JP2007031414A - Therapeutic agent of mucous membrane damage of digestive tract by using il-13 signaling inhibition as mechanism, and method for screening medicament - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a therapeutic agent of the mucous membrane damage of the digestive tract, and a method for screening the agent. <P>SOLUTION: The therapeutic agent of the mucous membrane damage of the digestive tract by using the inhibition of signaling by IL-13 as a mechanism is provided. An IL-13Rα2 receptor protein or a solubilized IL-13Rα2 protein, an anti-IL-13 antibody, or a protein binding with the IL-13 and deactivating the IL-13, a solubilized IL-13Rα1 receptor, an anti-IL-13Rα1 receptor, an IL-13Rα1 receptor antagonist, an IL-13 precursor gene or an antisense oligonucleotide or siRNA against the IL-13Rα1 gene, an aptamer against the IL-13 or IL-13Rα1, and the like are cited as the active ingredient of the therapeutic agent of the mucous membrane damage of the digestive tract. The method for screening a material having therapeutic effects of the mucous membrane damage of the digestive tract, and the kit therefor are also provided. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a therapeutic agent for gastrointestinal mucosa injury and a screening method for the therapeutic agent for gastrointestinal mucosa injury.

  The gastrointestinal mucosa is known to be damaged by various causes such as physical factors, stress, infection, drugs, radiation exposure, and autoimmune reaction. It is known that gastrointestinal mucosal damage caused by drugs is caused by, for example, side effects of non-steroidal anti-inflammatory drugs and multiple anticancer drugs. In addition, gastrointestinal mucosal injury may occur in the treatment of cancer by radiation.

  By performing treatment that promotes the repair and regeneration of injured mucosa, it is expected to promote early healing and to form a robust regenerative tissue. For gastric ulcers and gastritis, a drug treatment that promotes the repair and regeneration of injured gastrointestinal mucosa is performed using a drug having an action of promoting wound healing. Examples of such therapeutic agents are sucralfate, aldioxa, azulene sulfonate sodium, L-glutamine, gefarnate, cefalcon, teprenone, rebamipide, eguarene sodium, solcoseryl, polaprezinc.

  However, there are many unclear points regarding the mechanism of healing and regeneration of damaged gastrointestinal mucosa. Unlike in the case of gastric ulcer and gastritis, no pharmacotherapy has been established for injured mucosa of the small intestine, large intestine, and esophagus to promote repair and regeneration of the damaged mucosa. Research on clinical application of some of the above gastric / duodenal mucosal regeneration promoters, various cell growth factors such as KGF, GM-CSF, and EGF, and heparin that assists the activity of cell growth factors is underway. However, drug therapy has not yet been established. Therefore, in the technical field of gastrointestinal mucosal injury therapeutic agents, the creation of a drug that promotes the repair / regeneration of damaged mucosa is an unsolved problem.

  Helper T cells (Th cells) are immunocompetent cells that play a role in regulating the immune system, and their importance for gastrointestinal immunity is already known. Among Th cells, Th1 cells are activated by IL-12 and produce interferon-γ (IFN-γ), IL-2, IL-12, cytotoxic CD8-positive T cells, NK cells, neutrophils It is involved in cellular immunity by activating spheres and macrophages. Th2 cells are activated by a mechanism different from Th1 cells and produce IL-4, IL-5, IL-6, IL-10, IL-13, promote B cell differentiation and antibody production, I am involved in immunity. The immune reaction processes activated by Th1 cells and Th2 cells and cytokines involved in these processes are called Th1 type and Th2 type, respectively.

  Th1 and Th2 cells differentiate from Th0, a common progenitor cell. Th1-type cytokines inhibit Th2 cell activation, and Th2-type cytokines inhibit Th1 cell activation. From these facts, a relationship is considered between Th1 cells and Th2 cells, where one becomes dominant and the other becomes inferior.

  IL-4 and IL-13 are major immunoregulatory molecules involved in Th2-type immune activity, and are produced by Th2 cells, natural killer T (NKT) cells, mast cells, neutrophils, and the like. IL-4 and IL-13 play an important role in Th2 cell differentiation and antibody production by B cells.

  Although it is known that IL-4 and IL-13 have a common physiological action, this commonality is thought to be caused by sharing the receptor molecules of these cytokines. FIG. 1 is a schematic diagram relating to receptors for these cytokines. Charamonte et al. State that the most general understanding of these cytokine receptors is as follows (Chraramonte et al., J Exp Med 2003, 197: 687-701). Type 1 IL-4 receptor is a complex of IL-4 receptor α chain (IL-4Rα) and cytokine receptor common γ chain (γc), and IL-4 binds to this receptor. However, IL-13 does not bind. In the type 2 IL-4 receptor, IL-4Rα and IL-13 receptor α1 chain (IL-13Rα1) form a complex, and IL-4 and IL-13 can bind together. This receptor produces an intracellular signal when IL-4 or IL-13 binds. IL-4 receptor antagonists suppress the respiratory inflammatory response stimulated by IL-13 as well as IL-4 (Tomkinson et al., J Immunol 2001, 166: 5792-5800). IL-13Rα1 binds to tyrosine kinase 2 and regulates the signal of JAK (Janus kinase) -STAT (signal transducer and activator of transcription) system (Umeshita-Suyama et al., Int Immunol 2000, 12: 1499- 1509). In addition, there is a type of IL-13 receptor in which IL-13 receptor does not form a complex with IL-4Rα receptor, and IL-13 receptor sends a signal into the cell by binding to IL-13 It has also been suggested (Mattes et al., J Immunol 2001, 167: 1683-1692).

  IL-13 receptor protein includes IL-13 receptor α2 chain (IL-13Rα2) in addition to the above-mentioned IL-13Rα1. It is thought that IL-4 does not bind to this receptor. The affinity of IL-13 for IL-13Rα2 is higher than the affinity for IL-13Rα1 contained in the IL-4 type 2 receptor. IL-13Rα2 exists as a soluble protein in vivo and is known to exist away from the cell membrane. IL-13Rα2 is thought not to signal intracellularly even when IL-13 binds. That is, IL-13Rα2 is understood to be a decoy receptor that suppresses IL-13 binding to the IL-4 type 2 receptor by trapping IL-13 (Taube et al., J Immunol 2002, 169: 6482-6489).

  IL-4 and IL-13 are known to exhibit various physiological actions in addition to the regulation of immunocompetent cell activation. IL-4 and IL-13 are widely known to be important factors in the development of allergic airway inflammation and airway hyperresponsiveness. The onset of airway allergic reaction and interstitial pneumonia is not due to activation of B cells and antibody production, but is thought to be related to the action of IL-13 on epithelial and stromal cells (Wills- Karp et al., Science 1998, 282: 2258-2261). IL-13 effects include neutrophil accumulation in the airway epithelium, bronchial epithelial mucosal hyperproliferation, increased mucin production and hyaluronic acid accumulation, and increased fiber production. (Zhu et al., Am J Respir Crit Care Med 2001, 164: S67-70).

