Application of composition of astemizole and terfenadine in preparation of medicine for treating gastric cancer

Abstract

The invention belongs to the field of medicines, and particularly relates to application of an astemizole and terfenadine composition in preparation of a medicine for treating gastric cancer. It is found that the antihistamine drug terfenadine can increase the inhibition effect of astemizole on gastric cancer AGS and HGC27 cells and significantly inhibit proliferation of the gastric cancer AGS and HGC27 cells, and terfenadine and astemizole can inhibit migration of the gastric cancer cells, induce apoptosis of the gastric cancer cells and retard the cycle of the gastric cancer cells. The effect of inhibiting gastric cancer cell proliferation is better than that of a clinically common anticancer drug pentafluorouracil (5-Fu), and the compound has a good application prospect in treating gastric cancer.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to the application of a combination of astemizole and terfenadine in preparing a drug for treating gastric cancer. Background Art

[0002] Cancer remains a major health problem in the world today, and the global cancer burden is on the rise. Gastrointestinal cancer is one of the cancers with extremely high morbidity and mortality rates, with an estimated 5.1 million new cases and 3.6 million deaths worldwide in 2020, accounting for more than a quarter (26.7%) of the global cancer incidence and more than a third (36.5%) of all cancer-related deaths. Gastric cancer is one of the most deadly causes of cancer-related death. Although patients with gastric cancer who are detected early have a better prognosis, the survival rate of patients with advanced and recurrent gastric cancer who cannot undergo surgery is still very low, so there is an urgent need to develop new drugs to treat gastric cancer.

[0003] The development of classic drugs includes target discovery and validation, high-throughput screening to identify lead compounds, and optimization of lead compounds through medicinal chemistry methods. Preclinical drug research includes analyzing the efficacy of compounds in animal models, pharmacology (absorption, distribution, metabolism, elimination), toxicology, specificity and drug interaction studies. Therefore, traditional drug development methods have the disadvantages of high cost and high risk. In this case, the new use of old drugs is a promising drug discovery strategy for cancer treatment. This definition is to use drugs that have been approved for the treatment of non-malignant tumors to discover their potential anti-tumor targets. Compared with the development of drugs from scratch, the new use of old drugs, as an ideal alternative, greatly shortens the time, reduces investment, and improves the success rate of preclinical drug discovery.

[0004] Terfenadine and astemizole are second-generation antihistamines for treating allergic symptoms. The present invention finds that the antihistamines terfenadine and astemizole can inhibit the proliferation and migration of gastric cancer cells, reduce the mitochondrial membrane potential of gastric cancer cells, and induce apoptosis of gastric cancer cells; at the same time, terfenadine and astemizole can block the gastric cancer cell cycle at the G0 / G1 phase or the G2 / M phase; they have a good effect in treating gastric cancer, and their effect of inhibiting the proliferation of gastric cancer cells is better than 5-Fu, and have a good application prospect. Summary of the invention

[0005] In view of the above technical problems, the purpose of the present invention is to provide a pharmaceutical composition composed of terfenadine and astemizole and a new use thereof in preparing a drug for treating gastric cancer. Specifically, the present invention includes the following contents:

[0006] In a first aspect, the present invention provides a pharmaceutical composition, wherein the active ingredients of the pharmaceutical composition consist of terfenadine and astemizole.

[0007] Preferably, the structural formulas of terfenadine and astemizole are shown in the following formulas (I) and (II), respectively:

[0008]

[0009] In a second aspect, the present invention provides use of the pharmaceutical composition described in the first aspect in the preparation of a drug for preventing or treating gastric cancer.

[0010] Preferably, the pharmaceutical composition inhibits the proliferation of gastric cancer cells.

[0011] Preferably, the pharmaceutical composition inhibits the migration of gastric cancer cells.

[0012] Preferably, the pharmaceutical composition induces apoptosis of gastric cancer cells.

