Composition of mitochondrial autophagy inhibitor and immune checkpoint inhibitor and application

Abstract

The invention discloses a composition of a mitochondrial autophagy inhibitor and an immune checkpoint inhibitor and application, and belongs to the technical field of biological medicine. The invention provides a brand-new combined treatment strategy. In-vivo experiments prove that in melanoma, colon cancer and liver cancer models ineffective by single use of the immune checkpoint inhibitor, the curative effect of the immune checkpoint inhibitor can be significantly enhanced by combining the mitochondrial autophagy inhibitor, which is represented by synergistic inhibition of tumor volume and weight and significant increase of CD8 + T cell infiltration in a tumor microenvironment. The invention provides an effective scheme for overcoming the drug resistance of tumor immunotherapy, and the application prospect is wide.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a combination of a mitochondrial autophagy inhibitor and an immune checkpoint inhibitor and use thereof. BACKGROUND

[0002] Immune checkpoint inhibitors (ICIs), including antibodies targeting programmed death-1 (PD-1), programmed death-ligand 1 (PD-L1), cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), have made revolutionary progress in the treatment of various malignancies by relieving the inhibition of the immune system by tumors (Morad G, Helmink BA, Sharma P, Wargo JA. Hallmarks of response, resistance, and toxicity to immune checkpoint blockade. Cell. 2021 Oct 14;184(21):5309-5337. doi: 10.1016 / j.cell.2021.09.020.). Although immune checkpoint inhibitors have achieved significant therapeutic effects in clinical applications, only 10%-20% of patients benefit from immune checkpoint inhibitor (ICI) therapy, and are often accompanied by varying degrees of immune-related adverse events (Wang SJ, Dougan SK, Dougan M. Immune mechanisms of toxicity from checkpoint inhibitors. Trends Cancer. 2023 Jul;9(7):543-553. doi: 10.1016 / j.trecan.2023.04.002.). Therefore, developing therapeutic strategies that can reverse ICI resistance and improve their efficacy has become an urgent need in the field of tumor immunotherapy.

[0003] Mitophagy is a highly conserved cellular process in eukaryotic cells that selectively removes dysfunctional or excess mitochondria through autophagy. This process effectively regulates mitochondrial number and maintains cellular energy metabolism stability (Lu Y, Li Z, Zhang S, Zhang T, Liu Y, Zhang L. Cellular mitophagy:Mechanism, roles in diseases and small molecule pharmacological regulation. Theranostics. 2023 Jan 1;13(2):736-766. doi: 10.7150 / thno.79876.). However, research has shown that tumor cells can hijack this self-protective mechanism to promote their survival and development. Under the stress of the tumor microenvironment (such as hypoxia and nutrient deficiency), enhanced mitophagy helps tumor cells clear stress-damaged mitochondria, thereby maintaining energy supply, reducing reactive oxygen species (ROS)-mediated cell damage, and ultimately leading to resistance to chemotherapy, radiotherapy, and immune attacks (Panigrahi DP, Praharaj PP, Bhol CS, Mahapatra KK, Patra S, Behera BP, Mishra SR, Bhutia SK. The emerging, multifaceted role of mitophagy in cancer and cancer therapeutics. Semin Cancer Biol. 2020 Nov;66:45-58. doi: 10.1016 / j.semcancer.2019.07.015.). This suggests that high levels of mitophagy may be one of the potential mechanisms by which tumor cells achieve resistance to ICI drugs.

[0004] Currently, there is a lack of effective strategies to reverse tumor ICI resistance by specifically inhibiting mitophagy. Therefore, this paper proposes a novel treatment strategy that combines mitophagy inhibitors with immune checkpoint inhibitors. This strategy has significant theoretical and practical implications for overcoming the limitations of current immunotherapy and expanding the patient population that can benefit from it. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of the prior art by providing a composition and application of a mitophagy inhibitor and an immune checkpoint inhibitor, thereby overcoming problems such as low sensitivity and low efficacy in some tumor immunotherapies.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0007] In a first aspect, the present invention provides the use of a combination of a mitophagy inhibitor and an immune checkpoint inhibitor in the preparation of a medicament for treating tumors that are insensitive to or resistant to immune checkpoint inhibitors.

