt thereof according to [3], wherein the modified oligonucleotide comprises a base sequence that is 100% complementary to any eight or more consecutive bases in any of the following base sequences from the 5' end of sequence number 2 of the sequence listing: positions 172-187, 201-225, 227-244, 252-277 or 287-302, and the full-length base sequence of the modified oligonucleotide is more than 85% complementary to the equivalent portion of the base sequence of sequence number 2 of the sequence listing. [5] A modified oligonucleotide or a pharmaceutically permissible salt thereof, which is a modified oligonucleotide or a pharmaceutically permissible salt thereof having activity of inhibiting myotropic hormone expression, wherein the base sequence of the modified oligonucleotide is any base sequence selected from the group consisting of or consisting of 17 or 18 consecutive bases containing the base sequence, and the full-length base sequence of the modified oligonucleotide consists of a base sequence that is 100% complementary to the equal-length portion of the base sequence of sequence number 1: CCAGAATTATCCCGCT (complementary sequence of positions 16238-16253 of sequence number 1) (sequence number 275 in the sequence list) TCCAGAATTATCCCGC (complementary sequence of positions 16239-16254 of sequence number 1) (sequence number 276 in the sequence list) AGTTTTTACCAGTCAT (complementary sequence of positions 16256-16271 of sequence number 1) (sequence number 277 in the sequence list) CCAGTTTTTACCAGTC (complementary sequence of positions 16258-16273 of sequence number 1) (sequence number 278 in the sequence list) GACTTTTGGGAGTAGT (complementary sequence of positions 16289-16304 of sequence number 1) (sequence number 279 in the sequence list) AGACTTTTGGGAGTAG (complementary sequence of positions 16290-16305 of sequence number 1) (sequence number 229 in the sequence list) and GAAGTCTTCCCACAAT (complementary sequence of positions 16316-16331 of sequence number 1) (sequence number 231 of the sequence list). [6] A modified oligonucleotide or a pharmaceutically permissible salt thereof, having the activity of inhibiting myotropic hormone expression, wherein the base sequence of the modified oligonucleotide is selected from any of the following base sequences, or is a base sequence consisting of 17 or 18 consecutive bases containing the base sequence, and the full-length base sequence of the modified oligonucleotide is composed of a base sequence that is 100% complementary to the equal-length portion of the base sequence of sequence number 2: TTATCCCGCTCCCTGA (complementary sequence of positions 203-218 of sequence number 2) (sequence number 273 in the sequence list) or ATTATCCCGCTCCCTG (complementary sequence of positions 204-219 of sequence number 2) (sequence number 280 of the sequence list). [7] A modified oligonucleotide or a pharmaceutically permissible salt thereof according to any one of [1] to [6], wherein the modified oligonucleotide is single-stranded. [8] A modified oligonucleotide or a pharmaceutically permissible salt thereof according to any one of [1] to [7], wherein at least one nucleoside constituting the modified oligonucleotide comprises a modified sugar. [9] The modified oligonucleotide or its pharmaceutically permissible salt according to [8], wherein the modified sugar is a bicyclic sugar.

[0010] The modified oligonucleotide or its pharmaceutically permissible salt according to [9], wherein the bicyclic sugar is selected from the group consisting of the sugar moieties of LNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz] and ALNA[Trz].

[0011] The modified oligonucleotide or its pharmaceutically permissible salt according to [9] or

[10] , wherein the bicyclic sugar is the sugar moiety of ALNA[Ms].

[0012] The modified oligonucleotide or its pharmaceutically permissible salt according to [8], wherein the modified sugar is a substituted sugar.

[0013] The modified oligonucleotide or pharmaceutically permissible salt thereof according to any one of [1] to

[12] , wherein at least one nucleoside constituting the modified oligonucleotide comprises a modified base.

[0014] The modified oligonucleotide or its pharmaceutically permissible salt according to

[13] , wherein the modified base is 5-methylcytosine.

[0015] The modified oligonucleotide or pharmaceutically permissible salt thereof according to any one of [1] to

[14] , wherein at least one nucleoside internucleotide constituting the modified oligonucleotide is a modified nucleoside internucleotide.

[0016] The modified oligonucleotide or its pharmaceutically permissible salt according to

[15] , wherein the modified nucleoside internucleotide is a thiophosphate nucleoside internucleotide.

[0017] The modified oligonucleotide or a pharmaceutically permissible salt thereof according to any one of [1] to

[16] , wherein the modified oligonucleotide comprises: 1) Interval segments, 2) 5' wing segment, and 3) 3' wing segment, The spacer segment is located between the 5' wing segment and the 3' wing segment, and the sugar portion of the nucleosides constituting the 5' wing segment and the 3' wing segment is entirely a modified sugar.

[0018] A pharmaceutical composition comprising any one of the modified oligonucleotides described in [1] to

[17] or a pharmaceutically permissible salt thereof.

[0019] The pharmaceutical composition described in

[18] is used to treat or prevent muscle diseases.

[0020] According to the pharmaceutical composition described in

[19] , the muscle disease is selected from the group consisting of myasthenia gravis, inclusion body myositis, amyotrophic lateral sclerosis, disuse atrophy and sarcopenia.

[0021] According to the pharmaceutical composition described in

[20] , the muscle disease is muscular dystrophy.

[0022] According to the pharmaceutical composition described in

[21] , wherein the muscular dystrophy is selected from the group consisting of Duchenne muscular dystrophy, Behringer's muscular dystrophy and myofascitis.

[0023] According to the pharmaceutical composition described in

[19] , the muscle disease is Duchenne muscular dystrophy.

[0024] According to the pharmaceutical composition described in

[19] , the muscle disease is Behringer's muscular dystrophy.

[0025] According to the pharmaceutical composition described in

[19] , the muscle disease is myoglobinosis.

[0026] A method for treating or preventing a muscle disease, comprising administering to a subject an effective amount of any of the modified oligonucleotides described in any one of [1] to

[17] or a pharmaceutically permissible salt thereof.

[0027] Use of any of the modified oligonucleotides or pharmaceutically permissible salts thereof, as described in any one of [1] to

[17] , for the treatment or prevention of muscle diseases.

[0028] Use of any of the modified oligonucleotides or pharmaceutically permissible salts thereof described in any one of [1] to

[17] in the manufacture of pharmaceutical compositions for the treatment or prevention of muscle diseases.

[0029] The modified oligonucleotide or pharmaceutically permissible salt thereof, as described in any one of [1] to

[17] , for use in the manufacture of pharmaceutical compositions for the treatment or prevention of muscle diseases. Invention Effects The modified oligonucleotides of the present invention have excellent inhibitory activity against MRLN expression. Detailed Implementation It should be understood that the foregoing description and the following detailed description are merely illustrative and not intended to limit the claimed invention. In this specification, the singular form is used in conjunction with the plural form unless otherwise specified. The use of the terms "including" and other terms such as "includes" and "included" in this specification is not intended to be limiting. The section headings used in this specification are for structural purposes only and should not be construed as limiting the subject matter. Not limited thereto, all documents or portions thereof cited in this application, including patents, patent applications, reports, books, and papers, relating to the documents discussed herein, in their entirety, are expressly incorporated herein by reference. (definition) Unless otherwise specified, the nomenclature, steps, and methods used in analytical chemistry, organic synthetic chemistry, and related medical and pharmaceutical chemistry described herein are well-known and commonly used in the art. Standard methods can be used for the chemical synthesis and chemical analysis used in this specification. Where permissible, all patents, applications, publications, and other publications mentioned in the entirety of this specification, GenBank accessions and related sequence information available in databases such as the National Center for Biotechnology Information (NCBI), and other data relating to the documents discussed herein, in whole or in part, are incorporated by reference. In addition, this specification is filed together with an electronic serial list, the information of which is incorporated herein by reference in its entirety. Unless otherwise specified, the following terms have the following meanings. A "base" refers to a heterocyclic portion that can pair with bases in other nucleic acids. "Continuous bases" refers to bases that are adjacent to each other. "Base sequence" refers to the order of consecutive bases. "Nucleoside" refers to a molecule formed by the linkage of a sugar and a base. "Linked nucleosides" refers to nucleosides linked together in a continuous sequence by internucleotide bonds. A nucleotide is a molecule in which a phosphate group is bonded to the sugar portion of a nucleoside. The sugar portion of naturally occurring nucleotides is either ribose or deoxyribose. "Oligonucleotides" are polymers of nucleosides linked together by internucleotide bonds. "Nucleoside bond" refers to the chemical bond between nucleosides. "Modified nucleoside bonds" refers to the substitution or arbitrary change of naturally occurring nucleoside bonds (i.e., phosphodiester nucleoside bonds). For example, there are thiophosphate nucleoside bonds, but it is not limited to these. "Modified bases" refers to any base other than adenine, cytosine, guanine, thymidine, or uracil. For example, 5-methylcytosine is used, but it is not limited to this. "Modified nucleosides" independently refer to nucleosides that have modified sugars or modified bases. "Modified oligonucleotides" refer to oligonucleotides containing at least one modified nucleoside and / or a modified nucleoside bond. "Sugar" or "sugar portion" refers to the natural sugar portion or the modified sugar portion. "Modified sugars" refer to sugars that have undergone substitution or alteration from natural sugars. Examples of modified sugars include substituted sugars or bicyclic sugars. "Substitute sugars" refer to furans in RNA or DNA other than naturally occurring sugars. However, this does not include bicyclic sugars.

