Use of epalrestat in the manufacture of a preparation for inhibiting influenza virus

Abstract

The application discloses use of eprosartan in preparation of a preparation for preventing, treating, alleviating or inhibiting influenza virus proliferation, alone or in combination with other preparations. The eprosartan and the composition containing the eprosartan are safe, can well inhibit influenza virus proliferation, and have application prospects in treating influenza.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmacy, and relates to a use of eptastigmine in preparation of an agent for inhibiting influenza virus. BACKGROUND

[0002] Influenza virus is a representative virus of Orthomyxoviridae, which is divided into four genera of A, B, C and D, among which A, B and C types of influenza virus can infect humans. According to the statistics of WHO, 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 cases of severe illness and about 1 million deaths worldwide.

[0003] Influenza A virus (IAV) is a respiratory pathogen with significant economic and public health significance due to its high morbidity and mortality. Its viral genome consists of eight segmented single-stranded negative-strand RNAs, which can encode 10 essential proteins, including PB2, PB1, PA, HA, NP, NA, M1, M2, NS1, NEP / NS2 and various non-essential accessory proteins such as PB1-F2, PA-X, etc. Seasonal influenza A virus is usually of two subtypes of H1N1 and H3N2, which have caused repeated epidemics of varying severity for decades. In the United States, influenza causes more than 200,000 people to be hospitalized for treatment each year, and 3,000 to 49,000 people to die each year in non-epidemic seasons. Due to frequent antigenic drift and antigenic shift, new strains from other species can cause human influenza pandemic, such as "swine flu" H1N1 and avian influenza H5N1 in 2009. Since humans have little immunity to them, they can spread rapidly worldwide, posing a major threat to global health.

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

[0005] Drugs are another important tool to prevent and control human influenza. The drugs currently in clinical use for the treatment of influenza virus infection can be divided into the first generation M2 ion channel inhibitors, the second generation neuraminidase inhibitors (NAI) and the third generation cap-dependent endonuclease inhibitors. As an RNA virus with high mutation rate, influenza virus has rapidly evolved into multiple drug-resistant strains under the high-pressure screening of drugs since the first generation of anti-influenza drugs was introduced more than 50 years ago. Among the epidemic strains, the proportion of strains resistant to existing drugs is considerable. Given the limitations of human influenza virus vaccine application and the rapid generation of drug resistance, the necessity of screening new small molecule inhibitors of influenza virus is increasingly prominent.

[0006] Triptonide, NSC 165677, CAS No. 38647-11-9, is a Wnt signaling inhibitor, and its molecular structure is as follows:

[0007]

[0008] Brequinar, NSC 368390, DUP785, CAS No. 96187-53-0, is a potent inhibitor of dihydroorotate dehydrogenase and has potent activity against a broad spectrum of viruses, and its molecular structure is as follows:

[0009]

[0010] Voxelotor, GBT 440, CAS No. 1446321-46-5, is a sickle hemoglobin (HbS) polymerization inhibitor, and its molecular structure is as follows:

[0011]

[0012] Mizoribine, NSC 289637, HE 69, CAS No. 50924-49-7, is an immunosuppressant, and its molecular structure is as follows:

[0013]

[0014] Epalrestat, ONO2235, CAS No. 82159-09-9, is an aldose reductase inhibitor that effectively improves the symptoms of diabetic neuropathy and delays the progression of the disease. Its molecular structure is as follows:

[0015]

[0016] There is no report on the use of the aforementioned drugs in the treatment of influenza. SUMMARY

[0017] To solve the problems in the prior art, the present application provides a pharmaceutical composition, wherein the pharmaceutical active ingredient of the pharmaceutical composition comprises active substance a and active substance b;

[0018] The active substance a is epalrestat, any one of the pharmaceutically acceptable salts of epalrestat and the prodrugs of epalrestat, a combination of any two or a combination of three;

[0019] The active substance b is buquiner, any one of the pharmaceutically acceptable salts of buquiner and the prodrugs of buquiner, a combination of any two or a combination of three;

[0020] The structural formula of the epalrestat is:

[0021]

[0022] The structural formula of the buquiner is:

[0023]

[0024] In some embodiments, the molar ratio of the active substance a and the active substance b is 1:0.015-1.5 (for example, any one of 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 or a range between any two of them).

