Use of triptolide in preparing a preparation for inhibiting influenza virus

Abstract

The present invention discloses the use of triptolide in preparing a preparation for use alone or in combination with other preparations to prevent, treat, slow down, or inhibit the proliferation of influenza viruses. Triptolide and compositions containing triptolide have good safety, can effectively inhibit the proliferation of influenza viruses, and have application prospects in treating influenza.

Description

Technical Field

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

[0002] Influenza virus is a representative member of the Orthomyxoviridae family and is divided into four genera: A, B, C, and D. All three influenza virus types, A, B, and C, can infect humans. According to WHO statistics, an estimated 5% to 10% of adults and 20% to 30% of children contract influenza each year, resulting in 3 to 5 million severe cases and approximately 1 million deaths worldwide.

[0003] Influenza A virus (IAV) is a respiratory pathogen of significant economic and public health importance due to its high rates of severe illness and mortality. Its viral genome consists of eight segments of single-stranded, negative-sense RNA encoding ten essential proteins: PB2, PB1, PA, HA, NP, NA, M1, M2, NS1, NEP / NS2, and several nonessential accessory proteins, such as PB1-F2 and PA-X. Seasonal influenza A viruses, typically of the H1N1 and H3N2 subtypes, have caused recurrent epidemics of varying severity for decades. In the United States, influenza causes over 200,000 hospitalizations annually and, during the off-season, 3,000 to 49,000 deaths. Frequent antigenic drift and antigenic shift have allowed novel strains from other species to cause human influenza pandemics, such as the 2009 "swine flu" H1N1 and avian influenza H5N1. Because humans have little immunity to these viruses, they can spread rapidly worldwide, posing a significant threat to global health.

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

[0005] Drugs are another important tool for preventing and controlling human influenza. Currently available clinically for the treatment of influenza virus infection can be divided into first-generation M2 ion channel inhibitors, second-generation neuraminidase inhibitors (NAIs), and third-generation cap-dependent endonuclease inhibitors. Influenza viruses, as RNA viruses with high mutation rates, have rapidly evolved into multiple drug-resistant strains under high-pressure drug screening over the past 50 years since the advent of the first generation of anti-influenza drugs. Among circulating strains, strains resistant to existing drugs now account for a significant proportion. Given the limitations of human influenza virus vaccines and the rapid emergence of drug resistance, the need to screen for novel small-molecule influenza virus inhibitors is becoming increasingly prominent.

[0006] Triptonide, also known as PG 492, NSC 165677, and Triptonide in English, with a CAS number of 38647-11-9, is commonly used as a Wnt signaling inhibitor. Its molecular structure is as follows:

[0007]

[0008] Brequinar, also known as Bipenquinate, NSC 368390, DUP785, and Brequinar, with a CAS number of 96187-53-0, is a potent inhibitor of dihydroorotate dehydrogenase, with potent activity against a broad spectrum of viruses. Its molecular structure is as follows:

[0009]

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

[0011]

[0012] Mizoribine, also known as Bredinin, NSC 289637, HE 69, and Mizoribine in English, has a CAS number of 50924-49-7. Its conventional active use is as an immunosuppressant. Its molecular structure is as follows:

[0013]

[0014] Epalrestat, also known as ONO2235, Epalrestat, and with a CAS number of 82159-09-9, is a conventionally active aldose reductase inhibitor, effectively improving symptoms of diabetic neuropathy and slowing disease progression. Its molecular structure is as follows:

[0015]

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

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

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

[0019] The active substance a is any one or a combination of triptolide and a prodrug of triptolide;

[0020] The active substance b is any one of vorseluoto, a pharmaceutically acceptable salt of vorseluoto, and a prodrug of vorseluoto, a combination of any two, or a combination of all three;

[0021] The active substance c is any one of epalrestat, a pharmaceutically acceptable salt of epalrestat, and a prodrug of epalrestat, a combination of any two, or a combination of all three;

[0022] The active substance d is any one of buquinar, a pharmaceutically acceptable salt of buquinar, and a prodrug of buquinar, a combination of any two of them, or a combination of all three;

[0023] The active substance e is any one of mizoribine, a pharmaceutically acceptable salt of mizoribine, and a prodrug of mizoribine, a combination of any two, or a combination of all three; the structural formula of triptolide is:

[0024]

[0025] The structural formula of the voseluoto is:

[0026]

[0027] The structural formula of epalrestat is:

[0028]

[0029] The structural formula of the buquina is:

[0030]

[0031] The structural formula of the mizoribine is:

[0032]

[0033] 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 1:0.3-30 (for example, any ratio of 1:0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or a range between any two ratios);

[0034] When the second active substance is active substance c, the molar ratio of the first active substance to the second active substance is 1:0.1-13 (for example, any ratio of 1:0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or a range between any two ratios);

[0035] When the second active substance is active substance d, the molar ratio of the first active substance to the second active substance is 1:0.02-2 (for example, any ratio of 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 ratios);

[0036] When the second active substance is active substance e, the molar ratio of the first active substance to the second active substance is 1:0.8-80 (for example, any ratio of 1:0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or a range between any two ratios).

