Application of astemizole in the preparation of drugs for treating Tfh-related autoimmune diseases

Abstract

The present invention discloses the use of astemizole in preparing a drug for treating Tfh-related autoimmune diseases, in particular, in preparing a drug for treating systemic lupus erythematosus. The present invention first proposes the effect of astemizole on Tfh cell induction and differentiation, and at the same time, astemizole is used to treat lupus mice induced by imiquimod. After treatment, the disease phenotype of the mice is effectively alleviated, confirming that astemizole can be used as an effective drug for regulating Tfh cell differentiation and alleviating lupus.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to a new pharmaceutical use of astemizole, in particular to its application in preparing a drug for treating Tfh-related autoimmune diseases. Background Art

[0002] Systemic lupus erythematosus (SLE) is a classic autoimmune disease characterized by overactivation of the immune system, recruitment of autoreactive and inflammatory T cells, elevated levels of proinflammatory cytokines, formation of nuclear autoantibodies and immune complexes, and involvement of one or more organs including the skin, kidneys, joints, and nervous system. Currently, drugs for the treatment of SLE mainly control autoimmune responses by deeply suppressing the immune system, or reduce peripheral tissue damage by suppressing systemic inflammation. Traditional drugs such as antimalarial drugs, triptolide, glucocorticoids, and immunosuppressants are non-targeted drugs. Most patients can be stable or in a low-activity state, but they cannot be completely cured. At the same time, some patients have low responsiveness to drugs, no response, and long-term use of drugs has serious side effects. Existing SLE drugs still cannot meet clinical treatment needs.

[0003] Astemizole (AST) is a highly effective and long-lasting selective histamine H1 receptor (H1R) antagonist. In addition to its conventional use in the treatment of allergic diseases such as urticaria, it is also used in the anti-tumor and anti-viral fields. There are currently no reports of its use in the treatment of autoimmune diseases.

[0004] The present invention explores the application of astemizole in the treatment of autoimmune diseases and conducts off-label exploration of "new uses for old drugs", which is not only safe but also greatly reduces the cost and risk of new drug research and development for related diseases. Summary of the invention

[0005] The purpose of the present invention is to provide a new use of astemizole in preparing a drug for treating autoimmune diseases, in particular a new use of astemizole in preparing a drug for treating SLE.

[0006] The present invention uses astemizole for the first time to treat lupus-like mice induced by imiquimod (IMQ) by oral gavage. Astemizole can significantly reduce the enlargement of spleen and lymph nodes in IMQ-induced lupus model mice, reduce the level of peripheral blood anti-dsDNA antibodies, inhibit the proportion of germinal center B cells (Germinal center B cells, GC B), memory B cells (Memory B cells), follicular helper T cells (Follicular helper T cells, Tfh) and effector T cells (Effector T cells) in lymph node cells, and effectively alleviate the disease progression of IMQ-induced lupus model mice. The experimental results show that astemizole can inhibit the induced differentiation of Tfh, inhibit the production of GCB cells, and reduce the generation of autoantibodies, thereby exerting the therapeutic effect of SLE.

[0007] To this end, the present invention proposes the use of astemizole in the preparation of a drug for treating Tfh-related autoimmune diseases. The Tfh-related autoimmune disease is systemic lupus erythematosus.

[0008] Specifically, the systemic lupus erythematosus described in the present invention is an autoimmune disease characterized by T and B lymphocyte disorders and the production of a large number of autoantibodies, which is different from other common lupus erythematosus.

[0009] Furthermore, astemizole has an inhibitory effect on Tfh-induced differentiation and its mediated germinal center reaction, and the use of drugs that inhibit Tfh-induced differentiation in the preparation of drugs for treating Tfh-related autoimmune diseases is proposed. The drug is astemizole, and the disease is systemic lupus erythematosus.

[0010] Furthermore, the present invention also provides a drug for treating systemic lupus erythematosus, wherein the active ingredient of the drug comprises astemizole.

[0011] Furthermore, the drug also includes a pharmaceutically acceptable carrier to prepare a preparation.

[0012] Preferably, the preparation is a powder, tablet, injection, capsule or oral solution.

