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

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of fosaprepitant in preparation of a medicine for treating non-small cell lung cancer. The invention relates to new application of fosaprepitant, in particular to application of fosaprepitant in preparation of a medicine for treating non-small cell lung cancer and a medicine composition for treating non-small cell lung cancer. The medicine composition for treating the non-small cell lung cancer can be prepared into tablets, capsules, electuary, drops, granules, ointment or injection by adding universal medicinal auxiliary components into fosaprepitant with the purity of 98% (mass percent) or above. Pharmacological experiment results show that fosaprepitant can obviously inhibit occurrence and development of non-small cell lung cancer. Therefore, fosaprepitant can be used for preparing the medicine for treating the non-small cell lung cancer. The invention provides a new drug source for the treatment of non-small cell lung cancer.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to application of fosaprepitant in preparing a medicine for treating non-small cell lung cancer. Background Art

[0002] Lung cancer ranks second in cancer deaths, among which non-small cell lung cancer (NSCLC) is one of the malignant tumors with extremely poor prognosis, and lung adenocarcinoma accounts for about 40% of them. For unresectable locally advanced or metastatic solid tumors, anticancer drugs have always been the mainstream treatment strategy. However, the currently available anticancer targets are still mainly limited to the receptor tyrosine kinase family (RTKs), and the limitation of available targets has become the main constraint on the development of anti-tumor drugs. More and more research results show that abnormal expression of FAK (focal adhesion kinase) is closely related to the development of NSCLC. Some small molecule compounds with strong binding to FAK target protein can inhibit the further development of NSCLC and improve the prognosis of NSCLC by affecting the overexpression or activation of FAK. Therefore, through the virtual screening of small molecule compounds targeting FAK, fosaprepitant was selected as a research drug, and it is planned to study the topic of fosaprepitant inhibiting lung adenocarcinoma through FAK-related pathways, in order to further explore the mechanism of fosaprepitant inhibiting lung adenocarcinoma through FAK-related pathways, which is of great significance for the development of new small molecule targeted anticancer drugs. Currently, virtual screening to find novel and effective active pharmaceutical substances for the treatment of NSCLC has become a research hotspot.

[0003] Fosaprepitant (FOS) is a precursor of aprepitant and a neurokinin-1 receptor antagonist developed for the prevention of chemotherapy-induced nausea and vomiting (CINV). The chemical structure is as follows.

[0004]

[0005] Chemical structure of the compound Fosaprepitant Fosaprepitant is an effective drug for preventing chemotherapy nausea and vomiting in clinical practice, but there are few reports on its use in the treatment of lung cancer, and research activity is not high. So far, in terms of new uses of old drugs, there has been no systematic research report on fosaprepitant in inhibiting the progression of non-small cell lung cancer. Summary of the invention

[0006] In view of this, the present invention provides the use of fosaprepitant in preparing a drug for treating non-small cell lung cancer. Experiments show that fosaprepitant can significantly inhibit the occurrence and development of non-small cell lung cancer and can be used to prepare a drug for treating non-small cell lung cancer.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The use of fosaprepitant in the preparation of a drug for treating non-small cell lung cancer, wherein the structural formula of fosaprepitant is as follows: .

[0008] Furthermore, fosaprepitant is used for any one or more of the following: (I) Inhibit FAK protein phosphorylation; (II) preparing drugs or reagents for inhibiting FAK protein phosphorylation.

[0009] Furthermore, the inhibition of FAK protein phosphorylation includes inhibiting tyrosine phosphorylation at site 397, site 576 / 577, and site 925 of the FAK protein.

[0010] Furthermore, the FAK protein includes FAK protein in non-small cell lung cancer cells.

[0011] Furthermore, the non-small cell lung cancer cells include one or more of A549 and H1299 cells.

[0012] Furthermore, the drug is used for: (i) inhibiting the cell viability of non-small cell lung cancer cells; and / or (ii) inhibiting the proliferation of non-small cell lung cancer cells; and / or (iii) Promote apoptosis of non-small cell lung cancer cells.

[0013] Furthermore, the drug is used to inhibit the tumor size and weight of non-small cell lung cancer.

[0014] Furthermore, the drug comprises fosaprepitant or its geometric isomers, pharmaceutically acceptable salts, solvates or hydrates.

