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  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
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  • 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
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  • 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
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  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61P25/00—Drugs for disorders of the nervous system
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  • A61P25/00—Drugs for disorders of the nervous system
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  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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  • 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
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  • C12N2310/00—Structure or type of the nucleic acid
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  • C12N2310/11—Antisense
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  • C12N2310/00—Structure or type of the nucleic acid
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  • C12N2310/00—Structure or type of the nucleic acid
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/34—Spatial arrangement of the modifications
  • C12N2310/341—Gapmers, i.e. of the type ===---===
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/35—Nature of the modification
  • C12N2310/352—Nature of the modification linked to the nucleic acid via a carbon atom
  • C12N2310/3525—MOE, methoxyethoxy

Definitions

• the present invention provides antisense oligonucleotides targeting ATN1 mRNA or pre-mRNA, or pharmaceutically acceptable salts thereof, or hydrates thereof, the antisense oligonucleotides, pharmaceutically acceptable salts thereof, or them. It relates to a pharmaceutical composition containing a hydrate of
• DPLA Dentate red nucleus pallidus atrophy
• myoclonus involuntary movements called myoclonus, epilepsy, and progressive intellectual deterioration in children, and ataxia, ataxia, and progressive intellectual decline in adults.
• Choreoathetosis is a progressive disease that causes choreoathetosis, dementia, personality changes, etc.
• DRPLA is a disease (repeat disease) that develops due to abnormal expansion of repeat sequences present in the genome of a specific gene, resulting in functional abnormalities in RNA and proteins.
• DRPLA is known as a disease in which proteins show toxicity.
• DRPLA is divided into an adult type that develops after 20 years of age and a pediatric type that develops before 20 years of age, depending on the time of onset. .
• the present invention provides the following antisense oligonucleotides targeting ATN1 mRNA or pre-mRNA, or pharmaceutically acceptable salts or hydrates thereof, the antisense oligonucleotides, or pharmaceutically acceptable
• the present invention provides a pharmaceutical composition and the like containing a salt or a hydrate thereof.
• Oligonucleotides or pharmaceutically acceptable salts or hydrates thereof Positions 397 to 416, 759 to 778, 1127 to 1146, 1148 to 1173, 1182 to 1213, 1488 to 1525, and 1586 of the base sequence of SEQ ID NO: 471 ⁇ 1629th, 1686th ⁇ 1705th, 1768th ⁇ 1787th, 1870th ⁇ 1908th, 2097th ⁇ 2118th, 2151st ⁇ 2170th, 2239th ⁇ 2258th, 2287th ⁇ 2306th, 2317th ⁇ 2336th 2633rd to 2652nd, 2702nd to 2745th, 2832nd to 2866th, 2959th to 2978th, 3298th to 3317th, 3635th to 3654th, 3671st to 3690th, 3691st to 3710th, (7), which is complementary to a nucleic acid comprising at least 15 consecutive bases in a target region selected from the group consisting of positions 3750
• An antisense oligonucleotide or a pharmaceutically acceptable salt or hydrate thereof (10-3) 397th to 416th, 759th to 778th, 1127th to 1146th, 1148th to 1167th, 1151st to 1170th, 1154th to 1173rd, and 1182nd of the nucleotide sequence of SEQ ID NO: 471 ⁇ 1201, 1185 ⁇ 1204, 1194 ⁇ 1213, 1488 ⁇ 1507, 1491 ⁇ 1510, 1506 ⁇ 1525, 1586 ⁇ 1605, 1589 ⁇ 1608, 1595 ⁇ 1614 1598th to 1617th, 1601st to 1620th, 1604th to 1623rd, 1607th to 1626th, 1610th to 1629th, 1686th to 1705th, 1768th to 1787th, 1870th to 1889th, 1871st to 1890th, 1874th to 1893rd, 1877th to 1896th, 1886th to 1905th, 1889th to 1908th, 2097th
• the antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to (5), which is complementary to a nucleic acid comprising at least 15 consecutive bases in a target region selected from the group consisting of positions 1 to 4223, or them. hydrate.
• (12'-5) at least 15 in the target region selected from the group consisting of positions 1581 to 1624, positions 2703 to 2740, positions 3633 to 3655, and positions 4177 to 4199 of the nucleotide sequence of SEQ ID NO: 471 or a pharmaceutically acceptable salt or hydrate thereof according to (5), which is complementary to a nucleic acid comprising consecutive bases of (12'-6) Positions 1581 to 1600, 1582 to 1601, 1584 to 1603, 1585 to 1604, 1587 to 1606, 1588 to 1607, 1594 of the base sequence of SEQ ID NO: 471 1613th, 1596th to 1615th, 1597th to 1616th, 1602nd to 1621st, 1603rd to 1622nd, 1605th to 1624th, 2703rd to 2722nd, 2707th to 2726th, 2709th and up 2728, 2710-2729, 2712-2731, 2713-2732, 2715-2734, 2716-2735, 2718-2737, 2719-27
• a nucleic acid comprising consecutive bases of (12'-7) comprising at least 15 contiguous bases in the target region selected from the group consisting of positions 1587-1616, positions 2712-2735, and positions 4180-4199 of the nucleotide sequence of SEQ ID NO:471
• the antisense oligonucleotide according to (3) or (4) which comprises a nucleotide sequence having a sequence identity of 90% or more to the nucleotide sequence selected from the group consisting of 158, and 160 to 464, or a pharmaceutical thereof acceptable salts or hydrates thereof.
• (19-2) (i) a base selected from the group consisting of SEQ ID NOS: 21, 164-171, 173-177, 179-184, 187-214, 216, 219-222, 233-234, and 350-355 arrangement, (ii) 1 or a number in the base sequence selected from the group consisting of SEQ ID NOs: 21, 164-171, 173-177, 179-184, 187-214, 216, 219-222, 233-234, and 350-355 bases are added, deleted, or substituted, or and the antisense oligo according to (12'-3) or (12'-4), comprising a nucleotide sequence having a sequence identity of 90% or more to a nucleotide sequence selected from the group consisting of 350 to 355 A nucleotide or a pharmaceutically acceptable salt or hydrate thereof.
• the antisense oligonucleotide according to any one of (14) to (20), which comprises the nucleotide sequence of (i), or a pharmaceutically acceptable salt or hydrate thereof.