[0025] In some embodiments, the molar ratio of the active substance a and 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 pharmaceutical salt of the buquineran is selected from the group consisting of an aluminum salt, a zinc salt, an amine salt, an ammonium salt, a sodium salt, a calcium salt, a potassium salt, a magnesium salt, a silver salt, a lithium salt, a hydrochloride salt, a sulfate salt, a citrate salt, a benzenesulfonate salt, a hydrobromide salt, a hydrofluoride salt, a phosphate salt, an acetate salt, a propionate salt, a succinate salt, an oxalate salt, a malate salt, a succinate salt, a fumarate salt, a maleate salt, a tartrate salt, a trifluoroacetate salt, an aspartate salt, a taurinate salt, a gluconate salt, a fructate salt, a salicylate salt, a nitrate salt, a p-toluenesulfonate salt, a methanesulfonate salt, a benzoate salt, a citrate salt, a lactate salt, a citrate salt, a fumarate salt, and an ascorbate salt of the buquineran.

[0028] In some embodiments, the composition further comprises an adjuvant.

[0029] The second aspect of the present application provides a use of a biomaterial in the preparation of a product for improving a health condition, alone or in combination with other agents;

[0030] The biomaterial is the pharmaceutical composition or the active substance a according to the first aspect of the present application.

[0031] The active substance a is any one of the following: an eptastatin, a pharmaceutically acceptable salt of the eptastatin, and a prodrug of the eptastatin, a combination of any two of them, or a combination of all three of them.

[0032] The eptastatin has the following structural formula:

[0033]

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

[0035] U1: the improvement of the health condition is the prevention of influenza, the treatment of influenza, the alleviation of influenza, or the inhibition of the proliferation of influenza virus;

[0036] U2: the improvement of the health condition is the reduction of the damage of influenza virus to the lung;

[0037] U3: the improvement of the health condition is the reduction of the damage of influenza virus to the body by inhibiting the RNA-dependent RNA polymerase of the influenza virus;

[0038] U4: the improvement of the health condition is the reduction of the damage of influenza virus to the body by inhibiting the neuraminidase of the influenza virus.

[0039] In some embodiments, the product is selected from a pharmaceutical product.

[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 H1N1 type influenza virus, and the influenza is influenza caused by H1N1 type influenza virus.

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

[0043] Figure 1 Statistical results of the relationship between inoculation MOI, drug concentration and OD 450 are shown.

[0044] Figure 2 Statistical results of virus infection titers under the intervention of five drugs are shown.

[0045] Figure 3 Fitting curves of the CC 50 of five drugs are shown.

[0046] Figure 4 Fitting curves of the EC 50 of five drugs are shown.

[0047] Figure 5 Results of virus challenge protection experiments under the intervention of three drugs are shown.

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

[0049] Figure 7 Results of the effects of epalrestat on the neuraminidase activity of influenza virus are shown.

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

[0051] Figure 9 Results of molecular docking of epalrestat and influenza virus neuraminidase N1 are shown.

[0052] Figure 10 Results of the inhibition of virus protein expression by five drugs are shown.

[0053] Figure 11 Effects of siRNA interference drugs targeting genes on influenza virus replication are shown.

[0054] Figure 12 Statistical results of virus content under the intervention of the combination of five drugs are shown. DETAILED DESCRIPTION

[0055] In order to make the purpose, technical solutions and advantages of the present application clearer, the embodiments of the present application will be further described in detail below with reference to the drawings.

[0056] Example 1: Protective experiment of influenza virus infection

[0057] I. Establishment of positive control method

[0058] Phosphorothioate oseltamivir (purchased from MCE company, the same below) was used as a positive control drug to establish a method for screening anti-influenza virus small molecule compounds by CCK-8 cell viability.

[0059] First, fill PBS 100 μL in the periphery of a week in each well of a transparent 96-well cell culture plate to prevent edge effect. Then, MDCK cells were plated in the well plate at a density of 1 x 10 4 / well, and the medium was DMEM medium (purchased from Sigma-Aldrich company, the same below) containing 10 v / v% fetal bovine serum (purchased from WISENT company, the same below), and the amount used per well was 100 μL. Incubate at 37°C, 5% CO2, and when the cell density in the well plate grows to 90%, proceed with virus infection.

[0060] Use a multichannel pipette to discard the medium in the well, and use PBS to wash the cells twice to wash away the residual serum in the medium to prevent its influence on virus infection. After rinsing once with Opti-MEM medium (purchased from Gibco company, the same below) added with TPCK trypsin (final concentration 0.5 μg / mL), infect the cells with influenza virus WSN strain (H1N1 type) (NCBI Taxonomy ID: 382835) at MOI 0, MOI 0.01, MOI 0.1, MOI 1, MOI 5 and MOI 10, respectively, add 100 μL Opti-MEM medium containing 0.5 μg / mL TPCK trypsin per well, and incubate at 37°C, 5% CO2 for one hour to allow virus adsorption, then discard the virus liquid. Then add Opti-MEM medium (containing 0.1 v / v% DMSO, containing 0.5 μg / mL TPCK trypsin) containing 400 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL and 25 μg / mL phosphorothioate oseltamivir to the wells inoculated with different MOI, respectively, and use Opti-MEM medium (containing 0.5 μg / mL TPCK trypsin) containing 0.1 v / v% DMSO as negative control, and incubate at 37°C, 5% CO2 for 24 h before detecting the cell viability in each well.

