Application of Pik3cb inhibitor in treatment of Sjogren's disease

Abstract

The invention discloses an application of a Pik3cb inhibitor in treatment of Sjogren's disease. Experiments prove that the Pik3cb inhibitor TGX-221 can reduce the number, activation and chemotaxis of NOD mouse B cells of a Sjogren's disease model mouse, plasma cell differentiation and antibody secretion, improve the immune microenvironment of the submaxillary gland of the Sjogren's disease model mouse, reduce the infiltration degree of submaxillary gland lymphocytes and promote the salivary secretion function; and after the Pik3cb is over-expressed, the symptom and pathology of the Sjogren's disease are aggravated. Therefore, the Pik3cb inhibitor can be used for treating the Sjogren's disease. The invention provides a new thought for research and development of medicines for treating the Sjogren's disease, and has important application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of Pik3cb inhibitors in the treatment of Sjögren's disease. Background Technology

[0002] Sjögren's disease (SjD) is a systemic autoimmune disease characterized by both immune overactivity and immunodeficiency, primarily affecting the salivary and lacrimal glands. Typical clinical manifestations include dry eyes and dry mouth. Sjögren's disease has a high incidence and is clinically challenging to treat. Current treatment strategies are mainly limited to symptomatic treatment (such as stimulating salivation or using artificial saliva substitutes) or the use of immunosuppressants and glucocorticoids. Common treatments have side effects such as increased susceptibility to infection, tumor induction, osteoporosis, hormone dependence, and treatment resistance, and are ineffective in 20%-30% of patients. There is an urgent need to explore a safe, effective, and etiologically targeted treatment method.

[0003] The pathogenesis of Sjögren's disease is complex, involving an imbalance in the regulation of innate and adaptive immunity. Significant upregulation of type I interferon (IFN) signaling drives early disease progression, inducing overexpression of B-cell activating factor (BAFF). BAFF is key to linking innate immunity and B-cell activation, promoting the survival, differentiation, and autoantibody production of autoreactive B cells. Simultaneously, T-cell subset polarization drives disease progression—initially, glandular damage is primarily mediated by Th1 cells (secreting IFN-γ) and Th17 cells. As the disease progresses, follicular helper T (Tfh) cells regulate excessive B-cell activation, gradually leading to a dominance of B lymphocytes. Salivary gland epithelial cells, among others, mediate the recruitment of B cells and Tfh cells to exocrine glands via the CXCL13 (B-cell chemokine)-CXCR5 axis, promoting the formation of ectopic germinal center (GC)-like structures. Tfh cells maintain the stability of these structures and regulate B-cell differentiation into plasma cells and memory B cells. These ectopic germinal centers support somatic hypermutation and class switching of B cells and are a key site for the production of high-affinity autoantibodies such as anti-Ro / SSA and anti-La / SSB antibodies. These accumulated immune abnormalities ultimately lead to exocrine gland lymphocyte infiltration and dysfunction, increasing the risk of B-cell lymphoma in severely ill patients.

[0004] The Pik3cb gene encodes the PIK3CB protein (also known as P110β), the catalytic subunit of phosphatidylinositol 3-kinase (PI3K). The PI3K / AKT / mTOR signaling pathway is a crucial intracellular signal transduction pathway, playing vital biological roles in cell growth, survival, proliferation, apoptosis, autophagy, and immune regulation. The role of Pik3cb in the pathogenesis and treatment of SjD has not yet been reported. Summary of the Invention

[0005] The purpose of this invention is to find a more effective way to treat Sjögren's disease.

[0006] This invention first protects the use of Pik3cb inhibitors in the preparation of products for the treatment of Sjögren's disease; said use is achieved by injecting or taking Pik3cb inhibitors orally.

[0007] In any of the above applications, the product may specifically be a drug.

[0008] In any of the applications described above, the injection method can be intraperitoneal injection or intravenous injection. Intraperitoneal injection is preferred.

