WO2015016282A1 - Composition pharmaceutique pour prévenir ou traiter un syndrome gastro-intestinal induit par un rayonnement - Google Patents

Composition pharmaceutique pour prévenir ou traiter un syndrome gastro-intestinal induit par un rayonnement Download PDF

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WO2015016282A1
WO2015016282A1 PCT/JP2014/070127 JP2014070127W WO2015016282A1 WO 2015016282 A1 WO2015016282 A1 WO 2015016282A1 JP 2014070127 W JP2014070127 W JP 2014070127W WO 2015016282 A1 WO2015016282 A1 WO 2015016282A1
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tlr3
radiation
mice
pharmaceutical composition
rip1
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智 植松
直紀 武村
審良　静男
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国立大学法人東京大学
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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
    • A61K31/33—Heterocyclic compounds
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4178—1,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
    • 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
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/35—Animals modified by environmental factors, e.g. temperature, O2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the present invention relates to a preventive or therapeutic agent for gastrointestinal syndrome that may be caused by accidental exposure or radiation therapy.
  • Ionizing radiation causes various symptoms depending on the sensitivity of each organ to radiation.
  • the intestinal epithelium When exposed to 5 Gy or more, the intestinal epithelium is damaged, causing diarrhea due to decreased absorption and enteritis due to bacterial infection, resulting in subacute death.
  • gastrointestinal syndrome gastrointestinal syndrome; GIS
  • crypts containing intestinal epithelial stem cells are susceptible to damage.
  • the tumor suppressor gene p53 When radiation damages DNA in the cells of the intestinal crypts, the tumor suppressor gene p53 is activated and repairs the DNA damage. If the damage is severe and cannot be repaired, cell death is induced in a p53-dependent manner, and the cells of the crypt are killed, making it impossible to supply epithelial cells.
  • Non-patent Documents 1 and 2 Non-patent Documents 1 and 2.
  • crypt cells do not undergo p53-mediated cell death upon irradiation, but also lose their DNA repair function. Therefore, p53 ⁇ / ⁇ crypt cells are reproductive death or mitotic death independent of p53 (Non-patent Documents 1 and 2). Therefore, a therapeutic agent for GIS must not cause loss of DNA repair activity by p53, but there is no such therapeutic agent, and there has been no effective therapeutic method for GIS.
  • An object of the present invention is to provide a method for treating or preventing radiation digestive tract-induced syndrome.
  • TLR3-deficient mice are resistant to whole body irradiation with 10 Gy gamma rays. Irradiation with 10 Gy gamma rays induces p53-dependent cell death in crypt cells of the small intestine. We leaked RNA from cells destroyed by this cell death, and activated TLR3-dependent signaling by binding to TLR3 of crypt epithelial cells, resulting in a wider spectrum. The mechanism by which cell death was induced, crypts disappeared, and gastrointestinal syndrome progressed was clarified (FIG. 10).
  • the present inventors added (R) -2- (3-chloro-6-fluorobenzo [b] thiophene-2-carboxamido) -3-phenylpropanoic acid, which is an inhibitor of the interaction between TLR3 and double-stranded RNA. After administration, it was confirmed that the gastrointestinal syndrome induced after 10 Gy gamma irradiation could be suppressed as in TLR3-deficient mice.
  • a pathway for inducing cell death via IFN is known as a signal transduction pathway of TLR3.
  • a mouse (Trif ⁇ / ⁇ ) lacking a gene involved in this pathway is known.
  • Necrostatin-1 As a signal transduction pathway of TLR3 that induces cell death, the pathway shown in FIG. 12 is also known. Therefore, administration of Necrostatin-1 (Nec-1), which is known to specifically inhibit RIP1, all improved mortality, severity of diarrhea, and weight loss. It was confirmed that death was also suppressed. This strongly suggested that radiation-induced crypt cell death was induced by the pathway shown in FIG. 12, and that gastrointestinal syndrome could be prevented or treated by inhibiting signal transduction of this pathway. .
  • Nec-1 Necrostatin-1
  • a pharmaceutical composition for preventing or treating radiation-induced gastrointestinal syndrome comprising a Toll-like receptor (TLR) 3 inhibitor;
  • TLR3 inhibitor is a substance that inhibits the interaction between TLR3 and double-stranded RNA;
  • the radiation according to [2], wherein the substance that inhibits the interaction between TLR3 and double-stranded RNA is a compound represented by the following formula (I), a salt thereof, or a solvate thereof:
  • a pharmaceutical composition for preventing or treating inducible gastrointestinal syndrome [Where: n is an integer from 1 to 3; Ar 1 is an optionally substituted phenyl, indole, or naphthalene; Ar 2 is optionally substituted indole-2-yl or naphthalenyl; X 2 is —NR a —, —O—, or —S—; Z 1 and Z 2
  • the Toll-like receptor (TLR) 3 inhibitor is a group consisting of a double-stranded nucleic acid having an RNAi effect, an antisense nucleic acid, a ribozyme, a miRNA and a nucleic acid encoding the same, which suppresses the expression of the TLR3 gene.
  • the inhibitor for any of RIP1, RIP3 and FADD is Necrostatin-1, a salt thereof, or a solvate thereof, for prevention or treatment of radiation-induced gastrointestinal syndrome according to [9] above A pharmaceutical composition; [11] The inhibitor of any of RIP1, RIP3, and FADD is a dominant negative mutant of any of RIP1, RIP3, and FADD, or an antibody to any of RIP1, RIP3, and FADD, [9]
  • the inhibitor of any of RIP1, RIP3, and FADD is a double-stranded nucleic acid having an RNAi effect, an antisense nucleic acid, a ribozyme, a miRNA, and the like that inhibits the expression of any of the genes RIP1, RIP3, and FADD.
  • the pharmaceutical composition for prevention or treatment of radiation-induced gastrointestinal tract syndrome which is a nucleic acid selected from the group consisting of nucleic acids encoding this; [13]
  • a screening method for a preventive or therapeutic agent for radiation-induced gastrointestinal syndrome Contacting the candidate compound with a cell expressing TLR3 and incubating; Measuring the inhibition of binding between TLR3 and double-stranded RNA by the candidate compound, or the inhibition of TLR3-dependent and RIP1-mediated cell death induction signaling by the candidate compound, About.
  • intestinal crypt cell death can be suppressed while maintaining the DNA repair ability of p53, and gastrointestinal syndrome due to ionizing radiation can be treated or prevented. Since the pharmaceutical composition according to the present invention is effective before administration or after irradiation, it is useful even if it is administered prior to radiation treatment or after exposure to an accident. is there.
