New medical uses of β-blockers

Abstract

The present invention discloses a new medical use of β-blockers in the treatment of malignant pleural effusion, belonging to the field of medical technology. Clinical findings, in vivo experiments and in vitro cell tests show that β-blockers can improve malignant pleural effusion, reduce the proportion of tumor-associated macrophages in the tumor microenvironment, inhibit their immunosuppressive functions, and reverse the damage of macrophages to the anti-tumor effect of NK cells. The application of β-blockers in the treatment of tumor patients with malignant pleural effusion provides a new treatment strategy for improving the prognosis. At the same time, β-blockers have been widely used clinically, and their safety and toxic and side effects have been clarified, which can significantly reduce the scientific research investment and the medical burden of patients, and have important application value.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a new pharmaceutical use of β-blockers. Background Art

[0002] Malignant pleural effusion (MPE) is associated with poor prognosis in cancer patients, with a median survival of 4 - 9 months. The immunosuppressive microenvironment of malignant pleural effusion affects the efficacy of anti-tumor therapy and promotes tumor growth.

[0003] The occurrence and development of tumors are closely related to the immune microenvironment. The tumor microenvironment consists of cancer cells, immune cells, fibroblasts, endothelial cells, and extracellular matrix, etc. Tumor-associated macrophages (TAMs) are one of the most abundant tumor infiltrates in the tumor microenvironment and play a key role in regulating the tumor immunosuppressive microenvironment and tumor occurrence and progression. TAMs have high plasticity and can be polarized into pro-inflammatory and anti-tumor M1 macrophages, or polarized into anti-inflammatory and pro-tumor M2 macrophages. It is generally believed that most TAMs exhibit the phenotype and function of M2 macrophages, promoting angiogenesis, the occurrence, development, and metastasis of tumor cells, and inhibiting the immune response of cytotoxic T cells.

[0004] β-blockers, also known as β-adrenergic receptor blockers, mainly regulate the cardiovascular system, metabolic system, and bronchial smooth muscle by blocking the binding of β-receptors to catecholamines. Currently, commonly used β-blockers include dozens of drugs such as propranolol, metoprolol, and bisoprolol.

[0005] Currently, β-blockers have been widely used clinically, and their safety, drug metabolism characteristics, and side effects have been clarified, and they are also applicable to pediatric medications. Reports on the effect of β-blockers on malignant pleural effusion are still lacking. In the present invention, we found that β-blockers can inhibit the immunosuppressive function of tumor-associated macrophages and reverse the inhibitory effect of macrophages on the anti-tumor effect of NK cells; at the same time, the safe and effective "new use of old drugs" can significantly reduce the research investment and the medical burden of patients, and has important application value. Summary of the Invention

[0006] The object of the present invention is to provide a new pharmaceutical use of β-blockers. Clinical observations and in vitro cell experiments have found that β-blockers can promote the absorption of malignant pleural effusion, improve the ability of NK cells to kill tumor cells by reducing the proportion of tumor-associated macrophages in the tumor microenvironment and reducing their immunosuppressive function.

[0007] In the first aspect of the present invention, the application of β-blockers in the treatment of malignant pleural effusion is discovered.

[0008] Furthermore, the malignant pleural effusion includes pleural effusion caused by primary lung malignancies and secondary malignancies invading the pleura. The aforementioned β-blockers include propranolol, metoprolol, bisoprolol, etc.

[0009] On the other hand, the present invention discovers the application of β-blockers in regulating the functions of tumor-associated macrophages. Experiments of the present invention prove that β-blockers can inhibit the proportion of TAM in the tumor microenvironment, reduce its immunosuppressive function, and reverse the damaging effect of macrophages on the anti-tumor effect of NK cells.

[0010] In vivo experiments show that after oral administration of β-blockers, malignant pleural effusion is improved mainly by regulating the proportion of tumor-associated macrophages and their immunosuppressive functions. The results of in vitro experiments are consistent with the in vivo results.

