Application of glutathione in relieving toxic and side effects of bevacizumab immune system

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a novel method for relieving toxic and side effects of bevacizumab. Based on preclinical research, the combination of glutathione and bevacizumab relieves toxic and side effects on immune cells when bevacizumab is independently used. Experimental data comparison shows that compared with bevacizumab single drug treatment, the combined glutathione can significantly reduce the inhibition effect on immune cell activity, so that the cell proliferation activity is repaired, and meanwhile, the disordered cell cycle and the abnormally expressed cell factor level tend to be normalized. The invention provides a novel treatment scheme and thought for targeted treatment of tumor patients with the VEGF monoclonal antibody bevacizumab, is also beneficial to research on the toxic action mechanism of drugs and optimization of the treatment scheme, and has important potential clinical experimental research value.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and more specifically, relates to the application of glutathione in alleviating the immune system side effects of bevacizumab. Background Art

[0002] Targeted therapy is a cancer treatment method that aims to treat tumors by interfering with the growth, division, and spread of cancer cells. Targeted therapy is sometimes also referred to as "molecular targeted therapy", "molecular targeted treatment", "precision medicine", or similar names. Currently, targeted therapy is a focus in the development of many anti-cancer drugs. It is the cornerstone of precision medicine and a form of medical treatment that uses human gene and protein information to prevent, diagnose, and treat diseases.

[0003] Bevacizumab is a targeted therapy drug and is classified as an "anti-angiogenic drug". It inhibits tumor growth by blocking the blood supply to the tumor, rather than directly attacking cancer cells like traditional chemotherapy drugs. Specifically, bevacizumab is a recombinant humanized monoclonal antibody that can specifically bind to vascular endothelial growth factor (VEGF) and prevent the interaction between VEGF and its receptor. Vascular endothelial growth factor (VEGF) is a protein that promotes new blood vessel formation. Cancer cells secrete VEGF to stimulate their own blood supply, thereby obtaining nutrients and rapidly proliferating. Bevacizumab inhibits the growth of tumors by blocking the action of VEGF and cutting off the blood supply to the tumor. This unique mechanism has achieved remarkable efficacy in the treatment of various cancers. Bevacizumab is usually used in combination with chemotherapy drugs and is widely used in the treatment of various cancers, including but not limited to lung cancer, breast cancer, colorectal cancer, gastric cancer, etc.

[0004] In clinical use, bevacizumab may cause various adverse reactions. The types of these adverse reactions are diverse and the manifestations are complex, including but not limited to: injection site reactions, skin reactions, respiratory system reactions, nervous system reactions, cardiovascular system reactions, liver reactions, kidney reactions, and immune system reactions, etc. These adverse reactions may, in mild cases, affect the quality of life of patients, and in severe cases, may lead to treatment interruption or even endanger life. The specific mechanism of bevacizumab-induced adverse reactions is not fully understood, and current research mainly focuses on the following aspects: 1. Since bevacizumab is an antibody, it may cause immune-related reactions. 2. Bevacizumab inhibits angiogenesis and may affect normal physiological functions. 3. Bevacizumab may cause adverse reactions by affecting the VEGF-related signaling pathway.

[0005] The mechanism of action of bevacizumab adverse reactions has not been fully elucidated. Existing studies suggest that its immunomodulatory effects may be involved in the formation process of drug adverse reactions. As an anti-angiogenic drug, bevacizumab diffuses into the tumor microenvironment through the systemic circulation after intravenous administration. During drug delivery, immune cells in the peripheral circulation and the targeted area will come into contact with it. This ligand-receptor interaction may lead to abnormal immune cell function through signal pathway interference, thereby triggering a series of clinical reactions. Specifically, the immunosuppressive state may induce immune-related complications such as infections, persistent inflammatory responses, immune memory dysfunction, and hypersensitivity reactions.

[0006] In-depth exploration of the regulatory mechanism of bevacizumab on the immune system has dual value: on the one hand, it can reveal the principle of drug side effects and provide a theoretical basis for the development of targeted protective measures; on the other hand, by optimizing the treatment plan, the drug safety can be effectively improved, and ultimately help patients obtain better treatment effects, which is of great significance for improving the clinical practice of tumor treatment.

