CN117298109A

CN117298109A - Application of indacaterol in preparing medicines for preventing and/or treating lung cancer

Info

Publication number: CN117298109A
Application number: CN202311250277.5A
Authority: CN
Inventors: 孙建国; 余永新; 殷辰睿; 简春利; 李令臣
Original assignee: Second Affiliated Hospital Army Medical University
Current assignee: Second Affiliated Hospital Army Medical University
Priority date: 2023-09-26
Filing date: 2023-09-26
Publication date: 2023-12-29

Abstract

The invention provides application of indacaterol in preparing a medicine for preventing and/or treating lung cancer, and belongs to the technical field of medicines. The application of indacaterol in preparing medicines for preventing and/or treating lung cancer and the application of indacaterol in preparing medicines for enhancing the curative effect of radiotherapy in treating lung cancer. In vitro cell experiments and in vivo animal experiments show that indacaterol not only has the effect of inhibiting the growth of lung cancer tumor cells, but also can enhance the curative effect of radiotherapy, and has good drug safety. The technical scheme of the invention provides a new thought for clinical treatment of lung cancer and has a certain medical value.

Description

Application of indacaterol in preparing medicines for preventing and/or treating lung cancer

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of indacaterol in preparation of medicines for preventing and/or treating lung cancer.

Background

Lung cancer is the leading cause of cancer death worldwide, with a high rate of death. About 85% of lung cancer patients have non-small cell lung cancer (NSCLC). Radiation Therapy (RT) is an effective way to be widely used in the clinical management of NSCLC. However, NSCLC cells exhibit an inherent or acquired resistance to RT, which results in treatment failure and induction of local recurrence of NSCLC. Thus, there is an urgent need for new and more effective methods for enhancing radiation therapy for treating NSCLC to increase patient survival.

Indacterol (Indacaterol) is a monohydroxy quinoline that is a maleate salt for the treatment of chronic obstructive pulmonary disease. It has the actions of beta-adrenergic agonists and bronchodilators. It is a member of the quinolones, monohydroxy quinolines, indanones, secondary alcohols and secondary amino compounds. Indacaterol is a β2-adrenergic receptor agonist. The mechanism of action of indacaterol is to act as an adrenergic β2-receptor agonist in the treatment of asthma, and thus indacaterol is the only ultra-long acting inhaled β2 agonist (LABA) approved by the FDA for alleviating symptoms in COPD patients. However, there is no report on indacaterol treatment of lung cancer at present.

Disclosure of Invention

Therefore, the invention aims to provide the application of indacaterol in preparing the medicines for preventing and/or treating the lung cancer, the indacaterol can effectively inhibit the growth of the lung cancer cells, and simultaneously enhance the curative effect of radiotherapy, thereby providing a new thought for the clinical treatment of the lung cancer.

The invention provides application of indacaterol in preparing a medicine for preventing and/or treating lung cancer.

The invention provides application of indacaterol in preparing a medicine for enhancing radiotherapy curative effect in treating lung cancer.

Preferably, the radiotherapy comprises X-ray irradiation.

Preferably, the lung cancer comprises non-small cell lung cancer.

Preferably, the cell line of non-small cell lung cancer comprises at least one of the following: h460 cell line, H1299 cell line and PC9 cell line.

Preferably, the drug is a drug that inhibits proliferation and growth of cells of non-small cell lung cancer.

Preferably, the drug is a cell cycle arrest in the G0/G1 phase of a tissue lung cancer cell.

Preferably, the indacaterol has a structural formula shown in formula I:

preferably, in the medicine, the concentration of indacaterol is above 1.2558 mg/mL.

Preferably, the dosage form of the medicine comprises injection and powder for injection.

The invention provides application of indacaterol in preparing medicines for pre-treating lung cancer. According to the invention, in-vitro cell experiments and in-vivo animal experiments are respectively carried out to verify the drug effect of indacaterol on lung cancer, and in-vitro cell experiment results show that the proliferation of lung cancer cell lines can be inhibited by lower indacaterol drug concentration, meanwhile, the cloning of the lung cancer cells by indacaterol has remarkable inhibition effect, and the indacaterol blocks the cell cycle of the lung cancer cells to the G0/G1 phase, so that the proliferation of the lung cancer cells is inhibited; the in vivo animal experiment result shows that the indacaterol single drug can effectively inhibit the tumor growth in the mouse body and prolong the survival period of the mouse. It can be seen that indacaterol has the potential of treating lung cancer and provides a new idea for clinical medication.

Meanwhile, the indacaterol with an effective dose has low toxicity to normal cells, does not influence the weight and survival state of mice, is an approved drug on the market, and has good drug safety.

