HK1184189B - Prophylactic or therapeutic agent for fibrosis - Google Patents

Description

Prophylactic or therapeutic agent for fibrosis

Technical Field

The present invention relates to a therapeutic agent for fibrosis. Specifically, the present invention relates to small interfering RNA (siRNA) targeting a gene encoding Transforming Growth Factor (TGF) -beta 1, and a pharmaceutical product using the same.

Background

Pulmonary fibrosis refers to a condition in which lung tissue is fibrotic due to the accumulation of excess collagen and other extracellular matrix. In pulmonary fibrosis, idiopathic pulmonary fibrosis is a chronic refractory disease with poor prognosis with an average median survival time of 3 years and a 5-year survival rate of 20-40%. Although steroid drugs and immunosuppressants are used as treatments for pulmonary fibrosis, there is no effective treatment method for improving prognosis, and therefore, development of a new therapeutic agent is required.

In recent years, it has become clear that many diseases are caused by the onset of the disease, and many gene involvement has been reported in pulmonary fibrosis (patent documents 1 to 4 and non-patent documents 1 to 6). TGF- β 1 (patent documents 1 to 2 and non-patent documents 2 to 6), Smad3 (non-patent document 1), MCP-1 (patent document 3), and the like have been reported as main factors involved in pulmonary fibrosis.

On the other hand, nucleic acids, particularly siRNA, cause degradation of mRNA of a gene identical or almost identical to a specific sequence present in a cell, hindering expression of a target gene (RNA interference). Thus, the function of blocking the expression of a target gene by RNA interference is useful in reducing or treating disease symptoms caused by abnormal expression of a specific gene or genome. Also, as for pulmonary fibrosis-associated genes, there have been reports of attempts to suppress the expression of the genes using siRNA (patent documents 1 to 4 and non-patent documents 1 to 6).

However, the techniques reported so far only show the inhibitory effect of siRNA sequences on disease-related genes in experimental animals (mice, rats), but do not sufficiently show specific effects on human genes. Further, the inhibitory effect of siRNA is only shown at 200nM (non-patent document 1) and 20 to 500nM (non-patent document 5), and there is no nucleic acid molecule that can efficiently inhibit the expression of pulmonary fibrosis-associated gene at low concentration.

Several dozen kinds of sirnas targeting TGF- β 1 gene have been reported so far (patent documents 1, 5 to 7), but since there are numerous combinations in which the total length of TGF- β 1 gene is 2346 bases (genbank accession No. nm — 000660.3) and a sequence of about 20 mers is selected from the total length of the gene, it is not easy to design dsRNA or siRNA molecules that can efficiently suppress the expression of the gene.

Patent document

Patent document 1: international publication No. 2007/109097 pamphlet

Patent document 2: international publication No. 2003/035083 pamphlet

Patent document 3: japanese laid-open patent publication No. 2007-119396

Patent document 4: japanese Kokai publication No. 2009-516517

Patent document 5: international publication No. 2009/061417 pamphlet

Patent document 6: international publication No. 2008/109548 pamphlet

Patent document 7: international publication No. 2007/79224 pamphlet

Non-patent document

Non-patent document 1: WangZ, GaoZ, ShiY, SunY, LinZ, JiangH, HouT, WangQ, YuanX, Zhuux, WuH, JinY, JPlastReconstrathestSurg, 1193-

Non-patent document 2: HwangM, KimHJ, NohHJ, ChangYC, ChaeYM, KimKH, JeonJP, LeeTS, OhHK, LeeYS, Parkkk, ExpMolPathol,48-54,81,2006

Non-patent document 3: takabatake Y, Isakay, Mizuim, Kawachih, Shimizu F, ItoT, horiM, ImaiE, Gene ther,965-973,12,2005-

Non-patent document 4: XuW, WanglW, ShiJZ, GongZJ, HeapatiliareyPancreatDisint, 300-

Non-patent document 5: LiuXJ, RuanCM, GongXF, LiXZ, WangHL, WangMW, YInJQ, BiotechnolLett,1609-

Non-patent document 6: fuyuan Dailang, Finishenzi, Yuntanjiao, Yuanzu, Bintanjiang, Qianshan, sangye Heshan, Zhongxi Ying, J.J. respirator school, 185,46,2008

Disclosure of Invention

The present invention relates to a technique for providing siRNA effective for treating fibrosis and a pharmaceutical using the same.

The present inventors have conducted intensive studies to solve the above problems, and as a result, have found that a specific siRNA targeting a human TGF- β 1 gene can effectively inhibit the expression of TGF- β 1 in human cells, and further can improve the symptoms of pulmonary fibrosis without inducing an interferon response in the lung of a pulmonary fibrosis model mouse.

That is, the present invention relates to the following items 1) to 19).

1) An siRNA comprising a target sequence of 17 to 23 consecutive bases selected from the group consisting of the bases having the base numbers 1285 to 1318, 1398 to 1418, 1434 to 1463, 1548 to 1579, 1608 to 1628, 1700 to 1726, 1778 to 1798, 1806 to 1826 and 1887 to 1907 in the sequence No. 1, wherein the total length is 30 nucleotides or less.

2) The siRNA according to the above 1), wherein the siRNA is selected from the following (a) to(s).