[0007] Glutathione (glutathione, r-glutamyl cysteingl + glycine, GSH) is a tripeptide compound formed by glutamic acid, cysteine, and glycine connected by γ-amide bonds, and its active sulfhydryl group (-SH) endows its core function. As a biomolecule widely present in human cells, GSH neutralizes free radicals through the antioxidant defense mechanism, uses the sulfhydryl group to bind to toxins to achieve the detoxification function, and cooperates to maintain the balance of the immune system. This substance is not only used as a clinical detoxification drug, but also plays an important role in the development of functional foods, including the research and application of anti-aging preparations, immune-enhancing products, and tumor adjuvant treatment products. Summary of the Invention

[0008] Aiming at the above existing technical problems, the purpose of the present invention is to provide the application of glutathione in alleviating the immune system toxic and side effects of bevacizumab. The present invention uses the combination of glutathione and bevacizumab to alleviate the inhibitory effect of bevacizumab on the proliferation activity of immune cells in the central immune system and the peripheral immune system, and restores the normal cell cycle of immune cells and the activity of matrix metalloproteinase-9 (MMP-9) in immune cells to normal.

[0009] In order to achieve the above purpose, the present invention is realized through the following technical solutions:

[0010] The present invention provides the application of glutathione in alleviating the immune system toxic and side effects of bevacizumab.

[0011] Preferably, the immune system includes bone marrow cells in the central immune system and splenic lymphocytes in the peripheral immune system.

[0012] Preferably, the combined concentration of bevacizumab is 2.5 mg / mL, and the working concentration of glutathione during combination is 10 mM.

[0013] Preferably, the toxic and side effects of bevacizumab on the immune system are specifically manifested as a significant decrease in the proliferative activity of immune cells, disruption of the normal cell cycle of immune cells, and abnormal up-regulation of the activity of matrix metalloproteinase-9 (MMP-9) in immune cells.

[0014] Preferably, the combination of glutathione and bevacizumab can restore the proliferative activity of immune cells inhibited by single-agent bevacizumab treatment, restore the normal cell cycle, and down-regulate the activity of cellular MMP-9.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] By combining glutathione with bevacizumab, the present invention can effectively inhibit the immune-related adverse reactions caused by single-agent bevacizumab treatment, thereby improving the treatment tolerance of tumor patients. Experimental data show that compared with the single use of bevacizumab, the combined regimen can improve the immune cell activity (such as the proliferative ability of immune cells) of patients, and restore the expression level of abnormally up-regulated cytokines to the physiological range, and restore the disrupted cell cycle to normal. This combined treatment regimen provides a new technical path for antagonizing drug side effects, and at the same time establishes a verifiable model for clinical research on reducing the toxicity risk associated with tumor treatment, and has significant clinical transformation value. Description of the Drawings

[0017] Figure 1 It is a comparison chart of the proliferative activity of splenic lymphocytes between the single-agent bevacizumab group and the glutathione combination group.

[0018] Figure 2 It is a comparison chart of the proliferative activity of bone marrow cells between the single-agent bevacizumab group and the glutathione combination group.

[0019] Figure 3 It is a comparison chart of the MMP-9 gelatinase activity of splenic lymphocytes between the single-agent bevacizumab group and the glutathione combination group.

[0020] Figure 4 It is a comparison chart of the MMP-9 gelatinase activity of bone marrow cells between the single-agent bevacizumab group and the glutathione combination group.

[0021] Figure 5 It is a comparison chart of the cell cycle distribution of splenic lymphocytes between the single-agent bevacizumab group and the glutathione combination group.

[0022] Figure 6 It is a comparison chart of the cell cycle distribution of bone marrow cells between the single-agent bevacizumab group and the glutathione combination group. Detailed Embodiments

[0023] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments. However, the embodiments 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 technical field.

[0024] Example 1 Changes in the cell viability of immune cells intervened by bevacizumab alone and in combination with glutathione.

[0025] Take spleen, tibia, and femoral bone marrow tissue samples from SD rats and place them in PBS preservation solution containing 4% double antibodies.