[0037] In some embodiments, when the second active substance is active substance b, the molar ratio of the first active substance to the second active substance is 1:2-5;

[0038] When the second active substance is active substance c, the molar ratio of the first active substance to the second active substance is 1:0.5-2;

[0039] When the second active substance is active substance d, the molar ratio of the first active substance to the second active substance is 1:0.1-0.5;

[0040] When the second active substance is active substance e, the molar ratio of the first active substance to the second active substance is 1:5-15.

[0041] In some embodiments, the pharmaceutically acceptable salt of vorseluoto is selected from the group consisting of hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate, trifluoroacetate, aspartate, taurate, gluconate, fructonate, salicylate, nitrate, p-toluenesulfonate, methanesulfonate, benzoate, citrate, lactate, citrate, fumarate, and ascorbate of vorseluoto;

[0042] 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;

[0043] The pharmaceutically acceptable salt of buquinar 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, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate, trifluoroacetate, aspartate, taurate, gluconate, fructose, salicylate, nitrate, p-toluenesulfonate, methanesulfonate, benzoate, citrate, lactate, citrate, fumarate and ascorbate of buquinar;

[0044] The pharmaceutically acceptable salt of mizoribine is selected from the group consisting of hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate, trifluoroacetate, aspartate, taurate, gluconate, fructonate, salicylate, nitrate, p-toluenesulfonate, methanesulfonate, benzoate, citrate, lactate, citrate, fumarate and ascorbate of mizoribine.

[0045] In some embodiments, the composition further contains an auxiliary material.

[0046] A second aspect of the present invention provides a use of a biomaterial in the preparation of a product for use alone or in combination with other agents to improve health conditions;

[0047] The biological material is the pharmaceutical composition or active substance a described in the first aspect of the present invention;

[0048] The active substance a is any one or a combination of triptolide and a prodrug of triptolide;

[0049] The structural formula of the triptolide is:

[0050]

[0051] The use is selected from any one of the following U1, U2, U3 and U4 or a combination thereof;

[0052] U1: The improvement in health status is to prevent influenza, treat influenza, slow down influenza or inhibit the proliferation of influenza virus;

[0053] U2: The improvement in health status is reducing damage to the lungs caused by influenza virus, and the second active substance is active substance b, active substance c, or active substance e;

[0054] 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;

[0055] U4: The improvement of health status is to reduce the damage of influenza virus to the body by inhibiting influenza virus neuraminidase, and the second active substance is active substance c.

[0056] In some embodiments, the product is selected from pharmaceuticals.

[0057] In some embodiments, the influenza virus is influenza A virus and the influenza is influenza A.

[0058] In some embodiments, the influenza virus is an H1N1 influenza virus, and the influenza is influenza caused by an H1N1 influenza virus.

[0059] In some embodiments, the influenza is selected from human influenza, avian influenza, and swine influenza. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 The inoculation MOI, drug concentration and OD are shown. 450 Statistics of the relationship between them.

[0061] Figure 2 The statistics of viral infection titers under the intervention of 5 drugs are shown.

[0062] Figure 3 Five drug CCs are shown 50 The fitting curve of .

[0063] Figure 4 EC values ​​for five drugs are shown. 50 The fitting curve of .

[0064] Figure 5 The results of the virus attack and protection experiment under the intervention of three drugs are shown.

[0065] Figure 6 The results show the effects of four drugs on influenza A virus polymerase activity.

[0066] Figure 7 Results showing the effect of epalrestat on influenza virus neuraminidase activity.

[0067] Figure 8 The hydrogen bond analysis results of the molecular docking model are shown.

[0068] Figure 9 The molecular docking results of epalrestat and influenza virus neuraminidase N1 are shown.

[0069] Figure 10 The inhibition of viral protein expression by five drugs is shown.

[0070] Figure 11 Shown is the effect of siRNA interference drug target gene on influenza virus replication.

[0071] Figure 12 The figure shows the statistics of viral content under the intervention of 5 drug combinations. DETAILED DESCRIPTION

[0072] To make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0073] Example 1: Influenza virus infection protection experiment

[0074] 1. Establishment of positive control method

[0075] Using oseltamivir phosphate (purchased from MCE, the same below) as a positive control drug, a method for indirectly screening anti-influenza virus small molecule compounds based on CCK-8 cell viability was established.

[0076] First, fill each well of a transparent 96-well cell culture plate with 100 μL of PBS around the periphery to prevent edge effects. Then, MDCK cells were plated at 1×10 4 Cells were plated at a density of 1 / well in a microplate using 100 μL of DMEM (Sigma-Aldrich) supplemented with 10% v / v fetal bovine serum (WISENT). The cells were cultured at 37°C with 5% CO2. Viral infection was performed after the cells reached a density of 90%.

