Nephrotoxicity reducing agent

Abstract

To provide: a nephrotoxicity reducing agent for a pharmaceutical composition containing an antisense oligomer; and a method for reducing nephrotoxicity of the pharmaceutical composition.SOLUTION: In an embodiment, the present invention relates to a nephrotoxicity reducing agent for a pharmaceutical composition including an antisense oligomer. The nephrotoxicity reducing agent includes a sugar alcohol and is used in an amount such that the concentration of the sugar alcohol in the pharmaceutical composition is 1-400 mg / mL.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an agent for reducing the nephrotoxicity of a pharmaceutical composition containing an antisense oligomer, a method for reducing the nephrotoxicity of the pharmaceutical composition, a pharmaceutical composition containing an antisense oligomer and having reduced nephrotoxicity, and the like. [Background technology]

[0002] Antisense oligomers are nucleic acids that hybridize sequence-specifically to target mRNA and pre-mRNA. Antisense oligomers exert their effects through degradation of mRNA and pre-mRNA, exon skipping, exon inclusion, translation inhibition, etc., and are used as therapeutic agents for several diseases. For example, JP 2015-91229 A (Patent Document 1) discloses antisense nucleic acids that can treat Fukuyama muscular dystrophy and the like by normalizing the abnormal splicing of the fukutin gene harboring an insertion mutation of an SVA retrotransposon.

[0003] However, it has been known that antisense oligomers accumulate in the kidney (Non-Patent Document 1, Figure 2), that administration of morpholino oligomers results in nephrotoxicity (Non-Patent Document 2, Figure 3, Table 5), and that administration of morpholino oligomers results in the formation of basophilic substances in renal tubules (Non-Patent Document 3, Figure 1). A method for avoiding this nephrotoxicity and the appearance of basophilic substances in renal tubules was unknown. Meanwhile, it has been known that for oligonucleotides that have higher toxicity in aggregate form than in monomer form, multimer formation in therapeutic oligonucleotide formulations can be suppressed by heating with a chemical species such as mannitol that disrupts aggregation (Patent Document 2), and that sugars and sugar alcohols in liquid formulations suppress oligonucleotide aggregation (Patent Document 3). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special opening 2015-91229 [Patent Document 2] International Publication No. 2002 / 036767 [Patent Document 3] International Publication No. 2010 / 009038

Non-licensed literature

[0005]

Non-patent document 1

Non-patent document 2

Non-patent document 3

[0006] It has been known that pharmaceutical compositions containing antisense oligomers have the problem of nephrotoxicity. In this situation, it is desired to provide an improved pharmaceutical composition containing an antisense oligomer. [Means for solving the problem]

[0007] That is, the present invention provides agents for reducing the nephrotoxicity of pharmaceutical compositions containing the antisense oligomers described below, methods for reducing the nephrotoxicity of the pharmaceutical compositions, pharmaceutical compositions containing antisense oligomers with reduced nephrotoxicity, and the like. (1) A nephrotoxicity-reducing agent for a pharmaceutical composition containing an antisense oligomer, which contains a sugar alcohol and is used in an amount such that the concentration of the sugar alcohol in the pharmaceutical composition is 1 mg / mL to 400 mg / mL. (2) The nephrotoxicity-reducing agent according to (1), which is used in an amount such that the sugar alcohol concentration in the pharmaceutical composition becomes 2.5 mg / mL to 200 mg / mL. (3) The nephrotoxicity-reducing agent according to (1) or (2), wherein the concentration of the antisense oligomer in the pharmaceutical composition is 0.5 mg / mL to 200 mg / mL. (4-1) A nephrotoxicity-reducing agent for a pharmaceutical composition containing an antisense oligomer, which contains a sugar alcohol and is used in an amount such that the weight ratio of the sugar alcohol to the antisense oligomer is 0.05 to 30. (4-2) A nephrotoxicity-reducing agent for a pharmaceutical composition containing an antisense oligomer, which contains a sugar alcohol and is used in an amount such that the weight ratio of the sugar alcohol to the antisense oligomer is 0.05 to 90. (4-3) The nephrotoxicity-reducing agent according to (4-1) or (4-2), wherein the pharmaceutical composition and the nephrotoxicity-reducing agent are administered separately. (5) The nephrotoxicity-reducing agent according to any one of (4-1) to (4-3), which is used in an amount such that the weight ratio of the sugar alcohol to the antisense oligomer is 0.1 to 13.3. (6) The nephrotoxicity-reducing agent according to any one of (1) to (5), wherein the sugar alcohol is selected from the group consisting of mannitol, sorbitol, and a combination thereof. (7) The nephrotoxicity-reducing agent according to any one of (1) to (6), wherein the antisense oligomer is a morpholino oligomer. (8) The nephrotoxicity-reducing agent according to any one of (1) to (7), wherein the antisense oligomer is a phosphorodiamidate morpholino oligomer. (9) The 5' end of the antisense oligomer has the following chemical formulas (1) to (2):

[0008] [ka]