[0026] The following operations are completed under sterile conditions: The spleen tissue is mechanically dispersed through a 200-mesh cell sieve to obtain a single-cell suspension; after truncating both ends of the bone tissue, the bone marrow cavity is rinsed with PBS containing 4% double antibodies, and the obtained bone marrow fluid is filtered through a 200-mesh cell sieve.

[0027] Centrifuge at 1500 rpm for 5 min, and use Tris-NH4Cl red blood cell lysis buffer to remove the red blood cell components in the spleen and bone marrow suspensions.

[0028] After washing the cell pellet with PBS, resuspend it in RPMI-1640 complete medium containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin. Remove the adherent cells by culturing for 12 h to obtain a high-purity lymphocyte population.

[0029] Collect the cells cultured overnight, centrifuge, and resuspend them in SF medium for cell counting.

[0030] Experimental grouping design: Five groups are set for spleen lymphocytes and bone marrow cells respectively: blank control group (Untreated); low-dose bevacizumab group (0.25 mg / mL); clinical-dose bevacizumab group (2.5 mg / mL); low-dose combination group (0.25 mg / mL + 10 mM GSH); clinical-dose combination group (2.5 mg / mL + 10 mM GSH). The seeding density per well is 2×10 5 cells / well, set 3 replicates, and pre-culture at 37 °C and 5% CO2 for 30 min.

[0031] Experimental material information: Bevacizumab (Bevacizumab, national drug approval number S20200013) was purchased from Innovent Biologics, Inc. (Suzhou), stored at 4 °C, and a low-concentration working solution was prepared with PBS before use; glutathione (Aladdin) powder was dissolved in PBS to prepare a 10 mM mother liquor, and this concentration covers the human physiological supplementation range (1 - 10 mM).

[0032] After pre-culturing the cells, add the corresponding medicinal solution according to the grouping plan, and continue to incubate at 37°C and 5% CO2 for 20 h.

[0033] After 20 h, terminate the culture, centrifuge to collect the supernatant, and store it at -80°C for later use (samples for zymography analysis).

[0034] MTT detection procedure: Add 50 μL of 10% MTT solution to each well and incubate at 37°C for 4 h; centrifuge to discard the supernatant, add 100 μL of DMSO to fully dissolve the formazan crystals; measure the absorbance value (OD value) at a wavelength of 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0035] The MTT method is a method for detecting cell survival and growth. The detection principle is that succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-violet crystalline formazan and deposit it in the cells, while dead cells do not have this function. However, in the presence of substances with reducing activity, the formation of formazan can be enhanced, and the optical density (OD) value increases. Dimethyl sulfoxide (DMSO) can dissolve the formazan in the cells, and the light absorption value is measured at a wavelength of 570 nm using an ELISA reader, which can indirectly reflect cell viability.

[0036] Experimental results: As Figure 1 、 Figure 2 shown, compared with the non-intervention group, treatment with bevacizumab alone significantly inhibited the cell proliferation activity of splenic and bone marrow lymphocytes in a concentration-dependent manner. After the combined application of glutathione, the cell proliferation activity in each dose group was significantly increased, confirming that glutathione can effectively antagonize the immunosuppressive effect of bevacizumab and restore the impaired immune cell proliferation activity of immune cells.

[0037] Example 2 Detection of changes in MMP-9 activity in immune cells after intervention with bevacizumab alone and in combination with glutathione.

[0038] The zymography method was used to detect changes in the enzymatic activity of matrix metalloproteinase MMP-9. As one of the lymphocyte activation markers, the activity level of MMP-9 can reflect the functional state of immune cells. The specific operation is as follows: Mix the supernatant of splenic lymphocytes and bone marrow cells in different treatment groups with an equal volume of sample buffer, and load 20 μL per well for vertical electrophoresis (constant voltage of 100 V). After electrophoresis, wash the gel twice with the eluent (1 h each time), and then transfer it to the active incubation solution and incubate overnight at 37°C. Finally, stain with Coomassie Brilliant Blue R-250 and decolorize until the background is transparent, and collect the enzymolysis band images using a Bio-Rad gel imaging system.