  Regarding fibrosis, Th2-type cytokines, especially IL-13, have been shown to play a major role in granuloma development and liver fibrosis in schistosomiasis (Chraramonte et al., J Clin Invest 1999, 104: 777-785). Increased release of IL-4 and IL-13 during nematode infection in the gastrointestinal tract, increased release of histamine and acetylcholine due to effects on the nervous system and direct action on mucosal cells, increased mucosal permeability and glucose It is involved in decreased absorption and is widely involved in host-side defense responses (Madden et al., J Immunol. 2004, 172: 5616-5621).

  IL-13 suppresses the immune surveillance mechanism against cancer (Berzofsky et al., J Clin Invest 2004, 113: 1515-1525). In addition, IL-4 and IL-13 exert a proliferation promoting action by directly acting on immune cell-derived cancer cells (Oshima et al., J Biol Chem 2000, 275: 14375-14380). In addition, effects on cancer cells derived from other than immune cells have been reported. IL-4 has been shown to proliferate pancreatic cancer cells (Prokopchuk et al., Br J Cancer 2005, 92: 921-928). On the other hand, IL-4 is known to suppress the growth of colon cancer and breast cancer cells. It has been suggested that IL-13 produced from NKT cells shows cytotoxicity against colon cancer-derived cell lines (Fuss et al., J Clin Invest 2004, 113: 1490-1497).

  Studies have also been conducted on the possibility that Th2-type cytokines are involved in cell rotation of gastrointestinal tissues other than cancer. HIV infection increases IL-13 gene expression, but it has been reported that administration of IL-13 to macaques infected with retrovirus (SIV) causes atrophic damage to the epithelium of the small intestine (Zou et al., AIDS Res Hum Retroviruses 1998, 14: 775-83).

  There are reports that IL-13 production is increased and decreased in lamina propria cells of ulcerative colitis (Fuss et al., J Clin Invest 2004, 113: 1490-1497; Kadivar et al., Inflamm Bowel Dis 2004, 10: 593-598), the relationship between IL-13 and ulcerative colitis is not completely understood.

  A group of Dohi researchers, one of the inventors of the present invention, studied the role of Th2-type cytokines on pathological changes due to inflammation in the gastrointestinal tract (Dohi et al., J Exp Med 1999, 189: 1169- 1180; Dohi et al., Gastroenterology 2000, 119: 724-733). When colitis was induced with trinitrobenzene sulfate (TNBS) in IFN-γ-deficient mice with predominantly Th2-type immune responses, inflammation accompanied by fibrosis of epithelial tissues and atrophic changes occurred extensively. This marked inflammatory response in Th2-dominant mice was attributed to differences in immune system regulation and the involvement of IL-4. On the other hand, in mice where the Th1-type immune response was dominant, acute ulcer was caused by administration of TNBS into the large intestine, but the injury was limited to the site of ulcer.

  Another group of studies has shown that IL-13, produced by NKT cells, is the primary agent against extensive colonic epithelial injury caused by colitis with the sensitizer oxazolone. (Heller et al., Immunity 2002, 17: 629-638).

In addition, Dohi's research group investigated the effects of Th1 and Th2 on gastrointestinal inflammation using adoptive transfer of CD4 ⁺ CD45RB ^hi T cells with gene mutations in T cell-deficient mice. If the Th2 dominance IFN-gamma deficient CD4 ⁺ CD45RB ^hi T cells were adoptively transferred the atrophy of the small intestine villi, metaplasia goblet cells, and colitis mild occurs (Dohi et al., Gastroenterology 2003 , 124: 672-682). On the other hand, when adoptive transfer of Th1-dominant IL-4 deficient CD4 ⁺ CD45RB ^hi T cells, hypertrophic erosive gastritis and duodenitis accompanied by apoptosis of surface epithelial cells, and severe colon inflammation were observed. (Dohi et al., Am J Pathol 2004, 165: 1257-1268).

  These studies suggest that IL-4 and IL-13 may be involved in atrophic changes of the small intestinal villi that occur with infection and inflammation. However, these studies have not clarified the mechanism of action of IL-4 and IL-13 on immune gastrointestinal tract cell rotation and the role played by gastrointestinal mucosal repair and regeneration.

  Therefore, in order to investigate the role of IL-4 and IL-13 in the repair and regeneration of the gastrointestinal mucosa, Dohi's research group caused damage that did not cause an inflammatory reaction by irradiation, and repaired and recovered from the injury. The dynamics of Th2-type cytokines during the process were examined (Kawashima et al., 33rd Annual Meeting of the Immunological Society of Japan, Title No. 3-B-W32-19-P, Annual Meeting of the Immunological Society of Japan, Record of Annual Meeting 2003, 33: 225 Kawashima et al., 33rd Annual Meeting of the Immunological Society of Japan, Title No. 1-H-W13-13-O / P, Annual Meeting of the Immunological Society of Japan, Academic Meeting Records 2004, 34: 115).

  Irradiation of 3 Gy radiation to BALB / c mice caused damage to the small intestinal epithelium. Jejunal specimens 1 day after irradiation showed villous damage and lamina propria lymphocyte reduction. Immunohistochemical examination showed a wide range of apoptotic cells, and proliferation that still shows repair and regeneration. Few cells showed a signal. On the other hand, 3 days after irradiation, the number of apoptosis-positive cells decreased, and epithelial cell division and staining images of cell proliferation markers were observed in the basal layer, indicating that regeneration started from the construction of villi.

  At that time, the mRNA expression of cytokines in the mucosal intrinsic stroma cells was measured on the first and third days after irradiation, and an increase in IFN-γ expression, which is known to increase after irradiation, was observed. It was. In addition, increased expression of IL-4 and IL-13 was observed. The lamina propria cells 3 days after 3 Gy irradiation were isolated, and the cell composition ratio was examined by flow cytometry. As a result, 3 days after irradiation, the total number of lamina propria cells decreased, and CD4-positive cells including Th cells almost disappeared. On the other hand, natural killer (NK) cells were also known to release IL-13 and tolerate radiation, but in this study, NK cells remain 3 days after irradiation, and the composition ratio is It increased to about 3/4 of lamina propria cells. In addition, IL-13 mRNA expression was confirmed in NK cells. These results indicate that NK cells were the source of IL-13 production after irradiation.

  Receptor genes in mucosal intrinsic stroma cells were examined 1 day, 3 days, and 5 days after irradiation. IL-4Rα and IL-13Rα1 mRNA did not show increased expression, but IL-13Rα2 MRNA expression increased. The increase in the expression level was highest after 3 days compared to 1 day and 5 days later. IL-13Rα2 was expressed in the basement membrane and myofibroblasts.

In addition, IL-4Rα-deficient (IL-4Rα ^{− / −} ) mice with impaired Th2-type immune activity were examined for intestinal tissue after irradiation. On the first day of irradiation, IL-4Rα ^{− / −} mice showed jejunal mucosal injury as well as wild type BALB / c mice. On the other hand, on the third day of irradiation, although regeneration started in wild-type BALB / c mice, regeneration did not occur in IL-4Rα ^-/- mice as in the first day after irradiation. It was.

  These results suggest that the Th2-type cytokines IL-4 and IL-13 are involved in the repair and regeneration of damaged gastrointestinal mucosa. On the other hand, it was an interesting finding that the expression of IL-13Rα2, a decoy receptor of IL-13, increased in mucosal intrinsic stroma cells after irradiation in BALB / c mice. It was not clear whether it played a role.

