Arsenic trioxide combined copper ion and carrier composition and application thereof in liver cancer treatment

Abstract

The invention relates to the technical field of biology, in particular to arsenic trioxide combined copper ions, a carrier and application. Recognition and killing of tumors by inducing tumor cells ICD and activating an autoimmune system are one of effective strategies for solving clinical PD-1 antibody application pain spots at present. The invention clarifies the mechanism and curative effect of inducing tumor cells to generate ICD by combining low-concentration ATO with Cu < 2 + > and sensitizing PD-1 antibody immunotherapy, side effects possibly caused by large-dose application of ATO are avoided to a certain extent, and a new strategy is provided for improving the clinical benefit of the PD-1 antibody and improving the clinical practicability of ATO.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a combination of arsenic trioxide, copper ions and a carrier composition and application thereof. BACKGROUND

[0002] Hepatocellular Carcinoma (HCC) is the third leading cause of cancer-related death worldwide, and the number of new cases is expected to increase from 9057000 in 2020 to 1400000 in 2040. The high heterogeneity and drug resistance of liver cancer make its efficacy a clinical pain point. In recent years, immunotherapy, especially the emergence of immune checkpoint inhibitors (ICIs), has provided a new effective choice for the treatment of advanced HCC. At present, PD-1 / PD-L1 inhibitors have become an important choice for first-line and second-line treatment of advanced HCC. However, PD-1 inhibitors failed to achieve the pre-set overall survival endpoint in multiple clinical trials, KEYNOTE-240 and CheckMate 459 trials, suggesting the limitations of monotherapy. At the same time, not all patients can benefit from ICIs treatment, and the objective response rate (ORR) of PD-1 inhibitor monotherapy for advanced HCC is about 20% (angro B, Sarobe P, Hervás-Stubbs S, Melero I. Advances in immunotherapy for hepatocellular carcinoma. Nat Rev Gastroenterol Hepatol. 2021 Aug;18(8):525-543. doi: 10.1038 / s41575-021-00438-0. Epub 2021 Apr 13. PMID: 33850328; PMCID: PMC8042636. Table 1 first part). This means that most patients are insensitive to monotherapy ICIs, and there is primary drug resistance. In addition, for patients who initially respond, some will develop acquired drug resistance over time, leading to disease progression. Tumor cells can escape the therapeutic effect of immune checkpoint inhibitors through various mechanisms, including adaptive changes in the tumor microenvironment (TME), exhaustion of immune cells, and defects in tumor antigen presentation or recognition. Immunogenic Cell Death (ICD) is a special form of cell death that activates the host's anti-tumor immune response by releasing a series of molecular patterns (such as damage-associated molecular patterns, DAMPs) under specific stress conditions. This death is different from traditional immune-inert cell death (such as apoptosis), and it can convert dying tumor cells into in situ vaccines, triggering T cell-mediated adaptive immune responses, thereby effectively eliminating tumor cells and preventing recurrence. By inducing ICD in tumor cells, it can be self-converted into an effective tumor antigen, which is one of the effective strategies to solve the current ICI treatment dilemma.

[0003] As a traditional medicine, arsenic trioxide (ATO) has been widely recognized for its significant efficacy in the treatment of acute promyelocytic leukemia (APL) and potential anticancer activity in various solid tumor cell lines. The mechanisms of action include induction of apoptosis, inhibition of cell proliferation, angiogenesis, and differentiation. For example, ATO can exert its effects by increasing oxidative stress levels, and the thioredoxin system (TrxR and Trx) is one of the key targets of ATO in tumor therapy. In the gastric cancer cell line SGC7901, ATO was shown to alter the metabolic profile of the cells and induce cell death. In human chondrosarcoma cells, ATO activated miRNA-125b through demethylation, induced mesenchymal-epithelial transition (MET), and thus inhibited cell migration and invasion. In addition, ATO also down-regulated E2F1 expression in lung adenocarcinoma cells, thereby inhibiting tumor growth. Despite the potential efficacy of ATO in hepatocellular carcinoma, its clinical application is limited by severe systemic toxicity, low bioavailability, rapid renal clearance, and high toxicity to normal cells. Studies have shown that ATO as a single drug has not shown significant benefits in the treatment of solid tumors.

[0004] Several studies have confirmed that ATO can be used as an effective inducer of tumor ICD. However, the induction of ICD by ATO is significantly dependent on concentration, and high levels of ATO will cause a series of adverse reactions to normal cells and the body. Therefore, how to effectively induce ICD in tumor cells by using lower doses of ATO and reduce its side effects on the body is a pressing problem.

[0005] In recent years, copper ions and their complexes have received extensive attention as potential anticancer drugs because of their attractive redox properties in cancer cells, generating ROS through Fenton and Fenton-like reactions to induce apoptosis; meanwhile, excessive accumulation of intracellular copper ions also induces copper death, high concentrations of copper ions bind to fatty acylases in cells, leading to the accumulation of fatty acylated proteins and the loss of iron-sulfur cluster proteins, and ultimately causing metabolic disorders and cell death. However, as an essential trace element, excessive or insufficient levels of copper ions can be toxic to normal cells. As a means of tumor therapy, current copper ion delivery is not tumor-specific, and high levels of copper ions can cause significant side effects. The stability, bioavailability, and effective accumulation of copper complexes at tumor sites in the body also need to be further optimized.

