WO2008018517A1 - Agent thérapeutique et/ou préventif pour une maladie s'accompagnant d'une croissance cellulaire exagérée, et polynucléotide utile en tant que principe actif - Google Patents

Agent thérapeutique et/ou préventif pour une maladie s'accompagnant d'une croissance cellulaire exagérée, et polynucléotide utile en tant que principe actif Download PDF

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WO2008018517A1
WO2008018517A1 PCT/JP2007/065553 JP2007065553W WO2008018517A1 WO 2008018517 A1 WO2008018517 A1 WO 2008018517A1 JP 2007065553 W JP2007065553 W JP 2007065553W WO 2008018517 A1 WO2008018517 A1 WO 2008018517A1
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seq
base sequence
sequence represented
tak1
sense strand
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PCT/JP2007/065553
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English (en)
Japanese (ja)
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Koichi Seto
Yuji Matsuda
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Zeria Pharmaceutical Co., Ltd.
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Publication of WO2008018517A1 publication Critical patent/WO2008018517A1/fr

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C12N15/1137—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 enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
    • C12Y207/11025—Mitogen-activated protein kinase kinase kinase (2.7.11.25), i.e. MAPKKK or MAP3K
    • 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.

Definitions

  • the present invention provides a therapeutic and / or prophylactic agent for diseases associated with cell hyperproliferation, and a polynucleotide useful as an effective component thereof.
  • TGF- ⁇ -activated kinase 1 is a stress response kinase that is known to be activated by inflammatory site forces such as tumor necrosis factor-a (TNF—a). Signals from each site force-in receptor are transmitted to TAK1-binding protein 2 (TAB2) and TAB3 via TNF-a receptor-associating factor 2 (TRAF2) or TRAF6. As a result, TAB2 / 3 is ubiquitinated (Ub) by these TRAF molecules, and the TAK1-activated bow I metal is bow I.
  • TNF—a tumor necrosis factor-a
  • TAB2 / 3 is ubiquitinated (Ub) by these TRAF molecules, and the TAK1-activated bow I metal is bow I.
  • Activated TAK1 is known as MAPKKK, which activates transcription factor AP-1 through JNK and p38, which are mitogen-activated protein kinases (MAPK). It is also known to be an important protein kinase that activates the transcription factor NF- ⁇ through I ⁇ B kinase (IKK) (Non-patent Document 1).
  • TAK1 Inhibition of TAK1 suppresses activation of NF- ⁇ and suppresses the occurrence of ear edema (Non-patent Document 2).
  • TAK1 is induced by the dominant negative mutant strength STGF- ⁇ . It is also known to suppress the production of fibrotic mediator molecules such as collagen (Non-Patent Document 3).
  • fibrotic mediator molecules such as collagen
  • TAK1 is a kinase that constitutes the aforementioned signal transduction system related to inflammation and tissue fibrosis, and has been considered as a drug target for diseases accompanied by inflammation, particularly rheumatism and inflammatory bowel disease.
  • Non-patent Documents 3 to 5 It is known that signals via TAK1 are involved in apoptosis induction (Non-patent Documents 3 to 5).
  • Non-patent Document 6 there is a seemingly contradictory report (Non-patent Document 6) that apoptosis is induced in cells derived from knockout mice of TAK1, and it is not always clear how TAK 1 is involved in apoptosis induction. is not.
  • Generally cancerous The vesicles are thought to be resistant to apoptosis.
  • it may affect normal cells and cause strong side effects, which is undesirable.
  • Zaralenones are known as TAK1 inhibitors (Patent Document 1).
  • zearalenones have various actions such as the inhibitory action of other kinases (eg, tyrosine kinase, MEK1 kinase), and therefore TAK1 is specifically It cannot be inhibited.
  • RNAi RNA interference
  • Non-patent Document 7 RNA interference method has been developed as one of the techniques for specifically suppressing the expression of a specific gene.
  • RNAi was originally discovered in 1998 as a life phenomenon in which the expression of homologous RNA is strongly suppressed by the introduction of double-stranded RNA.
  • Attempts have been made to suppress the expression of specific genes targeted by the introduction of (dsRNA).
  • dsRNA double-stranded RNA
  • siRNA This is called small in terfering RNA (siRNA)).
  • This siRNA dissociates into single-stranded RNA (ssRNA), and the ssRNA specifically recognizes and cleaves the target mRNA as a complex RISC (RNA-induced silencing complex), thereby suppressing the expression of the mRNA. Is done.
  • RISC RNA-induced silencing complex
  • siRNA is introduced instead of dsRNA (Non-patent Document 8).
  • a method for treating a disease by suppressing the expression of a specific gene using siRNA has been studied! (Non-patent Document 9).
  • TAK1-specific inhibitors such as siRNA can be used for the treatment and / or prevention of diseases associated with cell hyperproliferation, and in particular, TAK1-specific inhibitors have a mechanism different from the induction of apoptosis. It has been reported that it can be used for the treatment and / or prevention of diseases associated with cell hyperproliferation! /.
