Pharmaceutical composition for anti-tumor and drug delivery system

Abstract

The application provides application of glutamine metabolism regulation and metal ions in antitumor. The application also provides a pharmaceutical composition for antitumor and a drug delivery system. The drug delivery system of the application comprises a glutamine metabolism regulator and a metal organic framework material encapsulating the glutamine metabolism regulator. The pharmaceutical composition of the application comprises a glutamine metabolism regulator and metal ions, or comprises the drug delivery system of the application.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of glutamine metabolism regulation and the combined use of metal ions in anti-tumor therapy. Background Technology

[0002] Malignant tumors have become the leading cause of death among humans. With the occurrence and development of tumors, their energy demands increase rapidly, leading to a unique tumor microenvironment in the tumor lesion area. Within this microenvironment, metabolic competition exists between tumor cells and immune cells. Highly proliferating tumor cells absorb large amounts of nutrients from the microenvironment to fuel their rapid growth and division, limiting the nutrient supply to immune cells. This inhibits immune cell proliferation, differentiation, and immune response, promotes immune escape, and results in a low immune response rate, severely hindering the effectiveness of anti-tumor immunotherapy. Therefore, new methods or approaches are urgently needed to improve anti-tumor efficacy. Summary of the Invention

[0003] This invention discovers that, under conditions of glutamine deficiency or starvation, the superimposed treatment with zinc ions can create a tumor microenvironment in which immune activation and metabolic disorders coexist. This therapeutic activation of innate immunity and nutritional restriction can synergistically promote tumor cell death, thereby enhancing the anti-tumor effect.

[0004] According to one aspect of the present invention, the use of a glutamine metabolism regulator and a metal ion in combination in the preparation of a medicament for the prevention and / or treatment of tumors is provided.

[0005] In some embodiments, the glutamine metabolism regulator includes at least one of a glutaminase inhibitor, a glutamine transporter inhibitor, or an exogenous glutamine limiter.

[0006] In some embodiments, the glutaminase inhibitor includes substances that reduce glutaminase expression and / or substances that reduce glutaminase activity or function.

[0007] In some embodiments, the substance that reduces glutaminase expression includes agents that inhibit or reduce the expression level of the glutaminase gene.

[0008] In some embodiments, the substance that reduces the activity or function of glutaminase includes an antibody or antigen-binding molecule thereof that specifically binds to the glutaminase and / or a small molecule compound that inhibits the activity or function of the glutaminase.

[0009] In some embodiments, the glutaminase inhibitor comprises a GLS1 inhibitor. In some embodiments, the glutaminase inhibitor comprises at least one of Telaglenastat (CB-839), BPTES, Glutaminase C-IN-1 (Compound 968), IPN60090, or a pharmaceutically acceptable salt thereof.

[0010] In some embodiments, glutamine transporter inhibitors include at least one of V-9302, GPNA, BCH, sulfasalazine, or a pharmaceutically acceptable salt thereof.

[0011] In some embodiments, the exogenous glutamine restriction includes reducing the supply of exogenous glutamine through dietary control or glutamine antagonists. In some embodiments, the glutamine antagonists include at least one of Sirpiglenastat (DPR-104), DON, JHU-083, or a pharmaceutically acceptable salt thereof.

[0012] In some embodiments, the glutaminase inhibitor and / or glutamine transporter inhibitor includes at least one of small molecule compounds, peptides, peptide mimics, matrix analogs, aptamers, siRNA (short interfering RNA), dsRNA (double-stranded RNA), miRNA (microRNA), shRNA (short hairpin RNA), ribozymes, or sgRNA (small guide RNA).

[0013] In some embodiments, the metal ion includes a transition metal ion. In some embodiments, the metal ion includes a divalent transition metal cation. In some embodiments, the metal ion includes zinc ions (Zn). 2+ ), cobalt ions (Co) 2+ Platinum ions (Pt) 2+ ), copper ions (Cu) 2+ ), iron ions (Fe 2+ ) or nickel ions (Ni 2+ At least one of the following.

[0014] In some embodiments, the metal ion may be derived from at least one of the nitrate, chloride, sulfate, gluconate, acetate, lactate, citrate, fumarate, etc., of the corresponding metal ion, or its pharmaceutically acceptable hydrate or crystal form.

[0015] In some embodiments, the dosage of the glutamine metabolism regulator is such that the subject is in a state of glutamine deficiency or glutamine starvation.

[0016] In some embodiments, the amount of the glutamine metabolism regulator is 0.1-1000 μM, preferably 0.1-100 μM, and more preferably 0.1-20 μM. In some specific embodiments, the amount of the glutaminase metabolism regulator can be 0.1 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 5 μM, 6 μM, 8 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 75 μM, 100 μM, 125 μM, 150 μM, 175 μM, 200 μM, 300 μM, 400 μM, 500 μM, 800 μM, 1000 μM, or any value between them.

[0017] In some embodiments, the amount of the glutaminase inhibitor is 0.1-1000 μM, preferably 0.1-100 μM, and more preferably 0.1-20 μM. In some specific embodiments, the amount of the glutaminase inhibitor can be 0.1 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 5 μM, 6 μM, 8 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 75 μM, 100 μM, 125 μM, 150 μM, 175 μM, 200 μM, 300 μM, 400 μM, 500 μM, 800 μM, 1000 μM, or any value between them.

[0018] In some specific embodiments, the amount of the glutaminase inhibitor CB-839 is 0.1-20 μM, for example 0.1 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 5 μM, 6 μM, 8 μM, 10 μM, 20 μM.

[0019] In some embodiments, the amount of metal ions used is 50 μM to 500 μM, preferably 75 μM to 200 μM. In some specific embodiments, the amount of metal ions used is 50 μM, 75 μM, 100 μM, 125 μM, 150 μM, 175 μM, 200 μM, 300 μM, 400 μM, 500 μM, or any value between them.

[0020] In some embodiments, the glutamine metabolism regulator and the metal ion are administered simultaneously or sequentially.

[0021] In some embodiments, the glutamine metabolism regulator and the metal ions are administered in combination via a metal-organic framework delivery system.

[0022] In some embodiments, the metal-organic framework material encapsulates the glutamine metabolism regulator.

[0023] In some embodiments, the metal-organic framework material is a ZIF material.

[0024] In some embodiments, the ZIF material includes at least one of ZIF-8, ZIF-90, and ZIF-78.

[0025] In some embodiments, the prevention and / or treatment of tumors includes at least one of inhibiting tumor cell proliferation, killing tumor cells, inhibiting tumorigenesis, reducing glutathione levels in tumor cells, inducing oxidative stress in tumor cells, or remodeling the tumor microenvironment in which immune activation and metabolic disorders coexist.

[0026] In some embodiments, the tumor includes solid tumors and hematomas. In some embodiments, the tumor includes glutamine-dependent tumors.

[0027] In some embodiments, the tumor includes at least one of liver cancer, glioma, colorectal cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, kidney cancer, melanoma, head and neck cancer, bladder cancer, central nervous system cancer, cervical cancer, endometrial cancer, leukemia, esophageal cancer, prostate cancer, or lymphoma.

[0028] In some embodiments, the glioma includes at least one of glioblastoma, astrocytoma, oligodendroglioma, or ependymoma.

[0029] In some embodiments, the combined use of glutamine metabolism regulation and metal ions activates the cGas-STING signaling pathway, serving as an innate immune activator for the prevention and / or treatment of tumors.

[0030] According to another aspect of the present invention, a drug delivery system is provided, the drug delivery system comprising a glutamine metabolism regulator and a metal-organic framework material encapsulating the glutamine metabolism regulator.

[0031] In some embodiments, the metal ions in the metal-organic framework material include transition metal ions.

[0032] In some embodiments, the metal ions include transition metal divalent cations.