[0009] In any of the above applications, the Pik3cb inhibitor may be a Pik3cb gene inhibitor or a Pik3cb protein inhibitor.

[0010] In any of the above applications, the Pik3cb protein inhibitor may specifically be TGX-221, which is a Pik3cb-specific inhibitor.

[0011] In any of the above-described applications, the treatment of Sjögren's disease may manifest as at least one of the following: improving salivary gland secretory function, improving salivary gland structure, improving the inflammatory microenvironment of the gland, inhibiting the activation of pathogenic immune cell populations, inhibiting the differentiation of pathogenic immune cell populations, inhibiting the chemotaxis of pathogenic immune cell populations, and reducing the number of pathogenic immune cell populations.

[0012] This invention also protects the use of Pik3cb as a target for the treatment of Sjögren's disease; said use is achieved by inhibiting Pik3cb.

[0013] In the above applications, the inhibition of Pik3cb is achieved by injection or oral administration of any of the aforementioned Pik3cb inhibitors. The injection method can be intraperitoneal injection or intravenous injection; intraperitoneal injection is preferred.

[0014] This invention also protects the application of any of the above-described Pik3cb inhibitors, which can be at least one of the following: improving salivary gland secretion function, improving salivary gland structure, improving the glandular inflammatory microenvironment, inhibiting the activation of pathogenic immune cell populations, inhibiting the differentiation of pathogenic immune cell populations, inhibiting the chemotaxis of pathogenic immune cell populations, and reducing the number of pathogenic immune cell populations.

[0015] In any of the above-described applications, the improvement in salivary gland secretion function can manifest as an increase in salivary flow rate.

[0016] In any of the above-described applications, the improvement in salivary gland structure may manifest as a reduction in lymphocyte infiltration foci. Further, the reduction in lymphocyte infiltration foci may manifest as a decrease in the area and / or number of lymphocyte infiltration foci.

[0017] In any of the above-described applications, the improvement of the glandular inflammatory microenvironment can manifest as the downregulation of pro-inflammatory factors and / or the upregulation of anti-inflammatory factors. Specifically, the pro-inflammatory factors can be IFN-γ and / or IL-6. The anti-inflammatory factor can be IL-10.

[0018] In any of the above-described applications, the pathogenic immune cell population is B cells and / or plasma cells.

[0019] The Pik3cb mentioned above is also known as P110β, which is the catalytic subunit of the phosphatidylinositol 3-kinase (PI3K).

[0020] The GeneID of any of the Pik3cb proteins mentioned above is 74769 (mouse) or 5291 (human).

[0021] Sjögren's disease (SjD) is a systemic autoimmune disease primarily affecting exocrine glands such as the salivary glands. Research conducted by the inventors of this application shows that the Pik3cb inhibitor TGX-221 can inhibit the number, activation, and chemotaxis of B cells, as well as plasma cell differentiation and antibody secretion in NOD mice (a SjD model mouse), improve the inflammatory microenvironment of the submandibular glands in NOD mice, reduce the degree of lymphocyte infiltration in the submandibular glands, and enhance salivary secretion function in NOD mice; while overexpression of Pik3cb exacerbates the symptoms and pathology of SjD. Therefore, inhibiting Pik3cb is a key aspect of intervention in SjD, and Pik3cb can serve as a target for the treatment of Sjögren's disease. This invention provides a reference for the translational application of PIK3cb inhibitors in the treatment of SjD and offers new ideas for the development of molecular products for treating SjD. This invention has significant application value. Attached Figure Description

[0022] Figure 1The Pik3cb inhibitor TGX-221 improves salivary flow rate in NOD mice; This indicates that p < 0.05. This means p < 0.0001.

[0023] Figure 2 H&E staining of the submandibular gland shows that the Pik3cb inhibitor TGX-221 can improve the submandibular gland structure in NOD mice and reduce the area and number of lymphocyte infiltration foci (indicated by arrows); Scale bar: 500 or 1000 micrometers (low magnification), 100 micrometers (magnified view).

