Use of sodium metatungstate in the preparation of a medicament for treating or preventing influenza virus infection

Abstract

This application belongs to the field of pharmaceutical technology. More specifically, it relates to the use of sodium metatungstate in the preparation of drugs for treating or preventing influenza virus infection. Experiments have shown that sodium metatungstate has a broad-spectrum anti-influenza virus effect and can effectively inhibit influenza virus strains of different serotypes such as H1N1 and H3N2 on cell models. In addition, it also has an inhibitory effect on oseltamivir-resistant virus strains. The study on the anti-influenza virus mechanism of sodium metatungstate found that sodium metatungstate does not directly inactivate influenza virus; although it causes agglutination of the virus during the virus adsorption process, it does not affect the occurrence of adsorption, nor does it affect the process of the virus being endocytosed into the cytoplasm. However, sodium metatungstate will block the nuclear entry of influenza virus vRNP, thereby inhibiting virus replication.

Description

Technical Field

[0001] This application belongs to the field of pharmaceutical technology, and more specifically, relates to the application of sodium metatungstate in the preparation of drugs for treating or preventing influenza virus infection. Background Art

[0002] Influenza virus is a virus of the family Orthomyxoviridae. The acute respiratory infectious disease caused by the influenza virus is called influenza, also known as the flu. Its main characteristics are suddenly occurring symptoms such as fever, sore throat, cough, nasal congestion, muscle pain, headache, fatigue, etc. Influenza viruses are divided into four types: influenza A virus (IAV), influenza B virus (IBV), influenza C virus (ICV), and influenza D virus (IDV). Among them, IAV and IBV are the main pathogens causing influenza. The genomes of IAV and IBV consist of 8 negative-sense single-stranded viral RNA gene segments (vRNAs), while ICV and IDV only have 7 vRNA gene segments. These gene segments can encode transcripts of 10 basic viral proteins and some accessory proteins. IAV is an enveloped virus. The viral envelope consists of a lipid bilayer containing three viral transmembrane proteins: hemagglutinin (HA), neuraminidase (NA), and ion channel protein (M2). The viral envelope is supported by the underlying matrix protein (M1). Inside the virus, the viral core is mainly composed of 8 viral ribonucleoproteins (vRNPs). Each vRNP consists of vRNA and the proteins bound to it. The vRNA is wrapped by multiple viral nucleoproteins (NPs) and binds to a single copy of the heterotrimeric viral polymerase composed of PB1, PB2, and PA proteins.

[0003] Currently, the prevention and treatment of influenza mainly rely on vaccines and antiviral drugs. Due to the easy mutation of influenza viruses, there are sometimes situations where seasonal vaccines have little effect on newly emerging pandemic viruses. In such cases, a matching vaccine must be produced each time. Antiviral drugs have always been a key tool in the fight against influenza viruses. Although many drugs targeting different target types have been developed and good therapeutic effects have been achieved in treatment, such as amantadine, oseltamivir, arbidol, etc. However, there are still some deficiencies, such as the emergence of drug-resistant strains for some drugs, relatively large drug side effects, and limited drug action coverage. Therefore, we urgently need to formulate new antiviral strategies to guide the development of anti-influenza drugs and improve the therapeutic effects of drugs. Finding highly effective anti-influenza drugs against wild-type and drug-resistant strains is of great significance for combating current and future influenza outbreaks caused by different influenza strains.

[0004] Sodium metatungstate, also known as sodium metatungstate monohydrate or sodium polytungstate, has been shown to have anti-human immunodeficiency virus activity by inhibiting the ATP hydrolase activity on the surface of extracellular and viral particles to inhibit virus infection. In addition, sodium metatungstate also has a variety of biological activities, such as inhibiting the activities of acetylcholinesterase and butyrylcholinesterase; having an anti-inflammatory effect on macrophages, inhibiting the release of TNF-α and nitric oxide by LPS-treated cells; it can also be used as an effective inhibitor of NTPDases, inhibiting tumor growth in mice. However, so far, no relevant reports on the anti-influenza virus activity of sodium metatungstate have been found. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present application provides the use of sodium metatungstate or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating or preventing influenza virus infection, thereby providing a safe and effective small molecule compound for the clinical treatment of influenza.

[0006] To achieve the above object, the present application provides the use of sodium metatungstate or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating or preventing influenza virus infection.

[0007] Preferably, the influenza virus is selected from one or more of influenza A virus and influenza B virus.

[0008] More preferably, the influenza A virus is selected from at least one of the virus strains of H1N1 subtype, H2N2 subtype and H3N2 subtype.

[0009] Generally speaking, compared with the prior art through the above technical solutions conceived by the present application, the following

[0010] Advantages:

[0011] (1) Sodium metatungstate is a small molecule compound. Experiments in the present application have shown that it has antiviral activity against a variety of influenza viruses on cells A549 and MDCK. Its CC 50 (half lethal concentration) in MDCK cells and A549 cells is greater than 500 μM. Sodium metatungstate can dose-dependently inhibit the replication of influenza virus on the two cell lines. Its EC 50 on MDCK is 0.82 μM, and its EC 50 (half inhibitory concentration) in A549 cells is 0.52 μM. By calculation, the selectivity index (SI) of sodium metatungstate in MDCK cells and A549 cells is greater than 609.76 and greater than 961.54 respectively.

[0012] (2) Experimental evidence in this application shows that sodium metatungstate has a broad-spectrum antiviral effect on a variety of influenza viruses and drug-resistant strains, with a dose-dependent effect. Through the study of the anti-influenza virus mechanism of sodium metatungstate, it is found that sodium metatungstate does not directly inactivate influenza viruses; although it causes virus agglutination during the virus adsorption process, it does not affect the adsorption or the process of the virus being endocytosed into the cytoplasm. However, sodium metatungstate blocks the nuclear entry of influenza virus vRNP, thereby inhibiting virus replication.

