CN114931578A

CN114931578A - Application of montelukast in preparation of medicines for treating systemic lupus erythematosus

Info

Publication number: CN114931578A
Application number: CN202210528034.2A
Authority: CN
Inventors: 陆前进; 赵俊鹏; 印慧琪; 李黎明
Original assignee: Institute of Dermatology and Skin Disease Hospital of CAMS
Current assignee: Institute of Dermatology and Skin Disease Hospital of CAMS
Priority date: 2022-05-16
Filing date: 2022-05-16
Publication date: 2022-08-23

Abstract

The invention discloses an application of montelukast in preparing a medicine for treating systemic lupus erythematosus. The first research of the invention shows that the montelukast has certain potential in treating the systemic lupus erythematosus, and the new application of the montelukast which is an old medicine can greatly shorten the research and development cost and research and development risk. The invention firstly researches the utility and mechanism of the montelukast for treating the systemic lupus erythematosus, researches the influence of the montelukast on Tfh induced differentiation and B cell antibody secretion, and provides an experimental basis for the treatment of the systemic lupus erythematosus.

Description

Application of montelukast in preparation of medicines for treating systemic lupus erythematosus

Technical Field

The invention belongs to the field of biological medicines, and relates to a novel medicinal application of a G Protein-Coupled Receptor (GPCR) -Cysteinyl leukotriene Receptor (Cysteinyl leukotriene Receptor 1, CYSLTR1) antagonist montelukast, in particular to an application of montelukast in preparing a medicament for treating systemic lupus erythematosus.

Background

Lupus erythematosus is an autoimmune connective tissue disease, the most typical symptom is butterfly-shaped erythema, which is mostly seen in women in 15-40 years old in childbearing age. Lupus erythematosus is a disease spectrum disease, and can be classified into subtypes such as Systemic Lupus Erythematosus (SLE), subacute cutaneous lupus erythematosus, discoid lupus erythematosus, neonatal lupus erythematosus, deep lupus erythematosus, drug-induced lupus erythematosus, and the like.

SLE is the most severe of the various forms of lupus erythematosus. Most patients develop typical butterfly or disk erythematous skin lesions with multiple systemic lesions. A small number of patients may develop from other types of lupus erythematosus. Some SLE patients also have other types of connective tissue diseases, such as dermatomyositis, Sjogren's syndrome, scleroderma, etc., which form an overlap syndrome.

SLE is an autoimmune disease mainly characterized by T, B lymphocyte disorder and a large amount of autoantibodies, and at present, SLE cannot be completely cured and has a long and repeated course of disease, often affects multiple tissues and organs of the whole body such as skin, kidney and central nervous system, and seriously harms life. SLE has complex mechanism, has not yet definite etiology, is better developed in women of child-bearing age, the main treatment means is glucocorticoid and immunosuppressant, most patients can be in stable or low activity state, but can not be cured completely, and simultaneously, the problems of low reactivity and no response to medicines and great side effect of long-term taking of the medicines exist. Therefore, treatment of SLE has been a major and difficult point of research.

Montelukast (Montelukast) as an antagonist of a G Protein-Coupled Receptor (GPCR) -Cysteinyl leukotriene Receptor (Cysteinyl leucotriene Receptor 1, CYSLTR1), can specifically inhibit Cysteinyl leukotriene in airways, is used for clinical treatment of asthma and allergic rhinitis, and is proved to relieve inflammatory and immune skin diseases such as atopic dermatitis, rheumatoid arthritis and the like. The study reports that montelukast can mediate migration of Th17 and inhibit secretion of IL-17, so that the experimental model of multiple sclerosis, namely experimental autoimmune encephalomyelitis, is relieved. In addition, montelukast can inhibit NF-kappa b signal pathway, thereby reducing the secretion of IL-6, IL-8 and MMP-3, and relieving the inflammatory response of rheumatoid arthritis.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to solve the technical problem that the traditional SLE treatment medicine has great side effects, provides a new application of the montelukast medicine in the treatment of SLE, and provides an experimental basis and a new treatment means for the treatment of SLE.

In order to achieve the purpose, the Montelukast is used for the first time for carrying out intraperitoneal injection treatment on MRL/Lpr mice, and the Montelukast is found to be capable of remarkably reducing the content of anti-dsDNA, ANA and urine protein in peripheral blood of the mice. Pathological staining of kidney and skin tissue showed a significant reduction in inflammatory cell infiltration and tissue damage compared to the drug-free group, with a significant reduction in spleen plasma cells, plasmablasts and Th1 cells. The experimental result shows that montelukast can inhibit the differentiation of Tfh, inhibit the proliferation of plasma cells, plasmablasts and Th1 cells, and reduce the generation of autoantibodies, thereby exerting the treatment effect of SLE.

