Application of enbopividone in preparation of medicine for resisting Chikungunya virus infection

Abstract

The invention relates to the technical field of medicines, in particular to application of enboviramide in preparation of a medicine for resisting chikungunya virus infection. The enbopividone belongs to a cyanamide dye compound, is a very effective medicine for treating enterobiasis at present, and is low in side effect and good in safety.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to the application of enbopirovinium in the preparation of anti-chikungunya virus infection drugs. Background Art

[0002] Pyrvinium pamoate, English name: Pyrvinium pamoate, chemical name: 4-[(3-carboxyl-2-hydroxynaphthalene-1-yl)methyl]-3-hydroxynaphthalene-2-carboxylic acid, is currently a very effective drug for the treatment of enterobiasis. Pyrvinium pamoate can interfere with the respiratory enzyme system of the worm, inhibit oxygen uptake, inhibit aerobic respiration, increase anaerobic glycolysis, and interfere with the absorption of exogenous glucose by the worm, deplete endogenous reserves, and cause the worm to die. Its therapeutic dose is 2.5 mg / kg. In addition, studies have shown that Pyrvinium pamoate can also promote apoptosis of cells infected with viruses, thereby inhibiting viral replication. Therefore, Pyrvinium pamoate has a high application value.

[0003] Chikungunya virus (CHIKV) is a single positive-strand RNA virus belonging to the genus Alphavirus of the family Togaviridae. Infection with chikungunya virus can cause chikungunya fever (CHIKF), which is characterized by fever, rash, and joint pain. In a small number of infected people, it can cause central nervous system infection, leading to death or serious sequelae. In recent years, the spread of CHIKV has gradually expanded, posing a huge threat to human life and health, and also placing a heavy burden on healthcare systems around the world. So far, there are no approved vaccines and antiviral drugs for human prevention and treatment. Therefore, there is an urgent need to develop safe and effective anti-CHIKV drugs. Summary of the invention

[0004] The present invention aims to provide a new use of enbopirovinium.

[0005] The invention provides the use of enbopirovinium in preparing a drug for resisting CHIKV infection.

[0006] The chemical structural formula of the compound Enbopirovinium is as follows:

[0007]

[0008] The present invention utilizes the experimental operation system of CHIKV-infected susceptible cells to screen candidate small molecule drugs that can inhibit CHIKV infection from a clinically approved drug small molecule library, and screens out enbopirovinium that can effectively inhibit CHIKV infection and has low cytotoxicity, and can be used as a potential CHIKV drug with application prospects.

[0009] The application of the present invention is characterized in that the dosage form of the above-mentioned drug is a dosage form for administration through the gastrointestinal tract.

[0010] The application of the present invention is characterized in that the dosage form of the above-mentioned drug is selected from powders, tablets, granules, capsules, solutions, emulsions, and suspensions.

[0011] The application of the present invention is characterized in that the dosage form of the above-mentioned drug is a dosage form for administration via a route other than the gastrointestinal tract.

[0012] The application described in the present invention is characterized in that the administration route dosage form of the above-mentioned drug is selected from injection dosage form, respiratory tract administration dosage form, nasal drops, mucosal administration dosage form or cavity administration dosage form. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 .The effect of Enbopirovinium on protecting HuH-7 cells against CHIKV infection;

[0014] That is, CHIKV infected HuH-7 cells, and Enbopivian or solvent DMSO was added at the same time. After 24 hours, the virus infection of the cells was detected by immunofluorescence technology, and the inhibition rate of the compound on CHIKV infection at a concentration of 10 μM was calculated based on the number of positive clones in each well.

[0015] Figure 2 .Toxic effect of Enbopirovinium on HuH-7 cells;

[0016] That is, HuH-7 cells were treated with different concentrations of enbopirovinium and solvent DMSO, and CCK8 reagent was added after 24 hours to detect the absorbance at 450nm.

[0017] Figure 3 .Inhibitory activity of different concentrations of enbopirovinium against CHIKV in the HuH-7 cell infection model.

[0018] That is, CHIKV infected HuH-7 cells, and different concentrations of enbopirovinium or solvent DMSO were added at the same time. After 24 hours, the virus infection of cells was detected by immunofluorescence technology, and the inhibition rate of CHIKV infection by the compound at different concentrations was calculated according to the number of positive clones in each well.

[0019] Figure 4 .The onset time of Enbopirovac inhibition of CHIKV.

[0020] That is, after CHIKV infects HuH-7 cells, enbopirovinium (4 μM) or solvent DMSO is added at different time periods. After the drug action time ends, the cells are cultured for another 12 hours, and the virus infection of the cells is detected by immunofluorescence technology. The inhibition rate of the compound on CHIKV infection at different time periods is calculated based on the number of positive clones in each well.