[0009] The nephrotoxicity-reducing agent according to any one of (1) to (8), wherein the nephrotoxicity-reducing agent is any one of the groups shown below. (10) The agent for reducing nephrotoxicity according to any one of (1) to (9), wherein the antisense oligomer contains four consecutive purine bases in its base sequence. (11) The nephrotoxicity-reducing agent according to (10), wherein at least two of the four consecutive purine bases are guanine. (12) The agent for reducing nephrotoxicity according to any one of (1) to (11), wherein the antisense oligomer comprises a base sequence selected from the group consisting of SEQ ID NOs: 1 to 10. (13) The nephrotoxicity-reducing agent according to any one of (1) to (12), wherein the antisense oligomer has a target sequence ranging from positions 115937 to 115981 of the base sequence shown in SEQ ID NO: 11. (14) A nephrotoxicity-reducing agent described in any of (1) to (12), wherein the antisense oligomer does not target the range of positions 115937 to 115981 of the base sequence shown in SEQ ID NO: 11. (15-1) A method for reducing the nephrotoxicity of a pharmaceutical composition containing an antisense oligomer, the method comprising adding a sugar alcohol in an amount such that the concentration of the sugar alcohol in the pharmaceutical composition becomes 1 mg / mL to 400 mg / mL. (15-2) The method according to (15-1), wherein the sugar alcohol is added in an amount such that the concentration of the sugar alcohol in the pharmaceutical composition becomes 2.5 mg / mL to 200 mg / mL. (15-3) The method according to (15-1) or (15-2), wherein the concentration of the antisense oligomer in the pharmaceutical composition is 0.5 mg / mL to 200 mg / mL. (16-1) A method for reducing the nephrotoxicity of an antisense oligomer in a subject to which the antisense oligomer has been administered, the method comprising administering a sugar alcohol to the subject, wherein the sugar alcohol is administered in an amount such that the weight ratio of the sugar alcohol to the antisense oligomer is 0.05 to 30. (16-2) A method for reducing the nephrotoxicity of an antisense oligomer in a subject to which the antisense oligomer has been administered, the method comprising administering a sugar alcohol to the subject, wherein the sugar alcohol is administered in an amount such that the weight ratio of the sugar alcohol to the antisense oligomer is 0.05 to 90. (16-3) The method according to (16-1) or (16-2), wherein the antisense oligomer and the sugar alcohol are administered separately. (16-4) The method according to any one of (16-1) to (16-3), wherein the sugar alcohol is used in an amount such that the weight ratio of the sugar alcohol to the antisense oligomer is 1 is 0.1 to 13.3. (17-1) A pharmaceutical composition with reduced nephrotoxicity, comprising an antisense oligomer, the pharmaceutical composition comprising a nephrotoxicity-reducing agent containing a sugar alcohol at a concentration of 1 mg / mL to 400 mg / mL. (17-2) The pharmaceutical composition according to (17-1), comprising a nephrotoxicity-reducing agent containing a sugar alcohol at a concentration of 2.5 mg / mL to 200 mg / mL. (17-3) The pharmaceutical composition according to (17-1) or (17-2), wherein the concentration of the antisense oligomer in the pharmaceutical composition is 0.5 mg / mL to 200 mg / mL. (18) The method or pharmaceutical composition according to any one of (15-1) to (17-3), wherein the sugar alcohol is selected from the group consisting of mannitol, sorbitol, and a combination thereof. (19) The method or pharmaceutical composition according to any one of (15-1) to (18), wherein the antisense oligomer is a morpholino oligomer. (20) The method or pharmaceutical composition according to any one of (15-1) to (19), wherein the antisense oligomer is a phosphorodiamidate morpholino oligomer. (21) The 5' end of the antisense oligomer has the following chemical formulas (1) to (2):

[0010] [ka]

[0011] The method or pharmaceutical composition according to any one of (15-1) to (20), wherein the compound is any one of the groups represented by the formula (15-1) to (20). (22) The method or pharmaceutical composition according to any one of (15-1) to (21), wherein the antisense oligomer contains four consecutive purine bases in its base sequence. (23) The method or pharmaceutical composition according to (22), wherein at least two of the four consecutive purine bases are guanine. (24) The method or pharmaceutical composition according to any one of (15-1) to (23), wherein the antisense oligomer comprises a base sequence selected from the group consisting of SEQ ID NOs: 1 to 10. (25) The nephrotoxicity-reducing agent according to any one of (15-1) to (24), wherein the antisense oligomer has a target sequence ranging from positions 115937 to 115981 of the base sequence shown in SEQ ID NO: 11. (26) A nephrotoxicity-reducing agent described in any of (15-1) to (24), wherein the antisense oligomer does not target the range of positions 115937 to 115981 of the base sequence shown in SEQ ID NO: 11. [Effects of the Invention]

[0012] The present invention provides an agent for reducing the nephrotoxicity of a pharmaceutical composition containing an antisense oligomer, and a method for reducing the nephrotoxicity of the pharmaceutical composition. [Brief explanation of the drawings]

[0013] [Figure 1] The absorbance measured at 620 nm under the conditions shown in Table 11 is shown. [Figure 2] The absorbance measured at 620 nm under the conditions shown in Table 12 is shown. [Figure 3] The absorbance measured at 620 nm under the conditions shown in Table 13 is shown. [Figure 4] The absorbance measured at 620 nm under the conditions shown in Table 14 is shown. [Figure 5] The absorbance measured at 620 nm under the conditions shown in Table 15 is shown. [Figure 6] The absorbance measured at 620 nm under the conditions shown in Table 16 is shown. DETAILED DESCRIPTION OF THE INVENTION

[0014] In one embodiment, the present invention relates to an agent for reducing the nephrotoxicity of a pharmaceutical composition containing an antisense oligomer. As used herein, the term "nephrotoxicity-reducing agent" refers to an agent for reducing the nephrotoxicity that may be present in a pharmaceutical composition containing an antisense oligomer or the phenomena that cause such toxicity (e.g., accumulation of basophilic substances). High concentrations of administered antisense oligomers are distributed and accumulated in the kidney during excretion, which can lead to the development of basophilic substances in the kidney. Nephrotoxicity refers to tissue damage or impaired renal function caused by extensive accumulation of antisense oligomers in the kidney. Renal tissue damage is known to result in, for example, dilation and necrosis of renal tubules, and impaired renal function is widely known to result in, for example, elevated blood urea nitrogen (BUN) and blood creatinine (Cre) levels. Reducing the nephrotoxicity of antisense oligomers can provide pharmaceutical compositions containing high doses of antisense oligomers and methods for treating diseases using such pharmaceutical compositions.

