Use of an imidazole derivative for the preparation of a formulation for inhibiting influenza virus

Abstract

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

Description

TECHNICAL FIELD

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

[0002] Influenza virus is a representative virus of Orthomyxoviridae, and 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 statistical data 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 problems such as 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 a first active substance and a second active substance;

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

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

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

[0021] The active substance c is vasoactive intestinal peptide, any one of a pharmaceutical salt of vasoactive intestinal peptide and a prodrug of vasoactive intestinal peptide, a combination of any two or a combination of three;

[0022] The structural formula of the imidazole is:

[0023]

[0024] The structural formula of the epalrestat is:

[0025]

[0026] The structural formula of the vasoactive intestinal peptide is:

[0027]

[0028] In some embodiments, when the second active substance is active substance b, the molar ratio of the first active substance to the second active substance is 1:0.02-2 (for example, 1:0.02, 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, 1.6, 1.7, 1.8, 1.9, 2, or a range between any two of the above values);

[0029] when the second active substance is active substance c, the molar ratio of the first active substance to the second active substance is 1:0.3-0.6.

[0030] In some embodiments, when the second active substance is active substance b, the molar ratio of the first active substance to the second active substance is 1:0.1-0.2.

[0031] when the second active substance is active substance c, the molar ratio of the first active substance to the second active substance is 1:0.3-0.6.

[0032] In some embodiments, the pharmaceutically acceptable salt of imidazolium is selected from the group consisting of hydrochloride, sulfate, citrate, besylate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate, trifluoroacetate, aspartate, taurate, gluconate, fructate, salicylate, nitrate, p-toluenesulfonate, methanesulfonate, benzoate, citrate, lactate, citrate, fumarate, and ascorbate;

[0033] 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;

[0034] The pharmaceutically acceptable salt of vasoactive intestinal peptide is selected from the group consisting of hydrochloride, sulfate, citrate, besylate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate, trifluoroacetate, aspartate, taurate, gluconate, fructate, salicylate, nitrate, p-toluenesulfonate, methanesulfonate, benzoate, citrate, lactate, citrate, fumarate, and ascorbate of vasoactive intestinal peptide.

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

[0036] The second aspect of the present application provides use of a biomaterial in the preparation of a product for improving health condition, alone or in combination with other preparations;

[0037] The biomaterial is the pharmaceutical composition or active substance a according to the first aspect of the present application.

[0038] The active substance a is imidazole ribin, any one of a pharmaceutically acceptable salt of imidazole ribin and a prodrug of imidazole ribin, a combination of any two or a combination of three;

[0039] The imidazole ribin has the following structural formula:

[0040]

[0041] The use is selected from any one or a combination of U1, U2, U3 and U4 as follows:

[0042] U1: the improved health condition is prevention, treatment, alleviation or inhibition of influenza virus proliferation;

[0043] U2: the improved health condition is reduction of lung damage caused by influenza virus;

[0044] U3: the improved health condition is reduction of damage to the body caused by influenza virus by inhibiting influenza virus RNA-dependent RNA polymerase;

[0045] U4: the improved health condition is reduction of damage to the body caused by influenza virus by inhibiting influenza virus neuraminidase, and the second active substance is active substance b.

[0046] In some embodiments, the product is selected from a pharmaceutical product, a health product, a feed, a feed additive, a food and a food additive.

[0047] In some embodiments, the influenza virus is influenza A virus, and the influenza is influenza A.

[0048] In some embodiments, the influenza virus is H1N1 influenza virus, and the influenza is influenza caused by H1N1 influenza virus.

[0049] In some embodiments, the influenza is selected from human influenza, avian influenza and swine influenza. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 Statistical relationships between inoculation MOI, drug concentration and OD 450 are shown.

[0051] Figure 2 Statistical relationships between virus infection titers under five drug interventions are shown.

[0052] Figure 3 Fitting curves of CC 50 of five drugs are shown.

