Application of FOXP3 specific regulator 430C10 in preparation of medicine for treating tissue inflammatory diseases caused by IFN-gamma

Abstract

The invention discloses an application of an FOXP3 specific regulator 430C10 in preparation of a medicine for treating tissue inflammatory diseases caused by IFN-gamma (interferon-gamma). Animal experiments prove that the FOXP3 specific regulator 430C10 can obviously improve tissue inflammation and disease progression caused by IFN-gamma in an IPEX mouse model caused by FOXP3 p.V408M mutation, a mouse enteritis model induced by dextran sodium sulfate salt and a scleroderma mouse model induced by bleomycin; meanwhile, an in-vitro experiment proves that the FOXP3 specific regulator 430C10 obviously inhibits the generation of IFN-gamma in regulatory T cells under an inflammatory condition, and further proves that the 430C10 has a remarkable treatment effect on all tissue inflammatory diseases caused by IFN-gamma, has a relatively good clinical application prospect and application benefits, and expands a new application field for the FOXP3 specific regulator 430C10.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the use of a FOXP3 specific modulator 430C10 in the preparation of a drug for treating tissue inflammatory diseases caused by IFN-γ. Background Technology

[0002] Regulatory T cells (Tregs) are a special subset of CD4+ T cells with inhibitory activity, playing important but distinct roles in immune homeostasis, the tumor microenvironment, and autoimmune diseases. In type I inflammatory responses, Tregs secrete large amounts of type II interferon γ (IFN-γ) and exhibit dysfunction, thereby losing control over Th1 inflammation. Furthermore, the accumulation of IFN-γ+ Tregs can further promote Th1 inflammatory responses. (Li, Lu et al. 2016, Kitz and Dominguez-Villar 2017, Huang, Tian et al. 2024, Liu, Zhang et al. 2024).

[0003] Immune dysregulation, poly-endocrinopathy, enteropathy, X-linked syndrome (IPEX) is a rare, X-linked autoimmune disease caused by the deletion or mutation of the Treg lineage-determining transcription factor FOXP3. More than 70 FOXP3 mutations have been identified that can lead to IPEX, with varying degrees of disease severity depending on the mutation, and even the same mutation can present different clinical phenotypes. Most mutations enhance Th2 and Th17-mediated immune responses, accompanied by elevated serum autoantibody and IgE levels. Due to individual patient differences, different types of immune responses, and the unclear pathological mechanisms caused by FOXP3 mutations, the clinical treatment of IPEX still faces significant challenges (Huang, Liu et al. 2020). However, in our recent study, combining single-cell sequencing results from patients and a gene mutation knock-in mouse model, we found that IPEX syndrome caused by the FOXP3p.V408M mutation is a Th1-type inflammatory response induced by IFN-γ+ Tregs.

[0004] Inflammatory bowel diseases (IBD) are a group of chronic inflammatory bowel diseases of unknown cause, primarily including Crohn's disease (CD) and ulcerative colitis (UC). Their pathogenesis involves genetic susceptibility, gut microbiota dysbiosis, and abnormal immune responses, particularly the Th1 / Th17 axis and regulatory T cells. Imbalance Crohn's disease is traditionally considered to be primarily Th1 cell-mediated inflammation. Th1 cells activate macrophages by secreting cytokines such as IFN-γ and tumor necrosis factor-α (TNF-α), amplifying the inflammatory response. (Sakuraba, Sato et al. 2009, Strober and Fuss 2011, Geremia, Biancheri et al. 2014, Liu, van Sommeren et al. 2015). Meanwhile, Treg cells play a crucial negative regulatory role in maintaining intestinal immune homeostasis. In IBD patients, although the number of Tregs may not decrease or may even increase, their suppressive function may be impaired. (Jaeger, Gamini et al. 2021, Kosinsky, Gonzalez et al. 2024). Recent studies have found that Treg cells possess functional plasticity and can differentiate into Th1-like Treg cells with high IFN-γ expression under specific inflammatory conditions, losing their inhibitory capacity and participating in the inflammatory process. (Li, Lu et al. 2016, Kitz and Dominguez-Villar 2017, Levine, Mendoza et al. 2017, Huang, Tian et al. 2024, Liu, Zhang et al. 2024). In a mouse model of DSS-induced colitis, the number of Th1-like Tregs expressing IFN-γ increased in the intestinal lamina propria, and their upregulation preceded that of conventional Th1 cells, indicating that Th1-like Tregs enhance the initial stage of inflammation by promoting Th1 cell development. (Hovhannisyan, Treatman et al. 2011, Di Giovangiulio, Rizzo et al. 2019). In summary, Th1 inflammation (through cytokines such as IFN-γ) and impaired Treg cell function play important roles in the pathogenesis of IBD (especially Crohn's disease), and Th1-like Tregs may be a key step in the Th1 tilting process during intestinal inflammation.

