Drug combination composition for treating tumor diseases and application

Abstract

The invention discloses an anti-tumor drug combination composition. The drug combination composition comprises a TLR7 / 8 agonist and an MDSC inhibitor. The combined medicine disclosed by the invention can be used for effectively treating cancers, and the effect of the combined medicine is obviously better than that of independent TLR7 / 8 agonist treatment and independent MDSC inhibitor treatment, so that the combined medicine and the MDSC inhibitor have a synergistic effect after being used together, and the occurrence of acquired drug resistance can be overcome, and the combined medicine has a good clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a combined drug composition for treating tumor diseases and its application. Background Art

[0002] Tumors are one of the leading causes of death worldwide, and their occurrence and development are closely related to complex immune escape mechanisms. In recent years, immunotherapy has become an important research direction in tumor treatment, which recognizes and eliminates tumor cells by mobilizing the anti-tumor potential of the body's immune system. However, existing single immunotherapy methods have limited efficacy in certain tumor types and are easily affected by the immunosuppression of the tumor microenvironment, leading to drug resistance or poor treatment effects in some patients. Therefore, exploring multi-target combined therapy to enhance anti-tumor immune response has gradually become an important research direction.

[0003] Tumor microenvironment (TME) is one of the key factors that determine the effectiveness of immunotherapy. Improving the immune microenvironment is the key to the success of tumor immunotherapy. The response rate and efficacy of immunotherapy can be effectively improved by relieving immunosuppression, activating effector immune cells, enhancing antigen presentation and metabolic regulation.

[0004] TLR7 / 8 agonists are a class of immunostimulants that can activate dendritic cells and macrophages. They can significantly enhance the body's immune response, activate effector T cells and inhibit tumor growth by upregulating type I interferon and inflammatory factors. However, there are a large number of myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment. These immunosuppressive cells significantly inhibit the activity of effector T cells and natural killer cells by secreting inhibitory factors (such as ARG1, IL-10) and depleting immune nutrients (such as L-arginine), becoming an important factor hindering the effect of immunotherapy. Therefore, MDSCs inhibitors can relieve the immunosuppressive state in the tumor microenvironment and restore the anti-tumor activity of effector immune cells by reducing the number of MDSCs or inhibiting their immunosuppressive function.

[0005] Existing studies have shown that although the use of TLR7 / 8 agonists or MDSCs inhibitors alone has certain anti-tumor effects, their effects are often limited and it is difficult to overcome the complex tumor immune escape mechanism. Based on this, the present invention innovatively proposes the combined use of TLR7 / 8 agonists and MDSCs inhibitors to produce synergistic anti-tumor effects by targeting different immune regulatory pathways in the tumor microenvironment. The combined use of drugs can not only significantly enhance the efficiency of antigen presentation and the activity of effector T cells, but also effectively inhibit the function of MDSCs and its inhibitory effect on the immune system, thereby comprehensively improving the tumor microenvironment and improving the therapeutic effect. The research results of the composition in animal tumor models showed that the combined use of drugs has obvious anti-tumor effects, can significantly inhibit tumor growth and metastasis, and has good safety and tolerability. The above research results provide a new strategy for the treatment of refractory tumors, and have important clinical translation value, which promotes the further development of the field of tumor immunotherapy. Summary of the invention

[0006] In view of this, the present invention provides a combined drug composition for treating tumor diseases and its application, the specific scheme is as follows: In one aspect, the present invention provides an anti-tumor combination drug composition, which comprises a TLR7 / 8 agonist and an MDSCs inhibitor.

[0007] Preferably, the active ingredients of the combination pharmaceutical composition consist of a TLR7 / 8 agonist and an MDSCs inhibitor.

[0008] Preferably, the combination composition further comprises a pharmaceutically acceptable carrier.

[0009] Preferably, the TLR7 / 8 agonist includes one or more of resiquimod, imiquimod, LHC165, BNT411, NJH395, and NKTR-262.

[0010] Preferably, the MDSCs inhibitor includes one or more of ibrutinib, acalabrutinib, and adranib.

[0011] Preferably, the administration of the TLR7 / 8 agonist and MDSCs inhibitor includes one or more of intratumoral injection, peritumoral injection, intravenous injection or oral administration.

[0012] In another aspect, the present invention provides an application of an anti-tumor combined drug composition, wherein the combined drug composition is used for preparing an anti-tumor drug.

[0013] Preferably, the tumor includes one or more of colon cancer, breast cancer and gastric cancer.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. Synergistic anti-tumor effect: The combined drug of the present invention has excellent effects in tumor treatment. Compared with the use of TLR7 / 8 agonists or MDSCs inhibitors alone, it can significantly inhibit tumor growth and metastasis. In animal experiments, such as the CT26 tumor-bearing mouse model, the growth of tumor volume in the combined drug group was significantly slowed down, and the survival time was significantly prolonged, which strongly proved the synergistic effect of the combination of the two, providing a more effective treatment strategy for conquering tumors.

