Application of Iguratimod in preparation of medicine for treating inflammatory bowel disease

Abstract

The invention discloses an application of Iguratimod or a pharmaceutical salt thereof in preparation of a medicine for preventing or treating inflammatory bowel diseases. The iguratimod provided by the invention has a remarkable treatment effect on the inflammatory bowel disease as a small molecule compound, and has the characteristics of low in-vivo and in-vitro toxicity and obvious treatment effect. Therefore, Iguratimod can be used as a therapeutic drug for the inflammatory bowel disease which is a major disease, has important clinical application value, and provides a scientific basis for further exploring the IBD pathogenesis mechanism.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to the use of iguratimod in preparing a medicine for treating inflammatory bowel disease. Background Art

[0002] Inflammatory bowel disease (IBD) is a type of autoimmune disease caused by multiple factors, including genetic susceptibility, immune dysregulation, infection, environmental exposure, and dietary habits. IBD clinical symptoms often include persistent and recurrent diarrhea, abdominal pain, bloody stools, and varying degrees of systemic symptoms. Based on clinical phenotype and lesion characteristics, IBD can be divided into ulcerative colitis (UC) and Crohn's disease (CD). UC lesions are confined to the large intestine, presenting as diffuse intestinal mucosal inflammation that extends from the rectum to part of the colon. In contrast, CD can involve the entire digestive tract, with inflammatory damage not limited to the mucosal tissue but progressing to deep longitudinal and transverse ulcers, accompanied by mucosal edema and intestinal wall thickening.

[0003] The pathogenesis of IBD is essentially an imbalance in the homeostatic network of the gut microbiota, immune system, and epithelial barrier. Although current clinical treatments aim to alleviate inflammatory responses and induce mucosal healing, including traditional immunosuppressants such as glucocorticoids and azathioprine, as well as newer targeted drugs such as anti-TNF-α monoclonal antibodies, the JAK inhibitor tofacitinib, and the sphingosine 1-phosphate receptor modulator ozanimod, the overall remission rate is only 20-30%. Of greater concern is the potential for long-term immunosuppressive therapy to increase the risk of opportunistic infections and malignancies, highlighting the urgent need to develop novel therapeutic strategies.

[0004] The physical and chemical barrier formed by the intestinal epithelium not only effectively blocks direct contact between external antigens and the intestinal immune system but also maintains a balanced intestinal immune microenvironment through dynamic regulatory mechanisms. The integrity of this barrier function is a key component of intestinal homeostasis, and its impairment is closely associated with the development and progression of IBD. Intestinal epithelial cells (IECs), composed of a single layer of polarized epithelial cells, maintain intestinal mucosal immune tolerance through chemical and physical barriers, as well as interactions between IECs, immune cells, and intestinal microbes. Increased intestinal epithelial permeability is not only a consequence of intestinal damage in IBD but also plays a driving role in the early stages of disease onset. Intestinal epithelial permeability can be divided into three transepithelial pathways: the pore-forming pathway, the leaky pathway, and the non-selective pathway caused by epithelial cell damage. The apical junction complex (AJC) regulates selective transepithelial exchange of substances and maintains low intestinal permeability. The AJC is composed of tight junctions, adherens junctions, and desmosomal junctions, each of which performs its own function to maintain a selective epithelial barrier. Adherens junctions, composed of transmembrane proteins E-cadherin and nectin, and cytoplasmic proteins catenins, connect to the cytoskeleton to maintain mechanical adhesion between cells. Transmembrane desmosomal proteins, called desmosomes, connect to intermediate filaments to maintain the mechanical strength of epithelial junctions. Tight junction proteins are core components controlling paracellular material exchange. Adherens junctions retain a 15-20 nm gap, while transmembrane tight junction proteins completely seal the intercellular space, regulating epithelial permeability through their selective channeling. The tight junction protein family includes the transmembrane proteins OCLN, CLDNs, JAMs, and tricellulin, which form a linear barrier, and the cytoplasmic scaffold proteins ZOs, cingulin, and afadin, which connect to the cytoskeleton. Inflammatory factors upregulated in IBD lesions induce changes in the expression of specific claudin family proteins, affecting the pore-forming pathway. For example, upregulation of claudin-2 increases cation permeability in the pore-forming pathway; endocytosis of occludin increases the leakage pathway; and downregulation of tricellulin increases paracellular permeability for macromolecules. Although current clinical treatments for IBD aim to alleviate abnormal inflammatory responses, therapeutic strategies that restore epithelial barrier function have shown IBD-alleviating effects in clinical trials in recent years. Therefore, therapeutic drugs that regulate tight junction proteins to improve the epithelial barrier are of great significance for the early treatment of IBD. Summary of the Invention

[0005] The present invention aims to provide a use of iguratimod in preparing a medicine for treating inflammatory bowel disease.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of the present invention provides a use of iguratimod or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating inflammatory bowel disease.