[0005] Systemic sclerosis (SSC) is an autoimmune disease characterized by progressive fibrosis of the skin and internal organs, microvascular complications, and the production of autoantibodies. Current research has found that in the early stages of SSC progression, impaired Treg function in patients leads to enhanced Th1, Th2, and Th17-mediated inflammation, thereby promoting SSC pathogenesis. (Frantz, Auffray et al. 2018, Zhu, Du et al. 2019, Gaydosik, Tabib et al. 2021). In the early stages of SSc, IFN-γ is produced in large quantities by T cells and NK cells, amplifying endothelial-immune cell interactions through the JAK-STAT axis and exacerbating vascular damage. Simultaneously, it synergistically stimulates fibroblasts with low concentrations of TGF-β, and is associated with Raynaud's phenomenon and fingertip ulcers. Although high doses of IFN-γ theoretically inhibit collagen synthesis, in vivo it actually promotes the inflammation-fibrosis feedforward loop and is positively correlated with MRSS, while its level decreases in the late stages, suggesting a bidirectional regulatory role of IFN-γ throughout disease evolution. (Jin, Zheng et al. 2022, Truchetet, Brembilla et al. 2023) Current research indicates that compared to normal skin tissue, SSc patients have higher levels of IFN-γ production in resident Treg cells and Tconv cells, suggesting that IFN-γ plays an important role in the progression of SSc (MacDonald, Dawson et al. 2015).

[0006] In addition, there are many autoimmune diseases caused by high levels of IFN-γ and impaired Treg cell function, including but not limited to multiple sclerosis, type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, Sjögren's syndrome, and vitiligo.

[0007] Past treatments for IPEX syndrome have largely involved bone marrow transplantation combined with long-term immunosuppressant therapy (such as rapamycin), which has resulted in numerous problems, including immune rejection and organ toxicity. Similarly, past treatments for enteritis have primarily involved glucocorticoids combined with broad-spectrum immunosuppressants. Current treatments for systemic sclerosis mainly consist of immunosuppressant therapy (such as mufitalimumab, methotrexate, cyclophosphamide, rituximab, and tocilizumab) and autologous hematopoietic stem cell transplantation. However, this broad-spectrum immunosuppression weakens the host's overall defenses, increases the incidence of sepsis and malignancies, fails to precisely treat local inflammation, and cannot restore dysfunctional Treg cells, leading to high relapse rates and long-term complications after drug withdrawal. Therefore, there is an urgent need to develop next-generation immunotherapies that are more targeted and safer to address the shortcomings of existing immunosuppressant therapies. Summary of the Invention

[0008] The purpose of this invention is to provide the application of FOXP3 specific modulator 430C10 in the preparation of drugs for treating inflammatory diseases of tissues caused by IFN-γ, in order to solve the many shortcomings of existing immunosuppressant therapies in the treatment of diseases such as inflammatory bowel disease and systemic sclerosis.

[0009] To solve the above-mentioned technical problems, the first solution provided by the present invention is: the application of FOXP3 specific modulator 430C10 in the preparation of drugs for treating tissue inflammatory diseases caused by IFN-γ. The CAS number of FOXP3 specific modulator 430C10 is 1340734-18-0, and its structural formula is shown in Formula I: (I).

[0010] Among them, inflammatory diseases caused by IFN-γ include IPEX syndrome, inflammatory bowel disease, systemic sclerosis, multiple sclerosis, type I diabetes, rheumatoid arthritis, systemic lupus erythematosus, Sjögren's syndrome, and vitiligo caused by FOXP3 p.V408M mutation.

[0011] In some embodiments, the aforementioned FOXP3-specific regulator 430C10 has at least one of the following functions: A1) reducing the production of IFN-γ in T cells in vivo; A2) Increase the ratio of Treg / Th1 cells in the body; A3) Enhances the inhibitory function of Treg cells.

[0012] In some embodiments, the above-mentioned drug further includes pharmaceutically acceptable excipients, which include at least one of diluents, wetting agents, binders, disintegrants, aqueous solvents, solubilizers, pH adjusters, isotonic adjusters, and isotonic adjusters.

[0013] In some embodiments, the dosage form of the above-mentioned drug includes at least one of oral formulations, injectable formulations, inhaled formulations, and topical formulations. Oral formulations include at least one of tablets, capsules, pills, powders / granules, oral liquids / syrups, and soft capsule microemulsions; injectable formulations include at least one of aqueous / oil-based formulations, lyophilized powder for injection, liposome injections, emulsions, and microspheres; inhaled formulations include at least one of dry powder inhalers, nebulized solutions, and metered-dose aerosols; and topical formulations include at least one of patches, pastes, ointments, creams, gels, oils, microneedles, films, poultices, sprays, and dressings.

[0014] To solve the above-mentioned technical problems, the second solution provided by the present invention is: the application of FOXP3 specific regulator 430C10 in the preparation of preparations that inhibit intestinal inflammation, lung inflammation and skin fibrosis, wherein the structural formula of FOXP3 specific regulator 430C10 is the aforementioned Formula I.

