WO2015016282A1 - Pharmaceutical composition for preventing or treating radiation-induced gastrointestinal syndrome - Google Patents

Abstract

　The invention addresses the problem of providing a method for treating or preventing gastrointestinal syndrome due to ionizing radiation. The invention provides a pharmaceutical composition for preventing or treating radiation-induced gastrointestinal syndrome including a Toll-like receptor (TLR) 3 inhibitor or a TLR3-dependent and RIP1-mediated cell death-inducing signal transmission inhibitor.

Description

Pharmaceutical composition for preventing or treating radiation-induced gastrointestinal syndrome

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. 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. In this radiation-induced gastrointestinal syndrome (gastrointestinal syndrome; GIS), crypts containing intestinal epithelial stem cells are susceptible to damage. 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.

Therefore, p53 was considered as a therapeutic target for GIS. However, it has been reported that deficiency of p53 does not protect mice from GIS (Non-patent Documents 1 and 2). In p53 ^{− / −} mice, 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.

As a result of repeated studies to solve the above problems, the present inventors have found that 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).

Furthermore, 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. (R) -2- (3-chloro-6-fluorobenzo [b] thiophene-2-carboxamido) -3-phenylpropanoic acid suppressed the onset of gastrointestinal syndrome more effectively when administered before irradiation, Even after administration, the deterioration of gastrointestinal syndrome was significantly suppressed.

Further, as shown in FIG. 11, 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. , Irf3 ^{− / −} , and Infar ^{− / −} mice) confirmed that this pathway was not involved in radiation-induced crypt cell death (FIG. 4).

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. .

Thus, the present invention
[1] A pharmaceutical composition for preventing or treating radiation-induced gastrointestinal syndrome, comprising a Toll-like receptor (TLR) 3 inhibitor;
[2] The pharmaceutical composition for prevention or treatment of radiation-induced gastrointestinal tract syndrome according to [1] above, wherein the TLR3 inhibitor is a substance that inhibits the interaction between TLR3 and double-stranded RNA;
[3] 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 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. ];
[4] The pharmaceutical composition for prevention or treatment of radiation-induced gastrointestinal syndrome according to [3], wherein the compound (I) is represented by the following formula (II):

[Where:
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. ];
[5] The radiation-inducing property according to [3], wherein the compound (II) is (R) -2- (3-chloro-6-fluorobenzo [b] thiophene-2-carboxamido) -3-phenylpropanoic acid A pharmaceutical composition for preventing or treating gastrointestinal syndrome;
[6] The pharmaceutical composition for prevention or treatment of radiation-induced gastrointestinal syndrome according to [1], wherein the Toll-like receptor (TLR) 3 inhibitor is a dominant negative mutant of TLR3 or an anti-TLR3 antibody. object;
[7] 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 pharmaceutical composition for prevention or treatment of radiation-induced gastrointestinal syndrome according to [1], which is a selected nucleic acid;
[8] A pharmaceutical composition for preventing or treating radiation-induced gastrointestinal syndrome, comprising an inhibitor of TLR3-dependent and RIP1-mediated cell death induction signaling;
[9] The radiation-induced gastrointestinal syndrome according to [8], wherein the TLR3-dependent and RIP1-mediated cell death induction signaling inhibitor is any one of RIP1, RIP3 and FADD. A pharmaceutical composition for the prevention or treatment of
[10] 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] A pharmaceutical composition for preventing or treating radiation-induced gastrointestinal tract syndrome according to claim 1;
[12] 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 according to [9] above, 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.

