Application of vocerotropine in preparation of preparation for inhibiting influenza virus

Abstract

The invention discloses an application of vocerotropine 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 vocerotropine and the composition containing the vocerotropine 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 vosaroton in the preparation of a preparation for inhibiting influenza virus. Background Art

[0002] Influenza virus is a representative virus of the family Orthomyxoviridae, which 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 5% to 10% of adults and 20% to 30% of children are infected with influenza every year, resulting in 3 to 5 million severe cases and about 1 million deaths globally.

[0003] Influenza A virus (IAV), as a respiratory pathogen of great economic and public health significance due to its high morbidity and mortality rates, has a viral genome composed of 8 segments of single-stranded negative-sense RNA and can encode 10 essential proteins, including PB2, PB1, PA, HA, NP, NA, M1, M2, NS1, NEP / NS2, and multiple non-essential accessory proteins such as PB1-F2, PA-X, etc. Seasonal influenza A viruses are usually the H1N1 and H3N2 subtypes, which have caused recurrent 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. Due to frequent antigenic drift and antigenic 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 globally, 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 have not achieved significant results due to problems such as low vaccination rates, rapid virus mutation rates, and inconsistent vaccine protection.

[0005] Drugs are another important tool for preventing and controlling human influenza. Currently, drugs used in clinical treatment of 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 past 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 drug 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 active substance a and active substance b;

[0018] The active substance a is any one of vosarotin, a medicinal salt of vosarotin, and a prodrug of vosarotin, any combination of two of them, or a combination of three of them;

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

[0020] The structural formula of the vosarotin is as follows:

[0021]

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

[0023]

[0024] In some embodiments, the molar ratio of the active substance a to the active substance b is 1:0.04 - 4 (for example, any ratio among 1:0.04, 0.05, 0.06, 0.07, 0.80, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4 or the range between any two ratios).

[0025] In some embodiments, the molar ratio of the active substance a to the active substance b is 1:0.3 - 0.5.

[0026] In some embodiments, the medicinal salt of the vosarotin is selected from hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate, trifluoroacetate, aspartate, taurine salt, gluconate, fructoate, salicylate, nitrate, p - toluenesulfonate, mesylate, benzoate, citrate, lactate, citrate, fumarate, and ascorbate of vosarotin;

[0027] The medicinal salt of the epalrestat is selected from aluminum salt, zinc salt, amine salt, ammonium salt, sodium salt, calcium salt, potassium salt, magnesium salt, silver salt, and lithium salt of epalrestat.

[0028] In some embodiments, the composition further contains excipients.

[0029] 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;

[0030] The biomaterial is the pharmaceutical composition or active substance a described in the first aspect of the present invention;

[0031] The active substance a is any one of vosaroton, the medicinal salt of vosaroton and the prodrug of vosaroton, any combination of two of them or a combination of three of them;

[0032] The active substance b is epalrestat, any one of the medicinal salt of epalrestat and the prodrug of epalrestat, any combination of two of them or a combination of three of them;

[0033] The structural formula of vosaroton is:

[0034]

[0035] The use is selected from any one or a combination of the following U1, U2, U3 and U4;

[0036] U1: The improvement of health conditions is to prevent influenza, treat influenza, slow down influenza or inhibit the proliferation of influenza virus;

[0037] U2: The improvement of health conditions is to reduce the damage of influenza virus to the lungs;

[0038] 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;

[0039] U4: The improvement of health conditions is to reduce the harm of influenza virus to the body by inhibiting the neuraminidase of influenza virus.

[0040] In some embodiments, the product is selected from drugs, health products, feeds, feed additives, foods and food additives.

[0041] In some embodiments, the influenza virus is influenza A virus and the influenza is influenza A.

[0042] In some embodiments, the influenza virus is H1N1 influenza virus and the influenza is influenza caused by H1N1 influenza virus.

[0043] In some embodiments, the influenza is selected from human influenza, avian influenza and swine influenza. Description of the Drawings

[0044] Figure 1 Shows the statistics of the relationship between the inoculation MOI, drug concentration and OD 450 among them.

[0045] Figure 2 Shows the statistics of virus infection titers under the intervention of 5 drugs.

[0046] Figure 3 Shows the CC 50 fitting curve of 5 drugs.

[0047] Figure 4 Shows the EC 50 fitting curve of 5 drugs.

[0048] Figure 5 Shows the results of the virus challenge protection experiment under the intervention of 3 drugs.

[0049] Figure 6 Shows the results of the effects of 4 drugs on the polymerase activity of influenza A virus.