Kawashima et al., 33rd Annual Meeting of the Immunological Society of Japan, Title No. 3-B-W32-19-P, Annual Meeting of the Immunological Society of Japan, Record of Annual Meeting 2003, 33: 225 Kawashima et al., 33rd Annual Meeting of the Immunological Society of Japan, Title No. 1-H-W13-13-O / P, Annual Meeting of the Immunological Society of Japan, Record of Annual Meeting 2004, 34: 115

  An object of the present invention is to provide a therapeutic agent for gastrointestinal mucosa injury and a screening method for the therapeutic agent for gastrointestinal mucosa injury.

  The present invention provides a therapeutic agent for gastrointestinal mucosal injury having an action mechanism that inhibits signaling by IL-13. Preferably, the active ingredient of the therapeutic agent for gastrointestinal mucosa injury is not limited, but is a protein using IL-13Rα2 receptor protein or solubilized IL-13Rα2 protein or a part thereof, an anti-IL-13 antibody, or IL-13 Proteins that bind to and inactivate, solubilized IL-13Rα1 receptor, anti-IL-13Rα1 receptor antibody, IL-13Rα1 receptor antagonist, antisense oligonucleotide to IL-13 precursor gene or siRNA or IL-13 It is selected from the group consisting of aptamers, antisense oligonucleotides to IL-13Rα1 gene or siRNA or aptamers to IL-13Rα1. The therapeutic agent for gastrointestinal mucosa injury of the present invention is useful for treating gastrointestinal mucosal injury by promoting regeneration of the damaged gastrointestinal mucosa.

  In another aspect, the present invention relates to a method for assaying whether a test substance has a therapeutic effect on gastrointestinal mucosal injury, wherein the IL-13Rα2 receptor is present in the presence and absence of the test substance. A method comprising measuring a gene expression level or an amount of IL-13Rα2 protein is provided. In yet another aspect, the present invention relates to a method for assaying whether a test substance has a therapeutic effect on gastrointestinal mucosal injury, wherein IL-13 precursor is present in the presence and absence of the test substance. Includes measuring somatic gene expression or IL-13 expression, or IL-13Rα1 gene expression or IL-13Rα1 protein, or measuring the binding of a candidate substance to IL-13 or IL-13Rα1 Provide a method.

  In yet another aspect, the invention relates to IL-13Rα2, or an oligonucleotide or polynucleotide encoding IL-13Rα2, or a cell expressing IL-13Rα2, or an anti-IL-13Rα2 antibody, or IL-13, or IL-13-encoding oligonucleotide or polynucleotide, or IL-13-expressing cell, or anti-IL-13 antibody, or IL-13Rα1, or an oligonucleotide or polynucleotide encoding IL-13Rα1, or IL-13Rα1 Provided is a kit for screening a cell having a therapeutic effect on gastrointestinal mucosal injury, comprising an expressing cell or an anti-IL-13Rα1 antibody.

  The substance found by the screening method of the present invention is useful as a therapeutic agent for gastrointestinal mucosal injury. These therapeutic agents for gastrointestinal mucosal damage increase IL-13Rα2 expression, trap IL-13, suppress IL-13 expression, or IL-13 and IL-13Rα1. It is considered that the therapeutic effect of gastrointestinal mucosal injury can be exerted by inhibiting the function of IL-13 by inhibiting the binding of IL-13R or suppressing the expression of IL-13Rα1.

  Signaling by IL-13, a cytokine, is performed through the IL-13Rα1 receptor. That is, IL-13 released from the cell into the intercellular space binds to the IL-13Rα1 receptor on the cell membrane and generates a signal that regulates the activation of intracellular signal transduction pathways such as the JAK-STAT system. On the other hand, the IL-13Rα2 receptor was known to be a decoy receptor that binds to IL-13 but does not generate a signal into the cell. IL-13Rα2 functions to suppress IL-13 signaling by trapping IL-13 and decreasing the amount of IL-13 binding to IL-13Rα1. IL-13Rα2 is produced in vivo and is present on the cell membrane, but it also exists in the intercellular space as a soluble form and functions, so even when this receptor protein is administered exogenously. The effect can be demonstrated.

  The present inventors have newly clarified that IL-13Rα2 is essential for the regeneration process by inhibiting the process of regeneration of the gastrointestinal mucosa damaged by IL-13. Based on this, a therapeutic method was invented in which IL-13 is trapped and IL-13 signaling is suppressed by administering a pharmaceutical composition containing IL-13Rα2 as an active ingredient. That is, the present invention is characterized in that the mechanism of action is to inhibit signaling by IL-13. IL-13Rα2 is a 380 amino acid receptor protein (Caput et al., J Biol Chem 1996, 271: 16921-16926), and the base sequence of the gene is known.

  As long as it shows the same action as IL-13Rα2, in place of IL-13Rα2 full-length protein, solubilized IL-13Rα2 protein or a protein using a part of IL-13Rα2 and a partial amino acid sequence is treated for gastrointestinal mucosal injury of the present invention. You may use for the active ingredient of the pharmaceutical composition for. Soluble IL-13Rα2 protein exists outside the living body (Donaldson et al., J Immunol 1998, 161: 2317-2324). As a method for producing a soluble IL-13Rα2 protein, an adenoviral vector incorporating a gene up to amino acid 345 which is the extracellular region of human IL-13Rα2 has been prepared (Trieu et al., Cancer Res 2004). , 64: 3271-3275). In addition, a fusion protein in which human immunoglobulin G is bound to a mouse IL-13Rα2 receptor is known (Chiaramonte et al., J Immunol 1999, 162: 920-930).

The mechanism of action of IL-13Rα2 is to trap IL-13 so that IL-13 does not bind to IL-13Rα1. It is easily presumed that the same effect can be realized even when anti-IL-13 antibody is administered. Therefore, an anti-IL-13 antibody can be used in place of IL-13Rα2 as an active ingredient of the pharmaceutical composition for gastrointestinal mucosal injury treatment of the present invention.
IL-13 is a cytokine synthesized as a precursor protein consisting of 132 amino acids and then cleaved by an enzyme to become a 112 amino acid mature protein having four glycosylation sites (Minty et al., Nature 1993). 362: 248-250; McKenzie et al., Proc Natl Acad Sci 1993, 3735-3739), the base sequence of the gene is known, and an anti-IL-13 antibody has already been prepared. The antibody may be a monoclonal antibody or a polyclonal antibody.

  In addition, as an active ingredient of the pharmaceutical composition for gastrointestinal mucosal injury treatment of the present invention, solubilized IL-13Rα1 whose amino acid sequence has been modified so as not to generate intracellular signaling in order to trap IL-13 Can be used. As an example, a protein that traps IL-4 and IL-13 has been made using a partial structure of the receptor (extracellular domain) (US Pat. No. 5,844,099).

  Furthermore, in order to reduce the amount of IL-13 binding to IL-13Rα1, the production amount of IL-13 may be decreased. Accordingly, the pharmaceutical composition for treatment of gastrointestinal mucosal injury of the present invention comprises an antisense oligonucleotide for IL-13 precursor gene, which suppresses the expression of IL-13 precursor, and an IL-13 precursor gene. Aptamers against siRNA or IL-13 can also be used.

