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WO2025206092A1 - Composé et composition pharmaceutique pour réguler l'expression de la myoréguline - Google Patents

Composé et composition pharmaceutique pour réguler l'expression de la myoréguline

Info

Publication number
WO2025206092A1
WO2025206092A1 PCT/JP2025/012308 JP2025012308W WO2025206092A1 WO 2025206092 A1 WO2025206092 A1 WO 2025206092A1 JP 2025012308 W JP2025012308 W JP 2025012308W WO 2025206092 A1 WO2025206092 A1 WO 2025206092A1
Authority
WO
WIPO (PCT)
Prior art keywords
modified oligonucleotide
base sequence
pharmaceutically acceptable
seq
acceptable salt
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/JP2025/012308
Other languages
English (en)
Japanese (ja)
Inventor
大輔 飯嶋
俊平 村田
謙 氏家
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Mitsubishi Tanabe Pharma Corp
Original Assignee
Mitsubishi Tanabe Pharma Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Mitsubishi Tanabe Pharma Corp filed Critical Mitsubishi Tanabe Pharma Corp
Publication of WO2025206092A1 publication Critical patent/WO2025206092A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the present invention relates to a compound for reducing at least one of the pre-mRNA level, mRNA level, and protein level of Myoregulin (MRLN) in an animal, and a pharmaceutical composition containing the compound.
  • MRLN Myoregulin
  • MRLN Myoregulin
  • DMD Duchenne muscular dystrophy
  • MRLN siRNA which suppresses MRLN mRNA expression
  • MRLN antisense oligonucleotides to DMD model mice (mdx mice) and sarcoglycanopathy model mice (sarcoglycan ⁇ knockout mice) to reduce MRLN gene expression resulted in a decrease in creatine kinase levels, a clinical indicator of muscular dystrophy and caused by muscle damage and muscle cell death, in both model mice (Patent Document 1).
  • Sarcoglycanopathy is a general term for limb girdle muscular dystrophy (LGMD) types 2C to 2F.
  • suppressing the expression level of MRLN can increase the amount of calcium uptake into the sarcoplasmic reticulum, making it effective in treating or preventing muscle diseases such as muscular dystrophy, which are accompanied by muscle damage, muscle cell death, and/or decreased muscle contractility due to abnormal calcium influx into the cytoplasm.
  • MRLN antisense oligonucleotides that suppress the expression level of MRLN for the treatment of muscle diseases.
  • the MRLN antisense oligonucleotides reported to date were designed based on the base sequence of the mouse MRLN gene (Patent Document 1), making their clinical application for the treatment or prevention of human muscle disease patients difficult.
  • the present invention provides compounds for inhibiting MRLN expression.
  • a modified oligonucleotide consisting of 12 to 30 linked nucleosides or a pharmaceutically acceptable salt thereof, which has an activity of inhibiting Myoregulin expression, wherein the modified oligonucleotide is located at positions 15 to 41, 62 to 108, 448 to 471, 649 to 666, 848 to 863, 1104 to 1119, 1232 to 1254, 1691 to 1719, 1735 to 1751, 2195 to 2211, 3195 to 3211, 3215 to 3215, 3216 to 3217, 3218 to 3219, 3220 to 3221, 3222 to 3222, 3223 to 3224, 3225 to 3225, 3226 to 3227, 3228 to 3229, 3230 to 3231, 3232 to 3232, 3233 to 3234, 3235 to 3235, 3236 to 3237, 3238 to 3239, 3240 to 3241, 3242 to 3243, 3244 to 3245, 3246 to 3248, 3248 to 3249,
  • the modified oligonucleotide is selected from the group consisting of positions 1232 to 1254, 1691 to 1719, 1735 to 1751, 2195 to 2211, 2289 to 2308, 3431 to 3446, 4127 to 4142, 4156 to 4171, 4500 to 4515, 5522 to 5537, 5727 to 5742, 5772 to 5803, 5998 to 6013, 6272 to 6287, 6763 to 6778, 8013 to 8028, 98
  • the modified oligonucleotide or a pharmaceutically acceptable salt thereof according to [1] comprising a base sequence that is 100% complementary to any 8 or more consecutive bases in any of the base sequences at positions 46 to 9864, 14963 to 14978, 16235 to 16254, 16256 to 16273, 16281 to 16306, or 16316 to 16331, and the full-length base sequence of the modified oligonucleotide is 85% or more complementary to an equal-length portion of the base sequence of SEQ ID
  • a modified oligonucleotide consisting of 12 to 30 linked nucleosides, or a pharmaceutically acceptable salt thereof, which has activity of inhibiting Myoregulin expression, wherein the modified oligonucleotide comprises a base sequence that is 100% complementary to any 8 or more consecutive bases in any of the base sequences at 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 from the 5' end of the base sequence of SEQ ID NO: 2 in the Sequence Listing, and the full-length base sequence of the modified oligonucleotide is 85% or more complementary to an equal-length portion of the base sequence of SEQ ID NO: 2 in the Sequence Listing.
