Precipitation inhibitor

Abstract

To provide a precipitation inhibitor for antisense oligomers in the urine for a subject to whom an antisense oligomer-containing drug composition has been administered, and provide a method for inhibiting the precipitation of antisense oligomers in the urine in a subject to whom the drug composition has been administered.SOLUTION: In an embodiment, the present invention relates to a precipitation inhibitor for antisense oligomers in the urine for a subject to whom an antisense oligomer-containing drug composition has been administered, wherein the precipitation inhibitor contains a sugar that is not glucose and is used in an amount that gives a sugar concentration in the drug composition of 0.5-3000 mg / mL.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an agent for inhibiting the precipitation of antisense oligomers in urine in a subject to which a pharmaceutical composition containing an antisense oligomer, including a sugar, has been administered, a method for inhibiting the precipitation of antisense oligomers in urine in a subject to which the pharmaceutical composition has been administered, and a pharmaceutical composition containing the precipitation inhibitor. [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] In this situation, it would be desirable to provide pharmaceutical compositions containing improved antisense oligomers. [Means for solving the problem]

[0007] That is, the present invention provides an agent for inhibiting the precipitation of antisense oligomers in urine in a subject administered a pharmaceutical composition containing an antisense oligomer, the antisense oligomer containing a sugar other than glucose as described below; a method for inhibiting the precipitation of antisense oligomers in urine in a subject administered the pharmaceutical composition; and a pharmaceutical composition containing the precipitation inhibitor. (1) An agent for inhibiting the precipitation of antisense oligomers in urine of a subject to which a pharmaceutical composition containing an antisense oligomer has been administered, the agent containing a sugar other than glucose and used in an amount such that the concentration of the sugar in the pharmaceutical composition is 0.5 mg / mL to 3000 mg / mL. (2) The precipitation inhibitor according to (1), which is used in an amount that results in a sugar concentration in the pharmaceutical composition of 2.6 mg / mL to 1694.1 mg / mL. (3) The precipitation inhibitor according to (1) or (2), wherein the concentration of the antisense oligomer in the pharmaceutical composition is 0.05 mg / mL to 2000 mg / mL. (4-1) An agent for inhibiting the precipitation of antisense oligomers in urine of a subject administered a pharmaceutical composition containing an antisense oligomer, the agent containing a sugar other than glucose and used in an amount such that the weight ratio of the sugar to the antisense oligomer is 0.1 to 100. (4-2) The precipitation inhibitor according to (4-1), wherein the pharmaceutical composition and the precipitation inhibitor are administered separately. (5) The precipitation inhibitor according to (4-1) or (4-2), which is used in an amount such that the weight ratio of the sugar to the antisense oligomer is 1 to 53. (6) The precipitation inhibitor according to any one of (1) to (5), wherein the sugar is a disaccharide sugar. (7) The precipitation inhibitor according to any one of (1) to (6), wherein the sugar is sucrose. (8) The precipitation inhibitor according to any one of (1) to (6), wherein the sugar is trehalose. (9) The precipitation inhibitor according to any one of (1) to (8), wherein the antisense oligomer is a morpholino oligomer. (10) The precipitation inhibitor according to any one of (1) to (9), wherein the antisense oligomer is a phosphorodiamidate morpholino oligomer. (11) The 5' end of the antisense oligomer has the following chemical formulas (1) to (2):

[0008] [ka]