[0006] Although ICIs show promise in the treatment of advanced HCC, not all patients benefit from them. The objective response rate (ORR) of PD-1 inhibitors as monotherapy in advanced HCC is about 20%. This means that most patients are insensitive to monotherapy with ICIs, and there is primary drug resistance. For example, Nivolumab and Pembrolizumab as monotherapy failed to achieve the pre-set overall survival (OS) endpoint in some clinical trials, and the KEYNOTE-240 and CheckMate459 trials further highlighted the limitations of monotherapy. Even patients who initially respond will develop acquired resistance over time, leading to disease progression. The liver, as an organ with natural immune tolerance, has a unique microenvironment that challenges HCC immunotherapy. The tumor microenvironment of HCC is usually rich in immunosuppressive cells (such as regulatory T cells Tregs, myeloid-derived suppressor cells MDSCs) and immunosuppressive molecules (such as TGF-β, IL-10), which together form a powerful immunosuppressive network, making it difficult for T cells to effectively recognize and eliminate tumor cells. This highly immunosuppressive microenvironment is an important reason for the limited efficacy of ICIs. One of the effective strategies to solve the current treatment dilemma of ICIs is to induce ICD in tumor cells and make them into effective tumor antigens.

[0007] A number of studies have confirmed that ATO can be an effective inducer of tumor ICD. However, ATO often causes a series of adverse reactions in clinical use, such as ECG changes, liver function abnormalities, peripheral neuropathy, and gastrointestinal reactions, etc., which limit its clinical application. However, the induction of ICD by ATO shows a significant concentration dependence. At the local concentration of the tumor that effectively induces ICD, its systemic toxicity may have caused significant harm to the patient. In fact, too high a level of ATO will directly cause cell death before inducing sufficient ICD in tumor cells, effectively activating the host's anti-tumor immunity, and exerting efficacy. In addition, ATO has low bioavailability in the body and is rapidly cleared by the kidneys, which results in insufficient drug concentration in the tumor site, making it difficult to effectively induce ICD and exert therapeutic effects. In order to make up for this deficiency, higher doses of the drug may be required, which will exacerbate its systemic toxicity problems. SUMMARY

[0008] The purpose of the present application is to overcome the deficiencies in the prior art and provide a kind of application. The present application aims to provide an anti-tumor strategy for improving the immune microenvironment of liver cancer by low-dose ATO combined with copper ions to induce immunogenic death of liver cancer cells. By destroying the redox dynamic balance of ATO, depleting intracellular GSH, and simultaneously reducing intracellular pH, the Cu 2+The mediated Fenton reaction further expands ROS, enhances the induction of immunogenic death of liver cancer cells, and reshapes the immune microenvironment of liver cancer, thereby providing a new treatment approach for liver cancer treatment.

[0009] The technical solution of the present application to solve the above technical problems is as follows: Application of arsenic trioxide combined with copper ions and carriers in the preparation of antitumor drugs.

[0010] Preferably, the tumor is liver cancer.

[0011] Preferably, the application of arsenic trioxide combined with copper ions, carriers and PD-1 antibodies in the preparation of antitumor drugs. Preferably, the tumor is liver cancer.

[0012] Preferably, the application of arsenic trioxide combined with copper ions and carriers in the preparation of antitumor drugs.

[0013] Preferably, the carrier is an ES carrier. Preferably, the tumor is liver cancer.

[0014] Preferably, the amount of arsenic trioxide is 0.8-5 mg / kg, and the amount of copper ions is 0.02-0.1 mg / kg.

[0015] Preferably, the amount of arsenic trioxide is 2-5 mg / kg, and the amount of copper ions is 0.05-0.1 mg / kg.

[0016] Preferably, the amount of the carrier is 20-100 mg / kg.

[0017] Arsenic trioxide combined with copper ions also includes other pharmaceutically acceptable excipients.

[0018] The copper ions are selected from copper salts of ionizable copper ions, preferably CuCl2.