[0008] The structure of Iguratimod (DE3834204C2) is shown below:

[0009]

[0010] The dosage of iguratimod in the animal model is 5-25 mg / kg.

[0011] The drug for preventing or treating inflammatory bowel disease uses iguratimod or a pharmaceutically acceptable salt thereof as the sole active ingredient.

[0012] The pharmaceutically acceptable salt is an acid addition salt formed by iguratimod with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.

[0013] The second aspect of the present invention provides a pharmaceutical preparation prepared from iguratimod or a pharmaceutically acceptable salt thereof and medically acceptable excipients.

[0014] The dosage form of the pharmaceutical preparation is selected from liquid medicine, tablet or capsule.

[0015] The pharmaceutical preparation can be administered orally, intravenously, or intraperitoneally.

[0016] The third aspect of the present invention provides a pharmaceutical composition prepared from iguratimod or a pharmaceutically acceptable salt thereof and other drugs for treating inflammatory bowel disease.

[0017] The other drugs for treating inflammatory bowel disease are selected from glucocorticoids, azathioprine, anti-TNF-α monoclonal antibody, tofacitinib, ozanimod, etc.

[0018] The present invention demonstrates the effectiveness of iguratimod in treating inflammatory bowel disease through both whole animal and cell-based studies. Specifically, iguratimod significantly increased occludin protein expression in intestinal organoids derived from patients with CD and effectively reduced paracellular leakage of FITC-dextran macromolecules (4kD). BrdU staining demonstrated that iguratimod effectively maintained intestinal epithelial cell proliferation under TNFα induction. Occludin immunofluorescence analysis demonstrated that iguratimod effectively ameliorated TNFα-induced epithelial structural damage in intestinal organoids. Mechanistic studies revealed that iguratimod effectively mitigated IFNγ / TNFα-induced occludin internalization and degradation in HT29 intestinal epithelial cells by reducing MLCK expression and inhibiting downstream p-MLC2 phosphorylation.

[0019] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0020] The iguratimod provided by the present invention, as a small molecule compound, has a significant therapeutic effect on inflammatory bowel disease, with low in vitro and in vivo toxicity and significant therapeutic effect. Therefore, iguratimod can be used as a therapeutic drug for inflammatory bowel disease, a major disease, and has important clinical application value. It also provides a scientific basis for further exploration of the pathogenesis of IBD. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram showing that iguratimod increases the expression of tight junction protein Occludin and alleviates the inflammation-related phenotype of PDO in vitro.

[0022] Figure 2 Schematic diagram of how iguratimod inhibits MLCK / p-MLC2 and maintains occludin expression.

[0023] Figure 3 This is a schematic diagram of the results of guratimod alleviating the pathological changes of DSS-induced acute colitis damage in mice.

[0024] Figure 4 This is a schematic diagram showing that iguratimod has no significant toxicity to various organs of mice with DSS-induced IBD.

[0025] Figure 5 This is a schematic diagram of the results of elamod alleviating DSS-induced intestinal inflammatory response.

[0026] Figure 6 This is a schematic diagram of the results of iguratimod alleviating TNBS-induced IBD pathological changes. DETAILED DESCRIPTION

[0027] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the following specific description is illustrative rather than restrictive and should not be used to limit the scope of protection of the present invention.

[0028] Example 1

[0029] Iguratimod alleviates inflammation-related phenotypes in in vitro patient-derived organoids (PDOs):

[0030] Materials: Iguratimod was purchased from MCE, USA; TNFα recombinant protein was purchased from Peprotech; LCL-161 was purchased from MCE, USA; FITC-Dextran was purchased from Merck, USA; antibodies against E-cadherin, Occludin, and GAPDH were purchased from Cell Signaling Technology, USA; antibody Desmoglein 2 was purchased from Abcam; and immunofluorescence secondary antibodies were purchased from ThermoFisher, USA.