[0015] To solve the above-mentioned technical problems, the third solution provided by the present invention is: the application of FOXP3 specific regulator 430C10 in the preparation of a formulation that inhibits dermal lymphocyte infiltration, wherein the structural formula of the FOXP3 specific regulator 430C10 is the aforementioned Formula I.

[0016] To solve the above-mentioned technical problems, the fourth solution provided by the present invention is: the application of FOXP3 specific regulator 430C10 in the preparation of a preparation that inhibits the differentiation of Th1-like Treg cells and the secretion of related cytokines, wherein the structural formula of the FOXP3 specific regulator 430C10 is the aforementioned Formula I.

[0017] The beneficial effects of this invention are as follows: Unlike existing technologies, this invention provides a novel use of the FOXP3-specific modulator 430C10 in the preparation of drugs for treating FOXP3 p.V408M type IPEX syndrome, inflammatory bowel disease, and scleroderma, through FOXP3V408M transgenic mice, DSS-induced mouse enteritis models, and bleomycin-induced scleroderma models. This invention has studied and confirmed that 430C10 can be used to treat FOXP3 p.V408M type IPEX syndrome, inflammatory bowel disease, and scleroderma: it can significantly reduce inflammation in the lungs, liver, and intestinal tissues of FOXP3V408M transgenic mice; it can significantly reduce colonic shortening and / or intestinal wall thickening in the DSS-induced enteritis model, reduce intestinal lamina propria lymphocyte infiltration, and slow down weight loss in mice; it can significantly reduce dermal thickening and / or inhibit dermal lymphocyte infiltration in the bleomycin-induced scleroderma model; and it can effectively inhibit the immune response induced by Th1-like Treg cells. Therefore, this invention is of great significance for developing 430C10 as a new drug for the treatment of FOXP3 p.V408M type IPEX syndrome, inflammatory bowel disease, scleroderma, and many other autoimmune diseases caused by impaired Treg cell function and IFN-γ. It has great application and economic value. Attached Figure Description

[0018] Figure 1 This is a diagram showing the experimental results of improving tissue inflammation in V408M mice in Example 1 of this invention: Figure 1 In the table, A represents the histopathological changes in the colon, lung, and liver tissues of mice in each group (Bar=100μm); B represents the inflammation score and inflammatory cell infiltration of histopathological sections of mice in each group (n=3 per group) (Note: *p<0.05, **p<0.01). Figure 2 This is a graph showing the experimental results of reducing the Th1 inflammatory response in V408M mice in Example 1 of this invention: Figure 2 In the figure, A is a flow cytometry analysis of IFN-γ and IL-17A production by Treg and CD4+ Tconv cells in each group of mice, and IFN-γ production by CD8+ T cells; B is a statistical graph of the flow cytometry analysis results in A (n=6 for each group) (Note: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Figure 3 This is a diagram showing the experimental results of inhibiting IFN-γ production in V408M Treg cells in Example 2 of this invention: Figure 3In the image, A represents the flow cytometry analysis (left) and statistical graph (right) of IFN-γ production by wild-type Treg cells and V408M Treg cells with or without 430C10 treatment (n=4 per group); B represents the flow cytometry analysis (left) and statistical graph (right) of IFN-γ production by wild-type Tconv cells and V408M Tconv cells with or without 430C10 treatment (n=4 per group) (Note: ****p<0.0001). Figure 4 This is a graph showing the experimental results of improving the enteritis phenotype in Example 3 of the present invention: Figure 4 In the table, A shows the weight changes of mice in each group during the modeling period (n=6 per group); B shows the representative results of colorectal endoscopy in each group (n=6 per group); C shows the representative results of colorectal length in each group (left) and the length statistics (right) (n=6 per group); D shows the histopathological changes of colon tissue in each group (Bar=100μm, left) and the statistics (right) (n=6 per group) (Note: ***p<0.001, ****p<0.0001); Figure 5 This is a graph showing the experimental results of improving the enteritis phenotype in Example 3 of the present invention: Figure 5 In the diagram, A represents the CD4 count of mice in each group. + FOXP3 + Treg cells and CD4 + FOXP3 - Tconv generates a flow cytometry plot of IFN-γ (top), and IFN-γ + In FOXP3 + and FOXP3 - The proportion of IFN-γ + Treg cells and IFN-γ + Statistical chart of the absolute number of Tconv cells in the colon (below) (n=6 per group) (Note: *p<0.05, ***p<0.001); Figure 6 This is a diagram showing the experimental results of inhibiting the induction of Th1-like Treg cell differentiation in Example 4 of this invention: Figure 6 In the figure, A represents the flow cytometry (left) and statistical graph (right) of IFN-γ production by wild-type Treg cells and Th1-like Treg cells under conditions of 430C10 treatment with or without (n=3 per group) (Note: ****p<0.0001). Figure 7 This is a diagram showing the experimental results of improving bleomycin-induced skin fibrosis in a mouse model of scleroderma in Example 5 of this invention: Figure 7In the middle, A shows the Masson staining results of mouse skin tissue in each group (left, Bar=100μm), and the statistical graph of dermal thickness and fibrosis degree (right, n=6 per group); B shows the dermal inflammatory cell infiltration and statistical graph of skin tissue pathological sections in each group (n=6 per group); C shows the α-SMA immunohistochemical results and statistical graph of skin tissue pathological sections in each group (n=6 per group) (Note: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Detailed Implementation

[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the application; the terms “comprising” and “having”, and any variations thereof, in the specification, claims, and foregoing description of the drawings are intended to cover non-exclusive inclusion.

