Method for reducing toxicity of antisense nucleic acids

Abstract

To reduce the strong hepatotoxicity of antisense nucleic acids that incorporate an artificial nucleobase (e.g., LNA).SOLUTION: 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.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an antisense nucleic acid having an activity of inhibiting or controlling the expression of a target gene by an antisense effect, and more particularly to a crosslinked antisense nucleic acid with reduced toxicity by the antisense nucleic acid. More specifically, the present invention relates to a crosslinked antisense nucleic acid that enables both maintenance or enhancement of the antisense effect and reduction of side effects by additionally adding and / or inserting a 2'-modified nucleic acid into the base sequence of the crosslinked nucleic acid.

Background Art

[0002] In recent years, oligonucleotides have been developed as pharmaceuticals called nucleic acid drugs. 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 an oligonucleotide (antisense nucleic acid (ASO)) complementary to a partial sequence of the mRNA (sense strand) of the target gene into cells.

[0003] Most of the pipelines in which clinical trials are being conducted for ASO are occupied by PS-type ASO. PS-type ASO is obtained by substituting one of the oxygen atoms of the phosphate group of the phosphodiester bond, which is a natural nucleic acid bond, with a sulfur atom to phosphorothioate (PS-modify). PS modification has been found to be improved in terms of nuclease resistance (Non-Patent Document 1), uptake into cells (Non-Patent Document 2), etc., but is known to cause various side effects such as hepatotoxicity (Non-Patent Document 3).

[0004] As a representative example of PS-type ASO, mipomersen sodium (trade name: Kynamro), developed by Ionis Pharmaceuticals as a therapeutic agent for hypercholesterolemia with a Gapmer structure called the second-generation nucleic acid structure, can be mentioned. It was approved in the United States in 2013 as the world's first systemic nucleic acid drug for homozygous familial hypercholesterolemia, but various side effects such as hepatotoxicity of PS-type ASO were recognized. Also, in Europe, it has not been approved due to side effects (Non-Patent Document 4).

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

[0006] Recently, in cross-linked antisense nucleic acids, attempts have been made to reduce hepatotoxicity by adding chemical modifications to the gap portion formed (Patent Document 1).

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Summary of the Invention

Problems to be Solved by the Invention

[0009] As described above, among antisense nucleic acids that exhibit effects clinically, those incorporating artificial nucleobases (LNA) are useful, but many of them exhibit strong hepatotoxicity. On the other hand, as a method for avoiding this toxicity, it is conceivable to reduce the number of LNAs to be introduced or change the sequence of the antisense nucleic acid itself, but in many cases, the activity as a pharmaceutical agent decreases. Therefore, an object of the present invention is to provide a crosslinked antisense nucleic acid and a medicine that reduce hepatotoxicity and maintain or enhance the activity of the antisense nucleic acid.

Means for Solving the Problems

[0010] Therefore, as a result of research aimed at providing the above-described antisense nucleic acid medicine, the present inventors have found that by additionally adding and / or inserting specific artificial nucleobases while maintaining the sequence of the antisense nucleic acid and the number of LNAs, the toxicity caused by the antisense nucleic acid can be reduced, and the present invention has been completed.

[0011] That is, the present invention is as follows. [1]A cross-linked antisense nucleic acid having a gap region consisting of 15 to 15 deoxyribonucleic acids 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 in at least one of the wing regions, additionally 1 to 4 2'-modified nucleic acids are added and / or inserted. [2]The 2'-modified nucleic acid has the following structural formula:

[0012] [Chemical formula]

[0013] [In the formula, R 1 and R 2 are 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] [Chemical formula]

[0015] (In the formula,

[0016] [Chemical formula]

[0017] is a bonding point with an adjacent nucleic acid, or OH; and X is S or O) and; R 4 is H, or a bonding point with an adjacent nucleic acid; and B represents a residue of a nucleobase which may have a protecting group or a modifying group) The cross-linked antisense nucleic acid according to [1], having [3]R 1 is H, and R 2 is methyl, the crosslinked antisense nucleic acid according to [2]. [4] The crosslinked antisense nucleic acid according to any one of [1] to [3], wherein the number of 2'-modified nucleic acids added and / or inserted in each of the [4] wing regions is 1 or 2. [5] The 2',4'-modified nucleic acid is as follows:

