Use of encenicline in the preparation of a drug for resisting infection of bunyavirus

Abstract

The present application relates to the technical field of medicine, and relates to application of empentiv in preparation of a drug for resisting infection of Bungomkenya virus. Empentiv belongs to cyanamide dye compounds, is a very effective drug for treating oxyuriasis at present, has low side effects and good safety.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of pyrvinium embonate in preparation of a drug for resisting Chikungunya virus infection. BACKGROUND

[0002] Pyrvinium embonate, English name: Pyrvinium embonate, chemical name: 4-[(3-carboxy-2-hydroxynaphthalen-1-yl)methyl]-3-hydroxynaphthalene-2-carboxylic acid, is a very effective drug for treating enterobiasis at present. Pyrvinium embonate can interfere with the respiratory enzyme system of the worm body, inhibit oxygen uptake, increase anaerobic glycolysis, and can interfere with the absorption of exogenous glucose by the worm body, deplete endogenous reserves, and cause the worm body to die, and its therapeutic dose is 2.5 mg / kg. And studies have shown that pyrvinium embonate can also promote the apoptosis of cells infected with viruses, thereby inhibiting viral replication, so pyrvinium embonate has high application value.

[0003] Chikungunya virus (CHIKV) is a single-stranded RNA virus, belonging to the alphavirus genus of the Togaviridae family, and its infection can cause Chikungunya fever (CHIKF) characterized by fever, rash, and joint pain. In a small number of infected individuals, central nervous system infection can lead to death or severe sequelae. In recent years, the transmission range of CHIKV has gradually expanded, posing a great threat to human life and health and placing a heavy burden on the world's health care system. So far, there is no approved vaccine or antiviral drug for human use, so there is an urgent need to develop safe and effective anti-CHIKV drugs. SUMMARY

[0004] The application aims to provide a new use of pyrvinium embonate.

[0005] The application provides application of pyrvinium embonate in preparation of a drug for resisting CHIKV infection.

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

[0007]

[0008] The application utilizes an experimental operation system of CHIKV-infected susceptible cells to screen candidate small molecule drugs that can inhibit CHIKV infection from a clinically approved small molecule drug library, and pyrvinium embonate is screened out as a potential CHIKV drug with application prospects because it can effectively inhibit CHIKV infection and has relatively small cytotoxicity.

[0009] The application is characterized in that the dosage form of the drug is a gastrointestinal administration dosage form.

[0010] The application is characterized in that the administration form of the drug is selected from the group consisting of powder, tablet, granule, capsule, solution, emulsion, suspension.

[0011] The application is characterized in that the administration form of the drug is selected from the group consisting of injection administration form, respiratory administration form, nasal drop, mucous membrane administration form or cavity administration form.

[0012] The application is characterized in that the administration form of the drug is selected from the group consisting of injection administration form, respiratory administration form, nasal drop, mucous membrane administration form or cavity administration form. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 . Effect of empramptuzumab on the protection of HuH-7 cells against CHIKV infection

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

[0015] Figure 2 . Toxicity of empramptuzumab on HuH-7 cells

[0016] That is, HuH-7 cells are treated with different concentrations of empramptuzumab and solvent DMSO, respectively. After 24 hours, CCK8 reagent is added, and the absorbance at 450 nm is detected.

[0017] Figure 3 . Inhibition activity of different concentrations of empramptuzumab on CHIKV in the HuH-7 cell infection model

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

[0019] Figure 4 . Time period for empramptuzumab to inhibit CHIKV

[0020] That is, after CHIKV infects HuH-7 cells, empramptuzumab (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. The infection of the virus on the cells is detected by immunofluorescence technology. The inhibition rate of the compound on CHIKV infection at different time periods is calculated according to the number of positive clones in each well.