[0021] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0022] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional in this technical field, and the reagents and materials used in the following embodiments are all commercially available. The test models used in the following embodiments include: FOXP3 V408M The knock-in mouse model (hereinafter referred to as V408M mouse) was constructed by Cyagen Biosciences on the C57BL / 6J genetic background using CRISPR / Cas9-mediated genome engineering technology to study the Treg cell dysfunction and IPEX syndrome caused by this mutation, thereby providing a key tool for the research of IPEX treatment strategies.

[0023] The DSS-induced enteritis model is one of the most commonly used and well-established animal models for studying IBD, and it is widely used in mechanistic studies, drug screening, and immune modulation research of ulcerative colitis (UC). DSS disrupts the tight junctions between intestinal epithelial cells through high negative charge, leading to impaired intestinal barrier function and activation of the immune system.

[0024] Bleomycin-induced scleroderma models are among the most commonly used animal models for studying scleroderma. By administering bleomycin locally or systemically, skin and visceral fibrosis is induced, mimicking key pathological features of human scleroderma, including inflammation, vascular lesions, and fibrosis.

[0025] Since clinical patient samples are not readily available, those skilled in the art use the above animal models to study the pathological process and pathogenesis of the aforementioned diseases. The following examples use the V408M mouse model, the DSS-induced acute enteritis model, and the bleomycin-induced scleroderma model to demonstrate the therapeutic effects and mechanisms of action of 430C10 in these diseases.

[0026] The specific experimental methods and results of each embodiment are described in detail below.

[0027] Example 1 I. Experimental Methods 1. Group intervention Male V408M mice, aged 8-12 weeks and weighing 25g±2g, were randomly divided into two groups of six mice each. Six same-sex, same-week-old C57BL / 6J mice were designated as a wild-type healthy control group. Mice were housed in an SPF-grade animal facility and provided with standard feed and water. The grouping and treatment protocols are as follows: (1) Wild-type healthy control group (WT+Vehicle): Wild-type mice were given the corresponding amount of CMC-Na solution by gavage daily for a total of 21 days.

[0028] (2) V408M mutant control group (V408M+Vehicle): V408M mice were given the corresponding amount of CMC-Na solution by gavage daily for a total of 21 days.

[0029] (3) V408M mutation treatment group (V408M+50mpk): V408M mice were given 10 mg•mL of a solution prepared with CMC-Na. -1 The 430C10 suspension, at 50 mg / kg -1 Mice were administered the dose by gavage daily for a total of 21 days.

[0030] All treatments begin with the first gavage (day 1), once a day for 21 consecutive days.

[0031] 2. Observation Indicators See histopathology.

[0032] 3. Histopathology After recording the above data on day 21, the mice were euthanized, and spleen, peripheral lymph nodes, and mesenteric lymph node tissues were collected from each group of mice for flow cytometry analysis. Colon, lung, and liver tissues from three mice in each group were fixed in 4% formalin solution, embedded in paraffin, sectioned, and stained with hematoxylin-eosin (H&E). Three high-power fields were randomly selected from each section for pathological scoring, and the average score was calculated.

[0033] 4. Statistical Methods Experimental data were analyzed using GraphPad Prism, and all data are expressed as mean ± standard error of mean (SEM). Multiple comparisons between groups were performed using one-way ANOVA and Tukey ANOVA. A p-value <0.05 was considered statistically significant.

[0034] II. Experimental Results 1,430C10 can improve tissue inflammation in V408M mice. Histopathological findings of mice in each group are as follows: Figure 1 As shown in -A, H&E staining of tissue sections indicated that the V408M mutation caused colitis, lung injury, and mild liver injury in mice, along with increased lymphocyte infiltration. However, treatment with 50 mg kg⁻¹ of 430C10 significantly reduced tissue inflammation scores and lymphocyte infiltration in V408M mice (*p<0.05, **p<0.01), indicating that at this dose, 430C10 has a significant ameliorative effect on tissue inflammation induced by the FOXP3 p.V408M mutation. Figure 1 -B).

[0035] 2. 430C10 can reduce Th1 inflammatory response in V408M mice. Furthermore, flow cytometry analysis showed that, compared with the WT+Vehicle group, the V408M+Vehicle group mice had higher levels of Treg cells and CD4+ in their lymphatic organs, including the spleen, peripheral lymph nodes (pLN), and mesenteric lymph nodes (mLN). + Tconv cells and CD8 + The proportion of IFN-γ+ cell subsets in T cells was significantly increased; compared with the V408M+Vehicle group, the proportion of IFN-γ+ cells in all T cells was significantly decreased in the V408M+50mpk treatment group (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).Figure 2 -A and B).