[0015] The presence or absence of a nephrotoxicity-reducing effect can be determined, for example, by measuring blood urea nitrogen (BUN) and blood creatinine (Cre) levels using, for example, the urease, GIDH, and enzymatic methods, respectively, as described in the Examples. For example, a nephrotoxicity-reducing effect can be determined to be present when the BUN and / or Cre levels are reduced by, for example, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more when the nephrotoxicity-reducing agent is administered compared to when the agent is not administered.

[0016] In one embodiment, the nephrotoxicity-reducing agent of the present invention may consist of or contain a sugar alcohol. Non-limiting examples of sugar alcohols include mannitol, sorbitol, glycerol, xylitol, arabitol, erythritol, galactitol, fucitol, iditol, inositol, volemitol, and lactitol. In one embodiment, the sugar alcohol is selected from the group consisting of mannitol, sorbitol, glycerol, and combinations thereof, preferably mannitol, sorbitol, and combinations thereof.

[0017] When the nephrotoxicity-reducing agent of the present invention contains components other than sugar alcohols, the nephrotoxicity-reducing agent may be formulated by appropriately blending a pharmaceutically acceptable carrier or additive (and, optionally, the nephrotoxicity-reducing agent of the present invention) with the sugar alcohol. Specifically, the agent may be formulated as oral preparations such as tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, or emulsions; or parenteral preparations such as injections, infusions, suppositories, ointments, or patches. Parenteral preparations are preferred. Injectable preparations may be lyophilized. The proportion of the carrier or additive may be appropriately determined based on the range commonly used in the pharmaceutical field. The carrier or additive that can be blended is not particularly limited, and examples include various carriers such as water, saline, other aqueous solvents, and aqueous or oily bases, as well as various additives such as excipients, binders, pH adjusters, disintegrants, absorption enhancers, lubricants, colorants, flavorings, and fragrances.

[0018] Examples of additives that can be incorporated into tablets, capsules, etc. include binders such as gelatin, corn starch, tragacanth, and gum arabic; excipients such as crystalline cellulose; bulking agents such as corn starch, gelatin, and alginic acid; lubricants such as magnesium stearate; sweeteners such as sucrose, lactose, and saccharin; and flavorings such as peppermint, saffron oil, and cherry. When the dosage unit is a capsule, the above-mentioned materials may further contain a liquid carrier such as an oil or fat. Sterile compositions for injection can be prepared according to conventional pharmaceutical practices (e.g., by dissolving or suspending the active ingredient in a solvent such as water for injection or natural vegetable oil). Aqueous solutions for injection include, for example, physiological saline, isotonic solutions containing glucose or other adjuvants (e.g., sodium chloride), and the like. Appropriate solubilizing agents, such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., polysorbate 80), are used. TM , HCO-50) and the like. As the oily liquid, for example, sesame oil, soybean oil, and the like are used, and they may be used in combination with solubilizing agents such as benzyl benzoate, benzyl alcohol, and the like. In addition, they may be blended with buffers (e.g., phosphate buffer, sodium acetate buffer), soothing agents (e.g., benzalkonium chloride, procaine hydrochloride, and the like), stabilizers (e.g., human serum albumin, polyethylene glycol, and the like), preservatives (e.g., benzyl alcohol, phenol, and the like), antioxidants, and the like. Furthermore, they may be prepared as lyophilized preparations.

[0019] As used herein, an antisense oligomer may be any of an oligonucleotide, a morpholino oligomer, or a peptide nucleic acid (PNA) oligomer (hereinafter, these will also be referred to as "the antisense oligonucleotide described herein," "the antisense morpholino oligomer described herein," or "the antisense peptide nucleic acid oligomer described herein," respectively).

[0020] An antisense oligonucleotide is an antisense oligomer whose constituent units are nucleotides, and such nucleotides may be any of ribonucleotides, deoxyribonucleotides, or modified nucleotides.

[0021] A modified nucleotide refers to a ribonucleotide or deoxyribonucleotide in which all or part of the nucleic acid base, sugar moiety, and phosphate linkage moiety that constitute the ribonucleotide or deoxyribonucleotide have been modified.

[0022] Examples of nucleic acid bases include adenine, guanine, hypoxanthine, cytosine, thymine, uracil, and modified bases thereof. Examples of such modified bases include pseudouracil, 3-methyluracil, dihydrouracil, 5-alkylcytosine (e.g., 5-methylcytosine), 5-alkyluracil (e.g., 5-ethyluracil), 5-halouracil (e.g., 5-bromouracil), 6-azapyrimidine, 6-alkylpyrimidine (e.g., 6-methyluracil), 2-thiouracil, 4-thiouracil, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, 1-methyladenine, 1-methylhypoxanthine, 1-methyluracil, 1-methylpyridine ... Examples of amino acids that may be used include xanthine, 2,2-dimethylguanine, 3-methylcytosine, 2-methyladenine, 2-methylguanine, N6-methyladenine, 7-methylguanine, 5-methoxyaminomethyl-2-thiouracil, 5-methylaminomethyluracil, 5-methylcarbonylmethyluracil, 5-methyloxyuracil, 5-methyl-2-thiouracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, 2-thiocytosine, purine, 2,6-diaminopurine, 2-aminopurine, isoguanine, indole, imidazole, and xanthine, but are not limited to these.

[0023] Modifications of the sugar moiety include, for example, modifications of the 2'-position of ribose and modifications of other parts of the sugar. Modifications of the 2'-position of ribose include, for example, substitution of the -OH group at the 2'-position of ribose with -OR, -R, -R'OR, -SH, -SR, -NH2, -NHR, -NR2, -N3, -CN, -F, -Cl, -Br, or -I. Here, R represents alkyl or aryl, and R' represents alkylene.

[0024] Modifications of other sugar moieties include, but are not limited to, substitution of the O at the 4' position of ribose or deoxyribose with S, and cross-linking of the 2' and 4' positions of the sugar, such as LNA (Locked Nucleic Acid) or ENA (2'-O,4'-C-Ethylene-bridged Nucleic Acids).