[0050] Figure 7 Shows the results of the effect of epalrestat on the neuraminidase activity of influenza virus.

[0051] Figure 8 Shows the results of the hydrogen bond analysis of the molecular docking model.

[0052] Figure 9 Shows the molecular docking results of epalrestat and influenza virus neuraminidase N1.

[0053] Figure 10 Shows the situation of 5 drugs inhibiting virus protein expression.

[0054] Figure 11 Shows the effect of siRNA interfering with drug target genes on influenza virus replication.

[0055] Figure 12 Shows the statistics of virus content under the intervention of combined use of 5 drugs. Detailed implementation manners

[0056] 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.

[0057] Example 1: Influenza virus infection protection experiment

[0058] I. Establishment of positive control method

[0059] 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.

[0060] First, 100 μL of PBS was filled in each well around the periphery of the transparent 96-well cell culture plate to prevent edge effects. Then, MDCK cells were seeded into the wells at a density of 1×10 4 / well, and the culture medium was DMEM medium (purchased from Sigma-Aldrich, the same below) containing 10 v / v% fetal bovine serum (purchased from WISENT, the same below), with a dosage of 100 μL per well. The cells were cultured at 37 °C and 5% CO 2 2. After the cell density in the well plate grew to 90%, virus infection was carried out.

[0061] The culture medium in the wells was discarded using a multi-channel pipette, and the cells were washed 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), the cells were infected 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. 100 μL of Opti-MEM medium containing 0.5 μg / mL TPCK trypsin was added to each well. After the virus adsorbed for one hour at 37 °C and 5% CO 2 2, the virus solution was discarded. Then, 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 was added to the wells inoculated with different MOIs respectively. Opti-MEM medium (containing 0.1 v / v% DMSO and 0.5 μg / mL TPCK trypsin) was used as a negative control. After culturing for 24 h at 37 °C and 5% CO 2 2, the cell viability in each well was detected.

[0062] The liquid in the well plate was completely discarded and replaced with 100 μL / well of DMEM medium. Then, 10 μL of CCK-8 solution (from the cell viability kit Cell Counting Kit-8, purchased from MCE, the same below) was added to each well. After culturing for 1 h at 37 °C and 5% CO 2 2, it was immediately placed on ice and the OD of each well was detected using a microplate reader. 450 For each combination of MOI and oseltamivir concentration, 3 wells were repeated. The absorbance of each well was detected 3 times and the average value was taken. GraphPad Prism 8.0.2 was used to analyze the data.

[0063] The CCK-8 detection results are asFigure 1 As 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 drug's inhibitory ability 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 drug's inhibitory ability against the virus.

[0064] II. Primary screening, secondary screening and data analysis

[0065] 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 FDA L1300-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 method in subsection I was used to detect the cell viability, and 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 as a result, 26 compounds showed strong cell protection ability.

[0066] Example 2: Virus titer determination experiment

[0067] I. Identification of the antiviral ability of compounds

[0068] 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 2Cultured under the conditions, 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, the cells were washed with PBS, and each well was replaced 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). A 0.1 v / v% DMSO aqueous solution was set as the negative control. After 24 h, 50 μL of the culture supernatant was collected into a 1.5 mL EP tube, frozen at -80 °C for later use, and then the plaque titration experiment was carried out.

[0069] II. Plaque Titration Experiment

[0070] MDCK cells were seeded in a 12-well cell culture plate, and the medium was 1×DMEM medium containing 0.5 μg / mL TPCK trypsin. After the cells grew confluent, the cells were washed twice with PBS to remove the residual medium serum. The cells were rinsed once with 1×DMEM medium containing 0.5 μg / mL TPCK trypsin and set aside for later use.

[0071] The culture supernatant in each well of Subsection I was melted on ice, and 2-fold serial dilutions were performed respectively using 1×DMEM medium containing 0.5 μg / mL TPCK trypsin. 100 μL of the virus solution was taken and added to the well plate for adsorption, and cultured at 37 °C and 5% CO 2 conditions, and the culture plate was shaken evenly every 10 min to make the virus solution evenly cover the cells. The low melting point agarose was dissolved in ddH 2 O to prepare a 2 w / w% agarose solution, heated and melted, and then placed in a 50 °C water bath for insulation. At the same time, an equal volume of 2×DMEM medium was incubated in a 37 °C water bath for insulation. An equal volume of the 2 w / w% agarose solution and 2×DMEM medium were mixed to prepare a 1×DMEM 1% agarose solution. After the virus infection was completed, the virus solution in the well plate was aspirated, and 1 mL of 1×DMEM 1 w / w% agarose solution was added to each well. The well plate was placed in a biosafety cabinet for 20 min until the agarose solidified, and then inverted and placed in a cell culture incubator. After culturing at 37 °C and 5% CO 2 conditions for 48 h, 1 mL of formalin fixative was added to each well. After fixing overnight, the fixative and agarose were discarded, and the number of plaque formations was counted and the virus titer was calculated. 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.