[0008] Preferably, the immune checkpoint inhibitor is selected from: anti-PD-1 antibody, anti-PD-L1 antibody or anti-CTLA-4 antibody.

[0009] Preferably, the immune checkpoint inhibitor is selected from: nivolumab, pembrolizumab, camrelizumab, atezolizumab, durvalumab, ipilimumab, or tesimumab.

[0010] Preferably, the mitophagy inhibitor is a small molecule inhibitor selected from: Mdivi-1, Liensinine, U0126, or pharmaceutically acceptable salts, solvates, or crystal forms thereof.

[0011] Preferably, the mitophagy inhibitor is Mdivi-1.

[0012] Preferably, the immune checkpoint inhibitor is an anti-PD-L1 antibody.

[0013] Preferably, the tumor includes liver cancer, melanoma, colon cancer, lung cancer, and breast cancer.

[0014] In a second aspect, the present invention provides a pharmaceutical composition for treating tumors, the pharmaceutical composition comprising the following active ingredients in parts by weight: 1-3 parts of an immune checkpoint inhibitor and 4-16 parts of a mitophagy inhibitor, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody and the mitophagy inhibitor is Mdivi-1.

[0015] The pharmaceutical composition is available in the form of tablets, capsules, pills, granules, oral liquids, or injections. Preferably, when the pharmaceutical composition is available in the form of an injection, the daily dosage is 0.4 mg / kg for anti-PD-L1 antibody and 1.5 mg / kg for Mdivi-1.

[0016] The present invention has the following beneficial effects:

[0017] (1) Synergistic effect, significantly enhancing anti-tumor efficacy: This invention is the first to demonstrate that in animal models of melanoma, colon cancer and liver cancer that are insensitive to ICI monotherapy, the combination of the mitochondrial autophagy inhibitor Mdivi-1 can significantly enhance the therapeutic effect of anti-PD-L1 antibody. The results showed that the tumor volume and weight in the combination treatment group were significantly lower than those in any single-drug group and the control group, demonstrating a strong synergistic anti-tumor effect.

[0018] (2) Effective reversal of immune checkpoint inhibitor resistance: The core advantage of this invention lies in its ability to overcome ICI resistance in tumors. Experimental results show that in models where anti-PD-L1 antibody alone is ineffective, the combination with Mdivi-1 produces a strong tumor-suppressive effect, which provides a novel and effective solution for overcoming the challenge of clinical ICI resistance.

[0019] (3) Reshaping the tumor immune microenvironment and activating anti-tumor immunity: This invention not only directly inhibits tumor growth, but more importantly, by inhibiting mitophagy, it may disrupt the metabolic adaptation of tumor cells, thereby stimulating a stronger anti-tumor immune response. Immunohistochemical results showed that CD8+ in the tumor microenvironment of the combined treatment group... + The significant increase in T-cell infiltration indicates that the combined strategy successfully transformed "immunosuppressive" "cold" tumors into "immunoactivated" "hot" tumors, fundamentally enhancing the body's own anti-cancer ability.

[0020] (4) It provides a novel paradigm of combined therapy: This invention ingeniously combines the fields of "targeting tumor cell metabolic adaptation" (mitochondrial autophagy inhibition) and "activating systemic immunity" (immune checkpoint inhibition), opening up a new direction for tumor immunotherapy. This strategy is different from simple drug stacking, but is a precise intervention based on a deep understanding of tumor drug resistance mechanisms.

[0021] (5) Potential safety advantages: In the examples, no significant additional toxicity was observed in mice due to the combination of drugs, suggesting that the regimen may have good tolerability while effectively improving efficacy, but this conclusion needs to be confirmed by further toxicological experiments.