Therefore, the invention claims the application of montelukast in preparing a medicament for treating systemic lupus erythematosus and the new application of the medicament.

Specifically, the treatment of systemic lupus erythematosus is an autoimmune disease which is mainly characterized by T, B lymphocyte disorder and a large amount of autoantibody production, and is different from other types of lupus erythematosus.

Preferably, the Montelukast is used in a dose of 0.2mg/kg to 0.4 mg/kg.

Further, the invention also provides a medicament for treating the systemic lupus erythematosus, and the active ingredient of the medicament comprises montelukast.

Still further, the medicament further comprises a pharmaceutically acceptable carrier.

Further, the medicine is powder, tablets, injection, capsules or oral liquid.

Further, the invention provides a pharmaceutical composition for treating systemic lupus erythematosus, which comprises montelukast.

Has the advantages that:

at present, the main treatment means of SLE are glucocorticoid and immunosuppressant, most of patients can be in a stable or low-activity state, but the disease cannot be completely cured, and the problems of low response and no response to the medicine and great side effect of the medicine taken for a long time exist. The montelukast is used as an old medicine for clinically treating asthma and allergic rhinitis, and has small toxic and side effects. The first research of the invention shows that the montelukast has certain potential in treating SLE, and the new application of the montelukast which is an old drug can greatly shorten the research and development cost and research and development risk. The invention firstly explores the effect and mechanism of the montelukast in treating SLE, researches the influence of the montelukast on Tfh induced differentiation and B cell antibody secretion, and provides an experimental basis for the treatment of SLE.

Drawings

The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.

FIG. 1 is a mouse Pristane (Pristane) -induced lupus expression levels of kidney and spleen mRNA of Montelukast target CYSLTR1 in MRL/lpr mice.

FIG. 2 is montelukast on human peripheral blood

Effect of CD4+ T cells on Treg and Tfh induced differentiation.

Figure 3 is that montelukast reduces the incidence of skin damage in lpr mice.

FIG. 4 shows that montelukast can reduce the levels of lpr mouse urine protein, peripheral blood anti-dsDNA, and ANA.

Fig. 5 shows that montelukast can alleviate pathological damage to the skin and kidney of lpr mice.

Fig. 6 shows that montelukast reduces the deposition of the rpr mouse kidney IgG2a and C3 immune complex.

FIG. 7 is a flow cytometry analysis of the spleen of an lpr mouse.

Figure 8 is that montelukast can inhibit antibody secretion by B cells in vitro.

Detailed Description

The invention will be better understood from the following examples.

1. Experimental Material

1.1 experimental drugs and reagents: montelukast (MCE), cyclophosphamide (Sigma-Aldrich), DMSO (Sigma-Aldrich), anti-dsDNA ELISA kit (jianglaibo), ANA ELISA kit (CUSABIO), flow antibody (BD pharmingen, Biolegend, eBioscience).

1.2 Experimental animals

MRL/lpr mice are adopted in the experiment, the week age is about 10-12 weeks, the weight is 30-40g, the MRL/lpr mice are provided by Shanghai slke laboratory animal finite liability company, and pristane (pristane) -induced lupus mice are provided by an immunological skin disease basic transformation key laboratory of Chinese academy of medicine science. C57 mouse, week old about 8-12 weeks, weight 20-25g, supplied by Nanjing Watson Biotech, Inc. Feeding conditions are as follows: room temperature is 18-20 ℃, humidity is 50-60%, light and shade are alternated (12h), luminosity is moderate, and ventilation and cleanness are good. All experiments were approved and conducted as directed by the ethical committee of the dermatologic hospital, national academy of medicine (the institute of dermatology, national academy of medicine).

2. Experimental methods

2.1 real-time fluorescent quantitative PCR (qPCR) detection of Montelukast target CYSLTR1 in mouse kidney and spleen mRNA expression level

According to the instructions, Trizol (Invitrogen) is used for extracting total RNA of kidney and spleen of a pristine-induced lupus mouse and an lpr mouse, and Novozam is used

III RT Supermix for qPCR (+ gDNA wiper) -RNA reverse transcription reagent reverse transcription to cDNA. The expression of CYSLTR1 was detected by qPCR using the ChamQ Universal SYBR qPCR Master Mix and primers in Roche LightCycler 96. The primer sequences are as follows: CYSLTR1 upstream primer: 5'-CCTCTCCGTGTGGTCTATTATGT-3', downstream primer: 5'-ATGCAAACGAACCTGGCTTTT-3' are provided.