[0021] Figure 5Effect of empentivir on CHIKV binding process

[0022] That is, CHIKV infects HuH-7 cells, empentivir or solvent DMSO is added, incubated on ice for 2h, the supernatant is discarded, washed with ice PBS for 3 times, then TRIzol lysis solution is added to extract total RNA, and the amount of CHIKV RNA bound to the cell surface is detected by RT-qPCR to calculate the effect of the compound on CHIKV binding at different concentrations.

[0023] Figure 6 Effect of empentivir on CHIKV membrane fusion process

[0024] That is, DiD-labeled CHIKV infects HuH-7 cells, incubated on ice for 2h, the supernatant is discarded, and fresh medium containing empentivir, NH4CL or solvent DMSO is added, and the DiD luminescence intensity is detected on a multifunctional enzyme label instrument to evaluate the effect of empentivir on CHIKV membrane fusion process.

[0025] Figure 7 Protective effect of empentivir on CHIKV infection of wild type C57BL / 6 mice

[0026] That is, after CHIKV infects wild type C57BL / 6 mice, empentivir is given by gavage, and the body weight change of the mice is observed Figure 7 A) and the survival rate Figure 7 B), to evaluate the protective effect of empentivir on CHIKV infected mice. DETAILED DESCRIPTION

[0027] In order to more clearly illustrate the present application, the present application will be further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the specific description below is illustrative rather than limiting, and should not limit the protection scope of the present application.

[0028] The empentivir used in the embodiments of the present application can be obtained by market purchase.

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

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

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

[0032] 3. Human hepatoma cell line HuH-7 cells, purchased from Shanghai Cell Institute of Chinese Academy of Sciences, and preserved by the Biomedical Defense Teaching and Research Office of the Navy Military Medical University of the People's Liberation Army.

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

[0034] 5. Cell digestion solution contains 0.25% trypsin and is prepared with phosphate buffer.

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

[0036] 7. Mouse anti-CHIKV monoclonal antibody is prepared by the Eastern Regional Center for Disease Control.

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

[0038] II. Experimental method:

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

[0040] The human liver cancer cell line HuH-7 cells were subcultured in T25 cell culture bottles with complete DMEM medium, inoculated in 96-well plates at 50,000 cells per well, 100 μL of DMEM medium, and cultured for 12 h. Then 50 μL of complete DMEM medium containing CHIKV was added to each well, with a multiplicity of infection (MOI) of 1; 50 μL of complete DMEM medium containing FDA small molecule drugs was also added, with a final drug concentration of 10 μM, and each concentration was repeated in 3 wells, and the same concentration of solvent DMSO was used as a control without drugs. The plates were incubated in a 37°C, 5% CO2 incubator. After 24 h, the infection of the cells by the virus was detected by immunofluorescence, as follows: the culture medium was removed from the plates, 100 μL of methanol was added to each well, and the plates were placed in a -20°C refrigerator for 30 min. Then the plates were removed, the methanol was aspirated, and each well was washed once with phosphate buffered saline (PBS). Then 100 μL of 3% bovine serum albumin (BSA)-PBS was added, and the plates were slowly shaken at room temperature for 2 h. The 3% BSA-PBS was removed from the plates, 100 μL of 1% BSA-PBS containing anti-CHIKV monoclonal antibody (antibody 1:2000 dilution) was added to each well, and the plates were slowly shaken at room temperature for 2 h. The anti-CHIKV monoclonal antibody working solution was removed from the plates, each well was washed 3 times with PBS, and then 100 μL of 1% BSA-PBS containing fluorescein Alexa Fluor 488-labeled anti-mouse IgG (fluorescein antibody 1:2000 dilution) was added. The plates were slowly shaken at room temperature in the dark for 2 h. The fluorescein antibody working solution was removed from the plates, 100 μL of DAPI nuclear staining solution was added to each well, and the plates were slowly shaken at room temperature in the dark for 10 min. The DAPI nuclear staining solution was removed from the plates, each well was washed 3 times with PBS, and the fluorescence distribution of the cells in each well was photographed using a cell imaging and analysis system (BioTek Cytation 5 Imaging Reader). The inhibition rate of each compound on CHIKV infection at a concentration of 10 μM was calculated based on the number of positive clones in each well, and the inhibition rate % = [(number of positive clones in the same concentration of DMSO well - number of positive clones in the drug-treated well) / number of positive clones in the same concentration of DMSO well] x 100. As shown in Table 1, at 10 μM, enpirotrifenum completely inhibited the infection of CHIKV. Figure 1