[0077] Use a multichannel pipette to discard the culture medium in the wells, and wash the cells twice with PBS to wash away the residual serum in the culture medium to prevent it from affecting viral infection. After rinsing once with Opti-MEM medium (purchased from Gibco, the same below) supplemented with TPCK trypsin (final concentration 0.5 μg / mL), the cells were infected with influenza virus WSN strain (H1N1 type) (NCBI Taxonomy ID: 382835) at MOI 0, MOI 0.01, MOI 0.1, MOI 1, MOI 5 and MOI 10, respectively. 100 μL of Opti-MEM medium containing 0.5 μg / mL TPCK trypsin was added to each well, and the virus was adsorbed for one hour at 37°C and 5% CO2, and the virus solution was discarded. Then, Opti-MEM medium (containing 0.1 v / v% DMSO and 0.5 μg / mL TPCK trypsin) with concentrations of 400 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL and 25 μg / mL oseltamivir phosphate was added to the wells inoculated with different MOIs, respectively. Opti-MEM medium containing 0.1 v / v% DMSO (containing 0.5 μg / mL TPCK trypsin) was used as a negative control. After culturing for 24 hours at 37°C and 5% CO2, the cell viability in each well was detected.

[0078] 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, the same below) to each well. Incubate at 37°C and 5% CO2 for 1 hour. Immediately place on ice and use a microplate reader to measure the OD value of each well. 450 Each combination of MOI and oseltamivir concentration was repeated in 3 wells, the absorbance of each well was detected 3 times and the average value was taken, and the data were analyzed using GraphPad Prism 8.0.2.

[0079] CCK-8 test results Figure 1 As shown. This shows that at the same MOI infection, different oseltamivir concentration groups exhibited a good gradient of cell activity. As the oseltamivir concentration increased, cell activity increased, indicating that the drug's ability to inhibit the virus improved. At the same time, between groups infected with the same drug concentration and different MOIs, a clear gradient of cell activity was also observed. With increasing MOI, cell viability decreased significantly. Under the same drug inhibitory ability, the more virus was inoculated, the lower the cell viability, indicating that this method has good sensitivity. This method can use OD values ​​to reflect the drug's ability to inhibit the virus.

[0080] 2. Primary screening, rescreening and data analysis

[0081] Based on the experimental results in Section 1, an MOI of 0.01 was selected for the screening experiment. Cell plating and virus infection were performed as in Section 1. After virus infection, 2,697 compounds from an FDA-approved drug library (test compounds, small molecule library FDA L1300-Z417864 purchased from Selleck) were added at a final concentration of 30 μM. Opti-MEM medium (containing 0.5 μg / mL TPCK trypsin) with 0.1% v / v DMSO was used as the solvent. Each compound was incubated in triplicate. A positive control of oseltamivir and a negative control of 0.1% v / v DMSO aqueous solution were included. An uninfected cell served as a blank control. All other steps and parameters were carried out in parallel. After 24 hours of incubation at 37°C and 5% CO₂, cell viability was assessed using the method described in Section 1. Absorbance measurements were taken three times for each well and the average was calculated. Sixty-seven compounds with known strong virus activity (higher OD values) in each plate demonstrated protective effects against virus infection compared to the control group. The same method was then used to rescreen the aforementioned 67 compounds, and the results showed that 26 of them showed strong cell protective ability.

[0082] Example 2: Virus titer determination experiment

[0083] 1. Identification of the antiviral ability of compounds

[0084] A549 cells were cultured at 1×10 5 Cells were plated at a density of 1 mL / well in a 12-well cell culture plate using F12K medium (purchased from Multicell, the same below) containing 10 v / v% fetal bovine serum. 1 mL was added to each well and cultured at 37°C under 5% CO2. After the cell density in the plate grew to 90%, the cells were infected with WSN (H1N1) virus at an MOI of 0.01. After one hour of viral adsorption, the cells were washed with PBS and replaced with 1 mL of Opti-MEM medium (containing 0.125 μg / mL TPCK trypsin and 0.1 v / v% DMSO) per well containing 30 μM of the test compound (19 of the 26 compounds obtained in Example 1). A 0.1 v / v% DMSO aqueous solution was used as a negative control. After 24 hours, 50 μL of the culture supernatant was collected in a 1.5 mL EP tube and frozen at -80°C until use in plaque titration experiments.

[0085] 2. Plaque titration experiment

[0086] MDCK cells were plated in a 12-well cell culture plate in 1× DMEM medium containing 0.5 μg / mL TPCK trypsin. Once the cells were confluent, they were washed twice with PBS to remove residual serum. Rinse once with 1× DMEM medium containing 0.5 μg / mL TPCK trypsin before use.

