Application of sodium bicarbonate in preparation of tumor immune drug enhancer
By activating the necroptosis pathway of TNF-α and inhibiting the NF-κB pathway using sodium bicarbonate, the problem of the inability of existing TNF-α anti-tumor drugs to specifically activate it is solved, achieving specific killing and growth inhibition of tumor cells, and providing a safe and effective tumor treatment option.
Patent Information
- Application Number
- CN202511986796.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-26
- Publication Date
- 2026-02-17
Smart Images
Figure CN121534077A_ABST
Abstract
Description
Technical Field
[0001] This invention pertains to pharmaceutical uses, specifically novel pharmaceutical uses of sodium bicarbonate, particularly its application in the preparation of enhancers for tumor immunotherapy drugs. Background Technology
[0002] Sodium bicarbonate has the molecular formula NaHCO3. Its pharmacological effects include: (1) treating metabolic acidosis by increasing the concentration of carbonate ions in the plasma, neutralizing hydrogen ions, and thus correcting acidosis; (2) alkalizing urine by increasing the pH value after the carbonate concentration in the urine increases, making it less likely for uric acid, sulfonamides, and hemoglobin to crystallize or aggregate in the urine; and (3) neutralizing stomach acid by rapidly neutralizing or buffering gastric acid without directly affecting gastric acid secretion. Therefore, the rapid increase in gastric pH relieves symptoms caused by high gastric acid.
[0003] Tumor necrosis factor α (TNF-α) is a major anti-tumor cytokine secreted by cytotoxic T cells. TNF-α has been used as an anti-tumor drug to treat tumors; however, its anti-tumor efficacy is unpredictable and it can even promote tumor growth. Later studies revealed that TNF-α has multiple functions: it can activate the nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) pathway, promoting tumor cell growth, and it can also activate the necroptosis pathway, leading to tumor cell death. The anti-tumor activity of TNF-α depends on its activation of the necroptosis pathway. Specifically activating the TNF-α-mediated necroptosis pathway is key to TNF-α's anti-tumor effect; discovering drugs that specifically activate the TNF-α-mediated necroptosis pathway without activating the NF-κB pathway has significant implications for tumor treatment. Summary of the Invention
[0004] One object of this invention is to provide the application of sodium bicarbonate in the preparation of tumor immunomodulatory agents. Sodium bicarbonate has the English name sodium bicarbonate and the molecular formula NaHCO3. TNF-α activates the NF-κB signaling pathway, enhances the inflammatory response, and promotes tumor cell growth. Under the action of sodium bicarbonate, the TNF-α-mediated NF-κB signaling pathway is inhibited, and the TNF-α-mediated receptor-interacting serine / threonine-protein kinase (RIPK)-dependent necroptosis signaling pathway is activated, leading to tumor cell death. This invention demonstrates the novelty, innovability, and practicality of sodium bicarbonate in the preparation of antitumor immunomodulators.
[0005] The application of sodium bicarbonate in the preparation of tumor necrosis factor α antitumor drugs.
[0006] The application of sodium bicarbonate in activating the tumor-killing effect of tumor necrosis factor α.
[0007] The application of sodium bicarbonate in inhibiting the tumor-promoting effect of tumor necrosis factor α.
[0008] The prepared drug contains pharmaceutical excipients or carriers permitted by the formulation.
[0009] The drug can be administered by injection or orally.
[0010] The administration of the drug via injection includes intratumoral injection.
[0011] The drug is formulated in the form of liquid, granules, tablets, powders, capsules, sustained-release preparations, droplets, or injections.
[0012] The concentration of sodium bicarbonate is 60-600 mM.
[0013] The beneficial effects of this invention are: (1) A new use of sodium bicarbonate in the preparation of TNF-α antitumor enhancers was developed; (2) Sodium bicarbonate is the most important buffer system for maintaining the pH of the human body and has good safety. (3) Therefore, sodium bicarbonate can be used to prepare drugs for treating tumors, which can reduce the cost of treatment. Attached Figure Description
[0014] Figure 1 The growth rate of SK-HEP-1 cells after treatment with TNF-α, 80 mM sodium bicarbonate, and TNF-α + 80 mM sodium bicarbonate.
[0015] Figure 2 The expression levels of pIκB and IκB proteins after treating SK-HEP-1 cells with TNF-α, 80 mM sodium bicarbonate, and TNF-α + 80 mM sodium bicarbonate.
[0016] Figure 3 Nuclear translocation of NF-κB after treatment of SK-HEP-1 cells with TNF-α, 80 mM sodium bicarbonate, and TNF-α + 80 mM sodium bicarbonate.