(a) siRNA comprising sense sequence represented by SEQ ID NO. 2 and antisense sequence represented by SEQ ID NO. 3

(b) siRNA comprising sense sequence represented by SEQ ID NO. 4 and antisense sequence represented by SEQ ID NO. 5

(c) siRNA comprising sense sequence represented by SEQ ID NO. 6 and antisense sequence represented by SEQ ID NO. 7

(d) siRNA comprising sense sequence represented by SEQ ID NO. 8 and antisense sequence represented by SEQ ID NO. 9

(e) siRNA comprising sense sequence represented by SEQ ID NO. 10 and antisense sequence represented by SEQ ID NO. 11

(f) siRNA comprising sense sequence represented by SEQ ID NO. 12 and antisense sequence represented by SEQ ID NO. 13

(g) siRNA comprising sense sequence represented by SEQ ID NO. 14 and antisense sequence represented by SEQ ID NO. 15

(h) siRNA comprising sense sequence represented by SEQ ID NO. 16 and antisense sequence represented by SEQ ID NO. 17

(i) siRNA comprising sense sequence represented by SEQ ID NO. 18 and antisense sequence represented by SEQ ID NO. 19

(j) siRNA comprising sense sequence represented by SEQ ID NO. 20 and antisense sequence represented by SEQ ID NO. 21

(k) siRNA comprising sense sequence represented by SEQ ID NO. 22 and antisense sequence represented by SEQ ID NO. 23

(l) siRNA comprising sense sequence represented by SEQ ID NO. 24 and antisense sequence represented by SEQ ID NO. 25

(m) siRNA comprising the sense sequence represented by SEQ ID NO. 26 and the antisense sequence represented by SEQ ID NO. 27

(n) siRNA comprising sense sequence represented by SEQ ID NO. 28 and antisense sequence represented by SEQ ID NO. 29

(o) siRNA comprising the sense sequence represented by SEQ ID NO. 30 and the antisense sequence represented by SEQ ID NO. 31

(p) siRNA comprising sense sequence represented by SEQ ID NO. 32 and antisense sequence represented by SEQ ID NO. 33

(q) siRNA comprising the sense sequence represented by SEQ ID NO. 34 and the antisense sequence represented by SEQ ID NO. 35

(r) siRNA comprising the sense sequence represented by SEQ ID NO. 36 and the antisense sequence represented by SEQ ID NO. 37

(s) siRNA comprising the sense sequence represented by SEQ ID NO. 54 and the antisense sequence represented by SEQ ID NO. 55

3) The siRNA according to 1) or 2), wherein 1 to 10 consecutive nucleotides from the end of the sense strand of the siRNA excluding the overhang nucleotide are converted into DNA.

4) The siRNA according to 1) to 3), wherein 1 to 10 nucleotides that are continuous from the terminal side of the 5' -end of the antisense strand of the siRNA are converted into DNA.

5) The siRNA according to 1) to 4), wherein 1 to 10 consecutive nucleotides excluding the overhang nucleotide from the terminal side of the 3 'end of the sense strand of the siRNA are converted into DNA, and 1 to 10 consecutive nucleotides from the terminal side of the 5' end of the antisense strand are converted into DNA.

6) The siRNA according to 1) to 5) above, wherein the 5' end of the antisense strand is monophosphorylated or monothiophosphorylated.

7) A pharmaceutical composition comprising the siRNA of any one of 1) to 6).

8) A TGF-. beta.1 gene expression inhibitor comprising the siRNA of any one of the above 1) to 6) as an active ingredient.

9) A preventive or therapeutic agent for fibrosis, comprising the siRNA of any one of 1) to 6) above as an active ingredient.

10) A prophylactic or therapeutic agent for pulmonary fibrosis or lung cancer, comprising the siRNA of any one of the above 1) to 6) as an active ingredient.

11) Use of the siRNAs of 1) to 6) above for producing a TGF-. beta.1 gene expression inhibitor.

12) Use of the siRNA of 1) to 6) above for producing a preventive or therapeutic agent for fibrosis.

13) Use of the siRNA of 1) to 6) for producing a prophylactic or therapeutic agent for pulmonary fibrosis or lung cancer.

14) siRNA of the above 1) to 6) for inhibiting expression of TGF-. beta.1 gene.

15) siRNA of the above 1) to 6) for preventing or treating fibrosis.

16) siRNA of the above 1) to 6) for use in the prevention or treatment of pulmonary fibrosis or lung cancer.

17) A method for inhibiting TGF-. beta.1 gene expression, which comprises administering the siRNA of 1) to 6) above to a human or an animal.

18) A method for preventing or treating fibrosis, characterized in that the siRNA of 1) to 6) above is administered to a human or an animal.

19) A method for preventing or treating pulmonary fibrosis or lung cancer, characterized in that the siRNA of 1) to 6) above is administered to a human or an animal.

The siRNA of the present invention can efficiently inhibit or inhibit the expression of TGF- β 1 at a low concentration, and is therefore useful as a pharmaceutical for preventing or treating fibrosis.

Drawings

FIG. 1 is a graph showing the inhibition rate of TGF-. beta.1 mRNA expression by siRNA.

FIG. 2 is a graph showing the inhibition rate of TGF-. beta.1 mRNA expression by chimeric siRNA.

FIG. 3 is a graph showing the inhibition rate of TGF-. beta.1 mRNA expression by a chimeric siRNA to which a phosphate or phosphorothioate is bonded.

FIG. 4 is a graph showing the expression inhibition ratio of TGF-. beta.1 in a pulmonary fibrosis model.

Fig. 5 is an optical microscope photograph (magnification: 5 times) of a lung tissue section (h.e. staining and masson trichrome staining).

Detailed Description

The target sequence of the siRNA of the present invention is 17 to 23 consecutive bases selected from the group consisting of the bases having the base numbers 1285 to 1318, 1398 to 1418, 1434 to 1463, 1548 to 1579, 1608 to 1628, 1700 to 1726, 1778 to 1798, 1806 to 1826 and 1887 to 1907 in the sequence No. 1, but the 17 to 23 consecutive bases selected from each group are preferably 19 to 23 bases, and more preferably 21 bases.

The nucleotide sequence shown in SEQ ID NO. 1 is the nucleotide sequence of TGF-. beta.1 mRNA, and the sequence information thereof is registered in GenBank as GenBank accession No. NM-000660.3.

