Application of epalrestat in preparation of preparation for inhibiting influenza virus

Abstract

The invention discloses application of epalrestat 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 epalrestat and the composition containing the epalrestat are good in safety, can well inhibit influenza virus proliferation, and have application prospects for treating influenza.

Description

Technical Field

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

[0002] Influenza virus is a representative virus of the Orthomyxoviridae family, which is divided into four genera: A, B, C, and D. Among them, influenza virus types A, B, and C can 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 each year, resulting in 3 to 5 million severe cases and about 1 million deaths worldwide.

[0003] Influenza A virus (IAV) is a respiratory pathogen of great economic and public health significance because of its ability to cause high rates of severe illness and mortality. Its viral genome consists of 8-segmented single-stranded negative-sense RNA, which 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 and PA-X. Seasonal influenza A viruses are usually of two subtypes, H1N1 and H3N2, and have caused repeated epidemics of varying severity for decades. In the United States, influenza causes more than 200,000 hospitalizations each year and 3,000 to 49,000 deaths each year during the non-epidemic season. Because of their frequent antigenic drift and antigenic shift, new strains from other species have caused human influenza pandemics, such as the "swine flu" H1N1 and avian influenza H5N1 in 2009. Since humans have almost no immunity to them, they can spread rapidly around the world, 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 significant results due to problems such as low vaccination rates, rapid virus mutation rates and uneven vaccine protection.

[0005] Drugs are another important tool for preventing and controlling human influenza. Currently, the drugs that have entered clinical use for the treatment of influenza virus infection can be divided into first-generation M2 ion channel inhibitors, second-generation neuraminidase inhibitors (NAIs), and third-generation cap-dependent endonuclease inhibitors. Influenza virus is an RNA virus with a high mutation rate. Since the advent of the first generation of anti-influenza drugs more than 50 years ago, it has rapidly evolved into a variety of drug-resistant strains under high-pressure drug screening. Among the epidemic strains, strains resistant to existing drugs already account for a considerable proportion. In view of the limitations of the application of human influenza virus vaccines and the rapid development of drug resistance, the necessity of screening new influenza virus small molecule inhibitors has become increasingly prominent.

[0006] Triptonide, also known as PG 492, NSC 165677, English name Triptonide, CAS number 38647-11-9, conventional active use is Wnt signal inhibitor, molecular structure is as follows:

[0007]

[0008] Brequinar, also known as Bipenquinate, NSC 368390, DUP785, English Brequinar, CAS No. 96187-53-0, is a potent inhibitor of dihydroorotate dehydrogenase in conventional active use, with potent activity against a broad spectrum of viruses. The molecular structure is as follows:

[0009]

[0010] Voxelotor, also known as GBT 440, has a CAS number of 1446321-46-5. Its conventional active use is as a sickle hemoglobin (HbS) polymerization inhibitor. Its molecular structure is as follows:

[0011]

[0012] Mizoribine, also known as Bredinin, NSC 289637, HE 69, English Mizoribine, CAS No. 50924-49-7, conventional active use is immunosuppressant, molecular structure is as follows:

[0013]

[0014] Epalrestat, also known as ONO2235, Epalrestat, CAS No. 82159-09-9, is a conventional active use as an aldose reductase inhibitor, which effectively improves the symptoms of diabetic neuropathy and delays the progression of the disease. The molecular structure is as follows:

[0015]

[0016] There are no reports on the use of the aforementioned drugs in the treatment of influenza. Summary of the invention

[0017] In order to solve the problems existing in the prior art, the first aspect of the present invention provides a pharmaceutical composition, wherein the pharmaceutical active ingredients of the pharmaceutical composition include active substance a and active substance b;

[0018] The active substance a 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;

[0019] The active substance b 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 all three;

[0020] The structural formula of epalrestat is:

[0021]

[0022] The structural formula of the buquina is:

[0023]

[0024] In some embodiments, the molar ratio of the active substance a to the active substance b is 1:0.015-1.5 (for example, 1:0.015, 0.016, 0.017, 0.018, 0.019, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, any ratio 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 1:0.1-0.2.

[0026] In some embodiments, the pharmaceutically acceptable salt of epalrestat is selected from the group consisting of aluminum salt, zinc salt, amine salt, ammonium salt, sodium salt, calcium salt, potassium salt, magnesium salt, silver salt and lithium salt of epalrestat;

[0027] 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.

[0028] In some embodiments, the composition further contains an auxiliary material.

[0029] A second aspect of the present invention provides a use of a biomaterial in the preparation of a product for use alone or in combination with other agents to improve health conditions;

[0030] The biological material 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 epalrestat, a pharmaceutically acceptable salt of epalrestat and a prodrug of epalrestat, a combination of any two or a combination of all three;

[0032] The structural formula of epalrestat is:

[0033]

[0034] The use is selected from any one or a combination of the following U1, U2, U3 and U4;

[0035] U1: The improvement in health status is to prevent influenza, treat influenza, slow down influenza or inhibit the proliferation of influenza virus;

[0036] U2: The improvement in health status is to reduce the damage of influenza virus to the lungs;

[0037] 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;

[0038] U4: The improvement of health status is to reduce the damage of influenza virus to the body by inhibiting influenza virus neuraminidase.