  • Non-Patent Document 1 Sakurai et al., The Journal of Biological Chemistry 274 (15): 10641-10 648 (1999)
  • Non-Patent Document 2 Tsuji et al., The Journal of Biological Chemistry 278 (20): 18485-1849 0 (2003)
  • Non-Patent Document 3 Ono et al., Biochemical and Biophysical Research Communications 30 7: 332-337 (2003)
  • Non-Patent Document 4 Kimura et al., The Journal of Biological Chemistry 275 (23): 17647-17 652 (2000)
  • Non-Patent Document 5 Edlund et al., Molecular Biology of the Cell 14: 529-544 (2003)
  • Non-Patent Document 6 Sayama et al., The Journal of Biological Chemistry (manuscript M601065200 as the Internet on June 5, 2006) (Published on)
  • Non-Patent Document 7 Hammond, FEBS Letters 579: 5822-5829 (2005)
  • Non-Patent Document 8 Hutvagner, FEBS Letters 579: 5850-5857 (2005)
  • Non-Patent Document 9 Manfredsson et al., Gene Therapy 13: 517-524 (2006)
  • Patent Document 1 Pamphlet of International Publication No. WO02 / 48135
  • the present invention provides a therapeutic and / or prophylactic agent for diseases associated with cell hyperproliferation, and a polynucleotide useful as an effective component thereof.
  • siRNAs have the potential to inhibit cell proliferation, and therefore that TAK1-specific inhibitors may be effective in the treatment and / or prevention of diseases involving cell overgrowth. I found it.
  • the present inventors further elucidated that cell growth-related genes are involved as the mechanism of action, and accompanied by cell overgrowth by a mechanism different from the induction of apoptosis that may cause side effects. It has been found that a disease can be treated and / or prevented, and thus can be useful for the treatment and / or prevention of the disease in a subject in which induction of apoptosis is not desired. Based on the above findings, the present inventors have completed the present invention.
  • the present invention is as follows:
  • a therapeutic and / or prophylactic agent for a disease associated with cell hyperproliferation comprising a TAK1-specific inhibitor.
  • a TAK1-specific inhibitor comprising a TAK1-specific inhibitor.
  • TAK1-specific inhibitor is an RNAi-inducible nucleic acid, an antisense nucleic acid, a dominant negative mutant, or an expression vector thereof.
  • RNAi-inducing nucleic acid is siRNA
  • a sense strand comprising the base sequence represented by any one of SEQ ID NOs: 8 to 13 and an antisense strand comprising the complementary sequence thereof, the sense strand and / or the 5 ′ end of the antisense strand and / Or a double-stranded RNA that may have an overhang at the 3 ′ end and has an activity of suppressing the expression of TAK1; or
  • siRNA is any of the following (a) to (f):
  • RNA a double-stranded RNA composed of a sense strand consisting of the base sequence represented by SEQ ID NO: 3 and an antisense strand consisting of a complementary sequence of the base sequence represented by SEQ ID NO: 10;
  • RNA a double-stranded RNA composed of a sense strand consisting of the base sequence represented by SEQ ID NO: 4 and an antisense strand consisting of a complementary sequence of the base sequence represented by SEQ ID NO: 11;
  • RNA comprising a sense strand consisting of the base sequence represented by SEQ ID NO: 5 and an antisense strand consisting of a complementary sequence of the base sequence represented by SEQ ID NO: 12;
  • a suppressant of progression from inflammatory diseases to cancer comprising a TAK1-specific inhibitor.
  • siRNA which is any of the following (a) or (b)! /:
  • a sense strand comprising the base sequence represented by any one of SEQ ID NOs: 8 to 13 and an antisense strand comprising the complementary sequence thereof, the sense strand and / or the 5 ′ end of the antisense strand and / Or a double-stranded RNA that may have an overhang at the 3 ′ end and has an activity of suppressing the expression of TAK1; or
  • siRNA which is any of the following (a) to (f):
  • RNA a double-stranded RNA composed of a sense strand consisting of the base sequence represented by SEQ ID NO: 1 and an antisense strand consisting of a complementary sequence of the base sequence represented by SEQ ID NO: 8;
  • a double-stranded RNA composed of a sense strand consisting of the base sequence represented by SEQ ID NO: 2 and an antisense strand consisting of a complementary sequence of the base sequence represented by SEQ ID NO: 9;
  • RNA a double-stranded RNA composed of a sense strand consisting of the base sequence represented by SEQ ID NO: 3 and an antisense strand consisting of a complementary sequence of the base sequence represented by SEQ ID NO: 10;
  • RNA a double-stranded RNA composed of a sense strand consisting of the base sequence represented by SEQ ID NO: 4 and an antisense strand consisting of a complementary sequence of the base sequence represented by SEQ ID NO: 11;
  • RNA comprising a sense strand consisting of the base sequence represented by SEQ ID NO: 5 and an antisense strand consisting of a complementary sequence of the base sequence represented by SEQ ID NO: 12;
  • Double-stranded RNA composed of a sense strand consisting of the base sequence represented by SEQ ID NO: 6 and an antisense strand consisting of a complementary sequence of the base sequence represented by SEQ ID NO: 14
  • a siRNA expression vector comprising a polynucleotide encoding the siRNA of [17] or [18].
