WO2012050181A1 - 線維症予防又は治療剤 - Google Patents
線維症予防又は治療剤 Download PDFInfo
- Publication number
- WO2012050181A1 WO2012050181A1 PCT/JP2011/073628 JP2011073628W WO2012050181A1 WO 2012050181 A1 WO2012050181 A1 WO 2012050181A1 JP 2011073628 W JP2011073628 W JP 2011073628W WO 2012050181 A1 WO2012050181 A1 WO 2012050181A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- sirna
- seq
- sequence shown
- sense
- antisense
- Prior art date
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1136—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against growth factors, growth regulators, cytokines, lymphokines or hormones
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
- C12N2310/141—MicroRNAs, miRNAs
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/31—Chemical structure of the backbone
- C12N2310/315—Phosphorothioates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2320/00—Applications; Uses
- C12N2320/30—Special therapeutic applications
Definitions
- 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) - ⁇ 1 and a pharmaceutical using the same.
- siRNA small interfering RNA
- TGF Transforming Growth Factor
- Pulmonary fibrosis refers to a condition in which lung tissue has become fibrotic due to accumulation of excess collagen and other extracellular matrix.
- pulmonary fibrosis idiopathic pulmonary fibrosis is a chronic intractable disease with a poor prognosis with an average median survival of 3 years and a 5-year survival rate of 20 to 40%.
- Steroids and immunosuppressants are used to treat pulmonary fibrosis, but there are currently no effective treatments that improve prognosis, and the development of new therapeutics is desired. Yes.
- Patent Documents 1 to 4 and Non-Patent Documents 1 to 6 have been clarified that many diseases develop due to genes, but many genes have been reported to participate in pulmonary fibrosis.
- TGF- ⁇ 1 Patent Literature 1-2, Non-Patent Literature 2-6), Smad3 (Non-Patent Literature 1), MCP-1 (Patent Literature 3), etc. have been reported as major factors involved in pulmonary fibrosis. Yes.
- nucleic acids particularly siRNA
- RNA interference causes degradation of mRNA of a gene that is identical or nearly identical to a specific sequence present in a cell, and inhibits expression of a target gene (RNA interference). Therefore, the target gene expression inhibition function by RNA interference is useful in reducing or treating a disease symptom caused by abnormal expression of a specific gene or gene group.
- pulmonary fibrosis-related genes there have been reports of attempts to suppress the expression of the genes using siRNA (Patent Documents 1-4 and Non-Patent Documents 1-6).
- siRNA sequences on disease-related genes in experimental animals (mouse and rat), but not sufficient effects specific to human genes.
- the suppression effect of siRNA also shows the effect in 200 nM (nonpatent literature 1) and 20-500 nM (nonpatent literature 5), it can suppress the expression of a pulmonary fibrosis related gene efficiently in low concentration.
- the nucleic acid molecule is not shown.
- siRNA targeting the TGF- ⁇ 1 gene several tens have been reported so far (Patent Documents 1 and 5-7), but the total length of the TGF- ⁇ 1 gene is 2346 bases (GenBank Accession No NM_000660.3), and there are innumerable combinations for selecting a sequence of about 20 mer from the entire length of the gene, and dsRNA and siRNA molecules that can more efficiently suppress the expression of the gene are designed from among the combinations. That is not easy.
- the present invention relates to providing an siRNA effective for treating fibrosis and a medicine using the same.
- the present inventors have found that a specific siRNA targeting the human TGF- ⁇ 1 gene can effectively suppress the expression of TGF- ⁇ 1 in human cells, It was found that the symptoms of pulmonary fibrosis can be improved without inducing an interferon response in the lungs of pulmonary fibrosis model mice.
- the present invention relates to the following 1) to 19).
- siRNA formed from the sense sequence shown in SEQ ID NO: 2 and the antisense sequence shown in SEQ ID NO: 3 (B) siRNA formed from the sense sequence shown in SEQ ID NO: 4 and the antisense sequence shown in SEQ ID NO: 5 (C) siRNA formed from the sense sequence shown in SEQ ID NO: 6 and the antisense sequence shown in SEQ ID NO: 7 (D) siRNA formed from the sense sequence shown in SEQ ID NO: 8 and the antisense sequence shown in SEQ ID NO: 9 (E) siRNA formed from the sense sequence shown in SEQ ID NO: 10 and the antisense sequence shown in SEQ ID NO: 11 (F) siRNA formed from the sense sequence shown in SEQ ID NO: 12 and the antisense sequence shown in SEQ ID NO: 13 (G) siRNA formed from the sense sequence shown in SEQ ID NO: 14 and the antisense sequence shown in SEQ ID NO: 15 (H) siRNA formed from the sense sequence shown in SEQ ID NO: 16 and the antisense sequence shown in SEQ ID NO:
- siRNA of 1) -3) above wherein 1 to 10 nucleotides continuous from the 5 ′ end of the antisense strand of the siRNA are converted to DNA.
- 1 to 10 consecutive nucleotides excluding overhanging nucleotides from the 3 ′ end of the sense strand of the siRNA are converted into DNA, and 1 to 10 consecutive from the 5 ′ end of the antisense strand
- the siRNA of the above 1) -4) wherein the nucleotide is converted to DNA.
- the siRNA of the above 1) to 5) wherein the 5 ′ end of the antisense strand is monophosphorylated or monothiophosphorylated.
- a pharmaceutical composition comprising the siRNA of any one of 1) to 6) above.
- a fibrosis preventive or therapeutic agent comprising the siRNA of any one of 1) to 6) above as an active ingredient.
- a preventive or therapeutic agent for pulmonary fibrosis or lung cancer comprising the siRNA of any one of 1) to 6) as an active ingredient.
- the siRNA of the present invention can efficiently suppress or inhibit the expression of TGF- ⁇ 1 at a low concentration, it is useful as a medicament for the prevention or treatment of fibrosis.
- the graph which shows the expression suppression rate of TGF- (beta) 1 mRNA by siRNA The graph which shows the expression suppression rate of TGF- (beta) 1 mRNA by chimera type siRNA.
- Light micrograph (magnification: 5 times) of lung tissue section H.E. staining and Masson trichrome staining).
- the sequences targeted by the siRNA of the present invention are the bases from base numbers 1285 to 1318, bases from 1398 to 1418, bases from 1434 to 1463, bases from 1548 to 1579, bases from 1608 to 1628 in SEQ ID NO: 1.
- the continuous 17 to 23 bases selected from are preferably 19 to 23 bases, and more preferably 21 bases.
- the base sequence represented by SEQ ID NO: 1 is the base sequence of TGF- ⁇ 1 mRNA, and the sequence information is registered in GenBank as GenBank Accession No. NM — 000660.3.
- the siRNA of the present invention is obtained by hybridizing an antisense strand, which is a sequence complementary to the target sequence of TGF- ⁇ 1 mRNA, and a sense strand, which is a sequence complementary to the antisense strand, It has the activity of cleaving TGF- ⁇ 1 mRNA (RNA interference action), and has the ability to block translation of the mRNA, that is, the ability to inhibit the expression of the TGF- ⁇ 1 gene.
- the nucleotide length of the siRNA of the present invention may be the same or different in the sense strand and the antisense strand, and the total length is 30 nucleotides or less, preferably 25 nucleotides or less, more preferably 23 nucleotides. Or alternatively 21 nucleotides.
- both ends of the sense strand and the antisense strand may be blunt ends, or the 3 ′ side of each strand may be an overhang (protruding end).
- blunt end means that the end region of the sense strand and the end region of the antisense strand that is paired with the end region of the double-stranded RNA are paired without forming a single-stranded portion. Means a structure.
- “overhang” is also called a dangling end, because there is no pairing base in the terminal region of the sense strand in the terminal part of the double-stranded RNA or the terminal region of the antisense strand. It means a structure in which a double strand cannot be formed and a single strand portion (protruding end) exists.
- the number of bases of the protruding terminal portion is 1 to 10 nucleotides, preferably 1 to 4 nucleotides, and more preferably 1 to 2 nucleotides. Note that the length of the protruding end is irrelevant between the two chains, and may be different from each other.
- the nucleotide at the protruding terminal portion may be RNA or DNA, and is preferably a base complementary to the target TGF- ⁇ 1 mRNA, but may be a base that is not complementary as long as it retains the RNA interference ability.
- the siRNA of the present invention is not only one double-stranded RNA composed of two separate strands, but may also be a double-stranded RNA formed by one strand taking a stem-loop structure. That is, the siRNA of the present invention includes RNA having a loop composed of 2 to 4 nucleotides at the 5 ′ end of the sense strand and the 3 ′ end of the antisense strand, the 3 ′ end of the sense strand and the 5 ′ end of the antisense strand. In addition, RNA having a loop composed of 2 to 4 nucleotides is also included.
- RNA in which a loop composed of 2 to 4 nucleotides is formed at both ends of the 5 'end of the sense strand and the 3' end of the antisense strand and the 3 'end of the sense strand and the 5' end of the antisense strand is also included.
- the siRNA of the present invention and the target sequence are desirably the same, but may be substantially the same, that is, a homologous sequence as long as the RNA interference can be induced.
- the antisense strand sequence of the siRNA of the present invention and the target sequence hybridize there may be one or several (eg, 2, 3, 4) mismatches. That is, the siRNA of the present invention has one or several bases substituted, added or deleted to the target sequence and can induce RNA interference, or 85% or more, preferably 90%, with the target sequence. % Or more, preferably 95% or more, more preferably 98% or more, and those capable of inducing RNA interference are included.
- the hybridization conditions in this case are in-vivo conditions when the siRNA of the present invention is administered in vivo and used as a pharmaceutical, and moderate when the siRNA of the present invention is used as a reagent in vitro.
- Stringent conditions or advanced stringent conditions such as 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, hybridization conditions at 50 ° C. to 70 ° C. for 12 to 160 hours. Is mentioned. These conditions are well known to those skilled in the art and are described in Sambrook et al. (Molecular Cloning: A Laboratory Manual second edition, Cold Spring Harbor Laboratory Press, New York, USA, 1989).
- the sequence identity may be calculated by the Lippman-Pearson method (Lipman-Pearson method; Science, 227, 1435, (1985)) or the like.
- genetic information processing software Genetyx-Win (Ver. 5.1.1; It is calculated by performing analysis with a unit size to compare (ktup) of 2 using a software development) homology analysis (Search homology) program.
- the siRNA of the present invention may be one in which any nucleotide of either the sense strand or the antisense strand is converted to DNA (hybrid type), the sense strand and / or the antisense strand as long as the above-described RNA interference can be induced.
- some nucleotides are converted to DNA (chimeric type).
- conversion of RNA nucleotides into DNA means conversion of AMP to dAMP, GMP to dGMP, CMP to dCMP, and UMP to dTMP.
- the hybrid type is preferably one in which the nucleotide of the sense strand is converted to DNA.
- Examples of the chimeric type include those obtained by converting some nucleotides on the downstream side (3 ′ end side of the sense strand, 5 ′ end side of the antisense strand) into DNA. Specifically, the nucleotides on the 3 ′ end side of the sense strand and the 5 ′ end side of the antisense strand are both converted to DNA, either the 3 ′ end side of the sense strand or the 5 ′ end side of the antisense strand And those obtained by converting nucleotides into DNA.