[0015] Furthermore, the medicine is in the form of tablets, capsules, granules, drops, lyophilized products, granules, ointments or injections.

[0016] Furthermore, the drug also includes one or more excipients, and the excipients include one or more of a carrier, a solvent, an emulsifier, a dispersant, a wetting agent, a binder, a polymer, a stabilizer, and a colorant.

[0017] The invention provides the use of fosaprepitant in inhibiting FAK protein phosphorylation.

[0018] In some specific embodiments of the present invention, the above-mentioned application of inhibiting FAK protein phosphorylation includes inhibiting tyrosine phosphorylation at sites 397, 576 / 577 and 925 of FAK protein.

[0019] In some specific embodiments of the present invention, the FAK protein used above includes FAK protein in non-small cell lung cancer cells.

[0020] In some specific embodiments of the present invention, the non-small cell lung cancer cells used above include one or more of A549 and H1299 cells.

[0021] The present invention also provides the use of fosaprepitant in treating non-small cell lung cancer.

[0022] The present invention also provides the use of fosaprepitant in any of the following items: (a) preparing drugs or reagents for inhibiting FAK protein phosphorylation; (b) preparing a drug or reagent for inhibiting cell viability of non-small cell lung cancer cells; (c) preparing drugs or reagents for inhibiting the proliferation of non-small cell lung cancer cells; (d) preparing drugs or reagents for promoting apoptosis of non-small cell lung cancer cells; (e) Preparation of drugs or agents for inhibiting tumor growth.

[0023] In some specific embodiments of the present invention, the above-mentioned drug or agent comprises an inert, non-toxic, pharmacologically suitable excipient.

[0024] In some specific embodiments of the present invention, the above-mentioned medicine or agent is in the form of tablets, capsules, granules, drops, lyophilized products, granules, ointments or injections.

[0025] In some specific embodiments of the present invention, the excipients of the above-mentioned drugs or agents are carriers, solvents, emulsifiers, dispersants, wetting agents, adhesives, stabilizers, colorants, and flavors.

[0026] In some specific embodiments of the present invention, the above-mentioned application of inhibiting FAK protein phosphorylation includes inhibiting tyrosine phosphorylation at sites 397, 576 / 577 and 925 of FAK protein.

[0027] In some specific embodiments of the present invention, the above-mentioned applied FAK protein includes FAK protein in non-small cell lung cancer cells.

[0028] The present invention also provides the use of fosaprepitant in preparing a pharmaceutical composition for treating non-small cell lung cancer.

[0029] In some specific embodiments of the present invention, the pharmaceutical composition described above comprises an inert, non-toxic, pharmacologically suitable excipient.

[0030] In some specific embodiments of the present invention, the pharmaceutical composition described above is a powder containing a therapeutically effective dose of fosaprepitant as an active ingredient.

[0031] The preferred technical solution of the present invention further comprises one or more pharmaceutically acceptable inert and non-toxic excipients.

[0032] Preferably, the excipient is a carrier, a solvent, an emulsifier, a dispersant, a wetting agent, a binder, a stabilizer, a colorant or a fragrance.

[0033] Preferably, the medicine is in the form of tablets, capsules, granules, drops, lyophilized products, granules, ointments or injections.

[0034] Preferably, the fosaprepitant, pharmaceutically acceptable salt, solvate or hydrate is used as the sole active ingredient.

[0035] In the above technical solution, the pharmaceutical composition is prepared into tablets, capsules, granules, drops, lyophilized products, granules, ointments or injections by adding pharmaceutical auxiliary ingredients with a mass fraction of more than 98% of fosaprepitant.

[0036] Beneficial Effects The present invention provides a use of fosaprepitant with high efficiency in resisting non-small cell lung cancer: fosaprepitant can significantly inhibit the proliferation, metastasis and invasion ability of small cell lung cancer, and can promote the apoptosis of non-small cell lung cancer.