  • a medicament comprising the siRNA of [17] or [18]! /, Comprising the siRNA expression vector of [19] above.
  • FIG. 1 is a view showing suppression of TAK1 mRNA expression in HeLa cells by siRNA having the nucleotide sequence represented by SEQ ID NO: 1 as a sense strand.
  • non-silencing [FIG. 2] A view showing suppression of expression of TAK1 mRNA in HeLa cells by siRNA having the nucleotide sequence represented by SEQ ID NO: 2 as a sense strand.
  • non-silencing [FIG. 3] A diagram showing suppression of expression of TAK1 mRNA in HeLa cells by siRNA having the nucleotide sequence represented by SEQ ID NO: 3 as a sense strand. non-silencing [FIG.
  • FIG. 4 A diagram showing suppression of expression of TAK1 mRNA in HeLa cells by siRNA having the nucleotide sequence represented by SEQ ID NO: 4 as a sense strand.
  • FIG. 5 A view showing suppression of expression of TAK1 mRNA in HeLa cells by siRNA having the nucleotide sequence represented by SEQ ID NO: 5 as a sense strand.
  • non-silencing FIG. 6
  • siRNA having the nucleotide sequence represented by SEQ ID NO: 1 as the sense strand It is a figure which shows the expression suppression of TAK1 mRNA in HT-29 cell.
  • FIG. 7 is a view showing inhibition of growth of HT-29 cells by siRNA having the nucleotide sequence represented by SEQ ID NO: 1 as a sense strand.
  • FIG. 8 shows the induction of apoptosis in HT-29 cells by Asiatic acid.
  • FIG. 9 is a diagram showing non-induction of apoptosis in HT-29 cells by siRNA having the nucleotide sequence represented by SEQ ID NO: 1 as a sense strand.
  • FIG. 10 is a view showing suppression of TAK1 mRNA expression in C2C12 cells by siRNA having the nucleotide sequence represented by SEQ ID NO: 7 as a sense strand.
  • siRNA having the nucleotide sequence represented by SEQ ID NO: 7 as a sense strand.
  • the present invention provides a therapeutic and / or prophylactic agent for diseases associated with cell hyperproliferation, comprising a TAK1-specific inhibitor.
  • the TAK1-specific inhibitor is not particularly limited as long as it is a substance that can specifically suppress the expression or function of TAK1, and examples of such an inhibitor include RNAi-inducible nucleic acid, antisense nucleic acid. , Dominant negative mutants.
  • the present invention also provides such inhibitors.
  • the RNAi-inducible nucleic acid for TAK1 refers to a polynucleotide that can induce RNA interference when introduced into a cell, and is preferably a RNA or RNA-DNA chimeric molecule.
  • the RNAi effect is a phenomenon in which RNA having a double-stranded structure containing the same nucleotide sequence as mRNA or a partial sequence thereof suppresses the expression of the mRNA.
  • the double-stranded structure may be composed of different strands, or it may be a double strand given by a single RNA stem loop structure! /.
  • RNAi-inducible nucleic acids include siRNA and miRNA.
  • the RNAi-inducing nucleic acid may be siRNA.
  • SiRNA against TAK1 is T It can target any part of AK1.
  • the siRNA molecule against TAK1 is not particularly limited as long as RNAi can be induced, but may be, for example, 2;! -27 bases long, preferably 2;!-25 bases long.
  • the siRNA against TAK1 can be a duplex comprising a sense strand and an antisense strand.
  • the siRNA against TAK1 may have an overhang at the 5 ′ end and / or the 3 ′ end of one or both of the sense strand and the antisense strand.
  • the overhang can be formed by the addition of 1 to several (eg, 1, 2 or 3) bases at the 5, and / or 3 ′ end of the sense and / or antisense strand.
  • the siRNA against TAK1 is a double-stranded RNA composed of a sense strand containing a base sequence represented by any of SEQ ID NOs: 8 to 13 and an antisense strand containing a complementary sequence thereof It can be.
  • the double-stranded RNA may have the above-described overhang at the 5 ′ end and / or the 3 ′ end of the sense strand and / or the antisense strand.
  • siRNA can be any of the following (a) to (f):
  • Double-stranded RNA which may have an overhang;
  • (d) It is composed of a sense strand containing the base sequence represented by SEQ ID NO: 11 and an antisense strand containing the complementary sequence, and is located at the 5 ′ end and / or 3 ′ end of the sense strand and / or antisense strand.
  • Double-stranded RNA which may have an overhang;
  • the overhang can be similar to that described above.
  • siRNA in the above (f) examples include the following (fl) and (f2):
  • (f2) It is composed of a sense strand containing the base sequence represented by SEQ ID NO: 15 and an antisense strand containing its complementary sequence, and is located at the 5 ′ end and / or 3 ′ end of the sense strand and / or antisense strand. Has an overhang! /, Even! /, Double stranded RNA.
  • siRNA having an overhang examples include the following (a ') to (f')!