According to the present invention, 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). (c) Weight loss of Tlr3 ^{+ / +} and Tlr3 ^{− / −} mice 5 days after TBI with or without poly I: C pretreatment. n = 5, results are mean ± standard error; ^* p <0.05, ^** p <0.01 (unpaired two-tailed Student's t-test). (d) and (e) are the white blood cell counts in blood and BM on day 0 and day 5 after TBI, respectively. n = 3; results are mean ± standard error. (f) H & E staining of tibia on day 0 and day 5. The scale bar is 200 μm. The results are representative of 3 independent experiments. Tlr3 ^{− / −} mice avoid GIS by reducing radiation-induced crypt cell death. (a) Kaplan-Meier survival analysis of Tlr3 ^{+ / +} and Tlr3 ^{− / −} mice subjected to TBI and allogeneic BMT. n = 5; ^** p <0.01 (log rank test). (b) Severity of diarrhea in Tlr3 ^{+ / +} and Tlr3 ^{− / −} mice. n = 5; results are mean ± standard error; ^* p <0.05, ^** p <0.01 (unpaired two-tailed Student's t-test). (c) Weight loss on day 5 of Tlr3 ^{+ / +} and Tlr3 ^{− / −} mice. 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 μm. 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). (e) H & E staining of small intestinal crypts on day 0 and day 3 of Tlr3 ^{+ / +} and Tlr3 ^{− / −} mice. Scale bar is 100 μm. An asterisk indicates a microcolony. The right panel shows the number of microcolonies. n = 3; results are mean ± standard error; ^** p <0.01 (unpaired two-tailed Student's t-test). (f) Immunohistochemical detection of BrdU in the small intestinal crypts on day 0 and day 3 of Tlr3 ^{+ / +} and Tlr3 ^{− / −} mice. Scale bar is 100 μm. The right panel shows the number of cells that have incorporated BrdU. 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 μm. The results are representative of 3 independent experiments. Stimulation of TLR3 with a ligand directly induces cell death in the small intestinal crypt. (a) RT-PCR (upper) and quantitative real-time PCR (lower) of Tlr3 mRNA in the small intestine of Tlr3 ^{+ / +} and Tlr3 ^{− / −} mice. Cr: crypts. LP: Mucosal intrinsic cells. M: Size marker. SP: Spleen. V: Villi. W: Whole intestine. n = 3; results are mean ± standard error. (b) Representative images of Tlr3 ^{+ / +} organoids and Tlr3 ^{− / −} organoids. The crypts were incubated with poly I: C after 6 days in culture. Scale bar is 100 μm. (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). (d) TUNEL staining of Tlr3 ^{+ / +} organoid and Tlr3 ^{− / −} organoid. TUNEL-stained epithelium exhibits green fluorescence. Nuclei were stained with DAPI (blue). The box is an enlarged view of the crypt-like domain. Scale bar is 100 μm. (e) 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 μm. The right panel shows the number of TUNEL positive cells. Results are mean ± standard error; ^** p <0.01 (unpaired two-tailed Student's t-test). The results are representative of 3 independent experiments. TLR3 mediates radiation-induced crypt cell death via the TRIF-RIP1 pathway. (a) TUNEL staining of small intestinal crypts of Tlr3 ^{− / −} mice, Inf3 ^{− / −} mice, and Ifnar3 ^{− / −} mice 6 hours after TBI and allogeneic BMT. TUNEL-stained epithelium exhibits green fluorescence. Nuclei were stained with DAPI (blue). Scale bar is 100 μm. 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). (b) H & E staining of small intestinal crypts on day 3 of Tlr3 ^{− / −} mice, Inf3 ^{− / −} mice, and Ifnar3 ^{− / −} mice. Scale bar is 100 μm. 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). (c) TUNEL staining of small intestinal crypts 6 hours after TBI and allogeneic BMT of mice treated or not treated with Nec-1. TUNEL-stained epithelium exhibits green fluorescence. Nuclei were stained with DAPI (blue). Scale bar is 100 μm. The right panel shows the number of TUNEL positive cells. n = 5; results are mean ± standard error; ^** p <0.01 (unpaired two-tailed Student's t-test). (d) TUNEL staining of small intestinal crypts 3 days after mice treated with or without Nec-1. Scale bar is 100 μm. An asterisk indicates a microcolony. The right panel shows the number of microcolonies. n = 5; results are mean ± standard error; ^** p <0.01 (unpaired two-tailed Student's t-test). The results are representative of 3 independent experiments. TLR3-mediated crypt cell death after TBI is p53-dependent. (a) TUNEL staining of small intestinal crypts of p53 ^{+ / +} mice and p53 ^{− / −} mice 6 hours after TBI and allogeneic BMT. TUNEL-stained epithelium exhibits green fluorescence. Nuclei were stained with DAPI (blue). Scale bar is 100 μm. The lower panel shows the number of TUNEL positive cells. 