Use of small molecule drug indisulam in the preparation of a drug for treating autoimmune diseases

Abstract

The application discloses application of a small-molecule drug indisulam in preparation of a drug for treating autoimmune diseases, and further verifies safety of the molecule, long-term continuous use of the drug does not cause weight loss of animals, and weight change has no difference with normal control mice; tissue sections of multiple organs show that the drug has no obvious toxic side effects on each organ. In addition, the drug can promote differentiation of regulatory T cells, but does not affect functions of other immune cells. Therefore, the drug is used for treating autoimmune diseases, and good effect is found.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to the application of small molecule drug indisulam in the preparation of a drug for treating autoimmune diseases. BACKGROUND

[0002] The formation of tumors is related to the immune system, and the function of immune cells is inhibited or exhausted, which is the key reason for tumor occurrence, escape, metastasis and recurrence. On the contrary, autoimmune diseases are caused by the hyperfunction or ineffective inhibition of immune cells in the body, leading to the attack of normal tissue cells by immune cells, and patients often need to take immunosuppressive drugs for life, which can inhibit the function of normal immune cells, thereby greatly increasing the risk of infection and tumor in patients. In the human body, there is a group of naturally occurring immune regulatory T cells that can timely and moderately inhibit the function of other immune cells, thereby maintaining the immune homeostasis of the body, maintaining the ability to eliminate external pathogenic microorganisms and tumor cells, and not causing autoimmune diseases.

[0003] Small molecule drugs have the advantages of wide use and mature theory. According to statistics, the number of small molecule drugs can account for 98% of the total in commonly used drugs. The structure of small molecule drugs has good spatial dispersion, and its chemical properties determine its good drug performance and pharmacokinetic properties. These characteristics make small molecule drugs have great advantages in drug research and development and other drug fields. Small molecule drugs are usually signal transduction inhibitors that can specifically block the signal transduction pathways necessary for tumor growth and proliferation, thereby achieving the purpose of treatment. In simple terms, small molecule drugs, as traditional drugs, can be taken orally and can act on intracellular targets, while large molecule drugs generally act on cell surface targets and inhibit protein interactions, with strong specificity, but generally cannot be taken orally and are difficult to enter cells. Small molecule drug indisulam has good tumor inhibition effect in preclinical research, and has passed the second phase of clinical research, but has no anti-tumor effect in the third phase of clinical trials. Whether small molecule drug indisulam is used for the treatment of other diseases has not been reported. Therefore, it is urgent to develop other therapeutic uses of small molecule drug indisulam. SUMMARY

[0004] Therefore, one of the purposes of the present application is to provide the application of small molecule drug indisulam in the preparation of a drug for treating autoimmune diseases.

[0005] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0006] 1. The application of small molecule drug indisulam in the preparation of a drug for treating autoimmune diseases.

[0007] Preferably, the autoimmune disease is autoimmune encephalomyelitis.

[0008] Preferably, the small molecule drug indisulam is used for preparing a medicament for increasing the proportion of regulatory T cells in autoimmune diseases.

[0009] Preferably, the small molecule drug indisulam is used for preparing a medicament for improving the spinal cord tissue lesions in autoimmune disease patients.

[0010] The present application discloses the use of a small molecule drug indisulam in the preparation of a medicament for treating autoimmune diseases. The safety of the molecule is further verified, and long-term continuous use of the drug does not cause weight loss in animals, and the weight change is not different from that of normal control mice. The tissue sections of multiple organs show that the drug has no obvious toxic and side effects on each organ. In addition, the drug can promote the differentiation of regulatory T cells. Therefore, we use the drug to treat autoimmune diseases, and find good effects. BRIEF DESCRIPTION OF DRAWINGS

[0011] In order to make the purpose, technical scheme and beneficial effects of the present application clearer, the present application provides the following drawings for illustration:

[0012] Figure 1 The weight change of the control group and the experimental group on the first day of injection and 29 days after injection;

[0013] Figure 2 The effect of the small molecule drug indisulam on mouse organs;

[0014] Figure 3 The proportion of regulatory T cells (Treg) in CD4 T cells in the spleen (SP) and lymph nodes (LN) of the Indisulam treatment group of mice;

[0015] Figure 4 The autoimmune encephalomyelitis score of the HPCD injection group and the Indisulam treatment group of mice, and the H&E staining results of the spinal cord tissue of the two groups of mice.

[0016] Figure 5 The proportion of regulatory T cells in the spleen of autoimmune encephalomyelitis mice after HPCD injection and Indisulam treatment. DETAILED DESCRIPTION

[0017] The present application will be further described below in conjunction with the drawings and specific examples, so that those skilled in the art can better understand the present application and implement it. The examples are not limiting to the present application.

