Application of omega-3 polyunsaturated fatty acid in preparation of synergist of immune checkpoint inhibitor

Abstract

The invention provides application of omega-3 polyunsaturated fatty acid in preparation of a synergist of an immune checkpoint inhibitor, and relates to the technical field of medical chemistry. The EPA can obviously down-regulate expression of PD-L1, can effectively improve the drug sensitivity of the PD-1 inhibitor, obviously enhances the anti-liver cancer effect of the PD-1 inhibitor, improves immune response and delays the progress of liver cancer caused by various disease causes. A combined treatment strategy is provided for liver cancer immunotherapy, immunotherapy response is enhanced, and the risk of immune drug resistance and super-progression is reduced, so that universal treatment synergy and safety improvement of liver cancer related to various etiologies are realized, and the application has important potential in clinical liver cancer immunotherapy.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry technology, specifically relating to the application of ω-3 polyunsaturated fatty acids in the preparation of potentiators for immune checkpoint inhibitors. Background Technology

[0002] Hepatocellular carcinoma (HCC) is a type of malignant tumor with significant etiological heterogeneity, mainly including virus-associated HCC and metabolic-associated fatty liver cancer. With the continuous rise in the incidence of metabolic diseases, the overlapping of multiple etiologies has become an important characteristic of HCC development. HCCs with different etiological backgrounds exhibit significant differences in immune microenvironment and treatment response. Existing research shows that virus-associated HCC has a certain sensitivity to immunotherapy, but acquired resistance easily develops during treatment, making it difficult to maintain efficacy long-term. Metabolic-associated fatty liver cancer, on the other hand, is generally insensitive to immunotherapy and may even induce tumor hyperprogression, severely limiting the application of immunotherapy in this type of patient. These differences suggest that metabolic abnormalities have become a crucial determinant of the efficacy of immunotherapy for HCC. However, current immunotherapy techniques mainly focus on directly activating the immune response, failing to systematically regulate metabolic imbalances in the tumor and its microenvironment. This makes it difficult to simultaneously address issues such as immune tolerance, insufficient efficacy, and the risk of hyperprogression, especially failing to meet the treatment needs of HCCs with different etiologies.

[0003] Eicosapentaenoic acid (EPA) is a member of the omega-3 polyunsaturated fatty acid family, mainly found in foods such as deep-sea fish oil. It possesses various biological activities, including scavenging free radicals, inhibiting inflammatory responses, protecting the cardiovascular system, and enhancing memory. However, the properties of EPA in immunotherapy, especially in liver cancer immunotherapy, have not been systematically studied. There are no studies on the synergistic effect of EPA on immunotherapy, nor have there been any explorations into its potential use as an immunotherapy enhancer in tumor treatment. Therefore, there is an urgent need to develop a drug with synergistic effects on liver cancer prevention and treatment, employing a liver cancer immunotherapy enhancement strategy centered on metabolic intervention. This strategy aims to reshape the tumor metabolic state and immune metabolic microenvironment, relieve metabolism-related immunosuppression, enhance the immunotherapy response, and reduce the risk of immune resistance and hyperprogression. This would achieve universal therapeutic enhancement and improved safety for liver cancers of various etiologies, thereby improving the overall effectiveness of liver cancer prevention and treatment. Summary of the Invention

[0004] To address the aforementioned technical problems, the primary objective of this invention is to provide the application of ω-3 polyunsaturated fatty acids in the preparation of synergists for PD-1 inhibitors.

[0005] A second objective of this invention is to provide the use of ω-3 polyunsaturated fatty acids in the preparation of drugs that enhance the anti-hepatocellular carcinoma activity of PD-1 inhibitors.

[0006] A third objective of this invention is to provide the application of ω-3 polyunsaturated fatty acids in the preparation of PD-L1 inhibitors. The EPA of this invention can significantly downregulate PD-L1 expression and can be used as a PD-L1 inhibitor for the prevention and / or treatment of liver cancer.

[0007] The fourth objective of this invention is to provide the application of EPA in the in vitro inhibition of PD-L1 expression in liver cancer cells.

[0008] The fifth objective of this invention is to provide an agent for the prevention and treatment of liver cancer.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of ω-3 polyunsaturated fatty acids in the preparation of PD-L1 inhibitors, wherein the ω-3 polyunsaturated fatty acids include EPA.

[0010] This invention also provides the application of EPA in the in vitro inhibition of PD-L1 expression in liver cancer cells.

[0011] The present invention also provides the use of ω-3 polyunsaturated fatty acids in the preparation of synergists for PD-1 inhibitors, wherein the ω-3 polyunsaturated fatty acids include EPA.