[0031] Experimental methods:

[0032] Establish patient-derived intestinal epithelial organoid culture: According to the literature (Molecular and Functional Characterization of Human Intestinal Organoids and Monolayers for Modeling Epithelial Barrier. Inflamm Bowel Dis, 2023, 29(2): 195-206), patient-derived intestinal crypts were collected to construct in vitro organoid culture, expanded and cultured for 4 days, and differentiated for 2 days to obtain differentiated epithelial cell structures.

[0033] Figure 1This is a schematic diagram showing how iguratimod increases tight junction protein occludin expression and alleviates PDO inflammatory phenotypes in vitro. PDO culture process: Patient tissue biopsies are washed with sterile PBS and cut into small pieces. EDTA is added to a final concentration of 10 mM and dissociated on a rotary shaker at 4°C for 30 minutes. After dissociation, vortex at maximum speed for 30 seconds, repeat 2-4 times, and allow to settle. The supernatant is aspirated and resuspended in 10 ml of DMEM (containing 0.1% BSA). The supernatant is filtered through a 70 μm filter and centrifuged at 400 g for 3 minutes at 4°C. A 10 μl sample is collected and observed under a microscope. If a large number of crypts, rich in adult intestinal stem cells, are visible, the isolation is successful and subsequent culture is performed. During the first three days of expanded culture after isolation or passaging (Expended Culture), PDOs existed as proliferative intestinal stem cells. Starting from the fourth day, after using iWAP2 to block Wnt3a signaling for 36 hours, the proportion of stem cells in the ileal organoids decreased, the proportion of absorptive intestinal epithelial cells (Enterocytes) and goblet cells increased, and the cells differentiated into a state dominated by epithelial cells (Enterocytes enriched). Bright field observation of PDO morphology showed that the luminal structure disappeared and the budding increased significantly ( Figure 1 This demonstrates that patient-derived organoids have effectively differentiated into an in vitro model that closely resembles the composition and structure of human terminal ileum epithelial cells.

[0034] To examine the effect of iguratimod on the expression of junction proteins in patient-derived organoids, 150 organoids were plated in 24-well plates during the expansion phase, cultured for 4 days, and differentiated for 2 days. Iguratimod (at concentrations of 15, 30, and 60 μM) was then added. After 6 hours of culture, the organoids were harvested and protein was extracted. The expression of intercellular junction proteins was assayed by Western Blot, using GAPDH as an internal control. Protein expression was assayed 6 hours after iguratimod treatment, revealing that iguratimod significantly increased the expression of the tight junction protein Occludin, and that the adhesion link protein E-cadherin and the desmosomal protein Desmoglein2 also showed an upward trend ( Figure 1 (As shown in B). Figure 1 The abbreviation is IGU.

[0035] The effect of iguratimod on the proliferation of patient-derived organoids induced by TNFα / LCL-161 was tested: 20 organoids were plated per well in a 96-well plate during the expansion phase and cultured for two days. The cells were pretreated with 30 μM iguratimod for 2 hours, and then stimulated with 50 ng / ml TNFα and 5 μM LCL-161 for 18 hours. BrdU staining was performed to detect cells that retained the ability to proliferate. The results are shown in Figure 2. Figure 1As shown in Figure C, PDO disintegrates and loses its proliferative capacity under the combined effects of TNFα and the IAP inhibitor LCL-161. BrdU assays for cell proliferation revealed a significant decrease in the number of cells remaining proliferative 18 hours after induction, a finding partially alleviated by iguratimod treatment. This suggests that iguratimod has a protective effect on alleviating inflammation-induced damage to epithelial cell viability and epithelial barrier structure.