  • RNA a double-stranded RNA composed of a sense strand consisting of the base sequence represented by SEQ ID NO: 1 and an antisense strand consisting of a complementary sequence of the base sequence represented by SEQ ID NO: 8;
  • (b ′) a double-stranded RNA composed of a sense strand consisting of the base sequence represented by SEQ ID NO: 2 and an antisense strand consisting of a complementary sequence of the base sequence represented by SEQ ID NO: 9;
  • Double-stranded RNA composed of a sense strand consisting of the base sequence represented by SEQ ID NO: 3 and an antisense strand consisting of a complementary sequence of the base sequence represented by SEQ ID NO: 10;
  • (d ′) a double-stranded RNA comprising a sense strand consisting of the base sequence represented by SEQ ID NO: 4 and an antisense strand consisting of a complementary sequence of the base sequence represented by SEQ ID NO: 11;
  • RNA a double-stranded RNA comprising a sense strand consisting of the base sequence represented by SEQ ID NO: 5 and an antisense strand consisting of a complementary sequence of the base sequence represented by SEQ ID NO: 12;
  • (f ′) (fl ′) a double-stranded RNA composed of a sense strand consisting of the base sequence represented by SEQ ID NO: 6 and an antisense strand consisting of a complementary sequence of the base sequence represented by SEQ ID NO: 14;
  • (f 2 ′) a double-stranded RNA comprising a sense strand consisting of the base sequence represented by SEQ ID NO: 7 and an antisense strand consisting of a complementary sequence of the base sequence represented by SEQ ID NO: 15.
  • 1 to several siRNAs are present at the 5 'end and / or 3' end of the base sequence represented by any of SEQ ID NOs: 8 to 13;
  • a double-stranded RNA composed of a sense strand containing a base sequence to which 1 to 3, more preferably 1 or 2 bases are added and / or deleted, and an antisense strand containing its complementary sequence It can be.
  • the double-stranded RNA may have the above-described overhang at the 5 ′ end and / or the 3 ′ end of the sense strand and / or the antisense strand.
  • Addition and / or deletion of one to several bases at the 5 ′ end and / or 3 ′ end of the base sequence represented by any one of SEQ ID NOs: 8 to 13 is referred to as retaining TAK1 expression suppression activity. From the viewpoint, it can be achieved so as to ensure the identity of the sense strand encoding the TAK1 gene and the partial base sequence of the antisense strand. For example, since the nucleotide sequence of the TAK1 gene is published by GenBank Accession No. NM-003188, 1 to several bases are added and / or so as to ensure the above identity with reference to such information. Or it can be deleted.
  • the antisense nucleic acid against TAK1 is composed of a nucleotide sequence capable of hybridizing with the transcription product under the physiological conditions of cells expressing the transcription product (mRNA or initial transcription product) of TAK1, and is in a hybridized state.
  • the type of the antisense nucleic acid may be DNA, RNA, or a DNA / RNA chimera.
  • the length of the antisense nucleic acid is not particularly limited as long as it can specifically hybridize with the TA K1 transcript. It is about 15 bases long and is complementary to the entire transcript sequence.
  • the sequence may contain From the viewpoint of ease of synthesis, antigenicity problems, etc., for example, oligonucleotides comprising about 15 bases or more, preferably about 15 to about 30 bases, more preferably about 18 bases to about 30 bases are exemplified.
  • antisense nucleic acids hybridize with TAK1 transcripts to inhibit translation and bind to double-stranded DNA to form a triplex. It may be capable of inhibiting transcription to mRNA.
  • the antisense nucleic acid may preferably include a nucleotide sequence represented by SEQ ID NOs:! To 15! /, A deviation (U is T when the antisense nucleic acid is DNA).
  • the dominant negative mutant of TAK1 protein refers to one whose function has been reduced by introducing a mutation into TAK1 protein.
  • the dominant negative mutant can indirectly inhibit its function by competing with natural TAK1.
  • Dominant negative mutants can be generated by introducing mutations into the nucleic acid encoding TAK1. Examples of the mutation include an amino acid mutation (eg, deletion, substitution or addition of one or more amino acids) that causes a decrease in the function of the functional site.
  • the dominant negative mutant for example, those already reported (eg, see Biochemical and Biophysical Research Communication 307: 332-337 (2003)) can be used.
  • the TAK1-specific inhibitor can also be provided as an expression vector.
  • the present invention also provides such an expression vector.
  • the expression vector of the present invention may comprise a polynucleotide encoding a TAK1-specific inhibitor and a promoter operably linked to the polynucleotide.
  • the promoter that can be included in the expression vector of the present invention is not particularly limited as long as it allows the expression of the factor under its control, and may be appropriately selected depending on the type of factor.
  • the polIII promoter eg, tRNA promoter, U6 promoter, HI promoter
  • mammalian promoter eg, CMV promoter, CAG promoter, SV40 promoter
  • the expression vector of the present invention further includes a selection marker gene (a gene that imparts resistance to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, phosphinothricin, a gene that complements an auxotrophic mutation, etc.) ) May further be included.
  • a selection marker gene a gene that imparts resistance to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, phosphinothricin, a gene that complements an auxotrophic mutation, etc.
  • the backbone of the expression vector of the present invention is not particularly limited as long as it can produce a TAK1-specific inhibitor in mammalian cells such as humans.