[0009] The precipitation inhibitor according to any one of (1) to (10), wherein the precipitation inhibitor is one of the groups shown below. (12) The precipitation inhibitor according to any one of (1) to (11), wherein the antisense oligomer contains four consecutive purine bases in its base sequence. (13) The precipitation inhibitor according to (12), wherein at least two of the four consecutive purine bases are guanine. (14) The precipitation inhibitor according to any one of (1) to (13), wherein the antisense oligomer comprises a base sequence selected from the group consisting of SEQ ID NOs: 1 to 12. (15-1) A method for suppressing precipitation of an antisense oligomer in urine in a subject to which a pharmaceutical composition containing an antisense oligomer has been administered, the method comprising adding a sugar other than glucose in an amount such that the sugar concentration in the pharmaceutical composition becomes 0.5 mg / mL to 3000 mg / mL. (15-2) The method according to (15-1), wherein the sugar is added in an amount such that the sugar concentration in the pharmaceutical composition becomes 2.6 mg / mL to 1694.1 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 0.05 mg / mL to 2000 mg / mL. (16-1) A method for suppressing precipitation of an antisense oligomer in urine in a subject to which an antisense oligomer has been administered, the method comprising administering a sugar other than glucose to the subject, in an amount such that the weight ratio of the sugar to the antisense oligomer is 0.1 to 100. (16-2) The method according to (16-1), wherein the antisense oligomer and the sugar are administered separately. (16-3) The method according to (16-1) or (16-2), wherein the sugar is used in an amount such that the weight ratio of the sugar to the antisense oligomer is 1 to 53. (17-1) A pharmaceutical composition comprising an antisense oligomer, the pharmaceutical composition comprising an inhibitor of antisense oligomer precipitation in urine of a subject to which the pharmaceutical composition is administered, the inhibitor containing a sugar other than glucose, at a concentration of 0.5 mg / mL to 3000 mg / mL. (17-2) The pharmaceutical composition according to (17-1), comprising a sugar-containing precipitation inhibitor at a concentration of 2.6 mg / mL to 1694.1 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 0.05 mg / mL to 2000 mg / mL. (18) The method or pharmaceutical composition according to any one of (15-1) to (17-3), wherein the sugar is a disaccharide sugar. (19) The method or pharmaceutical composition according to any one of (15-1) to (18), wherein the sugar is sucrose. (20) The method or pharmaceutical composition according to any one of (15-1) to (18), wherein the sugar is trehalose. (21) The method or pharmaceutical composition according to any one of (15-1) to (20), wherein the antisense oligomer is a morpholino oligomer. (22) The method or pharmaceutical composition according to any one of (15-1) to (21), wherein the antisense oligomer is a phosphorodiamidate morpholino oligomer. (23) 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 (22), wherein the compound is any one of the groups represented by the formula (15-1) to (22). (24) The method or pharmaceutical composition according to any one of (15-1) to (23), wherein the antisense oligomer contains four consecutive purine bases in its base sequence. (25) The method or pharmaceutical composition according to (24), wherein at least two of the four consecutive purine bases are guanine. (26) The method or pharmaceutical composition according to any one of (15-1) to (25), wherein the antisense oligomer comprises a base sequence selected from the group consisting of SEQ ID NOs: 1 to 12. [Effects of the Invention]

[0012] The present invention provides an agent for inhibiting the precipitation of antisense oligomers in urine in a subject to which a pharmaceutical composition containing an antisense oligomer has been administered, and a method for inhibiting the precipitation of antisense oligomers in urine in a subject to which the pharmaceutical composition has been administered. [Brief explanation of the drawings]

[0013] [Figure 1] The absorbance measured at 620 nm under the conditions shown in Table 4 is shown. [Figure 2] The absorbance measured at 620 nm under the conditions shown in Table 5 is shown. [Figure 3] The absorbance measured at 620 nm under the conditions shown in Table 6 is shown. [Figure 4] The absorbance measured at 620 nm under the conditions shown in Table 7 is shown. [Figure 5] The absorbance measured at 620 nm under the conditions shown in Table 8 is shown. [Figure 6] The absorbance measured at 620 nm under the conditions shown in Table 9 is shown. [Figure 7] The absorbance measured at 620 nm under the conditions shown in Table 10 is shown. [Figure 8] The absorbance measured at 620 nm under the conditions shown in Table 11 is shown. [Figure 9] This shows hematoxylin-eosin stained images of the kidneys after administration of PMO No. 8 administration solution (50 mg / mL PMO No. 8) at 10 mL / kg. [Figure 10] This shows hematoxylin-eosin stained images of the kidneys after administration of 10 mL / kg of PMO No. 8 administration solution (50 mg / mL PMO No. 8, 20 mg / mL sucrose). [Figure 11] This shows hematoxylin-eosin stained images of the kidneys after administration of 10 mL / kg of PMO No. 8 administration solution (50 mg / mL PMO No. 8, 50 mg / mL sucrose). [Figure 12] This shows hematoxylin-eosin stained images of the kidneys after administration of 10 mL / kg of PMO No. 8 administration solution (50 mg / mL PMO No. 8, 100 mg / mL sucrose). [Figure 13] The absorbance measured at 620 nm under the conditions shown in Table 15 is shown. [Figure 14] The absorbance measured at 620 nm under the conditions shown in Table 16 is shown. [Figure 15] The absorbance measured at 620 nm under the conditions shown in Table 17 is shown. [Figure 16] The absorbance measured at 620 nm under the conditions shown in Table 18 is shown. [Figure 17] The absorbance measured at 620 nm under the conditions shown in Table 19 is shown. [Figure 18] The absorbance measured at 620 nm under the conditions shown in Table 20 is shown. [Figure 19] The absorbance measured at 620 nm under the conditions shown in Table 21 is shown. DETAILED DESCRIPTION OF THE INVENTION

[0014] In one embodiment, the present invention relates to an agent for inhibiting the precipitation of antisense oligomers in urine of a subject administered a pharmaceutical composition containing an antisense oligomer. As used herein, the term "precipitation inhibitor" refers to an agent for inhibiting the precipitation of antisense oligomers in the urine of a subject administered a pharmaceutical composition containing an antisense oligomer. If the inhibition of precipitation reduces the accumulation of antisense oligomers in the kidneys in vivo, this is expected to lead to reduced nephrotoxicity. Nephrotoxicity refers to tissue damage or reduced renal function caused by increased accumulation of administered antisense oligomers in the kidneys. Furthermore, if precipitation in body fluids such as blood is inhibited in vivo, the concentration of antisense oligomers in body fluids is expected to be uniform, leading to stabilized efficacy and / or reduced toxicity.