- nucleotide length to be converted is preferably an arbitrary length up to a nucleotide corresponding to 1/2 of the RNA molecule, for example, 1 to 13 nucleotides, preferably 1 to 10 nucleotides from the end.
- suitable chimeric siRNAs have, for example, nucleotide lengths of 19 to 23, respectively, and overhanging nucleotides are removed from the 3 ′ end side of the sense strand.
- the number of DNA conversions of the sense strand (excluding overhanging nucleotides) and the antisense strand is more preferably the same.
- nucleotide (ribonucleotide, deoxyribonucleotide) of the siRNA of the present invention may be a nucleotide analog in which sugar, base and / or phosphate are chemically modified as long as it can induce RNA interference.
- nucleotide analogs with modified bases include 5-position-modified uridine or cytidine (eg, 5-propynyluridine, 5-propynylcytidine, 5-methylcytidine, 5-methyluridine, 5- (2-amino) propyl Uridine, 5-halocytidine, 5-halouridine, 5-methyloxyuridine, etc.); 8-position modified adenosine or guanosine (eg, 8-bromoguanosine, etc.); Deazanucleotide (eg, 7-deaza-adenosine, etc.); O-and N-alkylated nucleotides (for example, N6-methyladenosine etc.) and the like can be mentioned.
- 5-position-modified uridine or cytidine eg, 5-propynyluridine, 5-propynylcytidine, 5-methylcytidine, 5-methyluridine, 5- (2-amino) propyl Uridine
- nucleotide analogues modified with sugar include, for example, 2′-OH of ribonucleotide is H, OR, R, halogen atom, SH, SR, NH 2 , NHR, NR 2 , or CN (where , R represents an alkyl group having 1-6 carbon atoms, an alkenyl group, or an alkynyl group) and the like, and the 5′-terminal phosphorus substituent in which the 5′-terminal —OH is monophosphorylated or monothiophosphorylated. An oxidized form and a 5 'terminal monothiophosphorus form are mentioned.
- nucleotide analogs modified with phosphate include those in which the phosphoester group that binds adjacent ribonucleotides is replaced with a phosphothioate group.
- the siRNA of the present invention apart from the nucleotide analogues described above, contains the first to sixth nucleotides from the 5 ′ end side or 3 ′ end side of the sense strand (5 ′ end, 3 ′ end, or an internal portion other than the end).
- a specific substituent or a functional molecule may be bonded directly or via a linker to at least one of the base and sugar), and is 1st to 6th, preferably 1st to 4th from the 5 ′ end side of the sense strand It is preferable that the substituent or the functional molecule is bound to at least one of the nucleotides.
- examples of the substituent include an amino group; a mercapto group; a nitro group; an alkyl group having 1 to 40 carbon atoms (preferably 2 to 20, more preferably 4 to 12); and 1 to 40 carbon atoms (preferably Is an aminoalkyl group having 2 to 20 and more preferably 4 to 12); a thioalkyl group having 1 to 40 carbon atoms (preferably 2 to 20 and more preferably 4 to 12); and 1 to 40 carbon atoms (preferably 2 to 2).
- RNA interference effect can be remarkably enhanced.
- an aminoalkoxyl group having 1 to 40 carbon atoms (preferably 2 to 20, more preferably 4 to 12); 1 to 40 carbon atoms (preferably 2 to 20 carbon atoms)
- Preferably 4 to 12) thioalkoxyl group mono or dialkylamino group having 1 to 40 carbon atoms (preferably 2 to 20 and more preferably 4 to 12 carbon atoms); 1 to 40 carbon atoms (preferably Alkylthio group having 2 to 20 carbon atoms, more preferably 4 to 12); polyethylene oxide group having 2 to 40 carbon atoms (preferably 2 to 20 carbon atoms, more preferably 4 to 12 carbon atoms); 3 to 39 carbon atoms (preferably 3 to 21 carbon atoms) More preferred examples include polypropylene oxide groups of 3 to 12). By binding these substituents, the RNA interference effect can be remarkably enhanced.
- RNA including tRNA
- aptamers modified nucleotides, low molecular organic / inorganic materials, cholesterol, dendrimers, lipids, polymer materials, and the like.
- sugar examples include monosaccharides such as glucose, galactose, glucosamine, and galactosamine, and oligosaccharides or polysaccharides obtained by arbitrarily combining these.
- proteins existing in the living body proteins having a pharmacological action, proteins having a molecular recognition action, and the like can be used.
- proteins having a pharmacological action proteins having a molecular recognition action, and the like can be used.
- Examples of the protein include importin b protein, avidin, and an antibody.
- DNA having a base length of 5 to 50 preferably DNA having a base length of 5 to 25.
- peptide examples include octaarginine peptide R8, nuclear localization signal peptide sequences (HIV-1, Tat, SV40T antigen, etc.), nuclear export signal peptides (HIV-1, Rev, MAPKK, etc.), cell membrane fusion peptides, etc. Is mentioned.
- modified nucleotide examples include those obtained by modifying a phosphate skeleton such as phosphorothioate type and boranophosphate type DNA / RNA; 2′-modified nucleotides such as 2′-OMe modified RNA and 2′-F modified RNA; LNA (Locked Nucleic Acid) and ENA (2'-O, 4'-C-Ethylene-bridged nucleic acids) and other modified nucleotides, such as PNA (peptide nucleic acid) and morpholino nucleotides. Examples include different modified nucleotides (see WO 2008/140126 pamphlet and WO 2009/123185 pamphlet).
- Examples of the low molecular organic / inorganic materials include fluorescent substances such as Cy3 and Cy5; biotin; quantum dots; gold fine particles and the like.
- a polyamide amine dendrimer etc. are mentioned, for example.
- Examples of the lipid include a double chain having two hydrophobic groups described in International Publication No. 2009/123185, in addition to linoleic acid, DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine) and the like. Lipids.
- Examples of the polymer material include polyethylene glycol and polyethyleneimine.
- the group having a functional molecule may be a residue of the functional molecule itself, or a group in which one functional group of the bifunctional linker is bonded to the residue of the functional molecule. Also good. That is, in the former case, the functional molecule is directly bonded to a predetermined site of the sense strand RNA, and in the latter case, the functional molecule is attached to a predetermined site of the sense strand RNA via a bifunctional linker. Are connected.
- the bifunctional linker is not particularly limited as long as it is a linker containing two functional groups.
- N-succinimidyl-3- (2-pyridyldithio) propionate, N-4-maleimidobutyric acid, S- (2-pyridyldithio) cysteamine, iodoacetoxysuccinimide, N- (4-maleimidobutyryloxy) succinimide, N- [5- (3′-maleimidopropylamide) -1-carboxypentyl] iminodiacetic acid, N -(5-aminopentyl) -iminodiacetic acid etc. can be used.
- the binding site of the substituent or functional molecule or the linker linking these in the sense strand RNA these constitute the hydroxyl group of the phosphate portion of the predetermined nucleotide of the sense strand RNA. It is preferable that the hydrogen atom is bonded to a hydrogen atom.
- siRNA of the present invention include double-stranded RNA molecules formed from a sense sequence and an antisense sequence shown in the following (a) to (s).
- the chimera type is preferably one in which any number of nucleotides 1 to 8 from the 3 ′ end of the sense strand and 1 to 6 nucleotides from the 5 ′ end of the antisense strand are continuously converted to DNA.
- the case of (l) above is exemplified as follows.
- the method for producing the siRNA of the present invention is not particularly limited, but can be synthesized by a known production method, for example, in vitro chemical synthesis, or by a transcription system using a promoter and RNA polymerase. Chemical synthesis can be performed by a nucleic acid synthesizer using amidite resin containing nucleic acid molecules as siRNA constituents as raw materials. In the synthesis by the transcription system, double-stranded RNA can be synthesized by an in vitro transcription method in which hairpin RNA is trimmed.
- the siRNA of the present invention thus obtained can effectively suppress the expression of TGF- ⁇ 1 in human alveolar epithelium-derived cells at the mRNA level, as shown in the Examples below. Interferon reaction is not induced in the lungs, and the symptom of pulmonary fibrosis is improved. Therefore, the siRNA of the present invention and the expression vector capable of expressing the siRNA in the administration subject are useful as a medicament (pharmaceutical composition) for administration to humans or animals. Specifically, it is useful as a pharmaceutical agent for suppressing the expression of TGF- ⁇ 1 gene, a pharmaceutical agent for preventing or treating diseases caused by overexpression of TGF- ⁇ 1, for example, fibrosis, ie, a prophylactic or therapeutic agent for fibrosis. is there.
- lung fibrosis diseases that cause lung fibrosis include interstitial pneumonia, cystic fibrosis, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), Idiopathic interstitial pneumonia, which is an interstitial pneumonia that cannot be identified for a variety of reasons, including inflammatory lung disease, pulmonary infection, radioactive pneumonitis, drug-induced interstitial pneumonia, and interstitial pneumonia associated with collagen disease Among the IIPs), idiopathic pulmonary fibrosis (IPF) is particularly preferred.
- COPD chronic obstructive pulmonary disease
- ARDS acute respiratory distress syndrome
- Idiopathic interstitial pneumonia which is an interstitial pneumonia that cannot be identified for a variety of reasons, including inflammatory lung disease, pulmonary infection, radioactive pneumonitis, drug-induced interstitial pneumonia, and interstitial pneumonia associated with collagen disease Among the IIPs), idiopathic pulmonary fibrosis (IPF) is particularly preferred.
- Idiopathic interstitial pneumonia includes clinicopathological disorders: idiopathic pulmonary fibrosis (IPF), nonspecific interstitial pneumonia (NSIP), idiopathic organized pneumonia (COP / BOOP), acute Includes interstitial pneumonia (AIP), exfoliative interstitial pneumonia (DIP), interstitial lung disease associated with respiratory bronchiolitis (RB-ILD), lymphocytic interstitial pneumonia (LIP), etc. .
- IPF idiopathic pulmonary fibrosis
- NIP nonspecific interstitial pneumonia
- COP / BOOP idiopathic organized pneumonia
- AIP interstitial pneumonia
- DIP exfoliative interstitial pneumonia
- RB-ILD interstitial lung disease associated with respiratory bronchiolitis
- LIP lymphocytic interstitial pneumonia
- TGF- ⁇ 1 promotes invasion and metastasis of cancer, particularly lung adenocarcinoma (Mol Cell Biochem (2011) 355: 309.314, Cancer Genomics Proteomics 2010, 7, 217), and TGF- ⁇ 1 It has been reported that over-expression at the lesion site of normal tissue after cancer treatment and that the TGF- ⁇ 1 pathway can be targeted to suppress damage to the normal tissue (The Oncologist 2010; 15: 350.359)
- the siRNA of the present invention and the expression vector capable of expressing the siRNA in the administration subject are also useful as a preventive or therapeutic agent for cancer, particularly lung cancer.
- the siRNA of the present invention When used as a medicament, it can be used as it is, but it may be complexed with highly branched cyclic dextrin or cycloamylose.
- the highly branched cyclic dextrin is produced by allowing a branching enzyme to act on amylopectin, and refers to a glucan having a degree of polymerization of 50 to 5000 having an inner branched cyclic structure portion and an outer branched structure portion.