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. Scale bar is 100 μm. The lower panel shows the number of p53 positive cells. n = 3; results are mean ± standard error. ND is not detected. (d) 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). (e) TUNEL staining of small intestinal crypts 6 hours after administration of poly I: C in p53 ^{+ / +} mice and p53 ^{− / −} mice. TUNEL-stained epithelium exhibits green fluorescence. Nuclei were stained with DAPI (blue). Scale bar is 100 μm. The right panel shows the number of TUNEL positive cells. n = 3; results are mean ± standard error; results are representative of 3 independent experiments. Intestinal microbiota is not associated with crypt cell death after TBI. (a) TUNEL staining of small intestine crypts of SPF mice and GF mice 6 hours after TBI and BMT. TUNEL-stained epithelium exhibits green fluorescence. Nuclei were stained with DAPI (blue). Scale bar is 100 μm. The right panel shows the number of TUNEL positive cells. n = 3; results are mean ± standard error. (b) H & E staining of crypts on day 3 of SPF and GF mice. Scale bar is 100 μm. The right panel shows the number of microcolonies. n = 3; results are mean ± standard error. The results are representative of two independent experiments. Intracellular RNA leaks from irradiated crypt cells in a p53-dependent manner, which induces TLR3-mediated crypt cell death. (a) Transiently transfect HEK293 cells with human TLR3 expression vector and ISRE-luciferase reporter plasmid (HEK293-hTLR3) and incubate with homogenate of irradiated small intestine or treated with pronase, DNase, or RNase did. Reporter activity was measured with Triplicate. Results are mean ± standard error; ^* p <0.05 (unpaired two-tailed Student's t-test). (b) Quantification of the size distribution of total RNA in the culture supernatant of crypt chorionic organoids of p53 ^{+ / +} and p53 ^{− / −} mice one day after irradiation. (c) Representative images of crypt villi organoids of Tlr3 ^{+ / +} and Tlr3 ^{− / −} mice one day after irradiation. The crypts were RNase treated or untreated. The scale bar is 200 μm. The right panel shows the survival rate. Survival was measured by triplicate. Results are mean ± standard error. NS is not significantly different. ^* p <0.05 (unpaired two-tailed Student's t-test). (d) HEK293-hTLR3 cells were incubated with medium alone (Med), purified RNA, or poly I: C for 12 hours. Reporter activity was measured by triplicate. Results are mean ± standard error. ^* p <0.05, ^** p <0.01 (unpaired two-tailed Student's t-test). (e) Representative images (left) and viability (right) of in vitro cultured crypts 2 days after treatment with Tlr3 ^{+ / +} and Tlr3 ^{− / −} mice with Med or purified RNA. The scale bar is 200 μm. Survival was measured by triplicate. Results are mean ± standard error. ^* p <0.05, ^** p <0.01 (unpaired two-tailed Student's t-test). The results are representative of 3 independent experiments. Inhibition of TLR3 binding to RNA improves radiation-induced GIS. (a) Kaplan-Meier survival analysis of mice treated with TLR3 / dsRNA binding inhibitors prior to TBI and allogeneic BMT. n = 6; ^** p <0.01 (log rank test). (b) and (c) Diarrhea severity and weight loss in mice pre-treated or untreated with TLR3 / dsRNA binding inhibitors, respectively. n = 6; results are mean ± standard error; ^* p <0.05, ^** p <0.01 (unpaired two-tailed Student's t-test). (d) TUNEL staining of mouse small intestinal crypts 6 hours after treatment with TLR3 / dsRNA binding inhibitor. Scale bar is 100 μm. 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). (e) H & E staining of small intestinal crypts of mice 3 days after treatment with TLR3 / dsRNA binding inhibitor. Scale bar is 100 μm. An asterisk indicates a microcolony. The right panel shows the number of microcolonies. n = 5; results are mean ± standard error; ^** p <0.01 (unpaired two-tailed Student's t-test). The results are representative of 3 independent experiments. Post-treatment with TLR3 / dsRNA binding inhibitors improves radiation-induced GIS. (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). (b) and (c) The severity and weight loss of diarrhea in mice post-treated or untreated with TLR3 / dsRNA binding inhibitors, respectively. 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.