  In addition to these, IL-13 binds to a receptor that generates a signal in the cell. An antagonist, antibody, or solubilization that inhibits the function of this receptor or suppresses the binding of IL-13. IL-13 signaling can be suppressed by using a receptor protein. These substances can be used as active ingredients of the pharmaceutical composition for the treatment of gastrointestinal mucosal injury of the present invention. IL-13Rα1 is a 427 amino acid receptor protein (Aman et al., J Biol Chem 1996, 271: 29265-29270), and the base sequence of the gene is known. An antibody that suppresses the function of IL-13Rα1 has been produced (Patent WO2003080675-A2). The antibody may be a monoclonal antibody or a polyclonal antibody as long as it suppresses the function of IL-13Rα1.

  Furthermore, IL-13 signaling can also be suppressed by suppressing the expression of IL-13Rα1 receptor, which is the target of IL-13. Therefore, an antisense oligonucleotide that suppresses the expression of IL-13Rα1, an siRNA for IL-13Rα1 gene, or an aptamer for IL-13Rα1 can be used in the pharmaceutical composition for gastrointestinal mucosal injury treatment of the present invention. .

  The IL-13Rα2 receptor protein or solubilized IL-13Rα1 receptor protein used in the present invention can be produced recombinantly by a method known in the art using a known gene sequence. The sequence of IL-13Rα1 is described in GenBank NM_001560, and the sequence of IL-13Rα2 is described in GenBank NM_000640. DNA encoding these receptor proteins can be incorporated into an appropriate expression vector and introduced into either eukaryotic or prokaryotic cells to express the desired protein. Examples of host cells that can be used to express these proteins include, but are not limited to, prokaryotic hosts such as E. coli and Bacillus subtilis, and eukaryotic hosts such as yeast, fungi, insect cells and mammalian cells. Is mentioned.

  The vector includes a promoter region that drives expression of a gene encoding IL-13Rα2 receptor protein or solubilized IL-13Rα1 receptor protein, and further includes transcriptional and translational control sequences such as TATA box, capping sequence, CAAT sequence , 3 ′ non-coding region, enhancer, and the like. Examples of promoters include bla promoter, cat promoter, lacZ promoter when used in prokaryotic hosts, herpesvirus TK promoter, SV40 early promoter, yeast glycolysis when used in eukaryotic hosts. Examples include enzyme gene sequence promoters. Examples of vectors include, but are not limited to, pBR322, pUC118, pUC119, λgt10, λgt11, pMAM-neo, pKRC, BPV, vaccinia, SV40, 2-micron, and the like. In addition, vectors can be constructed to secrete and express these receptor proteins using signal sequences or to express these proteins in the form of a fusion protein with another protein. The construction of such expression vectors is well known in the art.

  Vectors constructed to express genes encoding IL-13Rα2 receptor protein or solubilized IL-13 Rα1 receptor protein can be used for transformation, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, It can be introduced into an appropriate host cell by calcium phosphate precipitation, direct microinjection or the like. These receptor proteins can be obtained by growing cells containing the vector in an appropriate medium to produce the desired protein, and recovering and purifying the desired recombinant protein from the cells or medium. Purification can be performed using size exclusion chromatography, HPLC, ion exchange chromatography, immunoaffinity chromatography, and the like.

  The anti-IL-13 antibody or anti-IL-13 Rα1 receptor antibody used in the present invention may be a polyclonal antibody or a monoclonal antibody. Polyclonal antibodies can be obtained by immunizing animals using IL-13 or IL-13Rα1 receptor as a sensitizing antigen and collecting antisera according to methods well known in the art. Monoclonal antibodies are immunized using IL-13 or IL-13 Rα1 receptor as a sensitizing antigen according to methods well known in the art, and the resulting immune cells are removed and fused with myeloma cells. It can be obtained by cloning a hybridoma producing an antibody and culturing the hybridoma.

  The monoclonal antibodies of the present invention include, in addition to antibodies produced by hybridomas, recombinant antibodies produced by transformants transformed with expression vectors containing antibody genes, chimeric antibodies, CDR-grafted antibodies, and these Antibody fragments and the like are included.

  Recombinant antibodies are obtained by cloning a cDNA encoding an antibody from a hybridoma that produces a monoclonal antibody that binds to IL-13 or IL-13 Rα1 receptor, and inserting this into an expression vector for animal cells, plant cells, etc. Can be produced by culturing the transformant. A chimeric antibody is an antibody composed of a heavy chain variable region and a light chain variable region of an antibody derived from an animal, and a heavy chain constant region and a light chain constant region of an antibody derived from another animal. Examples of antibody fragments that can bind to IL-13 or IL-13Rα1 receptor include Fab, F (ab ′) 2, Fab ′, scFv, and diabody.

  An antisense oligonucleotide or siRNA against IL-13 precursor gene or IL-13Rα1 gene can be recombinantly produced by a method known in the art using a known gene sequence. The IL-13 sequence is described in GenBank U31120 (precursor gene, complete cds), and the IL-13Rα1 sequence is described in GenBank NM — 001560.

  An antisense oligonucleotide for IL-13 precursor gene or IL-13Rα1 gene is an oligonucleotide that can specifically bind to mRNA encoding IL-13 precursor or IL-13Rα1 and inhibit its translation. Antisense oligonucleotides include antisense RNA and antisense DNA. An siRNA is a double-stranded RNA that can cause RNA interference. Aptamers represent nucleic acid ligands that bind to specific molecules and may be RNA or DNA. Aptamers can be obtained using a method called SELEX. RNA and DNA may be chemically modified. Various nucleic acid modifications are known to enhance the stability or cellular uptake of antisense oligonucleotides and siRNAs, any of which can be used in the present invention.

  When an antisense oligonucleotide or siRNA for IL-13 precursor gene or IL-13Rα1 gene or an aptamer for IL-13 or IL-13Rα1 is used as a therapeutic agent for gastrointestinal mucosal injury, for example, antisense oligonucleotide or siRNA Alternatively, aptamers can be administered directly to a subject, or vectors expressing antisense oligonucleotides or siRNA or aptamers can be administered and these expression vectors can be administered. Methods for introducing antisense oligonucleotides, siRNA, aptamers or vectors that express them are well known in the art.

  A protein that binds to and inactivates IL-13 can be identified by measuring the binding of the candidate protein to IL-13 and the inactivation of IL-13 by the candidate protein according to a known method. In addition, IL-13 Rα1 receptor antagonists and kinase inhibitors that bind to IL-13Rα1 receptor can be identified by measuring the binding between the candidate substance and IL-13 Rα1 receptor according to a known method. it can.

  The therapeutic agent for gastrointestinal mucosal injury of the present invention can be formulated by methods known to those skilled in the art. For example, a pharmaceutically acceptable carrier or medium, specifically, sterile water or saline, vegetable oil, emulsifier, suspension, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative And can be formulated in combination with a binder or the like as appropriate.