[0018]

Chemical formula

[0019] [wherein, 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]

Chemical formula

[0021] (wherein,

[0022]

Chemical formula

[0023] is a bonding point with an adjacent nucleic acid, or OH; and X is S or O) ; R 7 is H, or a bonding point with an adjacent nucleic acid; and B represents a residue of a nucleobase 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 crosslinked antisense nucleic acid according to any one of [1] to [5], comprising 2 to 4 2′,4′-modified nucleic acids. [7]The crosslinked antisense nucleic acid according to any one of [1] to [6], wherein X is a sulfur atom. [8]The crosslinked 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 medicament with reduced toxicity by an antisense nucleic acid, comprising the crosslinked antisense nucleic acid according to any one of [1] to [8]. [Advantages of the Invention]

[0024] According to the present invention, by adding and / or inserting a 2′-modified nucleic acid into the wing region composed of the crosslinked nucleic acid in the crosslinked antisense nucleic acid, the toxicity generated by the conventional crosslinked antisense nucleic acid can be reduced, and an antisense nucleic acid with improved medicinal efficacy can be provided. [Brief Description of the Drawings]

[0025]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

[0026] The present invention relates to a cross-linked antisense nucleic acid containing a 2'-modified nucleic acid with reduced toxicity, and a therapeutic agent containing the cross-linked antisense nucleic acid. The present invention will be described in detail below.

[0027] (1) Cross-linked antisense nucleic acid The cross-linked antisense nucleic acid of the present invention includes a gap region composed of deoxyribonucleic acid, and wing regions having 2',4'-modified nucleic acids at the 5' and 3' ends of the gap region, respectively. In each of the wing regions, an additionally 2'-modified nucleic acid is added and / or inserted.

[0028] The cross-linked antisense nucleic acid of the present invention has a structure composed of a gap region (a region composed of "DNA", and the binding mode connecting each nucleic acid is not limited, but can be a phosphorothioate bond or a phosphodiester bond, preferably a phosphorothioate bond) (the length can be arbitrary, preferably 8 to 10 bases) consisting of a base sequence complementary to the base sequence of the target nucleic acid, and wing regions (for example, in FIG. 1, the two regions composed of "LNA" are underlined) located at both ends of the gap region. Compared with a conventional antisense nucleic acid ("L6 D10"), it has a structure in which a 2'-modified nucleic acid ("MCE") is additionally added and / or inserted at the end or inside of the wing region (for example, in FIG. 2, MCE is shown in lowercase).

[0029] The wing region constituting the crosslinked antisense nucleic acid of the present invention contains 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, the type and number of 2',4'-modified nucleic acids are not limited as long as they contribute to the effect (see, for example, W. Brad Wan and Punit P. Seth, J. Med. Chem. 2016, 59, 9645-9667). Also, the bonding mode for linking each nucleic acid is not limited either, but it can be a phosphorothioate bond or a phosphodiester bond, preferably a phosphorothioate bond. Note that the "phosphorothioate bond" is obtained by substituting one of the oxygen atoms of the phosphate group of the phosphodiester bond, which is a natural nucleic acid bond, with a sulfur atom to form a phosphorothioate (PS).

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

[0031] [Chemical formula]

[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 has the structure:

[0033] [Chemical formula]

[0034] (In the formula,

[0035] [Chemical formula]

