Application of buquinalene in preparation of preparation for inhibiting influenza virus

Abstract

The invention discloses an application of buquinatraz in preparation of a preparation which is independently used or used in combination with other preparations to prevent and treat influenza, slow down influenza or inhibit influenza virus proliferation. The buquinaldine and the composition containing the buquinaldine are good in safety, can well inhibit influenza virus proliferation, and have application prospects for treating influenza.

Description

Technical Field

[0001] The present invention belongs to the field of pharmacy and relates to the use of buquinolate in the preparation of a preparation for inhibiting influenza virus. Background Art

[0002] Influenza virus is a representative virus of the family Orthomyxoviridae and is divided into four genera: A (alpha), B (beta), C (gamma), and D (delta). Among them, influenza A, B, and C viruses can all infect humans. According to WHO statistics, it is estimated that every year 5% to 10% of adults and 20% to 30% of children are infected with influenza, resulting in 3 to 5 million severe cases and about 1 million deaths globally.

[0003] Influenza A virus (IAV), because it can cause high morbidity and mortality rates, has become a respiratory pathogen of great economic and public health significance. Its viral genome consists of 8 segments of single-stranded negative-strand RNA and can encode 10 essential proteins, including PB2, PB1, PA, HA, NP, NA, M1, M2, NS1, NEP / NS2, and a variety of non-essential accessory proteins such as PB1-F2, PA-X, etc. Seasonal influenza A viruses are usually the H1N1 and H3N2 subtypes and have caused repeated epidemics of varying severity for decades. In the United States, influenza causes more than 200,000 hospitalizations every year and 3,000 to 49,000 deaths during non-epidemic seasons. Because of its frequent antigen drift and antigen shift, novel strains from other species can cause human influenza pandemics, such as the "swine flu" H1N1 and avian influenza H5N1 in 2009. Since humans have little immunity to them, they can spread rapidly worldwide, posing a major threat to global health.

[0004] Influenza virus is one of the major threats to global public health security. Vaccines have achieved good results in the prevention and control of animal influenza, but the prevention and control of human influenza has not achieved remarkable results due to problems such as low vaccination rates, fast virus mutation rates, and inconsistent vaccine protection.

[0005] Drugs are another important tool for preventing and controlling human influenza. Currently, drugs that have entered clinical use for treating influenza virus infections can be classified into first-generation M2 ion channel inhibitors, second-generation neuraminidase inhibitors (NAIs), and third-generation cap-dependent endonuclease inhibitors. As an RNA virus with a high mutation rate, influenza virus has rapidly evolved into multiple drug-resistant strains under the high-pressure screening of drugs in the more than fifty years since the advent of the first-generation anti-influenza drugs. Among the prevalent strains, drug-resistant strains to existing drugs already account for a considerable proportion. Given the limitations of the application of human influenza virus vaccines and the rapid emergence of drug resistance, the necessity of screening new small-molecule inhibitors of influenza virus has become increasingly prominent.

[0006] Triptonide, also known as PG 492, NSC 165677, with the English name Triptonide, CAS number 38647-11-9, and its conventional active use is as a Wnt signaling inhibitor. The molecular structural formula is as follows:

[0007]

[0008] Brequinar, also known as Bipenquinate, NSC 368390, DUP785, with the English name Brequinar, CAS number 96187-53-0, and its conventional active use is as a potent inhibitor of dihydroorotate dehydrogenase, with potent activity against a broad spectrum of viruses. The molecular structural formula is as follows:

[0009]

[0010] Voxelotor, also known as GBT 440, with the English name voxelotor, CAS number 1446321-46-5, and its conventional active use is as an inhibitor of sickle hemoglobin (HbS) polymerization. The molecular structural formula is as follows:

[0011]

[0012] Mizoribine, also known as Bredinin, NSC 289637, HE 69, with the English name Mizoribine, CAS number 50924-49-7, and its conventional active use is as an immunosuppressant. The molecular structural formula is as follows:

[0013]

[0014] Epalrestat, also known as ONO2235, Epalrestat, with the English name Epalrestat, CAS number 82159-09-9, and its conventional active use is as an aldose reductase inhibitor, effectively improving the related symptoms of diabetic neuropathy and delaying the progression of the disease. The molecular structural formula is as follows:

[0015]

[0016] There has been no report on the application of the aforementioned drugs in the treatment of influenza. Summary of the Invention

[0017] To solve the problems existing in the prior art, a first aspect of the present invention provides a pharmaceutical composition, and the pharmaceutically active ingredients of the pharmaceutical composition include a first active substance and a second active substance;

[0018] The first active substance is active substance a; the second active substance is active substance b or active substance c;

[0019] The active substance a is any one of buquineran, a medicinal salt of buquineran and a prodrug of buquineran, a combination of any two or a combination of three;

[0020] The active substance b is vosaroxin, any one of a medicinal salt of vosaroxin and a prodrug of vosaroxin, a combination of any two or a combination of three;

[0021] The active substance c is mizoribine, any one of a medicinal salt of mizoribine and a prodrug of mizoribine, a combination of any two or a combination of three;

[0022] The structural formula of the buquineran is as follows:

[0023]

[0024] The structural formula of the vosaroxin is as follows:

[0025]

[0026] The structural formula of the mizoribine is as follows:

[0027]

[0028] In some embodiments, when the second active substance is active substance b, the molar ratio of the first active substance to the second active substance is 1:2 - 180 (for example, any ratio among 1:2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180 or the range between any two ratios);

[0029] When the second active substance is active substance c, the molar ratio of the first active substance to the second active substance is 1:5 - 400 (for example, any ratio among 1:5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400 or the range between any two ratios).

[0030] In some embodiments, when the second active substance is active substance b, the molar ratio of the first active substance to the second active substance is 1:15 - 20;

[0031] When the second active substance is active substance c, the molar ratio of the first active substance to the second active substance is 1:30 - 50.