  For oral administration, tablets, pills, dragees, capsules, liquids, gels, syrups are prepared by mixing the therapeutic agent for gastrointestinal mucosal injury of the present invention with a pharmaceutically acceptable carrier well known in the art. , Slurries, suspensions and the like. As the carrier, those conventionally known in the art can be widely used. For example, excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid and the like. Water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone and other binders, dry starch, sodium alginate, agar powder, laminaran powder, carbonic acid Disintegrating agents such as sodium hydride, calcium carbonate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose; disintegrating inhibitors such as sucrose, stear cocoa butter, hydrogenated oil; quaternary ammonium salts, La Absorption accelerators such as sodium rilsulfate; humectants such as glycerin and starch; adsorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid; abundant such as purified talc, stearate, boric acid powder, polyethylene glycol An agent or the like can be used. Furthermore, the tablet can be a tablet coated with a normal coating, for example, a sugar-coated tablet, a gelatin-encapsulated tablet, an enteric-coated tablet, a film-coated tablet, a double tablet, or a multilayer tablet, if necessary.

  For parenteral administration, the therapeutic agent for gastrointestinal mucosal injury of the present invention can be formulated according to usual pharmaceutical practice using a pharmaceutically acceptable vehicle well known in the art.

  Water-soluble vehicles for injection include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and appropriate solubilizing aids. An agent such as alcohol, specifically ethanol, polyalcohol such as propylene glycol, polyethylene glycol, a nonionic surfactant such as polysorbate 80 (TM), HCO-50 may be used in combination.

  Oily vehicles include sesame oil and soybean oil, and benzyl benzoate and benzyl alcohol may be used in combination as solubilizing agents. Moreover, you may mix | blend with buffer, for example, phosphate buffer, sodium acetate buffer, a soothing agent, for example, procaine hydrochloride, stabilizer, for example, benzyl alcohol, phenol, antioxidant. The prepared injection solution is usually filled into a suitable ampoule.

  Suitable administration routes of the therapeutic agent for gastrointestinal mucosal injury of the present invention include, but are not limited to, oral, rectal, transmucosal, or enteral administration, or intramuscular, subcutaneous, intramedullary, intrathecal, direct intraventricular, Intravenous, intravitreal, intraperitoneal, intranasal, or intraocular injection is included. The administration route and administration method can be appropriately selected depending on the age and symptoms of the patient.

  The particularly preferred administration route and administration method of the therapeutic agent for gastrointestinal mucosal injury of the present invention include a preparation containing the therapeutic agent for gastrointestinal mucosal injury of the present invention, preferably a sustained-release preparation, during or after the affected area, for example, peritoneal cavity. It is to be administered within.

  The dose of the therapeutic agent for gastrointestinal mucosal injury of the present invention can be selected, for example, in the range of 0.0001 mg to 1000 mg per kg of body weight at a time. Alternatively, for example, the dose can be selected in the range of 0.001 to 100000 mg / body per patient, but is not necessarily limited to these values. The dose and administration method vary depending on the weight, age, symptoms, etc. of the patient, but can be appropriately selected by those skilled in the art.

The method of the present invention for examining whether or not a test substance has a therapeutic effect on gastrointestinal mucosal injury can be performed, for example, by any of the following methods:
-A test substance is administered to IL-4R-deficient mice and the increase in IL-13Rα2 expression level in the gastrointestinal tissue after irradiation is measured (mRNA, protein). An anti-IL-13Rα2 antibody is used when the amount of protein in the digestive tract tissue is measured by Western blot or ELISA.
-Applying a test substance to the culture medium of fibroblasts and myofibroblasts, and measuring the increase in IL-13Rα2 expression level.
-Binding of IL-13 and test substance is measured by fixing IL-13 to a detection plate for surface plasmon analysis and perfusing the test substance.
-Binding of IL-13 and test substance is measured by immunoprecipitation using an anti-IL-13 antibody.
-A test substance is administered to IL-4R-deficient mice, and the decrease in IL-13 expression level in the gastrointestinal tissue after irradiation is measured (mRNA, protein). An anti-IL-13 antibody is used when the amount of protein in the digestive tract tissue is measured by Western blot or ELISA.
-Apply a test substance to the culture medium of NK cells, and measure that IL-13 expression level decreases.
-Apply a mixture of IL-13 and test substance to a reaction solution in which activated IL-13 binds to IL-13Rα1 or IL-13Rα2, and measure that competitive inhibition of binding is reduced. .
-Measure the binding between the test substance and IL-13Rα1 (radioligand receptor binding experiment). Cultured monocytes that express IL-13Rα1 and lack IL-4Rα, or cell membrane specimens thereof are used.
-Measure that the test substance suppresses the activation of Tyr2 in cultured monocytes in which IL-13 expresses IL-13Rα1 and lacks IL-4Rα.
-A test substance is administered to a mouse, and the decrease in the expression level of IL-13Rα1 in the digestive tract tissue is measured (mRNA, protein). An anti-IL-13Rα1 antibody is used when the amount of protein in the gastrointestinal tissue is measured by Western blot or ELISA.

  EXAMPLES The present invention will be described below in more detail with reference to examples, but the present invention is not limited to these examples.

Correlation between regeneration of damaged small intestinal mucosa and expression of IL-13Rα2 receptor In previous studies, IL-13Rα2 expression increased at the time of repair / regeneration after radiation damage to the gastrointestinal mucosa, It has been suggested that IL-4 and IL-13, Th2-type cytokines, are involved in repair and regeneration. However, they have not been able to elucidate these roles.

Therefore, in this example, in order to elucidate the role of these Th2-type cytokines on tissue repair / regeneration, IL-4Rα-deficient mice (IL-4Rα ^{−) in which} regeneration of the damaged gastrointestinal mucosa after irradiation is delayed. We investigated the dynamics of IL-4 and IL-13 receptor expression in ^/- ), and revealed that IL-13α2 receptor expression has a corresponding relationship with tissue repair and regeneration. IL-4Rα ^{− / −} is a mouse with impaired Th2-type physiology because IL-4 lacks a receptor to act on cells.

IL-4Rα ^{− / −} mice (Jackson Laboratory, Bar Harbor, ME, USA) with BALB / c system as background were used as experimental animals. BALB / c mice (Japan Clare, Tokyo) were used as wild type (WT) strains. The whole body was irradiated with 3 Gy using X-ray irradiation equipment (MBR-1520-R, Hitachi Medical, Tokyo).

  As a jejunum tissue specimen, the jejunum at a position 3 cm from the gastroduodenal boundary was collected, fixed with 5% glacial acetic acid ethanol, and a paraffin-embedded section was prepared. To prepare a specimen for immunohistochemical staining with an anti-bromodeoxyuridine (BrdU) antibody, BrdU (1 mg / mouse) was intraperitoneally administered 1 h before collection of the jejunum. This specimen was also subjected to immunohistochemical staining with an anti-ssDNA antibody.

First, WT and IL- reported by Kawashima et al., (The 33rd Annual Meeting of the Immunological Society of Japan, Presentation No. 3-B-W32-19-P, Annual Meeting of the Immunological Society of Japan, Academic Meeting Record 2003, 33: 225) The differences in the regeneration of intestinal mucosal tissue after irradiation of 4Rα ^{− / −} mice are listed (FIGS. 2, 3, and 4).

FIG. 2 is a representative image of a specimen in which paraffin-embedded sections are stained with hematoxylin and eosin (HE). In the following text and figures, the results of mice not irradiated with radiation are shown as day 0. One day after irradiation (day 1), intestinal epithelial cells were damaged in both WT and IL-4Rα ^{− / −} , and hyalination of villi and a decrease in HE staining were observed. Three days after irradiation (day 3), the degree of injury decreased with WT, and cells in the epithelium and lamina propria increased, whereas IL-4Rα ^-/- showed mucosal injury and crypts similar to day 1. It was confirmed that atrophy was observed.