[0036] is a binding point with an adjacent nucleic acid or OH; and X is S or O) ; R 7 is H or a binding point with an adjacent nucleic acid; and B represents a residue of a nucleobase which may have a protecting group or a modifying group] may have. As described above, B represents a residue of a nucleobase which may have a protecting group or a modifying group. Examples of the nucleobase include natural nucleobases such as adenine, cytosine, guanine, uracil, thymine, 5-methylcytosine, etc., and unnatural nucleobases 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, 3-deazaadenine, etc. The protecting group or modifying group introduced onto the nucleobase may be such that the amino group is protected by 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 by an alkyl group such as a cyanoethyl group, 2-(4-nitrophenyl)ethyl group, 2-nitrobenzyl group, acyloxymethyl group, etc., or an acyl group such as a diphenylcarbamoyl group. In the case of uracil and thymine bases, the N3 position may be protected by an alkyl group or an acyl group. Further, the carbon atoms of the above natural and unnatural nucleobases may contain substituents such as 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, etc., or fluorescent functional groups, biotinyl groups, amino groups, spin labels, etc. attached thereto.

[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 conformations, N-type and S-type. Since the mRNA targeted by the aforementioned ASO mainly has an N-type sugar chain and an A-type helical structure, it is important for the sugar chain of ASO to also be N-type from the viewpoint of enhancing the affinity for RNA. Modified nucleic acids such as LNA (Locked Nucleic Acid; 2’-O,4’-C-methylene-bridged nucleic acid (2’,4’-BNA / LNA)) were developed based on this concept. For example, in LNA, by bridging the oxygen at the 2’ position and the carbon at the 4’ position with a methylene group, its conformation is fixed to the N-type, and no fluctuation between conformations occurs. Therefore, an oligonucleotide synthesized by incorporating several units of LNA has extremely high binding affinity and sequence specificity for RNA, and excellent heat resistance and nuclease resistance compared to oligonucleotides synthesized from conventional natural nucleic acids. Since other artificial nucleic acids also have such properties, they can 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 the toxicity of the cross-linked antisense nucleic acid can be reduced, but is 1 to 10 bases, preferably 1 to 5 bases, more preferably 1 to 3 bases.

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

[0040] [Chemical formula]

[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 has the structure:

[0042] [Chemical formula]

[0043] (wherein

[0044] [Chemical formula]

[0045] is a binding point to an adjacent nucleic acid or OH; and X is S or O) ; R 4 is H or a binding point to an adjacent nucleic acid; B represents a residue of a nucleobase which may have a protecting group or a modifying group) It may have. Here, the "alkyl group" refers to a linear or branched saturated aliphatic hydrocarbon group having 1 to 20 carbon atoms, and examples thereof include 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 "aralkyl group" refers to a group in which the above alkyl group is further substituted with an aryl group, and examples thereof include a benzyl group and a diphenylmethyl group. The "alkenyl group" refers to a linear or branched unsaturated aliphatic hydrocarbon having 2 to 20 carbon atoms and containing 1 to 3 carbon-carbon double bonds, and examples thereof include an allyl group and a homoallyl group. The "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, and examples thereof include a propargyl group. The "aryl group" refers to a monovalent residue derived from a compound containing benzene or a structure in which two or more benzenes are condensed or bonded, and examples thereof include a phenyl group and a naphthyl group. The "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), and examples thereof include a 2,2,2-trifluoroethyl group, a cyanomethyl group, a 2,2,2-trichloroethyl, a methoxypropyl group, and a methylthiopropyl group. The "aralkyl group having a substituent" refers to a group in which the above aralkyl 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, and examples thereof include a 4-chlorobenzyl group, a 4-nitrobenzyl group, and a 2-nitrobenzyl group. The "alkenyl group having a substituent" is, for example, a dichloroallyl group. The "aryl group having a substituent" is, for example, a 4-nitrophenyl group.

[0046] In one embodiment of the present invention, the substituent R in the above structural formula 1 is H, and R2 A modified nucleic acid having a methyl group is preferred. To date, the inventors have reported that such a modified nucleic acid, that is, an RNase H-dependent antisense oligonucleotide introduced with 2'-O-(2-N-methylcarbamoylethyl) (MCE) nucleic acid, reduces hepatotoxic effects as compared with an antisense oligonucleotide having a conventional 2'-O-methyloxyethyl (MOE) modification (see Masaki, Y., et al., Nucleic Acid Therapeutics, 28, 307-311 (2018)). It should be noted that the antisense oligonucleotides used here are such that all of the modified nucleic acids in the wing region are either all MCE or all MOE and do not include cross-linked nucleic acids such as LNA, which is different from the present invention.