[0013] (3) Experimental evidence in this application shows that sodium metatungstate exerts its antiviral effect at an early stage of the influenza virus life cycle. Sodium metatungstate has the characteristics of good antiviral effect, low cytotoxicity, simple preparation, and low cost, which has great potential for drug research and development and treatment. Brief Description of the Drawings

[0014] Figure 1 shows the inhibition rate and cell viability of different concentrations of sodium metatungstate on PR8 influenza virus in MDCK;

[0015] Figure 2 shows the effect of different concentrations of sodium metatungstate on the titer of progeny virus of infectious PR8 influenza virus in MDCK;

[0016] Figure 3 shows the inhibition rate and cell viability of different concentrations of sodium metatungstate on PR8 influenza virus in A549;

[0017] Figure 4 shows the effect of different concentrations of sodium metatungstate on the titer of progeny virus of infectious PR8 influenza virus in A549;

[0018] Figure 5 Content A shows the antiviral effect of sodium metatungstate on PR8, H3N2, or oseltamivir-resistant PR8 (PR8(Ose)) virus in A549 and MDCK cells; the level of virus replication is represented by the expression level of NP protein; Content B is the quantitative analysis of the fluorescence intensity of Figure 5 Content A, and the relative fluorescence intensity is plotted against the concentration of sodium metatungstate;

[0019] Figure 6 Content A shows the effect of adding sodium metatungstate at different times after cell infection with influenza virus on the replication of PR8 influenza virus by detecting the expression level of NP protein using indirect immunofluorescence assay; Content B is the quantitative analysis of the number of infected cells in Content A, reflecting the effect of drug administration at different times on the level of virus replication; Content C shows the expression level of NP protein after infection detected by Western blot, reflecting the effect of sodium metatungstate administration at different times on the replication of influenza virus;

[0020] Figure 7 is a graph showing the relationship between the concentration of sodium metatungstate and the influenza virus titer;

[0021] Figure 8 Content A: While A549 cells were infected with the virus at 4°C, sodium metatungstate was added. The control cells were not treated with the drug. After 1 hour, indirect immunofluorescence was used to detect the NP protein to observe the adsorption of the virus to the cells. Content B: While A549 cells were infected with the virus at 4°C, sodium metatungstate was added. The control cells were not treated with the drug. After 1 hour, the cells were fixed, dehydrated, coated with a conductive layer, and observed for virus adsorption on the cell surface by scanning electron microscopy;

[0022] Figure 9 Content A: A549 was infected with PR8 virus at 37°C and treated with sodium metatungstate (100 μM). The cells were fixed at different time points and detected for NP by IFA. Content B: Statistical analysis was performed on the number of endocytosed viruses (in the cytoplasm) in each cell 3 hours after virus infection in Content A, and the number of endocytosed viruses in each cell was counted. Content C: Statistical analysis was performed on the nuclear entry of vRNA in each cell 3 hours and 5 hours after virus infection in Content A, and the number of cells with nuclear entry of vRNA was counted. Specific embodiments

[0023] In order to make the objectives, technical solutions, and advantages of the present application clearer, the present application will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0024] Experimental results of the present application show that sodium metatungstate can effectively inhibit the replication of influenza virus within the non-toxic range and can be further developed into a drug for treating or preventing influenza virus infection diseases, having broad application prospects. The English name of sodium metatungstate is Sodium metatungstate, and it has the structure shown in Structural Formula I:

[0025]

[0026] This application determined the anti-influenza virus activity of sodium metatungstate in two cell lines, namely human lung epithelial cell line A549 and canine kidney epithelial cell line MDCK. The results showed that this small molecule compound has significant antiviral activity, and the antiviral effect shows a dose-dependent effect. Sodium metatungstate has a broad-spectrum anti-influenza virus effect and can effectively inhibit influenza virus strains of different serotypes such as H1N1 and H3N2 on the cell model. In addition, it also has an inhibitory effect on oseltamivir-resistant virus strains. The study on the anti-influenza virus mechanism of sodium metatungstate found that sodium metatungstate has no direct inactivating effect on influenza virus; although it can cause virus agglutination during the virus adsorption process, it does not affect the occurrence of adsorption or the process of virus endocytosis into the cytoplasm; however, sodium metatungstate will block the nuclear entry of influenza virus vRNP, thereby inhibiting virus replication. Sodium metatungstate disclosed in this application is a new type of anti-influenza virus drug, which has the advantages of good safety, selection index, and broad-spectrum anti-influenza virus, and can be used to develop drugs for the treatment or prevention of influenza virus infection, with broad application prospects.

[0027] In some embodiments, the technical solution adopted in this application is:

[0028] Use of sodium metatungstate or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating or preventing influenza virus infection:

[0029] 1. Evaluate the toxicity, anti-PR8 virus activity of sodium metatungstate in a cell model and calculate its selection index, and the steps are as follows:

[0030] (1) Seed human alveolar macrophage cell line A549 and canine kidney epithelial cell line MDCK into a 96-well plate at an appropriate density until confluent monolayers are formed.

[0031] (2) If the antiviral effect is to be detected, simultaneously infect with an appropriate concentration of PR8 influenza virus.

[0032] (3) Simultaneously add sodium metatungstate diluted to different concentration gradients with culture medium and incubate for 48 h.

[0033] (4) Detect the cell viability of the drug-treated group and the untreated group to detect the cytotoxicity of sodium metatungstate.

[0034] (5) Detect the influenza virus neuraminidase activity in the supernatant of the drug-treated group and the untreated group to evaluate the antiviral activity of sodium metatungstate.

[0035] (6) Detect the progeny infectious virus particles in the supernatant of the drug-treated group and the untreated group to evaluate the antiviral activity of sodium metatungstate.

[0036] (7) Calculate the selection index of sodium metatungstate on the two cell lines.