  • modified oligonucleotide or a pharmaceutically acceptable salt thereof according to [3], wherein the modified oligonucleotide comprises a base sequence that is 100% complementary to any 8 or more consecutive bases in any of the base sequences at positions 172 to 187, 201 to 225, 227 to 244, 252 to 277, or 287 to 302 from the 5' end of the base sequence of SEQ ID NO: 2 in the Sequence Listing, and the full-length base sequence of the modified oligonucleotide is 85% or more complementary to the equal-length portion of the base sequence of SEQ ID NO: 2 in the Sequence Listing.
  • a modified oligonucleotide or a pharmaceutically acceptable salt thereof having an activity of suppressing Myoregulin expression wherein the base sequence of the modified oligonucleotide is CCAGAATTATCCCGCT (complementary sequence of positions 16238 to 16253 of SEQ ID NO: 1) (SEQ ID NO: 275 in the Sequence Listing), TCCAGAATTATCCCGC (complementary sequence of positions 16239 to 16254 of SEQ ID NO: 1) (SEQ ID NO: 276 in the Sequence Listing), AGTTTTTACCAGTCAT (complementary sequence of positions 16256 to 16271 of SEQ ID NO: 1) (SEQ ID NO: 277 in the Sequence Listing), CCAGTTTTACCAGTC (complementary sequence of positions 16258 to 16273 of SEQ ID NO: 1) (SEQ ID NO: 278 in the Sequence Listing), GACTTTTTGGGAGTAGT (complementary sequence of positions 16289 to 16304 of SEQ ID NO: 1) (SEQ ID NO:
  • a modified oligonucleotide or a pharmaceutically acceptable salt thereof having an activity of suppressing Myoregulin expression wherein the base sequence of the modified oligonucleotide is TTATCCCGCTCCCTGA (complementary sequence of positions 203 to 218 of SEQ ID NO: 2) (SEQ ID NO: 273 in the Sequence Listing) or ATTATCCCGCTCCCTG (complementary sequence of positions 204 to 219 of SEQ ID NO: 2) (SEQ ID NO: 280 in the Sequence Listing)
  • a modified oligonucleotide or a pharmaceutically acceptable salt thereof which is any of the base sequences shown below, or a base sequence consisting of 17 or 18 consecutive bases including said base sequence, and wherein the full-length base sequence of the modified oligonucleotide is a base sequence that is 100% complementary to the equal-length portion of the base sequence of SEQ ID NO: 2.
  • modified oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [1] to [6], wherein the modified oligonucleotide is single-stranded.
  • the bicyclic sugar is selected from the group consisting of LNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], and ALNA[Trz] sugar moieties.
  • the modified oligonucleotide or a pharmaceutically acceptable salt thereof according to [9] or [10] wherein the bicyclic sugar is the sugar moiety of ALNA[Ms].
  • the modified internucleoside bond is a phosphorothioate internucleoside bond.
  • the modified oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [1] to [16], wherein all of the sugar moieties of the nucleosides constituting the 5' wing segment and the 3' wing segment are modified sugars.
  • a pharmaceutical composition comprising the modified oligonucleotide according to any one of [1] to [17] or a pharmaceutically acceptable salt thereof.
  • the pharmaceutical composition according to [20] wherein the muscle disease is muscular dystrophy.
  • Base sequence means the order of consecutive bases.
