JP2011155914A - Chemically modified sirna as lipid-abnormal disease-treating medicine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new modified siRNA molecule which is high in binding affinity with the target RNA of apolipoprotein B (ApoB) and has high stability in living bodies, and can exhibit an excellent RNA-interfering effect in the living bodies, and to provide a medicine composition containing the modified siRNA molecule as an active ingredient. <P>SOLUTION: The modified siRNA decreases expression of ApoB gene, wherein (a) the modified siRNA molecule has an antisense chain RNA comprising a base sequence complementary to the sequence of a part of the ApoB gene and a modified sense chain RNA having a base sequence complementary to the antisense chain RNA, and (b) the modified sense chain RNA is a modified oligoribonucleotide comprising totally 19 to 23 ribonucleotides and ribonucleotide analogues. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a modified siRNA capable of effectively suppressing in vivo expression of an apolipoprotein B (hereinafter also referred to as “ApoB”) gene. More specifically, it has excellent binding affinity with the target RNA, stability in serum and in vivo, and exhibits excellent RNA interference effect in vivo, thereby significantly suppressing ApoB expression in vivo. It relates to modified siRNA that can be. The modified siRNA can effectively prevent or treat a disease associated with ApoB expression.

  Apolipoprotein B (ApoB) is a glycoprotein that plays an integral role in lipid assembly and secretion, and in the transport and receptor-mediated uptake and delivery of another class of lipoproteins. The importance of such ApoB spans various functions from absorption and processing of dietary lipids to regulation of circulating lipoprotein levels (Non-patent Document 1). This latter characteristic is related to atherosclerosis sensitivity, and it is known that atherosclerosis sensitivity is highly related to the ambient concentration of ApoB-containing lipoprotein (Non-patent Document 1). .

  There are two forms of ApoB in mammals. ApoB-100 is a full-length protein containing 4536 amino acid residues and is synthesized exclusively in human liver (Non-patent Document 1). ApoB-48 is a deletion protein having 2152 amino acid residues in the N-terminal region of ApoB-100 and is synthesized in the small intestine of all mammals (Non-patent Document 1).

  The medical significance of ApoB in mammals has been demonstrated in studies using transgenic mice such as human ApoB overexpressing mice (Non-Patent Documents 2 to 3) or ApoB knockout mice (Non-Patent Documents 2 and 4). .

  Moreover, the increase in the plasma level of lipoprotein Lp (a) containing ApoB-100 is caused by atherosclerosis, hypercholesterolemia (Non-patent document 5), myocardial infarction (Non-patent document 6), and thrombosis (non-patent document). It has also been reported that it is associated with an increased risk of morbidity such as literature 7). The plasma concentration of the Lp (a) is strongly influenced by genetic factors and exhibits resistance to pharmaceutical therapy and diet therapy (Non-Patent Documents 8 to 9). The improvement is called apheresis. Plasma exchange therapy is the most effective method (Non-patent Document 10).

  One of the diseases caused by the expression of ApoB is known as familial hypercholesterolemia (FH) homozygote. The disease is a genetic disease associated with coronary artery disease due to hypercholesterolemia, cutaneous xanthoma, and juvenile arteriosclerosis, which is marked from the time of birth, and is an intractable disease in which no drugs such as statins succeed (non- Patent Document 11). The only treatment is plasma exchange therapy called LDL apheresis. However, such a treatment method requires a large-scale treatment by extracorporeal circulation once a week, so that the physical and mental burden is very large. In addition, there are many cases in which arteriosclerosis has progressed before reaching the age at which plasma exchange therapy can be started (Non-patent Document 11).

  Nucleic acid drugs such as antisense to ApoB and double-stranded RNA (siRNA) have been developed for the treatment of diseases caused by the expression of ApoB (Non-patent Document 12, Patent References 1-2).

  In particular, the RNA interference (RNAi) method using siRNA introduces two short strands of about 20-25 base pairs due to the action of Dicer in the cytoplasm when double strand RNA of about 100 base pairs is introduced into the cell. It is broken down into strand RNA, and then forms RNA / protein complex with multiple proteins (this complex is called RICS: RNA-induced silencing complex) and binds to the homologous site of mRNA produced from the target gene. This is a method that utilizes the strong suppression of gene expression.

  Today, the mainstream method is to use chemically synthesized double-stranded RNA of 21 bases with a dangling end of 2 bases at the 3 'end. It has been reported that the RNA interference effect is about 100 times higher than siRNA consisting of 21 bases in length, and is attracting attention. (Refer nonpatent literature 13).

  The RNA interference method using such synthetic RNA is relatively easy to prepare a sample, easy to handle, and has a very powerful effect, so that it can be used not only in the life science field but also in the biobusiness field. It is getting a lot of attention.

  However, even with this superior RNA interference method, in order to ensure the RNA interference effect of siRNA in vivo and enable clinical application, its in vivo stability, cell transduction, cell In reality, there are still many issues to be overcome such as internal localization, gene expression suppression effect, and target specificity.

Special Table 2006-522586 Special table 2009-507499

Davidson and Shelness, Annul Rev. Nutr., 2000, 20, 169-193 Kim and Young, J. Lipid Res., 1998, 39, 703-723 Nishina et al., J. Lipid Res., 1990, 31, 859-869 Farese et al., Proc. Natl. Acad. Sci. U. S. A., 1995, 92, 1774-1778 Seed et al., N. Engl. J. Med., 1990, 322, 1494-1499) Sandkamp et al., Clin. Chew., 1990, 36, 20-23 Nowak-Gottl et al., Pediatrics, 1997, 99, Eli Katan and Beynen, Am. J. Epidemiol., 1987, 125, 387-399 Vessby et al., Atherosclerosis, 1982, 44, 61-71 Hajjar and Nachman, Annul Rev. Med., 1996, 47, 423-442 Makino H, Harada-shiba M, Ther Apher Dial 2003, 7: 397-401 Soutschek J et al., Nature 2004, 432, 173-178 J. Rossi et.al. Nature Biotech., 23, 222-226 (2005)

  The present invention provides a novel modified siRNA molecule that has high binding affinity with target RNA of apolipoprotein B (ApoB) and stability in vivo, and can exhibit excellent RNA interference effects in vivo. Objective. A further object of the present invention is to provide a pharmaceutical composition comprising such a modified siRNA molecule as an active ingredient, particularly a pharmaceutical composition for preventing or treating a disease associated with the expression of ApoB such as dyslipidemia. .

  As mentioned above, RNA interference is an effective method that can specifically suppress the expression of the target gene. However, in order for siRNA to be practically applicable as a nucleic acid drug, it must be bound to the target RNA. It is necessary to satisfy many requirements such as affinity, stability in blood and in vivo. For this reason, even if excellent effects are observed in in vitro tests, there are many that are not actually applicable in vivo, and early development of siRNA that is effective in vivo as a nucleic acid drug is required. .

  The present inventors pay attention to such problems, and intensively study to develop siRNA molecules that have excellent binding affinity with target RNA and stability in vivo including blood, and are effective as nucleic acid drugs. As a result, we found that a modified siRNA molecule in which a part of the nucleotide in the sense strand was replaced with a specific nucleotide analog had excellent properties suitable for the above purpose, and actually expressed mRNA expression of ApoB in vivo. It was confirmed that it effectively suppressed and significantly reduced serum cholesterol levels. From these findings, the present inventors are convinced that the modified siRNA molecule is extremely effective as a practicable nucleic acid drug, particularly as a therapeutic agent for dyslipidemia including familial hypercholesterolemia designated as intractable disease. Thus, the present invention has been developed.

  This invention is completed based on said knowledge, Comprising: The following aspect is included.

(I) Modified siRNA that decreases the expression of apolipoprotein B gene
(I-1) A modified siRNA that decreases the expression of apolipoprotein B (hereinafter referred to as “ApoB”) gene,
(A) The modified siRNA molecule has an antisense strand RNA consisting of a base sequence complementary to a partial sequence of the ApoB gene, and a modified sense strand RNA having a base sequence complementary to the antisense strand RNA. ,
(B) The modified sense strand RNA is a modified oligoribonucleotide consisting of a total of 19 to 23 ribonucleotides and a ribonucleotide analog,
(C) The ribonucleotide analog is represented by the following general formula (1)

（式中、Ｂは置換基を有していてもよい芳香族複素環基若しくは芳香族炭化水素環基；Ｘはシングルボンドまたは置換基を有する窒素原子；Ｙは酸素原子、硫黄原子またはＢＨ_３；ｍは０〜２の整数；及びｎは０〜３の整数を意味する）
で示される構造を有するものであることを特徴とする、上記修飾siRNA。
（I-2）一般式（１）で示されるリボヌクレオチドアナログが、式中、Ｘはシングルボンド、ｎは１、及びｍは０であるものである、（I-1）に記載する修飾siRNA。
（I-3）アンチセンス鎖RNAが、配列番号１に示されるApoB遺伝子の10167〜10187の領域の塩基配列に相補的な塩基配列を有する塩基長21〜23のオリゴリボヌクレオチドである（I-1）または（I-2）に記載する修飾siRNA。
（I-4）修飾センス鎖RNAが、配列番号４または５の塩基配列を有する塩基長21〜23の修飾オリゴリボヌクレオチドである、（I-1）乃至（I-3）のいずれかに記載する修飾siRNA。

(Wherein B is an aromatic heterocyclic group or aromatic hydrocarbon ring group which may have a substituent; X is a single bond or a nitrogen atom having a substituent; Y is an oxygen atom, a sulfur atom or BH _3. M represents an integer of 0 to 2; and n represents an integer of 0 to 3).
The modified siRNA above, which has a structure represented by
(I-2) The modified siRNA according to (I-1), wherein the ribonucleotide analog represented by the general formula (1) is a compound in which X is a single bond, n is 1, and m is 0 .
(I-3) The antisense strand RNA is an oligoribonucleotide having a base length of 21 to 23 having a base sequence complementary to the base sequence of the region 10167 to 10187 of the ApoB gene shown in SEQ ID NO: 1 (I- The modified siRNA described in 1) or (I-2).
(I-4) The modified sense strand RNA is a modified oligoribonucleotide having a base length of 21 to 23 having the base sequence of SEQ ID NO: 4 or 5, according to any one of (I-1) to (I-3) Modified siRNA.