[0032] In some embodiments, the pharmaceutically acceptable salts of buquinarone are selected from aluminum salts, zinc salts, amine salts, ammonium salts, sodium salts, calcium salts, potassium salts, magnesium salts, silver salts, lithium salts, hydrochloride salts, sulfate salts, citrate salts, benzenesulfonate salts, hydrobromide salts, hydrofluoride salts, phosphate salts, acetate salts, propionate salts, succinate salts, oxalate salts, malate salts, succinate salts, fumarate salts, maleate salts, tartrate salts, trifluoroacetate salts, aspartate salts, taurine salts, gluconate salts, fructoate salts, salicylate salts, nitrate salts, p-toluenesulfonate salts, mesylate salts, benzoate salts, citrate salts, lactate salts, citrate salts, fumarate salts and ascorbate salts of buquinarone;

[0033] The pharmaceutically acceptable salts of voclosporin are selected from hydrochloride salts, sulfate salts, citrate salts, benzenesulfonate salts, hydrobromide salts, hydrofluoride salts, phosphate salts, acetate salts, propionate salts, succinate salts, oxalate salts, malate salts, succinate salts, fumarate salts, maleate salts, tartrate salts, trifluoroacetate salts, aspartate salts, taurine salts, gluconate salts, fructoate salts, salicylate salts, nitrate salts, p-toluenesulfonate salts, mesylate salts, benzoate salts, citrate salts, lactate salts, citrate salts, fumarate salts and ascorbate salts of voclosporin;

[0034] The pharmaceutically acceptable salts of mizoribine are selected from hydrochloride salts, sulfate salts, citrate salts, benzenesulfonate salts, hydrobromide salts, hydrofluoride salts, phosphate salts, acetate salts, propionate salts, succinate salts, oxalate salts, malate salts, succinate salts, fumarate salts, maleate salts, tartrate salts, trifluoroacetate salts, aspartate salts, taurine salts, gluconate salts, fructoate salts, salicylate salts, nitrate salts, p-toluenesulfonate salts, mesylate salts, benzoate salts, citrate salts, lactate salts, citrate salts, fumarate salts and ascorbate salts of mizoribine.

[0035] In some embodiments, the composition further contains excipients.

[0036] The second aspect of the present invention provides the use of a biomaterial in the preparation of a product for use alone or in combination with other preparations to improve health conditions;

[0037] The biomaterial is the pharmaceutical composition or active substance a described in the first aspect of the present invention;

[0038] The active substance a is any one of buquinate, a medicinal salt of buquinate, and a prodrug of buquinate, a combination of any two, or a combination of three;

[0039] The structural formula of the buquinate is:

[0040]

[0041] The use is selected from any one or a combination of the following U1, U2, and U3;

[0042] U1: The improvement of health conditions is to prevent influenza, treat influenza, slow down influenza, or inhibit the proliferation of influenza virus;

[0043] U2: The improvement of health conditions is to reduce the damage of influenza virus to the lungs, and the second active substance is active substance c;

[0044] U3: The improvement of health conditions is to reduce the harm of influenza virus to the body by inhibiting the RNA-dependent RNA polymerase of influenza virus.

[0045] In some embodiments, the product is selected from pharmaceuticals, health products, feeds, feed additives, foods, and food additives.

[0046] In some embodiments, the influenza virus is an influenza A virus, and the influenza is influenza A.

[0047] In some embodiments, the influenza virus is an H1N1 influenza virus, and the influenza is influenza caused by the H1N1 influenza virus.

[0048] In some embodiments, the influenza is selected from human influenza, avian influenza, and swine influenza. Description of the Drawings

[0049] Figure 1 Shows the statistical situation of the relationship between the inoculation MOI, drug concentration, and OD 450 Therebetween.

[0050] Figure 2 Shows the statistical situation of the virus infection titer under the intervention of 5 drugs.

[0051] Figure 3 Shows the fitting curves of 5 drugs CC 50 Thereof.

[0052] Figure 4 shows the fitting curves of 5 drugs EC 50

[0053] Figure 5 shows the results of the virus challenge protection experiment under 3 drug interventions

[0054] Figure 6 shows the results of the effects of 4 drugs on the polymerase activity of influenza A virus

[0055] Figure 7 shows the results of the effect of epalrestat on the neuraminidase activity of influenza virus

[0056] Figure 8 shows the results of the hydrogen bond analysis of the molecular docking model

[0057] Figure 9 shows the molecular docking results of epalrestat and influenza virus neuraminidase N1

[0058] Figure 10 shows the situation of 5 drugs inhibiting virus protein expression

[0059] Figure 11 shows the effect of siRNA interfering with drug target genes on influenza virus replication

[0060] Figure 12 shows the statistical situation of virus content under the intervention of combined use of 5 drugs Detailed implementation manners

[0061] To make the objectives, technical solutions and advantages of the present invention clearer, the following will further describe the embodiments of the present invention in detail with reference to the accompanying drawings

[0062] Example 1: Protective experiment against influenza virus infection

[0063] 1. Establishment of positive control method

[0064] Use oseltamivir phosphate (purchased from MCE Company, the same below) as the positive control drug to establish a method for indirectly screening anti-influenza virus small molecule compounds by CCK-8 cell viability

[0065] First, fill 100 μL of PBS in each well around the periphery of a transparent 96-well cell culture plate to prevent edge effects. Then seed MDCK cells into the wells at a density of 1×10 4 / well, and the culture medium is DMEM medium (purchased from Sigma-Aldrich Company, the same below) containing 10 v / v% fetal bovine serum (purchased from Wisent Company, the same below), with a dosage of 100 μL per well. Incubate at 37°C and 5% CO​2 Cultivate under the following conditions, and perform virus infection after the cell density in the well plate grows to 90%.

[0066] Use a multi-channel pipette to discard the culture medium in the wells, wash the cells twice with PBS to wash away the residual serum in the culture medium to prevent it from affecting virus infection. After rinsing once with Opti-MEM medium (purchased from Gibco, the same below) supplemented with TPCK trypsin (final concentration 0.5 μg / mL), infect the cells with influenza virus WSN strain (H1N1 type) (NCBI Taxonomy ID: 382835) at MOI 0, MOI 0.01, MOI 0.1, MOI 1, MOI 5, and MOI 10 respectively. Add 100 μL of Opti-MEM medium containing 0.5 μg / mL TPCK trypsin to each well, and incubate at 37 °C, 5% CO 2 After allowing the virus to adsorb for one hour under the conditions, discard the virus solution. Then, add Opti-MEM medium (containing 0.1 v / v% DMSO and 0.5 μg / mL TPCK trypsin) containing oseltamivir phosphate at concentrations of 400 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL, and 25 μg / mL to the wells inoculated with different MOIs respectively. Use Opti-MEM medium (containing 0.1 v / v% DMSO and 0.5 μg / mL TPCK trypsin) as the negative control, and incubate at 37 °C, 5% CO 2 After culturing for 24 h under the conditions, detect the cell viability in each well.