[0019] The beneficial effects of the present application are: by inducing ICD of tumor cells, activating the recognition and killing of the immune system to the tumor is one of the effective strategies to solve the pain points of clinical application of PD-1 antibody. This patent clarifies that low concentration of ATO combined with Cu 2+ Inducing ICD of tumor cells, sensitizing the mechanism and efficacy of PD-1 antibody immunotherapy, to some extent, avoids the side effects that may be caused by high-dose application of ATO, and provides a new strategy for improving the clinical benefit of PD-1 antibody and increasing the clinical practicability of ATO. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1The ability of different concentrations of ATO to induce ICD in human liver cancer Huh7 cells and mouse liver cancer Hepa1-6 cells was evaluated, n = 3, one-way ANOVA test, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001; Figure 2 The inhibitory effect of ATO on liver cancer cells and the toxicity of normal liver cells had significant concentration dependence; Figure 3 The effect of different concentrations of Cu 2+ on liver cancer cell viability; Figure 4 The effect of different concentrations of ES on liver cancer cell viability; Figure 5 Fixed ES 50 nM, different concentrations of Cu 2+ on liver cancer cell viability; Figure 6 ATO combined with Cu 2+ on liver cancer cell viability, Huh7 human liver cancer cells, Hepa1-6 mouse liver cancer cells, n = 6, one-way ANOVA test, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001; Figure 7 ATO combined with Cu 2+ Intracellular HMGB1 diffuses from the nucleus to the intracellular; Figure 8 Secretion level of HMGB1 in cell supernatant, n = 3, one-way ANOVA test, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001; Figure 9 Changes in extracellular ATP secretion levels and intracellular ATP levels, n = 3, one-way ANOVA test, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001; Figure 10 Cell surface CRT expression level, n = 3, one-way ANOVA test, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001; Figure 11 Effect of ATO on intracellular GSH levels, n = 3, one-way ANOVA test, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001; Figure 12 Effect of ATO on intracellular free Cu2+ The influence of horizontal, n = 3, using one-way ANOVA test, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001; Figure 13 The influence of ATO on intracellular pH, n = 3, using one-way ANOVA test, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001; Figure 14 Flow cytometry detection of tumor cell ROS, ·OH level, n = 3, using one-way ANOVA test, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001; Figure 15 Flow cytometry detection of DC maturation level in vitro, CD80, CD86 are DC cell maturation related surface molecules, both of which are highly expressed on the surface of DC cells, indicating DC maturation, n = 3, using one-way ANOVA test, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001; Figure 16 Preventive vaccine experiment of mouse subcutaneous tumor, divided into control group, ATO vaccine group, ATO + Cu 2+ Vaccine group, subcutaneous tumor modeling was performed 7 days after injection of vaccine, and continuous observation was made for 20 days, n = 6, using one-way ANOVA test, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001; Figure 17 Therapeutic vaccine experiment of mouse subcutaneous tumor, divided into control group, ATO vaccine group, ATO + Cu 2+ Vaccine group, subcutaneous tumor modeling was performed 7 days after injection of vaccine, and continuous observation was made for 20 days, n = 6, using one-way ANOVA test, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001; Figure 18 Observation of therapeutic effect of mouse subcutaneous tumor, divided into control group, ATO treatment group, ES + Cu 2+ Treatment group, ATO + ES + Cu 2+ Treatment group, n = 6, using one-way ANOVA test, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001; Figure 19 Observation of therapeutic effect of mouse subcutaneous tumor experiment, divided into control group, ATO + Cu 2+Treatment group, PD-1 antibody treatment group, ATO + Cu 2+ + PD-1 antibody group, n = 6, one-way ANOVA test, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001; Figure 20 Ki67 staining and relative quantification analysis of subcutaneous tumor tissue, n = 3, one-way ANOVA test, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001; Figure 21 CD8 + T cell percentage, n = 3, one-way ANOVA test, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001; Figure 22 Percentage of mature DC in subcutaneous tumor tissue, n = 3, one-way ANOVA test, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001; Figure 23 Percentage of MDSC in subcutaneous tumor tissue, n = 3, one-way ANOVA test, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001; Figure 24 Percentage of Treg in subcutaneous tumor tissue, n = 3, one-way ANOVA test, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001; Figure 25 Percentage of PD-L1 tumor cells in subcutaneous tumor tissue, n = 3, one-way ANOVA test, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001; Figure 26 Mouse weight change, n = 6; Figure 27 HE staining of main organs of mice; Figure 28 Evaluation of serum liver and kidney function indexes of mice, AST: glutamic-oxaloacetic transaminase, ALT: glutamic-pyruvic transaminase, BUN: creatinine, CREA: urea nitrogen, n = 3, one-way ANOVA test, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001. DETAILED DESCRIPTION

[0021] The present invention will be further described in detail below with reference to embodiments, but is not limited thereto.

[0022] Example 1: Cellular experiments: 1. Cell viability assay Human hepatocellular carcinoma Huh7 cell line, mouse hepatocellular carcinoma Hepa1-6 cell line, human hepatic stellate cell line LX-2 cell line, and human hepatic cell line THLE-2 cell line were treated for 24 hours with ATO (0-40 μM), CuCl2 (0-10000 nM), ES (0-10000 nM), and fixed ES (50 nM) with CuCl2 (0-10000 nM). A blank control group, a 10 μM ATO group, a copper ion group (50 nM ES + 500 nM CuCl2), and a combined group (10 μM ATO + 50 nM ES + 500 nM CuCl2) were also set up. The viability of hepatocellular carcinoma cells after different treatments was detected using a CCK8 cell viability assay kit to observe the killing effect on hepatocellular carcinoma cells.

[0023] 2. Detection of cell immunogenic death ICD detection of liver cancer cells includes the detection of HMGB1, CRT, and ATP secretion. (1) HMGB1 detection: qualitative and quantitative detection were performed by immunofluorescence staining and ELISA, respectively. For immunofluorescence staining, Huh7 cells in the blank control group, ATO group, copper ion group and combined group after 24 hours of treatment were stained with immunofluorescence to observe the localization of HMGB1 protein in the cells. If ICD occurs, HMGB1 can be observed to escape from the cell nucleus to the cytoplasm. For ELISA quantification, in addition to the above grouping, NAC group (5mM NAC), NAC combined group (10μM ATO + 50 nM ES + 500 nM CuCl2 + 5mM NAC), TTM group (20μM TTM) and TTM combined group (10μM ATO + 50 nM ES + 500 nM CuCl2 + 20μM TTM) were set up. The cell culture supernatant of each group was collected, and after centrifugation, the cell debris precipitate was discarded. The concentration of HMGB1 secreted by cells in the cell culture supernatant was detected according to the ELISA operation procedure.

