Combination use of specific selenium compound and immune checkpoint inhibitor for treatment of liver cancer

Abstract

The invention relates to a combination therapy for treating liver cancer. The combination therapy mainly comprises combined application of L-selenomethionine and an immune checkpoint inhibitor (especially an anti-PD1 antibody). The composition can effectively inhibit tumor growth and enhance the tumor killing ability of an immune system. By reducing the number of tumor infiltration neutrophils and weakening the immunosuppression function of the neutrophils, the L-selenomethionine significantly improves the anti-tumor immune response. The invention provides a safe and effective treatment choice, the curative effect of the immune checkpoint inhibitor is remarkably improved, and a new treatment hope is brought to patients.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and biomedicine, specifically relating to the combined use of a particular selenium compound and an immune checkpoint inhibitor for the treatment of liver cancer. Background Technology

[0002] Hepatocellular carcinoma (HCC) is one of the most common types of liver cancer and a leading cause of cancer-related deaths worldwide. Despite the availability of various treatment options, including surgery, chemotherapy, targeted therapy, and immunotherapy, efficacy for patients with advanced HCC remains limited. Immune checkpoint inhibitors (such as anti-programmed death protein-1, anti-PD1) have shown significant efficacy in some cancer types, but their antitumor response rates remain low in certain cases when used alone.

[0003] Therefore, research on compounds that synergize with immune checkpoint inhibitors has significant clinical implications. Summary of the Invention

[0004] The purpose of this invention is to provide a combination therapy for treating liver cancer, comprising the combined use of L-selenomethionine and immune checkpoint inhibitors (especially anti-PD1 antibodies). This invention provides a novel cancer treatment method that can effectively inhibit tumor growth and enhance the immune response, thereby offering more effective treatment options for liver cancer patients.

[0005] In a first aspect of the invention, there is provided the use of a pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of liver cancer, wherein the pharmaceutical composition comprises component (a) a therapeutically effective amount of selenomethionine and component (b) a therapeutically effective amount of an immune checkpoint inhibitor.

[0006] In another preferred embodiment, the liver cancer includes primary liver cancer and / or secondary liver cancer.

[0007] In another preferred embodiment, the liver cancer includes hepatocellular carcinoma, intrahepatic cholangiocarcinoma, or mixed-type liver cancer.

[0008] In another preferred embodiment, the liver cancer includes diffuse liver cancer, massive liver cancer, nodular liver cancer, or small liver cancer.

[0009] In another preferred embodiment, the liver cancer is primary liver cancer.

[0010] In another preferred embodiment, the liver cancer is primary hepatocellular carcinoma.

[0011] In another preferred embodiment, the selenomethionine is L-selenomethionine.

[0012] In another preferred embodiment, the immune checkpoint inhibitor includes PD1 antibody and / or PD-L1 antibody.

[0013] In another preferred embodiment, the mass ratio of the selenomethionine to the immune checkpoint inhibitor is 10:1 to 1:10, preferably 5:1 to 1:5, for example 2:1, 1.5:1, 8:7, 4:3, 1:1, 1:1.5, 3:4, 7:8, 1:2, 1:3.

[0014] In another preferred embodiment, the selenomethionine (preferably L-selenomethionine) enhances the antitumor effect by reducing tumor-infiltrating neutrophils and affecting their immunosuppressive function.

[0015] In another preferred embodiment, the liver cancer is a liver cancer in which neutrophils account for more than 60% of the total number of CD45-positive immune cells in the tumor, preferably more than 65%.

[0016] In another preferred embodiment, the pharmaceutical composition is also used for one or more purposes selected from the group consisting of:

[0017] (a) Reduce the number of tumors or reduce the quality or volume of tumors;

[0018] (b) Reduce the liver-to-body weight ratio;

[0019] (c) Reduce the number of neutrophils infiltrating the tumor;

[0020] (d) Reduce the expression of classical genes in neutrophils in tumors;

[0021] (e) weakens the immunosuppressive function in tumors;

[0022] (f) Downregulate genes related to the PD1 pathway.

