Application of astemizole and terfenadine combination in the preparation of drugs for treating gastric cancer

Abstract

The present invention belongs to the field of medicine and specifically relates to the use of a combination of astemizole and terfenadine in the preparation of a drug for treating gastric cancer. The present invention finds that the antihistamine drug terfenadine can enhance the inhibitory effect of astemizole on gastric cancer AGS and HGC27 cells, significantly inhibiting the proliferation of AGS and HGC27 gastric cancer cells. Furthermore, terfenadine and astemizole can inhibit the migration of gastric cancer cells, induce apoptosis of gastric cancer cells, and block the cell cycle. The effect of terfenadine and astemizole in inhibiting the proliferation of gastric cancer cells is superior to that of the commonly used clinical anticancer drug 5-fluorouracil (5-FU), and the present invention has good application prospects in the treatment of gastric cancer.

Description

Technical Field

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

[0002] Cancer remains a major global health concern, with the global cancer burden increasing. Gastrointestinal cancer is one of the most common cancers with the highest morbidity and mortality rates, with an estimated 5.1 million new cases and 3.6 million deaths worldwide in 2020. This represents over a quarter (26.7%) of global cancer incidence and over a third (36.5%) of all cancer-related deaths. Gastric cancer is one of the most lethal causes of cancer-related death. While patients with early-stage gastric cancer have a better prognosis, the survival rate for patients with inoperable advanced and recurrent gastric cancer remains low, necessitating the development of new therapeutics for the treatment of gastric cancer.

[0003] Classic drug development involves target discovery and validation, high-throughput screening to identify lead compounds, and optimization of lead compounds through medicinal chemistry methods. Preclinical drug research involves analyzing compound efficacy 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 context, repurposing old drugs is a promising drug discovery strategy for cancer treatment. This is defined as the use of drugs that have been approved for the treatment of non-malignant tumors to discover their potential anti-tumor targets. Compared with de novo drug development, repurposing old drugs is an ideal alternative that greatly shortens 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 arrest 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 in inhibiting the proliferation of gastric cancer cells is better than 5-Fu, and have good application prospects. Summary of the Invention

[0005] In response to the above technical problems, the present invention aims to provide a pharmaceutical composition comprising terfenadine and astemizole and its novel use 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 preparing 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 prepared into any dosage form of tablets, sprays, granules, capsules, oral solutions, injections, and suspensions.

[0014] The beneficial effects of the present invention are as follows: the present invention discovered that the combination of the antihistamine terfenadine and astemizole can inhibit the proliferation of gastric cancer cells, that terfenadine increases the anti-proliferation effect of astemizole on gastric cancer AGS and HGC27 cells, that terfenadine and astemizole can inhibit gastric cancer cell migration, reduce mitochondrial membrane potential, and induce apoptosis of gastric cancer cells; and that terfenadine and astemizole can simultaneously arrest gastric cancer cells in the G0 / G1 phase or the G2 / M phase, thereby having a significant effect in treating gastric cancer, and that the effect of the combination in treating gastric cancer is superior to that of 5-Fu, thereby having good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0016] Figure 2 Evaluation of the cytotoxicity of terfenadine and astemizole combined in 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 in gastric cancer cells AGS and HGC27 induced by terfenadine and astemizole

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

[0024] To make the technical means, creative features, objectives and effects of the present invention easier to understand, the present invention is further described below in conjunction with specific embodiments. However, the scope of protection 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 HGC27, MKN45, MGC803, and SGC7901 cells were obtained from the genetic resource collection of our 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. Gastric cancer MGC803 and SGC7901 cells were grown in DMEM (high glucose) medium containing 10% fetal bovine serum, 1% penicillin, and streptomycin. All cells were grown in a cell culture incubator at 5% CO2 and 37°C. When 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 8,000 cells / well, and the 96-well plate was placed in a cell culture incubator and cultured for 24 hours.

[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 complete, carefully aspirate the supernatant, add 100 μL of DMSO solution, and place the 96-well plate on a horizontal shaker at a speed of 120 r / min for about 20 minutes. Use an enzyme reader to measure the absorbance value at a wavelength of 490 nm. 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 after 48 hours is 50 The IC values ​​of astemizole for AGS, HGC27, MKN45, MGC803, and SGC7901 cells after 48 h were 5.14 μM, 3.95 μM, 5.01 μM, 6.24 μM, and 9.30 μM, respectively. 50 They were 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 hours.

[0033] (2) Astemizole was diluted to a 0.5 μM solution using RPMI1640 complete medium. Terfenadine solutions at 0 μM, 0.5 μM, 1.25 μM, and 2.5 μM were added simultaneously. The medium in the 96-well plate was aspirated and various concentrations of terfenadine and astemizole were added. The treated 96-well plate was then 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 incubation, carefully aspirate the supernatant and add 100 μL of DMSO solution. Place the 96-well plate on a horizontal shaker at 120 rpm for approximately 20 min. Measure the absorbance at 490 nm using a microplate reader. Calculate the inhibitory rate of the drug at different concentrations on the cells.