The siRNA of the present invention is formed by hybridizing an antisense strand, which is a sequence complementary to the target sequence of TGF- β 1mRNA, with a sense strand, which is a sequence complementary to the antisense strand, and has an activity of cleaving the TGF- β 1mRNA (RNA interference effect), and an ability to prevent translation of the mRNA, that is, an ability to inhibit expression of the TGF- β 1 gene.

The sense strand and the antisense strand may be the same nucleotide length or different nucleotide lengths in terms of the nucleotide length of the siRNA of the present invention, and the total length thereof is 30 nucleotides or less, preferably 25 nucleotides or less, more preferably 23 nucleotides or less, or 21 nucleotides.

The sense strand and the antisense strand may have smooth ends at both ends, or each strand may have an overhang (protruding end) at the 3' -side. Here, the "smooth end" refers to a structure in which the end region of the double-stranded RNA, the end region of the sense strand and the end region of the antisense strand that is paired with the sense strand are paired without forming a single-stranded portion. The term "overhang" is also referred to as a overhang, and refers to a structure in which a single-stranded portion (protruding end) is present because no paired base is present in the terminal region of the sense strand of the terminal portion of the double-stranded RNA or the terminal region of the antisense strand that is paired with the sense strand.

The number of bases in the overhang terminal portion is 1 to 10 nucleotides, preferably 1 to 4 nucleotides, and more preferably 1 to 2 nucleotides. It should be noted that the length of the protruding end is irrelevant between the two strands, and the two strands may be different lengths from each other. The nucleotide at the overhang terminal part may be RNA or DNA, and is preferably a base complementary to the mRNA of TGF-. beta.1 as a target, but may be a non-complementary base as long as the RNA interference ability is maintained.

The siRNA of the present invention may be 1 double-stranded RNA composed of 2 individual strands, or may be a double-stranded RNA in which 1 strand is formed by using a stem-loop structure. That is, the siRNA of the present invention includes RNA in which a loop consisting of 2 to 4 nucleotides is formed at the 5 '-end of the sense strand and the 3' -end of the antisense strand, and RNA in which a loop consisting of 2 to 4 nucleotides is formed at the 3 '-end of the sense strand and the 5' -end of the antisense strand. Further, the RNA includes RNA in which a loop consisting of 2 to 4 nucleotides is formed at the 5 '-end of the sense strand and the 3' -end of the antisense strand, and at both ends of the 3 '-end of the sense strand and the 5' -end of the antisense strand.

The siRNA of the present invention is preferably identical to the target sequence, but may have substantially the same, i.e., homologous, sequence as long as the RNA interference is induced. Specifically, as long as the antisense strand sequence of the siRNA of the present invention hybridizes to the target sequence, there may be 1 or several (e.g., 2, 3, 4) mismatches. That is, the siRNA of the present invention includes siRNA that can induce RNA interference by substituting, adding, or deleting 1 or several bases with respect to a target sequence, or siRNA that has 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more sequence homology with the target sequence and can induce RNA interference.

In this case, the hybridization conditions include in vivo conditions when the siRNA of the present invention is administered in vivo and used as a pharmaceutical product, and moderately stringent or highly stringent conditions when the siRNA of the present invention is used as a reagent in vitro, and examples of such conditions include 400mM NaCl, 40mM PIPESPH value 6.4, 1mM EDTA, and hybridization conditions at 50 ℃ to 70 ℃ for 12 to 160 hours. These conditions are well known to those skilled in the art and are described in Sambrookettal, molecular cloning, laboratory Manual diagnosis, Cold spring harbor laboratory Press, New York, USA, 1989.

The sequence homology can be calculated by the Lipman-Pearson method (Science,227,1435, (1985)) or the like, for example, by searching for 2 from Unitsizococcomache (ktup) using the homology analysis (Searchomology) program of genetic information processing software Genetyx-Win (Ver.5.1.1; software development).

The siRNA of the present invention includes siRNA (hybrid type) in which all nucleotides of either the sense strand or the antisense strand are converted into DNA, and siRNA (chimeric type) in which a part of the nucleotides of the sense strand and/or the antisense strand are converted into DNA, as long as the RNA interference can be induced.

Here, the conversion from RNA nucleotide to DNA refers to the conversion from AMP to dAMP, from GMP to dGMP, from CMP to dCMP, and from UMP to dTMP.

As the hybrid type, siRNA in which the nucleotide of the sense strand is converted into DNA is preferable. Examples of chimeric forms include sirnas in which a part of nucleotides on the downstream side (the 3 '-end side of the sense strand and the 5' -end side of the antisense strand) is converted into DNA. Specifically, there are siRNA in which nucleotides on the 3 '-terminal side of the sense strand and nucleotides on the 5' -terminal side of the antisense strand are converted into DNA together, and siRNA in which nucleotides on either the 3 '-terminal side of the sense strand or the 5' -terminal side of the antisense strand are converted into DNA. The converted nucleotide length is preferably any length up to a nucleotide corresponding to 1/2 in the RNA molecule, and examples thereof include 1 to 13 nucleotides, preferably 1 to 10 nucleotides from the end. From the viewpoint of RNA interference effect, stability of RNA molecule, safety, and the like, examples of preferable chimeric siRNA include siRNA having nucleotide lengths of 19 to 23 nucleotides, respectively, and continuously converting nucleotides having a length of 1 to 10 nucleotides, preferably 1 to 8 nucleotides, more preferably 1 to 6 nucleotides excluding a overhang nucleotide from the 3 '-terminal side of the sense strand, and 1 to 10 nucleotides, preferably 1 to 8 nucleotides, more preferably 1 to 6 nucleotides from the 5' -terminal side of the antisense strand into DNA in an arbitrary number (see table 2 described later). In addition, at this time, it is more preferable that the number of DNA transitions of the sense strand (excluding the overhang nucleotide) and the antisense strand are the same.