[0023] In another preferred embodiment, the classical genes in the neutrophils are selected from the group consisting of Camp, Ltf, MPO, NGP, or combinations thereof.

[0024] In another preferred embodiment, the PD1 pathway-related genes are selected from the group consisting of Lck, Pdcd1, Cd274, CTLA4, or combinations thereof.

[0025] In another preferred embodiment, the dosage form of the pharmaceutical composition or the drug is liquid, solid, or semi-solid.

[0026] In another preferred embodiment, the dosage form of the pharmaceutical composition or the drug includes tablets, granules, capsules, oral liquids, or injections.

[0027] In another preferred embodiment, the injectable includes an intravenous injectable, an intraperitoneal injectable, or a subcutaneous injectable.

[0028] In another preferred embodiment, the component (a) in the pharmaceutical composition accounts for 1-99 wt% of the total weight of the pharmaceutical composition, more preferably 10-90 wt%, and more preferably 30-70 wt%.

[0029] In another preferred embodiment, the component (b) comprises 1-99 wt% of the total weight of the pharmaceutical composition, more preferably 10-90 wt%, and more preferably 30-70 wt%.

[0030] In another preferred embodiment, the pharmaceutical composition further includes other drugs for the prevention and / or treatment of liver cancer.

[0031] In a second aspect of the invention, a pharmaceutical composition for the prevention and / or treatment of liver cancer is provided, comprising component (a) a therapeutically effective amount of selenomethionine and component (b) a therapeutically effective amount of an immune checkpoint inhibitor, wherein the mass ratio of the selenomethionine to the immune checkpoint inhibitor is 1:1 to 1:1.5, for example, 1:1, 1:1.2, 1:1.3, 1:1.4, 3:4, or 7:8.

[0032] In another preferred embodiment, the selenomethionine is L-selenomethionine.

[0033] In another preferred embodiment, the immune checkpoint inhibitor includes PD1 antibody and / or PD-L1 antibody.

[0034] In another preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0035] In another preferred embodiment, the dosage form of the pharmaceutical composition is liquid, solid, or semi-solid.

[0036] In another preferred embodiment, the dosage form of the pharmaceutical composition includes tablets, granules, capsules, oral liquids, or injections.

[0037] In another preferred embodiment, the injectable includes an intravenous injectable, an intraperitoneal injectable, or a subcutaneous injectable.

[0038] In another preferred embodiment, the total content of component (a) and component (b) is 1 to 99 wt% of the total weight of the composition, more preferably 5 to 90 wt%.

[0039] In another preferred embodiment, the pharmaceutical composition further includes other drugs for the prevention and / or treatment of liver cancer.

[0040] In a third aspect of the invention, a medicine box is provided, comprising:

[0041] (i) a first container, and a therapeutically effective amount of selenomethionine or a drug containing component (a) located in the first container; and

[0042] (ii) a second container, and a therapeutically effective amount of an immune checkpoint inhibitor or a drug containing component (b) located in the second container.

[0043] The mass ratio of the selenomethionine to the immune checkpoint inhibitor is 1:1 to 1:1.5, for example, 1:1, 1:1.2, 1:1.3, 1:1.4, 3:4, or 7:8.

[0044] In another preferred embodiment, the selenomethionine is L-selenomethionine.

[0045] In another preferred embodiment, the immune checkpoint inhibitor includes PD1 antibody and / or PD-L1 antibody.

[0046] In another preferred embodiment, the medicine box also contains instructions for administration of a drug containing or comprising component (a) and component (b) in combination to prevent and / or treat liver cancer.

[0047] In another preferred embodiment, the specification states that the ingredient (a) or the drug containing ingredient (a) located in the first container and the ingredient (b) or the drug containing ingredient (b) located in the second container are administered simultaneously or separately.