[0039] In some embodiments, the product is selected from the group consisting of medicines, health products, feeds, feed additives, foods and food additives.

[0040] In some embodiments, the influenza virus is an influenza A virus and the influenza is influenza A.

[0041] In some embodiments, the influenza virus is an H1N1 influenza virus, and the influenza is influenza caused by an H1N1 influenza virus.

[0042] In some embodiments, the influenza is selected from human influenza, avian influenza, and swine influenza. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The inoculation MOI, drug concentration and OD are shown. 450 Statistics on the relationship between them.

[0044] Figure 2 The statistics of viral infection titers under the intervention of 5 drugs are shown.

[0045] Figure 3 Five drugs CC are shown 50 The fitting curve of .

[0046] Figure 4 EC values ​​of 5 drugs are shown 50 The fitting curve of .

[0047] Figure 5 The results of the virus attack protection experiment under the intervention of three drugs are shown.

[0048] Figure 6 The results of the effects of four drugs on the polymerase activity of influenza A virus are shown.

[0049] Figure 7 Results showing the effect of epalrestat on influenza virus neuraminidase activity.

[0050] Figure 8 The results of hydrogen bond analysis of the molecular docking model are shown.

[0051] Fig. 9 The molecular docking results of epalrestat and influenza virus neuraminidase N1 are shown.

[0052] Fig.10 The inhibition of viral protein expression by five drugs is shown.

[0053] Fig.11 Shown is the effect of siRNA interference drug target gene on influenza virus replication.

[0054] Fig.12 The statistics of viral content under the intervention of 5 drug combinations are shown. DETAILED DESCRIPTION

[0055] In order to make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0056] Example 1: Influenza virus infection protection experiment

[0057] 1. Establishment of positive control method

[0058] Using oseltamivir phosphate (purchased from MCE, the same below) as a positive control drug, a method for indirectly screening anti-influenza virus small molecule compounds using CCK-8 cell viability was established.

[0059] First, fill each well of a transparent 96-well cell culture plate with 100 μL of PBS to prevent edge effects. Then, MDCK cells were plated at 1×10 4 The cells were plated at a density of 1 / well in a well plate, and the culture medium was DMEM culture medium (purchased from Sigma-Aldrich, hereinafter the same) containing 10 v / v% fetal bovine serum (purchased from WISENT, hereinafter the same), and the amount per well was 100 μL. The cells were cultured at 37°C and 5% CO2, and virus infection was performed after the cell density in the well plate grew to 90%.

[0060] Use a multichannel pipette to discard the culture medium in the wells, and 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 culture 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, and add 100 μL of Opti-MEM culture medium containing 0.5 μg / mL TPCK trypsin to each well. After adsorbing the virus for one hour at 37°C and 5% CO2, the virus solution is discarded. Then, Opti-MEM medium (containing 0.1 v / v% DMSO and 0.5 μg / mL TPCK trypsin) with concentrations of 400 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL and 25 μg / mL oseltamivir phosphate were added to the wells inoculated with different MOIs, respectively. Opti-MEM medium (containing 0.1 v / v% DMSO) was used as a negative control. After culturing for 24 h at 37°C and 5% CO2, the cell viability in each well was detected.

[0061] Discard the liquid in the well plate, replace with 100 μL / well DMEM medium, add 10 μL CCK-8 solution (from Cell Counting Kit-8, purchased from MCE, the same below) to each well, incubate at 37°C, 5% CO2 for 1 hour, and immediately place on ice to detect the OD of each well using a microplate reader. 450Each MOI and each concentration of oseltamivir were repeated in 3 wells, the absorbance of each well was detected 3 times and the average value was taken, and the data were analyzed using GraphPad Prism 8.0.2.

[0062] CCK-8 test results Figure 1 As shown. 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 ability to inhibit the virus increased. At the same time, between the drug groups with the same concentration, the different MOI infection groups also showed an obvious cell activity gradient. As the MOI increased, the cell viability decreased significantly. Under the same drug inhibition ability, the more viruses were inoculated, the lower the cell viability, indicating that this method has good sensitivity. This method can use OD values ​​to reflect the drug's ability to inhibit the virus.

[0063] 2. Primary screening, secondary screening and data analysis

[0064] According to the experimental results of Section 1, MOI 0.01 was selected for screening experiments. The cell plating and virus infection methods were the same as Section 1. After virus infection, 2697 compounds in the FDA-approved drug library were replaced with a final concentration of 30 μM (the test compounds, small molecule library FDA L1300-Z417864 purchased from Selleck), and Opti-MEM medium containing 0.1 v / v% DMSO (containing 0.5 μg / mL TPCK pancreatin) was used as the solvent. Each compound was repeated 3 wells. At the same time, oseltamivir was set as a positive control, 0.1 v / v% DMSO aqueous solution was used as a negative control, and the experiment without virus infection was used as a blank control group. Other steps and parameters were processed in parallel. After culturing for 24 hours at 37°C and 5% CO2, the cell viability was detected using the method in Section 1. The absorbance of each well was detected 3 times and the average value was taken. Compared with the control group, 67 compounds with strong known virus activity (larger OD value) in each plate were selected to show a protective effect on cells after virus infection. The same method was then used to rescreen the aforementioned 67 compounds, and the results showed that 26 of them showed strong cell protection ability.