[0035] The results are as follows Figure 2 As shown, the present invention found that terfenadine can increase the effect of astemizole in inhibiting gastric cancer cells AGS and HGC27 cells. 50 At different concentrations, astemizole (0.5μM or 1.25μM) and terfenadine (2.5μM) had weak inhibitory effects on gastric cancer cells AGS or HGC27. However, the combination of astemizole and terfenadine significantly enhanced the inhibitory effect on gastric cancer cells compared to either astemizole or terfenadine alone. Therefore, terfenadine can enhance 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 at G0 / G1 or G2 / M phase

[0037] (1) When gastric cancer AGS and HGC27 cells reached the logarithmic growth phase, they were seeded in a 6-well plate at a density of 500,000 cells / well. The seeded cells were then 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, the cells were centrifuged and the supernatant was discarded. The cells were washed twice with PBS, 100 μL of RNase A solution was added, and the cells were placed in a 37°C water bath for incubation for 30 min. After incubation, 400 μL of PI dye solution was added and pipetted evenly. The cells were transferred to a 4°C refrigerator for incubation for about 30 min, and the DNA content was detected and analyzed using a flow cytometer.

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

[0045] (2) Terfenadine and astemizole were prepared into different concentrations using complete culture medium. The culture medium in the 24-well plate was discarded, and 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.

[0046] (3) Discard the cell culture medium containing drugs, wash the cells twice with PBS, add 500 μL DAPI staining solution, stain for 5 minutes, discard the staining solution, wash once with PBS, add an appropriate amount of anti-fluorescence quenching mounting medium, observe under an inverted fluorescence microscope, take pictures, and record.

[0047] The results are as follows Figure 4 As shown, after treatment with different concentrations of terfenadine and astemizole, AGS and HGC27 cells showed bright blue fluorescence, indicating shrinkage of the nucleus and the appearance of apoptotic bodies. Terfenadine and astemizole can induce apoptosis in gastric cancer AGS and HGC27 cells.

[0048] 2. Cell Apoptosis Experiment

[0049] (1) AGS and HGC27 cells in the logarithmic phase were seeded into 6-well plates at a density of 500,000 cells / well. The cell suspension in the 6-well plates was placed in a cell culture incubator and incubated 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 then placed in a cell culture incubator and incubated 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 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 minutes, 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 in the results, both terfenadine and astemizole could induce apoptosis in 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 placed in a cell culture incubator and cultured 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. The medium containing the drugs is 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. 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 in the figure, 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 in 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 into a 24-well plate at a density of 30,000 cells / well. The cells were evenly distributed and placed in a cell culture incubator for 24 hours.

[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 placed in a cell culture incubator for another 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 a cell culture incubator for incubation 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 reached the logarithmic growth phase, the cells were digested, harvested with culture medium containing 1% FBS, and counted. Terfenadine and astemizole were diluted to different concentrations in cell culture medium containing 1% FBS, and the cell density was adjusted to 30,000 cells / well. 600 μL of cell culture medium containing 20% ​​FBS was added to the lower chamber of the Transwell chamber, and the cells were carefully placed in a cell culture incubator and incubated for 48 h.

[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 and fixed for 40 minutes.

[0066] (3) After fixation, the cells were washed twice with PBS, stained with 1% crystal violet staining solution for 20 min, and then washed 2-3 times with PBS. The cells in the upper chamber of the transwell were carefully wiped off with a cotton swab, and the cells that migrated under the membrane were placed on an inverted microscope for photography and 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 into 6-well plates at a density of 500,000 cells / well. The cells were evenly distributed and placed in a cell culture incubator for 24 h.

[0070] (2) Terfenadine and astemizole were diluted to 5 μM and 10 μM solutions using complete cell culture medium. The cell culture medium in the 6-well plate was discarded, and the culture medium containing terfenadine and astemizole was added. 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 pre-cooled low-temperature high-speed centrifuge, centrifuged at 4°C, 12,000 rpm for 10 min, and the supernatant was transferred to a new centrifuge tube. The protein concentration was determined using a BCA quantitative kit, and loading buffer was added to the remaining protein. The protein was denatured in a 100°C metal bath for 10 min. The protein was cooled to room temperature, centrifuged, and stored at -20°C.

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

[0074] (6) After blocking, wash the membrane three times with TBST for 10 min each time, place the PVDF membrane in the corresponding primary antibody, and prepare the primary antibody with antibody diluent at a ratio of 1:1000. Place the PVDF membrane in a 4°C refrigerator overnight and incubate 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 using ECL luminescent solution on an exposure instrument, photographed, and statistically analyzed using Image J.

[0076] The results are as follows Figure 9As shown, after 48 hours of treatment with terfenadine and astemizole, gastric cancer AGS and HGC27 cells significantly reduced the expression of cell cycle-related proteins CDK4, CDK6, and p-Rb, and also decreased the expression of key proteins in the PI3K / AKT cell signaling pathway. The experimental results indicate 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-described embodiments merely illustrate the implementation methods of the present invention. Although the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art may make other modifications without departing from the scope of the present invention, and such modifications are within the scope of protection of the present invention.

Claims

1. Use of a pharmaceutical composition in the preparation of a drug for preventing or treating gastric cancer; characterized in that: The active ingredients in the pharmaceutical composition consist of terfenadine and astemizole.

2. The use according to claim 1, characterized in that The pharmaceutical composition inhibits the proliferation of gastric cancer cells.

3. The use according to claim 1, characterized in that The pharmaceutical composition inhibits the migration of gastric cancer cells.

4. The use according to claim 1, wherein The pharmaceutical composition induces apoptosis of gastric cancer cells.

5. The use according to claim 1, characterized in that The pharmaceutical composition is added with pharmaceutically acceptable carriers and / or excipients to be prepared into any dosage form of tablets, sprays, granules, capsules, oral solutions, injections, and suspensions.

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