Application of combination of copper ion carrier illlisemox carrying copper ions and vitamin K3 in preparation of liver cancer treatment medicine

Abstract

The invention discloses application of a combination of a copper ion carrier ilisemox carrying copper ions and vitamin K3 in preparation of a medicine for treating liver cancer. Through high-throughput screening of metabolites having a synergistic effect with copper death (copper ion carrier ES combined with copper ions), preliminary screening shows that vitamin K3 can cooperatively kill liver cancer cells with ES + Cu < 2 + >, and further in-vivo and in-vitro various experiments prove that vitamin K3 can obviously promote liver cancer cell copper death, so that the vitamin K3 can obviously promote liver cancer cell copper death. Vitamin K3 and ES + Cu < 2 + > are combined for use, so that liver cancer can be effectively inhibited, and a remarkable synergistic killing effect on liver cancer is achieved. Therefore, the invention provides a strategy for synergistically treating the liver cancer by jointly using the vitamin K3 and the ES + Cu < 2 + >, and the strategy can be used for manufacturing liver cancer biological medicines, liver cancer chemical medicine raw material medicines and preparations.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology. More specifically, it relates to the application of copper ion carrier elixol loaded with copper ions in combination with vitamin K3 in the preparation of drugs for the treatment of liver cancer. Background Technology

[0002] Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer in clinical practice. According to authoritative domestic and international guidelines, liver cancer is generally classified into early-stage, intermediate-stage, and advanced-stage liver cancer. Radical treatments for early-stage liver cancer (single tumor without vascular invasion) mainly include surgical resection, local ablation, and liver transplantation. Due to donor scarcity, surgery and ablation have become the mainstream options, with radiofrequency ablation (RFA) being widely used due to its minimally invasive advantages. Studies have confirmed that RFA has a local control rate comparable to surgery for lesions <3cm in diameter. Its principle is to induce coagulative necrosis of the tumor through high-frequency current heating. However, lesions near important blood vessels or organs are prone to incomplete ablation due to the "thermal deposition effect," increasing the risk of local recurrence; heat loss from tumors near blood vessels may cause abnormal morphology of the ablation zone, thus affecting the tumor inactivation effect; in addition, radiofrequency energy may also induce vascular endothelial damage and thrombosis. This further limits the application of radiofrequency ablation (RFA) in liver cancer. For tumors in such high-risk locations, anatomical liver resection, while improving the radical cure rate by widening the resection margin, often results in patients with cirrhosis or tumors located in the porta hepatis losing the opportunity for surgery due to insufficient remaining liver volume. Transcatheter arterial chemoembolization (TACE) is the standard treatment for intermediate-stage liver cancer, but due to the tumor's dual blood supply (hepatic artery + portal vein) and the formation of collateral circulation, TACE rarely achieves complete ischemic necrosis of the tumor. The objective response rate of a single TACE treatment is only 35%, and the median survival after TACE is only about 12 months. Surgery is also one of the main treatment methods for intermediate-stage liver cancer, and surgical resection is a commonly used treatment. However, intermediate-stage liver cancer has a high incidence of microvascular invasion (MVI) and satellite lesions, and liver resection often cannot completely remove these micro-lesions. Moreover, intermediate-stage liver cancer is highly invasive and has a high potential for recurrence and metastasis. Therefore, the one-year recurrence rate after liver resection for intermediate-stage liver cancer can be as high as 50% or more. Furthermore, due to the large size of mid-stage liver cancer tumors, their proximity to important blood vessels, and the need to maintain postoperative liver function, it is difficult to ensure sufficient surgical margins. In addition, over 50% of liver cancer patients are diagnosed with advanced-stage liver cancer at initial diagnosis. Currently, the main treatment for advanced-stage liver cancer is targeted therapy, including first-line drugs such as sorafenib and lenvatinib, and second-line drugs such as regorafenib and cabozantinib. However, the median survival time for targeted therapy is only 10-13 months. Among these, the control rate of targeted therapy for patients with portal vein tumor thrombosis (PVTT) is less than 40%, with a median survival of only 8.1 months.Given the limitations of current clinical treatment options, it is crucial to develop new treatment modalities.