[0030] "Bicyclic sugars" refer to furanoses modified by cross-linking two different carbon atoms out of the four carbon atoms forming the furanose ring.

[0031] "Single-stranded oligonucleotides" are oligonucleotides that do not hybridize with their complementary strands.

[0032] "Equal-length portion" refers to a portion of the base sequence of a second nucleic acid having the same length as the base sequence of the first nucleic acid. In one embodiment, the first nucleic acid is a modified oligonucleotide, and the second nucleic acid is MRLN pre-mRNA or MRLN mRNA.

[0033] "Complementarity" refers to the ability of the bases of the first and second nucleic acids to pair. In one embodiment, adenine is complementary to thymidine or uracil. In one embodiment, cytosine is complementary to guanine. In one embodiment, 5-methylcytosine is complementary to guanine.

[0034] "100% complementarity" means that the base sequence of the first nucleic acid is completely complementary to the base sequence of the second nucleic acid. In one embodiment, the first nucleic acid is a modified oligonucleotide, and the target nucleic acid is the second nucleic acid.

[0035] "Mismatch" refers to a situation where the bases of the first nucleic acid cannot pair with the corresponding bases of the second nucleic acid or the target nucleic acid.

[0036] "Target nucleic acid," "target RNA," and "target RNA transcript" refer to nucleic acids, RNA, and RNA transcripts that are modified with oligonucleotides and can serve as targets, respectively. In one embodiment, the target nucleic acid includes a region of MRLN mRNA or MRLN pre-mRNA.

[0037] "Pharmaceutically permissible salt" refers to the physiologically and pharmaceutically permissible salt of the modified oligonucleotide of the present invention, such as salts formed on thiophosphates or phosphate diesters or on functional groups (e.g., amino groups) within the modified bases with inorganic ions such as metal ions.

[0038] "Improvement" means a reduction in at least one indicator, sign, or symptom of a related disease, disorder, or condition. The severity of the indicator can be determined by subjective or objective scales known to those skilled in the art.

[0039] "Animal" means human beings, or not limited to these, including non-human animals such as mice, rats, rabbits, dogs, cats, and pigs, and not limited to these, including non-human primates such as monkeys and chimpanzees.

[0040] "Effective amount" refers to the amount of the modified oligonucleotide of the present invention sufficient to achieve the desired physiological outcome for the individual requiring the medication. The effective amount can vary between individuals depending on the health and physical condition of the individual to be treated, the classification group of the individual to be treated, the formulation of the composition, the evaluation of the individual's medical condition, and other relevant factors.

[0041] "Individual" refers to a human or non-human animal chosen for treatment or therapy.

[0042] "Prevention" refers to delaying or preventing the onset or occurrence of a disease, disorder, or undesirable health condition, or one or more symptoms associated with that disease, disorder, or undesirable health condition, for a period ranging from a few minutes to indefinite. Prevention means reducing the risk of developing a disease, disorder, or undesirable health condition.

[0043] "Treatment" means to alleviate or eliminate a disease, disorder or undesirable health condition, or one or more symptoms associated with the disease, disorder or undesirable health condition, or to partially eliminate or completely resolve one or more causes of the disease, disorder or undesirable health condition itself. (Detailed Implementation) That is, providing a compound for inhibiting the expression of MRLN, a method of using the compound, and a pharmaceutical composition containing the compound.

[0045] (1) Modified oligonucleotides The modified oligonucleotides of this invention (hereinafter sometimes referred to as "compounds of this invention" or "modified oligonucleotides of this invention") are modified oligonucleotides with activity of inhibiting MRLN expression. Inhibition of MRLN expression (level) in this invention refers to the inhibition of at least one of the following: the level of pre-mRNA transcribed from the MRLN gene, the level of mRNA spliced ​​from the pre-mRNA, and the level of MRLN protein translated from the mRNA (sometimes collectively referred to as "MRLN expression level"). There are no particular limitations on the MRLN pre-mRNA, which may contain sequence variants, such as the sequence shown in GenBank accession number NC_000010.11: c59753455-59736692 (Sequence No. 1 in the sequence listing). Similarly, there are no particular limitations on the MRLN mRNA, which may contain sequence variants, such as the mRNA shown in GenBank accession number NM_001304731.2 (Sequence No. 2 in the sequence listing). It should be noted that the T in sequence No. 1 of the RNA sequence must be replaced with U. In addition, MRLN mRNA and pre-mRNA can contain various splicing variants, single nucleotide polymorphisms (SNPs), and other sequence variants.

[0046] The degree of inhibition of MRLN expression by the compounds of the present invention can be any degree, as long as at least one of the pre-mRNA level, mRNA level, and protein level of MRLN is reduced compared to the state without administration of the compound, and the result is a degree of prevention and / or improvement of symptoms associated with muscle disease that can be confirmed. Specifically, for example, in the in vitro MRLN expression assay method described later, after contacting cells with the compounds of the present invention, the MRLN expression level is reduced to 70% or less, preferably 50% or less, more preferably 40% or less, further preferably 30% or less, and particularly preferably 20% or less compared to the state without contact or contact with a negative control. For example, when RD cells (human rhabdomyosarcoma cells) are treated with 100 nM of the compounds of the present invention by lipid transfection, the MRLN expression level can be reduced to 50% or less. In addition, when RD cells are treated with 100 nM of the compound of the present invention using lipid transfection or with 1000 nM of the compound of the present invention using Gymnosis, the MRLN expression level can be reduced to less than 50% in either case.

[0047] As a method for verifying the activity of the compounds of the present invention, any method that can verify the inhibition of MRLN expression by the compounds of the present invention can be used, such as the method described in the "Evaluation Method of Compounds of the Present Invention" described later.

[0048] The compounds of the present invention are modified oligonucleotides consisting of 12 to 30 linked nucleosides, preferably 16 to 20 linked nucleosides, more preferably 16 to 18 linked nucleosides, and particularly preferably 16, 17 or 18 linked nucleosides, which have the activity of inhibiting MRLN expression. The modified oligonucleotide only needs to contain the following positions from the 5' end of the sequence of sequence number 1 in the sequence listing: positions 15-41, 62-108, 448-471, 649-666, 848-863, 1104-1119, 1232-1254, 1691-1719, 1735-1751, 2195-2211, 2289-2310, 2479-2512, 2616-2631, 2692-2716, 2751-2766, etc. Positions 2893-2909, 2979-2995, 3431-3447, 3564-3585, 3601-3617, 3721-3736, 4074-4089, 4127-4142, 4156-4171, 4277-4292, 4305-4322, 4500-4515, 4613-4628, 5039-5055, 5063-5079, 5103-5119, 5522-5540 Positions 5727-5742, 5772-5803, 5998-6013, 6272-6288, 6695-6711, 6763-6778, 7224-7247, 8013-8028, 9844-9864, 14649-14665, 14903-14920, 14951-14995, 16113-16129, 16235-16254, 16256-16273, 16281-1 Any 8 or more, preferably 12 or more, more preferably 14 or more, further preferably 16 or more and / or 20 or less, preferably 18 or less, more preferably 17 or less, further preferably 16 or less, and especially preferably 16 consecutive 100% complementary base sequences (hereinafter sometimes referred to as "MRLN complementary base sequences") from any of the following base sequences: 6306, 16316-16336, 16514-16536, 16578-16593, 16610-16637 or 16724-16739.