[0022] (6) Flexible approach and broad application prospects: This strategy is expected to be extended to a variety of solid tumors that are not sensitive to ICI treatment and has broad clinical application prospects. Attached Figure Description

[0023] Figure 1 Effects of combined drug therapy on mouse melanoma (A: Comparison of mouse melanoma weight; B: Comparison of mouse melanoma volume; C: CD8) + T cell contrast diagram; where * represent P values < 0.05, ** represents a p-value < 0.01, *** represents a p-value < 0.001).

[0024] Figure 2 Effects of combined drug therapy on colon cancer in mice (A: Comparison of colon cancer weight in mice; B: Comparison of colon cancer volume in mice; C: CD8) + T cell contrast diagram; where * represent P values < 0.05,** represents a p-value < 0.01, *** represents a p-value < 0.001).

[0025] Figure 3 Effects of combined drug therapy on mouse liver cancer (A: Comparison of mouse liver cancer weight; B: Comparison of mouse liver cancer volume; C: CD8) + T cell comparison chart; where * represents P value < 0.05, ** represents P value < 0.01, and *** represents P value < 0.001). Detailed Implementation

[0026] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0027] The terminology used in this invention, unless otherwise specified, generally has the meanings commonly understood by those skilled in the art. The invention is further described in detail below with reference to specific embodiments, accompanying drawings, and data. It should be understood that these embodiments are merely illustrative and not intended to limit the scope of the invention in any way. Various processes and methods not described in detail in the following embodiments are conventional methods known in the art. Unless otherwise specified, the materials, reagents, apparatuses, instruments, equipment, etc., used in the following examples are commercially available.

[0028] Example 1: Treatment of melanoma with the combination of mitochondrial autophagy inhibitor Mdivi-1 and anti-PD-L1 antibody

[0029] Experimental Methods: Wild-type mouse melanoma cells were subcutaneously inoculated into the forelimbs of 4-6 week old male C57BL / 6 mice to establish an animal model of melanoma resistant to immune checkpoint inhibitor treatment. One week later, the mice were randomly divided into four groups: control group, anti-PD-L1 antibody alone group, Mdivi-1 alone group, and anti-PD-L1 antibody + Mdivi-1 group, with 4 mice in each group. Anti-PD-L1 antibody: 100 μg / mouse, intraperitoneally injected twice a week; Mdivi-1: intraperitoneally injected once a day (20 mg / kg). Tumor volume was measured every 3 days (the formula for calculating tumor volume is: volume = 1 / 2 × long diameter × short diameter). 2 The measurement results are shown in Table 1.

[0030] Table 1. Effects of combined drug therapy on melanoma weight and volume in mice.

[0031] Result: As Figure 1 As shown, after two weeks of treatment, the tumor size and CD8 count were significantly different in the group treated only with anti-PD-L1 antibodies compared to the control group. +The lack of significant difference in T cells indicates that this tumor model is insensitive to anti-PD-L1 antibody treatment. However, the combination of Mdivi-1 and anti-PD-L1 antibody significantly inhibited tumor growth, reduced tumor size, and reduced CD8 activity. + The increase in T cells indicates that Mdivi-1 can significantly enhance the killing effect of anti-PD-L1 antibody on melanoma resistant to secondary immunotherapy.

[0032] Example 2: Treatment of colon cancer with the mitochondrial autophagy inhibitor Mdivi-1 combined with an anti-PD-L1 antibody

[0033] Experimental Methods: A colon cancer model resistant to immune checkpoint inhibitors was established by subcutaneously inoculating the forelimbs of 4-6 week old male BALB / c mice with mouse colon cancer cells. One week later, the mice were randomly divided into four groups: control group, anti-PD-L1 antibody alone group, Mdivi-1 alone group, and anti-PD-L1 antibody + Mdivi-1 group, with five mice in each group. Anti-PD-L1 antibody: 100 μg / mouse, administered intraperitoneally twice weekly; Mdivi-1: 20 mg / kg, administered intraperitoneally once daily. Tumor volume was measured every 3 days (the formula for calculating tumor volume is: volume = 1 / 2 × long diameter × short diameter). 2 The measurement results are shown in Table 2.

