Methods for reducing toxicity of antisense nucleic acids

Abstract

The purpose of the present invention is to reduce the strong hepatotoxicity of antisense nucleic acids that incorporate an artificial nucleobase (e.g., LNA). The present invention provides a bridged antisense nucleic acid for which antisense nucleic acid-induced toxicity is reduced by the supplemental addition to the wing region of a specific artificial nucleobase (e.g., MCA), while retaining the sequence of the antisense nucleic acid and the number of artificial nucleobases (e.g., LNA). The present invention also provides an antisense nucleic acid drug having a remarkably-reduced hepatotoxicity.

Description

[Technical field]

[0001] The present invention relates to a bridged antisense nucleic acid that has an activity of inhibiting or controlling the expression of a target gene by its antisense effect and reduces the toxicity of the antisense nucleic acid. More specifically, the present invention relates to a bridged antisense nucleic acid that can maintain or enhance the antisense effect while reducing side effects by additionally adding and / or inserting a 2'-modified nucleic acid into the base sequence of the bridged nucleic acid. [Background technology]

[0002] In recent years, oligonucleotides have been developed as pharmaceuticals known as nucleic acid drugs, and in particular, from the viewpoint of high selectivity for target genes, the development of nucleic acid drugs using the antisense method has been actively promoted. The antisense method is a method of selectively inhibiting or controlling the expression of a protein encoded by a target gene by introducing into cells an oligonucleotide (antisense nucleic acid (ASO)) that is complementary to a partial sequence of the mRNA (sense strand) of the target gene.

[0003] PS-type ASOs make up the majority of pipelines undergoing clinical trials as ASOs. PS-type ASOs are phosphorothioated (PS-conjugated) by replacing one of the oxygen atoms of the phosphate group of the phosphodiester bond, a natural nucleic acid bond, with a sulfur atom. PS-conjugation has been shown to improve nuclease resistance (Non-Patent Document 1) and uptake into cells (Non-Patent Document 2), but is known to cause various side effects such as hepatotoxicity (Non-Patent Document 3).

[0004] One representative example of PS-type ASO is mipomersen sodium (trade name: Kynamro), developed by Ionis Pharmaceuticals as an anticholesterolemia therapeutic drug with a gapmer structure, which is called a second-generation nucleic acid structure. In 2013, it was approved in the United States as the world's first systemically administered nucleic acid drug for homozygous familial hypercholesterolemia, but various side effects such as liver toxicity of PS-type ASO were recognized. In addition, it has not been approved in Europe due to side effects (Non-Patent Document 4).

[0005] In addition to the above, cross-linked artificial nucleic acids such as 2',4'-BNA (also known as LNA), cEt, ENA, and AmNA, which have improved binding affinity to target RNA, are known as ASOs. All of these are characterized by being 2',4'-modified, and are expected to improve the efficacy of ASOs due to their high binding strength to target RNA, improved activity, and shortened ASOs (Non-Patent Document 5).

[0006] Recently, attempts have been made to reduce liver toxicity in bridged antisense nucleic acids by chemically modifying the gap portion that constitutes them (Patent Document 1). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2018 / 155450 [Non-patent literature]

[0008] [Non-Patent Document 1] Stein, CA, et al., Nucleic Acids Res., 16, 3209-3221 (1988) [Non-Patent Document 2] Zhao, Q., et al., Antisense Res. Dev., 3, 53-66 (2009), doi:10.1089 / ard.1993.3.53 [Non-Patent Document 3] Agrawal S., Trends Biotechnol., 14, 376-87 (1996) [Non-Patent Document 4] European Medicines Agency, EMA / 792736 / 2012, EMEA / H / C / 002429, http: / / www.buenavistainv.com / dbimages / Isis%20Presentation.pdf [Non-Patent Document 5] Satoshi Kobiga and Yuya Kasahara, Japanese Pharmacological Journal, 148, 100-104 (2016) Summary of the Invention [Problem to be solved by the invention]

