Therapeutic drug for inflammatory diseases, and screening method for therapeutic drug

Abstract

To provide a therapeutic drug for inflammatory diseases, particularly inflammatory bowel disease, that has no serious side effects such as immunosuppression.SOLUTION: From an analysis of the mechanism of action of sulfasalazine, it has been revealed that an anti-inflammatory action occurs by increasing intracellular glutamate. An xCT inhibitor or a glutamine synthetase inhibitor that increases intracellular glutamate has been found to be effective in the treatment of inflammatory bowel disease. Furthermore, it is considered that a compound that suppresses the expression of xCT or a glutamine synthesis-inhibiting enzyme also has a therapeutic effect.SELECTED DRAWING: Figure 5D

Description

[Technical field]

[0001] The present invention relates to a therapeutic agent for inflammatory diseases, particularly inflammatory bowel diseases, and a method for screening such therapeutic agents. [Background technology]

[0002] Inflammatory bowel disease, typified by ulcerative colitis and Crohn's disease, is a disease in which chronic inflammation occurs in the intestines. In Japan, the number of patients has been rapidly increasing since the 1990s, with the number of patients with ulcerative colitis exceeding 200,000 and Crohn's disease exceeding 70,000. Inflammatory bowel disease develops at a young age and repeats periods of remission and relapse, requiring medical support throughout one's life. The cause of the onset of inflammatory bowel disease is still unknown, but it is known that it worsens due to increased cytokine expression by immune cells, deterioration of the intestinal environment due to disturbance of the intestinal flora, and a decrease in the barrier function of the intestinal epithelium.

[0003] Various therapeutic drugs have been developed for inflammatory bowel disease. Depending on the severity, 5-aminosalicylic acid (5-ASA), which suppresses inflammation, 5-ASA preparations such as sulfasalazine, which is metabolized in the body to 5-ASA, steroids, immunosuppressants, and biological preparations such as anti-TNF-α antibody preparations are used. If medical treatment is ineffective or if medical treatment is difficult due to side effects, surgical treatment is performed. However, surgical treatment is very invasive, and it is desirable to induce remission through medical treatment if possible. Furthermore, since steroids, immunosuppressants, and biological preparations used in cases of moderate to severe symptoms all have severe side effects such as immunosuppression, the development of therapeutic drugs without severe side effects is expected.

[0004] Currently, there is a therapeutic drug called sulfasalazine (salazosulfapyridine) that is administered to patients with relatively mild inflammatory bowel disease. Sulfasalazine is thought to be effective against inflammatory bowel disease by accumulating in the submucosal connective tissue of the intestinal tract and exerting an anti-inflammatory effect. In addition, sulfasalazine is known to be a specific inhibitor of cystine / glutamate antiporter (xCT, SLC7A11) in vitro (Non-Patent Document 1). xCT is an amino acid transporter that exists in the cell membrane and exchanges and transports extracellular cystine with intracellular glutamate. Cystine taken up by xCT is involved in the synthesis of the antioxidant glutathione, and has been shown to be involved in maintaining the survival of cancer cells, and therefore has been shown to have a therapeutic effect on several cancers, including brain tumors and breast cancer (Non-Patent Document 2).

[0005] However, it was not clear how xCT is involved in inflammatory bowel disease. It has been reported that administration of glutamine leads to improvement of symptoms in inflammatory bowel disease, but the relationship with xCT, which is a cystine / glutamate antiporter, is unclear. The present inventors analyzed the mechanism of action of xCT and found that xCT inhibition exhibits anti-inflammatory effects, and further found that intracellular retention of glutamate by xCT inhibition, not glutamine, induces anti-inflammatory effects, thus completing the present invention. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Gout PW. et al., Leukemia, 2001, Vol.15(10), pp.1633-1640. [Non-Patent Document 2] Chung, WJ et al., J. Neuroscience, 2005,Vol.25(31),pp.7101-7110. [Non-Patent Document 3] SatoH. et al., J Biol Chem, 2005, Vol.280(45), pp.37423-37429. [Non-Patent Document 4] WirtzS. et al., Nat Protoc, 2017, Vol.2(7), pp.1295-1309. [Non-Patent Document 5] Erben U. et al., Int J Clin Exp Pathol, 2014, Vol.7(8), pp.4557-4576. [Non-Patent Document 6] Shiomi Y. et al,. InflammatoryBowel Diseases, 2011, Vol.17(11), pp.2261-2274. [Non-Patent Document 7] Villar VH. et al., NatChem Biol , 2023, Vol.19(3), pp.292-300. Summary of the Invention [Problem to be solved by the invention]