[0013] Preferably, the pharmaceutical composition is added with pharmaceutically acceptable carriers and / or excipients to be made into any dosage form of tablets, sprays, granules, capsules, oral liquids, injections, and suspensions.

[0014] The beneficial effects of the present invention are as follows: the present invention finds that the antihistamine terfenadine and astemizole can inhibit the proliferation of gastric cancer cells in combination, terfenadine increases the anti-proliferation effect of astemizole on gastric cancer AGS and HGC27 cells, terfenadine and astemizole can inhibit the migration of gastric cancer cells, reduce mitochondrial membrane potential, and induce apoptosis of gastric cancer cells; at the same time, terfenadine and astemizole can block gastric cancer cells in the G0 / G1 phase or the G2 / M phase, have a significant effect of treating gastric cancer, and the effect of treating gastric cancer is better than 5-Fu, and has a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Evaluation of cytotoxicity of terfenadine and astemizole on different gastric cancer cell lines;

[0016] Figure 2 Evaluation of the cytotoxicity of terfenadine combined with astemizole on gastric cancer AGS and HGC27 cell lines;

[0017] Figure 3 Effects of terfenadine and astemizole on the cell cycle of gastric cancer cells AGS and HGC27;

[0018] Figure 4 Effects of terfenadine and astemizole on apoptosis of gastric cancer cells AGS and HGC27;

[0019] Figure 5 Flow cytometric analysis of apoptosis induced by terfenadine and astemizole in gastric cancer cells AGS and HGC27

[0020] Figure 6Terfenadine and astemizole inhibited the proliferation of gastric cancer cells AGS and HGC27;

[0021] Figure 7 Terfenadine and astemizole reduce mitochondrial membrane potential in gastric cancer cells AGS and HGC27;

[0022] Figure 8 Terfenadine and astemizole inhibited the migration of gastric cancer cells AGS and HGC27;

[0023] Fig. 9 Terfenadine and astemizole regulate the PI3K / AKT cell signaling pathway and the expression of cell cycle-related proteins. DETAILED DESCRIPTION

[0024] In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the present invention is further described below in conjunction with specific implementation methods. However, the protection scope of the present invention is not limited to the following embodiments.

[0025] The human gastric cancer cell line AGS described in the following examples was obtained from ATCC, and the HGC27, MKN45, MGC803, and SGC7901 cells were obtained from the genetic resources of this laboratory.

[0026] Example 1 Inhibitory Effects of Terfenadine and Astemizole on Gastric Cancer Cells

[0027] (1) Gastric cancer AGS, HGC27, and MKN45 cells were grown in RPMI1640 medium containing 10% fetal bovine serum, 1% penicillin and streptomycin, and gastric cancer MGC803 and SGC7901 cells were grown in DMEM (high glucose) medium containing 10% fetal bovine serum, 1% penicillin and streptomycin. The cells were grown in a cell culture incubator at 5% CO2 and 37°C. When the gastric cancer AGS, HGC27, MKN45, MGC803, and SGC7901 cells were in the logarithmic growth phase, the cells were seeded in a 96-well plate at a density of 8000 cells / well, and the 96-well plate was placed in a cell culture incubator and cultured for 24 h.

[0028] (2) Terfenadine and astemizole were diluted to different concentration gradients using RPMI1640 or DMEM complete medium. The medium in the 96-well plate was aspirated and medium containing different concentrations of terfenadine and astemizole was added. 5-Fu, a drug commonly used in the clinical treatment of gastric cancer, was used as a positive control. The treated 96-well plate was placed in a cell culture incubator for 48 hours. In the time gradient experiment, the placement time was 0, 24, 48, and 72 hours, respectively.