  • Nucleoside refers to a molecule consisting of a sugar and a base.
  • Linked nucleosides means nucleosides that are linked by internucleoside linkages in a contiguous sequence.
  • Nucleotide refers to a molecule in which a phosphate group is attached to the sugar portion of a nucleoside. Naturally occurring nucleotides have a ribose or deoxyribose sugar portion.
  • Internucleoside bond refers to the chemical bond between nucleosides.
  • Modified internucleoside linkage refers to a substitution or any change from a naturally occurring internucleoside linkage (i.e., a phosphodiester internucleoside linkage). For example, but not limited to, a phosphorothioate internucleoside linkage.
  • Modified base refers to any base other than adenine, cytosine, guanine, thymidine, or uracil. For example, but not limited to, 5-methylcytosine.
  • Modified nucleoside means a nucleoside having, independently, a modified sugar or modified base.
  • Modified oligonucleotide means an oligonucleotide containing at least one modified nucleoside and/or modified internucleoside linkage.
  • Substituted sugar means a furanosyl sugar other than the natural sugars of RNA or DNA, but does not include bicyclic sugars.
  • Bicyclic sugar means a furanosyl modified by bridging two different carbon atoms among the four carbon atoms forming the furanosyl ring.
  • “Isometric portion” refers to a portion of the base sequence of a second nucleic acid that has a length equal to the length of the base sequence of a first nucleic acid.
  • the first nucleic acid is a modified oligonucleotide and the second nucleic acid is MRLN pre-mRNA or MRLN mRNA.
  • “Complementary” refers to the capacity for pairing between the bases of a first nucleic acid and a second nucleic acid.
  • adenine is complementary to thymidine or uracil.
  • cytosine is complementary to guanine.
  • 5-methylcytosine is complementary to guanine.
  • 100% complementary means that the base sequence of a first nucleic acid is completely complementary to the base sequence of a second nucleic acid.
  • the first nucleic acid is a modified oligonucleotide and the target nucleic acid is the second nucleic acid.
  • mismatch refers to a case where a base in a first nucleic acid cannot pair with the corresponding base in a second nucleic acid or target nucleic acid.
  • Target nucleic acid refers to a nucleic acid, RNA, and RNA transcript, respectively, that can be targeted by a modified oligonucleotide.
  • the target nucleic acid comprises a region of MRLN mRNA or MRLN pre-mRNA.
  • “Pharmaceutically acceptable salt” means a physiologically and pharmaceutically acceptable salt of the modified oligonucleotide of the present invention, including salts formed with inorganic ions such as metal ions on the phosphorothioate or phosphodiester, or on functional groups (e.g., amino groups) in the modified base.
  • “Individual” means a human or non-human animal selected for treatment or therapy.
  • the base sequence is a base sequence consisting of 17, 18, 19, or 20, preferably 17 or 18, consecutive bases, including the complementary base sequence consisting of 16 consecutive bases.
  • 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 SEQ ID NO: 2.
  • the base sequence consisting of 17, 18, 19, or 20, preferably 17 or 18, consecutive bases is a base sequence in which 1 to 4, preferably 1 and/or 2, nucleosides are linked by internucleoside bonds at the 5' or 3' end of the complementary base sequence consisting of 16 consecutive bases.
  • the nucleosides and/or internucleoside bonds may be modified nucleosides and/or modified internucleoside bonds.
  • 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 SEQ ID NO: 1.
  • TTATCCCGCTCCCTGA complementary sequence of positions 203 to 218 of SEQ ID NO: 218 of SEQ ID NO: 2
  • ATTATCCCGCTCCCTG complementary sequence of positions 204 to 219 of SEQ ID NO: 2
  • SEQ ID NO: 280 in the Sequence Listing or a base sequence comprising 17, 18, 19, or 20, preferably 17 or 18, consecutive bases containing the base sequence.
  • 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 isolength portion of SEQ ID NO: 2.
  • 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 SEQ ID NO: 1.
