Application of combination of glutathione and PD-1 monoclonal antibody teralizard in relieving toxic and side effects of teralizard on immune system

Abstract

The invention belongs to the technical field of medicines, and more specifically relates to a novel method for relieving toxic and side effects of PD-1 monoclonal antibody teracil. Based on preclinical research, the invention finds that glutathione can recover inhibition on immune cells caused by tiralizumab; the combination of glutathione and tiralizumab relieves the toxic and side effects of tiralizumab to immune cells when tiralizumab is independently used. Experimental data show that compared with independent use of the tiralizumab, the combined use of the glutathione and the tiralizumab weakens the inhibition of the tiralizumab on immune cells, so that the cell activity and the cell factor activity are recovered. The invention provides a new therapeutic scheme and thought for PD-1 monoclonal antibody teraleib immunotherapy of tumor patients, is also beneficial to research of adverse drug reaction mechanisms, provides a method for research of drug safety, and has important clinical experimental research potential value.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and more specifically, relates to the application of glutathione and PD-1 monoclonal antibody tislelizumab in alleviating the toxic and side effects of tislelizumab on the immune system. Background Art

[0002] Cancer is a major problem in today's medical field. There are various treatments for different cancers, such as surgery, chemotherapy, radiotherapy, targeted therapy, immunotherapy, etc. At present, immunotherapy is developing more and more rapidly, and tumor immunotherapy is the most promising research direction in the field of tumor treatment.

[0003] Tislelizumab is a humanized anti-PD-1 monoclonal antibody that works by blocking the interaction between PD-1 (programmed death receptor-1) and PD-L1 (programmed death ligand-1). Under normal circumstances, PD-1 is expressed on T cells, while PD-L1 is expressed on certain types of immunosuppressive cells, such as tumor cells and certain immunosuppressive cells. This interaction inhibits the activity of T cells, allowing tumor cells to escape the surveillance and attack of the immune system. By blocking this interaction, tislelizumab can restore the activity of T cells and activate T cells to attack tumor cells. Tislelizumab has been approved in China for the treatment of the following cancers: 1. Relapsed or refractory classical Hodgkin's lymphoma after at least two lines of systemic chemotherapy 2. Advanced or metastatic urothelial carcinoma, including patients who have failed platinum-containing chemotherapy 3. First-line advanced squamous non-small cell lung cancer (NSCLC) patients, combined with chemotherapy 4. Advanced hepatocellular carcinoma, first-line monotherapy or combined with lenvatinib.

[0004] The adverse reactions of tislelizumab mainly include immune-related adverse reactions and drug-related adverse reactions. Immune-related adverse reactions involve multiple organ systems, such as skin, intestines, liver, endocrine system, etc., and manifest as symptoms such as rash, diarrhea, elevated liver enzymes, and abnormal thyroid function. Drug-related adverse reactions mainly include injection site reactions, fatigue, headache, etc. In clinical practice, timely identification and treatment of these adverse reactions are crucial to ensure the safety of treatment.

[0005] The mechanism of adverse reactions of tislelizumab is not fully understood, but it may be related to affecting the immune system. The drug enters the human body through intravenous injection and is transported through blood vessels to the location of the tumor. During transportation, the drug will come into contact with immune cells in the blood vessels, and after reaching the tumor, it will come into contact with immune cells in the tumor microenvironment. After the drug and immune cells come into contact, a reaction will occur. This reaction may affect the function of immune cells, thereby causing some adverse drug reactions. For example, if immune cells are suppressed, it may cause adverse reactions such as increased risk of infection, chronic inflammation, loss of immune memory, allergic reactions, and immune-related diseases.

[0006] Therefore, in-depth research on the effects and mechanisms of tilelizumab on immune cells, and thus finding means to reduce or alleviate adverse drug reactions, can provide new ideas and new evidence for the study of adverse drug reactions of tilelizumab, improve drug safety, and improve the prognosis of patients taking the drug, which has great social significance.

[0007] Glutathione (glutathione, r-glutamyl cysteingl + glycine, GSH) is a tripeptide containing γ-amide bonds and sulfhydryl groups. It is composed of glutamic acid, cysteine ​​and glycine and exists in almost every cell of the body. Glutathione can help maintain normal immune system function and has antioxidant and integrated detoxification effects. The sulfhydryl group on cysteine ​​is its active group (so it is often abbreviated as G-SH), which is easy to combine with certain drugs, toxins, etc., giving it an integrated detoxification effect. Glutathione can not only be used in medicine, but also as a base material for functional foods. It is widely used in functional foods such as delaying aging, enhancing immunity, and anti-tumor. Summary of the invention

[0008] In view of the above existing technical problems, the primary purpose of the present invention is to provide the application of glutathione and PD-1 monoclonal antibody tislelizumab in alleviating the toxic and side effects of tislelizumab on the immune system. The present invention uses glutathione and tislelizumab in combination to alleviate the inhibitory effect of tislelizumab on immune cells of the central immune system and immune cells of the peripheral immune system, and alleviates the inhibition of tislelizumab on the activity of MMP-9 in central and peripheral immune cells.