[0087] In each well of Section 1, thaw the culture supernatant on ice and dilute it two-fold using 1× DMEM medium containing 0.5 μg / mL TPCK trypsin. Add 100 μL of the virus solution to the plate for adsorption. Incubate at 37°C and 5% CO2, shaking the plate every 10 minutes to ensure even coverage of the cells. Dissolve low-melting-point agarose in ddH2O to create a 2 w / w% agarose solution. Heat to dissolve and then incubate in a 50°C waterbath. Simultaneously, prepare an equal volume of 2× DMEM medium and incubate in a 37°C waterbath. Mix equal volumes of the 2 w / w% agarose solution and 2× DMEM medium to create a 1% agarose solution in 1× DMEM. After infection, aspirate the virus solution from the plate and add 1 mL of 1× DMEM 1 w / w% agarose solution to each well. Place the plate in a biosafety cabinet for 20 minutes until the agarose solidifies, then invert and place it in a cell culture incubator. After incubation at 37°C, 5% CO₂ for 48 hours, add 1 mL of formalin fixative to each well. After overnight fixation, discard the fixative and agarose, count the number of plaques formed, and calculate the viral titer. Titer refers to the number of biologically active viral particles per unit volume of liquid. Titer is quantitatively expressed as the highest 2-fold serial dilution at which plaques are visible, expressed in Log₂ pfu / mL.

[0088] Compared with the DMSO control, the titers of five compounds showed a significant decrease. The five compounds are triptolide (code: T18-9), buquina (code: B18-5), vorseloto (code: V15-24), mizoribine (code: M9-26) and epalrestat (code: E16-1). The five compounds reduced the viral titer by 99.99% (P < 0.001), 99.22% (P < 0.001), 99.84% (P < 0.001), 79.25% (P < 0.01) and 95.00% (P < 0.001), respectively. See the titer statistics chart for details. Figure 2 .

[0089] Example 3: Compound Median Toxic Concentration CC 50 Determination

[0090] A transparent 96-well cell culture plate was filled with 100 μL of PBS in each well to prevent edge effects, and A549 cells were then plated at 1×10 4Cells were plated at a density of 1 μg / well in a 96-well cell culture plate. The culture medium was Opti-MEM medium, 100 μL per well, and cultured at 37°C and 5% CO2. After the cell density in the well plate grew to 90%, the culture medium was discarded, and the test compounds, triptolide, brequinar, vorseloto, mizoribine, and epalrestat, were dissolved in Opti-MEM medium, respectively, with a final concentration gradient of 0.1 μM, 1 μM, 10 μM, 100 μM, 500 μM, 1000 μM, 5000 μM, and 10000 μM. They were added to different wells at a volume of 100 μL per well, and each concentration was repeated for 3 wells. After culture at 37°C and 5% CO2 for 24 hours, the cell activity was detected. Each well was replaced with 100 μL of DMEM medium containing 10 v / v% fetal bovine serum, and then 10 μL of CCK-8 solution was added to each well. After incubation at 37°C and 5% CO2 for 1 hour, the wells were immediately placed on ice and the OD of each well was measured using a microplate reader. 450 The absorbance of each well was measured 3 times and the average value was taken. The 0.1 v / v% DMSO aqueous solution well was used as the negative control and its absorbance was set to 100%. The cell viability of the experimental groups with different concentrations was calculated. GraphPad Prism 8.0.2 was used to analyze the data. The CC of the compound was calculated by nonlinear regression and Nonlin fitting. 50 .

[0091] The fitting curve results are shown in Figure 3 CC of triptolide, brequinar, vorseloto, mizoribine, and epalrestat 50 They were 1.304 mM, 558.4 μM, 344.2 μM, >10 mM and 310.4 μM respectively.

[0092] Example 4: Compound half effective concentration EC 50 Determination

[0093] A549 cells were cultured at 1×10 4Cells were plated at a density of 100 μL / well in a 24-well cell culture plate using F12K medium supplemented with 10% v / v fetal bovine serum (FBS). 500 μL of cells were plated per well. After cells reached 90% growth, they were infected with influenza A virus (WSN) at an MOI of 0.01. After incubation at 37°C and 5% CO₂ for one hour to allow viral adsorption, the cells were replaced with 500 μL / well of Opti-MEM medium (containing 0.125 μg / mL TPCK trypsin and 0.1% v / v DMSO) containing various concentrations of the test compounds (triptolide, brequinar, vorseloto, mizoribine, and epalrestat). Each compound was plated at a final concentration gradient of 0.01 μM, 0.1 μM, 1 μM, and 10 μM. A 0.1% v / v DMSO solution was also used as a negative control. After incubation at 37°C and 5% CO₂ for 24 hours, the supernatant was discarded from each well, and total RNA was extracted from each well.

[0094] After determining the RNA concentration, the HiscriptR RIIQ RT SuperMix for qPCR reverse transcription kit (Vazyme, R223-01) was used according to the instructions. Genomic DNA was removed from the total RNA in each well, and 1 μg of RNA was used as a template. 5Seg_vRNA-RT (SEQ ID NO. 1) in Table 1 was used as a primer to reverse transcribe the mRNA into cDNA.