(II) Pharmaceutical composition (II-1 ) A pharmaceutical composition comprising the modified siRNA described in any one of (I-1) to (I-4) as an active ingredient.
(II-2) The pharmaceutical composition according to (II-1), which is a preventive or therapeutic agent for a disease associated with the expression of ApoB.
(II-3) The pharmaceutical composition according to (II-2), wherein the disease is any one selected from the group consisting of dyslipidemia, coronary artery disease, coronary heart disease, and atherosclerosis.
(II-4) The above dyslipidemia is familial hypercholesterolemia, familial mixed hyperlipidemia, familial type III hyperlipidemia, acquired hyperlipoproteinemia, high LDL cholesterolemia, The pharmaceutical composition according to (II-3), which is secondary hyperlipidemia such as statin-resistant hypercholesterolemia, hyper-Lp (a) or nephrotic syndrome.

  The modified siRNA of the present invention has excellent binding affinity with the target ApoB mRNA, and also has excellent stability in blood. For this reason, the expression of ApoB can be reliably suppressed in vivo, and diseases caused by the expression of ApoB, particularly dyslipidemia, can be effectively prevented and treated. The modified siRNA of the present invention is also characterized by low biological toxicity and high safety as shown in Experimental Example 2 described later. Therefore, the modified siRNA is useful as a highly effective active ingredient of a pharmaceutical composition for preventing or treating a disease caused by the expression of ApoB. That is, according to the present invention, a pharmaceutical composition comprising the above-described modified siRNA as an active ingredient, specifically, effective for effectively preventing and treating diseases associated with the expression of ApoB, particularly dyslipidemia. Pharmaceutical compositions can be provided.

In Experimental Example 1, unmodified siRNA (siApoB-1) and modified siRNA (siBNA-1, siBNA-2) were transfected into mouse hepatocytes, respectively, and the results of examining the influence of ApoB on mRNA expression are shown. The horizontal axis indicates the concentration of unmodified siRNA (siApoB-1) and modified siRNA (siBNA-1, siBNA-2). The result of measuring the amount of ApoB mRNA expression in the liver after administering (intravenously) unmodified siRNA (siApoB-1) and modified siRNA (siBNA-2) to the mouse as siRNA, respectively. A group of mice (control group) administered with physiological saline instead of siRNA is indicated as “Saline” (same in FIGS. 3 to 8). Unmodified siRNA (siApoB-1) and modified siRNA (siBNA-2) were each administered as an siRNA to mice (intravenous injection), and changes in serum total cholesterol levels over time (Day 0, Day 1, Day 2) were measured. Results are shown. Unmodified siRNA (siApoB-1) and modified siRNA (siBNA-2) were each administered as an siRNA to mice (intravenous injection), and changes in serum VLDL cholesterol levels over time (Day 0, Day 1, Day 2) were measured. Results are shown. Unmodified siRNA (siApoB-1) and modified siRNA (siBNA-2) were administered (intravenous injection) to mice as siRNA, respectively, and changes in serum LDL cholesterol levels over time (Day 0, Day 1, Day 2) were measured. Results are shown. Unmodified siRNA (siApoB-1) and modified siRNA (siBNA-2) were administered (intravenous injection) to mice as siRNA, respectively, and changes in serum HDL cholesterol levels over time (Day 0, Day 1, Day 2) were measured. Results are shown. Unmodified siRNA (siApoB-1) and modified siRNA (siBNA-2) were administered (intravenous injection) to the mouse as siRNA, respectively, and time-dependent ((Figure A) Day0, (Figure B) Day1, (Figure C) Day2) ) Shows the results of lipoprotein analysis (cholesterol level divided into 20 fractions) of the collected serum. As a siRNA, unmodified siRNA (siApoB-1) and modified siRNA (siBNA-2) were administered (intravenous injection) to mice, respectively, and from the serum collected over time (Day 0, Day 1, Day 2), the degree of liver damage [( A) ALT and (B) AST] were measured. The result of having measured the stability in serum about siApoB-1, siBNA-1, and siBNA-2 is shown (Experimental example 3).

(I) Modified siRNA
The modified siRNA of the present invention is a modified siRNA that decreases the expression of the human ApoB gene. The modified siRNA is composed of an antisense strand RNA consisting of a base sequence complementary to a partial sequence (target sequence) of the human ApoB gene and a modified sense strand RNA having a base sequence complementary to the antisense strand RNA. Soybeans are formed into double strands.

(I-1) ApoB gene and its target sequence The gene to which the present invention suppresses gene expression by the RNA interference effect is the ApoB gene. The origin is not limited, but preferably includes mammals, more preferably laboratory animals such as mice and rats, and particularly preferably humans.

  The nucleotide sequence of the human ApoB gene (mRNA) is NCBI accession number NM_000384 (the cDNA nucleotide sequence is shown in “SEQ ID NO. 1”, the same applies hereinafter), and the nucleotide sequence of the mouse ApoB gene (mRNA) used in the experimental examples. Can refer to NCBI accession number BC141357 (the base sequence of cDNA is shown in “SEQ ID NO: 2”, the same applies hereinafter).

  The target sequence in the gene is not particularly limited as long as it is a sequence capable of suppressing gene expression due to RNA interference effect, and can be appropriately determined by a known method, specifically using NCBI BLAST search or the like. . For example, a region consisting of 19 to 30 bases following the base “AA” in the exon part 50 to 100 bases downstream from the start codon of the coding region (ORF) of the ApoB gene, and having a GC content of around 50% Can be the target sequence. Employing such a complementary strand to the target sequence has empirically revealed in the art that an excellent RNA interference effect can be obtained. For example, the target sequence can be set according to the manual (Dicer Substrate RNAi Design) of IDT (Integrated DNA Technologies, INC). Recently, (i) the 5 ′ end of the antisense strand RNA is an A / U pair, (ii) the 5 ′ end of the sense strand RNA is a G / C pair, and (iii) the antisense strand RNA 5 'It has a high RNA interference effect by designing double-stranded RNA that has about 5 A / U pairs on the end side and (vi) no more than 9 G / C pairs in the double strand 2 It has been reported that single-stranded RNA can be designed (Ui-Tei et. Al, Nucleic Acids Res., 32, 936-948 (2004)).

  As a more preferred target sequence, a base sequence having at least a region of 10167 to 10187, a base sequence having a region of 1375 to 1395, a base sequence having a region of 7305 to 7325, 8142 to 8162, of human ApoB mRNA (SEQ ID NO: 1) A base sequence having the above region, a base sequence having the region of 12224-12244, a base sequence having the region of 12443-12463, and a base sequence having the region of 13104-13124. Preferably, it is a base sequence having a region of at least 10167-10187.

  In the case of mouse ApoB mRNA (SEQ ID NO: 2), it is a base sequence having at least a region of 1015 to 1035.

(I-2) Antisense strand RNA
The antisense strand RNA constituting the modified siRNA of the present invention is an oligoribonucleotide having a base sequence complementary to the target sequence in the ApoB gene (mRNA).

  The number of ribonucleotides constituting the oligoribonucleotide is not particularly limited as long as it exhibits an RNA interference effect, and can be usually 19 to 30, preferably 21 to 27, more preferably 21 to 23. .

  The modified siRNA of the present invention is such that the antisense strand and a sense strand described later are hybridized so as to have a dangling end of about 2 to 5 bases at both 3 ′ ends to form a double strand It may be. For example, when the antisense strand RNA and the modified sense strand RNA are both composed of 23 ribonucleotides (or ribonucleotides and ribonucleotide analogs), the antisense strand RNA 3 'end and the modified sense strand RNA A dangling end composed of about 2 to 5 ribonucleotides may be formed at the 3 ′ end. That is, for example, in the case of a double-stranded siRNA having a dangling end composed of two ribonucleotides, the 3rd to 23rd base sequences from the 3 ′ end side of the antisense strand RNA are modified sense strands described later. It is complementary to the 1st to 21st nucleotide sequence from the 5 ′ end of RNA and hybridizes therewith. The base constituting the dangling end is not particularly limited, and may be complementary to the base of the target sequence or not.