[0025] Modifications of the phosphate linkage moiety include, for example, substitution of a phosphodiester bond with a phosphorothioate bond, a phosphorodithioate bond, an alkylphosphonate bond, a phosphoramidate bond, or a boranophosphate bond (see, for example, Enya et al: Bioorganic & Medicinal Chemistry, 2008, 18, 9154-9160) (see, for example, Republished Patent Publication No. 2006 / 129594 and Republished Patent Publication No. 2006 / 038608).

[0026] In this specification, the alkyl is preferably a 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. The alkyl may be substituted, and examples of such substituents include halogen, alkoxy, cyano, and nitro, and the alkyl may be substituted with 1 to 3 of these.

[0027] In this specification, the cycloalkyl is preferably a cycloalkyl having a carbon number of 3 to 12. Specific examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, and cyclododecyl.

[0028] In this specification, halogen includes fluorine, chlorine, bromine, and iodine.

[0029] In this specification, examples of alkoxy include 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, etc. In particular, alkoxy having 1 to 3 carbon atoms is preferred.

[0030] In this specification, the aryl is preferably an aryl having 6 to 10 carbon atoms. Specific examples include phenyl, α-naphthyl, and β-naphthyl. Phenyl is particularly preferred. The aryl may be substituted, and examples of such substituents include alkyl, halogen, alkoxy, cyano, and nitro, and the aryl may be substituted with 1 to 3 of these.

[0031] In this specification, the alkylene is preferably a 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.

[0032] As used herein, acyl includes straight-chain or branched-chain alkanoyl or aroyl. Examples of alkanoyl include formyl, acetyl, 2-methylacetyl, 2,2-dimethylacetyl, propionyl, butyryl, isobutyryl, pentanoyl, 2,2-dimethylpropionyl, and hexanoyl. Examples of aroyl include benzoyl, toluoyl, and naphthoyl. Such aroyl may be substituted at any substitutable position, and may be substituted with alkyl.

[0033] The antisense oligonucleotides described herein can be easily synthesized using various automated synthesizers (e.g., AKTA oligopilot plus 10 / 100 (GE Healthcare)), or can be produced by outsourcing to a third party (e.g., Promega or Takara).

[0034] The antisense morpholino oligomer described herein is an antisense oligomer having a group represented by the following general formula as a constituent unit.

[0035] [ka]

[0036] (Wherein, Base represents a nucleic acid base; W represents a group represented by any of the following formulas:

[0037] [ka]

[0038] (Wherein, X is —CHR 1 , -O-CH2R 1 , -S-CH2R 1 , -NR 2 R 3 , or F; R 1 represents H, alkyl; R 2 and R 3 are the same or different and represent H, alkyl, cycloalkyl, or aryl; Y1 is O, S, CH2, or NR 1 represents; Y2 is O, S, or NR 1 represents; Z represents O or S. In this specification, the morpholino oligomer is preferably an oligomer having a group represented by the following formula as a constituent unit (phosphorodiamidate morpholino oligomer (hereinafter referred to as "PMO")).

[0039] [ka]

[0040] (In the formula, Base, R 2 , R 3 has the same meaning as above.) Morpholino oligomers can be produced, for example, according to the methods described in WO 1991 / 009033 or WO 2009 / 064471. In particular, PMOs can be produced according to the methods described in WO 2009 / 064471 or WO 2013 / 100190.

[0041] The antisense peptide nucleic acid oligomer described herein is an antisense oligomer having a group represented by the following general formula as a constituent unit.

[0042] [ka]

[0043] (In the formula, Base has the same meaning as defined above.) Peptide nucleic acid oligomers can be produced, for example, according to the following documents: 1)PE Nielsen, M. Egholm, RH Berg, O. Buchardt,Science, 254, 1497 (1991)2)M. Egholm, O. Buchardt, PE Nielsen, RH Berg,JACS, 114, 1895 (1992) 3) KL Dueholm, M. Egholm, C. Behrens, L. Christensen, HF Hansen, T. Vulpius, KH Petersen, RH Berg, PE Nielsen, O. Buchardt, J. Org. Chem., 59, 5767 (1994) 4)L. Christensen, R. Fitzpatrick, B. Gildea, KH Petersen, HF Hansen, T. Koch, M. Egholm,O. Buchardt, PE Nielsen, J. Coull, RH Berg, J. Pept. Sci., 1, 175 (1995)5)T. Koch, HF Hansen, P. Andersen, T. Larsen, HG Batz, K. Otteson, H. Orum, J. Pept. Res., 49, 80 (1997) The antisense oligomers described herein may be in the form of a pharmaceutically acceptable salt thereof, in the form of a hydrate, or in the form of a hydrate of a pharmaceutically acceptable salt thereof.

[0044] Examples of pharmaceutically acceptable salts of the antisense oligomers described herein include alkali metal salts such as sodium salts, potassium salts, and lithium salts, alkaline earth metal salts such as calcium salts and magnesium salts; metal salts such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, and cobalt salts; ammonium salts; t-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, N-benzyl-phenethylamine salts, Examples of suitable salts include organic amine salts such as piperazine salts, tetramethylammonium salts, and tris(hydroxymethyl)aminomethane salts; hydrohalide salts such as hydrofluoride, hydrochloride, hydrobromide, and hydroiodide; inorganic acid salts such as nitrate, perchlorate, sulfate, and phosphate; lower alkane sulfonate salts such as methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate; arylsulfonate salts such as benzenesulfonate and p-toluenesulfonate; organic acid salts such as acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, and maleate; and amino acid salts such as glycine salt, lysine salt, arginine salt, ornithine salt, glutamate, and aspartate. These salts can be prepared by known methods. Alternatively, the antisense oligomers described herein may be in the form of their hydrates.