[0036] To test the effect of iguratimod on the paracellular permeability pathway of patient-derived organoids: paired patient lesion-derived organoids and non-lesion-derived organoids were plated at 50 cells / well in a 4-well confocal chamber, cultured for 4 days of expansion and differentiation for 2 days, and then stained with 1 mg / ml FITC-Dextran for 30 minutes. The fluorescence intensity in the chamber was observed under a confocal microscope. Figure 1 As shown in Figure D, BF indicates brightfield observation of PDO morphology, and merge indicates the superposition of brightfield and fluorescence observation channels. PDO-N, PDO-I, and PDO-I+IGU represent control organoids, patient-derived organoids derived from adjacent lesions, and patient-derived organoids treated with iguratimod (abbreviated as IGU), respectively. The luminal structure of PDO is naturally suitable for monitoring barrier function. Using FITC-dextran 4kDa to characterize tight junction-mediated paracellular permeability, we observed increased intraluminal FITC fluorescence intensity and increased permeability in lesion-derived PDO, which was alleviated after iguratimod treatment, reducing the permeability of the epithelial paracellular leakage pathway.

[0037] The effect of iguratimod on the barrier structure of patient-derived organoids induced by TNFα / LCL-161 was examined: 50 organoids were plated per well in a 4-well confocal microwell. After 4 days of expansion and 2 days of differentiation, 30 μM iguratimod was added for 2 hours. After 18 hours of stimulation with TNFα 50 ng / ml, the cells were fixed and permeabilized. The cells were stained with Occludin (1:200) at 4°C overnight and incubated with secondary antibody (AF488 Mouse anti-Rabbit) (1:500) at room temperature for two hours. The cells were observed under a confocal microscope. The results are shown in Figure 2. Figure 1 As shown in E, TNFα was used to induce PDO to simulate inflammatory damage. Treatment with 50 ng / ml rhTNFα for 18 hours induced the collapse of the PDO monolayer epithelial structure and loss of intercellular connections. Treatment with iguratimod helped maintain normal epithelial cell arrangement and occludin expression.

[0038] Example 2

[0039] Iguratimod inhibits MLCK / p-MLC2 to maintain occludin membrane localization

[0040] Materials: Recombinant IFNγ and TNFα were purchased from Peprotech, USA; antibodies ZO-1 and p-MLC2 were purchased from Cell Signal Technology, USA; antibody JAM-A was purchased from Proteintech, China; and cytoplasm and cell membrane separation kit was purchased from Beyotime, China.

[0041] Experimental methods:

[0042] Detection of the effect of iguratimod on the expression of connexins in HT-29 cells induced by IFNγ / TNFα: HT-29 cells were cultured at 1.5×10 5 Cells were plated in 12-well plates and pretreated with iguratimod (15, 30, and 60 μM, respectively) for 2 h, stimulated with IFNγ 20 ng / ml for 48 h, and then co-stimulated with TNFα 40 ng / ml for 4 h. Proteins were extracted and the expression of intercellular junction proteins was detected by Western Blot. Figure 2 As shown in A, Figure 2 This is a schematic diagram showing that iguratimod inhibits MLCK / p-MLC2 and maintains Occludin expression. Figure 2 Abbreviated as IGU in

[15] . IFNγ combined with TNFα stimulation significantly reduced occludin protein expression, while having no significant effect on other connexins, E-cadherin, ZO-1, JAM-A, and Desmoglein 2, consistent with the PDO screening results. Iguratimod treatment effectively alleviated the inflammatory injury-induced decrease in occludin expression.

[0043] The effect of iguratimod on the expression of MLCK and Occludin in HT-29 cells induced by IFNγ / TNFα was examined: HT-29 cells were cultured at 1.5×10 5 Cells were plated in 12-well plates and pretreated with iguratimod (15, 30, and 60 μM, respectively) for 2 h. IFNγ 20 ng / ml was added for stimulation for 48 h. TNFα 40 ng / ml was then added for co-stimulation for 4 h. Cell proteins were extracted using a membrane separation kit, and the expression of cytoplasmic and membrane proteins was detected by Western Blot. Figure 2As shown in Figure B, after IFNγ / TNFα induction, cytoplasmic and membrane proteins were isolated and analyzed by Western blot for protein expression. Inflammatory cytokine induction increased MLCK expression in the whole cell, and membrane-localized expression was also significantly upregulated. Following iguratimod pretreatment, the induced increase in MLCK expression gradually decreased with increasing drug concentrations. Meanwhile, tight junction protein occludin, which is almost exclusively distributed on the cell membrane, experienced a sharp decrease in membrane expression after IFNγ / TNFα induction, impairing tight junctions. However, with increasing iguratimod concentrations, occludin's cell membrane localization gradually recovered and increased.