2.2 peripheral blood of human

CD4+ Treg and Tfh induced differentiation

To study montelukast on human CD4 ⁺

Effects of directed differentiation of T cells into Treg and Tfh cells. In the experiment, Ficoll (Cytiva) density gradient method is used for separating Peripheral Blood Mononuclear Cells (PBMC) of healthy people, and EasySep is used ^TM Human

CD4T Cell Iso Kit (Stemcell) extraction

CD4+ T cells divided into Treg group and Tfh group. Treg group: 5 μ g/ml anti-CD3 (R)&D Systems) and 2. mu.g/ml anti-CD28 (R)&D Systems) is stimulated and activated, 5ng/ml TGF-beta (Peprotetin) and 10ng/ml IL-2(Peprotetin) are added for Treg induced differentiation; tfh group: 5ng/ml of TGF-beta (Peprotatin), 20ng/ml of IL-6 (Peprotatin), 10ng/ml of IL-12 (Peprotatin) and 20ng/ml of IL-21 (Peprotatin) are added after stimulation and activation of 5 mu g/ml of anti-CD3 and 2 mu g/ml of anti-CD28 to perform Tfh induced differentiation. Treg and Tfh directed induced differentiation was performed in vitro in three conditions, DMSO (negative control), low montelukast concentration (1. mu.M) and high montelukast concentration (10. mu.M), respectively.

2.3 therapeutic Effect of Montelukast on SLE model mice MRL/lpr

2.3.1 mice were dosed

MRL/lpr mice were grouped as required: a negative control group (PBS + DMSO), a Cyclophosphamide (CTX) group, a montelukast low-concentration drug group (2.5mg/kg/d), and a montelukast high-concentration drug group (5mg/kg/d), wherein 7 to 9 drugs are contained in each group. SLE model mice MRL/lpr were injected intraperitoneally with montelukast administered daily to the high and low concentration drug groups (5.0mg/kg/d, 2.5mg/kg/d) and to the negative control group (PBS + DMSO) (200. mu.l/d), and the cyclophosphamide group once a week at a dose of 30 mg/kg.

2.3.2 mouse skin lesions, urine protein and autoantibody monitoring

The skin lesions of the mice were observed every week, photographed by a camera and kept, and urine protein was detected using ulite protein urine test paper. The ELISA kit is used for detecting the content of anti-dsDNA (Jianglaibio) and ANA (CUSABIO) in peripheral blood serum every two weeks.

2.3.3 histopathological and immunofluorescence analysis

To analyze skin and kidney inflammatory cell infiltration and pathological injury, mice were sacrificed after anesthesia at 19 weeks of age, skin and kidney tissues were collected and fixed with 4% paraformaldehyde, and then HE staining was performed to analyze inflammatory cell infiltration. Frozen sections of kidney were directly immunofluorescent stained with IgG2a (Abcam) and indirectly immunofluorescent stained with C3(Abcam) and corresponding fluorescent secondary antibodies.

2.3.4 flow cytometry

Mice were sacrificed after 19 weeks of age under anesthesia, spleens were removed, ground and filtered to prepare single cell suspensions, and cell surface, intracellular cytokine and nuclear transcription factor of T, B cells were labeled with corresponding antibodies as shown in tables 1 and 2.

TABLE 1

TABLE 2

Panel 1 group 2.5 x 10^6 spleen cells were plated in 24-well plates and 2. mu.l of ionomycin Golgi blocker (BD pharmingen) was plated per well for 5 h. Then, 1ml of PBS 500g was added and centrifuged for 5min, and after discarding the supernatant, the flow antibody was added and incubated at 4 ℃ for 45 min. After the primary membrane staining is finished, 1ml PBS 500g is added for centrifugation for 5min for washing, and APC-Streptavidin is added after washing, and incubation is carried out for 45min at 4 ℃. Then, the mixture was centrifuged for 5min with the addition of 500g of 1ml PBS, and washed. After membrane staining, a nucleotomy membrane reagent (Thermo) was added, 500. mu.l/tube of the disrupted membrane was fixed, incubated at 4 ℃ for 2h, and centrifuged at 500g of wash buffer 1ml for 5min to wash after disrupting the membrane. After washing, the above-mentioned antibodies (IFN-. gamma., IL-4, IL-17, FOXP3) which require the staining of the cell membrane and the nuclear membrane were added thereto, and incubated at 4 ℃ for 1.5 hours. After incubation, 1ml PBS 500g is added for centrifugation for 5min, the supernatant is discarded, the residual PBS is vortex suspended and precipitated, and the flow type machine is used.