[0029] (3) After continuing to culture in the cell culture incubator for a period of time, add 10 μL of 5 mg / mL MTT solution filtered with a 0.22 μm filter membrane, and continue to incubate the 96-well plate in the cell culture incubator for 4 hours. After the incubation is completed, carefully aspirate the supernatant, add 100 μL of DMSO solution, place the 96-well plate on a horizontal shaker at a speed of 120 r / min for about 20 minutes, and measure the absorbance value at a wavelength of 490 nm using an enzyme reader. Calculate the inhibition rate of different concentrations of drugs on cells, and use SPSS software to fit the IC values ​​of drugs on different cancer cells. 50 value.

[0030] The results are as follows Figure 1 As shown, the present invention found that terfenadine and astemizole can inhibit the growth of gastric cancer AGS, HGC27, MKN45, MGC803 and SGC7901 cells in a concentration-dependent manner, and can inhibit the growth of AGS and HGC27 cells in a time-dependent manner. Among them, the IC of terfenadine on AGS, HGC27, MKN45, MGC803, SGC7901 cells at 48h 50 The IC values ​​of astemizole for AGS, HGC27, MKN45, MGC803 and SGC7901 cells for 48 h were 5.14 μM, 3.95 μM, 5.01 μM, 6.24 μM and 9.30 μM, respectively. 50 They are 8.47 μM, 5.37 μM, 17.53 μM, 11.13 μM and 27.44 μM.

[0031] Example 2 Terfenadine enhances the inhibitory effect of astemizole on gastric cancer AGS and HGC27 cells

[0032] (1) Gastric cancer AGS and HGC27 cells were grown in RPMI1640 medium containing 10% fetal bovine serum, 1% penicillin and streptomycin in a cell culture incubator at 5% CO2 and 37°C. When the gastric cancer AGS and HGC27 cells were in the logarithmic growth phase, the cells were seeded in a 96-well plate at a density of 8000 cells / well and the 96-well plate was placed in a cell culture incubator and cultured for 24 h.

[0033] (2) Astemizole was diluted to a 0.5 μM solution using RPMI1640 complete medium. At the same time, 0 μM, 0.5 μM, 1.25 μM, and 2.5 μM terfenadine solutions were added. The medium in the 96-well plate was aspirated, and different concentrations of terfenadine and astemizole were added. The treated 96-well plate was placed in a cell culture incubator for 48 h.

[0034] (3) Add 10 μL of 5 mg / mL MTT solution and continue to incubate the 96-well plate in the cell culture incubator for 4 h. After the incubation is complete, carefully aspirate the supernatant, add 100 μL of DMSO solution, place the 96-well plate on a horizontal shaker at 120 r / min for about 20 min, and measure the absorbance value at a wavelength of 490 nm using an ELISA reader. Calculate the inhibition rate of different drug concentrations on the cells.

[0035] The results are as follows Figure 2 As shown, the present invention found that terfenadine can increase the inhibitory effect of astemizole on gastric cancer cells AGS and HGC27 cells. 50 At the same concentration, astemizole (0.5 μM or 1.25 μM) and terfenadine (2.5 μM) had a weak inhibitory effect on gastric cancer cells AGS or HGC27, but compared with astemizole or terfenadine alone, the combination of astemizole and terfenadine significantly enhanced the inhibitory effect on gastric cancer cells. Therefore, terfenadine can increase the inhibitory effect of astemizole on gastric cancer AGS and HGC27 cells, and the combination of the two has a significant inhibitory effect on gastric cancer.

[0036] Example 3 Terfenadine and astemizole can arrest gastric cancer AGS and HGC27 cells in G0 / G1 or G2 / M phase

[0037] (1) When gastric cancer AGS and HGC27 cells grow to the logarithmic growth phase, they are seeded in a 6-well plate at a density of 500,000 cells / well and the seeded cells are cultured in a cell culture incubator for 24 hours.

[0038] (2) The culture medium was aspirated, and 4 mL of complete culture medium containing different concentrations of terfenadine and astemizole was added. The treated cells were placed in a cell culture incubator and cultured for 24 h.