(I-3) Modified sense strand RNA
The modified sense strand RNA is a modified RNA that can hybridize with the antisense strand RNA to form a double strand. That is, the modified sense strand RNA is RNA containing a base sequence complementary to the antisense strand RNA. The modified sense strand RNA may not necessarily be a base sequence that is 100% complementary to the entire length of the sense strand RNA as long as it hybridizes with the antisense strand RNA to form a double strand. Preferably, it has a sequence complementary to 85% or more, more preferably 90% or more, of the antisense strand RNA.

  The modified sense strand RNA is a modified oligoribonucleotide composed of a ribonucleotide and a ribonucleotide analog. The total number of ribonucleotides and ribonucleotide analogs constituting such a modified sense strand RNA is not particularly limited. For example, 19-30, preferably 21-27, more preferably, as with antisense strand RNA. 21 to 23 can be mentioned. The number of ribonucleotides in the antisense strand and the total number of ribonucleotides and ribonucleotide analogs in the modified sense strand may be different from each other, but are preferably the same number.

  In the modified siRNA of the present invention, both the antisense strand RNA and the modified sense strand RNA are preferably composed of 23 bases including the dangling end.

  Examples of the ribonucleotide analog constituting the modified sense strand RNA include those having a structure represented by the following general formula (1).

（式中、Ｂは置換基を有していてもよい芳香族複素環基若しくは芳香族炭化水素環基；Ｘはシングルボンドまたは置換基を有する窒素原子；Ｙは酸素原子、硫黄原子またはＢＨ_３；ｍは０〜２の整数；及びｎは０〜３の整数を意味する）。

(Wherein B is an aromatic heterocyclic group or aromatic hydrocarbon ring group which may have a substituent; X is a single bond or a nitrogen atom having a substituent; Y is an oxygen atom, a sulfur atom or BH _3. M represents an integer of 0 to 2; and n represents an integer of 0 to 3).

In the present invention, the term “nucleotide analog” refers to an unnatural type of “nucleotide” in which a purine or pyrimidine base and a sugar are bonded, and an aromatic heterocycle and aromatic hydrocarbon ring other than purine and pyrimidine. A non-natural type in which a sugar can be combined with a substance that can be substituted with a purine or pyrimidine base. In the former non-natural type, the nucleotide is a phosphothioate group (in formula (1), where Y is sulfur) instead of the usual phosphate group (in the case where Y is an oxygen atom in formula (1)). And those having a linking group such as a boranophosphate group (when Y is BH ₃ in formula (1)). The latter non-natural type includes those in which the nucleotide has a linking group selected from the group consisting of a phosphate group, a phosphothioate group and a boranophosphate group.

  In the above general formula (1), the aromatic heterocyclic group represented by “B” refers to a carbon atom that is a constituent atom of a hydrocarbon ring as one or more hetero atoms such as a nitrogen atom, a sulfur atom, or an oxygen atom. The term refers to any group of 5 to 20-membered rings having a substituted structure and exhibiting aromaticity, and includes a single ring and a condensed ring. Specific examples include pyrimidine or purine nucleobase and pyrimidine or purine nucleobase which may have one or more substituents selected from the following α group. Here, for pyrimidine or purine nucleobase, any base that is generally known as a component of nucleic acid (for example, guanine, adenine, cytosine, thymine, uracil) and other bases of nucleic acid components similar to these can be used or substituted. Includes chemical structure. Others, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridazine, indolizine, indole, isoindole, isoquinoline, quinoline, naphthyridine, quinoxaline, Also included are quinazoline, pteridine, carbazole, phenanthridine, acridine, perimidine, phenazine, phenalazine, phenothiazine, furazane, phenoxazine, pyrrolidine, pyrroline, imidazolidine, imidazoline, pyrazolidine and the like. Preferred is a pyrimidine or purine nucleobase, a pyrimidine or purine nucleobase which may have one or more substituents selected from the following α group, specifically, a purin-9-yl group, A 2-oxo-pyrimidin-1-yl group, or a purin-9-yl group or a 2-oxo-pyrimidin-1-yl group having a substituent selected from the following group α is preferred.

  α group: hydroxyl group, hydroxyl group protected with a protecting group for nucleic acid synthesis, alkoxy group having 1 to 5 carbon atoms, mercapto group, mercapto group protected with a protecting group for nucleic acid synthesis, alkylthio group having 1 to 5 carbon atoms, amino Group, an amino group protected with a protecting group for nucleic acid synthesis, an amino group substituted with an alkyl group having 1 to 5 carbon atoms, an alkyl group having 1 to 5 carbon atoms, and a halogen atom. Here, a group suitable as “optionally substituted purine nucleobase” is 6-aminopurin-9-yl (ie, adeninyl), 6 in which the amino group is protected with a protecting group for nucleic acid synthesis. -Aminopurin-9-yl, 2,6-diaminopurin-9-yl, 2-amino-6-chloropurin-9-yl, 2-amino-6-amino group protected with a protecting group for nucleic acid synthesis Chloropurin-9-yl, 2-amino-6-fluoropurin-9-yl, 2-amino-6-fluoropurin-9-yl, 2-amino-6 in which the amino group is protected with a protecting group for nucleic acid synthesis -Bromopurin-9-yl, 2-amino-6-bromopurin-9-yl, amino-protected 2-amino-6-hydroxypurin-9-yl (ie, guanylyl) wherein the amino group is protected with a protecting group for nucleic acid synthesis The amino group is a protecting group for nucleic acid synthesis. Protected 2-amino-6-hydroxypurin-9-yl, 6-amino-2-methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl, 6-amino-2-fluoropurine -9-yl, 2,6-dimethoxypurin-9-yl, 2,6-dichloroprolin-2-yl or 6-mercaptopurin-9-yl group, more preferably 6-benzoylaminopurine- 9-yl, adenynyl, 2-isobutyrylamino-6-hydroxypurin-9-yl or guaninyl group.

  Further, a group suitable as “an optionally substituted pyrimidine nucleobase” is 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl (ie, cytosynyl), and an amino group is a nucleic acid. 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl protected with a synthetic protecting group, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl, 4-amino-2-oxo-5-chloro-1, wherein the amino group is protected with a protecting group for nucleic acid synthesis -Dihydropyrimidin-1-yl, 2-oxo-4-methoxy-1,2-dihydropyrimidin-1-yl, 2-oxo-4-mercapto-1,2-dihydropyrimidin-1-yl, 2-oxo- 4-hide Xyl-1,2-dihydropyrimidin-1-yl (ie uracinyl), 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl (ie thyminyl) or 4-amino- 5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl (ie, 5-methylcytosinyl) group, more preferably 2-oxo-4-benzoylamino-1,2-dihydropyrimidine- 1-yl, cytosynyl, thyminyl, uracinyl, 2-oxo-4-benzoylamino-5-methyl-1,2-dihydropyrimidin-1-yl, or 5-methylcytosinyl group.

  Among the “optionally substituted purine or pyrimidine nucleobases”, more preferably, 6-aminopurin-9-yl (ie, adenyl), the amino group is protected with a protecting group for nucleic acid synthesis. 6-aminopurin-9-yl, 2,6-diaminopurin-9-yl, 2-amino-6-chloropurin-9-yl, 2-amino- in which the amino group is protected with a protecting group for nucleic acid synthesis 6-chloropurin-9-yl, 2-amino-6-fluoropurin-9-yl, 2-amino-6-fluoropurin-9-yl, amino group in which the amino group is protected with a protecting group for nucleic acid synthesis, 2-amino -6-bromopurin-9-yl, 2-amino-6-bromopurin-9-yl, 2-amino-6-hydroxypurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis (ie, Guaninyl), the amino group is the nucleus Synthetic protecting groups protected 2-amino-6-hydroxypurin-9-yl, 6-amino-2-methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl, 6-amino 2-fluoropurin-9-yl, 2,6-dimethoxypurin-9-yl, 2,6-dichloropurin-9-yl, 6-mercaptopurin-9-yl, 2-oxo-4-amino-1 , 2-dihydropyrimidin-1-yl (ie, cytosynyl), 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl in which the amino group is protected with a protecting group for nucleic acid synthesis, 2-oxo- 4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidine- in which the amino group is protected with a protecting group for nucleic acid synthesis 1-yl 4-amino-2-oxo-5-chloro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-methoxy-1,2-dihydropyrimidin-1-yl, 2-oxo-4-mercapto- 1,2-dihydropyrimidin-1-yl, 2-oxo-4-hydroxy-1,2-dihydropyrimidin-1-yl (ie, uracinyl), 2-oxo-4-hydroxy-5-methyl-1,2 -Dihydropyrimidin-1-yl (ie, thyminyl), 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl (ie, 5-methylcytosinyl), or an amino group is a nucleic acid synthesis 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl protected with a protecting group of

