Application of spirolactone in preparation of synergist of immune checkpoint inhibitor

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses application of spirolactone in preparation of a synergist of an immune checkpoint inhibitor. The application finds that spirolactone can enhance the effect of the immune checkpoint inhibitor on treating liver cancer, has no toxic or side effect on animals, and improves the tumor killing ability of infiltrating NK cells. Animal experiments show that the progress of the liver cancer can be remarkably inhibited by combining the spirolactone with the immune checkpoint inhibitor, and the tumor killing capability of infiltrating NK cells can be improved by combining administration, so that the spirolactone can be used as an auxiliary medicine for clinically improving or enhancing the immune checkpoint to treat the liver cancer (HCC).

Description

Technical Field

[0001] The present application relates to the field of biomedical technology, and in particular to the use of spironolactone in the preparation of a potentiator of an immune checkpoint inhibitor. Background Art

[0002] Targeting the PD-1 / L1 checkpoint is currently the most effective immunotherapy in clinical practice. PD-1 is mainly expressed in activated T cells, B cells, NK cells and myeloid cells. Once it binds to the ligand PD-L1 / 2, it weakens the response of immune cells. In the tumor microenvironment, PD-L1 / 2 is generally expressed in tumor cells, but their expression is heterogeneous in different types of tumors. The interaction between PD-L1 / 2 on the tumor surface and PD-1 on the surface of infiltrating lymphocytes is considered to be the main mechanism of tumor immune escape and is an ideal target for immunotherapy.

[0003] However, in clinical practice, immune checkpoint therapy targeting PD1 / L1 has also exposed some problems, such as the damage to the body caused by side effects caused by immunotherapy, and the co-evolution of tumors during immunotherapy will further aggravate resistance to immunotherapy.

[0004] Although immunotherapy has significant therapeutic effects in tumors such as lymphoma and melanoma, it has poor therapeutic effects on the vast majority of patients with hepatocellular carcinoma (HCC), which is related to the diverse causes of HCC and its ability to escape immune surveillance. Therefore, finding new targets for HCC treatment and optimizing combined treatment plans have become major problems that need to be solved urgently in the field of HCC research. Summary of the invention

[0005] The purpose of the present application is to overcome the deficiencies of the above-mentioned prior art and provide an application of spironolactone in the preparation of a synergist of immune checkpoint inhibitors. The present application uses spironolactone to enhance the effect of immune checkpoint inhibitors in treating liver cancer and has no toxic side effects on animals.

[0006] To achieve the above purpose, the technical solution adopted by this application is:

[0007] The present application provides the use of spironolactone in the preparation of a synergist of immune checkpoint inhibitors for the treatment of liver cancer.

[0008] The inventors of the present application have found through extensive research and experiments that spironolactone can enhance the effect of immune checkpoint inhibitors in treating liver cancer, significantly reduce the liver tumor load, liver weight ratio and the proportion of tumor areas on the same liver lobe of animals, have no toxic side effects on animals, and improve the tumor killing ability of infiltrating NK cells (increase the proportion of NK cell killer cytokines, among which killer cytokines include IFN-γ + IFN-α + 、Granzyme B+ ). It can be seen that spironolactone can be used as an auxiliary drug to clinically improve or enhance immune checkpoints in the treatment of hepatocellular carcinoma (HCC).

[0009] As a preferred embodiment of the application described in the present application, the immune checkpoint inhibitor includes PD-1 monoclonal antibody.

[0010] As a preferred embodiment of the application described in the present application, the immune checkpoint inhibitor is a PD-1 monoclonal antibody.

[0011] Animal experiments have shown that spironolactone can significantly enhance the effect of PD-1 monoclonal antibody in treating liver cancer. The liver tumor load, liver weight ratio and proportion of tumor area on the same liver lobe of animals are significantly reduced, effectively inhibiting the progression of liver cancer with no toxic side effects on animals.

[0012] Spironolactone is a clinical potassium-sparing diuretic and blood pressure-lowering drug. The experimental results of this application show that spironolactone can be used as an auxiliary drug for clinical improvement or enhancement of immune checkpoint treatment of HCC.