[0053] Figure 4 Fitting curves of EC 50 of five drugs are shown.

[0054] Figure 5 The results of virus challenge protection experiments under three drug interventions are shown.

[0055] Figure 6 The results of the effects of four drugs on the polymerase activity of influenza A virus are shown.

[0056] Figure 7 The results of the effects of epalrestat on the neuraminidase activity of influenza virus are shown.

[0057] Figure 8 The results of hydrogen bond analysis of the molecular docking model are shown.

[0058] Figure 9 The results of molecular docking of epalrestat and influenza virus neuraminidase N1 are shown.

[0059] Figure 10 The results of the inhibition of viral protein expression by five drugs are shown.

[0060] Figure 11 The effects of siRNA interference drugs targeting genes on influenza virus replication are shown.

[0061] Figure 12 The statistical results of viral content under the intervention of the combination of five drugs are shown. DETAILED DESCRIPTION

[0062] 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.

[0063] Example 1: Protective experiment of influenza virus infection

[0064] I. Establishment of positive control method

[0065] Phosphorothioate oseltamivir (purchased from MCE Company, same below) was used as a positive control drug to establish a method for indirectly screening anti-influenza virus small molecule compounds by CCK-8 cell viability.

[0066] First, fill PBS 100 μL in the periphery of a transparent 96-well cell culture plate every well to prevent edge effects. Then, MDCK cells are plated in the well plate at a density of 1 × 10 4 cells / well, and the medium is DMEM medium (purchased from Sigma-Aldrich Company, same below) containing 10 v / v% fetal bovine serum (purchased from WISENT Company, same below) at 100 μL per well. Incubate at 37°C, 5% CO2, and when the cell density in the well plate grows to 90%, perform virus infection.

[0067] The culture medium in the wells was discarded using a multi-channel pipette, and the cells were washed twice with PBS to remove residual serum in the culture medium to prevent its influence on virus infection. After rinsing once with Opti-MEM medium (purchased from Gibco, same below) supplemented with TPCK trypsin (final concentration 0.5 μg / mL), the cells were infected with influenza virus WSN strain (H1N1 type) (NCBI Taxonomy ID: 382835) at MOI 0, MOI 0.01, MOI 0.1, MOI 1, MOI 5 and MOI 10, respectively, 100 μL of Opti-MEM medium containing 0.5 μg / mL TPCK trypsin was added to each well, and the virus was allowed to adsorb for one hour at 37°C, 5% CO2, and then the virus solution was discarded. Then Opti-MEM medium (containing 0.1 v / v% DMSO, containing 0.5 μg / mL TPCK trypsin) containing oseltamivir phosphate at concentrations of 400 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL and 25 μg / mL was added to the wells inoculated at different MOIs, respectively, and Opti-MEM medium containing 0.1 v / v% DMSO (containing 0.5 μg / mL TPCK trypsin) was used as a negative control, and the cell viability in each well was detected after 24 h of culture at 37°C, 5% CO2.

[0068] The liquid in the well plate was discarded and replaced with 100 μL / well of DMEM medium, and 10 μL of CCK-8 solution (from the Cell Counting Kit-8, purchased from MCE, same below) was added to each well, and after incubation at 37°C, 5% CO2 for 1 h, the plate was immediately placed on ice and the OD 450 of each well was detected using a microplate reader, 3 wells were repeated for each combination of MOI and oseltamivir concentration, and the absorbance of each well was detected 3 times and averaged, and the data was analyzed using GraphPad Prism 8.0.2.

[0069] The CCK-8 detection results are shown in Table 1. Figure 1 As can be seen from the above table, at the same MOI, different concentrations of oseltamivir groups showed a good gradient of cell activity, and with the increase of oseltamivir concentration, the cell activity increased, indicating that the drug's ability to inhibit the virus improved. At the same time, among the groups of the same concentration of drug, different MOI infection groups also showed a significant gradient of cell activity, and with the increase of MOI, the cell viability decreased significantly, and under the same drug inhibition ability, the more virus inoculated, the lower the cell viability, indicating that this method has good sensitivity. This method can use OD value to reflect the drug's ability to inhibit the virus.