[0036] Example 1 The results above consistently show that when using 50 mg•kg -1 At oral doses, 430C10 treatment can effectively improve Th1 inflammatory responses and tissue damage caused by increased IFN-γ secretion in T cells due to FOXP3 p.V408M mutation, and is worthy of widespread application.

[0037] Example 2 I. Experimental Methods 1. Group intervention Two male, 8-10 week old C57BL / 6 wild-type mice and V408M mice were randomly selected. The mice were housed in a specific sterile environment with temperature control (20-26℃) and provided with standard feed and water. The treatment protocol is as follows: CD4+CD25-CD62L+ naïve T cells were isolated from mouse spleen and differentiated under Treg cell polarization conditions (anti-CD3 / CD28, IL2, TGF-β); CD4+CD25-FOXP3-Tconv cells were also isolated from mouse spleen.

[0038] (1) Wild-type Treg control group (WT Treg+DMSO group): The corresponding amount of DMSO solvent was added to the Treg in vitro differentiation medium, and the naïve CD4+ T cells isolated from the spleen of wild-type mice were differentiated for 4 days and then detected by flow cytometry.

[0039] (2) Wild-type Treg drug-treated group (WT Treg+430C10 group): 1 μM of 430C10 dissolved in DMSO was added to the Treg in vitro differentiation medium. Naïve CD4+ T cells isolated from the spleen of wild-type mice were differentiated and then detected by flow cytometry 4 days later.

[0040] (3) Mutant Treg control group (V408M Treg+DMSO group): The corresponding amount of DMSO solvent was added to the Treg in vitro differentiation medium, and the naïve CD4+ T cells isolated from the spleen of V408M mutant mice were differentiated for 4 days and then detected by flow cytometry.

[0041] (4) Mutant Treg drug treatment group (V408M Treg+430C10 group): 1 μM of 430C10 dissolved in DMSO was added to the Treg in vitro differentiation medium. Naïve CD4+ T cells isolated from the spleen of V408M mutant mice were differentiated for 4 days and then detected by flow cytometry.

[0042] (5) Wild-type Tconv control group (WT Tconv+DMSO group): Add the corresponding amount of DMSO solvent to the culture medium of Tconv cells isolated from the spleen of wild-type mice, and perform flow cytometry detection after 24 hours of treatment.

[0043] (6) Wild-type Tconv drug-treated group (WT Tconv+430C10 group): 1 μM of 430C10 dissolved in DMSO was added to the culture medium of Tconv cells isolated from the spleen of wild-type mice and the cells were treated for 24 hours before flow cytometry detection.

[0044] (7) Mutant Tconv control group (V408M Tconv+DMSO group): Add the corresponding amount of DMSO solvent to the culture medium of Tconv cells isolated from the spleen of V408M mutant mice, and perform flow cytometry detection after 24 hours of treatment.

[0045] (8) Mutant Tconv drug treatment group (V408M Tconv+430C10 group): 1 μM of 430C10 dissolved in DMSO was added to the culture medium of Tconv cells isolated from the spleen of V408M mutant mice and the cells were treated for 24 hours before flow cytometry detection.

[0046] 2. Observation Indicators Tconv cells were collected on day 1 and Treg cells were collected on day 4. After stimulation with PMA, Ionomycin and GolgiStop for 4 hours, respectively, flow cytometry staining of cytokines was performed. The flow cytometry plots of IFN-γ production by Tconv and Treg cells with and without 430C10 stimulation were detected by flow cytometry.

[0047] 3. Statistical Methods Experimental data were analyzed using GraphPad Prism, and all data are expressed as mean ± standard error of mean (SEM). Multiple comparisons between groups were performed using one-way ANOVA and Tukey ANOVA. A p-value <0.05 was considered statistically significant.

[0048] II. Experimental Results 1,430C10 can inhibit IFN-γ production in V408M Treg cells but not V408M Tconv cells. Treg cells in each treatment group secreted IFN-γ as follows: Figure 3 As shown in -A, the study found that treatment with 1 μM of 430C10 increased IFN-γ levels in V408M Treg cells. + The proportion of Treg cells was significantly suppressed (****p<0.0001) Figure 3 -A).

[0049] IFN-γ secretion from Tconv cells in each treatment group was as follows: Figure 3 As shown in Figure B, the study found that treatment with 1 μM of 430C10 reduced IFN-γ in Tconv cells that did not express FOXP3. + The proportion of cells was not affected. Figure 3 -B).

[0050] Based on the experimental results of Example 2, it is evident that the FOXP3 p.V408M mutation induces increased IFN-γ production by Treg cells, thereby causing enhanced Th1 inflammatory responses and tissue damage in V408M mice. Treatment with 430C10 significantly reduces tissue damage and lymphocyte infiltration in the colon, lungs, and liver of V408M mice, demonstrating significant therapeutic efficacy. Furthermore, it can significantly inhibit IFN-γ production in a Treg-dependent manner, making it worthy of widespread application.