[0007] An objective of the present invention is to provide a therapeutic agent for an inflammatory disease that does not have serious side effects such as immunosuppression, and a screening method thereof. (1) A therapeutic and / or preventive agent for an inflammatory disease, comprising as an active ingredient a compound that increases intracellular glutamate. It has been suggested that glutamine in the intestinal epithelium is important in inflammatory diseases, such as inflammatory bowel disease. However, the present inventors have demonstrated that glutamic acid, not glutamine, is important, and that increasing intracellular glutamic acid leads to the treatment and prevention of inflammatory bowel disease.

[0008] (2) The therapeutic and / or prophylactic agent for an inflammatory disease according to (1), wherein the compound that increases intracellular glutamate is an xCT inhibitor or a glutamine synthetase inhibitor. Compounds that increase intracellular glutamate include inhibitors of xCT, which is a cystine / glutamate antiporter responsible for the export of glutamate from cells, and inhibitors of glutamine synthetase, an enzyme that synthesizes glutamine from glutamate.

[0009] (3) The therapeutic and / or prophylactic agent for inflammatory diseases according to (1), wherein the compound that increases intracellular glutamate is a compound that reduces the expression of xCT or glutamine synthetase. Not only inhibitors of xCT and glutamine synthetase, but also reduction in the expression of xCT and glutamine synthetase similarly exerts therapeutic and preventive effects against inflammatory diseases.

[0010] (4) The therapeutic and / or prophylactic agent for an inflammatory disease according to any one of (1) to (3), wherein the inflammatory disease is inflammatory bowel disease. Among inflammatory bowel diseases, the compound is particularly effective against inflammatory bowel diseases for which a large amount of data has been presented and analyzed in this specification.

[0011] (5) A method for screening therapeutic and / or prophylactic agents for inflammatory bowel disease, comprising adding a candidate compound and selecting the candidate compound using glutamic acid, cystine, or cysteine ​​as an indicator. By screening for compounds that increase intracellular glutamate or decrease cystine in the medium, it is possible to screen for therapeutic / prophylactic drugs for inflammatory bowel disease. For example, in screening for xCT inhibitors, it is possible to add a candidate compound to the medium of cells expressing xCT and directly measure the increase in intracellular glutamate, as well as to analyze the decrease in glutamate concentration in the medium or the increase in cystine in the medium, and select compounds that can be therapeutic drugs. Mass screening is possible if detection dye-reactive cystine analogs or fluorescently labeled cystine are used as indicators. In addition, for compounds that inhibit glutamine synthetase, candidate compounds can be added to an in vitro system using recombinant proteins, and the reaction products glutamine and ADP can be used as indicators for screening.

[0012] (6) A method for screening therapeutic and / or preventive agents for inflammatory bowel disease, comprising adding a candidate compound to a culture medium of cultured cells, analyzing the expression level of xCT or glutamine synthetase, and selecting a candidate compound by using a decrease in the expression level as an indicator. Compounds that can be used as therapeutic or preventive agents for inflammatory bowel disease may be those that reduce the expression levels of xCT or glutamine synthetase. The reduction in the expression levels can be achieved by analyzing mRNA or protein. [Brief description of the drawings]