[0034] Table 2. Effects of combined drug therapy on colon cancer weight and volume in mice.

[0035] Result: As Figure 2 As shown, after two weeks of treatment, the tumor size and CD8 count were significantly different in the group treated only with anti-PD-L1 antibodies compared to the control group. + The lack of significant difference in T cells indicates that this tumor model is insensitive to anti-PD-L1 antibody treatment. However, the combination of Mdivi-1 and anti-PD-L1 antibody significantly inhibited tumor growth, reduced tumor size, and reduced CD8 activity. + The increase in T cells indicates that Mdivi-1 can significantly enhance the killing effect of anti-PD-L1 antibody on secondary immunotherapy-resistant colon cancer.

[0036] Example 3: Treatment of liver cancer with the combination of mitochondrial autophagy inhibitor Mdivi-1 and anti-PD-L1 antibody

[0037] Experimental Methods: A liver cancer model resistant to immune checkpoint inhibitors was established by subcutaneously inoculating the forelimbs of 4-6 week old male C57BL / 6 mice with mouse liver cancer cells. One week later, the mice were randomly divided into four groups: control group, anti-PD-L1 antibody alone group, Mdivi-1 alone group, and anti-PD-L1 antibody + Mdivi-1 group, with 5 mice in each group. Anti-PD-L1 antibody: 75 μg / mouse, administered intraperitoneally twice weekly; Mdivi-1: 20 mg / kg, administered intraperitoneally once daily. Tumor volume was measured every 3 days (the formula for calculating tumor volume is: volume = 1 / 2 × long diameter × short diameter). 2 The measurement results are shown in Table 3.

[0038] Table 3. Effects of combined drug therapy on the weight and volume of liver cancer in mice.

[0039] Result: As Figure 3 Figure 3 As shown, after two weeks of treatment, the tumor size and CD8 count were significantly different in the group treated only with anti-PD-L1 antibodies compared to the control group. + The lack of significant difference in T cells indicates that this tumor model is insensitive to anti-PD-L1 antibody treatment. However, the combination of Mdivi-1 and anti-PD-L1 antibody significantly inhibited tumor growth, reduced tumor size, and reduced CD8 activity. + The increase in T cells indicates that Mdivi-1 can significantly enhance the killing effect of anti-PD-L1 antibody on liver cancer resistant to secondary immunotherapy.

[0040] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. However, the above description is merely a specific embodiment of the present invention, and the technical features of the present invention are not limited thereto. Any other embodiments derived by those skilled in the art without departing from the technical solution of the present invention should be covered within the patent scope of the present invention.

Claims

1. Use of a combination of a mitochondrial autophagy inhibitor and an immune checkpoint inhibitor in the preparation of a medicament for treating a tumor that is insensitive or resistant to the immune checkpoint inhibitor.

2. Use according to claim 1, characterized in that, The immune checkpoint inhibitor is selected from an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody.

3. Use according to claim 2, characterized in that, The immune checkpoint inhibitor is selected from nivolumab, pembrolizumab, camrelizumab, atezolizumab, durvalumab, ipilimumab, or tremelimumab.

4. Use according to claim 1, characterized in that, The mitochondrial autophagy inhibitor is a small molecule inhibitor selected from Mdivi-1, Liensinine, U0126, or a pharmaceutically acceptable salt, solvate, or crystalline form thereof.

5. Use according to claim 4, characterized in that, The mitochondrial autophagy inhibitor is Mdivi-1.

6. Use according to claim 5, characterized in that, The immune checkpoint inhibitor is an anti-PD-L1 antibody.

7. Use according to claim 1, characterized in that, The tumor comprises a liver cancer, a melanoma, a colon cancer, a lung cancer, a breast cancer.

8. A pharmaceutical composition for treating a tumor, characterized by, The pharmaceutical composition contains the active ingredients in the following mass fractions: 1-3 parts of the immune checkpoint inhibitor and 4-16 parts of the mitochondrial autophagy inhibitor, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody and the mitochondrial autophagy inhibitor is Mdivi-1.

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