[0024] (2) CRT detection. The expression of CRT on the surface of live cells was detected by flow cytometry. Huh7 cells from the blank control group, ATO group, copper ion group, combined group, NAC group, NAC combined group, TTM group and TTM combined group were collected and cultured for 24 hours. Cell CRT protein was labeled with AF488 fluorescent antibody and PI dye was used to distinguish between dead and live cells. The average fluorescence intensity of 488 nm excitation and 525 nm emission light in live cells was distinguished by flow cytometry. The relative expression of CRT on the cell membrane surface of each group was then determined.

[0025] (3) ATP detection. ATP was quantified relatively using an ATP detection kit. Huh7 cells and cell culture supernatants were collected from the blank control group, ATO group, copper ion group, combined group, NAC group, NAC combined group, TTM group, and TTM combined group after 24 hours of culture. The Huh7 cells were lysed to detect the intracellular ATP content, and the ATP content in the culture supernatant was measured simultaneously. The ATP secretion of cells in each group after treatment was compared.

[0026] 3. Intracellular GSH detection A blank control group, a 10 μM ATO group, a copper ion group (50 nM ES + 500 nM CuCl2), and a combined group (10 μM ATO + 50 nM ES + 500 nM CuCl2) were set up. Human hepatocellular carcinoma Huh7 cell line and mouse hepatocellular carcinoma Hepa1-6 cell line were treated for 24 hours, respectively. The intracellular GSH content was detected using a GSH detection kit.

[0027] 4. Detection of intracellular free copper ions A blank control group, a 10 μM ATO group, a copper ion group (50 nM ES + 500 nM CuCl2), and a combined group (10 μM ATO + 50 nM ES + 500 nM CuCl2) were set up. Human hepatocellular carcinoma Huh7 cell line and mouse hepatocellular carcinoma Hepa1-6 cell line were treated for 24 hours, respectively. The intracellular free copper ion level was detected using a cell copper ion detection kit.

[0028] 5. Detection of intracellular pH in live cells A blank control group and a 10 μM ATO group were set up. Cytoplasmic pH was measured using a green fluorescent intracellular pH quantification kit. HCC cells were collected and washed twice with Hank's Balanced Salt Solution (HBSS), then resuspended in diluted pH probe working solution. The cell suspension was incubated at 37°C for 40 min and then centrifuged at 300 × g for 5 min. After discarding the supernatant, the cell pellet was washed three times with HBSS, and after cell counting, it was seeded into 96-well plates at a density of 10,000 cells per well. Fluorescence intensity was detected using a fluorescence spectrophotometer: excitation wavelengths were 440 nm and 488 nm, and emission wavelength was 535 nm. The fluorescence intensity ratio at 488 nm to 440 nm was calculated. A standard curve was plotted using pH standard buffer according to the kit instructions.

[0029] 6. In vitro DC maturation experiment (1) Isolation and culture of mouse bone marrow dendritic cells (BMDCs) Bone marrow cells (BMDCs) were isolated from the femur and tibia of mice. Fresh femurs and tibias were isolated from 4-week-old mice and the bone marrow cavity was flushed approximately 10 times with RPMI 1640 medium until the cavity turned white (indicating that the bone marrow cells were largely washed away). The resulting cell suspension was filtered through a 70 μm cell sieve to remove tissue debris and other impurities; then, it was centrifuged at 400 × g for 5 minutes to collect the cell pellet. Red blood cell lysis buffer was added to the collected cell pellet, and the mixture was incubated at room temperature for 5 minutes to remove residual red blood cells. After washing the cells with phosphate-buffered saline (PBS), they were seeded into BMDC-specific medium for culture. On days 4 and 7 of cell culture, half the volume of fresh BMDC-specific medium was replaced to maintain the necessary nutrient environment for cell growth. On day 10, BMDCs in suspension and semi-adherent states were collected for subsequent experiments.

[0030] (2) Co-culture of BMDCs with Hepa1-6 cells and detection of BMDC maturity Collected BMDCs and treated Hepa1-6 cells (ATO, copper ion, or a combination thereof) were counted separately, then mixed at a 1:1 ratio and seeded into 6-well plates for co-culture for 24 hours. After co-culture, cells in suspension and semi-adherent states were collected, blocked with Fc, and then CD11c, CD80, and CD86 flow cytometry staining antibodies diluted with cell staining buffer were added to the cells. The cells were incubated at 4°C in the dark for 40 minutes. After washing the cells with PBS to remove unbound antibodies, the expression levels of CD11c, CD80, and CD86 on the cell surface were detected by flow cytometry to analyze the maturity of BMDCs (Note: CD11c is a DC cell-specific marker, and CD80 and CD86 are DC cell maturation-related surface molecules; elevated expression levels usually indicate increased DC cell maturity).

[0031] 7. Detection of Reactive Oxygen Species (ROS) and Hydroxyl Radicals (·OH) in Cells ROS and ·OH in cells were detected by flow cytometry. Four groups were established: a blank control group, an ATO group, a copper ion group, a combined group, a NAC group, a NAC combined group, a TTM group, and a TTM combined group. After culturing Huh7 cells for 24 hours, cell pellets were collected. Total ROS in the cells was labeled with 2',7'-dichlorodifluorofluorescein diacetate (DCFH-DA), and ·OH in the cells was labeled with hydroxyphenylfluorescein (HPF). The average fluorescence intensity of cells excited at 488 nm and emitted at 525 nm was detected by flow cytometry, which represented the relative content of total ROS and ·OH in each group.