[0013] [Figure 1A] FIG. 1 shows the results of an analysis of the effects of dextran sulfate sodium (DSS) administration on body weight (left) and disease activity index (right) in wild-type and xCT-deficient mice. [Figure 1B] FIG. 1 shows intestinal shortening in wild-type and xCT-deficient mice in a mouse model of DSS colitis. [Figure 1C] Graph showing histological findings (right) and tissue scores (left) of a mouse model of DSS colitis in wild-type and xCT-deficient mice. [Figure 1D] FIG. 1 shows the results of analyzing the expression of inflammatory cytokines in wild-type and xCT-deficient mice in a DSS colitis mouse model. [Figure 1E] FIG. 1 shows the results of analyzing intestinal permeability in wild-type and xCT-deficient mice. [Figure 2A] A schematic diagram of the experimental system for evaluating immune function via bone marrow transplantation. [Figure 2B] FIG. 1 shows the results of an analysis of the effect on body weight of a mouse model of DSS colitis in which bone marrow from xCT-deficient mice was transplanted. [Figure 2C] FIG. 1 shows the results of analyzing the effect on intestinal shortening in mice transplanted with bone marrow from xCT-deficient mice and induced DSS-induced colitis. [Figure 2D] FIG. 1 shows the results of analyzing the histological findings (right) and tissue score (left) of mice in which bone marrow from xCT-deficient mice was transplanted and DSS-induced colitis was induced. [Figure 2E] This figure shows the results of analyzing the expression of inflammatory cytokines in a mouse model of DSS colitis after transplantation of bone marrow from xCT-deficient mice. [Figure 3A] A schematic diagram of the experimental system in which wild-type and xCT-deficient mice are symbiotically reared and the intestinal microbiota is evaluated. [Figure 3B] Figure 1 shows the results of analyzing the intestinal flora of a mouse model of DSS colitis in a symbiotic feeding experiment. [Figure 3C] FIG. 1 shows the results of an analysis of intestinal metabolites in a mouse model of DSS colitis in a symbiotic feeding experiment. [Figure 3D] FIG. 1 shows the results of an analysis of the effect on body weight of wild-type and xCT-deficient mice in a symbiotic rearing experiment. [Figure 3E] Figure 1 shows the results of analyzing intestinal shortening in wild-type and xCT-deficient mice in a symbiotic breeding experiment. [Figure 3F] Figure showing the results of analyzing the histological findings (right) and tissue scores (left) of wild-type and xCT-deficient mice in a symbiotic breeding experiment. [Figure 4A] A schematic diagram of the experimental system for conditioned mice in which xCT is specifically deleted in the intestinal epithelium using the Cre / LoxP system. [Figure 4B] FIG. 1 shows the results of analyzing body weight (left) and disease activity index (right) in a DSS colitis model using conditional knockout mice lacking xCT in the intestinal epithelium. [Figure 4C] FIG. 1 shows the results of an analysis of intestinal shortening in a DSS colitis model using conditional knockout mice lacking xCT in the intestinal epithelium. [Figure 5A] Schematic diagram showing the function of xCT and intracellular glutamine synthesis. [Figure 5B] FIG. 1 shows the results of analyzing the effects of the glutamine synthetase inhibitor methionine sulfoximine (MSO) on body weight (left) and disease activity index (right) in a mouse model of DSS colitis. [Figure 5C] FIG. 1 shows the effect of MSO on intestinal shortening in a mouse model of DSS colitis. [Figure 5D]FIG. 1 is a schematic diagram showing the sites of action of the xCT inhibitor sulfasalazine and the glutamine synthetase inhibitor methionine sulfoximine, and glutamate retention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] In the course of analyzing the mechanism of action of sulfasalazine, the present inventors have found that the retention of glutamate in the intestinal epithelium alleviates the symptoms of inflammatory bowel disease. As will be described in detail below, they have found that xCT inhibitors, which suppress the excretion of glutamate from the intestinal epithelium, and glutamine synthetase inhibitors, which synthesize glutamine from glutamate, are effective in retaining glutamate in intestinal epithelial cells.