[0017] Figure 4 The expression levels of pRIPK1, RIPK1, pRIPK3, RIPK3, mixed lineage kinase domain-like protein (MLKL) and pMLKL were determined after SK-HEP-1 cells were treated with TNF-α, 80 mM sodium bicarbonate, and TNF-α + 80 mM sodium bicarbonate.
[0018] Figure 5 The growth rate of HeLa cells after treatment with TNF-α, 70 mM sodium bicarbonate, and TNF-α + 70 mM sodium bicarbonate.
[0019] Figure 6 Nuclear translocation of NF-κB after treatment of HeLa cells with TNF-α, 70 mM sodium bicarbonate, and TNF-α + 70 mM sodium bicarbonate.
[0020] Figure 7 The expression levels of pRIPK1, RIPK1, pRIPK3, RIPK3, pMLKL, and MLKL proteins after HeLa cells were treated with TNF-α, 70 mM sodium bicarbonate, and TNF-α + 70 mM sodium bicarbonate.
[0021] Figure 8 The growth rate of A549 cells after treatment with TNF-α, 80 mM sodium bicarbonate, and TNF-α + 80 mM sodium bicarbonate.
[0022] Figure 9 The growth rate of RKO cells after treatment with TNF-α, 80 mM sodium bicarbonate, and TNF-α + 80 mM sodium bicarbonate.
[0023] Figure 10 The study aimed to determine the volume changes of xenografts in the control group, TNF-α group, sodium bicarbonate group, and TNF-α+sodium bicarbonate group in a mouse model of human hepatocellular carcinoma.
[0024] Figure 11The weights of xenografts in the control group, TNF-α group, sodium bicarbonate group, and TNF-α+sodium bicarbonate group were measured in a mouse model of human hepatocellular carcinoma.
[0025] Figure 12 The size of xenografts in the control group, TNF-α group, sodium bicarbonate group, and TNF-α+sodium bicarbonate group were compared in a mouse model of human hepatocellular carcinoma.
[0026] Figure 13 The study aimed to determine the volume changes of xenografts in the control group, TNF-α group, sodium bicarbonate group, and TNF-α+sodium bicarbonate group in a mouse model of human cervical cancer.
[0027] Figure 14 The weights of xenografts in the control group, TNF-α group, sodium bicarbonate group, and TNF-α+sodium bicarbonate group were measured in a mouse model of human cervical cancer.
[0028] Figure 15 The size of xenografts in the control group, TNF-α group, sodium bicarbonate group, and TNF-α+sodium bicarbonate group were compared in a mouse model of human cervical cancer. Detailed Implementation
[0029] The present invention will be further described in conjunction with the accompanying drawings and embodiments.
[0030] TNF-α exerts different effects on tumor cells by inducing various signaling pathways: activating the NF-κB pathway promotes inflammation and tumor growth; activating the necroptosis pathway leads to tumor cell death. In the absence of sodium bicarbonate, TNF-α primarily activates the NF-κB pathway, promoting inflammation and tumor growth; while in the presence of sodium bicarbonate, TNF-α primarily activates the necroptosis pathway, leading to tumor cell death. This clearly demonstrates the role of sodium bicarbonate in enhancing the antitumor activity of TNF-α and its theoretical basis, possessing novelty and practicality.
[0031] Example 1: Sodium bicarbonate activates TNF-α to kill human liver cancer cells.
[0032] Experimental Methods: All tumor cell lines were derived from the Cancer Institute of Zhejiang University. Human hepatocellular carcinoma cells (SK-HEP-1) were cultured in RPMI-1640 medium (containing 10% fetal bovine serum and 20 mM sodium bicarbonate). Cells were divided into a control group, a TNF-α (#HZ1014, Proteintech, USA) group (25 ng / mL), a sodium bicarbonate group (0.67%; 80 mM), and a sodium bicarbonate + TNF-α group (TNF-α, 25 ng / mL; sodium bicarbonate, 0.67%; 80 mM).
[0033] The number of cells in each group was measured at 0, 24, 48, and 72 hours. Cells were digested with trypsin and counted under a microscope using a cell counting chamber.
[0034] SK-HEP-1 cells were seeded in small glass dishes. After 48 hours of treatment, cells from each group were harvested, fixed with 4% paraformaldehyde for 15 minutes, and then permeated with 1% BSA containing 0.3% Triton X-100 and blocked for 1 hour. Cells were then incubated overnight at 4°C with NF-κB (#8242, CST, USA) primary antibody. Cells were washed three times with PBS, incubated with fluorescent secondary antibody for 2 hours, and washed three times with PBS. Nuclei were stained with Hoechst for 15 minutes and washed three times with PBS. Cells were then imaged using a laser confocal microscope.