[0009] As described above, antisense nucleic acids that incorporate artificial nucleic acid bases (LNA) are useful for clinically effective antisense nucleic acids, but many of these exhibit strong hepatotoxicity. On the other hand, as a method for avoiding this toxicity, it is possible to reduce the number of LNAs to be introduced or to change the sequence of the antisense nucleic acid itself, but this often results in a decrease in activity as a pharmaceutical. Therefore, the present invention aims to provide a crosslinked antisense nucleic acid and a pharmaceutical that reduce hepatotoxicity and maintain or enhance the activity of the antisense nucleic acid. [Means for solving the problem]

[0010] Therefore, the inventors conducted research with the aim of providing such antisense nucleic acid pharmaceuticals, and as a result, they discovered that the toxicity of antisense nucleic acids can be reduced by adding and / or inserting specific artificial nucleic acid bases while maintaining the sequence of the antisense nucleic acid and the number of LNAs, thereby completing the present invention.

[0011] That is, the present invention is as follows. [1] A bridged antisense nucleic acid having a gap region consisting of 5 to 15 bases of deoxyribonucleic acid, and wing regions consisting of 2 to 10 2',4'-modified nucleic acids at the 5' and 3' ends of the gap region, respectively, wherein an additional 1 to 4 2'-modified nucleic acids have been added and / or inserted into at least one of the wing regions. [2] The 2'-modified nucleic acid has the following structural formula:

[0012] [ka]

[0013] [In the formula, R 1 and R 2 is independently selected from the group consisting of H, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aralkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, and a substituted or unsubstituted aryl group; R 3 is H, or the structure:

[0014] [ka]

[0015] (In the formula,

[0016] [ka]

[0017] is the point of attachment to the adjacent nucleic acid, or OH; and X is S or O. and; R 4 is H or a point of attachment to an adjacent nucleic acid; and B represents a residue of a nucleic acid base which may have a protecting group or a modifying group. The crosslinked antisense nucleic acid according to [1], [3] R 1 is H and R 2 The bridged antisense nucleic acid according to [2], wherein [4] The bridged antisense nucleic acid according to any one of [1] to [3], wherein one or two 2'-modified nucleic acids are added and / or inserted in each of the wing regions. [5] The 2',4'-modified nucleic acid is

[0018] [ka]

[0019] [In the formula, R 5 and R 8 are each independently selected from the group consisting of H, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aralkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, and a substituted or unsubstituted aryl group; R 6 is H, or the structure:

[0020] [ka]

[0021] (In the formula,

[0022] [ka]

[0023] is the point of attachment to the adjacent nucleic acid, or OH; and X is S or O. and; R 7 is H or a point of attachment to an adjacent nucleic acid; and B represents a residue of a nucleic acid base which may have a protecting group or a modifying group. The crosslinked antisense nucleic acid according to any one of [1] to [4], selected from the group consisting of: [6] The bridged antisense nucleic acid according to any one of [1] to [5], which comprises 2 to 4 2',4'-modified nucleic acids. [7] The bridged antisense nucleic acid according to any one of [1] to [6], wherein X is a sulfur atom. [8] The bridged antisense nucleic acid according to any one of [1] to [7], wherein the deoxyribonucleic acid has a length of 8 to 10 bases. [9] An antisense nucleic acid drug comprising the crosslinked antisense nucleic acid according to any one of [1] to [8] above, and having reduced toxicity caused by the antisense nucleic acid. Effect of the Invention

[0024] According to the present invention, by adding and / or inserting a 2'-modified nucleic acid into the wing region of a bridged antisense nucleic acid, which is composed of a bridged nucleic acid, it is possible to reduce the toxicity caused by conventional bridged antisense nucleic acids and provide an antisense nucleic acid with improved medicinal efficacy. [Brief description of the drawings]

[0025] [Figure 1] The results of purification and analysis of the synthesized GTCTGTGCATCTCTCC (SEQ ID NO: 1) by reversed-phase HPLC are shown. [Diagram 2] The results of purification and analysis of the synthesized GTcTGTGCATCTCtCC (sequence number 2) by reversed-phase HPLC are shown. [Diagram 3] The results of purification and analysis of the synthesized GTCtGTGCATCTcTCC (SEQ ID NO: 3) by reverse-phase HPLC are shown. [Figure 4] The results of purification and analysis of the synthesized cGTCTGTGCATCTCTCCt (SEQ ID NO: 4) by reverse-phase HPLC are shown. [Diagram 5] The results of purification and analysis of the synthesized CGtCTGTGCATCTCtCCT (SEQ ID NO: 5) by reverse-phase HPLC are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The present invention relates to a bridged antisense nucleic acid comprising a 2'-modified nucleic acid, which has reduced toxicity, and a therapeutic agent comprising the bridged antisense nucleic acid.The present invention is described in detail below.