[0047] The cross-linked 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 thereof 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. Further, when a 2'-modified nucleic acid is added, its position is at both ends of each wing region composed of 2',4'-modified nucleic acids, and it may be introduced into the gap region by substituting the deoxyribonucleotide residue in the gap region. On the other hand, when a 2'-modified nucleic acid is inserted, it is not limited as long as it is between any 2',4'-modified nucleic acids. Further, the nucleic acid sequences of the 5'-side wing region and the 3'-side wing region after the addition and / or insertion of the 2'-modified nucleic acid may be the same or different.

[0048] The crosslinked antisense nucleic acid of the present invention can be easily and routinely produced through well-known techniques of solid-phase synthesis. For example, it may be carried out according to the synthesis method using the phosphoramidite method in the literature of Masaki et al. (supra) reported for RNase H-dependent antisense oligonucleotides introduced with MCE. Alternatively, it may be carried out by applying a liquid-phase synthesis method such as 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 a solid-phase synthesis method. The crosslinked antisense nucleic acid of the present invention is synthesized in vitro and does not contain an antisense composition of biological origin or a gene vector construct designed to direct in vivo synthesis of an antisense molecule. Further, in order to assist uptake, distribution, and / or absorption, the molecule of the present invention may be mixed, encapsulated, conjugated, or bound as a conjugate with other molecules, molecular structures, or mixtures of compounds, for example, as liposomes, receptor-targeting molecules, oral, rectal, topical, or other formulations.

[0049] The crosslinked antisense nucleic acid of the present invention has reduced hepatotoxicity compared to the antisense oligonucleic acid before such modification by additionally adding and / or inserting at least one 2'-modified nucleic acid in the wing region (see Example 2). The term "reduced" means that the numerical value of the marker that is an indicator of hepatotoxicity when the crosslinked antisense nucleic acid of the present invention is administered is lower compared to when the antisense oligonucleic acid before modification is administered. For example, after administration of the crosslinked 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, 5% or less, compared to the serum concentrations measured under the same conditions except using the above-mentioned antisense oligonucleic acid before modification.

[0050] (2) Therapeutic agent As demonstrated in Example 2 described below, the cross-linked antisense nucleic acid of the present invention has reduced toxicity and, furthermore, maintains its activation as an antisense nucleic acid. According to the present invention, it can be provided as a therapeutic agent (medicine, preparation, or pharmaceutical composition) in the same manner as commonly used antisense nucleic acids. When the cross-linked antisense nucleic acid of the present invention is used as a therapeutic agent, the type of genetic disease is not particularly limited. The "deoxyribonucleic acid" constituting the gap region incorporated into the cross-linked antisense nucleic acid can target non-coding RNAs (microRNAs, ribosomal RNAs, tRNAs, etc.), mRNAs, single-stranded DNAs, etc. present in vivo. Therefore, using all or a partial sequence of these as the "target nucleic acid", an antisense nucleic acid containing a single-stranded nucleic acid consisting of a base sequence that is completely or sufficiently complementary to the base sequence of the target nucleic acid can be used as a therapeutic agent for treating and / or preventing genetic diseases corresponding to the target nucleic acid. Also, the length of the deoxyribonucleic acid is generally appropriately about 10 bases, but is not particularly limited as long as it is in the range of 5 to 15 bases. Preferably it is 8 to 10 bases, more preferably 10 bases. Furthermore,

[0051] In this specification, "(a single-stranded nucleic acid) consisting of a base sequence that is (sufficiently complementary to the base sequence of the target nucleic acid)" means 50% or more and less than 100% of the base sequence of the target nucleic acid, 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 that can pair with the base sequence. More specifically, in a nucleic acid strand consisting of a base sequence that is completely complementary to the base sequence of the target nucleic acid, for example, when 1 or 2 to 4 bases are substituted with other bases and as a result, the nucleotide residue at the substituted position cannot pair (in this case, the position where the substitution with other bases is made is referred to as a "mismatch site"); in a nucleic acid strand consisting of a base sequence that is completely complementary to the base sequence of the target nucleic acid, for example, when 1 or 2 to 4 bases are deleted and as a result, the nucleotide residue at the deleted position cannot pair, etc. can be mentioned.