[0051] Example 3 I. Experimental Methods 1. Group intervention Male, 8-week-old C57BL / 6J mice weighing 21g±2g were randomly divided into 3 groups of 6 mice each. The mice were housed in an SPF-grade animal facility and provided with standard feed. The grouping and treatment protocols are as follows: (1) Healthy control group (Water+Vehicle): Normal drinking water was provided, and mice were given 100 μL of CMC-Na solution by gavage daily for a total of 7 days.

[0052] (2) Enteritis model group (3% DSS+Vehicle): Mice were given 3% DSS aqueous solution in drinking water and were given 100 μL of CMC-Na solution by gavage daily for a total of 7 days.

[0053] (3) Enteritis treatment group (3% DSS + 50mpk): 3% DSS aqueous solution was provided in drinking water, and 10mg•mL of solution prepared with CMC-Na was added. -1 The 430C10 suspension solution, at 50 mg / kg -1 Mice were administered the dose by gavage daily for a total of 7 days.

[0054] All treatments begin one day before the first gavage (day 0), with gavage administered once daily and body weight measured once daily for 7 consecutive days.

[0055] 2. Observation Indicators The mice were monitored for changes in body weight daily, and colorectal endoscopy was performed on the mice after the modeling was completed.

[0056] 3. Histopathology After recording the above data on day 7, the mice were euthanized, and colonic tissue was collected from each group of mice for flow cytometry analysis. The proximal colonic tissue from each group was fixed in 4% formalin solution, embedded in paraffin, sectioned, and stained with H&E. Three high-power fields were randomly selected from each section for pathological scoring, and the average score was calculated.

[0057] 4. Statistical Methods Experimental data were analyzed using GraphPad Prism, and all data are expressed as mean ± standard error of mean (SEM). Multiple comparisons between groups were performed using one-way ANOVA and Tukey ANOVA. A p-value <0.05 was considered statistically significant.

[0058] II. Experimental Results 1,430C10 can improve the enteritis phenotype. The weight monitoring data of mice in each group are as follows: Figure 4 As shown in -A, 50mg kg -1 Treatment with 430C10 at specific concentrations significantly alleviated weight loss in mice induced by enteritis (***p<0.001). Endoscopic colorectal examination revealed no mucosal damage in the Water+Vehicle group, while the 3%DSS+Vehicle group exhibited mucosal edema and superficial ulcers. The intestinal condition in the 3%DSS+50mpk group was similar to that of the control group. Figure 4 -B).

[0059] Regarding colorectal length, the 3% DSS+Vehicle group showed a significant reduction in length and thickening of the intestinal wall compared to the healthy control group. Treatment with 430C10 significantly improved these symptoms, bringing the results closer to those of the healthy control group (***p<0.001, ****p<0.0001). Figure 4 -C). In the H&E staining results of mouse colon tissue sections, at 50 mg•kg... -1 Treatment with 430C10 at this dose significantly reduced intestinal tissue inflammation scores (****p<0.0001), indicating that 430C10 at this dose has a good therapeutic effect on DSS-induced acute enteritis. Figure 4 -D).

[0060] 2. 430C10 can reduce Th1 inflammatory response in mice with enteritis. Furthermore, flow cytometry analysis showed that the IFN-γ levels in Treg cells and CD4+ Tconv cells in the colon tissue of mice in the 3% DSS+Vehicle group were significantly higher than those in the Water+Vehicle group. +The proportions of all cell subsets were significantly increased; compared with the 3%DSS +Vehicle group, the proportion of IFN-γ+ cells in each T cell group was significantly decreased in the 3%DSS +50mpk treatment group (*p<0.05, **p<0.01, ***p<0.001). Figure 5 -A).

[0061] Example 3 consistently demonstrates that, at an oral dose of 50 mg kg⁻¹, 430C10 treatment effectively reduces IFN-γ. + The infiltration of Treg cells in the colon, thereby treating DSS-induced enteritis, is worthy of widespread application.

[0062] Example 4 I. Experimental Methods 1. Group intervention Three to five male, 8-10 week old C57BL / 6 wild-type mice were randomly selected. The mice were housed in a specific sterile environment with temperature control (20-26℃) and provided with standard feed and water. The treatment protocol is as follows: CD4+CD25-CD62L+ naïve T cells were isolated from the spleen and peripheral lymph nodes of mice and differentiated under Treg cell polarization conditions (anti-CD3 / CD28, IL2, TGF-β). On the fourth day, Th1 cell polarization conditions (anti-CD3 / CD28, IL2, IL12, IL27, IFN-γ, TGF-β) were applied.

[0063] (1) Wild-type control group (Ctrl+DMSO group): Four days after Treg-induced differentiation, TCR stimulation was given and the corresponding amount of DMSO solvent was added. Flow cytometry was performed one day later.

[0064] (2) Wild-type drug-treated group (Ctrl+430C10 group): Four days after Treg-induced differentiation, TCR stimulation was given and 1 μM of 430C10 dissolved in DMSO was added. Flow cytometry was performed one day later.