[0013] Furthermore, the present invention also provides a pharmaceutical composition for treating systemic lupus erythematosus, which comprises astemizole.

[0014] When used, the dose of astemizole is 50 mg / kg / day for oral administration to mice.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] (1) Astemizole is an old drug for the clinical treatment of allergic diseases with low toxicity and side effects. The present invention proposes for the first time that astemizole be used to treat Tfh-related autoimmune diseases, especially systemic lupus erythematosus, with a low dosage and significant effect. The new use of astemizole as an old drug can greatly reduce the cost and risk of new drug research and development.

[0017] (2) The present invention proposes for the first time the efficacy of astemizole in treating systemic lupus erythematosus and the inhibitory effect of astemizole on Tfh-induced differentiation and its mediated germinal center reaction, providing a solid foundation for the treatment of Tfh-related autoimmune diseases such as SLE. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0019] Figure 1 It is the effect of astemizole on T and B cell subsets in peripheral blood mononuclear cells (PBMC) of SLE patients.

[0020] Figure 2 Astemizole is a promising candidate for the treatment of primary CD4 + The influence of various T cell subsets.

[0021] Figure 3 Astemizole inhibits human peripheral blood naïve CD4 + In vitro directed differentiation process of T cells into Tfh cells.

[0022] Figure 4 Astemizole can reduce the enlargement of spleen and lymph nodes in IMQ-induced lupus mice.

[0023] Figure 5 Astemizole can reduce the content of Anti-dsDNA in the peripheral blood of IMQ-induced lupus mice.

[0024] Figure 6 Flow cytometric analysis of lymph node cells from IMQ-induced lupus mice with astemizole. DETAILED DESCRIPTION

[0025] The experimental methods described in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. described in the following examples are all commercially available unless otherwise specified.

[0026] In the following examples, the protein immunoblotting detection, the flow cytometry, the hematoxylin-eosin staining, the immunofluorescence analysis and other experimental methods are all conventional experimental methods well known to those skilled in the art. If the experimental method does not specify specific conditions, it is usually performed under conventional conditions.

[0027] In the following examples, the experimental materials used are as follows:

[0028] 1. Experimental drugs and reagents: Astemizole (purchased from Jiangsu Kewei Biological Co., Ltd.); DMSO (purchased from Sigma-Aldrich); Anti-dsDNA ELISA kit (purchased from Wuhan Huamei Bioengineering Co., Ltd. CUSABIO); functional antibodies and flow cytometry antibodies (purchased from BD pharmingen, Biolegend, eBioscience); Trizol reagent (purchased from Invitrogen); EasySep TM Mouse CD4 + T Cell Isolation Kit (purchased from Stemcell); RIPA protein lysis buffer (purchased from Beyotime); EasySep TM Human naïve CD4 + T Cell Isolation Kit Ⅱ (purchased from Stemcell).

[0029] 2. Experimental Animals:

[0030] (1) Pristane-induced lupus mice, i.e., pristane-modeled lupus mice: C57BL / 6J mice were injected intraperitoneally with 500 μL of pristane per mouse to induce modeling. The mice were approximately 6-8 weeks old and weighed 20-30 g. They were provided by the Key Laboratory of Basic Translational Immunological Skin Diseases, Chinese Academy of Medical Sciences.

[0031] (2) C57BL / 6J mice: about 8-12 weeks old, weighing 20-25 g, provided by Nanjing Watson Biotechnology Co., Ltd., used for IMQ modeling.

[0032] The breeding conditions of the animals used in the above experiments were: room temperature 18-20℃, humidity 50-60%, alternating light and dark (12 h), moderate light intensity, and good ventilation and cleanliness.

[0033] All experiments were approved and performed in accordance with the guidelines of the Ethics Committee of the Dermatology Hospital of Chinese Academy of Medical Sciences (Institute of Dermatology, Chinese Academy of Medical Sciences).

[0034] Example 1 Effects of astemizole on T and B cell subsets in PBMC of SLE patients.