[0011] Cell experiments show that after in vitro induction of M2 macrophages and treatment with β-blockers, the immunosuppressive function decreases, and the damaging effect of macrophages on the anti-tumor effect of NK cells is inhibited.

[0012] The present invention discovers for the first time the role of β-blockers in treating malignant pleural effusion. The present invention discovers for the first time that it exerts an anti-tumor effect by regulating the proportion and functions of macrophages in the tumor microenvironment. Description of the Drawings

[0013] Figure 1 Shows the changes in pleural effusion before and after oral administration of propranolol in Example 1.

[0014] Figure 2 Shows the changes in the proportions of immune cells TAM, MDSC, CD8, NK and the expression changes of immunosuppressive function molecules PD-L1 and ARG-1 of macrophages in malignant pleural effusion before (left bar graph) and after (right bar graph) oral administration of metoprolol in Example 2.

[0015] Figure 3 Shows the changes in the proportions of immune cells TAM, MDSC, CD8, NK after in vitro treatment of mononuclear cells in malignant pleural effusion with propranolol for 24 h in Example 3.

[0016] Figure 4 Shows the changes in the proportion of TAM after in vitro induction of monocytes into M2 macrophages and treatment with propranolol for 24 h and 48 h in Example 4.

[0017] Figure 5 Shows the expression changes of macrophage PD-L1 and ARG-1 after in vitro induction of monocytes into M2 macrophages and treatment with propranolol for 24 h in Example 5.

[0018] Figure 6Effect of propranolol treatment for 48 h on the killing of lung cancer cell A549 by NK cells by macrophages induced from in vitro monocytes in Example 6.

[0019] (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns indicates no significant statistical significance). Specific implementation manners

[0020] The above content of the present invention will be further described in detail below through examples, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following examples. Those skilled in the art can make various changes and modifications to the present invention, and any technology implemented based on the above content of the present invention belongs to the scope of the present invention.

[0021] Inclusion criteria for malignant pleural effusion: Pathological diagnosis is clear, and the patient has not received local thoracic treatment or systemic radiotherapy, chemotherapy, or immunotherapy.

[0022] Treatment of pleural effusion samples: Filter the collected pleural effusion through a 70-μm filter membrane, centrifuge at 3000 rpm for 10 min, and wash the precipitate 3 times with PBS. After resuspending the cells with PBS, add them to lymphocyte separation medium with a volume of 1 / 2 (note not to disturb the liquid interface), centrifuge at 2500 rpm for 25 min (acceleration rate is 5, deceleration rate is 5), aspirate the white film layer (mononuclear cells), and wash 3 times with PBS.

[0023] Flow cytometry detection of immune cell subsets and macrophage immunosuppressive function molecules in pleural effusion: After collecting the cells, wash them 2 times with PBS at 4°C. First, stain with a live / dead dye for 15 min, then centrifuge at 1500 rpm at 4°C for 5 min, and discard the supernatant. Resuspend the cells with flow cytometry loading buffer and stain the surface antigens (add flow cytometry antibodies according to the number of cells), incubate in the dark on ice for 15 min, centrifuge at 1500 rpm at 4°C for 5 min, and discard the supernatant. For intracellular antigen (ARG-1) staining, continue the following steps: Add a fixative (4% paraformaldehyde) and fix for half an hour, centrifuge at 1500 rpm at 4°C for 5 min, and discard the supernatant. Add 1 mL of 1X permeabilization agent and permeabilize on ice for half an hour in the dark. Centrifuge at 1500 rpm at 4°C for 5 min, discard the supernatant, then add the corresponding flow cytometry antibody, and incubate in the dark on ice for 15 min. Wash once with PBS, resuspend, and detect on the machine.