[0072] 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), voxelotor (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 .

[0073] Example 3: Median cytotoxic concentration CC of the compound 50 Determination

[0074] Fill 100 μL of PBS into each well around the periphery of a transparent 96-well cell culture plate to prevent edge effects, and then seed A549 cells in 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 2. After the cell density in the well plate grows to 90%, discard the culture medium. Dissolve the test compounds triptonide, buquinate, voxelotor, 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, and repeat 3 wells for each concentration. Culture at 37 °C and 5% CO 2 2 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 2 for 1 h, immediately place on ice and use an enzyme-linked immunosorbent assay (ELISA) reader to detect the OD 450 of each well. 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 50 of the compound by non-linear regression and Nonlin fitting.

[0075] See the fitting curve results in Figure 3 . The CC 50They were 1.304 mM, 558.4 μM, 344.2 μM, >10 mM, and 310.4 μM, respectively.

[0076] Example 4: Median effective concentration EC of the compound 50 Determination

[0077] A549 cells were seeded in a 24-well cell culture plate at a density of 1×10 4 / well, and the culture medium was F12K medium containing 10 v / v% fetal bovine serum, 500 μL per well. After the cells grew to 90%, they were infected with influenza A virus WSN at an MOI of 0.01. After culturing for 1 hour at 37°C and 5% CO 2 conditions to allow virus adsorption, the medium was replaced 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. The final concentrations of each compound were set at gradient concentrations of 0.01 μM, 0.1 μM, 1 μM, and 10 μM. At the same time, 0.1 v / v% DMSO aqueous solution was set as a negative control. After culturing for 24 h at 37°C and 5% CO 2 conditions, the supernatant was discarded from each well, and the total RNA in the cells of each well was extracted separately.

[0078] After measuring the RNA concentration, according to the instructions of the HiscriptR RIIQ RT SuperMix for qPCR reverse transcription kit (Vazyme, R223-01), genomic DNA was removed from the total RNA in each well. Using 1 μg of RNA as a template and 5Seg_vRNA-RT (SEQ ID NO.1) in Table 1 as a primer, the mRNA in it was reverse transcribed into cDNA.

[0079] According to the WSN strain reference gene sequence in NCBI, fluorescence quantitative PCR primers were designed 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. Each reaction used 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 fluorescence quantitative PCR reaction program 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 shown as 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 .

[0080] 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.

[0081] Table 1. Primer sequences

[0082]

[0083] Example 5: Organ plaque titration

[0084] 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 inhaling isoflurane. 1.5×10 4 pfu of influenza A virus WSN was inhaled through the nasal cavity. They were anesthetized and administered 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 5 v / v% DMSO aqueous solution, and the dose was 25 μL per mouse.

[0085] On the 3rd day after virus challenge, 3 mice from each group were anesthetized by inhaling isoflurane and then euthanized by cervical dislocation. The intact lung tissues were taken and placed in 2 mL EP tubes, 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 steel beads were added. They were ground at 4°C and 30 Hz for 5 min. Then they were centrifuged at 9000 r / min and 4°C for 5 min, and the supernatant was taken for plaque titration experiment according to the method in Example 2.

[0086] The results are shown in Figure 5 . Compared with the control group, the lung virus titers of vosaroxin, mizoribine, and epalrestat decreased by 71.74% (P < 0.01), 76.74% (P < 0.001), and 84.13% (P < 0.001), respectively.

[0087] Example 6: Determination of influenza virus polymerase activity

[0088] I. Synthesis and source of plasmids

[0089] (1) Renilla luciferase control reporter gene vector pRL-TK

[0090] 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.

[0091] (2) Plasmids encoding RNA polymerase components

[0092] Using the pCAGGS plasmid as the backbone, the coding sequences of the basic polymerase 1 (PB1) gene (see GenBank accession number LC333183.1), basic polymerase 2 (PB2) gene (see GenBank accession number LC333182.1), PA protein gene (see GenBank accession number LC333184.1), and nucleoprotein (NP) gene (see GenBank accession number LC333186.1) of the influenza virus WSN strain (full name A / WSN / 1933 strain, H1N1 subtype) 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.

[0093] (3) Firefly luciferase reporter gene vector pHH21-SC09NS F-Luc

[0094] 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 subtype), 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 NS vRNA end of this vector to initiate the expression of firefly luciferase.