[0015] Inhibitors of xCT include sulfasalazine, 5-ASA, imidazole ketone elastin, sorafenib, and regorafenib, which are already used as treatments for inflammatory bowel disease, as well as L-glutamic acid, L-cystine, L-α-aminoadipic acid, L-α-aminopimelic acid, L-homocysteic acid, L-β-N-oxalyl-L-α,β-diaminopropionic acid (β-L-ODAP), L-alanosine, ibotenic acid, L-serine-O-sulfate, ( Examples of such agents include RS)-4-bromo homoibotenic acid, quisqualic acid, (S)-4-carboxyphenylglycine, RS-4-Br-Homo-IBO, 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid (AMPA), arachidonylcyclopropylamide (ACPA), N-acetylamino-3-chloro-N-(2-diethylaminoethyl)benzamide (NACPA), TFMIH, NEIH, and CPPG. In addition, examples of such agents include antibodies, aptamers, and compounds that specifically bind to xCT. In addition, based on the following experimental results and the fact that xCT is not expressed in a steady state but is expressed when inflammatory bowel disease develops, siRNA, shRNA, and the like that suppress the expression of xCT are also considered to be effective.

[0016] It has also been reported that the variant isoform of CD44 (CD44v) stabilizes xCT on the surface of the cell membrane, thereby increasing the uptake of cystine from outside the cell and the export of glutamate from inside the cell. Therefore, it is possible to destabilize xCT and increase intracellular glutamate by inhibiting the binding with CD44v. Examples of such drugs include anti-CD44v antibodies, aptamers that specifically bind to CD44v, and siRNA or shRNA against CD44v.

[0017] In addition, examples of compounds that maintain the intracellular concentration of glutamic acid by inhibiting glutamine synthetase include methionine sulfoximine (MSO), which is also used in the following experiments, and other glutamic acid analogs, such as L-glutamic acid-γ-methyl ester, α-methyl glutamic acid, β-hydroxyglutamic acid, glutamic acid-γ-monohydroxamate, 2-amino-4-phosphonobutyric acid, L-glutamic acid-γ-monoethyl ester, L-glutamic acid dimethyl ester, L-glutamic acid-di-t-butyl ester, monophloroglutamic acid, L-glutamic acid diethyl ester, and D-glutamic acid. In addition, siRNA, shRNA, and the like that suppress the expression of glutamine synthetase are also considered to be effective. Treatment of inflammatory bowel disease by retaining glutamic acid in the intestinal epithelium has a mechanism of action that is different from that of currently used steroids, antibody drugs, and the like, and is therefore considered to have a synergistic effect when used in combination with existing drugs. In the following experiments, the analysis was performed by intraperitoneal administration, but it is preferable to deliver drugs to the intestinal epithelium using a drug delivery system that acts on the intestinal epithelium, such as the use of enema preparations and enteric-coated tablets. In addition to inhibiting xCT and glutamine synthetase, glutamate may also be administered directly. For example, glutamate can be delivered to the intestinal epithelium by using the above-mentioned drug delivery system and administering it in a dosage form that delivers glutamate to the intestine so that it can be taken up by the intestinal epithelium.

[0018] Inflammatory bowel disease is a disease that alternates between remission and active periods. It is also known that it is a multifactorial disease in which genetic and environmental factors are involved in the onset of the disease. Therefore, it is possible to maintain the remission period or prevent onset by administering the above-mentioned drugs as preventive drugs or supplements to those at high risk of developing the disease, such as those with a family history of inflammatory bowel disease. For example, if glutamate is administered as a supplement to high-risk groups in the remission period or before onset, it is highly likely to maintain the remission period and prevent onset. As mentioned above, xCT, which excretes glutamate outside the cells, is not expressed in the steady state, so if it is taken up from the intestinal epithelium during remission or before onset, it is not excreted very much, and it is thought that the glutamate concentration in the intestinal epithelial cells can be maintained. Glutamate is one of the non-essential amino acids synthesized in the body and is also contained in food, so it is thought that there will be no side effects even if it is continuously taken as a supplement. In addition, xCT inhibitors and glutamine synthetase inhibitors can be taken as supplements in doses similar to those of therapeutic drugs or in lower doses to provide a preventive effect.