[0033] In some embodiments, the metal ion includes at least one selected from zinc ions, cobalt ions, platinum ions, copper ions, iron ions, or nickel ions.

[0034] In some embodiments, the metal-organic framework material is a ZIF (zeolite imidazole framework) material.

[0035] In some embodiments, the ZIF material includes at least one of ZIF-8, ZIF-90, and ZIF-78.

[0036] In some embodiments, the glutamine metabolism regulator includes at least one of a glutaminase inhibitor, a glutamine transporter inhibitor, or an exogenous glutamine limiter.

[0037] In some embodiments, the glutaminase inhibitor includes substances that reduce glutaminase expression and / or substances that reduce glutaminase activity or function.

[0038] In some embodiments, the substance that reduces glutaminase expression includes agents that inhibit or reduce the expression level of the glutaminase gene.

[0039] In some embodiments, the substance that reduces the activity or function of glutaminase includes an antibody or antigen-binding molecule thereof that specifically binds to the glutaminase and / or a small molecule compound that inhibits the activity or function of the glutaminase.

[0040] In some embodiments, the glutaminase inhibitor comprises a GLS1 inhibitor. In some embodiments, the glutaminase inhibitor comprises at least one of Telaglenastat (CB-839), BPTES, Glutaminase C-IN-1 (Compound 968), IPN60090, or a pharmaceutically acceptable salt thereof.

[0041] In some embodiments, glutamine transporter inhibitors include at least one of V-9302, GPNA, BCH, sulfasalazine, or a pharmaceutically acceptable salt thereof.

[0042] In some embodiments, the exogenous glutamine restriction includes reducing the supply of exogenous glutamine through dietary control or glutamine antagonists. In some embodiments, the glutamine antagonists include at least one of Sirpiglenastat (DPR-104), DON, JHU-083, or a pharmaceutically acceptable salt thereof.

[0043] In some embodiments, the glutaminase inhibitor and / or glutamine transporter inhibitor includes at least one of small molecule compounds, peptides, peptide mimics, matrix analogs, aptamers, siRNA (short interfering RNA), dsRNA (double-stranded RNA), miRNA (microRNA), shRNA (short hairpin RNA), ribozymes, or sgRNA (small guide RNA).

[0044] According to another aspect of the present invention, a method for preparing the drug delivery system of the present invention is provided, the method comprising the following steps: mixing a first solution containing a metal inorganic salt, an organic ligand and a first organic solvent with a second solution containing a glutamine metabolism regulator and a second organic solvent, and then reacting the mixture to produce the drug delivery system.

[0045] In some embodiments, the inorganic metal salt is selected from one or more of the chloride, nitrate, acetate, carbonate, sulfate, or perchlorate salts of the metal ion.

[0046] In some embodiments, the organic ligand includes imidazole ligands, preferably 2-methylimidazole.

[0047] In some embodiments, the first organic solvent is selected from one or more of methanol, ethanol, acetonitrile, and acetone.

[0048] In some embodiments, the second organic solvent is selected from one or more of DMSO, N,N-dimethylformamide, or N,N-dimethylacetamide.

[0049] In some embodiments, the mass ratio of the glutamine metabolism regulator to the metal inorganic salt is 1:(2-50), for example 1:2, 1:4, 1:5, 1:6, 1:8, 1:10, 1:15, 1:20, 1:25, 1:30, 1:40, 1:50 or any value between them.

[0050] Preferably, the mass ratio of the glutamine metabolism regulator to the organic ligand is 1:(5-100), for example, 1:5, 1:8, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100 or any value between them.

[0051] In some embodiments, the reaction temperature is between 10°C and 35°C, for example, 10°C, 15°C, 20°C, 25°C, 27°C, 30°C, 32°C, 35°C, or any value between them. In some embodiments, the reaction temperature is between 20°C and 30°C.

[0052] In some embodiments, the reaction time is from 0.5 hours to 24 hours, for example, 0.5 hours, 1 hour, 2 hours, 5 hours, 8 hours, 10 hours, 12 hours, 18 hours, 20 hours, 24 hours, or any value between them. In some embodiments, the reaction time is from 0.5 hours to 5 hours.

[0053] According to another aspect of the present invention, a pharmaceutical composition for the prevention and / or treatment of tumors is provided, the pharmaceutical composition comprising a glutamine metabolism regulator and metal ions, or comprising the drug delivery system of the present invention or the drug delivery system obtained by the preparation method of the present invention.

[0054] In some embodiments, the glutamine metabolism regulator includes at least one of a glutaminase inhibitor, a glutamine transporter inhibitor, or an exogenous glutamine limiter.

[0055] In some embodiments, the glutaminase inhibitor includes substances that reduce glutaminase expression and / or substances that reduce glutaminase activity or function.

[0056] In some embodiments, the substance reducing glutaminase expression includes agents that inhibit or reduce the expression level of the glutaminase gene. In some embodiments, the substance reducing the activity or function of glutaminase includes antibodies or antigen-binding molecules thereof that specifically bind to the glutaminase and / or small molecule compounds that inhibit the activity or function of the glutaminase.

[0057] In some embodiments, the glutaminase inhibitor comprises a GLS1 inhibitor. In some embodiments, the glutaminase inhibitor comprises at least one of Telaglenastat (CB-839), BPTES, Glutaminase C-IN-1 (Compound 968), IPN60090, or a pharmaceutically acceptable salt thereof.

[0058] In some embodiments, glutamine transporter inhibitors include at least one of V-9302, GPNA, BCH, sulfasalazine, or a pharmaceutically acceptable salt thereof.

[0059] In some embodiments, the exogenous glutamine restriction includes reducing the supply of exogenous glutamine through dietary control or glutamine antagonists. In some embodiments, the glutamine antagonist comprises a glutamine structural analog. In some embodiments, the glutamine antagonist comprises at least one of Sirpiglenastat (DPR-104), DON, JHU-083, or a pharmaceutically acceptable salt thereof.

[0060] In some embodiments, the glutaminase inhibitor and / or glutamine transporter inhibitor includes at least one of siRNA (short interfering RNA), dsRNA (double-stranded RNA), miRNA (microRNA), shRNA (short hairpin RNA), ribozyme, or sgRNA (small guide RNA).

[0061] In some embodiments, the metal ion includes a transition metal ion. In some embodiments, the metal ion includes a divalent transition metal cation. In some embodiments, the metal ion includes zinc ions (Zn). 2+ ), cobalt ions (Co) 2+ Platinum ions (Pt) 2+ ), copper ions (Cu) 2+ ), iron ions (Fe 2+ ) or nickel ions (Ni 2+ At least one of the following.

[0062] In some embodiments, the metal ion may be derived from at least one of the nitrate, chloride, sulfate, gluconate, acetate, lactate, citrate, fumarate, etc., of the corresponding metal ion, or its pharmaceutically acceptable hydrate or crystal form.

[0063] In some embodiments, the dosage of the glutamine metabolism regulator is such that the subject is in a state of glutamine deficiency or glutamine starvation.

[0064] In some embodiments, the amount of metal ions used is 50 μM to 500 μM, preferably 75 μM to 200 μM. In some specific embodiments, the amount of metal ions used is 50 μM, 75 μM, 100 μM, 125 μM, 150 μM, 175 μM, 200 μM, 300 μM, 400 μM, 500 μM, or any value between them.

[0065] In some embodiments, the amount of the glutamine metabolism regulator is 0.1-1000 μM, preferably 0.1-100 μM, and more preferably 0.1-20 μM. In some specific embodiments, the amount of the glutaminase metabolism regulator can be 0.1 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 5 μM, 6 μM, 8 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 75 μM, 100 μM, 125 μM, 150 μM, 175 μM, 200 μM, 300 μM, 400 μM, 500 μM, 800 μM, 1000 μM, or any value between them.