[0037] 2. Evaluate the antiviral effect of sodium metatungstate against multiple influenza virus strains in a cell model, and the steps are as follows:

[0038] (1) Seed the human alveolar macrophage cell line A549 and the canine kidney epithelial cell line MDCK at an appropriate density in a 96-well plate until a confluent monolayer is formed;

[0039] (2) Infect with an appropriate concentration of PR8, H3N2, or oseltamivir-resistant PR8 (PR8(Ose)) virus;

[0040] (3) Add sodium metatungstate diluted to different concentration gradients with culture medium and incubate for 24 h;

[0041] (4) Detect the expression of influenza virus NP protein by immunofluorescence assay (IFA);

[0042] (5) Perform quantitative analysis on the fluorescence intensity of NP protein.

[0043] 3. Evaluate the stage of action of sodium metatungstate in the influenza virus life cycle in a cell model, and the steps are as follows:

[0044] 3.1 Preliminary exploration of the stage of action of sodium metatungstate in the influenza virus life cycle

[0045] (1) Seed the human alveolar macrophage cell line A549 at an appropriate density in a culture well until a confluent monolayer is formed;

[0046] (2) Infect with an appropriate concentration of PR8 influenza virus;

[0047] (3) Add sodium metatungstate at 0 h, 2 h, 4 h, 6 h, and 8 h post-infection respectively and incubate until 10 h post-infection;

[0048] (4) Detect the expression of influenza virus NP protein by immunofluorescence assay (IFA);

[0049] (5) Count the number of infected cells according to the expression of NP protein, calculate the proportion of cells occupied, and further calculate the virus replication level.

[0050] 3.2 Specific exploration of the accurate time period of action of sodium metatungstate in the influenza virus life cycle

[0051] (1) Seed the human alveolar macrophage cell line A549 at an appropriate density in a culture well until a confluent monolayer is formed;

[0052] (2) Infect with an appropriate concentration of PR8 influenza virus at 4 °C;

[0053] (3) Add sodium metatungstate at 0 min, 20 min, 40 min, and 60 min post-infection or during the -60 - 0 min period post-infection respectively and incubate until 8 h post-infection;

[0054] (4) Detect the expression of influenza virus NP protein and reference GAPDH protein in cells by Western blotting.

[0055] 4 Evaluate the direct inactivating effect of sodium metatungstate on influenza virus strains, and the steps are as follows:

[0056] (1) Seed MDCK at an appropriate density into culture wells until a confluent monolayer is formed.

[0057] (2) Mix the drug and the virus and incubate at 37 °C for 1 hour. Dilute the mixed solution serially 10-fold into 8 gradients and then use it to infect cells.

[0058] (3) Detect the virus titer by the TCID 50 method and compare it with the control group to determine the effect of the drug on virus activity.

[0059] 5 Explore the effect of sodium metatungstate on the process of influenza virus adsorption to cells, and the steps are as follows:

[0060] 5.1 Observe the adsorption of the virus under a laser confocal fluorescence microscope

[0061] (1) Seed the human alveolar macrophage cell line A549 at an appropriate density into a glass-bottomed culture dish until a confluent monolayer is formed.

[0062] (2) Infect with an appropriate concentration of PR8 influenza virus at 4 °C.

[0063] (3) Detect the distribution of influenza virus NP by immunofluorescence assay (IFA).

[0064] 5.2 Observe the adsorption of the virus under a scanning electron microscope

[0065] (1) Seed the human alveolar macrophage cell line A549 at an appropriate density onto a cell culture slide until a confluent monolayer is formed.

[0066] (2) Infect with an appropriate concentration of PR8 influenza virus at 4 °C.

[0067] (3) Directly observe the virus adsorbed on the cell surface with a scanning electron microscope.

[0068] 6 Explore the effect of sodium metatungstate on the endocytosis of influenza virus and the entry of vRNP into the nucleus, and the steps are as follows:

[0069] (1) Seed the human alveolar macrophage cell line A549 at an appropriate density into a glass-bottomed culture dish until a confluent monolayer is formed.

[0070] (2) Infect with an appropriate concentration of PR8 influenza virus.

[0071] (3) Add the sodium metatungstate drug solution simultaneously and incubate for 3 h and 5 h;

[0072] (4) Detect the distribution of influenza virus NP by immunofluorescence assay (IFA);

[0073] (5) Count the number of virus particles in the cytoplasm and the proportion of cells with infected nuclei.

[0074] The present invention provides the use of sodium metatungstate or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating or preventing influenza virus infection. In some embodiments, the influenza virus is selected from one or more of influenza A virus and influenza B virus. The influenza A virus is selected from at least one of virus strains of H1N1 subtype, H2N2 subtype, and H3N2 subtype, including some virus strains resistant to existing drugs. Currently marketed antiviral drugs for treating influenza include neuraminidase inhibitors (oseltamivir, zanamivir, peramivir) and RNA polymerase inhibitors (baloxavir).

[0075] The influenza viruses described in the embodiments of the present application include: influenza virus strains H1N1 (A / Puerto Rico / 8 / 1934; PR8), H1N1 oseltamivir-resistant strain (A / Puerto Rico / 8 / 1934, H274Y oseltamivir-resistant), and H3N2 (A / human / Hubei / 3 / 2005).

[0076] The present application also provides the use of sodium metatungstate or a pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting influenza virus replication in vitro, and the use of sodium metatungstate or a pharmaceutically acceptable salt thereof as the sole active ingredient in the preparation of a drug for treating or preventing influenza virus infection.

[0077] The anti-influenza evaluation model of the present application is mainly an in vitro model. The in vitro model mainly uses various influenza-sensitive cell lines or influenza pathology-related cell lines to evaluate the drug. Its advantages are that it can provide a large number of cells with the same genetic traits as the research object, is easy to operate, can eliminate the influence of other external factors, and can detect drug toxicity, effective concentration, and selection index, providing more basis for later mechanism research.