[0065] Example 2: Virus titer determination experiment

[0066] 1. Identification of antiviral ability of compounds

[0067] A549 cells were cultured at 1×10 5 / well in a 12-well cell culture plate, the culture medium is F12K culture medium (purchased from Multicell, the same below) containing 10v / v% fetal bovine serum, 1mL per well, cultured at 37°C, 5% CO2, after the cell density in the well plate grows to 90%, the cells are infected with WSN (H1N1) virus at MOI 0.01, one hour after virus adsorption, the cells are washed with PBS, and each well is replaced with 1mL Opti-MEM culture medium (containing 0.125μg / mL TPCK trypsin, containing 0.1v / v% DMSO) containing 30μM test compounds (19 compounds are selected from the 26 compounds obtained in Example 1), and a 0.1v / v% DMSO aqueous solution is set as a negative control. After 24h, 50μL of the culture supernatant is collected in a 1.5mL EP tube, frozen at -80°C for use, and then a plaque titration experiment is performed.

[0068] 2. Plaque titration experiment

[0069] MDCK cells were plated in a 12-well cell culture plate in a 1×DMEM medium containing 0.5 μg / mL TPCK trypsin. After the cells were fully grown, the cells were washed twice with PBS to remove the residual serum in the culture medium. The cells were rinsed once with 1×DMEM medium containing 0.5 μg / mL TPCK trypsin for later use.

[0070] The culture supernatant in each well of Section 1 was melted on ice, and 1×DMEM medium containing 0.5μg / mL TPCK trypsin was used to dilute it 2-fold, and 100μL of virus solution was added to the well plate for adsorption. The well plate was cultured at 37℃ and 5% CO2, and the culture plate was shaken evenly every 10min to make the virus solution evenly cover the cells. The low melting point agarose was dissolved in ddH2O to prepare a 2w / w% agarose solution, heated and melted, and placed in a 50℃ water bath to keep warm. At the same time, an equal volume of 2×DMEM culture medium was taken and kept warm in a 37℃ water bath. An equal volume of 2w / w% agarose solution and 2×DMEM culture 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 1mL of 1×DMEM 1w / w% agarose solution was added to each well. The well plate was placed in a biosafety cabinet for 20min. After the agarose solidified, it was inverted and placed in a cell culture incubator. After 48 hours of incubation at 37°C and 5% CO2, 1 mL of formalin fixative was added to each well. After overnight fixation, the fixative and agarose were discarded, and the number of plaques formed was counted and the virus titer was calculated. The titer refers to the number of biologically active virus particles per unit volume of liquid. The titer is quantitatively expressed as the highest dilution factor of the 2-fold dilution at which plaques can be seen, and the unit is Log2pfu / mL.

[0071] Compared with the DMSO control, the titers of five compounds showed a significant decrease. The five compounds are triptolide (code: T18-9), buquina (code: B18-5), vorseloto (code: V15-24), mizoribine (code: M9-26) and epalrestat (code: E16-1). The five compounds reduced the viral 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 statistics chart for details. Figure 2 .

[0072] Example 3: Compound half toxic concentration CC 50 Determination

[0073] A transparent 96-well cell culture plate was filled with 100 μL of PBS in each well to prevent edge effects, and then A549 cells were plated at 1×10 4 / well density was plated in a 96-well cell culture plate, the culture medium was Opti-MEM medium, 100 μL per well, and cultured at 37°C, 5% CO2. After the cell density in the well plate grew to 90%, the culture medium was discarded, and the test compounds triptolide, brequinar, vorseloto, mizoribine and epalrestat were dissolved in Opti-MEM medium, respectively, and the final concentrations were set to gradient concentrations of 0.1 μM, 1 μM, 10 μM, 100 μM, 500 μM, 1000 μM, 5000 μM, 10000 μM, and added to different wells, respectively, with an addition amount of 100 μL per well, and each concentration was repeated for 3 wells, and the cell activity was detected after culturing for 24 hours at 37°C and 5% CO2. Each well was replaced with 100 μL of DMEM medium containing 10 v / v% fetal bovine serum, and then 10 μL of CCK-8 solution was added to each well. After incubation at 37°C and 5% CO2 for 1 h, the well was immediately placed on ice and the OD of each well was measured using an enzyme-labeled instrument. 450 The absorbance of each well was measured 3 times and the average value was taken. The well with 0.1 v / v% DMSO aqueous solution was used as the negative control and its absorbance was set to 100%. The cell viability of the experimental groups with different concentrations was calculated. GraphPad Prism 8.0.2 was used to analyze the data. The CC of the compounds was calculated by nonlinear regression and Nonlin fitting. 50 .