[0066] In some embodiments, the amount of the glutaminase inhibitor is 0.1-1000 μM, preferably 0.1-100 μM, and more preferably 0.1-20 μM. In some specific embodiments, the amount of the glutaminase inhibitor can be 0.1 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 5 μM, 6 μM, 8 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 75 μM, 100 μM, 125 μM, 150 μM, 175 μM, 200 μM, 300 μM, 400 μM, 500 μM, 800 μM, 1000 μM, or any value between them.

[0067] In some specific embodiments, the amount of the glutaminase inhibitor CB-839 is 0.1-20 μM, for example 0.1 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 5 μM, 6 μM, 8 μM, 10 μM, 20 μM.

[0068] In some embodiments, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0069] In some embodiments, the pharmaceutically acceptable excipients include one or more of pharmaceutical carriers, diluents, adjuvants, and excipients.

[0070] In some embodiments, the pharmaceutical composition is a tablet, capsule, solution, granule, pill, powder, ointment, pill, suspension, powder, injection, suppository, cream, spray, patch, sustained-release formulation, controlled-release formulation, or targeted formulation.

[0071] In some embodiments, the tumor includes solid tumors and hematomas. In some embodiments, the tumor includes glutamine-dependent tumors.

[0072] In some embodiments, the tumor includes at least one of liver cancer, glioma, colorectal cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, kidney cancer, melanoma, head and neck cancer, bladder cancer, central nervous system cancer, cervical cancer, endometrial cancer, leukemia, esophageal cancer, prostate cancer, or lymphoma.

[0073] In some embodiments, the glioma includes at least one of glioblastoma, astrocytoma, oligodendroglioma, or ependymoma.

[0074] According to another aspect of the present invention, a method for preventing and / or treating tumors is provided, the method comprising administering to a subject an effective amount of a glutamine metabolism regulator and metal ions.

[0075] In some embodiments, the glutamine metabolism regulator includes at least one of a glutaminase inhibitor, a glutamine transporter inhibitor, or an exogenous glutamine limiter.

[0076] In some embodiments, the glutaminase inhibitor includes substances that reduce glutaminase expression and / or substances that reduce glutaminase activity or function.

[0077] In some embodiments, the substance reducing glutaminase expression includes agents that inhibit or reduce the expression level of the glutaminase gene. In some embodiments, the substance reducing the activity or function of glutaminase includes antibodies or antigen-binding molecules thereof that specifically bind to the glutaminase and / or small molecule compounds that inhibit the activity or function of the glutaminase.

[0078] In some embodiments, the glutaminase inhibitor comprises a GLS1 inhibitor. In some embodiments, the glutaminase inhibitor comprises at least one of Telaglenastat (CB-839), BPTES, Glutaminase C-IN-1 (Compound 968), IPN60090, or a pharmaceutically acceptable salt thereof.

[0079] In some embodiments, glutamine transporter inhibitors include at least one of V-9302, GPNA, BCH, sulfasalazine, or a pharmaceutically acceptable salt thereof.

[0080] In some embodiments, the exogenous glutamine restriction includes reducing the supply of exogenous glutamine through dietary control or glutamine antagonists. In some embodiments, the glutamine antagonists include at least one of Sirpiglenastat (DPR-104), DON, JHU-083, or a pharmaceutically acceptable salt thereof.

[0081] In some embodiments, the glutaminase inhibitor and / or glutamine transporter inhibitor includes at least one of siRNA (short interfering RNA), dsRNA (double-stranded RNA), miRNA (microRNA), shRNA (short hairpin RNA), ribozyme, or sgRNA (small guide RNA).

[0082] In some embodiments, the metal ion includes a transition metal ion. In some embodiments, the metal ion includes a divalent transition metal cation. In some embodiments, the metal ion includes zinc ions (Zn). 2+ ), cobalt ions (Co) 2+ Platinum ions (Pt) 2+ ), copper ions (Cu)2+ ), iron ions (Fe 2+ ) or nickel ions (Ni 2+ At least one of the following.

[0083] In some embodiments, the metal ion may be derived from at least one of the corresponding metal ion's hydrochloride, sulfate, phosphate, nitrate, gluconate, acetate, lactate, citrate, fumarate, etc., or their pharmaceutically acceptable hydrates or crystal forms.

[0084] In some embodiments, the dosage of the glutamine metabolism regulator is such that the subject is in a state of glutamine deficiency or glutamine starvation.

[0085] In some embodiments, the amount of the glutamine metabolism regulator is 0.1-1000 μM, preferably 0.1-100 μM, and more preferably 0.1-20 μM. In some specific embodiments, the amount of the glutaminase metabolism regulator can be 0.1 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 5 μM, 6 μM, 8 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 75 μM, 100 μM, 125 μM, 150 μM, 175 μM, 200 μM, 300 μM, 400 μM, 500 μM, 800 μM, 1000 μM, or any value between them.

[0086] In some embodiments, the amount of the glutaminase inhibitor is 0.1-1000 μM, preferably 0.1-100 μM, and more preferably 0.1-20 μM. In some specific embodiments, the amount of the glutaminase inhibitor can be 0.1 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 5 μM, 6 μM, 8 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 75 μM, 100 μM, 125 μM, 150 μM, 175 μM, 200 μM, 300 μM, 400 μM, 500 μM, 800 μM, 1000 μM, or any value between them.

[0087] In some specific embodiments, the amount of the glutaminase inhibitor CB-839 is 0.1-20 μM, for example 0.1 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 5 μM, 6 μM, 8 μM, 10 μM, 20 μM.

[0088] In some embodiments, the amount of metal ions used is 50 μM to 500 μM, preferably 75 μM to 200 μM. In some specific embodiments, the amount of metal ions used is 50 μM, 75 μM, 100 μM, 125 μM, 150 μM, 175 μM, 200 μM, 300 μM, 400 μM, 500 μM, or any value between them.

[0089] In some embodiments, the glutamine metabolism regulator and the metal ion are administered simultaneously or sequentially.

[0090] In some embodiments, the prevention and / or treatment of tumors includes at least one of inhibiting tumor cell proliferation, killing tumor cells, inhibiting tumorigenesis, reducing glutathione levels in tumor cells, inducing oxidative stress in tumor cells, or remodeling the tumor microenvironment in which immune activation and metabolic disorders coexist.

[0091] In some embodiments, the tumor includes solid tumors and hematomas. In some embodiments, the tumor includes glutamine-dependent tumors.

[0092] In some embodiments, the tumor includes at least one of liver cancer, glioma, colorectal cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, kidney cancer, melanoma, head and neck cancer, bladder cancer, central nervous system cancer, cervical cancer, endometrial cancer, leukemia, esophageal cancer, prostate cancer, or lymphoma.

[0093] In some embodiments, the glioma includes at least one of glioblastoma, astrocytoma, oligodendroglioma, or ependymoma. Attached Figure Description

[0094] Figure 1 The crystal violet staining results of different tumor cells treated with zinc ions in combination with different culture media according to some embodiments of the present invention.

[0095] Figure 2 The present invention provides statistical results of CCK8 cell experiments involving zinc ion treatment in combination with different culture media for different tumor cells according to some embodiments of the present invention. Specifically, A represents the results for LN229 glioma cells (ZnCl2 125 μM), B represents the results for LN18 glioma cells (ZnCl2 125 μM), C represents the results for HT29 colon cancer cells (ZnCl2 150 μM), D represents the results for ASPC pancreatic cancer cells (ZnCl2 125 μM), E represents the results for huh7 liver cancer cells (ZnCl2 150 μM), and F represents the results for hepG2 liver cancer cells (ZnCl2 150 μM).

[0096] Figure 3The WB results of HuH-7 cell line treated with zinc ions in combination with different culture media according to some embodiments of the present invention.