- the inner branched cyclic structure portion is a cyclic structure portion formed by an ⁇ -1,4-glucoside bond and an ⁇ -1,6-glucoside bond
- the outer branched structure portion is the inner branched cyclic structure.
- a non-cyclic structure portion bonded to the portion A non-cyclic structure portion bonded to the portion.
- Preferred forms of highly branched cyclic dextrin are those in which the degree of polymerization of the inner branched cyclic structure portion of the glucan is 10 to 100, the degree of polymerization of the outer branched structure portion of the glucan is 40 or more, and the outer branched structure. Examples are those in which the degree of polymerization of each unit chain in the portion is 10 to 20 on average.
- highly branched cyclic dextrin is commercially available, and a commercially available product can be used in the present invention.
- Cycloamylose is a cyclic ⁇ -1,4-glucan in which glucose is bonded by ⁇ -1,4 bonds, and has a three-dimensional and deep hollow portion inside the helix structure.
- the degree of polymerization of glucose in cycloamylose used in the present invention is not particularly limited, and examples thereof include 10 to 500, preferably 10 to 100, and more preferably 22 to 50.
- Cycloamylose can be prepared from glucose using an enzyme such as amylomaltase.
- cycloamylose is marketed and can use a commercial item in this invention (refer international publication 2009/61003 pamphlet).
- the siRNA of the present invention can be formulated as a pharmaceutical composition by a conventional method using one or more pharmaceutically acceptable carriers or excipients.
- the pharmaceutical composition may be a composition in any dosage form such as pulmonary administration, nasal administration, oral administration, rectal administration, injection, etc., and administration may be systemic or local.
- the dosage form can be any solution, suspension, emulsion, tablet, pill, pellet, capsule, powder, sustained release formulation, suppository, aerosol, spray, etc. But you can.
- an active ingredient in nasal administration, can be dissolved in an appropriate solvent (physiological saline, alcohol, etc.), and the solution can be delivered by injecting into the nose or nasally.
- the active ingredient may be packaged in a pressurized pack with a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas.
- a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas.
- it can be conveniently delivered in the form of an aerosol spray ejected from a nebulizer.
- the dosage unit can be determined by providing a valve to deliver a metered amount.
- administration as a powder inhaler is also possible.
- the active ingredient can be formulated as a solvent for parenteral administration (ie intravenous or intramuscular administration), for example by bolus injection or continuous infusion, preferably Hanks's solution, Ringer's solution, physiological saline, etc. Can be formulated as a physiologically compatible buffer.
- the solvent may contain prescribing agents such as suspending and stabilizing agents and / or dispersing agents.
- the active ingredient can be in powder form for reconstitution with a suitable excipient, eg, sterile, pyrogen-free water, before use.
- injectable formulations may be presented in unit dosage form, for example in ampoules or multi-dose containers, with the addition of preservatives.
- the therapeutic agents of the invention can be in the form of tablets, granules, powders, emulsions, capsules, syrups, aqueous or oily suspensions, or elixirs, for example.
- the composition can be coated to delay dispersion and absorption in the gastrointestinal tract and thereby provide a sustained action.
- Pharmaceutically acceptable carriers or excipients include, but are not limited to, liquids (eg, water, oil, saline, aqueous dextrose, ethanol, etc.), solids (eg, acacia gum, gelatin, starch, Glucose, lactose, sucrose, talc, sodium stearate, glycerol monostearate, keratin, colloidal silica, dried skim milk, glycerol, etc.).
- the therapeutic agent of the present invention includes adjuvants, preservatives, stabilizers, thickeners, lubricants, colorants, wetting agents, emulsifiers, pH buffering agents and the like that are blended in ordinary pharmaceutical compositions. You may contain a suitable thing.
- 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 dose of the pharmaceutical composition of the present invention is not particularly limited as long as an effective amount is applied.
- the dose is preferably 0.0001 to 100 mg, more preferably 0.002 to 1 mg per kg of body weight.
- an expression vector capable of expressing the siRNA within the administration subject can also be used.
- an appropriate gene therapy vector such as an adenovirus vector, an adeno-associated virus (AAV) vector, or a lentivirus vector.
- Example 1 Production of siRNA siRNA molecules targeting the TGF- ⁇ 1 gene shown in Table 3 below were designed, and each siRNA oligonucleotide was synthesized by a chemical synthesis method and purified by an HPLC purification method and used.
- siRNA the oligonucleotides shown in Table 3 were used, and when the cells became 40% confluent, they were introduced into the cells using Lipofectamine 2000 (Invitrogen). Specifically, 2.0 ⁇ L Lipofectamine 2000 per well was added to 98 ⁇ L OPTI-MEM (Invitrogen) and incubated at room temperature for 5 minutes (solution A). 0.625 ⁇ L of 0.2 ⁇ M siRNA solution was added to 99.375 ⁇ L OPTI-MEM (B solution). The A and B solutions were mixed and incubated for 20 minutes at room temperature. After incubation, AB mixture was added to each well of the 12-well plate. The final siRNA concentration was 0.1 nM.
- RNA sample was stored in a ⁇ 80 ° C. refrigerator until the next treatment.
- RNA concentration ( ⁇ g / mL) in the total RNA sample extracted from the cultured cells was calculated from the measured absorbance at 260 nm (control: TE buffer). Based on this value, the amount of solution corresponding to 0.1 ⁇ g of RNA in each sample was placed in a 96-well plate. Distilled water was added to this to make a total volume of 12 ⁇ L, and further 2 ⁇ L of gDNA Wipeout Buffer contained in QuantiTect Reverse Transcription Kit (QIAGEN) was added, vortexed, incubated at 42 ° C. for 2 minutes, and then cooled to 4 ° C.
- QIAGEN QuantiTect Reverse Transcription Kit
- Quantiscript Reverse Transcriptase contained in QuantiTect Reverse Transcription Kit (QIAGEN), 4 ⁇ L of Quantiscript RT Buffer, and 1 ⁇ L of RT Primer Mix, mixed, and incubated at 42 ° C. for 15 minutes. Subsequently, in order to inactivate Quantiscript Reverse Transcriptase, it was heated at 95 ° C. for 3 minutes and then cooled to 4 ° C. This preparation solution (cDNA preparation stock solution) was diluted 5-fold with TE buffer to obtain a cDNA solution for PCR targeting the target gene (TGF- ⁇ 1).
- the cDNA preparation stock solution was diluted 50 times with TE buffer, and GAPDH was selected as an internal standard gene to obtain a PCR cDNA solution.
- a control sample (siRNA non-administered) cDNA preparation stock solution was diluted 1, 10, 100, and 1000 times with TE buffer to obtain a sample for a PCR calibration curve targeting TGF- ⁇ 1.
- the control sample cDNA preparation stock solution was diluted 10, 100, 1000, and 10000 times with TE buffer to obtain a sample for a PCR calibration curve targeting GAPDH.
- siRNA numbers of d, p, r, f, c, g, q, j, n, i, h, e, a, k, l, o are 40% or more even at a concentration of 0.1 nM. It was found to have TGF- ⁇ 1 expression suppression efficiency. In particular, siRNAs with siRNA numbers of l and o showed a suppression efficiency of 80% or more even at 0.1 nM. Furthermore, this sequence showed a remarkable inhibitory effect even at 0.01 nM.
- Example 3 Preparation of Chimeric siRNA Based on the siRNA number (l), a chimeric siRNA molecule targeting the TGF- ⁇ 1 gene shown in Table 5 below was designed, and each chimeric siRNA oligonucleotide was synthesized by a chemical synthesis method. Synthesized and purified by HPLC purification method.
- TGF- ⁇ 1 expression suppression effect was evaluated in the same manner as in Example 2 using the oligonucleotides shown in Table 5 (concentration: 10 nM or 0.1 nM). The results are shown in FIG.
- Example 5 Synthesis of Phosphoric Acid or Thiophosphate Binding Chimera siRNA Based on the siRNA number (l), a phosphoric acid or thiophosphate binding chimeric siRNA molecule targeting the TGF- ⁇ 1 gene shown in Table 7 below was designed. Each chimeric siRNA oligonucleotide was synthesized by a chemical synthesis method and purified by an HPLC purification method.
- TGF- ⁇ 1 expression suppression effect was evaluated in the same manner as in Example 2 using the oligonucleotides shown in Table 7. The results are shown in FIG.
- siRNA molecules having DNA in the overhang portion targeting the TGF- ⁇ 1 gene shown in Table 9 below were designed, and siRNA Oligonucleotides were synthesized by chemical synthesis and purified by HPLC purification.
- TGF- ⁇ 1 expression suppression effect was evaluated in the same manner as in Example 2 using the oligonucleotides shown in Table 9. The results are shown in Table 10.
- Example 9 Evaluation of efficacy in pulmonary fibrosis model (in vivo test) Based on siRNA number (q) (SEQ ID NO: 34 and 35) which is a common sequence between mouse and human, chimera siRNA (q-C8: sense strand (5 ′ ⁇ 3 ′): CCAAGGGCUACCAtgccaact (SEQ ID NO: 52), anti Sense strand (5 ′ ⁇ 3 ′): ttggcaUGGUAGCCCUUGGGC (SEQ ID NO: 53) was designed, and chimeric siRNA oligonucleotides were synthesized and purified by HPLC purification in the same manner as in Example 3. This was used as a test substance.
- siRNA number SEQ ID NO: 34 and 35
- chimera siRNA q-C8: sense strand (5 ′ ⁇ 3 ′): CCAAGGGCUACCAtgccaact (SEQ ID NO: 52), anti Sense strand (5 ′ ⁇ 3 ′): ttggca
- the efficacy was evaluated in a bleomycin pulmonary fibrosis model mouse.
- pentobarbital Dainippon Sumitomo Pharma Co., Ltd.
- mice C57BL / 6NCrSlc (SLC), female, 13 weeks old
- ALZET TM osmotic pump model 2001, DURECT Corporation
- the ALZET TM osmotic pump was pre-injected with 200 ⁇ L of a bleomycin physiological saline solution of about 10 mg / mL, and bleomycin was manufactured by Nippon Kayaku Co., Ltd.
- test substance was dissolved in distilled water (Otsuka Pharmaceutical Co., Ltd.) on days 3, 7, and 14 after implantation of the ALZET TM osmotic pump, and 100 ⁇ g / body each time was MicroSprayer TM (model IA-1C, PENN CENTURY, Inc.) Was administered intratracheally.