[0044] The effect of iguratimod on IFNγ / TNFα-induced p-MLC2 expression in HT29 cells was detected: 1000 HT-29 cells / well were plated in a 4-well confocal chamber, stimulated with IFNγ 20ng / ml for 24h, and then co-stimulated with TNFα 40ng / ml for 4h, fixed with 4% PFA for 30min, permeabilized with 0.5% Triton-X100 for 30min, blocked with 3% goat serum for 1h, incubated with p-MLC2 primary antibody overnight, and incubated with secondary antibody (AF488 Mouse anti-Rabbit) (1:500) at room temperature for two hours, and detected under a confocal microscope. The results are shown in Figure 2. Figure 2 As shown in middle C, the phosphorylation level of p-MLC2 increased significantly under IFNγ / TNFα induction, especially along the stress fibers, indicating that the contractility of epithelial cells was enhanced, affecting the tight junction barrier function. The phosphorylation level of p-MLC2 was significantly inhibited after treatment with igurumod.

[0045] Example 3

[0046] Effects of iguratimod on dextran sodium sulfate (DSS)-induced ulcerative colitis (UC)-like IBD model

[0047] Materials: C57BL / 6 mice were purchased from the Experimental Animal Center of Shanghai Jiao Tong University, and dextran sodium sulfate was purchased from MP Biotechnology Co., Ltd., USA.

[0048] Experimental methods:

[0049] Establishment of DSS-induced IBD model: A DSS-induced IBD model was established according to the literature (Chemically induced mouse models of acute and chronic intestinal inflammation. Nat Protoc 2017, 12, 1295–1309). 40 male C57BL / 6 mice were randomly divided into five groups: vehicle control group (Vehicle group), 3% DSS group (DSS model group), 250 mg / kg 5-aminosalicylic acid group (5-ASA control group), iguratimod low-dose group (5 mg / kg) and iguratimod high-dose group (25 mg / kg), with 8 mice in each group. Figure 3 The abbreviation is IGU.

[0050] Detection of relevant indicators of the IBD model: daily monitoring of mouse body weight changes, and 10 days after modeling, the mice were killed, and colon tissue was obtained to measure the length of the mouse colon. Hematoxylin-eosin (H&E) staining was used to evaluate intestinal tissue structure, Ki67 staining was used to detect the proportion of intestinal proliferative epithelial cells, and Alcian blue staining-periodic acid-Schiff staining (AB-PAS staining) was used to detect the intestinal chemical barrier. Colon tissue protein was extracted for occludin and MLCK expression detection; intestinal epithelial cells in the colon tissue were isolated, and protein was extracted for occludin and MLCK expression detection; heart, liver, spleen, lung, and kidney tissue specimens were isolated, and H&E staining was used to analyze the tissue structure.

[0051] The results are as follows Figure 3 As shown, Figure 3 The following is a schematic diagram showing the pathological changes of iguratimod in alleviating DSS-induced acute colitis in mice. Figure 3 As shown in Figure A, iguratimod can slow down weight loss in mice with DSS-induced IBD. Following DSS induction, the body weight of mice in the DSS model group decreased significantly compared to the vehicle group. Both the low-dose and high-dose iguratimod groups were able to effectively slow weight loss (p<0.001), demonstrating that iguratimod can effectively slow down weight loss in mice with DSS-induced IBD.

[0052] The results of the effect of iguratimod on colon length in DSS-induced IBD mice are as follows Figure 3As shown in FIG. 6B, the length of the colon of the mice was shortened after DSS induction. The length of the colon of the mice in the DSS model group was significantly shortened compared with the Vehicle group. The length of the colon of the mice in the low-dose and high-dose IL-17A antibody groups was effectively reduced compared with the DSS model group (p<0.001), indicating that IL-17A antibody can effectively slow down the shortening of the colon of the mice with DSS-induced IBD.