[0037] The fosaprepitant provided by the present invention has the use of high efficiency against non-small cell lung cancer and has potential significant economic effects: the preparation prepared by fosaprepitant or its pharmaceutically acceptable salt or acceptable carrier has the application prospect of a drug for treating non-small cell lung cancer, and is developed and manufactured in accordance with the national innovative drug approval method, and is expected to become a new drug for treating non-small cell lung cancer with high efficiency and low toxicity and containing known active ingredients with independent intellectual property rights in my country, and has clear industrialization prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0039] Figure 1 The IC50 results of fosaprepitant for non-small cell lung cancer; Figure 2 These are the experimental results of the effect of fosaprepitant on the proliferation ability of non-small cell lung cancer; Figure 2 A shows the EDU results of the effect of fosaprepitant on the proliferation ability of non-small cell lung cancer; Figure 2 B shows the cloning results of the effect of fosaprepitant on the proliferation ability of non-small cell lung cancer; Figure 3The results of fosaprepitant on apoptosis of non-small cell lung cancer; Figure 4 The results of fosaprepitant on the nude mouse subcutaneous transplanted tumor model; Figure 4 A shows the results of fosaprepitant on the nude mouse subcutaneous transplanted tumor model; Figure 4 B shows the results of the nude mouse transplanted tumor mass in each group; Figure 4 C shows the tumor growth curve; Figure 4 D shows the mass curve of nude mice; Figure 5 The results are the effects of fosaprepitant on non-small cell lung cancer cells; Figure 5 A shows the effect of fosaprepitant on p-FAK (Y397), p-FAK (Y576 / 577) and p-FAK (Y925) proteins in non-small cell lung cancer cells; Figure 5 B shows the results of fosaprepitant on the expression of p-FAK (Y397) protein in subcutaneous transplanted tumor tissues of nude mice. DETAILED DESCRIPTION

[0040] The present invention discloses the use of fosaprepitant in the preparation of a drug for treating non-small cell lung cancer. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0041] The first technical problem to be solved by the present invention is to provide a new use of fosaprepitant, namely, the use of fosaprepitant in the preparation of a drug for treating non-small cell lung cancer.

[0042] To solve the above problems, the first aspect of the present invention provides a technical solution: use of fosaprepitant in preparing a pharmaceutical composition for treating non-small cell lung cancer. The pharmaceutical composition comprises an inert, non-toxic, pharmacologically suitable excipient.

[0043] The second aspect of the present invention provides a pharmaceutical composition for treating non-small cell lung cancer, wherein the composition contains a therapeutically effective dose of fosaprepitant as an active ingredient.

[0044] Preferably, the pharmaceutical composition further comprises one or more inert, non-toxic, pharmacologically suitable excipients.

[0045] Preferably, the excipient is a carrier, a solvent, an emulsifier, a dispersant, a wetting agent, a binder, a stabilizer, a colorant, or a flavor.

[0046] Preferably, the pharmaceutical composition is in the form of tablets, capsules, granules, drops, lyophilized products, granules, ointments or injections.

[0047] In the above technical solution, the pharmaceutical composition for treating non-small cell lung cancer can be prepared into tablets, capsules, granules, drops, lyophilized products, granules, ointments or injections by adding common pharmaceutical auxiliary ingredients to fosaprepitant with a purity of more than 98% (mass percentage).

[0048] The pharmaceutical composition according to the invention can act systemically and / or locally and, for this purpose, can be administered in a suitable manner, for example, by oral, parenteral, pulmonary or nasal routes. The composition according to the invention can be administered in dosage forms suitable for these administration routes.

[0049] Suitable for oral administration are administration forms which act according to the state of the art and release the pharmaceutical composition of the invention rapidly and / or in a modified manner, and which comprise the pharmaceutical composition of the invention in crystalline and / or amorphous and / or dissolved form, such as tablets (uncoated or coated tablets, which, for example, have a coating which resists gastric juice or which delays dissolution or does not dissolve, release according to the composition of the invention), tablets which break rapidly in the mouth, or films, films / lyophilisates, capsules (for example hard or soft capsules), sugar-coated tablets, granules, pellets, powders, emulsions, suspensions, aerosols or solutions.

[0050] Parenteral administration can avoid the absorption step (e.g. intravenously, intra-arterially, intracardially, intraspinally or intralumbarly or intraarticularly) or simultaneously include absorption (e.g. intramuscularly, subcutaneously, intradermally, percutaneously or intraperitoneally). Suitable dosage forms suitable for parenteral administration are particularly preparations for injection and infusion in the form of solutions, suspensions, emulsions, lyophilizates or sterile powders.