  The degree of apoptosis was observed by immunohistochemical staining using anti-single-stranded DNA (ssDNA) antibody on paraffin-embedded sections. Fig. 3A shows a representative sample, and Fig. 3B shows the average value and standard deviation of the number of ssDNA positive cells generated per crypt or villi calculated from the samples of 4 mice in each group. Show.

Epithelial cells of the intestinal mucosa are always born, proliferate in the crypts, migrate to the villi and stop growing, and old ones undergo apoptosis and detach. Reflecting this, ssDNA positive cells were observed in the villi on Day 0. On Day 1 after irradiation, both WT and IL-4Rα ^{− / −} increased ssDNA positive cells, indicating that apoptosis occurred in a wide range. On Day 3, the difference was that ssDNA positive cells decreased in WT but IL-4Rα ^-/- continued to have the same broad apoptosis as in Day 1.

The degree of tissue regeneration was examined by immunohistochemical staining using an antibody against BrdU, a marker for DNA synthesis. FIG. 4A shows a representative sample, and FIG. 4B shows the average value and standard deviation of the number of BrdU positive cells per crypt or villi calculated from the samples of 4 mice in each group. On Day 0, BrdU-positive cells were observed in the crypts with proliferative zones. On Day 1 after irradiation, BrdU-positive cells decreased for both WT and IL-4Rα ^{− / −} . On Day 3, it was shown that BrdU positive cells increased in WT compared to day 0, and active regeneration occurred. On the other hand, in IL-4Rα ^{− / −} , the number of BrdU positive cells was similar to that of day 0, and regeneration was not so active.

The above is summarized as follows. Before 3 Gy irradiation (day 0), cell rotation of WT and IL-4Rα ^{− / −} jejunal mucosa was similar to WT. On day 1 after irradiation, both WT and IL-4Rα ^{− / −} showed tissue damage and extensive apoptosis, and no regeneration occurred. On day 3 after irradiation, WT decreased apoptosis and thrived regeneration, reducing tissue injury, whereas IL-4Rα ^-/- continued apoptosis and injury, with less regeneration. It wasn't thriving. Therefore, it was confirmed that IL-4Rα ^{− / −} delayed the repair and regeneration of damaged mucosa.

Newly, in this Example, IL-4 and IL in IL-4Rα ^{− / −} small intestinal lamina propria mononuclear cells (LPMC) that are delayed in the regeneration of injured jejunal mucosa as shown above -13 receptor mRNA expression levels were measured and compared with WT results.

  The entire small intestine was collected, cut longitudinally, and washed with phosphate buffered saline (PBS). The specimens were treated with 30 min 2 mM ethylenediaminetetraacetic acid (EDTA) PBS to remove epithelial cells. The residue was digested with collagenase (Sigma Blend Collagenase Type E, Sigma-Aldrich, MO, USA) for 20 min and separated in a Percoll gradient to obtain LPMC. The mRNA expression level was measured by quantitative PCR using SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK) and a real-time PCR apparatus (ABI PRISM 7700 sequence detector, Applied Biosystems, Foster City, CA, USA). CDNA was prepared by reverse transcription from total RNA prepared from large intestine tissue using Rneasy Mini Kit (Qiagen, Hilden, Germany). Using GAPDH as a proofreading gene, the number of threshold cycles was calculated using software attached to the sequencer (ver 1.7, Applied Biosystems, Foster City, CA, USA).

FIG. 5A shows mRNA expression in LPMC before irradiation (day 0). Expression of γc, IL-13α1, and IL-13α2 was observed in both WT and IL-4Rα ^{− / −} , and the levels were not significantly different. IL-4Rα was not observed in IL-4Rα ^{− / −} .

FIG. 5B shows the change over time in the relative expression level, where the average expression level of mRNA before irradiation (day 0) in LPMC is 1. Each point represents the value of one individual, and the short horizontal line represents the average value of 5 animals. In WT, an increase in IL-13Rα2 mRNA expression was observed after irradiation with a peak at day 3, but this increase was not observed in IL-4Rα ^{− / −} . In addition, WT showed an increase in trefoil factor 1 (TFF1) mRNA expression that was related to cell proliferation / differentiation and mucin formation after irradiation, peaking on day 3, but this increase was observed in IL-4Rα ^-/-. Was not recognized. Regarding IL-13α1, γc and IL-4Rα, neither WT nor IL-4Rα ^{− / −} increased after irradiation.

Furthermore, jejunal paraffin-embedded specimens were immunohistochemically stained with anti-IL-13Rα2 antibody. A representative example is shown in FIG. In WT, transient increase in IL-13Rα2-positive cells was observed in the lamina propria, mainly on day 3. On the other hand, although IL-13Rα2 mRNA expression was confirmed in IL-4Rα ^{− / −} before irradiation, no significant increase in IL-13Rα2 positive cells occurred by day 7.

The above results revealed the relationship between regeneration and IL-13Rα2 expression in the intestinal mucosa damaged by radiation. In WT, the expression of IL-13Rα2 increased at the peak of regeneration. Furthermore, IL-4Rα ^{− / −} , which was not regenerated at the same time, did not show increased expression of IL-13Rα2. These results indicate that there is a correspondence between regeneration and increased expression of IL-13Rα2.

Expression of IL-13Rα2 by fibroblasts and myofibroblasts by IL-4 and IL-13
IL-13Rα2, whose expression increases after irradiation in WT, was observed in fibroblasts and fibroblasts (Kawashima et al., 33rd Annual Meeting of the Immunological Society of Japan, No. 3-B-W32-19- P, Annual Meeting of the Immunological Society of Japan / Academic Meeting Record 2003, 33: 225). In this example, it was shown that these cells express IL-13Rα2 upon stimulation with IL-4 and IL-13.

3-day-old WT mouse small intestine was minced finely in 10 mM EDTA PBS and cultured in Dulbecco's modified Eagle medium (DMEM) containing 20% fetal calf serum (FCS). After 6 days, the cells were subcultured, and 10 days later, 2.5 × 10 ⁴ cells were poured into 24 well plates. Some were stained with anti-α smooth muscle actin (αSMA) antibody to confirm cell organization. About 50% of these cells were fibroblasts and about 50% were myofibroblasts. After overnight culture, the medium was replaced with a medium containing IL-4 or IL-13. After 48 hours, total RNA was extracted, and IL-13Rα2 mRNA expression level was measured by quantitative RT-PCR.

  FIG. 7 shows the average value of the relative expression level of IL-13Rα2 mRNA with the value of no stimulation (control) as 1. IL-4 and IL-13 alone increased IL-13Rα2 mRNA expression, and showed a potentiating effect when combined. The results indicate that IL-4 and IL-13 stimulate IL-13Rα2 mRNA by stimulating fibroblast and myofibroblast type 2 IL-4 receptors (complex of IL-4Rα and IL-13Rα1). The possibility of expression is suggested.