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

[0062] The CCK-8 detection results are shown in Table 1. Figure 1 As can be seen from the above table, when infected with the same MOI, different concentrations of oseltamivir groups showed a good gradient of cell activity, and with the increase of the concentration of oseltamivir, the cell activity increased, indicating that the drug inhibition ability to virus was improved. At the same time, between the same concentration of drug groups, different MOI infection groups also showed a significant gradient of cell activity, and with the increase of MOI, the cell activity decreased significantly, and under the same drug inhibition ability, the more virus inoculated, the lower the cell activity, indicating that this method has good sensitivity. The method can use OD value to reflect the inhibition ability of the drug to the virus.

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

[0064] According to the experimental results of section one, MOI 0.01 was selected for screening experiment, and the cell plating and virus infection method were the same as section one. After infection with virus, 2697 kinds of compounds (test compounds, small molecule library FDA L1300-Z417864 purchased from Selleck company) in the FDA approved drug library with a final concentration of 30 μM were replaced, and 0.1 v / v % DMSO was used as the solvent. Opti-MEM medium (containing 0.5 μg / mL TPCK trypsin) containing 0.1 v / v % DMSO was used as the solvent, each compound was repeated in 3 wells, and oseltamivir positive control was set up, 0.1 v / v % DMSO aqueous solution was used as negative control, and uninfected virus experiment was used as blank control group, and other steps and parameters were processed in parallel. After incubation at 37°C, 5% CO2 for 24 h, the method in section one was used to detect the cell activity, and the absorbance of each well was detected 3 times and the average value was taken. Compared with the control group, 67 kinds of compounds with known strong virus activity (large OD value) in each plate showed protective effect on cells after virus infection. Then the same method was used to screen the 67 kinds of compounds, and the results showed that 26 kinds of compounds showed strong cell protection.

[0065] Example 2: Virus titer determination experiment

[0066] I. Identification of compound antiviral ability

[0067] A549 cells were seeded in 96-well plates at 1×10 5The density of the holes was laid in 12-hole cell culture plates, the culture medium was F12K culture medium containing 10v / v% fetal bovine serum (purchased from Multicell, same below), 1 mL per hole, 37°C, 5% CO2 culture condition, after the cell density in the hole plate grew to 90%, the cells were infected with WSN (H1N1) virus at MOI 0.01, after virus adsorption for one hour, the cells were washed with PBS, 1 mL of Opti-MEM culture medium containing 30 μM of the tested compound (19 compounds selected from 26 compounds obtained in Example 1) was added to each hole, 0.1v / v% DMSO aqueous solution was set as a negative control, 24 hours later, 50 μL of the culture supernatant was collected in a 1.5 mL EP tube and stored in -80°C for standby, and then the plaque titration experiment was carried out.

[0068] II. Plaque titration experiment

[0069] MDCK cells were laid in 12-hole cell culture plates, the culture medium was 1×DMEM culture medium containing 0.5 μg / mL TPCK trypsin, after the cells grew, the cells were washed twice with PBS to remove the residual culture medium serum. 1×DMEM culture medium containing 0.5 μg / mL TPCK trypsin was used for one time of wetting for standby.

[0070] The culture supernatant in each hole of Section I was thawed on ice, 2-fold dilution was carried out using 1×DMEM culture medium containing 0.5 μg / mL TPCK trypsin, 100 μL of the virus liquid was added to the hole plate for adsorption, 37°C, 5% CO2 culture condition, the hole plate was shaken uniformly every 10 minutes to make the virus liquid uniformly cover the cells. Low-melting-point agarose was dissolved in ddH2O to prepare a 2w / w% agarose solution, after heating and dissolving, it was placed in a 50°C water bath for standby, and an equal volume of 2×DMEM culture medium was placed in a 37°C water bath for standby. Equal volumes of the 2w / w% agarose solution and the 2×DMEM culture medium were mixed to prepare a 1×DMEM 1% agarose solution. After the virus infection was completed, the virus liquid in the hole plate was aspirated, 1 mL of the 1×DMEM 1w / w% agarose solution was added to each hole, the hole plate was placed in a biological safety cabinet for 20 minutes to allow the agarose to solidify, and then it was placed in a cell culture box upside down. After 48 hours of 37°C, 5% CO2 culture, 1 mL of formalin fixing solution was added to each hole, after overnight fixation, the fixing solution and the agarose were discarded, the number of plaque formation was counted, and the virus titer was calculated. The titer refers to the number of virus particles with biological activity per unit volume of liquid, the titer quantitative expression method is the highest dilution multiple of 2-fold dilution that can see the plaque, the unit is Log2 pfu / mL.