  • TLR3 exacerbates radiation-induced GIS.
  • (a) Kaplan-Meier survival analysis of Tlr3 + / + and Tlr3 ⁇ / ⁇ mice after TBI with or without pretreatment with poly I: C. n 5; * p ⁇ 0.05, ** p ⁇ 0.01 (log rank test).
  • (b) Severity of diarrhea after TBI with or without pretreatment with poly I: C and Tlr3 + / + and Tlr3 ⁇ / ⁇ mice. n 5, results are mean ⁇ standard error; * p ⁇ 0.05, ** p ⁇ 0.01 (unpaired two-tailed Student's t-test).
  • n 5; results are mean ⁇ standard error; ** p ⁇ 0.01 (unpaired two-tailed Student's t-test).
  • (d) 0 and 6 hour TUNEL staining of the small intestine of Tlr3 + / + and Tlr3 ⁇ / ⁇ mice. TUNEL-stained epithelium exhibits green fluorescence. Nuclei were stained with DAPI (blue). Scale bar is 100 ⁇ m. The right panel shows the number of TUNEL positive cells. n 4; results are mean ⁇ standard error; ** p ⁇ 0.01 (unpaired two-tailed Student's t-test).
  • n 3; results are mean ⁇ standard error; ** p ⁇ 0.01 (unpaired two-tailed Student's t-test).
  • g H & E staining of the small intestine of Tlr3 + / + and Tlr3 ⁇ / ⁇ mice on day 0 and day 5. Scale bar is 100 ⁇ m. The results are representative of 3 independent experiments. Stimulation of TLR3 with a ligand directly induces cell death in the small intestinal crypt.
  • RT-PCR upper
  • quantitative real-time PCR lower
  • LP Mucosal intrinsic cells.
  • M Size marker.
  • SP Spleen.
  • V Villi.
  • W Whole intestine.
  • n 3; results are mean ⁇ standard error.
  • (c) Survival rate of Tlr3 + / + organoid and Tlr3 ⁇ / ⁇ organoid after poly I: C treatment. n 4; results are mean ⁇ standard error; * p ⁇ 0.05 (unpaired two-tailed Student's t-test).
  • TUNEL staining of Tlr3 + / + organoid and Tlr3 ⁇ / ⁇ organoid exhibits green fluorescence. Nuclei were stained with DAPI (blue). The box is an enlarged view of the crypt-like domain. Scale bar is 100 ⁇ m.
  • TUNEL staining of small intestinal crypts 6 hours after poly I C administration of Tlr3 + / + mice and Tlr3 ⁇ / ⁇ mice. TUNEL-stained epithelium exhibits green fluorescence. Nuclei were stained with DAPI (blue). Scale bar is 100 ⁇ m. The right panel shows the number of TUNEL positive cells.
  • TLR3 mediates radiation-induced crypt cell death via the TRIF-RIP1 pathway.
  • n 4; results are mean ⁇ standard error; ** p ⁇ 0.01 (unpaired two-tailed Student's t-test).
  • (b) H & E staining of small intestinal crypts on day 3 of Tlr3 ⁇ / ⁇ mice, Inf3 ⁇ / ⁇ mice, and Ifnar3 ⁇ / ⁇ mice. Scale bar is 100 ⁇ m. Asterisk indicates microchrony. The right panel shows the number of microcolonies. n 3-5; results are mean ⁇ standard error; ** p ⁇ 0.01 (unpaired two-tailed Student's t-test).
  • TLR3-mediated crypt cell death after TBI is p53-dependent.
  • n 3; results are mean ⁇ standard error; * p ⁇ 0.05 (unpaired two-tailed Student's t-test).
  • (b) Quantitative real-time PCR of mRNA encoding Bax and PUMA in the small intestine of p53 + / + and p53 ⁇ / ⁇ mice. n 3; results are mean ⁇ standard error; * p ⁇ 0.05, ** p ⁇ 0.01 (unpaired two-tailed Student's t-test).
  • c Immunohistochemical detection of p53 in the small intestine crypts of Tlr3 + / + and Tlr3 ⁇ / ⁇ mice 0 or 6 hours after TBI and allogeneic transplantation.
  • Quantitative real-time PCR of mRNA encoding Bax and PUMA in the small intestine of Tlr3 + / + and Tlr3 ⁇ / ⁇ mice. n 3; results are mean ⁇ standard error; * p ⁇ 0.05, ** p ⁇ 0.01 (unpaired two-tailed Student's t-test).
  • n 6; results are mean ⁇ standard error; * p ⁇ 0.05, ** p ⁇ 0.01 (unpaired two-tailed Student's t-test).
  • TUNEL staining of mouse small intestinal crypts 6 hours after treatment with TLR3 / dsRNA binding inhibitor. Scale bar is 100 ⁇ m. The right panel shows the number of TUNEL positive cells. n 5; results are mean ⁇ standard error; * p ⁇ 0.05 (unpaired two-tailed Student's t-test).
  • (a) Kaplan-Meier survival analysis of mice treated with TLR3 / dsRNA binding inhibitors after TBI and allogeneic BMT. n 6; * p ⁇ 0.05 (log rank test).
  • n 4-6; results are mean ⁇ standard error; * p ⁇ 0.05 (unpaired two-tailed Student's t-test). The results are representative of two independent experiments.
  • Pathological mechanism of GIS When ionized gamma rays damage the DNA of intestinal crypt cells, p53 induces cell cycle arrest for DNA repair. When DNA damage is irreparable, p53 initiates cell death. When intracellular RNA is released from cells due to p53-mediated cell death by gamma irradiation, extensive crypt cell death is induced through TLR3, intestinal epithelial cell supply is insufficient, and chorionic epithelium is Destroyed and leads to death by GIS. The signal transduction pathway of TLR3 is shown. The signal transduction pathway of TLR3 is shown.
  • TLR3 inhibitor One embodiment of the pharmaceutical composition for preventing or treating radiation-induced gastrointestinal syndrome according to the present invention comprises a Toll-like receptor 3 (TLR3) inhibitor.
  • TLR3 inhibitor may be any substance as long as it inhibits intestinal crypt cell death induction by TLR3-dependent signaling, for example, all or one of the interactions between TLR3 and double-stranded RNA. It can be a substance that inhibits the part. Examples of such substances include low molecular weight compounds, anti-TLR3 antibodies, dominant negative mutants of TLR3, and the like.
  • a low molecular weight compound that inhibits the interaction between TLR3 and double-stranded RNA is not particularly limited.
  • compound (I) described in International Publication WO2012 / 099785 is known.