[0027] (1) Cross-linked antisense nucleic acid The bridged antisense nucleic acid of the present invention comprises a gap region consisting of deoxyribonucleic acid, and wing regions having 2',4'-modified nucleic acids at the 5' and 3' ends of the gap region, respectively, and is characterized in that an additional 2'-modified nucleic acid has been added and / or inserted into each of the wing regions.

[0028] The bridged antisense nucleic acid of the present invention is a conventional antisense nucleic acid ("L6") having a structure consisting of a gap region (a region consisting of "DNA", the binding mode for linking each nucleic acid to another is not limited, but may be a phosphorothioate bond or a phosphodiester bond, preferably a phosphorothioate bond) (which may be of any length, preferably 8 to 10 bases) consisting of a base sequence complementary to the base sequence of a target nucleic acid, and wing regions (for example, in FIG. 1, the two regions consisting of "LNA" are underlined) located on both ends of the gap region. D10"), a 2'-modified nucleic acid ("MCE") has been additionally added and / or inserted at the end or within the wing region (for example, MCE is shown in lowercase in Figure 2).

[0029] The wing region constituting the crosslinked antisense nucleic acid of the present invention includes or consists of a 2',4'-modified nucleic acid, and as long as it has the effect of reducing the toxicity of the crosslinked antisense nucleic acid and contributes to the effect, the type and number of 2',4'-modified nucleic acids are not limited (see, for example, W. Brad Wan and Punit P. Seth, J. Med. Chem. 2016, 59, 9645-9667). The bond mode for linking each nucleic acid to another is also not limited, but may be a phosphorothioate bond or a phosphodiester bond, and is preferably a phosphorothioate bond. Note that the "phosphorothioate bond" is a phosphorothioate (PS) bond formed by replacing one of the oxygen atoms of the phosphate group of a phosphodiester bond, which is a natural internucleic acid bond, with a sulfur atom.

[0030] According to the present invention, the 2',4'-modified nucleic acid can be, but is not limited to, the following structural formula:

[0031] [ka]

[0032] [In the formula, R 5 and R 8 are each independently selected from the group consisting of H, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aralkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, and a substituted or unsubstituted aryl group; R 6 is H, or the structure:

[0033] [ka]

[0034] (In the formula,

[0035] [ka]

[0036] is the point of attachment to the adjacent nucleic acid, or OH; and X is S or O. and; R 7 is H or a point of attachment to an adjacent nucleic acid; and B represents a residue of a nucleic acid base which may have a protecting group or a modifying group. The above B represents a residue of a nucleic acid base which may have a protecting group or a modifying group. Examples of the nucleic acid base include natural nucleic acid bases such as adenine, cytosine, guanine, uracil, thymine, and 5-methylcytosine, as well as non-natural nucleic acid bases such as 2-thiouracil, 2-thiothymine, 2-thiocytosine, 2-thio-5-methylcytosine, 2,4-diaminopurine, 6-thioguanine, uracil-5-yl, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine. The protecting group or modifying group introduced onto the nucleic acid base may be an amino group protected with one or two acyl groups, or an amidine-type protecting group. The oxygen atom of the keto group may be tautomerized to a hydroxyl group and protected with an alkyl group such as a cyanoethyl group, a 2-(4-nitrophenyl)ethyl group, a 2-nitrobenzyl group, or an acyloxymethyl group, or an acyl group such as a diphenylcarbamoyl group. In the case of a uracil base and a thymine base, the N3 position may be protected with an alkyl group or an acyl group. In addition, the carbon atoms of the above natural nucleobases and non-natural nucleobases may contain an amino group, a halogen group, an acyl group, a cyano group, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, an aralkyl group, an alkoxy group, an alkylthio group, an acylamino group, a carbamoyl group, a carboxy group, an acyloxy group, or the like, or may contain a fluorescent functional group, a biotinyl group, an amino group, a spin label, or the like.