[0071] Compared with the DMSO control, the titers of five compounds showed significant decline. The five compounds were triptolide (code: T18-9), buqinna (code: B18-5), votalolot (code: V15-24), imidazole ribin (code: M9-26) and epalrestat (code: E16-1), which respectively 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). The titer statistical chart is shown in Figure 2 .

[0072] Example 3: Compound median toxic concentration CC 50 determination

[0073] The periphery of a transparent 96-well cell culture plate was filled with PBS 100 μL per well to prevent edge effects, and then A549 cells were plated in the 96-well cell culture plate at a density of 1×10 4 / well, and the culture medium was Opti-MEM medium, 100 μL per well, and the culture was carried out 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, buqinna, votalolot, imidazole ribin and epalrestat were dissolved in Opti-MEM medium, and the final concentration was set to be gradient concentration 0.1 μM, 1 μM, 10 μM, 100 μM, 500 μM, 1000 μM, 5000 μM, 10000 μM, which were added to different wells, 100 μL per well, each concentration was repeated 3 wells, and the cell activity was detected after 24 h culture at 37°C, 5% CO2. Each well was replaced with 100 μL of DMEM medium containing 10 v / v% fetal bovine serum, and 10 μL of CCK-8 solution was added to each well, and after 1 h culture at 37°C, 5% CO2, it was immediately placed on ice and used to detect the OD 450 of each well with a microplate reader. The absorbance of each well was detected 3 times and the average value was taken, and the absorbance of the 0.1 v / v% DMSO water solution well was set as 100% as the negative control, and the cell viability of the experimental groups at different concentrations was calculated, and the data was analyzed by GraphPad Prism 8.0.2, and the CC 50 .

[0074] The fitting curve results are shown in Figure 3 . The CC 50 of triptolide, buqinna, votalolot, imidazole ribin and epalrestat were 1.304 mM, 558.4 μM, 344.2 μM and, >10 mM and 310.4 μM, respectively.

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

[0076] A549 cells were plated in 24-well cell culture plates at a density of 1 x 10 4 / well in F12K medium containing 10 v / v% fetal bovine serum, 500 μL per well, and were allowed to grow to 90% confluence. The cells were then infected with influenza A virus WSN at an MOI of 0.01, and were incubated at 37°C in 5% CO2 for one hour to allow virus adsorption. The medium was then replaced with Opti-MEM medium (containing 0.125 μg / mL TPCK trypsin, 0.1 v / v% DMSO) containing different concentrations of the test compounds (triptolide ketone, brequinar, votalolot, imidazole and epalrestat) at 500 μL / well. The final concentration of each compound was set to 0.01 μM, 0.1 μM, 1 μM and 10 μM, and 0.1 v / v% DMSO aqueous solution was set as a negative control. After incubation at 37°C in 5% CO2 for 24 hours, the supernatant was discarded from each well, and total RNA was extracted from the cells in each well.

[0077] After determining the RNA concentration, the total RNA in each well was subjected to genomic DNA removal according to the instructions of the HiscriptR RIIQ RT SuperMix for qPCR Reverse Transcription Kit (Vazyme, R223-01). 1 μg of 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 in each well into cDNA.

[0078] According to the WSN strain reference gene sequence in NCBI, the fluorescent quantitative PCR primers were designed using Primer Premier 5, and 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 5 μL of 2x ChamQ Universal SYBR qPCR Master Mix, 1 μL of cDNA template, 0.4 μL of each upstream and downstream primer, and 3.2 μL of ddH2O. Each reaction was repeated 3 times. The fluorescent quantitative PCR reaction program was 95°C pre-denaturation for 10 min; 95°C denaturation for 15 s; 60°C annealing for 1 min, a total of 40 cycles of denaturation and annealing; melting curve: 95°C, 15 s; 60°C, 1 min; 95°C, 10 s. 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_hGAPDH)}. The data were analyzed using GraphPad Prism 8.0.2, and the EC 50 .

[0079] The fitting curve results are shown in Figure 4 The EC 50 of triptolide, brinzolamide, vasoactive intestinal peptide, imidazole and epalrestat were 0.6564 μM, 0.1323 μM, 2.213 μM, 5.003 μM and 0.8847 μM, respectively.