[0049] The compounds of the present invention are modified oligonucleotides consisting of 12 to 30 linked nucleosides, preferably 16 to 20 linked nucleosides, more preferably 16 to 18 linked nucleosides, and particularly preferably 16, 17 or 18 linked nucleosides, which have the activity of inhibiting MRLN expression. The modified oligonucleotide only needs to contain the following positions from the 5' end of the sequence starting from sequence number 1 in the sequence listing: positions 1232-1254, 1691-1719, 1735-1751, 2195-2211, 2289-2308, 3431-3446, 4127-4142, 4156-4171, 4500-4515, 5522-5537, 5727-5742, 5772-5803, 5998-6013, 6272-6287, and 6763-677. The modified oligonucleotide may be any 8 or more, preferably 12 or more, more preferably 14 or more, further preferably 16 or more and / or 20 or less, preferably 18 or less, more preferably 17 or less, further preferably 16 or less, and particularly preferably 16 consecutive base sequences of 100% complementary bases from any of the following positions: 8th position, 8013-8028th position, 9846-9864th position, 14963-14978th ​​position, 16235-16254th position, 16256-16273rd position, 16281-16306th position, or 16316-16331st position.

[0050] The compounds of the present invention are modified oligonucleotides having the activity of inhibiting MRLN expression, consisting of 12 to 30 linked nucleosides, preferably 16 to 20 linked nucleosides, more preferably 16 to 18 linked nucleosides, and particularly preferably 16, 17, or 18 linked nucleosides. The modified oligonucleotide may contain at least 8, preferably 12, more preferably 14, further preferably 16 and / or less than 20, preferably less than 18, more preferably less than 17, further preferably less than 16, and particularly preferably 16 consecutive bases that are 100% complementary to the sequence of position 16256 to 16273 from the 5' end of the sequence in sequence number 1 of the sequence listing.

[0051] The compounds of the present invention are modified oligonucleotides consisting of 12 to 30 linked nucleosides, preferably 16 to 20 linked nucleosides, more preferably 16 to 18 linked nucleosides, and particularly preferably 16, 17 or 18 linked nucleosides, which have the activity of inhibiting MRLN expression. The modified oligonucleotide can be any oligonucleotide containing at least 8, preferably 12, more preferably 14, further preferably 16 and / or less than 20, preferably less than 18, more preferably less than 17, further preferably less than 16, and particularly preferably less than 16 consecutive bases that are 100% complementary to any one of the following base sequences starting from the 5' end of the sequence number 2 in the sequence listing: positions 15-41, 62-91, 100-129, 160-225, 227-244, 252-277, 287-307, 485-507, 549-564, 581-608, or 695-710.

[0052] The compound of the present invention is a modified oligonucleotide having the activity of inhibiting MRLN expression, consisting of 12 to 30 linked nucleosides, preferably 16 to 20 linked nucleosides, more preferably 16 to 18 linked nucleosides, and particularly preferably 16, 17, or 18 linked nucleosides. This modified oligonucleotide is only required to contain any 8 or more, preferably 12 or more, more preferably 14 or more, further preferably 16 or more and / or 20 or less, preferably 18 or less, more preferably 17 or less, further preferably 16 or less, and particularly preferably 16 consecutive bases that are 100% complementary to any one of the following base sequences from the 5' end of sequence number 2 in the sequence listing: positions 172 to 187, 201 to 225, 227 to 244, 252 to 277, or 287 to 302.

[0053] The modified oligonucleotides of the present invention are modified oligonucleotides consisting of 12 to 30 linked nucleosides, preferably 16 to 20 linked nucleosides, more preferably 16 to 18 linked nucleosides, and particularly preferably 16, 17, or 18 linked nucleosides. If the full-length base sequence of this modified oligonucleotide is 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 100% complementary to the equivalent-length portion of the base sequence of sequence number 1 or 2 in the sequence listing, then in addition to the aforementioned MRLN complementary base sequence, it may also have additional sequences at its 5' end and / or 3' end. Furthermore, as long as the modified oligonucleotide of the present invention is a modified oligonucleotide with activity of inhibiting MRLN expression, the additional sequences can be any sequence.

[0054] The full-length base sequence of the modified oligonucleotide of the present invention has at least 85% complementarity with the equivalent-length portion in sequence number 1 or 2 of the sequence listing, preferably at least 90%, more preferably at least 95%, and even more preferably at 100%. That is, the base sequence of the modified oligonucleotide of the present invention is preferably completely complementary to the equivalent-length portion of the modified oligonucleotide's base sequence in the base sequence of MRLN pre-mRNA or mRNA, but it may also have one or more mismatched bases. A base sequence with at least 85%, preferably at least 90%, and more preferably at least 95% complementarity can be used. The aforementioned mismatched bases can be continuous.

[0055] The percentage of complementarity between the modified oligonucleotides of this invention and MRLN pre-mRNA or mRNA can be determined conventionally using BLAST (basic local alignment search tools) and Power BLAST (Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656) procedures known in the art. For example, the percentage of homology, sequence consistency, or complementarity can be determined using the default settings of the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 489) via the GAP program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.).

[0056] Specifically, the base sequence of the modified oligonucleotide of the present invention consists of positions 15-30, 21-36, 22-37, 24-39, 26-41, 62-77, 69-84, 76-91, 91-106, 92-107, 93-108, 448-463, 449-464, 456-471, 649-664, 650-665, 651-666, 848-863, 1104-1119, 1232-1247, 1233-1248, and 1239-125, starting from the 5' end of the sequence of sequence number 1 in the sequence listing. 4th position, 1691-1706th position, 1704-1719th position, 1735-1750th position, 1736-1751st position, 2195-2210th position, 2196-2211th position, 2289-2304th position, 2290-2305th position, 2293-2308th position, 2295-2310th position, Positions 2497-2512, 2616-2631, 2692-2707, 2700-2715, 2701-2716, 2751-2766, 2893-2908, 2894-2909, 2979-2994, 2980-2995, 3431~ 3446th position, 3432-3447th positions, 3564-3579th positions, 3569-3584th positions, 3570-3585th positions, 3601-3616th positions, 3602-3617th positions, 3721-3736th positions, 4074-4089th positions, 4127-4142nd positions, 4156-4171st positions Positions 4277-4292, 4305-4320, 4306-4321, 4307-4322, 4500-4515, 4613-4628, 5039-5054, 5040-5055, 5063-5078, 5064-5079, 51 03~5118, 5104~5119, 5522~5537, 5525~5540, 5727~5742, 5772~5787, 5773~5788, 5774~5789, 5775~5790, 5776~5791, 5780~5 795th position, 5781-5796th positions, 5782-5797th positions, 5784-5799th positions, 5785-5800th positions, 5786-5801st positions, 5788-5803rd positions, 5998-6013th positions, 6272-6287th positions, 6273-6288th positions, 6695-6710th positions.Positions 6696-6711, 6763-6778, 7224-7239, 7232-7247, 8013-8028, 9844-9859, 9845-9860, 9846-9861, 9848-9863, 9849-9864, 14649-14664, 14650-14665, 14903-14918, 14904-14919, 14905-14920, 14951-14966, 14952-14967, 1495 3~14968, 14956~14971, 14957~14972, 14958~14973, 14959~14974, 14960~14975, 14961~14976, 14962~14977, 14963~14978, 14964~14979, 14965~14980, 14966~14981, 14967~14982, 14968~14983, 14980~14995, 16113~16128, 161 Positions 14-16129, 16235-16250, 16236-16251, 16237-16252, 16238-16253, 16239-16254, 16256-16271, 16258-16273, 16281-16296, 16282-16297, 16283-16298, 16285-16300, 16287-16302, 16289-16304, 16290-16305, 16291-16306, 16 A base sequence consisting of 16 consecutive complementary bases comprising at least 85%, preferably at least 90%, more preferably at least 95%, and particularly preferably 100% complementary bases from any one of the following positions: 316-16331, 16321-16336, 16514-16529, 16517-16532, 16521-16536, 16578-16593, 16610-16625, 16612-16627, 16614-16629, 16616-16631, 16622-16637, or 16724-16739. Alternatively, it may be a base sequence consisting of 17, 18, 19, or 20, preferably 17 or 18, consecutive bases comprising a base sequence of 16 complementary bases. Furthermore, the full-length base sequence of the modified oligonucleotide is at least 85% of the same length as the portion of the base sequence in Serial Number 1.86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher, preferably 90% or higher, more preferably 95% or higher, and even more preferably 100% complementary base sequences.