[0074] The fitting curve results can be found in Figure 3 CC of triptolide, brequinar, vorseloto, mizoribine, and epalrestat 50 They are 1.304mM, 558.4μM, 344.2μM and >10mM and 310.4μM respectively.

[0075] Example 4: Compound half effective concentration EC 50 Determination

[0076] A549 cells were cultured at 1×10 4 / well were plated in a 24-well cell culture plate, the culture medium was F12K culture medium containing 10v / v% fetal bovine serum, 500μL per well, after the cells grew to 90%, the cells were infected with influenza A virus WSN at MOI 0.01, and cultured at 37°C and 5% CO2 for one hour to allow the virus to adsorb, then replaced with Opti-MEM culture medium (containing 0.125μg / mL TPCK trypsin, 0.1v / v% DMSO) 500μL / well containing different concentrations of the test compounds (triptolide, brequinar, vorseloto, mizoribine and epalrestat). The final concentration of each compound was set to a gradient concentration of 0.01μM, 0.1μM, 1μM and 10μM, and a 0.1v / v% DMSO aqueous solution was set as a negative control. After culturing for 24h at 37°C and 5% CO2, the supernatant was discarded from each well, and the total RNA in each well was extracted.

[0077] After determining the RNA concentration, the HiscriptR RIIQ RT SuperMix for qPCR reverse transcription kit (Vazyme, R223-01) was used according to the instructions to remove genomic DNA from the total RNA in each well, and 1 μg RNA was used as a template and 5Seg_vRNA-RT (SEQ ID NO.1) in Table 1 was used as a primer to reverse transcribe the mRNA into cDNA.

[0078] According to the reference gene sequence of WSN strain in NCBI, Primer Premier 5 was used to design fluorescent quantitative PCR primers. The upstream and downstream primers are shown in Table 1 as 5Seg_NP qPCR-F (SEQ ID NO.2) and 5Seg_NP qPCR-R (SEQ ID NO.3). Each reaction used 2×ChamQ Universal SYBR qPCR Master Mix 5μL, cDNA template 1μL, upstream and downstream primers 0.4μL each, ddH2O3.2μL, and each reaction was repeated 3 times. The fluorescent quantitative PCR reaction program was 95℃ pre-denaturation for 10min; 95℃ denaturation for 15s; 60℃ annealing for 1min, denaturation and annealing for 40 cycles; melting curve: 95℃, 15s; 60℃, 1min; 95℃, 10s. The hGAPDH gene was used as an internal reference, and the internal reference gene primers are shown in Table 1 as qPCR-hGAPDH-F (SEQ ID NO.4) and qPCR-hGAPDH-R (SEQ ID NO.5). The 0.1 v / v% DMSO aqueous solution group was used as a negative control, and the viral RNA content of the viral NP segment was calculated using the formula 2^-{ΔΔCt=[Ct(EG_vNP)-Ct(EG_hGAPDH)]-Ct(DMSO_vNP)-Ct(DMSO_hGAP DH)}. GraphPad Prism 8.0.2 was used to analyze the data, and the EC values ​​of the compounds were calculated by nonlinear regression and Nonlin fitting. 50 .

[0079] The fitting curve results can be found in Figure 4 EC of triptolide, brequinar, vorseloto, mizoribine, and epalrestat 50 They are 0.6564μM, 0.1323μM, 2.213μM, 5.003μM and 0.8847μM respectively.

[0080] Table 1. Primer sequences

[0081]

[0082] Example 5: Organ plaque titration

[0083] The mice were divided into four groups: vorseloto, mizoribine, epalrestat and 5% DMSO, with 8 mice in each group. After weighing, they were anesthetized with isoflurane inhalation at 1.5×10 4 pfu of influenza A virus WSN were inhaled through the nasal cavity, anesthetized in the same way and administered through the nasal cavity every day. The dosages 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 with 5v / v% DMSO aqueous solution, with a dosage of 25 μL per mouse.

[0084] On the third day after the challenge, three mice in each group were euthanized by isoflurane inhalation anesthesia and cervical dislocation, and the complete lung tissue was taken in a 2 mL EP tube and frozen 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, and ground at 4 ° C and 30 Hz for 5 min. Then 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.

[0085] Results Figure 5 Compared with the control group, the virus titers in the lungs of the vorseloto, mizoribine, and epalrestat groups decreased by 71.74% (P < 0.01), 76.74% (P < 0.001), and 84.13% (P < 0.001), respectively.

[0086] Example 6: Influenza virus polymerase activity assay

[0087] 1. Synthesis and Source of Plasmid

[0088] (1) Renilla luciferase control reporter gene vector pRL-TK

[0089] The pRL series vectors are Renilla luciferase reporter vectors developed by Promega (Cat. No. E2241), and the TK promoter drives Renilla luciferase.