  • n is an integer from 1 to 3; Ar 1 is an optionally substituted phenyl, indole, or naphthalene; Ar 2 is optionally substituted indole-2-yl or naphthalenyl; X 2 is —NR a —, —O—, or —S—; Z 1 and Z 2 are each independently ⁇ NR a , ⁇ O, or ⁇ S; X 3 is —NR a R b , —OR c , or —SR d ; Each R a is independently hydrogen, an alkyl group, or a nitrogen protecting group; R b is hydrogen or an alkyl group; R c is independently hydrogen, an alkyl group, or a hydroxyl protecting group; and R d represents hydrogen, an alkyl group, or a thiol protecting group. ]
  • the compound (I) is represented by the following formula (II).
  • X 1 and X 2 are each independently —NR a —, —O—, or —S—; Z 1 and Z 2 are each independently ⁇ NR a , ⁇ O, or ⁇ S;
  • X 3 is —NR a R b , —OR c , or —SR d ;
  • R 1 , R 2 , R 3 and R 4 are each independently hydrogen, an alkyl group, —OR c , halogen, —NR a R b , or SR d , provided that at least one of R 1 to R 4 Is not hydrogen;
  • R b is hydrogen or an alkyl group;
  • R c is independently hydrogen, an alkyl group, or a hydroxyl protecting group; and
  • R d represents hydrogen, an alkyl group, or a thiol protecting group.
  • an anti-TLR3 antibody may be used as a substance that inhibits the interaction between TLR3 and double-stranded RNA.
  • the anti-TLR3 antibody is not particularly limited, and can be, for example, an antibody that binds to a binding site with a double-stranded RNA in TLR3.
  • “antibody” includes antibody fragments, and anti-TLR3 antibodies include monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain antibodies, Fab fragments, F (ab ′ ) 2 antibodies, scFv, bispecific antibodies, synthetic antibodies and the like. These antibodies can be prepared according to methods known to those skilled in the art.
  • monoclonal antibodies can be obtained by isolating antibody-producing cells from non-human mammals immunized with TLR3, fusing them with myeloma cells, etc. to produce hybridomas, and purifying the antibodies produced by the hybridomas. Can do.
  • Polyclonal antibodies can be obtained from the sera of animals immunized with TLR3.
  • TLR3 used for immunization may be derived from humans or other animals, may be full length or a fragment, and can be appropriately determined by those skilled in the art. In the case of a fragment, for example, it may be a fragment containing a binding site for double-stranded RNA in TLR3.
  • a non-human monoclonal antibody that efficiently inhibits the binding between TLR3 and double-stranded RNA can also be produced by a gene recombination method. For example, after preparing total RNA from a hybridoma producing the anti-TLR3 monoclonal antibody using standard techniques, preparing mRNA encoding the anti-TLR3 antibody using a commercially available kit, and then synthesizing cDNA using reverse transcriptase Then, DNA encoding an anti-TLR3 antibody can be obtained. An anti-TLR3 antibody can be expressed by transfecting an appropriate host cell with an expression vector containing such DNA and culturing under an appropriate condition.
  • DNA encoding the CDR region can also be obtained by PCR using the above cDNA as a template.
  • a human antibody or a humanized antibody can also be prepared by a gene recombination method according to a conventional method using DNA encoding such CDR region. For example, a DNA encoding a CDR region derived from a non-human antibody and a DNA designed to link the framework region of a human antibody are synthesized by PCR, and further linked to a DNA encoding a human antibody constant region. Thus, DNA encoding a human antibody can be obtained.
  • Such DNA is expressed by a known method (a method using a restriction enzyme, etc.) and an expression vector (eg, plasmid, retrovirus, adenovirus, adeno-associated virus (AAV), plant virus such as cauliflower mosaic virus or tobacco mosaic virus, cosmid) , YAC, EBV-derived episome) and the expression vector is transfected into an appropriate host cell to obtain a transformant.
  • the expression vector may further contain a promoter that regulates the expression of the antibody gene, a replication origin, a selection marker gene, and the like. The promoter and origin of replication can be appropriately selected depending on the type of host cell and vector.
  • a human antibody of an anti-TLR3 antibody can be expressed by culturing the transformant under appropriate conditions.
  • host cells include eukaryotic cells such as mammalian cells (CHO cells, COS cells, myeloma cells, HeLa cells, Vero cells, etc.), insect cells, plant cells, fungal cells (Saccharomyces, Aspergillus, etc.), E. coli (E. Coli), prokaryotic cells such as Bacillus subtilis can be used.
  • eukaryotic cells such as mammalian cells (CHO cells, COS cells, myeloma cells, HeLa cells, Vero cells, etc.), insect cells, plant cells, fungal cells (Saccharomyces, Aspergillus, etc.), E. coli (E. Coli), prokaryotic cells such as Bacillus subtilis can be used.
  • the expressed antibody can be isolated and purified by appropriately combining known methods (for example, affinity columns using protein A, other chromatography columns, filters, ultrafiltration, salting out, dialysis, etc.). it can.
  • the anti-TLR3 antibody of the present invention is a low molecular antibody such as a Fab fragment, F (ab ′) 2 antibody, scFv, etc.
  • the antibody can be expressed by the above method using a DNA encoding the low molecular antibody, Alternatively, the antibody can be prepared by treating with an enzyme such as papain or pepsin.
  • a dominant negative mutant of TLR3 may be used as a TLR3 inhibitor.
  • a vector containing a gene encoding a dominant negative mutant may be administered to express the mutant in vivo.
  • Such vectors can be prepared by those skilled in the art according to conventional methods.
  • a method for inserting a DNA encoding a dominant negative mutant of TLR3 into a cloning site such as a plasmid, a plasmid having a backbone sequence of an adenoviral vector, and a sequence homologous thereto, and a dominant negative mutant of TLR3
  • a method of preparing a viral vector by homologous recombination by introducing both a shuttle plasmid arranged at both ends of DNA to be introduced into cells or E. coli.
  • TLR3 inhibitors also include substances that suppress all or part of TLR3 expression.
  • examples of such substances include double-stranded nucleic acids having an RNAi effect, antisense nucleic acids, ribozymes, and nucleic acids encoding them.
  • the RNAi effect is a sequence-specific gene expression suppression mechanism induced by a double-stranded nucleic acid.
  • the target specificity is very high, and it is highly safe because it uses a gene expression suppression mechanism that originally exists in vivo.
  • Examples of the double-stranded nucleic acid having an RNAi effect include siRNA.