[0097] Figure 4 The images show crystal violet staining results of different tumor cells treated with zinc ions in combination with different culture media and CB-839 drug in some embodiments of the present invention. Specifically, A represents LN229 glioma cells, B represents LN18 glioma cells, C represents HT29 colon cancer cells, D represents ASPC pancreatic cancer cells, E represents huh7 liver cancer cells, and F represents hepG2 liver cancer cells.

[0098] Figure 5 The WB results of HuH-7 cell lines under zinc ion treatment combined with different culture media treatment and CB-839 drug treatment are presented according to some embodiments of the present invention.

[0099] Figure 6 Transmission electron microscope images of ZIF8 and CB-839@ZIF8 according to some embodiments of the present invention.

[0100] Figure 7 The images show crystal violet staining results of different tumor cells treated with ZIF8 and CB-839@ZIF8 in combination with different culture media according to some embodiments of the present invention. A represents the results for LN229 glioma cells, B represents the results for LN18 glioma cells, C represents the results for HT29 colon cancer cells, D represents the results for ASPC pancreatic cancer cells, E represents the results for huh7 liver cancer cells, and F represents the results for hepG2 liver cancer cells.

[0101] Figure 8 The images show crystal violet staining results of different tumor cells treated with ZIF8 and CB-839@ZIF8 in combination with different culture media according to some embodiments of the present invention. A represents the results for LN18 glioma cells, and B represents the results for huh7 liver cancer cells.

[0102] Figure 9 The WB results of ZIF8 and CB-839@ZIF8 treatment of huh7 cell lines in different culture media are presented according to some embodiments of the present invention. Detailed Implementation

[0103] Abnormally active cell proliferation is one of the prominent characteristics of tumors. To achieve rapid proliferation, in addition to requiring continuous proliferation signals and evading growth inhibition, tumor cells also need to alter their metabolic pathways to provide sufficient substances and energy for cell division. These alterations in tumor cell metabolic pathways are mainly mediated by the abnormal expression and activation of metabolic enzymes. It is generally believed that these metabolic enzymes primarily participate in cell cycle regulation by providing metabolites. However, increasing evidence in recent years suggests that the non-canonical functions of metabolic enzymes can regulate a variety of complex cellular activities and the development of diseases.

[0104] Glutamine is a major nutrient that tumor cells take up from the tumor microenvironment, promoting their proliferation. Besides tumor cells, immune cells (macrophages and lymphocytes) also require large amounts of glutamine to maintain their proliferation and immune response. When tumor cells' utilization of glutamine is blocked, T cells tend to exhibit high proliferation and a longevity phenotype, thereby inhibiting immune evasion. Glutaminase (GLS), as the key rate-limiting enzyme in glutamine catabolism, catalyzes the hydrolysis of glutamine into glutamate, providing the necessary carbon source for the rapid proliferation of malignant tumors to drive the tricarboxylic acid cycle, and also maintaining intracellular redox homeostasis by supporting glutathione biosynthesis.

[0105] CB-839 (Telaglenastat), a first-in-class, highly selective, orally administered small-molecule GLS1 allosteric inhibitor, works by specifically binding to the allosteric site of the active GLS1 tetramer, blocking substrate metabolic flux and thus inducing lethal oxidative stress while cutting off tumor energy supply. Current consensus in the academic community is that although CB-839 exhibits potent antitumor activity in c-Myc-driven or KEAP1 / NRF2-mutated tumor models, its clinical application is focusing on precisely overcoming drug resistance and improving therapeutic gain through combination therapy with targeted therapy or immunotherapy due to the metabolic compensation mechanisms of the tumor microenvironment.

[0106] This invention reveals that innate immune signaling and metabolic disorders induce glutathione depletion in tumor cells under glutamine starvation, coupled with zinc ion treatment, leading to cellular oxidative stress. Therefore, glutamine starvation combined with zinc ion treatment can create a tumor microenvironment characterized by both immune activation and metabolic disorders. This therapeutic activation of innate immunity and nutritional restriction synergistically promotes tumor cell death.

[0107] Exemplary experimental results of this invention show that zinc ion superimposed on glutamine deficiency can inhibit the death of various tumor cells, including glioma, liver cancer, colorectal cancer, and pancreatic cancer. This type of cell death can be reversed by the zinc ion chelator EDTA, glutamine replenishment, or the addition of the antioxidant acetylcysteine ​​(NAC).

[0108] Numerous specific details are set forth in the following description to provide a full understanding of the invention. However, the invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0109] The terminology used in one or more embodiments of this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the one or more embodiments of this specification. The singular forms “a,” “described,” and “the” as used in one or more embodiments of this specification and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used in one or more embodiments of this specification refers to and includes any or all possible combinations of one or more associated listed items.

[0110] It should be understood that although the terms first, second, etc., may be used to describe various information in one or more embodiments of this specification, such information should not be limited to these terms. These terms are only used to distinguish information of the same type from one another. For example, first may also be referred to as second without departing from the scope of one or more embodiments of this specification, and similarly, second may also be referred to as first. Depending on the context, the word "if" as used herein may be interpreted as "when," "when," or "in response to a determination."

[0111] The term "multiple" can refer to two or more, and "at least one" can refer to one, two or more.

[0112] The term "and / or" is merely a description of the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. Additionally, in this invention, the character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0113] The term "glutaminase (GLS)" refers to a key intracellular enzyme responsible for hydrolyzing glutamine (Gln) into glutamate and ammonia. It is the rate-limiting enzyme in the first step of glutamine metabolism. There are two types of GLS: GLS1 and GLS2.

[0114] The term "glutaminase inhibitor" refers to a small molecule compound or biological agent that selectively inhibits the activity of glutaminase, thereby blocking the catabolism of glutamine by inhibiting glutaminase. In some embodiments, the glutaminase inhibitor includes substances that reduce glutaminase expression and / or substances that reduce glutaminase activity or function.

[0115] In some embodiments, the substance reducing glutaminase expression includes agents that inhibit or reduce the expression level of the glutaminase gene. In some embodiments, the substance reducing glutaminase expression is selected from at least one of the following (i)-(vi): (i) agents that inhibit the transcription of the glutaminase gene into mRNA; (ii) agents that block or inhibit the translation level of glutaminase mRNA; (iii) agents that promote the degradation of glutaminase mRNA; (iv) agents that specifically recognize and cleave the guide nucleic acid of the glutaminase gene; (v) agents for partially or completely knocking out the glutaminase gene; and (vi) agents for reducing the level of glutaminase protein. In some embodiments, the substance reducing glutaminase expression includes substances that silence, knock down, or knock out the glutaminase gene. In some embodiments, the reagents for reducing glutaminase expression include: antisense oligonucleotides (e.g., ASO), small interfering RNA (siRNA), microRNA (miRNA), double-stranded RNA (dsRNA), small hairpin RNA (shRNA), nucleic acid aptamers, ribozymes, and gene editors. In some embodiments, the gene editor includes a zinc finger protein gene editing system, a TALEN gene editing system, or a CRISPR / Cas9 gene editing system.

[0116] In some embodiments, the substance that reduces the activity or function of glutaminase includes: an antibody or antigen-binding molecule thereof that specifically binds to the glutaminase and / or a small molecule compound that inhibits the activity or function of the glutaminase.

[0117] In some embodiments, the glutaminase inhibitor comprises a GLS1 inhibitor. In some embodiments, the glutaminase inhibitor comprises at least one of Telaglenastat (CB-839), BPTES, Glutaminase C-IN-1 (Compound 968), IPN60090, or a pharmaceutically acceptable salt thereof.

[0118] The structural formula for Telaglenastat (CB-839) is shown below: .

[0119] The structural formula for BPTES is shown below: .

[0120] The structural formula of Glutaminase C-IN-1 (Compound 968) is shown below: .