[0051] Suitable for another route of administration are, for example, pharmaceutical forms for inhalation, such as powder inhalers or nebulizers, or pharmaceutical forms which can be taken nasally, in the form of drops, solutions or sprays.

[0052] The pharmaceutical composition according to the invention can be converted into the administration form described. This can be done in a manner known per se by mixing with inert, nontoxic, pharmacologically suitable excipients. These excipients include in particular carriers (e.g. microcrystalline cellulose, lactose, mannitol, starch), solvents (e.g. liquid polyethylene glycol), emulsifiers and dispersants or wetting agents (e.g. sodium lauryl sulfate, polyoxysorbitan oleate, propylene glycol), binders (e.g. polyvinyl pyrrolidone), synthetic and natural polymers (e.g. albumin), stabilizers (e.g. antioxidants, such as ascorbic acid), colorants (e.g. inorganic pigments, such as iron oxides) and masking flavors and odors.

[0053] The effective dose of fosaprepitant can vary according to the route of administration, the patient's age and weight, the severity of the disease, and other relevant factors. The recommended dose for oral administration is 200-800 mg / time, 1-2 times a day; the recommended dose for parenteral administration is 100-400 mg / time, once a day.

[0054] The technical scheme of the present invention is: to study its effect on non-small cell lung cancer through in vitro cell experiments (CCK8, EDU, clone formation and flow cytometry) and in vivo animal experiments, observe the therapeutic effect of fosaprepitant on non-small cell lung cancer, and thus provide experimental basis for fosaprepitant to treat non-small cell lung cancer.

[0055] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0056] The experimental materials and instrument information involved in the specific embodiments are as follows: 1. Experimental Animals BALB / c athymic nude mice, 25 females, 3-5 weeks old, weighing 18-25 g, clean grade, provided by Shanghai Slack Experimental Animal Co., Ltd. During the experiment, the temperature in the breeding room was 20±3.2°C, the relative humidity was 65-75%, and food and water were available freely.

[0057] 2. Drugs and reagents: 2.1 Medicines Fosaprepitant dimeglumine, white powder, content ≥98%, provided by Shanghai MCE Company, stored in a refrigerator at 4℃. Dissolve in PBS before use and prepare immediately.

[0058] 2.2 Reagents Cell lines: A549 and H1299 (human lung adenocarcinoma cell lines) were purchased from the Cell Bank / Stem Cell Bank of the Chinese Academy of Sciences in Shanghai, China; fetal bovine serum was produced by Gibco; RPMI-1640 medium was purchased from Procell; CCK8 was purchased from APExBIO; EdU detection kit was purchased from Haibi Yuntian Biotechnology Co., Ltd.; apoptosis detection kit was purchased from Nanjing Formase Biotechnology Co., Ltd.; Matrigel was purchased from BD Biotechnology, USA; FAK(3285 S), Phospho-FAK (Tyr397)(3283 S), Phospho-FAK (Tyr576 / 577)(3281S), Phospho-FAK (Tyr925)(3284 S) CST, USA.

[0059] 2.3 Instruments Electronic balance, Shanghai Ohaus Instrument Co., Ltd.; vernier caliper (HAMILTION); constant temperature cell culture incubator, Thermo Corporation, USA; biosafety operating table, Suzhou Antai Air Technology Co., Ltd.; multi-function microplate reader (SpectraMax M5), Molecular Devices, USA; inverted microscope, Olympus Corporation, Japan.

[0060] The statistical analysis method involved in the specific embodiment is as follows: GraphPad Prism 8.0 software was used to perform statistical analysis and plot the experimental data. The Student's t test was used to compare the two groups, and the measurement data were expressed as mean ± standard error (mean ± SD). The count data were analyzed using the chi-square test. When P < 0.05, it was considered statistically significant. Each group of experiments was repeated three times or more.