[0048] In another preferred embodiment, the specification indicates one or more application features selected from the group consisting of:

[0049] (a) The dosage of the selenomethionine is 0.1-10 mg / kg, preferably 1-5 mg / kg, and more preferably 1-2 mg / kg;

[0050] (b) The application frequency of the selenomethionine is 1-7 consecutive days per week, preferably 5-7 consecutive days per week;

[0051] (c) The application time of the selenoamino acid is 1-20 weeks, preferably 2-12 weeks, and more preferably 4-8 weeks;

[0052] (d) The dosage of the immune checkpoint inhibitor is 1-30 mg / kg, preferably 1-10 mg / kg, and more preferably 5-10 mg / kg;

[0053] (e) The immune checkpoint inhibitor is administered 1-5 times per week, preferably 1-2 times per week, for example once every 3-4 days.

[0054] (f) The immune checkpoint inhibitor is administered for 1-20 weeks, preferably 2-12 weeks, and more preferably 4-8 weeks.

[0055] In a fourth aspect of the present invention, a method for preventing and / or treating liver cancer is provided, comprising the steps of:

[0056] The pharmaceutical composition described in the second aspect of the present invention, or the medicine box described in the third aspect of the present invention, is applied to the recipient as needed.

[0057] In another preferred embodiment, the application includes injection.

[0058] In another preferred embodiment, the injection includes intravenous injection, intraperitoneal injection, or subcutaneous injection.

[0059] In another preferred embodiment, the object includes a human or a non-human mammal.

[0060] In another preferred embodiment, the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.

[0061] In another preferred embodiment, the selenomethionine and the immune checkpoint inhibitor are administered simultaneously or separately.

[0062] In another preferred embodiment, the dosage of the selenomethionine is 0.1-10 mg / kg, preferably 1-5 mg / kg, and more preferably 1-2 mg / kg.

[0063] In another preferred embodiment, the selenomethionine is applied for 1-7 consecutive days per week, more preferably 5-7 consecutive days per week.

[0064] In another preferred embodiment, the application time of the selenoamino acid is 1-20 weeks, more preferably 2-12 weeks, and even more preferably 4-8 weeks.

[0065] In another preferred embodiment, the dosage of the immune checkpoint inhibitor is 1-30 mg / kg, preferably 1-10 mg / kg, and more preferably 5-10 mg / kg.

[0066] In another preferred embodiment, the immune checkpoint inhibitor is administered 1-5 times per week, preferably 1-2 times per week, for example once every 3-4 days.

[0067] In another preferred embodiment, the immune checkpoint inhibitor is administered for 1-20 weeks, more preferably 2-12 weeks, and even more preferably 4-8 weeks.

[0068] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0069] Figure 1 The method and results of constructing a liver cancer model are shown. (a) Fluid dynamic transfection induces liver tumor formation; (b) Drug administration regimen; (c) HE staining and immunofluorescence staining 14 days later show that the liver cancer model was successfully constructed.

[0070] Figure 2 The effects of L-selenomethionine alone, PD-1 alone, and combination therapy are shown. (a) Hepatocellular carcinoma modeling and drug administration regimen; (b) Optical photographs of tumor number in liver tissue in different groups; (c) Analysis of tumor number in liver tissue in different groups; (d) Analysis of liver weight ratio in liver tissue in different groups.

[0071] Figure 3 The number of neutrophils infiltrating the tumor was shown in different groups.

[0072] Figure 4 The graph shows the analysis of the number of neutrophils infiltrating the tumor in different groups.

[0073] Figure 5 The images show the distribution of neutrophils infiltrating the tumor in different groups, as revealed by immunofluorescence staining of tissue sections.

[0074] Figure 6 The combination therapy group showed a reduction in tumor-infiltrating neutrophils. (a) Gene volcano plot of expression in the combination therapy group; (b) REACTOME differential gene annotation analysis plot; (c) KEGG differential gene annotation analysis plot.