[0021] Figure 5.Effects of Enbopirovinium on the binding process of CHIKV

[0022] That is, CHIKV infected HuH-7 cells, and enbopirovinium or solvent DMSO was added at the same time, incubated on ice for 2 hours, the supernatant was discarded, washed three times with ice PBS, and then TRIzol lysis buffer was added to extract total RNA. RT-qPCR was used to detect the amount of CHIKV RNA bound to the cell surface, so as to calculate the effect of compounds at different concentrations on CHIKV binding.

[0023] Figure 6 .Effects of Enbopirovinium on CHIKV membrane fusion process

[0024] That is, DiD-labeled CHIKV infected HuH-7 cells, incubated on ice for 2 hours, discarded the supernatant, replaced with fresh culture medium containing enbopironium, NH4CL or solvent DMSO, and detected the DiD luminescence intensity on a multifunctional microplate reader to evaluate the effect of enbopironium on the CHIKV membrane fusion process.

[0025] Figure 7 .Protective effect of Enbopirovinium on wild-type C57BL / 6 mice infected with CHIKV

[0026] That is, after CHIKV infected wild-type C57BL / 6 mice, they were given enbopirovinium by gavage and the weight changes of the mice were observed ( Figure 7 A) and survival rate ( Figure 7 B), Evaluation of the protective effect of enbopirovinium on CHIKV-infected mice. DETAILED DESCRIPTION

[0027] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0028] The enbopirovinium used in the embodiments of the present invention can be purchased commercially.

[0029] 1. Viruses, drugs, reagents and other materials

[0030] 1. Virus: CHIKV, isolated from patient samples in Ruili, Yunnan in 2019.

[0031] 2. Compounds: 2580 kinds of US FDA chemical drug molecule library, purchased from Selleck Company, USA.

[0032] 3.Human hepatoma cell line HuH-7 cells were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences and preserved by the Department of Biomedical Protection, Naval Medical University of the Chinese People's Liberation Army.

[0033] 4. DMEM cell culture medium is a product of Hyclone Company, USA. 10% fetal bovine serum, non-essential amino acids, ampicillin and streptomycin (100 μL / ml each) were added when used. The culture medium additives were all products of Thermo Fisher Company, USA.

[0034] 5. Cell digestion solution, containing 0.25% trypsin, prepared with phosphate buffer.

[0035] 6.CCK8 cell activity and proliferation detection kit is a product of Dojindo Company of Japan.

[0036] 7. Mouse anti-CHIKV monoclonal antibody was prepared by the Eastern Theater Command Center for Disease Control and Prevention.

[0037] 8. Alexa Fluor 488-labeled anti-mouse IgG was a product of Thermo Fisher, USA.

[0038] 2. Experimental methods:

[0039] (I) Screening of anti-CHIKV drugs from the FDA drug small molecule library containing 2580 small molecule compounds

[0040] Human liver cancer cell line HuH-7 cells were subcultured in T25 cell culture flasks with complete DMEM culture medium, inoculated in 96-well plates, 50,000 cells per well, 100 μL DMEM culture medium, and cultured for 12 hours. Subsequently, 50 μL of complete DMEM culture medium containing CHIKV was added to each well, and the virus infection multiplicity (MOI) was 1; at the same time, 50 μL of complete DMEM culture medium containing FDA small molecule chemical drugs was added, and the final drug concentration was 10 μM. Each concentration was repeated 3 wells, and the same concentration of solvent DMSO was used as a control without drug addition. Cultured in a 37°C, 5% CO2 incubator. After 24 hours, the virus infection of cells was detected by immunofluorescence technology. The specific operation was as follows: the culture medium in the culture plate was removed, 100 μL of methanol was added to each well, and the culture plate was placed in a -20°C refrigerator. After 30 minutes, the culture plate was taken out, the methanol was removed, and each well was washed once with phosphate buffered saline (PBS), and then 100 μL of PBS containing 3% bovine serum albumin (BSA) (hereinafter referred to as 3% BSA-PBS) was added, and the plate was placed on a horizontal shaker and slowly shaken at room temperature for 2 hours. The 3% BSA-PBS in the culture plate was removed, and 100 μL of 1% BSA-PBS containing anti-CHIKV monoclonal antibody (antibody 1:2000 dilution) was added to each well, and the plate was slowly shaken at room temperature for 2 hours. The anti-CHIKV monoclonal antibody working solution in the culture plate was removed, and each well was washed 3 times with PBS, and then 100 μL of Alexa Fluor was added. 488-labeled anti-mouse IgG in 1% BSA-PBS (fluorescein antibody 1:2000 dilution), shake slowly at room temperature in the dark for 2 hours, remove the fluorescein antibody working solution in the culture plate, add 100 μL of DAPI nuclear staining solution to each well, shake slowly at room temperature in the dark for 10 minutes, remove the DAPI nuclear staining solution in the culture plate, wash each well with PBS 3 times, take pictures of the fluorescence distribution of cells in each well using a cell imaging and analysis system (BioTek Cytation 5Imaging Reader), calculate the inhibition rate of CHIKV infection of each compound at a concentration of 10 μM based on the number of positive clones in each well, inhibition rate % = [(number of positive clones in DMSO wells with the same concentration - number of positive clones in drug-added wells) / number of positive clones in DMSO wells with the same concentration] × 100. Figure 1 As shown, at 10 μM, enbopirovinium completely inhibited CHIKV infection.