[0009] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0010] The present invention provides the use of glutathione and PD-1 monoclonal antibody tislelizumab in combination for alleviating the toxic and side effects of tislelizumab on the immune system.

[0011] Preferably, the immune system is bone marrow cells of the central immune system and spleen lymphocytes of the peripheral immune system.

[0012] Preferably, the combined concentration of glutathione is 10 mM, and the combined concentration of tislelizumab is 10 mg / mL.

[0013] Preferably, the toxic side effects of tislelizumab on the immune system are inhibition of the cell viability of immune cells and inhibition of the MMP-9 activity of immune cells.

[0014] Preferably, the glutathione combined with tislelizumab can restore the cell viability of suppressed immune cells and the MMP-9 activity of suppressed immune cells.

[0015] Preferably, based on the preclinical studies of the present invention on the combined use of glutathione and tislelizumab, a new method is provided for the clinical use of tislelizumab, which can alleviate the toxic and side effects of tislelizumab on immune cells.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The advantages of using glutathione in combination with tislelizumab in the present invention are multifaceted. By combining glutathione, the adverse reactions caused by the influence on the immune system during the treatment of tumors with tislelizumab can be significantly reduced, thereby improving the quality of life of patients. Experimental data show that compared with the use of tislelizumab alone, the combined use of glutathione improves the function of the patient's immune cells and restores the affected cytokines. The present invention provides a new treatment plan for tumor patients, helps to study the mechanism of adverse drug reactions, reduces drug side effects, and has important clinical experimental research value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a bar graph of spleen lymphocyte viability after intervention with tislelizumab alone and in combination with glutathione

[0019] Figure 2 This is a bar graph of bone marrow cell viability after intervention with tislelizumab alone and in combination with glutathione

[0020] Figure 3 This is the change of MMP-9 activity in spleen lymphocytes after intervention with tislelizumab alone and in combination with glutathione

[0021] Figure 4 This is the change of MMP-9 activity in bone marrow cells after intervention with tislelizumab alone and in combination with glutathione DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0023] Example 1 Intervention of tislelizumab alone and in combination with glutathione on changes in immune cell viability

[0024] The spleen, tibia, and femur were removed from SD rats and placed in a tube containing 4% double antibody in PBS.

[0025] Under sterile conditions, the spleen was gently ground into a single cell suspension through a 200-mesh cell sieve; the ends of the bones were cut off, and the bone marrow was flushed out of the bone marrow cavity with PBS containing 4% double antibody through a sterile syringe, and then filtered through a 200-mesh cell sieve.

[0026] The cells were centrifuged at 1500 rpm for 5 minutes, and the erythrocytes in the spleen and bone marrow cell suspensions were treated with Tris-NH4Cl erythrocyte lysis buffer.

[0027] The cell pellet was washed with PBS and cultured overnight in RPMI-1640 medium containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin to remove adherent cells and obtain purer lymphocytes.

[0028] The cell suspension cultured overnight was centrifuged, and then SF was added to resuspend and mix well before cell counting.

[0029] Splenic lymphocytes and bone marrow cells were divided into five groups: untreated group (Untreated), low-concentration tislelizumab group (1 mg / mL), clinical intravenous concentration tislelizumab group (10 mg / mL), low-concentration tislelizumab group and glutathione (10 mM) group (1 mg / mL+GSH), clinical intravenous concentration tislelizumab group and glutathione (10 mM) group (10 mg / mL+GSH). Three replicate wells were set for each group, and the cell concentration in each well was set to 2x10 5 The cells were placed in a 37°C, 5% CO2 cell culture incubator for 30 minutes.

[0030] Tislelizumab, with national medicine standard S20190045, was purchased from BeiGene (Shanghai) Biotechnology Co., Ltd. Tislelizumab was taken out from 4°C and prepared with PBS to prepare a low concentration group. Glutathione was purchased from Aladdin, and the powder was dissolved in PBS to prepare a 10mM drug solution. Under normal circumstances, the range of glutathione supplementation for humans is 1-10mM.

[0031] The cells were taken out of the incubator and drug solutions were added in order according to the groups. After adding the drugs, the cells were placed in a 37°C, 5% CO2 cell incubator for 20 hours.

[0032] After 20 h, the cells were taken out, centrifuged and the supernatant was taken out and saved for gelatin zymogram analysis.

