Application of salmeterol in preparation of medicine for treating non-small cell lung cancer

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of salmeterol in preparation of a medicine for treating non-small cell lung cancer. It is found for the first time that the compound salmeterol is an effective inhibitor of asparagine synthetase (ASNS), and the compound salmeterol inhibits the biological activity and / or protein expression level of the ASNS by directly targeting the ASNS, so that a synthetic pathway of endogenous asparagine of cells is blocked. On the basis, salmeterol directly targets ASNS, inhibits the activity and / or expression level of ASNS, blocks synthesis of endogenous asparagine, and further inhibits tumor cell proliferation and induces tumor cell apoptosis. The new application of salmeterol as the ASNS inhibitor is innovatively found, a new mechanism, a new medicine and a new scheme are provided for treatment of the non-small cell lung cancer, and the salmeterol has important clinical application prospects and scientific value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of salmeterol in preparation of a drug for treating non-small cell lung cancer. BACKGROUND

[0002] Lung cancer is one of the malignant tumors with the highest mortality rate worldwide, among which non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancer cases, and the main subtypes include lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). Although certain progress has been made in existing treatment methods (such as chemotherapy, targeted therapy and immunotherapy), the overall prognosis of NSCLC patients is still poor, and there are problems such as low response rate and easy drug resistance. Therefore, developing new and effective NSCLC treatment strategies is still a technical problem to be solved in the field.

[0003] Salmeterol is a known long-acting beta2 adrenergic receptor agonist, and its chemical structure contains a long aliphatic side chain, which gives it high fat solubility and enables it to bind firmly to the receptor, thereby achieving bronchodilation effects for 12-24 hours. Based on this property, salmeterol is widely used in the clinic for the treatment of asthma and chronic obstructive pulmonary disease (COPD), and its safety and pharmacokinetic characteristics have been fully verified. However, prior to the present disclosure, the application of salmeterol in the field of tumor treatment, particularly its specific target and molecular mechanism as an anticancer drug, was not clear.

[0004] On the other hand, asparagine synthetase (ASNS) is a key enzyme that catalyzes the conversion of aspartic acid to asparagine, responsible for maintaining the homeostasis of intracellular asparagine, and is essential for cell growth and proliferation. Numerous studies have confirmed that ASNS is abnormally highly expressed in various malignant tumors including NSCLC, and is closely related to tumor proliferation, invasion and resistance to certain chemotherapeutic drugs. Therefore, ASNS has been recognized as a highly potential target for anticancer drugs. However, as of the filing date of the present application, no small molecule inhibitor specifically targeting ASNS has been approved for marketing worldwide. SUMMARY

[0005] In order to solve the problems in the background art, the present application provides a new medical use of salmeterol, which provides an innovative application for the treatment of non-small cell lung cancer (NSCLC).

[0006] The first aspect of the present application aims to provide the use of salmeterol in the preparation of a drug for treating tumors.

[0007] The second aspect of the present application aims to provide the use of salmeterol in the preparation of an asparagine synthetase inhibitor.

[0008] The third aspect of the present application aims to provide a method for inhibiting asparagine synthetase in vitro.

[0009] The fourth aspect of the present application aims to provide the use of salmeterol in the preparation of a chemotherapeutic drug sensitizer.

[0010] The fifth aspect of the present application aims to provide a pharmaceutical composition.

[0011] In order to achieve the above-mentioned objects of the present application, the technical solutions adopted by the present application are as follows: The first aspect of the present application provides the use of salmeterol in the preparation of a drug for treating tumors.

[0012] In some embodiments of the present application, the salmeterol has a chemical formula of C 25 H 37 NO4, a CAS No. of 89365-50-4, and a structural formula as shown below: .

[0013] In some embodiments of the present application, the salmeterol includes a pharmaceutically acceptable salt.

[0014] In some embodiments of the present application, the pharmaceutically acceptable salt includes an acid addition salt and a base addition salt.