[0067] Discard all the liquid in the well plate, replace it with 100 μL / well of DMEM medium, and then add 10 μL of CCK-8 solution (from the Cell Counting Kit-8 for cell viability, purchased from MCE, the same below) to each well. Incubate at 37 °C, 5% CO 2 After culturing for 1 h under the conditions, immediately place it on ice and use an enzyme-linked immunosorbent assay reader to detect the OD of each well 450 , repeat each combination of MOI and each concentration of oseltamivir in 3 wells, measure the absorbance of each well 3 times and take the average value, and analyze the data using GraphPad Prism 8.0.2.

[0068] The CCK-8 detection results are as Figure 1As shown in the figure. It can be seen that when infected with the same MOI, different concentrations of oseltamivir groups showed a good cell activity gradient. As the concentration of oseltamivir increased, the cell activity increased, indicating that the inhibitory ability of the drug against the virus improved. At the same time, among the drug groups with the same concentration, different MOI infection groups also showed an obvious cell activity gradient. As the MOI increased, the cell viability decreased significantly. Under the same drug inhibitory ability, the more virus inoculated, the lower the cell viability, indicating that this method has good sensitivity. This method can use the OD value to reflect the inhibitory ability of the drug against the virus.

[0069] II. Primary screening, secondary screening and data analysis

[0070] According to the experimental results in subsection I, MOI 0.01 was selected for the screening experiment. The cell seeding and virus infection methods were the same as those in subsection I. After infecting the virus, they were respectively changed to 2,697 compounds in the FDA-approved drug library with a final concentration of 30 μM (test compounds, the small molecule library FDAL1300-Z417864 was purchased from Selleck). Opti-MEM medium containing 0.1 v / v% DMSO (containing 0.5 μg / mL TPCK trypsin) was used as the solvent. Each compound was repeated in 3 wells. At the same time, oseltamivir positive control was set, and 0.1 v / v% DMSO aqueous solution was used as the negative control. The experiment without virus infection was used as the blank control group, and other steps and parameters were processed in parallel. After culturing at 37 °C and 5% CO 2 For 24 h under the conditions, the cell viability was detected using the method in subsection I. The absorbance of each well was detected 3 times and the average value was taken. Compared with the control group, 67 compounds with stronger known virus activity (larger OD value) in each plate showed a protective effect on the cells after virus infection. Then, the same method was used to perform a secondary screening on the aforementioned 67 compounds, and the results showed that 26 compounds showed strong cell protection ability.

[0071] Example 2: Virus titer determination experiment

[0072] I. Identification of antiviral ability of compounds

[0073] A549 cells were seeded in a 12-well cell culture plate at a density of 1×10 5 / well. The culture medium was F12K medium containing 10 v / v% fetal bovine serum (purchased from Multicell, the same below), 1 mL per well. At 37 °C and 5% CO 2Cultivate under the specified conditions. After the cell density in the well plate grows to 90%, infect the cells with WSN (H1N1) virus at an MOI of 0.01. After the virus adsorbs for one hour, wash the cells with PBS, and replace the medium in each well with 1 mL of Opti-MEM medium (containing 0.125 μg / mL TPCK trypsin and 0.1 v / v% DMSO) containing 30 μM of the test compound (selected from 19 out of the 26 compounds obtained in Example 1). Set 0.1 v / v% DMSO aqueous solution as the negative control. After 24 h, collect 50 μL of the culture supernatant into a 1.5 mL EP tube, freeze it at -80 °C for later use, and then perform the plaque titration experiment.

[0074] II. Plaque Titration Experiment

[0075] Seed MDCK cells in a 12-well cell culture plate with the medium being 1×DMEM medium containing 0.5 μg / mL TPCK trypsin. After the cells grow to confluence, wash the cells twice with PBS to remove the residual medium and serum. Rinse once with 1×DMEM medium containing 0.5 μg / mL TPCK trypsin and set aside.

[0076] Thaw the culture supernatant in each well of Section I on ice, and perform 2-fold serial dilutions respectively using 1×DMEM medium containing 0.5 μg / mL TPCK trypsin. Take 100 μL of the virus solution and add it to the well plate for adsorption. Incubate at 37 °C, 5% CO 2 conditions, and shake the culture plate evenly every 10 min to ensure that the virus solution uniformly covers the cells. Dissolve low melting point agarose in ddH 2 O to prepare a 2 w / w% agarose solution, heat and melt it, and keep it in a 50 °C water bath. At the same time, take an equal volume of 2×DMEM medium and keep it in a 37 °C water bath. Mix an equal volume of the 2 w / w% agarose solution and 2×DMEM medium to prepare a 1×DMEM 1% agarose solution. After the virus infection is completed, aspirate the virus solution in the well plate, add 1 mL of 1×DMEM 1 w / w% agarose solution to each well, place the well plate in the biosafety cabinet for 20 min until the agarose solidifies, and then invert it and place it in the cell culture incubator. Incubate at 37 °C, 5% CO 2 conditions for 48 h. Then add 1 mL of formalin fixative to each well, fix overnight, discard the fixative and agarose, count the number of plaque formations, and calculate the virus titer. The titer refers to the number of biologically active virus particles in a unit volume of liquid. The quantitative representation method of the titer is the highest dilution multiple of the 2-fold serial dilution at which plaques can be seen, with the unit Log 2 pfu / mL.

[0077] Compared with the DMSO control, the titers of 5 compounds showed a significant decrease. These five compounds are triptonide (code: T18-9), buquinate (code: B18-5), vosaroxin (code: V15-24), mizoribine (code: M9-26), and epalrestat (code: E16-1). These 5 compounds decreased the virus titer by 99.99% (P < 0.001), 99.22% (P < 0.001), 99.84% (P < 0.001), 79.25% (P < 0.01), and 95.00% (P < 0.001) respectively. See the titer statistical chart in Figure 2 。

[0078] Example 3: Median cytotoxic concentration CC of the compound 50 Determination