[0035] Cells from different time points were collected, and proteins were extracted. The protein content of pIκB (#2859, CST, USA), IκB (#4814, CST, USA), pRIPK1 (#44590, CST, USA), RIPK1 (#3493, CST, USA), pRIPK3 (#93654, CST, USA), RIPK3 (#10188, CST, USA), pMLKL (#91689, CST, USA), and MLKL (#14993, CST, USA) was detected by Western Blot.
[0036] Experimental results: See Figure 1 , Figure 2 , Figure 3 and Figure 4 . Figure 1 The results showed that, compared to the control group, TNF-α did not alter the growth of human liver cancer cells, sodium bicarbonate could inhibit the growth of liver cancer cells, and sodium bicarbonate combined with TNF-α could kill human liver cancer cells. Figure 2 The results showed that sodium bicarbonate can inhibit TNF-α activation of the NF-κB pathway. Figure 3 The results showed that sodium bicarbonate could partially inhibit TNF-α-induced NF-κB nuclear translocation. Figure 4 Sodium bicarbonate induced TNF-α activation of the RIPK-related necrosis pathway, leading to necrosis of human liver cancer cells.
[0037] Example 2: Sodium bicarbonate activates TNF-α to kill human cervical cancer cells.
[0038] Experimental Methods: All tumor cell lines were derived from the Cancer Institute of Zhejiang University. Human cervical cancer cells (HeLa) were cultured in RPMI-1640 medium (containing 10% fetal bovine serum and 20 mM sodium bicarbonate). Cells were divided into a control group, a TNF-α group (25 ng / mL), a sodium bicarbonate group (0.59%; 70 mM), and a sodium bicarbonate + TNF-α group (TNF-α, 25 ng / mL; sodium bicarbonate, 0.59%; 70 mM).
[0039] The number of cells in each group was measured at 0, 24, 48, and 72 hours. Cells were digested with trypsin and counted under a microscope using a cell counting chamber.
[0040] HeLa cells were seeded in small glass dishes. After 24 hours of treatment, cells from each group were harvested, fixed with 4% paraformaldehyde for 15 minutes, and then permeated with 1% BSA containing 0.3% Triton X-100 and blocked for 1 hour. Cells were then incubated overnight at 4°C with NF-κB primary antibody. Cells were washed three times with PBS, incubated with fluorescent secondary antibody for 2 hours, and washed three times with PBS. Nuclei were stained with Hoechst for 15 minutes and washed three times with PBS. Cells were then imaged using a laser confocal microscope.
[0041] Cells from each group were collected at 24-hour time points, and proteins were extracted. The protein content of pRIPK1, RIPK1, pRIPK3, RIPK3, pMLKL, and MLKL was detected by Western blotting.
[0042] Experimental results: See Figure 5 , Figure 6 and Figure 7 . Figure 5 The results showed that, compared to the control group, TNF-α did not alter the growth of human cervical cancer cells, sodium bicarbonate could inhibit the growth of cervical cancer cells, and sodium bicarbonate combined with TNF-α could kill human cervical cancer cells. Figure 6 The results showed that sodium bicarbonate could partially inhibit TNF-α-induced NF-κB nuclear translocation and block the TNF-α-activated NF-κB pathway. Figure 7 Sodium bicarbonate was shown to induce TNF-α to activate the RIPK-related necrosis pathway, thereby inducing necrosis in human cervical cancer cells.
[0043] Example 3: Sodium bicarbonate activates TNF-α to kill human lung adenocarcinoma cells.
[0044] Experimental Methods: All tumor cell lines were derived from the Cancer Institute of Zhejiang University. Human lung adenocarcinoma cells (A549) were cultured in RPMI-1640 medium (containing 10% fetal bovine serum and 20 mM sodium bicarbonate). Cells were divided into a control group, a TNF-α group (25 ng / mL), a sodium bicarbonate group (0.67%; 80 mM), and a sodium bicarbonate + TNF-α group (TNF-α, 25 ng / mL; sodium bicarbonate, 0.67%; 80 mM). Cell counts were measured at 0, 24, 48, and 72 h. Cells were digested with trypsin and counted under a microscope using a cell counting chamber.