[0019] The present invention will be explained below with reference to the following data. [Effect of xCT deficiency on DSS-induced colitis] xCT-deficient mice (Non-Patent Document 3) and wild-type (C57BL / 6, WT) mice were induced with inflammatory bowel disease by dextran sulfate sodium (DSS) and used for analysis. - / -) were given 3% DSS as drinking water for 7 days to induce inflammatory bowel disease, and body weight change, disease activity index score (Non-Patent Document 4), and histological findings of the large intestine were analyzed (Figure 1). Figure 1A shows body weight change (left) and disease activity index (right). Weight loss was suppressed in xCT-deficient mice compared to wild-type mice, and significant differences (P<0.0001) were observed especially on the 6th and 7th days after DSS administration. Furthermore, the disease activity index was significantly lower in xCT-deficient mice compared to wild-type mice, indicating resistance to DSS enteritis. In the figures in this specification, significant differences are indicated by *: P<0.05, **: P<0.01, ***: P<0.001, and ****: P<0.0001.

[0020] In addition, shortening of the intestinal tract was observed in the DSS colitis model. Mice were dissected on the 7th day after DSS administration, and the length of the intestinal tract was analyzed. Significant inhibition of shortening of the intestinal tract was observed in xCT-deficient mice (Figure 1B). Furthermore, colon tissue was fixed and microscopically observed by HE staining (Figure 1C). Compared with wild-type mice, the mucosal tissue of the intestinal epithelium was relatively well preserved in xCT-deficient mice, and the tissue score (Non-Patent Document 5), which indicates the degree of inflammation, was also significantly lower.

[0021] Similarly, the mice were fed 3% DSS and then dissected on the 6th day, and the expression of inflammatory cytokines in the colon was analyzed by RT-PCR (Figure 1D). Specifically, the collected colonic tissue was homogenized with a bead homogenizer, and mRNA was extracted using ISOGEN (Nippon Gene) and ReliaPrep™ RNA Miniprep Systems (Promega). Reverse transcription to cDNA was performed using ReverTra Ace™ qPCR RT Master Mix (TOYOBO), and analysis was performed by RT-PCR using THUNDERBIRD™ SYBR qPCR Mix (TOYOBO). β-actin was used as an internal control, and the relative quantification between samples was evaluated. The expression of the inflammatory cytokines Il-6, Il-1β, Cxcl2, Il-10, and Ly6G, known as a neutrophil marker, which were analyzed, was suppressed in xCT-deficient mice.

[0022] Intestinal permeability was analyzed. FITC-labeled dextran (Sigma-Akdrich) was administered via tail vein injection before inducing colitis by giving 3% DSS to the drinking water and 3 days after the start of induction, and intestinal permeability was analyzed. The mice were euthanized 4 hours after administration, and the amount of FITC-dextran in the serum was measured using a multimode plate reader (Molecular Devices) (Figure 1E). On the 3rd day after induction of DSS-induced colitis, the amount of FITC-dextran penetrating into the serum of wild-type mice was significantly increased compared to xCT-deficient mice. On the other hand, the amount of FITC-dextran in the serum of xCT-deficient mice was low, indicating that intestinal permeability was maintained at a low level.

[0023] [Effect of xCT deficiency on immune responses] As mentioned above, although the cause of inflammatory bowel disease is unknown, there are reports that immune responses, deterioration of the intestinal environment, and impaired barrier function of the intestinal epithelium are associated with the worsening of symptoms. Therefore, we decided to analyze immune responses, intestinal environment, and barrier function of the intestinal epithelium. First, immune function was evaluated by bone marrow transplantation. Bone marrow from xCT-deficient mice or wild-type mice was transplanted into wild-type mice, DSS-induced enteritis was induced, and immune function was evaluated (Figure 2A). Bone marrow was removed from wild-type and xCT-deficient mice with a leukocyte cell surface marker of CD45.1 positive as donor mice, and the bone marrow was transplanted into wild-type mice with a CD45.2-positive background that had been pretreated with radiation. After 13 to 15 weeks, the engraftment of the transplanted leukocytes was confirmed, enteritis was induced with 3% DSS, and immune function was evaluated. The transplantation efficiency was 96-97%.