[0053] The results of the alleviation of intestinal barrier injury induced by DSS by IL-17A antibody are shown in FIG. 6C. Figure 3 As shown in FIG. 6C, IL-17A antibody alleviates the histological structure and intestinal barrier injury of the mice with DSS-induced IBD. After DSS induction, the epithelial structure of the intestinal tract of the mice was severely damaged, the intestinal crypt was lost, and a large number of inflammatory cells infiltrated between the crypt and the basal layer. The positive drug 5-aminosalicylic acid partially improved the inflammation, the number of crypts partially rose, and the distance between the crypts was wide; however, the length of the crypt was shortened, and the bottom was not directly attached to the mucosal muscle layer. The intestinal tissue structure of the mice in the drug treatment group injected with IL-17A antibody was significantly alleviated. Immunohistochemistry of the colon of the mice showed that proliferative cells (ki67 + ) were mainly expressed in the crypt part, and the distribution gradually decreased toward the top of the villus, consistent with the crypt-villus axis. The differentiation degree gradually increased, and the stemness gradually decreased in the process of migration from the crypt to the top of the villus. In the DSS model group, the crypt was lost, the expression of ki67 of the epithelial cells was reduced, and the infiltration of immune cells between the lower layer of the crypt and the mucosal muscle layer was increased; after administration of IL-17A antibody, the proportion of ki67 + cells in the crypt increased, and the expression of ki67 in the high-dose IL-17A antibody group was widely distributed along the crypt-villus axis, showing the characteristics of tissue repair. A large number of mucin secreted by goblet cells maintains the chemical barrier of the intestinal tract. In the DSS-induced model group, due to the damage to the crypt structure, the secretory epithelial cells were lost, and the secretion of mucin was reduced. Only a small amount of neutral mucin expression (dark purple) was observed by AB-PAS staining. In the 5-ASA group and the low-dose IL-17A antibody group, the expression of mucin was significantly increased. In the high-dose IL-17A antibody group, the proportion of acid mucin was increased, and the chemical barrier was repaired. H&E, ki67, and AB-PAS staining showed that IL-17A antibody effectively improved the damage to the intestinal tissue structure of the mice induced by DSS and repaired the chemical barrier of the intestinal mucosa.

[0054] The results of the inhibition of the expression of MLCK and the increase in the expression of Occludin in the colon of the mice with DSS-induced IBD by IL-17A antibody are shown in FIG. 6D. Figure 3 As shown in FIG. 6D, after the intestinal epithelial cells of the mice were separated, the expression of the proteins was detected by WB. The expression of MLCK protein in the intestinal epithelial cells was significantly increased after DSS modeling, and the expression of Occludin protein was significantly reduced. IL-17A antibody significantly inhibited the expression of MLCK protein and increased the expression of Occludin protein, thereby improving the tight junction of the intestinal epithelial cells.

[0055] The results of iguratimod inhibiting the expression of p-MLC2 in the colon of DSS-induced IBD mice are as follows Figure 3 As shown in Figure E, immunohistochemistry was used to detect the expression of p-MLC2 in intestinal epithelial cells. As shown in the figure, after DSS-induced modeling, the phosphorylation level of p-MLC2 increased with the increase of MLCK expression. Immunohistochemical analysis specifically showed that the expression of p-MLC2 in intestinal epithelial cells increased. The high-dose iguratimod group showed a significant decrease in the phosphorylation level of p-MLC2 in the intestinal epithelium. Semi-quantitative analysis of the immunohistochemical results was performed using ( Figure 3 (right side of Figure E).

[0056] The results showed that iguratimod had no significant toxicity to various organs of DSS-induced IBD mice. Figure 4 As shown, Figure 4 This figure shows the results showing that iguratimod had no significant toxicity to various organs in mice with DSS-induced IBD. HE staining confirmed that iguratimod had no significant damage to the heart, liver, kidney, lung, spleen, and other organs of the mice.

[0057] Example 4

[0058] Iguratimod alleviates DSS-induced intestinal inflammatory response

[0059] Materials: CBA kits were purchased from BD, and antibodies Anti-CD45-APC-AF750, Anti-CD11b-FITC, Anti-CX3CR1-PerCP-Cy5.5, Anti F4 / 80-APC, Anti-CD3-FITC, Anti-CD4-PerCP-Cy5.5, Anti-CD8-PE-Cy7, and Anti-Ly6c-PE were purchased from BioLegend.

[0060] Peripheral blood serum was collected from the vehicle control group (Vehicle group), 3% DSS group (DSS model group), iguratimod low-dose group (5 mg / kg) and iguratimod high-dose group (25 mg / kg) of DSS-induced ulcerative colitis mice, and the concentrations of inflammatory factors in peripheral blood serum were detected according to the CBA kit operating procedures.