- the administration volume was 75 ⁇ L / body on the 3rd and 7th days after the start of bleomycin administration, and 50 ⁇ L / body on the 14th day.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Endocrinology (AREA)
- Epidemiology (AREA)
- Pulmonology (AREA)
- Oncology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
1)配列番号1の塩基番号1285~1318番の塩基、1398~1418番の塩基、1434~1463番の塩基、1548~1579番の塩基、1608~1628番の塩基、1700~1726番の塩基、1778~1798番の塩基、1806~1826番の塩基及び1887~1907番の塩基よりなる群から選ばれる連続する17~23塩基を標的配列とし、全長が30ヌクレオチド以下であるsiRNA。
2)siRNAが、以下の(a)~(s)から選ばれる上記1)のsiRNA。
(a)配列番号2に示されるセンス配列と配列番号3に示されるアンチセンス配列とから形成されるsiRNA
(b)配列番号4に示されるセンス配列と配列番号5に示されるアンチセンス配列とから形成されるsiRNA
(c)配列番号6に示されるセンス配列と配列番号7に示されるアンチセンス配列とから形成されるsiRNA
(d)配列番号8に示されるセンス配列と配列番号9に示されるアンチセンス配列とから形成されるsiRNA
(e)配列番号10に示されるセンス配列と配列番号11に示されるアンチセンス配列とから形成されるsiRNA
(f)配列番号12に示されるセンス配列と配列番号13に示されるアンチセンス配列とから形成されるsiRNA
(g)配列番号14に示されるセンス配列と配列番号15に示されるアンチセンス配列とから形成されるsiRNA
(h)配列番号16に示されるセンス配列と配列番号17に示されるアンチセンス配列とから形成されるsiRNA
(i)配列番号18に示されるセンス配列と配列番号19に示されるアンチセンス配列とから形成されるsiRNA
(j)配列番号20に示されるセンス配列と配列番号21に示されるアンチセンス配列とから形成されるsiRNA
(k)配列番号22に示されるセンス配列と配列番号23に示されるアンチセンス配列とから形成されるsiRNA
(l)配列番号24に示されるセンス配列と配列番号25に示されるアンチセンス配列とから形成されるsiRNA
(m)配列番号26に示されるセンス配列と配列番号27に示されるアンチセンス配列とから形成されるsiRNA
(n)配列番号28に示されるセンス配列と配列番号29に示されるアンチセンス配列とから形成されるsiRNA
(o)配列番号30に示されるセンス配列と配列番号31に示されるアンチセンス配列とから形成されるsiRNA
(p)配列番号32に示されるセンス配列と配列番号33に示されるアンチセンス配列とから形成されるsiRNA
(q)配列番号34に示されるセンス配列と配列番号35に示されるアンチセンス配列とから形成されるsiRNA
(r)配列番号36に示されるセンス配列と配列番号37に示されるアンチセンス配列とから形成されるsiRNA
(s)配列番号54に示されるセンス配列と配列番号55に示されるアンチセンス配列とから形成されるsiRNA
3)前記siRNAのセンス鎖の3’末端の末端側からオーバーハングヌクレオチドを除いた連続する1~10ヌクレオチドがDNAに変換された上記1)又は2)のsiRNA。
4)前記siRNAのアンチセンス鎖の5’末端の末端側から連続する1~10ヌクレオチドがDNAに変換された上記1)-3)のsiRNA。
5)前記siRNAのセンス鎖の3’末端の末端側からオーバーハングヌクレオチドを除いた連続する1~10ヌクレオチドがDNAに変換され、且つアンチセンス鎖の5’末端の末端側から連続する1~10ヌクレオチドがDNAに変換された上記1)-4)のsiRNA。
6)アンチセンス鎖の5’末端がモノリン酸化又はモノチオリン酸化された上記1)~5)のsiRNA。
7)上記1)~6)のいずれかのsiRNAを含有する医薬組成物。
8)上記1)~6)のいずれかのsiRNAを有効成分として含有するTGF-β1遺伝子発現抑制剤。
9)上記1)~6)のいずれかのsiRNAを有効成分として含有する線維症予防又は治療剤。
10)上記1)~6)のいずれかのsiRNAを有効成分として含有する肺線維症若しくは肺がんの予防又は治療剤。
11)TGF-β1遺伝子発現抑制剤の製造のための、上記1)~6)のsiRNAの使用。
12)線維症予防又は治療剤の製造のための、上記1)~6)のsiRNAの使用。
13)肺線維症若しくは肺がんの予防又は治療剤の製造のための、上記1)~6)のsiRNAの使用。
14)TGF-β1遺伝子発現抑制のための、上記1)~6)のsiRNA。
15)線維症予防又は治療のための、上記1)~6)のsiRNA。
16)肺線維症若しくは肺がんの予防又は治療のための、上記1)~6)のsiRNA。
17)上記1)~6)のsiRNAをヒト又は動物に投与することを特徴とする、TGF-β1遺伝子発現抑制方法。
18)上記1)~6)のsiRNAをヒト又は動物に投与することを特徴とする、線維症予防又は治療方法。
19)上記1)~6)のsiRNAをヒト又は動物に投与することを特徴とする、肺線維症若しくは肺がんの予防又は治療方法。
配列番号1で示される塩基配列は、TGF-β1のmRNAの塩基配列であり、当該配列情報はGenBankに、GenBank Accession No. NM_000660.3として登録されている。
また、センス鎖及びアンチセンス鎖の両端は、平滑末端でもよいし、それぞれの鎖の3’側がオーバーハング(突出末端)であっても良い。ここで、「平滑末端」とは、2本鎖RNAの末端部分において、センス鎖の末端領域とそれに対合するアンチセンス鎖の末端領域が、1本鎖部分を形成することなく対合している構造を意味する。また、「オーバーハング」は、ダングリングエンドとも称され、2本鎖RNAの末端部分のセンス鎖の末端領域又はそれに対合するアンチセンス鎖の末端領域において、対合する塩基が存在しないために2本鎖を形成できず、1本鎖部分(突出末端)が存在している構造を意味する。
突出末端部分の塩基数は1~10ヌクレオチドであり、好ましくは1~4ヌクレオチドであり、さらに好ましくは1~2ヌクレオチドである。尚、突出末端の長さは二つの鎖の間で無関係であり、互いに異なる長さであっても良い。突出末端部分のヌクレオチドはRNAでも、DNAでもよく、標的であるTGF-β1のmRNAに相補的な塩基が好ましいが、上記RNA干渉能を保持する限り相補的でない塩基であってもよい。
尚、この場合のハイブリダイズ条件は、本発明のsiRNAを生体内に投与して医薬として用いる場合は、生体内の条件であり、本発明のsiRNAを試薬としてin vitroで用いる場合は、中度のストリンジェントな条件又は高度なストリンジェントな条件であり、このような条件として、例えば、400mM NaCl、40mM PIPES pH6.4、1mM EDTA、50℃~70℃で12~160時間でのハイブリゼーション条件が挙げられる。これらの条件については、当業者に周知であり、Sambrook et al.(Molecular Cloning: A Laboratory Manual second edition, Cold Spring Harbor Laboratory Press, New York, USA, 1989)に記載されている。
また、配列同一性は、リップマン-パーソン法(Lipman-Pearson法;Science, 227, 1435, (1985))等によって計算すればよく、例えば、遺伝情報処理ソフトウェアGenetyx-Win(Ver.5.1.1;ソフトウェア開発)のホモロジー解析(Search homology)プログラムを用いて、Unit size to compare(ktup)を2として解析を行なうことにより算出される。
ここで、RNAヌクレオチドのDNAへの変換とは、AMPをdAMPへ、GMPをdGMPへ、CMPをdCMPへ、UMPをdTMPへ変換することを意味する。
ハイブリッド型としては、センス鎖のヌクレオチドをDNAに変換したものが好ましい。キメラ型としては、下流側(センス鎖の3’末端側、アンチセンス鎖の5’末端側)の一部のヌクレオチドをDNAに変換したものが挙げられる。具体的には、センス鎖の3’末端側及びアンチセンス鎖の5’末端側のヌクレオチドを共にDNAに変換したもの、センス鎖の3’末端側又はアンチセンス鎖の5’末端側の何れかのヌクレオチドをDNAに変換したものが挙げられる。また、変換するヌクレオチド長は、RNA分子の1/2に相当するヌクレオチドまでの任意長とするのが好ましく、例えば末端から1~13ヌクレオチド、好ましくは1~10ヌクレオチドが挙げられる。RNA干渉効果、RNA分子の安定性、安全性等の点から、好適なキメラ型siRNAとしては、例えばヌクレオチド長がそれぞれ19~23であり、センス鎖の3’末端側からオーバーハングヌクレオチドを除いた1~10、好ましくは1~8、より好ましくは1~6のヌクレオチド及びアンチセンス鎖の5’末端側から1~10、好ましくは1~8、より好ましくは1~6のヌクレオチドを任意数、連続してDNAに変換したものが挙げられる(後記〔表2〕参照)。また、この場合、センス鎖(オーバーハングヌクレオチドを除く)とアンチセンス鎖のDNA変換数は同一であるのがより好ましい。
また、糖が修飾されたヌクレオチド類似体としては、例えば、リボヌクレオチドの2’-OHが、H、OR、R、ハロゲン原子、SH、SR、NH2、NHR、NR2、もしくはCN(ここで、Rは炭素数1-6のアルキル基、アルケニル基又はアルキニル基を示す)等によって置換された2’位糖置換体、5’末端の-OHがモノリン酸化又はモノチオリン酸化された5’末端リン酸化体、5’末端モノチオリン酸化体が挙げられる。
リン酸塩が修飾されたヌクレオチド類似体としては、隣接するリボヌクレオチドを結合するホスホエステル基を、ホスホチオエート基で置換したものが挙げられる。
ここで、置換基としては、一例として、アミノ基;メルカプト基;ニトロ基;炭素数1~40(好ましくは2~20、更に好ましくは4~12)のアルキル基;炭素数1~40(好ましくは2~20、更に好ましくは4~12)のアミノアルキル基;炭素数1~40(好ましくは2~20、更に好ましくは4~12)のチオアルキル基;炭素数1~40(好ましくは2~20、更に好ましくは4~12)のアルコキシル基;炭素数1~40(好ましくは2~20、更に好ましくは4~12)のアミノアルコキシル基;炭素数1~40(好ましくは2~20、更に好ましくは4~12)のチオアルコキシル基;炭素数1~40(好ましくは2~20、更に好ましくは4~12)のモノ若しくはジアルキルアミノ基;炭素数1~40(好ましくは2~20、更に好ましくは4~12)のアルキルチオ基;炭素数2~40(好ましくは2~20、更に好ましくは4~12)のポリエチレンオキサイド基;炭素数3~39(好ましくは3~21、更に好ましくは3~12)のポリプロピレンオキサイド基等を挙げることができる。これらの置換基を結合させることによって、RNA干渉効果を顕著に増強させることが可能になる。
上記センス鎖RNAにおける、置換基若しくは機能性分子又はこれらを連結するリンカーの結合部位については、特に限定されるものではないが、これらがセンス鎖RNAの所定のヌクレオチドのリン酸部分の水酸基を構成する水素原子と置換されて結合していることが好ましい。
化学的合成は、siRNA構成要素である核酸分子を含むアミダイド樹脂を原料として、核酸合成装置により合成することが可能である。
転写系による合成は、ヘアピン型RNAをトリミングする試験管内転写法により2本鎖RNAを合成することが可能である。
従って、本発明のsiRNA及び当該siRNAを投与対象内で発現可能な発現ベクターは、ヒト又は動物に投与するための医薬(医薬組成物)として有用である。具体的には、TGF-β1遺伝子発現抑制のための医薬、TGF-β1の過剰発現に起因する疾患、例えば線維症の予防・治療のための医薬、すなわち線維症の予防又は治療剤として有用である。
ここで、肺の線維化を来たす疾患としては、間質性肺炎、嚢胞性線維症、慢性閉塞性肺疾患(COPD: Chronic obstructive pulmonary disease)、急性呼吸促迫症候群 (ARDS: Acute respiratory distress syndrome)、炎症性肺疾患、肺感染症、放射性肺臓炎、薬剤性間質性肺炎、膠原病に伴う間質性肺炎など多岐にわたるが、原因が特定できない間質性肺炎である特発性間質性肺炎(IIPs)のうち特発性肺線維症(IPF)が特に好ましい。特発性間質性肺炎(IIPs)には臨床病理学的疾患として、特発性肺線維症(IPF)、非特異的間質性肺炎(NSIP)、特発性器質化肺炎(COP/BOOP)、急性間質性肺炎(AIP)、剥離性間質性肺炎(DIP)、呼吸細気管支炎に伴う間質性肺疾患(RB-ILD)、リンパ球性間質性肺炎(LIP)等が包含される。
また、TGF-β1は、がん、特に肺腺がんの浸潤や転移を促進すること(Mol Cell Biochem (2011) 355:309.314、Cancer Genomics Proteomics 2010, 7, 217)、TGF-β1が、がん治療後の正常組織の損傷部位で過剰発現し、TGF-β1経路を標的とすることで当該正常組織の損傷を抑制できること(The Oncologist 2010;15:350.359)等が報告されていることから、本発明のsiRNA及び当該siRNAを投与対象内で発現可能な発現ベクターは、がん、特に肺がんの予防又は治療剤としても有用である。
シクロアミロースは、グルコースがα-1,4結合により結合した環状α-1,4-グルカンであり、へリックス構造の内側に立体的で奥行きのある空洞部分を有している。本発明に使用されるシクロアミロースにおけるグルコースの重合度としては、特に制限されるものではないが、例えば10~500、好ましくは10~100、更に好ましくは22~50が例示される。シクロアミロースは、アミロマルターゼ等の酵素を利用して、グルコースから調製することができる。また、シクロアミロースは市販されており、本発明では市販品を使用することもできる(国際公開第2009/61003号パンフレット参照)。
この場合の発現ベクターは、例えば、本発明のsiRNAをコードすることができるDNAを、適当な遺伝子治療用ベクター、例えば、アデノウイルスベクター、アデノ随伴ウイルス(AAV)ベクター、又はレンチウイルスベクター等に挿入することにより構築することができる。
実施例1 siRNAの作製
下記表3に示されるTGF-β1遺伝子を標的とするsiRNA分子を設計し、各siRNAオリゴヌクレオチドを化学合成法によって合成し、HPLC精製法にて精製して使用した。
(1)細胞
ヒト肺胞上皮由来のA549細胞(DSファーマバイオメディカル株式会社)を用いた。
1×105個の細胞を10%牛胎児血清含有のD-MEM培地(Dulbecco's modified Eagle's Medium, 100unit/mLペニシリン, 100μg/mLストレプトマイシン含有,12ウェルプレート)に播種する。37℃、5%CO2条件下で一晩培養後、40%コンフルエントとなったA549細胞の培地を無血清培地に交換した。