[0032] Example 2: Animal experiments 1. Whole-cell tumor vaccine experiment The efficacy of whole-cell tumor vaccines is the gold standard for testing whether intracellular tumor development (ICD) occurs in vivo. Vaccine trials are divided into two parts: preventive vaccines and therapeutic vaccines. The difference lies in whether the vaccine is injected before or after subcutaneous tumor formation in mice.

[0033] (1) Construction of whole-cell tumor vaccine Hepa1-6 cells were treated with 10 μM ATO or a combination of 10 μM ATO + 50 nM ES + 500 nM CuCl2 for 24 hours, and the cytotoxic, low-viability tumor cells were collected as ATO whole-cell vaccines or ATO-CuCl2 vaccines. 2+ Whole-cell vaccine. After counting the collected cells, the cell concentration was adjusted to 2000 W / ml using PBS. Each mouse in the experimental group was subcutaneously injected with 100 μl of the corresponding vaccine. The whole-cell vaccine was prepared fresh for each use.

[0034] (2) Observation of the efficacy of whole-cell vaccine The experiment was divided into a blank control group, an ATO vaccine group, and a combined vaccine group.

[0035] In the preventative vaccine experiment, C57BL / 6J mice were first subcutaneously injected into the right groin with 100 μl of physiological saline, ATO whole-cell vaccine, and ATO-Cu, respectively. 2+ For whole-cell vaccines, 200W Hepa1-6 cells were injected into the left groin of mice 7 days after vaccination. The growth of subcutaneous tumors in mice was observed. On the 20th day after subcutaneous tumor inoculation, mice were sacrificed and subcutaneous tumors were removed. The weight of subcutaneous tumors was measured and a curve of subcutaneous tumor volume change was plotted.

[0036] The therapeutic vaccine experiment first involved inoculating 200W Hepa1-6 cells into the left groin of C57BL / 6J mice. On the fourth day after inoculation with the subcutaneous tumor, 100μl of physiological saline, ATO whole-cell vaccine, and ATO-Cu were subcutaneously injected into the right groin of the mice, respectively. 2+ Whole-cell vaccine was administered, and the growth of subcutaneous tumors in mice was observed. On day 15 after vaccination, mice were sacrificed and subcutaneous tumors were removed. The weight of the subcutaneous tumors was measured, and a curve showing the change in subcutaneous tumor volume was plotted.

[0037] 2. ATO combined with Cu 2+ In vivo anti-tumor efficacy observation Using C57BL / 6J mice, the mice were first injected with 2×10⁻⁶ ions into the left inguinal region. 6 A subcutaneous hepatocellular carcinoma tumor model was constructed using Hepa1-6 PBS. When a clearly visible tumor mass was observed, mice were randomly divided into four treatment groups, each receiving the following interventions: Control group: daily intraperitoneal injection of 100 μL sterile saline; ATO group: daily intraperitoneal injection of ATO 4 mg / kg (the concentration range of low-dose ATO administered via in vitro cell therapy is 5-10 μM. Based on theoretical estimation, combined with ATO's protein binding, tissue distribution, rapid metabolism (short half-life), first-pass effect, and relevant literature reports, the theoretical in vivo concentration range for mice to achieve effective tissue concentration is calculated to be 0.7-5 mg / kg. We selected 4 mg / kg for verification in practical applications), administered for 10 consecutive days; Copper ion group: daily intraperitoneal injection of 40 mg / kg ES and 0.06 mg / kg CuCl2, administered for 10 consecutive days; ATO-Cu 2+ Group: Mice were administered the above-mentioned doses daily via intraperitoneal injection of ATO, ES, and CuCl2 for 10 consecutive days. From the start of administration, the health status, tumor size, and body weight of the mice were continuously monitored. On day 14 of treatment, the tumor-bearing mice were euthanized; subsequently, their subcutaneous tumors were carefully dissected and their weight recorded.

[0038] 3. ATO-Cu2+ The efficacy of combined PD-1 antibody therapy in anti-tumor treatment and its impact on the tumor immune microenvironment. Using C57BL / 6J mice, the mice were first injected with 2×10⁻⁶ ions into the left inguinal region. 6 A subcutaneous hepatocellular carcinoma tumor model was established using Hepa1-6 PBS. When a clearly visible tumor mass was observed, mice were randomly divided into four treatment groups, each receiving the following interventions: Control group: daily intraperitoneal injection of 100 μL sterile saline; ATO-Cu 2 Group ⁺: Daily intraperitoneal injection of a mixed drug solution (total volume 100 μL) containing 4 mg / kg ATO, 40 mg / kg ES, and 0.06 mg / kg CuCl2; Group PD-1: Intraperitoneal injection of anti-mouse PD-1 antibody, 10 mg / kg per mouse (100 μL injection volume each time), once every 3 days; ATO-Cu 2 ⁺+PD-1 combination group: ATO-Cu was administered concurrently with the above dosage and dosing regimen. 2 ⁺ A mixture of drug solution and anti-mouse PD-1 antibody was administered. From the start of administration, the health status, tumor size, and weight of the mice were continuously monitored. On days 10-11 of treatment, the tumor-bearing mice were euthanized; subsequently, the subcutaneous tumors were carefully dissected and separated, and the tumor weight was recorded. The collected tumor tissue was subjected to KI67 immunohistochemical staining and immune microenvironment flow cytometry analysis. Simultaneously, the major organs (heart, lungs, liver, spleen, and kidneys) and serum were collected from the mice. HE staining was used to observe whether the treatment caused damage to the major organs, and serum liver and kidney function indicators were measured to determine if there was acute liver and kidney toxicity.

