Use of GppNHp in the preparation of drugs for treating systemic lupus erythematosus

Abstract

The present invention provides the use of GppNHp in the preparation of a medicament for treating systemic lupus erythematosus, belonging to the technical field of medicine. By using the necroptosis inhibitor GppNHp, the present invention can significantly alleviate various symptoms of lupus model mice, such as significantly reducing the volume and weight of the spleen and lymph nodes, slowing down kidney injury, reducing urinary protein, decreasing the concentration of tumor necrosis factor (TNF-α), and increasing the concentration of complement C3. The preparation provided by the present invention can effectively improve the symptoms of lupus mice and is expected to improve the quality of life of SLE patients.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the application of GppNHp in the preparation of drugs for treating systemic lupus erythematosus. Background Art

[0002] Systemic lupus erythematosus (SLE) is a complex autoimmune disease that occurs mostly in young women. Its pathological mechanism mainly involves overactive B-cell and T-cell signal transduction, as well as abnormal release of cytokines. Currently, the treatment of SLE mainly relies on widely used non-specific hormones and immunosuppressants. Although these treatment methods can control the disease condition and relieve symptoms to a certain extent, long-term use will bring a series of side effects, such as osteoporosis, increased risk of infection, kidney damage, etc.

[0003] The existing treatment methods mainly include: 1. Glucocorticoids: used to quickly control the inflammatory response, but long-term use will cause serious side effects. 2. Antimalarial drugs (such as hydroxychloroquine): used to treat mild to moderate SLE and have an immunomodulatory effect, but the treatment effect is limited. 3. Immunosuppressants (such as cyclophosphamide, etc.): used to treat severe SLE, but may lead to over-inhibition of the immune system and other serious side effects. 4. Biological agents (such as belimumab): targeting specific immune cells or cytokines. Although the curative effect is significant, the cost is high and side effects such as infection may occur. The common problem with these treatment regimens is that they cannot fundamentally regulate or correct the immune abnormalities that cause SLE, and most are accompanied by serious side effects, which limits their long-term use.

[0004] Although the existing treatment methods have played a role in controlling the symptoms of SLE, there are still significant deficiencies in terms of safety, persistence of efficacy, and cost-effectiveness. Especially long-term reliance on hormone and immunosuppressant treatments may not only lead to various complications, but also for some patients, the treatment effect gradually weakens. There is an urgent need to develop a new treatment method to more safely and effectively regulate the immune system of patients, reduce the side effects of long-term treatment, and improve the quality of life of patients. Necroptosis is a form of regulated cell death that has been found to be closely related to the pathogenesis of various autoimmune diseases in recent years. Therefore, it is particularly important to study a necroptosis inhibitor as an intervention drug for the treatment of SLE. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide the application of GppNHp in the preparation of drugs for treating systemic lupus erythematosus. By using the necroptosis inhibitor GppNHp, the present invention significantly alleviates various symptoms of lupus model mice, including reducing the volume and weight of the spleen and lymph nodes, slowing down kidney damage, decreasing the urine protein concentration, and regulating related immune inflammatory response indexes. The present invention provides a safe and effective preparation, which is expected to improve the quality of life of patients.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides the application of GppNHp in the preparation of drugs for treating systemic lupus erythematosus.

[0008] Preferably, the concentration of GppNHp is 5-15 μg / μL.

[0009] Preferably, the solvent of GppNHp is phosphate buffer solution.

[0010] Preferably, the usage method of GppNHp is injection.

[0011] Preferably, the usage frequency of GppNHp is 1-5 days / time.

[0012] The present invention also provides a medicament for treating systemic lupus erythematosus, which comprises GppNHp and its pharmaceutically acceptable carrier.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides the application of GppNHp in the preparation of drugs for treating systemic lupus erythematosus. By using the necroptosis inhibitor GppNHp, the present invention can significantly alleviate various symptoms of SLE, such as significantly reducing the volume and weight of the spleen and lymph nodes, slowing down kidney damage, decreasing the concentrations of urine protein and inflammatory factors (tumor necrosis factor α (TNF-α)), and increasing the concentration of complement C3. The preparation provided by the present invention can effectively improve the symptoms of lupus mice and is expected to improve the quality of life of SLE patients. Description of the Drawings