[0041] (II) Toxicity of enpirotrifenum to cells

[0042] ​Respectively, the culture of human hepatoma cell line HuH-7 cells were inoculated in 96-well plates, 50000 cells per well, 100 μL of culture medium, 12 h later, the original culture medium was removed, 100 μL of complete DMEM medium containing gradient dilution of enpirotriflumuron was added to each well, the final concentration of enpirotriflumuron was 0.0064, 0.032, 0.16, 0.8, 4, 20 and 100 μM 7 gradients, each concentration was repeated 3 wells, and the same concentration of solvent DMSO was used as a control without drug. Placed in a 37℃, 5% CO2 incubator for culture. After 24 h, 10 μL of CCK8 cell activity and proliferation detection reagent was added to each well, placed in a 37℃, 5% CO2 incubator, 30 min later the multi-function enzyme marker was used to detect the absorbance of each well at 450 nm wavelength, and the cytotoxicity of the drug was evaluated according to the difference in 450 nm absorbance between the drug-treated wells and the solvent wells. For example Figure 2 It was shown that enpirotriflumuron had little cytotoxicity, and its CC50 was greater than 20 μM.

[0043] (Three) Inhibition of CHIKV by enpirotriflumuron in a cell infection model

[0044] The passaged human hepatoma cell line HuH-7 cells were inoculated in 96-well plates, 50000 cells per well, 100 μL of culture medium, cultured for 12 h, the original culture medium was removed, then 50 μL of complete DMEM medium containing CHIKV was added to each well, the virus MOI (multiplicity of infection) was 1; At the same time, 50 μL of complete DMEM medium containing enpirotriflumuron was added, the final concentration of the drug was 0.0064, 0.032, 0.16, 0.8, 4, 20 and 100 μM 7 concentration gradients, each concentration was repeated 3 wells, and the same concentration of solvent DMSO was used as a control without drug. Placed in a 37℃, 5% CO2 incubator for culture. After 24 h, the virus infection of cells was detected by immunofluorescence technology, and the specific operation was the same as described in (One). The inhibition rate of the compound at different concentrations on CHIKV infection was calculated according to the number of positive clones in each well, and the inhibition rate % = [(the number of positive clones in the same concentration of DMSO well - the number of positive clones in the drug-treated well) / the number of positive clones in the same concentration of DMSO well] x 100. The results are shown in Figure 3 As shown, enpirotriflumuron can significantly inhibit the infection of CHIKV on HuH-7 cells, and its EC50 is 0.1307 μM.

[0045] (Four) Time window experiment to detect the onset stage of enpirotriflumuron against CHIKV

[0046] To determine the onset stage of the anti-CHIKV effect of envofenamide, we designed a drug action time window experiment. The human hepatoma cell line HuH-7 cells were seeded in 96-well plates at 50,000 cells per well in 100 μL of culture medium and incubated for 12 h. The original culture medium was removed, and then 100 μL of complete DMEM culture medium containing CHIKV was added to each well at a MOI (multiplicity of infection) of 1, and the cells were infected with the virus for 2 h. The drug action time period was set (the time point of adding the virus was 0, the time points before this were negative, and the time points after this were positive): envofenamide (4 μM) was added at -2, 0, 2, 4, 6, 8, and 10 h, and the same concentration of DMSO was used as a control. The drug action time was 2 h. After 12 h of continuous culture, immunofluorescence detection was performed according to the method described in (I). According to the number of positive clones in each well, the inhibition rate of the compound on CHIKV infection was calculated: % inhibition = [(number of positive clones in the same concentration of DMSO well - number of positive clones in the drug well) / number of positive clones in the same concentration of DMSO well] x 100. According to the inhibition rate, the onset stage of the anti-CHIKV effect of envofenamide was determined. The results, as shown in Table 1, showed that envofenamide had an antiviral effect at each time period of viral infection, and the antiviral effect was best in the early stage. Figure 4