[0037] As is well known, the sugar moiety of natural nucleic acids (DNA and RNA) has a five-membered ring consisting of four carbon atoms and one oxygen atom, and the sugar moiety takes two types of conformation, N-type and S-type. Since the mRNA targeted by the ASO described above mainly has an N-type sugar chain and an A-type helical structure, it is important that the sugar chain of the ASO also takes the N-type from the viewpoint of increasing affinity for RNA. Modified nucleic acids such as LNA (Locked Nucleic Acid; 2'-O,4'-C-methylene-bridged nucleic acid (2',4'-BNA / LNA)) have been developed based on this concept. For example, in LNA, the oxygen at the 2' position and the carbon at the 4' position are bridged by a methylene group, so that the conformation is fixed to the N type and there is no fluctuation between conformations. Therefore, oligonucleotides synthesized by incorporating several units of LNA have extremely high binding strength and sequence specificity to RNA, and also exhibit excellent heat resistance and nuclease resistance, compared to oligonucleotides synthesized with conventional natural nucleic acids. Other artificial nucleic acids also have such properties and can therefore be used in the present invention.According to the present invention, the 2',4'-modified nucleic acid is preferably LNA.

[0038] As described above, the number of 2'-modified nucleic acids constituting the wing region is not limited as long as it is a number that can reduce the toxicity of the crosslinked antisense nucleic acid, but is 1 to 10 bases, preferably 1 to 5 bases, and more preferably 1 to 3 bases.

[0039] According to the present invention, the 2'-modified nucleic acid added and / or inserted into the wing region may have, but is not limited to, the following structural formula:

[0040] [ka]

[0041] [In the formula, R 1 and R 2is independently selected from the group consisting of H, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aralkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, and a substituted or unsubstituted aryl group; R 3 is H, or the structure:

[0042] [ka]

[0043] (In the formula,

[0044] [ka]

[0045] is the point of attachment to the adjacent nucleic acid, or OH; and X is S or O. and; R 4 is H or a point of attachment to an adjacent nucleic acid; B represents a residue of a nucleic acid base which may have a protecting group or a modifying group. Here, the term "alkyl group" refers to a linear or branched saturated aliphatic hydrocarbon group having 1 to 20 carbon atoms, such as a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, and a cyclohexyl group. The term "aralkyl group" refers to a group in which the above-mentioned alkyl group is further substituted with an aryl group, such as a benzyl group and a diphenylmethyl group. The term "alkenyl group" refers to a linear or branched unsaturated aliphatic hydrocarbon group having 2 to 20 carbon atoms and containing 1 to 3 carbon-carbon double bonds, such as an allyl group and a homoallyl group. The term "alkynyl group" refers to a linear or branched unsaturated aliphatic hydrocarbon group having 2 to 20 carbon atoms and containing 1 to 3 carbon-carbon triple bonds, such as a propargyl group. The term "aryl group" refers to a monovalent residue derived from a compound or derivative thereof that contains benzene or a structure in which two or more benzenes are condensed or bonded, such as a phenyl group, naphthyl group, etc. The term "alkyl group having a substituent" refers to a group in which the above alkyl group is further substituted with an amino group, a halogen group, an acyl group, a cyano group, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, an aralkyl group, an alkoxy group, an alkylthio group, an acylamino group, a carbamoyl group, a carboxy group, or an acyloxy group, such as a 2,2,2-trifluoroethyl group, a cyanomethyl group, a 2,2,2-trichloroethyl group, a methoxypropyl group, or a methylthiopropyl group. The term "substituted aralkyl group" refers to the above aralkyl group further substituted with an amino group, a halogen group, an acyl group, a cyano group, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, an aralkyl group, an alkoxy group, an alkylthio group, an acylamino group, a carbamoyl group, a carboxy group, or an acyloxy group, such as 4-chlorobenzyl group, 4-nitrobenzyl group, and 2-nitrobenzyl group. The term "substituted alkenyl group" refers to, for example, a dichloroallyl group. The term "substituted aryl group" refers to, for example, a 4-nitrophenyl group.