[0039] Results and Analysis (i) ATO exhibits concentration-dependent effects on ICD induced by liver cancer cells. Immunogenic cell death (ICD) primarily manifests as the release of damage-associated molecular patterns (DAMPs) such as HMGB1, CRT, and ATP. Figure 1 After treating human hepatocellular carcinoma Huh7 cells and mouse hepatocellular carcinoma Hepa1-6 cells with 0, 5, 10, and 20 μATO for 24 hours, the cell membrane surface CRT level, cell culture supernatant HMGB1 content, and ATP content showed a significant concentration-dependent increase, while the intracellular ATP content showed a concentration-dependent decrease. This indicates that ATO has a significant concentration-dependent effect on ICD induced by hepatocellular carcinoma cells.

[0040] (ii) The inhibitory effect of ATO on liver cancer cells and its toxicity to normal hepatocytes are significantly concentration-dependent. By observing the effects of different concentrations of ATO on hepatocellular carcinoma cells and normal human hepatocytes, this study aimed to determine whether the inhibitory effect of ATO on hepatocellular carcinoma cells and its toxicity to normal hepatocytes was concentration-dependent. Results are as follows:Figure 2 After 24 hours of treatment, the IC50 values ​​of ATO for inhibiting the viability of the two tumor cell lines were 14.81 μM and 14.79 μM, respectively, and the IC50 values ​​for inhibiting the viability of the two normal hepatocyte cell lines were 17.96 μM and 9.35 μM, respectively. In the 5-15 μM range, ATO showed a rapid increase in cytotoxicity to THLE-2 cells; therefore, 10 μM ATO was chosen as the concentration for subsequent experiments, as it exhibited relatively low cytotoxicity to normal hepatocytes while effectively inducing ICD in liver cancer cells. Furthermore, several existing studies using ATO as an ICD inducer have employed higher concentrations such as 25 μM and 15 μM to induce sufficient cell death. (See Chen J, Jin Z, Zhang S, Zhang Efficacy of PD-1 Inhibitors in Hepatocellular Carcinoma byInducing Immunogenic Cell Death via the ROS / ERS Pathway. Immun Inflamm Dis. 2025 Jun;13(6):e70214.) (III) Observation of the effects of different concentrations of copper ions and copper ion carriers on liver cancer cells The results of treating HCC cells with different concentrations of CuCl2 for 24 hours are as follows: Figure 3 Because free copper ions have difficulty directly entering cells, CuCl2 alone has almost no inhibitory effect on HCC cells in the 0-10000 nM range. We used ES as a copper ion carrier to introduce copper ions into the cell, such as... Figure 4 , Figure 5 Treatment of HCC cells with different concentrations of ES alone showed almost no effect on HCC cell viability within the range of 0-5000 nM. However, when the ES concentration was fixed at 50 nM and different concentrations of CuCl2 were added, HCC cell viability increased with increasing CuCl2 concentration. 2The increase in ⁺ concentration followed by a decrease indicates that ES successfully introduced Cu into the cell. 2 ⁺ and played an anti-HCC role.

[0041] (iv) Low concentrations of ATO combined with copper ions significantly enhance the killing effect on liver cancer cells. The effects of 24-hour treatment with 10 μM ATO, 50 nM ES + 500 nM CuCl2, and 10 μM ATO + 50 nM ES + 500 nM CuCl2 on the viability of human and mouse hepatocellular carcinoma cell lines were compared and observed. Figure 6 As shown, at lower concentrations, ATO or Cu 2 + After treatment of human or mouse hepatocellular carcinoma cells, cell viability remained at approximately 80%. When the two treatments were used in combination, the killing efficiency significantly increased (Huh7 cell line, ATO group average 77.469%, Cu...). 2+ The average value of the group was 84.652%, and the average value of the combined group was 40.644%, n=6; for the Hepa1-6 cell line, the average value of the ATO group was 79.908%, and the average value of Cu was... 2+ The average value of the group was 85.051%, and the average value of the combined group was 42.432%, n=6;).

[0042] (v) Low-dose ATO combined with Cu 2+ Enhanced copper-catalyzed Fenton reaction amplifies ROS and induces immunogenic cell death in liver cancer cells. 1. Low-dose ATO combined with Cu 2+ It can enhance the immunogenic death of liver cancer cells. Immunogenic cell death (ICD) primarily manifests as the release of damage-associated molecular patterns (DAMPs) such as HMGB1, CRT, and ATP. Figure 7 As shown, immunofluorescence staining revealed that after ATO treatment, intracellular HMGB1 was observed to migrate from the nucleus to the cytoplasm in liver cancer cells. Combined with ATO and Cu... 2+ A more significant HMGB1 overflow was then observed. For example... Figure 8 ELISA analysis showed that the level of HMGB1 secreted by tumor cells increased in the cell supernatant after ATO treatment, while Cu alone... 2+ HMGB1 levels remained unchanged after treatment, but the combined ATO and Cu treatment... 2+ HMGB1 levels subsequently increased significantly, an increase that could be partially reversed by the copper ion chelator TTM, but almost completely reversed by the oxidative stress inhibitor NAC. Similarly... Figure 9 As shown, ATP secretion in the cell supernatant was similar to that of HMGB1, and a certain degree of reduction in intracellular ATP levels was also observed. Furthermore, as... Figure 10Flow cytometry analysis revealed that the level of calreticulin (CRT) on the cell surface showed a trend similar to that of HMGB1 and ATP secretion. This indicates that low-dose ATO can induce ICD in liver cancer cells, and that Cu alone... 2+ ATO and Cu cannot induce ICD. 2+ The combination significantly increased the occurrence of ICD in liver cancer cells, which could be reversed by copper chelators and was significantly associated with oxidative stress.