[0080] Table 1. Primer sequences

[0081]

[0082] Example 5: Organ plaque titration

[0083] The mice were divided into four groups of vasoactive intestinal peptide, imidazole, epalrestat and 5% DMSO, 8 in each group. After weighing, the mice were anesthetized with isoflurane inhalation, and 1.5x10 4 pfu of influenza A virus WSN was inhaled through the nose, and the mice were anesthetized and administered intranasally in the same way 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 5 v / v % DMSO aqueous solution, and the dosage was 25 μL per mouse.

[0084] On the 3rd day after challenge, 3 mice from each group were anesthetized by isoflurane inhalation and then euthanized by cervical dislocation. The whole lung tissues were taken 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 a grinding steel ball was added. The mixture was ground at 4°C and 30 Hz for 5 min. Then, the mixture was centrifuged at 9000 r / min and 4°C for 5 min. The supernatant was taken and subjected to plaque titration test according to the method of Example 2.

[0085] The results are shown in Table 1. Figure 5 Compared with the control group, the lung virus titers of vosalotol, mizoribine and epalrestat decreased by 71.74% (P<0.01), 76.74% (P<0.001) and 84.13% (P<0.001), respectively.

[0086] Example 6: Determination of influenza virus polymerase activity

[0087] I. Synthesis and source of plasmids

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

[0089] The pRL series vector is a Renilla luciferase reporter vector developed by Promega Company (product number: E2241), and the Renilla luciferase is driven by the TK promoter.

[0090] (2) RNA polymerase component plasmid

[0091] The coding sequence of the basic polymerase 1 (PB1) gene (see GenBank No. LC333183.1), the coding sequence of the basic 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 luciferase reporter gene vector pHH21-SC09NS F-Luc was constructed, which can produce negative-sense RNA containing 176 bases of the 3' end of NS vRNA derived from the influenza virus SC09 strain (full name A / Sichuan / 1 / 2009 strain, H1N1 type), firefly lucase, a stop codon (TAA), and 179 bases of the 5' end of the SC09NS vRNA, wherein the non-coding sequences of the NS vRNA ends on the vector can be recognized and combined by the influenza virus polymerase to initiate the expression of firefly lucase.

[0094] The construction method of the plasmid vectors of the foregoing (2) and (3) is described in the following documents:

[0095] 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.

[0096] II. Principle of enzyme activity determination

[0097] The influenza virus RNA polymerase is composed of three subunits PB2, PB1 and PA, and is an RNA-dependent RNA polymerase. It plays an important role in viral genome transcription together with the NP protein. In order to explore the effect of drugs on the activity of influenza virus polymerase, a dual luciferase reporter assay was used.

[0098] In the dual-luciferase reporter system Dual-Glo® Reporter System (purchased from Promega Company, item 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 transcriptional regulatory element of the target gene is constructed into a luciferase expression vector to construct a reporter gene plasmid, so that this sequence regulates the transcription and expression of luciferase; 2. The reporter gene plasmid is transfected into cells, which are lysed after being given different treatments, and the substrate luciferin is added. Luciferase can catalyze luciferin to emit fluorescence; 3. The high and low fluorescence values obtained can be used to judge the effect of different treatment groups on the transcriptional regulatory element; 4. To avoid errors caused by differences in transfection efficiency when plasmids are transfected into cells, a Renilla luciferase reporter gene plasmid is usually introduced 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, and is little affected by influenza virus inhibitors. The expression of Firefly luciferase is affected by the content or activity of RNA polymerase, and is further affected by influenza virus inhibitors. The ratio of the fluorescence produced by the two luciferases can be used to quantitatively or semi-quantitatively determine the strength of the inhibitory effect of the drug on influenza virus RNA polymerase.

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

[0101] 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 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 a GloMax 96 microplate luminometer (Promega) to determine the luciferase activity of the transfected cells using a dual luciferase reporter gene assay system. The Firefly luciferase reacts with the substrate LAR II to produce a fluorescence signal, which is detected by the instrument and then quenched by the Stop & Glo reagent to activate the Renilla luciferase to produce fluorescence. The effect of the drug on the activity of the influenza virus polymerase can be analyzed.

[0102] Data processing: First, the ratio of Firefly luciferase / Renilla luciferase for each tube was calculated, and then the ratio of the control group was taken as unit 1, and the relative luciferase activity of the different treatment groups, i.e., the activity of the influenza virus polymerase, was obtained.