[0039] (3) The cells were removed, the culture medium was discarded, trypsin was added for digestion and the cells were collected, the cells were washed twice with PBS, and then 1 mL of pre-cooled 70% anhydrous ethanol was added and placed in a -20°C refrigerator overnight.

[0040] (4) After fixation, centrifuge the cells and discard the supernatant. Wash the cells twice with PBS, add 100 μL of RNase A solution, and incubate them in a 37°C water bath for 30 min. After incubation, add 400 μL of PI dye solution and blow evenly. Transfer them to a 4°C refrigerator and incubate for about 30 min. Perform DNA content detection and analysis using a flow cytometer.

[0041] The results are as follows Figure 3As shown, terfenadine can block the cell cycle of AGS cells at the G0 / G1 phase and HGC27 cells at the G2 / M phase; astemizole can block the cell cycle of both gastric cancer AGS and HGC27 cells at the G0 / G1 phase.

[0042] Example 4 Terfenadine and astemizole can induce apoptosis in gastric cancer AGS and HGC27 cells

[0043] 1.DAPI staining experiment

[0044] (1) When AGS and HGC27 cells reached the logarithmic growth phase, they were seeded in a 24-well plate at a density of 30,000 to 50,000 cells / well. The cells were evenly pipetted and placed in a cell culture incubator for 24 h.

[0045] (2) Terfenadine and astemizole were prepared into different concentrations using complete culture medium, the culture medium in the 24-well plate was discarded, cell culture medium containing different concentrations of terfenadine and astemizole was added, and the treated cells were placed in a cell culture incubator and incubated for 48 h.

[0046] (3) The cell culture medium containing drugs was discarded, the cells were washed twice with PBS, 500 μL DAPI staining solution was added, and the cells were stained for 5 min. The staining solution was discarded, the cells were washed once with PBS, an appropriate amount of anti-fluorescence quenching mounting medium was added, and the cells were observed under an inverted fluorescence microscope, photographed, and recorded.

[0047] The results are as follows Figure 4 As shown, after AGS and HGC27 cells were treated with different concentrations of terfenadine and astemizole, the cells showed bright blue fluorescence, indicating that the cell nucleus was shrunken and apoptotic bodies appeared. Terfenadine and astemizole can induce apoptosis of gastric cancer AGS and HGC27 cells.

[0048] 2. Cell apoptosis experiment

[0049] (1) AGS and HGC27 cells in the logarithmic phase were seeded in a 6-well plate at a density of 500,000 cells / well, and the cell suspension in the 6-well plate was placed in a cell culture incubator for further incubation for 24 h.

[0050] (2) The cell culture medium was discarded, and 4 mL of complete culture medium containing different concentrations of terfenadine and astemizole was added. The treated cells were continued to be incubated in a cell culture incubator for 48 h.

[0051] (3) Discard the cell culture medium, wash once with PBS, add about 1 mL of EDTA-free trypsin solution to each well for digestion, collect the digested cells, add 300 μL of 1× Bingding buffer, blow the cell suspension evenly, take 100 μL of the cell suspension and place it in a flow cytometer, add 5 μL of Annexin V / Alexa Fluor 488 solution, mix well, and incubate at room temperature in the dark for 5 min, add 10 μL of 20 μg / mL PI solution, and quickly add 400 μL of PBS solution, blow evenly, and detect and analyze on a flow cytometer.

[0052] The results are as follows Figure 5 As shown, both terfenadine and astemizole could induce apoptosis of gastric cancer AGS and HGC27 cells in a concentration-dependent manner.

[0053] Example 5: Terfenadine and astemizole inhibit the proliferation of gastric cancer AGS and HGC27 cells

[0054] (1) When gastric cancer AGS and HGC27 cells grew to the logarithmic growth phase, the cells were seeded in a 24-well plate at a density of 500 cells / well, and the cell suspension was cultured in a cell culture incubator for 24 h.