[0024] xCT-deficient mouse-derived bone marrow transplantation mice (in the figure, xCT - / - Although body weight loss was observed on day 9 in xCT-deficient mice (WT → WT) compared to wild-type bone marrow transplantation mice (WT → WT in the figure) (Fig. 2B), there were no significant differences between the two in colon length (Fig. 2C), tissue staining images and histology scores (Fig. 2D), or expression of inflammatory cytokines (Fig. 2E). This indicates that the resistance of xCT-deficient mice to DSS colitis is not due to immune function derived from the blood lineage of the bone marrow.

[0025] [Effects of xCT deficiency on the intestinal environment] Next, we examined the possibility that the intestinal environment of xCT-deficient mice confers resistance to inflammation against DSS-induced colitis. After giving birth to pregnant wild-type and xCT-deficient mice, they were symbiotically reared for 4 weeks until weaning to homogenize the intestinal microbiota, and then genotyped and reared for 4 months after weaning. Fecal samples were collected before DSS administration, and the intestinal microbiota and intestinal metabolites were analyzed (Fig. 3A). In xCT-deficient mice, the proportion of the Bacillales order of the Firmicutes phylum, and further subdivision analysis showed an increase in the proportion of some of the genera Lachnospira and Clostridium (Fig. 3B). These bacteria produce lactic acid and butyric acid, which contribute to anti-inflammatory effects, but analysis of intestinal metabolites showed no difference in lactic acid and butyric acid (Fig. 3C). Mice symbiotically reared under the same conditions were administered 1% DSS for 6 days to analyze the effects of DSS administration. In xCT-deficient mice, weight loss was significantly suppressed from the 5th day of DSS administration (Fig. 3D), intestinal shortening was significantly suppressed (Fig. 3E), and histological scores were significantly reduced (Fig. 3F). Even after symbiotic feeding, xCT-deficient mice showed resistance to inflammation caused by DSS colitis. From the above, although differences were observed in some intestinal bacteria in xCT-deficient mice, it is considered that the differences are not sufficient to affect intestinal metabolites that contribute to inflammation resistance.

[0026] [Effects of xCT deficiency on intestinal epithelial function] Next, we investigated whether the resistance of xCT-deficient mice to DSS-induced colitis was due to a decrease in the barrier function of the intestinal epithelium. Conditional knockout mice lacking xCT specifically in the intestinal epithelium were generated using the Cre / LoxP system. Conditional knockout mice (Slc7a11 F / F) of xCT were generated using frozen sperm purchased from the European Mouse Mutant Archive (EMMA) and crossed with Villin-Cre mice expressing the DNA recombinase enzyme Cre in the intestinal epithelium. Flox / Flox Only xCT is not lost, and Flox / Flox In addition, xCT was deleted in the intestinal epithelium carrying Villin-Cre, which expresses Cre in the intestine (Figure 4A).

[0027] In the conditional knockout mice lacking xCT in the intestinal epithelium, weight loss was significantly suppressed from the sixth day of DSS administration in the DSS colitis model, and an anti-inflammatory effect was observed (Fig. 4B, left). In terms of disease activity index, a significant anti-inflammatory effect was observed in the conditional knockout mice from the fourth day of DSS administration (Fig. 4B, right). Intestinal shortening was also significantly suppressed in the conditional knockout mice (Fig. 4C). These results suggest that the anti-inflammatory effect of xCT deficiency is mediated by intestinal epithelial cells.

[0028] The function of xCT in the intestinal epithelium is to take up cystine into the cells and to excrete glutamate out of the cells (Figure 5A). It has been suggested that glutamine in the intestinal epithelium is important in inflammatory bowel disease (Non-Patent Document 6). Experimental results using xCT-deficient mice suggest that intracellular glutamate is increased, but the increase in glutamine is unknown. Therefore, we inhibited the activity of glutamine synthetase, which synthesizes glutamine from glutamate, and performed analysis. It has been reported that methionine sulfoximine (MSO), an inhibitor of glutamine synthetase, increases glutamate and decreases glutamine in plasma and cells.