[0015] The "pharmaceutically acceptable acid addition salt" refers to a salt formed with an inorganic acid and an organic acid, which retains the biological effectiveness and properties of the free base and is not biologically or otherwise undesirable, and the inorganic acid includes, but is not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., and the organic acid includes, but is not limited to, acetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, camphoric acid, camphor 10 sulfonic acid, decanoic acid, hexanoic acid, octanoic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane 1,2 disulfonic acid, ethanesulfonic acid, 2 hydroxyethanesulfonic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptanoic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2 oxoglutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methane sulfonic acid, etc.

[0016] "Pharmaceutically acceptable base addition salt" refers to those salts which retain the biological effectiveness and properties of the free acids and which are not biologically or otherwise undesirable. These salts are prepared from addition of inorganic or organic acids to the free base. Salts derived from inorganic acids include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic acids include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion-exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, ethylenediamine, glucosamine, methylglucosamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0017] In some embodiments of the present application, the tumor comprises lung cancer; preferably, the lung cancer comprises non-small cell lung cancer.

[0018] In some embodiments of the present application, the tumor comprises lung cancer; preferably, the lung cancer comprises non-small cell lung cancer.

[0019] In some embodiments of the present application, the drug comprises a pharmaceutically acceptable excipient; In some embodiments of the present application, the pharmaceutically acceptable excipient comprises at least one of a solvent, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure adjusting agent, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending agent, a coating material, an aromatic agent, an anti-adhesion agent, an integrating agent, a penetration enhancer, a pH adjusting agent, a buffer, a plasticizer, a surfactant, an antifoaming agent, a thickening agent, an inclusion agent, a humectant, an absorbent, a diluent, a flocculating agent and a deflocculating agent, a filter aid, a release retarder, a carrier.

[0020] The above pharmaceutically acceptable excipients are generally recognized as suitable for this use and as non-active ingredients of a medicament. A compendium of pharmaceutically acceptable excipients can be found in Handbook of Pharmaceutical Excipients, 2ndEdition, Edited by A. Wade and P. J. Weller; published by American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994; Chinese Pharmacopoeia-Pharmaceutical Excipients, and the like.

[0021] In some embodiments of the present application, the dosage form of the drug includes a gastrointestinal administration dosage form, or a non-gastrointestinal administration dosage form.

[0022] In some embodiments of the present application, the gastrointestinal administration dosage form includes at least one of a powder, a tablet, a granule, a capsule, a sustained-release agent, a solution, a dry suspension, an effervescent tablet, an emulsion, a suspension, a syrup, a drop, a chewable tablet. In some embodiments of the present application, the non-gastrointestinal administration dosage form includes at least one of an injection administration dosage form, a respiratory administration dosage form, a skin administration dosage form, a mucous membrane administration dosage form, a cavity administration dosage form.

[0023] In some embodiments of the present application, the dosage form of the drug is a dosage form suitable for children or a dosage form suitable for adults.

[0024] In some embodiments of the present application, the administration subject of the drug is a mammal. In some embodiments of the present application, the mammal includes a human.

[0025] In a second aspect of the present application, there is provided a use of salmeterol in the preparation of an asparagine synthetase inhibitor.

[0026] In some embodiments of the present application, salmeterol can directly target and inhibit asparagine synthetase, thereby blocking the key metabolic pathway of tumor cells.

[0027] In a third aspect of the present application, there is provided a method for inhibiting asparagine synthetase in vitro, comprising the following steps: treating a sample with salmeterol.

[0028] In some embodiments of the present application, the sample is a cell, which includes but is not limited to a tumor cell.

[0029] In some embodiments of the present application, a person skilled in the art can adjust the treatment dose and treatment time of salmeterol according to the specific type of cell, and the specific dose and time do not limit the present application.

[0030] In a fourth aspect of the present application, there is provided a use of salmeterol in the preparation of a chemotherapeutic drug sensitizer.

[0031] In some embodiments of the present application, salmeterol can enhance the efficacy of other chemotherapeutic drugs and / or act as a drug sensitizer for reversing drug resistance of tumor cells.

[0032] In some embodiments of the present application, the chemotherapeutic drug includes a platinum drug, including but not limited to cisplatin, carboplatin, cycloplatin, nedaplatin, oxaliplatin, and lobaplatin.