[0015] The presence or absence of a precipitation inhibitory effect can be determined by adding an antisense oligomer and a precipitation inhibitor to a solvent simulating urine, and examining whether the precipitation inhibitor reduces the increase in absorbance caused by the precipitation of the antisense oligomer, as described in the Examples. For example, after adding the antisense oligomer and the precipitation inhibitor to the solvent (e.g., 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, or 90 minutes), the absorbance can be measured at 37 ° C. In this case, when the precipitation inhibitor is added, the time until precipitation is observed (an increase in absorbance at 620 nm is observed) or the absorbance at 620 nm is reduced by, for example, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more, compared to when the precipitation inhibitor is not added, the precipitation inhibitory effect can be determined. Examples of solvents that mimic urine include aqueous solutions containing 20 nmol / mL to 9000 nmol / mL, 50 nmol / mL to 3600 nmol / mL, or 100 nmol / mL to 1800 nmol / mL of KCl and 15 nmol / mL to 2400 nmol / mL, 40 nmol / mL to 900 nmol / mL, or 77 nmol / mL to 462 nmol / mL of NaCl.

[0016] From the results of the Examples of the present application, it can be assumed that there may be a linear relationship between the amount of sugar administered to a subject and the urinary concentration, and therefore the sugar concentration in a solvent simulating urine (hereinafter also referred to as "urinary concentration") can be converted to the sugar concentration in a formulation as follows. For example, based on the results when 500 mg / kg of mannitol was administered to cynomolgus monkeys, the urinary concentration can be converted to the sugar administration amount using the following calculation formula, and the sugar administration amount can then be converted to the concentration in the formulation at the time of administration (at the time of use) based on the body weight and the volume of the administration solution.

[0017] Glucose dose (mg / kg) = urinary concentration (mg / mL) / estimated urinary concentration in cynomolgus monkeys (127.5 (mg / mL)) × dose to cynomolgus monkeys (500 (mg / kg)) D-mannitol concentration in formulation (mg / mL) = D-mannitol dose (mg / kg) × body weight (kg) ÷ volume of solution administered (mL) The body weight can be assumed to be, for example, 3 to 80 kg, and the volume of the administered solution can be assumed to be, for example, 50 to 200 mL.

[0018] Similarly, the concentration of anti-inflammatory oligomers in a solvent simulating urine as described in the Examples can be converted to the concentration of anti-inflammatory oligomers in the formulation.

[0019] In one embodiment, the precipitation inhibitor of the present invention may consist of or contain a sugar (excluding glucose). The sugar may be, for example, a sugar of disaccharide or more, or may be a disaccharide sugar. Non-limiting examples of sugars include disaccharides such as sucrose, lactose, lactulose, trehalose, maltose, and isomaltose; trisaccharides such as raffinose, melezitose, and maltotriose; and monosaccharides such as galactose, mannose, fructose, ribose, xylose, arabinose, and lyxose. In one embodiment, the sugar is or contains sucrose. In another embodiment, the sugar is or contains trehalose.

[0020] When the precipitation inhibitor of the present invention contains components other than sugar, the precipitation inhibitor may be formulated by appropriately blending a pharmaceutically acceptable carrier or additive (and optionally the precipitation inhibitor of the present invention) with the sugar. Specifically, the precipitation inhibitor may be formulated as oral preparations such as tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, and emulsions; or parenteral preparations such as injections, infusions, suppositories, ointments, and patches. Parenteral preparations are preferred. Injections may be freeze-dried preparations. The blending ratio of the carrier or additive may be appropriately set 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, physiological 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.

[0021] 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, or 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). Examples of aqueous solutions for injection include physiological saline, isotonic solutions containing glucose or other adjuvants (e.g., sodium chloride), and the like. Suitable 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.

[0022] 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).

[0023] An antisense oligonucleotide is an antisense oligomer whose constituent units are nucleotides, and such nucleotides may be any of ribonucleotides, deoxyribonucleotides, or modified nucleotides.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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).

[0028] 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).

[0029] 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.

[0030] 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.

[0031] In this specification, halogen includes fluorine, chlorine, bromine, and iodine.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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).

[0037] The antisense morpholino oligomer described herein is an antisense oligomer having a group represented by the following general formula as a constituent unit.

[0038] [ka]

[0039] (Wherein, Base represents a nucleic acid base; W represents a group represented by any of the following formulas:

[0040] [ka]

[0041] (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")).

[0042] [ka]

[0043] (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.

[0044] 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.

[0045] [ka]

[0046] (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.

[0047] 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.

[0048] 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).