[0003] Liver cancer cells require higher concentrations of copper than normal cells to support tumor growth. Copper death, a recently discovered regulated form of cell death, holds promise as a novel therapeutic strategy for liver cancer because the liver plays a central role in copper metabolism, and imbalances in copper metabolism can adversely affect the liver. Copper death is triggered by excessive intracellular copper accumulation, leading to mitochondrial protein aggregation, which in turn results in the loss of iron-sulfur cluster proteins, thereby disrupting the tricarboxylic acid (TCA) cycle and causing cell death. Enhancing copper death has become a promising therapeutic strategy. Reducing copper proliferation through copper chelators or promoting copper death using copper ion carriers provides new avenues for liver cancer treatment. Commonly used copper compounds in clinical practice include copper chelators and copper ion carriers. Copper chelators, such as tetrathiomolybdate (TTM), can inhibit copper uptake, reduce the tumorigenicity of liver cancer cell lines, inhibit glycolysis, and hinder tumor progression. Penicillamine and trientine also have anti-angiogenic and tumor cell proliferation-inhibiting effects, and can also reduce PD-L1 expression in tumor tissues and stimulate anti-cancer immune responses. Copper ion carriers such as dithiothreitol (DSF) and elesclomol (ES) can induce copper death in hepatocellular carcinoma (HCC) cells. ES can transport excess copper to mitochondria, triggering oxidative stress and leading to cell death. As an anticancer drug dependent on the transport of extracellular copper, the safety and cytotoxicity of ES have been validated in preclinical and clinical trials, making it worthy of attention in HCC treatment. However, the effect of ES-induced copper death in tumor cells is limited when used alone, and copper ion carriers require improved treatment modalities to achieve satisfactory therapeutic effects. Combination therapy is an important strategy for improving the efficacy of HCC treatment. Previous studies have shown that combining copper ion carriers with targeted drugs, such as lenvatinib, can enhance antitumor effects; sorafenib combined with copper ion carriers has significant synergistic cytotoxicity against HCC cells. Combined with chemotherapy and radiotherapy, HCC patients with high CRG scores are more sensitive to certain chemotherapeutic drugs, and the combination of copper compounds with chemotherapeutic drugs can reduce tumor cell proliferation. Although copper compounds and combination therapies have shown potential in HCC treatment, many challenges remain. The fact that some clinical trial results have not yet been published affects the accurate assessment of their efficacy; poor clinical efficacy, defects in experimental design and methodology, issues with drug safety and side effects, undesirable drug properties, and limited commercial feasibility all restrict the development of these treatment options.