[0061] Figure 5 This is a schematic diagram of the results of iguratimod alleviating DSS-induced intestinal inflammatory response. Figure 5As shown in FIG. 2A, the expression levels of IL-6, MCP-1, IFNγ and TNFα in the peripheral blood of the DSS-induced model group were significantly increased, indicating that the mice had a severe reaction. However, the expression of inflammatory factors in the peripheral blood was significantly inhibited in the low-dose and high-dose IL-23p19 antibody treatment groups. The expression level of IL-6 in the low-dose IL-23p19 antibody treatment group was significantly reduced (p<0.01). The expression levels of MCP-1 (p<0.0001), IFNγ (p<0.05) and TNFα (p<0.01) in the peripheral blood of the high-dose IL-23p19 antibody treatment group were significantly reduced, indicating that the IL-23p19 antibody had a significant overall in vivo anti-inflammatory effect.

[0062] The terminal colon segments of about 0.5 cm in length were collected from the DSS-induced mice with ulcerative colitis in the solvent control group (Vehicle group), 3% DSS group (DSS model group) and high-dose IL-23p19 antibody treatment group (25 mg / kg), and total RNA was extracted by Trizol for the analysis of inflammatory factors by fluorescent quantitative PCR. The results are shown in FIG. 2B. Figure 5 As shown in FIG. 2B, the expression levels of TNFα, IL-6, IFNγ, IL1β, IL12b, IL21, IL22, iNOS and CXCL1 in the intestinal tissue of the DSS model group were significantly increased, indicating that the immune response of the intestinal tissue of the mice was abnormally activated. However, the transcription and expression of inflammatory factors in the intestinal tissue were significantly inhibited in the high-dose IL-23p19 antibody treatment group. The expression levels of TNFα (p<0.01), IL-6 (p<0.05), IFNγ (p<0.05), IL1β (p<0.01), IL12b (p<0.01), IL21 (p<0.01), IL22 (p<0.05), iNOS (p<0.01) and CXCL1 (p<0.0001) in the peripheral blood were significantly reduced, indicating that the IL-23p19 antibody significantly relieved the abnormally activated immune response of the intestinal tissue.

[0063] The terminal colon segments of about 1 cm in length were collected from the DSS-induced mice with ulcerative colitis in the solvent control group (Vehicle group), 3% DSS group (DSS model group), low-dose IL-23p19 antibody treatment group (5 mg / kg) and high-dose IL-23p19 antibody treatment group (25 mg / kg). The intestinal tissue of 6 mice in each treatment group was collected and combined as one sample. The intestinal tissue immune cells were isolated according to the literature (Wen Zhenliang et al., 2019, Flow cytometric detection of intestinal lamina propria mononuclear phagocytes and cytokine secretion). Staining was performed according to the staining protocol recommended by Biolegend, and detection was performed on a BD-FortessaX20. The results are shown in FIG. 2C. Figure 5 As shown in FIG. 2C, the analysis of the myeloid and lymphoid immune cell clusters in the intestinal lamina propria of the mice before and after modeling and treatment showed that the pro-inflammatory CD3 + CD8 +cells (p<0.05) and Ly6c + CX3CR1 int (p<0.05) cell population ratio, and Figure 5 The results in B are consistent, indicating that elamod significantly alleviates the immune response of intestinal tissue.

[0064] Example 5

[0065] Iguratimod alleviates trinitrobenzenesulfonic acid (TNBS)-induced Crohn's disease (CD)-like IBD model

[0066] Materials: BALB / C mice were purchased from the Experimental Animal Center of Shanghai Jiao Tong University, and TNBS and tribromoethanol were purchased from Dalian Meilun Biotechnology Co., Ltd.

[0067] The TNBS-induced IBD model was established by enema of 50% TNBS-50% EtOH (final TNBS concentration was 2.5%). According to the literature (Nature protocols, 12(7), 1295–1309.), 40 male BALB / C mice were randomly divided into five groups: vehicle control group (Vehicle group), 50% TNBS-EtOH group (model group), 3 mg / kg tacrolimus treatment group (TAC control group), low-dose iguratimod group (5 mg / kg) and high-dose iguratimod group (25 mg / kg), with 8 mice in each group.