siRNAとして、上記表3に示すオリゴヌクレオチドを用い、細胞が40%コンフルエントとなった時点で、Lipofectamine2000 (インビトロジェン社)を用いて上記細胞への導入を行った。
具体的には、各ウェルあたり2.0μL Lipofectamine2000を98μL OPTI-MEM(インビトロジェン)に添加し、5分間室温でインキュベーションした(A溶液)。
0.2μM siRNA溶液0.625μLを99.375μL OPTI-MEMに添加した(B溶液)。A及びB溶液を混和し、室温で20分間インキュベーションした。インキュベーション後、12-ウェルプレート各ウェルにAB混液を添加した。siRNA最終濃度が、0.1nMとなるように添加した。
(i)サイトカイン処理
siRNA及びLipofectamine混液を添加6時間後、培地を0.1%BSA(牛血清アルブミン)及びサイトカイン(1ng/mL IL-1β及び1ng/mL TNF-α)含有D-MEM培地に交換し、12時間培養した。培養後上清をサンプリングした。
細胞中全RNA抽出には、自動核酸抽出装置QuickGene-810(富士フイルム株式会社)とQuickGene-810の専用キットであるQuickGene RNA cultured cell kitS(富士フイルム株式会社)を用いた。細胞を1.0mL PBSで洗浄し、細胞溶解液0.5mLを添加し、細胞中に含まれる全RNAの抽出を行った。0.5mL溶解液(LRC、メルカプトエタノール添加済み)を添加した12ウェルプレートをシーソー型振盪機で5分間撹拌した。ピペッティングにより液を5-6往復させてよく混ぜ、液をエッペンドルフチューブへ移した。エタノールを420μL添加し、ボルテックスミキサーで15秒間撹拌後、QuickGene-810で処理を行なった。QuickGene-810での処理中、DNase(RQ1 RNase-free DNase,Promega)を添加した。抽出した全RNAサンプルは、次処理を行うまで-80℃冷蔵庫にて保管した。
培養細胞から抽出された全RNAサンプル中のRNA濃度(μg/mL)を、260nmにおける吸光度の測定値から計算した(対照:TEバッファー)。この値をもとに、各サンプルにおいてRNA量が0.1μgに相当する溶液量を96ウェルプレートに入れた。これに蒸留水を加えて全量12μLとし、さらにQuantiTect Reverse Transcription Kit(QIAGEN)に含まれるgDNA Wipeout Buffer 2μLを加え、ボルテックス混和後、42℃で2分間インキュベートし、その後4℃に冷却した。これにQuantiTect Reverse Transcription Kit(QIAGEN)に含まれるQuantiscript Reverse Transcriptase 1μL、Quantiscript RT Buffer 4μL、及びRT Primer Mix 1μLを加え、混和し、42℃で15分間インキュベートした。続いてQuantiscript Reverse Transcriptaseを失活させるため95℃で3分間加熱した後、4℃に冷却した。
この調製液(cDNA調製原液)をTEバッファーで5倍希釈し、標的遺伝子(TGF-β1)をターゲットとしたPCR用cDNA溶液とした。
また、cDNA調製原液をTEバッファーで50倍希釈し、内部標準遺伝子としてGAPDHを選定しPCR用cDNA溶液とした。なお、コントロールサンプル(siRNA非投与)のcDNA調製原液をTEバッファーで1、10、100、及び1000倍希釈し、TGF-β1をターゲットとしたPCR検量線用サンプルとした。同様にコントロールサンプルcDNA調製原液をTEバッファーで10、100、1000、及び10000倍希釈し、GAPDHをターゲットとしたPCR検量線用サンプルとした。
TGF-β1に由来するcDNA産物2.5μLを鋳型として、12.5μL QuantiFast SYBR Green PCR Master Mix (QIAGEN)と、ヒト由来TGF-β1遺伝子又はヒト由来GAPDH遺伝子の2.5μL QuantiTect Primer Assay (QIAGEN)を用いて、滅菌蒸留水にて最終容量25μLとしたPCR反応溶液を調製した。調製した溶液は、Applied Biosystems 7500 (Life Technologies Japan)にて、95℃で5分間加熱した後、1)95℃,10秒、2)60℃,35秒のサイクルを40回繰り返した後に、95℃から60℃に徐冷し、熱解離測定を行った。PCR増幅過程に由来するCt(Threshold Cycle)値を基に、GAPDH遺伝子のCt値による各標的遺伝子の増幅割合を補正し、標的遺伝子のmRNA抑制効果を評価した。結果を図1及び表4に示す。
siRNA番号(l)を基に、下記表5に示したTGF-β1遺伝子を標的とするキメラ型siRNA分子を設計し、各キメラ型siRNAオリゴヌクレオチドを化学合成法によって合成し、HPLC精製法にて精製して使用した。
表5に示すオリゴヌクレオチド(濃度:10nM又は0.1nM)を用いて実施例2と同様の方法で、TGF-β1の発現抑制効果を評価した。結果を図2及び表6に示す。
siRNA番号(l)を基に、下記表7に示したTGF-β1遺伝子を標的とするリン酸又はチオリン酸結合キメラ型siRNA分子を設計し、各キメラ型siRNAオリゴヌクレオチドを化学合成法によって合成し、HPLC精製法にて精製して使用した。
表7に示すオリゴヌクレオチドを用いて実施例2と同様の方法でTGF-β1の発現抑制効果を評価した。結果を図3及び表8に示す。
siRNA番号(l)を基に、下記表9に示したTGF-β1遺伝子を標的とするオーバーハング部分にDNAを有するsiRNA分子を設計し、siRNAオリゴヌクレオチドを化学合成法によって合成し、HPLC精製法にて精製して使用した。
表9に示すオリゴヌクレオチドを用いて実施例2と同様の方法でTGF-β1の発現抑制効果を評価した。結果を表10に示す。
マウスとヒトの共通配列であるsiRNA番号(q)(配列番号34及び35)を基に、キメラ型siRNA(q-C8:センス鎖(5'→3'):CCAAGGGCUACCAtgccaact(配列番号52)、アンチセンス鎖(5'→3'):ttggcaUGGUAGCCCUUGGGC(配列番号53)を設計し、実施例3と同様にしてキメラ型siRNAオリゴヌクレオチドを合成し、HPLC精製法にて精製した。これを被験物質として用いて、ブレオマイシン肺線維症モデルマウスにて効力の評価を行った。
マウス(C57BL/6NCrSlc(SLC)、メス、13週齢)にペントバルビタール(大日本住友製薬株式会社製)をマウス腹腔内に投与後、麻酔下のマウス背部皮下にALZETTM osmotic pump(model 2001, DURECT Corporation)を埋め込み作成した。なお、ALZETTM osmotic pumpには約10mg/mLのブレオマイシン生理食塩水溶液200μLをあらかじめ注入しており、ブレオマイシンは日本化薬株式会社製を使用した。
被験物質は、ALZETTM osmotic pump埋め込み後、3、7、14日目に蒸留水(大塚製薬株式会社)で溶解し、毎回100μg/bodyをMicroSprayerTM(model IA-1C、PENN CENTURY, Inc.)を用いて気管内投与した。投与容量は、ブレオマイシン投与開始後3、7日目は75μL/body、14日目は50μL/bodyで行った。
なお、比較対照として、ALZETTM osmotic pumpに生理食塩水のみ200μLを注入して、被験物質として蒸留水を投与した群、およびALZETTM osmotic pumpに約10mg/mLのブレオマイシン生理食塩水溶液200μLを注入して、被験物質として蒸留水を投与した群を設定した。
採取したBALFは、遠心分離(2000rpm、4℃、10分間)し、上清のTGF-β1蛋白量をELISA法にて測定した。結果を図4に示す。図中、SALは、生理食塩水(saline)、DWは、蒸留水(distilled water)、BLMは、ブレオマイシン(bleomycin)を示す。
10%中性緩衝ホルマリン固定液で固定した肺組織は、パラフィン包埋、組織切片作製後、H.E.(Hematoxilin-Eosin)染色およびマッソントリクローム染色を行った。組織図を図5に示す。
Claims (19)
- 配列番号1の塩基番号1285~1318番の塩基、1398~1418番の塩基、1434~1463番の塩基、1548~1579番の塩基、1608~1628番の塩基、1700~1726番の塩基、1778~1798番の塩基、1806~1826番の塩基及び1887~1907番の塩基よりなる群から選ばれる連続する17~23塩基を標的配列とし、全長が30ヌクレオチド以下であるsiRNA。
- siRNAが、以下の(a)~(s)から選ばれる請求項1記載のsiRNA。
(a)配列番号2に示されるセンス配列と配列番号3に示されるアンチセンス配列とから形成されるsiRNA
(b)配列番号4に示されるセンス配列と配列番号5に示されるアンチセンス配列とから形成されるsiRNA
(c)配列番号6に示されるセンス配列と配列番号7に示されるアンチセンス配列とから形成されるsiRNA
(d)配列番号8に示されるセンス配列と配列番号9に示されるアンチセンス配列とから形成されるsiRNA
(e)配列番号10に示されるセンス配列と配列番号11に示されるアンチセンス配列とから形成されるsiRNA
(f)配列番号12に示されるセンス配列と配列番号13に示されるアンチセンス配列とから形成されるsiRNA
(g)配列番号14に示されるセンス配列と配列番号15に示されるアンチセンス配列とから形成されるsiRNA
(h)配列番号16に示されるセンス配列と配列番号17に示されるアンチセンス配列とから形成されるsiRNA
(i)配列番号18に示されるセンス配列と配列番号19に示されるアンチセンス配列とから形成されるsiRNA
(j)配列番号20に示されるセンス配列と配列番号21に示されるアンチセンス配列とから形成されるsiRNA
(k)配列番号22に示されるセンス配列と配列番号23に示されるアンチセンス配列とから形成されるsiRNA
(l)配列番号24に示されるセンス配列と配列番号25に示されるアンチセンス配列とから形成されるsiRNA
(m)配列番号26に示されるセンス配列と配列番号27に示されるアンチセンス配列とから形成されるsiRNA
(n)配列番号28に示されるセンス配列と配列番号29に示されるアンチセンス配列とから形成されるsiRNA
(o)配列番号30に示されるセンス配列と配列番号31に示されるアンチセンス配列とから形成されるsiRNA
(p)配列番号32に示されるセンス配列と配列番号33に示されるアンチセンス配列とから形成されるsiRNA
(q)配列番号34に示されるセンス配列と配列番号35に示されるアンチセンス配列とから形成されるsiRNA
(r)配列番号36に示されるセンス配列と配列番号37に示されるアンチセンス配列とから形成されるsiRNA
(s)配列番号54に示されるセンス配列と配列番号55に示されるアンチセンス配列とから形成されるsiRNA - 前記siRNAのセンス鎖の3’末端の末端側からオーバーハングヌクレオチドを除いた連続する1~10ヌクレオチドがDNAに変換された請求項1又は2に記載のsiRNA。
- 前記siRNAのアンチセンス鎖の5’末端の末端側から連続する1~10ヌクレオチドがDNAに変換された請求項1~3の何れか1項に記載のsiRNA。
- 前記siRNAのセンス鎖の3’末端の末端側からオーバーハングヌクレオチドを除いた連続する1~10ヌクレオチドがDNAに変換され、且つアンチセンス鎖の5’末端の末端側から連続する1~10ヌクレオチドがDNAに変換された請求項1~4の何れか1項に記載のsiRNA。
- アンチセンス鎖の5’末端がモノリン酸化又はモノチオリン酸化された請求項1~5の何れか1項に記載のsiRNA。
- 請求項1~6のいずれか1項に記載のsiRNAを含有する医薬組成物。
- 請求項1~6のいずれか1項に記載のsiRNAを有効成分として含有するTGF-β1遺伝子発現抑制剤。
- 請求項1~6のいずれか1項に記載のsiRNAを有効成分として含有する線維症予防又は治療剤。
- 請求項1~6のいずれか1項に記載のsiRNAを有効成分として含有する肺線維症若しくは肺がんの予防又は治療剤。
- TGF-β1遺伝子発現抑制剤の製造のための、請求項1~6のいずれか1項に記載のsiRNAの使用。
- 線維症予防又は治療剤の製造のための、請求項1~6のいずれか1項に記載のsiRNAの使用。
- 肺線維症若しくは肺がんの予防又は治療剤の製造のための、請求項1~6のいずれか1項に記載のsiRNAの使用。
- TGF-β1遺伝子発現抑制のための、請求項1~6のいずれか1項に記載のsiRNA。
- 線維症予防又は治療のための、請求項1~6のいずれか1項に記載のsiRNA。
- 肺線維症若しくは肺がんの予防又は治療のための、請求項1~6のいずれか1項に記載のsiRNA。
- 請求項1~6のいずれか1項に記載のsiRNAをヒト又は動物に投与することを特徴とする、TGF-β1遺伝子発現抑制方法。
- 請求項1~6のいずれか1項に記載のsiRNAをヒト又は動物に投与することを特徴とする、線維症予防又は治療方法。
- 請求項1~6のいずれか1項に記載のsiRNAをヒト又は動物に投与することを特徴とする、肺線維症若しくは肺がんの予防又は治療方法。
Priority Applications (31)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PL11832611T PL2628798T3 (pl) | 2010-10-14 | 2011-10-14 | Środek profilaktyczny lub terapeutyczny przeciw włóknieniu |
MX2013003698A MX348555B (es) | 2010-10-14 | 2011-10-14 | Agente preventivo o terapeutico contra la fibrosis. |
DK11832611.5T DK2628798T3 (da) | 2010-10-14 | 2011-10-14 | Profylaktisk eller terapeutisk middel mod fibrose |
BR112013006541A BR112013006541A2 (pt) | 2010-10-14 | 2011-10-14 | agente preventivo ou terapêutico para fibrose |
NZ609440A NZ609440A (en) | 2010-10-14 | 2011-10-14 | Preventative or therapeutic agent for fibrosis |
KR1020137007962A KR101853799B1 (ko) | 2010-10-14 | 2011-10-14 | 섬유증 예방 또는 치료제 |
SG2013024757A SG189245A1 (en) | 2010-10-14 | 2011-10-14 | Preventive or therapeutic agent for fibrosis |
KR1020187011751A KR102167225B1 (ko) | 2010-10-14 | 2011-10-14 | 섬유증 예방 또는 치료제 |