(Pharmaceutical composition)
[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.
The 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.

[Small molecule compound that inhibits the interaction between TLR3 and double-stranded RNA]
A low molecular weight compound that inhibits the interaction between TLR3 and double-stranded RNA is not particularly limited. For example, compound (I) described in International Publication WO2012 / 099785 is known.

[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 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. ]

More specifically, the compound (I) is represented by the following formula (II).

[Where:
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. ]

The disclosure of International Publication No. WO2012 / 099785 is hereby incorporated by reference in its entirety.
The present inventors can administer (R) -2- (3-chloro-6-fluorobenzo [b] thiophene-2-carboxamido) -3-phenylpropanoic acid either before or after irradiation. It has been demonstrated to suppress radiation-induced intestinal crypt cell death and suppress the onset and exacerbation of gastrointestinal syndrome. The compound has been sold as a reagent so far.

〔antibody〕
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.
In the present specification, “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. For example, 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.
If a non-human monoclonal antibody that efficiently inhibits the binding between TLR3 and double-stranded RNA can be obtained, it 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.

In addition, 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.
Next, a human antibody of an anti-TLR3 antibody can be expressed by culturing the transformant under appropriate conditions.
Examples of 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.

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.
When the anti-TLR3 antibody of the present invention is a low molecular antibody such as a Fab fragment, F (ab ′) 2 antibody, scFv, etc., it 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.

[Dominant negative mutant]
As a TLR3 inhibitor, a dominant negative mutant of TLR3 may be used. In this case, 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. For example, 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 And 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.

[Double-stranded nucleic acid having RNAi effect, antisense nucleic acid, ribozyme]
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. 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. In general, 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. . For example, 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. 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. When 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.
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.

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.

[Inhibitor of TLR3-dependent and RIP1-mediated cell death induction signaling]
One embodiment of the pharmaceutical composition for preventing or treating radiation-induced gastrointestinal syndrome according to the present invention comprises the inhibitor of TLR3-dependent and RIP1-mediated cell death induction signaling shown in FIG.
For example, known inhibitors for RIP1 (receptor interacting protein 1), RIP3 (receptor interacting protein 3), and FADD (Fas-associated protein with Death Domain) may be used.
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.
The terms 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. For example, intravenous injection such as infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, enema, oral enteric solvent, etc. can be selected, and the administration method should be selected appropriately depending on the age and symptoms of the patient Can do.

In the pharmaceutical composition of the present invention, 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. For example, 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, preservative, stabilizer, buffer, suspension , Isotonizing agents, binders, disintegrants, lubricants, fluidity promoters, but flavoring agents and the like without limitation.

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.

When 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. In addition, 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.

Moreover, 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.

[Prevention method, treatment method]
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). For example, if irradiation of the gastrointestinal region is unavoidable, such as full irradiation of the abdomen.
In the case of radiotherapy, it is possible to prevent the digestive tract syndrome prophylactically by administering the pharmaceutical composition of the present invention prior to irradiation. In addition, as demonstrated by the present inventors, 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. By administering to the gastrointestinal tract, it is possible to suppress digestive tract syndrome.
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).

[Screening method]
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.

In the screening method according to the present invention, subsequently,
(i) inhibition of binding between TLR3 and double-stranded RNA by a candidate compound, or
(ii) The candidate compound is evaluated by measuring TLR3-dependent and RIP1-mediated cell death induction signaling inhibition by the candidate compound.
These methods can also be appropriately performed by those skilled in the art according to a known method or a method analogous thereto.

Hereinafter, the present invention will be specifically described based on examples, but the present invention is not limited thereto. Those skilled in the art can change the present invention into various modes without departing from the meaning of the present invention, and such changes are also included in the scope of the present invention.

(Materials and methods)
〔mouse〕
Wild-type BALB / c mice (female, 8-12 weeks old) under SPF or GF conditions were purchased from CLEA Japan. Tlr2 ^-/- , Tlr3 ^-/- , Tlr4 ^-/- , Tlr5 ^-/- , Tlr7 ^-/- , Tlr9 ^-/- , Trif ^-/- , Irf ^-/- mice are 8 generations in the BALB / c background This was produced by backcrossing (References 12, 23-28). Ifnar ^-/- mice (BALB / c) (reference 29) and p53 ^-/- mice (BALB / c) (reference 30) are provided by heterozygote-deficient mice (S. Miyatake and N. Mori, respectively) Received). 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 treatment]
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.

(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.

[Radiation treatment]
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). In some experiments, mice were treated with 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. Unless otherwise noted, 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.

[White blood cell count in blood and BM]
Blood was collected from the retroorbital vein on days 0 and 5 after irradiation. Blood was treated with erythrocyte lysis buffer (Sigma-Aldrich) to remove erythrocytes. Thereafter, the cells were suspended in PBS, an aliquot was mixed with Turk solution, and leukocytes were counted using a blood cell system.
BM cells were collected from the femur by flowing Hanks' balanced salt solution using a 28-gauge needle and syringe on days 0 and 5 after irradiation. Red blood cell lysis and white blood cell count were performed as described above.