[0039] Experimental results: As Figure 3As shown, in splenic lymphocytes, treatment with bevacizumab alone caused a significant increase in MMP-9 activity, and the morphology of the enzymatic digestion band showed abnormal diffusion. After combination with glutathione, not only the activity decreased, but the band morphology was also consistent with that of the non-intervention group. As Figure 4 shown, in bone marrow cells, the MMP-9 activity in the bevacizumab alone treatment group (2.5 mg / mL) was significantly enhanced compared with the non-intervention group, while the activity returned to near the baseline level after combination with glutathione (10 mM); the above results indicate that the combination of glutathione can reverse the abnormal up-regulation of MMP-9 activity and the abnormal diffusion of enzyme conformation in immune cells caused by bevacizumab.

[0040] Example 3 Detection of cell cycle changes in immune cells intervened by bevacizumab alone and in combination with glutathione.

[0041] Take 1×10 6 cells and place them in a 1.5 mL tube, wash twice with PBS at 4°C (1500 rpm, 5 min).

[0042] Slowly add 200 μL of pre-cooled 70% ethanol along the wall, mix well, repeat three times, and finally make up the volume to 1 ml. Fix overnight at -20°C.

[0043] Take out the fixed cell suspension, centrifuge at 1500 rpm at 4°C for 5 min, and discard the supernatant.

[0044] Add 1 mL of pre-cooled PBS and wash twice to remove residual ethanol (1500 rpm, 5 min).

[0045] Add 100 μL of PI staining solution (50 μg / mL) to each tube, incubate at 37°C in the dark for 30 min, then perform flow cytometry detection and save the data after completion.

[0046] Experimental results: As Figure 5 、 Figure 6 shown, in splenic lymphocytes and bone marrow cells, compared with the non-intervention group, single-agent treatment with bevacizumab increased the G2 / M phase of cells and blocked cell mitosis. After combination with glutathione, the proportion of cells in the G2 / M phase returned to normal, indicating that the combination of glutathione can restore the normal cell cycle of immune cells disrupted by bevacizumab.

[0047] The above results indicate that single-agent treatment with bevacizumab can simultaneously inhibit the cell proliferation vitality of central (spleen) and peripheral (bone marrow) immune cells, disrupt the normal cell cycle, and abnormally up-regulate the MMP-9 activity of cells. While the combination of glutathione can effectively restore the proliferation vitality of inhibited immune cells, restore the normal cell cycle, down-regulate the abnormal MMP-9 enzyme activity, and correct the abnormal enzyme conformation, suggesting that this combination regimen has a synergistic effect on repairing the functional integrity of immune cells.

[0048] The advantages of using glutathione to alleviate the immune toxic and side effects of bevacizumab in the present invention are manifold. By combining glutathione with bevacizumab for drug administration, the present invention can effectively inhibit the immune-related adverse reactions caused by single-agent treatment with bevacizumab, thereby enhancing the treatment tolerance of tumor patients. Experimental data show that, compared with the single use of bevacizumab, the combined use regimen can improve the activity of immune cells in patients (such as the proliferative activity of immune cells), restore the expression levels of abnormally up-regulated cytokines to the physiological range, and restore the disrupted cell cycle to normal. This combined treatment regimen provides a new technical approach for antagonizing drug side effects, which has practical significance for exploring the mechanism of drug toxicity and reducing treatment-related toxic and side reactions, and provides an important reference basis for subsequent clinical research.

Claims

1. Application of glutathione in alleviating immune system side effects of bevacizumab.

2. The application according to claim 1, wherein The immune system includes bone marrow cells of the central immune system and splenic lymphocytes of the peripheral immune system.

3. The application according to claim 1, characterized in that, The combined concentration of bevacizumab is 2.5 mg / mL, and the working concentration of glutathione during combination is 10 mM.

4. The application according to claim 1, characterized in that, The immune system side effects of bevacizumab are specifically manifested as a significant decrease in the proliferative activity of immune cells, disruption of the normal cell cycle of immune cells, and abnormal up-regulation of the activity of matrix metalloproteinase-9 (MMP-9) in immune cells.

5. The application according to claim 1, characterized in that The combination of glutathione and bevacizumab can restore the proliferative activity of immune cells inhibited by single-agent bevacizumab treatment, restore the normal cell cycle, and down-regulate the activity of cellular MMP-9.

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