[0046] In one embodiment of the present invention, the substituent R 1 is H and R2 is a methyl group. The present inventors have reported that RNase H-dependent antisense oligonucleotides incorporating such modified nucleic acids, i.e., 2'-O-(2-N-methylcarbamoylethyl) (MCE) nucleic acids, have reduced hepatotoxicity compared to antisense oligonucleotides having conventional 2'-O-methyloxyethyl (MOE) modifications (see Masaki, Y., et al., Nucleic Acid Therapeutics, 28, 307-311 (2018)). Note that the antisense oligonucleotides used here have all modified nucleic acids in the wing region that are MCE or MOE, and do not include bridged nucleic acids such as LNA, which is different from the present invention.

[0047] The crosslinked antisense nucleic acid of the present invention is one in which a 2'-modified nucleic acid (typically, MCE) is additionally added and / or inserted into the wing region, and the number of such nucleic acids is not limited, but may be 1 to 8, for example, 1 to 5, 1 to 4, 1 to 3, 2 to 5, 2 to 4, 3 to 5, 6, 5, 4, 3, 2, or 1. When a 2'-modified nucleic acid is added, the position is at both ends of each wing region composed of a 2',4'-modified nucleic acid, and the nucleic acid may be introduced into the gap region by substituting a deoxyribonucleotide residue in the gap region. On the other hand, when a 2'-modified nucleic acid is inserted, the position is not limited as long as it is between any of the 2',4'-modified nucleic acids. Furthermore, the nucleic acid sequences of the 5'-side wing region and the 3'-side wing region after the 2'-modified nucleic acid is added and / or inserted may be the same or different.

[0048] The bridged antisense nucleic acid of the present invention can be easily and routinely produced through the well-known technique of solid-phase synthesis. For example, it may be produced according to the synthesis method using the phosphoramidite method described in the literature by Masaki et al. (supra) in which MCE-introduced RNase H-dependent antisense oligonucleotides were reported. Alternatively, it may be produced by applying the liquid phase synthesis method by the improved H-phosphonate method described by Reese et al. (see Colin B. Reese and Hongbin Yan, J. Chem. Soc., Perkin Trans., 1, 2002, 2619-2633) or a similar method to the solid phase synthesis method. The bridged antisense nucleic acid of the present invention is synthesized in vitro and does not include antisense compositions of biological origin or gene vector constructs designed to direct the in vivo synthesis of antisense molecules. The molecules of the invention may also be mixed, encapsulated, conjugated, or combined with other molecules, molecular structures, or mixtures of compounds, for example, in liposomes, receptor-targeted molecules, oral, rectal, topical, or other formulations to aid uptake, distribution, and / or absorption.

[0049] The bridged antisense nucleic acid of the present invention has at least one 2'-modified nucleic acid additionally added and / or inserted in the wing region, and thus has reduced hepatotoxicity compared to the unmodified antisense oligonucleic acid (see Example 2). The term "reduced" refers to the fact that the value of a marker that is an indicator of hepatotoxicity when the bridged antisense nucleic acid of the present invention is administered is lower than that when the unmodified antisense oligonucleic acid is administered. For example, after administration of the bridged antisense nucleic acid, the serum concentrations of ALT (alanine aminotransferase), AST (aspartate aminotransferase) and LDH (lactate dehydrogenase), which are indicators of hepatotoxicity, are 60% or less, for example, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, or 5% or less, of the serum concentrations measured under the same conditions except that the unmodified antisense oligonucleic acid is used.

[0050] (2) Therapeutic drugs As demonstrated in Example 2 described later, the crosslinked antisense nucleic acid of the present invention has reduced toxicity and maintains its activity as an antisense nucleic acid. According to the present invention, it can be provided as a therapeutic drug (medicine, formulation, or pharmaceutical composition) in the same way as a commonly used antisense nucleic acid. When the crosslinked antisense nucleic acid of the present invention is used as a therapeutic drug, the type of genetic disease is not particularly limited. The "deoxyribonucleic acid" constituting the gap region incorporated into the crosslinked antisense nucleic acid can target non-coding RNA (microRNA, ribosomal RNA, tRNA, etc.), mRNA, single-stranded DNA, etc. present in the living body, so that the entire sequence or a part of the sequence of these can be used as a "target nucleic acid" and an antisense nucleic acid containing a single-stranded nucleic acid consisting of a base sequence completely or sufficiently complementary to the base sequence of the target nucleic acid can be used as a therapeutic drug for treating and / or preventing a genetic disease corresponding to the target nucleic acid. In addition, the length of the deoxyribonucleic acid is generally appropriate to be about 10 bases, but is not particularly limited as long as it is in the range of 5 to 15 bases. It is preferably 8 to 10 bases, more preferably 10 bases. Furthermore,