[0024] Mononuclear cell sorting: Pour healthy human peripheral blood into a 50 mL centrifuge tube, and add saline to 30 mL per tube. Slowly pour in 15 mL of lymphocyte separation solution and centrifuge at 2500 rpm for 25 min (acceleration speed is 5, deceleration speed is 5). Use a pipette to aspirate the buffy coat layer, which is human peripheral blood mononuclear cells (PBMCs), and wash it 3 times with PBS. Resuspend the PBMCs with magnetic sorting Buffer, perform cell counting, centrifuge at 1500 rpm at 4 °C for 5 min, and discard the supernatant. According to the instructions, add 80 μL of magnetic sorting buffer and 20 μL of CD14 Beads (buffer:Beads = 4:1) to every 1X10 7 cells, mix well and incubate at 4 °C in the dark for 20 min, shaking and mixing well every 5 min. After incubation, add 5 mL of magnetic sorting Buffer to wash the cells, centrifuge at 1500 rpm for 5 min, discard the supernatant, add 1 mL of magnetic sorting Buffer to resuspend the cells, and pipette to mix well. Fix the magnetic sorting column on the magnetic field, add 2 mL of magnetic sorting Buffer to rinse the column, and add the cell suspension to the column until it all passes through. Wash the column 2 times with magnetic sorting Buffer, add 5 mL of magnetic sorting Buffer to the column, take the column out of the magnetic field, quickly push out the liquid to the centrifuge tube away from the magnetic field with a piston, centrifuge at 1500 rpm for 5 min, and wash 2 times with PBS. Resuspend the cells with 5 mL of 10% RPMI 1640 medium, that is, the CD14 + monocyte mixture.

[0025] Cell immunofluorescence detection of the expression of macrophage PD-L1 and ARG-1: Fix the cells at room temperature with 4% paraformaldehyde for 0.5 h, and wash 3 times with PBS; permeabilize the cells with 1% Triton X100 for 10 min, and wash 3 times with PBS; block with goat serum for 1 h, and then wash 3 times with PBS. Then add the diluted rabbit primary antibodies (anti-PD-L1, anti-ARG-1) to each slide and incubate overnight at 4 °C. The next day, after rewarming for 1 h, wash 5 times with PBS, add the secondary antibody Alexa Fluor 488-AffiniPure Donkey Anti-Rabbit IgG (1:500), incubate at room temperature for 1 h, and wash 3 times with PBS; add DAPI (1:1000) and incubate at room temperature for 5 min. Observe and take pictures under a fluorescence microscope (Perkinelmer, Vectra), and analyze the data using InForm image analysis software.

[0026] Example 1

[0027] The inventor found in clinical practice that for lung cancer patients with malignant pleural effusion complicated with cardiovascular diseases, after taking propranolol at the clinical application dose, the changes in malignant pleural effusion were shown by chest CT imaging. The dose of propranolol taken orally was the clinical application dose; the patients with malignant pleural effusion were untreated and had not undergone systematic clinical cancer treatment. (As Figure 1 shown) The results showed that after taking propranolol orally, the amount of malignant pleural effusion in the patients decreased significantly.

[0028] Example 2

[0029] For patients with malignant pleural effusion complicated with cardiovascular diseases, 3 days after taking metoprolol at the clinical application dose, the changes in the proportion of immune cell subsets in the pleural effusion and the expression changes of the immunosuppressive function molecules PD-L1 and ARG-1 of macrophages were detected by flow cytometry. The pleural effusion before and after taking metoprolol was collected, and mononuclear cells in the pleural effusion were obtained according to the above-mentioned pleural effusion treatment method. The TAM (CD14 + CD63 + )、MDSC (HLA-DR - CD11b + CD33 + ), CD8 (CD3 + CD8 + ), NK (CD3 - CD56 + ) proportion changes in the pleural effusion before and after treatment and the expression changes of PD-L1 and ARG-1 of macrophages were detected; the data were analyzed by Flowjo software. (As Figure 2 shown)

[0030] The results showed that after taking the β-blocker (metoprolol), the proportion of tumor-associated macrophages in the malignant pleural effusion decreased significantly, and the immunosuppressive function was reduced.