[0075] Figure 7 The graph shows the enrichment analysis of the differentially expressed gene REACTOME in the combination therapy group.

[0076] Figure 8 The study showed that L-selenomethionine affected the immunosuppressive function of the PD1 pathway in the combination therapy group. (a) Gene volcano plot of expression in the combination therapy group; (b) REACTOME differential gene annotation analysis plot; (c) KEGG differential gene annotation analysis plot.

[0077] Figure 9 The graph shows the enrichment analysis of the differentially expressed gene REACTOME in the combination therapy group. Detailed Implementation

[0078] Through extensive and in-depth research, the inventors have discovered for the first time a combination therapy with significant anti-tumor effects, combining L-selenomethionine with immune checkpoint inhibitors (especially PD1 antibodies), particularly suitable for treating liver cancer. This combination not only directly inhibits tumor growth but also significantly improves anti-cancer efficacy by reducing the number of tumor-infiltrating neutrophils and weakening their immunosuppressive function, providing a new treatment option for patients with primary liver cancer. This invention was completed based on this discovery.

[0079] the term

[0080] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0081] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0082] selenomethionine

[0083] Selenomethionine (SeMet) is a naturally occurring selenium-containing amino acid with potential antioxidant, immunomodulatory, and anticancer properties.

[0084] use

[0085] Existing technologies have attempted to combine L-selenomethionine with anti-PD1 therapy to treat lung cancer, but the synergistic effect is poor, sometimes even less effective than L-selenomethionine alone. This invention, however, reveals that in a primary mouse liver cancer model, L-selenomethionine effectively inhibits tumor growth and exhibits a significant synergistic anti-tumor effect with anti-PD1 therapy. This anti-tumor effect is primarily achieved by reducing tumor-infiltrating neutrophils (TANs) and influencing their immunosuppressive function.

[0086] Pharmaceutical Compositions and Administration

[0087] On the other hand, the present invention also provides a pharmaceutical composition comprising (a) a therapeutically effective amount of selenomethionine; (b) a therapeutically effective amount of an immune checkpoint inhibitor; and (c) a pharmaceutically acceptable carrier or excipient, wherein the mass ratio of selenomethionine to the immune checkpoint inhibitor is 1:1 to 1:1.5.

[0088] The dosage of each of the two active ingredients of the present invention is typically 10 micrograms to 100 milligrams per dose, preferably 100 to 1000 micrograms per dose. For the purposes of the present invention, a therapeutically effective dose is an active ingredient administered to an individual at a dose of about 0.01 mg / kg to 50 mg / kg, preferably 0.05 mg / kg to 10 mg / kg body weight. Furthermore, the two active ingredients of the present invention can be used alone or in combination with other therapeutic agents (e.g., formulated into the same pharmaceutical composition or packaged in a common kit in different containers).

[0089] Pharmaceutical compositions may also contain pharmaceutically acceptable carriers. The term "pharmaceutically acceptable carrier" refers to a carrier used for the administration of a therapeutic agent. This term refers to pharmaceutical carriers that do not induce antibodies harmful to the individual receiving the composition and do not cause excessive toxicity after administration. These carriers are well known to those skilled in the art. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, adjuvants, and combinations thereof.

[0090] Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerin, and ethanol. Additionally, these carriers may contain auxiliary substances such as wetting agents or emulsifiers, pH buffers, etc.

[0091] Typically, therapeutic compositions can be formulated as injectable preparations, such as liquid solutions or suspensions; they can also be formulated as solid forms, such as lyophilized powders, that are suitable for reconstitution into solutions or suspensions prior to injection and are carried by a liquid carrier.

[0092] Once formulated into the compositions of the present invention, they can be administered via conventional routes, including (but not limited to): intratumoral, intramuscular, intravenous, subcutaneous, intradermal, intraperitoneal, or local administration. The objects to be prevented or treated can be animals, such as mammals; especially humans.