[0061] The present invention will be further described below in conjunction with embodiments: Example 1: CCK8 experiment (1) When the density of A549 and H1299 in the T25 flask reaches 80-90%, wash them twice with PBS and then digest and centrifuge them. Resuspend them in 1 mL of complete culture medium and count the cells. Prepare a total tube with a final concentration of 3 × 104 cells / mL. Add PBS to the wells on the outermost circle of the 96-well plate in advance to prevent the cell suspension in the middle from evaporating. Use a pipette to draw 100 μL of the suspension containing 3,000 cells into the wells and place them in a constant temperature incubator for culture.

[0062] (2) After 24 hours, the original culture medium was replaced with a culture medium containing different concentrations of fosaprepitant prepared in advance by complete culture medium, and A549 and H1299 cells were treated with drugs. A549 cells were treated with a concentration gradient of 0μM, 25μM, 50μM, 75μM, 100μM, 125μM, 150μM, 200μM, 250μM, and 300μM; H1299 cells were treated with a concentration gradient of 0μM, 25μM, 50μM, 75μM, 100μM, 125μM, 150μM, 200μM, 250μM, and 300μM. Three sub-wells were set for each cell type and each concentration gradient, and the cells were placed in an incubator to detect the absorbance values ​​of fosaprepitant at 48 hours and 72 hours after treatment.

[0063] (3) Add 10 μL of CCK8 solution to each well and record the absorbance at 450 nm and 630 nm using a multifunctional microplate reader after 2 hours. The percentage of relative cell viability is calculated as follows: [OD(450 nm)-OD(630 nm)] / AVERAGE(ODDMSO450 nm-ODDMSO630 nm)*100.

[0064] result: Fosaprepitant can inhibit the cell viability of non-small cell lung cancer cells. Figure 1 As shown in A (corresponding data in Table 1), fosaprepitant can inhibit the proliferation of non-small cell lung cancer cells A549 and H1299. The IC50 values ​​of fosaprepitant for each cell line were calculated using Graphpad software. The IC50 values ​​of A549 and H1299 at 48 hours were 35.05μM and 52.99μM, respectively, and the IC50 values ​​at 72 hours were 19.70μM and 40.97μM, respectively. It can be seen that fosaprepitant can inhibit the proliferation of non-small cell lung cancer cells.

[0065] Table 1

[0066] Example 2: EDU experiment (1) Digested A549 and H1299 cells were plated into six-well plates. When the cells grew to 50% of the bottom of the plate, fosaprepitant (the concentrations for A549 were 0 μM, 15 μM, and 25 μM, respectively; the concentrations for H1299 were 0 μM, 15 μM, and 25 μM, respectively) was added for 48 hours.

[0067] (2) Digest the cells and centrifuge them. Use a pipette to take 1 mL of complete culture medium to mix the precipitate and then count the cells. Prepare a total tube with a final concentration of 3 × 104 cells / mL. Add PBS to the outermost circle of wells in the 96-well plate to prevent the cell suspension in the middle from evaporating. Use a pipette to take 100 μL of the suspension containing 3,000 cells and add it to the wells. Place the wells in a constant temperature incubator for culture.

[0068] (3) Aspirate the original culture medium and add 100 μL of culture medium containing EdU diluted with complete culture medium (the ratio of EdU to complete culture medium is 1:1000) to each well and incubate for 2 hours.

[0069] (4) Aspirate the original culture medium, place the 96-well plate on a shaker, and wash the cells three times with 50 μL PBS solution per well for 5 minutes each time to wash away the residual unbound EdU reagent.

[0070] (5) Add 50 μL of 4% paraformaldehyde in PBS into the wells along the side walls to fix the cells and incubate at room temperature for 30 min.

[0071] (6) Aspirate the fixative and add washing solution (PBS containing 0.3% BSA) to each well and wash three times for 5 minutes each time to remove the residual fixative.

[0072] (7) Aspirate the washing solution, add 50 μL of permeabilization solution (PBS containing 0.9% TritonX-100) and permeabilize for 15 min. Aspirate the permeabilization solution and wash with the washing solution three times for 5 min each time to wash away the residual permeabilization solution.

[0073] (8) Aspirate the washing solution, add 50uL Click reaction solution, incubate at room temperature in the dark for 30min, and wash three times with washing solution.

[0074] (9) Aspirate the washing solution, add 50uL Hoechst 33342 solution to stain the cell nucleus, incubate at room temperature in the dark for 10 minutes, and wash three times with washing solution.