[0090] (2) RNA polymerase component plasmid

[0091] Using the pCAGGS plasmid as the backbone, the coding sequence of the alkaline polymerase 1 (PB1) gene (see GenBank No. LC333183.1), the coding sequence of the alkaline polymerase 2 (PB2) gene (see GenBank No. LC333182.1), the coding sequence of the PA protein gene (see GenBank No. LC333184.1), and the coding sequence of the nucleoprotein (NP) gene (see GenBank No. 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, which were named pCAGGS-WSNPB1, pCAGGS-WSN PB2, pCAGGS-WSN PA, and pCAGGS-WSN NP, respectively.

[0092] (3) Firefly luciferase reporter gene vector pHH21-SC09NS F-Luc

[0093] A firefly luciferase reporter gene vector pHH21-SC09NS F-Luc was constructed to produce a negative-sense RNA containing 176 bases at the 3' end of NSvRNA derived from 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 SC09NS vRNA, wherein influenza virus polymerase can recognize and bind to the non-coding sequence at the end of NS vRNA on the vector to initiate the expression of firefly luciferase.

[0094] The construction methods of the plasmid vectors of (2) and (3) above refer to the following documents:

[0095] Luo W, Zhang J, Liang L, et al., 2018. Phospholipid scramblase 1interacts with influenza A virus NP,impairing its nuclear import and therebysuppressing virus replication. PLoS pathogens, 14(1):e1006851.

[0096] 2. Principle of enzyme activity determination

[0097] Influenza virus RNA polymerase is composed of three subunits: PB2, PB1, and PA. It is an RNA-dependent RNA polymerase that works with NP protein to perform the important function of viral genome transcription. In order to explore the effects of drugs on the activity of influenza virus polymerase, a dual luciferase reporter experiment was used.

[0098] In the dual luciferase reporter system In the Reporter System (purchased from Promega, catalog number: E1960), firefly luciferase is used as a reporter gene for gene expression, and Renilla luciferase is used as an internal reference gene. 1. The target gene transcriptional regulatory element is constructed into an expression vector with 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 the cells are lysed after different treatments, and the substrate luciferin is added. Luciferase can catalyze luciferin to emit fluorescence; 3. The fluorescence value obtained by the detection can determine the effect of different treatment groups on the transcriptional regulatory element; 4. In order to avoid errors caused by differences in efficiency when plasmids are transfected into cells, a Renilla luciferase reporter gene plasmid is usually transferred as an internal reference to correct the transfection efficiency between different samples.

[0099] The expression of Renilla luciferase is constitutive and can be used as a control. It is little affected by influenza virus inhibitors. The expression of firefly luciferase is affected by the content or activity of RNA polymerase, and then 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 strength of the drug's inhibitory ability on influenza virus RNA polymerase.

[0100] The polymerase expression plasmids (pCAGGS-PA, pCAGGS-PB1, pCAGGS-PB2, and pCAGGS-NP) of four WSNs (A / WSN / 33 (WSN, H1N1)) were constructed to express them in cells and form the influenza virus RNA polymerase complex.

[0101] Four polymerase expression plasmids, firefly luciferase reporter gene vector pHH21-SC09NS F-Luc and sea renilla luciferase control reporter gene vector pRL-TK were co-transfected into HEK293T cells, and drugs were added to the cells at the same time. After transfection, the cells were lysed and loaded into the GloMax 96 microplate luminometer (Promega) to measure the luciferase activity of the transfected cells using the dual luciferase reporter gene assay system (firefly luciferase reacts with the substrate LARⅡ to produce a fluorescent signal, which is detected by the instrument and quenched with Stop&Glo reagent, while activating sea renilla luciferase to produce fluorescence) to analyze the effect of drugs on influenza virus polymerase activity.

[0102] Data processing: First, calculate the ratio of Firefly luciferase / Renilla luciferase in each tube, and then take the ratio of the control group as 1 to obtain the relative luciferase activity of different treatment groups, that is, the activity of influenza virus polymerase.

[0103] 2. Activity determination

[0104] A 12-well cell culture plate was coated with poly-lysine, and then HEK293T cells were plated in the 12-well cell culture plate. The culture medium was DMEM containing 10% FBS. After the cells grew to 80%, 500ng pCAGGS-WSN PB2, 500ng pCAGGS-WSN PB1, 500ng pCAGGS-WSN PA, 500ng pCAGGS-WSN NP plasmids, 200ng pHH21-SC09NSF-Luc and 10ng pRL-TK plasmids were transfected into the cells at the same time. 10h after transfection, the culture medium containing plasmids and transfection reagents was discarded and replaced with HEK293T cell culture medium containing 20μM and 30μM of the test compounds (triptolide, brequinar, vorseloto and imidazoribine), respectively, and then cultured for another 26h. After discarding the cell culture supernatant, wash once with PBS, use 250μL Passive Lysis Buffer to lyse the cells in each well, centrifuge at 12000r / min for 5min, and take 20μL of the supernatant for use The polymerase complex activity was detected using the Reporter System.