[0078] The present application uses the canine kidney epithelial cell line MDCK and the human lung epithelial cell line A549, which are closely related to influenza pathology, to quantitatively analyze the in vitro anti-influenza effect of sodium metatungstate and calculate its selection index. Subsequently, the infection stage of its action is evaluated to further explore its effects on virus adsorption, endocytosis into cells, and vRNP nuclear entry in the early stage of the influenza virus life cycle.

[0079] In some embodiments, the drug comprises sodium metatungstate or a pharmaceutically acceptable salt thereof, and further comprises a pharmaceutically acceptable excipient. The excipient is selected from one or more of a solvent, a dispersant, a diluent, a filler, a wetting agent, a binder, a disintegrant, a lubricant, a preservative, a suspending agent, an emulsifier, an excipient, a flavoring agent, and a carrier. The drug is a tablet, a capsule, a granule, a dropping pill, a liquid preparation, an extract, a suppository, a gel, an aerosol, or a patch. The liquid preparation includes, but is not limited to, an oral liquid and an injection.

[0080] The applications described in this application are selected from: avoiding or reducing the infection rate of influenza virus; inhibiting the growth of influenza virus; inhibiting the replication of influenza virus; inhibiting the inflammatory effect caused by influenza virus; increasing the survival rate of an organism infected with influenza virus; delaying the rate of body weight loss caused by influenza virus infection; improving or avoiding cell damage in the body caused by influenza virus infection; improving or avoiding alveolar structure damage in the body caused by influenza virus infection; improving or avoiding myocardial tissue damage in the body caused by influenza virus infection; or eliminating, improving, or avoiding otitis media in the body caused by influenza virus infection, at least one of which.

[0081] The term "pharmaceutically acceptable excipient" refers to a component that does not interfere with the biological activity efficacy of the compound sodium metatungstate and is not significantly toxic to the body at a therapeutically effective concentration of its administration, including any one or a combination of at least two of a solvent, a dispersant, a diluent, a filler, a wetting agent, a binder, a disintegrant, a lubricant, a preservative, a suspending agent, an emulsifier, an excipient, a flavoring agent, etc., and a carrier. The use of the foregoing components for pharmaceutically active substances is well known in the art. Each component can be used alone or in combination with a variety of others.

[0082] Experimental materials:

[0083] (1) Cell lines and viruses required for the experiment

[0084] Cell lines used:

[0085] Both MDCK and A549 cells were purchased from ATCC;

[0086] Virus strains used:

[0087] Influenza virus strains H1N1 (A / Puerto Rico / 8 / 1934; PR8), H1N1 oseltamivir-resistant strain (A / Puerto Rico / 8 / 1934, H274Y oseltamivir-resistant), and H3N2 (A / human / Hubei / 3 / 2005) were all provided by the Microbial (Virus) Strain Preservation Center of the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0088] (2) Drugs required for the experiment

[0089] Sodium metatungstate (CAS No.: 12141-67-2) used in the embodiments of the present application was purchased from Shanghai Macklin Biochemical Co., Ltd.; during cell experiments, the drug was dissolved in sterile deionized water.

[0090] (3) Reagents required for the experiment:

[0091] DMEM medium was purchased from Gibco;

[0092] Fetal bovine serum was purchased from Meilunbio;

[0093] TPCK-trypsin and MUNANA were purchased from Sigma;

[0094] Cell Titer-Glo assay kit was purchased from Promega;

[0095] Mouse-derived anti-NP protein primary antibody was purchased from Sino biological;

[0096] Anti-mouse fluorescent secondary antibody Alexa Fluor 488 was purchased from Cell Signaling Technology;

[0097] DAPI staining solution was purchased from Roche.

[0098] (4) Instruments required for the experiment:

[0099] Biological safety cabinet (AC2-4S1) was purchased from ESCO;

[0100] Cell incubator (Heracell VIOS160i) was purchased from Thermo;

[0101] Fluorescence inverted microscope (Eclipse Ti2-U) was purchased from NiKon;

[0102] Multifunctional microplate reader (Varioskan LUX) was purchased from Thermo;

[0103] Laser confocal fluorescence microscope (TCS SP8) was purchased from Leica;

[0104] Field emission scanning electron microscope (Ultra-55) was purchased from Zeiss.

[0105] Example 1

[0106] Evaluation of the cytotoxicity and anti-influenza virus activity of sodium metatungstate in MDCK and A549 cells

[0107] 1 Cell culture

[0108] After the cryopreserved and revived cells were passaged three times, they were expanded in DMEM medium containing 10% fetal bovine serum and double antibiotics (100 U / mL penicillin and 100 μg / mL streptomycin), and the seeding density was not less than 1.5×10 5 ce1l / mL, and the passage density was not higher than 5×10 5 cell / mL.

[0109] Cytotoxicity detection of sodium metatungstate

[0110] MDCK and A549 cells were seeded in 96-well cell culture plates at 1.5×10 4 cells / well (volume 100 μL), and cultured in an incubator at 37°C and 5% CO 2 for about 16 h to allow the cells to adhere and form a monolayer. Different concentrations of sodium metatungstate drugs were prepared with DMEM basal medium and added to the corresponding cell wells at 100 μL / well. Eight concentration gradients were set for the drugs, and 3 replicate wells were set for each gradient concentration, and their final concentrations were 0.2 μM, 0.7 μM, 2.1 μM, 6.2 μM, 18.5 μM, 55.6 μM, 166.7 μM, and 500 μM. They were cultured in an incubator containing 5% CO 2 at 37°C for 48 h.

[0111] After discarding the medium, 100 μL / well of Cell Titer-Glo detection reagent was added, and the culture plate was incubated on a shaker at room temperature for 10 minutes. Then, the incubated reagent was transferred to a black-walled and clear-bottom 96-well plate at 80 μL / well, and the chemiluminescence was measured using a microplate reader, and the readings were recorded.