Comparison of jejunal IL-4 and IL-13 mRNA expression between WT and IL-4Rα ^-/-
In WT, IFN-γ, IL-13 and IL-4 mRNAs increase after irradiation (Kawashima et al., 33rd Annual Meeting of the Immunological Society of Japan, No. 3-B-W32-19-P, Annual meeting and academic meeting record 2003, 33: 225). In this example, IL-13, IL-4 and IFN-γ mRNA expression levels were newly measured in IL-4Rα ^{− / −} mice and compared with WT mRNA expression.

The mRNA expression level in the small intestinal mucosal lamina propria mononuclear cells was measured by the same method as the cytokine receptor mRNA measurement of Example 1. A band showing the mRNA expression level of each individual is shown in FIG. Before irradiation (day 0), IL-4Rα ^{− / −} small intestine showed a large amount of IFN-γ mRNA expression, indicating that Th1-type immune activity was active. In IL-4Rα ^{− / −} , IL-13 and IL-4 mRNAs were highly expressed compared to WT.

IL-4R [alpha] in Day 1 and day 3 ^{- / -} the IL-4 mRNA expression levels showed no significant increase compared to day 0, but was slightly less than the expression level in WT, whose expression was confirmed . On day 1 and day 3 after irradiation, IL-4Rα ^{− / −} mRNA expression levels of IFN-γ and IL-13 were increased, indicating high expression similar to WT.

IL-13 production after irradiation is performed by NK cells (Kawashima et al., 33rd Annual Meeting of the Immunological Society of Japan, Title No. 1-H-W13-13-O / P, Annual Meeting of the Immunological Society of Japan, Record of the Annual Meeting) 2004, 34: 115). NK cells are activated by IFN, a Th1-type cytokine, and various stress signals (Long et al., J Exp Med. 2002, 196: 1399-1402), so IL-13 production through NK cells May also occur when there is an impairment in Th2-type immune function such as IL-4Rα ^{− / −} .

Considering that IL-4Rα ^{− / −} did not show an increase in IL-13Rα2 mRNA expression in Example 1, in IL-2Rα ^{− / −} , fibroblasts and myofibroblasts induced by IL-4 and IL-13 in Example 2 It was strongly suggested that the increase in IL-13Rα2 mRNA expression occurs in the IL-4 receptor (composed of IL-4Rα and IL-13α1).

In Example 1, since IL-4Rα ^{− / −} mice did not undergo regeneration and increase in IL-13Rα2 on day 3 after irradiation, the correspondence between intestinal mucosal tissue regeneration and IL-13Rα2 was shown.

IL-13Rα2 is a decoy receptor that does not cause intracellular signals even when IL-13 binds. By trapping IL-13, IL-13Rα2 is mediated by receptors other than IL-13Rα2 on the cell surface. It suppresses signal generation in activated cells (activation of intracellular JAK / STAT pathways, etc.). In this example, it was shown that IL-13 was also produced in IL-4Rα ^{− / −} mice. Therefore, IL-13Rα 2 does not increase IL-4Rα ^{− / −} , and IL-13 signaling to cells It was suggested that this could not be suppressed.

Improvement of gastrointestinal mucosal regeneration delay in IL-4Rα1-/-mice by IL-13Rα2 administration Based on the results of Example 1, there is a correspondence between regeneration of jejunal mucosa damaged by irradiation and IL-13Rα2 production It has been shown. In addition, the results of Example 3 indicate that IL-4Rα ^{− / −} mice in which regeneration after irradiation was delayed did not increase IL-13Rα2 and thus could not suppress IL-13 signaling to cells. It was done. Here, if regeneration occurs by increasing the expression of IL-13Rα2 in IL-4Rα ^{− / −} mice, it is indicated that failure to suppress the action of IL-13 is the cause of no regeneration. Therefore, in this example, it was examined whether or not regeneration occurs by administering IL-13Rα2 protein to IL-4Rα ^{− / −} mice.

  The IL-13Rα2 cDNA was cloned from the WT small intestine 3 days after irradiation, and a fragment of amino acids 1-332 corresponding to the binding site was cloned into 5'-GCGCAGATCTCCGGATCCGTCTGGCCCTGTGTAAC-3 '(SEQ ID NO: 1) and 5'-GCGCAGATCTTCCGGATCCGTCTGGCCCTGTGTAACCTTCCCAAC-3' ( It was amplified by PCR using SEQ ID NO: 2) as a primer. The spacer sequence GSGADPEE was subcloned at the 3 ′ end. This fragment was ligated to the NheI and BamH1 sites of the CD5 leader-IgG1 vector (CD5lneg1) containing human Ig. After confirming the sequence, DNA containing IL-13Rα2-Ig was recloned with the GFP marker sequence into the retroviral expression vector pMXs. To obtain large amounts of IL-13Rα2-Ig chimeric protein, retrovirus was infected into mouse myeloma SP2 / 0 cell line and cells expressing GFP were concentrated by sorting. The chimeric protein was extracted from the supernatant of cells cultured in a CELLine flask (Becton-Dickinson, Franklin Lakes, NJ, USA) and purified using protein A Sepharose choromatography (Pharmacia, Uppsala, Sweden).

For IL-4Rα ^-/- mice, control human Ig (h-Ig) or IL-13Rα2-Ig 300μg, 3 Gy before irradiation, before irradiation (day 0), after irradiation day 1, after irradiation day 2 was administered intraperitoneally. Other methods were the same as those in Example 1.

  FIG. 9 shows representative examples of HE staining, immunohistochemical staining of ssDNA, which is a marker of apoptosis, and BrdU, which is a marker of cell proliferation, on a jejunal paraffin-embedded specimen on irradiation day 3. FIG. 10 shows the average value and standard deviation of the number of ssDNA positive cells and the number of BrdU positive cells in each group.

On day 3 after irradiation, WT recovered from mucosal injury, apoptosis was low, and regeneration was vigorous.In IL-4Rα ^-/- mice, mucosal injury continued, many cells showing apoptosis, and regeneration was There were few. In the group administered IL-4Rα ^{− / −} with control h-Ig, there was no effect of administration on injury, apoptosis, and regeneration. In the group where IL-13Rα2 was administered to IL-4Rα ^{− / −} , as in WT, there was little mucosal damage, apoptotic cells decreased, and regeneration was also thriving.

In IL-4Rα ^-/- , both regeneration of damaged mucosa and increase of IL-13Rα2 did not occur after irradiation, but regeneration occurred by supplementing IL-13Rα2 in mice of this strain. It was shown that the increase in IL-13Rα2 is not a secondary reaction associated with regeneration, but a process necessary for the gastrointestinal mucosa to undergo regeneration. This result shows that IL-13Rα2 has an effect of enhancing regeneration on the gastrointestinal mucosa where regeneration is delayed.

In addition, the fact that IL-13Rα2 which is a decoy receptor has an effect of enhancing regeneration indicates that IL-13 signaling suppresses regeneration. Since IL-4Rα ^-/- mice lack the IL-4Rα receptor, IL-13 signaling is transmitted through ILs other than type 2 IL-4 receptors (consisting of IL-4Rα and IL-13Rα1). This was suggested to be caused by the -13 receptor.

In this example, IL-13Rα2 administration almost completely improved the delay in regeneration of IL-4Rα ^{− / −} , so it was revealed that IL-13 is a critical factor that suppresses regeneration. It was.