The invention provides application of indacaterol in preparing a medicine for improving radiation sensitivity in treating lung cancer. According to the invention, in vivo animal experiments are carried out to verify the drug effect of indacaterol on improving the sensitivity of lung cancer radiotherapy, and experimental results show that compared with a radiotherapy group, the radiotherapy combined indacaterol treatment group shows a remarkable difference in inhibiting tumor-bearing volume increase, so that the indacaterol has the drug effect of resisting lung cancer, can further improve the sensitivity of tumors on radiotherapy by combining with a radiotherapy method, can more effectively control tumor growth, and prolongs the survival period of mice. Therefore, the invention can provide a certain guiding significance for clinical treatment.

Drawings

FIG. 1 shows the results of CCK-8 cell proliferation assays for different non-small cell lung cancer (NSCLC) cell lines;

FIG. 2 shows the results of panel clone formation for different non-small cell lung cancer (NSCLC) cell lines, wherein A is the observation of panel clone formation and B is the statistics of cell clone formation;

FIG. 3 is a graph showing the effect of indacaterol administration on cell cycle of various non-small cell lung cancer (NSCLC) cell lines;

FIG. 4 is a graph showing statistics of different cell cycles for different non-small cell lung cancer (NSCLC) cell lines following indacaterol administration;

FIG. 5 is a flow chart of a scheme for in vivo administration of indacaterol;

FIG. 6 is a graph showing the effect of indacaterol administration on tumor volume in mice;

FIG. 7 is a graph showing tumor growth in each mouse after in vivo administration of indacaterol, wherein A is the control result and B is the indacaterol result;

FIG. 8 is a graph comparing tumor volumes in mice after in vivo administration of indacaterol;

FIG. 9 is a graph showing the change in body weight of mice after in vivo administration of indacaterol;

FIG. 10 is a graph showing survival of mice following in vivo administration of indacaterol;

FIG. 11 is a flow chart of a therapeutic regimen for radiation therapy in combination with indacaterol administration;

FIG. 12 is a graph showing the effect of radiation therapy in combination with indacaterol administration on tumor volume in mice;

FIG. 13 is a graph showing tumor growth in mice of each treatment group, wherein A is the control group, B is the indacaterol single drug treatment group, C is the radiation treatment group, and D is the radiation treatment combined indacaterol treatment group;

FIG. 14 is a graph showing comparison of tumor volumes in mice in different treatment groups;

FIG. 15 is a graph showing the change in body weight of mice in different treatment groups after in vivo administration;

figure 16 is a graph showing survival of mice following in vivo dosing in mice in different treatment groups.

Detailed Description

In the present invention, indacaterol is a monohydroxyquinoline composed of 5- [ (1R) -2- [ (5, 6-diethyl-2, 3-dihydro-1H-inden-2-yl) amino group]-1-hydroxyethyl group]-8-hydroxy-1H-quinolin-2-one having a molecular weight of 392.49g/mol and a chemical formula C, also referred to as QAB149 ₂₄ H ₂₈ N ₂ O ₃ The CAS number is: 312753-06-3. The structural formula of the indacaterol is preferably shown as formula I: in the examples of the present invention, the indacaterol was purchased from selleck biotechnology limited, usa.

In the present invention, the lung cancer preferably includes non-small cell lung cancer. The cell line of non-small cell lung cancer preferably comprises at least one of the following: h460 cell line, H1299 cell line and PC9 cell line. In the examples of the invention, indacaterol acts on H460 cell line IC ₅₀ A value of 15.91 (+ -0.1) μm; when acting on H1299 cell line, IC ₅₀ A value of 11.2 (+ -0.3) μm; when acting on PC9 cell line, IC ₅₀ The value was 5.9 (+ -0.2) μm. IC when indacaterol acts on murine lung cancer cell line LLC ₅₀ The value was 10.3 (+ -0.3) μm. It can be seen that a lower concentration of indacaterol inhibits proliferation of NSCLC cell lines, preferably a drug that inhibits proliferation of cells of non-small cell lung cancer. The plate cloning experiment shows that the proliferation capacity of the cells of H460 and H1299 and PC9 cell strain after being treated by indacaterol is obviously inhibited. Indicating that indacaterol has remarkable inhibiting effect on the clone formation of NSCLC cells. The PI method cell cycle experiment shows that indacaterol blocks the cell cycle of NSCLC cells to the G0/G1 phase, thereby inhibiting the proliferation of tumor cells. In vivo animal experiments show that compared with the control group, the indacaterol treatment group not onlyCan inhibit tumor growth, and prolong animal survival time.

The preparation method of the medicine is not particularly limited, and the preparation method of the medicine known in the art can be adopted.

In the present invention, the radiotherapy preferably comprises X-ray irradiation. The dose of the X-ray irradiation is preferably 20Gy. Use of radiation therapy in combination with indacaterol for the treatment of lung cancer. In the embodiment of the invention, compared with a pure radiation treatment group, the radiation treatment and indacaterol combined treatment group can obviously inhibit the growth of tumor-bearing, has no obvious influence on the weight of mice, and prolongs the survival time of the mice, thus indicating that the indacaterol administration can enhance the treatment effect of radiotherapy.

