Combination drugs and applications for treating and / or preventing lung cancer

Abstract

The present invention belongs to the field of medical technology, and specifically relates to a combination drug and application for treating and / or preventing lung cancer. The combination drug of the present invention is a combination drug of honokiol liposomes and immune checkpoint inhibitors administered separately or simultaneously. Among them, the immune checkpoint inhibitor is a PD-1 antibody. Experiments have shown that the combination drug of the present invention can more effectively inhibit lung cancer than immune checkpoint inhibitors used alone through the synergistic effect of honokiol liposomes and PD-1 antibodies. And honokiol liposomes can reduce the infiltration of immunosuppressive cells and tumor-promoting M2 macrophages derived from the bone marrow of the lungs, and combine with PD-1 inhibitors to achieve obvious synergistic therapeutic effects.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a combined drug for treating and / or preventing lung cancer and its application. Background Art

[0002] Lung cancer is the most common cancer in the world, accounting for 11.6% of all cancer patients, and ranks high in cancer mortality. Non-small-cell lung cancer (NSCLC) accounts for more than 80% of lung cancer, with a five-year survival rate of less than 20%. Drug resistance within one year of treatment is one of the main reasons for the low survival rate. Immune checkpoint inhibitors, especially programmed cell death protein 1 (PD-1) / programmed death-ligand 1 (PD-L1) antibodies, have become the first-line and second-line standard treatment for epidermal growth factor receptor (EGFR) / anaplastic lymphomakinase (ALK)-negative advanced NSCLC and the standard treatment after synchronous chemoradiotherapy for locally advanced NSCLC, and have shown promising results in adjuvant / neoadjuvant therapy. However, immune checkpoint inhibition therapy is not effective for every patient. Due to the complexity of the tumor microenvironment, myeloid-derived suppressor cells (MDSC) limit the effect of immune checkpoint inhibitors by inhibiting T cell function. In current preclinical studies, chemotherapy drugs (such as gemcitabine) are used to regulate MDSC infiltration and enhance the inhibitory effect of PD-1 antibodies (such as nivolumab) on lung cancer, but chemotherapy drugs have strong toxic side effects and require high physical strength and physical conditions of patients; or vascular inhibitors (such as bevacizumab) are used to reduce circulating MDSC infiltration, but this method has an unstable effect on MDSC in the tumor microenvironment and cannot directly enhance the effect of PD-1 antibodies. In other preclinical studies on mouse lung cancer models, drugs that inhibit MDSC have not yet entered clinical research, and development takes longer and is more complicated.

[0003] Magnolia officinalis is an important traditional Chinese medicinal material, and honokiol is an important medicinal ingredient. It has a wide range of pharmacological effects, such as anti-inflammatory, vasoconstriction, and anti-tumor. Due to its low toxicity, the anti-tumor effect of honokiol has become a research hotspot in recent years. Honokiol is more stable and easier to absorb when encapsulated in nanoliposomes. At present, honokiol liposomes have also shown certain efficacy in tumor-related clinical trials, but its role in tumor immunity has rarely been reported. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention aims to provide a combined drug and application for treating and / or preventing lung cancer.

[0005] Wherein, the combined drug is a combined drug of magnolol liposomes and immune checkpoint inhibitors administered separately or simultaneously.

[0006] Wherein, the immune checkpoint inhibitor is a PD-1 antibody.

[0007] Preferably, the PD-1 antibody is PD-1 antibody (RMP1-14, BioXCell), pembrolizumab, nivolumab, sintilimab, carrelizumab, tislelizumab, durvalumab, atezolizumab or sugemalimab.

[0008] Preferably, the PD-1 antibody is PD-1 antibody (RMP1-14, BioXCell).

[0009] The PD-1 antibody also includes preparations made with pharmaceutically acceptable excipients, such as injections, lyophilized preparations, capsules, etc.