[0079] Fill 100 μL of PBS into each well around the periphery of a transparent 96-well cell culture plate to prevent edge effects. Then seed A549 cells into the 96-well cell culture plate at a density of 1×10 4 / well. The culture medium is Opti-MEM medium, 100 μL per well, and culture at 37 °C and 5% CO 2 conditions. After the cell density in the well plate grows to 90%, discard the culture medium. Dissolve the test compounds triptonide, buquinate, vosaroxin, mizoribine, and epalrestat in Opti-MEM medium respectively, and set the final concentrations at gradient concentrations of 0.1 μM, 1 μM, 10 μM, 100 μM, 500 μM, 1000 μM, 5000 μM, and 10000 μM. Add them to different wells respectively, with an addition amount of 100 μL per well. Repeat each concentration in 3 wells and culture at 37 °C and 5% CO 2 conditions for 24 h, and then detect their cell viability. Replace the medium in each well with 100 μL of DMEM medium containing 10 v / v% fetal bovine serum, and then add 10 μL of CCK-8 solution to each well. After culturing at 37 °C and 5% CO 2 conditions for 1 h, immediately place on ice and use an enzyme-linked immunosorbent assay (ELISA) reader to detect the OD of each well 450 , measure the absorbance of each well 3 times and take the average value. Use the 0.1 v / v% DMSO aqueous solution well as the negative control, set its absorbance as 100%, calculate the cell viability of different concentration experimental groups, analyze the data using GraphPad Prism 8.0.2, and calculate the CC of the compound by non-linear regression and Nonlin fitting 50 。

[0080] See the fitting curve results in Figure 3 。The CC of triptonide, buquinate, vosaroxin, mizoribine, and epalrestat 50They were 1.304 mM, 558.4 μM, 344.2 μM, >10 mM, and 310.4 μM, respectively.

[0081] Example 4: Median effective concentration EC of the compound 50 Determination

[0082] Seed A549 cells at a density of 1×10 4 / well in a 24-well cell culture plate. The culture medium is F12K medium containing 10 v / v% fetal bovine serum, 500 μL per well. After the cells grow to 90%, infect the cells with influenza A virus WSN at an MOI of 0.01. Incubate at 37 °C and 5% CO 2 After culturing for 1 hour to allow the virus to adsorb, replace it with Opti-MEM medium (containing 0.125 μg / mL TPCK trypsin and 0.1 v / v% DMSO) containing different concentrations of the test compounds (triptonide, buquinar, vosaroxin, mizoribine, and epalrestat), 500 μL / well. Set gradient concentrations of 0.01 μM, 0.1 μM, 1 μM, and 10 μM for the final concentration of each compound. At the same time, set 0.1 v / v% DMSO aqueous solution as the negative control. Incubate at 37 °C and 5% CO 2 After culturing for 24 h, discard the supernatant from each well and extract the total RNA from the cells in each well.

[0083] After measuring the RNA concentration, operate according to the instructions of the HiscriptR RIIQ RT SuperMix for qPCR reverse transcription kit (Vazyme, R223-01). Remove genomic DNA from the total RNA in each well. Use 1 μg of RNA as a template and reverse transcribe the mRNA into cDNA with 5Seg_vRNA-RT (SEQ ID NO.1) in Table 1 as the primer.

[0084] According to the WSN strain reference gene sequence in NCBI, design fluorescence quantitative PCR primers using Primer Premier 5. The upstream and downstream primers are shown as 5Seg_NP qPCR-F (SEQ ID NO.2) and 5Seg_NP qPCR-R (SEQ ID NO.3) in Table 1. Use 5 μL of 2×ChamQ Universal SYBR qPCR Master Mix, 1 μL of cDNA template, 0.4 μL of each upstream and downstream primer, and ddH 23.2 μL of O3, and each reaction was repeated 3 times. The reaction procedure for fluorescence quantitative PCR was pre-denaturation at 95°C for 10 min; denaturation at 95°C for 15 s; annealing at 60°C for 1 min, and the denaturation and annealing were cycled 40 times in total; melting curve: 95°C for 15 s; 60°C for 1 min; 95°C for 10 s. The hGAPDH gene was used as an internal reference, and the primers for the internal reference gene were as shown in qPCR-hGAPDH-F (SEQ ID NO.4) and qPCR-hGAPDH-R (SEQ ID NO.5) in Table 1. The 0.1 v / v% DMSO aqueous solution group was used as a negative control, and the formula 2^-{ΔΔCt = [Ct(EG_vNP)-Ct(EG_hGAPDH)] - Ct(DMSO_vNP)-Ct(DMSO_hGAPDH)} was used to calculate the viral RNA content of the viral NP segment. GraphPad Prism 8.0.2 was used to analyze the data. After non-linear regression, Nonlin fitting was used to calculate the EC 50 .

[0085] The results of the fitting curve are shown in Figure 4 . The EC 50 of triptonide, buquinate, vosaroton, mizoribine and epalrestat were 0.6564 μM, 0.1323 μM, 2.213 μM, 5.003 μM and 0.8847 μM, respectively.

[0086] Table 1. Primer sequences

[0087]

[0088] Example 5: Organ plaque titration

[0089] The mice were divided into four groups: vosaroton, mizoribine, epalrestat and 5% DMSO, with 8 mice in each group. After weighing, they were anesthetized by inhalation of isoflurane. 1.5×10 4 pfu of influenza A virus WSN was inhaled through the nasal cavity. They were anesthetized and administered drugs through the nasal cavity in the same way every day. The administration doses of the three drugs were 1.52 mg / kg body weight, 3 mg / kg body weight, and 0.24 mg / kg body weight, respectively. The control group was administered a 5 v / v% DMSO aqueous solution with a dose of 25 μL per mouse.

[0090] On the 3rd day after virus challenge, 3 mice from each group were euthanized by cervical dislocation after being anesthetized by inhalation of isoflurane. The intact lung tissue was taken and placed in a 2 mL EP tube, and stored at -80°C. After thawing, 1 mL of PBS buffer containing 1% penicillin-streptomycin double antibody was added to each tube, and grinding beads were added. It was ground at 4°C and 30 Hz for 5 min. Then it was centrifuged at 9000 r / min at 4°C for 5 min, and the supernatant was taken for plaque titration experiment according to the method in Example 2.

[0091] The results are shown in Figure 5 . Compared with the control group, the lung virus titers of vosarotinib, mizoribine, and epalrestat decreased by 71.74% (P < 0.01), 76.74% (P < 0.001), and 84.13% (P < 0.001), respectively.

[0092] Example 6: Determination of influenza virus polymerase activity

[0093] I. Synthesis and source of plasmids

[0094] (1) Renilla luciferase control reporter gene vector pRL-TK

[0095] The pRL series vectors are Renilla luciferase reporter vectors developed by Promega (product number: E2241), and the Renilla luciferase is driven by the TK promoter.