[0057] Specifically, the base sequence of the modified oligonucleotide of the present invention consists of positions 1232-1247, 1233-1248, 1239-1254, 1691-1706, 1704-1719, 1735-1750, 1736-1751, 2195-2210, 2196-2211, 2289-2304, 2290-2305, and 2293-2308, starting from the 5' end of the base sequence of sequence number 1 in the sequence listing. Positions 3431-3446, 4127-4142, 4156-4171, 4500-4515, 5522-5537, 5727-5742, 5772-5787, 5773-5788, 5774-5789, 5775-5790, 5776-5791, 5780-5795, 5781-5796, 5782-5797, 5784-5799, 5785-580 0th digit, 5786~5801st digit, 5788~5803rd digit, 5998~6013th digit, 6272~6287th digit, 6763~6778th digit, 8013~8028th digit, 9846~9861st digit, 9848~9863rd digit, 9849~9864th digit, 14963~14978th ​​digit, 16235~16250th digit, 16236~16251st digit, 16237~16252nd digit, 16238~16253rd digit, 16239~1st digit A base sequence comprising at least 85%, preferably at least 90%, more preferably at least 95%, and particularly preferably 100% complementary 16 consecutive bases from any one of the following positions: 6254, 16256-16271, 16258-16273, 16281-16296, 16282-16297, 16283-16298, 16289-16304, 16290-16305, 16291-16306, or 16316-16331. Alternatively, a base sequence comprising 17, 18, 19, or 20, preferably 17 or 18, consecutive bases including the complementary 16 consecutive bases. In addition, the full-length base sequence of the modified oligonucleotide is a base sequence that is 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 100% complementary to the same length portion of the base sequence of sequence number 1.

[0058] Specifically, the modified oligonucleotide of the present invention comprises a base sequence consisting of 16 consecutive bases that are at least 85%, preferably at least 90%, more preferably at least 95%, and particularly preferably 100% complementary to any one of the base sequences from positions 16256 to 16271 or 16258 to 16273, starting from the 5' end of the base sequence of sequence number 1 in the sequence listing. Alternatively, it may consist of 17, 18, 19, or 20, preferably 17 or 18, consecutive bases comprising the base sequence consisting of the complementary 16 consecutive bases. In addition, the full-length base sequence of the modified oligonucleotide is a base sequence that is 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 100% complementary to the base sequence in sequence number 1.

[0059] Specifically, the modified oligonucleotide of the present invention comprises positions 15-30, 21-36, 22-37, 24-39, 26-41, 62-77, 69-84, 76-91, 100-115, 102-117, 112-127, 113-128, 114-129, 160-175, 161-176, ..., starting from the 5' end of the sequence of sequence number 2 in the sequence listing. Positions 162-177, 165-180, 166-181, 167-182, 168-183, 169-184, 170-185, 171-186, 172-187, 173-188, 174-189, 175-190, 176-191, 177-192, 189-204, 199-214, 200-215, 201-216 Positions 202-217, 203-218, 204-219, 206-221, 207-222, 208-223, 209-224, 210-225, 227-242, 229-244, 252-267, 253-268, 254-269, 256-271, 258-273, 260-275, 261-276, 262-277 A base sequence comprising at least 85%, preferably at least 90%, more preferably at least 95%, and particularly preferably 100% complementary 16 consecutive bases from any one of the following positions: positions 287-302, 292-307, 485-500, 488-503, 492-507, 549-564, 581-596, 583-598, 585-600, 587-602, 593-608, or 695-710. Alternatively, a base sequence comprising 17, 18, 19, or 20, preferably 17 or 18, consecutive bases including the complementary 16 consecutive bases. In addition, the full-length base sequence of the modified oligonucleotide is a base sequence that is 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 100% complementary to the base sequence of the same length portion in Serial No. 2.

[0060] Specifically, the base sequence of the modified oligonucleotide of the present invention consists of positions 172-187, 201-216, 203-218, 204-219, 206-221, 207-222, 208-223, 209-224, 210-225, and 227-228, starting from the 5' end of the base sequence of sequence number 2 in the sequence listing. A base sequence comprising at least 85%, preferably at least 90%, more preferably at least 95%, and particularly preferably 100% complementary 16 consecutive bases from any one of the following positions: position 42, positions 229-244, positions 252-267, positions 253-268, positions 254-269, positions 260-275, positions 261-276, positions 262-277, or positions 287-302. Alternatively, a base sequence comprising 17, 18, 19, or 20, preferably 17 or 18, consecutive bases including the complementary 16 consecutive bases. In addition, the full-length base sequence of the modified oligonucleotide is a base sequence that is 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 100% complementary to the base sequence of the same length portion in Serial No. 2.

[0061] In such modified oligonucleotides, the base sequence consisting of 17, 18, 19, or 20, preferably 17 or 18, consecutive bases is a sequence of 1 to 4, preferably 1 and / or 2, nucleosides linked by a nucleoside internucleotide bond at the 5' or 3' end of the complementary 16-base sequence. This nucleoside and / or nucleoside internucleotide bond can be a modified nucleoside and / or a modified nucleoside internucleotide bond.

[0062] As a specific example of the base sequence of the modified oligonucleotide of the present invention, the following base sequence can be cited, or a base sequence consisting of 17, 18, 19, or 20, preferably 17 or 18, consecutive bases containing the specified base sequence can be cited. Furthermore, the full-length base sequence of the modified oligonucleotide is a base sequence that is 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 100% complementary to the base sequence of the same length portion in Serial No. 1. CCAGAATTATCCCGCT (complementary sequence of positions 16238-16253 of sequence number 1) (sequence number 275 in the sequence list) TCCAGAATTATCCCGC (complementary sequence of positions 16239-16254 of sequence number 1) (sequence number 276 in the sequence list) AGTTTTTACCAGTCAT (complementary sequence of positions 16256-16271 of sequence number 1) (sequence number 277 in the sequence list) CCAGTTTTTACCAGTC (complementary sequence of positions 16258-16273 of sequence number 1) (sequence number 278 in the sequence list) GACTTTTGGGAGTAGT (complementary sequence of positions 16289-16304 of sequence number 1) (sequence number 279 in the sequence list) AGACTTTTGGGAGTAG (complementary sequence of positions 16290-16305 of sequence number 1) (sequence number 229 in the sequence list) or GAAGTCTTCCCACAAT (complementary sequence of positions 16316-16331 of sequence number 1) (sequence number 231 of the sequence list).

[0063] As another example of the specific base sequence of the modified oligonucleotide of the present invention, the following base sequences can be cited, or a base sequence consisting of 17, 18, 19, or 20, preferably 17 or 18, consecutive bases containing such a base sequence can be cited. Furthermore, the full-length base sequence of the modified oligonucleotide is a base sequence that is 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 100% complementary to the base sequence of the same length portion in Serial No. 2. TTATCCCGCTCCCTGA (complementary sequence of positions 203-218 of sequence number 2) (sequence number 273 in the sequence list) or ATTATCCCGCTCCCTG (complementary sequence of positions 204-219 of sequence number 2) (sequence number 280 of the sequence list).