[0095] The construction methods of the plasmid vectors in (2) and (3) above can be found in the following literature:

[0096] 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.

[0097] II. Principle of enzyme activity assay

[0098] 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.

[0099] In the Dual-Luciferase Reporter System (purchased from Promega, catalog number: E1960), firefly luciferase is used as the reporter gene for gene expression, while 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 values obtained, the effects of different treatment groups on this transcriptional regulatory element can be judged; 4. To avoid errors caused by differences in plasmid transfection efficiency when transfecting cells, a reporter gene plasmid of Renilla luciferase is usually transfected as an internal reference to correct the transfection efficiency between different samples.

[0100] The expression of Renilla luciferase is constitutive and can be used as a control, and it is little affected by influenza virus inhibitors. The expression of firefly luciferase is affected by the content or activity of RNA polymerase and is thus affected 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.

[0101] Construct four 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.

[0102] Co-transfect the four 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 activity of influenza virus polymerase.

[0103] Data processing: First, calculate the ratio of Firefly luciferase / Renilla luciferase in each tube, and then take the ratio of the control group as unit 1 to obtain the relative luciferase activity of different treatment groups, that is, the activity of influenza virus polymerase.

[0104] II. Activity measurement

[0105] Coat a 12-well cell culture plate with polylysine, and 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%, co-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 Reporter System for detecting the activity of the polymerase complex.

[0106] Under the action of compounds with gradient concentrations, using 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, vosaroxin, 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 vosaroxin 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).

[0107] Thus, these four drugs can all inhibit the RNA polymerase activity of influenza virus, which helps to inhibit influenza virus.

[0108] Example 7: Detection of influenza virus neuraminidase activity

[0109] I. Cell detection experiment

[0110] According to the instructions, a white non-bottom-transparent 96-well plate was used to reduce light loss and the influence between wells. Influenza A virus WSN at 3 × 10 7 pfu / mL 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. The procedure was repeated in 3 wells and incubated at 37 °C for 20 min (at this time, the final concentration of the compound was 33.33 μM); 25 μL of 1000×NA-XTD TM Substrate (purchased from Thermofisher) diluted 1:1000 (volume ratio) 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 GLOMAX96 microplate luminometer. The detection time for each well was 1 s, and the measurement was repeated 3 times and the average value was taken.

[0111] The neuraminidase activity is shown in Figure 7A. Thus, epalrestat showed inhibitory activity against influenza A virus neuraminidase.

[0112] 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 per well and the average value taken after measuring 3 times. The data were analyzed using the software GraphPad Prism 8.0.2, and the IC 50 of the compound was calculated by non-linear regression and Nonlin fitting.

[0113] The fitting curve is shown 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.

[0114] II. Molecular docking experiment

[0115] The protein-ligand structure was downloaded from the RCSB website (https: / / www.rcsb.org / ), 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 is from the Pubchem website (https: / / pubchem.ncbi.nlm.nih.gov / ). Ligand pretreatment 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.

[0116] Then, using AutoDockTools-1.5.7 software, the protein model was hydrogenated, the docking box was set and saved. 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.

[0117] Using AutoDock vina software, with E16-1 as the ligand, it was 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 AutoDockTool software, the results are as shown in Figure 8 (B~F). Among them, in models 1, 2, 3, 6, and 7, E16-1 forms two hydrogen bonds with the neuraminidase binding pocket. 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, model 1 has the lowest binding energy, as shown in Figure 8 (A), which is -6.5 kcal / mol.

[0118] 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.

[0119] Example 8: Detection of the Inhibition of Influenza Virus Protein Expression by Compounds

[0120] 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. The cells were cultured at 37 °C under 5% CO 2 conditions. 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, 3 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.

[0121] 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.

[0122] 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 3 times at high speed at room temperature on a shaker with PBST solution for 10 min each to wash away the unbound primary antibody. The secondary antibody was prepared at a ratio of 1:5000 (volume ratio), and the dilution buffer was PBS. After incubating at room temperature in the dark for 1 h, the membrane was washed 3 times in the dark, 10 min each time. The results were imaged and analyzed using an Odyssey CLX near-infrared scanning detector.

[0123] For the internal reference GAPDH, the primary antibody was a rabbit polyclonal GAPDH 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 monoclonal PB1 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).

[0124] For the influenza virus nucleoprotein NP, the primary antibody was a mouse monoclonal NP 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).

[0125] 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 show an obvious decrease while the virus titer decreased significantly.