[0070] II. Preliminary screening, rescreening and data analysis

[0071] According to the experimental results of section one, MOI 0.01 was selected for screening experiment, 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 FDA approved drug library with a final concentration of 30 μM were replaced, 0.1 v / v % DMSO-containing Opti-MEM medium (containing 0.5 μg / mL TPCK trypsin) was used as a solvent, each compound was repeated 3 holes, oseltamivir positive control was set, 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 24 h culture at 37℃, 5% CO2, the cell viability was detected by the method in section one, the absorbance of each well was detected for 3 times and the average value was taken. Compared with the control group, 67 kinds of compounds with known strong virus activity (high OD value) in each plate showed protective effect on cells after virus infection. Then the same method was used to rescreen the 67 kinds of compounds, and the results showed that 26 kinds of compounds showed strong cell protection.

[0072] Example 2: Virus titer determination experiment

[0073] I. Identification of compound antiviral ability

[0074] A549 cells were plated in 12-well cell culture plates at a density of 1×10 5 / mL per well, and the culture medium was F12K medium containing 10 v / v % fetal bovine serum (purchased from Multicell, same below), 1 mL per well, 37℃, 5% CO2 culture, when the cell density in the well plate grew to 90%, the cells were infected with WSN (H1N1) virus at MOI 0.01, after one hour of virus adsorption, the cells were washed with PBS, 1 mL of Opti-MEM medium (containing 0.125 μg / mL TPCK trypsin, containing 0.1 v / v % DMSO) containing 30 μM test compound (19 kinds of compounds selected from 26 kinds of compounds obtained in example 1) was replaced for each well, 0.1 v / v % DMSO aqueous solution was set as negative control, 50 μL of culture supernatant was collected in 1.5 mL EP tube after 24 h, and then stored at -80℃ for use, and then plaque titration experiment was carried out.

[0075] II. Plaque titration experiment

[0076] MDCK cells were plated in 12-well cell culture plates, the culture medium was 1×DMEM 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 medium containing 0.5 μg / mL TPCK trypsin was used for washing once for standby.

[0077] In Section 1, the culture supernatant from each well was thawed on ice and serially diluted 2-fold with 1×DMEM medium containing 0.5 μg / mL TPCK trypsin. 100 μL of the virus solution was added to each well to allow adsorption, and the plate was incubated at 37°C and 5% CO2, with the plate shaken evenly every 10 min to ensure uniform virus coverage. Low-melting-point agarose was dissolved in ddH2O to prepare a 2 w / w agarose solution, which was then heated to dissolve and incubated in a 50°C water bath. Simultaneously, an equal volume of 2×DMEM medium was incubated in a 37°C water bath. An equal volume of the 2 w / w agarose solution and 2×DMEM medium was mixed to prepare a 1×DMEM 1% agarose solution. After virus infection, the virus solution was aspirated from the wells, and 1 mL of 1×DMEM 1 w / w agarose solution was added to each well. The plate was placed in a biosafety cabinet for 20 min to allow the agarose to solidify, then inverted and placed in a cell culture incubator. After incubation at 37℃ and 5% CO2 for 48 h, 1 mL of formalin fixative was added to each well. After fixation overnight, the fixative and agarose were discarded, and the number of plaques was counted and the virus titer was calculated. Titer refers to the number of biologically active virus particles per unit volume of liquid. The titer is quantitatively expressed as the highest serial dilution of 2-fold to which plaques can be seen, with units of Log2pfu / mL.

[0078] Compared with the DMSO control, the titers of five compounds showed a significant decrease. These five compounds were triptolide (code: T18-9), buquina (code: B18-5), vocerotropin (code: V15-24), imidazolidinedione (code: M9-26), and epalrestat (code: E16-1). These 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. Figure 2 .