In addition, as for the siRNA of the present invention, as long as the above-mentioned RNA interference can be induced, the nucleotide (ribonucleotide, deoxyribonucleotide) thereof may be a nucleotide analog in which sugar, base and/or phosphate is chemically modified. Examples of the nucleotide analogs in which the base is modified include modified uracil or cytosine at the 5-position (e.g., 5-propynyluracil, 5-propynylcytosine, 5-methylcytosine, 5-methyluracil, 5- (2-amino) propyluracil, 5-halogenocytosine, 5-halogenouracil, 5-methoxyuracil, etc.); adenine or guanine modified at position 8 (e.g., 8-bromoguanine, etc.); deazanucleotides (e.g., 7-deazaadenine, etc.); o-and N-alkylated nucleotides (e.g., N6-methyladenine, etc.), and the like.

Examples of the sugar-modified nucleotide analogs include those in which 2' -OH of ribonucleotide is replaced by H, OR, R, halogenAtom, SH, SR, NH₂、NHR、NR₂Or CN (wherein R represents an alkyl group having 1 to 6 carbon atoms, an alkenyl group or an alkynyl group), a sugar substituent at the 2 '-position substituted by CN, a 5' -end phosphorylate in which-OH at the 5 '-end is monophosphorylated or monothiophosphorylated, and a 5' -end monothiophosphorylate.

Examples of the phosphate-modified nucleotide analogs include nucleotide analogs in which a sulfate group, which bonds adjacent ribonucleotides, is substituted with a phosphorothioate group.

In addition, unlike the nucleotide analogs described above, the siRNA of the present invention may have a specific substituent or functional molecule bonded to at least one of nucleotides No. 1 to 6 (5 ' end, 3 ' end, or internal base or sugar other than end) from the 5' end side or 3 ' end side of the sense strand, either directly or via a linker, and preferably the substituent or functional molecule is bonded to at least one of nucleotides No. 1 to 6, preferably No. 1 to 4, from the 5' end side of the sense strand.

Here, as the substituent, an amino group; a mercapto group; a nitro group; an alkyl group having 1 to 40 (preferably 2 to 20, and more preferably 4 to 12) carbon atoms; an aminoalkyl group having 1 to 40 carbon atoms (preferably 2 to 20 carbon atoms, and more preferably 4 to 12 carbon atoms); a thioalkyl group having 1 to 40 (preferably 2 to 20, more preferably 4 to 12) carbon atoms; an alkoxy group having 1 to 40 (preferably 2 to 20, and more preferably 4 to 12) carbon atoms; an aminoalkoxy group having 1 to 40 (preferably 2 to 20, more preferably 4 to 12) carbon atoms; a thioalkoxy group having 1 to 40 carbon atoms (preferably 2 to 20 carbon atoms, and more preferably 4 to 12 carbon atoms); a mono-or dialkylamino group having 1 to 40 (preferably 2 to 20, more preferably 4 to 12) carbon atoms; an alkylmercapto group having 1 to 40 (preferably 2 to 20, more preferably 4 to 12) carbon atoms; a polyethylene oxide group having 2 to 40 carbon atoms (preferably 2 to 20 carbon atoms, and more preferably 4 to 12 carbon atoms); polypropylene oxide groups having 3 to 39 carbon atoms (preferably 3 to 21 carbon atoms, more preferably 3 to 12 carbon atoms). By bonding these substituents, the RNA interference effect can be significantly enhanced.

Examples of the functional molecule include sugars, proteins, peptides, amino acids, DNA, RNA (including tRNA), aptamers, modified nucleotides, low-molecular organic/inorganic materials, cholesterol, dendrimers, lipids, and polymer materials. By adding a functional molecule, an excellent RNA interference effect and a useful effect based on the functional molecule can be achieved at the same time.

Examples of the sugar include monosaccharides such as glucose, galactose, glucosamine, and galactosamine, and oligosaccharides and polysaccharides obtained by arbitrarily combining these monosaccharides.

The protein may be a protein present in the body, a protein having a pharmacological action, a protein having a molecular recognition action, or the like, and examples of the protein include an import protein b protein, avidin, an antibody, or the like.

Specifically, the DNA may be DNA having a base length of 5 to 50, preferably 5 to 25.

Examples of the peptides include octa-arginine peptide R8, nuclear localization signal peptide sequences (HIV-1Tat, SV40T antigen, etc.), nuclear export signal peptides (HIV-1Rev, MAPKK, etc.), cell membrane fusion peptides, and the like. Examples of the modified nucleotide include modified nucleotides obtained by modifying a phosphate backbone of phosphorothioate-type or boranophosphate-type DNA/RNA; 2 ' -modified nucleotides such as 2 ' -OMe-modified RNA and 2 ' -F-modified RNA; modified nucleotides formed by crosslinking sugar molecules of nucleotides such as LNA (LockedNucleicic acid), ENA (2 '-O, 4' -C-Ethylene-bridged nucleotides) and the like; modified nucleotides having different basic skeletons, such as PNA (peptide nucleic acid) and morpholino nucleotide (see WO 2008/140126 and WO 2009/123185).

Examples of the low-molecular organic/inorganic material include fluorescent materials such as Cy3 and Cy 5; biotin; quantum dots; gold particles, and the like. Examples of the dendrimer include polyamidoamine dendrimers. Examples of the lipid include linoleic acid, DOPE (1, 2-Dioleoyl-sn-glycerol-3-phosphoethanomine), and the like, and in addition, a double-stranded lipid having 2 hydrophobic groups as described in pamphlet of international publication No. 2009/123185. Examples of the polymer material include polyethylene glycol and polyethyleneimine.