[0024] Figure 3 The ELISA results show that Pik3cb inhibitors reduce the levels of inflammatory factors IL-6 and IFN-γ in the submandibular glands of NOD mice and increase the levels of the anti-inflammatory factor IL-10. This indicates that p < 0.05. This indicates that p < 0.001. This means p < 0.0001.

[0025] Figure 4 TSA staining of the submandibular gland shows the effect of Pik3cb inhibitors on CD19 in the submandibular gland of NOD mice. + Effects of B cells: Inhibitors can reduce CD19 in the submandibular gland. + B cell infiltration; among them, CD19 + B cells (pink), cell nuclei stained with DAPI (blue), MERGE indicates overlapping images; scale bar: 50 micrometers.

[0026] Figure 5 Immunofluorescence staining of spleen to show that Pik3cb inhibitors reduce the number of plasma cells in the red pulp of the spleen of NOD mice; scale bar: 50 micrometers.

[0027] Figure 6 The ELISA results show that Pik3cb inhibitors reduce BAFF levels in the spleen of NOD mice. This indicates that p < 0.05. This indicates that p < 0.01. This indicates that p < 0.001.

[0028] Figure 7The results of ELISA show that Pik3cb inhibitors reduce CXCL13 levels in the submandibular glands of NOD mice; This indicates that p < 0.05. This indicates that p < 0.01. This indicates that p < 0.001.

[0029] Figure 8 TSA staining of the submandibular gland shows that Pik3cb inhibitors reduce PIK3CB expression in submandibular gland B cells of NOD mice. PIK3CB (red), CD19 + B cells (pink), cell nuclei stained with DAPI (blue), MERGE indicates overlapping images; scale bar: 50 micrometers. Detailed Implementation

[0030] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0032] The animal experiments described below were approved by the Animal Care and Use Committee of Medcona Biotechnology Co., Ltd., approval number: MDKN-2022-094.

[0033] Implementation examples

[0034] I. Experimental Materials and Methods 1. Laboratory animals NOD / Ltj mice are non-obese diabetic (NOD) mice and a classic animal model for studying SjD. The inhibitor group, overexpression group, DMSO control group, and PBS control group were all 16-week-old female NOD mice, and 16-week-old female ICR mice were used as the ICR group, i.e., the normal group.

[0035] All mice were purchased from Beijing Huafukang Biotechnology Co., Ltd., and were housed in a specific pathogen-free animal facility with free access to feed and drinking water.

[0036] 2. Group processing Inhibitor group (i.e., TGX-221 treatment group): TGX-221 (Selleck Chemicals, USA), a specific inhibitor of Pik3cb, was dissolved in 0.5% dimethyl sulfoxide (DMSO) to obtain a 10 mM TGX-221 solution; then, NOD mice were intraperitoneally injected with 0.1 ml of TGX-221 solution at a dose of 10 mg / kg body weight once daily for 4 weeks.

[0037] Overexpression group (i.e., Pik3cb overexpression group): Using AAV9 adenovirus as a vector, the coding gene for Pik3cb was inserted (using the CD19 promoter) to obtain the PIK3CB overexpression vector. Then, 0.1 ml of PBS buffer containing the PIK3CB overexpression vector was injected into the spleen of NOD mice (the injection dose of the PIK3CB overexpression vector was 5 × 10⁻⁶). 11 (vg / mouse) mediates B cell-specific overexpression of PIK3CB. NOD mice overexpressing Pik3cb were obtained through testing, referred to as PIK3CB overexpression mice.

[0038] DMSO control group: NOD mice were intraperitoneally injected with 0.1 ml of 5% DMSO once daily for 4 weeks.

[0039] PBS control group: NOD mice were injected intraperitoneally with 0.1 ml of PBS buffer.

[0040] ICR group (i.e. normal group): ICR mice that did not receive any treatment.