[0004] Increasing evidence suggests a close relationship between the metabolic processes of liver cancer cells and their sensitivity to copper death. Vitamins are essential trace elements for maintaining metabolic processes, and vitamin K3, as a coagulation cofactor, participates in the coagulation process. Its anti-tumor effects have become a research focus in recent years. Vitamin K3 has inhibitory or cytotoxic effects on the growth of various tumor cells. Vitamin K3 inhibits tumor cell growth and promotes tumor cell death by increasing oxidative stress levels in tumor cells, promoting apoptosis, inhibiting the cell cycle, and promoting autophagy. Vitamin K is used in clinical anti-tumor treatment, primarily for hematologic and digestive system tumors. Combined with other chemotherapy drugs, it can improve the prevention and treatment of tumors. With the development of new vitamin K derivatives and formulations, vitamin K shows promising application prospects in tumor treatment. Vitamin K3 mainly functions in the liver, increasing the oxidative stress levels of tumor cells. It is a routinely used clinical drug with good biocompatibility in patients, facilitating clinical application. Previous studies have shown that vitamin K3 can induce apoptosis in liver cancer cells. However, there are currently no reports on the role of copper ion carriers combined with vitamin K3 in liver cancer treatment. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide the application of copper ion carrier irismo combined with vitamin K3 in the preparation of liver cancer treatment drugs.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution: Liver cancer cells require higher concentrations of copper than normal cells to support tumor growth. Copper death, a recently discovered regulated form of cell death, holds promise as a novel therapeutic strategy for liver cancer. Ilisimor, as a copper ion carrier, can induce copper death and has been extensively studied in various solid tumors in recent years. However, the use of ilisimor alone or in combination with chemotherapy has not translated into clinical prognostic benefits. Given that copper death is closely linked to metabolic pathways such as glycolysis and amino acid metabolism, these metabolic changes may affect cellular sensitivity to copper toxicity. Therefore, this invention utilizes high-throughput screening to induce copper death (copper ion carrier ES combined with copper ions, ES+Cu). 2+ Metabolites with synergistic effects have been preliminarily found to contain vitamin K3, which can react with ES+Cu. 2+ It synergistically kills liver cancer cells. In vivo and in vitro experiments (the coefficient of drug interaction (CDI) in in vitro cell experiments ranged from 0.207 to 0.671, all less than 0.7), suggesting that vitamin K3 synergistically kills liver cancer cells with ES+Cu. 2+(Vitamin K3 has a significant effect on killing tumor cells) Multiple experiments have confirmed that vitamin K3 can significantly promote copper death in liver cancer cells. Vitamin K3 and ES+Cu 2+ Combined use can effectively and synergistically inhibit liver cancer. Therefore, this invention proposes the combined use of vitamin K3 and ES+Cu. 2+ A synergistic treatment strategy for liver cancer, targeting this major and complex disease. Vitamin K3 and ES+Cu 2+ It can be used in the manufacture of biopharmaceuticals for liver cancer, as well as raw materials and formulations for chemical drugs for liver cancer.

[0007] Therefore, this invention provides the application of the copper ion carrier irismo, carrying copper ions in combination with vitamin K3, in the preparation of a drug for treating liver cancer. Specifically, it describes the application of the copper ion carrier irismo, copper ions, and vitamin K3 in combination for the preparation of a drug for treating liver cancer.

[0008] Furthermore, the drug treats liver cancer by synergistically inhibiting the proliferation of liver cancer cells and synergistically promoting apoptosis of liver cancer cells.

[0009] Furthermore, the drug treats liver cancer by synergistically promoting copper death in liver cancer cells.

[0010] The present invention also provides a liver cancer treatment drug, wherein the liver cancer treatment drug contains the copper ion carrier irismo, copper ions and vitamin K3.

[0011] Further, the molar ratio of the copper ion carrier ilismo, copper ions and vitamin K3 is 1:(1-25):(160-510) (preferably 1:25:500).

[0012] Furthermore, the liver cancer treatment drug also includes pharmaceutically acceptable excipients.

[0013] Furthermore, the dosage form of the drug is an injection.

[0014] Liver cancer is one of the most common malignant tumors in China and globally. Liver cancer patients often have elevated serum copper levels and copper overload in cancerous tissue. Targeted copper transport via copper carriers can induce copper death in liver cancer cells. This invention reveals that copper ion carriers alone have limited effect in inducing copper death in liver cancer, and vitamin K3 alone is not ideal for treating liver cancer. However, the combined use of both can effectively kill liver cancer cells by promoting copper death. Specifically, the copper ion carrier ES, carrying copper ions, and vitamin K3 used in combination can achieve synergistic treatment of liver cancer.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of the copper ion carrier irismo in combination with vitamin K3 in the preparation of a drug for treating liver cancer. This invention utilizes high-throughput screening and copper death (copper ion carrier ES combined with copper ions, ES+Cu) 2+Metabolites with synergistic effects; preliminary screening showed that vitamin K3 can interact with ES+Cu. 2+ Vitamin K3 synergistically kills liver cancer cells. Further in vivo and in vitro experiments confirmed that vitamin K3 can significantly promote copper cell death in liver cancer cells. (The text also mentions vitamin K3 and ES+Cu, but this appears unrelated to the preceding sentence and may be a separate, incomplete thought.) 2+ The combined use of vitamin K3 and ES+Cu can effectively inhibit liver cancer and has a significant synergistic killing effect on liver cancer. Therefore, this invention proposes the combined use of vitamin K3 and ES+Cu. 2+ Strategies for synergistic treatment of liver cancer. Attached Figure Description