[0033] In a fifth aspect of the present application, a pharmaceutical composition is provided, comprising Salmeterol and a chemotherapeutic drug.

[0034] In some embodiments of the present application, the Salmeterol comprises a pharmaceutically acceptable salt.

[0035] In some embodiments of the present application, the chemotherapeutic drug comprises a platinum drug, including but not limited to: cisplatin, carboplatin, cycloplatin, nedaplatin, oxaliplatin, lobaplatin.

[0036] The beneficial effects of the present application are: The present application first discovers the new use of Salmeterol as an effective inhibitor of asparagine synthetase (ASNS), which has the following advantages: 1. Discovering new uses of marketed drugs, avoiding high risks and long periods of new drug development: The present application uses Salmeterol, which has been widely used in clinical practice and has been fully verified in terms of safety and pharmacokinetic characteristics, as a new type of non-small cell lung cancer (NSCLC) treatment drug. This greatly reduces the risk and time required for new drug development, providing a fast and accessible innovative treatment option for NSCLC patients.

[0037] 2. Providing a completely new anticancer mechanism, targeting ASNS: The present application first discovers that Salmeterol can directly target and inhibit asparagine synthetase (ASNS), thereby blocking the key metabolic pathway of tumor cells. ASNS is a target point that is abnormally highly expressed in NSCLC and is closely related to tumor proliferation, invasion and drug resistance.

[0038] 3. Solving the dilemma of "having a target but no drug": Although ASNS has been identified as a highly potential anticancer target, there has been no small molecule inhibitor specifically targeting ASNS approved for marketing worldwide. The present application successfully solves the technical problem of "having a target but no drug" by using the existing drug Salmeterol.

[0039] 4. Expanding the treatment range: Unlike targeted therapy that is only effective for specific genetic mutations, the mechanism of Salmeterol targeting ASNS may be applicable to a wider group of NSCLC patients, including those who are insensitive or resistant to existing therapies.

[0040] 5. Expanding the application field of Salmeterol: The present application overturns the understanding of the pharmacological effects of Salmeterol, expanding it from a drug only used for respiratory diseases to a drug with significant antitumor activity, and its antitumor activity does not depend on its known target ADRB2, tapping its great potential in the field of tumor treatment. BRIEF DESCRIPTION OF DRAWINGS

[0041] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 Tumor volume map (left) and tumor growth curve (right) results for salmeterol treatment of LLC lung cancer cells.

[0042] Figure 2 The simulated docking results of salmeterol and ASNS.

[0043] Figure 3 The results of the Biacore experiment on salmeterol and ASNS.

[0044] Figure 4 The results show that salmeterol regulates ASNS enzyme activity.

[0045] Figure 5 The therapeutic effect of salmeterol on tumor cells with knocked-out ASNS.

[0046] Figure 6 The therapeutic effect of salmeterol combined with chemotherapy drugs. Detailed Implementation

[0047] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0048] The relevant sequences involved in this invention are shown in Table 1.

[0049]

[0050] Example 1: Results of Salmeterol treatment for LLC lung cancer cells 1. Experimental Methods Ten female C57BL / 6J mice were purchased from Vital River Pharmaceuticals, Beijing. All mice were housed under specific pathogen-free conditions and used according to protocols approved by the Animal Welfare Institute Care and Use Committee of Tsinghua University. Mice were initially randomly assigned to groups based on age and weight, and were 6–8 weeks old at the time of injection.