[0105] Under the action of gradient concentrations of compounds, the luciferase ratio of the 0.1 v / v% DMSO aqueous solution group was taken as 100%, and the polymerase activity of the five compounds that inhibited influenza virus replication in Example 2 was measured. The results are shown in Figure 6 As shown, triptolide, buquina, vorseloto and mizoribine can significantly inhibit the activity of influenza virus polymerase, and the polymerase activity of the 30μM group is lower than that of the 20μM group, indicating that the inhibitory effect is dose-dependent. Compared with the control group, the polymerase activity of triptolide 20μM group and 30μM group decreased by 99.51% and 99.57%, respectively (P<0.001); compared with the control group, the polymerase activity of buquina 20μM group and 30μM group decreased by 98.96% and 99.19%, respectively (P<0.001); compared with the control group, the polymerase activity of vorseloto 20μM group and 30μM group decreased by 48.04% and 70.52%, respectively (P<0.001); compared with the control group, the polymerase activity of mizoribine 20μM group and 30μM group decreased by 18.55% and 28.11%, respectively (P<0.01).

[0106] It can be seen that the four drugs can inhibit the activity of influenza virus RNA polymerase and help inhibit influenza virus.

[0107] Example 7: Detection of influenza virus neuraminidase activity

[0108] 1. Cell Detection Experiment

[0109] According to the instructions, use a white non-transparent bottom 96-well plate to reduce light loss and interference between wells. 7 pfu / mL of influenza A virus WSN using NA-XTD TM After the Assay Buffer (purchased from Thermofisher) was diluted 1:100 (volume ratio), 25 μL was added to each well. The test compound (epalrestat) was diluted to 100 μM using the aforementioned Assay Buffer, 25 μL was added to each well, repeated for 3 wells, and incubated at 37°C for 20 min (the final concentration of the compound was 33.33 μM at this time); 25 μL of 1000×NA-XTD diluted 1:1000 (volume ratio) was added to each well. TM Substrate (purchased from Thermofisher), incubate at room temperature for 30 min; add 60 μL NA-XTD to each well TM Accelerator (purchased from Thermofisher), 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 performed 3 times to obtain the average value.

[0110] Neuraminidase activity Figure 7 A. It can be seen that epalrestat exhibits inhibitory activity against influenza A virus neuraminidase.

[0111] Then, the half maximal inhibitory concentration (IC) of epalrestat on neuraminidase was measured. 50 ), that is, the concentration of the compound required to inhibit the enzyme activity by half, to evaluate the inhibitory activity of the compound on the enzyme. 7 pfu / mL of influenza A virus WSN using NA-XTD TM After dilution with Assay Buffer 1:100, add 25 μL to each well, and use Assay Buffer to perform gradient dilution of the compound to be tested. Set the gradient to 1000000nM, 200000nM, 100000nM, 40000nM, 20000nM, 8000nM, 1600nM, 320nM, 64nM, 12.8nM, 2.56nM, 0.512nM and 0.1024nM. Repeat 3 wells for each concentration, that is, the final concentration of the test is 333333.33nM, 66666.67nM, 33333.33nM, 13333.33nM, 6666.67nM, 2666.67nM, 533.33nM, 106.67nM, 21.33nM, 4.27nM, 0.85nM, 0.17nM and 0.03nM, incubate at 37°C for 20min, then add 25μL NA-XTD to each wellTM Substrate, incubate at room temperature for 30 min; finally, add 60 μL NA-XTD to each well TM Accelerator, using GLOMAX 96 microplate luminometer to detect luminescence value, detection time of each well is 1s, and the average value is obtained by 3 measurements. The data are analyzed by GraphPad Prism 8.0.2 software, and the IC of the compound is calculated by nonlinear regression and Nonlin fitting. 50 .

[0112] Fitting curve see Figure 7 B. It can be seen that epalrestat exhibits concentration-dependent inhibitory activity on neuraminidase. After curve fitting, the drug concentration required to inhibit neuraminidase by half, i.e., IC 50 It is 17.36μM.

[0113] 2. Molecular docking experiment

[0114] The protein ligand structure was downloaded from the RCSB website (https: / / www.rcsb.org / ), PBD ID: 6D96, the protein is the X-ray diffraction structure of influenza virus A / BrevigMission / 1 / 1918 (H1N1) neuraminidase (NA) expressed in HEK-293E cells, and the ligand E16-1 structure is from the Pubchem website (https: / / pubchem.ncbi.nlm.nih.gov / ). PyMOL software was used for ligand pretreatment to remove water molecules, calcium ions, and residual ligands in the model, and to remove repeated peptide chains.

[0115] Then, the protein model was hydrogenated using AutoDockTools-1.5.7 software, and the docking box was set and saved. The chemical bonds of the ligands were set to be rigid and flexible and saved as pdbqt format files. Molecular docking was performed using the AutoDock vina command with the following 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". The output docking results were visualized, and binding energy and hydrogen bond analysis were performed using AutoDockTools-1.5.7 and PyMOL 2.5.

[0116] Using AutoDock vina software, E16-1 was used as the ligand and docked into the active pocket of influenza virus N1 subtype neuraminidase. A total of 9 docking models were output ( Figure 8 (A)) was analyzed by AutoDockTool software for hydrogen bonding. The results are as follows Figure 8 (B-F), where E16-1 in models 1, 2, 3, 6, and 7 all formed two hydrogen bonds with the neuraminidase binding pocket. The docking results were visualized using PyMOL software. Fig. 9 As shown, A is the binding pocket, and B to 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 As shown in (A), it is -6.5 kcal / mol.