  • siRNA When siRNA is used in mammalian cells, it is usually a double-stranded RNA of about 19 to 30 bases, and preferably about 21 to 25 bases, but a longer double strand that can be cleaved by an enzyme (Dicer) to become siRNA. It may be a strand RNA.
  • a double-stranded nucleic acid having an RNAi effect has one base sequence complementary to a part of the target nucleic acid and the other complementary sequence.
  • a double-stranded nucleic acid having an RNAi effect generally has two protruding bases (overhangs) at the 3 ′ end of each other, but may be of a blunt end type having no overhangs. .
  • a 25-base blunt-end RNA has the advantage of minimizing interferon-responsive gene activation, preventing off-target effects from the sense strand, and being very stable in serum for in vivo use Also suitable for.
  • a double-stranded nucleic acid having an RNAi effect can be designed according to a known method based on the base sequence of the target gene.
  • the double-stranded nucleic acid having an RNAi effect may be a double-stranded RNA or a DNA-RNA chimera-type double-stranded nucleic acid, and may be an artificial nucleic acid or a nucleic acid subjected to various modifications. There may be.
  • An antisense nucleic acid has a base sequence complementary to a target gene (typically mRNA that is a transcription product), and generally has a length of 10 to 100 bases, preferably 15 to 30 bases. Single-stranded nucleic acid.
  • a target gene typically mRNA that is a transcription product
  • Single-stranded nucleic acid By introducing an antisense nucleic acid into a cell and hybridizing to a target gene, gene expression is inhibited.
  • the antisense nucleic acid may not be completely complementary to the target gene as long as the effect of inhibiting the expression of the target gene is obtained.
  • Antisense nucleic acids can be appropriately designed by those skilled in the art using known software or the like.
  • the antisense nucleic acid may be any of DNA, RNA, DNA-RNA chimera, and may be modified.
  • Ribozymes are nucleic acid molecules that catalytically hydrolyze target RNA, and are composed of an antisense region having a sequence complementary to the target RNA and a catalytic center region responsible for the cleavage reaction.
  • the ribozyme can be appropriately designed by those skilled in the art according to known methods. Ribozymes are generally RNA molecules, but DNA-RNA chimeric molecules can also be used.
  • a nucleic acid encoding any one of the above-described double-stranded nucleic acid, antisense nucleic acid, and ribozyme having the RNAi effect can also be used as a TLR3 expression inhibitor of the present invention.
  • a vector containing such a nucleic acid is introduced into a cell, a double-stranded nucleic acid, an antisense nucleic acid, and a ribozyme having an RNAi effect are expressed in the cell, and each exerts a TLR3 expression suppressing effect.
  • RNA As a nucleic acid encoding a double-stranded nucleic acid having an RNAi effect, a DNA encoding each of the double strands may be used, or a single-stranded nucleic acid formed by connecting double-stranded nucleic acids via a loop is encoded. DNA may be used. In the latter case, the single-stranded RNA obtained by transcription in the cell has a complementary structure hybridized in the molecule and takes a hairpin type structure. This RNA is called shRNA (short hairpin RNA). When shRNA moves into the cytoplasm, the loop part is cleaved by the enzyme (Dicer) to form double-stranded RNA and exert RNAi effect.
  • Dicer enzyme
  • the TLR3 inhibitor contained in the pharmaceutical composition according to the present invention may be a pharmaceutically acceptable salt or solvate regardless of whether it is a low molecular compound, protein, peptide or nucleic acid.
  • One embodiment of the pharmaceutical composition for preventing or treating radiation-induced gastrointestinal syndrome comprises the inhibitor of TLR3-dependent and RIP1-mediated cell death induction signaling shown in FIG.
  • known inhibitors for RIP1 receptor interacting protein 1
  • RIP3 receptor interacting protein 3
  • FADD Fas-associated protein with Death Domain
  • the present inventors have improved the lethality, the severity of diarrhea, and the weight loss when irradiated with Necrostatin-1, a specific inhibitor for RIP1, and also suppressed cell death of small intestinal crypts Confirmed that it will be.
  • the inhibitor of TLR3-dependent and RIP1-mediated cell death induction signaling may be a dominant negative mutant of either RIP1, RIP3, or FADD, or an antibody to any of RIP1, RIP3, and FADD Good. Further, it may be a double-stranded nucleic acid having an RNAi effect, an antisense nucleic acid, a ribozyme, a miRNA, and a nucleic acid encoding the same, which inhibit the expression of any of RIP1, RIP3 and FADD genes.
  • negative dominant mutant antibody, double-stranded nucleic acid having RNAi effect, antisense nucleic acid, ribozyme, miRNA, and nucleic acid encoding the same are the same as those used for the TLR3 inhibitor, The description is omitted here.
  • the pharmaceutical composition of the present invention can be administered orally or parenterally, systemically or locally.
  • intravenous injection such as infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, enema, oral enteric solvent, etc.
  • the administration method should be selected appropriately depending on the age and symptoms of the patient Can do.
  • a pharmaceutically acceptable carrier such as a preservative and a stabilizer may be added.
  • the pharmaceutically acceptable carrier is not particularly limited as long as it is pharmacologically and pharmaceutically acceptable.
  • pharmaceutically acceptable organic solvents such as water, saline, phosphate buffer, dextrose, glycerol, ethanol, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate , Water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin , Mannitol, sorbitol, lactose, surfactant, excipient, flavor
  • the pharmaceutical composition of the present invention can be formulated into a normal medical preparation form.
  • the medical preparation is appropriately prepared using the carrier.
  • the form of the medical preparation is not particularly limited, and is appropriately selected depending on the purpose of treatment. Typical examples thereof include tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, suppositories, injections (solutions, suspensions, emulsions) and the like. These preparations may be produced by a commonly used method.
  • the pharmaceutical composition of the present invention contains a nucleic acid
  • it can be formulated by encapsulating the nucleic acid in a carrier such as a liposome, polymer micelle, or cationic carrier.
  • a nucleic acid carrier such as protamine may also be used. It is also preferable to target the affected area by binding an antibody or the like to these carriers. It is also possible to increase the retention in blood by binding cholesterol or the like to the nucleic acid.
  • the nucleic acid when the pharmaceutical composition of the present invention contains a nucleic acid encoding siRNA or the like and is expressed in cells after administration, the nucleic acid may be a viral vector such as retrovirus, adenovirus, Sendai virus, It can also be inserted into non-viral vectors such as liposomes and administered into cells.
  • a viral vector such as retrovirus, adenovirus, Sendai virus
  • the amount of the active ingredient contained in the pharmaceutical composition of the present invention can be appropriately determined by those skilled in the art according to the type of the active ingredient.