• (22) 468th to 487th, 474th to 493rd, 583rd to 602nd, 586th to 605th, 619th to 638th, 770th to 789th, 774th to 793th of the base sequence of SEQ ID NO: 471 775-794, 778-797, 804-823, 851-870, 1160-1179, 1162-1181, 1170-1189, 1173-1192, 1205th to 1224th, 1210th to 1229th, 1216th to 1235th, 1217th to 1236th, 1219th to 1238th, 1385th to 1404th, 1441st to 1460th, 1818th to 1837th, 1902th ⁇ 1921st, 1905th ⁇ 1924th, 1908th ⁇ 1927th, 1914
• SEQ. a base sequence selected from the group consisting of 146 and 159;
• SEQ. A nucleotide sequence in which one or several nucleotides are added, deleted, or substituted in a nucleotide sequence selected from the group consisting of 159, or
• a gapmer comprising an antisense oligonucleotide comprising a central gap region and two wing regions (5' wing region and 3' wing region) adjacent to the 5' end side and 3' end side of the gap region is the antisense oligonucleotide according to (26) or a pharmaceutically acceptable salt or hydrate thereof.
• the antisense oligonucleotide is composed of, from the 5′ side to the 3′ side, a 5′ wing region with a length of 5 nucleotides, a gap region with a length of 10 nucleotides, and a 3′ wing region with a length of 5 nucleotides. (27-1), or a pharmaceutically acceptable salt or hydrate thereof.
• one or more of the bond between the 2nd and 3rd nucleosides, the bond between the 3rd and 4th nucleosides, and the bond between the 4th and 5th nucleosides from the 5′ side of the 5′ wing region is a phosphodiester bond and/or a bond between the first and second nucleosides, a bond between the second and third nucleosides, and a bond between the third and fourth nucleosides from the 5' side of the 3' wing region
• the bond between the 2nd and 3rd nucleosides and the bond between the 4th and 5th nucleosides from the 5' side of the 5' wing region is a phosphodiester bond, and/or the 5' of the 3' wing region
• the bond between the 1st and 2nd nucleosides and the bond between the 3rd and 4th nucleosides from the side are phosphodiester bonds, and the bonds between other nucleosides are all phosphorothioate bonds, according to (29)
• the bond between the 2nd and 3rd nucleosides, the bond between the 3rd and 4th nucleosides, and the bond between the 4th and 5th nucleosides from the 5' side of the 5' wing region are phosphodiester bonds; and/or the linkages between the 1st and 2nd nucleosides, the 2nd and 3rd nucleosides, and the 3rd and 4th nucleosides from the 5' side of the 3' wing region are phosphodiester bond and all other internucleoside bonds are phosphorothioate bonds, or a pharmaceutically acceptable salt or hydrate thereof according to (29).
• the wing region comprises a 2'-OMe (2'-O- CH3 ) group and/or a 2'-O-MOE ( 2' -O- CH2CH2OCH3 ) group, ( 27-1 ) ) to (31), or a pharmaceutically acceptable salt or hydrate thereof.
• a pharmaceutical composition comprising the antisense oligonucleotide according to any one of (1) to (32) or a pharmaceutically acceptable salt or hydrate thereof.
• antisense oligonucleotides targeting ATN1 mRNA or pre-mRNA, pharmaceutically acceptable salts thereof, or hydrates thereof, and the antisense oligonucleotides, pharmaceutically acceptable salts thereof, or Compositions and the like containing these hydrates are provided.
• the present invention since it directly inhibits the mutated ATN1 that is the causative gene of DRPLA, it is possible to provide a therapeutic agent for DPLA with high therapeutic satisfaction.
• antisense oligonucleotides can be designed targeting the mutant ATN1, which is the causative gene of DRPLA. Therefore, according to a preferred embodiment of the present invention, therapeutic agents for DRPLA with less side effects can be provided. According to one aspect of the invention, it is also possible to provide a tailored treatment based on the individual patient's genetic information.
• the present invention relates to positions 1 to 84, positions 142 to 168, positions 197 to 224, positions 286 to 317, positions 324 to 370, and positions 391 to 434 of the nucleotide sequence of SEQ ID NO: 471.
• SEQ ID NO: 471 is the sequence of human atrophin-1 (ATN1) mRNA (Gen Bank: NM_001007026).
• the sequence of SEQ ID NO: 471 is a base sequence containing 4355 bases, and the region consisting of positions 1691 to 1747 of SEQ ID NO: 1 is a repeat region containing the CAG/CAA repeat sequence.
• the target sequences of the antisense oligonucleotides of the invention do not contain repeat regions containing CAG/CAA repeat sequences.
• the antisense oligonucleotides of the invention comprise or consist of at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, such as 20 contiguous bases in the target region. is complementary to a nucleic acid that is
• an antisense oligonucleotide that is "complementary" to a nucleic acid is not limited to an antisense oligonucleotide that forms Watson-Crick base pairs with the nucleic acid of interest. Also includes antisense oligonucleotides that form a (wobble base pair).
• the Watson-Crick base pair means a base pair in which hydrogen bonds are formed between adenine - thymine, adenine - uracil, and guanine - cytosine
• the fluctuation base pair means guanine - uracil, inosine - It refers to base pairs in which hydrogen bonds are formed between uracil, inosine-adenine, and inosine-cytosine.
• the "complementary base sequence" may not have 100% complementarity with the target base sequence.
• Non-complementary bases of bases, 4 bases, or 5 bases may be included, and base sequences that are 1 base, 2 bases, 3 bases, 4 bases, or 5 bases shorter than the target base sequence may be
• an antisense oligonucleotide that is "complementary" to a nucleic acid is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementarity.
• Complementarity can be readily determined by one skilled in the art, for example, by aligning two sequences, counting the number of bases that form Watson-Crick base pairs or wobble base pairs between the sequences, and determining the base pairs. It can be calculated by dividing the number of bases formed by the total number of bases in the sequence and multiplying this by 100.
• stringent conditions may be low stringent conditions, moderately stringent conditions, or highly stringent conditions.
• Low stringent conditions are, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 32°C.