  • examples thereof include plasmid vectors and virus vectors. It is done.
  • vectors suitable for administration to mammals include adenovirus and retrovirus.
  • Virus vectors such as virus, adeno-associated virus, herpes virus, vaccinia virus, box virus, polio virus, Sindbis virus, Sendai virus, etc.
  • the agent of the present invention may contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carriers include sucrose, starch, mannitol, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate and other excipients, cellulose, methyl cellulose, Binders such as hydroxypropylcellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, starch, starch, carboxymethylcellulose, hydroxypropyl starch, sodium glycol glycol starch, sodium hydrogen carbonate, calcium phosphate, citrate Disintegrants such as calcium, lubricants such as magnesium stearate, air mouth gill, talc, sodium lauryl sulfate, fragrances such as citrate, menthol, glycyllysine ammonium salt, glycine, orange powder, benzoate Preservatives such as sodium, sodium hydrogen sulfite, methyl paraben, propyl para
  • a formulation suitable for oral administration is a solution obtained by dissolving an effective amount of a TAK1-specific inhibitor in a diluent such as water or physiological saline, and an effective amount of the TAK1-specific inhibitor as a solid or granule.
  • a diluent such as water or physiological saline
  • an effective amount of the TAK1-specific inhibitor as a solid or granule.
  • Suitable formulations for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions. This may contain antioxidants, buffers, antibacterial agents, tonicity agents and the like. Aqueous and non-aqueous sterile suspensions are also included, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives and the like.
  • the preparation can be sealed in a unit dose or multiple doses like ampoules and vials.
  • the active ingredient and a pharmaceutically acceptable carrier can be lyophilized and stored in a state that may be dissolved or suspended in a suitable sterile vehicle immediately before use.
  • the dosage of the agent of the present invention varies depending on the type of active ingredient 'activity, the severity of the disease, the animal species to be administered, the drug acceptability of the administration target, body weight, age, etc.
  • the amount of active ingredient per day can be from about 0.001 to about 1000 mg / kg.
  • the agent of the present invention may further contain a substance that promotes the introduction of a TAK1-specific inhibitor into cells.
  • substances include high molecular weight polymers such as polycationic reagents (Lipofectamine, Lipofectamine 2000, HiPerFect, FuGENE, DharmaFECT, TransIT), polyethylene glycol, polylysine, and collagen.
  • the agent of the present invention may be useful for the treatment and / or prevention of diseases associated with cell hyperproliferation.
  • the disease associated with cell overgrowth is not particularly limited as long as it proliferates beyond the range of normal cell growth and becomes aggravated.
  • malignant tumors i.e. cancer
  • benign tumors etc.
  • inflammatory diseases that are partly or that are concerned about progress to cancer.
  • malignant tumors include cancer in any tissue (eg, digestive system, respiratory system, genital system, skin), but digestive cancer (eg, colon cancer, small intestine cancer, rectal cancer).
  • Duodenal cancer, gastric cancer, esophageal cancer, liver cancer are preferred.
  • Benign tumors include those that originate from epithelial cells (eg, papilloma, adenoma, polyp, cystadenoma), those that originate from non-epithelial cells (eg, fibroma, myxoma, lipoma, chondroma, Osteoma, rhabdomyosarcoma, leiomyoma, hemangioma), and nematodes and polyps are preferred.
  • Examples of inflammatory diseases that are concerned about progress to cancer include inflammatory bowel diseases (eg, ulcerative colitis, Crohn's disease), rheumatism, fibrosis, hepatitis, and the like. preferable.
  • the agent of the present invention may also be useful for suppressing the progression from inflammatory diseases to cancer.
  • Inflammatory diseases and cancer can be the same as described above.
  • Suppression of progression to cancer can be, for example, prevention of canceration, or suppression or delay of progression of disease state.
  • the agent of the present invention may also have a feature that by inhibiting TAK1 specifically, cell proliferation can be suppressed without inducing apoptosis that may cause side effects. Therefore, the agent of the present invention is required to treat and / or prevent a disease associated with cell hyperproliferation, and induction of apoptosis is not desirable! / It is also preferable that the subject is the subject of administration. Les.
  • Apoto Subjects who are not desired to induce cis include, for example, patients receiving treatment with immunosuppressive drugs (eg, cyclosphamide, azathioprine, cyclosporine), diseases that are not desired to induce apoptosis (eg, viruses, bacteria, etc.) Patients suffering from infections caused by various pathogens) and patients who have undergone organ transplantation.
  • immunosuppressive drugs eg, cyclosphamide, azathioprine, cyclosporine
  • diseases that are not desired to induce apoptosis eg, viruses, bacteria, etc.
  • the present invention also provides a cell in which TAK1 expression is specifically suppressed or a tissue containing the same.
  • the cell or tissue of the present invention may be a cell or tissue with suppressed proliferation.
  • the cell or tissue of the present invention may be a cell or tissue derived from the aforementioned tumor and / or tissue (eg, digestive system).
  • the cell or tissue of the present invention is a cell in which TAK1 expression is transiently suppressed, or a cell in which TAK1 expression is permanently suppressed (eg, a modified cell of a genome containing TAK1), or such It can be a tissue containing cells.