[0112] The cell survival rate at different concentrations of sodium metatungstate was calculated according to the following formula:

[0113] Survival rate (%) = drug treatment group / untreated control group * 100%

[0114] The results are as Figure 1 and Figure 3 (legend circle line) shown. After MDCK and A549 cells were treated with sodium metatungstate at the highest concentration of 500 μM for 48 h, the cell viability was significantly decreased compared with the control group, but neither decreased to 50% of the control group activity, indicating that sodium metatungstate has a certain weak toxicity to MDCK and A549 cells at this concentration, and its half-toxic concentration CC 50 is greater than 500 μM.

[0115] 3 Antiviral activity of sodium metatungstate against influenza virus strain H1N1(PR8)

[0116] 3.1 Experimental principle: MUNANA (4-methylumbelliferyl-a-N-acetyl-neuraminate) is a specific substrate for influenza virus neuraminidase. The catalytic product generated under the action of neuraminidase can produce fluorescence at 450 nm when irradiated with excitation light at 370 nm. The fluorescence intensity represents the level of virus neuraminidase, so it can reflect the amount of virus in the supernatant of cultured cells.

[0117] 3.2 Seed MDCK and A549 cells at 1.5×10 4 cells / well (volume 100 μL) into 96-well cell culture plates, and culture them in an incubator at 37 °C and 5% CO 2 for about 16 h to allow the cells to adhere and form a monolayer. Add PR8 virus at 0.02 MOI (for MDCK) or 0.1 MOI (for A549) to the infection group, and at the same time add drugs at various gradient concentrations (the final concentration starts from 500 μM and is serially diluted 3-fold into 8 gradients, with 3 replicates for each gradient, and the total volume of the culture medium is 100 μL). After culturing for 2 h, discard the culture medium, wash twice with PBS, and add 100 μL of drug solution or DMEM basal medium at the same final concentration as above, and continue to culture in an incubator at 37 °C until 48 hpi. Take the supernatant of each experimental well for the detection of neuraminidase level and virus titer.

[0118] 3.3 Add 20 μL of the substrate 40 μmol / L MUNANA prepared with buffer (32.5 mM MES, 4 mM CaCl 2 , pH 6.5) to a black-bottom opaque 96-well microplate, then add 40 μL of the culture supernatant of each experimental well, incubate at 37 °C in the dark for 60 min, add the reaction termination solution (0.14 M NaOH, 83% ethanol) at 60 μL / well, and measure the fluorescence value on a microplate reader (excitation wavelength 370 nm, emission wavelength 450 nm).

[0119] 3.4 Calculate the inhibition rate of the drug on influenza virus replication in each detection well

[0120] Inhibition rate (%) = [1 - (drug-treated well - blank control) / (virus control well - blank control)] * 100%

[0121] 3.5 Seed MDCK cells at 1.5×10 4 cells / well (volume 100 μL) into 96-well cell culture plates, and culture them in an incubator at 37 °C and 5% CO 2Cultivate for about 16 h in an incubator to make the cells adhere and grow into a monolayer. Prepare DMEM basal medium containing TPCK-trypsin with a final concentration of 2 μg / mL, and dilute the supernatant solution collected in 3.2 by 10-fold gradient, with a total of 8 dilution degrees, and set 6 replicates for each dilution degree. Add 100 μL / well of the dilution solution into the cell wells, and add DMEM basal medium containing TPCK-trypsin to the normal cell control. After culturing in a 37 °C incubator for 2 h, aspirate the culture medium and wash twice with PBS. Add 100 μL / well of DMEM maintenance medium (2% fetal bovine serum) containing TPCK-trypsin with a final concentration of 2 μg / mL to all wells and continue to culture in the incubator.

[0122] The results are as Figure 1 and Figure 3 (legend triangle line) shows that sodium metatungstate significantly inhibits influenza virus replication and shows a dose-dependent relationship. Its half-maximal effective concentration (EC 50 in MDCK and A549 cells is 0.82 μM and 0.52 μM respectively.

[0123] The results are as Figure 2 and Figure 4 shown. Sodium metatungstate inhibits the production of progeny infectious virus particles in a dose-dependent manner on both MDCK and A549 cells.

[0124] 3.6 Calculation of drug selectivity index

[0125] The drug selectivity index (SI) is used to judge the safety range of drug effects. A selectivity index greater than 1.00 is effective, and the larger the index, the larger the safety range. The calculation formula is: SI = CC 50 / EC 50

[0126] Combined with the above data, sodium metatungstate is greater than 609.76 and greater than 961.54 in MDCK cells and A549 cells respectively, and belongs to a safe and highly effective anti-influenza virus drug.

[0127] Example 2

[0128] Evaluation of the anti-multiple influenza virus activities of sodium metatungstate in MDCK and A549 cells

[0129] Seed MDCK or A549 cells at 1.5×10 4 cells / well into a 96-well cell culture plate, and culture at 37 °C, 5% CO 2Cultivate for about 16 h in an incubator to allow the cells to adhere and grow into a monolayer. Prepare sodium metatungstate at different concentrations with DMEM basal medium and add it to the corresponding cell wells. A total of 5 concentration gradients are set, and their final concentrations are 2.22 μM, 6.67 μM, 20 μM, 60 μM, and 180 μM respectively. At the same time, add virus solutions with 0.02 and 0.1 MOI to MDCK and A549 cells respectively to a total volume of 100 μL. Each type of cell is infected with H1N1 (PR8) virus strain, H3N2 virus strain, and oseltamivir-resistant PR8 virus strain (PR8(Ose)), and cultivate in a 37 °C cell incubator for 24 h. Detect the expression of influenza virus NP by indirect immunofluorescence assay (IFA).