[0051] Salmeterol was purchased from Aladdin (S346847-50). The powder was dissolved in DMSO, and the stock solution was dispensed in 200 μL portions. The dissolved stock solution was diluted to 50 mg / ml before subcutaneous injection, and then administered to mice via intratumoral injection in 0.1 mL volumes. For the model to validate the anti-tumor effect of salmeterol established in C57BL / 6J, LLC tumor cells were resuspended in sterile PBS at a density of 1 x 10 7 cells per milliliter, and then injected subcutaneously into the flank of the mouse on the left leg in a volume of 0.1 milliliters. Next, 10 mice were randomly divided into two groups, LLC-vector + DMSO group and LLC-vector + salmeterol group. Starting from the first day of subcutaneous injection of cells, the tumor formation was observed every 3 days. When the tumor volume reached 100 mm 3 on the 7th day, salmeterol was injected around the tumor from the 7th day, and salmeterol was diluted to 50 mg / ml, then the mice were treated with intratumoral injection in a volume of 0.1 milliliters, twice a week for two weeks. The experiment was terminated immediately when the tumor volume approached the ethical endpoint, and the mice were sacrificed 25 days after implantation, the tumor was removed, weighed and photographed. The tumor volume of the mice was measured every three days during the entire experimental period until the end of the experiment.

[0052] 2. Experimental results The results are shown in Figure 1 Salmeterol significantly inhibited the LLC tumor volume.

[0053] Example 2 Salmeterol can be used as an ASNS inhibitor 1. Salmeterol and ASNS simulation binding experiment First stage: preparation (molecular pretreatment) 1. Preparation of receptor protein (ASNS) Step 1.1: Obtain and screen protein structure Access the PDB database. Enter "Asparagine Synthetase" and "Homosapiens" in the search box.

[0054] Select a structure with high resolution, no mutation or containing co-crystallized ligand. Select PDB ID: 6GQ3 to download the.pdb format file of the structure.

[0055] Step 1.2: Clean and optimize protein structure Open the downloaded PDB file using PyMOL or ADT.

[0056] Remove water molecules: There are usually a large number of water molecules in protein crystal structures, which may interfere with docking.

[0057] Remove existing ligands and small molecules: Remove molecules such as AMP and aspartic acid in the structure to leave the binding pocket.

[0058] Completing residues and side chains: Check if there are missing atoms or residues in the structure and complete them using SWISS-MODEL.

[0059] Save the cleaned protein chain as a new PDB file, named ASNS_protein.pdb.

[0060] Step 1.3: Prepare the receptor file in PDBQT format Launch AutoDock Tools (ADT).

[0061] Click File ->Read ->Molecule to load ASNS_protein.pdb.

[0062] Click Edit ->Hydrogens ->Add to add polar hydrogens to the protein.

[0063] Click Edit ->Charges ->Compute Gasteiger to calculate Gasteiger charges for the protein.

[0064] Click File ->Save ->Write PDBQT to save the processed protein as ASNS_protein.pdbqt file. This file contains atomic coordinates, charges, and atom type information.

[0065] 2. Preparation of the ligand molecule (salmeterol) Step 2.1: Obtain the ligand structure Visit the PubChem database. Search for "Salmeterol" (CID: 5152).

[0066] Download the SDF or MOL2 format file of its 3D conformation.

[0067] Step 2.2: Prepare the ligand file in PDBQT format Launch ADT.

[0068] Click Ligand ->Input ->Open to load the downloaded Salmeterol SDF file.

[0069] ADT will automatically detect the chemical properties of the molecule. Click Ligand ->Torsion Tree ->Detect Root to automatically detect the rotatable bonds of the molecule.

[0070] Confirm that the number of rotatable bonds is reasonable (the long-chain part of the salmeterol molecule has high flexibility).

[0071] Save the ligand as salmeterol.pdbqt by clicking Ligand ->Output ->Save as PDBQT.

[0072] Stage 2: Set up docking parameters and run 1. Define the binding pocket (Grid Box) Step 3.1: Determine the docking center In ADT, load ASNS_protein.pdbqt.

[0073] Key step: Determine the region for docking. Choose the known active site of ASNS. Determine the center based on literature or the position of the original ligand (e.g. AMP) in the 6GDI structure.

[0074] In ADT, select Grid ->Grid Box.

[0075] Step 3.2: Set Grid Box parameters A cubic box will appear, representing the space for docking search.

[0076] Move the center of the cubic box to the predicted active pocket center by adjusting the x, y, z coordinates of the Center Grid Box.

[0077] Adjust the Number of points in x, y, z-dimension (set to 60, 60, 60) and Spacing (set to 0.375 Å) to ensure that the cubic box is large enough to completely cover the active pocket, while providing sufficient space for ligand rotation and translation.