[0016] Figure 1 This is for high-throughput screening of metabolites that exhibit synergistic effects with the copper ion carrier ES. Among them, Figure 1 Figure AB shows the survival ratio of different metabolites combined with copper ion carrier ES carrying copper ions (Cu / ES) versus Cu / ES alone in the liver cancer cell lines Huh7 (A) and SK-Hep1 (B); C shows the chemical structural formulas of vitamin K3 and the copper ion carrier Elesclomol. Each dot in Figure AB represents a metabolite.

[0017] Figure 2 The effect of copper ions loaded onto the copper ion carrier ES in combination with vitamin K3 on the proliferation and apoptosis of liver cancer cells. Among other things, Figure 2 The middle section (A) shows the effect of vitamin K3 on copper death in different hepatocellular carcinoma cell lines as detected by cell proliferation assay; (B) shows the effect of vitamin K3 on copper death in hepatocellular carcinoma cells as detected by colony formation assay, with the left image representing colony formation and the right image showing statistical data on colony formation; (C) shows the effect of vitamin K3 and Cu / ES on apoptosis in hepatocellular carcinoma cells as detected by flow cytometry, with the left image showing flow cytometry analysis and the right image showing statistical data on apoptosis rates. Cu / ES represents the simultaneous application of copper ions carried by the copper ion carrier ES. Ctrl represents the control group, Vitamin K3 represents the vitamin K3 monotherapy group, Cu / ES represents the group treated with copper ions carried by the copper ion carrier ES, and Vitamin K3+Cu / ES represents the group treated with vitamin K3 combined with Cu / ES.

[0018] Figure 3 The effect of vitamin K3 on copper cell death in liver cancer cells was observed using Western blotting and immunofluorescence. Among other things, Figure 3In the diagram, A represents the Western blotting (WB) assay for vitamin K3; BE represents the immunofluorescence assay for the aggregation of DLAT in Huh7 (B&D) and SK-Hep1 (C&E) liver cancer cells by vitamin K3, as well as the co-localization of DLAT with mitochondria. B&C are representative immunofluorescence images, and D&E are statistical graphs of DLAT signal. Ctrl represents the control group, Vitamin K3 represents the vitamin K3 monotherapy group, Cu / ES represents the group treated with copper ions carried by the copper ion carrier ES, and Vitamin K3+Cu / ES represents the group treated with vitamin K3 combined with Cu / ES.

[0019] Figure 4 The effect of vitamin K3 in synergy with the copper ion carrier ES on the growth of liver cancer in vivo. Among them, Figure 4 A shows the growth curve of subcutaneous tumors in the Huh7 hepatocellular carcinoma cell line; B shows images of subcutaneous tumors in each group at the experimental endpoint; C shows a statistical chart of subcutaneous tumor quality at the experimental endpoint; D shows a representative image of in situ tumor formation in mouse livers; E shows a statistical chart of in situ tumor quality in mouse livers; F shows changes in mouse body weight during single-drug or combination therapy. Ctrl represents the control group, Vitamin K3 represents the vitamin K3 monotherapy group, Cu / ES represents the group treated with copper ions carried by the copper ion carrier ES, and Vitamin K3+Cu / ES represents the group treated with vitamin K3 combined with Cu / ES. Detailed Implementation

[0020] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0021] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0022] Example 1: High-throughput screening of drugs with synergistic effects on copper death in liver cancer cells. Copper death is closely linked to metabolic pathways, such as glycolysis and amino acid metabolism, and these metabolic changes may affect cellular sensitivity to copper toxicity. Abnormal accumulation of metabolites may also act as signaling molecules, activating intracellular death signaling pathways and accelerating the cell death process. Based on this, we used the copper ion carrier irismo to deliver copper ions (ES+Cu). 2+ High-throughput screening, combining Cu / ES with metabolites (mentioned below), revealed that vitamin K3 can synergistically kill liver cancer cells with ES. The specific method is as follows: (1) Liver cancer cells Huh7 and SK-Hep1 were pre-coated into 96-well plates with 3500 cells per well. The next day, after cell attachment, Cu / ES (15 nM / 15 nM) was added, along with a metabolite from the Cherry Pick Library (30 μM). Only one FDA-approved drug was added to each well. Cell viability was assessed using a CCK8 assay 48 hours after treatment. Cell viability was compared between the dual-drug combination (Cu / ES combined with each metabolite) and the Cu / ES-only group. After screening, we obtained the promoting or inhibiting effects of each metabolite on copper death, ultimately identifying five metabolites that significantly promoted copper death in liver cancer cells.