Panel 2 group 2.0 x 10^6 spleen cells were added to flow tubes, 0.5. mu.l Mouse Fc block (Biolegend) was added per tube, incubated at room temperature for 15min, and flow antibody incubation was continued. After membrane staining for 45min at 4 ℃, adding 1ml PBS 500g, centrifuging for 5min, washing, discarding supernatant, carrying out vortex suspension precipitation on residual PBS, and performing flow-type machine.

2.3.5 Effect of Montelukast on in vitro antibody secretion by B cells

C57 mice were aged about 8-12 weeks, sacrificed after anesthesia, spleens were removed under sterile conditions, ground and filtered to prepare single cell suspensions, CD19+ B cell sorting was performed using easy Sep Mouse CD19 positive Selection Kit II (Stemcell), and the sorted CD19+ B cell pellet was resuspended in DMEM complete medium (10% FBS + 1% penicillin/streptomycin + 55. mu.M. beta. -mercaptoethanol) and added to cell culture plates at 1.0 × 10^6 cells/well. In addition, addition of 100ng/ml R848(Sigma) and 30ng/ml IL-4(Peprotetin) per well induced initial B cell differentiation into plasma cells and antibody secretion. A blank control group (without adding R848 and IL-4), a negative control group (with adding R848 and IL-4) and a montelukast drug group (10 mu.M) were set, and cell supernatants were collected after 66 hours and used LEGENDplex ^TM Mouse Immunoglobulin Isotyping Panel (6-plex) (Biolegend) detects IgG1, IgG2a, IgG2b, IgG3, IgA, IgM.

3. Results of the experiment

3.1 Montelukast target CYSLTR1 renal, spleen mRNA expression levels in Pristane-induced lupus murine models and MRL/lpr mice. As shown in FIG. 1, the mRNA expression of CYSLTR1 in Pristane-induced lupus mouse model, MRL/lpr mouse kidney and spleen was significantly higher than that of C57 mouse, and there was a statistical difference (P < 0.05;. P < 0.01;. P < 0.001;. P < 0.0001), suggesting that CYSLTR1 may promote the development of SLE and antagonize CYSLTR1 may have the effect of treating SLE.

3.2 Montelukast to human CD4 ⁺

Effects of T cell directed differentiation into Treg and Tfh cells. FIG. 2 ML: montelukast low concentration (Montelukast low concentration 1 μ M); MH: montelukast high control (Montelukast high concentration 10. mu.M), Negative control and Untreated represent Negative controls. As shown in fig. 2(A, C), montelukast did not affect

CD4+ T cells induced differentiation into Treg (CD25+ CD127low) cells; FIG. 2(B, D) Montelukast inhibition

Induction of differentiation of CD4+ T cells into Tfh (CXCR5+ PD-1+) cells (, P)<0.05，n＝3)。

3.3 Montelukast reduces the incidence of lpr skin lesions. FIG. 3: NC stands for a negative control group, Montelukast5mg/kg stands for a Montelukast high-concentration drug group, and Montelukast 2.5mg/kg stands for a Montelukast low-concentration drug group. At 19 weeks of age, there was no difference in body weight between the groups, but 6 mice in the negative control group showed skin lesions (6/9, 66.7%) (fig. 3), while only 2 mice in both the high and low concentration groups of montelukast showed skin lesions (high concentration: 2/7, 28.6%; low concentration: 2/9, 22.2%), and no mice in the CTX group showed skin lesions, indicating that montelukast decreased the incidence of lpr skin lesions.