[0103] II. Determination of activity

[0104] The 12-well cell culture plates were coated with polylysine, and then HEK293T cells were plated in the 12-well cell culture plates with DMEM containing 10% FBS. When the cells grew to 80%, 500 ng pCAGGS-WSN PB2, 500 ng pCAGGS-WSN PB1, 500 ng pCAGGS-WSN PA, 500 ng pCAGGS-WSN NP plasmids, 200 ng pHH21-SC09NSF-Luc and 10 ng pRL-TK plasmids were transfected into the cells at the same time. Ten hours after transfection, the culture medium containing plasmids and transfection reagents was discarded, and the culture medium containing 20 μM and 30 μM of the test compounds (triptolide, brequinar, votalin and mizoribine) was replaced, and then the cells were cultured for another 26 hours. After the cell culture supernatant was discarded, the cells were washed once with PBS, and then the cells were lysed with 250 μL Passive Lysis Buffer per well. After centrifugation at 12000 r / min for 5 min, 20 μL of the supernatant was taken for the detection of the activity of the polymerase complex by using the Dual-Glo® Luciferase Assay System. Reporter System.

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

[0106] It can be seen that the four drugs can inhibit the activity of influenza virus RNA polymerase, which helps to inhibit the influenza virus.

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

[0108] I. Cell detection experiment

[0109] According to the instructions, white non-bottom transparent 96-well plates were used to reduce light loss and the influence between wells. 3 x 10 7 pfu / mL of influenza A virus WSN was used to detect the activity of neuraminidase. The activity of neuraminidase was detected by using NA-XTD TM After dilution of Assay Buffer (purchased from Thermofisher) at 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, and 25 μL was added to each well. The test was repeated in triplicate, and incubation was performed at 37°C for 20 min (at this time, the final concentration of the compound was 33.33 μM). 25 μL of 1:1000 (volume ratio) diluted 1000 x NA-XTD was added to each well. TM Substrate (purchased from Thermofisher) was incubated at room temperature for 30 min. 60 μL of NA-XTD was added to each well. TM Accelerator (purchased from Thermofisher) was immediately used to detect the luminescence value using a GLOMAX 96 microplate luminometer. The detection time was 1 s per well, and the measurement was repeated 3 times to obtain the average value.

[0110] Neuraminidase activity is shown in Figure 7 A. Thus, epalrestat showed inhibition of influenza A virus neuraminidase.

[0111] Subsequently, the half maximal inhibitory concentration (IC 50 ) of epalrestat on neuraminidase was detected, i.e., the concentration of the compound required to inhibit enzyme activity by half, to evaluate the inhibitory activity of the compound on the enzyme. 3 x 10 7 pfu / mL of influenza A virus WSN was used to detect the activity of neuraminidase. The activity of neuraminidase was detected by using NA-XTD TM After dilution of Assay Buffer (purchased from Thermofisher) at 1:100 (volume ratio), 25 μL was added to each well. The test compound was gradient-diluted using Assay Buffer, and the gradient was set to 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. Each concentration was repeated in triplicate, i.e., the final concentration was 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 was added to each well.TM Substrate, incubate at room temperature for 30 minutes; finally, add 60 μL NA-XTD to each well TM Accelerator, using GLOMAX 96 microplate luminometer to detect luminescence values, detection time per well 1 s, measurement 3 times to take the average value. Data were analyzed using GraphPad Prism 8.0.2 software, and IC values ​​of compounds were 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, PBD ID: 6D96, was downloaded from the RCSB website (https: / / www.rcsb.org / ). This protein is the X-ray diffraction structure of influenza virus A / BrevigMission / 1 / 1918 (H1N1) neuraminidase (NA) expressed in HEK-293E cells. The ligand E16-1 structure was obtained from Pubchem (https: / / pubchem.ncbi.nlm.nih.gov / ). Ligand pretreatment was performed using PyMOL software to remove water molecules, calcium ions, and residual ligands from the model, as well as to remove duplicate peptide chains.

[0115] The protein model was then hydrogenated using AutoDockTools 1.5.7, and the docking box was set and saved. The ligand chemical bonds were set to rigid and flexible, and the data was saved as a pdbqt file. 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 docking results were visualized, and binding energy and hydrogen bond analysis was performed using AutoDockTools 1.5.7 and PyMOL 2.5.

[0116] Using AutoDock vina software, E16-1 as ligand, dock into the active pocket of influenza virus N1 subtype neuraminidase, a total of 9 docking models were output Figure 8 (A)), hydrogen bond analysis was performed using AutoDockTool software, and the results are shown in Figure 8 (B-F). Figure 9 Visual analysis of the docking results was performed using PyMOL software, and the results are shown in Figure 8 (A), which is -6.5 kcal / mol.

[0117] The docking site of model 1 was analyzed, and the results are shown in Figure 9 (G), the E16-1 molecule was combined with influenza virus N1 subtype neuraminidase Arg292 and Arg371 sites by hydrogen bond (yellow dotted line). In addition, the binding site that appeared multiple times in other docking models was Arg118.