[0045] Experimental results: See Figure 8 Compared to the control group, TNF-α did not alter the growth of human lung adenocarcinoma cells, sodium bicarbonate inhibited the growth of lung adenocarcinoma cells, and the combination of sodium bicarbonate and TNF-α killed human lung adenocarcinoma cells.
[0046] Example 4: Sodium bicarbonate activates TNF-α to kill human colon adenocarcinoma cells.
[0047] Experimental Methods: All tumor cell lines were derived from the Cancer Institute of Zhejiang University. Human colon adenocarcinoma cells (RKO) were cultured in RPMI-1640 medium (containing 10% fetal bovine serum and 20 mM sodium bicarbonate). Cells were divided into a control group, a TNF-α group (25 ng / mL), a sodium bicarbonate group (0.67%; 80 mM), and a sodium bicarbonate + TNF-α group (TNF-α, 25 ng / mL; sodium bicarbonate, 0.67%; 80 mM). Cell counts were measured at 0, 24, 48, and 72 h. Cells were digested with trypsin and counted under a microscope using a cell counting chamber.
[0048] Experimental results: See Figure 9 Compared to the control group, TNF-α did not alter the growth of human colon adenocarcinoma cells, the sodium bicarbonate group inhibited the growth of colon adenocarcinoma cells, and the combination of sodium bicarbonate and TNF-α killed human colon adenocarcinoma cells.
[0049] Example 5: Sodium bicarbonate can alter the action of TNF-α and inhibit the growth of human hepatocellular tumors.
[0050] Experimental Methods: After receiving NOD / CID mice, six mice were divided into two cages and fed normally for three days to allow them to acclimatize to the environment. During this period, the water was changed daily, and feed was added every three days. Before inoculating with tumor cells, the mice were weighed, and mice with similar weights were selected. SK-HEP-1 cells (Zhejiang University Cancer Institute) were cultured until the cells were in the logarithmic growth phase and 80% of the cells had reached confluence in the culture flask. After trypsin digestion of the cells for 5 minutes, fresh culture medium containing 10% fetal bovine serum was added to neutralize the trypsin, and the cells were centrifuged at 1000 rpm for 5 minutes. The cell pellet was collected. The cells were washed three times with pre-chilled PBS solution, centrifuged for 5 minutes each time, to completely remove serum components from the culture medium. The cells were resuspended in pre-chilled serum-free culture medium, counted, and the cell density was adjusted to 4 × 10⁶ cells / mL. 7 / mL. Keep the cell suspension on ice until animal inoculation. Inoculate subcutaneously with 100 μL of cell suspension (4 × 10⁹ / mL) using a 1 mL syringe. 6 The sample was applied to the armpit of the upper limb of NOD / SCID mice. Each mouse was then marked with a 5% picric acid / anhydrous ethanol solution.
[0051] The following day, mice that had been inoculated with tumor cells were randomly divided into groups of six. Ten days later, the tumors were clearly palpable (approximately the size of a mung bean). Every other day, the control group received a subcutaneous injection of 100 µL of saline around the tumor, while the TNF-α group received an injection of 100 µL of TNF-α (120 × 10⁻⁶). 4 U / Kg); Sodium bicarbonate group injected 100µL of NaHCO3 (Sigma-Aldrich, USA) to a final concentration of 1.68%; Sodium bicarbonate + TNF-α group injected 100µL of TNF-α (120×10⁻⁶ U / kg); 4 A mixture of sodium bicarbonate (U / Kg) and sodium bicarbonate (Sigma-Aldrich, USA).
[0052] The mouse's weight and tumor size were measured every other day. Tumor size was measured using calipers, taking into account both the maximum diameter (L) and the minimum diameter (W). Tumor volume was calculated as V = L × W × W / 2.
[0053] The experiment ended after 37 days. The tumor was carefully isolated, weighed, and photographed.
[0054] Experimental results: See Figure 10 , Figure 11 and Figure 12 . Figure 10 The results showed that the growth rate of xenografts in mice in the sodium bicarbonate + TNF-α group was significantly slower than that in the TNF-α group. Figure 11 The results showed that the tumor weight in the sodium bicarbonate + TNF-α group was significantly smaller than that in the TNF-α group. Figure 12 The results showed that, in conjunction with sodium bicarbonate, TNF-α could inhibit the growth of human hepatocellular carcinoma.
[0055] Example 6: Sodium bicarbonate can alter the function of TNF-α and inhibit the growth of human cervical tumors.