[0049] [ka]

[0050] Hereinafter, the groups represented by (1), (2), and (3) above will be referred to as "group (1)," "group (2)," and "group (3)," respectively.

[0051] 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.

[0052] 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 12; (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 12; or (iii) a base sequence selected from the group consisting of SEQ ID NOs: 1 to 12 in which one or more bases have been added, deleted, or substituted.

[0053] 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.

[0054] 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.

[0055] In one embodiment, the antisense oligomer described herein targets the range of positions 115937 to 115981 of the genomic sequence (SEQ ID NO: 13) 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: 13 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: 13 include antisense oligomers containing a nucleotide sequence selected from the group consisting of SEQ ID NOs: 8 to 12.

[0056] 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.

[0057] The pharmaceutical compositions described herein may be formulated by appropriately blending a pharmaceutically acceptable carrier or additive (and optionally the sugar of the present invention). The pharmaceutically acceptable carrier or additive and formulation of the pharmaceutical composition are the same as those described for the precipitation inhibitor of the present invention, except that the antisense nucleic acid is an active ingredient.

[0058] In one embodiment, the precipitation inhibitor of the present invention is used and / or added to the pharmaceutical composition described herein in an amount such that the sugar concentration in the pharmaceutical composition is 0.5 mg / mL to 3000 mg / mL, for example, 2.6 mg / mL to 1694 mg / mL. In one embodiment, the precipitation inhibitor of the present invention is used and / or added to the pharmaceutical composition in an amount such that the sugar concentration in the pharmaceutical composition is 0.5 mg / mL or more, 1 mg / mL or more, 1.5 mg / mL or more, 2 mg / mL or more, 2.5 mg / mL or more, 3 mg / mL or more, 5 mg / mL or more, 10 mg / mL or more, 20 mg / mL or more, 30 mg / mL or more, 40 mg / mL or more, 50 mg / mL or more, or 100 mg / mL or more. Furthermore, the precipitation inhibitor of the present invention is used and / or added to the pharmaceutical composition in an amount such that the sugar concentration in the pharmaceutical composition is 3000 mg / mL or less, 2500 mg / mL or less, 2500 mg / mL or less, 2000 mg / mL or less, 1500 mg / mL or less, or 1000 mg / mL or less, for example.

[0059] In one embodiment, the concentration of the antisense oligomer in the pharmaceutical compositions described herein is 0.05 mg / mL to 2000 mg / mL, for example, 0.3 mg / mL to 350 mg / mL. In one embodiment, the concentration of the antisense oligomer in the pharmaceutical compositions described herein may be 0.05 mg / mL or more, 0.1 mg / mL or more, 0.2 mg / mL or more, 0.3 mg / mL or more, 0.4 mg / mL or more, 0.5 mg / mL or more, 1 mg / mL or more, 5 mg / mL or more, or 10 mg / mL or more, and may be 2000 mg / mL or less, 1500 mg / mL or less, 1000 mg / mL or less, 750 mg / mL or less, 500 mg / mL or less, 400 mg / mL or less, 350 mg / mL or less, 300 mg / mL or less, or 250 mg / mL or less.

[0060] In one embodiment, the present invention relates to a precipitation inhibitor for a pharmaceutical composition containing an antisense oligomer, the precipitation inhibitor comprising a saccharide, the saccharide being used in an amount such that the weight ratio of the saccharide relative to the antisense oligomer is 0.1 to 100, for example, 1 to 53. The pharmaceutical composition containing an antisense oligomer and the saccharide are as described above. In one embodiment, the precipitation inhibitor of the present invention is used in an amount such that the weight ratio of the saccharide relative 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, 1.2 or more, 2 or more, or 5 or more. In one embodiment, the precipitation inhibitor of the present invention is used in an amount such that the weight ratio of the saccharide relative to the antisense oligomer is 100 or less, 53 or less, 40 or less, 30 or less, 25 or less, 20 or less, 15 or less, or 10 or less.

[0061] In one embodiment, the precipitation inhibitor of the present invention is contained in the pharmaceutical composition described herein and administered together with the pharmaceutical composition described herein.For example, the antisense oligomer described herein may be lyophilized, optionally together with a carrier such as glucose, and dissolved in a solvent such as water for injection to form a pharmaceutical composition, which may then be mixed with the precipitation inhibitor described herein, and the amount may then be adjusted optionally with a solvent to administer to a subject.In addition, for example, the antisense oligomer described herein may be lyophilized together with the precipitation inhibitor of the present invention, which may then be dissolved in a solvent such as water for injection to form a pharmaceutical composition, and the amount may then be adjusted optionally with a solvent to administer to a subject.