[0118] Example 8: Detection of compound inhibition of influenza virus protein expression

[0119] A549 cells were plated in a 12-well cell culture plate at a density of 1×10 5 cells per well, and the culture medium was F12K medium containing 10 v / v% fetal bovine serum. The cells were cultured at 37°C and 5% CO2, and when the cell density in the well plate reached 90%, the cells were infected with WSN (H1N1) virus at an MOI of 0.01. After one hour of virus adsorption, each compound to be tested was placed in 3 wells, and 1 mL of Opti-MEM medium containing 5 μM, 10 μM, and 20 μM of the compound to be tested (triptolide ketone, brequinar, votalolot, imidazole and ipraglifast) was added, respectively. 0.1 v / v% DMSO aqueous solution was set as a negative control, and Oseltamivir phosphate was set as a positive control. After 24 hours, 50 μL of culture supernatant was collected in a 1.5 mL EP tube and frozen at -80°C.

[0120] After discarding the cell culture supernatant, the cells were washed once with PBS, and 100 μL of 1× SDS lysis buffer, 1 μL of PMSF solution, and 0.1 μL of ribozyme were added to each well. After lysis at room temperature for 5 min, the lysis solution was collected in a 1.5 mL EP tube and denatured at 95°C for 10 min for Western Blot detection.

[0121] The above protein sample was added to the 10% SDS-PAGE gel loading well at 15 μL / well, electrophoresis was carried out at 80 V, and after the protein sample began to separate through the stacking gel, the voltage was changed to 120 V until the end of electrophoresis. 0.45 μm nitrocellulose membrane (NC membrane) was placed in NC membrane equilibration solution for 30 s, and then eblot L1 rapid wet transfer membrane instrument was used to complete the membrane transfer. After blocking with 5% skim milk solution at room temperature for 1 h, the residual blocking solution was washed with PBS solution, and the primary antibody was diluted 1:1000 with PBS and incubated at room temperature for 1 h on a shaker. After incubation, the secondary antibody was washed with PBST solution at room temperature for 10 min at high speed on a shaker, repeated 3 times to wash away the unbound primary antibody. The secondary antibody was prepared at a dilution of 1:5000 (volume ratio) in PBS, and incubated at room temperature for 1 h in the dark. The membrane was washed 3 times in the dark for 10 min each time, and imaged using an Odyssey CLX near-infrared scanning detector and the results were analyzed.

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

[0123] For the influenza virus nucleoprotein NP, the primary antibody was mouse NP monoclonal antibody (laboratory self-made), and the secondary antibody was Dylight 680 labeled goat anti-mouse IgG (Li-COR Bioscience, Cat. No. 926-68070).

[0124] The WB results of each group and the virus titer are shown in Figure 10 As can be seen from the gray value analysis of protein expression, no viral protein could be detected in the triptolide ketone group; the NP protein and PB1 protein in the buquinolate group showed a significant gradient decrease; the NP protein and PB1 protein in the vosalotolot group decreased; the NP protein in the imidazole ridine group showed no significant decrease, and the PB1 protein showed a gradient decrease; the NP protein in the epalrestat group also showed no significant decrease, and the PB1 protein showed a gradient decrease. The NP protein content in the positive control oseltamivir group showed no significant decrease, but the virus titer decreased significantly.

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

[0126] According to the human gene sequence of the target gene of Boceprevir (Gene name: DHODH, Gene ID: 231, GenBank No. NM_001361.5) and the human gene sequence of the target gene of Imidapril (Gene name: IMPDH2, GenBank No. NM_001410759.1, Gene ID: 1723) and the human gene sequence of the target gene of Epalrestat (Gene name: AKR1B1, GenBank No. NM_001628.4, Gene ID: 3615), siRNAs were designed respectively, and the RNA sequence complementary to the target sequence in each siRNA was shown in si_231 (SEQ ID NO. 6), si_1723 (SEQ ID NO. 7), si_3615 (SEQ ID NO. 8) in Table 2 respectively, and the sequence shown in si_NC (SEQ ID NO. 9) was used as a negative control. The siRNAs and the negative control si_NC were transfected into A549 cells to interfere with the expression of the related genes using the reverse transfection method in the instructions of Lipofectamine TM RNAiMAX (purchased from ThermoFisher Scientific, item number 13778030). One group of cells was collected for cell samples at 36 h after infection and Western Blot experiment was performed to detect the interference efficiency of protein level. Another group of cells was infected with influenza A virus WSN at 36 h after infection with MOI of 0.01, and 1 mL / well of Opti-MEM medium containing 0.125 μg / mL TPCK trypsin was used 1 h after infection. The cell culture supernatant was collected 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.