• “moderately stringent conditions” are, for example, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 50% formamide at 42°C or 5 x SSC, 1% SDS, 50 mM Tris-HCl (pH 7.0). 5), 50% formamide, 42°C.
• “Highly stringent conditions” are, for example, conditions of 5 ⁇ SSC, 5 ⁇ Denhardt's solution, 0.5% SDS, 50% formamide, 50 ° C. or 0.2 ⁇ SSC, 0.1% SDS, 65 ° C. be. Under these conditions, it can be expected that base sequences with higher sequence identity can be efficiently obtained as the temperature is raised. However, multiple factors such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration can be considered as factors affecting the stringency of hybridization, and those skilled in the art can appropriately select these factors. Similar stringency can be achieved by
• a commercially available kit for hybridization for example, AlkPhos Direct Labeling and Detection System (GE Healthcare) can be used.
• the membrane was washed with a primary washing buffer containing 0.1% (w/v) SDS at 55°C. After washing, hybridization can be detected.
• DIG digoxigenin
• a commercially available reagent e.g., PCR labeling mix (Roche Diagnostics), etc.
• Hybridization can be detected using a DIG nucleic acid detection kit (Roche Diagnostics) or the like.
• the identity of the nucleotide sequence is determined using the algorithm BLAST (Basic Local Alignment Search Tool) by Carlin and Arthur (Proc. Natl. Acad. Sci. USA 872264-2268, 1990; Proc Natl Acad Sci USA 90: 5873, 1993).
• BLAST Basic Local Alignment Search Tool
• Programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (Altschul SF, et al: J Mol Biol 215: 403, 1990).
• BLAST and Gapped BLAST programs use the default parameters of each program.
• the antisense oligonucleotides of the present invention are, for example, 15 nucleotides or longer, 16 nucleotides or longer, 17 nucleotides or longer, 18 nucleotides or longer, 19 nucleotides or longer, 20 nucleotides or longer, 21 nucleotides or longer, 22 nucleotides or longer, 23 nucleotides or longer, 24 nucleotides or longer, 25 nucleotides or longer, 26 nucleotides or longer, 27 nucleotides or longer, 28 nucleotides or longer, 29 nucleotides or longer, or 30 nucleotides long.
• Antisense oligonucleotides of the present invention are, for example, It may consist of 17-25 nucleotides, 18-25 nucleotides, 19-25 nucleotides, 20-25 nucleotides, 16-24 nucleotides, 17-23 nucleotides, 18-22 nucleotides, 19-21 nucleotides, such as 20 nucleotides.
• Examples of pharmaceutically acceptable salts of the antisense oligonucleotides of the invention include alkali metal salts such as sodium, potassium and lithium salts; alkaline earth metal salts such as calcium and magnesium salts; salts, metal salts such as iron salts, zinc salts, copper salts, nickel salts, cobalt salts; ammonium salts; t-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, Organic amine salts such as tetramethylammoni
• inorganic acid salts such as , perchlorate, sulfate, phosphate; lower alkanesulfonates such as methanesulfonate, trifluoromethanesulfonate, ethanesulfonate; benzenesulfonate, p-toluene arylsulfonates such as sulfonates; organic acid salts such as acetates, malate, fumarate, succinate, citrate, tartrate, oxalate, maleate; glycinates, lysines salts, amino acid salts such as arginine salts, ornithine salts, glutamates, aspartates, and the like.
• the antisense oligonucleotides of the invention may be in their hydrate form.
• the antisense oligonucleotides of the present invention are composed of nucleotides, which may be ribonucleotides, deoxyribonucleotides, or modified nucleotides.
• Modified nucleotides refer to those in which all or part of the nucleobases, sugar moieties, and phosphate-binding moieties that make up ribonucleotides or deoxyribonucleotides have been modified.
• Nucleic acid bases include, for example, adenine, guanine, hypoxanthine, cytosine, thymine, uracil, or modified bases thereof.
• modified bases include pseudouracil, 3-methyluracil, dihydrouracil, 5-alkylcytosine (eg, 5-methylcytosine), 5-alkyluracil (eg, 5-ethyluracil), 5-halouracil (eg, , 5-bromouracil), 6-azapyrimidine, 6-alkylpyrimidine (e.g., 6-methyluracil), 2-thiouracil, 4-thiouracil, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxy methylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, 1-methyladenine, 1-methylhypoxanthine, 2,2-dimethylguanine, 3-methylcytosine, 2-methyladenine, 2-methylguanine, N
• thymine “T” and uracil “U” are interchangeable and either “T” or “U” essentially affects the activity of the antisense oligonucleotides of the invention. does not occur, the nucleotide sequences shown in this specification are indicated by the same SEQ ID NO, including the case where "T” is "U”.
• sequences containing modified bases are represented by the same SEQ ID numbers as sequences not containing modified bases. For example, “cytosine” and “methylcytosine” are interchangeable, and “cytosine” is " “Methylcytosine” is indicated by the same sequence number.
• Modifications of the sugar moiety can include, for example, modifications at the 2' position of ribose and modifications at other parts of the sugar.
• Modifications at the 2'-position of ribose include, for example, -OH group at the 2'-position of ribose with -OR, -OROR, -R, -R'OR, -SH, -SR, -NH 2 , -NHR, - NR 2 , —N 3 , —CN, —F, —Cl, —Br, —I, for example —OMe (—O—CH 3 ) or —Omethoxyethyl (—O—MOE: —O—CH 2 CH 2 OCH 3 ) can be mentioned.
• R represents alkyl, cycloalkyl, acyl or aryl.
• R' represents alkylene.
• Modifications of other parts of the sugar include, for example, those in which the 4'-position of ribose or deoxyribose is replaced with S, those in which the 2'-position and 4'-position of the sugar are crosslinked, for example, LNA (Locked Nucleic Acid ) or ENA (2′-O,4′-C-Ethylene-bridged Nucleic Acids), but are not limited thereto.
• LNA Locked Nucleic Acid
• ENA (2′-O,4′-C-Ethylene-bridged Nucleic Acids
• Modifications of the phosphate binding moiety include, for example, phosphodiester linkages, phosphorothioate linkages, phosphorodithioate linkages, alkylphosphonate linkages, phosphoramidate linkages, boranophosphate linkages (e.g., Enya et al: Bioorganic & Medicinal Chemistry , 2008, 18, 9154-9160) (see, eg, Republished Patent Publication No. 2006/129594 and Republished Patent Publication No. 2006/038608).