  In general formula (I), the aromatic hydrocarbon ring group represented by “B” is a monovalent substituent obtained by removing one hydrogen atom from an aromatic hydrocarbon ring having 6 to 20 carbon atoms. Means a single ring or a condensed ring. Specifically, for example, phenyl, indenyl, naphthyl, pentarenyl, azulenyl, heptaenyl, biphenylenyl, indacenyl, fluorenyl, phenanthryl, anthryl and the like can be used as the base part of the nucleic acid component for the purpose of the present invention. Includes structure. In addition, the aromatic hydrocarbon ring is a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, an amino group, an amino group protected with a protecting group for nucleic acid synthesis, a halogen atom, a lower alkyl group, an alkoxy group, a carboxyl group, The aromatic hydrocarbon group which may be substituted with one or more groups such as aryloxy group, nitro group, trifluoromethyl, phenyl group and the like may be substituted, for example, 4-hydroxy Phenyl, 2-hydroxyphenyl, 4-aminophenyl, 2-aminophenyl, 2-methylphenyl, 2,6-dimethylphenyl, 2-chlorophenyl, 4-chlorophenyl, 2,4-dichlorophenyl, 2,5-dichlorophenyl, 2 -Bromophenyl, 4-methoxyphenyl, 4-chloro-2-nitrophenyl, 4-nitrophenyl, , 4-dinitrophenyl, biphenyl, and the like. The aromatic hydrocarbon ring group which may be substituted is preferably a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, an amino group, an amino group protected with a protecting group for nucleic acid synthesis, or a lower alkoxy group. Or the phenyl group substituted by the nitro group, a phenyl group, etc. are mentioned.

  The protecting group of the α group “mercapto group protected with a protecting group for nucleic acid synthesis” is not particularly limited as long as it can stably protect a mercapto group during nucleic acid synthesis, Specifically, it refers to a protecting group that is stable under acidic or neutral conditions and can be cleaved by a chemical method such as hydrogenolysis, hydrolysis, electrolysis, and photolysis. In addition to those mentioned above, “disulfide-forming groups” such as alkylthio groups such as methylthio, ethylthio and tert-butylthio, arylthio groups such as benzylthio, etc. can be mentioned, preferably “aliphatic acyl groups” or An “aromatic acyl group”, more preferably a benzoyl group or a benzyl group.

  Examples of the “C 1-5 alkoxy group” in the α group include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, s-butoxy, tert-butoxy, and n-pentoxy. Preferably, it is a methoxy or ethoxy group.

  Examples of the “C 1-5 alkylthio group” in the α group include methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, s-butylthio, tert-butylthio, and n-pentylthio. Is a methylthio or ethylthio group.

  Examples of the “amino group substituted with an alkyl group having 1 to 5 carbon atoms” in the α group include, for example, methylamino, ethylamino, propylamino, isopropylamino, butylamino, isobutylamino, s-butylamino, tert-butyl Amino, dimethylamino, diethylamino, dipropylamino, diisopropylamino, dibutylamino, diisobutylamino, di (s-butyl) amino, di (tert-butyl) amino can be mentioned, preferably methylamino, ethylamino , Dimethylamino, diethylamino or diisopropylamino group.

  Examples of the “C 1-5 alkyl group” in the α group include methyl, ethyl, propyl, isopropyl, isopropyl, butyl, isobutyl, s-butyl, tert-butyl, n-pentyl, and the like. Preferred is a methyl or ethyl group.

  Examples of the “halogen atom” in the α group include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom, and preferably a fluorine atom or a chlorine atom.

  The protecting group of the “amino group protected with a protecting group for nucleic acid synthesis” in the α group is not particularly limited as long as it can stably protect an amino group during nucleic acid synthesis. Refers to protecting groups that are stable under acidic or neutral conditions and can be cleaved by chemical methods such as hydrogenolysis, hydrolysis, electrolysis and photolysis, for example, formyl, acetyl, propionyl, butyryl, isobutyryl , Pentanoyl, pivaloyl, valeryl, isovaleryl, octanoyl, nonanoyl, decanoyl, 3-methylnonanoyl, 8-methylnonanoyl, 3-ethyloctanoyl, 3,7-dimethyloctanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl , Hexadecanoyl, 1-methylpentadecanoyl, 14-methylpenta Alkylcarbonyl groups such as canoyl, 13,13-dimethyltetradecanoyl, heptadecanoyl, 15-methylhexadecanoyl, octadecanoyl, 1-methylheptadecanoyl, nonadecanoyl, nonadecanoyl, icosanoyl and heinacosanoyl, succinoyl, glutaroyl, adipoyl Carboxylated alkylcarbonyl groups such as, halogeno lower alkylcarbonyl groups such as chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl, lower alkoxy lower alkylcarbonyl groups such as methoxyacetyl, (E) -2-methyl- “Aliphatic acyl groups” such as unsaturated alkylcarbonyl groups such as 2-butenoyl; arylcarbonyl groups such as benzoyl, α-naphthoyl, β-naphthoyl, 2- Lomobenzoyl, halogenoarylcarbonyl groups such as 4-chlorobenzoyl, 2,4,6-trimethylbenzoyl, lower alkylated arylcarbonyl groups such as 4-toluoyl, lower alkoxylated arylcarbonyl groups such as 4-anisoyl, Carboxylated arylcarbonyl groups such as 2-carboxybenzoyl, 3-carboxybenzoyl, 4-carboxybenzoyl, nitrated arylcarbonyl groups such as 4-nitrobenzoyl, 2-nitrobenzoyl; 2- (methoxycarbonyl) benzoyl An “aromatic acyl group” such as a lower alkoxycarbonylated arylcarbonyl group, an arylated arylcarbonyl group such as 4-phenylbenzoyl; methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl, “Lower alkoxycarbonyl group” such as sobutoxycarbonyl; “Lower alkoxycarbonyl group substituted with halogen or tri-lower alkylsilyl group” such as 2,2,2-trichloroethoxycarbonyl, 2-trimethylsilylethoxycarbonyl; Vinyl “Alkenyloxycarbonyl group” such as oxycarbonyl, aryloxycarbonyl; 1-2 “lower” such as benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl An aralkyloxycarbonyl group in which the aryl ring may be substituted with an alkoxy or nitro group ”can be mentioned, preferably an“ aliphatic acyl group ”or“ aromatic acyl group ”, more preferably Benzoyl It is.

  In the general formula (I), when “X” is “a nitrogen atom having a substituent”, the substituent includes a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, A sulfonyl group, a silyl group, and a functional molecular unit substituent can be mentioned.

  Here, the “alkyl group” refers to a linear or branched alkyl group having 1 to 20 carbon atoms, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, tert-butyl. , N-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl Straight chain having 1 to 6 carbon atoms such as 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl In addition to branched alkyl groups (in this specification, these are also referred to as lower alkyl groups), heptyl, octyl, nonyl Decyl include straight or branched chain alkyl group having 7 to 20 carbon atoms, preferably a straight or branched chain alkyl group having 1 to 6 carbon atoms described above.

  The “alkenyl group” refers to a linear or branched alkenyl group having 2 to 20 carbon atoms, and includes ethenyl, 1-propenyl, 2-propenyl, 1-methyl-2-propenyl, 1-methyl-1-propenyl. 2-methyl-1-propenyl, 2-methyl-2-propenyl, 2-ethyl-2-propenyl, 1-butenyl, 2-butenyl, 1-methyl-2-butenyl, 1-methyl-1-butenyl, 3 -Methyl-2-butenyl, 1-ethyl-2-butenyl, 3-butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl, 1-ethyl-3-butenyl, 1-pentenyl, 2-pentenyl 1-methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-pentenyl, 1-methyl-3-pentenyl, 2-methyl-3-pentenyl, 4-pentenyl, 1-methyl C2-C6 linear or branched alkenyl such as ru-4-pentenyl, 2-methyl-4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl In addition to the groups (in the present specification, these are also referred to as lower alkenyl groups), geranyl, farnesyl, and the like are included, and preferably the above-described linear or branched alkenyl group having 2 to 6 carbon atoms. is there.

  The “cycloalkyl group” refers to a cycloalkyl group having 3 to 10 carbon atoms, and examples thereof include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbornyl, adamantyl, and the like, preferably cyclopropyl, A cycloalkyl group having 3 to 8 carbon atoms such as cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like. In addition, the “cycloalkyl group” includes a heterocyclic group in which one or more methylenes on the ring of the cycloalkyl group are substituted with an oxygen atom, a sulfur atom, or a nitrogen atom substituted with an alkyl group, Examples thereof include a tetrahydropyranyl group.