[Histological analysis]
After irradiation or poly I: C treatment, the jejunum tissue was excised from the small intestine 4 cm below the gastroduodenal boundary at 0, 6, 72, and 120 hours, washed with PBS, and fixed overnight with 10% formalin. And embedded in paraffin. 5 μm sections were stained with hematoxylin-eosin (H & E) and observed with an optical microscope using BZ-II Image Analysis Application (BZ-9000, Keyence). Crypt survival was assessed 3 days after irradiation by a modified microcolony assay (Ref. 9). Briefly, the number of “regenerative crypts” in jejunal sections was counted based on histological appearance. Here, 10 or more chromophilic non-panate cells and those containing at least one panate cell were defined as regenerating crypts. On days 0 and 5 after irradiation, 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.

[Immunohistochemical analysis]
To label S phase cells in the proliferating phase in the small intestine, mice were injected intraperitoneally with BrdU (5-bromo-2'-deoxyuridine; 50 mg / kg body weight) one hour prior to sacrifice. 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.
To detect p53 protein accumulation, sections were treated with mouse anti-p53 antibody (x100, Pab 240, Abcam) at 4 ° C overnight and in citrate buffer (pH 6.0) at 121 ° C for 15 minutes. 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.

[RT-PCR and quantitative real-time PCR]
According to the method of Fujimoto et al. (Reference 31) and Sato et al. (Reference 10), 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. for 30 minutes to remove epithelial cells, and then washed well with fresh PBS. The small intestine fragment was stirred continuously at 37 ° C for 60 minutes with 0.425 mg / ml Liberase T-flex (Roche) and 100 mg / ml DNase I (Roche) in RPMI 1640 supplemented with 10% fetal calf serum. And digested. Suspended cells were subjected to density gradient centrifugation at 40% and 75% (vol / vol) Percoll. Cells collected from the interface were washed and used as mucosal intrinsic cells. Total RNA was isolated using Trizol reagent (Invitrogen) according to the instructions for use. Subsequently, RQ1 RNase-free DNase (Promega) was added to the total RNA sample 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 μg 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.

[Preparation of small RNA fraction and large RNA fraction]
Total 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).

[In vitro crypt culture]
Small intestinal crypts were isolated as described above. The crypts were mounted on Matrigel (Bd Biosciences) and seeded in 48 well flat bottom plates to contain 200 crypts in 100 ml Matrigel. Each well contains 2% fetal bovine serum, 100 U / ml penicillin, 100 mg / ml streptomycin, 10 mM HEPES, Glutamax (Invitrogen), 1 × N2 (Invitrogen), 1 × B27 (Invitrogen), 1 mM N-acetyl Cysteine (Nakalai Tesque), 10 mM Y-27632 (Stemgent) and growth factors (50 ng / ml epidermal growth factor, 100 ng / ml noggin, and 1 mg / ml R-spondin-1) (all R & D Systems) Advanced DMEM / F12 (Invitrogen) containing was overlaid. The medium was added every other day, and all mediums were changed after 4 days of culture. At that time, Y-27632 was not added, nor was it added to the subsequent culture. On days 5-6 of culture, 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. In order to evaluate the cell killing effect on cultured crypts by irradiation or RNA, cell viability was measured using MTT (3- (4,5-dimethyl-2-thiazolyl) using MTT cell count kit (Nacalai Tesque). -2,5-diphenyltetrazolium bromide). The crypts were cultured with 500 mg / ml MTT for 1 hour and collected on Matrigel with Cell Recovery Solution (BD Bioscience). Formazan precipitated in the crypts was dissolved and the absorbance at 570 nm was measured. The survival rate of crypt cells was calculated according to the following formula.
Viability (%) = (value of treated crypt cells / value of untreated crypt cells) × 100
For whole mount analysis, 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. One day after the irradiation, the supernatant was collected, and 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.

[Preparation of 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. For enzyme digestion assays, 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. .

[Reporter assay]
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. Cells were then incubated with 20 mg / ml purified RNA or 0.5 mg / ml poly I: C complexed with Lipofectamine 2000 reagent, or intestinal homogenate. After 12 hours of culture, 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 ⁺ spleen tree cell culture]
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-α4, and IFN-β were measured by quantitative real-time PCR.

〔Statistical analysis〕
Statistical significance was assessed by a two-sided test with unpaired Student's st-test. Differences in survival rate between groups after irradiation were evaluated by Kaplan-Meier plot and log-rank tests. A p-value of less than 0.05 was considered statistically significant.