• alkyl is preferably linear or branched alkyl having 1 to 6 carbon atoms. Specific examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl and isohexyl. be done.
• the alkyl may be substituted, and examples of such substituents include halogen, alkoxy, cyano, and nitro, and may be substituted with 1 to 3 of these.
• cycloalkyl having 3 to 12 carbon atoms is preferred as cycloalkyl.
• Specific examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl and cyclododecyl.
• halogen includes fluorine, chlorine, bromine, and iodine.
• alkoxy refers to linear or branched alkoxy having 1 to 6 carbon atoms, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert. -butoxy, n-pentyloxy, isopentyloxy, n-hexyloxy, isohexyloxy and the like.
• Alkoxy having 1 to 3 carbon atoms is particularly preferred.
• aryl having 6 to 10 carbon atoms is preferred as aryl.
• Specific examples include phenyl, ⁇ -naphthyl, and ⁇ -naphthyl. Phenyl is particularly preferred.
• the aryl may be substituted, and examples of such substituents include alkyl, halogen, alkoxy, cyano, and nitro, which may be substituted 1-3 times.
• alkylene is preferably linear or branched alkylene having 1 to 6 carbon atoms.
• Specific examples include methylene, ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, 2-(ethyl)trimethylene, and 1-(methyl)tetramethylene.
• acyl can include linear or branched alkanoyl or aroyl.
• Alkanoyl includes, for example, formyl, acetyl, 2-methylacetyl, 2,2-dimethylacetyl, propionyl, butyryl, isobutyryl, pentanoyl, 2,2-dimethylpropionyl, hexanoyl and the like.
• Aroyl includes, for example, benzoyl, toluoyl and naphthoyl. Such aroyl may be optionally substituted at any substitutable position or may be substituted with alkyl.
• the present invention provides that the antisense oligonucleotide comprises a central gap region and two wing regions flanking the 5′ and 3′ ends of the gap region (5′ wing region and 3′ wing region, respectively). are also described as gapmers.
• a gap region is a region recognized by RNase H and is composed of deoxyribonucleotides with unmodified sugar moieties.
• the wing region contains at least one modified nucleotide, for example, all of which are composed of modified nucleotides (eg, ribonucleotides modified at the 2' position of ribose).
• the nucleosides of the 5' and 3' wing regions each have at least one, such as two or more, three or more, four or more, five or more sugar moiety modifications, such as a 2'-OMe group. and/or contain a 2'-O-MOE group, for example all of the nucleosides in the 5' and 3' wing regions may contain a 2'-OMe group and/or a 2'-O-MOE group. .
• the nucleosides of the 5' wing region and 3' wing region may contain modifications in the base portion, such as containing at least one methylcytosine.
• the length of the gap region is not limited, but may be, for example, 5-15, 8-12, 9-11 or 10 bases long.
• the length of the 5' wing region and the 3' wing region is not limited, but may be, for example, each independently 2-10, 3-8, 4-6 or 5 bases long.
• the gap region is 10 bases long and the 5' and 3' wing regions are each 5 bases long.
• gapmers of the invention comprise modifications of one or more phosphate binding moieties, such as phosphorothioate linkages, such as one or more, two or more, three or more, four or more, five internucleotide linkages. One or more, ten or more, fifteen or more, such as all may be phosphorothioate linkages. In one embodiment, the gapmers of the present invention have all phosphorothioate linkages between the nucleosides.
• the gapmers of the invention have 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more , or 10 or more (eg, 2-6), eg, in the 5′ wing region and/or the 3′ wing region, and all other internucleoside linkages are phosphorothioate linkages.
• one of the linkages between the 2nd and 3rd nucleosides, the linkages between the 3rd and 4th nucleosides, and the linkages between the 4th and 5th nucleosides from the 5' side of the 5' wing region are phosphodiester bonds, and/or the bond between the first and second nucleosides, the bond between the second and third nucleosides, and the third and fourth nucleosides from the 5′ side of the 3′ wing region
• One or more of the internucleoside linkages are phosphodiester linkages and all other internucleoside linkages are phosphorothioate linkages.
• the bond between the second and third nucleosides and the bond between the fourth and fifth nucleosides from the 5′ side of the 5′ wing region are phosphodiester bonds, and/or The bond between the 1st and 2nd nucleosides and the bond between the 3rd and 4th nucleosides from the 5' side are phosphodiester bonds, and all other bonds between nucleosides are phosphorothioate bonds.
• the linkages between the 2nd and 3rd nucleosides, the 3rd and 4th nucleosides, and the 4th and 5th nucleoside linkages from the 5' side of the 5' wing region are phosphodiester and/or the bond between the 1st and 2nd nucleosides, the 2nd and 3rd nucleosides, and the 3rd and 4th nucleosides from the 5' side of the 3' wing region
• a phosphodiester bond, and all other internucleoside bonds are phosphorothioate bonds.
• the bond between the 2nd and 3rd nucleosides from the 5′ side of the 5′ wing region and the bond between the 3rd and 4th nucleosides from the 5′ side are phosphodiester bonds, and/or the 3′ wing region
• the bonds between the 2nd and 3rd nucleosides and the bonds between the 3rd and 4th nucleosides from the 5′ side of are phosphodiester bonds, and the bonds between other nucleosides are all phosphorothioate bonds.
• the linkage between the 3rd and 4th nucleosides and the linkage between the 4th and 5th nucleosides from the 5' side of the 5' wing region is a phosphodiester linkage, and/or the 3' wing region
• the bonds between the first and second nucleosides and the bonds between the second and third nucleosides from the 5' side of are phosphodiester bonds, and the bonds between other nucleosides are all phosphorothioate bonds.
• the bond between the 5' second and third nucleosides of the 5' wing region is a phosphodiester bond and/or the 5' third and fourth nucleosides of the 3' wing region
• the internucleoside linkages are phosphodiester linkages, and all other internucleoside linkages are phosphorothioate linkages.