[0012] The present invention also provides the use of ω-3 polyunsaturated fatty acids in the preparation of drugs that enhance the anti-hepatocellular carcinoma activity of PD-1 inhibitors, wherein the ω-3 polyunsaturated fatty acids include EPA.

[0013] As one implementation method, the EPA is used orally in combination with the PD-1 inhibitor administered intravenously.

[0014] The present invention also provides an agent for the prevention and treatment of liver cancer, the agent comprising a therapeutically effective amount of EPA and a therapeutically effective amount of a PD-1 inhibitor.

[0015] In one embodiment, the formulation comprises 3-8% EPA by mass and a PD-1 inhibitor at a dosage of 2-6 mg / kg.

[0016] In one embodiment, the PD-1 inhibitor includes a PD-1 neutralizing antibody.

[0017] As one implementation, the formulation also includes a pharmaceutically acceptable carrier.

[0018] In one embodiment, the formulation is an oral formulation and / or an injectable formulation.

[0019] The advantages of this invention compared to existing technologies are as follows: This invention has demonstrated in cells and in mice that EPA significantly downregulates PD-L1 expression, and can be used as a PD-L1 inhibitor in the treatment of liver cancer.

[0020] In this invention, EPA can not only reverse immune resistance, but also has a sensitizing effect on immunotherapy. It can effectively improve the efficacy of immune checkpoint inhibitors (PD-1 inhibitors), significantly enhance the effect of immune checkpoint inhibitors in the fight against liver cancer, improve immune response, and delay the progression of liver cancer.

[0021] In this invention, EPA can significantly enhance the response to immunotherapy and reduce the risk of immune resistance and hyperprogression, thereby achieving universal therapeutic efficacy and improved safety for liver cancers of various etiologies. Attached Figure Description

[0022] Figure 1 The effect of EPA treatment on PD-L1 expression in Hepa1-6 liver cancer cells was investigated. Figure 2 The effect of EPA on PD-L1 expression in liver cancer tissue in mice (high-pressure hydrodynamic model); Figure 3 This image shows the results of EPA sensitization to PD-1 inhibitors (hepatocellular carcinoma subcutaneous tumor model). Image A shows tumors sensitized to PD-1 inhibitors after feeding tumor-forming mice. Images B and C are statistical graphs of tumor size and composition. Figure 4 The graph shows the results of EPA sensitization to PD-1 neutralizing antibody (metabolic fatty liver disease-related hepatocellular carcinoma model). AC represents the liver inhibition effect in mice compared to the control group and the group using EPA alone, while DF represents the anti-cancer effect of using PD-1 neutralizing antibody alone and the combination of PD-1 neutralizing antibody and EPA. Detailed Implementation

[0023] This invention provides the application of ω-3 polyunsaturated fatty acids in the preparation of PD-L1 inhibitors, wherein the ω-3 polyunsaturated fatty acids include EPA. This invention demonstrates that EPA exhibits a high inhibitory effect on PD-L1 expression in liver cancer cells. To further verify the inhibitory effect of EPA on PD-L1 expression, this invention uses a C57 / BL6 mouse model as the experimental subject to investigate the role of EPA in PD-L1 expression in mice. This invention constructs a liver cancer model by injecting CTNNB1, c-MYC plasmids, and SB100 plasmids into the liver of mice using a high-pressure hydrodynamic method. After tumor formation in the mice, the expression level of PD-L1 in the group fed a diet containing 5% EPA and the conventional diet group was detected, revealing that EPA inhibits PD-L1 expression in mouse liver cancer tissues. This invention, through studies on liver cancer cells and a mouse liver cancer model, has found that EPA has a significant inhibitory effect on PD-L1 expression.

[0024] This invention also provides the application of EPA in the in vitro inhibition of PD-L1 expression in liver cancer cells. In this invention, Hepal-6 liver cancer cells were used as the experimental subject, and were incubated with 0–300 μM EPA for 12–36 h. The preferred concentration of EPA was 100 μM, 200 μM, or 300 μM; the preferred incubation time was 24 h. After incubation, the expression level of PD-L1 was detected. As an optional implementation, the detection was performed using Western blot to detect the intracellular PD-L1 expression level. This invention constructs a cell model for inhibiting PD-L1 expression in liver cancer cells.

[0025] The present invention also provides the use of ω-3 polyunsaturated fatty acids in the preparation of synergists for PD-1 inhibitors, wherein the ω-3 polyunsaturated fatty acids include EPA.