[0015] 2. Overcoming the problem of drug resistance: In the process of tumor treatment, drug resistance is a major thorny issue. This combination drug composition can effectively overcome the occurrence of acquired drug resistance, and by simultaneously acting on different immune regulatory pathways in the tumor microenvironment, it reduces the possibility of tumor cells becoming resistant to a single drug, maintains the stability of the treatment effect, and brings new hope for long-term tumor treatment.

[0016] 3. Improve the tumor microenvironment: TLR7 / 8 agonists can activate dendritic cells and macrophages, upregulate type I interferon and inflammatory factors, and enhance the body's immune response; MDSCs inhibitors can reduce the number of MDSCs or inhibit their immunosuppressive function. The combined use of the two can comprehensively improve the tumor microenvironment, enhance antigen presentation efficiency and effector T cell activity, eliminate immunosuppressive factors, and create favorable conditions for the immune system to effectively identify and eliminate tumor cells.

[0017] 4. Good safety and tolerability: In animal experiments, the combination drug showed good safety and tolerability. During the treatment, there was no obvious abnormal fluctuation in the weight of the mice, and no serious adverse reactions were caused, indicating that the combination drug regimen has high feasibility in clinical applications. Patients can hope to maintain a good physical condition and improve their quality of life while obtaining therapeutic effects.

[0018] The combined drug of the present invention can effectively treat cancer, and the effect is significantly better than the single TLR7 / 8 agonist treatment and MDSCs inhibitor treatment used alone, indicating that the two have a synergistic effect after being used in combination, and can overcome the occurrence of acquired drug resistance, and have good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0020] Figure 1 The results of mouse tumor growth in Example 1 of the present invention are statistically shown: A is a tumor photo; B is a tumor volume curve; and C is a mouse survival curve.

[0021] Figure 2The figure shows the body weight of each group of mice during the treatment period in Example 1 of the present invention.

[0022] Figure 3 The flow cytometry analysis results of the tumor tissue collected in Example 2 of the present invention: A is the ratio of CD80+CD86+ DCs in the collected tumor tissue; B is the ratio of CD8+ T cells in the collected tumor tissue; C is the ratio of MDSCs in the collected tumor tissue.

[0023] Figure 4 In Example 2 of the present invention, flow cytometric analysis results of draining lymph nodes were collected. DETAILED DESCRIPTION

[0024] In order to be able to understand the characteristics and technical contents of the present invention in more detail, the implementation of the present invention is described in detail below in conjunction with the accompanying drawings, which are only for reference and explanation purposes and are not used to limit the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above-mentioned content of the present invention. In the following examples, unless otherwise indicated, the reagents used can be obtained from commercial channels.

[0025] The mouse-derived CT26 colon cancer cells used in the embodiments of the present invention were preserved by the Tumor Research Experimental Center of Nanjing Drum Tower Hospital; specific pathogen-free (SPF) grade BALB / c female mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.; R848 used in the present invention was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; Ibrutinib was purchased from MedChemexpress, USA.

[0026] The main reagents used in the examples of the present invention include RPMI 1640 culture medium purchased from Corning, USA; fetal bovine serum purchased from Gibco, USA; trypsin for cell digestion, PBS buffer, penicillin and streptomycin all purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.

[0027] The main equipment used in the embodiments of the present invention, including the clean bench, CO2 incubator, and high-speed low-temperature centrifuge, are all products of ThermoFisher Company of the United States; various micropipetters are products of Eppendorf Company of Germany; and the flow cytometer is a product of Beckman Coulter Co., Ltd. of the United States.

[0028] All materials, reagents and instruments used in the present invention are well known in the art, but do not limit the implementation of the present invention. Other reagents and equipment well known in the art can be applied to the implementation of the following embodiments of the present invention.

[0029] Example 1 In this example, CT26 cells were inoculated subcutaneously in 4-6 week old BALB / c mice to construct a mouse subcutaneous tumor implant model, and resiquimod (R848) and ibrutinib (Ibrutinib) were given for intervention. By observing and comparing the changes in tumor volume growth and the survival status of mice, the in vivo synergistic inhibitory effect of R848 and Ibrutinib on the growth of colon cancer cells was verified.

[0030] The construction process of the animal tumor model is as follows: digest and wash the CT26 cell line and adjust its concentration to 1×10 7 4-6 week old BALB / c mice were subcutaneously inoculated with 100ul containing 1×10 6 After the cells grew into tumors for 7 days, the mice were randomly divided into four groups (n=6): control group, R848 group, Ibrutinib group, and Ibrutinib + R848 combined group. Each group was given the corresponding drug or vehicle. The specific treatments are as follows: Control group: injected with an equal volume of normal saline, with the same frequency of administration as the single-drug groups, i.e., 100 ul of normal saline per mouse, injected every other day, for a total of 3 times.