Promotion of gastrointestinal mucosal regeneration in WT mice by IL-13Rα2 administration Example 4 showed the effect of IL-13Rα2 administration that normalizes delayed IL-4Rα ^{− / −} mouse gastrointestinal mucosal regeneration. The purpose of this example was to show the effect of IL-13Rα2 in enhancing the regeneration of damaged gastrointestinal mucosa in BALB / c mice, which are wild type (WT) strains.

  BALB / c mice were used as experimental animals. Using the same apparatus as in Example 1, X-rays with a dose of 12 Gy were whole-body irradiated. In the IL-13Rα2 administration group, the same IL-13Rα2-Ig 200 μg as used in Example 4 was administered on the day before irradiation, before irradiation (day 0), after irradiation day 1, after irradiation day 2, after irradiation day 3 And intraperitoneally. Human Ig (h-Ig) was administered to the control group. On day 4 after irradiation, the entire small intestine and the entire large intestine were taken out, cut vertically, and immersed in 10% formalin overnight, and then a paraffin-embedded specimen was prepared. HE staining was performed, and the number of regenerated crypt microcolony consisting of 10 cells or more was counted. The small intestine was divided into three, and the small intestine 1, small intestine 2, and small intestine 3 were formed from the top. Small intestine 1 consisted of jejunum, small intestine 2 consisted of jejunum and ileum, and small intestine 3 consisted of ileum.

  In the IL-13Rα2-administered group, the number of microcolonies in the regenerating crypts was large, and regeneration of the specimen mucosa was promoted. FIG. 11 shows the average value and standard deviation of the number of microcolonies of regenerating crypts in each region of the intestinal tract in the control group and the IL-13Rα2 administration group. In FIG. 11, * indicates that a significant difference was observed at 5% level between the administration groups by the Mann-Whitney U test. IL-13Rα2 obviously increased the number of microcolonies of regenerating crypts in three areas of the small intestine and large intestine.

In this example, by administering IL-13Rα2, which is a decoy receptor for IL-13, irradiation was performed not only in IL-4Rα ^{− / −} mice having immunological abnormalities but also in wild type mice. It was shown that regeneration of injured gastrointestinal mucosa was promoted. Therefore, treatments that suppress signaling by IL-13 are not limited to gastrointestinal diseases with special backgrounds such as immune abnormalities, and can be widely used for various pathological conditions that cause gastrointestinal epithelial injury. Indicated.

  The present invention is useful for the treatment of gastrointestinal mucosal injury.

FIG. 1 is a schematic diagram of IL-4 and IL-13 receptors. FIG. 2 shows representative examples of jejunal tissue images before and after irradiation in wild type (WT) and IL-4α-deficient (IL-4α ^{− / −} ) mice. Fig. 3 shows apoptosis before and after irradiation in the jejunum of wild type (WT) and IL-4α-deficient (IL-4α ^-/- ) mice by immunohistochemical staining using an anti-single-stranded DNA antibody. is there. FIG. 4 shows proliferating cells before and after irradiation in the jejunum of wild type (WT) and IL-4α-deficient (IL-4α ^{− / −} ) mice by immunohistochemical staining using an anti-bromodeoxyuridine antibody. is there. 5, IL-4.alpha. Deficient and wild-type ^{(WT) (IL-4α -} / -) shows the mRNA expression level of cytokine receptors before and after irradiation in the mouse small intestinal lamina propria. FIG. 6 shows the expression of IL-13Rα2 receptor before and after irradiation in the jejunum of wild type (WT) and IL-4α-deficient (IL-4α ^{− / −} ) mice by immunohistochemical staining. FIG. 7 shows the stimulating effect of IL-4 and IL-13 on IL-13Rα2 mRNA expression in cultured fibroblasts and myofibroblasts derived from wild-type mice. FIG. 8 shows the mRNA expression levels of cytokines before and after irradiation in the lamina propria of the wild type (WT) and IL-4α-deficient (IL-4α ^{− / −} ) mouse small intestinal mucosa. FIG. 9 shows the regeneration promoting effect of IL-13Rα2 intraperitoneal administration in the jejunum of IL-4α-deficient (IL-4α ^{− / −} ) mice by histology and immunohistochemical staining. FIG. 10 shows immunohistochemical staining of the apoptosis-suppressing and regeneration-promoting effects of IL-13Rα2 intraperitoneal administration in the jejunum of IL-4α-deficient (IL-4α ^{− / −} ) mice. FIG. 11 shows the effect of increasing the number of microcolonies of intestinal regenerating crypts by intraperitoneal administration of IL-13Rα2 in the small intestine and large intestine of wild-type mice after irradiation.

Claims (12)

A therapeutic agent for gastrointestinal mucosal injury whose mechanism of action is to inhibit IL-13 signaling. The therapeutic agent for gastrointestinal mucosal injury according to claim 1, comprising IL-13Rα2 receptor protein or a part thereof as an active ingredient. The therapeutic agent for gastrointestinal mucosal injury according to claim 1, comprising an anti-IL-13 antibody or a protein that binds and inactivates IL-13 as an active ingredient. The therapeutic agent for gastrointestinal mucosal injury according to claim 1, comprising an antisense oligonucleotide for siRNA of IL-13, an siRNA or an aptamer for IL-13 as an active ingredient. The therapeutic agent for gastrointestinal mucosal injury according to claim 1, comprising an anti-IL-13 Rα1 receptor antibody as an active ingredient. Effective solubilized IL-13 Rα1 receptor, IL-13 Rα1 receptor antagonist, kinase inhibitor binding to IL-13Rα1 receptor, antisense oligonucleotide to IL-13Rα1 gene, siRNA or aptamer to IL-13Rα1 receptor The therapeutic agent for gastrointestinal mucosa injury according to claim 1 as a component. A method for testing whether or not a test substance has a therapeutic effect on gastrointestinal mucosal injury, wherein the expression level of IL-13Rα2 receptor gene or IL-13Rα2 protein in cells in the presence and absence of the test substance A method comprising measuring the amount of. IL-13Rα2, or an oligonucleotide or polynucleotide encoding IL-13Rα2, or a cell that expresses IL-13Rα2, or a screening agent for a substance having a therapeutic effect on gastrointestinal mucosal injury, including anti-IL-13Rα2 antibody kit. A method for testing whether a test substance has a therapeutic effect on gastrointestinal mucosal injury, measuring the binding between a candidate substance and IL-13, or in the presence and absence of a test substance A method comprising measuring IL-13 precursor gene expression level or IL-13 protein level in a cell. A kit for screening a substance having a therapeutic effect on gastrointestinal mucosal injury, comprising IL-13, an oligonucleotide or polynucleotide encoding IL-13, or a cell expressing IL-13, or an anti-IL-13 antibody . A method for testing whether a test substance has a therapeutic effect on gastrointestinal mucosal injury, measuring the binding between a candidate substance and IL-13Rα1, or in the presence and absence of a test substance A method comprising measuring IL-13 Rα1 gene expression level or IL-13 Rα1 protein level in a cell. IL-13Rα1, or an oligonucleotide or polynucleotide encoding IL-13Rα1, or a cell that expresses IL-13Rα1, or a substance having a therapeutic effect on gastrointestinal mucosal injury, including anti-IL-13Rα1 antibody kit.