[0041] 3. Saliva flow rate detection Saliva flow rates (SFR) are a core indicator for assessing salivary gland secretory function, used to quantify the effect of the Pik3cb inhibitor TGX-221 on improving glandular secretory function. The simplified procedure is as follows: Pilocarpine injection (Beijing Shuanghe Pharmaceutical Co., Ltd.) at a concentration of 100 mg / mL is administered intraperitoneally, at a dose of 5 mg / kg. All saliva secreted within 10 minutes is collected. Saliva flow rate is expressed as "mg / 10min" and calculated using the formula: SFR = Total saliva weight (mg) ÷ Collection time (min).

[0042] 4. Histological analysis (1) Immediately after obtaining tissue samples from the submandibular glands (SMGs) or spleen, fix them with 4% paraformaldehyde, embed the samples in paraffin, cut them into 4μm thick sections, and then perform hematoxylin-eosin (H&E) staining.

[0043] (2) Multiplex immunofluorescence staining was performed using the tyramide signal amplification (TSA) method. Specifically, submandibular gland sections were incubated with primary antibodies, and then immunofluorescence staining was used to detect the expression of PIK3CB in submandibular gland B cells. The primary antibodies were rabbit anti-PIK3CB-Cy5 (dilution ratio 1:200; Solarbio, K016107P) and rabbit anti-CD19-Cy3 (dilution ratio 1:500; Solarbio, K003431P).

[0044] (3) Immunofluorescence staining was used to detect splenic plasma cells. Specifically, the sections were heated in Tris-EDTA buffer (pH 9.0, C1037, Solarbio, China) for antigen retrieval, incubated with 3% hydrogen peroxide, and then blocked with serum. The sections were then incubated with primary antibody overnight.

[0045] The primary antibody was rabbit anti-CD138-FITC (dilution ratio 1:100; Solarbio, K012955RR).

[0046] 5. Enzyme-linked immunosorbent assay (ELISA) Tissue lysates were extracted from the spleen or submandibular gland of mice, and the levels of B cell activating factor BAFF in the spleen and cytokines IL-6, IL-10, IFN-γ, and CXCL13 in the submandibular gland were detected using a mouse ELISA kit (Solarbio).

[0047] 6. Statistical Analysis All experimental data and graphs were analyzed using GraphPad Prism 10 software (GraphPad, USA). Quantitative data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using one-way ANOVA, and multiple comparisons were performed using the Tukey test or Dunnett test. A p-value < 0.05 was considered statistically significant.

[0048] II. Experimental Results 1. Pik3cb inhibitor TGX-221 improves salivary gland secretion function in NOD mice. (1) Using the DMSO control group or the PBS control group as controls, the effect of the Pik3cb inhibitor TGX-221 on the salivary gland function of NOD mice was evaluated.

[0049] Test results are shown Figure 1 The results showed that, compared with the DMSO control group or the PBS control group, TGX-221 treatment significantly increased salivary flow rate in NOD mice, i.e., improved salivary gland secretion function in NOD mice; while the salivary flow rate in the Pik3cb overexpression group was significantly reduced.

[0050] 2. Pik3cb inhibitor TGX-221 improves salivary gland structure in NOD mice. The effects of the Pik3cb inhibitor TGX-221 on salivary gland structure in NOD mice were assessed using H&E staining. Test results are shown Figure 2 The results showed that, compared with the DMSO or PBS control groups, the area and number of lymphocyte infiltration foci in the submandibular glands of NOD mice treated with TGX-221 were significantly reduced, while the area and number of lymphocyte infiltration foci in the submandibular glands of NOD mice in the Pik3cb overexpression group were significantly increased. This indicates that inhibiting Pik3cb can reduce the formation of lymphocyte infiltration foci in the submandibular glands of NOD mice and improve the salivary gland structure of NOD mice.