[0010] Wherein, the honokiol liposome includes but is not limited to the semi-finished product of honokiol liposome and the finished product of honokiol liposome. The semi-finished product of honokiol liposome refers to the raw material of honokiol that has been preliminarily treated with liposomes, such as liposome micropowder, and can be further prepared into other dosage forms, such as capsules, liposomes, tablets, injections, etc. The finished product of honokiol liposome refers to the finished dosage form of honokiol, and the prescription and preparation process include but are not limited to the technology disclosed in the invention patent with patent application number 200610021277.8.

[0011] Preferably, the honokiol liposome is honokiol liposome lyophilized powder.

[0012] Wherein, the mass ratio of the main drug of the honokiol liposome and the immune checkpoint inhibitor is 2:1 to 1:1.

[0013] Preferably, the mass ratio of the honokiol liposome to the main drug of the immune checkpoint inhibitor is 2:1.

[0014] Use of honokiol liposomes and immune checkpoint inhibitors as anticancer active ingredients in the preparation of drugs for treating and / or preventing lung cancer.

[0015] Wherein, the lung cancer also includes lung cancer metastasis.

[0016] Among them, in the use, the mass ratio of the main drugs of magnolol liposome and immune checkpoint inhibitor is 2:1 to 1:1.

[0017] Preferably, in the use, the mass ratio of the main drug of honokiol liposome and immune checkpoint inhibitor is 2:1.

[0018] Among them, in the use, the drug is a preparation prepared by adding honokiol liposomes and immune checkpoint inhibitors as anti-cancer active ingredients and adding pharmaceutically acceptable excipients or auxiliary ingredients.

[0019] Wherein, the preparations include but are not limited to injection preparations and freeze-dried preparations.

[0020] Preferably, the preparation is an injectable preparation.

[0021] Among them, in the use, the honokiol liposomes in the medicine reduce the infiltration of MDSC and / or M2 macrophages.

[0022] Note: The "main drug" mentioned in the present invention refers to the active ingredient of the drug, such as the main drug in honokiol liposome is honokiol, and the main drug of the immune checkpoint inhibitor is PD-1 antibody.

[0023] Beneficial effects: Experiments have shown that honokiol liposomes have a certain effect on tumor inhibition, while the anti-tumor effect of single-drug PD-1 antibody is not good. The tumor in the group of honokiol liposomes combined with PD-1 antibody is the smallest; and although honokiol liposomes alone and PD-1 antibody alone have a certain inhibitory effect on tumor nodules, the lung tumor nodules of mice in the honokiol liposome combined with PD-1 antibody group are significantly reduced, and the lung wet weight is also significantly reduced. It can be seen that in the combined drug of the present invention, honokiol liposomes can improve the efficacy of lung cancer immune checkpoint inhibitors and improve the tumor microenvironment. Honokiol liposomes and immune checkpoint inhibitors can play a synergistic anti-tumor effect, which is better than using honokiol liposomes or immune checkpoint inhibitors alone, so it can better treat lung cancer and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a graph showing the inhibition of lung cancer tumor growth by magnolol liposomes combined with PD-1 antibodies in Example 1; wherein A is a photo of a subcutaneous tumor in mice; B is a graph showing the tumor growth curve; and C is a graph showing the tumor weight of a subcutaneous tumor in mice.

[0025] Figure 2 This is a graph showing the inhibition of lung metastasis tumor growth by magnolol liposomes combined with PD-1 antibodies in Example 2; wherein A is a photo of mouse lung tumor nodules and HE staining images; B is a statistical graph of mouse lung wet weight; and C is a statistical graph of the number of mouse lung nodules.

[0026] Figure 3 This is the result of Example 3 that magnolol liposomes significantly reduced the infiltration of bone marrow-derived immunosuppressive cells (MDSCs) in the tumor microenvironment; A indicates that MDSCs are CD11b + Gr-1 + A is the result diagram of a group of cells; B is the flow cytometry analysis diagram (left) and statistical diagram (right) of MDSCs.

[0027] Figure 4 This is the result of Example 4 that magnolol liposomes significantly reduced the infiltration of M2 macrophages in the tumor microenvironment; A indicates that M2 macrophages are CD206-positive macrophages (CD11b + F4 / 80 + ) result diagram; B is the flow cytometry analysis diagram (left) and statistical diagram (right) of M2 macrophages.