  • the cell or tissue of the present invention may be a primary cultured cell or cell line, or a tissue containing them (eg, a tissue into which those cells have been transferred).
  • the cell or tissue of the present invention can be prepared, for example, by treating a cell or tissue accompanied by hyperproliferation with the above-mentioned TAK1-specific inhibitor.
  • the cells or tissues of the present invention may also be isolated from TAK1 knockout animals (see, eg, Sayama et al., The Journal of Biological Chemistry (published on the Internet on June 5, 2006 as manuscript M601065200)). S can.
  • a TAK1 knockout animal may be one that may involve cell overgrowth.
  • a TAK1 knockout animal may suffer from a disease involving cell overgrowth.
  • Such disease of TAK1 knockout animals can be carried out by a method known per se such as treatment of drugs such as carcinogens.
  • the present invention also provides such a TAK1 knockout animal that may involve cell overgrowth.
  • the cell or tissue of the present invention can be used, for example, for the development of a therapeutic and / or preventive drug for a disease associated with cell hyperproliferation, in particular, for the disease with reduced side effects, analysis of the cell growth mechanism, cell proliferation It may be useful for searching for regulatory genes.
  • Example 1 Inhibition of TAK1 expression in HeLa cells by RNAi
  • HeLa cells (Human Science Promotion Foundation) were cultured for 1 day in a DMEM medium containing 10% FBS (Invitrogen) at 37 ° C in a 5% CO environment at 50% confluence.
  • Use Lipofectine 2000 (Invitrogen) as the siRNA transfer reagent for the cultured cells, and the ratio of the transfer reagent to siRNA (10 M) at 1.5: 1 (V / V) siRNA against TAK1 (final 10 nM) was further cultured.
  • Cells to which only the introduction reagent was added were used as control containers.
  • Cells 72 hours after siRNA introduction were collected using a lysate contained in an RNA extraction kit / RNeasy mini kit (QIAG EN), and total RNA was extracted.
  • the nucleotide sequence of the siRNA used was as follows.
  • (AG) indicates 2 bases of overhang.
  • the sequences from which the two overhanging bases in SEQ ID NOs: 1 to 5 are deleted are shown as SEQ ID NOs: 8 to 12 respectively.
  • RNA was extracted using RNeasy mini kit (QIAGEN) according to the method attached to the kit. The obtained total RNA solution was measured for concentration and used as a cage for cDNA preparation.
  • cDNA was prepared by using 10 g of vertical RNA in a 50 1 reaction system, denatured at 72 ° C for 5 minutes, and then reacted at 42 ° C for 60 minutes using reverse transcriptase (M-MLV). It was. Using the obtained cDNA, Real time RT-PCR was performed to quantify the amount of TAK1 mRNA.
  • Real-time RT-PCR is performed with PRISM7900 by mixing vertical cDNA (4.0 ⁇ 1), TaqMan probe (1 ⁇ 5 ⁇ 1), Master Mix (l 5 1), and purified water (9.511 1). Comparative quantitative analysis was performed by the ⁇ Ct method.
  • Example 2 Inhibition of TAK1 expression in HT-29 cells by RNAi
  • HT-29 cells make human colon cancer cell line HT-29 cells (Dainippon Pharmaceutical Co., Ltd.) 50% confluent at 37 ° C, 5% CO using DMEM medium (Invitrogen) containing 10% FBS for 1 day. Intercultured. Use DharmaFECT4 (Dharmacon) as a siRNA transfer reagent in the cultured cells, and a ratio of transfer reagent to siRNA dOM) of 3: 1 (V / V) to siRNA against TAK1 of SEQ ID NO: 1 (final 10 nM) was further cultured. Cells 72 hours after siRNA introduction were collected using a lysate contained in an RNA extraction kit / RNeasy mini kit (QIAGEN), and total RNA was extracted.
  • DharmaFECT4 Dharmacon
  • RNA was extracted using RNeasy mini kit (QIAGEN) according to the method attached to the kit. The obtained total RNA solution was measured for concentration and used as a cage for cDNA preparation.
  • cDNA was prepared by using 10 g of vertical RNA in a 50 1 reaction system, denatured at 72 ° C for 5 minutes, and then reacted at 42 ° C for 60 minutes using reverse transcriptase (M-MLV). It was. Using the obtained cDNA, Real time RT-PCR was performed to quantify the amount of TAK1 mRNA.
  • Real-time RT-PCR is performed with PRISM7900 by mixing vertical cDNA (4.0 ⁇ 1), TaqMan probe (1 ⁇ 5 ⁇ 1), Master Mix (l 5 1), and purified water (9.511 1). Comparative quantitative analysis was performed by the ⁇ Ct method.
  • Example 3 Change in gene expression level in HT-29 cells knocked down with TAK1 using siRNA (DNA microarray)
  • HT-29 cells (Dainippon Pharmaceutical Co., Ltd.) were cultured in a DMEM medium (Invitrogen) containing 10% FBS for 1 day at 37 ° C in a 5% CO environment to become 50% confluent.