In the present invention, the concentration of indacaterol in the medicine is preferably 1.2558mg/mL or more. The dosage forms of the medicine preferably comprise injection and powder for injection. The preparation method of the medicine is not particularly limited, and the preparation method of the medicine known in the art can be adopted.

The application of indacaterol provided in the present invention to the preparation of a medicine for preventing and/or treating lung cancer is described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

In vitro experiments of indacaterol inhibiting growth of non-small cell lung cancer cell strain

1. CCK-8 cell proliferation assay

1. Experimental apparatus and reagent description

1.1 experimental reagents: indaterol (Indacaterol) (99.99%, selleck Biotechnology Co., ltd.) RPMI-1640 medium (Gibco, siemens Feishmanic technologies Co., ltd.), double antibody (penicillin and streptomycin) (Gibco, siemens Feishmanic technologies Co., ltd.), FBS NewZealand Origin (Invigetech, inc., U.S.), 0.25% trypsin (HyClone, siemens Feishmanic technologies Co., ltd.), cell Counting Kit-8 (Invigetech, inc., U.S.A.).

1.2 experiment consumable: 25cm ² Square gas cap miter cell culture flask (beijing kovich technologies limited), 15mL centrifuge tube sterilization (beijing kovich technologies limited Labselect), 3mL extended pasteur pipette (no tin resistant life technologies limited), dimethyl sulfoxide (DMSO) (sigma aldrich trade limited), 96-well cell culture plate (beijing kovich technologies limited Labselect), PBS (1×) dry powder (beijing kovich technologies limited Biosharp), giemsa staining (10×) (Shanghai bi cloud biotechnology limited), 6-well cell culture plate (beijing kovich technologies limited Labselect).

1.3 laboratory apparatus: full wavelength multifunctional enzyme labeling instrument (Simer Feishier technology Co.), ultra clean bench (Souzhou purification Equipment Instrument Co., ltd.), inverted microscope (Olympus Co., ltd.), LDZX-50KBS vertical pressure steam sterilizer (Shanghai Shen An medical instruments Co., ltd.), TG16-W desk-top high speed centrifuge (Hunan Xiang instruments laboratory instruments development Co., ltd.), TGL-16M high speed desk-top refrigerated centrifuge (Lu Xiangyi centrifuge instruments Co., ltd.), CO ₂ Incubator (Simer Feishier technology (China)) ZNCL-BS intelligent magnetic stirrer (Shanghai Zhongzhu instruments Co., ltd.) Milli-Q ultra pure water machine (U.S. Millipore Pharmacia).

1.4 preparation of Indaterol in vitro experiment working solution: 5mg of indacaterol dry powder is dissolved in 127.39 mu LDMSO solution, evenly mixed until the solution is clear, prepared into a mother solution with the concentration of 100mM, and the mother solution is stored at the temperature of minus 20 ℃ after being packaged. Diluting the mother solution 1000 times with complete culture medium according to experimental requirement to obtain working solution with concentration of 100 μm, and sequentially diluting with complete culture medium to obtain working solution with concentrations of 50 μm, 25 μm, 12.5 μm, 6.25 μm and 3.125 μm. The DMSO content is not more than one thousandth, so that the experimental result is not affected.

2. The experimental method comprises the following steps:

2.1 detection of cell Activity (CCK-8 method)

Experiment on the first day:

1) And culturing the cell strain with good growth state, and at least stably passaging for more than 3 generations for subsequent experiments.

2) Washing of already grown cells 25cm with PBS ² The flask was incubated with 1mL trypsin for 2min, complete medium was added to terminate the digestion, the cells were blown off, the cell suspension was pipetted into a new 15mL centrifuge tube, at 800rpm, and centrifuged for 5min.

3) After the cells were resuspended in complete medium, 20. Mu.L of the cell suspension was aspirated into a cell counter plate, counted on a cell counter, 3 sampling points were counted and finally averaged.

4) After the concentration was obtained, 5X 10 cells were inoculated per well ³ Individual cells were counted and seeded.

5) Setting an experimental group, a control group and blank holes, and adding 100 mu LPBS solution into each hole of the frame to prevent the complete culture medium from evaporating too fast due to excessive drying in the incubation process of the 96-hole cell culture plate.

4) Adjusting density, inoculating into 96-well plate, adding 100 μl of cell suspension into each well, and placing at 37deg.C under 5% CO ₂ Culturing in an incubator.

The loading layout is shown in table 1:

TABLE 1 96 well plate loading layout

The following experiment:

1) Complete medium in 96-well plates was pipetted off.

2) According to the experimental design, the candidate drug small molecules are prepared into the following concentration by using a complete culture medium: 100. Mu.M, 50. Mu.M, 25. Mu.M, 12.5. Mu.M, 6.25. Mu.M, 3.125. Mu.M.