JP2012538721A JP6022940B2 (ja) | 2010-10-14 | 2011-10-14 | 線維症予防又は治療剤 |
RU2013121802/10A RU2583290C2 (ru) | 2010-10-14 | 2011-10-14 | Профилактический или терапевтический агент для лечения фиброза |
EP19158202.2A EP3517614A1 (en) | 2010-10-14 | 2011-10-14 | Preventive or therapeutic agent for fibrosis |
EP11832611.5A EP2628798B1 (en) | 2010-10-14 | 2011-10-14 | Prophylactic or therapeutic agent for fibrosis |
AU2011314653A AU2011314653B2 (en) | 2010-10-14 | 2011-10-14 | Preventative or therapeutic agent for fibrosis |
CN201180044862.9A CN103119166B (zh) | 2010-10-14 | 2011-10-14 | 纤维症预防或者治疗剂 |
LTEP11832611.5T LT2628798T (lt) | 2010-10-14 | 2011-10-14 | Profilaktinis arba terapinis agentas nuo fibrozės |
CA2813163A CA2813163C (en) | 2010-10-14 | 2011-10-14 | Preventive or therapeutic agent for fibrosis |
US13/824,080 US8772262B2 (en) | 2010-10-14 | 2011-10-14 | Preventive or therapeutic agent for fibrosis |
ES11832611T ES2720135T3 (es) | 2010-10-14 | 2011-10-14 | Agente profiláctico o terapéutico para la fibrosis |
SI201131728T SI2628798T1 (sl) | 2010-10-14 | 2011-10-14 | Profilaktično ali terapevtsko sredstvo za fibrozo |
IL224791A IL224791A (en) | 2010-10-14 | 2013-02-18 | Prophylactic or medical cause of fibrosis |
ZA2013/01279A ZA201301279B (en) | 2010-10-14 | 2013-02-19 | Preventive or therapeutic agent for fibrosis |
HK13111550.4A HK1184189A1 (zh) | 2010-10-14 | 2013-10-15 | 纖維症預防或者治療劑 |
US14/272,898 US9273314B2 (en) | 2010-10-14 | 2014-05-08 | Preventive or therapeutic agent for fibrosis |
AU2015261583A AU2015261583C1 (en) | 2010-10-14 | 2015-11-25 | Preventative or therapeutic agent for fibrosis |
US15/000,204 US9637743B2 (en) | 2010-10-14 | 2016-01-19 | Preventive or therapeutic agent for fibrosis |
IL245995A IL245995B (en) | 2010-10-14 | 2016-06-02 | Prophylactic or medical agent for fibrosis |
US15/465,961 US10125366B2 (en) | 2010-10-14 | 2017-03-22 | Preventive or therapeutic agent for fibrosis |
US16/139,461 US20190010500A1 (en) | 2010-10-14 | 2018-09-24 | Preventive or therapeutic agent for fibrosis |
HRP20190722TT HRP20190722T1 (hr) | 2010-10-14 | 2019-04-17 | Profilaktički ili terapijski agent za fibrozu |
CY20191100643T CY1121945T1 (el) | 2010-10-14 | 2019-06-20 | Προφυλακτικος και θεραπευτικος παραγοντας της ινωσης |
US16/528,920 US20200291406A1 (en) | 2010-10-14 | 2019-08-01 | Preventive or therapeutic agent for fibrosis |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2010231946 | 2010-10-14 | ||
JP2010-231946 | 2010-10-14 |
Related Child Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US13/824,080 A-371-Of-International US8772262B2 (en) | 2010-10-14 | 2011-10-14 | Preventive or therapeutic agent for fibrosis |
US14/272,898 Continuation US9273314B2 (en) | 2010-10-14 | 2014-05-08 | Preventive or therapeutic agent for fibrosis |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2012050181A1 true WO2012050181A1 (ja) | 2012-04-19 |
Family
ID=45938402
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2011/073628 WO2012050181A1 (ja) | 2010-10-14 | 2011-10-14 | 線維症予防又は治療剤 |
Country Status (30)
Country | Link |
---|---|
US (6) | US8772262B2 (ja) |
EP (2) | EP2628798B1 (ja) |
JP (4) | JP6022940B2 (ja) |
KR (2) | KR101853799B1 (ja) |
CN (2) | CN103119166B (ja) |
AR (1) | AR083445A1 (ja) |
AU (1) | AU2011314653B2 (ja) |
BR (1) | BR112013006541A2 (ja) |
CA (1) | CA2813163C (ja) |
CO (1) | CO6660456A2 (ja) |
CY (1) | CY1121945T1 (ja) |
DK (1) | DK2628798T3 (ja) |
ES (1) | ES2720135T3 (ja) |
HK (1) | HK1184189A1 (ja) |
HR (1) | HRP20190722T1 (ja) |
HU (1) | HUE043891T2 (ja) |
IL (2) | IL224791A (ja) |
LT (1) | LT2628798T (ja) |
MX (1) | MX348555B (ja) |
MY (1) | MY165964A (ja) |
NZ (2) | NZ609440A (ja) |
PL (1) | PL2628798T3 (ja) |
PT (1) | PT2628798T (ja) |
RU (1) | RU2583290C2 (ja) |
SG (3) | SG10201907649QA (ja) |
SI (1) | SI2628798T1 (ja) |
TR (1) | TR201905060T4 (ja) |
TW (2) | TWI572715B (ja) |
WO (1) | WO2012050181A1 (ja) |
ZA (1) | ZA201301279B (ja) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2015093495A1 (ja) * | 2013-12-16 | 2015-06-25 | 株式会社ボナック | TGF-β1遺伝子発現制御のための一本鎖核酸分子 |
JP2017000155A (ja) * | 2010-10-14 | 2017-01-05 | 国立大学法人三重大学 | 線維症予防又は治療剤 |
WO2017043490A1 (ja) * | 2015-09-07 | 2017-03-16 | 協和発酵バイオ株式会社 | 自然免疫誘導効果が増強した二重鎖リボ核酸 |
US10337009B2 (en) | 2014-12-15 | 2019-07-02 | Bonac Corporation | Single-stranded nucleic acid molecule for inhibiting TGF-β1 expression |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
RU2714257C2 (ru) * | 2015-10-30 | 2020-02-13 | Бонак Корпорейшн | КОМПОЗИЦИЯ, СОДЕРЖАЩАЯ В СТАБИЛЬНОМ СОСТОЯНИИ ОДНОЦЕПОЧЕЧНУЮ МОЛЕКУЛУ НУКЛЕИНОВОЙ КИСЛОТЫ, КОТОРАЯ ПОДАВЛЯЕТ ЭКСПРЕССИЮ ГЕНА TGF-β1 |
Citations (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2003035083A1 (de) | 2001-10-26 | 2003-05-01 | Ribopharma Ag | Medikament zur behandlung einer fibrotischen erkrankung durch rna interferenz |
JP2007119396A (ja) | 2005-10-28 | 2007-05-17 | Hosokawa Funtai Gijutsu Kenkyusho:Kk | 核酸化合物封入ナノ粒子を含む経肺投与用医薬製剤 |
WO2007079224A2 (en) | 2005-12-30 | 2007-07-12 | Wei-Wu He | Sirna compositions promoting scar-free wound healing of skin and methods for wound treatment |
WO2007109097A2 (en) | 2006-03-16 | 2007-09-27 | Alnylam Pharmaceuticals, Inc. | RNAi MODULATION OF TGF-BETA AND THERAPEUTIC USES THEREOF |
WO2008109548A2 (en) | 2007-03-02 | 2008-09-12 | Mdrna, Inc. | Nucleic acid compounds for inhibiting tgfb gene expression and uses thereof |
WO2008140126A1 (ja) | 2007-05-09 | 2008-11-20 | Riken | 1本鎖環状rnaおよびその製造方法 |
JP2009516517A (ja) | 2005-11-21 | 2009-04-23 | ジヨンソン・アンド・ジヨンソン・リサーチ・ピーテイワイ・リミテツド | 多標的干渉rnaならびにそれらの使用および設計方法 |
WO2009061417A1 (en) | 2007-11-06 | 2009-05-14 | Sirnaomics, Inc. | Multi-targeted rnai therapeutics for scarless wound healing of skin |
WO2009061003A2 (en) | 2007-11-08 | 2009-05-14 | Otsuka Pharmaceutical Co., Ltd. | Nucleic acid complex and nucleic acid delivery composition |
WO2009123185A1 (ja) | 2008-03-31 | 2009-10-08 | 独立行政法人産業技術総合研究所 | Rna干渉効果が高い2本鎖脂質修飾rna |
JP2010172344A (ja) * | 2003-04-02 | 2010-08-12 | Dharmacon Inc | Rna干渉において使用するための修飾ポリヌクレオチド |
JP2010528985A (ja) * | 2007-05-04 | 2010-08-26 | エムディーアールエヌエー,インコーポレイテッド | アミノ酸脂質およびその使用 |
Family Cites Families (31)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE19956568A1 (de) | 1999-01-30 | 2000-08-17 | Roland Kreutzer | Verfahren und Medikament zur Hemmung der Expression eines vorgegebenen Gens |
US7829693B2 (en) | 1999-11-24 | 2010-11-09 | Alnylam Pharmaceuticals, Inc. | Compositions and methods for inhibiting expression of a target gene |
DE10100586C1 (de) | 2001-01-09 | 2002-04-11 | Ribopharma Ag | Verfahren zur Hemmung der Expression eines Ziegens |
WO2003035869A1 (de) | 2001-10-26 | 2003-05-01 | Ribopharma Ag | Verwendung einer doppelsträngigen ribonukleinsäure zur gezielten hemmung der expression eines vorgegebenen zielgens |