[0075] (10) After adding the anti-fluorescence quencher, store in a refrigerator at 4°C away from light.

[0076] (11) Observe the proportion of EdU-positive cells under a fluorescence microscope.

[0077] result: Fosaprepitant can inhibit the proliferation of non-small cell lung cancer cells. Figure 2 As shown in A (corresponding data in Table 2), 48 hours after the addition of fosaprepitant, the changes in cell proliferation ability were detected by EdU experiment. After the addition of fosaprepitant, the proportion of EdU-positive cells was significantly reduced, and the proportion of EdU-positive cells decreased with the increase of fosaprepitant concentration, which indicated that fosaprepitant could inhibit the proliferation ability of non-small cell lung cancer cells.

[0078] Table 2

[0079] Example 3: Clone formation experiment (1) Digest 80-90% A549 and H1299 cells in a T25 culture flask and plate an appropriate amount of cells in a six-well plate. When the cells grow to 50%-60% of the bottom of the plate, add fosaprepitant (solvent: PBS; the concentrations for A549 are 0μM, 15μM, and 25μM, respectively; the concentrations for H1299 are 0μM, 15μM, and 25μM, respectively) and treat for 48 hours.

[0080] (2) Digest the cells with trypsin and stop the digestion with complete medium. Count the cells after centrifugation and resuspend. Inoculate 3,000 cells per well into a six-well plate pre-added with 2% complete medium. Gently shake the six-well plate and place it in an incubator for 7-14 days.

[0081] (3) When the number of cells in most colonies is >50, the culture is terminated, the original culture medium is discarded, the cells are washed twice with PBS, 1 mL of methanol is slowly added to the wells, and fixed at room temperature for 30 minutes.

[0082] (4) Aspirate the methanol, dry the tube, add 1 mL of crystal violet staining solution, and stain overnight.

[0083] (5) Aspirate and discard the crystal violet, wash away the residual staining solution with PBS, let it dry, and take photos and count.

[0084] result: Fosaprepitant can inhibit the proliferation of non-small cell lung cancer cells. Figure 2 As shown in B (corresponding normalized data are shown in Table 3 ), after the addition of fosaprepitant, the number of cell clones decreased significantly, and the number of cell clones decreased with the increase of fosaprepitant concentration.

[0085] Table 3

[0086] Example 4: Flow cytometry apoptosis assay (1) Digest 80-90% A549 and H1299 cells in T25 culture flasks, plate an appropriate amount of cells in a six-well plate, and when the cells grow to 50%-60% of the bottom of the plate, add fosaprepitant (solvent: PBS; the concentrations for A549 are 0 μM, 15 μM, and 25 μM, respectively; the concentrations for H1299 are 0 μM, 15 μM, and 25 μM, respectively) and treat for 48 hours.

[0087] (2) Collect the original cell culture medium, aspirate 500 μL of EDTA-free trypsin and add it to the wells. Place the six-well plate in a constant temperature incubator to accelerate digestion. Use the original cell supernatant to stop digestion. Collect the cell suspension and centrifuge.

[0088] (3) Aspirate and discard the supernatant, wash the cells twice with PBS, and centrifuge again.

[0089] (4) Aspirate and discard PBS, use a pipette to draw 200 μL of Annexin V-FITC binding solution and add it to the EP tube, blow off the precipitate, gently blow the cells, and resuspend the cells.

[0090] (5) Pipette 5 μL Annexin V-FITC and 5 μL PI staining solution into the buffer solution respectively, gently pipette to mix, and incubate at 37°C for 20 minutes in the dark.

[0091] (6) Transfer the cell suspension to a flow cytometer, and perform statistical analysis on the data after testing.

[0092] result: Fosaprepitant can promote apoptosis of non-small cell lung cancer cells. Figure 3 As shown in Table 4, flow cytometry was used to detect the effect of fosaprepitant on apoptosis of non-small cell lung cancer cells 48 hours after addition of fosaprepitant. The results showed that fosaprepitant could increase the proportion of apoptotic cells, and the effect of fosaprepitant on apoptosis was concentration-dependent.