[0013] As a preferred embodiment of the application described in the present application, the dosage of spironolactone used in animal experiments is 20-30 mg / kg, and the dosage is preferably 25 mg / kg.

[0014] The animal model used in the animal experiment is: RIL-175-induced mouse HCC model or Hepa 1-6-induced mouse HCC model.

[0015] The method for constructing the animal model comprises the following steps:

[0016] After anesthesia, a small incision of about 1 cm was made along the midline of the mouse abdomen using sterile surgical scissors. The portal vein was then found and 2-3×10 liver cancer cells were directly injected into the mouse. 6 Hepa 1-6 or RIL-175 tumor cells were injected into the portal vein of the liver, and finally the inner and outer epithelial sutures of the mice were surgically sutured to construct the RIL-175-induced mouse HCC model or the Hepa 1-6-induced mouse HCC model.

[0017] Animal experiments using spironolactone within the above dosage range can better enhance the effect of immune checkpoint therapy in liver cancer.

[0018] The present application also provides an application of a preparation in the preparation of a synergist of an immune checkpoint inhibitor for the treatment of liver cancer. The preparation is prepared by adding an effective amount of spironolactone to a pharmaceutically acceptable carrier.

[0019] The use of a preparation containing an effective amount of spironolactone can significantly enhance the effect of immune checkpoint inhibitors in the treatment of liver cancer and improve the tumor killing ability of infiltrating NK cells (increasing the proportion of NK cell killer cytokines, among which killer cytokines include IFN-γ + IFN-α + 、Granzyme B + ).

[0020] As a preferred embodiment of the application described in the present application, the carrier includes at least one of a diluent, an excipient, a dispersant and a surfactant.

[0021] As a preferred embodiment of the application described in the present application, the dosage form of the preparation includes tablets, capsules, granules or injections.

[0022] The present application also provides the use of spironolactone combined with an immune checkpoint inhibitor in the preparation of a liver cancer drug, characterized in that the immune checkpoint inhibitor includes a PD-1 monoclonal antibody or a PD-L1 monoclonal antibody.

[0023] Animal experiments have shown that the combination of spironolactone and immune checkpoint inhibitors can significantly inhibit the progression of liver cancer without toxic side effects on animals. In addition, the combined administration increases the proportion of NK cell killer cytokines (killer cytokines include IFN-γ + IFN-α + 、Granzyme B + ), which enhances the tumor-killing ability of infiltrating NK cells.

[0024] As a preferred embodiment of the application described in the present application, the liver cancer drug also includes a pharmaceutically acceptable carrier.

[0025] The pharmaceutically acceptable carrier has certain physiological activity, but the addition of this component will not change the dominant position of the above-mentioned drug in the treatment process of the disease, but only play an auxiliary role, which is only the utilization of the known activity of the component and is a common auxiliary treatment method in the medical field. If the above-mentioned auxiliary component is used in combination with the drug of the present application, it still falls within the scope of protection of the present application.

[0026] Compared with the prior art, this application has the following beneficial effects:

[0027] The present application provides an application of spironolactone in the preparation of a synergist of an immune checkpoint inhibitor. Spironolactone can enhance the effect of immune checkpoint inhibitors in treating liver cancer, has no toxic side effects on animals, and improves the tumor killing ability of infiltrating NK cells. And through animal experiments, it is known that combining spironolactone with immune checkpoint inhibitors can significantly inhibit the progression of liver cancer, and combined administration can also improve the tumor killing ability of infiltrating NK cells (increasing the proportion of NK cell killing cytokines), indicating that spironolactone can be used as an auxiliary drug for clinical improvement or enhancement of immune checkpoint treatment of liver cancer (HCC). BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The results of spironolactone-enhanced PD-1 monoclonal antibody treatment in RIL-175-induced mouse HCC model ( Figure 1 A in the middle is a picture of liver tumor in wild-type mice after treatment with single drug (spironolactone or PD1 monoclonal antibody) / combination drug (spironolactone and PD1 monoclonal antibody) in the RIL-175-induced mouse HCC model, and the liver weight ratio and the ratio of tumor area on the same liver lobe; Figure 1 B is the cytotoxic cytokine (IFN-γ) of NK cells in the liver of mice bearing tumors + IFN-α + 、Granzyme B + ) ratio result);