[0095] Based on the WSN strain reference gene sequence from NCBI, Primer Premier 5 was used to design fluorescent quantitative PCR primers. The upstream and downstream primers are shown in Table 1 as 5Seg_NP qPCR-F (SEQ ID NO. 2) and 5Seg_NP qPCR-R (SEQ ID NO. 3). Each reaction used 5 μL of 2× ChamQ Universal SYBR qPCR Master Mix, 1 μL of cDNA template, 0.4 μL of each upstream and downstream primer, and 3.2 μL of ddH2O. Each reaction was repeated three times. The fluorescent quantitative PCR reaction program was pre-denaturation at 95°C for 10 min; denaturation at 95°C for 15 s; annealing at 60°C for 1 min, for a total of 40 cycles. Melting curve analysis was performed as follows: 95°C for 15 s; 60°C for 1 min; and 95°C for 10 s. The hGAPDH gene was used as an internal reference. 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). A 0.1 v / v% DMSO aqueous solution group was used as a negative control, and the viral RNA content in the viral NP segment was calculated using the formula 2^-{ΔΔCt=[Ct(EG_vNP)-Ct(EG_hGAPDH)]-Ct(DMSO_vNP)-Ct(DMSO_hGAP DH)}. Data were analyzed using GraphPad Prism 8.0.2, and the EC values ​​of the compounds were calculated by nonlinear regression and Nonlin fitting. 50 .

[0096] The fitting curve results are shown in Figure 4 ECs of triptolide, brequinar, vorselostat, mizoribine, and epalrestat 50 They are 0.6564μM, 0.1323μM, 2.213μM, 5.003μM and 0.8847μM respectively.

[0097] Table 1. Primer sequences

[0098]

[0099] Example 5: Organ plaque titration

[0100] The mice were divided into four groups: vorseloto, mizoribine, epalrestat and 5% DMSO, with 8 mice in each group. After weighing, they were anesthetized with isoflurane inhalation at a rate of 1.5×10 4 WSNs were anesthetized with pfu of influenza A virus and nasally inhaled. The three drugs were administered in the same way every day at doses of 1.52 mg / kg body weight, 3 mg / kg body weight, and 0.24 mg / kg body weight, respectively. The control group was administered with 5 v / v% DMSO aqueous solution at a dose of 25 μL per mouse.

[0101] On the third day after challenge, three mice from each group were euthanized by cervical dislocation after isoflurane inhalation anesthesia. Intact lung tissue was collected and placed in 2 mL EP tubes and frozen at -80°C. After thawing, 1 mL of PBS buffer containing 1% penicillin-streptomycin was added to each tube, followed by grinding with steel beads. The tubes were ground at 30 Hz at 4°C for 5 min. The supernatant was then centrifuged at 9000 rpm at 4°C for 5 min, and plaque titration was performed using the method described in Example 2.

[0102] Results see Figure 5 Compared with the control group, the viral titers in the lungs of the vorseloto, mizoribine, and epalrestat groups decreased by 71.74% (P < 0.01), 76.74% (P < 0.001), and 84.13% (P < 0.001), respectively.

[0103] Example 6: Influenza virus polymerase activity assay

[0104] 1. Synthesis and Source of Plasmids

[0105] (1) Renilla luciferase control reporter gene vector pRL-TK

[0106] The pRL series vectors are Renilla luciferase reporter vectors developed by Promega (Cat. No. E2241), and the TK promoter drives Renilla luciferase.

[0107] (2) RNA polymerase component plasmid

[0108] Using the pCAGGS plasmid as a backbone, the coding sequences of the alkaline polymerase 1 (PB1) gene (see GenBank No. LC333183.1), alkaline polymerase 2 (PB2) gene (see GenBank No. LC333182.1), PA protein gene (see GenBank No. LC333184.1), and 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.

[0109] (3) Firefly luciferase reporter gene vector pHH21-SC09NS F-Luc

[0110] A firefly luciferase reporter gene vector, pHH21-SC09NS F-Luc, was constructed to produce a negative-sense RNA containing 176 bases at the 3' end of NSvRNA derived from the influenza virus SC09 strain (full name: A / Sichuan / 1 / 2009 strain, H1N1 type), firefly luciferase, a stop codon (TAA), and 179 bases at the 5' end of SC09NS vRNA. Influenza virus polymerase can recognize and bind to the non-coding sequence at the end of the NS vRNA on the vector, thereby initiating the expression of firefly luciferase.

[0111] The construction methods of the plasmid vectors of (2) and (3) above are described in the following documents:

[0112] Luo W, Zhang J, Liang L, et al., 2018. Phospholipid scramblase 1interacts with influenza A virus NP,impairing its nuclear import and therebysuppressing virus replication. PLoS pathogens, 14(1):e1006851.

[0113] 2. Principle of Enzyme Activity Assay

[0114] Influenza virus RNA polymerase is composed of three subunits: PB2, PB1, and PA. It is an RNA-dependent RNA polymerase that works together with the NP protein to perform the important function of viral genome transcription. In order to explore the effect of drugs on the activity of influenza virus polymerase, a dual-luciferase reporter experiment was used to explore it.