  • the pharmaceutical composition of the present invention is administered to humans or non-human mammals (eg, mice, rats, rabbits, dogs, pigs, cows, horses, monkeys) and the like for the purpose of preventing or treating herpes virus infection. be able to.
  • the present invention also includes a method for preventing or treating radiation-induced gastrointestinal syndrome, which comprises administering the pharmaceutical composition according to the present invention described above.
  • Treatment or prevention of radiation-induced gastrointestinal syndrome includes alleviation or suppression of one or more symptoms related to radiation-induced gastrointestinal syndrome, prevention or delay of worsening or progression, prevention or delay of occurrence of symptoms, etc. Meaning, it can be evaluated by at least one of the presence or absence of diarrhea, the severity of symptoms, the weight, the survival rate, the degree of cell death of intestinal crypt cells, and the form.
  • the preventive or therapeutic method according to the present invention can be used for any gastrointestinal syndrome induced by radiation, but typically, exposure due to an accident or radiotherapy (especially treatment of peritoneal dissemination of ovarian cancer).
  • the pharmaceutical composition of the present invention alleviates the gastrointestinal syndrome even when administered after irradiation, so that the pharmaceutical composition of the present invention can be quickly used even in the event of an accidental exposure.
  • the prevention or treatment method of the present invention is used for humans or mammals other than humans (for example, mice, rats, rabbits, dogs, pigs, cows, horses, monkeys).
  • the present invention also provides a method for screening a medicament for the prevention or treatment of radiation-induced gastrointestinal syndrome.
  • One aspect of the screening method according to the present invention is: Contacting the candidate compound with a cell expressing TLR3 and incubating; Measuring the inhibition of binding between TLR3 and double-stranded RNA by the candidate compound, or the inhibition of TLR3-dependent and RIP1-mediated cell death induction signaling by the candidate compound.
  • the candidate compound may be any substance such as a low molecular compound, a high molecular compound, a peptide, a protein, a nucleic acid, a sugar, or a lipid.
  • a cell that expresses TLR3 is not particularly limited, and can be, for example, an epithelial cell that expresses TLR3.
  • Cell culture medium, candidate compound concentration, temperature, pH and other conditions in the step of incubating the candidate compound and TLR3-expressing cells in contact are appropriately selected by those skilled in the art depending on the type of cell and the type of candidate compound be able to.
  • mice were housed in standard cages with a light-dark cycle of 12 hours in a temperature-controlled room in the SPF environment of the Osaka University Infectious Animal Laboratory. Free access to standard laboratory mouse feed and drinking water.
  • GF mice were bred with vinyl isolators in laboratory animal facilities at the Institute of Medical Science, the University of Tokyo. The light / dark cycle was 12 hours, so that the autoclaved feed and sterilized water were always accessible.
  • Poly I C treatment
  • Poly I: C (poly I: C-HMW, Invivogen) was diluted with pathogen-free PBS without using a transfection reagent or a liposome carrier.
  • Poly I: C was administered by intraperitoneal injection in mice.
  • Nec-1 (Sigma-Aldrich) was dissolved in DMSO and diluted with pathogen-free PBS. Nec-1 was injected multiple times intraperitoneally in mice to achieve a significant protective effect on GIS.
  • TLR3 / dsRNA binding inhibitor (Treatment with TLR3 / dsRNA binding inhibitor) (R) -2- (3-Chloro-6-fluorobenzo [b] thiophene-2-carboxamido) -3-phenylpropanoic acid (Calbiochem), a TLR3 / dsRNA binding inhibitor, is dissolved in DMSO and free of pathogens Dilute with PBS. Inhibitors were administered multiple times by intraperitoneal injection in mice.
  • mice Female, 8-12 weeks old were exposed to 10 Gy TBI at a dose rate of 88 cGy / min using a 137 Cs irradiator (Gammacell 40 Exactor; MDS Nordion).
  • poly I C (20 mg / kg body weight) 1 hour before irradiation.
  • Nec-1 125 mg / kg body weight was administered 1 hour before, 2 hours, 24 hours, 48 hours, and 72 hours after irradiation.
  • TLR3 / dsRNA binding inhibitor (1 mg per mouse was administered 1 hour before or 1 hour after irradiation. Thereafter, the inhibitors were injected at 24 and 48 hours.
  • mice were subsequently injected once intravenously with 1 ⁇ 10 7 BM cells obtained from donor mice of matched strain, gender, and genotype. After irradiation, the mice were given 1,000 U / ml polymyxin B and 1 mg / ml neomycin in their drinking water. The severity of GIS was evaluated by observing weight loss and diarrhea. Diarrhea was scored daily on the hair near the anus and scored as 0 for no signs, 1 for light brown feces, and 2 for tar-like black feces.
  • chromophilic non-panate cells 10 or more chromophilic non-panate cells and those containing at least one panate cell were defined as regenerating crypts.
  • tibias were collected, fixed with 70% ethanol for 3 days, embedded in methyl methacrylate resin, and stained with H & E. Intestinal cell death was assessed by TUNEL staining.
  • Small intestine paraffin sections were treated with reagents from an in situ Cell Death Detection Kit, Fluorescein (Roche Diagnostics). Samples were prepared with ProLong Gold Antifade Reagent with DAPI (invitrogen) and observed under a fluorescence microscope using BZ-II Image Analysis Application. To determine the number of TUNEL positive cells per crypt, at least 50 crypts were counted.
  • mice were injected intraperitoneally with BrdU (5-bromo-2'-deoxyuridine; 50 mg / kg body weight) one hour prior to sacrifice.
  • BrdU 5-bromo-2'-deoxyuridine
  • Small intestine paraffin sections were prepared as described above. Endogenous peroxidase activity was stopped with 3% hydroperoxide for 10 minutes. Sections were treated with 2N HCl for 30 minutes, blocked with 10% goat serum (in PBS) for 30 minutes, and stained overnight at 4 ° C. with mouse anti-BrdU antibody (100 ⁇ , Ab-3, Lab Vision Corporation).
  • Sections were washed and incubated with biotin-conjugated goat anti-mouse IgG (1,500 ⁇ , Kirkegaard & Perry Laboratories) for 1 hour at 37 ° C. and then with streptavidin-conjugated horseradish peroxidase (Thermo Fisher Scientific) for 30 minutes at room temperature. Sections were visualized with 3,3′-diaminobenzidine and hydroperoxide and counterstained with hematoxylin. BrdU positive cells were counted in at least 50 crypts.