[0018] The small molecule drug indisulam was purchased from Shanghai Hainixing Biotech Co., Ltd. CAS: 165668-41-7.

[0019] Example 1, Establishment of the model of autoimmune encephalomyelitis (EAE)

[0020] Preparation of each group: 30 μΐ of 10 mg / ml myelin oligodendrocyte glycoprotein (MOG) + 100 μΐ of 1 x PBS + 100 μΐ of 5 mg / ml complete Freund's adjuvant (CFA) per mouse.

[0021] Emulsion configuration: connect two threaded syringes in a three-way valve, pull out the push rod of one of them, add PBS, MOG and CFA in turn, install the syringes, push the syringes through the switch three-way valve to remove the gas, push back and forth on the ice until completely emulsified, then transfer to a syringe, and store on ice (add a drop of emulsion to a beaker, the droplet will be stably maintained as a lump, slowly dissipate, indicating that the emulsification is complete).

[0022] Mouse immunization: anesthetize the mouse with isoflurane, and subcutaneously inject 100 μΐ of emulsion into the mouse back at the waist with a 2 ml syringe. Then use an insulin syringe to inject 100 μΐ of pertussis toxin (PTX: 2 μΐ of 100 mg / ml PTX + 98 μΐ of 1 x PBS) into the mouse's eye vein, and repeat the injection after 48 h.

[0023] Weigh and score: from the day of immunization, weigh the mice every other day, and weigh and score every day after the onset of symptoms (0 points for normal, 1 point for tail weakness, 2 points for limb weakness, 3 points for limb paralysis, 4 points for front limb paralysis, 5 points for death, and 0.5 points for intermediate state).

[0024] Standard for successful modeling: the mice begin to develop symptoms 7-14 days after sensitization, showing symptoms such as uncoordinated movement, limb paralysis, and other clinical manifestations of multiple sclerosis. The disease level standard is divided into 5 levels. Level 1: animal tail weakness; Level 2: tail weakness plus limb weakness; Level 3: mild limb paralysis; Level 4: severe limb paralysis, unable to recover after being turned over passively; Level 5: moribund state.

[0025] Example 2, Effect of small molecule drug indisulam on normal mouse weight or organs

[0026] Cyclodextrin configuration: dissolve hydroxypropyl β-cyclodextrin (pharmaceutical grade) in sterile normal saline at 0.2 g / ml.

[0027] Indisulam configuration: dilute Indisulam (100 mg / ml) with the above HPCD at a ratio of 1:19.

[0028] Injection: Take normal mice each other day intraperitoneal injection of 100 μl hydroxypropyl β-cyclodextrin (HPCD) (control group) or Indisulam (experimental group), four weeks in a row, before injection (day 1) and four weeks after injection (day 29) for each mouse weighing and statistics, results as shown in Figure 1 The results show that there is no significant difference in body weight and the ratio of body weight to the first day between the experimental group and the control group at 29 days after injection.

[0029] Then take the control group and the experimental group treated heart, liver, spleen, lung, kidney, large intestine and small intestine for H&E staining, H&E staining commissioned biological company, including dehydration, pruning, embedding, sectioning, staining, mounting, etc., finally microscopy, results as shown in Figure 2 The results show that there is no obvious difference between the experimental group and the control group, indicating that the drug has no obvious toxic side effects on each organ.

[0030] Example 3, the influence of small molecule drug indisulam on autoimmune diseases

[0031] After the modeling is successful, the drug is injected in the manner of example 2, and part of the brain and lumbar spinal cord is taken for H&E staining after 29 days. The remaining brain and spinal cord tissue is subjected to lymphocyte separation, and flow cytometry is used to analyze the cell condition.

[0032] The specific steps of flow cytometry (FACS) are as follows:

[0033] 1. Cell harvesting:

[0034] ① Lymph node cell acquisition: the mouse is killed by breaking the neck and placed on a foam board covered with 70% alcohol disinfection paper towels, the mouse skin is cut open, and two lymph nodes are taken from each of the axillary, axillary and inguinal region, ground and filtered in 1x PBS buffer, centrifuged (500g, 5min) and resuspended with 1x PBS buffer.

[0035] ② Spleen cell acquisition: take the spleen, the steps are the same as above, after grinding and filtering, centrifuge (500g, 5min), resuspend with 1ml ACK Lysing Buffer, lyse red blood cells at room temperature for 5min, centrifuge (500g, 5min) and resuspend with 1x PBS buffer.