[0064] As a base sequence for such a modified oligonucleotide, the following base sequences are preferably examples, or, more preferably, base sequences consisting of 17, 18, 19, or 20 consecutive bases containing that base sequence are examples. Furthermore, the full-length base sequence of the modified oligonucleotide is a base sequence that is 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 100% complementary to the base sequence of the same length portion in Serial No. 1. TCCAGAATTATCCCGC (complementary sequence of positions 16239-16254 of sequence number 1) (sequence number 276 in the sequence list) AGTTTTTACCAGTCAT (complementary sequence of positions 16256-16271 of sequence number 1) (sequence number 277 in the sequence list) CCAGTTTTTACCAGTC (complementary sequence of positions 16258-16273 of sequence number 1) (sequence number 278 in the sequence list) AGACTTTTGGGAGTAG (complementary sequence of positions 16290-16305 of sequence number 1) (sequence number 229 in the sequence list) or GAAGTCTTCCCACAAT (complementary sequence of positions 16316-16331 of sequence number 1) (sequence number 231 of the sequence list).

[0065] Furthermore, as the base sequence of such modified oligonucleotide, the following base sequences are further preferred, or a base sequence consisting of 17, 18, 19, or 20, preferably 17 or 18, consecutive bases containing such a base sequence can be cited. Additionally, the full-length base sequence of the modified oligonucleotide is a base sequence that is 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 100% complementary to the equivalent length portion of the base sequence of Serial Number 1. AGTTTTTACCAGTCAT (complementary sequence of positions 16256-16271 of sequence number 1) (sequence number 277 in the sequence list) CCAGTTTTTACCAGTC (complementary sequence of positions 16258-16273 of sequence number 1) (sequence number 278 of the sequence list) or AGACTTTTGGGAGTAG (complementary sequence of positions 16290-16305 of sequence number 1) (sequence number 229 of the sequence list).

[0066] Of the bases contained in the base sequence of these modified oligonucleotides, one or more cytosine bases may be the modified base 5-methylcytosine, as described later. Specifically, such modified oligonucleotide base sequences are, for example, the base sequences shown in Serial No. 213, Serial No. 214, Serial No. 218, Serial No. 219, Serial No. 228, Serial No. 229, or Serial No. 231, or base sequences consisting of 17, 18, 19, or 20, preferably 17 or 18, consecutive bases containing such a base sequence. Furthermore, the full-length base sequence of the modified oligonucleotide is a base sequence that is at least 90%, preferably at least 95%, and more preferably 100% complementary to the base sequence of the same length portion in Serial No. 1. Alternatively, it may be the base sequence shown in Serial No. 273 or Serial No. 274, or a base sequence consisting of 17, 18, 19, or 20, preferably 17 or 18, consecutive bases containing such a base sequence. The full-length base sequence of the modified oligonucleotide is a base sequence that is 90% or more, preferably 95% or more, more preferably 100% complementary to the base sequence of the same length portion in Serial No. 2. Preferably, it is a base sequence shown in Serial No. 214, Serial No. 218, Serial No. 219, Serial No. 229 or Serial No. 231, or a base sequence consisting of 17, 18, 19 or 20, preferably 17 or 18 consecutive bases containing that base sequence. Examples of full-length base sequences of the modified oligonucleotide include base sequences that are 90% or more, preferably 95% or more, more preferably 100% complementary to the base sequence of the same length portion in Serial No. 1. More preferably, it is a base sequence represented by Serial No. 218, Serial No. 219 or Serial No. 229, or a base sequence consisting of 17, 18, 19 or 20, preferably 17 or 18 consecutive bases containing the base sequence. Examples include base sequences in which the full-length base sequence of the modified oligonucleotide is 90% or more, preferably 95% or more, and more preferably 100% complementary to the base sequence in Serial No. 1.

[0067] The oligonucleotides modified in this invention can be double-stranded oligonucleotides, but single-stranded oligonucleotides are preferred.

[0068] The modified oligonucleotides of the present invention can be oligonucleotides in which at least one nucleoside of the oligonucleotide contains a modified base, or oligonucleotides in which at least one nucleoside of the oligonucleotide contains a modified sugar. Additionally, at least one internucleotide bond constituting the modified oligonucleotide can contain a modified internucleotide bond.

[0069] The modified oligonucleotides of the present invention preferably use modified oligonucleotides in which at least one nucleoside constituting the oligonucleotide contains a modifying sugar. Examples of modifying sugars include substituted sugars or bicyclic sugars. Modified oligonucleotides having such modifying sugars possess advantageous properties such as enhanced nuclease stability and increased binding affinity to target nucleic acids.

[0070] As substituted sugars, examples include, for instance, the sugar moiety of nucleosides modified with 5'-vinyl, 5'-methyl, 4'-S, 2'-F, 2'-OCH3 (2'-OMe), 2'-OCH2CH3, 2'-OCH2CH2F, or 2'-O(CH2)2OCH3 (2'-MOE).

[0071] As a bicyclic sugar, examples include, for instance, the sugar moiety of a nucleoside cross-linked between the 4' and 2' riboside ring atoms; specifically, examples include the sugar moiety of a nucleoside cross-linked with one of the following formulas: 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA). Other examples of bicyclic sugars include the sugar moiety of the cross-linked artificial nucleic acid ALNA disclosed in International Publication No. 2020 / 100826; specifically, the sugar moiety of ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], or ALNA[Trz], with the sugar moiety of ALNA[Ms] being preferred.

[0072] In one embodiment (ALNA[mU]), the nucleotide comprising the bicyclic sugar (the sugar moiety of ALNA[mU]) is a nucleotide represented by the following formula (I) (for example, refer to WO 2020 / 100826): [In the formula, B is a base;] R1, R2, R3, and R4 are each an independent hydrogen atom; R5 and R6 are each an independent hydrogen atom; m is 1; X is a group represented by the following formula (II-1). The following symbols in formula (II-1) indicate the binding sites with the 2'-amino group; [Chemistry 3] One of R7 and R8 is a hydrogen atom, and the other is an unsubstituted methyl group.

[0073] In one embodiment (ALNA[ipU]), the nucleotide comprising the bicyclic sugar (the sugar moiety of ALNA[ipU]) is a nucleotide having formula (I) as defined in ALNA[mU] above (for example, refer to International Publication No. 2020 / 100826), in which formula, B is a base; R1, R2, R3, and R4 are each an independent hydrogen atom; R5 and R6 are each an independent hydrogen atom; m is 1; X is a group represented by the following formula (II-1): One of R7 and R8 is a hydrogen atom, and the other is an unsubstituted isopropyl group.

[0074] In one embodiment (ALNA[Trz]), the nucleotide comprising the bicyclic sugar (the sugar moiety of ALNA[Trz]) is a nucleotide having formula (I) as defined in ALNA[mU] above (e.g., refer to International Publication No. 2020 / 100826), in which formula, B is a base; R1, R2, R3, and R4 are each an independent hydrogen atom; R5 and R6 are each an independent hydrogen atom; m is 1; X is a group represented by the following formula (II-2): A is 1,5-dimethyl-1,2,4-triazol-3-yl.

[0075] In one embodiment (ALNA[Oxz]), the nucleotide comprising the bicyclic sugar (the sugar moiety of ALNA[Oxz]) is a nucleotide having formula (I) as defined in ALNA[mU] above (e.g., refer to International Publication No. 2020 / 100826), in which formula, B is a base; R1, R2, R3, and R4 are each an independent hydrogen atom; R5 and R6 are each an independent hydrogen atom; m is 1; X is a group represented by the following formula (II-2), A is 5-methyl-1,2,4-oxadiazol-3-yl.

[0076] In one embodiment (ALNA[Ms]), the nucleotide containing the bicyclic sugar (ALNA[Ms]) is a nucleotide having formula (I) as defined in ALNA[mU] above (e.g., refer to WO 2020 / 100826), in which formula, B is a base; R1, R2, R3, and R4 are each an independent hydrogen atom; R5 and R6 are each an independent hydrogen atom; m is 1; X is a group represented by the following formula (II-3): M is a sulfonyl group substituted with an unsubstituted methyl group.

[0077] The modified oligonucleotides of this invention preferably use oligonucleotides in which at least one nucleoside contains a modified base. Modified oligonucleotides with such modified bases have advantageous properties such as enhanced nuclease stability and increased binding affinity to target nucleic acids. As a modified base, 5-methylcytosine can be cited as an example. 5-methylcytosine refers to cytosine modified by a methyl group bonded at the 5-position.