[0068] Detection of relevant indicators of the IBD model: The weight changes of mice were detected daily. The mice were killed 4 days after modeling, the colon tissue was taken to measure the length of the mouse colon, and the intestinal tissue structure was evaluated by hematoxylin and eosin (H&E) staining.

[0069] The results are as follows Figure 6 As shown, Figure 6 The following is a schematic diagram showing the results of iguratimod alleviating the pathological changes in TNBS-induced IBD mice. Figure 6 As shown in A, the effect of iguratimod on the body weight of TNBS-induced IBD mice is shown in Figure 6 As shown in Figure B, iguratimod can slow down the weight loss of mice with TNBS-induced IBD. After TNBS induction, the body weight of mice in the TNBS model group was significantly reduced compared with the vehicle group. Both the low-dose and high-dose iguratimod groups were able to effectively slow down the weight loss of mice (p<0.05), indicating that iguratimod can effectively slow down the weight loss of mice with TNBS-induced IBD.

[0070] The results of the effect of iguratimod on colon length in TNBS-induced IBD mice are as follows Figure 6As shown in Figure C, iguratimod alleviates TNBS-induced shortening of colon length in IBD mice. Following TNBS induction, the colon length of mice in the TNBS model group was significantly shortened compared to the vehicle group. Both the low-dose and high-dose iguratimod groups were able to effectively reduce the reduction in colon length (p<0.05), indicating that iguratimod can effectively mitigate TNBS-induced shortening of colon length in IBD mice.

[0071] Iguratimod alleviates TNBS-induced intestinal tissue damage Figure 6 As shown in D, H&E staining showed that the intestinal tissue of mice was severely damaged after TNBS enema, and the intestinal crypt structure was lost. After intraperitoneal injection of tacrolimus and iguratimod, the intestinal tissue structure of mice was significantly restored.

[0072] The iguratimod provided by the present invention can effectively treat DSS-induced ulcerative colitis in C57 mice and TNBS-induced Crohn's disease-like IBD, slowing down weight loss in mice, reducing colon shortening in mice, improving intestinal tissue damage in mice, and maintaining relatively intact tissue structure; it has an inhibitory effect on the immune response of mouse intestinal tissue and peripheral blood, and maintains occludin protein expression in intestinal epithelial cells by inhibiting the MLCK / p-MLC2 pathway, improving the intestinal epithelial barrier under inflammatory damage; and has no significant toxicity to various organs in DSS-induced IBD mice. In in vitro experiments, it was confirmed from IFNγ / TNFα-stimulated HT-29 cells that iguratimod protects the intestinal epithelial cell barrier function under inflammatory damage by inhibiting the MLCK / p-MLC2 pathway and maintaining occludin expression and membrane localization.

[0073] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with the present invention can make slight changes or modifications to equivalent embodiments using the above-mentioned technical contents without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the solution of the present invention.

Claims

1. Use of iguratimod or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating inflammatory bowel disease.

2. Use of iguratimod or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a medicament for preventing or treating inflammatory bowel disease, characterized in that: The structure of Iguratimod is shown below: 。 3. Use of iguratimod or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a medicament for preventing or treating inflammatory bowel disease, characterized in that: The drug for preventing or treating inflammatory bowel disease uses iguratimod or a pharmaceutically acceptable salt thereof as the sole active ingredient.

4. Use of iguratimod or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a medicament for preventing or treating inflammatory bowel disease, characterized in that: The pharmaceutically acceptable salt is an acid addition salt formed by iguratimod with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.

5. A pharmaceutical preparation, characterized in that The invention is prepared from iguratimod or its pharmaceutically acceptable salt and medically acceptable auxiliary materials.

6. The pharmaceutical preparation according to claim 5, characterized in that The dosage form of the pharmaceutical preparation is selected from liquid medicine, tablet or capsule.

7. The pharmaceutical preparation according to claim 5, characterized in that The pharmaceutical preparation can be administered orally, intravenously, or intraperitoneally.

8. A pharmaceutical composition, characterized in that The invention is prepared from iguratimod or its pharmaceutically acceptable salt and other drugs for treating inflammatory bowel disease.

9. The pharmaceutical composition according to claim 8, characterized in that The other drugs for treating inflammatory bowel disease are selected from glucocorticoids, azathioprine, anti-TNF-α monoclonal antibody, tofacitinib, and ozanimod.

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