[0127] For the internal reference GAPDH, the primary antibody was rabbit GAPDH polyclonal antibody (Proteintech, 10494-1-AP), and the secondary antibody was Dylight 680 labeled goat anti-rabbit IgG (Li-COR Bioscience, 926-68071). For the boceprevir target protein, the primary antibody was mouse DHODH monoclonal antibody (Proteintech, 67977-1-Ig), and the secondary antibody was Dylight 680 labeled goat anti-mouse IgG (Li-COR Bioscience, 926-68070). For the imidaprilin target protein, the primary antibody was mouse IMPDH2 monoclonal antibody (Proteintech, 67663-1-Ig), and the secondary antibody was Dylight 680 labeled goat anti-mouse IgG (Li-COR Bioscience, 926-68070). For the epalrestat target protein, the primary antibody was rabbit AKR1B1 polyclonal antibody (Invitrogen, PA5-29718), and the secondary antibody was Dylight 680 labeled goat anti-rabbit IgG (Li-COR Bioscience, 926-68071).

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

[0129]

[0130] After 36h of siRNA interference, the expression of boceprevir Target, imidaprilin Target and epalrestat Target genes all decreased Figure 11 On this basis, after infection with the virus, the virus titers slightly increased at 24h and 48h. The virus titers of boceprevir Target increased by 1.86 times and 4.18 times at 24h and 48h after infection, respectively; the virus titers of imidaprilin Target increased by 3.03 times and 6.87 times at 24h and 48h after infection, respectively; the virus titers of epalrestat Target increased by 3.39 times and 4.53 times at 24h and 48h after infection, respectively Figure 11 B).

[0131] After the target proteins in the cells treated with the three drugs were inhibited by siRNA, the virus titers increased, indicating that the three target proteins were involved in the action of the drugs in inhibiting the virus.

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

[0133] I. Preparation of drugs

[0134] The drug solutions of triptolide, brequinar, vorseloto, mizoribine and epalrestat were prepared in 0.1 v / v% DMSO (the solvent was 0.1 v / v% DMSO). The concentrations are shown in Table 3 below (all EC values ​​calculated in Example 4). 50 corresponding concentration).

[0135] Combine any two of the above five drugs (a total of ten combinations) with double the EC of each drug. 50 The corresponding concentrations of the stock solution 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 concentrations are shown in Table 3 below, Nos. 6-15. The solvent is 0.1 v / v% DMSO.

[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 test compounds, and the solution No. 16 was used as a negative control. Each sample was repeated 12 times. The other 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. The specific vRNA content is shown in column 3 of Table 3. The statistics of influenza virus NP gene vRNA content in the negative control, two single drugs, and the combination of the two single drugs are shown in Table 3. Figure 12 .

[0138] As can be seen, the viral load of the two drugs tested in most combinations was the lowest, significantly better than that of the individual drugs, and some showed significant synergistic effects. No combination of drugs was found to have a higher viral load than that of the individual drugs, and none of the drugs antagonized each other. Therefore, the combined use of the five drugs has promising application prospects.

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

[0140]

[0141]

[0142] It is understood from common technical knowledge that the present invention may be implemented by other embodiments that do not depart from its spirit or essential features. Therefore, the embodiments disclosed above are, in all respects, merely illustrative and not exclusive. All modifications within the scope of the present invention or equivalent to the scope of the present invention are intended to be 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 or a combination of two of epalrestat and a pharmaceutically acceptable salt of epalrestat; The active substance b is any one or a combination of two of buquinar and a pharmaceutically acceptable salt of buquinar; The structural formula of epalrestat is: The structural formula of the buquina is:

2. The pharmaceutical composition according to claim 1, wherein 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 2, wherein 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, wherein 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 brequinar 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, lithium salt, hydrochloride, sulfate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, maleate, tartrate, trifluoroacetate, aspartate, taurate, gluconate, fructose, salicylate, nitrate, p-toluenesulfonate, methanesulfonate, benzoate, citrate, lactate, fumarate and ascorbate of brequinar.

5. The pharmaceutical composition according to claim 1, wherein The composition further contains auxiliary materials.

6. Use of a biomaterial in the preparation of a medicament for use alone or in combination with other agents to improve health conditions; The biomaterial is the pharmaceutical composition or active substance a according to any one of claims 1 to 5; The active substance a is any one or a combination of two of epalrestat and a pharmaceutically acceptable salt of epalrestat; The structural formula of epalrestat is: The use is selected from any one of the following U1, U2, U3 and U4 or a combination thereof; U1: The improvement in health status is to prevent influenza, treat influenza, slow down influenza or inhibit the proliferation of influenza virus; U2: The improved health status refers to reducing the damage of influenza virus to the lungs; U3: The improvement of health status is to reduce the damage 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; The influenza virus is an H1N1 influenza virus, and the influenza is influenza caused by the H1N1 influenza virus.

7. 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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