[0056] Experimental Methods: After receiving NOD / SCID mice, six mice were divided into two cages and fed normally for three days to allow them to acclimatize to the environment. During this period, the water was changed daily, and feed was added every three days. Before inoculating with tumor cells, the mice were weighed, and mice with similar weights were selected. HeLa cells (Zhejiang University Cancer Institute) were cultured until the cells were in the logarithmic growth phase and 80% confluent in the culture flask. After trypsin digestion of the cells for 5 minutes, fresh culture medium containing 10% fetal bovine serum was added to neutralize the trypsin, and the cells were centrifuged at 1000 rpm for 5 minutes. The cell pellet was collected. The cells were washed three times with pre-chilled PBS solution, centrifuged for 5 minutes each time, to completely remove serum components from the culture medium. The cells were resuspended in pre-chilled serum-free culture medium, counted, and the cell density was adjusted to 102. 7 / mL. Keep the cell suspension on ice until animal inoculation. Inoculate subcutaneously with 100µL of cell suspension (1×10⁻⁶) using a 1mL syringe. 6 The sample was applied to the armpit of the upper limb of NOD / SCID mice. Each mouse was then marked with a 5% picric acid / anhydrous ethanol solution.
[0057] The next day, mice that had been inoculated with tumor cells were randomly divided into groups of 6. After 7 days, the tumors were clearly palpable (about the size of a mung bean). Every other day, the control group received a subcutaneous injection of 100 µL of saline around the tumor, while the TNF-α group received a injection of 100 µL of TNF-α (120 × 10⁻⁶). 4 U / Kg); Sodium bicarbonate group injected 100µL of NaHCO3 (Sigma-Aldrich, USA) to a final concentration of 1.68%; Sodium bicarbonate + TNF-α group injected 100µL of TNF-α (120×10⁻⁶ U / kg); 4 A mixture of sodium bicarbonate (U / Kg) and sodium bicarbonate (Sigma-Aldrich, USA).
[0058] The mouse's weight and tumor size were measured every other day. Tumor size was measured using calipers, taking into account both the maximum diameter (L) and the minimum diameter (W). Tumor volume was calculated as V = L × W × W / 2.
[0059] The experiment ended after 11 days. The tumor was carefully isolated, weighed, and photographed.
[0060] Experimental results: See Figure 13 , Figure 14 and Figure 15 . Figure 13 The results showed that the growth rate of xenografts in mice in the sodium bicarbonate + TNF-α group was significantly slower than that in the TNF-α group. Figure 14The results showed that the tumor weight in the sodium bicarbonate + TNF-α group was significantly smaller than that in the TNF-α group. Figure 15 The results showed that, in conjunction with sodium bicarbonate, TNF-α could inhibit the growth of human cervical tumors.
[0061] The above in vitro and in vivo examples illustrate that sodium bicarbonate can locally inhibit the NF-κB pathway of TNF-α, activate the RIPK pathway, and induce tumor cell necrosis. It can kill tumor cells, inhibit tumor growth, and can be applied to the clinical treatment of tumors. It is a safe and effective tumor treatment method.
Claims
1. Application of sodium bicarbonate in the preparation of enhancers for tumor immunotherapy drugs.
2. The application of sodium bicarbonate according to claim 1 in the preparation of tumor necrosis factor α antitumor drugs, characterized in that, The application of sodium bicarbonate in the preparation of tumor necrosis factor α antitumor drugs.
3. The application of sodium bicarbonate according to claim 1 in the preparation of an enhancer for tumor immunotherapy, characterized in that, The application of sodium bicarbonate in activating the tumor-killing effect of tumor necrosis factor α.
4. The application of sodium bicarbonate according to claim 1 in the preparation of an enhancer for tumor immunotherapy, characterized in that, The application of sodium bicarbonate in inhibiting the tumor-promoting effect of tumor necrosis factor α.
5. The application of sodium bicarbonate according to claim 1 in the preparation of an enhancer for tumor immunotherapy, characterized in that, The prepared drug contains pharmaceutical excipients or carriers permitted by the formulation.
6. The application of sodium bicarbonate according to claim 1 in the preparation of an enhancer for tumor immunotherapy, characterized in that, The drug can be administered by injection or orally.
7. The application of sodium bicarbonate according to claim 6 in the preparation of an enhancer for tumor immunotherapy, characterized in that, The administration of the drug via injection includes intratumoral injection.
8. The application of sodium bicarbonate according to claim 1 or 5 in the preparation of an enhancer for tumor immunotherapy, characterized in that, The drug is formulated in the form of liquid, granules, tablets, powders, capsules, sustained-release preparations, droplets, or injections.