[0078] Record the values of center_x, center_y, center_z and size_x, size_y, size_z. These will be used in the subsequent configuration file.

[0079] 2、Biacore experiment of ASNS and salmeterol (1) Sensor chip preparation and activation: Place the sensor chip in the Biacore instrument. Use the default program to flow through the chip surface. Use an EDC / NHS mixed solution to flow through the flow cell to activate the carboxyl groups on the chip surface, converting them to highly active O-acyl isourea esters.

[0080] (2) ASNS protein coupling (ligand immobilization): Dilute the recombinant ASNS protein to the appropriate concentration with the appropriate buffer (usually a coupling buffer, which can be different from the running buffer). Optimizing the coupling concentration is critical to achieve the desired response unit (RU) value while avoiding overloading that leads to poor binding. Slowly flow the diluted ASNS protein through the activated flow cell. The amino groups on the ASNS protein (such as lysine residues) will react with the activated carboxyl groups to form stable amide bonds, immobilizing the ASNS on the chip surface. Monitor the SPR signal (RU value) in real time.

[0081] (3) Deactivation of unreacted sites Use an ethanolamine solution to flow through the flow cell and react with the residual EDC / NHS activated groups on the chip surface to block these sites and prevent non-specific binding of subsequent analytes.

[0082] (4) Baseline establishment: Use HBS-EP+ as the running buffer to flow through the chip to establish a stable baseline signal.

[0083] (5) Salmeterol binding analysis: Gradient dilution of salmeterol: Dilute salmeterol with the running buffer in a series of gradients to prepare analyte solutions of different concentrations (from low to high: 0.015625 µM, 0.03125 µM, 0.0625 µM, 0.125 µM, 0.25 µM, 0.5 µM, 1 µM). Then flow the salmeterol solutions of different concentrations through the ASNS-immobilized flow cell in turn. Within 180s, the analyte (salmeterol) binds to the immobilized ligand (ASNS). The SPR signal increases with increasing concentration. Finally, stop injecting the analyte and only flow the running buffer through the flow cell. The immobilized ligand separates from the dissociated analyte, and the SPR signal gradually decreases.

[0084] (6) Data collection: Record the changes in SPR signal in real time during the binding and dissociation phases.

[0085] 3. Salmeterol regulation of ASNS enzyme activity experiment Preparation: Obtain high-purity recombinant human ASNS protein. Dissolve salmeterol in DMSO as a stock solution, and dilute it with reaction buffer to prepare a series of working concentrations.

[0086] Reaction system construction: In a non-transparent 96-well plate, set up different groups: blank control (no enzyme), positive control (with enzyme, only add DMSO solvent), and drug treatment group (with enzyme, add different concentrations of salmeterol).

[0087] Drug pre-incubation: Add 100 uL kinase buffer, 5 uM Salmeterol (or DMSO) to the well, and finally add 20 ug ASNS protein. Pre-incubate at room temperature for 15-30 minutes to allow drug to fully bind to enzyme. Start reaction: Add substrate mixture containing 100 uM ATP, 2 mM aspartate and glutamine to start the enzymatic reaction. Place the reaction plate in a 37°C incubator for 60 minutes.

[0088] Signal detection: According to the instructions of the asparagine content kit, add detection reagent to terminate the reaction and perform signal conversion. Finally use a multifunctional microplate reader to read the luminescence value of each well.

[0089] 4. Experimental results The results of the simulated binding experiment of Salmeterol and ASNS are shown in Figure 2 Salmeterol can stably bind to the ATP pocket of ASNS.

[0090] The results of the Biacore experiment of ASNS and Salmeterol are shown in Figure 3 Salmeterol can directly bind to ASNS.

[0091] The results of the experiment of Salmeterol regulating ASNS enzyme activity are shown in Figure 4 Salmeterol can directly inhibit the enzyme activity of ASNS.