[0065] (3) Th1-like control group (Stim+DMSO group): Four days after Treg-induced differentiation, Th1 polarization conditions were given and the corresponding amount of DMSO solvent was added. Flow cytometry was performed one day later.

[0066] (4) Th1-like drug addition group (Stim + 430C10 group): Th1 polarization conditions were given 4 days after Treg induction differentiation, and 1 μM of 430C10 dissolved in DMSO was added. Flow cytometry was performed 1 day later.

[0067] 2. Observation Indicators Treg cells were harvested on day 5 and stimulated for 4 hours with PMA, Ionomycin, and Golgi Stop, respectively. Flow cytometry staining of cytokines was then performed to detect the flow cytometry graphs of IFN-γ production in Treg cells with and without 430C10 stimulation.

[0068] 3. Statistical Methods Experimental data were analyzed using GraphPad Prism, and all data are expressed as mean ± standard error of mean (SEM). Multiple comparisons between groups were performed using one-way ANOVA and Tukey ANOVA. A p-value <0.05 was considered statistically significant.

[0069] II. Experimental Results 1,430C10 can inhibit the induced differentiation of Th1-like Treg cells. Treg cells in each treatment group secreted IFN-γ as follows: Figure 6 As shown in Figure A, the study found that under Th1 polarization conditions, Treg cells differentiated into IFN-γ+ Th1-like Treg cells; after treatment with 1 μM of 430C10, the proportion of IFN-γ+ Th1-like Treg cells was significantly inhibited (p < 0.0001). Figure 3 -A).

[0070] Based on the experimental results of Example 4, it can be seen that under Th1 inflammatory conditions, Treg cells are prone to transform into Th1-like Treg cells. With the increase of IFN-γ+ Treg subsets, 430C10 treatment can significantly reduce the transformation rate of Treg cells into Th1-like Treg cells, and reduce the proportion and absolute number of colon-infiltrating IFN-γ+ Treg cells in mice with enteritis in vivo, thereby effectively alleviating intestinal inflammation and weight loss. The therapeutic effect is significant and worthy of promotion and application.

[0071] Example 5 I. Experimental Methods 1. Group intervention Male C57BL / 6J mice, aged 6-8 weeks and weighing 20g±2g, were randomly divided into 4 groups of 6 mice each. The mice were housed in an SPF-grade animal facility and provided with standard feed and water. The grouping and treatment protocols are as follows: (1) Healthy control group (Ctrl+Vehicle): On the first day of modeling, mice were shaved 1*1 cm on the back and injected subcutaneously with 200 μL PBS, followed by injection every other day (i.e., odd-numbered days); on the second day, mice were given the corresponding amount of CMC-Na solution by gavage, followed by gavage every other day (i.e., even-numbered days).

[0072] (2) Healthy treatment group (Ctrl+430C10): On the first day of modeling, mice were shaved 1*1 cm on their backs and injected subcutaneously with 200 μL of PBS, followed by injection every other day (i.e., odd-numbered days); on the second day, they were administered 50 mg•kg via gavage. -1 Mice were administered a dose of 430C10 suspension by gavage daily, followed by gavage every other day (i.e., even-numbered days).

[0073] (3) Modeling control group (SSc+Vehicle): On the first day of modeling, mice were shaved on the back (1*1 cm) and then subcutaneously injected with 200 μL of 0.5 U•ml solution. -1 Bleomycin solution was administered, followed by injection every other day (i.e., odd-numbered days); on the second day, the corresponding amount of CMC-Na solution was administered by gavage, followed by gavage every other day (i.e., even-numbered days).

[0074] (4) Modeling treatment group (SSc+430C10): On the first day of modeling, mice were shaved on the back (1*1 cm) and then subcutaneously injected with 200 μL of 0.5 U•ml. -1 Bleomycin solution was administered by injection every other day (i.e., on odd-numbered days); on the second day, it was administered by gavage at a dose of 50 mg / kg. -1 Mice were administered a dose of 430C10 suspension by gavage daily, followed by gavage every other day (i.e., even-numbered days).

[0075] All treatments begin on the first subcutaneous injection (day 1) and continue for 42 days.

[0076] 2. Observation Indicators See histopathology.

[0077] 3. Histopathology On day 42, mice were euthanized, and skin tissue was collected from each group of mice. The tissue was fixed in 4% formalin solution, embedded in paraffin, sectioned, and stained with hematoxylin-eosin (H&E), Masson stain, and α-SMA immunohistochemical staining. Three high-power fields were randomly selected from each section to measure dermal thickness, inflammatory cell infiltration, and degree of fibrosis, and the average values ​​were taken.

[0078] 4. Statistical Methods Experimental data were analyzed using GraphPad Prism, and all data are expressed as mean ± standard deviation (SD). Multiple comparisons between groups were performed using one-way ANOVA and Tukey ANOVA. A p-value <0.05 was considered statistically significant.