US7423142B2 (en) | 2001-01-09 | 2008-09-09 | Alnylam Pharmaceuticals, Inc. | Compositions and methods for inhibiting expression of anti-apoptotic genes |
US8546143B2 (en) | 2001-01-09 | 2013-10-01 | Alnylam Pharmaceuticals, Inc. | Compositions and methods for inhibiting expression of a target gene |
US7767802B2 (en) | 2001-01-09 | 2010-08-03 | Alnylam Pharmaceuticals, Inc. | Compositions and methods for inhibiting expression of anti-apoptotic genes |
DE10163098B4 (de) | 2001-10-12 | 2005-06-02 | Alnylam Europe Ag | Verfahren zur Hemmung der Replikation von Viren |
US7745418B2 (en) | 2001-10-12 | 2010-06-29 | Alnylam Pharmaceuticals, Inc. | Compositions and methods for inhibiting viral replication |
US20050119202A1 (en) | 2001-10-26 | 2005-06-02 | Roland Kreutzer | Medicament to treat a fibrotic disease |
US20040121348A1 (en) | 2001-10-26 | 2004-06-24 | Ribopharma Ag | Compositions and methods for treating pancreatic cancer |
DE10230997A1 (de) | 2001-10-26 | 2003-07-17 | Ribopharma Ag | Medikament zur Erhöhung der Wirksamkeit eines Rezeptor-vermittelt Apoptose in Tumorzellen auslösenden Arzneimittels |
US7668257B2 (en) * | 2003-10-01 | 2010-02-23 | Samsung Electronics Co., Ltd. | Transmissions with reduced code rate in 8VSB digital television |
KR101147147B1 (ko) | 2004-04-01 | 2012-05-25 | 머크 샤프 앤드 돔 코포레이션 | Rna 간섭의 오프 타겟 효과 감소를 위한 변형된폴리뉴클레오타이드 |
JP2010519905A (ja) | 2007-03-02 | 2010-06-10 | エムディーアールエヌエー,インコーポレイテッド | Akt遺伝子の発現を抑制するための核酸化合物およびその使用 |
US20080299659A1 (en) | 2007-03-02 | 2008-12-04 | Nastech Pharmaceutical Company Inc. | Nucleic acid compounds for inhibiting apob gene expression and uses thereof |
JP2010519913A (ja) | 2007-03-02 | 2010-06-10 | エムディーアールエヌエー,インコーポレイテッド | Wnt遺伝子の発現を抑制するための核酸化合物およびその使用 |
US20080286866A1 (en) | 2007-03-02 | 2008-11-20 | Nastech Pharmaceutical Company Inc. | Nucleic acid compounds for inhibiting vegf gene expression and uses thereof |
US20080287383A1 (en) | 2007-03-02 | 2008-11-20 | Nastech Pharmaceutical Company Inc. | Nucleic acid compounds for inhibiting erbb gene expression and uses thereof |
EP2121925A2 (en) | 2007-03-02 | 2009-11-25 | MDRNA, Inc. | Nucleic acid compounds for inhibiting ras gene expression and uses thereof |
JP2010519907A (ja) | 2007-03-02 | 2010-06-10 | エムディーアールエヌエー,インコーポレイテッド | Vegfファミリー遺伝子の発現を抑制するための核酸化合物およびその使用 |
WO2008109449A1 (en) | 2007-03-02 | 2008-09-12 | Mdrna Inc. | Nucleic acid compounds for inhibiting bcl2 gene expression and uses thereof |
WO2008109381A2 (en) | 2007-03-02 | 2008-09-12 | Mdrna, Inc. | Nucleic acid compounds for inhibiting hif1a gene expression and uses thereof |
JP2010519911A (ja) | 2007-03-02 | 2010-06-10 | エムディーアールエヌエー,インコーポレイテッド | Myc遺伝子の発現を抑制するための核酸化合物およびその使用 |
EP2121923A1 (en) | 2007-03-02 | 2009-11-25 | MDRNA, Inc. | Nucleic acid compounds for inhibiting erbb family gene expression and uses thereof |
US20100105134A1 (en) | 2007-03-02 | 2010-04-29 | Mdrna, Inc. | Nucleic acid compounds for inhibiting gene expression and uses thereof |
WO2009082817A1 (en) * | 2007-12-27 | 2009-07-09 | Protiva Biotherapeutics, Inc. | Silencing of polo-like kinase expression using interfering rna |
US8188060B2 (en) * | 2008-02-11 | 2012-05-29 | Dharmacon, Inc. | Duplex oligonucleotides with enhanced functionality in gene regulation |
CN110042099A (zh) | 2010-03-24 | 2019-07-23 | 菲奥医药公司 | 皮肤与纤维化症候中的rna干扰 |
WO2011119871A1 (en) * | 2010-03-24 | 2011-09-29 | Rxi Phrmaceuticals Corporation | Rna interference in ocular indications |
AR083445A1 (es) | 2010-10-14 | 2013-02-27 | Univ Mie | siARN CONTRA LA FIBROSIS |
-
2011
- 2011-10-13 AR ARP110103808A patent/AR083445A1/es unknown
- 2011-10-14 TR TR2019/05060T patent/TR201905060T4/tr unknown
- 2011-10-14 CA CA2813163A patent/CA2813163C/en not_active Expired - Fee Related
- 2011-10-14 EP EP11832611.5A patent/EP2628798B1/en active Active
- 2011-10-14 CN CN201180044862.9A patent/CN103119166B/zh not_active Expired - Fee Related
- 2011-10-14 BR BR112013006541A patent/BR112013006541A2/pt not_active IP Right Cessation
- 2011-10-14 PT PT11832611T patent/PT2628798T/pt unknown
- 2011-10-14 DK DK11832611.5T patent/DK2628798T3/da active
- 2011-10-14 SI SI201131728T patent/SI2628798T1/sl unknown
- 2011-10-14 PL PL11832611T patent/PL2628798T3/pl unknown
- 2011-10-14 RU RU2013121802/10A patent/RU2583290C2/ru active
- 2011-10-14 KR KR1020137007962A patent/KR101853799B1/ko active IP Right Grant
- 2011-10-14 NZ NZ609440A patent/NZ609440A/en not_active IP Right Cessation
- 2011-10-14 SG SG10201907649QA patent/SG10201907649QA/en unknown
- 2011-10-14 ES ES11832611T patent/ES2720135T3/es active Active
- 2011-10-14 MY MYPI2013000567A patent/MY165964A/en unknown
- 2011-10-14 JP JP2012538721A patent/JP6022940B2/ja not_active Expired - Fee Related
- 2011-10-14 SG SG2013024757A patent/SG189245A1/en unknown
- 2011-10-14 SG SG10201508365RA patent/SG10201508365RA/en unknown
- 2011-10-14 LT LTEP11832611.5T patent/LT2628798T/lt unknown
- 2011-10-14 NZ NZ630501A patent/NZ630501A/en not_active IP Right Cessation
- 2011-10-14 WO PCT/JP2011/073628 patent/WO2012050181A1/ja active Application Filing
- 2011-10-14 CN CN201610411134.1A patent/CN106086022B/zh not_active Expired - Fee Related
- 2011-10-14 US US13/824,080 patent/US8772262B2/en not_active Expired - Fee Related
- 2011-10-14 EP EP19158202.2A patent/EP3517614A1/en not_active Withdrawn
- 2011-10-14 HU HUE11832611A patent/HUE043891T2/hu unknown
- 2011-10-14 MX MX2013003698A patent/MX348555B/es active IP Right Grant
- 2011-10-14 TW TW100137418A patent/TWI572715B/zh not_active IP Right Cessation
- 2011-10-14 AU AU2011314653A patent/AU2011314653B2/en not_active Ceased
- 2011-10-14 TW TW105110662A patent/TWI679023B/zh not_active IP Right Cessation
- 2011-10-14 KR KR1020187011751A patent/KR102167225B1/ko active IP Right Grant
-
2013
- 2013-02-18 IL IL224791A patent/IL224791A/en active IP Right Grant
- 2013-02-19 ZA ZA2013/01279A patent/ZA201301279B/en unknown
- 2013-04-11 CO CO13094380A patent/CO6660456A2/es unknown
- 2013-10-15 HK HK13111550.4A patent/HK1184189A1/zh not_active IP Right Cessation
-
2014
- 2014-05-08 US US14/272,898 patent/US9273314B2/en not_active Expired - Fee Related
-
2016
- 2016-01-19 US US15/000,204 patent/US9637743B2/en active Active
- 2016-06-02 IL IL245995A patent/IL245995B/en active IP Right Grant
- 2016-07-19 JP JP2016141580A patent/JP2017000155A/ja active Pending
-
2017
- 2017-03-22 US US15/465,961 patent/US10125366B2/en not_active Expired - Fee Related
- 2017-12-27 JP JP2017250450A patent/JP2018088923A/ja active Pending
-
2018
- 2018-09-24 US US16/139,461 patent/US20190010500A1/en not_active Abandoned
-
2019
- 2019-04-17 HR HRP20190722TT patent/HRP20190722T1/hr unknown
- 2019-06-20 CY CY20191100643T patent/CY1121945T1/el unknown
- 2019-08-01 US US16/528,920 patent/US20200291406A1/en not_active Abandoned
-
2020
- 2020-01-28 JP JP2020011342A patent/JP2020078314A/ja not_active Withdrawn