[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, pharmaceutically acceptable carriers, diluents, excipients, etc. can be included. The term "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and generally do not cause allergic reactions such as acute gastric peristalsis or similar adverse reactions when administered to a patient. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle administered together with a compound. Such pharmaceutical carriers may be inert liquids such as oils and water, including oils of petroleum, animal, vegetable, or synthetic origin such as arachis oil, soybean oil, mineral oil, sesame oil, etc. Water or physiological saline, as well as aqueous dextrose and glycerol aqueous solutions, are preferably used as carriers, particularly for injection solutions.

[0053] In a more specific embodiment of the present invention, a pharmaceutical composition is provided that comprises a therapeutically effective amount of a crosslinked antisense nucleic acid together with a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant, and / or carrier. Such compositions contain additives such as various buffers (e.g., Tris-HCl, acetate, phosphate), diluents of pH and ionic strength, detergents and solubilizers (e.g., Tween80, polysorbate 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol), and bulking substances (e.g., lactose, mannitol), etc. Also, the pharmaceutical composition may be prepared in a liquid form or may be a dry powder such as a lyophilized form.

[0054] The pharmaceutical composition provided according to the present invention is preferably administered by, but not limited to, injection, oral, pulmonary, or nasal routes. More preferably, it is delivered by intravenous, intraarterial, intraperitoneal, intramuscular, or subcutaneous administration routes.

[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 crosslinked 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 contain a suitable solvent for reconstituting the lyophilized components. The individual components of the kit are packaged in separate containers, and together with such containers, a notice in a form defined by a government agency that regulates the manufacture, use, or sale of pharmaceuticals or biological products, reflecting approval by an agency that regulates the manufacture, use, or sale for human administration, may be attached.

[0057] If 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. When used in vivo, the expression construct may be formulated into a pharmaceutically acceptable injectable composition. In this case, the container means may be a single inhaler, syringe, pipette, eye dropper, or other similar device, from which the formulation can be applied to the affected area of the animal, injected into the animal, or further applied to and mixed with other components of the kit.

[0058] Also, the components of the kit may be provided in a dry or lyophilized form. When a reagent or component is provided in a dry form, generally, reconstitution is performed by adding a suitable solvent. It is also contemplated that the solvent may be provided in a separate container means. Regardless of the number or type of containers, the kit of the present invention may include or be packaged together with a device for assisting in injecting / administrating or placing the final complex composition into the body of the animal. Such a device may be an inhaler, syringe, pipette, forceps, measuring spoon, eye dropper, or any medically approved delivery vehicle.

[0059] The present invention will be further specifically described by the following examples, but the present invention is not limited by these examples in any way.

Example

[0060] Example 1: Synthesis of Crosslinked Antisense Nucleic Acid The crosslinked antisense nucleic acids shown in Figures 1 to 6 targeting Factor XI were synthesized on a DNA automatic synthesizer by the solid-phase synthesis method using the phosphoramidite method known to those skilled in the art, and purified and analyzed by reverse-phase HPLC. The underlined part represents LNA, the capital letters represent DNA, and the small letters represent MCE nucleic acids. All nucleic acids are linked by phosphorothioate bonds.

[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) Note that the name of the cross-linked antisense nucleic acid is described together with the numbers of each of "L (LNA)", "D (deoxyribonucleic acid of factor XI gene)", and "M (MCE)". For example, "L4 M2 D10" is composed of 4 LNAs, 2 MCEs, and 10 DNAs, and the layout diagram of the nucleic acids is as shown above.