[0033] Add 50 μL of 10% MTT solution to each well and place in a 37°C, 5% CO2 cell culture incubator for 4 hours. Take out after 4 hours, centrifuge and discard the supernatant. Add 100 μL of DMSO to each well and blow evenly, and measure its light absorption (OD) value at a wavelength of 570 nm using an enzyme-linked immunosorbent assay.

[0034] The MTT method is a method for detecting cell survival and growth. The detection principle is that the succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple crystalline formazan and deposit it in the cells, while dead cells do not have this function. However, in the presence of reducing active substances, the formation of formazan can be increased and the optical density (OD) value can be increased. Dimethyl sulfoxide (DMSO) can dissolve the formazan in cells, and its light absorption value can be measured at a wavelength of 570nm using an enzyme-linked immunosorbent assay, which can indirectly reflect cell viability.

[0035] Experimental results: Figure 1 , 2 As shown in the figure, compared with unintervention cells, when tislelizumab intervened in immune cells alone, it significantly inhibited the cell viability of spleen lymphocytes and bone marrow cells, and it was concentration-dependent. After being used in combination with glutathione, this inhibitory effect on cell viability was alleviated, and compared with tislelizumab alone, the cell viability of immune cells was significantly improved. This shows that glutathione can alleviate the inhibitory effect of tislelizumab on immune cells and enhance the cell viability of suppressed immune cells.

[0036] Example 2: Changes in MMP-9 activity in immune cells detected by intervention with tislelizumab alone or in combination with glutathione

[0037] The activity of matrix metalloproteinase MMP-9 was determined by gelatin zymography. MMP-9 is one of the markers of lymphocyte activation. The supernatant of spleen lymphocytes and bone marrow cells after intervention was mixed with an equal volume of sample buffer and loaded, 20 μL per well. The gel was run at 100V for vertical electrophoresis. The gel was eluted twice with elution buffer for 1 hour each time, and incubated overnight in active incubation buffer. The gel was stained with Coomassie Brilliant Blue R-250, eluted until white bands appeared on the background, and photographed with the Bio-Rad gel imaging system.

[0038] Experimental results: Figure 3 As shown in the figure, compared with untreated cells, tislelizumab alone inhibited the activity of MMP-9 in spleen lymphocytes, and combined with glutathione significantly increased the activity of MMP-9 in the suppressed spleen lymphocytes. Figure 4As shown in the figure, compared with unintervention cells, the use of tislelizumab alone slightly inhibited the activity of MMP-9 in bone marrow cells and changed its morphology at the original concentration. The combination with glutathione increased the activity of MMP-9 in spleen lymphocytes that was inhibited and restored the morphology of the affected bands to normal. This shows that the combined use of glutathione can restore the activity and morphology of MMP-9 in immune cells affected by tislelizumab to normal.

[0039] The above results show that when tislelizumab is used alone, it has an inhibitory effect on the cell viability and cellular MMP-9 activity of immune cells in the central and peripheral immune systems. The combined use of tislelizumab and glutathione can alleviate the above inhibitory effects to a certain extent and restore the inhibited and affected cell viability and cellular MMP-9 activity.

[0040] The advantages of using glutathione in combination with tislelizumab in the present invention are multifaceted. By combining glutathione, the adverse reactions caused by the influence of immune cells when tislelizumab is used to treat tumors can be significantly reduced, and the quality of life of patients can be improved. Experimental data show that compared with the use of tislelizumab alone, the combined use of glutathione improves the function of patients' immune cells, and the affected cell viability and cytokines are restored. The present invention provides a new treatment plan for tumor patients, helps to study the mechanism of adverse drug reactions, reduces drug side effects, and has important clinical experimental research value.

Claims

1. The application of glutathione and PD-1 monoclonal antibody tislelizumab in alleviating the toxic and side effects of tislelizumab on the immune system.

2. The application according to claim 1, characterized in that: The immune cells are bone marrow cells of the central immune system and spleen lymphocytes of the peripheral immune system.

3. The application according to claim 1, characterized in that: The combined concentration of glutathione is 10 mM, and the combined concentration of tislelizumab is 1 mg / ml and 10 mg / ml.

4. The use according to claim 1, characterized in that: The toxic side effects of tislelizumab on immune cells include inhibiting the cell viability of immune cells and inhibiting the MMP-9 activity of immune cells.

5. The use according to claim 1, characterized in that: The glutathione combined with tislelizumab can restore the cell viability and cell MMP-9 activity of suppressed immune cells.

6. The use according to claim 1, characterized in that: The combination of glutathione and tislelizumab provides a new method for studying adverse drug reactions in clinical practice, and can alleviate the toxic and side effects of tislelizumab on immune cells.

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