(result)
TLR3 is an exacerbation factor of radiation-induced gastrointestinal syndrome (GIS).
To investigate the effect of TLR3 activation on GIS, BALB / c background Tlr3 ^{+ / +} and Tlr3 ^{− / −} mice were synthesized TLR3 prior to total body γ-irradiation (TBI). Treated with the ligand poly I: C (reference 7).
Since BALB / c mice are known to have high radiosensitivity, 10-Gy TBI was performed to evaluate radiosensitivity (Reference 8). No protective effect was observed with poly I: C treatment, which significantly increased the TBI sensitivity of Tlr3 ^{+ / +} mice (FIG. 1a). 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.

Next, the sensitivity of other TLR family-deficient mice to radiation-induced GIS was examined. However, unlike Tlr3 ^{− / −} mice, other TLR-deficient mice did not show changes in survival, diarrhea, and weight loss after TBI compared to wild type mice. Subsequently, HPS of Tlr3 ^{− / −} mice was examined. However, there was no difference in bone marrow (BM) suppression and leukopenia between Tlr3 ^{+ / +} and Tlr3 ^{− / −} mice after TBI (FIGS. 1d-f). Thus, TLR3 is specifically activated after TBI and exacerbates GIS.

TLR3 mediates crypt cell death in radiation-induced GIS.
To eliminate the possibility of hematopoietic syndrome (HPS), Tlr3 ^{+ / +} and Tlr3 ^-/- mice were subjected to bone marrow transplantation (BMT) immediately after TBI. 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. Compared to Tlr3 ^{+ / +} BMT mice, Tlr3 ^{− / −} BMT mice had significantly less diarrhea symptoms and weight loss (FIGS. 2b, c). Thus, with Tlr3 ^-/- mice, GIS was relaxed.

In addition, 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. As a result, 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.
Next, a TUNEL assay was performed to examine cell death of crypt epithelial cells in the small intestine. As shown in FIG. 2d, after 6 hours from TBI, the number of TUNEL positive cells per crypt in Tlr3 ^{− / −} mice was significantly less than in Tlr3 ^{+ / +} mice. Thus, TLR3 is involved in cell death of crypt cells after irradiation.
Three days after TBI, 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).

Next, we compared the histological expression of GIS in the small intestine of Tlr3 ^{+ / +} and Tlr3 ^{− / −} mice after TBI. 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. On day 5, 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). Examination of chorionic epithelial cell death showed little increase in TUNEL positive cells in both Tlr3 ^{+ / +} and Tlr3 ^{− / −} mice at 6 hours and 3 days after TBI. In addition, after 6 hours and 3 days after TBI, there was almost no accumulation of inflammatory cells in the small intestinal villi in both Tlr3 ^{+ / +} mice and Tlr3 ^{− / −} mice. Thus, neither radiation-induced epithelial cell death nor inflammation had an effect on villi integrity.

These results show that 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). Both the crypts of Tlr3 ^{+ / +} and Tlr3 ^{− / −} mice formed crypt-villus organoids as well. In the crypt chorionic organoids, the crypt domain surrounded the central tubular structure lined by the villi-like epithelium and budding was observed continuously. When poly I: C was administered, the crypt domain disappeared in Tlr3 ^{+ / +} organoids. In contrast, Tlr3 ^{− / −} organoids maintained the structure of the crypt villi organoids regardless of poly I: C stimulation.
Next, the cell viability of Tlr3 ^{+ / +} organoid and Tlr3 ^{− / −} organoid was evaluated by MTT assay (FIG. 3 c). After poly I: C treatment, the cell viability of Tlr3 ^{+ / +} organoids decreased dramatically, but the viability of Tlr3 ^{− / −} organoids did not change. Furthermore, whole mount analysis showed that poly I: C stimulation increased the number of TUNEL positive cells in the crypt domain of Tlr3 ^{+ / +} organoids, but not Tlr3 ^{− / −} organoids (FIG. 3d). ). Furthermore, the effect of poly I: C on small intestine crypts in vivo was investigated. Intraperitoneal administration of poly I: C induced the appearance of TUNEL positive crypt cells in Tlr3 ^{+ / +} mice, but not in Tlr3 ^{− / −} mice (FIG. 3e). Thus, ligand stimulation of TLR3 directly induces extensive cell death in the crypt epithelium.