[0062] In another embodiment, the precipitation inhibitor 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 precipitation inhibitor described herein may be administered separately to a subject. As used herein, "simultaneous" administration of the precipitation inhibitor and the pharmaceutical composition means that the precipitation inhibitor and the pharmaceutical composition are administered at the same time. As used herein, "sequential" administration of the precipitation inhibitor and the pharmaceutical composition means that they are administered at different times. Specifically, the precipitation inhibitor can be administered before or after the precipitation inhibitor. In this case, the interval between the administration of the precipitation inhibitor and the pharmaceutical composition is not limited, and may be, for example, several minutes, several hours, or even up to a day.

[0063] In this specification, subjects to which the pharmaceutical composition and / or deposition inhibitor 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.

[0064] The dosage when administering the pharmaceutical composition and / or the precipitation inhibitor can be adjusted taking into consideration the type of antisense oligomer contained in the pharmaceutical composition and the type of sugar contained in the precipitation inhibitor, the dosage form of the pharmaceutical composition and the precipitation inhibitor, 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 of the sugar can be, for example, For example, the daily dose can be within the range of 0.1 mg to 200 g / human, for example, 1 mg to 100 g / human, for example, 10 mg to 50 g / human, for example, 100 mg to 50 g / human, for example, 100 mg to 40 g / human, for example, 100 mg to 30 g / human, for example, 100 mg to 20 g / human, for example, 1 g to 20 g / human, for example, 2 g to 20 g / human, for example, 1 g to 10 g / human, for example, 100 mg to 1 g / human. The number and frequency of administration are not limited, and can be, for example, once to two or three times a day, with intervals of one to two or three days. Alternatively, for example, it can be administered once, followed by another administration a few days later, for a total of two administrations.

[0065] In one embodiment, the present invention relates to a method for inhibiting urinary precipitation of an antisense oligomer in a subject administered an antisense oligomer or a pharmaceutical composition containing an antisense oligomer, or a method for producing the pharmaceutical composition, the method comprising adding sugar to the pharmaceutical composition in an amount such that the sugar concentration in the pharmaceutical composition becomes 0.5 mg / mL to 3000 mg / mL. In this embodiment, the antisense oligomer, pharmaceutical composition, sugar, sugar concentration in the pharmaceutical composition, etc. are as described herein.

[0066] In one embodiment, the present invention relates to a method for inhibiting precipitation of an antisense oligomer in urine in a subject to which an antisense oligomer or a pharmaceutical composition containing the antisense oligomer has been administered, the method comprising administering a sugar or a precipitation inhibitor to the subject, wherein the sugar is administered in an amount such that the weight ratio of the sugar relative to the antisense oligomer is 0.1 to 100, where the weight ratio of the antisense oligomer is taken as 1. In this embodiment, the antisense oligomer, the pharmaceutical composition, the sugar, the precipitation inhibitor, the weight ratio of the sugar relative to the antisense oligomer is taken as 1, and the like are as described herein.

[0067] In one embodiment, the present invention relates to a pharmaceutical composition comprising an antisense oligomer and a sugar-containing precipitation inhibitor at a concentration of 0.5 mg / mL to 3000 mg / mL. In this embodiment, the antisense oligomer, pharmaceutical composition, sugar, and the sugar concentration in the pharmaceutical composition are as described herein. [Example]

[0068] Example 1: Preparation of antisense oligomers (PMOs) The antisense oligomers (PMO Nos. 1 to 12 (SEQ ID NOs: 1 to 12)) 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.

[0069] [Table 1]

[0070] "Group (1)" and "Group (2)" in the above table are as follows:

[0071] [ka]

[0072] Example 2: Preliminary study to evaluate the effect of sugar on inhibiting precipitation To set test conditions such as the concentrations of sugars and antisense oligomers in urine, D-mannitol, which corresponds to the reduced form of the monosaccharide D-mannose, and the antisense oligomer were administered to cynomolgus monkeys, and the urinary concentration shortly after administration was estimated from the plasma concentration transition and systemic clearance.

[0073] 20% D-mannitol was diluted to 50 mg / mL with saline and administered at 10 mL / kg to three male and three female cynomolgus monkeys aged 1 to 2 years. Plasma concentrations were measured at 0 hours (immediately after administration), 0.25 hours, 1 hour, 4 hours, 8 hours, and 24 hours after administration (Table 2, values ​​a, b, c, d, e, and f; each value is the average of three monkeys). The area under the plasma concentration-time curve (AUC 0-24 hr ), total body clearance (CL tot ) was calculated (Table 2, values ​​g and h). Furthermore, the plasma concentrations at 0 and 0.25 hours after administration (Table 2, values ​​a and b) were averaged to calculate the mean plasma concentration from 0 to 0.25 hours (Table 2, value i), and CL tot The amount of D-mannitol excreted per body weight up to 0.25 hours after administration was calculated using the data (Table 2, value h) (Table 2, value j). The urine volume up to 0.25 hours after administration was calculated based on the daily urine volume of a cynomolgus monkey (body weight: 5 kg), which is 375 mL (B Clark, DA Smith Pharmacokinetics and toxicity testing. Crit Rev Toxicol. 1984;12(4):343-85.) and estimated the urinary mannitol concentration 0.25 hours after administration as 3.91 mL (Table 2, value k).