[0121] The structural formula of IPN60090 is shown below: .

[0122] The term "glutamine transporter inhibitor" refers to small molecules or biological agents that target glutamine transporter proteins (such as ASCT2 / SLC1A5, SLC38A1 / A2, SLC7A11) on the cell membrane, thereby blocking glutamine transport and preventing cells from taking up glutamine.

[0123] In some embodiments, glutamine transporter inhibitors include at least one of V-9302, GPNA, BCH, sulfasalazine, or a pharmaceutically acceptable salt thereof.

[0124] The structural formula of V-9302 is shown below: .

[0125] The structural formula of GPNA is shown below: .

[0126] The structural formula of BCH is shown below: .

[0127] The structural formula of sulfasalazine is shown below: .

[0128] The term "exogenous glutamine restriction" refers to reducing the supply of exogenous glutamine through dietary control, environmental control, or glutamine antagonists, without completely cutting off supplementation, while maintaining a baseline glutamine level required for basal physiological metabolism. Glutamine antagonists are small molecules or biological agents with structures similar to glutamine that can competitively bind to the active sites of glutamine's metabolic enzymes or transport proteins, thereby blocking glutamine from participating in various downstream biological functions.

[0129] In some embodiments, the glutamine antagonist includes at least one of Sirpiglenastat (DPR-104), DON, JHU-083, or a pharmaceutically acceptable salt thereof.

[0130] The structure of Sirpiglenastat (DPR-104) is shown below: .

[0131] The structural formula of DON (6-diazo-5-oxonorleucine, L-6-Diazo-5-oxonorleucine) is shown below: .

[0132] The structural formula of JHU-083 is shown below: .

[0133] The term "glutamine deficiency" as used herein refers to a decrease in glutamine concentration, but not necessarily a complete drop to zero; it primarily refers to a physiological / pathological state in vivo, but can also refer to low-concentration conditions in vitro. In some embodiments, glutamine deficiency means that the subject's plasma glutamine concentration is below or approximately the lower limit of normal plasma glutamine concentration. Typically, the fasting plasma glutamine concentration in adults ranges from approximately 400 to 800 μmol / L (the specific range varies depending on the laboratory). In some embodiments, glutamine deficiency means that the subject's plasma glutamine concentration is below or approximately 400 μM (where "approximately" means a relative deviation from 400 μM not exceeding 5%), for example, approximately 400 μM, below 400 μM, below 350 μM, below 300 μM, below 250 μM, below 200 μM, below 150 μM, below 100 μM, below 50 μM, below 25 μM, below 20 μM, below 15 μM, below 10 μM, or below 5 μM. The term “glutamine starvation” used in this article refers to the near-complete removal of glutamine from the extracellular environment (0 mMGln), primarily in in vitro cell experimental models.

[0134] As used herein, the term "pharmaceuticalally acceptable excipient" refers to a carrier, diluent, adjuvant, and / or excipient used in a drug product that must be compatible with the other components of the formulation and harmless to the recipient. Some examples of materials that can serve as pharmaceutically acceptable excipients include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; malt; gelatin and other non-toxic, compatible lubricants such as sodium lauryl sulfonate and magnesium stearate; as well as colorants; release agents; coating agents; sweeteners; flavorings and fragrances; preservatives and antioxidants, which may also be used within the drug product at the discretion of the formulation engineer.

[0135] The term “subject” as used herein includes, but is not limited to, human and / or non-human animals, such as mammals like mice, rats, primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and / or dogs. The terms “human,” “patient,” and “subject” may be used interchangeably herein.

[0136] The term "prevention" as used in this article refers to preventive treatment of subclinical disease states, aimed at reducing the probability of clinical disease states occurring. "Prevention" can be divided into (a) primary prevention and (b) secondary prevention. Primary prevention is defined as treatment of subjects who have not yet presented with a clinical disease state, while secondary prevention is defined as prevention of the recurrence of the same or similar clinical disease state.

[0137] As used in this article, the term “treatment” refers to the treatment of a disease, symptom, or condition, including: a) suppressing the development of a disease, symptom, or condition; and / or, b) delaying or alleviating a disease, symptom, or condition.

[0138] As used herein, the term "effective amount" refers to a quantity sufficient to elicit a target biological response. As will be understood by those skilled in the art, the effective amount of the medicament of this invention can vary depending on factors such as the biological target, the pharmacokinetics of the drug, the disease being treated, the administration method, and the age, health status, and symptoms of the subject. Effective amounts include therapeutic effective amounts and prophylactic effective amounts.

[0139] The drugs provided by this invention can be administered via many routes, including but not limited to: oral administration, parenteral administration, inhalation administration, local administration, rectal administration, nasal administration, oral administration, administration via implantation, or other administration methods. For example, parenteral administration as used herein includes subcutaneous administration, intradermal administration, intravenous administration, intramuscular administration, intra-articular administration, intra-arterial administration, intra-synovial administration, intrasternal administration, or infusion techniques, etc.

[0140] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0141] Example 1. Materials The following examples involve the following chemical reagents: ZnCl2 (Sigma, 429430), CB-389 (Aladdin, C174237), Acetylcysteine ​​(MCE, HY-B0215), L-glutamine (Sigma, G8540-25G), EDTA (Sigma), Crystal Violet (Maclean, C805209), Non-Fat Powdered Milk (BBI, A600669), Anhydrous Methanol (Sinopharm Reagent, 800804190), CCK8 (Fode Biotechnology, FD3788), Zinc Nitrate Hexahydrate (Sigma-Aldrich, 228737-500G), 2-Methylimidazole (Sigma-Aldrich, M50850-500G), Concentrated Nitric Acid (aladdin, N433826-2.5L).

[0142] The WB experiments in the following examples involve the following primary antibodies: Pan-Akt (C67E7) Rabbit Monoclonal Antibody: Akt (pan) (C67E7) Rabbit Monoclonal Antibody (Cell Signaling Technology, 4691S), p-Akt (Ser473) (D9E) Rabbit Monoclonal Antibody: Phospho-Akt (Ser473) (D9E) Rabbit Monoclonal Antibody (Cell Signaling Technology, 4060S), TBK1 / NAK (D1B4) Rabbit Monoclonal Antibody: TBK1 / NAK (D1B4) Rabbit Monoclonal Antibody (Cell Signaling Technology, 3504S), p-TBK1 / NAK (Ser172) (D52C2) Rabbit Monoclonal Antibody: Phospho-TBK1 / NAK (Ser172) (D52C2) Rabbit Monoclonal Antibody (Cell Signaling Technology, 4691S), p-Akt (Ser473) (D9E) Rabbit Monoclonal Antibody (Cell Signaling Technology, 4060S), p-TBK1 / NAK (D1B4) Rabbit Monoclonal Antibody: ...691S), p-Akt (Ser473) (D9E) Rabbit Monoclonal Antibody (Cell Signaling Technology, 469 Signaling Technology, 5483S), β-actin (13E5) rabbit monoclonal antibody: beta-Actin (13E5) Rabbit Monoclonal Antibody (Cell Signaling Technology, 4970S), β-Tubulin Mouse Monoclonal Antibody: β-Tubulin Mouse mAb (ABCLONAL, AC021).

[0143] The WB experiments in the following examples involve the following secondary antibodies: Secondary antibodies: horseradish peroxidase conjugated goat anti-rabbit IgG (HRP-linked Antibody) (Cell Signaling Technology, 7074), horseradish peroxidase conjugated goat anti-mouse IgG (H+L) (abclonal, AS003).