[0096] (2) Plasmids of RNA polymerase components

[0097] Using the pCAGGS plasmid as the backbone, the coding sequences of the basic polymerase 1 (PB1) gene (see GenBank number LC333183.1), basic polymerase 2 (PB2) gene (see GenBank number LC333182.1), PA protein gene (see GenBank number LC333184.1), and nucleoprotein (NP) gene (see GenBank number LC333186.1) of the influenza virus WSN strain (full name A / WSN / 1933 strain, H1N1 type) were respectively transferred into the gene expression cassette of the pCAGGS plasmid to form recombinant plasmids capable of expressing PB1, PB2, PA protein, and NP of the influenza virus WSN strain, named pCAGGS-WSNPB1, pCAGGS-WSN PB2, pCAGGS-WSN PA, and pCAGGS-WSN NP, respectively.

[0098] (3) Firefly luciferase reporter gene vector pHH21-SC09NS F-Luc

[0099] Construct a firefly luciferase reporter gene vector pHH21-SC09NS F-Luc that can produce a negative-sense RNA containing 176 bases at the 3' end of the NS vRNA derived from the influenza virus SC09 strain (full name A / Sichuan / 1 / 2009 strain, H1N1 type), firefly luciferase, a stop codon (TAA), and 179 bases at the 5' end of the SC09NS vRNA. The influenza virus polymerase can recognize and bind to the non-coding sequence at the end of the NS vRNA on this vector to initiate the expression of firefly luciferase.

[0100] The construction methods of the plasmid vectors in (2) and (3) above can be found in the following literature:

[0101] Luo W, Zhang J, Liang L, et al., 2018. Phospholipid scramblase 1 interacts with influenza A virus NP, impairing its nuclear import and thereby suppressing virus replication. PLoS pathogens, 14(1): e1006851.

[0102] II. Principle of enzyme activity assay

[0103] The influenza virus RNA polymerase consists of three subunits, PB2, PB1, and PA. It is an RNA-dependent RNA polymerase and plays an important role in virus genome transcription together with the NP protein. To explore the effect of drugs on the activity of influenza virus polymerase, a dual-luciferase reporter assay was used.

[0104] In the Dual- Reporter System (purchased from Promega, catalog number: E1960), firefly luciferase is used as the reporter gene for gene expression, and Renilla luciferase is used as the internal reference gene. 1. The transcriptional regulatory element of the target gene is constructed into an expression vector carrying luciferase to construct a reporter gene plasmid, so that this sequence regulates the transcriptional expression of luciferase; 2. The reporter gene plasmid is transfected into cells, and after different treatments, the cells are lysed and the substrate luciferin is added. Luciferase can catalyze luciferin to emit fluorescence; 3. By detecting the fluorescence intensity, the effect of different treatment groups on this transcriptional regulatory element can be judged; 4. To avoid errors caused by differences in plasmid transfection efficiency, a Renilla luciferase reporter gene plasmid is usually transfected as an internal reference to correct the transfection efficiency between different samples.

[0105] The expression of Renilla luciferase is constitutive and can be used as a control, which is little affected by influenza virus inhibitors. The expression of firefly luciferase is affected by the content or activity of RNA polymerase and thus by influenza virus inhibitory drugs. The ratio of the fluorescence produced by the two luciferases can be used to quantitatively or semi-quantitatively determine the inhibitory ability of drugs on influenza virus RNA polymerase.

[0106] Construct 4 polymerase expression plasmids (pCAGGS-PA, pCAGGS-PB1, pCAGGS-PB2, pCAGGS-NP) of WSN (A / WSN / 33(WSN,H1N1)) to express intracellularly and form an influenza virus RNA polymerase complex.

[0107] Co-transfect the 4 polymerase expression plasmids, the firefly luciferase reporter gene vector pHH21-SC09NS F-Luc, and the Renilla luciferase control reporter gene vector pRL-TK into HEK293T cells, and add drugs to the cells simultaneously. After transfection, lyse the cells, load the samples onto a GloMax 96 microplate photometer (Promega), and use the dual luciferase reporter gene assay system to measure the luciferase activity of the transfected cells (the reaction of firefly luciferase with substrate LARⅡ produces a fluorescence signal, which is quenched by Stop&Glo reagent after being detected by the instrument, and at the same time, Renilla luciferase is activated to produce fluorescence) to analyze the effect of the drug on the influenza virus polymerase activity.

[0108] Data processing: First, calculate the ratio of Firefly luciferase / Renilla luciferase for each tube, and then use the ratio of the control group as unit 1 to obtain the relative luciferase activity of different treatment groups, which is the activity of the influenza virus polymerase.

[0109] II. Activity determination

[0110] Coat a 12-well cell culture plate with polylysine, then seed HEK293T cells in the 12-well cell culture plate. The culture medium is DMEM containing 10% FBS. After the cells grow to 80%, simultaneously transfect 500 ng of pCAGGS-WSN PB2, 500 ng of pCAGGS-WSN PB1, 500 ng of pCAGGS-WSN PA, 500 ng of pCAGGS-WSN NP plasmids, 200 ng of pHH21-SC09NS F-Luc, and 10 ng of pRL-TK plasmids into the cells. After 10 h of transfection, discard the culture medium containing the plasmids and transfection reagent, and replace it with HEK293T cell culture medium containing 20 μM and 30 μM of the test compounds (triptolide, buquinar, vosaroxin, and mizoribine) respectively, and then culture for another 26 h. After discarding the cell culture supernatant, wash once with PBS, lyse the cells in each well with 250 μL of Passive Lysis Buffer, centrifuge at 12000 r / min for 5 min, and take 20 μL of the supernatant to use the Dual- Reporter System for polymerase complex activity detection.

[0111] Under the action of gradient concentration compounds, taking the luciferase ratio of the 0.1 v / v% DMSO aqueous solution group as 100%, the polymerase activities of the 5 compounds that inhibit influenza virus replication in Example 2 were measured. The results are as Figure 6 shown. Triptonide, buquinate, vosaroton, and mizoribine can all significantly inhibit the polymerase activity of influenza virus, and the polymerase activity of the 30 μM group is lower than that of the 20 μM group, indicating that their inhibitory effects are dose-dependent. The polymerase activities of the 20 μM and 30 μM groups of triptonide decreased by 99.51% and 99.57% respectively compared with the control group (P < 0.001); the polymerase activities of the 20 μM and 30 μM groups of buquinate decreased by 98.96% and 99.19% respectively compared with the control group (P < 0.001); the polymerase activities of the 20 μM and 30 μM groups of vosaroton decreased by 48.04% and 70.52% respectively compared with the control group (P < 0.001); the polymerase activities of the 20 μM and 30 μM groups of mizoribine decreased by 18.55% and 28.11% respectively compared with the control group (P < 0.01).