[0079] Example 3: Half-maximal toxic concentration (CC) of compound 50 Measurement

[0080] To prevent edge effects, fill each well of a clear 96-well cell culture plate with 100 μL of PBS around the perimeter. Then, inoculate A549 cells at a rate of 1 × 10⁻⁶ cells / well. 4The cells were seeded in 96-well cell culture plates at a density of 1×10 450 The absorbance of each well was measured three times, and the average value was taken. The 0.1% v / v DMSO aqueous solution well was used as the negative control, and the absorbance thereof was set to 100%. The cell viability of the experimental groups at different concentrations was calculated, and the data were analyzed using GraphPad Prism 8.0.2. Through nonlinear regression, the CC 50 .

[0081] The fitting curve results are shown in Figure 3 The CC 50 of triptolide, brequinar, votalin, mizoribine and epalrestat were 1.304 mM, 558.4 μM, 344.2 μM and >10 mM and 310.4 μM, respectively.

[0082] Example 4: Determination of the half-effective concentration EC 50 of the compounds

[0083] A549 cells were seeded in 96-well cell culture plates at a density of 1×10 4The cells were seeded in 24-well cell culture plates at a density of 1 x 105 cells per well in 500 μL of F12K medium containing 10 v / v% fetal bovine serum. After the cells grew to 90%, the cells were infected with influenza A virus WSN at a MOI of 0.01. After adsorption for one hour at 37°C in a 5% CO2 incubator, the medium was replaced with 500 μL / well of Opti-MEM medium (containing 0.125 μg / mL TPCK trypsin, 0.1 v / v% DMSO) containing different concentrations of the test compound (triptolide, brivudine, votalictat, mizoribine and epalrestat). 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 24 hours of incubation at 37°C in a 5% CO2 incubator, the supernatant was discarded from each well, and the total RNA in each well was extracted.

[0084] After the RNA concentration was determined, the total RNA in each well was removed according to the instructions of the HiscriptR RIIQ RT SuperMix for qPCR Reverse Transcription Kit (Vazyme, R223-01), and 1 μg of RNA was used as a template. The mRNA in each well was reverse transcribed into cDNA using 5Seg_vRNA-RT (SEQ ID NO. 1) in Table 1 as a primer.

[0085] 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 .

[0086] The fitting curve results are shown in Figure 4 The EC 50 of triptolide, brequinar, vasoactive intestinal peptide, mizoribine and epalrestat were 0.6564 μM, 0.1323 μM, 2.213 μM, 5.003 μM and 0.8847 μM, respectively.

[0087] Table 1. Primer sequences

[0088]

[0089] Example 5: Organ plaque titration

[0090] The mice were divided into four groups of vasoactive intestinal peptide, mizoribine and 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, and the control group was administered 5 v / v % DMSO aqueous solution at a dose of 25 μL per mouse.

[0091] 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 tissue was taken and stored in 2 mL EP tube 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 tube was ground at 4°C and 30 Hz for 5 min. Then, the tube 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.

[0092] 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.

[0093] Example 6: Determination of influenza virus polymerase activity

[0094] I. Synthesis and source of plasmids

[0095] (1) Renilla luciferase control reporter vector pRL-TK

[0096] 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.

[0097] (2) RNA polymerase component plasmid

[0098] 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.

[0099] (3) Firefly luciferase reporter gene vector pHH21-SC09NS F-Luc

[0100] A luciferase reporter gene vector pHH21-SC09NS F-Luc was constructed, which could produce a 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 luciferase, a stop codon (TAA), and 179 bases of the 5' end of the SC09NS vRNA, wherein the non-coding sequence of the NS vRNA end on the vector could be recognized and combined by the influenza virus polymerase to initiate the expression of firefly luciferase.

[0101] The construction method of the plasmid vectors of the foregoing (2) and (3) is described in the following references:

[0102] 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.