[0117] The docking sites of model 1 were analyzed and the results were as follows Fig. 9 As shown in (G), the E16-1 molecule binds to the influenza virus N1 subtype neuraminidase Arg292 and Arg371 sites by hydrogen bonds (yellow dashed lines). In addition, the binding site that appears multiple times in other docking models is Arg118.

[0118] Example 8: Detection of Compounds Inhibiting Influenza Virus Protein Expression

[0119] A549 cells were cultured at 1×10 5 / well were plated in a 12-well cell culture plate, the culture medium was F12K culture medium containing 10v / v% fetal bovine serum, and cultured at 37°C and 5% CO2. 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. One hour after virus adsorption, 3 wells of each test compound were replaced with 1 mL of Opti-MEM culture medium (containing 0.125μg / mL TPCK trypsin, 0.1v / v% DMSO) containing 5μM, 10μM, and 20μM of the test compound (triptolide, brequinar, vorseloto, mizoribine, and epalrestat), respectively. A 0.1v / v% DMSO aqueous solution was set as a negative control, and Oseltamivir phosphate was used as a positive control. After 24h, 50μL of the culture supernatant was collected in a 1.5mL EP tube and frozen at -80°C.

[0120] After discarding the cell culture supernatant, wash the cells once with PBS, add 100 μL 1× SDS lysis buffer, 1 μL PMSF solution and 0.1 μL ribozyme to each well. After lysing at room temperature for 5 minutes, collect the lysate into a 1.5 mL EP tube and denature at 95°C for 10 minutes for Western Blot detection.

[0121] The above protein samples were added to the 10% SDS-PAGE gel loading wells at 15 μL / well, and electrophoresis was performed at 80V. After the protein samples began to separate through the stacking gel, the electrophoresis was changed to 120V until the electrophoresis was completed. A 0.45 μm nitrocellulose membrane (NC membrane) was placed in the NC membrane equilibrium solution for 30 seconds, and then the membrane was transferred using the eblot L1 fast wet transfer membrane transfer instrument. After blocking with 5% skim milk solution at room temperature for 1 hour, the residual blocking solution was washed with PBS solution, and the primary antibody was diluted with PBS1:1000 and incubated at low speed on a shaker for 1 hour at room temperature. After incubation, PBST solution was used to wash at high speed on a shaker at room temperature for 10 minutes, and repeated 3 times to wash away the unbound primary antibody. The secondary antibody was prepared at 1:5000 (volume ratio), the diluent was PBS, and after incubation at room temperature in the dark for 1 hour, the membrane was washed 3 times in the dark, each time for 10 minutes, and the Odyssey CLX near-infrared scanning detector was used to image and analyze the results.

[0122] For the internal reference GAPDH, the primary antibody was a rabbit GAPDH polyclonal antibody (Proteintech, Cat. No. 10494-1-AP), and the secondary antibody was a goat anti-rabbit IgG labeled with Dylight 680 (Li-COR Bioscience, Cat. No. 926-68071). For the influenza polymerase protein PB1, the primary antibody was a mouse PB1 monoclonal antibody (self-made in the laboratory), and the secondary antibody was a goat anti-mouse IgG labeled with Dylight 680 (Li-COR Bioscience, Cat. No. 926-68070).

[0123] For influenza virus nucleoprotein NP, the primary antibody was a mouse NP monoclonal antibody (prepared in the applicant's laboratory), and the secondary antibody was a Dylight 680-labeled goat anti-mouse IgG (Li-COR Bioscience, catalog number 926-68070).

[0124] WB results and virus titers of each group refer to Fig.10 . It can be seen that the gray value analysis of protein expression showed that no viral protein could be detected in the triptolide group; the NP protein and PB1 protein in the buquina group showed a significant gradient decrease; the NP protein and PB1 protein in the vorseloto group both decreased; the NP protein in the mizoribine group did not decrease significantly, and the PB1 protein showed a gradient decrease; the epalrestat group also showed no significant decrease in NP protein, while the expression of PB1 protein showed a gradient decrease. In the positive control oseltamivir group, the NP protein content did not decrease significantly, but the viral titer decreased significantly.