[0092] Example 3 Treatment effect of Salmeterol on tumors depends on ASNS 1. Construction of ASNS, ADRB1, ADRB2, ADRB3 knockout cell lines by Crispr Cas9 technology (1) Cells in the logarithmic growth phase were evenly plated into 100 mm cell culture dishes, and the number of cells was about 2.0~3.0×10 6 .

[0093] (2) Prepare transient transfection system: A solution: Lipo3000 10 uL, Opti-MEM 625 uL; B solution: ASNS knockout plasmid 10 ug, p3000 10 uL, Opti-MEM 625 uL.

[0094] (3) Mix A solution and B solution evenly, and stand at room temperature for 30 min. Slowly add the transfection mixture to the cell culture medium, and observe the cell state after transfection for 4-6 h. Replace the fresh culture medium according to the situation (4) After 24 h of transfection, the transfection efficiency of cells was observed by green fluorescence under a fluorescence microscope. After the cells were digested into a single cell suspension and resuspended with PBS, 300 μL of complete medium was added to each well of a 96-well plate, and positive monoclonal cells were sorted by flow cytometry.

[0095] (5) The cell plate was placed back into the incubator for culture. After 1-2 weeks, the single clone cell strain that grew up was selected, digested, and subcultured into a six-well plate for culture. Cell DNA and protein were collected for identification.

[0096] 2. Drug treatment Day 1: Cell plating Cell preparation: LL, A549, and H1975 cells in the logarithmic growth phase were trypsinized, centrifuged, resuspended with complete medium containing 10% FBS, and counted with a hemocytometer.

[0097] Cell plating: The cells were diluted to an appropriate concentration (usually 1 x 10 3 cells / mL), and 2 mL of the cell suspension was added to each well of a six-well plate, so that each well contained about 800-1000 cells.

[0098] Adherent culture: The six-well plate was gently shaken to distribute the cells evenly, and then placed in a cell incubator at 37°C and 5% CO2 for 24 hours to allow the cells to adhere completely.

[0099] Day 2: Drug treatment Grouping: Vehicle control group: DMSO was added to the medium at the same volume as the drug group.

[0100] Salmeterol treatment group: Salmeterol was added to the medium to a final concentration of 5 μM.

[0101] Each group had three replicate wells to ensure the reliability of the results.

[0102] Preparation of drug solution: The required volume was calculated according to the concentration of the salmeterol stock solution (10 mM). For example, 1 μL of the 10 mM stock solution was added to 2 mL of medium to prepare a 5 μM working solution.

[0103] Similarly, the control group was prepared with medium containing an equal amount of DMSO (e.g., 0.05%).

[0104] Medium change: The old medium in the six-well plate was carefully aspirated and replaced with 2 mL of the prepared drug-containing or DMSO-containing medium per well.

[0105] Continue incubation: Put the plate back into the incubator and continue incubation.

[0106] Day 3 to the end of incubation (about 10-14 days) Periodic medium change: Change the fresh medium containing drug (or DMSO) every 2-3 days to maintain the drug concentration and sufficient nutrition.

[0107] Observe the clones: During the incubation, observe the cell state and clone formation under the inverted microscope regularly. When the clones in the control group are clearly visible (white small dots can be seen with the naked eye, and each clone contains about > 50 cells under the microscope), the incubation can be terminated.

[0108] Termination of incubation and staining Wash: Carefully aspirate the medium and gently rinse the cells with PBS twice to wash away the residual medium and dead cells.

[0109] Fixation: Add 1 mL of 4% paraformaldehyde or pre-cooled methanol to each well and fix at room temperature for 15-20 minutes. This step is to fix the cell clones on the culture plate.

[0110] Discard the fixative: Carefully aspirate the fixative. If paraformaldehyde is used, wash with PBS 1-2 times.

[0111] Staining: Add 1 mL of 0.5% crystal violet staining solution to each well, ensuring complete coverage of the well bottom. Incubate at room temperature for 20-30 minutes.

[0112] Wash: Carefully aspirate the crystal violet staining solution. Carefully and repeatedly rinse the culture plate with deionized water or tap water at a slow flow rate until the background is clear and there is no purple residue.