[0103] II. Principle of enzyme activity determination

[0104] 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.

[0105] In the dual luciferase reporter system 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. In order 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] II. Determination of activity

[0111] 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, votalate 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 and used for luciferase activity detection using the Dual-Luciferase Reporter Assay System. Reporter System to detect the activity of the polymerase complex.

[0112] The polymerase activity of the five compounds that inhibited the replication of influenza virus in Example 2 was detected under the action of gradient concentrations of the compounds. The luciferase ratio of the 0.1 v / v% DMSO aqueous solution group was taken as 100%, and the results are shown in Table 1. Figure 6 As shown in Table 1, triptolide, brequinar, votalate and mizoribine can significantly inhibit the polymerase activity of influenza virus, and the polymerase activity of the 30 μM group is lower than that of the 20 μM group, indicating that the inhibition 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 votalate 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.

[0113] It can be seen that the four drugs can inhibit the RNA polymerase activity of influenza virus, which helps to inhibit influenza virus.

[0114] Example 7: Detection of the neuraminidase activity of influenza virus

[0115] I. Cell detection experiment

[0116] 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. 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 three times to obtain the average value.

[0117] Neuraminidase activity is described in Figure 7 A. As can be seen, epalrestat showed inhibition of influenza A virus neuraminidase.

[0118] 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 at 1:100, 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 the substrate and incubate at room temperature for 30 min; finally, add 60 μL of NA-XTD to each well. TM An accelerator was used to detect luminescence values ​​in a GLOMAX 96 microplate chemiluminescence analyzer. The detection time per well was 1 second, and three measurements were taken, with the average value calculated. Data were analyzed using GraphPad Prism 8.0.2 software. The IC50 values ​​of the compounds were calculated using nonlinear regression and nonlin fitting. 50 .

[0119] See fitted curve Figure 7 B. Therefore, epalrestat exhibits concentration-dependent inhibitory activity against neuraminidase. Curve fitting revealed the drug concentration required to inhibit neuraminidase to the extent that it does so, i.e., the IC50 concentration. 50 The value is 17.36 μM.

[0120] II. Molecular docking experiments

[0121] The protein ligand structure was downloaded from the RCSB website (https: / / www.rcsb.org / ), PBD ID: 6D96. This protein is the X-ray diffraction structure of the neuraminidase (NA) of influenza virus A / BrevigMission / 1 / 1918 (H1N1) expressed in HEK-293E cells. The structure of ligand E16-1 was obtained from the Pubchem website (https: / / pubchem.ncbi.nlm.nih.gov / ). Ligand pretreatment was performed using PyMOL software to remove water molecules, calcium ions, and residual ligands from the model, while also removing repeating peptide chains.

[0122] Then, using AutoDockTools-1.5.7 software, the protein model was hydrogenated, the docking box was set, and the data was saved. The stiffness and flexibility of the ligand chemical bonds were set and saved as a pdbqt file. Molecular docking was performed using the AutoDock vina command with the parameters "eceptor=6d96 pymol.pdbqt; ligand=E16-1.pdbqt; center_x=17.652; center_y=-9.22; center_z=15.293; size_x=15.0; size_y=15.0; size_z=15.0; out=6d96-E16-1_out.pdbqt". The docking results were visualized, and binding energy and hydrogen bond analysis were performed using AutoDockTools-1.5.7 and PyMOL 2.5.

[0123] 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 by 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.

[0124] 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.

[0125] Example 8: Detection of compound inhibition of influenza virus protein expression

[0126] A549 cells were plated in a 12-well cell culture plate at a density of 1×10 5 per well, and the culture medium was F12K medium containing 10 v / v% fetal bovine serum. The cells were cultured at 37°C, 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, 20 μM of the compound to be tested (triptolide ketone, brequinar, votalolot, imidazole and ipraglifast) was added, 0.125 μg / mL TPCK trypsin, 0.1 v / v% DMSO. The negative control was 0.1 v / v% DMSO aqueous solution, and the positive control was Oseltamivir phosphate. After 24 hours, 50 μL of culture supernatant was collected in a 1.5 mL EP tube and frozen at -80°C.