[0112] It can be seen that these four drugs can all inhibit the activity of influenza virus RNA polymerase and contribute to the inhibition of influenza virus.

[0113] Example 7: Detection of influenza virus neuraminidase activity

[0114] I. Cell detection experiment

[0115] According to the instructions, a white non-bottom-transparent 96-well plate was used to reduce light loss and the influence between wells. 3×10 7 pfu / mL of influenza A virus WSN was diluted 1:100 (volume ratio) with NA-XTD TM Assay Buffer (purchased from Thermofisher), and 25 μL was added to each well. Similarly, the test compound (epalrestat) was diluted to 100 μM with the aforementioned Assay Buffer, and 25 μL was added to each well. Three wells were repeated and incubated at 37 °C for 20 min (at this time, the final concentration of the compound was 33.33 μM); 25 μL of 1:1000 (volume ratio) diluted 1000×NA-XTD TM Substrate (purchased from Thermofisher) was added to each well and incubated at room temperature for 30 min; 60 μL of NA-XTD TM Accelerator (purchased from Thermofisher) was added to each well, and the luminescence value was immediately detected using a GLOMAX 96 microplate luminometer. The detection time for each well was 1 s, and the average value was taken after measuring 3 times.

[0116] The neuraminidase activity is referred to Figure 7A. Thus, epalrestat showed inhibitory activity against influenza A virus neuraminidase.

[0117] Subsequently, the half maximal inhibitory concentration (IC 50 ) of epalrestat against neuraminidase, that is, the concentration of the compound required when the enzyme activity is inhibited by half, was detected to evaluate the inhibitory activity of the compound against the enzyme. Influenza A virus WSN at 3×10 7 pfu / mL was diluted 1:100 with NA-XTD TM Assay Buffer, and 25 μL was added to each well. The test compound was serially diluted with Assay Buffer, and gradient dilutions of 1000000 nM, 200000 nM, 100000 nM, 40000 nM, 20000 nM, 8000 nM, 1600 nM, 320 nM, 64 nM, 12.8 nM, 2.56 nM, 0.512 nM, and 0.1024 nM were set. Each concentration was repeated in 3 wells, that is, the final concentrations detected were 333333.33 nM, 66666.67 nM, 33333.33 nM, 13333.33 nM, 6666.67 nM, 2666.67 nM, 533.33 nM, 106.67 nM, 21.33 nM, 4.27 nM, 0.85 nM, 0.17 nM, and 0.03 nM. After incubation at 37 °C for 20 min, 25 μL of NA-XTD TM Substrate was added to each well and incubated at room temperature for 30 min; finally, 60 μL of NA-XTD TM Accelerator was added to each well. The luminescence value was detected using a GLOMAX 96 microplate luminometer, with a detection time of 1 s for each well, and the average value was taken after measuring 3 times. The data was analyzed using GraphPad Prism 8.0.2 software. After non-linear regression and Nonlin fitting, the IC 50 of the compound was calculated.

[0118] See the fitted curve in Figure 7 B. Thus, epalrestat showed concentration-dependent inhibitory activity against neuraminidase. After curve fitting, the drug concentration required for epalrestat to inhibit neuraminidase by half, that is, the IC 50 was 17.36 μM.

[0119] II. Molecular docking experiment

[0120] The protein-ligand structure was downloaded from the RCSB website (https: / / www.rcsb.org / ), with PBD ID: 6D96. This protein is the X-ray diffraction structure of influenza virus A / Brevig Mission / 1 / 1918 (H1N1) neuraminidase (NA) expressed in HEK-293E cells. The ligand E16-1 structure was obtained from the Pubchem website (https: / / pubchem.ncbi.nlm.nih.gov / ). Ligand preprocessing was performed using PyMOL software to remove water molecules, calcium ions, and residual ligands in the model, and at the same time, duplicate peptide chains were removed.

[0121] Then, the AutoDockTools-1.5.7 software was used to hydrogenate the protein model, set the docking box, and save it. The chemical bonds of the ligand were set to be rigid and flexible and saved as a pdbqt format file. The AutoDock vina command was used for molecular docking, with the parameters "eceptor=6d96 pymol.pdbqt; ligand=E16-1.pdbqt; center_x=17.652; center_y=-9.22; center_z=15.293; size_x=15.0; size_y=15.0; size_z=15.0; out=6d96-E16-1_out.pdbqt". For the output docking results, AutoDockTools-1.5.7 and PyMOL 2.5 were used for visualization, binding energy, and hydrogen bond analysis.

[0122] Using the AutoDock vina software, E16-1 was used as the ligand and docked into the active pocket of influenza virus N1 subtype neuraminidase, and a total of 9 docking models were output Figure 8 (A). After hydrogen bond analysis using the AutoDockTool software, the results are as shown in Figure 8 (B~F). Among them, E16-1 forms two hydrogen bonds with the binding pocket of neuraminidase in models 1, 2, 3, 6, and 7. The docking results were visually analyzed using PyMOL software, and the results are as shown in Figure 9 . A is the binding pocket, and B~F are the binding forms of models 1, 2, 3, 6, and 7 respectively. Among them, the binding energy of model 1 is the lowest, as shown in Figure 8 (A), which is -6.5 kcal / mol.

[0123] Analysis of the docking site of model 1 showed that the results are as shown in Figure 9 (G). The E16-1 molecule binds to the Arg292 and Arg371 sites of influenza virus N1 subtype neuraminidase through hydrogen bonds (yellow dotted lines). In addition, the binding site that appears multiple times in other docking models is Arg118.

[0124] Example 8: Detection of the Inhibition of Influenza Virus Protein Expression by Compounds

[0125] A549 cells were seeded in a 12-well cell culture plate at a density of 1×10 5 / well. The culture medium was F12K medium containing 10 v / v% fetal bovine serum, and the cells were cultured at 37 °C and 5% CO 2 . After the cell density in the well plate grew to 90%, the cells were infected with WSN (H1N1) virus at an MOI of 0.01. After one hour of virus adsorption, for each test compound, three wells were changed to 1 mL of Opti-MEM medium (containing 0.125 μg / mL TPCK trypsin and 0.1 v / v% DMSO) containing 5 μM, 10 μM, and 20 μM of the test compound (triptonide, buquineran, vosaroxin, mizoribine, and epalrestat). An aqueous solution of 0.1 v / v% DMSO was set as the negative control, and the positive control was oseltamivir phosphate. After 24 h, 50 μL of the culture supernatant was collected into a 1.5 mL EP tube and stored at -80 °C.