[0093] When the pharmaceutical compositions of the present invention are used for actual treatment, various dosage forms of the pharmaceutical compositions may be used depending on the application. Preferably, they are intravenous preparations or intraperitoneal injections.

[0094] These pharmaceutical compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally by adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizers, and the formulation process can be carried out in the conventional manner depending on the dosage form.

[0095] For example, the preparation of the injection can be carried out as follows: the two active ingredients of the present invention are dissolved together with the base substance in sterile water (in which surfactants are dissolved), the osmotic pressure and pH are adjusted to physiological state, and suitable pharmaceutical additives such as preservatives, stabilizers, buffers, isotonic agents, antioxidants and thickeners can be added as appropriate, and then the solution is completely dissolved.

[0096] The pharmaceutical compositions of the present invention can also be administered in a sustained-release form. For example, the two active ingredients of the present invention can be incorporated into a pill or microcapsule carrying a sustained-release polymer, and then the pill or microcapsule can be surgically implanted into the tissue to be treated. Examples of sustained-release polymers include ethylene-vinyl acetate copolymers, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymers, lactic acid-glycolic acid copolymers, etc., and preferably biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers.

[0097] When the pharmaceutical composition of the present invention is used for actual treatment, the dosage of the two active ingredients can be reasonably determined according to the weight, age, sex, and symptom severity of each patient to be treated.

[0098] medicine box

[0099] In another aspect, the present invention also provides a medicine box comprising a first container and a therapeutically effective amount of selenomethionine or a drug containing component (a) located in the first container; a second container and a therapeutically effective amount of an immune checkpoint inhibitor or a drug containing component (b) located in the second container.

[0100] When selenomethionine and immune checkpoint inhibitors are not suitable to be combined to form a single drug composition, or when the administration frequencies of the two active ingredients are not synchronized, the drug can be administered to the patient in the form of a pillbox.

[0101] The medicine box of the present invention also includes an instruction manual.

[0102] The instructions state that ingredient (a) or a drug containing ingredient (a) located in the first container may be administered separately from or simultaneously with ingredient (b) or a drug containing ingredient (b) located in the second container.

[0103] The main advantages of this invention include:

[0104] (1) This invention discovered that L-selenomethionine, as an active ingredient, can effectively inhibit the growth of primary mouse liver cancer.

[0105] (2) This invention has discovered that when L-selenomethionine is used in combination with anti-PD1 therapy, it exhibits a synergistic anti-tumor effect and significantly enhances the immune system's ability to kill tumors.

[0106] (3) This invention has discovered that L-selenomethionine enhances the anti-tumor immune response by reducing the number of tumor-infiltrating neutrophils and affecting their immunosuppressive function.

[0107] (4) L-selenomethionine exhibits good safety and tolerability, and can enhance the effect of immunotherapy without significantly increasing toxicity.

[0108] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0109] Experimental methods

[0110] 1. Establishment of a Primary Mouse Model of Hepatocellular Carcinoma: All experimental animals were housed and used in accordance with the guidelines of the Animal Care and Use Committee of the Shanghai Institute of Nutrition and Health, and were approved by them. The animals used were in good health, and all experiments used male C57BL / 6J mice. The C57BL / 6 mice used in this study were purchased from Shanghai Silex Laboratory Animal Co., Ltd. Mice were housed in a temperature-controlled chamber following a 12-hour light-12-hour dark diurnal cycle. The mouse model of hepatocellular carcinoma was established using a hydrodynamic tail vein injection method. Briefly, at 8 weeks of age, 30 micrograms of the appropriate pT4 vector (pT4-NRas) were injected into the mice. G12V The plasmid (pT4-myr-AKT) and 3 μg of plasmid containing SB100X transposase were diluted in sterile filtered Ringer's solution to a total volume of 10% of the mouse body weight and injected into the lateral tail vein over 7 seconds. The DNA used for hydrodynamic tail vein injection was prepared using an endotoxin-free plasmid extraction kit (Tiangen Biotech, China).