[0014] Figure 1 It is a graph of the volume and weight results of the spleen and axillary lymph nodes of mice before and after treatment with the necroptosis inhibitor GppNHp, where A is the graph of the spleen volume of mice before and after treatment with the necroptosis inhibitor GppNHp, B is the graph of the spleen weight results of mice before and after treatment with the necroptosis inhibitor GppNHp, C is the graph of the axillary lymph node volume of mice before and after treatment with the necroptosis inhibitor GppNHp, and D is the graph of the axillary lymph node weight results of mice before and after treatment with the necroptosis inhibitor GppNHp;

[0015] Figure 2 It is a pathological HE staining diagram of the kidneys of mice before and after treatment with the necroptosis inhibitor GppNHp, and the scale bar is 20 μm;

[0016] Figure 3 It is a diagram showing the changes in the levels of autoantibodies (anti-dsDNA antibodies), complement C3, and tumor necrosis factor TNF-α before and after treatment with the necroptosis inhibitor GppNHp;

[0017] Figure 4 It is a diagram showing the changes in serum creatinine and urine protein concentrations before and after treatment with the necroptosis inhibitor GppNHp;

[0018] Figure 5 It is a diagram showing the viability of neutrophils in the peripheral blood of mice detected by flow cytometry before and after treatment;

[0019] Figures 1 to 5 In it, MRL / Mpj represents the normal mouse group, MRL / lpr + DMSO represents the lupus model mouse group without treatment, using only DMSO as a control, MRL / lpr + GppNHp represents the lupus model mouse group treated with the necroptosis inhibitor GppNHp, and MRL / lpr + CTX represents the lupus model mouse group treated with the first-line clinical drug cyclophosphamide (CTX). Detailed implementation mode

[0020] The present invention provides the application of GppNHp in the preparation of drugs for treating systemic lupus erythematosus.

[0021] In the present invention, GppNHp is dissolved in phosphate buffer, and sterile filtration is carried out to obtain a GppNHp injection solution with a concentration of 5 - 15 μg / μL. When the GppNHp injection solution is used for mice, it is injected at a dosage of 0.5 - 1.5 μL / g of mouse body weight and a frequency of 1 - 5 days / time, and continuously used for 8 - 15 times; the concentration of the GppNHp injection solution is preferably 8 - 12 μg / μL, more preferably 10 μg / μL, the concentration of the phosphate buffer is 0.001 - 0.02 M, preferably 0.005 - 0.015 M, more preferably 0.01 M, the pH of the phosphate buffer is 7.0 - 7.6, preferably 7.1 - 7.5, more preferably 7.2 - 7.4, the sterile filtration uses a filter membrane with a pore size of 0.22 μm, the dosage of GppNHp is preferably 0.8 - 1.2 μL / g of mouse body weight, more preferably 1 μL / g of mouse body weight, the frequency of use is preferably 2 - 4 days / time, more preferably 3 days / time, and the number of consecutive uses is preferably 9 - 12 times, more preferably 10 consecutive times.

[0022] The present invention also provides a medicament for treating systemic lupus erythematosus, and the medicament comprises GppNHp and its pharmaceutically acceptable carrier.

[0023] In the present invention, the medicament is preferably an injection containing GppNHp.

[0024] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they cannot be construed as limiting the protection scope of the present invention.

[0025] Embodiment

[0026] 1. Experimental materials and preparations

[0027] 1) Experimental animals: Male MRL / lpr mice aged 14 - 16 weeks (purchased from Cyagen Biosciences Inc.) were selected. Such mice are widely used in disease models due to their autoimmune characteristics similar to those of human systemic lupus erythematosus.

[0028] 2) Drug preparation: GppNHp (purchased from MCE, catalog number HY - 137167) was dissolved in phosphate - buffered saline (PBS, 0.01 M, pH 7.2 - 7.4) at a concentration of 10 μg / μL. All solutions were sterile - filtered through a 0.22 - μm filter membrane before use.

[0029] 2. Experimental design

[0030] 1) Grouping and treatment: The MRL / lpr mice were randomly divided into four groups. Among them, the treatment group was intraperitoneally injected with the necroptosis inhibitor GppNHp solution (MRL / lpr + GppNHp), and the control groups were respectively injected with an equal volume of DMSO (MRL / lpr + DMSO) and CTX (MRL / lpr + CTX, CTX was purchased from Sigma). Each injection was performed every 3 days, with a dose of 1 μL / g body weight each time, for a total of 10 times. Male MRL / Mpj mice aged 14 - 16 weeks (purchased from Cyagen Biosciences Inc.) were selected as the healthy control group without any treatment.

[0031] 2) Monitoring of physiological indicators: During the injection process, the body weight, food and water intake of the mice were monitored daily to evaluate the potential toxicity of the drug and its impact on the overall health of the animals.