[0062] Example 2: Evaluation of Hepatotoxicity and Activity of Cross-Linked Antisense Nucleic Acid (1) Administration and Feed Sampling A physiological saline solution (3 mg / mL) of the cross-linked antisense nucleic acid was subcutaneously administered to 5-week-old female C57BL / 6JJcl mice at 10 mL per 1 kg of body weight. 72 hours later, all the mice were anesthetized with isoflurane, and blood was collected from the heart using a 23G injection needle and a syringe containing 10,000 units / 10 mL of sodium heparin added, and plasma was collected. Then, after incising the abdominal aorta to cause exsanguination and death, the liver was collected. A part of the liver was trimmed.

[0063] (2) Quantitative PCR Analysis of Target Gene Expression The collected liver was homogenized using TRIzol reagent (Invitrogen, product number: 15596018) to extract total RNA. A part of the prepared sample was subjected to DNase treatment and purification 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) 15It was synthesized in a 20-μL reaction system using a primer. After cDNA synthesis, 20 μL of sterilized water was added to make a total of 40 μL. Quantitative measurement of the mRNA of each gene was performed 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, the serum concentrations of ALT (alanine aminotransferase), AST (aspartate aminotransferase), and LDH (lactate dehydrogenase), which are indicators of hepatotoxicity, were measured by the JSCC standardization method. From the results in Table 1 below, in L6 without the introduction of MCE D10 showed high values of ALT, AST, and LDH, while the other cross-linked antisense nucleic acids containing MCE had low values of ALT, AST, and LDH. Typically, when comparing the average values of AST, L4 M2 D10, L6 M2 D8, L6 M2 D10, and L6 TM2 The values of D10 were, respectively, L6 0.03 times, 0.03 times, 0.04 times, and 0.06 times that of D10, and the toxicity was reduced by about 100-fold. On the other hand, for FXI / GAPDH (%), which is an indicator of antisense activity, L4 M2 D10, L6 M2 D8, L6 M2 D10, and L6 TM2 The values of D10 were, respectively, L6 only reduced to 4.5 times, 8 times, 2.5 times, and 2 times that of D10, and it was revealed that hepatotoxicity was reduced while maintaining antisense activity.

[0065]

Table 1

[0066] Furthermore, in addition to the above, similar experiments were also conducted on four separately synthesized cross-linked antisense nucleic acids. For these cross-linked antisense nucleic acids, hepatotoxicity and activity were measured for AST and ALT.

[0067]

Table 2

[0068] Comparing Table 1 and Table 2, for L6 without the introduction of MCE shown in Table 1 D10 showed high values for ALT and AST, while for other cross-linked antisense nucleic acids containing MCE, ALT, AST, and LDH were low. Typically, when comparing the average values of AST, L4 M2 D10 mid, L5 M1 D10 5, L6 M1 D9 nat, L6 M2 D10 mm values are 0.04 times, 0.04 times, 0.02 times, 0.13 times that of L6 D10, on the order of about 10 - 100 times, and the toxicity is reduced. On the other hand, for FXI / GAPDH(%), an indicator of antisense activity, L4 M2 D10 mid, L5 M1 D10 5, L6 M1 D9 nat, L6 M2 D10 mm values are 1.5 times, 1.5 times, 3 times, 3 times that of L6 D10, and it was only reduced to this extent, and it was clarified that while maintaining the antisense activity, the hepatotoxicity was reduced.

Industrial Applicability

[0069] Since the crosslinked antisense nucleic acid of the present invention can significantly reduce hepatotoxicity while maintaining its activity, it can be a highly practical antisense nucleic acid medicine and is extremely valuable in industry.

[0070] All publications and patent documents cited in this specification are hereby incorporated by reference in their entirety. For the purpose of illustration, specific embodiments of the present invention have been described in this specification, but it will be readily understood by those skilled in the art that various modifications may be made without departing from the spirit and scope of the present 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 an additional 1 to 4 2'-modified nucleic acids are added and / or inserted into at least one of the wing regions.

2. The 2'-modified nucleic acid has the following structural formula: 【Chemistry 1】 [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: 【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 bridged antisense nucleic acid of claim 1, having the following structure:

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

4. The bridged antisense nucleic acid according to any one of claims 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 【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 4, selected from the group consisting of:

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

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

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

9. 9. 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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