[0111] 2. L-Selenomethionine and Anti-PD1 Intervention in Mice: Fourteen days after delivery of the oncogene, small tumors were observed in HE staining and immunofluorescence staining, at which point treatment was initiated. The dosage of L-selenomethionine was 2 mg / kg, with each mouse weighing approximately 25g. This translates to approximately 50ug of L-selenomethionine injected intraperitoneally per mouse daily. The PD1 antibody (Bio X Cell, InVivoMAb anti-mouse PD-1, RMP1-14) was administered to each mouse twice weekly at 200ug per injection. This dosage referenced most of the literature. The control group used PBS and an isotype control antibody against the PD1 antibody at the same injection dose.

[0112] 3. Flow Cytometry Analysis: Liver tumor tissue was removed from mice and washed with ice-cold phosphate-buffered saline to remove blood. After digestion using the Miltenyi Tumor Digestion Kit (130-096-730), the tumor tissue was sieved through a 70-micron cell separation sieve to obtain a single-cell suspension. The resulting cell suspension was centrifuged at 2000 rpm for 5 minutes, the supernatant was discarded, and red blood cells were lysed and resuspended in fresh PBS. The resulting single-cell suspension was then counted, and an appropriate number of cells were used for staining. Live / dead staining was used. TM After staining with a staining kit for fixing dead cells for 10 minutes, cells were incubated for 10 minutes with Fc receptor blocking antibodies anti-CD16 / CD32 to reduce non-specific binding. Then, anti-CD45 (30-F11, 1:200), anti-CD11b (M1 / 70, 1:200), and anti-Ly6G (1A8, 1:200) antibodies were added, and the cells were incubated on ice in the dark for 30 minutes. All antibodies were purchased from eBioScience. After staining, cells were washed twice with ice-cold PBS to remove unbound antibodies and both live and dead stains. Data were then acquired using a Cytoflex LX flow cytometer and analyzed using FlowJo software.

[0113] 4. RNA Sequencing: Tumors were immediately flash-frozen in liquid nitrogen and stored at -80°C to prevent RNA degradation. Total RNA was extracted using TRIzol reagent and quality-tested using an Agilent 2100 Bioanalyzer to ensure no significant RNA degradation. The total RNA was then processed using mRNA enrichment methods, reverse transcribed into cDNA, and subjected to end repair, A-tailing, and sequencing adapter ligation. Subsequent PCR amplification and quality control were performed. High-throughput sequencing was performed using the Illumina platform, with PE150 paired-end sequencing at a sequencing throughput of 6G. FastQC was used for quality control, removing low-quality reads and adapter contamination sequences. Clean reads were aligned to the reference genome using HISAT2 or STAR software, gene quantification was performed using feature counts software, and finally, differential expression analysis was performed using software such as DESeq2.

[0114] 5. Immunofluorescence staining: Mouse tumor tissues were first fixed in 4% paraformaldehyde for 24 hours, then embedded in paraffin and sectioned. The paraffin sections were stained to detect GFP (Abmart, 7G9) markers, neutrophil markers LY6G (Servicebio, GB11229), and S100A9 (R&D, AF2065) in the tumors. The sections were then incubated for 2 hours at room temperature in a dark, humidified chamber with the corresponding secondary antibodies (Thermo, donkey anti-mouse IgG (H+L) Alexa Fluor 488, donkey anti-goat IgG (H+L) Alexa Fluor 568, and chicken anti-rabbit IgG (H+L) Alexa Fluor 6471:500). Finally, the cell nuclei were counterstained with DAPI and the sections were mounted. Fluorescence images were acquired using an Olympus FV1000 confocal laser scanning microscope.