Here, the group having a functional molecule may be a residue of the functional molecule itself, or a group in which one functional group of a bifunctional linker is bonded to a residue of the functional molecule. In other words, in the former case, the functional molecule is directly bonded to the predetermined site of the sense strand RNA, and in the latter case, the functional molecule is bonded to the predetermined site of the sense strand RNA via a bifunctional linker. The bifunctional linker is not particularly limited as long as it is a linker having 2 functional groups, and examples thereof include N-hydroxysuccinimide ester of 3- (2-pyridyldithio) propionic acid, N-4-maleimidobutyric acid, S- (2-pyridyldithio) cysteamine, succinimidyl iodoacetate, N- (4-maleimidobutyryloxy) succinimide, N- [5- (3' -maleimidopropionamido) -1-carboxypentyl ] iminodiacetic acid, and N- (5-aminopentyl) -iminodiacetic acid.

The substituent or the functional molecule in the sense strand RNA or the bonding site of the linker connecting them is not particularly limited, but it is preferable that the substituent or the functional molecule is bonded by substituting a hydrogen atom of a hydroxyl group of a phosphate moiety of a predetermined nucleotide constituting the sense strand RNA.

Specific examples of the siRNA of the present invention include double-stranded RNA molecules comprising a sense sequence and an antisense sequence as shown in the following (a) to(s).

TABLE 1

Further, as the chimeric type, siRNA in which 1 to 8 nucleotides from the 3 '-terminal side of the sense strand and 1 to 6 nucleotides from the 5' -terminal side of the antisense strand are successively converted into DNA in an arbitrary number is preferably used, and for example, the case of using the above (l) is exemplified as follows.

TABLE 2

In the table, upper case letters represent RNA, lower case letters represent DNA, and underlining indicates the position of overhang.

The method for producing the siRNA of the present invention is not particularly limited, and the siRNA can be synthesized by a known production method, for example, by chemical synthesis in vitro or by a transcription system using a promoter and RNA polymerase.

Chemical synthesis can be performed using an Amidite resin containing a nucleic acid molecule as an siRNA component as a starting material, and using a nucleic acid synthesizer.

Synthesis by transcription System double-stranded RNA can be synthesized by in vitro transcription method in which hairpin RNA is trimmed.

As shown in examples described later, the siRNA of the present invention obtained in this way can effectively inhibit the expression of TGF- β 1 at the mRNA level in cells derived from human alveolar epithelial cells, and can exert an effect of improving the symptoms of pulmonary fibrosis without inducing an interferon response in the lungs of a pulmonary fibrosis model mouse.

Therefore, the siRNA of the present invention and the expression vector capable of expressing the siRNA in an administration subject are useful as a pharmaceutical product (pharmaceutical composition) for administration to a human or an animal. Specifically, the present invention is useful as a pharmaceutical for inhibiting the expression of TGF-. beta.1 gene, a pharmaceutical for preventing and treating diseases caused by the overexpression of TGF-. beta.1, for example, fibrosis, that is, a prophylactic or therapeutic agent for fibrosis.

Here, the diseases causing pulmonary fibrosis include various types of diseases, such as interstitial pneumonia, cystic fibrosis, Chronic Obstructive Pulmonary Disease (COPD), Acute Respiratory Distress Syndrome (ARDS), inflammatory lung diseases, pulmonary infection, radiation pneumonitis, interstitial pneumonia with collagen disease, and Idiopathic Pulmonary Fibrosis (IPF) among Idiopathic Interstitial Pneumonia (IIPs) having an indeterminate cause of interstitial pneumonia. Examples of the disease in clinical pathology in Idiopathic Interstitial Pneumonia (IIPs) include Idiopathic Pulmonary Fibrosis (IPF), nonspecific interstitial pneumonia (NSIP), idiopathic mechanized pneumonia (COP/BOOP), Acute Interstitial Pneumonia (AIP), Desquamative Interstitial Pneumonia (DIP), interstitial lung disease accompanied by respiratory bronchiolitis (RB-ILD), and Lymphocytic Interstitial Pneumonia (LIP).

In addition, TGF-. beta.1 has been reported to promote infiltration and metastasis of cancer, particularly lung adenocarcinoma (MolCellBiochem (2011)355:309.314, cancer genomics proteomics2010,7,217); TGF-. beta.1 is overexpressed in the damaged site of a normal tissue after cancer treatment, and damage to the normal tissue can be suppressed by targeting the TGF-. beta.1 pathway (TheOncoloist 2010; 15:350.359), and the like, so the siRNA of the present invention and an expression vector that can express the siRNA in a subject are also useful as a prophylactic or therapeutic agent for cancer, particularly lung cancer.

When the siRNA of the present invention is used as a pharmaceutical, it can be used as it is, but may form a complex with a highly branched cyclodextrin or cyclodextrin. Herein, the highly branched cyclodextrin refers to glucan having an inner branched cyclic moiety and an outer branched moiety and having a degree of polymerization of 50 to 5000, which is produced by reacting pullulan with a branching enzyme. Here, the inner branched cyclic moiety means a cyclic moiety formed by an α -1, 4-glycosidic bond and an α -1, 6-glycosidic bond, and the outer branched moiety is a non-cyclic moiety bonded to the inner branched cyclic moiety. Preferred examples of the highly branched cyclodextrin include a form in which the polymerization degree of the inner branched cyclic moiety of the glucan is 10 to 100, a form in which the polymerization degree of the outer branched moiety of the glucan is 40 or more, and a form in which the polymerization degree of each unit chain of the outer branched moiety is from 10 to 20 on average. Highly branched cyclodextrins are commercially available, and commercially available products can also be used in the present invention.

Cyclodextrin is a cyclic α -1, 4-glucan in which glucose is bonded by α -1,4 bonds, and has a three-dimensional and deep hollow portion inside a helical structure. The polymerization degree of glucose in the cyclodextrin used in the present invention is not particularly limited, and is, for example, 10 to 500, preferably 10 to 100, and more preferably 22 to 50. Cyclodextrins can be prepared from glucose using enzymes such as maltoglucosyltransferase. Cyclodextrin is commercially available, and a commercially available product can be used in the present invention (see pamphlet of international publication No. 2009/61003).