[0051] In the present specification, "(single-stranded nucleic acid) consisting of a base sequence sufficiently complementary (to the base sequence of the target nucleic acid)" may be a base sequence that can pair with 50% or more and less than 100%, preferably 60% or more and less than 100%, more preferably 70% or more and less than 100%, even more preferably 80% or more and less than 100%, and even more preferably 90% or more and less than 100% of the bases in the base sequence of the target nucleic acid. More specifically, examples of such a case include a case in which, in a nucleic acid chain consisting of a base sequence completely complementary to the base sequence of the target nucleic acid, 1 or 2 to 4 bases are replaced with other bases, and the nucleotide residue at the replaced position is unable to pair (in this case, the position replaced with the other base is called a "mismatch site"); a case in which, in a nucleic acid chain consisting of a base sequence completely complementary to the base sequence of the target nucleic acid, 1 or 2 to 4 bases are deleted, and the nucleotide residue at the deleted position is unable to pair.

[0052] When the crosslinked antisense nucleic acid of the present invention is used as a pharmaceutical composition, in addition to the crosslinked antisense nucleic acid as an active ingredient, a medicamentously acceptable carrier, diluent, excipient, etc. can be included. The term "pharmaceutical acceptable" refers to molecular entities and compositions that are physiologically tolerated and generally do not cause allergic or similar adverse reactions, such as acute gastric peristalsis, when administered to a patient. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Such pharmaceutical carriers may be inert liquids such as water and oils, including oils of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water or saline, as well as aqueous dextrose and glycerol solutions, are preferably used as carriers, particularly as injection solutions.

[0053] In a more specific embodiment of the present invention, a pharmaceutical composition is provided that includes a therapeutically effective amount of a crosslinked antisense nucleic acid together with a pharma- ceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant, and / or carrier. Such compositions include additives such as various buffers (e.g., Tris-HCl, acetate, phosphate), diluents of pH and ionic strength, detergents and solubilizers (e.g., Tween 80, polysorbate 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol), and bulking agents (e.g., lactose, mannitol). Pharmaceutical compositions may also be prepared in liquid form or in dry powder form, such as lyophilized form.

[0054] The pharmaceutical compositions provided according to the present invention are preferably administered by, but not limited to, injection, oral, pulmonary, or nasal routes, and more preferably delivered by intravenous, intraarterial, intraperitoneal, intramuscular, or subcutaneous routes of administration.

[0055] (3) Kit The present invention provides a kit for treating and / or preventing a patient having a genetic disease, the kit comprising at least one bridged antisense nucleic acid packaged in a suitable container and instructions for its use.

[0056] The contents of the kit may be lyophilized, and the kit may further comprise a suitable solvent for reconstituting the lyophilized components. Individual components of the kit may be packaged in separate containers with a notice in the form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceutical or biological products, reflecting approval by the agency of such manufacture, use, or sale for human administration.

[0057] When the components of the kit are provided in one or more liquid solutions, the liquid solution may be an aqueous solution, such as a sterile aqueous solution. For in vivo use, the expression construct may be formulated into a pharma- ceutically acceptable injectable composition. In this case, the container means may be a single inhaler, syringe, pipette, eyedropper, or other similar device from which the formulation may be applied to the affected area of ​​an animal, injected into an animal, or applied to and mixed with other components of the kit.

[0058] The components of the kit may also be provided in dry or lyophilized form. When a reagent or component is provided as a dry form, reconstitution is generally accomplished by the addition of a suitable solvent. It is contemplated that the solvent may also be provided in another container means. Regardless of the number or type of containers, the kit of the present invention may include or be packaged with a device to aid in the injection / administration or placement of the final complex composition in the animal's body. Such a device may be an inhaler, syringe, pipette, tweezers, measuring spoon, eye dropper, or any medically accepted delivery vehicle.