[0055] (2) After the cells adhere to the wall, the cell culture medium is discarded, and 1 mL of culture medium containing different concentrations of terfenadine and astemizole is slowly added. The cells are placed in a cell culture incubator and cultured for 7-10 days, with the drug-containing culture medium replaced every 3 days.

[0056] (3) After each cell clone has grown to contain at least 20 cells, the culture medium is carefully aspirated, and 500 μL of 4% paraformaldehyde is added to each well for fixation for 40 min. After fixation, the paraformaldehyde is aspirated, and the wells are washed twice with PBS, and 500 μL of 1% crystal violet staining solution is added for staining for 20 min. After staining, the wells are washed 2-3 times with PBS, photographed, and statistically analyzed using Image J.

[0057] The results are as follows Figure 6 As shown, after treating gastric cancer AGS and HGC27 cells with different concentrations of terfenadine and astemizole, the proliferation of cells was inhibited, and the number of cell clones formed by AGS and HGC27 cells was significantly reduced.

[0058] Example 6 Terfenadine and astemizole reduce mitochondrial membrane potential in gastric cancer AGS and HGC27 cells

[0059] (1) AGS and HGC27 cells in the logarithmic phase were seeded in a 24-well plate at a density of 30,000 cells / well. The cells were evenly perfused and placed in a cell culture incubator for 24 h.

[0060] (2) The supernatant cell culture medium was discarded, and 1 mL of cell culture medium containing different concentrations of terfenadine and astemizole was added. The treated cells were placed in a cell culture incubator and incubated for 48 h.

[0061] (3) The cell culture medium of the positive control group was aspirated, 1 mL of 10 μM positive drug CCCP was added, and the cells were incubated in the cell culture incubator for 40 min. The supernatant was discarded, and the cells were washed twice with staining buffer. 1 mL of TMRE staining solution was added and placed in the cell culture incubator for 20 min. The staining solution was aspirated, and the cells were washed twice with staining buffer. An appropriate amount of anti-fluorescence quenching mounting medium was added, and the cells were observed under an inverted fluorescence microscope and photographed for record.

[0062] The results are as follows Figure 7 As shown, after treating gastric cancer AGS and HGC27 cells with different concentrations of terfenadine and astemizole for 48 hours, the mitochondrial membrane potential of gastric cancer AGS and HGC27 cells was significantly decreased.

[0063] Example 7 Terfenadine and astemizole inhibit the migration of gastric cancer AGS and HGC27 cells

[0064] (1) When AGS and HGC27 cells grow to the logarithmic growth phase, digest the cells, collect the cells with a culture medium containing 1% FBS, and count them. Dilute terfenadine and astemizole to different concentrations with a cell culture medium containing 1% FBS, and adjust the cell density to 30,000 cells / well. Add 600 μL of cell culture medium containing 20% ​​FBS to the lower chamber of the Transwell chamber, and carefully place the cells in a cell culture incubator and incubate for 48 hours.

[0065] (2) The culture medium in the upper and lower chambers of the transwell chamber was discarded, the chamber was washed twice with PBS, and 500 μL of 4% paraformaldehyde fixative was added for 40 min.

[0066] (3) After fixation, wash twice with PBS, add 1% crystal violet staining solution and stain for 20 min, then wash 2-3 times with PBS, carefully wipe the cells in the upper chamber of the transwell with a cotton swab, place the cells migrating under the membrane on an inverted microscope and take pictures, and perform statistical analysis using Image J.

[0067] The results are as follows Figure 8 As shown, both terfenadine and astemizole could inhibit the cell migration of gastric cancer AGS and HGC27 cells in a concentration-dependent manner. After treatment with terfenadine and astemizole, the number of migrated AGS and HGC27 cells was significantly reduced.