[0031] R848 group: R848 was dissolved in 5% DMSO and 45% PEG-300 cosolvents, and diluted with saline (the final working concentration was 0.1 mg / ml, and the injection volume was about 100 ul / mouse). R848 group mice were given R848 alone, intratumorally injected, at a dose of 10 ug / mouse, every other day, for a total of 3 times.

[0032] Ibrutinib (Ib) group: Ibrutinib was dissolved in 5% DMSO and 45% PEG-300 cosolvents, and diluted with normal saline (the final working concentration was 0.2 mg / ml, and the injection volume was 100 ul / mouse). Mice in the Ibrutinib group were given Ibrutinib alone, intratumorally injected, at a dose of 20 ug / mouse, every other day, for a total of 3 times.

[0033] R848+Ibrutinib (RI) combination group: R848 and Ibrutinib were administered as above, with the same dosage and frequency as the single-drug groups, i.e., the drugs were administered every other day for a total of 3 times.

[0034] The growth changes of tumor volume and the survival status of mice were observed and compared. The subcutaneous tumor volume and mouse weight were measured every two days.

[0035] The results of mouse tumor growth were as follows Figure 1As shown in Figure 2, after the treatment, the combined treatment regimen significantly inhibited the tumor volume compared with R848 (10 ug / mouse) or Ibrutinib (20 ug / mouse) alone ( Figure 1 A to Figure 1 C). This indicates that combined drug use can produce a synergistic effect and further enhance the inhibitory effect on tumor growth.

[0036] Example 2 This example is treated with all the methods of Example 1. One week after the last treatment, the tumor tissue and draining lymph nodes are completely removed for immune response analysis to verify the immunological mechanism of the in vivo synergistic inhibitory effect of R848 and Ibrutinib on the growth of colon cancer cells.

[0037] Tumor-draining lymph nodes (TDLN) are key to activating naive T cells by tumor antigen-loaded dendritic cells (DC). The present invention prepares a single cell suspension of tumor-draining lymph nodes by mechanical grinding and washes it with PBS three times for later use. At the same time, the tumor tissue is cut into small pieces, digested with collagenase type IV, and gently stirred for 2 h at 37°C before preparing a single cell suspension of tumor by mechanical grinding. All cell samples are then resuspended in PBS precooled at 4°C, stained with specific antibodies in the dark at 4°C for 30 min, and then washed and analyzed by flow cytometry.

[0038] The flow cytometric analysis results of tumor tissues were as follows: Figure 3 As shown in Figure 2, compared with the control group, the proportion of tumor dendritic cells (Tumor-DC) in the combination group was significantly upregulated, almost three times that of the control group ( Figure 3 A), the expression of CD3+CD8+ T cells (Tumor-T) in the tumor of the combination group was significantly higher than that of all treatment groups ( Figure 3 B), the proportion of MDSCs in the combination group was significantly lower than that in the R848 group ( Figure 3 C) These results indicate that a strong cytotoxic T cell response was triggered in the tumor.

[0039] The flow cytometric analysis results of draining lymph nodes were as follows: Figure 4 As shown, the combination group also induced a significant increase in mature DCs in lymph nodes.

[0040] The above description is only a preferred embodiment of the present invention and is not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An anti-tumor combined drug composition, characterized in that: The combined drug composition comprises a TLR7 / 8 agonist and an MDSC inhibitor.

2. The anti-tumor combined drug composition according to claim 1, characterized in that: The active ingredients of the combined pharmaceutical composition consist of a TLR7 / 8 agonist and an MDSC inhibitor.

3. The anti-tumor combined drug composition according to claim 1, characterized in that: The combined pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

4. The anti-tumor combined drug composition according to claim 1, characterized in that: The TLR7 / 8 agonist includes one or more of resiquimod, imiquimod, LHC165, BNT411, NJH395, and NKTR-262.

5. The anti-tumor combined drug composition according to claim 1, characterized in that: The MDSC inhibitor includes one or more of ibrutinib, acalabrutinib, and adranib.

6. The anti-tumor combined drug composition according to claim 1, characterized in that: The administration of the TLR7 / 8 agonist and MDSC inhibitor includes one or more of intratumoral injection, peritumoral injection, intravenous injection or oral administration.

7. An application of an anti-tumor combined drug composition, characterized in that: The combined drug composition is used for preparing anti-tumor drugs.

8. The use according to claim 7, characterized in that: The tumor includes one or more of colon cancer, breast cancer and gastric cancer.

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