3) Adding into corresponding wells, each 100 μl, placing at 37deg.C, 5% CO ₂ In the incubator, culturing was continued for 48 hours.

Post-culture experiments:

1) Complete medium in 96-well plates was pipetted off.

2) The prepared CCK-8 working solution was added to the 96-well plate in an amount of 100. Mu.L per well.

3) Incubate in a constant temperature incubator for 1h.

4) Taking out the well incubated 96-well plate, standing at room temperature for 10min, and detecting the absorbance (OD value) of each well at 450nm on an enzyme-labeled instrument after the solution is stable. Cell growth curves were plotted according to OD values.

5) Cell viability was calculated according to formula I, IC was calculated using GraphPadPrism ₅₀ Values.

Cell viability (%) = (OD experimental well-OD blank well)/(OD control well-OD blank well) ×100% formula I

Wherein OD test group: CCK-8 working solution containing cells and small molecule drugs;

OD control group: CCK-8 working solution containing cells and no small molecule medicine;

OD blank: does not contain cells, CCK-8 working solution and micromolecular medicaments.

Results of CCK-8 cell proliferation experiments

Indamaterol IC acting on human different non-small cell lung cancer (NSCLC) cell lines ₅₀ The values are shown in FIG. 1, which shows, first, IC after 48h of its action on NSCLC cell lines ₅₀ The values are respectively: IC when acting on H460 cell line ₅₀ A value of 15.91 (+ -0.1) μm; when acting on H1299 cell line, IC ₅₀ A value of 11.2 (+ -0.3) μm; when acting on PC9 cell line, IC ₅₀ The value was 5.9 (+ -0.2) μm. IC when indacaterol acts on murine lung cancer cell line LLC ₅₀ The value was 10.3 (+ -0.3) μm. It can be seen that proliferation of NSCLC cell lines can be inhibited at lower indacaterol drug concentrations. Meanwhile, when indacaterol acts on human normal lung epithelial cell strain Beas-2b, IC thereof ₅₀ The value of 23.1 (+ -0.1) mu M is obviously higher than that of IC (integrated circuit) acting on 4 humanized NSCLC cell strains and 1 murine lung cancer cell strain ₅₀ The values (see Table 2) demonstrate that indacaterol can relieve the burden of normal cells of the body when it exerts an antitumor effect, and is suitable for the subsequent study.

TABLE 2 IC of different non-small cell lung cancer (NSCLC) cell lines ₅₀ Value of

2. Cell plate clone formation experiments

1. Experimental method

1) Taking cells in logarithmic growth phase, digesting with 0.25% trypsin, stopping the digestion with RPMI-1640 complete medium, gently blowing the cells, centrifuging for 5min at 800g, discarding the supernatant, and then re-suspending the cells in the complete medium for later use.

2) Diluting cell suspension, inoculating 300 cells in each hole, adding RPMI-1640 complete culture medium to complement the volume to 2mL, inoculating in a 6-hole cell culture plate, and gently shaking to disperse and uniformly distribute the cells. Setting a control group and an experimental group, wherein the experimental group is added with the corresponding IC of each cell strain ₅₀ Concentration of indacaterol.

3) After the cells are attached, indacaterol is added, after the medicine is acted for 48 hours, fresh RPMI-1640 complete culture medium is changed for continuous culture for 2 to 3 weeks, and liquid is changed every 72 hours.

4) It is often observed that the culture is terminated when macroscopic clones appear in the culture plate. The cell culture broth was discarded, carefully rinsed 2 times with PBS and fixed with 4% paraformaldehyde fixing solution for 15min at room temperature.

5) The fixative was removed, washed twice with PBS and stained with Giemsa for 30min.

6) The staining solution was slowly washed off with PBS, dried in air at room temperature, photographed under a mirror and counted.

7) The colony formation rate was calculated according to formula II:

clone formation rate = (number of clones/number of inoculated cells) ×100% formula II.

2. Results of cloning experiments

In general, in vitro single cells proliferate for more than 6 passages, and cell clusters formed by their progeny are called cell clones or colonies. Therefore, the cell clone formation assay can be used to detect the proliferation potential of single cells. To investigate the proliferative effect of indacaterol on humanized NSCLC cells, development ofCell clone formation experiments. As shown in FIG. 2, the 4 NSCLC cell lines were passed through IC of indacaterol, respectively ₅₀ After concentration treatment, all cell lines were significantly reduced compared to the normal control group. Wherein the proliferation capacity of the cells of the H460 and H1299 cell lines after treatment is obviously inhibited. The experimental results prove that the indacaterol has remarkable inhibition effect on the clone formation of NSCLC cells.

3. Cell cycle experiment (PI)

1. Experimental method

2) Cell concentration was adjusted to 1X 10 ⁵ The cells were seeded in 6-well cell culture plates and gently shaken to disperse and evenly distribute the cells. A control group and an experimental group were set, and indacaterol was added to the experimental group.