[0041] (II) Toxicity of Enbopirovinium to Cells

[0042] The cultured human liver cancer cell line HuH-7 cells were inoculated into 96-well plates, with 50,000 cells per well and 100 μL of culture medium. After 12 hours, the original culture medium was aspirated and 100 μL of complete DMEM culture medium containing gradiently diluted embopirine was added to each well. The final concentration of embopirine was 7 gradients of 0.0064, 0.032, 0.16, 0.8, 4, 20 and 100 μM. Each concentration was repeated 3 wells, and the same concentration of solvent DMSO was used as a control without drug. The plates were cultured in a 37°C, 5% CO2 incubator. After 24 hours, 10 μL of CCK8 cell activity and proliferation detection reagent was added to each well and the plates were placed in a 37°C, 5% CO2 incubator. After 30 minutes, the absorbance of each well at a wavelength of 450 nm was detected using a multifunctional microplate reader. The cytotoxicity of the drug was evaluated based on the difference in absorbance at 450 nm between wells treated with different concentrations of drugs and wells with solvents. Figure 2 The results showed that enbopirovinium had low cytotoxicity, with a CC50 greater than 20 μM.

[0043] (III) Inhibitory effect of enbopirovinium on CHIKV in cell infection model

[0044] The subcultured human liver cancer cell line HuH-7 cells were inoculated into 96-well plates, with 50,000 cells per well and 100 μL of culture medium. The plates were cultured for 12 hours, and the original culture medium was removed. Then, 50 μL of complete DMEM culture medium containing CHIKV was added to each well, and the virus MOI (multiplicity of infection) was 1; at the same time, 50 μL of complete DMEM culture medium containing enbopirovinium was added, and the final drug concentration was 7 concentration gradients of 0.0064, 0.032, 0.16, 0.8, 4, 20 and 100 μM, and each concentration was repeated 3 wells. The same concentration of solvent DMSO was used as a control without drug addition. The plates were cultured in an incubator at 37°C and 5% CO2. After 24 hours, the virus infection of the cells was detected by immunofluorescence technology. The specific operation was the same as described in (I). The inhibition rate of the compound on CHIKV infection at different concentrations was calculated based on the number of positive clones in each well. Inhibition rate % = [(the number of positive clones in the DMSO well with the same concentration - the number of positive clones in the drug-added well) / the number of positive clones in the DMSO well with the same concentration] × 100. Figure 3 As shown, enbopirovinium can significantly inhibit the infection of CHIKV to HuH-7 cells, with an EC50 of 0.1307 μM.

[0045] (IV) Time window experiment to detect the onset of action of enbopirovinium against CHIKV

[0046] In order to clarify the effective stage of enbopirovin against CHIKV, we designed a drug action time window experiment for detection. The subcultured human liver cancer cell line HuH-7 cells were inoculated in 96-well plates, with 50,000 cells per well and 100 μL of culture medium, and cultured for 12 hours. The original culture medium was aspirated, and then 100 μL of complete DMEM culture medium containing CHIKV was added to each well, the virus MOI (multiplicity of infection) was 1, and the virus was infected for 2 hours. The drug action time period was set (the time point of adding virus was recorded as 0, the time point before this was recorded as a negative value, and the time point after this was recorded as a positive value): enbopirovin (4 μM) was added at -2, 0, 2, 4, 6, 8, and 10 hours, respectively, and the same concentration of solvent DMSO was used as a control. The drug action time was 2 hours. After continuing to culture for 12 hours, immunofluorescence detection was performed, and the specific operation was the same as described in (I). The inhibition rate of the compound on CHIKV infection was calculated based on the number of positive clones in each well. Inhibition rate % = [(the number of positive clones in the wells with the same concentration of DMSO - the number of positive clones in the wells with drug addition) / the number of positive clones in the wells with the same concentration of DMSO] × 100. The onset stage of the effect of Enbopirovinium against CHIKV was determined based on the inhibition rate. The results are shown in Figure 4 As shown, enbopirovinium has antiviral effects at all time periods of viral infection, and its antiviral effect is best in the early stage.