Patent Citations (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2003035083A1 (de) | 2001-10-26 | 2003-05-01 | Ribopharma Ag | Medikament zur behandlung einer fibrotischen erkrankung durch rna interferenz |
JP2010172344A (ja) * | 2003-04-02 | 2010-08-12 | Dharmacon Inc | Rna干渉において使用するための修飾ポリヌクレオチド |
JP2007119396A (ja) | 2005-10-28 | 2007-05-17 | Hosokawa Funtai Gijutsu Kenkyusho:Kk | 核酸化合物封入ナノ粒子を含む経肺投与用医薬製剤 |
JP2009516517A (ja) | 2005-11-21 | 2009-04-23 | ジヨンソン・アンド・ジヨンソン・リサーチ・ピーテイワイ・リミテツド | 多標的干渉rnaならびにそれらの使用および設計方法 |
WO2007079224A2 (en) | 2005-12-30 | 2007-07-12 | Wei-Wu He | Sirna compositions promoting scar-free wound healing of skin and methods for wound treatment |
WO2007109097A2 (en) | 2006-03-16 | 2007-09-27 | Alnylam Pharmaceuticals, Inc. | RNAi MODULATION OF TGF-BETA AND THERAPEUTIC USES THEREOF |
WO2008109548A2 (en) | 2007-03-02 | 2008-09-12 | Mdrna, Inc. | Nucleic acid compounds for inhibiting tgfb gene expression and uses thereof |
JP2010528985A (ja) * | 2007-05-04 | 2010-08-26 | エムディーアールエヌエー,インコーポレイテッド | アミノ酸脂質およびその使用 |
WO2008140126A1 (ja) | 2007-05-09 | 2008-11-20 | Riken | 1本鎖環状rnaおよびその製造方法 |
WO2009061417A1 (en) | 2007-11-06 | 2009-05-14 | Sirnaomics, Inc. | Multi-targeted rnai therapeutics for scarless wound healing of skin |
WO2009061003A2 (en) | 2007-11-08 | 2009-05-14 | Otsuka Pharmaceutical Co., Ltd. | Nucleic acid complex and nucleic acid delivery composition |
WO2009123185A1 (ja) | 2008-03-31 | 2009-10-08 | 独立行政法人産業技術総合研究所 | Rna干渉効果が高い2本鎖脂質修飾rna |
Non-Patent Citations (16)
Title |
---|
CANCER GENOMICS PROTEOMICS, vol. 7, 2010, pages 217 |
HWANG M; KIM HJ; NOH HJ; CHANG YC; CHAE YM; KIM KH; JEON JP; LEE TS; OH HK; LEE YS, EXP MOL PATHOL, vol. 81, 2006, pages 48 - 54 |
JUTARO FUKUMOTO; SAIKO SUETSUGU; CHIKA HARADA; TOMONOBU KAWAGUCHI; NAOKI HAMADA; TAKASHIGE MAEYAMA; KAZUYOSHI KUWANO; YOICHI NAKAN, THE JOURNAL OF THE JAPANESE RESPIRATORY SOCIETY, vol. 46, 2008, pages 185 |
LAI, TC. ET AL.: "Small interfering RNAs (siRNAs) targeting TGF-betal mRNA suppress asbestos-induced expression of TGF-betal and CTGF in fibroblasts.", J. ENVIRON. PATHOL. TOXICOL. ONCOL., vol. 28, no. 2, 2009, pages 109 - 119 * |
LIPMAN-PEARSON, SCIENCE, vol. 227, 1985, pages 1435 |
LIU XJ; RUAN CM; GONG XF; LI XZ; WANG HL; WANG MW; YIN JQ, BIOTECHNOL LETT, 2005, pages 1609 - 1615 |
MOL CELL BIOCHEM, vol. 355, 2011, pages 309 - 314 |
MOORE, LD. ET AL.: "Silencing of transforming growth factor-§I in situ by RNA interference for breast cancer: implications for proliferation and migration in vitro and metastasis in vivo.", CLIN. CANCER RES., vol. 14, no. 15, 2008, pages 4961 - 4970 * |
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual second edition", 1989, COLD SPRING HARBOR LABORATORY PRESS |
See also references of EP2628798A4 |
TAKABATAKE Y; ISAKA Y; MIZUI M; KAWACHI H; SHIMIZU F; ITO T; HORI M; IMAI E, GENE THER, vol. 12, 2005, pages 965 - 973 |
THE ONCOLOGIST, vol. 15, 2010, pages 350 - 359 |
UI-TEI, K. ET AL.: "Functional dissection of siRNA sequence by systematic DNA substitution: modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect.", NUCLEIC ACIDS RES., vol. 36, no. 7, 2008, pages 2136 - 2151, XP002598044, DOI: doi:10.1093/nar/gkn042 * |
WANG Z; GAO Z; SHI Y; SUN Y; LIN Z; JIANG H; HOU T; WANG Q; YUAN X; ZHU X, J PLAST RECONSTR AESTHET SURG, vol. 60, 2007, pages 1193 - 1199 |
XU W; WANG LW; SHI JZ; GONG ZJ, HEPATOBILIARY PANCREAT DIS INT, vol. 8, 2009, pages 300 - 308 |
YU, W. ET AL.: "Inhibitive effect of specific stealth siRNAs on TGF-pl expression of mouse lung fibroblasts.", DI-SAN JUNYI DAXUE XUEBAO BIANJIBU, vol. 29, no. 21, 2007, pages 2038 - 2040 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2017000155A (ja) * | 2010-10-14 | 2017-01-05 | 国立大学法人三重大学 | 線維症予防又は治療剤 |
US10125366B2 (en) | 2010-10-14 | 2018-11-13 | Mie University | Preventive or therapeutic agent for fibrosis |
WO2015093495A1 (ja) * | 2013-12-16 | 2015-06-25 | 株式会社ボナック | TGF-β1遺伝子発現制御のための一本鎖核酸分子 |
US10337009B2 (en) | 2014-12-15 | 2019-07-02 | Bonac Corporation | Single-stranded nucleic acid molecule for inhibiting TGF-β1 expression |
WO2017043490A1 (ja) * | 2015-09-07 | 2017-03-16 | 協和発酵バイオ株式会社 | 自然免疫誘導効果が増強した二重鎖リボ核酸 |
Also Published As
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6865169B2 (ja) | P21遺伝子調節のためのrna干渉剤 | |
ES2969371T3 (es) | Interferencia por ARN para el tratamiento de trastornos de ganancia de función | |
JP2018088923A (ja) | 線維症予防又は治療剤 | |
JP2013143917A (ja) | 線維症予防又は治療剤 | |
JP6311148B2 (ja) | 線維症予防又は治療剤 | |
AU2015261583C1 (en) | Preventative or therapeutic agent for fibrosis | |
CN114144526B (zh) | Eph2a适配体及其用途 | |
TW201718854A (zh) | 供p21基因調控之RNA干擾劑 | |
BR112016000163B1 (pt) | Estrutura de oligo rna de dupla hélice, nanopartículas, composição farmacêutica e formulação liofilizada |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
WWE | Wipo information: entry into national phase |
Ref document number: 201180044862.9 Country of ref document: CN |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 11832611 Country of ref document: EP Kind code of ref document: A1 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2012538721 Country of ref document: JP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2011832611 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 12013500361 Country of ref document: PH |
|
WWE | Wipo information: entry into national phase |
Ref document number: 13824080 Country of ref document: US |
|
ENP | Entry into the national phase |
Ref document number: 2011314653 Country of ref document: AU Date of ref document: 20111014 Kind code of ref document: A Ref document number: 2813163 Country of ref document: CA Ref document number: 20137007962 Country of ref document: KR Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: MX/A/2013/003698 Country of ref document: MX |
|
WWE | Wipo information: entry into national phase |
Ref document number: 13094380 Country of ref document: CO |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
WWE | Wipo information: entry into national phase |
Ref document number: A201305964 Country of ref document: UA |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2013121802 Country of ref document: RU |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01A Ref document number: 112013006541 Country of ref document: BR |
|
WWE | Wipo information: entry into national phase |
Ref document number: 245995 Country of ref document: IL |
|
WWE | Wipo information: entry into national phase |
Ref document number: IDP00201605429 Country of ref document: ID |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01E Ref document number: 112013006541 Country of ref document: BR |
|
ENP | Entry into the national phase |
Ref document number: 112013006541 Country of ref document: BR Kind code of ref document: A2 Effective date: 20130322 |