[0093] Table 4

[0094] Example 5: CDX nude mouse transplant tumor model (1) Lung adenocarcinoma cell line A549 was used as the tumorigenic cell in this experiment. A549 cells with good growth were selected and expanded in a 10 cm dish. A549 cells were passaged one day before subcutaneous tumor formation and inoculated subcutaneously into nude mice when they were in the logarithmic growth phase.

[0095] (2) Digest the cells in the large dish, centrifuge, wash the cells twice with PBS, mix them by pipetting, centrifuge again, aspirate 1 mL of serum-free medium to resuspend the cells, mix them by pipetting, count the cells, and prepare a total tube with a final concentration of 4 × 107 cells / mL. Aspirate 3000 μL of the cell suspension and add the matrix gel at a volume ratio of 1:1. Mix them by pipetting and place on ice.

[0096] (3) In this experiment, 25 female nude mice aged 3-5 weeks were selected as experimental subjects. The inoculation site under the right armpit of the mice was disinfected with iodine, and 100 μL of the mixed solution was aspirated and inoculated subcutaneously.

[0097] (4) After the transplanted tumors grew, 25 nude mice were randomly divided into a blank control group (normal saline), a low-dose fosaprepitant group (FOS, 20 mg / kg), a high-dose fosaprepitant group (FOS, 60 mg / kg), a cisplatin group (CDDP, 2.5 mg / kg), and a VS6063 group (25 mg / kg). The tumor volume was measured with a vernier caliper once every two days, and the weight of the nude mice was weighed with an electronic balance. The tumor volume was calculated using the formula: V=L×D2 / 2, where L is the longest diameter of the transplanted tumor and D is the shortest diameter of the transplanted tumor. Administration of the drug was started after the tumor volume reached 80-100 mm3.

[0098] (5) Every two days, the low-dose group and high-dose group were intraperitoneally injected with 20 mg / kg and 60 mg / kg of fosaprepitant (FOS), respectively. The blank control group was injected with an equal amount of normal saline. The cisplatin group was intraperitoneally injected with 2.5 mg / kg cisplatin (CDDP). The VS6063 group was given 25 mg / kg VS6063. After 7 treatments, the nude mice were killed and the tumors were removed.

[0099] (6) Take photos and weigh the tumor.

[0100] result: Fosaprepitant can inhibit the growth of transplanted tumors in nude mice. After confirming the effect of fosaprepitant in vitro, the A549 cell line was inoculated subcutaneously in BALB / c nude mice, and a CDX nude mouse transplanted tumor model was constructed to verify the effect of fosaprepitant in vivo. After the tumor grew to about 80-100mm3, the mice randomly divided into 5 groups were injected intraperitoneally with 20mg / kg and 60mg / kg of fosaprepitant (FOS) in the low-dose group and high-dose group every two days, respectively. The blank control group was injected with an equal amount of normal saline, the cisplatin group was intraperitoneally injected with 2.5mg / kg cisplatin (CDDP), and the VS6063 group was given 25mg / kg VS6063. After 7 doses, the mice were killed. The results showed that the tumor size and weight of the fosaprepitant group were significantly smaller than those of the blank control group. The tumor size and weight of the high-dose group were not significantly different from those of the cisplatin group and were superior to the marketed FAK inhibitor-VS6063, ( Figure 4 A. Figure 4 B; corresponding data are shown in Table 5). The tumor volume was measured every two days during the experiment, and the recorded tumor volume was plotted as a line graph ( Figure 4 C; see Table 6 for the corresponding data). Figure 4 D (corresponding data are shown in Table 7), fosaprepitant had no effect on the mass of nude mice and had little in vivo toxicity.

[0101] Table 5

[0102] Table 6

[0103] Table 7

[0104] Example 6: Western blot (1) Lyse cell samples or tumor tissues with Bio-Tech RIPA buffer and centrifuge to obtain supernatant. Add 5× Loading Buffer and heat at 100℃ for 7 minutes. Store protein samples in a -80℃ refrigerator.

[0105] (2) Perform SDS-PAGE electrophoresis in constant voltage mode, set the voltage to 140 V for 70 min.

[0106] (3) Perform wet transfer. Perform transfer at a constant current of 320 mA for 1.5 hours.