[0047] (Five) Effect of envofenamide on CHIKV binding

[0048] ​The time window experiment showed that the best anti-viral effect of envofenamide was in the early stage of viral infection, so we first detected the effect of envofenamide on virus binding. The passaged human hepatoma cell line HuH-7 was inoculated in a 24-well plate, 200,000 cells per well, 100 μL of culture medium, and cultured for 12 h. The original culture medium was aspirated, and then 200 μL of complete DMEM culture medium containing CHIKV was added to each well, with a virus MOI (multiplicity of infection) of 10. At the same time, 200 μL of complete DMEM culture medium containing envofenamide was added, with drug final concentrations of 0.0064, 0.032, 0.16, 0.8, 4 and 20 μM in 6 concentration gradients, and the same concentration of solvent DMSO as the control without drug. The virus and drug mixture was incubated with Huh-7 cells on ice, and after 2 h the supernatant was discarded, washed with ice PBS three times, and then 500 μL of TRIzol lysis solution was added to each well and blown repeatedly. After the cells were completely lysed, the lysate was collected in a nuclease-free EP tube. 100 μL of chloroform was added to each EP tube, shaken for 10 s, and allowed to stand at room temperature for 15 min. After standing, high-speed refrigerated centrifugation (12000 rpm / min, 4°C) was performed to separate the layers. The upper colorless liquid was carefully aspirated (without aspirating the intermediate layer of protein precipitate and the lower red phenol-chloroform phase) and transferred to a new nuclease-free EP tube. 250 μL of isopropanol (pre-cooled in a -20°C refrigerator) was added to each EP tube, gently mixed until the liquid became clear, and allowed to stand at room temperature for 10 min. High-speed refrigerated centrifugation (12000 rpm / min, 4°C) was performed. The supernatant was discarded, 500 μL of 75% ethanol was added to each EP tube, and the precipitate was washed by screwing for 2 min. The centrifuge was refrigerated and centrifuged (7500 rpm / min, 4°C). The supernatant was discarded, the EP tube cap was opened, and the precipitate was air-dried at room temperature until it disappeared (10 μL of a pipette was used to aspirate as much liquid as possible to facilitate rapid air-drying of the RNA and prevent degradation). 20 μL of nuclease-free water was added to each EP tube, and the RNA was dissolved by gentle blowing. The concentration and purity of the extracted RNA were then detected using a microplate reader (the extracted RNA should be reverse transcribed into cDNA as soon as possible, and if not, it should be frozen at -80°C). A 20 μL reverse transcription system was then prepared (2 μg of RNA, 4 μL of 5×PrimeScript RT Master Mix, and DEPCH2O to a final volume of 20 μL). The prepared reverse transcription system was mixed and incubated in a 37°C water bath for 30 min. The cDNA obtained by reverse transcription was subjected to RT-qPCR using a Japanese TAKARA TB green kit. The reaction system (20 μL) was prepared as follows: primers (forward + reverse) 0.5 μL; DEPC water 9.5 μL; TB green + rox 9.5 μL; cDNA product 0.5 μL.Subsequent reaction procedure (two-step method, 40 cycles): pre-denaturation: 95°C, 30 s; denaturation 95°C, 5 s; annealing, elongation: 60°C, 30 s; dissolution reaction: 95°C, 5 s, 60°C, 1 min, 95°C, 15 s. The same reaction system and reaction procedure were repeated 3 times, and the average value was taken. The data were analyzed and the relative quantity (RQ) of CHIKV RNA bound to the cell surface was calculated and compared by the ΔΔCT method. The results are shown in Figure 4. Figure 5 As shown in Figure 4, empentivir did not affect the binding process of CHIKV.