[0045] The antisense oligomer described herein may have at its 5' end a group represented by any one of the following chemical formulae (1) to (3), preferably either (1) or (2).

[0046] [ka]

[0047] Hereinafter, the groups represented by (1), (2), and (3) above will be referred to as "group (1)," "group (2)," and "group (3)," respectively.

[0048] The base sequence of the antisense oligomer described herein is not limited, but may include, for example, four consecutive purine bases in the base sequence, and at least two of the four consecutive purine bases may be guanine.

[0049] In one embodiment, the antisense oligomer described herein comprises or consists of (i) a base sequence selected from the group consisting of SEQ ID NOs: 1 to 10; (ii) a base sequence having 80% or more, 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 sequence identity to a base sequence selected from the group consisting of SEQ ID NOs: 1 to 10; or (iii) a base sequence selected from the group consisting of SEQ ID NOs: 1 to 10 in which one or more bases have been added, deleted, or substituted.

[0050] As used herein, the "identity" of a nucleotide sequence refers to the percentage of identical bases when two nucleotide sequences are aligned. Sequence identity can be determined using FASTA (Science 227 (4693): 1435-1441, (1985)) or the BLAST (Basic Local Alignment Search Tool) algorithm by Carlin and Altschul (Proc. Natl. Acad. Sci. USA 872264-2268, 1990; Proc Natl Acad Sci USA 90: 5873, 1993). Programs based on the BLAST algorithm, such as blastn, blastx, tblastn, and tblastx, have been developed (Altschul SF, et al: J Mol Biol 215: 403, 1990). When analyzing a nucleotide sequence using blastn, the parameters are, for example, score = 100 and word length = 12. When using BLAST and Gapped BLAST programs, the default parameters of each program are used.

[0051] As used herein, "several" in a base sequence in which one or more bases have been added, deleted, or substituted means two, three, four, five, six, seven, eight, nine, or ten bases.

[0052] In one embodiment, the antisense oligomer described herein targets the range of positions 115937 to 115981 of the genomic sequence (SEQ ID NO: 11) of an insertion-mutated fukutin gene in which an SVA-type retrotransposon sequence (GenBank ACCESSION: AB185332) is inserted into the genomic sequence of the fukutin gene (GenBank ACCESSION: AB038490), or does not target this range. Examples of antisense oligomers that target the range of positions 115937 to 115981 of SEQ ID NO: 11 include antisense oligomers containing a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 7. Examples of antisense oligomers that do not target the range of positions 115937 to 115981 of SEQ ID NO: 11 include antisense oligomers containing a nucleotide sequence selected from the group consisting of SEQ ID NOs: 8 to 10.

[0053] In one embodiment, the antisense oligomer described herein is a conjugate to which a functional peptide, such as a cell-penetrating peptide (CPP), is attached. Known or commercially available functional peptides can be used herein. Functional peptides that can be used herein include, for example, the arginine-rich peptides disclosed in WO 2008 / 036127; or organ-targeting peptides, such as RXR and RBR, disclosed in WO 2009 / 005793; or peptides containing amino acid subunits disclosed in WO 2012 / 150960. Cell-penetrating peptides (CPPs) represent short peptide sequences of 10 to about 30 amino acids that can cross the cell membrane of mammalian cells and thus improve cellular drug delivery (see, for example, Hum Mol Genet. 2011 Aug 15; 20(16): 3151-3160; Pharmacology & Therapeutics 154 (2015) 78-86). Known or commercially available CPPs can be used herein. CPPs that can be used herein include those described in, for example, Pharmacology & Therapeutics 154 (2015) 78-86, Table 1 on page 80, such as TAT(48-60), penetratin, polyarginine, Oct4, WT1-pTj, DPV3, transportan, MAP, VP22, Rep1, KW, KFGF, FGF12, interferon β3 peptide, C105Y, TP2; and CPPs listed in Table 1 in paragraph

[0085] of the specification of JP2017-500856 (International Publication No. WO2015 / 089487), such as DPV10 / 6, DPV15b, YM-3, Tat, LR11, C45D18, Lyp-1, Lyp-2, BMV GAG, hLF1-22, C45D18, LR20, etc. CPPs are commercially available, for example, from Funakoshi Co., Ltd. Commercially available CPPs such as TAT ​​(Funakoshi Co., Ltd.) and penetratin (Funakoshi Co., Ltd.), or known CPPs, such as R8, can be used herein. Preferred CPPs that can be used herein include, for example, hLIMK, TAT, penetratin, R8, etc. (see International Publication Nos. WO 2016 / 187425, WO 2018 / 118662, WO 2018 / 118599, WO 2018 / 118627, EBioMedicine 45 (2019) 630-645, etc.). The CPP can be directly linked to the antisense oligomer herein, or can be linked via a linker that can link the CPP to the antisense oligomer. Known linkers can be used herein. Such linkers include, for example, those described in JP-T-2017-500856 (WO 2015 / 089487 pamphlet), WO 2015 / 089487 pamphlet, WO 2009 / 073809 pamphlet, WO 2013 / 075035 pamphlet, WO 2015 / 105083 pamphlet, WO 2014 / 179620 pamphlet, WO 2015 / 006740 pamphlet, WO 2017 / 010575 pamphlet, and the like.Preferred linkers that can be used herein include, for example, 4-maleimidobutyric acid, a linker that can be linked to the functional peptide or antisense oligomer of the present invention via a disulfide bond, etc. The conjugate of the present invention can be prepared by methods known to those skilled in the art.

[0054] The pharmaceutical compositions described herein may be formulated by appropriately blending a pharmaceutically acceptable carrier or additive (and optionally the sugar alcohol of the present invention). The pharmaceutically acceptable carrier or additive and formulation of the pharmaceutical composition are the same as those described for the nephrotoxicity-reducing agent of the present invention, except that the antisense nucleic acid is an active ingredient.