[0074] [Table 2]

[0075] The estimated urinary concentrations obtained in this manner were averaged, and the urinary concentration shortly after administration of 500 mg / kg of D-mannitol was estimated to be 127.5 mg / mL. Because D-mannitol is hardly distributed in the body and is rapidly excreted in the urine without being metabolized, it can be assumed that there is a linear relationship between the administered dose and the urinary concentration. Therefore, using the following calculation formula, the urinary concentration was converted into the D-mannitol administered dose, and the administered dose was converted into the concentration in the formulation at the time of administration (when used) using the body weight and the volume of the administered solution.

[0076] D-mannitol dose (mg / kg) = urinary concentration (mg / mL) / 127.5 (mg / mL) × 500 (mg / kg) D-mannitol concentration in formulation (mg / mL) = D-mannitol dose (mg / kg) × body weight (kg) ÷ volume of solution administered (mL) At this time, the body weight was set to 3 to 80 kg, and the volume of the administered solution was set to 50 to 200 mL.

[0077] Sucrose and other sugars described herein are not reabsorbed in the renal tubules and exhibit pharmacokinetics similar to that of D-mannitol (AW Winkler, J Parra. J Clin Invest. 1937 Nov;16(6):859-67.), and therefore can be converted to the concentration in the formulation at the time of administration (use) using the same conversion formula. For example, a sugar solution with an estimated urinary concentration of 135 mg / mL can be converted to the urinary concentration when 529.4 mg / kg of sugar is administered, and it is assumed that a formulation with a sugar concentration of 7.9 to 847.1 mg / mL will be administered (used).

[0078] In addition, the antisense oligomer of PMO No. 12 (SEQ ID NO: 12) was dissolved in saline at 1.6 mg / mL and administered at 10 mL / kg to three 1-year-old male cynomolgus monkeys. The plasma concentrations were measured at 0 hours (immediately after the end of administration), 0.25 hours, 0.5 hours, 1 hour, 2 hours, 6 hours, and 24 hours after administration (Table 3, values ​​a, b, c, d, e, f, g; each value is the average of three monkeys). The area under the plasma concentration-time curve (AUC 0-24 hr ), total body clearance (CL tot) was calculated (Table 3, values ​​h and i). Furthermore, the plasma concentrations at 0 and 0.25 hours after administration (Table 3, values ​​a and b) were averaged to calculate the mean plasma concentration from 0 to 0.25 hours (Table 3, value j), and CL tot The amount of antisense oligomer excreted per unit body weight up to 0.25 hours after administration was calculated using the data (Table 3, value i) (Table 3, value k). The urine volume up to 0.25 hours after administration was taken as 3.91 mL, which is the daily urine volume of a cynomolgus monkey (body weight: 5 kg) of 375 mL, and the urinary antisense oligomer concentration 0.25 hours after administration was estimated (Table 3, value l).

[0079] [Table 3]

[0080] Since it can be assumed from Table 3 that there is a linear relationship between the antisense oligomer dose and urinary concentration, the urinary concentration was converted to the antisense oligomer dose using the following formula, and the antisense oligomer dose was converted to the concentration in the formulation at the time of administration (time of use) based on the body weight and the volume of the administration solution.

[0081] Antisense oligomer dose (mg / kg) = urinary concentration (mg / mL) / estimated urinary concentration in cynomolgus monkeys (mg / mL) × dose to cynomolgus monkeys (mg / kg) Antisense oligomer concentration in formulation (mg / mL) = antisense oligomer dose (mg / kg) × body weight (kg) ÷ volume of administration (mL) The estimated urinary concentration in cynomolgus monkeys was the value obtained when 60 mg / kg of antisense oligomer was administered. The body weight ranged from 3 to 80 kg, and the administration volume was 50 to 200 mL. For example, an antisense oligomer aqueous solution with an estimated urinary concentration of 24.75 mg / mL can be converted to the urinary concentration obtained when 43.41 mg / kg of antisense oligomer was administered, and it is assumed that a formulation with an antisense oligomer concentration of 0.7 to 69.5 mg / mL will be administered (used). Example 3: Evaluation of precipitation suppression effect by sucrose Of the antisense oligomers shown in Table 1, PMO Nos. 8, 9, 10, and 11 were evaluated for their usefulness for pharmaceutical applications by assessing the amount of precipitation in the presence of ions simulating those found in urine and the inhibitory effect of sucrose on precipitation. Test Method Each antisense oligomer was dissolved in water for injection or aqueous sucrose solution and mixed with an aqueous solution containing potassium chloride and sodium chloride, simulating urine. The evaluation conditions (solution composition) are shown in Tables 4 to 11. 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 varied depending on the antisense oligomer dose and urine volume, conditions under which precipitation was observed were established for each sequence using water for injection as the medium. Measurements were then conducted under conditions in which only the medium was replaced with aqueous sucrose solution, and the absorbances obtained under both conditions were 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 an aqueous sucrose solution was used as the medium, it was determined that the solution had a precipitation-inhibiting effect if 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.