The siRNA of the present invention can be formulated into a pharmaceutical composition by a conventional method using 1 or more pharmaceutically acceptable carriers or excipients. The pharmaceutical composition may be in any form of administration, such as pulmonary administration, nasal administration, oral administration, rectal administration, or injection, and may be administered systemically or locally. The dosage form can be any dosage form suitable for use, such as liquid preparation, suspending agent, emulsion, tablet, pill, granule, capsule, powder, sustained-release preparation, suppository, aerosol, spray, etc., according to the administration route.

For example, for nasal administration, the active ingredient may be dissolved in an appropriate solvent (physiological saline, alcohol, etc.) and the solution may be administered by nasal injection or nasal instillation. Alternatively, for pulmonary or nasal administration, the active ingredient is conveniently delivered as an aerosol spray from a pressurized pack or nebulizer, with the use of a suitable nebulizer, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the unit dose of administration can be determined by providing a valve to deliver a metered dose. Further, the composition can be administered as a powder inhalant.

In the case of injection, the active ingredient may be formulated in a solvent for parenteral administration (i.e., intravenous or intramuscular administration) by, for example, bolus injection or continuous injection, and is preferably formulated in a physiologically compatible buffer such as hank's solution, ringer's solution, or physiological saline. The solvent may contain formulatable agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for reconstitution with a suitable vehicle such as sterilized water containing no exothermic substance before use. The preparation for injection may be provided by adding a preservative, for example, in the form of an ampoule or a unit dosage form in a multi-administration container.

For oral administration, the therapeutic agents of the present invention may be in the form of, for example, tablets, granules, powders, emulsions, capsules, syrups, aqueous or oily suspensions, or elixirs. In the case of tablet or pill forms, the compositions may be coated in order to delay dispersion and absorption in the gastrointestinal tract, thereby resulting in a sustained action over a long period of time.

The pharmaceutically acceptable carrier or excipient is not limited, and examples thereof include a liquid (e.g., water, oil, physiological saline, an aqueous glucose solution, ethanol, etc.), and a solid (e.g., gum arabic, gelatin, starch, glucose, lactose, sucrose, talc, sodium stearate, glycerol monostearate, keratin, colloidal silica, dried skim milk, glycerin, etc.). The therapeutic agent of the present invention may contain suitable additives such as adjuvants, preservatives, stabilizers, thickeners, lubricants, colorants, wetting agents, emulsifiers, pH buffers, and the like, which are generally incorporated in pharmaceutical compositions.

The pharmaceutical composition of the present invention may contain 0.001 to 50% by mass, preferably 0.01 to 10% by mass, and more preferably 0.1 to 1% by mass of the siRNA of the present invention.

The amount of the pharmaceutical composition of the present invention to be administered is not particularly limited as long as an effective amount is used, and is, for example, preferably 0.0001 to 100mg, and more preferably 0.002 to 1mg per 1kg body weight.

In the pharmaceutical composition of the present invention, an expression vector that can express the siRNA in a subject may be used instead of the siRNA of the present invention.

In this case, the expression vector can be constructed by inserting a DNA encoding the siRNA of the present invention into an appropriate vector for gene therapy, for example, an adenovirus vector, an adeno-associated virus (AAV) vector, a lentivirus vector, or the like.

Examples

The present invention will be described in further detail below with reference to examples, but the technical scope of the present invention is not limited to these examples.

EXAMPLE 1 preparation of siRNA

siRNA molecules targeting TGF-. beta.1 genes shown in Table 3 below were designed, and siRNA oligonucleotides were synthesized by a chemical synthesis method and purified by an HPLC purification method.

TABLE 3

Example 2 evaluation of TGF-. beta.1 expression inhibitory Effect (in vitro)

(1) Cells

A549 cells derived from human alveolar epithelial cells (DSPharmabiomedicalal Co., Ltd.) were used.

(2) Culture conditions

1 × 10⁵The individual cells were seeded in D-MEM medium (Dulbecco's ' smodifiedEagle's Medium containing 100unit/mL penicillin, 100. mu.g/mL streptomycin, 12-well plate) containing 10% fetal bovine serum. At 37 deg.C, 5% CO₂After overnight culture under the conditions of (1), the medium of the a549 cells which became 40% confluent was changed to a serum-free medium.

(3) Pretreatment and amount of siRNA added

When the cells were 40% confluent, the oligonucleotides shown in Table 3 were used as siRNA, and the cells were introduced into the cells using Lipofectamine2000 (Invitrogen).

Specifically, Lipofectamine2000 was added to 98. mu.LOPTI-MEM (Invitrogen) at 2.0. mu.L per well, and incubated at room temperature for 5 minutes (solution A).

0.625. mu.L of a 0.2. mu.MsiRNA solution was added to 99.375. mu.L of OPTI-MEM (B solution). The A and B solutions were mixed and incubated at room temperature for 20 minutes. After the culture, the AB mixture was added to each well of the 12-well plate. The siRNA was added so that the final concentration became 0.1 nM.

(4) Post-treatment

(i) Cytokine treatment

After adding the mixture of siRNA and Lipofectamine for 6 hours, the medium was changed to D-MEM medium containing 0.1% BSA (bovine serum albumin) and cytokines (1ng/mL IL-1. beta. and 1ng/mL TNF-. alpha.) and cultured for 12 hours. The supernatant after the culture was sampled.