  • mice anti-p53 antibody x100, Pab 240, Abcam
  • citrate buffer pH 6.0
  • the antigen was removed by heating.
  • Detection and visualization of the primary antibody was performed by the method described above.
  • Activated caspase 3 was detected by the method described above using a rabbit anti-cleavable caspase 3 antibody (100 ⁇ , 5A1E, Cell Signaling Technology).
  • the bound primary antibody was detected by reacting with biotin-conjugated goat anti-rabbit IgG (1,500 ⁇ , Kirkegaard & Perry Laboratories) at 37 ° C. for 1 hour and visualized by the method described above. Positive cells were counted in at least 50 crypts.
  • villi, crypts and mucosal intrinsic cells were prepared from the small intestine. Briefly, small intestine tissue fragments were incubated with 2 mM EDTA (in PBS) for 30 minutes on ice. After removing EDTA, the sample was vigorously shaken with cold PBS and centrifuged to obtain a supernatant fraction rich in villi and crypts. For isolation of mucosal intrinsic cells, small intestine fragments were treated with PBS containing 10% FCS and 10 mM EDTA at 37 ° C.
  • RNA sample was analyzed for RQ1 RNase-free DNase (Promega) and incubated at 37 ° C. for 45 minutes. After purification by phenol-chloroform extraction, total RNA was ethanol precipitated and resuspended in double distilled water. 2 ⁇ g of total RNA was reverse transcribed using SuperScript III First-Strand Synthesis SuperMix (Invitrogen) and using random hexamers as primers according to the instructions. Primer sequences for PCR are as follows.
  • the primer pair and Taq polymerase (Takara Shuzo) were subjected to PCR performed under the following conditions: 94 ° C for 5 minutes; 97 ° C for 30 seconds, 57 ° C for 30 seconds, 72 ° C for 30 seconds; 72 ° C for 7 minutes.
  • PCR products were separated by agarose gel electrophoresis.
  • Quantitative real-time PCR includes cDNA, Real-time PCR Master Mix (Toyobo), and final volume containing 18S rRNA-specific primers (Applied Biosystems), or Tlr3, Bax or Puma-specific primers (Applied Biosystems) as internal controls In 25 ml solution, it was performed using ABI Prism 7700 sequence detection system (Applied Biosystems). After incubating at 95 ° C. for 10 minutes, the resulting product was amplified at 35 ° C. for 15 seconds, 60 ° C. for 60 seconds, and 50 ° C. for 120 seconds under 35 cycles.
  • RNA was isolated as described above and further separated into small RNA ( ⁇ 200 nucleotides) and large RNA (> 200 nucleotides) fractions using the miRNeasy Mini Kit and RNeasy MinElute Cleanup Kit (Qiagen). The size of the separated RNA was confirmed with Agilent 2100 Bioanalyzer and Agilent RNA 6000 Pico Kit (Agilent Technology).
  • crypts were treated with 100 mg / ml purified RNA or poly I: C complexed with Lipofectamine 2000 reagent or exposed to 10 Gy radiation. For enzyme digestion assays, crypts were incubated with 5 mg / ml DNase-free RNase (Roche Diagnostics). Images of cultured crypts were taken with a light microscope and BZ-II Image Analysis Application.
  • Viability (%) (value of treated crypt cells / value of untreated crypt cells) ⁇ 100
  • cultured crypts were recovered from Matrigel, fixed directly with 4% paraformaldehyde, and stained using in situ Cell Death Detection Kit, Fluorescein. Nuclei were stained with 10 mg / ml DAPI (4 ′, 6′-diamidino-2-phenylindole). Images were taken with a fluorescence microscope and BZ-II Image Analysis Application. For quantification of RNA in the culture supernatant, SUPERase-In RNase Inhibitor (Ambion) was added at a concentration of 0.5 U / ml.
  • RNA extraction was performed using Trizol LS reagent (Invitrogen) according to the instruction manual.
  • the purified RNA was quantified using Agilent 2100 Bioanalyzer and Agilent RNA 6000 Pico Kit.
  • intestinal homogenate The small intestine was collected 6 hours after 10 Gy irradiation, washed thoroughly with sterilized PBS, and homogenized by centrifugation at 15,000 g for 3 minutes with DMEM. The supernatant was used as an intestinal homogenate.
  • intestinal homogenates were incubated with 10 U / ml pronase, 10 U / ml RNase-free-DNase, 5 mg / ml DNase-free RNase (both Roche Diagnostics) for 1 hour at 37 ° C. .
  • HEK293 cells (ATCC) were cultured in DMEM supplemented with 10% fetal calf serum in an incubator at 37 ° C. and 5% CO 2 .
  • HEK293 cells were transiently transfected with Lipofectamine 2000 reagent with ISRE-luciferase reporter plasmid and expression vector plasmid pUNO-hTLR3 or empty control vector pUNO-mcs (Invivogen). After 12 hours, cells were seeded in 96-well flat-bottom plates at 5 ⁇ 10 4 cells per well.
  • RNA Ribonucleic acid
  • RNA Ribonucleic acid
  • 0.5 mg / ml poly I C complexed with Lipofectamine 2000 reagent, or intestinal homogenate.
  • the cells were collected, lysed with passive lysis buffer (Promega), and luciferase activity was measured using Dual-Luciferase Reporter Assay System (Promega). Renilla luciferase plasmid driven by the TK promoter was used as an internal control.
  • CD8 + dendritic cells were purified from the spleen of wild type mice (female, 8-12 weeks old) by magnetic sorting using CD8 + Dendritic Cell Isolation Kit (Miltenyi Biotec). The purity of the sorted dendritic cells was usually> 95%.
  • CD8 + dendritic cells were seeded in 96-well round bottom plates at 5 ⁇ 10 4 cells per well, in 200 ml RPMI 1640 containing 10% fetal calf serum at 37 ° C., 5% CO Cultured in 2 incubators. Cells were pretreated with 100 mM TLR3 / dsRNA binding inhibitor 1 hour prior to stimulation with 10 mg / ml poly I: C. After 12 hours of incubation, expression levels of mRNA encoding IL-12p40, IFN- ⁇ 4, and IFN- ⁇ were measured by quantitative real-time PCR.
  • TLR3 is an exacerbation factor of radiation-induced gastrointestinal syndrome (GIS).