3) After cell attachment, indacaterol was added, after 48h of action, the medium was discarded, washed 3 times with PBS and digested with 0.25% trypsin, and after completion of the digestion, the cells were stopped and centrifuged.

4) 1mL of pre-chilled PBS was added, the cells were resuspended, and transferred to a 1.5mL centrifuge tube, the pelleted cells were centrifuged again, the supernatant carefully removed, and about 50. Mu.L of PBS remained to avoid pipetting away the cells.

5) 1mL 70% ethanol which is cooled is added, the mixture is gently blown and evenly mixed, and the mixture is fixed for 24 hours at 4 ℃.

6) 1000g centrifugal 5min, careful to absorb the supernatant, can remain about 50 u L70% ethanol, adding 1mL PBS, mixing, centrifugal precipitation of cells, careful to absorb the supernatant, can remain about 50 u L PBS.

7) 0.5mL of propidium iodide staining solution is added to each tube of cell sample, and cell precipitation is slowly and fully resuspended, and the cell precipitation is subjected to light-shielding warm bath at 37 ℃ for 30min.

8) Red fluorescence was detected with a flow cytometer at excitation wavelength 488nm while fluorescence scattering was detected and cell cycle was analyzed with ModFit LT software.

2. Experimental results

In order to further explore the proliferation influence process of indacaterol drug molecules on humanized NSCLC cell lines, cell cycle was detected, and the results are shown in FIG. 3 and FIG. 4, wherein 4 NSCLC cell lines respectively pass through indacaterol IC ₅₀ After concentration treatment, the proportion of the G0/G1 phase of the cell cycle of all cell lines is observed to be obviously increased, wherein the proportion is respectively reflected in that the G0/G1 phase of H460 cells is increased from 40.12+/-2.89% to 70.61 +/-2.46%; the G0/G1 phase of H1299 cells increased from 70.25 + -2.07% to 79.19+ -2.11%; the G0/G1 phase of PC9 cells was increased from 47.78.+ -. 5.09% to 55.29.+ -. 5.43%. Wherein the change in G0/G1 phase of H1299 cells is most pronounced, followed by the G0/G1 phase of H460 cells. From this, the results of the dosing group were significantly different from those of the control group, indicating that indacaterol could block the cell cycle of NSCLC cells to G0/G1 phase, thereby inhibiting proliferation of tumor cells.

Example 2

Verification of indacaterol for in vivo animal experiments

A C57 mouse subcutaneous tumor-bearing model is established by using a lung cancer cell strain LLC of the mouse, and whether the growth of tumors can be controlled after the indacaterol is injected into the abdominal cavity or not and the survival state and the survival period of the mouse are observed. To evaluate the in vivo safety and efficacy of indacaterol.

1. Experimental reagent Instrument description

1.1 experimental reagents: indantrole (99.99%, selleck Biotechnology Co., ltd.), polyethylene glycol (PEG-300) (. Gtoreq.97.0%, medChemExp Biotechnology Co., USA), tween-80 (Tween-80) (1.095 g/mL, BBI Life sciences Co., ltd.), DMEM high-sugar medium (Gibco, siemens Feishmanic technologies Co., ltd.), diabody (penicillin and streptomycin) (Gibco, siemens Feishmanic technologies Co., ltd.), FBS New Zealand Origin (Invigetech, ink Co., USA), 0.25% trypsin (HyClone, siemens), dimethyl sulfoxide (DMSO) (Sigma Aldrich Co., ltd.), tribromoethanol (97%, sigma Aldrich trade Co., ltd.), and iodophor (Shando Li Erkang medical technologies Co., ltd.).

1.2 experiment consumable: 25cm ² Square gas cap bevel cell culture flask (Labselect, beijing Gekko technology Co., ltd.), 15mL centrifuge tube sterilization (Labselect, beijing Gekko technology Co., ltd.), 3mL extended Papanicolaou pipette (Labselect, beijing Gekko technology Co., ltd.), PBS (1X) dry powder (Biosharp, beijing Gekko technology Co., ltd.).

1.3 laboratory apparatus: electronic balance (Shanghai precision scientific instruments Co., ltd.), pure water instrument (Millipore, USA), forceps (Beijing Xin moisturizing technologies Co., ltd.), surgical scissors (Beijing Xin moisturizing technologies Co., ltd.), insulin syringe (BD Co., USA), vernier caliper (Shanghai constant quantity with Co., ltd.), cell counter (Shanghai Rui Yu Biotechnology Co., ltd.), pet shaver (Deshi Electrical Co., ltd.), small animal radiation instrument X-red320 (PXI of national instruments Co., ltd.).