• the bond between the 3rd and 4th nucleosides from the 5' side of the 5' wing region is a phosphodiester bond and/or the 2nd and 3rd nucleosides from the 5' side of the 3' wing region
• the internucleoside linkages are phosphodiester linkages, and all other internucleoside linkages are phosphorothioate linkages.
• the bond between the 4th and 5th nucleosides from the 5' side of the 5' wing region is a phosphodiester bond and/or the 1st and 2nd nucleosides from the 5' side of the 3' wing region
• the internucleoside linkages are phosphodiester linkages, and all other internucleoside linkages are phosphorothioate linkages.
• the gapmers of the invention contain no modification of the phosphate linking moiety and all of the internucleotide linkages may be phosphodiester linkages.
• the antisense oligonucleotides of the present invention are positions 1 to 84, 142 to 168, 197 to 224, 286 to 317, and 324 to 370 of the nucleotide sequence of SEQ ID NO: 471.
• the antisense oligonucleotides of the present invention are 1 to 84, 142 to 168, 391 to 434, 697 to 780, and 962 to 980 of the nucleotide sequence of SEQ ID NO: 471.
• the antisense oligonucleotides of the present invention are at positions 1 to 69, 331 to 363, 398 to 427, 697 to 773, and 867 to 890 of the nucleotide sequence of SEQ ID NO: 471.
• the antisense oligonucleotides of the present invention are positions 1 to 69, 398 to 427, 697 to 773, 1132 to 1167, and 1272 to 1320 of the nucleotide sequence of SEQ ID NO: 471.
• nucleic acid comprising at least 15 contiguous bases in a target region selected from the group consisting of positions 3898, 3938-4032, and 4177-4225.
• the antisense oligonucleotides of the present invention are at positions 14 to 33, 34 to 53, 54 to 73, 148 to 167, and 397 to 432 of the nucleotide sequence of SEQ ID NO: 471.
• the antisense oligonucleotides of the present invention are positions 14 to 33, 34 to 53, 54 to 73, 148 to 167, and 397 to 416 of the nucleotide sequence of SEQ ID NO: 471.
• antisense oligonucleotides that are complementary to a nucleic acid containing at least 15 contiguous bases in the target sequence include: (i) SEQ ID NOs:1-6, 8, 9, 11, 14-24, 26, 29, 31-60, 66-67, 73-75, 79-82, 86-89, 92-111, 115-136 , 138-145, 147-158, and a base sequence selected from the group consisting of 160-464, (ii) SEQ.
• the antisense oligonucleotides of the present invention are at positions 14 to 33, 34 to 53, 398 to 427, 723 to 742, and 747 to 766 of the nucleotide sequence of SEQ ID NO: 471.
• the antisense oligonucleotides of the present invention are at positions 14-33, 34-53, 398-417, 399-418, 400-419 of the nucleotide sequence of SEQ ID NO: 471. , 401st to 420th, 402nd to 421st, 403rd to 422nd, 404th to 423rd, 405th to 424th, 406th to 425th, 407th to 426th, 408th to 427th, 723 1st to 742nd, 747th to 766th, 1148th to 1167th, 1289th to 1308th, 1484th to 1503rd, 1488th to 1507th, 1491st to 1510th, 1493th to 1512th, 1506th and up 1525, 1517-1536, 1531-1550, 1545-1564, 1578-1597, 1579-1598, 1580-1599, 1581-1600, 1582-1601 , 1583rd to 1602
• the antisense oligonucleotides of the present invention are at positions 14 to 33, 34 to 53, 54 to 73, 148 to 167, and 397 to 432 of the nucleotide sequence of SEQ ID NO: 471.
• the antisense oligonucleotides of the present invention are positions 14 to 33, 34 to 53, 54 to 73, 148 to 167, and 397 to 416 of the nucleotide sequence of SEQ ID NO: 471.
• the antisense oligonucleotides of the present invention are at positions 14-33, 34-53, 747-766, 1148-1167, 1289-1308 of the nucleotide sequence of SEQ ID NO: 471. , 1484-1564, 1578-1623, 1686-1705, 1748-1767, 1768-1787, 1794-1813, 1877-1905, 2151-2170, 2206 2225th, 2239th to 2272nd, 2287th to 2336th, 2337th to 2373rd, 2387th to 2406th, 2613th to 2632nd, 2633rd to 2668th, 2672nd to 2691st, 2699th and up 2748th, 2775th to 2809th, 2826th to 2860th, 3032nd to 3051st, 3052nd to 3071st, 3168th to 3187th, 3232nd to 3270th, 3408th to 3427th, 3590th to 3690th , 3691-3728, 3739-375
• the antisense oligonucleotides of the present invention are at positions 14-33, 34-53, 747-766, 1148-1167, 1289-1308 of the nucleotide sequence of SEQ ID NO: 471. , 1484-1503, 1488-1507, 1491-1510, 1493-1512, 1506-1525, 1517-1536, 1531-1550, 1545-1564, 1578 1597th to 1597th, 1586th to 1605th, 1589th to 1608th, 1592nd to 1611th, 1595th to 1614th, 1598th to 1617th, 1601st to 1620th, 1604th to 1623rd, 1686th and up 1705, 1748-1767, 1768-1787, 1794-1813, 1877-1896, 1880-1899, 1883-1902, 1886-1905, 2151-2170 , 2206-2225, 2239-2258, 2253-2272, 2287-2306, 2301-2320, 2317-2336, 2337-235
• the antisense oligonucleotides of the present invention are positive control antisense oligonucleotides for ATN-1 gene expression levels when administered to A204 cells, as measured according to the methods described in the Examples herein.
• the ratio (mean value) to the ATN-1 gene expression level when the nucleotides OMe-6 or NRH-71 are administered is selected from those that are 1.0 or less in any of the Examples.
• Examples of such antisense oligonucleotides include positions 397 to 416, positions 759 to 778, positions 1127 to 1146, positions 1148 to 1173, positions 1182 to 1213, and positions 1488 of the base sequence of SEQ ID NO: 471.
• the antisense oligonucleotides of the present invention are positive control antisense oligonucleotides for ATN-1 gene expression levels when administered to A204 cells, as measured according to the methods described in the Examples herein.
• the ratio (mean value) to the ATN-1 gene expression level when the nucleotides OMe-6 or NRH-71 are administered is selected from those that are 0.75 or less in any of the examples.