  • the base sequence of such a modified oligonucleotide is more preferably: AGTTTTTACCAGTCAT (complementary sequence of positions 16256 to 16271 of SEQ ID NO: 1) (SEQ ID NO: 277 in the Sequence Listing), CCAGTTTTTTACCAGTC (complementary sequence of positions 16258 to 16273 of SEQ ID NO: 1) (SEQ ID NO: 278 in the Sequence Listing) or AGACTTTTGGGGAGTAG (complementary sequence of positions 16290 to 16305 of SEQ ID NO: 1) (SEQ ID NO: 229 in the Sequence Listing) or a base sequence comprising 17, 18, 19, or 20, preferably 17 or 18, consecutive bases containing the base sequence.
  • 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 equal-length portion of the base sequence of SEQ ID NO: 1.
  • the full-length base sequence of the modified oligonucleotide is a base sequence that is 90% or more, preferably 95% or more, and more preferably 100% complementary to the base sequence of the isomeric portion in SEQ ID NO:1.
  • the base sequence may be the base sequence set forth in SEQ ID NO:273 or SEQ ID NO:274, or a base sequence comprising said base sequence and consisting of 17, 18, 19, or 20, preferably 17 or 18, consecutive bases.
  • the full-length base sequence of the s-modified oligonucleotide is 90% or more, preferably 95% or more, and more preferably 100% complementary to the base sequence of the isolength portion in SEQ ID NO: 2.
  • the modified oligonucleotide is 90% or more, preferably 95% or more, and more preferably 100% complementary to the equal-length portion of the base sequence in SEQ ID NO: 1.
  • the nucleoside containing a bicyclic sugar is a nucleoside having formula (I), as defined above in ALNA[mU], wherein: B is a base; R 1 , R 2 , R 3 and R 4 are each independently a hydrogen atom; R5 and R6 are each independently a hydrogen atom; m is 1; X is represented by the following formula (II-1): is a group represented by the formula: A nucleoside in which one of R7 and R8 is a hydrogen atom and the other is an unsubstituted isopropyl group (see, for example, WO 2020/100826).
  • the bicyclic (sugar portion of ALNA[Trz]) containing nucleoside is a nucleoside having formula (I) as defined above in ALNA[mU], wherein: B is a base; R 1 , R 2 , R 3 and R 4 are each independently a hydrogen atom; R5 and R6 are each independently a hydrogen atom; m is 1; X is represented by the following formula (II-2): is a group represented by the formula: A is a nucleoside in which A is a 1,5-dimethyl-1,2,4-triazol-3-yl group (see, for example, WO 2020/100826).
  • the nucleoside containing a bicyclic sugar is a nucleoside having formula (I), as defined above in ALNA[mU], wherein: B is a base; R 1 , R 2 , R 3 and R 4 are each independently a hydrogen atom; R5 and R6 are each independently a hydrogen atom; m is 1; X is represented by the following formula (II-2): is a group represented by the formula: A is a nucleoside in which A is a 5-methyl-1,2,4-oxadiazol-3-yl group (see, for example, WO 2020/100826).
  • the modified oligonucleotide of the present invention is preferably one in which at least one of the internucleoside linkages comprising it contains a modified internucleoside linkage.
  • Modified oligonucleotides having such modified internucleoside linkages have advantageous properties such as enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.
  • modified internucleoside linkages include phosphorothioate internucleoside linkages.
  • a phosphorothioate internucleoside linkage refers to a linkage between nucleosides in which the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom.
  • the modified oligonucleotides of the present invention can have a gapmer structure to achieve increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for target nucleic acids, and/or increased MRLN expression inhibitory activity.
  • a gapmer structure refers to a structure in which an internal region having multiple nucleosides that supports RNase H cleavage is located between external regions having one or more nucleosides.
  • the internal region can be referred to as a "gap segment.”
  • the external region can be referred to as a "wing segment.”
  • the wing segment located 5' from the gap segment can be referred to as a "5' wing segment.”
  • the wing segment located 3' from the gap segment can be referred to as a "3' wing segment.”
  • the sugar moieties of all nucleosides in the wing segment are modified sugars.
  • the sugar moieties of the nucleosides adjacent to the wing segment are sugar moieties of natural DNA.
  • the sugar moieties of nucleosides in the gap segment that are not adjacent to the wing segments may consist solely of natural DNA sugar moieties or may contain one or more modified sugars.