[0026] The present invention also provides the use of ω-3 polyunsaturated fatty acids in the preparation of drugs that enhance the anti-hepatocellular carcinoma activity of PD-1 inhibitors, wherein the ω-3 polyunsaturated fatty acids include EPA.

[0027] In this invention, the binding of PD-L1 to PD-1 has an immunosuppressive effect, and blocking the binding of PD-1 to PD-L1 on the surface of tumor cells is a core target of tumor immunotherapy. In this invention, the EPA is administered orally in combination with the PD-1 inhibitor administered intravenously. The PD-1 inhibitor includes a PD-1 neutralizing antibody.

[0028] This invention investigated the synergistic effect of EPA combined with PD-1 inhibitors. In this invention, revived Hepal-6 liver cancer cells were transplanted subcutaneously into mice. After tumor formation, the size of subcutaneous tumor tissue was measured in groups fed a diet containing 5% EPA, a conventional diet, a PD-1 neutralizing antibody group, and a combination of 5% EPA and PD-1 neutralizing antibody. The study found that EPA has a sensitizing effect on immunotherapy, effectively improving the efficacy of PD-1 inhibitors, enhancing the immune response, and significantly enhancing the anti-liver cancer effect of PD-1 inhibitors. The combination of EPA and PD-1 inhibitors significantly enhances the anti-tumor activity of PD-1 inhibitors.

[0029] This invention investigates the role of EPA in reversing the hyperprogression induced by PD-1 inhibitors in metabolically related fatty liver disease-associated hepatocellular carcinoma (HCC). A C57 / BL6 mouse model of HCC with metabolically related fatty liver disease was established using a high-fat diet, high-sugar water intake, and intraperitoneal injection of carbon tetrachloride. After tumor formation, the number of tumors on the liver was measured in groups fed a diet containing 5% EPA, a conventional diet, a PD-1 neutralizing antibody group, and a combination of 5% EPA and PD-1 neutralizing antibody. The study found that EPA has the effect of reversing the hyperprogression induced by PD-1 inhibitors in immunotherapy, effectively improving the efficacy of PD-1 inhibitors, and significantly enhancing the anti-hepatocellular carcinoma effect.

[0030] This invention also provides an agent for the prevention and treatment of liver cancer, the agent comprising a therapeutically effective amount of EPA and a therapeutically effective amount of a PD-1 inhibitor. In this invention, the agent comprises EPA at a mass ratio of 3-8% and a PD-1 inhibitor at a dosage of 2-6 mg / kg. As one embodiment, the PD-1 inhibitor comprises a PD-1 neutralizing antibody.

[0031] In this invention, the formulation further includes a pharmaceutically acceptable carrier, which includes buffers, diluents, excipients, or preservatives. The formulation is an oral formulation and / or an injectable formulation.

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of this invention.

[0033] Unless otherwise specified, the materials, reagents, etc. used in the following examples are commercially available. Unless otherwise specified, they are generally used under conventional conditions or under conditions recommended by the company.

[0034] C57 / BL6 mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., Hepa1-6 liver cancer cells were purchased from Fuheng Biotechnology, EPA was purchased from Aladdin, Western diet mouse food was purchased from Deitz, and PD1 neutralizing antibody was purchased from Selleck.

[0035] Example 1 1. Western blot-based detection of intracellular PD-L1 expression This invention first examined the effect of ω-3 PUFAs (eicosapentaenoic acid) (EPA) on PD-L1 expression at the cellular level.

[0036] (i) Recovery and passage of Hepa1-6 cells: Hepa1-6 cells were rapidly thawed from liquid nitrogen and centrifuged (1500 rpm, 5 min). The supernatant was removed, and the cells were resuspended in 10% fetal bovine serum medium and transferred to culture dishes. The cells were then incubated at 37°C. After 2-3 days of incubation, the cells were washed with PBS and digested with trypsin. The cells were then centrifuged (1500 rpm, 5 min), the supernatant was removed, and the cells were resuspended in culture medium.

[0037] (ii) Cell seeding: The cells were counted and distributed into each culture dish at an average density of 300,000 cells per dish. The cells were then incubated overnight at 37 degrees Celsius. (iii) Drug treatment: After Hepa1-6 cells were treated with 0 μM, 100 μM, 200 μM, and 300 μM EPA for 24 h, the cells were collected, lysed on ice with RIPA lysis buffer (strong), centrifuged at 12000 rpm for 5 min at 4 °C, and the supernatant was collected to extract total protein.

[0038] (iv) Western blot analysis of intracellular PD-L1 expression: Protein concentration was determined using the Bicinchoninic Acid (BCA) quantitative method, followed by polyacrylamide gel electrophoresis. After 2 hours of transfer, the membrane was blocked with 10% milk for 2 hours, then incubated with primary antibody overnight. After washing, secondary antibody was added and incubated for 2 hours. The results were observed using a chemiluminescence imaging system after incubation with both primary and secondary antibodies.