[0115] In the dual-luciferase reporter system In the Reporter System (purchased from Promega, Cat. No. E1960), firefly luciferase serves as a reporter gene for gene expression, while Renilla luciferase serves as an internal reference gene. 1. The target gene's transcriptional regulatory element is constructed into an expression vector carrying luciferase, creating a reporter plasmid. This sequence regulates the transcriptional expression of luciferase. 2. The reporter plasmid is transfected into cells, treated with various methods, and then lysed. The substrate luciferin is added, which catalyzes the luciferase to produce fluorescence. 3. The resulting fluorescence values ​​are used to determine the effect of different treatments on the transcriptional regulatory element. 4. To minimize errors caused by differences in transfection efficiency, a Renilla luciferase reporter plasmid is often added as an internal reference to correct for transfection efficiency between samples.

[0116] Renilla luciferase is constitutively expressed and can be used as a control. It is minimally affected by influenza virus inhibitors. Firefly luciferase expression is affected by the content or activity of RNA polymerase, and thus by influenza virus inhibitory drugs. The ratio of the fluorescence produced by the two luciferases can be used to quantitatively or semi-quantitatively determine the inhibitory ability of drugs on influenza virus RNA polymerase.

[0117] The polymerase expression plasmids (pCAGGS-PA, pCAGGS-PB1, pCAGGS-PB2, and pCAGGS-NP) of four WSNs (A / WSN / 33 (WSN, H1N1)) were constructed to express them in cells and form the influenza virus RNA polymerase complex.

[0118] 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. After transfection, the cells were lysed and loaded onto a GloMax 96-well microplate luminometer (Promega). Luciferase activity in the transfected cells was measured using a dual-luciferase reporter gene assay system. (Firefly luciferase reacts with the substrate LARⅡ to produce a fluorescent signal. After instrumental detection, the firefly luciferase is quenched with Stop & Glo reagent and the Renilla luciferase is activated to produce fluorescence.) This assay can be used to analyze the effects of drugs on influenza virus polymerase activity.

[0119] Data processing: First, calculate the ratio of Firefly luciferase / Renilla luciferase in each tube. Then, taking the ratio of the control group as 1, the relative luciferase activity of the different treatment groups, that is, the activity of influenza virus polymerase, can be obtained.

[0120] 2. Activity Determination

[0121] Polylysine was used to coat a 12-well cell culture plate, and then HEK293T cells were plated in the 12-well cell culture plate. The culture medium was DMEM containing 10% FBS. After the cells grew to 80%, 500ng pCAGGS-WSN PB2, 500ng pCAGGS-WSN PB1, 500ng pCAGGS-WSN PA, 500ng pCAGGS-WSN NP plasmids, 200ng pHH21-SC09NSF-Luc and 10ng pRL-TK plasmids were transfected into the cells at the same time. 10h after transfection, the culture medium containing plasmids and transfection reagents was discarded and replaced with HEK293T cell culture medium containing 20μM and 30μM test compounds (triptolide, brequinar, vorseloto and mizoribine), respectively, and then cultured for another 26h. After discarding the cell culture supernatant, wash once with PBS, use 250μL Passive Lysis Buffer to lyse the cells in each well, centrifuge at 12000r / min for 5min, and take 20μL supernatant for use. The polymerase complex activity was detected using the Reporter System.

[0122] Under the action of gradient concentrations of compounds, the luciferase ratio of the 0.1 v / v% DMSO aqueous solution group was taken as 100%, and the polymerase activity of the five compounds that inhibited influenza virus replication in Example 2 was determined. Figure 6 As shown, triptolide, buquinar, vorselotto, and mizoribine all significantly inhibited influenza virus polymerase activity, with the polymerase activity in the 30 μM group being lower than that in the 20 μM group, indicating a dose-dependent inhibitory effect. Compared with the control group, the polymerase activity of the 20 μM and 30 μM triptolide groups decreased by 99.51% and 99.57%, respectively (P < 0.001); the polymerase activity of the 20 μM and 30 μM buquinar groups decreased by 98.96% and 99.19%, respectively (P < 0.001); the polymerase activity of the 20 μM and 30 μM vorselotto groups decreased by 48.04% and 70.52%, respectively (P < 0.001); and the polymerase activity of the 20 μM and 30 μM mizoribine groups decreased by 18.55% and 28.11%, respectively (P < 0.01).

[0123] This shows that the four drugs can inhibit the activity of influenza virus RNA polymerase and help inhibit influenza virus.

[0124] Example 7: Detection of Influenza Virus Neuraminidase Activity

[0125] 1. Cell Detection Experiment

[0126] According to the instructions, use a white non-transparent bottom 96-well plate to reduce light loss and interference between wells. 7 pfu / mL of influenza A virus WSN using NA-XTD TM After diluting the 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. Repeat for 3 wells and incubate at 37°C for 20 min (the final concentration of the compound was 33.33 μM at this time). 25 μL of 1000×NA-XTD diluted at 1:1000 (volume ratio) was added to each well. TM Substrate (purchased from Thermofisher), incubate at room temperature for 30 min; add 60 μL NA-XTD to each well TM Accelerator (purchased from Thermofisher), and the luminescence value was immediately detected using a GLOMAX96 microplate luminometer. The detection time for each well was 1 s, and the measurement was performed 3 times to obtain the average value.