[0055] In one embodiment, the nephrotoxicity-reducing agent of the present invention is used and / or added to the pharmaceutical composition described herein in an amount such that the sugar alcohol concentration in the pharmaceutical composition is 1 mg / mL to 400 mg / mL, e.g., 2.5 mg / mL to 200 mg / mL. In one embodiment, the nephrotoxicity-reducing agent of the present invention is used and / or added to the pharmaceutical composition in an amount such that the sugar alcohol concentration in the pharmaceutical composition is 1 mg / mL or more, 2.5 mg / mL or more, 3 mg / mL or more, 4 mg / mL or more, 5 mg / mL or more, 10 mg / mL or more, 15 mg / mL or more, 20 mg / mL or more, 30 mg / mL or more, or 40 mg / mL or more. Furthermore, the nephrotoxicity-reducing agent of the present invention is used and / or added to the pharmaceutical composition in an amount such that the sugar alcohol concentration in the pharmaceutical composition is 400 mg / mL or less, 350 mg / mL or less, 300 mg / mL or less, 250 mg / mL or less, or 200 mg / mL or less.

[0056] In one embodiment, the concentration of the antisense oligomer in the pharmaceutical compositions described herein is 0.5 mg / mL to 200 mg / mL, for example, 6 mg / mL to 200 mg / mL. In one embodiment, the concentration of the antisense oligomer in the pharmaceutical compositions described herein may be 0.5 mg / mL or more, 1 mg / mL or more, 2 mg / mL or more, 3 mg / mL or more, 4 mg / mL or more, 5 mg / mL or more, or 6 mg / mL or more, and may be 200 mg / mL or less, 180 mg / mL or less, 150 mg / mL or less, 140 mg / mL or less, or 130 mg / mL or less.

[0057] In one embodiment, the present invention relates to a nephrotoxicity-reducing agent for a pharmaceutical composition containing an antisense oligomer, which contains a sugar alcohol and is used in an amount such that the weight ratio of the sugar alcohol to the antisense oligomer is 0.05 to 90, 0.05 to 30, for example, 0.1 to 13.3. The pharmaceutical composition containing an antisense oligomer and the sugar alcohol are as described above. In one embodiment, the nephrotoxicity-reducing agent of the present invention is used in an amount such that the weight ratio of the sugar alcohol to the antisense oligomer is 0.05 or more, 0.1 or more, 0.15 or more, 0.2 or more, 0.3 or more, 0.4 or more, 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, 0.9 or more, 1 or more, 2 or more, or 5 or more. In one embodiment, the nephrotoxicity-reducing agent of the present invention is used in an amount such that the weight ratio of sugar alcohol to antisense oligomer is 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 or less, 25 or less, 20 or less, 15 or less, 13.3 or less, or 10 or less.

[0058] In one embodiment, the nephrotoxicity-reducing agent of the present invention is contained in a pharmaceutical composition described herein and administered together with the pharmaceutical composition described herein. For example, a pharmaceutical composition prepared by lyophilizing an antisense oligomer described herein, optionally together with a carrier such as glucose, and dissolving the lyophilized antisense oligomer in a solvent such as water for injection may be mixed with a nephrotoxicity-reducing agent described herein, and then the amount may be adjusted with a solvent, and then administered to a subject. Alternatively, for example, a pharmaceutical composition prepared by lyophilizing an antisense oligomer described herein together with a nephrotoxicity-reducing agent of the present invention may be dissolved in a solvent such as water for injection to form a pharmaceutical composition, and then the amount may be adjusted with a solvent, and then administered to a subject.

[0059] In another embodiment, the nephrotoxicity-reducing agent of the present invention is not included in the pharmaceutical composition described herein and is administered separately (simultaneously or sequentially) from the pharmaceutical composition described herein. For example, a pharmaceutical composition prepared by dissolving a lyophilized antisense oligomer described herein in a solvent such as water for injection and the nephrotoxicity-reducing agent described herein may be administered to a subject separately. As used herein, "simultaneously" administering the nephrotoxicity-reducing agent and the pharmaceutical composition means that the nephrotoxicity-reducing agent and the pharmaceutical composition are administered at the same time. As used herein, "sequentially" administering the nephrotoxicity-reducing agent and the pharmaceutical composition means that they are administered at different times. Specifically, the nephrotoxicity-reducing agent can be administered before or after the nephrotoxicity-reducing agent. In this case, the interval between the administration of the nephrotoxicity-reducing agent and the pharmaceutical composition may be, but is not limited to, several minutes, several hours, or even up to a day.

[0060] In this specification, subjects to which the pharmaceutical composition and / or nephrotoxicity-reducing agent is administered include, but are not limited to, mammals, for example, primates such as humans, laboratory animals such as rats, mice, and brown rats, and livestock animals such as pigs, cows, horses, and sheep, and preferably humans.

[0061] The dosage when administering the pharmaceutical composition and / or the nephrotoxicity-reducing agent can be adjusted taking into consideration the type of antisense oligomer contained in the pharmaceutical composition and the sugar alcohol contained in the nephrotoxicity-reducing agent, the dosage form of the pharmaceutical composition and the nephrotoxicity-reducing agent, the condition of the subject such as age and weight, the route of administration, and the nature and symptoms of the disease. The amount of the antisense oligomer can be, for example, within the range of 0.1 mg to 10 g / human per day, for example, within the range of 1 mg to 1 g / human, for example, within the range of 10 mg to 100 mg / human. The amount can be, for example, within the range of 0.1 mg to 200 g per day per human, for example, within the range of 1 mg to 100 g per day per human, for example, within the range of 10 mg to 50 g per day per human, for example, within the range of 100 mg to 50 g per day per human, for example, within the range of 100 mg to 40 g per day per human, for example, within the range of 100 mg to 30 g per day per human, for example, within the range of 100 mg to 20 g per day per human, for example, within the range of 1 g to 20 g per human, for example, within the range of 2 g to 20 g per human, for example, within the range of 1 g to 10 g per human, for example, within the range of 100 mg to 1 g per human. The number and frequency of administration are not limited, but can be, for example, once to two or three times per day, with intervals of one to two or three days between each administration. Alternatively, for example, it can be administered once, followed by another administration a few days later, for a total of two administrations.