[0130] After discarding the supernatant, add PBS solution to wash the cells once and discard it. Add 50 μL / well of 4% paraformaldehyde and incubate at room temperature for 20 min to fix the cells. Add 100 μL / well of PBS solution to wash the cells twice, add 50 μL / well of PBS solution containing 0.3% Triton X-100, and incubate at room temperature for 20 min for cell permeabilization. After permeabilization, add 100 μL / well of PBS solution to wash the cells twice, add 50 μL / well of PBS solution containing 5% bovine serum albumin, and incubate in a 37 °C incubator for 1 h for blocking treatment. Add 100 μL / well of PBS solution to wash the cells twice, mix the mouse-derived anti-NP protein primary antibody and PBS solution at a ratio of 1:1000, then add 50 μL / well and incubate overnight in a 4 °C refrigerator. Add 100 μL / well of PBS solution to wash the cells three times, mix the anti-mouse fluorescent secondary antibody Alexa Fluor 488 and PBS at a ratio of 1:1000, add 50 μL / well and incubate for 1 h in a 37 °C dark environment. After incubation, add 100 μL / well of PBS solution to wash the cells twice, then observe the samples using a fluorescence inverted microscope, take and save the images, quantitatively analyze the fluorescence intensity of the NP protein using ImageJ software, and normalize the data with the virus control group as a reference.

[0131] The results are as Figure 5 shown in Content A. Sodium metatungstate inhibited the expression of NP protein of three virus strains, namely H1N1 (PR8) virus strain, H3N2 virus strain, and oseltamivir-resistant PR8 virus strain (PR8(Ose)), on MDCK and A549 cells, and showed a dose-dependent relationship.

[0132] Figure 5 Content B is Figure 5 after quantitatively analyzing the fluorescence intensity of Content A, plotting the relative fluorescence intensity against the concentration of sodium metatungstate, which reflects the dose-dependent antiviral effect of the drug. As Figure 5 shown in Content B, Figure 5The fluorescence intensity of content A can inhibit the expression of NP protein by nearly or more than 50% when the concentration is greater than 20 μM compared with the virus control group.

[0133] In summary, sodium metatungstate has a broad-spectrum antiviral effect against a variety of influenza viruses and also has an antiviral effect on influenza drug-resistant strains.

[0134] Example 3

[0135] Determine the stage of action of sodium metatungstate in the influenza virus life cycle

[0136] 1. Seed A549 cells at 6×10 3 cells / well into a 96-well cell culture plate and culture in an incubator at 37 °C and 5% CO 2 for 16 h. Add 0.5 MOI of PR8 virus solution to a total volume of 40 μL of culture medium (DMEM basal medium) and culture in an incubator at 37 °C and 5% CO2. At different time points after virus infection (0, 2, 4, 6, 8 h), add 10 μL of the drug solution to the corresponding cell wells to make the final concentration of sodium metatungstate 100 μM. Continue to culture in an incubator at 37 °C until 10 h post-infection. Detect the expression of influenza virus NP by indirect immunofluorescence assay (IFA). The protocol is the same as the IFA method in Example 2. Observe the samples using a fluorescence inverted microscope, take and save images. Calculate the number of infected cells based on the number of cells expressing the NP protein, calculate the total number of cells based on the number of cell nuclei stained with DAPI, calculate the proportion of infected cells as the number of infected cells / total number of cells, and normalize the data with reference to the proportion of infected cells in the virus control group to obtain the virus replication level.

[0137] The results are as Figure 6 shown in content A. Compared with the virus control group, the expression of NP protein decreased significantly when the drug was added at 0 - 10 hpi, and there was no significant difference in the expression of NP protein when the drug was administered at 2 - 10 hpi or more delayed time.

[0138] The results Figure 6 in content B are the statistics of the number of infected cells in Figure 6 content A, which also shows that adding the drug at 0 - 10 hpi can effectively reduce the number of virus-infected cells, while adding the drug at 2 - 10 hpi or more delayed time has no effect. These results indicate that the stage of action of sodium metatungstate is between 0 - 2 h in the influenza virus life cycle, belonging to its early stage.

[0139] 2. Seed A549 cells at 1.7×10 5 cells / well into a 12-well cell culture plate and culture in an incubator at 37 °C and 5% CO 2Cultivate for about 16 h in an incubator to make the cells adhere and grow into a monolayer. The drug solution, virus solution, DMEM basal medium, and cell culture plates are all placed in a 4°C refrigerator for pre-cooling for 30 min. Add 1 MOI virus at 4°C. For the -60 - 0 min group, simultaneously add a sodium metatungstate drug solution with a final concentration of 100 μM, and culture at 4°C for 1 h to allow the virus to adsorb to the cells. After infecting at 4°C for 1 h, discard the supernatant, add 1 mL of pre-warmed DMEM medium (recorded as 0 hpi at this time), and place it in an incubator at 37°C and 5% CO 2 Cultivate in an incubator. At different time points after infection (0 min, 20 min, 40 min, 60 min), add a sodium metatungstate drug solution with a final concentration of 100 μM to the corresponding cell wells. After 8 h of virus infection, detect the expression of virus NP protein and the internal reference protein GAPDH by Western Blot.

[0140] Add 50 μL / well of RIPA lysis buffer and place the cell culture plate on ice for incubation for 20 min to fully lyse the cells. Then collect the cell lysate, centrifuge at 12,000 rpm at 4°C for 10 min, transfer the supernatant to a new centrifuge tube, add Loading buffer (5×), and pipette to mix evenly. Boil in boiling water for 10 min to prepare the total cell protein sample.