1.4 preparation of reagents

1.4.1 Indamaterol injection preparation: the dose administered was 6.5mg/kg, the average body weight of the animals was: 19.32g, 100. Mu.L per mouse, requires co-solvents PEG-300 and Tween-80, and ddH because of low drug solubility ₂ O。

The working solution has the concentration of: 1.2558mg/mL;

mother liquor: 0.8163mg of the drug was dissolved in 32.5. Mu.L of DMSO solution (mother liquor concentration 25.116 mg/kg);

the in-vivo formula is as follows: the 32.5 mu LDMSO mother liquor was added to 260 mu LPEG-300 and mixed until clear. Then 32.5 mu LTwen-80 was added and mixed until clear. 325 mu LddH was added ₂ O, mixing until clear (ratio: 5% DMSO+40% PEG-300+5% Tween-80+50% ddH) ₂ O). The medicine injection is prepared according to the amount of 6.5 medicines each time.

The control group was formulated in the same formulation by adding DMSO in the same volume as the drug mother solution.

1.4.2 formulation of tribromoethanol anesthetic (2.5%): 1.25g was taken and 50mL ddH was added ₂ O, thoroughly mixed under ultrasound, and filtered with a 0.22 μm filter to prepare 2.5% anesthetic, and the injection dose was calculated as 0.1mL/10g per mouse.

1.5 laboratory animals

Female C57 mice, 6-8 weeks old, weighing 17-20g, purchased from Fukang Biotechnology Co., ltd., beijing, animal license number: SCXK (jing) 2019-0008. 6 experimental animals are kept in a clean animal house at 20-25 ℃ and humidity of 40-60% in one cage, so that sufficient drinking water and feed are ensured, and padding is replaced at regular time.

2. The experimental method comprises the following steps:

2.1 culture of mouse LLC lung cancer cell lines

Cell resuscitation: before each experiment, the experimental consumables are put into an ultra-clean bench for sterilization by ultraviolet irradiation for 30min. Preheating a water bath kettle at 37 ℃.

1) The frozen tube containing the cells was removed from the liquid nitrogen tank, immediately placed in warm water at 37℃and gently shaken with forceps to rapidly thaw the tube.

2) Putting the mixture into an ultra-clean bench for operation, sucking 1mL of DMEM high-sugar complete culture medium for uniform mixing as soon as possible after melting, transferring the mixture into a new 15mL centrifuge tube, adding 2mL of complete culture medium, uniformly mixing, and centrifuging for 5min at 800 g.

3) The supernatant was aspirated off with a 3mL extended Pasteur pipette, and 1mL complete medium was added to blow the cells apart and transferred to a new 25cm ² In the flask, 4mL was supplemented and shaken well.

4) Observing under an inverted microscope, determining cell morphology and proper dispersion degree, and placing into 37 ℃ and 5% CO ₂ Is cultured in an incubator of (a).

5) After 24h, the new complete medium was replaced.

Cell passage:

1) And (3) performing aseptic operation in the super clean bench, opening a bottle cap of the culture bottle, and pouring out the culture solution.

2) 1mL of PBS was added to the grown cells with a 3mL Pasteur pipette, the flask was gently shaken to allow the PBS to flow across all cell surfaces, and then poured off, and rinsing was repeated 2-3 times to remove the stock culture as much as possible.

3) 1mL pancreatin is added, after incubation for 2-3 min in a 37 ℃ incubator, the flask is placed under an inverted microscope for observation, and digestion is immediately stopped after cytoplasmatic retraction and cytoplasmatic enlargement of cells are found.

4) Digestion was stopped by adding 3mL of complete medium pre-heated to 37 ℃ and repeated pipetting with a 1mL pipette to disperse the cells. The blowing process is performed sequentially starting from one side of the flask bottom to the end of the other to ensure that all bottoms are blown.

5) All liquid was aspirated into a fresh 15mL centrifuge tube, placed in a centrifuge, and centrifuged for 5min at 800 g.

6) After centrifugation, the supernatant was discarded, 3mL of complete medium was aspirated, and the resuspended cells were gently blown.

7) 1mL of the cell suspension was aspirated and inoculated into a new 25cm ² The flask was filled with complete medium to 4mL. Gently shaking, observing under an inverted microscope, confirming the state, and concentrating at 37deg.C under 5% CO ₂ Culturing in an incubator.

Notice that:

1) All liquids should be preheated to 37℃before addition to the cell flask. All liquids should be adjusted to a pH of about 7.2 and should not exceed 7.4.

2) When the cells are digested and passaged, bubbles are not generated during blowing, the blowing force is not excessive, otherwise, the cells are damaged.

3) The residual amount of digestive juice is reduced as much as possible, and if too much digestive juice is used, the cells are damaged, and a plurality of complete culture mediums can be added for neutralization.

4) The time for placing the culture flask outside the super clean bench should be as short as possible.

2.2 construction of C57 mouse LLC subcutaneous tumor-bearing model

The purchased C57 female mice were first kept for 3-5 days to acclimatize. The pets were shaved Mao Qibei on the day before tumor inoculation, and the hair preparation position was located at the root of the right thigh surface. On the day of tumor bearing inoculation, marking the ear marks of the mice, weighing and recording in advance, and disinfecting tumor bearing parts by using iodophors before tumor bearing.