[0039] Experimental results are as follows Figure 1 As shown, EPA exhibits a concentration gradient that inhibits PD-L1 expression, and EPA demonstrates a good inhibitory effect on PD-L1 expression in liver cancer cells.

[0040] 2. Western blot-based detection of PD-L1 expression in mice (i) Constructing a liver cancer model using hydrodynamic methods: A liver cancer model was constructed by injecting the CTNNB1 plasmid, c-MYC plasmid, and SB100 plasmid into the liver of C57 / BL6 mice using a high-pressure hydrodynamic method.

[0041] (ii) Randomization and EPA treatment: After tumors formed in the mice, the mice were randomly divided into two groups. One group was fed Western diet rat food containing 5% EPA, and the other group was fed Western diet rat food as the NC group. The daily food intake was about 3-4 g. (iii) After feeding for 40 days, liver tissue was collected, cells were lysed on ice with RIPA lysis buffer (strong), centrifuged at 12,000 rpm for 5 min at 4°C, and the supernatant was collected to extract total protein.

[0042] (iv) Western blot analysis of intracellular PD-L1 expression: Protein concentration was determined using the Bicinchoninic Acid (BCA) quantitative method, followed by polyacrylamide gel electrophoresis. After 2 hours of transfer, the membrane was blocked with 10% milk for 2 hours, then incubated with primary antibody overnight. After washing, secondary antibody was added and incubated for 2 hours. The results were observed using a chemiluminescence imaging system after incubation with both primary and secondary antibodies.

[0043] Experimental results are as follows Figure 2 As shown, in the liver model constructed in the hydrodynamic model, EPA significantly inhibited the expression of PDL1, and EPA showed a significant inhibitory effect on PD-L1 expression in animals.

[0044] Example 2 In vivo combined inhibition of tumor growth experiment (mouse subcutaneous tumor model) Results on cells and in mice demonstrated that EPA significantly inhibited PD-L1 expression. Since PD-1 is a major target of immune checkpoint inhibitors, this suggests that EPA may not only reverse immune resistance but also sensitize the immune system. Therefore, this invention further investigated the synergistic effects of EPA combined with immune checkpoint inhibitors.

[0045] (i) Recovery and passage of Hepa1-6 cells: Hep1-6 cells were rapidly thawed from liquid nitrogen and centrifuged (1500 rpm, 5 min). The supernatant was removed, and the cells were resuspended in 10% fetal bovine serum medium and transferred to culture dishes. The cells were then incubated at 37°C. After 2-3 days of incubation, the cells were washed with PBS and digested with trypsin. The cells were then centrifuged (1500 rpm, 5 min), the supernatant was removed, and the cells were resuspended in culture medium.

[0046] (ii) Cell seeding: Count the cells and distribute them evenly into each culture dish at 300,000 cells per dish. Incubate overnight at 37 degrees Celsius. (iii) Collect the suspended cells by centrifugation (1500 rpm for 5 min), wash the cells twice with PBS (1500 rpm for 5 min), and resuspend them to 2×10⁻⁶ cells / min. 6 / ml, and inject 100 µL of the resuspended solution subcutaneously into mice; (iv) Random grouping and processing: After tumor formation was confirmed in mice, they were randomly divided into four groups: a control group, an EPA treatment group, a PD-1 neutralizing antibody group, and a combined PD-1 neutralizing antibody and EPA treatment group. The EPA group was fed a Western diet containing 5% EPA, while the control group was fed the same Western diet, with a daily intake of approximately 3–4 g. The PD-1 neutralizing antibody group received a single intravenous injection of PD-1 neutralizing antibody at a dose of 1 mg / kg. The combined PD-1 neutralizing antibody and EPA treatment group received approximately 3–4 g of a Western diet containing 5% EPA daily, along with a single injection of PD-1 neutralizing antibody at a dose of 1 mg / kg. All groups were treated for 21 days.

[0047] (vi) After the experiment, subcutaneous tumor tissue of mice was collected, photographed, weighed, and the data was collected and analyzed.

[0048] Experimental results are as follows Figure 3 As shown in Figure B, compared with the control group, the EPA group and the PD-1 inhibitor group alone inhibited the growth of liver cancer cells (inhibition rates: 60.97% and 71.96%, respectively). However, the combined group was significantly better than the control group and the single-drug group (inhibition rate: 86.21%). The experimental results indicate that although EPA or PD-1 neutralizing antibody alone showed good in vivo antitumor inhibitory activity, it was still inferior to the combined group. The above studies demonstrate that the combination of EPA and PD-1 neutralizing antibody can significantly enhance in vivo antitumor activity.