[0028] Note: In the figure, NS represents the normal saline group, liposome represents the empty liposome group, Lipo-HNK represents the honokiol liposome group, PD-1 represents the PD-1 antibody group, 2A3 represents the PD-1 antibody isotype control drug group, and Lipo-HNK+PD-1 represents the honokiol liposome combined with PD-1 antibody group. DETAILED DESCRIPTION

[0029] The present invention reduces the infiltration of MDSC and M2 macrophages for the first time through honokiol liposomes, effectively regulates the microenvironment of the tumor, and has a significant synergistic therapeutic effect with PD-1 antibodies. Specifically, honokiol liposomes reduce MDSC and M2 macrophages in the lung tumor microenvironment in a mouse lung cancer model through its active ingredient honokiol, and further enhance the body's anti-tumor immune response in combination with PD-1 antibodies, significantly reducing the number of lung tumor nodules in the mouse lung cancer model, and inhibiting the growth and metastasis of lung cancer.

[0030] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0031] Example 1: Honokiol liposomes combined with PD-1 antibodies significantly inhibit the growth of lung cancer

[0032] In this example, honokiol liposomes were combined with PD-1 antibodies to treat mouse lung cancer models to observe the therapeutic effect of the combination. The honokiol liposome freeze-dried powder used in the experiment was from a Chinese patent with a patent number of 200610021277.8; the PD-1 antibody was RMP1-14, BioXCell.

[0033] 1. Cell culture: Select mouse lung cancer cell line LL / 2 (Lewis lung cancer cell, purchased from American Type Culture Collection (ATCC)), resuspend in DMEM culture medium (Gibco-invitrogen, USA) complete medium (containing 10% fetal bovine serum, 100Μ / ml penicillin, 100mg / ml streptomycin), and then spread in a 100mm diameter culture dish to ensure that the cells are evenly distributed in the culture dish. The culture dish is placed in a 37℃ cell culture incubator with a CO2 concentration of 5%, and the cell growth status is observed under a microscope every day, and the culture medium is replaced regularly. When the tumor cells grow to 80-90%, they are collected and prepared for inoculation.

[0034] 2. Subcutaneous tumor model: LL / 2 lung cancer cells in the logarithmic growth phase were collected, and the method was similar to cell passage. The cells were washed twice with serum-free and antibiotic-free DMEM medium to prepare a cell suspension of 5×10^6 cells / ml. The cells were inoculated into the subcutaneous part of the right thigh of 6-8 week old female C57BL / 6 mice (all cultured in Specific Pathogen Free (SPF) animal rooms) at a density of 5×10^5 cells / mouse. When the subcutaneous tumor of the mouse grew to 100mm 3 , while ensuring that the tumor size among the groups was consistent, the patients were divided into (1) solvent group (normal saline group, NS); (2) empty liposome group (liposome); (3) honokiol liposome monotherapy group (Lipo-HNK); (4) PD-1 antibody control IgG antibody 2A3 group (2A3); (5) PD-1 antibody monotherapy group (PD1); (6) honokiol liposome and PD-1 antibody combination treatment group (Lipo-HNK+PD1), and then the drug treatment was carried out.

[0035] 3. Drug treatment: Take the lyophilized powder of honokiol liposomes and prepare it into a 2mg / mL isotonic solution with sterile water. According to the main drug honokiol concentration of 20mg / kg, it is administered through the tail vein once every two days; PD-1 antibody (RMP1-14, BioXCell) is administered once every four days by intraperitoneal injection according to the main drug PD-1 antibody concentration of 10mg / kg; both are administered for 20 days. The tumor size was measured and recorded with a vernier caliper every 3 days. The longest and shortest diameters of the subcutaneous tumor were measured each time (both in mm), and the tumor volume was calculated according to the formula volume = a×b 2 / 2 (where a is the longest diameter of the tumor and b is the shortest diameter of the tumor), and the tumor growth curve was drawn.