[0082] [Table 4]

[0083] [Table 5]

[0084] [Table 6]

[0085] [Table 7]

[0086] [Table 8]

[0087] [Table 9]

[0088] [Table 10]

[0089] [Table 11]

[0090] Test results For all of the tested antisense oligomers, when sucrose was used as the medium, the time until precipitation was observed was delayed and the increase in absorbance was suppressed, indicating that sucrose inhibits the precipitation of antisense oligomers. The respective results are shown in Figures 1 to 8.

[0091] These results indicate that sucrose inhibits the precipitation of antisense oligomers in urine. Example 4: Evaluation of the inhibitory effect of sucrose on precipitation in the kidney Of the antisense oligomers shown in Table 1, PMO No. 8 (SEQ ID NO: 8) was evaluated for its precipitation from urine in the kidney and inhibition of precipitation by sucrose in order to verify its further usefulness for pharmaceutical applications. (1) Evaluation method The antisense oligomer of PMO No. 8 was dissolved in water for injection containing 0.9% sodium chloride, and a solution of sucrose (Fujifilm Wako Pure Chemical Industries, Ltd.) dissolved in saline was added to prepare administration solutions containing each antisense oligomer and sucrose at the concentrations listed in Tables 12 and 13.

[0092] These solutions were administered into the tail vein of 6-week-old male C57BL / 6J mice (N=3) at the doses (10 mL / kg) listed in Table 12. The following day, some of the mice administered the antisense oligomer of PMO No. 8 were necropsied, and formalin-fixed, paraffin-embedded kidney specimens were prepared. Thin sections of the specimens were stained with hematoxylin and eosin and subjected to histopathological examination.

[0093] [Table 12]

[0094] (2) Evaluation results Histopathological examination revealed basophilic substances in the lumen of renal tubules in the kidneys of mice administered the control solution containing no sucrose. This change was thought to reflect the precipitation of nucleic acids. The amount of precipitate decreased depending on the sucrose concentration, demonstrating an inhibitory effect on precipitation. The results are shown in Table 13 and Figures 9, 10, 11, and 12.

[0095] [Table 13]

[0096] Example 5: Evaluation of the inhibitory effect of sucrose on precipitation in the kidney Of the antisense oligomers shown in Table 1, PMO No. 11 (SEQ ID NO: 11) was evaluated for its precipitation from urine in the kidney and inhibition of precipitation by sucrose in order to verify its further usefulness for pharmaceutical applications. (1) Evaluation method The antisense oligomer of PMO No. 11 was dissolved in physiological saline to prepare a control administration solution containing the antisense oligomer at the concentrations shown in Table 14 but without sucrose.

[0097] Next, the antisense oligomer of PMO No. 11 and sucrose were dissolved in physiological saline to prepare an administration solution containing the antisense oligomer and sucrose at the concentrations shown in Table 14.

[0098] These solutions were administered at 20 mL / kg into the tail vein of 6-week-old male C57BL / 6J mice (N=5). The following day, the mice were necropsied, and formalin-fixed, paraffin-embedded kidney specimens were prepared. Thin sections of the specimens were stained with hematoxylin and eosin and subjected to histopathological examination. Sucrose-containing solutions were judged to have suppressed precipitation if their kidney histopathological findings improved compared to those of mice administered solutions without sucrose. (2) Evaluation results The antisense oligomer tested improved the findings in histopathological examination of the kidneys of mice administered with a sucrose-containing administration solution, confirming that sucrose inhibits precipitation. The results are shown in Table 14.

[0099] [Table 14]

[0100] Example 6: Evaluation of precipitation suppression effect of sucrose Of the antisense oligomers shown in Table 1, PMO Nos. 1, 2, and 7 were evaluated for their usefulness for pharmaceutical applications by assessing the amount of precipitation in the presence of ions simulating those found in urine and the inhibitory effect of sucrose on precipitation.

[0101] Test Method Each antisense oligomer was dissolved in water for injection or aqueous sucrose solution and mixed with an aqueous solution containing potassium chloride and sodium chloride, simulating urine. The evaluation conditions (solution compositions) are shown in Tables 4-11 and 15-17. 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 varied depending on the antisense oligomer dose and urine volume, conditions under which precipitation was observed were established for each sequence using water for injection as the medium. Measurements were then conducted under the same conditions, with only the medium replaced with aqueous sucrose solution, and the absorbances obtained under both conditions were 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 an aqueous sucrose solution was used as the medium, it was determined that the solution had a precipitation-inhibiting effect if 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.