[0115] Example 1: Construction of a mouse liver cancer model

[0116] Hydrodynamic transfection (HVT) is a technique commonly used to construct mouse liver cancer models. It achieves efficient gene expression in the liver by rapidly injecting a large volume of DNA solution into the mouse tail vein. In this method, a plasmid carrying an oncogene is diluted in a sterile solution equivalent to 10% of the mouse's body weight and injected into the mouse via the tail vein within a very short time (usually less than 10 seconds). The large volume and rapid injection cause a temporary increase in intravascular pressure in the liver, allowing DNA to enter hepatocytes efficiently. To enhance the efficiency of gene integration and expression, the Sleeping Beauty transposon system is used, employing the SB100X transposase to integrate the target gene into the hepatocyte genome, thereby achieving long-term and stable gene expression. This method is simple and efficient, and can induce liver tumor formation in a short time; therefore, it is widely used in research on the occurrence, development, and treatment of liver cancer.

[0117] Example 2: Synergistic effect of L-selenomethionine and anti-PD1 therapy against liver cancer tumors.

[0118] like Figure 1 As shown, the liver cancer model uses NRAS. G12V A primary hepatocellular carcinoma model driven by +myr-AKT was established by delivering a G12V mutant version of NRAS and myristylated AKT via hydrodynamic transfection, resulting in a primary hepatocellular carcinoma model within approximately 8 weeks. Fourteen days after oncogene delivery, small tumors were observed in HE staining and immunofluorescence staining, at which point treatment was initiated. The dosage of L-selenomethionine was 2 mg / kg, with each mouse weighing approximately 25g. This translates to approximately 50ug of L-selenomethionine administered intraperitoneally per mouse daily. PD1 antibody was administered to each mouse twice weekly at a dose of 200ug.

[0119] Specifically, in a primary mouse model of liver cancer, L-selenomethionine (2 mg / kg) was administered once daily via intraperitoneal injection. Simultaneously, another group of mice were treated with anti-PD1 antibody twice weekly at a dose of 200 μg / mouse.

[0120] This dosage reference is based on the dosages found in most literature. The control group used PBS and the isotype control antibody IgG2a for PD1, with identical injection doses. The L-selenomethionine monotherapy group used L-selenomethionine and the isotype control antibody IgG2a for PD1, with identical injection doses. The anti-PD1 antibody monotherapy group used PBS and PD1 antibody, with identical injection doses. The combination therapy group used L-selenomethionine and anti-PD1 antibody, with identical injection doses.

[0121] like Figure 2As shown, after 8 weeks of model construction, phenotypic analysis of sacrificed mice revealed that L-selenomethionine treatment alone significantly reduced liver weight-to-body weight ratio and tumor number, with slightly better results than PD1 treatment alone. Combination therapy further reduced tumor number compared to monotherapy, indicating a synergistic effect between L-selenomethionine and PD1 antibody.

[0122] Example 3

[0123] like Figure 3-4 As shown in NRAS G12V In a +myr-AKT-driven primary liver cancer model, there is extensive infiltration of neutrophils. Neutrophils comprise over 60% of the CD45-positive immune cells in the tumor, and they are known to exacerbate tumor growth through immunosuppression in various ways. Treatment with L-selenomethionine significantly downregulated the number of neutrophils infiltrating the tumor, while no significant change was observed in the PD1 antibody treatment group. A significant downregulation was also observed in the combination therapy group.

[0124] like Figure 5 As shown, immunofluorescence staining of tissue sections revealed that the expression of neutrophil markers Ly6G and S100A9 in GFP-positive areas, i.e., cells infiltrating the tumor region, was consistent with the flow cytometry results, leading to the conclusion that L-selenomethionine can reduce neutrophil infiltration in tumors.