  • DMEM medium Invitrogen
  • DharmaFECT4 Dharmacon
  • the ratio of introduction reagent to siRNA (10 a M) is 3: 1 (V / V).
  • SiRNA against TAK1 of SEQ ID NO: 1 (final 10 nM) was introduced and further cultured.
  • Cells 72 hours after introduction were collected using a lysate contained in an RNA extraction kit / RNeasy mini kit (QIAGEN), and total RNA was extracted.
  • RNA For extraction of total RNA, use the RNeasy mini kit (QIAGEN) and the one attached to the kit. I went according to the law.
  • RNA was converted into a saddle shape, and a riboprobe was prepared according to the attached method using One-Cycle Target Labeling and Control Reagents (Affymetritas)!
  • the prepared riboprobe was hybridized (45 ° C, 16 hr) in a GeneChip (registered trademark) array (human Genome U133 Plus 2.0 Array), and then the array was washed with GeneChip (registered trademark) Fluidics Station 450 Then, a color reaction was carried out.
  • the obtained array was scanned with a GeneChip (registered trademark) Scanner 3000, and the fluorescence intensity was measured. Statistical comparison analysis was performed using a dedicated Workstation.
  • TAK1 expression was suppressed using siRNA for the gene whose expression level was reported to change in pre-cancer patients and cancer patients (Exp. Mol. Path. (2006) 80: 1-10). Compared to the cases, the expression level of each gene was reversed except for some DNA repair genes (UNG2). In order to maintain the cells in a normal state, it is desirable that the expression level of genes involved in tumorigenesis be suppressed without lowering the expression level of genes related to DNA repair. Since TAK1 suppression satisfies these conditions, It was suggested that siRNA can be used for the prevention and treatment of cell proliferative diseases such as cancer.
  • HT-29 cells (Dainippon Pharmaceutical Co., Ltd.) were cultured in a DMEM medium (Invitrogen) containing 10% FBS for 1 day at 37 ° C in a 5% CO environment to become 50% confluent.
  • DMEM medium Invitrogen
  • DharmaFECT4 Dharmacon
  • the ratio of introduction reagent to siRNA (10 11 M) is 3: 1 (V / V).
  • SiRNA against TAK1 SEQ ID NO: 1
  • Example 6 Detection of apoptosis in HT-29 cells knocked down by TAK1
  • Human colon cancer cells HT-29 (Dainippon Pharmaceutical Co., Ltd.) were cultured in a DMEM medium (Invitrogen) containing 10% FBS for 1 day at 37 ° C in a 5% CO environment to become 50% confluent.
  • DMEM medium Invitrogen
  • Use DharmaFECT4 (Dharmacon) as a siRNA transfer reagent to the cultured cells, and 311 ⁇ 8 (6 1 10 nM) at a ratio of 311 ⁇ 8 (10 ⁇ ) of SEQ ID NO: 1 to 311 ⁇ 8 (10 ⁇ ) ) was introduced and further cultured.
  • Cells 72 hours after siRNA introduction were collected from the culture vessel by trypsin treatment, and the number of cells was counted.
  • Human colon cancer cells HT-29 (Dainippon Pharmaceutical Co., Ltd.) were cultured in a DMEM medium (Invitrogen) containing 10% FBS for 1 day at 37 ° C in a 5% CO environment so as to be 80% confluent.
  • DMEM medium Invitrogen
  • Asiatic acid SIGMA
  • SIGMA Asiatic acid
  • the cells that have been introduced with siRNA and those that have been treated with Asiatic acid 3.0 ⁇ 10 3 were used per ELISA plate.
  • the 96-well plate was washed with the buffer supplied with the kit, a coloring reagent was added, and after 8 minutes, the absorbance at 405 nm (Reference 492 nm) was measured with a SUNRISE plate reader (TECAN).
  • Example 7 1 ⁇ Nyoji 2 (: Suppression of expression of Dingpachi 1 1 in 12 Hoso
  • C2C12 cells (Human Science Promotion Foundation) were cultured in DMEM medium (Invitrogen) containing 5% FBS at 37 ° C in a 5% CO environment so as to be 50% confluent.
  • DMEM medium Invitrogen
  • DharmaFECT4 Dharmacon
  • siRNA final 10 nM
  • mouse TAK1 a ratio of transfer reagent to siRNA (10 M) of 3: 1 (V / V).
  • cells 72 hours after siRNA introduction were collected using a lysate contained in an RNA extraction kit / RNeasy mini kit (QIAGEN), and total RNA was extracted.
  • the nucleotide sequence of the siRNA used was as follows.
  • (AG) indicates 2 bases of overhang.
  • a sequence obtained by deleting two bases of the overhang in SEQ ID NO: 7 is shown as SEQ ID NO: 15.
  • RNA was extracted using RNeasy mini kit (QIAGEN) according to the method attached to the kit. The obtained total RNA solution was measured for concentration and used as a cage for cDNA preparation.
  • cDNA was prepared by using 10 g of vertical RNA in a 50 1 reaction system, denatured at 72 ° C for 5 minutes, and then reacted at 42 ° C for 60 minutes using reverse transcriptase (M-MLV). It was.
  • R eal-time RT-PCR was performed to quantify the amount of TAK1 mRNA.