[0049] Example 3 In vivo efficacy trial of enhanced immune checkpoint therapy for liver cancer (metabolic fatty liver disease-associated liver cancer model) (i) A metabolic-related fatty liver disease-associated hepatocellular carcinoma model was established in C57 / BL6 mice by a combination of a high-fat diet, a high-sugar drinking water diet, and intraperitoneal injection of carbon tetrachloride. The specific procedure was as follows: 8-week-old male C57BL / 6J mice were fed a high-fat, high-sugar Western diet, and were given high glucose and fructose drinking water (23.1 g / L fructose, 18.9 g / L glucose) and intraperitoneal injection of 0.32 µg / ml CCl4 weekly. Tumors formed in about 6 months. (ii) After tumor formation was confirmed in mice, the mice were randomly divided into three groups: a control group and an EPA group, as well as a control group, a PD-1 neutralizing antibody group, and a PD-1 neutralizing antibody and EPA combined treatment group. The EPA group was fed a Western diet containing 5% EPA, while the control group was fed a Western diet with a daily intake of approximately 3-4 g. The PD-1 neutralizing antibody group received a single injection of PD-1 neutralizing antibody via tail vein at a dose of 4 mg / kg. The PD-1 neutralizing antibody and EPA combined treatment group received approximately 3-4 g of a Western diet containing 5% EPA daily, and simultaneously received a single injection of PD-1 neutralizing antibody at a dose of 4 mg / kg. All groups were treated for 40 days.

[0050] (iii) After the experiment, mouse liver tissue was collected, photographed, weighed, and the data was collected and analyzed.

[0051] The results are as follows Figure 4 As shown, EPA alone can inhibit the growth of liver cancer, with an inhibition rate of 55.56% (e.g., ...). Figure 4 (As shown in Figure B). PD-1 neutralizing antibodies are ineffective in treating liver cancer (e.g., Figure 4 As shown in Figure E), this indicates that PD-1 neutralizing antibodies are insensitive to immunotherapy. However, combination therapy with PD-1 neutralizing antibodies and EPA can reverse drug resistance, significantly inhibit the growth of liver cancer, and the inhibitory effect is higher than that of EPA alone, with an inhibition rate of 83.33% (as shown in Figure E). Figure 4 (As shown in Figure E). Meanwhile, compared to the control group and the single-agent group, the incidence of tumors in the combination therapy group was 50% (e.g., ...). Figure 4 (As shown in Figure F). The above results indicate that EPA has the effect of reversing the progression of hepatocellular carcinoma associated with metabolic-related fatty liver disease promoted by PD-1 neutralizing antibodies. In summary, the EPA of this invention can effectively improve the efficacy of PD-1 inhibitors and significantly enhance the anti-hepatocellular carcinoma effect, possessing potential clinical translational value for enhancing the efficacy of immunotherapy.

[0052] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. The application of ω-3 polyunsaturated fatty acids in the preparation of PD-L1 inhibitors, characterized in that, The ω-3 polyunsaturated fatty acids include EPA.

2. Application of EPA in inhibiting PD-L1 expression in liver cancer cells in vitro.

3. The application of ω-3 polyunsaturated fatty acids in the preparation of synergists for PD-1 inhibitors, characterized in that, The ω-3 polyunsaturated fatty acids include EPA.

4. The application of ω-3 polyunsaturated fatty acids in the preparation of drugs that enhance the anti-hepatocellular carcinoma activity of PD-1 inhibitors, characterized in that, The ω-3 polyunsaturated fatty acids include EPA.

5. The application according to claim 3 or 4, characterized in that, The EPA is administered orally in combination with the PD-1 inhibitor administered intravenously.

6. A preparation for preventing and treating liver cancer, characterized in that, The formulation comprises a therapeutically effective amount of EPA and a therapeutically effective amount of a PD-1 inhibitor.

7. The formulation according to claim 6, characterized in that, The formulation comprises 3-8% EPA by mass and a PD-1 inhibitor at a dosage of 2-6 mg / kg.

8. The formulation according to claim 6 or 7, characterized in that, The PD-1 inhibitors include PD-1 neutralizing antibodies.

9. The formulation according to any one of claims 6 to 8, characterized in that, The formulation also includes a pharmaceutically acceptable carrier.

10. The formulation according to any one of claims 6 to 8, characterized in that, The formulation is an oral formulation and / or an injectable formulation.

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