(ii) Total RNA extraction in cells

For total RNA extraction in cells, an automated nucleic acid extraction apparatus, QuickGene-810(Fujifilm corporation) and QuickGeneRNAculturedcellkings (Fujifilm corporation), which is a kit dedicated to the QuickGene-810, were used. The cells were washed with 1.0mL of LPBS, and 0.5mL of a cell lysate was added to extract total RNA contained in the cells. A12-well plate to which 0.5mL of a dissolving solution (LRC, mercaptoethanol was added) was added was stirred for 5 minutes using a seesaw shaker. The solution was thoroughly mixed by 5 to 6 reciprocations using a pipette, and the solution was transferred to an Eppendorf tube. Add 420. mu.L of ethanol, stir with vortex mixer for 15 seconds, and then treat with QuickGene-810. DNase (RQ1RNase-freeDNase, Promega) was added to the treatment with QuickGene-810. The extracted total RNA samples were stored in a freezer at-80 ℃ until the next treatment.

(iii) cDNA conversion of Total RNA

The RNA concentration (. mu.g/mL) in the total RNA sample extracted from the cultured cells was calculated from the measured value of the absorbance at 260nm (control: TE buffer). Based on this value, for each sample, a solution in an amount equivalent to 0.1. mu.g of RNA was added to a 96-well plate. Distilled water was added thereto so that the total amount was 12. mu.L, and 2. mu.L of gDNAWipeoutBuffer contained in QuantiTectPersereTranscriptionkit (QIAGEN) was further added thereto, followed by vortex mixing, incubation at 42 ℃ for 2 minutes, and thereafter cooling to 4 ℃. To this, QuantiscriptreTranscriptase 1. mu. L, QuantiscriptRTBuffer 4. mu.L and RTPrimerMix 1. mu.L contained in QuantiTectReverseTranscriptionkit (QIAGEN) were added, mixed, and cultured at 42 ℃ for 15 minutes. Then, in order to inactivate QuantiscriptReverseTranscriptase, the mixture was heated at 95 ℃ for 3 minutes and then cooled to 4 ℃.

The preparation (cDNA preparation stock solution) was diluted 5-fold with TE buffer to prepare a cDNA solution for PCR targeting the target gene (TGF-. beta.1).

The cDNA preparation stock solution was diluted 50-fold with TE buffer, and GAPDH was selected as an internal standard gene to prepare a cDNA solution for PCR. The cDNA preparation stock solutions of control samples (siRNA non-administered) were diluted 1, 10, 100, and 1000 times with TE buffer to prepare samples for PCR standard curve targeting TGF-. beta.1. Similarly, the control sample cDNA preparation stock solution was diluted 10, 100, 1000 and 10000 times with TE buffer solution to prepare samples for PCR standard curve targeting GAPDH.

(5) Method for measuring TGF-beta 1 expression level

Using 12.5. mu.L of QuantiFastSYBRGreenPCRMastermix (QIAGEN) and 2.5. mu.L of QuantiTectPrimerAssay (QIAGEN) derived from the human TGF-. beta.1 gene or from the human GAPDH gene, 2.5. mu.L of a cDNA product derived from TGF-. beta.1 was used as a template to prepare a PCR reaction solution with a final volume of 25. mu.L using sterile distilled water. The resulting solution was heated at 95 ℃ for 5 minutes using an applied biosystems7500(Life technologies Japan), and then a cycle of 1)95 ℃ for 10 seconds and 2)60 ℃ for 35 seconds was repeated 40 times, followed by slow cooling from 95 ℃ to 60 ℃ for thermal dissociation measurement. The amplification ratio of each target gene based on the Ct value of GAPDH gene was corrected based on the Ct (threshold cycle) value from the PCR amplification process, and the mRNA inhibitory effect of the target gene was evaluated. The results are shown in fig. 1 and table 4.

TABLE 4

It was found that siRNAs with siRNA numbers d, p, r, f, c, g, q, j, n, i, h, e, a, k, l, and o had an expression inhibition efficiency of TGF-. beta.1 of 40% or more even at a concentration of 0.1 nM. In particular, siRNAs with siRNA numbers l and o showed an inhibition efficiency of 80% or more even at 0.1 nM. Furthermore, the sequence showed significant inhibitory effect even at 0.01 nM.

Example 3 preparation of chimeric siRNA

Chimeric siRNA molecules targeting TGF- β 1 gene shown in table 5 below were designed based on siRNA number (l), and each chimeric siRNA oligonucleotide was synthesized by chemical synthesis and purified by HPLC purification method.

TABLE 5

In the table, the upper case letters represent RNA, the lower case letters represent DNA, and underlining indicates the position of overhang.

Example 4 evaluation of TGF-. beta.1 expression inhibitory Effect

The inhibitory effect of TGF-. beta.1 expression was evaluated in the same manner as in example 2 using the oligonucleotides shown in Table 5 (concentration: 10nM or 0.1 nM). The results are shown in fig. 2 and table 6.

TABLE 6

Example 5 Synthesis of chimeric siRNA having phosphate or phosphorothioate bonded thereto

Based on the siRNA number (l), chimeric siRNA molecules to which a phosphate or phosphorothioate is bonded and which are targeted by a TGF-. beta.1 gene shown in Table 7 below were designed, and various chimeric siRNA oligonucleotides were synthesized by a chemical synthesis method and purified by an HPLC purification method.

TABLE 7

In the table, upper case letters represent RNA, lower case letters represent DNA, and underlining indicates the position of overhang.

In addition, P-represents 5 'terminal phosphorylation, and PS-represents 5' terminal phosphorothioate

Example 6 evaluation of TGF-. beta.1 expression inhibitory Effect

Using the oligonucleotides shown in Table 7, the effect of inhibiting the expression of TGF-. beta.1 was evaluated in the same manner as in example 2. The results are shown in fig. 3 and table 8.

TABLE 8

Example 7 Synthesis of siRNA having DNA in overhang portion

siRNA molecules having DNA in the overhang portion targeting TGF-. beta.1 gene shown in Table 9 below were designed based on siRNA number (l), and siRNA oligonucleotides were synthesized by chemical synthesis and purified by HPLC purification.

TABLE 9

In the table, upper case letters represent RNA, lower case letters represent DNA, and underlining indicates the position of overhang.