[0127] Neuraminidase activity Figure 7 A. This indicates that epalrestat exhibits inhibitory activity against influenza A virus neuraminidase.

[0128] Then, the half maximal inhibitory concentration (IC) of epalrestat on neuraminidase was determined. 50 ), that is, the concentration of the compound required to inhibit the enzyme activity by half, to evaluate the inhibitory activity of the compound on the enzyme. 7 pfu / mL of influenza A virus WSN using NA-XTD TM After diluting the Assay Buffer 1:100, add 25 μL to each well. Use Assay Buffer to perform a gradient dilution of the test compound. Set the gradient to 1000000nM, 200000nM, 100000nM, 40000nM, 20000nM, 8000nM, 1600nM, 320nM, 64nM, 12.8nM, 2.56nM, 0.512nM and 0.1024nM. Repeat 3 wells for each concentration. The final detection concentration is 333333.33nM, 66666.67nM, 33333.33nM, 13333.33nM, 6666.67nM, 2666.67nM, 533.33nM, 106.67nM, 21.33nM, 4.27nM, 0.85nM, 0.17nM and 0.03nM, incubate at 37°C for 20min, then add 25μL NA-XTD to each 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 .

[0129] 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.

[0130] 2. Molecular Docking Experiment

[0131] 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.

[0132] 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 model 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.

[0133] Using AutoDock vina software, E16-1 was used as the ligand to dock into the active pocket of influenza virus N1 subtype neuraminidase, and a total of 9 docking models were output ( Figure 8 (A)), hydrogen bond analysis was performed using AutoDockTool software, and the results were as follows Figure 8 As shown in (B-F), E16-1 in models 1, 2, 3, 6, and 7 all forms two hydrogen bonds with the neuraminidase binding pocket. The docking results were visualized and analyzed using PyMOL software. Figure 9 As shown, A is the binding pocket, and B to F are the binding forms of models 1, 2, 3, 6, and 7, respectively. Among them, model 1 has the lowest binding energy, as shown in Figure 8 As shown in (A), it is -6.5 kcal / mol.

[0134] The docking sites of model 1 were analyzed and the results were as follows: Figure 9 As shown in (G), the E16-1 molecule binds to the influenza virus N1 subtype neuraminidase at Arg292 and Arg371 via hydrogen bonds (yellow dashed lines). In addition, the binding site that appears repeatedly in other docking models is Arg118.

[0135] Example 8: Detection of Compounds Inhibiting Influenza Virus Protein Expression

[0136] A549 cells were cultured at 1×10 5 Cells were plated at a density of 1 / well in a 12-well cell culture plate in F12K medium containing 10 v / v% fetal bovine serum and cultured at 37°C under 5% CO2. After the cell density in the well plate grew to 90%, the cells were infected with WSN (H1N1) virus at an MOI of 0.01. One hour after virus adsorption, three wells of each test compound were replaced with 1 mL of Opti-MEM medium (containing 0.125 μg / mL TPCK trypsin and 0.1 v / v% DMSO) containing 5 μM, 10 μM, and 20 μM of the test compound (triptolide, brequinar, vorseloto, mizoribine, and epalrestat), respectively. A 0.1 v / v% DMSO aqueous solution was used as a negative control, and oseltamivir phosphate was used as a positive control. After 24 hours, 50 μL of the culture supernatant was collected in a 1.5 mL EP tube and frozen at -80°C.

[0137] After discarding the cell culture supernatant, wash the cells once with PBS. Add 100 μL of 1× SDS lysis buffer, 1 μL of PMSF solution, and 0.1 μL of ribozyme to each well. Lyse the cells at room temperature for 5 minutes. Collect the lysate into a 1.5 mL EP tube and denature at 95°C for 10 minutes before Western blotting.

[0138] The protein samples were loaded onto a 10% SDS-PAGE gel at 15 μL / well and electrophoresed at 80 V. Once the protein samples began to separate through the stacking gel, the electrophoresis voltage was switched to 120 V until the end of the run. A 0.45 μm nitrocellulose membrane (NC membrane) was placed in NC membrane equilibration buffer for 30 seconds before transfer using an eblot L1 rapid wet transfer device. After blocking with 5% skim milk solution at room temperature for 1 hour, any remaining blocking solution was removed with PBS. The primary antibody was diluted 1:1000 in PBS and incubated on a shaker at low speed for 1 hour at room temperature. Following incubation, the membrane was washed three times with PBST at high speed for 10 minutes at room temperature on a shaker to remove any unbound primary antibody. A secondary antibody was prepared at a 1:5000 (volume ratio) in PBS and incubated at room temperature in the dark for 1 hour. The membrane was then washed three times for 10 minutes in the dark and imaged and analyzed using an Odyssey CLX near-infrared scanner.