[0125] Example 9: Effect of siRNA interference drug target gene on influenza virus replication

[0126] siRNAs were designed according to the human gene sequence buquina target gene (gene name DHODH, Gene ID: 231, GenBank No. NM_001361.5), the human gene sequence imidazolipin target gene (gene name IMPDH2, GenBank No. NM_001410759.1, Gene ID: 1723), and the human gene sequence epalrestat target gene (gene name AKR1B1, GenBank No. NM_001628.4, Gene ID: 3615), and the RNA sequences complementary to the target sequences in each siRNA were shown in Table 2, si_231 (SEQ ID NO. 6), si_1723 (SEQ ID NO. 7), and si_3615 (SEQ ID NO. 8), respectively, and the sequence shown in si_NC (SEQ ID NO. 9) was used as a negative control. Lipofectamine was used TM The reverse transfection method in the RNAiMAX (purchased from ThermoFisherScientific, catalog number 13778030) manual transfected siRNA and negative control si_NC into A549 cells to interfere with the expression of related genes. One group of cells collected cell samples 36 hours after infection and performed Western Blot experiments to detect the interference efficiency at the protein level. Another group of cells was infected with influenza A virus WSN at MOI 0.01 36 hours after infection, and 1 mL / well Opti-MEM medium containing 0.125 μg / mL TPCK pancreatin was used after infection for 1 hour. The cell culture supernatant was collected at 24 hours and 48 hours after infection, and the virus titer was determined by plaque titration experiments according to the method in Example 2.

[0127] For the internal reference GAPDH, the primary antibody was a rabbit GAPDH polyclonal antibody (Proteintech, Cat. No. 10494-1-AP), and the secondary antibody was a goat anti-rabbit IgG labeled with Dylight 680 (Li-COR Bioscience, Cat. No. 926-68071). For the target gene protein of brequina, the primary antibody was a mouse DHODH monoclonal antibody (Proteintech, Cat. No. 67977-1-Ig), and the secondary antibody was a goat anti-mouse IgG labeled with Dylight 680 (Li-COR Bioscience, Cat. No. 926-68070). For the target gene protein of mizoribine, the primary antibody was a mouse IMPDH2 monoclonal antibody (Proteintech, Cat. No. 67663-1-Ig), and the secondary antibody was a goat anti-mouse IgG labeled with Dylight 680 (Li-COR Bioscience, Cat. No. 926-68070). For the target gene protein of epalrestat, the primary antibody was rabbit AKR1B1 polyclonal antibody (Invitrogen, Catalog No. PA5-29718), and the secondary antibody was Dylight 680-labeled goat anti-rabbit IgG (Li-COR Bioscience, Catalog No. 926-68071).

[0128] Table 2. siRNA sequences (sequences of RNA chains complementary to target sequences)

[0129]

[0130] After 36h of siRNA interference, the expression levels of brequinar Target, mizoribine Target and epalrestat Target genes decreased ( Fig.11 A), on this basis, after infection with the virus, the virus titer increased slightly at 24h and 48h. The virus titer of buquina Target increased by 1.86 times and 4.18 times at 24h and 48h after infection, respectively; the virus titer of mizoribine Target increased by 3.03 times and 6.87 times at 24h and 48h after infection, respectively; the virus titer of epalrestat Target increased by 3.39 times and 4.53 times at 24h and 48h after infection, respectively ( Fig.11 B).

[0131] After the target proteins in cells treated with the three drugs were inhibited by siRNA, the viral titer increased, indicating that the aforementioned three target proteins were involved in the drug's inhibitory effect on the virus.

[0132] Example 10: Testing of the effect of drug combination in inhibiting virus

[0133] 1. Preparation of drugs

[0134] The drug solutions of triptolide, brequinar, vorseloto, mizoribine and epalrestat were prepared, and the solvent was 0.1 v / v% DMSO (the solvent was 0.1 v / v% DMSO). The concentrations were shown in Table 3, No. 1-5 (all EC values ​​calculated in Example 4). 50 corresponding concentration).

[0135] Any two of the above five drugs were combined (a total of ten combinations) with double the EC of each drug. 50 The stock solutions of corresponding concentrations were mixed in equal volumes to prepare a composite solution, in which the concentrations of the two drugs in the composite were both EC 50 The corresponding concentration. The solvent is 0.1 v / v% DMSO. The concentration of the stock solution in the preparation method is shown in No. 6-15 in Table 3 below.

[0136] The following negative control was set up, as shown in No. 16 in Table 3 below.

[0137] The drug solutions No. 1-15 in Table 3 were used as the test compounds, and the solution shown in No. 16 was used as the negative control. Each sample was repeated 12 times. The other method steps were the same as those in Example 4. The content of influenza virus NP gene vRNA in the corresponding wells of each test solution was determined. For specific vRNA content, see the third column of Table 3. For statistics on the content of influenza virus NP gene vRNA in the negative control, two single drugs, and the combination of the two single drugs in the drug combination, see Fig.12 .

[0138] It can be seen that the viral content of the two drugs in most combinations is the lowest, which is significantly better than that of single drugs, and some have obvious synergistic effects. No cases were found where the viral content of the combined drug was higher than that of the single drug, and there was no antagonism between these drugs. Therefore, the combined use of the five drugs has a better application prospect.

[0139] Table 3. Drug configuration and virus content statistics

[0140]

[0141]

[0142] It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are included in 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 epalrestat, a pharmaceutically acceptable salt of epalrestat and a prodrug of epalrestat, a combination of any two or a combination of all three; The active substance b 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 all three; The structural formula of epalrestat is: The structural formula of the buquina 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 1:0.015-1.

5.

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 1:0.1-0.

2.

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

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 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 epalrestat 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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