  The “aryl group” means a monovalent substituent having 6 to 14 carbon atoms obtained by removing one hydrogen atom from an aromatic hydrocarbon group, and examples thereof include phenyl, indenyl, naphthyl, phenanthrenyl, anthracenyl and the like. . Further, the aryl ring may be substituted with one or more groups such as a halogen atom, a lower alkyl group, a hydroxyl group, an alkoxy group, an aryloxy group, an amino group, a nitro group, trifluoromethyl, a phenyl group, Examples of the optionally substituted aryl group include 2-methylphenyl, 2,6-dimethylphenyl, 2-chlorophenyl, 4-chlorophenyl, 2,4-dichlorophenyl, 2,5-dichlorophenyl, and 2-bromo. Phenyl, 4-methoxyphenyl, 4-chloro-2-nitrophenyl, 4-nitrophenyl, 2,4-dinitrophenyl, biphenyl and the like can be mentioned. Preferable examples include a halogen atom, a phenyl group substituted with a lower alkoxy group nitro group, and a phenyl group.

  The “aralkyl group” means an alkyl group having 1 to 6 carbon atoms substituted with an aryl group, and includes benzyl, α-naphthylmethyl, β-naphthylmethyl, indenylmethyl, phenanthrenylmethyl, anthracenyl “Methyl group substituted with 1 to 3 aryl groups” such as methyl, diphenylmethyl, triphenylmethyl, α-naphthyldiphenylmethyl, 9-anthrylmethyl, 4-methylbenzyl, 2,4,6 -Trimethylbenzyl, 3,4,5-trimethylbenzyl, 4-methoxybenzyl, 4-methoxyphenyldiphenylmethyl, 4,4'-dimethoxytriphenylmethyl, 2-nitrobenzyl, 4-nitrobenzyl, 4-chlorobenzyl, “Lower alkyl, lower alkoxy, halogen, such as 4-bromobenzyl, 4-cyanobenzyl, etc. 1-phenethyl, 2-phenethyl, 1-naphthylethyl, 2-naphthylethyl, 1-phenylpropyl, “methyl group substituted with 1 to 3 aryl groups in which the aryl ring is substituted with an ano group” -Phenylpropyl, 3-phenylpropyl, 1-naphthylpropyl, 2-naphthylpropyl, 3-naphthylpropyl, 1-phenylbutyl, 2-phenylbutyl, 3-phenylbutyl, 4-phenylbutyl, 1-naphthylbutyl, 2 -Naphthylbutyl, 3-naphthylbutyl, 4-naphthylbutyl, 1-phenylpentyl, 2-phenylpentyl, 3-phenylpentyl, 4-phenylpentyl, 5-phenylpentyl, 1-naphthylpentyl, 2-naphthylpentyl, 3 -Naphthylpentyl, 4-naphthylpentyl, 5-naphthylpentyl, 1 Phenylhexyl, 2-phenylhexyl, 3-phenylhexyl, 4-phenylhexyl, 5-phenylhexyl, 6-phenylhexyl, 1-naphthylpentyl, 2-naphthylpentyl, 3-naphthylpentyl, 4-naphthylpentyl, 5- “C3-C6 alkyl group substituted with aryl group” such as naphthylpentyl, 6-naphthylpentyl, and the like are included. Preferably, “methyl group substituted with 1 to 3 aryl groups”, “methyl substituted with 1 to 3 aryl groups with aryl ring substituted with lower alkyl, lower alkoxy, halogen, cyano group” Group ”, more preferably 4-methoxyphenyldiphenylmethyl, 4,4′-dimethoxytriphenylmethyl.

  Examples of the “acyl group” include formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl, isovaleryl, octanoyl, nonanoyl, decanoyl, 3-methylnonanoyl, 8-methylnonanoyl, 3-ethyloctanoyl, 3,7-dimethyl Octanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, 1-methylpentadecanoyl, 14-methylpentadecanoyl, 13,13-dimethyltetradecanoyl, heptadecanoyl, 15-methylhexayl Alkylcarbonyl groups such as decanoyl, octadecanoyl, 1-methylheptadecanoyl, nonadecanoyl, icosanoyl and heinacosanoyl, succinoyl, glutaroyl, Carboxylated alkylcarbonyl groups such as poyl, halogeno lower alkylcarbonyl groups such as chloroacetyl, dichloroacetyl, trichloroacetyl and trifluoroacetyl, lower alkoxy lower alkylcarbonyl groups such as methoxyacetyl, aryls such as phenoxyacetyl group Oxy-lower alkylcarbonyl groups, “aliphatic acyl groups” such as unsaturated alkylcarbonyl groups such as (E) -2-methyl-2-butenoyl, and arylcarbonyls such as benzoyl, α-naphthoyl, β-naphthoyl Groups, halogenoarylcarbonyl groups such as 2-bromobenzoyl, 4-chlorobenzoyl, lower alkylated arylcarbonyl groups such as 2,4,6-trimethylbenzoyl, 4-toluoyl, low groups such as 4-anisoyl Alkoxylated arylcarbonyl groups, carboxylated arylcarbonyl groups such as 2-carboxybenzoyl, 3-carboxybenzoyl, 4-carboxybenzoyl, nitrated arylcarbonyl groups such as 4-nitrobenzoyl, 2-nitrobenzoyl; 2- ( A lower alkoxycarbonylated arylcarbonyl group such as methoxycarbonyl) benzoyl, an “aromatic acyl group” such as an arylated arylcarbonyl group such as 4-phenylbenzoyl, preferably formyl, acetyl, propionyl, Butyryl, isobutyryl, pentanoyl, pivaloyl, benzoyl and phenoxyacetyl groups.

  Examples of the “sulfonyl group” include an “aliphatic sulfonyl group” such as a sulfonyl group substituted with a linear or branched alkyl group having 1 to 6 carbon atoms such as methanesulfonyl and ethanesulfonyl, benzenesulfonyl, and p-toluenesulfonyl. Examples thereof include “aromatic sulfonyl groups” such as sulfonyl groups substituted by various aryl groups such as methanesulfonyl and p-toluenesulfonyl groups.

  Examples of the “silyl group” include “tri-lower alkylsilyl group” such as trimethylsilyl, triethylsilyl, isopropyldimethylsilyl, t-butyldimethylsilyl, methyldiisopropylsilyl, methyldi-t-butylsilyl, triisopropylsilyl, diphenylmethylsilyl, Examples thereof include “tri-lower alkylsilyl groups substituted with 1 to 2 aryl groups” such as butyldiphenylbutylsilyl, diphenylisopropylsilyl, phenyldiisopropylsilyl, and preferably trimethylsilyl, triethylsilyl, triisopropylsilyl , T-butyldimethylsilyl, t-butyldiphenylsilyl, and more preferably trimethylsilyl.

  “Functional molecular unit substituents” include labeled molecules (for example, fluorescent molecules, chemiluminescent molecules, molecular species containing radioactive isotopes, etc.), DNA and RNA cleaving active molecules, intracellular and nuclear translocation signal peptides, etc. Is included.

Among the ribonucleotide analogs (1), preferred are:
(A) a ribonucleotide analog wherein X is a single bond;
(B) X is a nitrogen atom having a substituent, and the substituent is a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, an alkenyl group having 1 to 5 carbon atoms, an aryl group having 6 to 14 carbon atoms, 1 to A C1-C5 aliphatic acyl group such as a methyl group, a methanesulfonyl group or a p-toluenesulfonyl group, an aromatic sulfonyl group or an acetyl group substituted with three aryl groups, or a phenoxyacetyl group And ribonucleotide analogs that are aromatic acyl groups such as benzoyl groups, and the substituent is a functional molecular unit substituent, which is a fluorescent or chemiluminescent labeling molecule, a nucleic acid cleavage active functional group, or an intracellular or nuclear A ribonucleotide analog that is an internalization signal peptide,
(C) B is 6-aminopurin-9-yl (ie, adeninyl), 6-aminopurin-9-yl having an amino group protected with a protecting group for nucleic acid synthesis, 2,6-diaminopurine-9- Yl, 2-amino-6-chloropurin-9-yl, 2-amino-6-chloropurin-9-yl, amino-protected 2-amino-6-fluoropurine-9 2-yl-6-fluoropurin-9-yl, 2-amino-6-bromopurin-9-yl, amino group is protected with a protecting group for nucleic acid synthesis, amino group is a protecting group for nucleic acid synthesis Protected 2-amino-6-bromopurin-9-yl, 2-amino-6-hydroxypurin-9-yl (ie, guaninyl), 2-amino- in which the amino group is protected with a protecting group for nucleic acid synthesis 6-hydroxypurin-9-yl, 6 Amino-2-methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl, 6-amino-2-fluoropurin-9-yl, 2,6-dimethoxypurin-9-yl, 2, 6-dichloropurin-9-yl, 6-mercaptopurin-9-yl, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl (ie, cytosynyl), amino group is a protecting group for nucleic acid synthesis Protected 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl, the amino group being a nucleic acid Synthetic protecting group protected 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl, 4-amino-2-oxo-5-chloro-1,2-dihydropyrimidine- 1-I 2-oxo-4-methoxy-1,2-dihydropyrimidin-1-yl, 2-oxo-4-mercapto-1,2-dihydropyrimidin-1-yl, 2-oxo-4-hydroxy-1,2 -Dihydropyrimidin-1-yl (ie uracinyl), 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl (ie thyminyl), 4-amino-5-methyl-2 -Oxo-1,2-dihydropyrimidin-1-yl (ie 5-methylcytosinyl) group or 4-amino-5-methyl-2-oxo-1, wherein the amino group is protected with a protecting group for nucleic acid synthesis A nucleotide analog which is 2-dihydropyrimidin-1-yl;
(D) B is benzoylaminopurin-9-yl, adenyl, 2-isobutyrylamino-6-hydroxypurin-9-yl, guaninyl, 2-oxo-4-benzoylamino-1,2-dihydropyrimidine- A ribonucleotide analog which is a 1-yl, cytosynyl, 2-oxo-5-methyl-4-benzoylamino-1,2-dihydropyrimidin-1-yl, 5-methylcytosinyl, uracinyl or thyminyl group,
(E) a ribonucleotide analog wherein m is 0 and n is 1.
(F) a ribonucleotide analog wherein Y is an oxygen atom,
Can be mentioned.