[0029] Figure 2 The results of spironolactone-enhanced PD-1 monoclonal antibody treatment of Hepa 1-6-induced mouse HCC model ( Figure 2 A in the middle is a picture of liver tumors in wild-type and Hepa 1-6-induced mouse HCC models after single-drug / combination therapy in the Hepa 1-6-induced mouse HCC model; Figure 2 (B) shows the ratio of mouse liver weight and tumor area on the same liver lobe in the Hepa 1-6-induced mouse HCC model;

[0030] Figure 3 The results of the toxicity test of spironolactone combined with PD-1 monoclonal antibody on liver tumor-bearing mice ( Figure 3 Figure A shows the ratio of different cells in the blood of the Hepa 1-6-induced mouse HCC model. Figure 3 (B) shows the HE staining result of mouse kidney pathology in Hepa 1-6-induced mouse HCC model). DETAILED DESCRIPTION

[0031] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0032] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0033] In this application, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0034] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified, and the components and raw materials used in each parallel experiment are of the same type.

[0035] The present application provides the use of spironolactone in the preparation of a synergist of immune checkpoint inhibitors for the treatment of liver cancer.

[0036] In some specific embodiments, the immune checkpoint inhibitor includes PD-1 monoclonal antibody or PD-L1 monoclonal antibody. Preferably, the immune checkpoint inhibitor is PD-1 monoclonal antibody.

[0037] In some specific embodiments, the dosage of spironolactone used in animal experiments is 20-30 mg / kg.

[0038] The present application also provides an application of a preparation in the preparation of a synergist of an immune checkpoint inhibitor for the treatment of liver cancer. The preparation is prepared by adding an effective amount of spironolactone to a pharmaceutically acceptable carrier.

[0039] In some specific embodiments, the carrier comprises at least one of a diluent, an excipient, a dispersant and a surfactant.

[0040] In some specific embodiments, the dosage form of the preparation includes tablets, capsules, granules or injections.

[0041] The present application also provides the use of spironolactone combined with an immune checkpoint inhibitor in the preparation of a liver cancer drug, wherein the immune checkpoint inhibitor includes a PD-1 monoclonal antibody or a PD-L1 monoclonal antibody. Preferably, the immune checkpoint inhibitor is a PD-1 monoclonal antibody.

[0042] In some specific embodiments, the liver cancer drug further comprises a pharmaceutically acceptable carrier.

[0043] Example 1: Spironolactone enhances the effect of PD-1 monoclonal antibody in treating mouse hepatocellular carcinoma (HCC)

[0044] 1. HCC induction and drug administration in mice:

[0045] Methods: For the HCC model induced by Hepa 1-6 or RIL-175, a small incision of about 1 cm was made along the midline of the abdomen of the anesthetized mouse using sterile surgical scissors. Then the portal vein was found and 2-3×10 liver cancer cells were directly injected into the mouse. 6 Hepa 1-6 or RIL-175 tumor cells were injected into the portal vein of the liver, and the inner and outer epithelial sutures of the mice were sutured surgically. One week after surgery, the mice were intraperitoneally injected with spironolactone (25 mg / kg), PD-1 monoclonal antibody (50 μg / 25 g), or a combination of the two. The mice were sacrificed 2 weeks after treatment to examine the differences in tumor burden in the mice.

[0046] 2. Flow cytometry was used to detect the expression of NK cell cytokines. The staining method was as follows (APC-Cy7-CD45.2, BV421-CD3, BV650-NK1.1, PE-Cy7-IFN-γ, Percp5.5-Tnf, PE-GranzymeB), and the results were analyzed using Flowjo.

[0047] Results: Figure 1 and Figure 2 shown.