1) And (3) adding a proper amount of PBS (phosphate buffer solution) to resuspend the LLC cells in the logarithmic growth phase after digestion and centrifugation, blowing uniformly, and sucking the LLC cells onto a cell culture plate for counting in a cell counter.

2) The cell suspension was adjusted to a concentration of 1X 10 with PBS ⁷ The volume of the solution is one per mL,and added to a 200. Mu.L EP tube in a volume of 100. Mu.L/tube to give a cell number of 1X 10 per tube ⁶ And (3) on ice for standby.

3) The skin around the right thigh of the mice was sterilized with iodophor, then cells in the EP tube were blown evenly and aspirated using an insulin syringe, and then tumor-bearing was performed subcutaneously on the mice that had been sterilized (see fig. 5).

4) Regular observations confirm whether tumor-bearing was successful. Tumor volumes were observed every two days after tumor growth, and weighed and recorded. And measuring the length and width of the transplanted tumor by using a vernier caliper, and finally calculating the tumor size according to a volume formula. Tumor volumes were calculated according to formula III.

Tumor volume= (length x width ² ) Formula III.

5) When the tumor volume of the mice reaches 30 cm to 60cm ³ In this case, 100. Mu.L of the drug solution was withdrawn with an insulin needle every day for intraperitoneal administration, and the administration was continued for 10 days, and the same solvent was administered in the same volume to the control group for intraperitoneal administration. Tumor volumes were measured at two days apart simultaneously, weighed and recorded.

6) The observed endpoint was reached when the tumor met any of three conditions:

tumor collapse; tumor volume up to 1000mm ³ The method comprises the steps of carrying out a first treatment on the surface of the The tumor diameter reached 20mm.

7) After reaching the observation end point, the mice are sacrificed by adopting a spinal dislocation method, and the mice are delivered to an animal experiment center for unified treatment of the corpses.

2.3 construction of C57 mouse LLC subcutaneous tumor-bearing radiotherapy model

The purchased C57 female mice are first kept for 3-5 days to adapt to the environment. The pet was shaved Mao Qibei on the day before tumor bearing, and the hair preparation position was located at the root of the right thigh surface. On the day of tumor bearing, marking the ear marks of the mice, weighing and recording in advance, and disinfecting tumor bearing parts by using iodophors before tumor bearing.

Radiotherapy information: x-ray, voltage 320Kv, current 12.5mA, height 40cm, dose rate: 1.7Gy/min, the experimental irradiation dose is 20Gy, and one irradiation is performed.

A total of 4 groups: control (Control), radiation therapy alone (IR), administration alone (indicaterol), and combination therapy (ir+indicaterol).

2.4 experimental protocol:

2) The cell suspension was adjusted to a concentration of 1X 10 with PBS ⁷ mu.L/mL and added to 200. Mu.L EP tube in a volume of 100. Mu.L/tube, giving a cell count of 1X 10 per tube ⁶ And (3) on ice for standby.

3) The skin around the right thigh of the mice was sterilized with iodophor, then cells in the EP tube were blown evenly and aspirated using an insulin syringe, and then tumor-bearing was performed subcutaneously on the mice that had been sterilized.

4) Regular observations confirm whether tumor-bearing was successful. Tumor volumes were observed every two days after tumor growth, and weighed and recorded. And measuring the length and width of the transplanted tumor by using a vernier caliper, and finally calculating the tumor size according to a formula III.

5) When the tumor volume of the mice reaches 200cm ³ When left and right, the treatment is performed. The indacaterol single medicine group is that 100 mu L of medicine solution is extracted by an insulin needle every day for intraperitoneal administration, and the administration is carried out for 10 consecutive days; the control group was given the same volume of the same solvent for intraperitoneal injection; single 20Gy X-ray irradiation is carried out on the pure radiotherapy treatment group; the radiotherapy and indacaterol combined treatment group is firstly subjected to single 20GyX radiation irradiation, and is subjected to intraperitoneal injection administration after 12 hours at intervals, and the administration is carried out for 10 consecutive days. Tumor volumes were measured, weighed and recorded every two days.

tumor collapse; tumor volume reaches 2000mm ³ The method comprises the steps of carrying out a first treatment on the surface of the The tumor diameter reached 20mm.

2.5 radiotherapy protocol:

1) Each mouse is injected with 200 mu L of tribromoethanol anesthetic, and after the mice are anesthetized, the mice are fixed on a paperboard by a disposable medical adhesive tape to expose the tumors on the thighs, so that the tumors of the mice are positioned on the same horizontal line;

2) Placing in a mouse irradiator, and exposing only the tumor part to an irradiation field;

3) Irradiating the mice with 20Gy X-rays;

3) After the irradiation is finished, the adhesive tape is removed, the mice are put back into the mouse cage, and the mice are observed until the mice recover to be normal.