[0126] After discarding the cell culture supernatant, the cells were washed once with PBS. Then, 100 μL of 1×SDS lysis buffer, 1 μL of PMSF solution, and 0.1 μL of ribonuclease were added to each well. After lysing at room temperature for 5 min, the lysate was collected into a 1.5 mL EP tube and denatured at 95 °C for 10 min for Western Blot detection.

[0127] The above protein samples were added to the wells of a 10% SDS-PAGE gel at 15 μL / well and electrophoresed at 80 V. After the protein samples began to separate through the stacking gel, the voltage was changed to 120 V until the electrophoresis was completed. A 0.45 μm nitrocellulose membrane (NC membrane) was placed in the NC membrane equilibration solution and allowed to stand for 30 s, and then the membrane transfer was completed using an eblot L1 rapid wet membrane transfer instrument. After blocking with a 5% skim milk solution at room temperature for 1 h, the residual blocking solution was washed away with PBS solution. The primary antibody was diluted 1:1000 with PBS and incubated at low speed at room temperature on a shaker for 1 h. After incubation, the membrane was washed at high speed at room temperature on a shaker with PBST solution for 10 min, and this was repeated 3 times to wash away the unbound primary antibody. The secondary antibody was prepared at a ratio of 1:5000 (volume ratio), and the diluent was PBS. After incubating at room temperature in the dark for 1 h, the membrane was washed 3 times in the dark, each time for 10 min. The results were imaged and analyzed using an Odyssey CLX near-infrared scanning detector.

[0128] For the internal reference GAPDH, the primary antibody was a rabbit-derived GAPDH polyclonal antibody (Proteintech, catalog number 10494-1-AP), and the secondary antibody was Dylight 680-labeled goat anti-rabbit IgG (Li-COR Bioscience, catalog number 926-68071). For the influenza polymerase protein PB1, the primary antibody was a mouse-derived PB1 monoclonal antibody (self-made in the laboratory), and the secondary antibody was Dylight 680-labeled goat anti-mouse IgG (Li-COR Bioscience, catalog number 926-68070).

[0129] For the influenza virus nucleoprotein NP, the primary antibody was a mouse-derived NP monoclonal antibody (self-made in the applicant's laboratory), and the secondary antibody was Dylight 680-labeled goat anti-mouse IgG (Li-COR Bioscience, catalog number 926-68070).

[0130] The WB results and virus titers of each group are shown in Figure 10 . It can be seen that through gray value analysis of the protein expression, the virus protein could not be detected in the triptonide group; both the NP protein and the PB1 protein showed obvious gradient decreases in the buquinolate group; both the NP protein and the PB1 protein decreased in the vosaroxin group; the NP protein did not show an obvious decrease in the mizoribine group, while the PB1 protein showed a gradient decrease; the NP protein also did not show an obvious decrease in the epalrestat group, while the expression level of the PB1 protein showed a gradient decrease. In the positive control oseltamivir group, the content of the NP protein did not decrease significantly while the virus titer decreased significantly.

[0131] Example 9: Effects of siRNA interfering with drug target genes on influenza virus replication

[0132] According to the human gene sequences of the buquinolate target gene (gene name DHODH, Gene ID: 231, GenBank number NM_001361.5), the mizoribine target gene (gene name IMPDH2, GenBank number NM_001410759.1, Gene ID: 1723), and the epalrestat target gene (gene name AKR1B1, GenBank number NM_001628.4, Gene ID: 3615), siRNAs were designed respectively. The RNA sequences complementary to the target sequences in each siRNA are shown as si_231 (SEQ ID NO.6), si_1723 (SEQ ID NO.7), and si_3615 (SEQ ID NO.8) in Table 2 respectively, and the sequence shown as si_NC (SEQ ID NO.9) was used as the negative control. Using Lipofectamine TMThe siRNA and negative control si_NC were transfected into A549 cells by the reverse transfection method described in the RNAiMAX (purchased from Thermo Fisher Scientific, catalog number 13778030) instruction manual to interfere with the expression of related genes. One group of cells was harvested 36 h after infection, and the cell samples were subjected to Western Blot assay to detect the interference efficiency at the protein level. Another group of cells was infected with influenza A virus WSN at an MOI of 0.01 36 h after infection. After 1 h of infection, the medium was changed to Opti-MEM medium containing 0.125 μg / mL TPCK trypsin at 1 mL / well. The cell culture supernatants were harvested at 24 h and 48 h after infection, respectively, and the plaque titration assay was performed according to the method in Example 2 to determine the virus titer.

[0133] For the internal reference GAPDH, the primary antibody was a rabbit polyclonal antibody against GAPDH (Proteintech, catalog number 10494-1-AP), and the secondary antibody was a Dylight 680-labeled goat anti-rabbit IgG (Li-COR Bioscience, catalog number 926-68071). For the protein of the buquinolate target gene, the primary antibody was a mouse monoclonal antibody against DHODH (Proteintech, catalog number 67977-1-Ig), and the secondary antibody was a Dylight 680-labeled goat anti-mouse IgG (Li-COR Bioscience, catalog number 926-68070). For the protein of the mizoribine target gene, the primary antibody was a mouse monoclonal antibody against IMPDH2 (Proteintech, catalog number 67663-1-Ig), and the secondary antibody was a Dylight 680-labeled goat anti-mouse IgG (Li-COR Bioscience, catalog number 926-68070). For the protein of the epalrestat target gene, the primary antibody was a rabbit polyclonal antibody against AKR1B1 (Invitrogen, catalog number PA5-29718), and the secondary antibody was a Dylight 680-labeled goat anti-rabbit IgG (Li-COR Bioscience, catalog number 926-68071).