[0036] ③ Brain and spinal cord cell acquisition: take the brain and spinal cord, the steps are the same as above, grind and filter the brain and spinal cord after cutting them into small pieces, centrifuge (500g, 5min) and resuspend with 50% Percoll, slowly add to the upper layer of 70% Percoll, centrifuge (18℃, 500g, 30min, slow rise and slow fall) and then take the middle layer, centrifuge (500g, 5min) and resuspend with 1x PBS buffer.

[0037] 2. Cell death staining: centrifuge the cell suspension (500g, 5min), discard the supernatant, resuspend each sample with 100 μl of dead-live staining solution (zombie yellow: 1xPBS buffer 1:1000), incubate at room temperature for 15min in the dark, centrifuge (500g, 5min) and resuspend with 1xPBS buffer.

[0038] 3. Cell surface staining: prepare the surface antibody premix solution according to the surface antibody: FACS Buffer (0.5% FBS-PBS) 1:400, centrifuge the suspension (500g, 5min), discard the supernatant, resuspend each sample with 50 μl of the surface antibody premix solution, incubate on ice for 30min in the dark, centrifuge (500g, 5min) and resuspend with FACS Buffer.

[0039] 4. Cytokine / transcription factor staining: centrifuge the cell suspension (500g, 5min), discard the supernatant, resuspend each sample with 50 μl of the corresponding cytokine / transcription factor fixation solution, incubate on ice for 30min in the dark, centrifuge (10000g, 1min), resuspend each sample with 200 μl of the corresponding Wash Buffer, centrifuge (10000g, 1min), resuspend each sample with 50 μl of the cytokine / transcription factor staining solution (cytokine antibody: cytokine Wash Buffer 1:200, transcription factor antibody: transcription factor Wash Buffer 1:100), incubate on ice for 30min in the dark, centrifuge (10000g, 1min), and resuspend each sample with 200 μl of the corresponding Wash Buffer. (If both cytokine and transcription factor are stained, use the transcription factor Kit separately)

[0040] 5. Sample collection: centrifuge the stained suspension (500g, 5min for unfixed, 10000g, 1min for fixed), discard the supernatant, resuspend with 200-500 μl of FACS Buffer, filter through a nylon mesh into a flow cytometry sample tube, and perform flow cytometry fluorescence detection using a flow cytometer.

[0041] The results are shown in Figure 3 The results show that the proportion of regulatory T cells (Treg) in CD4 T cells in the spleen (SP) and lymph nodes (LN) of Indisulam injection group mice is significantly increased.

[0042] H&E staining was entrusted to a biological company, and the mouse spinal cord was taken for histological section staining: including dehydration, trimming, embedding, sectioning, staining, and mounting, and finally microscopic examination. The results are shown in Figure 4The results show that the autoimmune encephalomyelitis (EAE) mice were injected with cyclodextrin and Indisulam every other day, and it was found that Indisulam can significantly reduce the occurrence of disease in model mice, and significantly improve the weight loss of mice, and the pathological changes of spinal cord tissue are also significantly better than those of the HPCD group.

[0043] Through flow cytometry analysis, it was found that compared with the HPCD treatment group, the proportion of regulatory T cells in the spleen of autoimmune encephalomyelitis mice treated with Indisulam was significantly increased.

[0044] The above results show that Indisulam can significantly increase the proportion of regulatory T cells in the spleen of autoimmune encephalomyelitis mice, thereby reducing the occurrence of disease in model mice, and significantly improving the weight loss of mice, and the pathological changes of spinal cord tissue are also significantly better than those of the HPCD group.

[0045] After the successful modeling of autoimmune encephalomyelitis mice was injected with HPCD and treated with Indisulam, the proportion of regulatory T cells in the spleen was detected, and the results are shown in Figure 5 The results show that the proportion of regulatory T cells in the spleen of the Indisulam treatment group is higher.

[0046] The above-described examples are only preferred embodiments for fully illustrating the present application, and the protection scope of the present application is not limited thereto. Any equivalent replacement or transformation made by those skilled in the art on the basis of the present application is within the protection scope of the present application. The protection scope of the present application is subject to the claims.

Claims

1. Use of the small molecule drug indisulam for the preparation of a medicament for the treatment of an autoimmune disease, characterized in that: The autoimmune disease is autoimmune encephalomyelitis.

2. Use of the small molecule drug indisulam for the preparation of a medicament for increasing the proportion of regulatory T cells in autoimmune diseases, characterized in that: The autoimmune disease is autoimmune encephalomyelitis. The autoimmune disease is autoimmune encephalomyelitis.

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