[0059] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples in any way. EXAMPLES

[0060] Example 1: Synthesis of bridged antisense nucleic acid The bridged antisense nucleic acids shown in Figures 1 to 6, which target factor XI as a gene, were synthesized on an automatic DNA synthesizer by solid-phase synthesis using the phosphoramidite method known to those skilled in the art, and were purified and analyzed by reversed-phase HPLC. The underlined parts represent LNA, the capital letters represent DNA, and the lowercase letters represent MCE nucleic acids. All nucleic acids are linked by phosphorothioates.

[0061] L6 D10: GTC TGTGCATCTC TCC (SEQ ID NO:1) L4 M2 D10: GT cTGTGCATCTCt CC (SEQ ID NO:2) L6 M2 D8: GTC tGTGCATCTc TCC (SEQ ID NO:3) L6 M2 D10:c GTC TGTGCATCTC TCC t (SEQ ID NO: 4) L6 TM2 D10: CG tCTGTGCATCTCt CCT (SEQ ID NO:5) L4 M2 D10 mid: G t C TGTGCATCTC T c C (SEQ ID NO:6) L5 M1 D10 5: GT cTGTGCATCTC TCC (SEQ ID NO:7) L6 M1 D9 nat: GTC TgTGCATCTC TCC (SEQ ID NO:8) L6 M2 D10 mm:t GTC TGTGCATCTC TCC c (SEQ ID NO: 9) The name of the bridged antisense nucleic acid is written with the number of "L (LNA)", "D (deoxyribonucleic acid of the factor XI gene)", and "M (MCE)". For example, "L4 M2 "D10" is composed of 4 LNAs, 2 MCEs, and 10 DNAs, and the nucleic acid arrangement is as shown above.

[0062] Example 2: Evaluation of hepatotoxicity and activity of bridged antisense nucleic acids (1) Administration and Feed Collection A saline solution of the cross-linked antisense nucleic acid (3 mg / mL) was subcutaneously administered at 10 mL per kg of body weight to 5-week-old female C57BL / 6JJcl mice. After 72 hours, all mice were anesthetized with isoflurane, and blood was collected from the heart using a 23G needle and a syringe containing 10,000 units / 10 mL of sodium heparin, and plasma was collected. The abdominal aorta was then incised to kill the mice by exsanguination, and the liver was then collected. A portion of the liver was trimmed.

[0063] (2) Quantitative PCR analysis of target gene expression The liver was homogenized using TRIzol reagent (Invitrogen, product number: 15596018) and total RNA was extracted. A portion of the prepared sample was treated with DNase and purified using the RNeasy Mini Kit (QIAGEN, product number: 74104). cDNA was synthesized using the 1st Strand cDNA Synthesis Kit for RT-PCR (AMV) (Roche, product number: 11483188001). Oligo-p(dT) 15The primers were used to synthesize the cDNA in a 20 μL reaction system. After cDNA synthesis, 20 μL of sterile water was added to make a total of 40 μL. The mRNA of each gene was quantified using LightCycler FastStart DNA Master SYBR Green I (Roche, product number: 12239264001).

[0064] (3) Measurement of ALT, AST, and LDH After administration of the cross-linked antisense nucleic acid, serum concentrations of ALT (alanine aminotransferase), AST (aspartate aminotransferase), and LDH (lactate dehydrogenase), which are indicators of liver toxicity, were measured using the JSCC standardized method. D10 showed high ALT, AST, and LDH values, whereas other cross-linking antisenses including MCE showed low ALT, AST, and LDH values. M2 D10, L6 M2 D8, L6 M2 D10 and L6 TM2 The values ​​of D10 are, respectively, L6 The toxicity was reduced by about 100 times compared to that of L4, 0.03 times, 0.03 times, 0.04 times, and 0.06 times that of D10. On the other hand, the FXI / GAPDH (%), which is an index of antisense activity, was significantly lower than that of L4. M2 D10, L6 M2 D8, L6 M2 D10 and L6 TM2 The values ​​of D10 are, respectively, L6 The antisense activity was reduced to only 4.5-fold, 8-fold, 2.5-fold, and 2-fold that of D10, demonstrating that the hepatotoxicity was reduced while maintaining the antisense activity.