[0127] 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 lysate was collected into a 1.5 mL EP tube and denatured at 95°C for 10 min for Western Blot detection.

[0128] 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 the NC membrane equilibration solution for 30 s, and then the membrane was transferred using eblot L1 rapid wet transfer membrane instrument. 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.

[0129] 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).

[0130] 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).

[0131] 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 boceprevir group showed a significant gradient decrease; the NP protein and PB1 protein in the vaborletot group decreased; the NP protein in the mizoribine 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.

[0132] Example 9: Effect of siRNA interference drug target gene on influenza virus replication

[0133] 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. The RNA sequence complementary to the target sequence in each siRNA is shown in Table 2 as si_231 (SEQ ID NO. 6), si_1723 (SEQ ID NO. 7), si_3615 (SEQ ID NO. 8), respectively. The sequence shown as 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, respectively, and the plaque titration experiment was performed according to the method in Example 2 to determine the virus titer.

[0134] For the internal control GAPDH, the primary antibody was a rabbit-derived GAPDH polyclonal antibody (Proteintech, catalog number 10494-1-AP), and the secondary antibody was Dylight 680-labeled goat anti-rabbit IgG (Li-COR Bioscience, catalog number 926-68071). For the target gene protein of buquina, the primary antibody was a mouse-derived DHODH monoclonal antibody (Proteintech, catalog number 67977-1-Ig), and the secondary antibody was Dylight 680-labeled goat anti-mouse IgG (Li-COR Bioscience, catalog number 926-68070). For the target gene protein of imidazoribine, the primary antibody was a mouse-derived IMPDH2 monoclonal antibody (Proteintech, catalog number 67663-1-Ig), and the secondary antibody was Dylight 680-labeled goat anti-mouse IgG (Li-COR Bioscience, catalog number 926-68070). For the target gene protein of epalrestat, the primary antibody was a rabbit-derived AKR1B1 polyclonal antibody (Invitrogen, catalog number PA5-29718), and the secondary antibody was Dylight 680-labeled goat anti-rabbit IgG (Li-COR Bioscience, catalog number 926-68071).

[0135] Table 2. siRNA sequences (sequences of RNA strands complementary to the target sequence)

[0136]

[0137] After 36 hours of siRNA interference, the expression levels of the buquina target, imidazolidin target, and epalrestat target genes all decreased. Figure 11 A) Based on this, viral titers slightly increased at 24h and 48h after infection. Buquina Target showed a 1.86-fold and 4.18-fold increase in viral titers at 24h and 48h post-infection, respectively; imidazolidin Target showed a 3.03-fold and 6.87-fold increase at 24h and 48h post-infection, respectively; and epalrestat Target showed a 3.39-fold and 4.53-fold increase at 24h and 48h post-infection, respectively. Figure 11 B).

[0138] After inhibiting the target proteins in cells treated with these three drugs using siRNA, the viral titer increased, indicating that the aforementioned three target proteins were involved in the drug's inhibitory effect on the virus.

[0139] Example 10: Test of the antiviral effect of drug combination

[0140] I. Drug Preparation

[0141] Prepare drug solutions of triptolide, buquina, vorselotor, mizoribine, and epalrestat in 0.1 v / v DMSO. The concentrations are shown in Table 3, numbers 1-5 below (all EC values ​​calculated in Example 4). 50 (Corresponding concentration).

[0142] Any two of the aforementioned five drugs can be combined (a total of ten combinations), using twice the EC50 of the single drug. 50 A composition solution is prepared by mixing equal volumes of stock solutions of corresponding concentrations. The concentrations of the two drugs in the composition are both EC. 50 Corresponding concentrations. The solvent is 0.1 v / v % DMSO. The stock solution concentrations in the preparation method are shown in items 6-15 of Table 3 below.