[0144] The following examples involve the following cell culture media and culture reagents: DMEM basic high-glucose medium (gibco, C11995500BT), DMEM high-glucose glutamine-free medium (gibco, 11960-044), RPMI Medium 1640 basic medium (gibco, C11875500BT), McCoy's 5A (containing GlutaPlus, containing HEPES) (Seville, G4541), trypsin (gibco, 25200-072), penicillin-streptomycin (gibco, 15140-122), serum (gibco, 10091148), dialysis fetal bovine serum (FBS) Dialyzed (LMAI Bio, LM1055), PBS (Seville, G4202).

[0145] Low-glutamine medium (0.5 mM Gln medium): For example, add 250 μL of 200 mM L-glutamine (sigma, G8540) stock solution to DMEM high-glucose glutamine-free (gibco, 11960-044) medium, and finally bring the volume up to 100 mL.

[0146] Electrophoresis buffer (10× stock solution, 1L): Tris base: 30.2 g, glycine: 188 g, SDS: 10 g, add distilled water to a final volume of 1L, and stir until completely dissolved. Usage concentration (1×): Take 100 mL of the 10× stock solution and dilute with 900 mL of distilled water.

[0147] Transfer buffer (10× stock solution, 1L): Tris base: 29g, glycine: 144g, add ddH2O to a final volume of 1L, stir until completely dissolved. Usage concentration (1×): Take 100 mL of 10× stock solution, add 700 mL of ddH2O, then add 200 mL of methanol, mix well, and use.

[0148] Crystal violet solution: 250 mL ddH2O, 250 mL anhydrous methanol, 2.5 g crystal violet, mix well and set aside at room temperature.

[0149] 5% skim milk powder: Weigh 2.5 g of skim milk powder using a balance, add PBST solution to make up to 50 mL, vortex to mix well and set aside.

[0150] 2. Cell Culture Cell lines: LN229 cells, LN18 cells, HepG2 cells, HuH-7 cells, HT29 cells, ASPC cells, etc.

[0151] Culture conditions: Cells were cultured in a medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution at 37°C in a 5% CO2 incubator. LN229 cells, HepG2 cells, LN18 cells, and HuH-7 cells were cultured using DMEM basic; HT29 cells were cultured using McCoy's 5A; and ASPC cells were cultured using RPMI Medium 1640 basic.

[0152] Cell passage: Once cells reach 80%-90% confluence, passage them. Digest the cells using a 0.05% trypsin-EDTA solution to separate them. After digestion, neutralize the trypsin with FBS-containing medium, then centrifuge and discard the supernatant. Resuspend the cells in fresh medium and seed them into new culture containers for further culture.

[0153] Cell cryopreservation and thawing: When cells reach full growth potential, they are cryopreserved using cryopreservation solution. Cryopreserved cells are placed at -80°C overnight, then transferred to liquid nitrogen for long-term storage. For thawing, the cryovials are rapidly heated in a 37°C water bath until completely thawed, then immediately transferred to culture medium containing FBS. The cryopreservation solution is removed by centrifugation, and the cells are then cultured in fresh culture medium.

[0154] 3. Western Blot Experiment (1) Sample preparation: Extract proteins from cells, lyse the samples using a lysis buffer (such as RIPA) containing protease inhibitors and phosphatase inhibitors, and determine the protein concentration by the BCA method to ensure consistent protein loading.

[0155] (2) SDS-PAGE electrophoresis: Add an equal amount of protein sample to a polyacrylamide gel and separate the proteins according to their molecular weight using an electric field.

[0156] (3) Transfer: The protein in the gel was transferred to the PVDF membrane by wet transfer at 90V for 90min.

[0157] (4) Blocking and antibody incubation: Block the membrane with 5% skim milk powder to reduce non-specific binding. Add specific primary antibody and incubate overnight on a shaker (4°C). Then, wash three times with TBST for 10 min each time. Incubate with secondary antibody (room temperature) on a shaker for 60 min. Detect the target protein through antigen-antibody specific reaction. Finally, wash three times with TBST.

[0158] (5) Signal detection and analysis: After incubation with HRP-labeled secondary antibody, protein bands were developed and detected using ECL chemiluminescent reagent. Finally, Western blotting images were acquired using an Amersham Imager 680 series ultrasensitive multi-functional imager.

[0159] 4. CCK8 test (1) Cell seeding: After digesting and counting the logarithmic growth phase cells, seed them at an appropriate density (7000 cells / well) in a 96-well plate with 100 μL of cell suspension per well. Incubate at 37°C in a 5% CO2 incubator for 24 hours.

[0160] (2) Drug treatment: According to the experimental design, different concentrations of the test drug or compound were added to each well and cultured for 12-60 h.

[0161] (3) CCK-8 detection: After the cells are cultured as required, the original culture medium is removed, and then CCK-8-cell culture medium mixture (containing 10% CCK-8) is added to each well. The cells are incubated at 37°C in the dark for 0.5-1h to allow the reaction to proceed fully.

[0162] (4) Absorbance measurement: The absorbance (OD value) of each well was measured at a wavelength of 450 nm using an ELISA reader.

[0163] (5) Data processing Cell viability is calculated using a formula. Cell viability = [(OD value of experimental wells - OD value of blank wells) / (OD value of control wells - OD value of blank wells)] × 100% 5. Crystal violet cell viability assay Cell seeding: Cells are evenly seeded in 24 or 48-well plates and cultured adherently for 12 h to allow the cells to reach 80-90% confluence.

[0164] Drug treatment: According to the experimental design, the cells were grouped and treated accordingly, and incubated at 37°C and 5% CO2 for 12-24 hours.

[0165] Cell cloning experiment: Discard the culture medium, add PBS to each well to wash the cells once, then add 1 mL of crystal violet solution for fixation and staining, shake on a shaker at room temperature for 30 min, discard the crystal violet solution, wash off excess staining agent several times with ddH2O, dry in a 65℃ oven, and finally scan and compare the cell counts between different experimental groups.

[0166] Example 1 Cell culture: Glioma cells (LN229, LN18), colon cancer cells (HT29), pancreatic cancer cells (ASPC), and liver cancer cells (HuH-7, HepG2) were cultured according to the methods described in "Materials and Methods" section "2. Cell Culture". Cell seeding: Cells were evenly seeded into 24 / 48-well plates and cultured adherently for 12 h to achieve 80-90% confluence.

[0167] Drug treatment: According to the experimental design, cells that have reached 80-90% confluence are grouped as follows and treated accordingly, and incubated at 37℃ and 5% CO2 for 24-60 hours (the treatment varies depending on the cell line).

[0168] (1) Normal complete culture medium group: control, ZnCl2 (75μM), ZnCl2 (100μM), ZnCl2 (125μM), ZnCl2 (150μM); (2) Glutamine starvation culture medium group (No Glutamine): control, ZnCl2 (75 μM), ZnCl2 (100 μM), ZnCl2 (125 μM), ZnCl2 (150 μM); (3) Glutamine replenishment culture medium group (No Glutamine + L-Glutamine): control, ZnCl2 (75μM), ZnCl2 (100μM), ZnCl2 (125μM); (4) Glutamine starvation supplemented with NAC medium group (No Glutamine + NAC): control, ZnCl2 (75 μM), ZnCl2 (100 μM), ZnCl2 (125 μM); (5) Glutamine starvation culture medium supplemented with EDTA (No Glutamine + EDTA): control, ZnCl2 (75 μM), ZnCl2 (100 μM), ZnCl2 (125 μM).

[0169] Among them, the normal complete culture medium group used DMEM basic high glucose medium with serum, the glutamine starvation culture medium group used DMEM high glucose glutamine-free medium with dialyzed fetal bovine serum, the glutamine replenishment culture medium group used L-glutamine directly added to the glutamine starvation culture medium (final concentration of L-glutamine was 2mM), and the glutamine starvation inhibitor group used EDTA or NAC directly added to the glutamine starvation culture medium (final concentration of EDTA was 20μM, and final concentration of NAC was 4mM).