[0065] [Table 1]

[0066] In addition to the above, similar experiments were carried out on four other cross-linked antisense nucleic acids that were synthesized separately. For these cross-linked antisense nucleic acids, the hepatotoxicity and activity for AST and ALT were measured.

[0067] [Table 2]

[0068] Comparing Table 1 and Table 2, the L6 without MCE shown in Table 1 D10 showed high ALT and AST values, whereas other cross-linking antisenses including MCE showed low ALT, AST, and LDH values. M2 D10 mid, L5 M1 D10 5, L6 M1 D9 nat, L6 M2 D10 The value of mm is L6 The toxicity was reduced by 0.04, 0.04, 0.02, and 0.13 times that of D10, on the order of about 10-100 times. On the other hand, the FXI / GAPDH (%) index, which is an index of antisense activity, was significantly higher than that of L4. M2 D10 mid, L5 M1 D10 5, L6 M1 D9 nat, L6 M2 D10 The value of mm is L6 The reductions were only 1.5-fold, 1.5-fold, 3-fold, and 3-fold compared to D10, demonstrating that the hepatotoxicity was reduced while maintaining the antisense activity. [Industrial Applicability]

[0069] The crosslinked antisense nucleic acid of the present invention can significantly reduce hepatotoxicity while maintaining its activity, and therefore has extremely high industrial utility as a highly practical antisense nucleic acid pharmaceutical.

[0070] All publications and patents cited herein are incorporated by reference in their entirety. Although specific embodiments of the invention have been described herein for purposes of illustration, those skilled in the art will readily understand that various modifications may be made without departing from the spirit and scope of the invention.

Claims

1. A bridged antisense nucleic acid having a gap region consisting of 5 to 15 bases of deoxyribonucleic acid, and wing regions consisting of 2 to 10 2',4'-modified nucleic acids at the 5' and 3' ends of the gap region, respectively, wherein 1 to 4 additional 2'-modified nucleic acids are added and / or inserted into at least one of the wing regions, and the 2'-modified nucleic acid has the following structural formula: 【Chemistry 1】 [In the formula, R 1 and R 2 are independently selected from the group consisting of H, substituted or unsubstituted alkyl groups, substituted or unsubstituted aralkyl groups, substituted or unsubstituted alkenyl groups, substituted or unsubstituted alkynyl groups, and substituted or unsubstituted aryl groups; R 3 is H, or the structure: 【Chemistry 2】 (In the formula, 【Chemistry 3】 is the point of attachment to the adjacent nucleic acid, or OH; and X is S or O. and R 4 is H or a point of attachment to an adjacent nucleic acid; and B represents a residue of a nucleic acid base which may have a protecting group or a modifying group. The above-mentioned bridged antisense nucleic acid, which has the following structure:

2. R 1 is H, and R 2 The bridged antisense nucleic acid of claim 1, wherein is methyl.

3. The bridged antisense nucleic acid according to claim 1 or 2, wherein one or two 2'-modified nucleic acids are added and / or inserted in each of the wing regions.

4. The 2',4'-modified nucleic acid is 【Chemistry 4】 [In the formula, R 5 and R 8 are each independently selected from the group consisting of H, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aralkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, and a substituted or unsubstituted aryl group; R 6 is H, or the structure: 【Chemistry 5】 (In the formula, 【Chemistry 6】 is the point of attachment to the adjacent nucleic acid, or OH; and X is S or O. and R 7 is H or a point of attachment to an adjacent nucleic acid; and B represents a residue of a nucleic acid base which may have a protecting group or a modifying group. The crosslinked antisense nucleic acid according to any one of claims 1 to 3, selected from the group consisting of:

5. The bridged antisense nucleic acid according to any one of claims 1 to 4, which comprises 1 to 4 2',4'-modified nucleic acids.

6. The bridged antisense nucleic acid according to any one of claims 1 to 5, wherein X is a sulfur atom.

7. The bridged antisense nucleic acid according to any one of claims 1 to 6, wherein the deoxyribonucleic acid is 8 to 10 bases in length.

8. 8. An antisense nucleic acid drug comprising the crosslinked antisense nucleic acid according to claim 1, and having reduced toxicity caused by the antisense nucleic acid.

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