2.6 statistical analysis

All data in this experiment were measured as mean ± standard deviation and analyzed and plotted using GraphpadPrism 8.0 software. Statistical analysis data, and matching t test is adopted for variable comparison between two groups; the comparison between the groups is two by two, then single factor analysis of variance is adopted, p < 0.05 indicates that the difference is statistically significant.

3. In vivo mouse experiment results

(1) Indantrole single drug efficacy evaluation experiment

During the experiment, partial oversized and undersized tumor mice and tumor-crushed mice are eliminated, and the rest mice are randomly divided into an experiment group and a control group, wherein each group comprises 6 mice. When the tumor volume of the mice reaches 30-60mm ³ At the beginning of the treatment, tumor volumes were measured and mice weights were weighed every 2 days, the protocol is shown in fig. 5 below, and day 0 represents the time at which treatment was initiated.

The results are shown in fig. 6 and 7, and when the observations were completed, the control group and the experimental group exhibited significant differences, indicating that indacaterol exerted the tumor growth inhibiting effect in mice. Meanwhile, according to the results shown in fig. 8, the tumor size of the experimental group is obviously smaller than that of the control group, so that the tumor of the experimental group is effectively controlled, and the in-vivo effectiveness of indacaterol is demonstrated.

To investigate the in vivo safety of indacaterol, mice were monitored for body weight, and the results are shown in fig. 9 below, in which the body weight of both experimental and control groups was steadily increased, and there was no statistical difference between the two groups. And the vital signs of the two groups of mice are stable in the observation time, and the mice in the administration group have no acute poisoning symptoms. According to the experimental results, the indacaterol can be used in mice with certain safety.

In vivo efficacy of indacaterol was then evaluated, and the experimental group clearly achieved a longer survival period than the control group. Demonstrating that not only the growth of the tumor in mice was controlled but also the survival of mice was significantly prolonged after administration (see figure 10).

(2) Indanterol enhanced radiotherapy efficacy experiment

In the experimental process, mice with oversized and undersized part of tumor and mice with tumor collapse are eliminated, and the rest mice are randomly divided into four groups, namely a Control group (Control), a pure radiotherapy group (IR), a pure administration group (Indacaterol) and a combined treatment group (IR+Indacaterol), wherein 6 mice are selected from each group. When the tumor volume of the mice reaches 200mm ³ On the left and right, treatment was started, tumor volumes were measured and weights of mice were weighed every 2 days, and the experimental protocol is shown in fig. 11 below, with day 0 representing the time at which treatment was started.

The results are shown in the following figures 12-14, when the observation is finished, the control group and the indacaterol single administration group show obvious differences, and then the single radiotherapy group and the combined treatment group also show obvious differences, which indicates that the indacaterol not only plays a role in inhibiting tumor growth, but also enhances the curative effect of the radiotherapy in the mice.

To investigate the in vivo safety of indacaterol, weight monitoring was performed on each group of mice, and as a result, as shown in fig. 15, the weight of each group of mice was a steady increase trend, and there was no statistical difference between groups. And the vital signs of each group of mice are stable in the observation time and have no acute poisoning symptoms. According to the experimental results, the indacaterol can be used in mice with certain safety.

The in vivo efficacy of indacaterol was then evaluated, and the experimental results of fig. 16 show that a longer survival period was significantly obtained in the indacaterol experimental group compared to the control group. And compared with a pure radiotherapy group, the combined treatment group also obtains longer survival time, which shows that after the combined effect of indacaterol and radiotherapy, the treatment effect is obviously enhanced.

The experiment proves that the medicine A has certain safety when being used in vivo, can exert the effect of enhancing the curative effect by combining with the radiotherapy, can more effectively control the tumor growth when being used by combining with the radiotherapy, and prolongs the survival period of mice. Therefore, the method can provide a certain guiding significance for clinical treatment.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. Application of indacaterol in preparing medicines for preventing and/or treating lung cancer is provided.

2. Application of indacaterol in preparing medicine for enhancing radiotherapy effect in treating lung cancer.

3. The use according to claim 2, wherein said radiotherapy comprises X-ray irradiation.

4. The use according to any one of claims 1 to 3, wherein the lung cancer comprises non-small cell lung cancer.

5. The use of claim 4, wherein the cell line of non-small cell lung cancer comprises at least one of: h460 cell line, H1299 cell line and PC9 cell line.

6. The use according to claim 1, wherein the medicament is a medicament for inhibiting proliferation and growth of cells of non-small cell lung cancer.

7. The use according to claim 6, wherein the drug is a cell cycle arrest in G0/G1 phase of a tissue lung cancer cell.

8. The use according to claim 1, wherein the indacaterol has the structural formula shown in formula I:

9. the use according to claim 1, wherein the concentration of indacaterol in the medicament is 1.2558mg/mL or more.

10. The use according to claim 1, wherein the pharmaceutical dosage form comprises an injection, a powder for injection.