[0051] 3. The Pik3cb inhibitor TGX-221 significantly improved the local inflammatory microenvironment of the submandibular gland in NOD mice. IL-6 levels reflect the activity of local inflammation in the gland, promoting B cell differentiation into plasma cells and producing large amounts of autoantibodies. IFN-γ, a typical Th1 cytokine, is a key pathogenic factor in the pathogenesis of Sjögren's disease. It can induce apoptosis of salivary gland epithelial cells, leading to impaired secretory function (manifested as dry mouth). It can also amplify signals, attracting more lymphocytes into the gland and exacerbating inflammatory infiltration in the submandibular gland. Higher IFN-γ levels generally indicate more severe glandular inflammation and poorer secretory function. IL-10 is a major anti-inflammatory / immunomodulatory cytokine. When responding to severe inflammation (such as excessive IFN-γ), the body produces IL-10 to attempt to suppress an excessive immune response. Based on this, to evaluate the effects of the Pik3cb inhibitor TGX-221 and Pik3cb overexpression on the local inflammatory microenvironment of the submandibular gland, the inventors of this application used ELISA to detect the expression levels of IL-6, IFN-γ, and IL-10 in the submandibular glands of various groups of mice.

[0052] Test results are shown Figure 3The results showed that the submandibular glands of mice in the PBS and DMSO control groups exhibited significant pro-inflammatory characteristics, characterized by high expression levels of IL-6 and IFN-γ and low levels of the anti-inflammatory factor IL-10. Pik3cb overexpression exacerbated this inflammatory feature, as evidenced by significant upregulation of IL-6 and IFN-γ. The high expression of IFN-γ and IL-6 reflects a strong Th1-type immune response and active chronic inflammation in the glandular region, a typical feature of SjD pathogenesis in NOD mice. Compared with the PBS, DMSO, and overexpression groups, treatment with the inhibitor TGX-221 significantly reversed this inflammatory trend; the expression levels of pro-inflammatory factors (IFN-γ and IL-6) in the submandibular glands of mice in the inhibitor group were significantly downregulated, while the level of the anti-inflammatory factor IL-10 was significantly increased.

[0053] These results indicate that the inhibitor TGX-221 can suppress overactive Th1 immune responses and B cell-mediated pro-inflammatory signaling, while enhancing local immunomodulatory protective mechanisms (IL-10) in the gland. This shift from a pro-inflammatory state to immune homeostasis may be an important biological basis for this treatment to alleviate glandular inflammatory damage and improve secretory function.

[0054] 4. The Pik3cb inhibitor TGX-221 significantly inhibits the differentiation of B cells and splenic plasma cells. B cell overactivation is a hallmark of SjD, manifested as hypergammaglobulinemia, massive antibody production, and salivary gland lymphocyte infiltration. Plasma cells, the main functional cells after terminal differentiation of B cells, produce large amounts of antibodies, a typical pathological feature of SjD. Therefore, the inventors of this application evaluated the effects of Pik3cb on submandibular gland B cells and splenic plasma cells.

[0055] Test results are shown Figure 4 and Figure 5 The results showed that, compared with the DMSO control group and the PBS control group, the number of submandibular gland B cells and plasma cells in the red pulp of the spleen (in the spleen, plasma cells are mainly concentrated in the red pulp) was significantly reduced in the TGX-221 treatment group; however, this inhibitory effect was significantly reversed in the overexpression group, that is, overexpression of Pik3cb led to a significant increase in the number of submandibular gland B cells and plasma cells in the red pulp of the spleen.

[0056] BAFF is a key regulator of B cell and plasma cell survival and differentiation; therefore, the inventors of this application also evaluated the effect of Pik3cb inhibitors on spleen BAFF. The test results are shown below. Figure 6 The results showed that, compared with the DMSO control group and the PBS control group, the TGX-221 treatment group significantly inhibited BAFF expression, while the Pik3cb overexpression group significantly increased BAFF levels.

[0057] Therefore, the Pik3cb inhibitor TGX-221 can effectively inhibit the survival and terminal differentiation dynamics of autoreactive B cells and plasma cells, thereby exerting an anti-autoimmune effect.