[0141] Prepare a 10% SDS-PAGE electrophoresis gel, add 10 μL of protein sample to each well, and perform gel electrophoresis. After electrophoresis, take out the lower gel and transfer the protein in the gel to a PVDF membrane. Place the PVDF membrane in a TBST solution containing 5% skim milk and block at room temperature for 1 h. Wash 3 times with TBST on a shaker, 10 min each time. Cut out the target band from the membrane according to the position of the Marker, add the corresponding primary antibody solution (diluted 1000 times with TBST), and incubate overnight on a shaker at 4°C. Wash 3 times with TBST, 10 min each time. Add the horseradish peroxidase-labeled secondary antibody solution (diluted 1000 times with TBST) and incubate on a shaker at room temperature for 1 h. Wash 3 times with TBST on a shaker, 10 min each time. Prepare the ECL chemiluminescent solution and drop it to cover the entire membrane, place it in a chemiluminescent imaging system for development, and then take a photo and save the result.

[0142] The results are as Figure 6As shown in content C, GAPDH protein is an internal reference protein in cells, and there are no significant differences in all groups, indicating that the protein loading amounts are consistent. Compared with the virus control group, administration at 0 min - 8 h and 20 min - 8 h can effectively reduce the expression level of NP protein. There are no significant differences in administration at 40 min - 8 h and 60 min - 8 h, while the level of NP protein is up-regulated instead during -60 min - 0 h. According to the content of NP protein reflecting the replication level of influenza virus, this result shows that adding sodium metatungstate for treatment during 0 - 20 min after virus adsorption has a good inhibitory effect on virus replication.

[0143] Example 4

[0144] Direct inactivating effect of sodium metatungstate on influenza virus

[0145] Dilute sodium metatungstate drug with DMEM basal medium to prepare 500, 50, and 5 μM solutions. For the virus-inactivated group, mix the PR8 virus solution (10 6.5 TCID 50 / mL) and the sodium metatungstate drug solution in a volume ratio of 4:1, and incubate in a 37°C incubator for 1 h. Dilute the virus-drug mixed solution 100-fold after 1 h. For the control group, incubate the above virus solution and drug solution in a 37°C incubator for 1 h respectively, dilute 100-fold after 1 h respectively and mix in a volume ratio of 4:1. Measure the influenza virus titer of the mixed solution in both groups according to the method 3.5 in Example 1.

[0146] After incubating different concentrations of sodium metatungstate with PR8 virus at 37°C for 1 h, detect the virus titer by the TCID 50 method to determine whether the virus infectivity is reduced. The results are shown in Figure 7 As shown. There are no significant differences in the virus titers between the virus-inactivated group and the control group, indicating that sodium metatungstate has no direct inactivating effect on influenza virus.

[0147] Example 5

[0148] Effect of sodium metatungstate on influenza virus adsorption to cells

[0149] 1. Seed A549 cells at 1.2×10 5 cells / dish into glass-bottom culture dishes (the diameter of the glass slide is 15 mm), and culture at 37°C, 5% CO 2Cultivate for about 16 h in an incubator to allow the cells to adhere and grow into a monolayer. The drug solution, virus solution, DMEM basal medium, and cell culture dishes are all placed in a 4 °C refrigerator for pre-cooling for 30 min. At 4 °C, add 10 MOI of the virus to the cells on the central glass slide in the dish, and simultaneously add sodium metatungstate drug solutions with final concentrations of 100 μM, 10 μM, and 1 μM respectively. The total volume of the culture medium is 100 μL, and incubate at 4 °C for 1 h to allow the virus to adsorb to the cells. Detect the distribution of influenza virus NP by indirect immunofluorescence assay (IFA) to locate the position of influenza virus on the cells.

[0150] After virus adsorption, discard the supernatant, add pre-cooled PBS solution to wash the cells once and then discard it. Add 1 mL / dish of 4% paraformaldehyde and incubate at room temperature for 20 min to fix the cells. In subsequent operations, except that the PBS solution needs to cover the entire culture dish, other solutions are added dropwise to the central glass slide in the dish. Add 1 mL / dish of PBS solution to wash the cells twice, add 200 μL / dish of PBS solution containing 0.3% Triton X-100, and incubate at room temperature for 20 min for cell permeabilization. After permeabilization, add 1 mL / dish of PBS solution to wash the cells twice, add 200 μL / dish of PBS solution containing 5% bovine serum albumin, and incubate in a 37 °C incubator for 1 h for blocking treatment. Add 1 mL / dish of PBS solution to wash the cells twice, mix the mouse-derived anti-NP protein primary antibody and PBS solution at a ratio of 1:1000, then add 200 μL / dish and incubate overnight in a 4 °C refrigerator. Add 1 mL / dish of PBS solution to wash the cells three times, mix the anti-mouse fluorescent secondary antibody Alexa Fluor 488 and PBS at a ratio of 1:1000, add 200 μL / dish and incubate in a 37 °C dark environment for 1 h. Add 1 mL / dish of PBS solution to wash the cells three times, mix the DAPI staining solution and PBS solution at a ratio of 1:5000, add 200 μL / dish and incubate in a dark environment at room temperature for 10 min. After incubation, add 1 mL / dish of PBS solution to wash the cells twice, and then observe the samples using a laser confocal fluorescence microscope, take pictures and save the images.

[0151] The results are as Figure 8 shown in Content A. Compared with the uniform adsorption of influenza virus on the cell surface in the virus control group, the virus adsorbed on the cell surface in a aggregated and non-uniform manner under sodium metatungstate treatment, and the degree of aggregation increased with the increase of drug concentration. This indicates that sodium metatungstate does not affect the adsorption of influenza virus to cells, but causes virus aggregation.

[0152] 2 Place the cell coverslips (18 mm in diameter) into the wells of a 12-well cell culture plate, and inoculate A549 cells at 7.34×10 4 cells / well onto the coverslips, and incubate at 37 °C, 5% CO 2Cultivate for about 16 h in an incubator to make the cells adhere and grow into a monolayer. The drug solution, virus solution, DMEM basal medium, and cell culture dish are all placed in a 4 °C refrigerator for pre-cooling for 30 min. Add 2 MOI of virus to the cells at the central glass slide of the dish at 4 °C, and at the same time add the drug solution of sodium metatungstate with a final concentration of 100 μM. The total volume of the culture medium is 400 μL, and culture at 4 °C for 1 h to allow the virus to adsorb to the cells. Observe and directly locate the position of influenza virus on the cells with the aid of a scanning electron microscope.