[0170] The results of the crystal violet cell survival experiment are as follows Figure 1 As shown. Figure 1 This indicates that zinc ion supplementation combined with glutamine deficiency or starvation can inhibit the death of various tumor cells, including glioma, liver cancer, colorectal cancer, and pancreatic cancer. Specifically, compared with the normal complete culture medium group, tumor cell death was significantly aggravated with increasing ZnCl2 concentration in the glutamine-starved culture medium group. Furthermore, the addition of glutamine supplementation, zinc ion chelators, or antioxidants further validated that zinc ion supplementation combined with glutamine deficiency or starvation can enhance the killing effect on tumor cells.

[0171] The results of the CCK8 test are as follows: Figure 2 As shown. With Figure 1 Similarly, Figure 2 The results from the study indicate that glutamine deficiency or starvation makes tumor cells highly sensitive to the toxicity of zinc ions, and that glutamine deficiency or starvation combined with zinc ions can enhance the killing effect on tumor cells.

[0172] Western blot results of HuH-7 cells are as follows: Figure 3 As shown, p-TBK1 S172 represents TBK1 kinase activation, a key molecule in innate immunity and cellular stress responses, while p-AKT S473 represents AKT / mTORC2 pathway activation, a core pathway regulating cell survival and metabolism. Western blot results in HuH-7 cells indicate that glutamine deficiency or starvation is a prerequisite for zinc ion-induced activation of the TBK1 and AKT pathways. Only under metabolic stress of glutamine deficiency or starvation can zinc ions dose-dependently activate p-TBK1 S172 and p-AKT S473. The regulatory trends of p-TBK1 S172 and p-AKT S473 in other tumor cell lines are consistent.

[0173] Example 2 Cell seeding: Cells were evenly seeded in 24-well plates and cultured adherently for 12 h to achieve 90-100% confluence.

[0174] Drug treatment: According to the experimental design, the cells were grouped as follows and treated accordingly, and incubated at 37°C and 5% CO2 for 8-24 hours (the incubation time varies depending on the cell line).

[0175] (1) Normal complete culture medium group: control, CB-839 (4μM), ZnCl2 (100μM or 125μM), Zn (100μM or 125μM) + CB-839 (4μM); (2) Glutamine starvation culture medium group (No Glutamine): control, CB-839 (4μM), ZnCl2 (100μM or 125μM), Zn (100μM or 125μM) + CB-839 (4μM); (3) Glutamine starvation supplemented with NAC medium group (No Glutamine + NAC): control, CB-839 (4μM), ZnCl2 (100μM or 125μM), Zn (100μM or 125μM) + CB-839 (4μM); (4) Glutamine starvation culture medium supplemented with EDTA (No Glutamine + EDTA): control, CB-839 (4μM), ZnCl2 (100μM or 125μM), Zn (100μM or 125μM) + CB-839 (4μM).

[0176] Among them, the normal complete culture medium group used DMEM basic high-glucose medium, the glutamine starvation medium group used DMEM high-glucose glutamine-free medium, and the glutamine starvation inhibitor group used EDTA or NAC directly added to the glutamine starvation medium (EDTA final concentration 20 μM, NAC final concentration 4 mM).

[0177] The results of crystal violet cell survival experiments on various tumor cells treated with zinc ions and CB-839 were as follows: Figure 4 As shown. Figure 4 The results from the study showed that, based on the fact that zinc ions combined with glutamine deficiency or starvation can inhibit the death of various tumor cells, treatment with lower concentrations of zinc ions combined with CB-839 can achieve better killing effects.

[0178] Western blot results of HuH-7 cells are as follows: Figure 5 As shown, zinc alone can increase phosphorylation at the TBK1 S172 site, promoting the cGas-STING signaling pathway, and the addition of CB-839 can further promote this innate immune activation pathway. Other tumor cell lines also showed similar effects.

[0179] Example 3 (1) Synthesis of ZIF-8 Weigh 297.5 mg of zinc nitrate hexahydrate and 660 mg of 2-methylimidazole, and dissolve them separately in 11.3 mL of methanol. Mix the two solutions and stir continuously at room temperature for 1 hour. Centrifuge (10,000 rpm for 10 minutes), wash, and collect the sample to obtain ZIF-8.

[0180] (2) Synthesis of CB-839@ZIF-8 drug delivery system Weigh 297.5 mg of zinc nitrate hexahydrate and 660 mg of 2-methylimidazole, and dissolve them separately in 11.3 mL of methanol. Dissolve 20 mg of CB-839 in 0.5 mL of DMSO. Mix the three solutions and stir continuously at room temperature for 1 hour. Centrifuge (10,000 rpm for 10 minutes), wash, and collect the sample to obtain CB-839@ZIF8.

[0181] Transmission electron microscopy experiment: The synthesized ZIF-8 and CB-839@ZIF-8 samples were diluted and dropped onto a carbon film. After drying in a cool place, the particle size and morphology of the samples were observed using a transmission electron microscope (HT7700). Figure 6 The results showed that CB-839@ZIF-8 has a crystalline morphology and a particle size significantly larger than ZIF-8, which to some extent corroborates the successful preparation of the drug delivery system CB-839@ZIF-8.

[0182] Determination of CB839 concentration: UV standard curve: Prepare solutions of different concentrations (5, 10, 15, 20, 25 μg / ml) and plot the standard curve. Measure the concentration of the supernatant using a UV spectrophotometer, and then calculate the CB839 encapsulation efficiency: Encapsulation efficiency = (W... 投药量 -W 上清 ) / W 投药量 The results showed that the CB-839 encapsulation rate of CB-839@ZIF-8 was 22.8%.

[0183] Example 4 The cell-killing effect of the CB-839@ZIF-8 drug delivery system was tested in a manner similar to that in Example 2. Details are shown below.

[0184] Cell seeding: Cells were evenly seeded in 48-well plates and cultured adherently for 12 hours to allow the cells to reach 80-90% confluence.

[0185] Drug treatment: According to the experimental design, the cells were grouped as follows and treated accordingly, and incubated at 37°C and 5% CO2 for 8-24 hours (the incubation time varies depending on the cell line).

[0186] (1) Normal complete culture medium group: CB-839 (4μM), ZnCl2 (100μM or 125μM), Zn (100μM or 125μM) + CB-839 (4μM), ZIF-8 (with Zn 2+ (based on 100μM or 125μM), CB-839@ZIF-8 (in Zn 2+ (Based on 100μM or 125μM). (2) Glutamine starvation culture medium group (No Glutamine): CB-839 (4μM), ZnCl2 (100μM or 125μM), Zn (100μM or 125μM) + CB-839 (4μM), ZIF-8 (with Zn 2+ (based on 100μM or 125μM), CB-839@ZIF-8 (in Zn 2+ (Based on 100μM or 125μM). (3) Low Glutamine medium group: CB-839 (4μM), ZnCl2 (100μM or 125μM), Zn (100μM or 125μM) + CB-839 (4μM), ZIF-8 (with Zn) 2+ (based on 100μM or 125μM), CB-839@ZIF-8 (in Zn 2+ (Based on 100μM or 125μM).

[0187] The normal complete culture medium group used DMEM basic high-glucose medium, the glutamine starvation medium group used DMEM high-glucose glutamine-free medium, and the low-glutamine medium group used low-glutamine medium (0.5 mM Gln medium). For the normal complete culture medium group and the glutamine starvation medium group, the ZnCl2 concentration was 100 μM for LN229 glioma cells, 100 μM for LN18 glioma cells, 125 μM for HT29 colon cancer cells, 100 μM for ASPC pancreatic cancer cells, 125 μM for huh7 liver cancer cells, and 100 μM for hepatocellular carcinoma cells. For the low-glutamine medium group, the ZnCl2 concentration was 135 μM for LN18 glioma cells and 140 μM for huh7 liver cancer cells.