  • the modified oligonucleotides of the present invention may exist in the form of their pharmaceutically acceptable salts.
  • Pharmaceutically acceptable salts may be any salt that retains the desired biological activity of the modified oligonucleotides of the present invention without imparting any undesirable toxicological effects thereto, but salts formed with inorganic ions such as metal ions are preferred. Specific examples include sodium salts and potassium salts, with sodium salts being preferred.
  • the modified oligonucleotides of the present invention may also exist in the form of ions.
  • modified oligonucleotides or pharmaceutically acceptable salts thereof of the present invention can be synthesized by conventional methods, for example, by the phosphoramidite method using amidites that are commercially available or can be synthesized by known methods.
  • the modified oligonucleotide or pharmaceutically acceptable salt thereof of the present invention may be conjugated to a conjugate group at one or more locations.
  • conjugate groups include proteins having affinity for biological molecules such as fatty acids, cholesterol, carbohydrates, phospholipids, and antibodies, as well as biotin, phenazine, vitamins, peptides, folates, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes.
  • the conjugated modified oligonucleotides are produced by known methods, and those that enhance their activity, tissue distribution, cellular distribution, or cellular uptake can be selected.
  • the conjugate group may be bound directly to the modified oligonucleotide or via a linker.
  • the method for evaluating the compound of the present invention may be any method that can verify the suppression of the expression level of MRLN in cells by the compound of the present invention. Specifically, for example, the following in vitro and in vivo MRLN expression measurement methods can be used.
  • MRLN-expressing cells any cells that express MRLN (hereinafter sometimes referred to as "MRLN-expressing cells"), such as RD cells (human rhabdomyosarcoma cells).
  • the intracellular mRNA level of MRLN can be assayed using various methods known in the art, including, for example, Northern blot analysis, competitive polymerase chain reaction (PCR), or quantitative real-time PCR.
  • PCR competitive polymerase chain reaction
  • Intracellular protein levels of MRLN can be assayed using a variety of methods known in the art, including, for example, immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assay, protein activity assay (e.g., caspase activity assay), immunohistochemistry, immunocytochemistry, or fluorescence-activated cell sorting (FACS).
  • immunoprecipitation Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assay, protein activity assay (e.g., caspase activity assay), immunohistochemistry, immunocytochemistry, or fluorescence-activated cell sorting (FACS).
  • An in vivo MRLN expression measurement method for evaluating the suppression of MRLN expression in cells by a compound of the present invention involves, for example, administering a compound of the present invention to an animal that expresses MRLN and analyzing the MRLN expression level in the cells.
  • composition containing a modified oligonucleotide or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable salt thereof can be used as a pharmaceutical composition.
  • the modified oligonucleotide of the present invention conjugated with a conjugate group or a pharmaceutically acceptable salt thereof can be used as a pharmaceutical composition.
  • the pharmaceutical composition may further contain a pharmaceutically acceptable carrier. That is, the pharmaceutical composition may contain the modified oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are selected appropriately depending on the dosage form of the pharmaceutical composition, but include, for example, excipients, lubricants, binders, and disintegrants in solid preparations, and solvents, solubilizers, suspending agents, isotonicity agents, buffers, and soothing agents in liquid preparations. Furthermore, conventional additives such as preservatives, antioxidants, colorants, sweeteners, adsorbents, and wetting agents can also be used as needed in appropriate amounts.
  • Dosage forms of pharmaceutical compositions for parenteral administration include injectable preparations (e.g., drip infusions, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, intracerebral administration preparations, and intraspinal administration preparations), topical preparations (e.g., ointments, poultices, lotions), suppositories, inhalants, eye preparations, eye ointments, nasal drops, ear drops, and liposomes.
  • injectable preparations e.g., drip infusions, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, intracerebral administration preparations, and intraspinal administration preparations
  • topical preparations e.g., ointments, poultices, lotions
  • suppositories e.g., inhalants, eye preparations, eye ointments, nasal drops, ear drops, and liposomes
  • an injectable formulation can be prepared by dissolving the modified oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof in a sterile aqueous solution.
  • sterile aqueous solutions include sterile saline, sterile water, and sterile phosphate-buffered saline.