[0078] The modified oligonucleotides of the present invention preferably use oligonucleotides in which at least one nucleotide internucleotide bond is included in the modified nucleotide internucleotide bond. Modified oligonucleotides having such modified nucleotide internucleotide bonds have advantageous characteristics such as enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.

[0079] As a modification of inter-nucleoside bonds, specifically, thiophosphate inter-nucleoside bonds can be cited as an example. Thiophosphate inter-nucleoside bonds refer to inter-nucleoside bonds in which a phosphodiester bond is modified by replacing one non-crosslinked oxygen atom with a sulfur atom.

[0080] The modified oligonucleotides of the present invention, in order to obtain increased resistance to degradation caused by nucleases, increased cellular uptake, increased binding affinity to target nucleic acids, and / or increased MRLN expression inhibitory activity, may have a gap-mer structure. A gap-mer structure refers to a structure in which an internal region of multiple nucleotides that assist RNase H-based cleavage is located between an external region containing one or more nucleotides. The internal region may be called a "spacer fragment." The external region may be called a "wing fragment." A wing fragment located 5' further to the side than the spacer fragment may be called a "5' wing fragment." A wing fragment located 3' further to the side than the spacer fragment may be called a "3' wing fragment." In the gap-mer, the sugar moiety of all nucleotides in the wing fragment is a modified sugar. In the spacer fragment, the sugar moiety of the nucleotides adjacent to the wing fragment (the 5' and 3' nucleotides of the spacer fragment) is the natural sugar moiety of DNA. In the nucleosides of the spacer fragments, the sugar portion of the nucleosides not adjacent to the wing fragments can be either the sugar portion of natural DNA or contain one or more modified sugars.

[0081] Examples of modified oligonucleotides having such a gap-mer structure include 1) a spacer fragment, 2) a 5' wing fragment, and 3) a 3' wing fragment, wherein the spacer fragment is located between the 5' wing fragment and the 3' wing fragment, and the sugar moiety of the nucleosides constituting the 5' wing fragment and the 3' wing fragment is entirely a modified sugar. Furthermore, the sugar moiety of ALNA[Ms] is preferably used as the modified sugar.

[0082] The modified oligonucleotides of the present invention can exist in the form of these pharmaceutically acceptable salts. As a pharmaceutically acceptable salt, any salt that maintains the desired biological activity of the modified oligonucleotides of the present invention without causing undesirable toxic effects is acceptable; salts formed with inorganic ions such as metal ions are preferred. Specifically, sodium salts and potassium salts are examples, with sodium salts being preferred. Furthermore, the modified oligonucleotides of the present invention can also exist in ionic form.

[0083] The modified oligonucleotides of the present invention or their pharmaceutically permissible salts can be synthesized by conventional methods, for example, by using phosphoramides that can be synthesized by commercially available or known methods.

[0084] The modified oligonucleotides of the present invention, or their pharmaceutically permissible salts, can be coupled to a coupling group at one or more sites. Examples of coupling groups include, specifically, fatty acids, cholesterol, glycosides, phospholipids, antibodies, proteins with affinity for molecules in vivo, biotin, phenazine, vitamins, peptides, folates, phenanthridine, anthraquinones, acridine, fluorescein, rhodamine, coumarins, and pigments. The aforementioned coupled modified oligonucleotides are manufactured using known methods, and modified oligonucleotides that enhance their activity, tissue distribution, cellular distribution, or cellular uptake can be selected. The coupling group can be directly bonded to the modified oligonucleotide or via a linker.

[0085] (Evaluation method of the compounds of this invention) As an evaluation method for the compounds of the present invention, any method that can verify the intracellular expression level of MRLN inhibited by the compounds of the present invention can be used. Specifically, for example, the in vitro and in vivo MRLN expression assay methods shown below can be used.

[0086] In the in vitro MRLN expression assay method used to evaluate the inhibition of intracellular MRLN expression of the compounds of the present invention, any cell can be used as long as it expresses MRLN (hereinafter sometimes referred to as "MRLN-expressing cells"), for example, RD cells (human rhabdomyosarcoma cells).

[0087] The method for contacting the compounds of the present invention with MRLN-expressing cells is not particularly limited, and methods commonly used for introducing nucleic acids into cells can be cited. Specifically, examples include lipid transfection, electroporation, and gymnosis. In lipid transfection or gymnosis, the compounds of the present invention can be used, for example, at a final concentration of 3, 10, 30, or 100 nM. As for the treatment time, an example is 48 hours.

[0088] Intracellular mRNA levels of MRLNs can be analyzed using various methods known in the art. Specifically, examples include Northern blotting analysis, competitive polymerase chain reaction (PCR), or quantitative real-time PCR.

[0089] Protein levels in MRLN cells can be analyzed using a variety of methods known in this field. Specifically, examples include immunoprecipitation, Western blotting, enzyme-linked immunosorbent assay (ELISA), quantitative protein analysis, protein activity analysis (e.g., caspase activity analysis), immunohistochemistry, immunocytochemistry, or fluorescence activated cell sorting (FACS).

[0090] In vivo MRLN expression assays for evaluating the inhibition of intracellular MRLN expression by the compounds of the present invention can be exemplified by administering the compounds of the present invention to animals expressing MRLN, and then analyzing the MRLN expression levels in those cells as described above.

[0091] (2) A pharmaceutical composition comprising a modified oligonucleotide or a pharmaceutically permissible salt thereof or a pharmaceutically permissible salt thereof. The modified oligonucleotides of the present invention, or pharmaceutically permissible salts thereof, can be used as pharmaceutical compositions. Additionally, the modified oligonucleotides of the present invention, or pharmaceutically permissible salts thereof, coupled to a coupling group, can be used as pharmaceutical compositions. The pharmaceutical compositions may further comprise a pharmaceutically permissible carrier. That is, the pharmaceutical composition may comprise the modified oligonucleotides of the present invention, or pharmaceutically permissible salts thereof, and a pharmaceutically permissible carrier.

[0092] As a pharmaceutically acceptable carrier, it can be appropriately selected according to the dosage form of the drug composition. Examples include excipients, lubricants, binders, and disintegrants in solid dosage forms, or solvents, dissolving aids, suspending agents, isotonic agents, buffers, and analgesics in liquid dosage forms. In addition, as needed, common preservatives, antioxidants, colorants, sweeteners, adsorbents, humectants, and other additives can also be used appropriately and in appropriate amounts.

[0093] Examples of dosage forms of pharmaceutical compositions for non-oral administration include injectable preparations (e.g., intravenous drip, intramuscular injection, subcutaneous injection, intradermal injection, intracerebral administration preparations, intraspinal administration preparations), topical preparations (e.g., ointments, pastes, lotions), suppositories, inhalers, eye preparations, eye ointments, nasal drops, ear drops, liposomes, etc. Methods for preparing dosage forms for non-oral administration are well known to those skilled in the art.

[0094] For example, injectable formulations can be prepared by dissolving the modified oligonucleotides of the present invention or their pharmaceutically permissible salts in a sterile aqueous solution. Examples of sterile aqueous solutions include sterile physiological saline, sterile water, and sterile phosphate-buffered saline. Additionally, dissolving agents, buffers, pH adjusters, isotonic agents, analgesics, preservatives, and stabilizers can be added as needed. Alternatively, lyophilized formulations for immediate use can also be prepared.

[0095] Dosage forms of pharmaceutical compositions for oral administration include solid or liquid dosage forms, specifically tablets, coated tablets, pills, granules, powders, capsules, syrups, emulsions, suspensions, injections, lozenges, etc. Methods for preparing dosage forms for oral administration are well known to those skilled in the art.

[0096] The modified oligonucleotides of the present invention, or their pharmaceutically permissible salts, or pharmaceutical compositions comprising them, can be used for the treatment or prevention of muscle diseases. In muscle diseases, it has been reported that increasing calcium uptake in the sarcoplasmic reticulum by inhibiting MRLN expression levels can lead to a decrease in cytoplasmic calcium levels and an increase in muscle contractility. Therefore, the modified oligonucleotides of the present invention, or pharmaceutically permissible salts thereof, or pharmaceutical compositions comprising them, are effective for the treatment or prevention of muscle diseases accompanied by muscle dysfunction, myocyte death, and / or decreased muscle contractility due to abnormal calcium influx into the cytoplasm.