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

[0050] Since empentivir did not affect the binding process of CHIKV, we then detected the effect of empentivir on viral membrane fusion. CHIKV was labeled with fluorescent dye DiD (DiD is a liposoluble fluorescent dye. When labeled on the viral envelope at a high concentration, fluorescence quenching occurs. After the viral envelope fuses with the cell membrane, the fluorescent molecules are uniformly dispersed, reducing the concentration and causing fluorescence de-quenching. Therefore, by observing the DiD fluorescence intensity, the membrane fusion of DiD-labeled molecules can be detected). 1 mL of CHIKV (5 x 10 7 PFU / ml) was added with 5 μL of DiD, mixed well and incubated in a 37°C incubator for 30 min.

[0051] The passaged human hepatoma cell line HuH-7 was inoculated in a 96-well plate at 50,000 cells per well, with 100 μL of culture medium, and cultured for 12 h. The original culture medium was then removed, and 100 μL of complete DMEM culture medium containing DiD-labeled CHIKV was added to each well, with a viral MOI (multiplicity of infection) of 10. After incubation on ice for 2 h, the supernatant was discarded, and 100 μL of complete DMEM culture medium containing empentivir (4 μM), NH4CL (50 mM) or DMSO was added. Detection was performed on a multifunctional enzyme labeler: temperature: 37°C; excitation wavelength: 644 nm; absorption wavelength: 665 nm; detection: 8 h, detection every 10 min, for a total of 48 times. By detecting the DiD fluorescence intensity, the membrane fusion of the virus was analyzed. The results are shown in Figure 5. Figure 6 As shown in Figure 5, both empentivir and NH4CL can significantly inhibit the membrane fusion process of CHIKV.

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

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

[0054] The empentivir dry powder was fully dissolved with corn oil solution, and was administered by gavage, the dose was 15 mg / Kg / day, once a day. A total of 24 6-week-old female C57BL / 6 mice were randomly divided into 3 groups, namely: 1, the drug administration group, the mice were administered by gavage on the day of virus attack, and then 50 μL of 2*10 5 PFU virus was administered by intranasal administration, 8 mice; 2, the control group, the mice were administered by gavage with corn oil on the day of virus attack, and then 50 μL of 2*10 5 PFU virus was administered by intranasal administration, 8 mice; 3, the blank control group, the mice were not subjected to virus attack and drug administration, 4 mice.

[0055] The body weight of the mice was measured from the day of virus attack (before infection), and the body weight of the mice was measured twice a day before gavage administration. The survival of the mice was observed twice a day (at the time of gavage administration and 12 hours after administration), and the changes in body weight and survival of the mice were recorded.

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

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

[0058] The above in vitro and in vivo experimental results show that empentivir has significant CHIKV activity, and can be used to prepare a drug for resisting CHIKV infection.

[0059] The foregoing generally describes the main features of the application and advantages of the application. It should be understood that the application is not limited in scope to the above embodiments and that the application can be practiced with various modifications and alterations without departing from the spirit and scope of the application. The application is limited only by the claims that follow and equivalents thereof.

Claims

1. The use of empentivir in the preparation of a drug for resisting the infection of the chikungunya virus.

2. Use according to claim 1, characterized in that: The drug for resisting the infection of the chikungunya virus is a drug composition containing empentivir as the only active ingredient or a drug composition containing empentivir.

3. Use according to claim 2, characterized in that: The drug composition containing empentivir refers to a drug composition containing empentivir and pharmaceutically acceptable excipient(s).

4. Use according to any one of claims 1 to 3, characterized in that: The drug is used for preventing or treating the infection of the chikungunya virus.

5. Use according to any one of claims 1 to 3, characterized in that: The administration form of the drug is selected from the group consisting of powder, tablet, granule, capsule, solution, emulsion and suspension.

6. Use according to any one of claims 1 to 3, characterized in that: The administration route of the drug is selected from the group consisting of injection administration and gastrointestinal administration.

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