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. Unlike other members of the TLR family, TLR3 does not use the adapter myeloid differentiation factor 88 (MyD88) (11, 12). Instead, 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. Consistent with survival and symptoms, crypt cell death was reduced in Trif ^{− / −} mice than in Trif ^{+ / +} mice (FIGS. 4a, b). Thus, 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). Furthermore, no significant differences in survival rate, symptoms, and crypt damage were observed between Ifnar ^{− / −} mice and Ifnar ^{+ / +} mice (FIGS. 4a and b). Thus, IRF3 and type 1 IFN were not involved in the cause of GIS. The N-terminal region of TRIF is essential for TRIF-mediated IRF3 activation, whereas the C-terminal region has been reported to be important for NF-κB activation and cell death. Receptor-interacting protein 1 (RIP1), a death domain with a kinase inside, binds to TRIF via the RIP homologous interaction motif domain in the C-terminal region, and activates NF-κB and cell death This signal is transmitted (References 13 and 14). To examine the involvement of RIP1 in GIS after TBI, 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. Furthermore, 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).

These results suggest that 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). In TLR3-mediated apoptosis, 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). In contrast, necroptosis is a programmed necrosis that is induced by a ligand-activated death domain receptor when caspase function is absent or inhibited (16). . In the crypts of Tlr3 ^{+ / +} mice, there was an increase in activated caspase 3-positive cells after TBI and poly I: C administration. In Tlr3 ^{− / −} mice, the number of activated caspase 3 positive cells was significantly reduced after TBI and poly I: C administration. Thus, 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). In fact, in p53 ^{− / −} mice after TBI, the induction of TUNEL positive crypt cells and Bax and Puma mRNA was almost completely suppressed (FIGS. 5a and b). Thus, crypt cell death 6 hours after TBI was p53-dependent. Interestingly, TUNEL positive crypt cells were still detected in Tlr3 ^{− / −} mice 6 hours after TBI (FIG. 2d). Six hours after TBI, accumulation of P53 protein in crypt cells and up-regulation of Bax and Puma mRNA were induced in the small intestine of Tlr3 ^{+ / +} and Tlr3 ^{− / −} mice to a significantly similar degree (FIG. 5c). D). Thus, signaling of p53-mediated cell death after TBI was not lost in Tlr3 ^{− / −} mice.
According to a recent report, TLR3 induction is regulated by p53 in several cancer cell lines (Reference 19). However, there was no significant difference in Tlr3 mRNA expression in the small intestine between p53 ^{+ / +} and p53 ^{− / −} mice regardless of TBI. Next, 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). These results indicate that TLR3 on crypt cells is not activated without p53-dependent events after irradiation.

Intestinal microbiota is not important for crypt cell death after TBI.
Next, it was examined which substances activate TLR3 to induce crypt cell death after TBI. 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).

Self RNA induces 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. Interestingly, the small intestine homogenate significantly stimulated ISRE reporter activity in HEK293 cells when transiently transfected with an expression plasmid encoding TLR3 (FIG. 7a). In addition, RNase treatment decreased the ability to stimulate ISRE reporter activity via TLR3 of small intestine homogenate, but not proteinase treatment or DNase treatment. Thus, it was suggested that small intestine homogenate RNA stimulates TLR3.

Next, using an in vitro crypt culture system, it was investigated whether radiation caused leakage of intracellular RNA. Small intestine crypts derived from p53 ^{+ / +} mice and p53 ^{− / −} mice were cultured, and morphological changes after gamma irradiation and organoid survival were examined. After gamma irradiation, the crypt domain disappeared from the p53 ^{+ / +} organoid, but not from the p53 ^{− / −} organoid. In the MTT assay, γ-irradiation significantly reduced the viability of p53 ^{+ / +} organoids, but did not reduce the viability of p53 ^{− / −} organoids. Thus, crypt cell death caused by gamma irradiation in vitro is p53 dependent.
Next, 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. Interestingly, 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).
In addition, we compared radiation-induced cell death of crypt villi organoids in Tlr3 ^{+ / +} and Tlr3 ^{− / −} mice. Compared to Tlr3 ^{+ / +} organoids, loss of crypt domains after irradiation in Tlr3 ^{− / −} organoids was clearly mitigated (FIG. 7c). In addition, the cell viability of Tlr3 ^{− / −} organoids was significantly higher than that of Tlr3 ^{+ / +} organoids. Interestingly, incubation of organoids with RNase improved Tlr3 ^{+ / +} organoid atrophy and increased survival to the same extent as Tlr3 ^{− / −} organoids.