[0079] II. Experimental Results 1,430C10 can improve the skin fibrosis score in a bleomycin-induced scleroderma mouse model. Masson staining results for each group of mice are as follows: Figure 7 As shown in Figure A, the staining results indicate that, compared to the Ctrl+Vehicle group, the SSc+Vehicle group showed a significant increase in dermal thickness and fibrosis, while the 50 mg•kg... -1 Treatment with 430C10 at specific concentrations reduced dermal thickness and fibrosis after modeling to levels seen in healthy controls (*p<0.05, **p<0.01, ***p<0.001). Pathological section results for each group of mice are shown below. Figure 7 As shown in Figure -B, the dermal inflammatory cell infiltration was significantly reduced in the SSc+430C10 group compared to the SSc+Vehicle group (*p<0.05). The α-SMA staining results of mouse tissue sections from each group are shown below. Figure 7 As shown in -C, compared to the Ctrl+Vehicle group, the α-SMA of the SSc+Vehicle group... + The number of myofibroblasts was significantly increased at 50 mg / kg. -1 Treatment with 430C10 at specific concentrations can significantly reduce α-SMA. + The number of myofibroblasts was significantly reduced, indicating that treatment with 430C10 significantly reduced the degree of dermal fibrosis in the scleroderma model (***p<0.001, ****p<0.0001).

[0080] Example 5 consistently demonstrates that 430C10 treatment at an oral dose of 50 mg kg⁻¹ effectively reduces the degree of skin fibrosis in a bleomycin-induced scleroderma model, and is worthy of widespread application.

[0081] Comprehensive analysis of experimental results Based on the experimental results of Examples 1 to 5 above, it can be seen that in FOXP3 p.V408M transgenic mice, both lymphoid and non-lymphoid tissues (including colon, lung, and liver) showed tissue inflammation caused by increased IFN-γ levels produced by T cells due to impaired Treg cell function. Treatment with the FOXP3-specific modulator 430C10 significantly reduced the level of IFN-γ secreted by T cells in V408M mice and significantly alleviated the histopathology of non-lymphoid tissues. In a DSS-induced acute enteritis mouse model, mice exhibited significant intestinal inflammation and increased colonic infiltration of IFN-γ+ Treg cells. Treatment with 430C10 significantly reduced weight loss, intestinal inflammation, and the number of IFN-γ+ Treg cells in the colon caused by enteritis. In a bleomycin-induced scleroderma mouse model, the model group exhibited dermal fibrosis and increased lymphocyte infiltration. Treatment with 430C10 significantly reduced the degree of dermal fibrosis and effectively reduced lymphocyte infiltration. In vitro differentiation experiments showed that 430C10 could inhibit the secretion of IFN-γ by Treg cells under inflammatory conditions.

[0082] This demonstrates that the FOXP3-specific modulator 430C10 used in this invention is a highly effective, safe, economical, and convenient FOXP3-specific modulator that can prevent / treat autoimmune diseases caused by impaired Treg cell function and high levels of IFN-γ, including but not limited to FOXP3 p.V408M type IPEX syndrome, inflammatory bowel disease, systemic sclerosis, etc. It can effectively solve the problems of poor efficacy and large side effects of existing immunosuppressants, as well as graft-versus-host reactions caused by hematopoietic stem cell transplantation, and has broad application prospects.

[0083] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0084] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The use of FOXP3-specific modulator 430C10 in the preparation of drugs for treating tissue inflammatory diseases caused by IFN-γ, characterized in that, The CAS number of the FOXP3 specific modulator 430C10 is 1340734-18-0, and its structural formula is shown in Formula I: （I）。 2. The application according to claim 1, characterized in that, The inflammatory diseases caused by IFN-γ include IPEX syndrome, inflammatory bowel disease, systemic sclerosis, multiple sclerosis, type I diabetes, rheumatoid arthritis, systemic lupus erythematosus, Sjögren's syndrome, and vitiligo caused by the FOXP3 p.V408M mutation.

3. The application according to claim 1, characterized in that, The FOXP3 specific modulator 430C10 has at least one of the following functions: A1) Reduces the production of IFN-γ in T cells in vivo; A2) Increase the ratio of Treg / Th1 cells in the body; A3) Enhances the inhibitory function of Treg cells.

4. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients, which include at least one of diluents, wetting agents, binders, disintegrants, aqueous solvents, solubilizers, pH adjusters, isotonic adjusters, and isotonic adjusters.

5. The application according to claim 4, characterized in that, The dosage form of the drug includes at least one of oral preparations, injectable preparations, inhaled preparations, and topical preparations.

6. Application of FOXP3 specific modulator 430C10 in the preparation of formulations that inhibit intestinal inflammation, lung inflammation, and skin fibrosis.

7. Application of FOXP3 specific modulator 430C10 in the preparation of formulations that inhibit dermal lymphocyte infiltration.

8. Application of FOXP3 specific regulator 430C10 in the preparation of formulations that inhibit Th1-like Treg cell differentiation and related cytokine secretion.

Images