[0023] The results are as follows Figure 1 As shown, high-throughput screening of drugs with a synergistic effect on copper death in liver cancer cells was found in Huh7 ( Figure 1 A) and SK-Hep1 ( Figure 1 B) Vitamin K in both cell types significantly promotes copper death in liver cancer cells. The structural formulas of vitamin K3 and the copper ion carrier elesclomol are shown below. Figure 1 As shown in C.

[0024] Example 2: Effects of copper ion carrier ES combined with vitamin K3 on the proliferation and apoptosis of liver cancer cells. (1) Cell proliferation assay to detect the synergistic inhibitory effect of Cu / ES and vitamin K3 on hepatocellular carcinoma cells. One day in advance, hepatocellular carcinoma cells Huh7, MHCC-97h, SK-Hep1, and SNU387 were seeded into 96-well plates, with 3500 cells per well. After cell adhesion, hepatocellular carcinoma cells were treated with Cu / ES (15nM / 15nM) alone or in combination with different concentrations of vitamin K3 (0, 2.5μM, 5μM, 7.5μM) for 48 hours. The viability of hepatocellular carcinoma cells was detected using a CCK8 assay kit. The drug interaction index (CDI) of each hepatocellular carcinoma cell was calculated at a vitamin K3 concentration of 7.5μM. The CDI value of Huh7 was 0.207, the CDI value of MHCC-97h was 0.671, the CDI value of SK-Hep1 was 0.533, and the CDI value of SNU387 was 0.332. The CDI value is used to assess the synergistic effect of two drugs: when CDI < 1, the two drugs have a synergistic effect; when CDI < 0.7, the synergistic effect is very significant.

[0025] (2) Colony formation assay to detect the effect of vitamin K3 on copper death in hepatocellular carcinoma cells. Huh7 hepatocellular carcinoma cells were digested and counted, then seeded into 12-well plates with 300 cells per well. Cells were cultured at 37°C for 5-7 days. After colony formation stabilized, the cells were divided into 4 groups, with 3 replicates per group. The cells were treated with single or combined drugs: control group (Ctrl), with an equal volume of DMSO; Cu / ES group, with CuCl2 (1 μM) and ES (40 nM); vitamin K3 group, with vitamin K3 at a concentration of 20 μM; and vitamin K3 combined with Cu / ES group, with CuCl2 (1 μM), ES (40 nM), and vitamin K3 at a concentration of 20 μM. After the cells reached the point where visible cell clones were formed, they were fixed and stained with crystal violet. Cell clone formation was observed, the number of clones was recorded, and statistical analysis was performed.

[0026] (3) Flow cytometry assay to detect the synergistic effect of vitamin K3 on copper death in liver cancer cells. Different liver cancer cells were treated with Cu / ES alone or in combination with vitamin K3 (drug concentration: CuCl2 1 μM, ES 40 nM, vitamin K3 concentration: 20 μM). After 48 h, cells were collected and apoptosis was detected using the Annexin V-FITC / PI apoptosis kit (Vazyme, A211-01).

[0027] The results are as follows Figure 2 As shown, we validated this in different hepatocellular carcinoma (HCC) cell lines. Cell proliferation assays, colony formation assays, and flow cytometry-based apoptosis assays revealed that the copper ion carrier irismo, combined with vitamin K3, significantly inhibited the proliferation of HCC cells and promoted their apoptosis, exhibiting a good synergistic effect. Figure 2 AC).