• Examples of such antisense oligonucleotides include positions 397 to 416, 1194 to 1213, 1506 to 1525, 1586 to 1626, 1870 to 1896 and 2097 of the base sequence of SEQ ID NO: 471.
• antisense oligonucleotides that are complementary to nucleic acids containing at least 15 contiguous bases in the target sequence include: a base sequence selected from the group consisting of 107, 116, 122, 126, 128, 130-134, 143, 145, 148, 151, 153, 154, 157, and 160-163; (ii) SEQ.
• antisense oligonucleotides containing or consisting of a base sequence having a sequence identity of 90% or more, such as the base sequence of (i) are exemplified.
• the antisense oligonucleotides of the present invention are positive control antisense oligonucleotides for ATN-1 gene expression levels when administered to A204 cells, as measured according to the methods described in the Examples herein. It is selected so that the ratio (mean value) to the ATN-1 gene expression level when OMe-6 or NRH-71, which are nucleotides, is administered is 0.5 or less in any of the Examples.
• Examples of such antisense oligonucleotides include positions 1598 to 1626, positions 1870 to 1890, positions 2097 to 2116, positions 2711 to 2736, positions 2841 to 2860, and 4170 of the base sequence of SEQ ID NO: 471.
• Antisense oligonucleotides consisting of 15-22 nucleotides complementary to a nucleic acid containing at least 15 contiguous bases in a target region selected from the group consisting of positions 1-4198 are included.
• the antisense oligonucleotides of the present invention are at positions 400-425, 1581-1624, 1878-1898, 2094-2113, 2629-2656 of the nucleotide sequence of SEQ ID NO: 471. , 2700-2741, 2839-2863, 2962-2981, 3302-3325, 3631-3658, 3687-3714, 3745-3764, 3980-4007, 4177 It is complementary to a nucleic acid containing at least 15 contiguous bases in a target region selected from the group consisting of positions 4199-4199 and 4204-4223.
• the antisense oligonucleotides of the present invention are 400-419, 403-422, 406-425, 1581-1600, 1582-1601 of the nucleotide sequence of SEQ ID NO: 471. , 1583-1602, 1584-1603, 1585-1604, 1587-1606, 1588-1607, 1590-1609, 1591-1610, 1592-1611, 1593 Rank ⁇ 1612th, Rank 1594th ⁇ 1613th, Rank 1596th ⁇ 1615th, Rank 1597th ⁇ 1616th, Rank 1599th ⁇ 1618th, Rank 1600th ⁇ 1619th, Rank 1602nd ⁇ 1621st, Rank 1603rd ⁇ 1622nd, Rank 1605 ⁇ 1624, 1878-1897, 1879-1898, 2094-2113, 2629-2648, 2637-2656, 2700-2719, 2701-2720, 2703-2722 , 2704-2723, 2706-2725,
• the antisense oligonucleotides of the present invention are positive control antisense oligonucleotides for ATN-1 gene expression levels when administered to A204 cells, as measured according to the methods described in the Examples herein.
• the ratio (mean value) to the ATN-1 gene expression level when the nucleotides OMe-6 or NRH-71 are administered is selected from those that are 1.0 or less in any of the examples.
• Examples of such antisense oligonucleotides include positions 400 to 425, positions 1581 to 1624, positions 1878 to 1898, positions 2637 to 2656, positions 2700 to 2741, and positions 2839 of the base sequence of SEQ ID NO: 471.
• Antisense oligonucleotides of 15-22 nucleotides that are complementary to nucleic acids containing at least 15 contiguous bases in the target region of choice are included.
• the antisense oligonucleotides of the present invention are positive control antisense oligonucleotides for ATN-1 gene expression levels when administered to A204 cells, as measured according to the methods described in the Examples herein. It is selected so that the ratio (mean value) to the ATN-1 gene expression level when OMe-6 or NRH-71, which are nucleotides, is administered is 0.75 or less in any of Examples.
• antisense oligonucleotides include targets selected from the group consisting of positions 1581 to 1624, positions 2703 to 2740, positions 3633 to 3655, and positions 4177 to 4199 of the nucleotide sequence of SEQ ID NO: 471.
• Antisense oligonucleotides of 15-22 nucleotides complementary to nucleic acids containing at least 15 contiguous bases in the region are included.
• An antisense oligonucleotide comprising or consisting of a nucleotide sequence having 90% or more sequence identity with the selected nucleotide sequence, such as the nucleotide sequence of
• the antisense oligonucleotides of the present invention are positive control antisense oligonucleotides for ATN-1 gene expression levels when administered to A204 cells, as measured according to the methods described in the Examples herein. It is selected so that the ratio (mean value) to the ATN-1 gene expression level when OMe-6 or NRH-71, which are nucleotides, is administered is 0.5 or less in any of the Examples.
• Examples of such antisense oligonucleotides include at least 15 antisense oligonucleotides in the target region selected from the group consisting of positions 1587 to 1616, positions 2712 to 2735, and positions 4180 to 4199 of the nucleotide sequence of SEQ ID NO: 471.
• Antisense oligonucleotides consisting of 15-22 nucleotides that are complementary to nucleic acids containing consecutive bases are included. Also, in the target region selected from the group consisting of positions 1587 to 1606, positions 1597 to 1616, positions 2712 to 2731, positions 2716 to 2735, and positions 4180 to 4199 of the nucleotide sequence of SEQ ID NO: 471 Antisense oligonucleotides consisting of 15-22 nucleotides that are complementary to nucleic acids containing at least 15 contiguous bases are included.
• antisense oligonucleotides complementary to a nucleic acid comprising at least 15 contiguous bases in the target sequence include: (i) bases selected from the group consisting of SEQ ID NOs: 170, 177, 196, 199, and 221; arrangement, (ii) a nucleotide sequence selected from the group consisting of SEQ ID NOS: 170, 177, 196, 199, and 221 with one or several bases added, deleted, or substituted, or (iii) SEQ ID NO: 170, 177, 196, 199, and 221.
• the present invention relates to positions 468 to 487, positions 474 to 493, positions 583 to 602, positions 586 to 605, positions 619 to 638, and positions 770 to 789 of the nucleotide sequence of SEQ ID NO: 471.