  • solubilizers, buffers, pH adjusters, isotonicity agents, soothing agents, preservatives, stabilizers, etc. can be added as needed. It can also be made into a lyophilized formulation for preparation immediately before use.
  • Dosage forms for oral administration of pharmaceutical compositions include solid or liquid dosage forms, specifically tablets, coated tablets, pills, fine granules, granules, powders, capsules, syrups, emulsions, suspensions, injections, and lozenges. Methods for preparing dosage forms for oral administration are well known to those skilled in the art.
  • the modified oligonucleotides of the present invention or pharmaceutically acceptable salts thereof, or pharmaceutical compositions containing them can be used for the treatment or prevention of muscle diseases. It has been reported that suppressing the expression level of MRLN in muscle diseases increases calcium uptake in the sarcoplasmic reticulum, thereby reducing the amount of calcium in the cytoplasm and increasing muscle contractility. Therefore, the modified oligonucleotides of the present invention or pharmaceutically acceptable salts thereof, or pharmaceutical compositions containing them, are effective in treating or preventing muscle disorders caused by abnormal calcium influx into the cytoplasm, muscle cell death, and/or muscle contractility reduction.
  • modified oligonucleotides or pharmaceutically acceptable salts thereof, or pharmaceutical compositions containing them, of the present invention reduce the amount of calcium in the cytoplasm and increase calcium uptake into the sarcoplasmic reticulum, thereby preventing muscle damage and muscle cell death in muscular dystrophies and/or maintaining or restoring muscle contractility.
  • the dosage of the modified oligonucleotide or pharmaceutically acceptable salt thereof of the present invention, or a pharmaceutical composition containing the same can be systemically administered, for example, orally, intravenously, or intra-arterially.
  • the dosage of the modified oligonucleotide or pharmaceutically acceptable salt thereof of the present invention, or a pharmaceutical composition containing the same can be varied as appropriate depending on the purpose of use, severity of the disease, and the age, weight, and sex of the patient, but can be, for example, 0.1 ng to 100 mg/kg/day of the modified oligonucleotide.
  • Example 1 Preparation of Modified Oligonucleotides
  • Modified oligonucleotides were synthesized and purified by Nippon Gene Co., Ltd./Nippon Gene Material Co., Ltd. using ALNA[Ms] amidite (synthesized by the method described in International Publication WO 2020/100826) according to a general solid-phase synthesis method for oligonucleotides.
  • the compounds were identified using a high-performance liquid chromatograph mass spectrometer.
  • the synthesized modified oligonucleotide compounds are shown in Tables 1-1 to 1-6 (16-residue modified oligonucleotides). Each nucleoside is represented by a single letter, and the internucleoside bond is a phosphorothioate bond.
  • the mature target start position indicates the MRLN mRNA 5' target site of the modified oligonucleotide (the position of SEQ ID NO: 2 in the Sequence Listing corresponding to the 3' end of the modified oligonucleotide), and the mature target end position indicates the MRLN mRNA 3' target site of the modified oligonucleotide (the position of SEQ ID NO: 2 in the Sequence Listing corresponding to the 5' end of the modified oligonucleotide).
  • Example 2 In vitro MRLN expression ratio test (Lipofection method) The modified oligonucleotides synthesized in Example 1 were mixed with lipofection reagent and added to a 384-well plate at 5 ⁇ L/well. RD cells suspended in 10% serum-containing medium were seeded on top of the mixture at 5 x 10 3 cells/45 ⁇ L/well and cultured in a CO 2 incubator. After 48 hours, the modified oligonucleotide-containing medium was removed from the cells and washed with phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • a SuperPrep Cell Lysis & RT Kit for qPCR (Toyobo) was used to prepare a cell lysate containing RNA, and an RT reaction was performed from the lysate to synthesize template cDNA.
  • Real-time PCR was performed using this cDNA to quantify MRLN mRNA expression levels.
  • the MRLN expression ratio was calculated as a percentage by comparing the MRLN mRNA level in cells to which the modified oligonucleotide was added at a final concentration of 100 nmol/L with the MRLN mRNA level in cells to which the modified oligonucleotide was not added, and is shown in Tables 1-1 to 1-6.