[0143] The following negative control was set up, as shown in Table 3, number 16.

[0144] Using solutions 1-15 in Table 3 as test compounds and solution 16 as a negative control, each sample was repeated 12 times. Other methods and procedures were the same as in Example 4. The content of influenza virus NP gene vRNA in the corresponding wells of each test solution was determined. See column 3 of Table 3 for specific vRNA content. For statistics on the influenza virus NP gene vRNA content in the negative control, two single drugs, and combinations of these two single drugs, see [link to table]. Figure 12 .

[0145] Therefore, it is evident that the viral loads of the two drugs in most combinations were the lowest, significantly better than those of the single drugs, and some showed significant synergistic effects. No cases were found where the viral loads of the combination drugs were higher than those of the single drugs, and there were no antagonistic interactions among these drugs. Therefore, the combination of five drugs shows better application prospects.

[0146] Table 3. Statistics on Drug Preparation and Virus Content

[0147]

[0148]

[0149] As is known from common technical knowledge, this invention can be implemented through other embodiments that do not depart from its spirit or essential characteristics. Therefore, the disclosed embodiments described above are merely illustrative in all respects and are not the only ones. All modifications within the scope of this invention or its equivalents are included in this invention.

Claims

1. A pharmaceutical composition, wherein the pharmaceutically active ingredient of the pharmaceutical composition comprises a first active substance and a second active substance; The first active substance is any one or a combination of two of imidazoribine and pharmaceutical salts of imidazoribine; The second active substance is any one or a combination of two of vocerotrope and vocerotrope medicinal salts; The structural formula of the imidazoribine is: ； The structural formula of the Vucelot is: 。 2. The pharmaceutical composition according to claim 1, characterized in that, The molar ratio of the first active substance to the second active substance is 1:0.04-4.

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

6.

4. The pharmaceutical composition according to any one of claims 1-3, characterized in that, The pharmaceutical salts of imidazoribine are selected from imidazoribine hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoric acid, phosphate, acetate, propionate, succinate, oxalate, malate, maleate, tartrate, trifluoroacetate, aspartate, taurine, gluconate, fructose, salicylate, nitrate, p-toluenesulfonate, methanesulfonate, benzoate, citrate, lactate, fumarate, and ascorbate. The pharmaceutical salts of Voselotto are selected from the hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoric acid, phosphate, acetate, propionate, succinate, oxalate, malate, maleate, tartrate, trifluoroacetate, aspartate, taurine, gluconate, fructose, salicylate, nitrate, p-toluenesulfonate, methanesulfonate, benzoate, citrate, lactate, fumarate, and ascorbate of Voselotto.

5. The pharmaceutical composition according to claim 1, characterized in that, The composition also contains excipients.

6. Use of a biomaterial in the preparation of a product for use alone or in combination with other formulations to improve health conditions; The biomaterial is the pharmaceutical composition according to any one of claims 1-5; The intended use is selected from any one or a combination of the following U1, U2 and U3; U1: The improvement of health status refers to preventing influenza, treating influenza, slowing down influenza, or inhibiting the proliferation of influenza virus; U2: The improvement in health status refers to reducing the damage of the influenza virus to the lungs; U3: The improvement in health status is achieved by reducing the damage of influenza virus to the body by inhibiting influenza virus RNA-dependent RNA polymerase.

7. The use as described in claim 6, characterized in that, The product is selected from pharmaceuticals.

8. The use as described in claim 6, characterized in that, The influenza virus is a type A influenza virus, and the influenza is type A influenza.

9. The use as described in claim 6, characterized in that, The influenza virus is the H1N1 influenza virus, and the influenza is influenza caused by the H1N1 influenza virus.

10. The use as described in claim 6, characterized in that, The influenza strains mentioned are human influenza, avian influenza, and swine influenza.

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