[0058] 5. The Pik3cb inhibitor TGX-221 inhibits B cell chemotaxis via CXCL13. The chemokine CXCL13, by binding to the B cell-specific receptor CXCR5, is crucial for B cell recruitment and chemotaxis, a process key to the formation of submandibular gland lymphocytic foci. Therefore, the expression of CXCL13 in the submandibular gland was further investigated. Results are shown below. Figure 7 The results showed that, compared with the DMSO and PBS control groups, the CXCL13 level was significantly reduced in the TGX-221 treatment group and significantly increased in the Pik3cb overexpression group. This indicates that the Pik3cb inhibitor downregulated the CXCL13 level in the submandibular gland, thereby inhibiting B cell chemotaxis towards the submandibular gland and ultimately reducing the formation of lymphocyte infiltration foci in the submandibular gland.

[0059] 6. The Pik3cb inhibitor TGX-221 downregulates PIK3CB expression in B cells of NOD mice. The applicant of this invention also used TSA to detect the effect of Pik3cb inhibitors on PIK3CB expression in NOD mouse B cells. The results are shown below. Figure 8 The results showed that in the PBS and DMSO control groups, the expression of PIK3CB in the submandibular glands of NOD mice was mainly concentrated in B cells; the expression of PIK3CB in B cells of the submandibular glands of NOD mice in the TGX-221 treatment group was significantly reduced, while the expression of PIK3CB in B cells of the submandibular glands of NOD mice in the overexpression group was significantly upregulated.

[0060] The above results suggest that inhibiting Pik3cb via TGX-221 can suppress the number, activation, differentiation, and chemotaxis of B cells, thereby improving the pathogenesis of SjD. Therefore, inhibiting Pik3cb is a key axis in the intervention of SjD.

[0061] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The use of Pik3cb inhibitors in the preparation of products for the treatment of Sjögren's disease; said use is achieved by injecting or orally administering Pik3cb inhibitors.

2. The application according to claim 1, characterized in that: The injection method is intraperitoneal injection or intravenous injection.

3. The application according to claim 1, characterized in that: The treatment for Sjögren's disease is manifested in at least one of the following: improving salivary gland secretory function, improving salivary gland structure, improving the inflammatory microenvironment of the gland, inhibiting the activation of pathogenic immune cell populations, inhibiting the differentiation of pathogenic immune cell populations, inhibiting the chemotaxis of pathogenic immune cell populations, and reducing the number of pathogenic immune cell populations.

4. The application of Pik3cb as a target for the treatment of Sjögren's disease; said application is achieved by inhibiting Pik3cb.

5. The application of Pik3cb inhibitors aims to improve salivary gland secretion function, improve salivary gland structure, improve the inflammatory microenvironment of the gland, inhibit the activation of pathogenic immune cell populations, inhibit the differentiation of pathogenic immune cell populations, inhibit the chemotaxis of pathogenic immune cell populations, and reduce the number of pathogenic immune cell populations.

6. The application according to claim 1 or 5, characterized in that: The Pik3cb inhibitor is a Pik3cb gene inhibitor or a Pik3cb protein inhibitor.

7. The application according to claim 6, characterized in that: The Pik3cb protein inhibitor is TGX-221.

8. The application according to claim 3 or 5, characterized in that: The improvement in salivary gland secretion function is manifested as an increase in salivary flow rate; The improvement in salivary gland structure is manifested in a reduction of lymphocyte infiltration foci; Furthermore, a reduction in lymphocyte infiltration foci is manifested as a decrease in the area and / or number of lymphocyte infiltration foci.

9. The application according to claim 3 or 5, characterized in that: The improvement of the glandular inflammatory microenvironment is manifested by the downregulation of pro-inflammatory factors and / or the upregulation of anti-inflammatory factors.

10. The application according to claim 3 or 5, characterized in that: The pathogenic immune cell population consists of B cells and / or plasma cells.

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