The above results suggested that endogenous RNA derived from crypt cells that died p53-dependently after γ-irradiation induces extensive cell death via TLR3.
Next, it was examined whether RNA isolated from the small intestine after TBI stimulates ISRE reporter activity via TLR3. As a result, irradiated small intestine-derived total RNA significantly increased ISRE reporter activity in a TLR3-dependent manner (FIG. 7d). As shown in FIG. 7b, a small RNA group of 200 nucleotides or less was detected from the culture supernatant of p53 ^{+ / +} organoid after γ-irradiation. Recently, siRNA (small interfering RNA) was shown to suppress angiogenesis via TLR3 (Reference 21). Therefore, we thought that small non-coding RNAs such as host-derived microRNAs are involved in TLR3-mediated crypt cell death after TBI. However, stimulation of ISRE reporter activity via TLR3 by small RNAs of 200 nucleotides or less was not observed (FIG. 7d).
Contrary to expectations, large RNAs of 4200 nucleotides or more significantly stimulated the ISRE reporter via TLR3. Next, we examined whether 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). After treatment with large RNA, the survival rate of Tlr3 ^{+ / +} organoids was significantly reduced, but the survival rate of Tlr3 ^{− / −} organoids was not reduced. Thus, it was suggested that large RNA derived from irradiated small intestinal cells induces crypt cell death via TLR3.

TLR3 / dsRNS binding inhibitors suppress mouse GIS.
The above results strongly suggested that endogenous RNA could induce TLR3-dependent crypt cell death in GIS. To elucidate the mechanism in vivo, (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 CD8α-positive splenic dendritic cells after poly I: C stimulation. This upregulation is completely dependent on TLR3 (Ref. 6). Treatment of 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.

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Claims (13)

1. A pharmaceutical composition for preventing or treating radiation-induced gastrointestinal syndrome, comprising a Toll-like receptor (TLR) 3 inhibitor.
2. The pharmaceutical composition for preventing or treating radiation-induced gastrointestinal syndrome according to claim 1, wherein the TLR3 inhibitor is a substance that inhibits the interaction between TLR3 and double-stranded RNA.
3. The radiation-induced gastrointestinal tract according to claim 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 a 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 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. ]
4. The pharmaceutical composition for preventing or treating radiation-induced gastrointestinal syndrome according to claim 3, wherein the compound (I) is represented by the following formula (II).
   

   

   [Where:
   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. ]
5. The radiation-induced gastrointestinal syndrome according to claim 3, wherein the compound (II) is (R) -2- (3-chloro-6-fluorobenzo [b] thiophene-2-carboxamido) -3-phenylpropanoic acid. A pharmaceutical composition for prevention or treatment.
6. The Toll-like receptor (TLR) 3 inhibitor is
   The pharmaceutical composition for preventing or treating radiation-induced gastrointestinal syndrome according to claim 1, which is a dominant negative mutant of TLR3 or an anti-TLR3 antibody.
7. The Toll-like receptor (TLR) 3 inhibitor is
   The radiation-inducible protein according to claim 1, which is a nucleic acid selected from the group consisting of a double-stranded nucleic acid having RNAi effect, an antisense nucleic acid, a ribozyme, a miRNA and a nucleic acid encoding the same, which inhibits the expression of the TLR3 gene. A pharmaceutical composition for preventing or treating gastrointestinal syndrome.
8. A pharmaceutical composition for preventing or treating radiation-induced gastrointestinal syndrome, comprising an inhibitor of TLR3-dependent and RIP1-mediated cell death induction signaling.
9. The prevention or treatment of radiation-induced gastrointestinal syndrome according to claim 8, wherein the inhibitor of TLR3-dependent and RIP1-mediated cell death induction signaling is any one of RIP1, RIP3 and FADD. Pharmaceutical composition.
10. The pharmaceutical composition for prevention or treatment of radiation-induced gastrointestinal syndrome according to claim 9, wherein the inhibitor of any of RIP1, RIP3 and FADD is Necrostatin-1, a salt thereof, or a solvate thereof.
11. 10. The radiation according to claim 9, wherein the inhibitor of any of RIP1, RIP3 and FADD is an antibody to any of RIP1, RIP3 and FADD, a dominant negative mutant, or any of RIP1, RIP3 and FADD. A pharmaceutical composition for preventing or treating inducible gastrointestinal syndrome.
12. 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, an miRNA, and the like that inhibits the expression of any of RIP1, RIP3, and FADD genes The pharmaceutical composition for prevention or treatment of radiation-induced gastrointestinal tract syndrome according to claim 9, which is a nucleic acid selected from the group consisting of:
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 inhibition of TLR3-dependent and RIP1-mediated cell death induction signaling by the candidate compound.

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