[0062] In one embodiment, the present invention relates to a method for reducing the nephrotoxicity of a pharmaceutical composition comprising an antisense oligomer, or a method for producing a pharmaceutical composition comprising an antisense oligomer with reduced nephrotoxicity, the method comprising adding a sugar alcohol to the pharmaceutical composition in an amount such that the sugar alcohol concentration in the pharmaceutical composition becomes 1 mg / mL to 400 mg / mL. In this embodiment, the antisense oligomer, pharmaceutical composition, sugar alcohol, and the concentration of the sugar alcohol in the pharmaceutical composition are as described herein.

[0063] In one embodiment, the present invention relates to a method for reducing the nephrotoxicity of an antisense oligomer in a subject to whom an antisense oligomer or a pharmaceutical composition containing the antisense oligomer has been administered, the method comprising administering a sugar alcohol or a nephrotoxicity-reducing agent to the subject, wherein the sugar alcohol is administered in an amount such that the weight ratio of the sugar alcohol relative to the antisense oligomer is 0.05 to 90 or 0.05 to 30, relative to the weight of the antisense oligomer being 1. In this embodiment, the antisense oligomer, the pharmaceutical composition, the sugar alcohol, the nephrotoxicity-reducing agent, the weight ratio of the sugar alcohol relative to the antisense oligomer being 1, and the like are as described herein.

[0064] In one embodiment, the present invention relates to a pharmaceutical composition with reduced nephrotoxicity comprising an antisense oligomer, the pharmaceutical composition comprising a nephrotoxicity-reducing agent comprising a sugar alcohol at a concentration of 1 mg / mL to 400 mg / mL. In this embodiment, the antisense oligomer, pharmaceutical composition, sugar alcohol, and the concentration of the sugar alcohol in the pharmaceutical composition are as described herein. [Example]

[0065] Example 1: Preparation of antisense oligomers (PMOs) The antisense oligomers (PMO Nos. 1 to 10 (SEQ ID NOs: 1 to 10)) shown in Table 1 were synthesized according to the method described in International Publication WO2013 / 100190. The theoretical molecular weight values ​​of each antisense oligomer and the values ​​actually measured by ESI-TOF-MS are also shown.

[0066] [Table 1]

[0067] "Group (1)" and "Group (2)" in the above table are as follows:

[0068] [ka]

[0069] Example 2: Evaluation of the nephrotoxicity-reducing effect of D-mannitol Of the antisense oligomers shown in Table 1, PMO Nos. 1, 2, 3, 4, 6, 7, and 10 (SEQ ID NOs: 1, 2, 3, 4, 6, 7, and 10) were subjected to a safety evaluation in the kidney to verify their usefulness for pharmaceutical applications. (1) Evaluation method Each of the antisense oligomers of PMO Nos. 1, 2, 6, 7, and 10 was dissolved in water for injection (Otsuka Pharmaceutical) containing 0.9% sodium chloride to prepare a control administration solution containing each antisense oligomer at the concentrations listed in Tables 2, 3, 6, 7, and 8 but without D-mannitol. Each of the antisense oligomers of PMO Nos. 3 and 4 was dissolved in 5% glucose (Otsuka Pharmaceutical) to prepare a control administration solution containing each antisense oligomer at the concentrations listed in Tables 4 and 5 but without D-mannitol.

[0070] Next, each antisense oligomer of PMO Nos. 1, 2, 6, 7, and 10 was dissolved in water for injection containing 0.9% sodium chloride, and a solution of 20% D-mannitol (Yoshindo) diluted with saline (Otsuka Pharmaceutical) or water for injection containing 0.9% sodium chloride was added to prepare administration solutions containing each antisense oligomer and D-mannitol at the concentrations listed in Tables 2, 3, 6, 7, and 8. Additionally, each antisense oligomer of PMO Nos. 3 and 4 (SEQ ID NOs: 3 and 4) was dissolved in a solution of 15% D-mannitol (Terumo) diluted with water for injection to prepare administration solutions containing each antisense oligomer and D-mannitol at the concentrations listed in Tables 4 and 5.

[0071] These solutions were administered into the tail vein of 6-week-old male C57BL / 6J mice at 4, 10, or 20 mL / kg. The following day, serum was collected from the mice, and blood urea nitrogen (BUN) levels were measured using the urease-GIDH method, and blood creatinine (Cre) levels were measured using an enzymatic method using a JCA-BM6050 or JCA-BM8060 automated biochemical analyzer (JEOL). Reduced nephrotoxicity was determined when both BUN and Cre levels were reduced in mice administered with the D-mannitol-containing solution compared to mice administered with a control solution that did not contain D-mannitol. (2) Evaluation results Each of the tested antisense oligomers showed a tendency to decrease both BUN and Cre levels in mice administered with a D-mannitol-containing solution, confirming that D-mannitol reduces nephrotoxicity. The results are shown in Tables 2, 3, 4, 5, 6, 7, and 8.

[0072] [Table 2]

[0073] [Table 3]

[0074] [Table 4]

[0075] [Table 5]

[0076] [Table 6]

[0077] [Table 7]

[0078] [Table 8]

[0079] These results demonstrate that D-mannitol reduces the nephrotoxicity caused by antisense oligomer administration. Example 3: Evaluation of the nephrotoxicity-reducing effect of D-sorbitol Of the antisense oligomers shown in Table 1, PMO Nos. 2 and 3 (SEQ ID NOs: 2 and 3) were subjected to a safety evaluation in the kidney to verify their usefulness for medical applications. (1) Evaluation method The antisense oligomer of PMO No. 2 was dissolved in physiological saline to prepare a control administration solution containing the antisense oligomer at the concentration listed in Table 9 but without D-sorbitol. The antisense oligomer of PMO No. 3 was dissolved in Dulbecco's PBS(-) (manufactured by Nissui) to prepare a control administration solution containing the antisense oligomer at the concentration listed in Table 10 but without D-sorbitol.