[0035] Peripheral blood was collected from healthy individuals and SLE patients to isolate PBMCs. The specific steps were as follows: (1) Prepare several 15 mL centrifuge tubes, number each centrifuge tube according to the peripheral blood sample information, and add an equal volume of Ficoll-Paque PREMIUM lymphocyte separation solution to each centrifuge tube; (2) Add an equal volume of 1× PBS buffer to each peripheral blood sample for dilution, and then slowly add it to the centrifuge tube containing Ficoll-Paque PREMIUM lymphocyte separation solution, paying attention to maintaining the annular boundary between Ficoll-Paque PREMIUM lymphocyte separation solution and peripheral blood sample dilution solution; (3) Balance the centrifuge tube in (2) and gently place it in a centrifuge. The centrifugation conditions are: 20°C, 1800 rpm (acceleration 8, deceleration 0), 25 min; (4) Carefully remove the centrifuged sample in (3), and use a Pasteur pipette to slowly aspirate the cloud layer into another new 15 mL centrifuge tube to avoid inhaling the upper serum and the lower Ficoll-Paque PREMIUM lymphocyte separation solution; (5) Add appropriate amount of 1× PBS buffer to each PBMC extracted in (4) and mix well. Place in a centrifuge. Centrifuge conditions: 20℃, 500g (acceleration 8, deceleration 9), 10 min; (6) Carefully remove the sample after centrifugation in (5), discard the supernatant and add about 5 mL of red blood cell lysis solution. Mix well and lyse the red blood cells at room temperature for 7 min. Add about 10 mL of 1× PBS buffer and centrifuge. Centrifuge conditions: 20℃, 500g, 10 min; (7) Carefully remove the sample after centrifugation in (6), discard the supernatant, and the cell pellet obtained is the PBMC isolated from human peripheral blood.

[0036] The isolated PBMCs were lysed with lysate and cultured with 2 µg / mL Anti-human CD3 and 1 µg / mL Anti-human CD28 antibodies, and treated with astemizole (10 µM) and vehicle control (Vehicle). After three days of culture, the cells were harvested and flow cytometry was performed using a BD Aria III flow cytometer to detect the proportion of each T and B cell subset.

[0037] The results are as follows Figure 1 As shown, CD4 + The proportion of T cells decreased significantly, among which the proportion of Tfh and Th2 cells decreased significantly (n=4, *: P <0.05,**: P <0.01); meanwhile CD19 +The proportion of B cells also decreased significantly, among which the proportions of memory B cells, plasmablasts and antibody-secreting cells all decreased (n=5, *: P <0.05,**: P <0.01). It can be seen that astemizole can significantly inhibit the abnormal increase of T and B cells in SLE patients during the disease process, especially it can significantly reduce the proportion of Tfh cells and antibody secreting cells (ASC).

[0038] Example 2 Effect of Astemizole on Primary CD4 in Spleens of Pristane-Induced Lupus Mice + The influence of various T cell subsets.

[0039] To study the effect of astemizole on primary CD4 + In this example, five Pristane-induced lupus mice were first killed, and the spleens were ground, filtered, and lysed, and the cells were counted. About 1×10 7 cells in sterile flow cytometry tubes using EasySep TM Mouse CD4 + CD4 T Cell Isolation Kit (Stemcell) was used for magnetic bead separation and extraction + T cells were sorted and counted again. A 24-well plate was pre-coated with 5 μg / mL Anti-mouse-CD3e and placed in a 37°C cell culture incubator for 4 h. + T cells were plated in coated 24-well plates, with approximately 1×10 6 Each well was stimulated and activated by adding 2 μg / mL Anti-mouse-CD28, and then added complete culture medium (Control: solvent control group) and 10 μM astemizole (drug intervention group) respectively, and cultured in a cell culture incubator at 37°C for 48 h. Note that the experiment needs to be completed on a clean bench to ensure a sterile environment and avoid cell contamination. After 48 h, the cells were collected and stained with flow cytometry antibodies and detected by flow cytometer BD Aria III.

[0040] The results are as follows Figure 2 As shown, the flow cytometry results showed that the proportions of Tfh and Th1 cells in the astemizole group were significantly lower than those in the solvent control group, and the proportion of Treg cells was significantly increased, among which the effect on the proportion of Tfh subsets was the most significant (n=5, *: P <0.05,**: P <0.01).

[0041] Example 3 Effect of astemizole on the differentiation of human peripheral blood naïve CD4+ T cells into Tfh.