  Here, preferred combinations of (a) to (f) are (a) + [(c) or (d)] + (e) + (f) and (b) + [(c) or (d)] + (E) + (f), more preferably a ribonucleotide analog represented by the following formula (2) or (3), particularly preferably a ribonucleotide analog represented by the following formula (3).

（式中、Ｂは前記と同意義、Ｒは水素原子または炭素数１〜６のアルキル基またはベンジル基を意味する。）

(In the formula, B is as defined above, and R represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a benzyl group.)

  In addition, as a C1-C6 alkyl group here, the C1-C6 linear or branched alkyl group mentioned above can be mentioned. A methyl group is preferred.

  Methods for producing such ribonucleotide analogs are already known. For example, referring to JP-A-10-304889, JP-A 2000-297097, WO2003 / 025173, WO2005 / 021570, etc., common technical knowledge in the industry. Can be synthesized based on

  The modified sense strand RNA contains one or more such ribonucleotide analogs. Preferably, it is a modified oligoribonucleotide comprising 2 to 15, more preferably 3 to 10, continuously or discontinuously. The position of the ribonucleotide analog in the modified sense strand RNA is not particularly limited, but is 10 bases downstream or upstream from the 5 ′ end or 3 ′ end, preferably 8 bases from the 5 ′ end or 3 ′ end, respectively. It can be located continuously or discontinuously in the downstream or upstream region.

(I-4) Modified siRNA
The modified siRNA of the present invention is formed by hybridizing the above-mentioned antisense strand RNA and the above-mentioned modified sense strand RNA to form a duplex. The modified siRNA of the present invention having a modified sense strand RNA is (a) a duplex is stably formed, and (b) an unmodified siRNA consisting of an antisense strand RNA and an unmodified sense strand, Increasing enzyme resistance to nucleases increases in vivo stability.

  Here, the latter (b) in vivo stability can be evaluated as an improvement in serum resistance (enzyme degradation resistance), in other words, an increase in serum half-life. Here, the serum half-life means the time required for the amount of siRNA to decrease to half in serum. Such serum half-life can be carried out according to the method described in Example 3 described later, and can be measured with reference to such description.

  Specifically, as shown in Experimental Example 3, the corresponding unmodified siRNA (shown as “siApoB-1” in FIG. 8) has a serum half-life of 138 minutes. The serum half-life of the modified siRNA is more than twice that (in FIG. 8, the serum half-life of the modified siRNA “siBNA-1” of the present invention is 267 minutes, and the serum half-life of “siBNA-2” is 1107 minutes). The preferred serum half-life of the modified siRNA of the invention is at least 3 times, more preferably at least 8 times the serum half-life of the corresponding unmodified siRNA.

  The modified siRNA of the present invention has a higher interfering RNA effect under in vivo conditions than the corresponding unmodified siRNA, and thus is superior in the effect of reducing the expression of the ApoB gene in vivo. For example, as shown in Experimental Example 2, the modified siRNA of the present invention was administered to the mouse intravenously at a rate of 10 pmol / g body weight on the first day and the third day, and then collected on the fourth day. It has an siRNA interference effect capable of inhibiting the expression of mRNA encoding ApoB by at least 20% (FIG. 2).

  The modified siRNA of the present invention can be synthesized using a known DNA synthesizer according to information on the target sequence of the ApoB gene and a known RNA synthesis technique. For example, the antisense strand RNA and the modified sense strand RNA of the present invention may be synthesized separately and synthesized by hybridizing them, or introduced into a microorganism or the like using an expression vector, and biological Can also be manufactured.

  Preferred modified siRNAs include a nucleotide sequence “3 ′” complementary to the nucleotide sequence of the 10167 to 10187 region of human ApoB mRNA (SEQ ID NO: 1) and the nucleotide sequence of the 1015 to 1035 region of mouse ApoB mRNA (SEQ ID NO: 2). Oligoribonucleotide having “-caguagugugacuuaugguua-5 ′” (SEQ ID NO: 3) as an antisense strand RNA, and at least one ribonucleotide of the complementary base sequence “gucaucacacugaauaccaau” (SEQ ID NO: 4), preferably the base sequence 2 or more of ribonucleotides located 10 bases downstream from the 5 ′ end or 10 bases upstream from the 3 ′ end, preferably 8 bases downstream from the 5 ′ end or 8 bases upstream from the 3 ′ end Alternatively, non-sequentially substituted with the aforementioned ribonucleotide analog having the corresponding base.

Specific examples of the base sequence of the modified sense strand of the modified siRNA include those having the following sequences.
5'-GTCATcacacugaauaccaaudTdT-3 '(SEQ ID NO: 5)
5'-GTcaTcacacugaaTacCaaudTdT-3 '(SEQ ID NO: 6)
(In the above sequence, the base indicated by capital letters means the base consisting of the nucleotide analog represented by the above formula (2) or (3)).

  The modified siRNA of the present invention is not easily degraded by nucleases and can exist in the living body for a long time after administration to the living body. Thereafter, the antisense strand RNA binds to the target mRNA, cleaves the mRNA, and suppresses protein synthesis at the translation level.

  From these facts, the modified siRNA of the present invention is a highly practical RNA interference agent that is stable in vivo and exhibits an excellent RNA interference effect. Such a modified siRNA is expected to be useful as an active ingredient of a pharmaceutical agent for preventing or treating a disease caused by the expression of ApoB.

  Here, diseases associated with the expression of ApoB include dyslipidemia, coronary artery disease, coronary heart disease, and atherosclerosis. Dyslipidemias include familial hypercholesterolemia, familial mixed hyperlipidemia, familial type III hyperlipidemia, acquired hyperlipoproteinemia, high LDL cholesterolemia, statin-resistant high cholesterol Mention may be made of secondary hyperlipidemia such as septicemia, hyper-Lp (a) blood, and nephrotic syndrome.

(II) Pharmaceutical Composition The pharmaceutical composition targeted by the present invention comprises such a modified siRNA as an active ingredient.

  The pharmaceutical composition according to the present invention can optionally contain a pharmacologically acceptable carrier depending on its administration form.

  The pharmaceutical compositions of the present invention can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration includes (a) oral administration, (b) pulmonary administration, eg, inhalation of powder or aerosol (including by nebulizer, intratracheal, intranasal), (c) epidermis, transdermal, intraocular and mucosa Topical administration (including vaginal and rectal delivery), or (d) intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, or intracranial (eg, intrathecal or intraventricular) administration Can be parenteral.

  Compositions and formulations for oral administration include, but are not limited to, powders (powder) or granules, microparticles, nanoparticles, suspensions or aqueous solutions in water or non-aqueous media, capsules ( Hard, soft), sachets, tablets and the like.

  Compositions and formulations for topical administration include transdermal patches, ointments, lotions, creams, gels, drops, sprays, suppositories, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like are necessary or desirable. Coated condoms, gloves and the like can also be used. Preferred topical formulations include those in which the oligonucleotide analogs of the present invention are mixed with topical delivery agents such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants.

  The pharmaceutical composition of the present invention includes, but is not limited to, liquids, emulsions, and liposome-containing preparations. These compositions can be prepared from a variety of components including, but not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Delivery of drugs to liver tissue is facilitated by carrier vehicle delivery including, but not limited to, cationic liposomes, cyclodextrins, porphyrin derivatives, branched dendrimers, polyethyleneimine polymers, nanoparticles and microspheres. (Dass CR. J Pharm Pharmacol 2002; 54 (1): 3-27).

  The pharmaceutical composition can be preferably used as a preventive or therapeutic agent for a disease associated with the expression of human ApoB, wherein the disease associated with the expression of human ApoB includes dyslipidemia, coronary artery disease, coronary heart disease And atherosclerosis. In addition, as mentioned above, dyslipidemia includes familial hypercholesterolemia, familial mixed hyperlipidemia, familial type III hyperlipidemia, acquired hyperlipoproteinemia, high LDL cholesterolemia, In addition to primary hyperlipidemia such as statin-resistant hypercholesterolemia and hyper-Lp (a), secondary hyperlipidemia such as nephrotic syndrome can be mentioned.

  Hereinafter, the present invention will be described in more detail with reference to examples, but the scope of the present invention is not limited to these examples.