[0080] Next, the antisense oligomer of PMO No. 2 was dissolved in a solution of D-sorbitol (Maruishi Pharmaceutical) dissolved in physiological saline to prepare an administration solution containing the antisense oligomer and D-sorbitol at the concentrations listed in Table 9. Furthermore, the antisense oligomer of PMO No. 3 was dissolved in a solution of D-sorbitol dissolved in water for injection to prepare an administration solution containing the antisense oligomer and D-sorbitol at the concentrations listed in Table 10.

[0081] These solutions were administered into the tail vein of 6-week-old male C57BL / 6J mice (N=3) at 4 or 10 mL / kg. The following day, serum was collected from the mice, and blood urea nitrogen (BUN) levels were measured using the urease-GIDH method, and blood creatinine (Cre) levels were measured using an enzymatic method using a JCA-BM8060 automatic biochemical analyzer (JEOL). Reduced nephrotoxicity was determined when both BUN and Cre levels were reduced in mice administered with the D-sorbitol-containing solution compared to mice administered with the D-sorbitol-free solution. (2) Evaluation results Each of the tested antisense oligomers showed a tendency to decrease both BUN and Cre levels in mice administered with a D-sorbitol-containing solution, confirming that D-sorbitol reduces nephrotoxicity. The results are shown in Tables 9 and 10.

[0082] [Table 9]

[0083] [Table 10]

[0084] Example 4: Evaluation of the precipitation-inhibiting effect of D-mannitol Test Method Among the antisense oligomers shown in Table 1, PMO Nos. 1, 2, 3, 5, 6, and 7 (SEQ ID NOs: 1, 2, 3, 5, 6, and 7) were evaluated for their usefulness for pharmaceutical applications, and the amount of precipitation in the presence of ions simulating those found in urine and the precipitation-inhibitory effect of D-mannitol were evaluated.

[0085] Each antisense oligomer was dissolved in physiological saline or D-mannitol solution and mixed with an aqueous solution containing potassium chloride and sodium chloride, which simulates urine. The evaluation conditions (solution composition) are shown in Tables 11 to 16. The absorbance of the mixed solution was measured at 620 nm using an Infinite F200 Pro plate reader (TECAN) at 37°C. Since the concentrations of the antisense oligomer, potassium chloride, and sodium chloride vary depending on the antisense oligomer dose and urine volume, conditions under which precipitation is observed were established for each sequence using water for injection as the medium. Furthermore, measurements were performed under conditions in which only the medium was replaced with D-mannitol solution, and the absorbance obtained under both conditions was compared. An increase in absorbance was considered to indicate precipitation, and the time until precipitation was observed and the increase in absorbance were evaluated. When the D-mannitol aqueous solution was used as the medium, the time until precipitation was observed was slower or the increase in absorbance was smaller than when water for injection was used as the medium, and it was determined that the solution had a precipitation-inhibiting effect.

[0086] [Table 11]

[0087] [Table 12]

[0088] [Table 13]

[0089] [Table 14]

[0090] [Table 15]

[0091] [Table 16]

[0092] Test results When D-mannitol was used as the medium for all of the tested antisense oligomers, the time until precipitation was observed was delayed and the increase in absorbance was suppressed, indicating that D-mannitol inhibits the precipitation of antisense oligomers. The respective results are shown in Figures 1 to 6.

[0093] These results demonstrate that D-mannitol inhibits the precipitation of antisense oligomers in urine.

Claims

1. A combination product comprising a pharmaceutical composition containing an antisense oligomer and a nephrotoxicity reducing agent containing mannitol, sorbitol, or a combination thereof, wherein the nephrotoxicity reducing agent is used in mixture with the pharmaceutical composition, and the concentration of mannitol, sorbitol, or a combination thereof in the resulting mixture is 1 mg / mL to 200 mg / mL.

2. The combination product according to Claim 1, wherein the concentration of mannitol, sorbitol, or a combination thereof in the mixed solution is 40 mg / mL to 200 mg / mL.

3. The combination product according to claim 1, wherein the concentration of mannitol, sorbitol, or a combination thereof in the mixed solution is 120 mg / mL to 200 mg / mL.

4. The combination product according to Claim 1, wherein the concentration of the antisense oligomer in the pharmaceutical composition is 0.5 mg / mL to 200 mg / mL.

5. The combination product according to claim 4, wherein the concentration of the antisense oligomer in the pharmaceutical composition is 9.9 mg / mL to 15 mg / mL.

6. The combination product according to claim 1, wherein the antisense oligomer is a morpholino oligomer.

7. The combination product according to claim 1, wherein the antisense oligomer is a phosphorodiamidate morpholino oligomer.

8. The 5' end of the antisense oligomer is of the following chemical formulas (1) to (2): 【Chemistry 1】 A combination product according to claim 1, which is based on any of the following.

9. The combination product according to claim 1, wherein the antisense oligomer contains four consecutive purine bases in its base sequence.

10. The combination according to claim 9, wherein at least two of the four consecutive purine bases are guanine.

11. The combination product according to claim 1, wherein the antisense oligomer comprises a base sequence selected from the group consisting of SEQ ID NOs: 1 to 10.

12. The combination product according to claim 1, wherein the antisense oligomer targets the range from position 115937 to position 115981 of the base sequence shown in Sequence ID No.

11.

13. The combination according to any one of claims 1 to 12, wherein the antisense oligomer does not target the range from position 115937 to position 115981 of the base sequence shown in Sequence ID No. 11.

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