[0188] The results of crystal violet cell survival experiments using the CB-839@ZIF-8 drug delivery system to treat various tumor cells are as follows: Figure 7 and Figure 8 As shown. Figure 7 AF and Figure 8 The results from the study indicate that, under conditions of glutamine deficiency or glutamine starvation, the ZIF-8-loaded CB-839 drug delivery system can effectively kill various types of tumor cells.

[0189] Figure 9 Western blot analysis of HuH-7 cells showed that both ZIF-8 and CB-839@ZIF-8 activated p-TBK1 S172 and p-AKT S473 under glutamine deficiency or starvation metabolic stress. ZIF-8 alone can activate the p-TBK1 phosphorylated cGAS-STING signaling pathway, and CB-839@ZIF-8 with added CB-839 can further promote this innate immune activation pathway. Other tumor cell lines also showed similar effects.

[0190] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make possible changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope defined in the claims of the present invention.

Claims

1. The application of the combined use of glutamine metabolism regulators and metal ions in the preparation of drugs for the prevention and / or treatment of tumors.

2. The application according to claim 1, characterized in that, The glutamine metabolism regulator includes at least one of glutaminase inhibitors, glutamine transporter inhibitors, or exogenous glutamine restriction. Preferably, the glutaminase inhibitor includes substances that reduce glutaminase expression and / or substances that reduce glutaminase activity or function; more preferably, the glutaminase inhibitor includes a GLS1 enzyme inhibitor; even more preferably, the glutaminase inhibitor includes at least one of CB-839, BPTES, Compound 968, IPN60090, or a pharmaceutically acceptable salt thereof. Preferably, the glutamine transporter inhibitor includes at least one of V-9302, GPNA, BCH, sulfasalazine, or a pharmaceutically acceptable salt thereof; Preferably, the exogenous glutamine restriction includes reducing exogenous glutamine supply through dietary control or glutamine antagonists; more preferably, the glutamine antagonist includes at least one of DPR-104, DON, JHU-083, or a pharmaceutically acceptable salt thereof; and / or The metal ions include transition metal ions; preferably, the metal ions include divalent transition metal cations; more preferably, the metal ions include at least one of zinc ions, cobalt ions, platinum ions, copper ions, iron ions, or nickel ions.

3. The application according to claim 1 or 2, characterized in that, The dosage of the glutamine metabolism regulator is such that the subject is in a state of glutamine deficiency or glutamine starvation; preferably, the dosage of the glutamine metabolism regulator is 0.1-1000 μM, more preferably 0.1-100 μM, and even more preferably 0.1-20 μM; and / or The amount of the metal ions used is 50 μM - 500 μM, preferably 75 μM - 200 μM, and / or The glutamine metabolism regulator and the metal ions are administered simultaneously or sequentially. Preferably, the glutamine metabolism regulator and the metal ions are administered in combination via a metal-organic framework delivery system; More preferably, the metal-organic framework material encapsulates the glutamine metabolism regulator; More preferably, the metal-organic framework material is a ZIF material; More preferably, the ZIF material includes at least one of ZIF-8, ZIF-90, and ZIF-78.

4. The application according to any one of claims 1-3, characterized in that, The prevention and / or treatment of tumors includes at least one of the following: inhibiting tumor cell proliferation, killing tumor cells, inhibiting tumorigenesis, reducing glutathione levels in tumor cells, inducing oxidative stress in tumor cells, or remodeling the tumor microenvironment in which immune activation and metabolic disorders coexist; and / or The tumor includes solid tumors and hematologic malignancies; preferably, the tumor includes glutamine-dependent tumors; more preferably, the tumor includes at least one of liver cancer, glioma, colorectal cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, kidney cancer, melanoma, head and neck cancer, bladder cancer, central nervous system cancer, cervical cancer, endometrial cancer, leukemia, esophageal cancer, prostate cancer, or lymphoma; and / or The regulation of glutamine metabolism and the combined use of metal ions activate the cGas-STING signaling pathway.

5. A drug delivery system comprising a glutamine metabolism regulator and a metal-organic framework material encapsulating the glutamine metabolism regulator.

6. The drug delivery system according to claim 5, characterized in that, The metal-organic framework material contains metal ions that include transition metal ions; preferably, the metal ions include divalent transition metal cations; more preferably, the metal ions include at least one of zinc ions, cobalt ions, platinum ions, copper ions, iron ions, or nickel ions; and / or The metal-organic framework material is a ZIF material; preferably, the ZIF material includes at least one of ZIF-8, ZIF-90, and ZIF-78; and / or The glutamine metabolism regulator includes at least one of glutaminase inhibitors, glutamine transporter inhibitors, or exogenous glutamine restriction. Preferably, the glutaminase inhibitor includes substances that reduce glutaminase expression and / or substances that reduce glutaminase activity or function; more preferably, the glutaminase inhibitor includes a GLS1 enzyme inhibitor; even more preferably, the glutaminase inhibitor includes at least one of CB-839, BPTES, Compound 968, IPN60090, or a pharmaceutically acceptable salt thereof. Preferably, the glutamine transporter inhibitor includes at least one of V-9302, GPNA, BCH, sulfasalazine, or a pharmaceutically acceptable salt thereof; Preferably, the exogenous glutamine restriction includes reducing the supply of exogenous glutamine through dietary control or glutamine antagonists; more preferably, the glutamine antagonists include at least one of DPR-104, DON, JHU-083, or a pharmaceutically acceptable salt thereof.

7. A method for preparing the drug delivery system according to claim 5 or 6, comprising the following steps: The drug delivery system is produced by mixing a first solution containing a metal inorganic salt, an organic ligand, and a first organic solvent with a second solution containing a glutamine metabolism regulator and a second organic solvent. Preferably, the inorganic metal salt is selected from one or more of the chloride, nitrate, acetate, carbonate, sulfate, or perchlorate salts of the metal ion; Preferably, the organic ligand comprises an imidazole ligand, and more preferably 2-methylimidazole; Preferably, the first organic solvent is selected from one or more of methanol, ethanol, acetonitrile, and acetone; Preferably, the second organic solvent is selected from one or more of DMSO, N,N-dimethylformamide, or N,N-dimethylacetamide; Preferably, the mass ratio of the glutamine metabolism regulator to the metal inorganic salt is 1:(2-50). Preferably, the mass ratio of the glutamine metabolism regulator to the organic ligand is 1:(5-100). Preferably, the reaction temperature is 10°C to 35°C, more preferably 20°C to 30°C; Preferably, the reaction time is 0.5 hours to 24 hours, and more preferably 0.5 hours to 5 hours.

8. A pharmaceutical composition for the prevention and / or treatment of tumors, comprising a glutamine metabolism regulator and a metal ion, or comprising the drug delivery system of claim 5 or 6 or the drug delivery system obtained by the preparation method of claim 7.

9. The pharmaceutical composition according to claim 8, characterized in that, The glutamine metabolism regulator and metal ion are the same as those defined in claim 2; and / or The dosage of the glutamine metabolism regulator is such that the subject reaches a state of glutamine deficiency or glutamine starvation; preferably, the dosage of the glutamine metabolism regulator is 0.1-1000 μM, more preferably 0.1-100 μM, and even more preferably 0.1-20 μM; and / or The amount of metal ions used is 50 μM - 500 μM, preferably 75 μM - 200 μM.

10. The pharmaceutical composition according to claim 8 or 9, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable excipients; Preferably, the pharmaceutically acceptable excipients include one or more of pharmaceutical carriers, diluents, adjuvants, and excipients; Preferably, the pharmaceutical composition is a tablet, capsule, solution, granule, pill, powder, ointment, pill, suspension, powder, injection, suppository, cream, spray, patch, sustained-release preparation, controlled-release preparation, or targeted preparation.

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