[0113] Dry and photograph: Invert the washed culture plate on absorbent paper and dry thoroughly at room temperature. After drying, use a scanner or camera to take a photo of the entire 6-well plate and save the image.

[0114] 3、Experimental results The experimental results are shown in Figure 5 The tumor cells with ASNS knocked out are not sensitive to Salmeterol, and after knocking out the original target points of Salmeterol, ADRB1, ADRB2 and ADRB3, the tumor cells are still sensitive to Salmeterol, indicating that Salmeterol can specifically inhibit ASNS and limit tumor growth. 1、Experimental method (1) Animals and cells Cell line: LLC mouse lung cancer cells.

[0115] Experimental animals: 6 to 8-week-old female C57BL / 6J mice.

[0116] (2) Tumor inoculation LLC cells in logarithmic growth phase were resuspended in sterile PBS to prepare a cell suspension with a concentration of 10 7 cells / ml.

[0117] Each mouse was subcutaneously inoculated with 100 μL of the cell suspension (i.e., 10 6 cells) on the right axillary abdomen.

[0118] (3) Grouping and administration After inoculation, tumor growth was monitored regularly. When the average tumor volume reached about 100 mm³, the mice were randomly divided into the following four groups: Control group (Vehicle Control): Peritumoral injection of an equal amount of solvent as the drug.

[0119] Salmeterol monotherapy group (Salmeterol): Peritumoral injection of salmeterol at a dose of 5 mg / kg, twice a week.

[0120] Cisplatin monotherapy group (Cisplatin): Intraperitoneal (i.p.) injection of cisplatin at a dose of 4 mg / kg, once every 5 days.

[0121] Combination therapy group (Combination): Peritumoral injection of salmeterol at 5 mg / kg, twice a week.

[0122] Intraperitoneal (i.p.) injection of cisplatin at 4 mg / kg, once every 5 days.

[0123] (4) Tumor measurement and data monitoring The length and width of the tumor were measured every 3 to 4 days using a vernier caliper.

[0124] The formula for calculating the tumor volume was: Volume (mm³) = (length x width x width) / 2.

[0125] At the same time, the changes in the body weight and health status (such as activity, hair, diet) of the mice were closely monitored as indicators for assessing drug toxicity.

[0126] 2、Experimental results The experimental results, as shown in Figure 6 , showed that the therapeutic effect of salmeterol on tumor volume was better than that of cisplatin, and the combination of salmeterol and cisplatin could achieve a synergistic effect, further reducing the tumor volume.

[0127] The embodiments of the present application are described in detail above with reference to the drawings, but the present application is not limited to the above-described embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present application. Furthermore, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of salmeterol in the manufacture of a medicament for treating a tumor.

2. The use according to claim 1, wherein: the salmeterol comprises a pharmaceutically acceptable salt thereof.

3. The use according to claim 2, wherein: the pharmaceutically acceptable salt comprises an acid addition salt and a base addition salt.

4. The use according to claim 1, wherein: the tumor comprises lung cancer; preferably, the lung cancer comprises non-small cell lung cancer.

5. The use according to claim 1, wherein: the medicament comprises a pharmaceutically acceptable excipient; preferably, the pharmaceutically acceptable excipient comprises at least one of a solvent, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure adjusting agent, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending agent, a coating material, an aromatic agent, an anti-adhesion agent, an integrating agent, a penetration enhancer, a pH adjusting agent, a buffer, a plasticizer, a surfactant, an antifoaming agent, a thickening agent, an inclusion agent, a humectant, an absorbent, a diluent, a flocculating agent and a deflocculating agent, a filter aid, a release retarder, a carrier.

6. The use according to claim 1, wherein: the dosage form of the medicament comprises a trans-gastrointestinal administration dosage form, or a non-trans-gastrointestinal administration dosage form.

7. Use of salmeterol in the manufacture of an asparagine synthetase inhibitor.

8. A method for inhibiting asparagine synthetase in vitro, comprising the steps of: treating a sample with salmeterol.

9. Use of salmeterol in the manufacture of a chemotherapeutic drug sensitizer.

10. A pharmaceutical composition comprising salmeterol and a chemotherapeutic drug.

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