[0048] in, Figure 1 Figure A shows liver tumor-bearing images of wild-type mice, liver weight ratio, and proportion of tumor area on the same liver lobe after treatment with single-drug (spironolactone or PD1 monoclonal antibody) / combination administration (spironolactone and PD1 monoclonal antibody) in the RIL-175-induced mouse HCC model.

[0049] Figure 1 B is the NK cell killing cytokine (IFN-γ) in the tumor-bearing liver of the RIL-175-induced mouse HCC model + IFN-α + 、Granzyme B + ) ratio results.

[0050] in, Figure 2 A in the middle shows liver tumor-bearing images of wild-type and Hepa 1-6-induced mouse HCC models after single-drug / combination therapy in the Hepa 1-6-induced mouse HCC model.

[0051] Figure 2 Figure B shows the ratio of mouse liver weight and the proportion of tumor area on the same liver lobe in the Hepa 1-6-induced mouse HCC model.

[0052] From the above results, it can be seen that compared with the control group (100μL IgG (0.05μg / μL) + 50μL DMSO) and the single-drug treatment group, the combination of the two drugs significantly inhibited the progression of mouse liver cancer (HCC), and the combined administration also enhanced the tumor killing ability of infiltrating NK cells.

[0053] Example 2: The combination therapy has no toxic side effects on liver tumor-bearing mice

[0054] Method: The kidneys and blood of the mice in the treatment group and the control group in Example 1 were taken for pathological HE staining and blood cell ratio detection.

[0055] Results: Figure 3 shown.

[0056] in, Figure 3 Figure A shows the ratio of different cells in the blood of the Hepa 1-6-induced mouse HCC model. Figure 3 Middle B shows the HE staining results of mouse kidney pathology in the Hepa 1-6-induced mouse HCC model.

[0057] Compared with the control group and the single-drug treatment group, the combination of dual drugs had no obvious toxic side effects on mice.

[0058] This application induces mouse liver cancer (HCC) in vivo, adopts a single PD-1 monoclonal antibody, a single spironolactone, and a combination of the two. Spironolactone can enhance the effect of immune checkpoint inhibitors in treating liver cancer, has no toxic side effects on animals, and improves the tumor killing ability of infiltrating NK cells. And through animal experiments, it is known that combining spironolactone with immune checkpoint inhibitors can enhance the therapeutic effect on mouse liver cancer. The results of liver tumors in vivo show that the combined administration of the two effectively inhibits the progression of liver cancer. The combined administration can also improve the tumor killing ability of infiltrating NK cells (increases the proportion of NK cell killing cytokines), and has no toxic side effects on mice. Spironolactone is a clinical potassium-sparing diuretic and blood pressure-lowering drug. The above experimental results suggest that spironolactone can be used as an adjuvant drug for clinical improvement or enhancement of immune checkpoint treatment of HCC.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. Application of spironolactone in the preparation of enhancers of immune checkpoint inhibitors for the treatment of liver cancer.

2. The use according to claim 1, characterized in that The immune checkpoint inhibitors include PD-1 monoclonal antibody or PD-L1 monoclonal antibody.

3. The use according to claim 1, characterized in that The immune checkpoint inhibitor is PD-1 monoclonal antibody.

4. The use according to claim 1, characterized in that The dosage of spironolactone used in animal experiments is 20-30 mg / kg.

5. Use of a preparation in the preparation of a synergist of an immune checkpoint inhibitor for the treatment of liver cancer, characterized in that: The preparation is prepared by adding an effective amount of spironolactone to a pharmaceutically acceptable carrier.

6. The use according to claim 5, characterized in that The carrier includes at least one of a diluent, an excipient, a dispersant and a surfactant.

7. The use according to claim 5, characterized in that The dosage form of the preparation includes tablets, capsules, granules or injections.

8. Use of spironolactone combined with immune checkpoint inhibitors in the preparation of liver cancer drugs, characterized in that: The immune checkpoint inhibitors include PD-1 monoclonal antibody or PD-L1 monoclonal antibody.

9. The use according to claim 8, characterized in that The liver cancer drug also includes a pharmaceutically acceptable carrier.

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