[0028] Example 3: Effect of Vitamin K3 on Copper Death Protein Levels in Liver Cancer Cells (1) Western blot analysis of the effect of vitamin K3 on copper death. The experiment was divided into four groups: control group (adding the same amount of ddH2O and DMSO as the experimental group), Cu / ES group alone, vitamin K3 group alone, and Cu / ES and vitamin K3 combined group (drug concentration: CuCl2 1 μM, ES 40 nM, vitamin K3 concentration: 20 μM). After 12 hours of drug treatment, proteins were extracted using a whole cell lysis kit (KGI Biotech, catalog number KGP250) according to the instructions. Protein concentration was determined using a BCA protein assay kit (Thermo Fisher, catalog number 23225). After SDS-PAGE electrophoresis, an equal amount of protein sample was transferred to a polyvinylidene fluoride membrane (PVDF membrane, Millipore, catalog number Immobilon-P PVDF). The membrane was first blocked with 5% skim milk powder for 1 hour, then incubated with primary antibody at 4°C overnight, and then incubated with secondary antibody at room temperature for 2 hours. Protein expression was detected using a secondary antibody coupled with horseradish peroxidase (HRP) and enhanced chemiluminescence detection technology.

[0029] (2) Immunofluorescence assay was used to detect the aggregation of DLAT in liver cancer cells and the co-localization of DLAT with mitochondria by vitamin K3. 5 × 10⁻⁶ cells were used. 4 Two Huh7 and SK-Hep1 cells were seeded on slides and treated with drugs alone or in combination: Control group (Ctrl) with an equal volume of DMSO; Cu / ES group with CuCl2 (1 μM) and ES (40 nM); Vitamin K3 group with Vitamin K3 (20 μM); and Vitamin K3 combined with Cu / ES group with CuCl2 (1 μM), ES (40 nM), and Vitamin K3 (20 μM). Two hours after drug administration, cells were stained with MitoTracker 100 nM for 40 minutes, washed twice with PBS, and then fixed with 4% paraformaldehyde for 15 minutes. Next, cells were treated with 0.5% Triton X-100 at room temperature for 15 minutes, followed by washing three times with PBS for 3 minutes each time. After three PBS washes, cells were blocked in a solution containing 5% goat serum for 1 hour. Next, the cells were incubated for 1 hour at room temperature with DLAT primary antibody (Cell Signaling Technology) pre-diluted overnight at 4°C and goat anti-rabbit IgG secondary antibody labeled with Alexa Fluor 546 (BOSTER, catalog number AR0009). The cell nuclei were stained with DAPI (Meilun Biotechnology, catalog number MA0128). Finally, the cells were observed and photographed using a confocal microscope.

[0030] Cu +It interacts with key intracellular proteins, especially lipoylated proteins associated with FDX1, such as DLAT. FDX1 is involved in regulating Cu. + The reduction or esterification of related proteins leads to the abnormal aggregation of proteins such as DLAT. DLAT aggregation interferes with normal cellular function and ultimately triggers cell death through the cuproporosis mechanism. The results are as follows... Figure 3 As shown, through in vitro experiments, we found that the combined use of vitamin K3 and Cu / ES can promote DLAT aggregation (Oligomers DLAT), reduce the expression level of liponylated DLAT, and at the same time, other regulatory proteins of copper death and liponylation pathway proteins are altered to varying degrees. Figure 3 A). We performed immunofluorescence experiments on different hepatocellular carcinoma cells and found the same changes as in Western blotting experiments, suggesting that the combined use of vitamin K3 and Cu / ES can increase DLAT expression in Oligomers and promote DLAT aggregation. Figure 3 (BE) indicates that vitamin K3 can promote copper death at the cellular level.