[0134] Table 2. siRNA sequences (sequences of the RNA strands complementary to the target sequences)

[0135]

[0136] After 36 h of siRNA interference, the expression levels of the buquinolate Target, mizoribine Target, and epalrestat Target genes all decreased ( Figure 11A), After being infected with the virus on this basis, the virus titer increased slightly at 24 h and 48 h. The virus titer of Buquinac Target increased by 1.86 times and 4.18 times respectively at 24 h and 48 h after infection; the virus titer of Mizoribine Target increased by 3.03 times and 6.87 times respectively at 24 h and 48 h after infection; the virus titer of Epalrestat Target increased by 3.39 times and 4.53 times respectively at 24 h and 48 h after being infected with the virus( Figure 11 B).

[0137] After inhibiting the target proteins in the cells treated with these three drugs by siRNA, the virus titer increased, indicating that the above-mentioned three target proteins were involved in the effect of the drugs in inhibiting the virus.

[0138] Example 10: Test on the effect of drug combination in inhibiting virus

[0139] I. Preparation of drugs

[0140] Prepare drug solutions of triptonide, buquinac, vosaroxin, mizoribine and epalrestat, and the solvent is 0.1 v / v% DMSO (the solvent is 0.1 v / v% DMSO). The concentrations are shown in Nos. 1-5 in Table 3 below (all are the EC 50 corresponding concentrations).

[0141] Any two of the above five drugs are combined (a total of ten combinations), and the stock solutions of the two drugs at twice the EC 50 corresponding concentrations are mixed in equal volumes to prepare a composition solution, and the concentrations of the two drugs in the composition are both the EC 50 corresponding concentrations. The solvent is 0.1 v / v% DMSO. The stock solution concentrations in the preparation method are shown in Nos. 6-15 in Table 3 below.

[0142] Set the following negative control, as shown in No. 16 in Table 3 below.

[0143] Using the liquid medicines in Nos. 1-15 in Table 3 as the test compounds respectively, and the solution shown in No. 16 as the negative control, with 12 replicates for each sample, and the other method steps are the same as those in Example 4, measure the content of influenza virus NP gene vRNA in the corresponding wells of each test solution. The specific content of vRNA is shown in the third column of Table 3. The statistical results of the content of influenza virus NP gene vRNA in the negative control, two single drugs and the combined use of these two single drugs in the drug combination are shown in Figure 12 .

[0144] It can be seen that the virus content tested for the two drugs in most of the compositions is the lowest, significantly better than that of the single drug, and some show obvious synergistic effects. It was not found that the virus content of the combined use of drugs was higher than that of the single drug, and there was no mutual antagonism between these drugs. Therefore, the combined use of the five drugs has better application prospects.

[0145] Table 3. Statistical Table of Drug Configuration and Virus Content

[0146]

[0147]

[0148] As is known by common technical knowledge, the present invention can be implemented by other embodiments that do not depart from its spiritual essence or essential features. Therefore, the above-disclosed embodiments are illustrative in all aspects and not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are encompassed by the present invention.

Claims

1. A pharmaceutical composition, wherein the pharmaceutical active ingredient of the pharmaceutical composition comprises a first active substance and a second active substance; The first active substance is active substance a; the second active substance is active substance b or active substance c; The active substance a is any one of buquinar, a pharmaceutically acceptable salt of buquinar and a prodrug of buquinar, a combination of any two or a combination of three; The active substance b is any one of vorseluoto, a pharmaceutically acceptable salt of vorseluoto and a prodrug of vorseluoto, a combination of any two or a combination of three; The active substance c is any one of mizoribine, a pharmaceutically acceptable salt of mizoribine and a prodrug of mizoribine, a combination of any two or a combination of all three; The structural formula of the buquina is: The structural formula of the Voseluoto is: The structural formula of the mizoribine is:

2. The pharmaceutical composition according to claim 1, characterized in that When the second active substance is active substance b, the molar ratio of the first active substance to the second active substance is 1:2-180; When the second active substance is active substance c, the molar ratio of the first active substance to the second active substance is 1:5-400.

3. The pharmaceutical composition according to claim 1 or 2, characterized in that When the second active substance is active substance b, the molar ratio of the first active substance to the second active substance is 1:15-20; When the second active substance is active substance c, the molar ratio of the first active substance to the second active substance is 1:30-50.

4. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that The pharmaceutically acceptable salt of buquinar is selected from aluminum salt, zinc salt, amine salt, ammonium salt, sodium salt, calcium salt, potassium salt, magnesium salt, silver salt, lithium salt, hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate, trifluoroacetate, aspartate, taurine, gluconate, fructose, salicylate, nitrate, p-toluenesulfonate, methanesulfonate, benzoate, citrate, lactate, citrate, fumarate and ascorbate of buquinar; The pharmaceutically acceptable salt of vorseluoto is selected from the group consisting of hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate, trifluoroacetate, aspartate, taurate, gluconate, fructose, salicylate, nitrate, p-toluenesulfonate, methanesulfonate, benzoate, citrate, lactate, citrate, fumarate and ascorbate of vorseluoto; The pharmaceutically acceptable salt of mizoribine is selected from the group consisting of hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate, trifluoroacetate, aspartate, taurine, gluconate, fructose, salicylate, nitrate, p-toluenesulfonate, methanesulfonate, benzoate, citrate, lactate, citrate, fumarate and ascorbate of mizoribine.

5. The pharmaceutical composition according to claim 1, characterized in that The composition further contains auxiliary materials.

6. Use of a biological material in the preparation of a product for use alone or in combination with other agents to improve health conditions; The biological material is the pharmaceutical composition or active substance a according to any one of claims 1 to 5; The active substance a is any one of buquinar, a pharmaceutically acceptable salt of buquinar and a prodrug of buquinar, a combination of any two or a combination of three; The structural formula of the buquina is: The use is selected from any one or a combination of the following U1, U2 and U3; U1: The improvement in health status is to prevent influenza, treat influenza, slow down influenza or inhibit the proliferation of influenza virus; U2: The improvement in health status is to reduce the damage of influenza virus to the lungs, and the second active substance is active substance c; U3: The improvement of health status is to reduce the harm of influenza virus to the body by inhibiting RNA polymerase that influenza virus RNA depends on.

7. The use according to claim 6, characterized in that The product is selected from medicines, health products, feeds, feed additives, foods and food additives.

8. The use according to claim 6, characterized in that The influenza virus is influenza A virus, and the influenza is influenza A.

9. The use according to claim 6, characterized in that The influenza virus is an H1N1 influenza virus, and the influenza is influenza caused by the H1N1 influenza virus.

10. The use according to claim 6, characterized in that The influenza is selected from human influenza, avian influenza, and swine influenza.

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