[0029] Wild-type mice were given 3% DSS in drinking water for 7 days to develop DSS-induced colitis. During the 7-day colitis induction period, MSO or the solvent PBS was administered intraperitoneally once a day. MSO was administered at 10 mg / kg / day, which has been reported to cause accumulation of glutamate / reduction of glutamine in blood and liver (Non-Patent Document 7). The effects on body weight and disease activity index are shown in Figure 5B. A significant inhibitory effect on weight loss was observed in the MSO-administered group (Figure 5B, left). A significant inhibitory effect on disease activity index was also observed in the MSO-administered group (Figure 5B, right). Furthermore, dissection was performed on the 7th day after the start of the experiment, and the length of the intestine was analyzed, and a significant inhibitory effect on intestinal shortening was observed in the MSO-administered group (Figure 5C).

[0030] As mentioned above, it was previously believed that glutamine is important in alleviating the symptoms of inflammatory bowel disease. However, the results of this experiment showed that it is important to retain glutamate, not glutamine, in the cells, and that retaining glutamate in the cells leads to anti-inflammatory effects. The above results indicate that inflammatory bowel disease can be treated by administering xCT inhibitors, glutamine synthetase inhibitors, or compounds that reduce the expression of xCT or glutamine synthetase.

[0031] In addition, as shown in the experiment using FITC-dextran (see Figure 1E), intestinal permeability was maintained by inhibiting the function of xCT and retaining intracellular glutamate. In other words, it is possible that the barrier function of cells can be enhanced by increasing the intracellular concentration of glutamate. Although it is necessary to wait for the results of future studies, xCT inhibitors and glutamine synthetase inhibitors may be effective against inflammatory diseases caused by disorders of the barrier mechanism of cells, such as atopic dermatitis. There have been no reports to date that the retention of glutamate suppresses inflammatory effects, and this suggests the possibility of providing new medicines for not only inflammatory bowel diseases but also inflammatory diseases caused by disorders of the barrier mechanism of cells.

[0032] A method for screening therapeutic agents for inflammatory diseases including inflammatory bowel disease may involve screening compounds that inhibit xCT or glutamine synthetase. For example, in screening for xCT inhibitors, cells expressing xCT may be used, focusing on the transporter function of xCT, and a candidate compound may be added to the medium to analyze whether the uptake of cystine or the excretion of glutamate in the medium is inhibited. In this case, a detection dye-reactive cystine analog or a fluorescently labeled cystine may be used as an indicator. Compounds that inhibit glutamine synthetase may be screened in an in vitro system using recombinant proteins, using the reaction products glutamine and ADP as indicators.

[0033] In addition, as described above, nucleic acid drugs such as siRNA that suppress the expression of xCT or glutamine synthetase are also thought to be effective, so nucleic acids that reduce the expression of xCT or glutamine synthetase may be designed and used as therapeutic drugs.

Claims

1. A therapeutic and / or preventive agent for inflammatory diseases, comprising as an active ingredient a compound that increases intracellular glutamate.

2. The compound that increases intracellular glutamate is 2. The therapeutic and / or prophylactic agent for inflammatory diseases according to claim 1, which is an xCT inhibitor or a glutamine synthetase inhibitor.

3. The compound that increases intracellular glutamate is 2. The therapeutic and / or prophylactic agent for inflammatory diseases according to claim 1, which is a compound that reduces the expression of xCT or glutamine synthetase.

4. The therapeutic and / or prophylactic agent for inflammatory diseases according to any one of claims 1 to 3, wherein the inflammatory disease is inflammatory bowel disease.

5. Add a candidate compound, A method for screening therapeutic and / or preventive agents for inflammatory bowel disease, which comprises selecting candidate compounds using glutamic acid, cystine, or cysteine ​​as an indicator.

6. Adding a candidate compound to the medium of the cultured cells; Analyzing the expression level of xCT or glutamine synthetase, A method for screening therapeutic and / or preventive agents for inflammatory bowel disease, which comprises selecting candidate compounds using a decrease in the expression level as an index.

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