[0097] Examples of such muscle diseases include, for instance, muscular dystrophy, inclusion body myositis, amyotrophic lateral sclerosis (ALS), disuse atrophy, or sarcopenia, with muscular dystrophy being the most preferred. Specifically, examples of muscular dystrophy include, for instance, Duchenne muscular dystrophy, Behringer's muscular dystrophy, or sarcopenic muscular disease, with Duchenne muscular dystrophy or sarcopenic muscular disease being the most preferred, and Duchenne muscular dystrophy being the most more preferred. Furthermore, sarcopenic muscular diseases include LGMD2C, LGMD2D, LGMD2E, and LGMD2F.

[0098] In particular, regarding muscular dystrophy, two mechanisms are known: 1) Due to the deficiency of the dystrophin-glycoprotein complex, membrane permeability is increased, leading to increased intracellular calcium levels in the cytoplasm, resulting in muscle dysfunction and myocyte death; 2) Due to the deficiency of the dystrophin-glycoprotein complex, nNOS (neuronal nitric oxide synthase) is reduced, while iNOS (inducible nitric oxide synthase) in the cytoplasm increases, leading to nitrosation of Rhinodine receptors, decreased intracellular calcium in the sarcoplasmic reticulum, and decreased muscle contractility. The modified oligonucleotides of the present invention, or pharmaceutically permissible salts thereof, or pharmaceutical compositions comprising them, can inhibit muscle dysfunction and myocyte death in muscular dystrophy and / or maintain or restore muscle contractility by reducing intracellular calcium levels in the cytoplasm and increasing calcium uptake in the sarcoplasmic reticulum.

[0099] Therefore, this invention relates to: Pharmaceutical compositions for the treatment or prevention of muscle diseases, comprising the modified oligonucleotides of the present invention or their pharmaceutically permissible salts; A method for treating or preventing muscle diseases, comprising the step of administering an effective amount of the modified oligonucleotide of the present invention or a pharmaceutically permissible salt thereof (or a pharmaceutical composition comprising the modified oligonucleotide or a pharmaceutically permissible salt thereof) to a subject. The use of the modified oligonucleotide or its pharmaceutically permissible salt (or a pharmaceutical composition containing the modified oligonucleotide or its pharmaceutically permissible salt) of the present invention in the manufacture of a pharmaceutical composition for the treatment or prevention of muscle diseases; The modified oligonucleotide of the present invention or a pharmaceutically permissible salt thereof (or a pharmaceutical composition containing the modified oligonucleotide or a pharmaceutically permissible salt thereof) is used in the manufacture of pharmaceutical compositions for the treatment or prevention of muscle diseases. The present invention relates to the use of modified oligonucleotides or pharmaceutically permissible salts thereof (or pharmaceutical compositions comprising such modified oligonucleotides or pharmaceutically permissible salts thereof) for the treatment or prevention of muscle diseases.

[0100] The “subject” as the intended recipient includes humans or other mammals (e.g., one or more of the following: mouse, guinea pig, hamster, rat, mouse, rabbit, pig, sheep, goat, cow, horse, cat, dog, marmoset, monkey, or chimpanzee).

[0101] Examples of administration methods for the modified oligonucleotides or their pharmaceutically permissible salts, or pharmaceutical compositions comprising them, according to the present invention include systemic administration such as oral, intravenous, or intra-arterial administration. The dosage of the modified oligonucleotides or their pharmaceutically permissible salts, or pharmaceutical compositions comprising them, according to the purpose of use, the severity of the disease, the patient's age, weight, sex, etc., can be appropriately varied, for example, from 0.1 ng to 100 mg / kg / day based on the amount of modified oligonucleotide.

[0102] The modified oligonucleotides of the present invention, or their pharmaceutically permissible salts, or pharmaceutical compositions comprising them, can be used alone or in combination with one or more other pharmaceutical agents.

[0103] Inclusion based on non-restricted disclosure and reference The compounds, compositions, and methods described in this specification are specifically described according to a particular embodiment. However, the following examples are merely illustrative of the compounds described in this specification and are not intended to limit their application. All references cited in this application are incorporated herein by reference in their entirety. Example

[0104] Example 1 Preparation of modified oligonucleotides The modified oligonucleotides were synthesized and purified using ALNA[Ms]phosphite (synthesized according to the method described in International Publication No. WO2020 / 100826) via solid-phase synthesis of oligonucleotides, using Nippon Gene Corporation / Nippon Gene Materials Corporation. It should be noted that the compounds were identified using high-performance liquid chromatography-mass spectrometry.

[0105] The synthesized modified oligonucleotide compounds are shown in Tables 1-1 to 1-6 (16-residue modified oligonucleotides). In the compound descriptions, each nucleoside is represented by a single letter, and the inter-nucleoside bonds are phosphate thioester bonds. The nucleosides are as follows: A = adenosine containing the ALNA[Ms] sugar moiety as the modified sugar, T = thymidine containing the ALNA[Ms] sugar moiety as the modified sugar, G = guanosine containing the ALNA[Ms] sugar moiety as the modified sugar, C = 5-methylcytidine containing the ALNA[Ms] sugar moiety as the modified sugar and containing 5-methylcytosine as the modified base, a = 2'-deoxyribonucleotide adenosine, t = 2'-deoxyribonucleotide thymidine, g = 2'-deoxyribonucleotide guanosine, c = 2'-deoxyribonucleotide cytidine. The premature target start position indicates the 5' target site of the modified oligonucleotide MRLN pre-mRNA (position 1 in the sequence listing corresponding to the 3' end of the modified oligonucleotide), and the premature target end position indicates the 3' target site of the modified oligonucleotide MRLN pre-mRNA (position 1 in the sequence listing corresponding to the 5' end of the modified oligonucleotide). The mature target start position indicates the 5' target site of the modified oligonucleotide MRLN mRNA (position 2 in the sequence listing corresponding to the 3' end of the modified oligonucleotide), and the mature target end position indicates the 3' target site of the modified oligonucleotide MRLN mRNA (position 2 in the sequence listing corresponding to the 5' end of the modified oligonucleotide).

[0106] [Table 1-1]

[0107] [Table 1-2]

[0108] [Table 1-3]

[0109] [Table 1-4]

[0110] [Table 1-5]

[0111] [Table 1-6]

[0112] Example 2 In vitro MRLN expression ratio assay (Lipofection method) The modified oligonucleotide synthesized in Example 1 was mixed with lipofection reagent and added to a 384-well plate at a rate of 5 μL / well. The solution was then sprayed onto the plate at 5 × 10⁻⁶ ppm. 3 Cells were seeded at 45 μL / well in RD cells suspended in medium containing 10% serum and cultured in a CO2 incubator. After 48 hours, the medium containing the modified oligonucleotides was removed from the cells, and the cells were washed with phosphate-buffered saline (PBS). Then, using the SuperPrep Cell Lysis & RT Kit for qPCR (Toyobo), cell lysates containing RNA were prepared, and RT reactions based on the lysates were performed to synthesize template cDNA. Real-time PCR was performed using this cDNA to quantify MRLN mRNA expression. Regarding MRLN expression ratios, the MRLN mRNA levels in cells supplemented with a final concentration of 100 nmol / L of modified oligonucleotides were compared with those in cells without the supplemented modified oligonucleotides, and the percentages are shown in Tables 1-1 to 1-6.

[0113] Example 3 In vitro MRLN expression ratio assay (Gymnosis method) RD cells suspended in a medium containing 2% serum were incubated at 5 × 10⁻⁶. 3Cells were seeded at 45 μL / well in 384-well plates and cultured in a CO2 incubator. After 72 hours, the medium was replaced with the same medium, and the modified oligonucleotide synthesized in Example 1 was added at 5 μL / well, followed by further CO2 incubation. After 72 hours, the medium containing the modified oligonucleotide was removed from the cells, and the cells were washed with phosphate-buffered saline (PBS). Then, using the SuperPrepCell Lysis & RT Kit for qPCR (Toyobo), cell lysates containing RNA were prepared, and RT reactions based on the lysates were performed to synthesize template cDNA. Real-time PCR was performed using this cDNA to quantify MRLN mRNA expression. Regarding MRLN expression ratios, the MRLN mRNA levels in cells supplemented with a final concentration of 1000 nmol / L of modified oligonucleotide were compared with those in cells without the added modified oligonucleotide, and the percentages are shown in Tables 1-1 to 1-6.