[0153] After virus adsorption, discard the solution, add 500 μL / well of pre-cooled 2.5% glutaraldehyde solution for fixation for 2 h. Add 1 mL / well of PBS solution to wash 2 times, add ethanol aqueous solutions with different volume ratios of 500 μL / well for gradient dehydration, in the order of 10%, 30%, 50%, 70%, 90%, 100%, 100%, and dehydrate for 10 min at each gradient, then air-dry naturally overnight at room temperature. Place it in a sputter coater for gold spraying on the cell surface, and observe and photograph and record it under a field emission scanning electron microscope. The results are as Figure 8 shown in Content B. Compared with the influenza virus adsorbed on the cell surface in a uniformly dispersed form in the virus control group, the virus adsorbed on the cell surface in an aggregated and non-uniform manner under the treatment of sodium metatungstate.

[0154] In summary, sodium metatungstate does not affect the adsorption of influenza virus on the cell surface, but causes the aggregation of the virus in a drug concentration-dependent manner during the adsorption process.

[0155] Example 6

[0156] Effect of sodium metatungstate on endocytosis of influenza virus and nuclear entry of vRNP

[0157] Inoculate A549 cells at 1.2×10 5 cells / dish into a bottom glass culture dish (the diameter of the glass slide is 15 mm), and cultivate in an incubator at 37 °C and 5% CO 2 for about 16 h to make the cells adhere and grow into a monolayer. Add 1 MOI of virus to the cells at the central glass slide of the dish, and at the same time add the drug solution of sodium metatungstate with a final concentration of 100 μM. The total volume of the culture medium is 200 μL, and cultivate at 37 °C for 3 h or 5 h. Detect the expression and distribution of influenza virus NP by indirect immunofluorescence assay (IFA), and the protocol is the same as the IFA method in Example 5. Observe the samples with a laser confocal fluorescence microscope, photograph and save the images, calculate the number of virus particles in the cytoplasm of a single cell according to the number of NP fluorescence dots in the cytoplasm; judge whether the nucleus is infected by the virus according to the expression of NP in the nucleus, and calculate the proportion of cells with infected nuclei as the number of cells with infected nuclei / total number of cells.

[0158] The results are as Figure 9As shown in Content A, at 3 h and 5 h post-infection, sodium metatungstate treatment allowed the influenza virus to still enter the cytoplasm, but there was no distribution of NP protein in the nucleus, indicating that the nuclear entry of vRNP was blocked.

[0159] The results are as Figure 9 shown in Content B. By counting Figure 9 the number of viruses in the cytoplasm of individual cells at 3 hpi in Content A, compared with the virus group, the number of viruses in the cytoplasm under sodium metatungstate treatment increased. The reason is likely that at 3 h after influenza virus infection, its life cycle has reached the middle and late stages, that is, the stage after vRNP enters the nucleus, so the number of viruses in the cytoplasm decreases, while under sodium metatungstate treatment, the virus remains in the cytoplasm because it cannot enter the nucleus. This result indicates that the endocytosis of the virus is not affected.

[0160] The results are as Figure 9 shown in Content C. By counting Figure 9 the proportion of cells with infected nuclei at 3 hpi and 5 hpi in Content A, compared with the virus group, the number of cells with infected nuclei under sodium metatungstate treatment was significantly reduced. This result indicates that the nuclear entry of virus vRNP is blocked.

[0161] In summary, sodium metatungstate does not affect the process of influenza virus being endocytosed into the cytoplasm, but blocks the nuclear entry of virus vRNP.

[0162] In this application, the toxicity of sodium metatungstate to cells was detected on the human lung epithelial cell line A549 and the canine kidney epithelial cell line MDCK. Also, the antiviral activity of sodium metatungstate in the two cell lines was determined within a concentration range that was completely non-toxic. The results showed that this small molecule compound has significant antiviral activity, and the antiviral effect shows a dose-dependent effect. In the cell model, sodium metatungstate can effectively inhibit different influenza virus strains, showing a broad-spectrum antiviral effect. The mechanism of action of sodium metatungstate against influenza virus indicates that sodium metatungstate acts in the early stage of influenza virus infection, especially when administered within 0 - 20 minutes after virus infection, which can effectively reduce the level of virus replication. Sodium metatungstate has no direct inactivating effect on influenza virus, and at the same time does not affect virus adsorption, but causes virus aggregation during the virus adsorption process. Sodium metatungstate does not affect the process of virus being endocytosed into the cytoplasm, but blocks the nuclear entry of virus vRNP, thereby inhibiting virus replication.

[0163] It is easy for those skilled in the art to understand that the above description is only a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent replacements, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. Use of sodium metatungstate or its pharmaceutically acceptable salt in the preparation of a drug for treating or preventing influenza virus infection; the sodium metatungstate or its pharmaceutically acceptable salt is the only antiviral active ingredient in the drug; The influenza virus is influenza A virus; the influenza A virus is selected from at least one of the virus strains of H1N1 subtype and H3N2 subtype.

2. The use according to claim 1, characterized in that The medicine comprises sodium metatungstate or a pharmaceutically acceptable salt thereof, and also comprises a pharmaceutically acceptable auxiliary material.

3. The use according to claim 2, characterized in that The auxiliary material is selected from one or more of solvents, dispersants, fillers, wetting agents, binders, disintegrants, lubricants, preservatives, suspending agents, emulsifiers and flavoring agents.

4. The use according to claim 1, characterized in that The medicine is in the form of tablets, capsules, granules, pills, liquid preparations, decoctions, suppositories, gels, aerosols or patches.

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