  • GIS radiation-induced gastrointestinal syndrome
  • Tlr3 + / + mice We also examined post-irradiation diarrhea and weight loss (references 1 and 2), which are the main symptoms of GIS. Symptoms of diarrhea in untreated Tlr3 + / + mice gradually worsened after 3 days of TBI, but poly I: C-treated Tlr3 + / + mice showed more severe symptoms from day 1 ( FIG. 1b). In addition, poly I: C treatment significantly promoted weight loss in Tlr3 + / + mice (FIG. 1c). In contrast to Tlr3 + / + mice, Tlr3 ⁇ / ⁇ mice had no effect of poly I: C treatment on diarrhea and weight loss after TBI. Thus, TLR3 stimulation with poly I: C exacerbates radiation-induced GIS. Interestingly, Tlr3 -/- mice, with or without poly I: C treatment, had milder GIS symptoms including mortality, diarrhea, and weight loss compared to Tlr3 + / + mice. there were.
  • TLR3 is specifically activated after TBI and exacerbates GIS.
  • TLR3 mediates crypt cell death in radiation-induced GIS.
  • HPS hematopoietic syndrome
  • Tlr3 + / + and Tlr3 -/- mice were subjected to bone marrow transplantation (BMT) immediately after TBI.
  • BMT bone marrow transplantation
  • TBI was lethal in Tlr3 + / + mice, but Tlr3 ⁇ / ⁇ mice survived under the same conditions (FIG. 2a). This indicated that irradiated Tlr3 ⁇ / ⁇ mice died of HPS.
  • Tlr3 ⁇ / ⁇ BMT mice had significantly less diarrhea symptoms and weight loss (FIGS. 2b, c).
  • FIGS. 2b, c diarrhea symptoms and weight loss
  • Tlr3 - / - and Tlr3 + / + mice that had received a BM cells from a mouse Tlr3 + / + Tlr3 got a BM cells from a mouse - / - were prepared mouse.
  • GIS was relaxed in Tlr3 ⁇ / ⁇ recipient mice regardless of donor cells. This indicated that TLR3 on non-BM cells greatly contributed to the cause of GIS, and that TLR3 on BM cells did not contribute.
  • a TUNEL assay was performed to examine cell death of crypt epithelial cells in the small intestine. As shown in FIG.
  • Tlr3 + / + mice had few cryptomicrocolony in the small intestine, which is an indicator of crypt regeneration, but Tlr3 -/- mice had very many cryptomicrocolony. ( Figure 2e).
  • Tlr3 ⁇ / + mice showed a normal intestinal structure as normal as Tlr3 + / + mice before TBI (FIG. 2g). This indicates that TLR3 is not involved in organ formation in the small intestine.
  • the small intestine of Tlr3 + / + mice showed signs of villous destruction such as blunting, fusion, and epithelial exposure, but in the small intestine of Tlr3 -/- mice The villi structure was preserved (Fig. 2g).
  • Tlr3 ⁇ / ⁇ mice have milder radiation-induced crypt cell death than Tlr3 + / + mice, and as a result, Tlr3 ⁇ / ⁇ mice regenerated crypts on day 3. Shows that normal re-epithelialization of the small intestinal villi occurs on day 5 and prevents GIS-induced death.
  • TLR3 directly induces cell death in the small intestine crypts. Since Tlr3 + / + and Tlr3 ⁇ / ⁇ mice differed in the extent of crypt cell death after TBI, we investigated whether TLR3 is directly involved in this phenomenon. As shown in FIG. 3a, crypt epithelial cells highly expressed Tlr3 mRNA. Thus, how poly I: C acts on crypt cells was examined using an in vitro crypt cell culture method (Reference 10). Small intestine crypts of Tlr3 + / + and Tlr3 ⁇ / ⁇ mice were cultured in a Matrigel-based culture system for 6 days (FIG. 3b).
  • TLR3 induces crypt cell death after TBI via the TRIF-RIP1 pathway.
  • TLR is composed of an extracellular domain, a transmembrane domain, and a cytoplasmic signaling domain known as the Toll / interleukin-1 receptor homology (TIR) domain (Ref. 4).
  • TLR recognizes the ligand through the extracellular domain and induces homotypic binding between the TIR domain of the receptor and the TIR domain of the adapter molecule to transmit a signal.
  • TLR3 does not use the adapter myeloid differentiation factor 88 (MyD88) (11, 12).
  • TLR3 recruits another adapter molecule, TIR domain-containing adapter-inducible interferon beta (TRIF) (TIR-containing adapter molecule-1; also known as TICAM-1), and type 1 interferon (IFN) It induces the activation of interferon regulatory factor 3 (IRF3) for gene transcription (Reference 12). Therefore, we investigated the role of TRIF in GIS. Similar to Tlr3 ⁇ / ⁇ mice, Trif ⁇ / ⁇ mice survived significantly longer after 10-Gy TBI compared to Trif + / + mice. Compared to Trif + / + mice, Trif -/- mice had significantly less diarrhea symptoms and weight loss.
  • TIR domain-containing adapter-inducible interferon beta TIR-containing adapter molecule-1; also known as TICAM-1
  • IFN type 1 interferon
  • IRF3 interferon regulatory factor 3
  • Trif ⁇ / ⁇ mice showed a phenotype similar to Tlr3 ⁇ / ⁇ mice after 10-Gy TBI. Therefore, further, IRF3 - / - mice and Ifnar - / - with the mouse was examined the involvement of the GIS of IRF3 type 1 IFN receptor (IFNAR). Interestingly, there were no significant differences between survival, diarrhea, weight loss, and crypt damage between Irf3 + / + and Irf3 ⁇ / ⁇ mice (FIGS. 4a, b).
  • mice were treated with necrostatin-1 (Nec-1) (reference 15), a RIP1-specific allosteric inhibitor.
  • Nec-1 treatment improved survival and diarrhea, but did not significantly differ in weight loss.
  • Nec-1 treatment significantly increased the number of TUNEL positive crypt cells 6 hours after TBI and increased the number of microcolony 3 days after TBI (FIGS. 4c, d).
  • TLR3 mediates radiation-induced crypt cell death through the TRIF-RIP1 pathway.
  • TLR3 has been thought to induce two different cell deaths in a cell type-specific manner, apoptosis and necrosis (Reference 16).
  • Apoptosis occurs through ligand stimulation of the death receptor and activation of caspase family members in response to intracellular damage (Ref. 16).
  • binding of TRIF and RIP1 induces downstream signaling through FADD (Fas-associated protein with death domain) and caspase 8, which cleaves effector caspase 3 and apoptosis is performed (References 17, 18).
  • necroptosis is a programmed necrosis that is induced by a ligand-activated death domain receptor when caspase function is absent or inhibited (16).
  • Tlr3 + / + mice there was an increase in activated caspase 3-positive cells after TBI and poly I: C administration.