  • R eal-time RT-PCR is performed with PRISM7900 by mixing vertical cDNA (4.0 ⁇ 1), TaqMan probe (1.5 ⁇ 1), Master Mix (l 5 1), and purified water (9.511 1). Comparative quantitative analysis was performed by the ⁇ Ct method.
  • TAK1 siRNA represented by SEQ ID NO: 7 (FIG. 10).
  • the agent of the present invention may be useful for the treatment and / or prevention of diseases associated with cell hyperproliferation.
  • the agent of the present invention cannot induce apoptosis that may cause side effects, and exhibits a mechanism of action different from the conventional one. Therefore, the agent of the present invention is useful for treatment and / or prevention of the disease in a subject in which induction of apoptosis is not desired. Can be useful.
  • the siRNA or the expression vector of the present invention is useful as a medicament, for example, for the treatment and / or prevention of a disease associated with cell overgrowth, particularly for the treatment and / or prevention of the disease in which apoptosis induction is not desired. obtain.
  • the cell or tissue of the present invention can be useful, for example, for the development of a therapeutic and / or prophylactic agent for a disease associated with cell hyperproliferation, in particular, the disease with reduced side effects.

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Abstract

La présente invention concerne un agent thérapeutique et/ou préventif pour une maladie qui s'accompagne d'une croissance cellulaire exagérée, en particulier un agent thérapeutique et/ou préventif pour ladite maladie ayant des effets secondaires réduits. Plus particulièrement, l'invention concerne un agent thérapeutique et/ou préventif pour une maladie qui s'accompagne d'une croissance cellulaire exagérée, contenant un inhibiteur spécifique de TAK1 ; un ARNsi contre le gène de TAK1 ; un vecteur d'expression d'ARNsi contenant un polynucléotide codant pour l'ARNsi ; un produit pharmaceutique contenant l'ARNsi ou le vecteur d'expression d'ARNsi ; une cellule présentant une capacité proliférative réduite, dans laquelle l'expression de TAK1 a été spécifiquement supprimée, ou un tissu contenant la cellule ; et similaires.
PCT/JP2007/065553 2006-08-09 2007-08-08 Agent thérapeutique et/ou préventif pour une maladie s'accompagnant d'une croissance cellulaire exagérée, et polynucléotide utile en tant que principe actif WO2008018517A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017059177A3 (fr) * 2015-09-30 2017-07-20 Vycellix, Inc. Administration améliorée de gènes dans des cellules tueuses naturelles, des cellules souches hématopoïétiques et des macrophages

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Publication number Priority date Publication date Assignee Title
WO2000023610A1 (fr) * 1998-10-21 2000-04-27 Chugai Seiyaku Kabushiki Kaisha Methode de criblage de composes empechant la transduction de signal de cytokines inflammatoires
JP2000197500A (ja) * 1998-02-06 2000-07-18 Tanabe Seiyaku Co Ltd ＴＡＫ１を標的とするＮＦ―κＢ活性化抑制薬及びその同定方法
JP2004292315A (ja) * 2000-12-14 2004-10-21 Chugai Pharmaceut Co Ltd Ｔａｋ１阻害剤
WO2006023210A2 (fr) * 2004-07-23 2006-03-02 Aveo Pharmaceuticals, Inc. Gene gp132: techniques et compositions de traitement du cancer

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JP2000197500A (ja) * 1998-02-06 2000-07-18 Tanabe Seiyaku Co Ltd ＴＡＫ１を標的とするＮＦ―κＢ活性化抑制薬及びその同定方法
WO2000023610A1 (fr) * 1998-10-21 2000-04-27 Chugai Seiyaku Kabushiki Kaisha Methode de criblage de composes empechant la transduction de signal de cytokines inflammatoires
JP2004292315A (ja) * 2000-12-14 2004-10-21 Chugai Pharmaceut Co Ltd Ｔａｋ１阻害剤
WO2006023210A2 (fr) * 2004-07-23 2006-03-02 Aveo Pharmaceuticals, Inc. Gene gp132: techniques et compositions de traitement du cancer

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CHOO M.K. ET AL.: "Blockade of transforming growth factor-beta-activated kinase 1 activity enhances TRAIL-induced apoptosis through activation of a caspase cascade", MOL. CANCER THER., vol. 5, no. 12, 2006, pages 2970 - 2976, XP003020773 *
CHOO M.K. ET AL.: "TAK-1mediated stress signaling pathways are essential for TNF-alpha-promoted pulmonary metastasis of murine colon cancer cells", INT. J. CANCER, vol. 118, no. 11, June 2006 (2006-06-01), pages 2758 - 2764, XP002465093 *
ONO K. ET AL.: "A dominant negative TAK1 inhibits cellular fibrotic responses induced by TGF-beta", BIOCHEM. BIOPHYS. RES., vol. 307, no. 2, 2003, pages 332 - 337, XP004435306 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017059177A3 (fr) * 2015-09-30 2017-07-20 Vycellix, Inc. Administration améliorée de gènes dans des cellules tueuses naturelles, des cellules souches hématopoïétiques et des macrophages

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