[0126] Example 9: Effect of siRNA interfering with drug target genes on influenza virus replication

[0127] 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 experiments 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 cells were 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, and the plaque titration experiment was performed according to the method in Example 2 to determine the virus titer.

[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 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-derived DHODH monoclonal antibody (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-derived IMPDH2 monoclonal antibody (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-derived AKR1B1 polyclonal antibody (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).

[0129] Table 2. siRNA sequences (sequences of the RNA strands complementary to the target sequences)

[0130]

[0131] 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 titers increased slightly at 24 h and 48 h. The virus titers of Buquinac Target increased by 1.86-fold and 4.18-fold at 24 h and 48 h after infection, respectively; the virus titers of Mizoribine Target increased by 3.03-fold and 6.87-fold at 24 h and 48 h after infection, respectively; the virus titers of Epalrestat Target increased by 3.39-fold and 4.53-fold at 24 h and 48 h after being infected with the virus, respectively ( Figure 11 B).

[0132] After inhibiting the target proteins in the cells treated with these three drugs by siRNA, the virus titer increased, indicating that the aforementioned three target proteins were involved in the effect of the drugs in inhibiting the virus.

[0133] Example 10: Test on the effect of drug combination in inhibiting virus

[0134] I. Preparation of drugs

[0135] 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 ECs calculated in Example 4 50 corresponding concentrations).

[0136] Mix any two of the aforementioned five drugs (a total of ten combinations) in equal volume with the stock solutions of the individual drugs at twice the EC 50 corresponding concentrations to prepare a composition solution. The concentrations of the two drugs in the composition are both the ECs of 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.

[0137] Set the following negative control, as shown in No. 16 in Table 3 below.

[0138] Using the liquid medicines in Nos. 1-15 in Table 3 as test compounds 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 in Example 4, determine the content of influenza virus NP gene vRNA in the corresponding wells of each test solution. See the third column of Table 3 for the specific vRNA content. 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 .

[0139] 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 drugs, and some show obvious synergistic effects. No cases where the virus content of the combined use of drugs is higher than that of the single drug were found, and there is no mutual antagonism between these drugs. Therefore, the combined use of the five drugs has better application prospects.

[0140] Table 3. Statistical Table of Drug Configuration and Virus Content

[0141] Number Drug formulation scheme vRNA content % 1 0.1323 μM Brequinar. 37.4 2 5.003 μM Mizoribine. 32.59 3 0.6564 μM Triptonide. 17.18 4 2.213 μM Voxelotor. 78.85 5 0.8847 μM Epalrestat. 18.77 6 0.2646 μM Brequinar + 10.006 μM Mizoribine 13.05 7 1.3128 μM Triptonide + 10.006 μM Mizoribine 1.51 8 0.2646 μM Brequinar + 1.3128 μM Triptonide 1.765 9 0.2646 μM Brequinar + 4.426 μM Voxelotor 3.65 10 1.3128 μM Triptonide + 1.7694 μM Epalrestat 16.72 11 4.426 μM Voxelotor + 1.3128 μM Triptonide 17.69 12 4.426 μM Voxelotor + 10.006 μM Mizoribine 7.62 13 4.426 μM Voxelotor + 1.7694 μM Epalrestat 29.46 14 1.7694 μM Epalrestat + 0.2646 μM Brequinar 4.87 15 1.7694 μM Epalrestat + 10.006 μM Mizoribine 2.56 16 0.01% DMSO 100

[0142] 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 ingredients of the pharmaceutical composition include active substance a and active substance b; The active substance a 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 b is any one of epalrestat, a pharmaceutically acceptable salt of epalrestat and a prodrug of epalrestat, a combination of any two or a combination of all three; The structural formula of the Voseluoto is: The structural formula of epalrestat is:

2. The pharmaceutical composition according to claim 1, characterized in that The molar ratio of the active substance a to the active substance b is 1:0.04-4.

3. The pharmaceutical composition according to claim 1 or 2, characterized in that The molar ratio of the active substance a to the active substance b is 1:0.3-0.

5.

4. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that 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 epalrestat is selected from aluminum salt, zinc salt, amine salt, ammonium salt, sodium salt, calcium salt, potassium salt, magnesium salt, silver salt and lithium salt of epalrestat.

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 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 b is any one of epalrestat, a pharmaceutically acceptable salt of epalrestat and a prodrug of epalrestat, a combination of any two or a combination of all three; The structural formula of the Voseluoto is: The use is selected from any one or a combination of the following U1, U2, U3 and U4; 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; 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; U4: The improvement of health status is to reduce the damage of influenza virus to the body by inhibiting influenza virus neuraminidase.

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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