Preparation Example Preparation of siBNA-1 and siBNA-2
(1) siRNA of apolipoprotein B (ApoB) (hereinafter also referred to as “siApoB-1”)
ApoB siRNA (siApoB-1) having the following base sequence was used.
siApoB-1 sense strand: 5'-gucatcacacugaauaccaaudTdT-3 '(SEQ ID NO: 7)
siApoB-1 antisense strand: 3′-dTdTcaguagugugacuuaugguua-5 ′ (SEQ ID NO: 8).

  Here, the antisense strand of siApoB-1 (SEQ ID NO: 8) is the base sequence of the 10167-10187 region of the human ApoB gene (SEQ ID NO: 1) and the mouse ApoB gene (SEQ ID NO: 2) except for the 2 bases on the 3 ′ end side. ) In the 1015 to 1035 region. That is, the base sequence of 21 residues from the 5 ′ end of the sense strand (SEQ ID NO: 7) of siApoB-1 (SEQ ID NO: 4) is the base sequence of the 10167-10187 region of the human ApoB gene (SEQ ID NO: 1) and the mouse ApoB gene This corresponds to the base sequence of the region 1015 to 1035 of (SEQ ID NO: 2).

(2) siBNA-1 and siBNA-2
The following formula:

で示されるBNA（2’-O,4’-C-bridged nucleic acid）を導入した修飾siRNAとして、下記のsiBNA-1及びsiBNA-2を合成した。BNAを大文字で示す。

The following siBNA-1 and siBNA-2 were synthesized as modified siRNAs into which BNA (2′-O, 4′-C-bridged nucleic acid) shown in FIG. BNA is shown in capital letters.

<SiBNA-1>
5'-GTCATcacacugaauaccaaudTdT-3 '(SEQ ID NO: 5)
3'-dTdTcaguagugugacuuaugguua-5 '(SEQ ID NO: 8)
<SiBNA-2>
5'-GTcaTcacacugaaTacCaaudTdT-3 '(SEQ ID NO: 6)
3'-dTdTcaguagugugacuuaugguua-5 '(SEQ ID NO: 8).

The above siBNA-1 and siBNA-2 can be synthesized with reference to the following documents:
1． S. Obika, et al, Tetrahedron Lett., 1997, 38, 8735-8738.
2. S. Obika, et al., Tetrahedron Lett., 1998, 39, 5401-5404.
3. S. Obika, et al., Bioorg. Med. Chem., 2001, 9, 1001-1011.
4. Koshkin, AA, et al., J. Tetrahedron 1998, 54,3607-3630
5. Singh, SK, et al., J. Chem. Commun. 1998, 455-456.

  The following Experimental Examples 1 to 3 were performed using siApoB-1, siBNA-1 and siBNA-2 prepared above.

Experimental Example 1 Expression evaluation of ApoB (in vitro test)
Mouse hepatocytes were transfected with siApoB-1, siBNA-1 and siBNA-2 at the respective concentrations prepared above, and the influence of ApoB on mRNA expression was examined.

(1) Experimental method
Two pieces of mouse hepatocytes (NMuLi cells) were seeded and confluent in a 10 cm dish.
(1) One day later, the cells were seeded on a 6-well plate at a rate of 600,000 cells / well. Thereafter, the cells were incubated at 37 ° C. for 36 hours in a 5% CO ₂ incubator until the whole became about 70 to 80% confluent.

  (2) 36 hours after the incubation, the cells were transfected with siApoB-1, siBNA-1 and siBNA-2 (hereinafter collectively referred to as “siRNA” for convenience) in the following manner.

  First, siRNA to be transfected was prepared at a concentration 4 times the final concentration. Specifically, 150 pmol of each siRNA and 7.5 μl of Lipofectamine RNAiMAX (Invitrogen) were placed in a tube and scaled up to a total of 3750 ul with Opti-MEM (Invitrogen). As a result, a 40 nM siRNA solution can be prepared. The tube was left at room temperature for 20 minutes. The 1 nM solution and the 0.1 nM solution of siRNA were prepared by further diluting the 40 nM solution 40 times and 400 times, respectively. In addition, siRNA 150 pmol and Lipofectamine RNAiMAX 7.5 μl were mixed and scaled up to a total of 375 μl with Opti-MEM. Ten tubes were prepared and allowed to stand at room temperature for 20 minutes, and then combined into 5 ml tubes.

Meanwhile, the 6-well plate was washed, and 1.5 ml of antibiotic-free D-MEM was placed in each well. Then, 500 μl of the siRNA solution prepared above was added to each well. When converted per well, the siRNA solutions of 100 nM, 10 nM, 1 nM, and 0.1 nM contain 800 pmol, 80 pmol, 8 pmol, and 0.8 pmol siRNA, respectively. The siRNA solution was added to each well and then incubated at 37 ° C. for 24 hours in a 5% CO ₂ incubator.

  (3) Thereafter, the plate after 24 hours from the incubation was washed, 1 ml of TRIzol Regent (invitrogen) was added to each well, and the cell lysate solution was placed in a 1.5 ml tube.

  Subsequently, total RNA was extracted using an AGPC (Acid Guanidium thiocyanate-Phenol-Chloroform extraction) method. The extracted total RNA was dissolved in 15 to 20 μl of RNase free water and incubated at 65 ° C. for 5 minutes. Thereafter, the absorbance of RNA was measured with a spectrophotometer (NanoDrop), and the concentration was calculated.

  After the calculation, 10 μg of each was taken into a 1.5 ml capacity tube, and cDNA was prepared using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). PCR is performed at 25 ° C for 10 minutes, then at 37 ° C for 120 minutes, then at 85 ° C for 10 seconds, and the temperature is lowered to 4 ° C. After preparing the cDNA, it was transferred to a 1.5 ml capacity tube, and a new 1.5 ml capacity tube diluted 50 times was prepared.

  Using this, Real-Time RT PCR was performed by the intercalator method using SYBR Green I (Applied Biosystems). The PCR condition was that the sample was set at 95 ° C. for 20 seconds, and then a cycle of 95 ° C. for 1 second and 60 ° C. for 20 seconds was performed 40 times. As a PCR apparatus, Step one plus real-time PCR system (Applied Biosystems) was used. The sequence of Primer used is shown below. Primer was used at 1 pmol for each sample.

ApoB- Forward Primer: TGGGCAACTTTACCTATGACTT (SEQ ID NO: 9)
ApoB-Reverse primer: AAGGAAATGGGCAACGATA (SEQ ID NO: 10)
GAPDH-Forward Primer: CAAAATGGTGAAGGTCGGTGTG (SEQ ID NO: 11)
GAPDH-ReversePrimer: ATTTGATGTTAGTGGGGTCTCG (SEQ ID NO: 12).

(2) Experimental results The expression level of ApoB mRNA in mouse hepatocytes transfected with siApoB-1, siBNA-1 and siBNA-2 at various concentrations (0, 0.1, 1, 10, 100 nM) was evaluated by the Relative CT Method. . In addition, the mRNA expression level of ApoB was relatively quantified using the result of the GPDDH mRNA expression level.

  The results are shown in FIG.

  From these results, it was confirmed that siApoB-1, siBNA-1 and siBNA-2 all suppress ApoB mRNA expression in mouse hepatocytes in a concentration-dependent manner. This confirms that siBNA-1 and siBNA-2 of the present invention can induce RNA interference in vitro (has an RNA interference effect) in the same manner as unmodified siRNA against ApoB (siApoB-1). It was done.

Experimental Example 2 Expression evaluation of ApoB (in vivo test)
SiApoB-1 and siBNA-2 were administered to mice as an siRNA (intravenous injection), respectively, (a) mRNA expression level of ApoB in the liver, (b) cholesterol level in blood, (c) degree of liver damage (ALT · AST) and (d) lipoprotein analysis.

(1) Experimental method (1-1) ApoB expression evaluation test A 6-week-old mouse C57BL6 / J (male: CLEA Japan) was used as a test animal. After two weeks of high-fat diet loading, blood was collected on day 0 and intravenously administered siBNA-1 or siApoB-1 at a rate of 10 pmol / g body weight. On the third day, blood was collected again and siBNA-1 or siApoB-1 was intravenously administered at a rate of 10 pmol / g body weight. Four days later, blood was collected from the inferior vena cava, then perfused from the superior mesenteric vein with PBS, and after completion, the liver was collected, washed with PBS, minced, snap-frozen with liquid nitrogen, and then −80 ° C. Saved with. The frozen liver sections were homogenized in 5 ml of TRIzol Regent (Invitrogen), and total RNA was extracted using the AGPC method.

  Using 10 μg of each, cDNA was prepared by High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). RNA was quantified using Real-Time PCR. The sequence of the used Primer is shown below. SYBR Green used was Applied Biosystems.

ApoB- Forward Primer: TGGGCAACTTTACCTATGACTT (SEQ ID NO: 9)
ApoB-Reverse primer: AAGGAAATGGGCAACGATA (SEQ ID NO: 10)
GAPDH-Forward Primer: CAAAATGGTGAAGGTCGGTGTG (SEQ ID NO: 11)
GAPDH-ReversePrimer: ATTTGATGTTAGTGGGGTCTCG (SEQ ID NO: 12).