• the antisense oligonucleotides of the invention inhibit the function of the target region.
• inhibiting the function of the target region means that the genomic RNA containing the target region bound to the target region and formed a double strand with the antisense oligonucleotide is cleaved by RNaseH. inhibiting replication of the genomic RNA containing the target region, inhibiting translation of the target region if it is translated, and inhibiting transcription of the genomic RNA containing the target region.
• RNA-dependent RNA polymerase is synthesized by RNA-dependent RNA polymerase using a portion of (-) strand RNA as a template that is synthesized by RNA-dependent RNA polymerase using (+) strand genomic RNA as a template.
• the antisense oligonucleotides of the present invention can be easily synthesized using various automated synthesizers (eg, AKTA oligopilot plus 10/100 (GE Healthcare)), or can be synthesized by third parties (eg, Promega). or Takara) or the like.
• automated synthesizers eg, AKTA oligopilot plus 10/100 (GE Healthcare)
• third parties eg, Promega). or Takara
• the present invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising an antisense oligonucleotide or a pharmaceutically acceptable salt or hydrate thereof.
• the pharmaceutical compositions of the invention may contain carriers that facilitate delivery of the antisense nucleotides.
• Such carriers are not particularly limited as long as they are pharmaceutically acceptable, and examples thereof include cationic carriers such as cationic liposomes and cationic polymers, and carriers utilizing virus envelopes. .
• Cationic liposomes include, for example, liposomes formed from 2-O-(2-diethylaminoethyl)carbamoyl-1,3-O-dioleoylglycerol and phospholipids as essential constituents (hereinafter, “liposome A” ), Oligofectamine (registered trademark) (manufactured by Invitrogen), Lipofectin (registered trademark) (manufactured by Invitrogen), Lipofectamine (registered trademark) (manufactured by Invitrogen), Lipofectamine 2000 (registered trademark) (manufactured by Invitrogen ), DMRIE-C (registered trademark) (manufactured by Invitrogen), GeneSilencer (registered trademark) (manufactured by Gene Therapy Systems), TransMessenger (registered trademark) (manufactured by QIAGEN), TransIT TKO (registered trademark) (manufactured by Mir
• cationic polymers examples include JetSI (registered trademark) (manufactured by Qbiogene) and Jet-PEI (registered trademark) (polyethyleneimine, manufactured by Qbiogene).
• Carriers using viral envelopes include, for example, GenomeOne (registered trademark) (HVJ-E liposome, manufactured by Ishihara Sangyo Co., Ltd.).
• GenomeOne registered trademark
• HVJ-E liposome manufactured by Ishihara Sangyo Co., Ltd.
• the antisense oligonucleotides of the present invention may be conjugated with lipids and the like in pharmaceutical compositions in order to facilitate delivery of the antisense oligonucleotides.
• it can be conjugated with cholesterol as described in Bijsterbosch, M.K. et al. (2000) Nucleic Acid Res., 28, 2717-2725.
• the pharmaceutical composition of the present invention may contain a pharmaceutically acceptable additive in addition to the antisense oligonucleotide or a pharmaceutically acceptable salt or hydrate thereof and optionally the above carrier.
• a pharmaceutically acceptable additive include, for example, emulsifying aids (e.g. C6-22 fatty acids and pharmaceutically acceptable salts thereof, albumin, dextran), stabilizers (e.g. cholesterol, phosphatidic acid, mannitol, sorbitol).
• tonicity agents e.g., sodium chloride, glucose, maltose, lactose, sucrose, trehalose
• pH adjusters e.g., hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, sodium hydroxide, potassium hydroxide, triethanolamine
• the content of the additive in the composition of the present invention is suitably 90% by weight or less, preferably 70% by weight or less, more preferably 50% by weight or less.
• the method for preparing the pharmaceutical composition of the present invention is not limited, it can be prepared, for example, by adding the antisense oligonucleotide of the present invention to a carrier dispersion and stirring appropriately. Moreover, the additive can be added in an appropriate step before or after the addition of the antisense oligonucleotide of the present invention.
• the aqueous solvent that can be used when adding the antisense oligonucleotide of the present invention is not particularly limited as long as it is pharmaceutically acceptable. For example, water for injection, distilled water for injection, electrolytes such as physiological saline liquid, glucose liquid, sugar liquid such as maltose liquid. Moreover, conditions such as pH and temperature in such a case can be appropriately selected by those skilled in the art.
• the pharmaceutical composition of the present invention can be, for example, a liquid formulation or a lyophilized formulation thereof.
• the freeze-dried formulation can be prepared by freeze-drying the composition of the present invention in the form of a liquid formulation by a conventional method. For example, after appropriately sterilizing the composition of the present invention in the form of a liquid formulation, a predetermined amount is dispensed into vials and pre-frozen under conditions within the range of about -40°C to -20°C. for about 2 hours, primary drying under reduced pressure within the range of about 0 ° C. to 10 ° C., then secondary drying under reduced pressure within the range of about 15 ° C. to 25 ° C. It can be freeze-dried. . Then, in general, the inside of the vial is replaced with nitrogen gas and capped to obtain a freeze-dried preparation of the composition of the present invention.
• the freeze-dried formulation of the pharmaceutical composition of the present invention can generally be redissolved by adding any suitable solution (redissolving solution) before use.
• suitable solution redissolving solution
• suitable solution include water for injection, physiological saline, and other general infusion solutions.
• the volume of this re-dissolution solution is not particularly limited depending on the application and the like, but it is suitable to be 0.5 to 2 times the volume of the liquid before freeze-drying or 500 mL or less.
• the dosage for administering the pharmaceutical composition of the present invention is determined by considering the type of the antisense oligonucleotide of the present invention contained, the dosage form, the patient's condition such as age and weight, the route of administration, and the nature and degree of the disease.
• the amount of the antisense oligonucleotide of the present invention is 0.01 mg to 20 mg per 1 kg body weight, preferably 0.03 mg to 10 mg per 1 kg body weight, More preferably 0.05 mg to 4 mg per kg of body weight, more preferably 0.1 mg to 2 mg per kg of body weight.
• Dosing frequency can be once every 1-3 days, once a week, or once every 2-3 weeks. This number may vary depending on the type of target disease, dosage form, and target molecule. Accordingly, a lower dose or administration frequency may be sufficient in some cases, and conversely, a higher dose or administration frequency may be required.