[0047] (V) Effect of Enbopirovinium on CHIKV Binding

[0048] The time window experiment showed that the antiviral effect of enbopirovinium was best in the early stage of viral infection, so we first detected the effect of enbopirovinium on virus binding. The subcultured human liver cancer cell line HuH-7 was inoculated in a 24-well plate, with 200,000 cells per well and 100 μL of culture medium. After culturing for 12 hours, the original culture medium was aspirated, and then 200 μL of complete DMEM culture medium containing CHIKV was added to each well, and the virus MOI (multiplicity of infection) was 10; at the same time, 200 μL of complete DMEM culture medium containing enbopirovinium was added, and the final drug concentration was 0.0064, 0.032, 0.16, 0.8, 4 and 20 μM 6 concentration gradients, and the equal concentration solvent DMSO was used as a control without drug. The virus and drug mixture was incubated with Huh-7 cells on ice, and the supernatant was discarded after 2 hours. After washing three times with ice PBS, 500 μL was added to each well. TRIzol lysis solution was repeatedly blown, and after the cells were completely lysed, the lysis solution was collected into the EP tube for nuclease removal; 100 μL of chloroform was added to each EP tube, shaken for 10 seconds, and allowed to stand at room temperature for 15 minutes; after standing, high-speed refrigerated centrifugation (12000rpm / min, 4°C) was performed to separate the layers; the upper colorless liquid was carefully aspirated with a micropipette (be careful not to aspirate the protein precipitate in the middle layer and the red phenol-chloroform phase in the lower layer), and transferred to a new EP tube for nuclease removal; 250 μL of isopropanol was added to each EP tube (isopropanol was pre-cooled in a -20°C refrigerator in advance), and gently mixed until the liquid became clear, and allowed to stand at room temperature for 10 minutes; high-speed refrigerated centrifugation was performed in a centrifuge (12000rpm / min, 4℃); discard the supernatant, add 500μL75% ethanol to each EP tube, spirally mix for 2min, wash the precipitate; centrifuge refrigerated centrifugation (7500rpm / min, 4℃); discard the supernatant, open the EP tube cap, dry at room temperature until the precipitate disappears (use a 10μL pipette to suck out all the liquid as much as possible to facilitate the rapid drying of RNA and avoid degradation), add 20μL of denuclease water to each EP tube, gently blow to completely dissolve the RNA; then use an ELISA reader to detect the concentration and purity of the extracted RNA (the extracted RNA needs to be reverse transcribed into cDNA as soon as possible. If reverse transcription is not performed, it needs to be frozen in a -80℃ refrigerator). Then prepare 20μL reverse transcription system (2μgRNA, 4μL 5×PrimeScript RT Master Mix, and then use DEPCH2O to make it 20μL), mix the prepared reverse transcription system and place it in a 37℃ water bath for 30min. The cDNA product obtained by reverse transcription was subjected to RT-qPCR using the Japanese TAKARA TB green kit, and the reaction system (20 μL system) was configured as follows: primer (forward + reverse) 0.5 μL; DEPC water 9.5 μL; TB green + rox 9.5 μL; cDNA product 0.5 μL.The subsequent reaction procedure (two-step method, 40 cycles): pre-denaturation: 95°C, 30s; denaturation 95°C, 5s; annealing and extension: 60°C, 30s; dissolution reaction: 95°C, 5s, 60°C, 1min, 95°C, 15s. The same reaction system and reaction procedure were repeated 3 times, and the average value was taken. The data were analyzed by the ΔΔCT method and the relative expression amount (RQ) of CHIKV RNA bound to the cell surface was calculated and compared. The results are shown in Figure 2. Figure 5 As shown, enbopirovinium did not affect the binding process of CHIKV.

[0049] (VI) Effect of Enbopirovinium on CHIKV membrane fusion

[0050] Since enbopironium did not affect the binding process of CHIKV, we then tested the effect of enbopironium on viral membrane fusion. CHIKV was labeled with the fluorescent dye DiD (DiD is a fat-soluble fluorescent dye. When the concentration of DiD is high, fluorescence quenching occurs. When the viral envelope fuses with the cell membrane, the fluorescent molecules diffuse evenly, reducing their concentration and causing fluorescence dequenching. Therefore, by observing the DiD fluorescence intensity, the membrane fusion of DiD-labeled molecules can be detected). 7 5 μL DiD was added to the culture medium (PFU / ml), mixed and incubated in a 37°C incubator for 30 min.