[0043] 2. Low-dose ATO disrupts intracellular redox balance, lowers intracellular pH, enhances the copper-catalyzed Fenton reaction, and amplifies ROS. Copper ions can catalyze the Fenton reaction within cells. In an acidic environment, this reaction converts H₂O₂, produced by cellular energy metabolism, into highly oxidizing ·OH, which is highly toxic to tumor cells and can induce intracellular cytotoxicity (ICD). Intracellular ·OH levels reflect the catalytic activity of the Fenton reaction within cells.

[0044] Glutathione (GSH) is one of the most common antioxidants in cells, and it plays an important role in maintaining intracellular copper homeostasis by binding to free Cu in the cell. 2+ , causing intracellular Cu 2+ It remains at a low level. When intracellular redox homeostasis is disrupted and reactive oxygen species (ROS) levels rise, they consume large amounts of intracellular GSH, leading to increased intracellular CO2 and Cu2+ levels. 2+ Insufficient GSH binding makes it difficult to maintain intracellular copper homeostasis. For example... Figure 11 When ATO was added, intracellular GSH levels decreased significantly, and at the same time... Figure 12 The intracellular copper ion level did not change significantly after the addition of ATO alone, but when copper ions were introduced in combination, the intracellular copper ion level increased significantly again compared with the introduction of copper ions alone, indicating that the redox balance disruption caused by ATO increased the accumulation of intracellular copper ions.

[0045] like Figure 13 As shown, we observed that when ATO was applied to the cells, the intracellular pH decreased significantly, which provided favorable acidic conditions for the copper ion-catalyzed Fenton reaction.

[0046] Finally, we measured the total ROS and ·OH in liver cancer cells treated with different methods, and the results are as follows: Figure 14 As shown, ATO treatment alone had almost no effect on intracellular ·OH, but when Cu was introduced into the cell... 2+ Subsequently, the ·OH level increased, while ATO and Cu... 2+The combined use significantly increased intracellular ·OH levels; however, the increase in ·OH was almost completely reversed upon the addition of the copper chelator TTM and the antioxidant NAC. Consistent with this trend, the use of ATO or Cu alone... 2+ Both can induce ROS production in liver cancer cells, and the combination of ATO and Cu 2+ It produces a stronger ROS, which can be partially reversed by the copper chelator TTM and almost completely reversed by the antioxidant NAC. Its overall trend is basically consistent with the occurrence of ICD in liver cancer cells. These results indicate that ATO and ATO combined with Cu... 2+ The induction of ICD in liver cancer cells is related to ROS production, while the combination of ATO and Cu... 2+ The amplification effect of ROS arises from the disruption of intracellular redox homeostasis by ATO, resulting in Cu 2+ ·OH is generated via the Fenton reaction.

[0047] 3. Low-dose ATO combined with Cu 2+ It can effectively induce the in vitro maturation of mouse dendritic cells (DCs). Dendritic cells (DCs) are the most potent antigen-presenting cells. DCs are closely related to the occurrence and development of tumors; a higher number of infiltrating DCs in most solid tumors generally indicates a better prognosis. When tumor cells develop intracellular antigenic divide (ICD), DCs can more effectively recognize tumor cells and differentiate into mature cells to perform antigen presentation. Figure 15 When bone marrow-derived dendritic cells (DCs) extracted and cultured from mouse bone marrow are compared with normal mouse liver cancer cells, ATO-treated liver cancer cells, and Cu... 2+ After co-culturing the treated hepatocellular carcinoma cells and the tumor cells treated with the combined drug, it was observed that the hepatocellular carcinoma cells treated with the combined drug had a significantly higher ability to induce dendritic cell maturation than the other groups, that is, the combined treatment could significantly induce hepatocellular carcinoma cells to undergo ICD.

[0048] (vi) ATO combined with Cu 2+ Induced whole-cell tumor vaccines can effectively exert anti-tumor effects. The gold standard for verifying whether tumor cells have undergone ICD in vivo is the efficacy of whole-cell tumor cells induced by ICD inducers. By pre-injecting whole-cell vaccines constructed from tumor cells that have already undergone ICD induction, the body's own immune system is activated to recognize the tumor, thereby preventing tumor development. Alternatively, by injecting cell vaccines after tumor formation, anti-tumor immunity is effectively strengthened, achieving a killing effect on existing tumors. Figure 16 As shown, by injecting a whole-cell vaccine induced by ATO or in combination with copper ions 7 days before subcutaneous tumor modeling, 20 days after subcutaneous tumor formation, compared with the control group, the tumor volume and weight of the ATO-induced vaccine group were significantly reduced, while the tumors in the combined group completely regressed. The treatment vaccine results are as follows...Figure 17 The ATO-induced vaccine group showed significant tumor shrinkage, while the combined treatment group experienced partial tumor regression, and the overall therapeutic effect was superior to that of the ATO vaccine. These results further demonstrate the effectiveness of ATO combined with Cu. 2+ It can effectively induce ICD in vitro, and the tumor cells induced to develop ICD can effectively activate the body's own immunity and exert a powerful anti-tumor effect.