[0031] Example 3

[0032] Collect mononuclear cells obtained by treating malignant pleural effusion, resuspend the cells with RPMI 1640 medium, adjust the cell density to 1X10 6 / mL and then plate them in 12-well plates. Add 2 μL of propranolol (concentration: 10 μM) to each well with 2 mL of cell suspension, and place them in an incubator at 37 °C and 5% CO 2 for 24 h. Detect TAM (CD14 + CD63 + )、MDSC (HLA-DR - CD11b + CD33 + )、CD8 (CD3 + CD8+ )), NK (CD3 - CD56 + ) ratio change; data were analyzed by Flowjo software. (As Figure 3 shown)

[0033] After treatment with β-blocker (propranolol) in vitro, the change in the proportion of immune cells in pleural effusion was consistent with that of oral β-blocker in vivo.

[0034] Example 4

[0035] The magnetically sorted CD14 + monocytes were adjusted to a concentration of 1X10 6 / mL with complete 1640 medium, plated at 1 mL per well in a 24-well plate, M-CSF factor was added to a concentration of 20 ng / mL, mixed well, and placed in a 5% CO 2 , and cultured in an incubator at 37 °C. At days 1, 3, and 5, half of the medium was changed, and at the same time, cytokines were supplemented to a concentration of 20 ng / mL. On day 7, all the cells in each well were changed to fresh medium, and cytokines were added in sufficient amounts to induce M2 macrophages. At the same time, 1 μL of propranolol (10 μM) was added to one group and treated for 24 h and 48 h; the proportion change of M2 macrophages (CD14 + CD163 + ) was detected by flow cytometry. (As Figure 4 shown)

[0036] The results showed that propranolol could inhibit the polarization of monocytes into M2 macrophages.

[0037] Example 5

[0038] Peripheral blood monocytes were induced to M2 macrophages in vitro, and after treatment with propranolol for 24 h, the changes in the expression levels of macrophage functional molecules PD-L1 and ARG-1 were detected by flow cytometry and immunofluorescence. (As Figure 5 shown)

[0039] The results showed that propranolol could reduce the immunosuppressive function of macrophages while reducing the proportion of M2 macrophages.

[0040] Example 6

[0041] The effect of β-blocker (propranolol) on the function of NK cells killing tumors damaged by M2 macrophages was detected by flow cytometry. First, the target cells were labeled with CSFE: 1X10 5Resuspend the A549 cells, add 1 μL of CSFE, incubate at 37°C for 20 min in the dark. Shake once in the middle, discard the supernatant, add 1 mL of complete 1640 medium and incubate at 37°C for 20 min, then wash twice with PBS. Divide the induced M2 macrophages into two groups, and add propranolol to one group for treatment for 48 h. Then, plate the cells in a 96-well plate according to the ratio of two groups of macrophages: NK cells (NK-92): A549 (labeled with CSFE) of 1:1:1, 5:5:1, 10:10:1, 20:20:1, and incubate for 6 h. Digest the cell mixture with trypsin, wash twice, add the PI working solution, and detect on the machine after incubating in the dark for 30 min. The cells that are double positive for CFSE and PI are the target cells killed, and dividing by the total number of target cells gives the killing rate. (As Figure 6 shown)

[0042] The results showed that propranolol could reverse the inhibitory effect of macrophages on the killing of tumors by NK cells.

[0043] The specific embodiments described in the present invention are only illustrative of the spirit of the present invention. Those skilled in the art of the technology described in the present invention can also make various improvements or modifications to the present invention without departing from the principle of the present invention, and these improvements and modifications should also be within the protection scope of the claims of the present invention.

Claims

1. Use of propranolol as the sole active agent in the preparation of a medicament for preventing or treating malignant pleural effusion, characterized in that, the malignant pleural effusion is a pleural effusion caused by primary lung malignancy or secondary malignancy invading the pleura.

2. The use according to claim 1, characterized in that, the medicament can reduce the proportion of tumor-associated macrophages in the tumor microenvironment and their immunosuppressive function.

Images