[0051] The subcultured human liver cancer cell line HuH-7 was inoculated into a 96-well plate, with 50,000 cells per well and 100 μL of culture medium. The culture was carried out for 12 hours, and the original culture medium was removed. Subsequently, 100 μL of complete DMEM culture medium containing DiD-labeled CHIKV was added to each well, and the virus MOI (multiplicity of infection) was 10. After incubation on ice for 2 hours, the supernatant was discarded, and 100 μL of complete DMEM culture medium containing 4 μM embropivirine, 50 mM NH4CL or DMSO was added. The plate was tested on a multifunctional microplate reader: temperature: 37°C; excitation wavelength: 644 nm; absorption wavelength: 665 nm; detection for 8 hours, once every 10 minutes, and 48 times in succession. The membrane fusion of the virus was analyzed by detecting the DiD fluorescence intensity. The results are shown in Figure 6 As shown, both enbopirovinium and NH4CL can significantly inhibit the membrane fusion process of CHIKV.

[0052] (VII) Enbopirovinium can effectively protect mice against CHIKV infection and reduce the mortality rate of mice

[0053] We first conducted a preliminary study on the virus challenge dose in mice, with a dose of 2*10 7PFU, 2*10 6 PFU, 2*10 5 PFU and 2*10 4 PFU 4 doses were intranasally infected into 6-week-old wild-type C57BL / 6 female mice. The weight of mice in all 4 groups began to decrease significantly on the 6th day, and mice died on the 9th day after infection with the first 2 doses, with a mortality rate of 87.5%. The above results show that CHIKV has a high mortality rate for wild-type C57BL / 6 mice and is easy to establish a mouse infection model.

[0054] Enbopirovinium powder was fully dissolved in corn oil solution and administered by gavage at a dose of 15 mg / kg / day, once a day. A total of 24 6-week-old female C57BL / 6 mice were randomly divided into 3 groups: 1. The drug administration group was first administered by gavage on the day of virus attack, followed by 50 μL 2*10 intranasal drops. 5 PFU virus, 8 mice; 2. Control group, corn oil was first gavaged on the day of virus attack, and 50μL 2*10 5 PFU virus, 8 mice; 3. Blank control group, no virus attack and no drug administration, 4 mice.

[0055] The mice were weighed starting from the day of virus attack (before virus infection) and then weighed every day before oral administration. The survival of the mice was observed twice a day (during oral administration and 12 hours after administration), and the changes in mouse weight and survival were recorded.

[0056] The weight changes of mice Figure 7 As shown in A: The recorded body weight values ​​of the DMSO group and the drug administration group decreased significantly from the 5th day after the virus attack, and the recorded body weight value of the drug administration group was higher than that of the DMSO group.

[0057] The survival of mice Figure 7 As shown in B: In the DMSO group, mice began to die on the 7th day after virus attack, and by the 12th day, 6 mice died, and the remaining 2 mice recovered to normal, with a mortality rate of 75%; in the drug administration group, mice began to die on the 10th day after virus attack, and by the 12th day, 4 mice died, and the remaining 4 mice recovered to normal, with a mortality rate of 50%. The results show that Enbopirovinium can effectively resist CHIKV infection in mice.

[0058] The above in vitro and in vivo experimental results all show that enbopirovinium has significant CHIKV activity and can be used to prepare drugs against CHIKV infection.

[0059] The above shows and describes the main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention. These changes and improvements fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. Application of Enbopirovinium in the preparation of drugs against Chikungunya virus infection.

2. The use according to claim 1, characterized in that: The anti-chikungunya virus infection drug uses the cyanamide dye compound embopironium as the only active ingredient, or a drug composition containing embopironium.

3. The use according to claim 2, characterized in that: The pharmaceutical composition containing the cyanamide dye compound embopyramide refers to a pharmaceutical composition composed of one or more pharmaceutically acceptable excipients of embopyramide.

4. The use according to any one of claims 1 to 3, characterized in that: The medicine is used for preventing or treating infection by Chikungunya virus.

5. The use according to any one of claims 1 to 3, characterized in that: The drug is in the form of a powder, tablet, granule, capsule, solution, emulsion, or suspension.

6. The use according to any one of claims 1 to 3, characterized in that: The drug administration route dosage form is selected from injection dosage form and gastrointestinal administration dosage form.

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