[0036] 4. Experimental results: In the mouse subcutaneous tumor model, after treating the mice at the end of the experiment (about 30 days), subcutaneous tumor nodules were taken. Lipo-HNK had a certain effect on inhibiting tumors, while the anti-tumor effect of single-drug PD-1 antibody was poor. The tumor in the Lipo-HNK combined with PD-1 antibody group was the smallest ( Figure 1 A); From the growth curve, the tumor growth curve of mice in the Lipo-HNK combined with PD-1 antibody group was the most gentle ( Figure 1 B); The tumors of mice in the Lipo-HNK combined with PD-1 antibody group were significantly lighter than those in the Lipo-HNK monotherapy group (0.48 g vs. 0.82 g) ( Figure 1 C), therefore, honokiol liposomes combined with PD-1 antibodies have obvious synergistic effects and can significantly inhibit the growth of lung cancer.

[0037] Example 2: Honokiol liposomes combined with PD-1 antibody significantly inhibit the metastasis of lung cancer

[0038] In this example, honokiol liposomes were combined with anti-PD-1 antibodies to treat a mouse lung cancer metastasis model, and the therapeutic effect of the combination was observed.

[0039] 1. Cell culture Reference Example 1.

[0040] 2. Lung metastasis model: Construction of mouse lung cancer metastasis model: LL / 2 lung cancer cells in the logarithmic growth phase were collected and washed twice with serum-free and antibiotic-free DMEM medium to prepare a cell suspension of 1×10^6 cells / mL. The cells were inoculated into the tail vein of 6-8 week old female C57BL / 6 mice at a density of 1×10^5 cells / mouse. Similar to the subcutaneous tumor model, the mice were divided into 6 groups.

[0041] 3. Drug treatment: On the 5th day after the inoculation of LL / 2 cells into the tail vein, the corresponding treatment was started, referring to the drug treatment part of Example 1.

[0042] 4. Experimental results: In the mouse lung metastasis model, at the end of the experiment (about 28 days), the mice were treated and the lungs were taken. Both macroscopic and HE staining showed that Lipo-HNK alone and PD-1 antibody alone had a certain inhibitory effect on tumor nodules. The number of lung tumor nodules in mice in the Lipo-HNK combined with PD-1 antibody group was significantly reduced (see Figure 2 A); The lung wet weight of mice in the combined treatment group was also significantly reduced ( Figure 2 B); When counting the tumor nodules in the mouse lungs, it was also found that Lipo-HNK alone (11) and PD-1 antibody alone (14.8) could inhibit the formation of lung tumor nodules to a certain extent, and Lipo-HNK combined with PD-1 antibody was more effective in inhibiting the formation of lung tumor nodules (0.2 in the combined group vs. 44.8 in the NS group) ( Figure 2 C), it can be seen that compared with the single use of honokiol liposomes and PD-1 antibodies, honokiol liposomes combined with PD-1 antibodies have a synergistic enhancement effect and can significantly inhibit the metastasis of lung cancer.

[0043] Example 3: Honokiol liposomes significantly reduce the infiltration of MDSC in the tumor microenvironment

[0044] PD-1 antibodies activate the immune response of T cells to tumor cells by blocking the binding of PD-1 to its ligand, awakening the patient's own anti-tumor effect. However, there is a group of immunosuppressive cells (such as MDSC or M2 macrophages) in the immune microenvironment of the tumor, which not only prevents T cells from attacking tumor cells, but also secretes growth factors to nourish tumor cells, promotes the formation of tumor blood vessels, and causes tumor cell metastasis and spread. Studies have reported that macrophages will also engulf PD-1 antibodies bound to T cells, resulting in the failure of immunotherapy. In order to further study the synergistic treatment mechanism of honokiol and PD-1 antibodies, the tumor microenvironment was further analyzed by flow cytometry, referring to Examples 3 and 4.