[0102] [Table 15] [Table 16] [Table 17]

[0103] Test results For all of the tested antisense oligomers, when sucrose was used as the medium, the time until precipitation was observed was delayed and the increase in absorbance was suppressed, indicating that sucrose inhibits the precipitation of antisense oligomers. The respective results are shown in Figures 13 to 15. These results indicate that sucrose inhibits the precipitation of antisense oligomers in urine.

[0104] Example 7: Evaluation of precipitation-inhibiting effect of trehalose Of the antisense oligomers shown in Table 1, PMO Nos. 1, 2, 7, and 8 were evaluated for their usefulness for pharmaceutical applications by assessing the amount of precipitation in the presence of ions simulating those found in urine and the inhibitory effect of trehalose on precipitation.

[0105] Test Method Each antisense oligomer was dissolved in water for injection or aqueous trehalose solution and mixed with an aqueous solution containing potassium chloride and sodium chloride, simulating urine. The evaluation conditions (solution composition) are shown in Tables 18 to 21. 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. Measurements were then conducted under conditions in which only the medium was replaced with aqueous trehalose solution, and the absorbances obtained under both conditions were 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 trehalose 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 precipitation-inhibiting effect was present.

[0106] [Table 18] [Table 19] [Table 20] [Table 21]

[0107] Test results For all of the tested antisense oligomers, when trehalose was used as the medium, the time until precipitation was observed was delayed and the increase in absorbance was suppressed, indicating that trehalose inhibits the precipitation of antisense oligomers. The respective results are shown in Figures 16 to 19.

[0108] These results demonstrate that trehalose inhibits the precipitation of antisense oligomers in urine.

Claims

1. An agent for inhibiting the precipitation of antisense oligomers in urine in a subject to which a pharmaceutical composition containing an antisense oligomer has been administered, the agent containing a sugar other than glucose, and used in an amount such that the concentration of the sugar in the pharmaceutical composition is 0.5 mg / mL to 3000 mg / mL.

2. The precipitation inhibitor according to claim 1, which is used in an amount such that the sugar concentration in the pharmaceutical composition is 2.6 mg / mL to 1694.1 mg / mL.

3. The precipitation inhibitor according to claim 1 or 2, wherein the concentration of the antisense oligomer in the pharmaceutical composition is 0.05 mg / mL to 2000 mg / mL.

4. An agent for inhibiting the precipitation of antisense oligomers in urine in a subject to which a pharmaceutical composition containing an antisense oligomer has been administered, the agent containing a sugar other than glucose and used in an amount such that the weight ratio of the sugar to the antisense oligomer is 0.1 to 100.

5. The precipitation inhibitor according to claim 4, which is used in an amount such that the weight ratio of sugar to antisense oligomer is 1 to 53.

6. The deposition inhibitor according to any one of claims 1 to 5, wherein the sugar is a disaccharide sugar.

7. The precipitation inhibitor according to any one of claims 1 to 6, wherein the sugar is sucrose.

8. The precipitation inhibitor according to any one of claims 1 to 6, wherein the sugar is trehalose.

9. The precipitation inhibitor according to any one of claims 1 to 8, wherein the antisense oligomer is a morpholino oligomer.

10. The precipitation inhibitor according to any one of claims 1 to 9, wherein the antisense oligomer is a phosphorodiamidate morpholino oligomer.

11. The 5' end of the antisense oligomer has the following chemical formulas (1) to (2): 【Chemical 1】 The deposition inhibitor according to any one of claims 1 to 10, wherein the precipitation inhibitor is any one of the groups shown below.

12. The precipitation inhibitor according to any one of claims 1 to 11, wherein the antisense oligomer contains four consecutive purine bases in its base sequence.

13. 13. The precipitation inhibitor according to claim 12, wherein at least two of the four consecutive purine bases are guanine.

14. The precipitation inhibitor according to any one of claims 1 to 13, wherein the antisense oligomer comprises a base sequence selected from the group consisting of SEQ ID NOs: 1 to 12.

15. A method for inhibiting precipitation of antisense oligomers in urine in a subject to whom a pharmaceutical composition containing an antisense oligomer has been administered, the method comprising adding a sugar other than glucose in an amount such that the concentration of the sugar in the pharmaceutical composition becomes 0.5 mg / mL to 3000 mg / mL.

16. A method for suppressing the precipitation of antisense oligomers in urine in a subject to whom an antisense oligomer has been administered, the method comprising administering a sugar other than glucose to the subject, in an amount such that the weight ratio of the sugar to the antisense oligomer is 0.1 to 100.

17. A pharmaceutical composition comprising an antisense oligomer, the pharmaceutical composition comprising an inhibitor of antisense oligomer precipitation in urine of a subject to which the pharmaceutical composition is administered, the inhibitor comprising a sugar other than glucose, at a concentration of 0.5 mg / mL to 3000 mg / mL.

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