[0107] (4) Immunoreaction and color development. The NC membrane was blocked with 5% BSA on a shaker at room temperature for 1 hour. After blocking, it was placed in a specific primary antibody diluted 1:1000 in 1xTBST and incubated overnight at 4°C on a shaker. After the primary antibody incubation, the membrane was washed 4 times for 5 minutes each time, and then incubated with a secondary antibody diluted 1:50000 on a shaker at room temperature for 1 hour. After the secondary antibody incubation, the membrane was washed 4 times for 5 minutes each time, and finally the ECL chemiluminescence method was used for color development.

[0108] result: Fosaprepitant can inhibit the phosphorylation of tyrosine at sites 397, 576 / 577, and 925 of the FAK protein without affecting the expression of total FAK protein, thereby inhibiting the proliferation of non-small cell lung cancer cells and promoting their apoptosis. Figure 5 As shown in A, the protein changes of FAK and p-FAK (Y397), p-FAK (Y576 / 577) and p-FAK (Y925) in A549 and H1299 cells were detected by Western blot. It was found that fosaprepitant could inhibit the phosphorylation of tyrosine at sites 397, 576 / 577 and 925 of FAK protein, and this inhibition was concentration-dependent. Figure 5 As shown in B, the protein changes of FAK and p-FAK (Y397), p-FAK (Y576 / 577) and p-FAK (Y925) in the blank control group and the low-dose, high-dose and VS6063 groups of fosaprepitant in the transplanted tumors were detected by Western blot. It was found that fosaprepitant can also significantly inhibit the protein changes of p-FAK (Y397), p-FAK (Y576 / 577) and p-FAK (Y925) in the transplanted tumors in vivo.

[0109] In summary, fosaprepitant can inhibit the proliferation ability of non-small cell lung cancer cells in vivo and in vitro.

[0110] Preparation Example 1 Take 20 g of the monomer compound of fosaprepitant, add 280 g of medicinal starch, mix the two thoroughly, and prepare 1000 capsules, each capsule weighing 0.3 g and containing 20 mg of fosaprepitant.

[0111] Preparation Example 2 Take 100 g of the monomer compound of fosaprepitant, add 200 g of medicinal starch, mix the two thoroughly, and prepare 1000 tablets, each weighing 0.3 g and containing 100 mg of fosaprepitant.

[0112] Preparation Example 3 Take 20 g of the monomer compound of fosaprepitant, add 100 mL of 1,2-propylene glycol, fully dissolve, dilute to 1000 mL with sterile water for injection, mix well and dispense into 1000 ampoules, each 1 mL, containing 20 mg of fosaprepitant.

[0113] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. The use of fosaprepitant in the preparation of a drug for treating non-small cell lung cancer, characterized in that: The structural formula of the fosaprepitant is as follows: 。 2. The use according to claim 1, characterized in that Fosaprepitant is used for any one or more of the following: (I) Inhibit FAK protein phosphorylation; (II) preparing drugs or reagents for inhibiting FAK protein phosphorylation.

3. The use according to claim 2, characterized in that The inhibiting of FAK protein phosphorylation includes inhibiting tyrosine phosphorylation at the 397th, 576 / 577 and 925th sites of the FAK protein.

4. The use according to claim 2, characterized in that The FAK protein includes the FAK protein in non-small cell lung cancer cells.

5. The use according to claim 4, characterized in that The non-small cell lung cancer cells include one or more of A549 and H1299 cells.

6. The use according to claim 1, characterized in that The drug is used to: (i) inhibiting the cell viability of non-small cell lung cancer cells; and / or (ii) inhibiting the proliferation of non-small cell lung cancer cells; and / or (iii) Promote apoptosis of non-small cell lung cancer cells.

7. The use according to claim 1, characterized in that The drug is used to inhibit the tumor size and weight of non-small cell lung cancer.

8. The use according to claim 1, characterized in that The drug includes fosaprepitant or its geometric isomers, pharmaceutically acceptable salts, solvates or hydrates.

9. The use according to claim 1, characterized in that The medicine is in the form of tablets, capsules, granules, drops, lyophilized products, granules, ointments or injections.

10. The use according to claim 9, characterized in that The drug further comprises one or more excipients, and the excipients include one or more of a carrier, a solvent, an emulsifier, a dispersant, a wetting agent, a binder, a polymer, a stabilizer, and a colorant.

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