[0045] 1. Sample preparation: Cut the lung tissue with tumor nodules into small pieces as possible, add 6-8mL of serum-free DMEM culture medium containing type I and type IV collagenase (1mg / mL) into a centrifuge tube, shake and mix in a 37°C incubator for digestion for 1 hour, filter the digested cell suspension with a 70μm cell mesh, take the filtrate, centrifuge at 1500rpm for 3 minutes; discard the supernatant, add 5mL of red blood cell lysis buffer, lyse on ice for 3 minutes, and then centrifuge at 1500rpm for 3 minutes; wash the cells with 3ml PBS buffer, centrifuge at 1200rpm for 3 minutes, and resuspend the cells with an appropriate amount of PBS buffer to prepare a tissue cell suspension.

[0046] 2. Staining: Count the prepared cell suspension, adjust the concentration of the cell suspension to 1×10^7 cells / mL, add 100μL PBS and place it in a flow tube, directly add the flow antibody of the membrane antigen (generally 1μl / 1×10^6 cells), incubate at 4℃ in the dark for 30 minutes, add 1mL PBS buffer to each tube to stop, centrifuge at 1200rpm for 3 minutes, aspirate the supernatant, add 500μL PBS buffer to resuspend, store at 4℃ in the dark and wait for detection.

[0047] 3. Experimental results: MDSC cells are CD45+CD11b+Gr1+ cells ( Figure 3 A); Flow cytometry results showed that Lipo-HNK could significantly reduce the infiltration of MDSC cells in the tumor microenvironment (Lipo-HNK group 12.2% vs. NS group 44.73%), and the combined treatment group also reduced MDSC infiltration to a certain extent (27.24%) ( Figure 3 B), the infiltrating MDSCs in the combined group accounted for 27.24% of the total immune cells, which was significantly lower than that in the PD-1 antibody monotherapy group (35.9%). n=5, *p<0.05, **p<0.01, ***p<0.001.

[0048] Example 4: Honokiol liposomes significantly reduce the infiltration of M2 macrophages in the tumor microenvironment

[0049] Sample preparation and staining were as in Example 3.

[0050] Experimental results: M2 macrophages are CD45+CD11b+F4 / 80+CD206+ cells ( Figure 4 A); Flow cytometry results showed that Lipo-HNK can significantly reduce the infiltration of MDSC cells in the tumor microenvironment (Lipo-HNK group 5.47% vs. NS group 18.28%), and the combined treatment group also significantly reduced MDSC infiltration (4.77%) ( Figure 4 B); The infiltrating M2 macrophages in the combined group accounted for 4.77% of the total macrophages, which was significantly lower than that in the PD-1 antibody monotherapy group (21.67%). n=5, *p<0.05, **p<0.01, ***p<0.001.

Claims

1. A combined drug for treating lung cancer, characterized in that: The combined drug is a combined drug of honokiol liposomes and immune checkpoint inhibitor PD-1 antibody RMP1-14 administered separately or simultaneously.

2. The combined drug according to claim 1, characterized in that: The honokiol liposome is honokiol liposome freeze-dried powder.

3. The combined drug according to claim 1 or 2, characterized in that: The mass ratio of the honokiol liposome to the main drug of the immune checkpoint inhibitor is 2:1 to 1:

1.

4. The combined drug according to claim 3, characterized in that: The mass ratio of the main drug is 2:

1.

5. The use of magnolia officinalis liposomes and immune checkpoint inhibitor PD-1 antibody RMP1-14 as anti-cancer active ingredients in the preparation of drugs for the treatment of lung cancer.

6. The use according to claim 5, characterized in that: The mass ratio of the main drug of honokiol liposome and immune checkpoint inhibitor is 2:1 to 1:

1.

7. The use according to claim 6, characterized in that: The mass ratio of the main drug is 2:

1.

8. The use according to any one of claims 5 to 7, characterized in that: The drug is a preparation prepared by using honokiol liposomes and immune checkpoint inhibitor PD-1 antibody RMP1-14 as anti-cancer active ingredients, and adding pharmaceutically acceptable auxiliary ingredients.

9. The use according to claim 8, characterized in that: The preparation is an injection preparation.

10. The use according to claim 5, characterized in that: The honokiol liposomes in the drug reduce the infiltration of MDSC and / or M2 macrophages.

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