• the dosage form of the pharmaceutical composition of the present invention is not particularly limited as long as it is a pharmaceutically acceptable dosage form, and can be selected according to the treatment method. Examples include administration, subcutaneous administration, oral administration, intra-tissue administration, transdermal administration, pulmonary administration, nasal administration, administration to the central nervous system, and the like. Examples of administration to the central nervous system include intrathecal, intracranial, eg, intraventricular or lateral ventricular, intraparenchymal, and subparietal (pia mater) administration. Moreover, the dosage form that the composition of the present invention can take is not particularly limited, and examples thereof include various injections, oral preparations, infusions, inhalants, ointments, lotions, poultices, and the like. .
• Subjects to which the antisense oligonucleotides or pharmaceutical compositions of the present invention are administered are, for example, mammals such as primates such as humans, laboratory animals such as rats, mice, and rats, and livestock animals such as pigs, cows, horses, and sheep. etc., preferably human.
• the present invention provides antisense oligonucleotides of the present invention, pharmaceutically acceptable salts or hydrates thereof, or pharmaceutical compositions, comprising the step of administering to a subject.
• the present invention relates to a method for treating and/or preventing nucleopallidal Louis body atrophy (DRPLA).
• the pharmaceutical composition in this embodiment, and the dose and route of administration of the pharmaceutical composition are as described herein.
• DRPLA dentate nucleopallidal Louis body atrophy
• DRPLA dentate nucleopallidal louis body atrophy or symptoms thereof (e.g., ataxia, myoclonus, epilepsy, and one or more of progressive intellectual regression, ataxia, choreoathetosis, dementia, or personality changes in adults), alleviation, amelioration, and remission.
• prevention of DRPLA includes reducing the risk of developing DRPLA or symptoms thereof.
• a “gapmer” is defined as an oligomeric compound, generally an oligonucleotide, having a central region of 2'-deoxyoligonucleotide flanked by two adjacent non-deoxyoligonucleotide segments. The central region is called the “gap” and the adjacent segments are called the "wings”.
• the gapmers used in this example have a 10 nucleotide gap flanked by two adjacent wings of 5 nucleotides. This is called a 5-10-5 gapmer.
• Example 1 Synthesis of gapmer (oligonucleotide)> Gapmers (oligonucleotides) used in this example are generally described in W.W. Brad Wan et al., Nucleic Acid Research, Vol. 42, No. 22 13456 (2014) and the like.
• Base is 5-methylcytosine (C), thymine (T), adenine (A), or guanine (G) and Me is methyl.
• a 20-mer subject gapmer (oligonucleotide) having a 2′-OMe group represented by formula (a) or a 2′-O-MOE group represented by formula (b) in the nucleoside of the wing is prepared by an automatic nucleic acid synthesizer. It was synthesized on a 1 ⁇ mol scale using NTS M-8-MX DNA/RNA (Nippon Techno Service Co., Ltd.). Standard phosphoramidite protocol for chain length extension (solid support: Glen UnySupport, DDTT ([(N,N-Dimethylaminomethylidene)amino]-3H-1,2,4-dithiazoline-3-thione) etc.
• the product was treated with deblocking solution-1 obtained from Fuji Film Wako Co., Ltd. to remove the DMTr group, and then treated with ammonia to cut out the desired product from the solid phase carrier.
• the target product was obtained by purifying the crude product obtained by distilling off the solvent by reverse phase HPLC.
• Gapmers (oligonucleotides) of interest were designed as antisense oligonucleotides (ASO) targeting human atrophin-1 (ATN1) (Gen Bank: NM_001007026, SEQ ID NO: 471).
• ASO antisense oligonucleotides
• ATN1 human atrophin-1
• Tables 1, 2, 3 and 4 show the sequences of the oligonucleotides in terms of DNA bases.
• the Cs in the gap and wing portions are actually methyl Cs
• the Ts in the wing portions are actually Ts.
• the C in the gap portion and the wing portion is actually a methyl C
• the T in the wing portion is actually a T
• the underlined bases in the 3′ wing portion are the bases that differ from SEQ ID NO:196.
• Test Example 1 Measurement of ATN-1 gene knockdown activity (inhibitory activity) of gapmer using A204 human rhabdomyosarcoma cell line 1 or Table 2 at a concentration of 1 ⁇ M or 2 ⁇ M of the test subject gapmer or positive control gapmer in Table 2 using SF Cell Line 4D-Nucleofector (TM) X KitS, according to the protocol attached to the kit, 4D-Nucleofector ( TM). The program used was DS-130. After transduction, cells were cultured overnight at 37° C.
• TM SF Cell Line 4D-Nucleofector
• T thymine
• NRH-71 the gap and wing Cs (cytosines) are actually methyl Cs, and the wing Ts are actually Ts.
• RNA was extracted according to the protocol attached to NucleoSpin(R) RNA (Takara Bio). The concentration of the extracted total RNA was measured using NanoDrop (Thermo Fisher). 200 ng of the extracted total RNA was subjected to reverse transcription (RT) reaction using a High Capacity cDNA Reverse Transcription Kit (applied biosystems) and random primers attached to the kit. Specifically, a reaction solution was prepared according to the protocol attached to the kit. The thermal cycler used was TaKaRa PCR Thermal Cycler Dice Touch (Takara Bio). The RT reaction program used is as follows.
• the RT reaction solution was qPCR was performed on the template to measure the RNA expression level of ATN1 endogenously expressed in A204 cells.
• ATN1 endogenously expressed in A204 cells.
• two sequences within ATN1 (exon 4 to exon 5, exon 6 to exon 7) was used as a template.
• a reaction solution was prepared according to the protocol attached to the kit.
• QuantStudio6Flex Real-Time PCR System (applied biosystems) was used. The qPCR program used is as follows.
• the ratio of inhibition to the ATN1 RNA expression level in cells to which only the introduction operation with 4D-Nucleofector (TM) was performed without the addition of gapmer was calculated from the ratio of the ATN1 RNA expression level at the time of gapmer introduction of the test substance. was analyzed for inhibitory activity of gapmers of The results are shown in Tables 5-9.

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