  The results were evaluated by Relative CT Method.

(1-2) Blood cholesterol level and ATL / AST measurement, lipoprotein analysis Blood collected during the test period is left at room temperature for about 20 minutes, then centrifuged at 4 ° C and 5000 rpm for 20 minutes to remove serum. separated. For each serum sample, serum total cholesterol level, VLDV cholesterol level, LDL cholesterol level, and HDL cholesterol level were measured using Wako's Cholesterol E-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). Similarly, ALT / AST was measured with Wako's transaminase CII-Test Wako to evaluate the degree of liver damage. Furthermore, for the analysis of lipoprotein, Skylight Biotech Co., Ltd. analyzed the cholesterol level of 20 fractions of lipoprotein by HPLC.

(2) Experimental results (2-1) ApoB expression evaluation Expression of ApoB mRNA in the liver in the control group administered with saline (Saline administration group) and the test group administered with siApoB-1 or siBNA-2, respectively The results of evaluation of the amount by the Relative CT Method are shown in FIG. The results are relatively quantified using the GAPDH results.

  As can be seen from this result, in vivo, siBNA-2 significantly suppressed the mRNA expression of ApoB (P <0.05) more than the unmodified siRNA against ApoB (siApoB-1). From this, it was confirmed that siBNA-2 exhibits a higher RNA interference effect in vivo than unmodified siRNA (siApoB-1), and functions as a small interfering RNA molecule effective in vivo.

(2-2) Cholesterol level in blood Total cholesterol level, VLDV cholesterol level, LDL cholesterol level in serum of control group (saline administration group) and test group (siApoB-1 administration group, siBNA-2 administration group), And the time-dependent change (Day0, Day1, Day2) of HDL cholesterol level is shown in FIGS.

  As can be seen from this result, from the first day after administration, the siBNA-2 administration group compared to the control group and siApoB-1 administration group, the total cholesterol level in serum and the VLDV cholesterol level called bad cholesterol It was also found that both LDL cholesterol levels were significantly reduced (FIGS. 3-5). In contrast, the HDL cholesterol level called good cholesterol did not decrease (FIG. 6). In this siBNA-2 administration group, changes in VLDV and LDL cholesterol levels (decrease with time; Day 1-2) and changes in HDL cholesterol levels (no change with time; Day 0 → 2) were determined by lipoprotein analysis (20 for lipoprotein). Time-lapse analysis of cholesterol levels in fractions: Day 0, Day 1, Day 2) was well reflected (FIGS. 7A to C).

  From this, it was confirmed that siBNA-2 actually functions as a small interfering RNA molecule in vivo and exerts an effect of improving dyslipidemia, that is, its effectiveness as a medicine.

  As described above, as shown in FIG. 3, the total cholesterol level in the siBNA-2 administration group was significantly lower than that in the control group. The cholesterol fractions are shown in Fig. 4 to Fig. 6. Among the total cholesterol, especially the bad VLDL and LDL fractions showed a decrease in cholesterol level, and the good HDL showed changes. I understand that there is no. This proves that siBNA-2 has the effect of specifically reducing only LDL and VLDL involved in ApoB. FIG. 7 shows the cholesterol level divided into 20 fractions, which shows that VLDL and LDL are specifically decreased.

  8A and 8B show the results of ALT and AST indicating the degree of liver damage. From these results, it was confirmed that the siBNA-2 administration group had a lower degree of liver damage, lower toxicity and higher safety than the control group (saline administration group) and the siApoB-1 administration group.

Experimental Example 3 Enzymatic degradation resistance test
For siApoB-1 and siBNA-1 and siBNA-2 produced in the preparation examples, the stability in serum was evaluated by the following method.

  First, 40 pmol of each siRNA (siApoB-1, siBNA-1, siBNA-2) was added to 100 μl of mouse serum, stirred and incubated at 37 ° C. Also. Immediately after the addition and stirring (before the start of incubation), 5 μl was taken out, placed in a 1.5 ml tube and snap-frozen with liquid nitrogen. The sample at this time was set to 0 min.

  Sampling was performed over time from the incubating serum (0-1500 minutes). All samples were stored at −80 ° C. after collection. Thawed at the time of measurement, 5 μl of Proteinase K (TaKaRa) and 5 μl of DMSO were added and incubated at 37 ° C. for 30 minutes. 3 ul of 6 × Sample Buffer was added thereto, and electrophoresis was performed using 20% TBE gel (Invitrogen) (first 75V, 15 minutes, then 110V for 120 minutes). After electrophoresis, the gel was put into 100 ml of the same TBE Buffer, and 10 μl of SYBR Gold (Invitrogen) was added and permeated for 20 to 40 minutes. Thereafter, images were taken with LAS-4000 mini manufactured by FujiFilm, and the expression level of ApoB mRNA was quantified by Image J from the darkness of the band in the gel photo image.

  The results are shown in FIG.

  As can be seen from this result, it was confirmed that the unmodified siRNA against ApoB (siApoB-1) rapidly decreased in serum and lacked RNA resistance (enzymatic degradation resistance) in serum (serum half-life) : 138 minutes). In contrast, modified siRNAs (siBNA-1, siBNA-2) in which a part of the siApoB-1 sense strand was replaced with BNA suppressed the decrease in activity caused by siApoB-1 ( Serum half-life: 267 minutes for siBNA-1, 1107 minutes for siBNA-2, serum resistance (resistance to enzymatic degradation), that is, modified siRNA (siBNA-1, siBNA-2 are not easily degraded by enzymes, other It can be seen that it can stay in the blood longer than that, leading to an increase in RNA interference effect (ApoB expression suppression effect), among the modified siRNA (siBNA-1, siBNA-2) In particular, siBNA-2 was particularly excellent in serum resistance (enzyme degradation resistance), and it was found that the RNA interference effect persists in vivo.

  SEQ ID NO: 3 is an oligoribonucleotide (anti-antigen) having a base sequence complementary to the base sequence of the 10167-10187 region of human ApoB mRNA (SEQ ID NO: 1) or the 1015-1035 region of mouse ApoB mRNA (SEQ ID NO: 2). Sense strand RNA), and SEQ ID NO: 4 shows a complementary base sequence. SEQ ID NOs: 5 and 6 show the base sequence of the modified sense strand of the modified siRNA. SEQ ID NOs: 7 and 8 show the sense strand and the antisense strand of ApoB siRNA, respectively. SEQ ID NOs: 9 and 10 respectively show a forward primer and a reverse primer for detecting ApoB mRNA by Real-Time PCR. SEQ ID NOs: 11 and 12 respectively represent a forward primer and a reverse primer for detecting GAPDH mRNA by Real-Time PCR.

Claims (8)

A modified siRNA that decreases the expression of the apolipoprotein B gene,
(A) The modified siRNA molecule comprises an antisense strand RNA having a base sequence complementary to a partial sequence of the apolipoprotein B gene, and a modified sense strand RNA having a base sequence complementary to the antisense strand RNA. Have
(B) The modified sense strand RNA is a modified oligoribonucleotide consisting of a total of 19 to 23 ribonucleotides and a ribonucleotide analog,
(C) The ribonucleotide analog is represented by the following general formula (1)

(Wherein B is an aromatic heterocyclic group or aromatic hydrocarbon ring group which may have a substituent; X is a single bond or a nitrogen atom having a substituent; Y is an oxygen atom, a sulfur atom or BH _3. M represents an integer of 0 to 2; and n represents an integer of 0 to 3).
The modified siRNA above, which has a structure represented by   The modified siRNA according to claim 1, wherein the ribonucleotide analog represented by the general formula (1) is one in which X is a single bond, n is 1, and m is 0.   The antisense strand RNA is an oligoribonucleotide having a base length of 21 to 23 having a base sequence complementary to the base sequence of the region 10167 to 10187 of the apolipoprotein B gene represented by SEQ ID NO: 1. Modified siRNA to be described.   The modified siRNA according to any one of claims 1 to 3, wherein the modified sense strand RNA is a modified oligoribonucleotide having a base length of 21 to 23 having the base sequence of SEQ ID NO: 4 or 5.   A pharmaceutical composition comprising the modified siRNA according to any one of claims 1 to 4 as an active ingredient.   The pharmaceutical composition according to claim 5, which is a prophylactic or therapeutic agent for a disease associated with expression of human apolipoprotein B.   The pharmaceutical composition according to claim 6, wherein the disease is any one selected from the group consisting of dyslipidemia, coronary artery disease, coronary heart disease, and atherosclerosis.   The above dyslipidemia is familial hypercholesterolemia, familial mixed hyperlipidemia, familial type III hyperlipidemia, acquired hyperlipoproteinemia, high LDL cholesterolemia, statin resistant hypercholesterolemia The pharmaceutical composition according to claim 7, wherein the composition is secondary hyperlipidemia such as symptom, hyper Lp (a) blood disease or nephrotic syndrome.

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