[0031] Example 4: Mouse model demonstrates the therapeutic effect of combined vitamin K3 and ES on liver cancer. (1) Nude mouse tumor model: 4-5 week old male BALB / c nude mice were housed in an SPF environment and subcutaneously inoculated with 1×10 7 Huh7 cells were inoculated, and drug administration began on the fifth day after inoculation, with administration every two days. Cu / ES was administered intraperitoneally at a dose of 0.045 / 30 mg / kg; Vitamin K3 was administered intraperitoneally at a dose of 10 mg / kg. Tumor volume, nude mouse weight, activity level, and diet were recorded. The experiment was terminated when the tumor volume reached 1000-1500 mm³ or when the nude mice exhibited abnormalities such as emaciation, decreased activity, or ulceration. At this point, the nude mice were euthanized by cervical dislocation, the tumor tissue was completely dissected, weighed, and photographed. Some tissue was fixed with 4% paraformaldehyde (for pathological sections), and some was flash-frozen in liquid nitrogen and stored at -80°C for subsequent molecular experiments.

[0032] (2) In situ liver tumorigenesis model: 4-5 week old male C57 / BL6 mice were housed in an SPF environment, and Hepa1-6 cells were inoculated in situ into the liver at a concentration of 1×10⁻⁶. 6 First, after intraperitoneal anesthesia with 1% sodium pentobarbital, an abdominal incision was made through the linea alba, and 1 × 10⁶ Hepa1-6 cells were injected into the liver parenchyma. 6A hepatocellular carcinoma model was established in mice. Survival and tumor growth were monitored throughout the experiment. Drug administration began on day 5 post-inoculation and continued every two days. Cu / ES was administered intraperitoneally at a dose of 0.045 / 30 mg / kg; Vitamin K3 was administered intraperitoneally at a dose of 10 mg / kg. Tumor volume, mouse weight, activity level, and diet were recorded. The experiment was terminated when mice exhibited emaciation, decreased activity, or ulceration. Mice were then euthanized by cervical dislocation, and the tumor tissue was completely dissected, weighed, and photographed. Some tissue was fixed with 4% paraformaldehyde (for pathological sections), and some was flash-frozen in liquid nitrogen and stored at -80°C for subsequent molecular experiments.

[0033] The results are as follows Figure 4 As shown, using a mouse subcutaneous tumor-bearing experiment, it was found that the combined use of Cu / ES and vitamin K3 can effectively inhibit the in vivo growth of liver cancer tumors. Figure 4 AC. Simultaneously, the same synergistic inhibitory effect was observed in a mouse hepatocellular carcinoma orthotopic model (AC). Figure 4 DE). Our analysis of mouse body weight during drug administration revealed no significant difference in weight among the groups, indicating that neither the use of copper ionophores, vitamin K3 alone nor in combination caused loss of appetite or emaciation in mice. Figure 4 F). Therefore, the combination of copper ion carrier ES, copper ions, and vitamin K3 proposed in this invention for the treatment of liver cancer is safe and effective in animals.

Claims

1. Application of copper ion carrier ilisimor in combination with vitamin K3 in the preparation of drugs for the treatment of liver cancer.

2. The application according to claim 1, characterized in that, The drug treats liver cancer by synergistically inhibiting the proliferation of liver cancer cells and synergistically promoting apoptosis of liver cancer cells.

3. The application according to claim 1, characterized in that, The drug treats liver cancer by synergistically promoting copper death in liver cancer cells.

4. The application according to claim 1, characterized in that, The molar ratio of the copper ion carrier ilismo, copper ions and vitamin K3 is 1:(1-25):(160-510).

5. The application according to claim 4, characterized in that, The molar ratio of the copper ion carrier ilismo, copper ions, and vitamin K3 is 1:25:

500.

6. A liver cancer treatment drug, characterized in that, It contains the copper ion carrier irismo, copper ions, and vitamin K3.

7. The liver cancer treatment drug according to claim 6, characterized in that, The molar ratio of the copper ion carrier ilismo, copper ions and vitamin K3 is 1:(1-25):(160-510).

8. The liver cancer treatment drug according to claim 7, characterized in that, The molar ratio of the copper ion carrier ilismo, copper ions, and vitamin K3 is 1:25:

500.

9. The liver cancer treatment drug according to any one of claims 6 to 8, characterized in that, It also includes pharmaceutically acceptable excipients.

10. The liver cancer treatment drug according to any one of claims 6 to 8, characterized in that, The drug is in the form of an injection.

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