3.4 Montelukast can reduce the content of the urine protein, peripheral blood anti-dsDNA and ANA of the lpr mouse. FIG. 4: DMSO represents a negative control group, CTX represents a cyclophosphamide drug group, Montelukast5mg/kg represents a Montelukast high concentration drug group, and Montelukast 2.5mg/kg represents a Montelukast low concentration drug group. As shown in FIG. 4, compared to the negative control group, the Montelukast drug group urine protein (FIG. 4B) (urine protein, M5 group vs PBS + DMSO group: 17.1 + -13.5 mg/dl vs 182.5 + -128.4 mg/dl, < P0.05), peripheral blood autoantibodies anti-dsDNA (FIG. 4C) (anti-dsDNA, M5 group vs PBS + DMSO group: 14.2 + -18.6 ng/ml vs 160.9 + -210.0 ng/ml, < P0.05), and ANA (FIG. 4D) (ANA, M5 group vs PBS + DMSO group: 37.3 + -20.1 ng/ml vs 83.1 + -43.1 ng/ml, < P0.05) were significantly decreased. The above results indicate that montelukast can significantly reduce the urinary protein and autoantibodies of lpr mice.

3.5 Montelukast relieves pathological damage to the skin and kidneys of lpr mice. FIG. 5: c57 represents a C57 mouse, CTX represents a cyclophosphamide drug group, DMSO represents a negative control group, M5 represents a montelukast high-concentration drug group (5mg/kg), and M2.5 represents a montelukast low-concentration drug group (2.5 mg/kg). HE histopathological staining is carried out on the skin and the kidney of the mouse, and the result shows that the infiltration of the lymphocyte on the skin and the kidney of the mouse of the montelukast medicament group is obviously reduced, the swelling hyperplasia degree of the glomerulus is reduced (figure 5),

3.6 Montelukast reduces the deposition of the Ipr mouse kidney IgG2a and C3 immune complex. FIG. 6: DMSO represents a negative control group, CTX represents a cyclophosphamide drug group, M5 represents a montelukast high-concentration drug group (5mg/kg), and M2.5 represents a montelukast low-concentration drug group (2.5 mg/kg). The mice kidneys were frozen sections and immunofluorescent stained with IgG2a and C3, showing that montelukast significantly reduced the deposition of IgG2a and C3 immunocomplexes in the lpr mice kidneys (fig. 6).

3.7lpr mouse spleen flow cytometry analysis. FIG. 7: control represents a negative control group, CTX represents a cyclophosphamide drug group, M5 represents a montelukast high-concentration drug group (5mg/kg), and M2.5 represents a montelukast low-concentration drug group (2.5 mg/kg). As shown in fig. 7, montelukast was found to significantly reduce the ratio of plasma cells, plasmablasts and Th1 cells (P < 0.05;. P < 0.01;. P < 0.001;. P < 0.0001) by flow cytometry analysis of isolated mouse spleen cells.

3.8 Montelukast inhibits antibody secretion by B cells in vitro. FIG. 8: BLANK stands for BLANK Control (without addition of R848 and IL-4), Control for negative Control (with addition of R848 and IL-4), Montelukast for Montelukast drug group (10. mu.M) (with addition of R848+ IL-4 and Montelukast). As shown in FIG. 8, the main secreted antibody of B cells after R848 and IL-4 stimulation for 66h was IgM, and the other types of antibodies were less secreted. Montelukast inhibits IgM secretion (P < 0.05;. P < 0.01) with a tendency to decrease IgG1, IgG2a, IgG2b, IgG3, IgA (P > 0.05). In conclusion, montelukast can inhibit antibody secretion by B cells in vitro.

The invention provides a thought and a method for applying montelukast in preparing a medicament for treating systemic lupus erythematosus, and a method and a way for realizing the technical scheme are many, the above description is only a preferred embodiment of the invention, and it should be noted that, for a person skilled in the art, a plurality of improvements and modifications can be made without departing from the principle of the invention, and the improvements and modifications should be regarded as the protection scope of the invention. All the components not specified in the present embodiment can be realized by the prior art.

Claims

1. Use of montelukast in the manufacture of a medicament for treating systemic lupus erythematosus.

2. The use of claim 1, wherein the systemic lupus erythematosus is an autoimmune disease characterized primarily by disorders of T-and B-lymphocytes, and massive autoantibody production.

3. The use according to claim 1, characterized in that the montelukast is used in a dose ranging from 0.2mg/kg to 0.4 mg/kg.

4. A medicament for the treatment of systemic lupus erythematosus, characterized in that its active ingredient comprises montelukast.

5. The agent for treating systemic lupus erythematosus according to claim 4, further comprising a pharmaceutically acceptable carrier.

6. The agent for treating systemic lupus erythematosus in claim 4, wherein the agent is a powder, a tablet, an injection, a capsule or an oral liquid.

7. A pharmaceutical composition for treating systemic lupus erythematosus comprising montelukast.