[0068] Example 8: Terfenadine and astemizole regulate the PI3K / AKT cell signaling pathway to inhibit the proliferation of gastric cancer AGS and HGC27 cells

[0069] (1) AGS and HGC27 cells grown to the logarithmic phase were seeded in a 6-well plate at a density of 500,000 cells / well. The cells were evenly pipetted and placed in a cell culture incubator for 24 h.

[0070] (2) Terfenadine and astemizole were diluted to 5 μM and 10 μM solutions with complete cell culture medium, the cell culture medium in the 6-well plate was discarded, the culture medium containing terfenadine and astemizole was added, and the treated cells were placed in a cell culture incubator and incubated for 48 h.

[0071] (3) Discard the cell culture medium, wash twice with PBS, add trypsin to digest and collect the cells, add RIRA lysis buffer containing 1% phosphatase inhibitor and 1% PMSF, and place on ice for 30 minutes, flicking every 10 minutes.

[0072] (4) After lysis, the centrifuge tube was placed in a precooled low-temperature high-speed centrifuge and centrifuged at 4°C, 12,000 r / min for 10 min. After centrifugation, the supernatant was transferred to a new centrifuge tube, and the protein concentration was determined using a BCA quantitative kit. The loading buffer was added to the remaining protein and placed in a 100°C metal bath for denaturation for 10 min. The protein was cooled to room temperature, centrifuged, and stored at -20°C.

[0073] (5) Prepare 10% SDS-PAGE gel, load the sample, run at a constant voltage of 60 V for 45 min, then run at a voltage of 120 V until the proteins are separated, and then transfer the proteins to the PVDF membrane at a constant current of 300 mA for 2 h. After the transfer, wash the membrane three times with TBST and block it with 5% skim milk for 2 h.

[0074] (6) After blocking, wash the membrane three times with TBST, each time for 10 min, and place the PVDF membrane in the corresponding primary antibody. The primary antibody is prepared with antibody diluent at a ratio of 1:1000. Place the PVDF membrane in a 4°C refrigerator and incubate overnight for 8-10 h.

[0075] (7) After incubation with the primary antibody, the strips were washed three times with TBST solution for 10 min each time. The secondary antibody was prepared with TBST solution at a ratio of 1:10,000. The strips were placed in the secondary antibody solution and incubated on a shaker for 1-2 h. The strips were then washed three times with TBST solution for 10 min each time. The strips were exposed with ECL luminescent solution on an exposure instrument, photographed, and statistically analyzed using Image J.

[0076] The results are as follows Fig. 9As shown, after 48 hours of treatment of gastric cancer AGS and HGC27 cells with terfenadine and astemizole, the expression of cell cycle-related proteins CDK4, CDK6, and p-Rb was significantly reduced, and the expression of key proteins in the PI3K / AKT cell signaling pathway was also reduced. The experimental results show that terfenadine and astemizole can inhibit the proliferation of gastric cancer AGS and HGC27 cells by regulating the PI3K / AKT cell signaling pathway.

[0077] The above-mentioned embodiments are only embodiments of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that for ordinary technicians in this field, other improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A pharmaceutical composition, characterized in that The active ingredients in the pharmaceutical composition consist of terfenadine and astemizole.

2. The pharmaceutical composition according to claim 1, characterized in that The structural formulas of terfenadine and astemizole are shown in the following formulas (I) and (II), respectively:

3. Use of the pharmaceutical composition according to claim 1 or 2 in the preparation of drugs for preventing or treating gastric cancer.

4. The use according to claim 3, characterized in that The pharmaceutical composition inhibits the proliferation of gastric cancer cells.

5. The use according to claim 3, characterized in that The pharmaceutical composition inhibits the migration of gastric cancer cells.

6. The use according to claim 3, characterized in that The pharmaceutical composition induces apoptosis of gastric cancer cells.

7. The use according to claim 3, characterized in that The pharmaceutical composition is added with pharmaceutically acceptable carriers and / or excipients to be made into any dosage form of tablets, sprays, granules, capsules, oral liquids, injections, and suspensions.

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