  • Example 3 In vitro MRLN expression ratio test (Gymnosis method) RD cells suspended in 2% serum-containing medium were seeded at 5 x 10 cells/45 ⁇ L/well in a 384-well plate and cultured in a CO2 incubator. After 72 hours, the medium was replaced with the same medium, and the modified oligonucleotides synthesized in Example 1 were added at 5 ⁇ L/well, followed by culture in a CO2 incubator. After 72 hours, the modified oligonucleotide-containing medium was removed from the cells and washed with phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • a SuperPrep Cell Lysis & RT Kit for qPCR (Toyobo) was used to prepare a cell lysate containing RNA, and an RT reaction was performed from the lysate to synthesize template cDNA.
  • Real-time PCR was performed using this cDNA to quantify MRLN mRNA expression levels.
  • the MRLN expression ratio was calculated as a percentage by comparing the MRLN mRNA level in cells to which the modified oligonucleotide was added at a final concentration of 1000 nmol/L with the MRLN mRNA level in cells to which the modified oligonucleotide was not added, and is shown in Tables 1-1 to 1-6.

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Abstract

Oligonucléotide modifié présentant une activité d'inhibition de l'expression de la myoréguline et contenant 12 à 30 nucléosides liés, ou sel pharmaceutiquement acceptable de l'oligonucléotide modifié. L'oligonucléotide modifié comporte une séquence de bases complémentaire à 100 % de huit bases consécutives ou plus dans une région spécifique d'une séquence de bases représentée par SEQ ID NO : 1 dans la liste de séquences. La séquence de bases pleine longueur de l'oligonucléotide modifié présente une complémentarité d'au moins 85 % avec une partie de longueur égale de la séquence de bases représentée par SEQ ID NO : 1 dans la liste de séquences.
PCT/JP2025/012308 2024-03-27 2025-03-27 Composé et composition pharmaceutique pour réguler l'expression de la myoréguline Pending WO2025206092A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016530882A (ja) * 2013-08-09 2016-10-06 アイオーニス ファーマシューティカルズ， インコーポレーテッドＩｏｎｉｓ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ，Ｉｎｃ． 筋強直性ジストロフィープロテインキナーゼ（ｄｍｐｋ）の発現を調節するための化合物及び方法
WO2017047816A1 (fr) * 2015-09-18 2017-03-23 田辺三菱製薬株式会社 Acide nucléique réticulé guna, procédé de production de ce dernier, et composé intermédiaire
JP2018511307A (ja) * 2015-04-03 2018-04-26 アイオーニス ファーマシューティカルズ， インコーポレーテッドＩｏｎｉｓ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ，Ｉｎｃ． Ｔｍｐｒｓｓ６発現を調節するための化合物及び方法
WO2020100826A1 (fr) * 2018-11-12 2020-05-22 田辺三菱製薬株式会社 Acide nucléique artificiel réticulé alna
WO2022014703A1 (fr) * 2020-07-17 2022-01-20 田辺三菱製薬株式会社 Agent pour prévenir ou traiter une maladie musculaire

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016530882A (ja) * 2013-08-09 2016-10-06 アイオーニス ファーマシューティカルズ， インコーポレーテッドＩｏｎｉｓ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ，Ｉｎｃ． 筋強直性ジストロフィープロテインキナーゼ（ｄｍｐｋ）の発現を調節するための化合物及び方法
JP2018511307A (ja) * 2015-04-03 2018-04-26 アイオーニス ファーマシューティカルズ， インコーポレーテッドＩｏｎｉｓ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ，Ｉｎｃ． Ｔｍｐｒｓｓ６発現を調節するための化合物及び方法
WO2017047816A1 (fr) * 2015-09-18 2017-03-23 田辺三菱製薬株式会社 Acide nucléique réticulé guna, procédé de production de ce dernier, et composé intermédiaire
WO2020100826A1 (fr) * 2018-11-12 2020-05-22 田辺三菱製薬株式会社 Acide nucléique artificiel réticulé alna
WO2022014703A1 (fr) * 2020-07-17 2022-01-20 田辺三菱製薬株式会社 Agent pour prévenir ou traiter une maladie musculaire

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