[0042] In order to study the effect of astemizole on the directional differentiation of human naïve CD4+ T cells into Tfh cells, this example first used Ficoll (Cytiva) density gradient centrifugation to separate PBMCs from healthy subjects, and then used EasySepTM Human naïve CD4 + T Cell Isolation Kit Ⅱ (Stemcell) was used to extract initial CD4 + T cells were then stimulated and activated with 5 μg / mL Anti-CD3 and 2 μg / mL Anti-CD28, and then 5 ng / mL TGF-β (Peprotetin), 20 ng / mL IL-6 (Peprotetin), 10 ng / mL IL-12 (Peprotetin), and 20 ng / mL IL-21 (Peprotetin) were added for Tfh induced differentiation. Each person's cells were divided into three groups, and DMSO (negative control group), low concentration of astemizole (1.25 μM), medium concentration of astemizole (2.5 μM), and high concentration of astemizole (5 μM) were added to perform Tfh directional differentiation in vitro.

[0043] The results are as follows Figure 3 As shown in Figure 2, astemizole (5 μM) can inhibit the expression of human peripheral blood naïve CD4 + T cells to Tfh (CD4 + CXCR5 + PD-1 + ) cells induced to differentiate (n=4, *: P <0.05,**: P <0.01).

[0044] Example 4 The intervention effect of astemizole on IMQ-induced SLE model mice.

[0045] In order to further clarify the role of astemizole in alleviating the pathological progression of SLE, we conducted an in vivo efficacy study of astemizole in an IMQ-induced lupus mouse model. The animal groups are shown in Table 1, and the IMQ modeling method is as follows: 8-10 week old C57BL / 6J female mice were taken, and about 15 mg of 5% imiquimod cream was applied to the inner edge of the left ear of each mouse, and the ointment was gently spread evenly. After the drug was observed to be absorbed until transparent, the mouse was put back into the cage. When applying the ointment, avoid rubbing the ointment on the bedding to reduce the amount of the ointment, and apply it three times a week.

[0046] At 9 weeks after modeling, the mice were killed, weighed, and blood was drawn to collect mouse serum. The spleen and lymph nodes were taken for photography, weighing, and flow cytometry.

[0047] Table 1 Grouping of IMQ-induced lupus mice

[0048] Grouping Mouse strains Modeling method Treatment Dosage Dosing frequency Control group C57BL / 6J PBS ear wipe Normal saline Oral gavage every day Model Group C57BL / 6J IMQ ear wipe Normal saline Oral gavage every day Low concentration group C57BL / 6J IMQ ear wipe AST 30 mg / kg Oral gavage every day High concentration group C57BL / 6J IMQ ear wipe AST 50 mg / kg Oral gavage every day

[0049] The lupus phenotypes of mice in each group at the end of treatment were as follows:

[0050] like Figure 4 As shown in the results, at the end of treatment, the spleen, lymph node volume and relative wet weight of the model group mice were significantly increased compared with those of the control group. High concentration of astemizole can reduce the enlargement of spleen and lymph nodes in IMQ-induced lupus model mice.

[0051] like Figure 5 As shown, high concentrations of astemizole at the end of treatment can reduce MRL / lpr Urine protein and peripheral blood Anti-dsDNA content of model mice.

[0052] like Figure 6 As shown, flow cytometry analysis of mouse lymph node cells showed that compared with the control group, the proportion of GC B, Memory B, Tfh and effector T cells in the lymph node cells of the model group mice increased, and high concentration of astemizole inhibited the increase in the proportion of GC B, Memory B, Tfh and effector T cells in the lymph nodes of IMQ-induced lupus mice (*: P <0.05,**: P <0.01).

[0053] Overall, AST (50 mg / kg / day) treatment effectively alleviated disease progression in IMQ-induced lupus model mice and can be used to treat systemic lupus erythematosus.

[0054] There are many methods and approaches to implement the technical solution of the present invention. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. The application of astemizole in the preparation of drugs for the treatment of systemic lupus erythematosus.

2. Use of a drug that inhibits Tfh-induced differentiation in the preparation of a drug for treating systemic lupus erythematosus, wherein the drug is astemizole.

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