[0125] RNA-seq was performed on tumor tissues of selenium-supplemented liver cancer mice, and volcano plots were obtained. Figure 6 As shown in Figure a, the expression of classic genes such as Camp, Ltf, MPO, and NGP in neutrophils showed a significant decrease, demonstrating that selenium supplementation exerted a significant effect on this cell group. REACTOME and KEGG differential gene annotation analysis ( Figure 6 Both the results (bc) and (bc) indicate the significant impact of selenium on the immune system, as evidenced by its high enrichment in the immune system. Reactome enrichment analysis of differentially expressed genes also reveals enrichment in the neutrophil degranulation pathway, consistent with previous findings regarding neutrophils. (Enrichment chord diagram) Figure 7 It can be seen that genes are enriched in this pathway.

[0126] Example 4

[0127] Compared with PD1 monotherapy, selenium-based anti-PD1 combination therapy showed significant downregulation of PD1 pathway-related genes such as Lck, Pdcd1, Cd274, and CTLA4. Figure 8(a) Because the PD1 signaling pathway mediates immunosuppression, its downregulation implies that selenium supplementation has a positive effect on anti-PD1 therapy. REACTOME and KEGG analysis ( Figure 8 Both β and β show that selenium supplementation has a significant impact on the immune system. RECATOME gene enrichment analysis was performed to compare differentially expressed genes between selenium anti-PD1 combination therapy and PD1 therapy alone. Figure 9 The study revealed changes in multiple T cell-related pathways, especially the PD1 pathway.

[0128] Summarize

[0129] The above experiments show that L-selenomethionine significantly inhibits tumor growth, and the tumor volume is significantly smaller compared to the control group. When L-selenomethionine is used in combination with anti-PD1 antibody, tumor growth is significantly inhibited, and the tumor volume is further reduced compared to the single treatment group. In all treatment groups, L-selenomethionine significantly reduced the number of tumor-infiltrating neutrophils and weakened their immunosuppressive function, thereby enhancing the anti-tumor immune response.

[0130] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. Use of a pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of liver cancer, wherein, The pharmaceutical composition comprises (a) a therapeutically effective amount of selenomethionine and (b) a therapeutically effective amount of an immune checkpoint inhibitor.

2. The use as described in claim 1, characterized in that, The liver cancer mentioned includes primary liver cancer and / or secondary liver cancer.

3. The use as described in claim 1, characterized in that, The selenomethionine is L-selenomethionine; the immune checkpoint inhibitor includes PD1 antibody and / or PD-L1 antibody.

4. The use as described in claim 1, characterized in that, The mass ratio of the selenomethionine to the immune checkpoint inhibitor is 10:1 to 1:10, preferably 5:1 to 1:

5.

5. The use as described in claim 1, characterized in that, The selenomethionine enhances the anti-tumor effect by reducing tumor-infiltrating neutrophils and affecting their immunosuppressive function.

6. The use as described in claim 1, characterized in that, The pharmaceutical composition or dosage form of the drug includes tablets, granules, capsules, oral liquids, or injections.

7. The use as described in claim 6, characterized in that, The injectables include intravenous injections, intraperitoneal injections, or subcutaneous injections.

8. A pharmaceutical composition for the prevention and / or treatment of liver cancer, comprising component (a) a therapeutically effective amount of selenomethionine and component (b) a therapeutically effective amount of an immune checkpoint inhibitor, wherein, The mass ratio of the selenomethionine to the immune checkpoint inhibitor is 1:1 to 1:1.

5.

9. The pharmaceutical composition according to claim 8, characterized in that, The selenomethionine is L-selenomethionine; the immune checkpoint inhibitor includes PD1 antibody and / or PD-L1 antibody.

10. A medicine box, comprising: (i) a first container, and a component (a) therein containing a therapeutically effective amount of selenomethionine or a drug containing component (a); and (ii) a second container, and a therapeutically effective amount of an immune checkpoint inhibitor or a drug containing component (b) located in the second container. The mass ratio of the selenomethionine to the immune checkpoint inhibitor is 1:1 to 1:1.5.

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