[0049] (vii) ATO combined with Cu 2+ Effectively enhances the in vivo anti-tumor effect of PD-1 antibodies and improves the tumor immune microenvironment. 1. ATO combined with Cu 2+ In vivo tumor-suppressing efficacy observation Previous studies have reported poor efficacy of ATO alone in vivo for the treatment of liver cancer. We first observed the combined use of ATO and Cu. 2+ Whether it can exert anti-tumor effects. After constructing subcutaneous hepatocellular carcinoma tumors in mice, ATO and Cu were injected intraperitoneally. 2+ Or in combination with ATO-Cu 2 + The result is as follows Figure 18 After intraperitoneal injection of ATO alone, the mean terminal tumor weight decreased by 21% and the mean terminal tumor volume decreased by 23% compared with the blank control group; after intraperitoneal injection of ES+Cu alone... 2+ After treatment, compared with the blank control group, the average terminal tumor weight decreased by 16% and the average terminal tumor volume decreased by 15%; combined with ATO-Cu 2+ After treatment, the average terminal tumor weight decreased by 43% and the average terminal tumor volume decreased by 44% compared with the blank control group.

[0050] 2. ATO combined with Cu 2+ In vivo antitumor efficacy of PD-1 sensitizing antibodies Given the current limitations in the efficacy of PD-1 antibody therapy for liver cancer, we introduced ATO combined with Cu, which can induce ICD in tumor cells. 2+ Treatment aims to enhance the body's sensitivity to PD-1 antibodies. Results of in vivo experiments in mice are as follows... Figure 19 As shown, ATO combined with Cu was administered via intraperitoneal injection. 2+ PD-1 antibody or combination therapy, ATO-Cu 2+ The combined PD-1 antibody treatment group showed significantly improved efficacy compared to other single-drug treatment groups, with a significant decrease in tumor weight and volume (compared to the blank control group, ATO-Cu) 2+ The average final tumor weight decreased by 35% and the average final tumor volume decreased by 37% in the group receiving the PD-1 antibody treatment; the average final tumor weight decreased by 47% and the average final tumor volume decreased by 46% in the PD-1 antibody group; and the average final tumor weight decreased by 85% and the average final tumor volume decreased by 90% in the combination therapy group. Figure 20Immunohistochemical staining of Ki67, a marker of tumor cell proliferation activity, revealed a significant decrease in tumor growth rate in the PD-1 combined group.

[0051] 3. ATO combined with Cu 2+ Improve the tumor immune microenvironment.

[0052] Flow cytometry was used to analyze changes in the tumor immune microenvironment after drug administration in mouse tumor tissues. The combined treatment group was compared with other treatment groups to identify CD8+ cells in the immune cell population that inhibit tumor growth. + The percentage of effector T cells and mature DCs was significantly increased. Figure 21 , 22 The percentages of Tregs and MDSCs, which promote tumor growth, decreased significantly. Figure 23 , 24 Furthermore, the level of PD-L1 on the surface of tumor cells was significantly decreased ( ). Figure 25 This indicates that low-dose ATO combined with Cu 2+ It effectively improves the tumor immunosuppressive microenvironment and enhances the efficacy of PD-1 antibodies.

[0053] 4. ATO combined with Cu 2+ It is safe and effective when used in vivo.

[0054] By recording the changes in mouse body weight during the above experiment, and collecting the major organ tissues and serum of the mice after the drug administration, such as... Figure 26 As shown, the mice's weight increased steadily and slowly over time, with no significant weight loss. Figure 27 HE staining of major organs (lung, heart, liver, spleen, and kidney) in mice showed no significant organ damage. Serum liver and kidney function tests are shown in [the table below]. Figure 28 Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine, and blood urea nitrogen levels in mice from each group were within the normal range, with no significant differences observed. These data indicate that low-dose ATO combined with Cu... 2+ It is safe and effective when used in vivo.

[0055] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. Application of arsenic trioxide combined with copper ions and carrier in the preparation of antitumor drugs.

2. The application according to claim 1, characterized in that, The carrier is an ES carrier.

3. The application according to claim 1, characterized in that, The tumor is liver cancer.

4. Application of arsenic trioxide combined with copper ions, carriers, and PD-1 antibodies in the preparation of antitumor drugs.

5. The application according to any one of claims 1 to 4, characterized in that, The dosage of arsenic trioxide is 0.8-5 mg / Kg, and the dosage of copper ions is 0.02-0.1 mg / Kg.

6. The application according to claim 5, characterized in that, The dosage of arsenic trioxide is 2-5 mg / Kg, and the dosage of copper ions is 0.05-0.1 mg / Kg.

7. The application according to any one of claims 1 to 4, characterized in that, The amount of the carrier used is 20-100 mg / kg.

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