  • Tlr3 ⁇ / ⁇ mice the number of activated caspase 3 positive cells was significantly reduced after TBI and poly I: C administration.
  • crypt cell death induced by the TLR3 pathway after TBI is apoptosis.
  • TLR3-mediated crypt cell death after TBI is p53-dependent.
  • p53 has been suggested to be a major causative factor of radiation-induced crypt cell death (Ref. 3). Ionizing radiation induces phosphorylation of p53 protein, thereby inhibiting ubiquitination and subsequent proteasome degradation (Ref. 3). Phosphorylation of p53 causes nuclear accumulation of transcriptionally activated p53, and as a result, induces pro-apoptotic genes such as Bax (Bcl-2 related X protein) and PUMA (p53-upregulated modulator of apoptosis).
  • Bax Bax
  • PUMA p53-upregulated modulator of apoptosis
  • TLR3 induction is regulated by p53 in several cancer cell lines (Reference 19).
  • poly I C-mediated cell death in p53 ⁇ / ⁇ mice was examined. Similar to p53 + / + mice, intraperitoneal injection of poly I: C induced crypt epithelial cell death in p53 ⁇ / ⁇ mice (FIG. 5e).
  • Intestinal microbiota is not important for crypt cell death after TBI.
  • TLR3 The mammalian gastrointestinal tract contains a variety of microorganisms that make up the gut microbiota. Since TLR3 is known as a viral dsRNA sensor (Refs. 4, 5, and 7), crypt cell death in sterile (GF) mice after TBI was examined. However, there was no difference between GF and SPF mice regarding crypt cell death 6 hours after TBI and regeneration after 3 days. This suggests that the gut microbiota is not involved in the activation of TLR3 after TBI (FIGS. 6a, b).
  • TLR3-dependent crypt cell death In addition to exogenous ligands derived from microorganisms, TLRs have been shown to induce inflammatory responses in response to endogenous ligands released from damaged tissues (Ref. 20). Endogenous ligands derived from these hosts are cellular components or inducible gene products such as intracellular constituents, extracellular matrix proteins, and nucleic acids that exacerbate tissue damage through TLR activation. Therefore, we hypothesized that endogenous molecules are involved in TLR3-mediated crypt cell death after TBI. First, 10-Gy post-TBI small intestine homogenate is interferon-mediated via TLR3. Stimulated response element) It was investigated whether reporter activity could be stimulated.
  • small intestine homogenate significantly stimulated ISRE reporter activity in HEK293 cells when transiently transfected with an expression plasmid encoding TLR3 (FIG. 7a).
  • RNase treatment decreased the ability to stimulate ISRE reporter activity via TLR3 of small intestine homogenate, but not proteinase treatment or DNase treatment.
  • small intestine homogenate RNA stimulates TLR3.
  • crypt cell death caused by gamma irradiation in vitro is p53 dependent.
  • total RNA derived from the culture supernatant was analyzed using an Agilent 2100 bioanalyzer, and the amount and size distribution of nucleic acids were measured.
  • RNA molecules of various lengths ranging from 25 to 1000 nucleotides were detected in p53 + / + organoid culture supernatants after gamma irradiation, but not in p53 -/- organoids (FIG. 7b).
  • RNA isolated from the small intestine after TBI stimulates ISRE reporter activity via TLR3.
  • irradiated small intestine-derived total RNA significantly increased ISRE reporter activity in a TLR3-dependent manner (FIG. 7d).
  • FIG. 7b a small RNA group of 200 nucleotides or less was detected from the culture supernatant of p53 + / + organoid after ⁇ -irradiation.
  • siRNA small interfering RNA
  • RNAs such as host-derived microRNAs are involved in TLR3-mediated crypt cell death after TBI.
  • stimulation of ISRE reporter activity via TLR3 by small RNAs of 200 nucleotides or less was not observed (FIG. 7d).
  • large RNAs induce crypt cell death in vitro. Large RNA induced destruction of crypt villi organoids in Tlr3 + / + mice, but not small RNA, and large RNA did not destroy crypt chorionic organoids in Tlr3 -/- mice (Fig. 7e).
  • TLR3 / dsRNS binding inhibitors suppress mouse GIS.
  • endogenous RNA could induce TLR3-dependent crypt cell death in GIS.
  • (R) -2- (3-chloro-6-fluorobenzo [b] thiophene-2-carboxamide) -3 is a competitive inhibitor of dsRNA-TLR3 binding -Phenylpropanoic acid was used (reference 22). It was also confirmed that this low molecular weight compound suppressed the upregulation of mRNA encoding IL-12p40 in CD8 ⁇ -positive splenic dendritic cells after poly I: C stimulation. This upregulation is completely dependent on TLR3 (Ref. 6).
  • mice with a TLR3 inhibitor prior to TBI significantly increased survival and improved diarrhea and weight loss (FIGS. 8a-c). Furthermore, pretreatment with TLR3 inhibitors significantly reduced crypt cell death and increased microcolony 6 hours and 3 days after TBI (FIG. 8d, e). Inhibitor treatment resulted in a significant increase in survival and GIS symptoms even when performed after TBI (FIGS. 9a-c). Thus, inhibition of TLR3 / dsRNA binding effectively reduced crypt cell death and protected mice from GIS.
  • Apoptosis a review of programmed cell death. Toxicol. Pathol. 35, 495-516 (2007). 19. Taura, M. et al. P53 regulates Toll-like receptor 3 expression and function in human epithelial cell lines.Mol. Cell Biol. 28, 6557-6567 (2008). 20. Takeuchi, O. & Akira, S. Pattern recognition receptors and inflammation. Cell 140, 805-820 (2010). 21. Kleinman, M. E. et al. Sequence- and target-independent angiogenesis suppression by siRNA via TLR3. Nature 452, 591-597 (2008). 22. Cheng, K., Wang, X. & Yin, H. Small-molecule inhibitors of the TLR3 / dsRNA complex.

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Abstract

L'invention pose le problème consistant à mettre en œuvre une méthode de traitement ou de prévention d'un syndrome gastro-intestinal dû à un rayonnement ionisant. L'invention concerne une composition pharmaceutique permettant de prévenir ou de traiter un syndrome gastro-intestinal induit par un rayonnement et comprenant un inhibiteur du récepteur du type Toll (TLR)3 ou un inhibiteur de transmission de signal induisant la mort cellulaire dépendant de TLR3 ou induite par RIP1.
PCT/JP2014/070127 2013-07-30 2014-07-30 Composition pharmaceutique pour prévenir ou traiter un syndrome gastro-intestinal induit par un rayonnement WO2015016282A1 (fr)

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