[0032] 3. Collection and analysis of biological samples

[0033] 1) Urine and blood samples: On the third day after the last injection in the experiment, urine and venous blood of the mice were collected. The urine was stored at -80 °C for subsequent analysis of urine protein concentration. The blood was added to a heparin-containing tube, left to stand at 25 °C for 30 min, then centrifuged at 3000 rpm at 4 °C for 15 min, and the supernatant was collected for biochemical analysis.

[0034] 2) Tissue samples: After blood was collected by enucleating the eyeballs, the mice were sacrificed, and the spleen, lymph nodes, kidneys and other related organs were collected for measurement of weight and volume. The results are shown in Figure 1 . Each organ was fixed in 4% paraformaldehyde and then subjected to HE staining to evaluate the pathological changes of the tissue. The results are shown in Figure 2 .

[0035] 4. Biomarker analysis

[0036] 1) Immunoassay: ELISA kits were used to measure the levels of TNF-α, complement C3 and anti-double-stranded DNA (dsDNA) antibodies in the serum. The results are shown in Figure 3 .

[0037] Renal function test: Serum creatinine and urine protein concentration were measured to evaluate the degree of kidney injury. The results are shown in Figure 4 .

[0038] 2) Flow cytometry analysis: Miltenyi Biotec kits (purchased from Miltenyi Biotec GmbH, Germany) were used to isolate peripheral blood neutrophils and detect the viability of the cells. The results are shown in Figure 5 .

[0039] 5. Data processing and statistical analysis

[0040] Statistical method: Graphpad Prism 8.0 software was used for statistical analysis. Measurement data were expressed as . The t-test was used for comparison between two groups. P < 0.05 indicates a statistically significant difference between the two.

[0041] 6. Experimental results and evaluation

[0042] As can be seen from Figure 1 , compared with the MRL / lpr + DMSO control group, after treatment with GppNHp, the volumes of the spleen and axillary lymph nodes of the mice were significantly smaller and the weights were significantly lighter, indicating that the use of GppNHp can significantly reduce the volumes and weights of the spleen and axillary lymph nodes. As can be seen from Figure 2It can be seen that the normal mouse group (MRL / Mpj) represents a healthy control: the structure of the glomerulus is relatively complete, the cells are arranged closely, and no obvious pathological features are observed. In the classical lupus model mouse group (MRL / lpr + DMSO), which was untreated and only used DMSO as a control, the structure of the glomerulus was significantly abnormal, with thickening of the glomerular basement membrane, disordered cell arrangement, and inflammatory cell infiltration, indicating severe pathological damage in the local kidney. In the mouse group treated with the necroptosis inhibitor GppNHp (MRL / lpr + GppNHp), compared with the untreated MRL / lpr + DMSO group, the structure of the glomerulus was improved, the thickening of the basement membrane was alleviated, the cells were arranged more closely, and the inflammatory infiltration was reduced, indicating that GppNHp treatment had a certain improvement effect on kidney pathological damage. CTX (cyclophosphamide) is a commonly used immunosuppressant for treating SLE patients clinically. The mouse group treated with CTX (MRL / lpr + CTX) was used as a positive control. Compared with the MRL / lpr + DMSO group, the pathological damage of the glomerulus was significantly reduced, and the tissue structure was improved close to normal, but there were still a small number of inflammatory cell infiltrations. From Figures 3 to 5 It can be seen that compared with the MRL / lpr + DMSO control group, after treatment with GppNHp, the urinary protein concentration and serum creatinine concentration decreased significantly, indicating that GppNHp can slow down kidney damage in lupus mice; after treatment with GppNHp, the concentrations of anti-dsDNA antibody and tumor necrosis factor TNF-α decreased, and the concentration of complement C3 increased. It can be seen that the necroptosis inhibitor GppNHp can correct the abnormal autoimmune response in lupus mice by regulating the activity of immune cells, thus significantly alleviating the lupus phenotype in mice, which provides an important theoretical and practical basis for developing new strategies for treating systemic lupus erythematosus and has broad application prospects.

[0043] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of GppNHp in the preparation of drugs for treating systemic lupus erythematosus.

2. The application according to claim 1, wherein The concentration of GppNHp is 5 - 15 μg / μL.

3. The application according to claim 2, characterized in that, The solvent of GppNHp is phosphate buffer solution.

4. The application according to claim 3, characterized in that The administration method of GppNHp is injection.

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