Example 8 evaluation of TGF-. beta.1 expression inhibitory Effect

Using the oligonucleotides shown in Table 9, the effect of inhibiting the expression of TGF-. beta.1 was evaluated in the same manner as in example 2. The results are shown in Table 10.

Watch 10

Example 9 evaluation of efficacy in pulmonary fibrosis model (in vitro test)

Chimeric siRNAs (q-C8: sense strand (5 '→ 3'): CCAAGGGCUACCAtgccaact (SEQ ID NO: 52) and antisense strand (5 '→ 3'): ttggcaUGGUAGCCCUUGGGC (SEQ ID NO: 53) were designed based on siRNA numbers (q) (SEQ ID NO: 34 and 35) which are common sequences between mice and humans, and chimeric siRNA oligonucleotides were synthesized in the same manner as in example 3 and purified by HPLC purification.

Subject: mice (C57BL/6NCrSlc (SLC), female, 13 weeks old) were intraperitoneally administered pentobarbital (manufactured by Sumitomo pharmaceutical Co., Ltd., Japan), and then the backs of the anesthetized mice were subcutaneously implanted with ALZET^TMOsmotic pump (model2001, duretrcorporation). Note that ALZET is used in^TM200. mu.L of a physiological saline solution of about 10mg/mL of bleomycin prepared by Kagaku Kogyo K.K. was injected into the osmotic pump in advance.

Subjecting the material to ALZET^TMAfter 3, 7 and 14 days after the immersion of the osmotic pump, the solution was dissolved in distilled water (Otsuka pharmaceutical Co., Ltd.) and micro spray was used^TM(modelIA-1C, PENNCCENTURY, Inc.) was administered intratracheally at 100 μ g/body each time. In terms of administration volume, bleomycin administration was performed at 75. mu.L/body on days 3 and 7 after the start of administration, and at 50. mu.L/body on day 14.

As a comparison control, two groups were set, namely, in ALZET^TMA group in which 200. mu.L of physiological saline alone was injected into the osmotic pump and distilled water was given as a test substance; and in ALZET^TM200. mu.L of a bleomycin physiological saline solution of about 10mg/mL was injected into the osmotic pump, and distilled water was given as a test substance.

In ALZET^TMOn day 21 after the osmotic pump was implanted, pentobarbital was administered into the abdominal cavity of the mouse, and the mice were anesthetized by exfoliationThe lower mouse had skin and muscle in the neck, exposing the trachea. After the carotid vein was exsanguinated and then sacrificed, 2mL of physiological saline (Otsuka pharmaceutical Co., Ltd.) was injected into the trachea in 3 times using an indwelling needle, and about 2mLBALF (broncholavelolarvoflavagefluid) was collected. Subsequently, the chest was opened, an incision was made in the left atrial appendage, and the right ventricle was perfused with about 1mL of physiological saline to remove the lung. For histological evaluation, the left lobe of the excised lung was immersed in 10% neutral buffered formalin (Wako pure chemical industries, Ltd.).

The collected BALF was centrifuged (2000rpm, 4 ℃ C., 10 minutes), and the amount of TGF-. beta.1 protein in the supernatant was measured by ELISA. The results are shown in FIG. 4. In the figure, SAL represents physiological saline (saline), DW represents distilled water (distilledwater), and BLM represents bleomycin (bleomycin).

Lung tissues fixed with 10% neutral buffered formalin fixative were paraffin-embedded and prepared into tissue sections, which were then stained with h.e. (Hematoxilin-Eosin) and masson trichrome. The structure is shown in FIG. 5.

As is clear from FIG. 4, in the test substance-administered group (BLM/TGF-. beta.1), the amount of TGF-. beta.1 in BALF was significantly decreased. This effect was not observed in the comparative control distilled water administration group (BLM/DW). Further, as is clear from the tissue map of FIG. 5, the degree of inflammation and fibrosis was reduced in the test substance-administered group.

Claims (12)

1. An siRNA selected from the group consisting of the following (l) to (o), wherein the siRNA has a total length of 30 nucleotides or less:

(l) siRNA comprising sense sequence represented by SEQ ID NO. 24 and antisense sequence represented by SEQ ID NO. 25

(o) siRNA comprising the sense sequence represented by SEQ ID NO. 30 and the antisense sequence represented by SEQ ID NO. 31.

2. The siRNA of claim 1, wherein consecutive 1-10 nucleotides from the terminal side of the 3' end of the sense strand of said siRNA excluding the overhang nucleotide are converted into DNA.

3. The siRNA of claim 1 or 2, wherein 1 to 10 consecutive nucleotides from the terminal side of the 5' terminus of the antisense strand of said siRNA are converted into DNA.

4. siRNA according to claim 1 or 2, wherein the 5' end of the antisense strand is monophosphorylated or monothiophosphorylated.

5. siRNA according to claim 3, wherein the 5' end of the antisense strand is monophosphorylated or monothiophosphorylated.

6. A pharmaceutical composition comprising the siRNA of any one of claims 1 to 5.

7. A TGF- β 1 gene expression inhibitor comprising the siRNA of any one of claims 1 to 5 as an active ingredient.

8. A preventive or therapeutic agent for fibrosis, comprising the siRNA of any one of claims 1 to 5 as an active ingredient.

9. A prophylactic or therapeutic agent for pulmonary fibrosis or lung cancer, comprising the siRNA of any one of claims 1 to 5 as an active ingredient.

10. Use of the siRNA of any one of claims 1 to 5 for producing an inhibitor of TGF- β 1 gene expression.

11. Use of the siRNA of any one of claims 1 to 5 for the manufacture of a prophylactic or therapeutic agent for fibrosis.

12. Use of the siRNA of any one of claims 1 to 5 for producing a prophylactic or therapeutic agent for pulmonary fibrosis or lung cancer.