Claims

1. A modified oligonucleotide or a pharmaceutically permissible salt thereof, comprising 12 to 30 linked nucleosides, having the activity of inhibiting myotropic hormone expression, wherein, The modified oligonucleotide comprises positions 15-41, 62-108, 448-471, 649-666, 848-863, 1104-1119, 1232-1254, 1691-1719, 1735-1751, 2195-2211, 2289-2310, 2479-2512, 2616-2631, 2692-2716, and 2... 751~2766, 2893~2909, 2979~2995, 3431~3447, 3564~3585, 3601~3617, 3721~3736, 4074~4089, 4127~4142, 4156~4171, 4277~4292, 4305~4322, 4500~4515, 4613~4628, 5039~5055, 5063~5079 Positions 5103-5119, 5522-5540, 5727-5742, 5772-5803, 5998-6013, 6272-6288, 6695-6711, 6763-6778, 7224-7247, 8013-8028, 9844-9864, 14649-14665, 14903-14920, 14951-14995, 16113-16129, ... The modified oligonucleotide has a 100% complementary sequence of any eight or more consecutive bases in any of the following base sequences: positions 16235-16254, 16256-16273, 16281-16306, 16316-16336, 16514-16536, 16578-16593, 16610-16637, or 16724-16739. The full-length base sequence of the modified oligonucleotide has more than 85% complementarity with the equivalent-length portion of the base sequence of sequence number 1 in the sequence listing.

2. The modified oligonucleotide or its pharmaceutically permissible salt according to claim 1, wherein, The modified oligonucleotide comprises positions 1232-1254, 1691-1719, 1735-1751, 2195-2211, 2289-2308, 3431-3446, 4127-4142, 4156-4171, 4500-4515, 5522-5537, 5727-5742, 5772-5803, 5998-6013, and 627, starting from the 5' end of the sequence of sequence number 1 in the sequence listing. The modified oligonucleotide has a 100% complementary sequence of any eight or more consecutive bases in any of the following base sequences: positions 2-6287, 6763-6778, 8013-8028, 9846-9864, 14963-14978, 16235-16254, 16256-16273, 16281-16306, or 16316-16331. The full-length base sequence of the modified oligonucleotide has more than 85% complementarity with the equivalent-length portion of the base sequence of sequence number 1 in the sequence listing.

3. A modified oligonucleotide or a pharmaceutically permissible salt thereof, comprising 12-30 linked nucleosides, having the activity of inhibiting myotropic hormone expression, wherein... The modified oligonucleotide comprises a base sequence that is 100% complementary to any eight or more consecutive bases in any of the following sequences starting from the 5' end of the sequence of sequence number 2 in the sequence listing: positions 15-41, 62-91, 100-129, 160-225, 227-244, 252-277, 287-307, 485-507, 549-564, 581-608, or 695-710. The full-length base sequence of the modified oligonucleotide has at least 85% complementarity to the equivalent-length portion of the base sequence of sequence number 2 in the sequence listing.

4. The modified oligonucleotide or its pharmaceutically permissible salt according to claim 3, wherein, The modified oligonucleotide comprises a base sequence that is 100% complementary to any eight or more consecutive bases in any of the following base sequences starting from the 5' end of sequence number 2 in the sequence listing: positions 172-187, 201-225, 227-244, 252-277, or 287-302. The full-length base sequence of the modified oligonucleotide is more than 85% complementary to the equivalent-length portion of the base sequence in sequence number 2 in the sequence listing.

5. A modified oligonucleotide or a pharmaceutically permissible salt thereof, wherein the modified oligonucleotide or a pharmaceutically permissible salt thereof is an activity of inhibiting myotropic hormone expression, the base sequence of the modified oligonucleotide being any base sequence selected from the group consisting of sequence numbers 275-279, 229 and 231 of the sequence listing, or a base sequence consisting of 17 or 18 consecutive bases containing the base sequence, the full-length base sequence of the modified oligonucleotide being composed of a base sequence that is 100% complementary to the equal-length portion of the base sequence of sequence number 1.

6. A modified oligonucleotide or a pharmaceutically permissible salt thereof, wherein the modified oligonucleotide or a pharmaceutically permissible salt thereof is an activity of inhibiting myotropic hormone expression, the base sequence of said modified oligonucleotide being any base sequence of sequence number 273 or 280 of the sequence listing, or a base sequence consisting of 17 or 18 consecutive bases containing said base sequence, the full-length base sequence of said modified oligonucleotide being composed of a base sequence that is 100% complementary to the equal-length portion of the base sequence of sequence number 2.

7. The modified oligonucleotide or its pharmaceutically permissible salt according to any one of claims 1 to 6, wherein, The modified oligonucleotide is single-stranded.

8. The modified oligonucleotide or its pharmaceutically permissible salt according to any one of claims 1 to 6, wherein, At least one nucleoside constituting the modified oligonucleotide contains a modified sugar.

9. The modified oligonucleotide or a pharmaceutically permissible salt thereof according to claim 8, wherein, The modified sugar is a bicyclic sugar.

10. The modified oligonucleotide or a pharmaceutically permissible salt thereof according to claim 9, wherein, The bicyclic sugar is selected from the group consisting of the sugar moieties of LNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], and ALNA[Trz].

11. The modified oligonucleotide or a pharmaceutically permissible salt thereof according to claim 10, wherein, The bicyclic sugar is the sugar portion of ALNA[Ms].

12. The modified oligonucleotide or a pharmaceutically permissible salt thereof according to claim 8, wherein, The modified sugar is a substituted sugar.

13. The modified oligonucleotide or its pharmaceutically permissible salt according to any one of claims 1 to 6, wherein, At least one nucleoside constituting the modified oligonucleotide contains a modified base.

14. The modified oligonucleotide or a pharmaceutically permissible salt thereof according to claim 13, wherein, The modified base is 5-methylcytosine.

15. The modified oligonucleotide or its pharmaceutically permissible salt according to any one of claims 1 to 6, wherein, At least one internucleotide bond constituting the modified oligonucleotide is a modified internucleotide bond.

16. The modified oligonucleotide or a pharmaceutically permissible salt thereof according to claim 15, wherein, The modified nucleoside inter-bond is a thiophosphate nucleoside inter-bond.

17. The modified oligonucleotide or its pharmaceutically permissible salt according to any one of claims 1 to 6, wherein, The modified oligonucleotide comprises: 1) Interval segments, 2) 5' wing segment, and 3) 3' wing segment, The spacer segment is located between the 5' wing segment and the 3' wing segment. The glycosides constituting the 5' and 3' wings are all modified sugars.

18. A pharmaceutical composition comprising any one of the modified oligonucleotides of claims 1 to 6 or a pharmaceutically permissible salt thereof.

19. The pharmaceutical composition according to claim 18, for the treatment or prevention of muscle diseases.

20. The pharmaceutical composition according to claim 19, wherein, The muscle diseases mentioned are selected from the group consisting of myofascial dystrophy, inclusion body myositis, amyotrophic lateral sclerosis, disuse atrophy, and sarcopenia.

21. The pharmaceutical composition according to claim 20, wherein, The muscle disease mentioned is muscular dystrophy.

22. The pharmaceutical composition according to claim 21, wherein, The muscular dystrophy was selected from the group consisting of Duchenne muscular dystrophy, Behringer's muscular dystrophy, and myofascitis.

23. The pharmaceutical composition according to claim 19, wherein, The muscle disease described is Duchenne muscular dystrophy.

24. The pharmaceutical composition according to claim 19, wherein, The muscle disease described is Benedict's muscular dystrophy.

25. The pharmaceutical composition according to claim 19, wherein, The muscle disease in question is myofascitis.

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