[0139] For the internal control GAPDH, the primary antibody was a rabbit GAPDH polyclonal antibody (Proteintech, Cat. No. 10494-1-AP), and the secondary antibody was a goat anti-rabbit IgG labeled with Dylight 680 (Li-COR Bioscience, Cat. No. 926-68071). For the influenza polymerase protein PB1, the primary antibody was a mouse PB1 monoclonal antibody (made in the laboratory), and the secondary antibody was a goat anti-mouse IgG labeled with Dylight 680 (Li-COR Bioscience, Cat. No. 926-68070).

[0140] The primary antibody against influenza virus nucleoprotein NP was a mouse NP monoclonal antibody (prepared in the applicant's laboratory), and the secondary antibody was a Dylight 680-labeled goat anti-mouse IgG (Li-COR Bioscience, Cat. No. 926-68070).

[0141] WB results and virus titers of each group are shown in Figure 10 Grayscale analysis of protein expression revealed no viral protein in the triptolide group; a significant decrease in both NP and PB1 proteins in the buquina group; and a decrease in both NP and PB1 proteins in the vorseloto group. In the mizoribine group, NP protein levels did not decrease significantly, while PB1 protein levels showed a gradual decrease. Similarly, in the epalrestat group, NP protein levels did not decrease significantly, while PB1 protein expression levels showed a gradual decrease. In the positive control, oseltamivir, NP protein levels did not decrease significantly, while viral titers decreased significantly.

[0142] Example 9: Effect of siRNA Interference on Drug Target Genes on Influenza Virus Replication

[0143] siRNAs were designed based on the human gene sequence of the target gene of buquina (gene name DHODH, Gene ID: 231, GenBank No. NM_001361.5), the human gene sequence of the target gene of mizoribine (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). The RNA sequences complementary to the target sequences in each siRNA are shown in Table 2 as si_231 (SEQ ID NO. 6), si_1723 (SEQ ID NO. 7), and si_3615 (SEQ ID NO. 8), respectively. The sequence shown in si_NC (SEQ ID NO. 9) was used as a negative control. Lipofectamine was used. TM siRNA and the negative control si_NC were transfected into A549 cells using the reverse transfection method described in the RNAiMAX (purchased from ThermoFisher Scientific, Cat. No. 13778030) instructions to interfere with the expression of relevant genes. Cell samples were collected from one group 36 hours after infection and Western blot analysis was performed to determine protein-level interference efficiency. Another group of cells was infected with influenza A virus WSN at an MOI of 0.01 36 hours after infection. One hour after infection, the cells were replaced with 1 mL / well of Opti-MEM medium containing 0.125 μg / mL TPCK trypsin. Cell culture supernatants were collected 24 and 48 hours after infection, and viral titers were determined by plaque titration using the same method as in Example 2.

[0144] For the internal control GAPDH, the primary antibody was a rabbit GAPDH polyclonal antibody (Proteintech, Cat. No. 10494-1-AP), and the secondary antibody was a Dylight 680-conjugated goat anti-rabbit IgG (Li-COR Bioscience, Cat. No. 926-68071). For the brequinar target protein, the primary antibody was a mouse DHODH monoclonal antibody (Proteintech, Cat. No. 67977-1-Ig), and the secondary antibody was a Dylight 680-conjugated goat anti-mouse IgG (Li-COR Bioscience, Cat. No. 926-68070). For the mizoribine target protein, the primary antibody was a mouse IMPDH2 monoclonal antibody (Proteintech, Cat. No. 67663-1-Ig), and the secondary antibody was a Dylight 680-conjugated goat anti-mouse IgG (Li-COR Bioscience, Cat. No. 926-68070). For the target gene protein of epalrestat, the primary antibody was a rabbit AKR1B1 polyclonal antibody (Invitrogen, Catalog No. PA5-29718), and the secondary antibody was a Dylight 680-labeled goat anti-rabbit IgG (Li-COR Bioscience, Catalog No. 926-68071).

[0145] Table 2. siRNA sequences (sequences of RNA chains complementary to target sequences)

[0146]

[0147] After 36 hours of siRNA interference, the expression levels of brequinar Target, mizoribine Target and epalrestat Target genes were all decreased ( Figure 11 A), on this basis, after infection with the virus, the virus titer increased slightly at 24h and 48h. The virus titer of buquina Target increased by 1.86 times and 4.18 times at 24h and 48h after infection, respectively; the virus titer of mizoribine Target increased by 3.03 times and 6.87 times at 24h and 48h after infection, respectively; the virus titer of epalrestat Target increased by 3.39 times and 4.53 times at 24h and 48h after infection, respectively ( Figure 11 B).

[0148] After the target proteins in cells treated with the three drugs were inhibited by siRNA, the viral titer increased, indicating that the aforementioned three target proteins were involved in the drug's effect of inhibiting the virus.

[0149] Example 10: Testing of the Virus Inhibition Effect of Drug Combinations

[0150] 1. Preparation of drugs

[0151] 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).

[0152] 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.

[0153] The following negative control was set up, as shown in No. 16 in Table 3 below.

[0154] 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 .

[0155] 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.

[0156] Table 3. Drug configuration and virus content statistics

[0157]

[0158]

[0159] 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 for inhibiting influenza A virus or treating influenza A, wherein the pharmaceutical active ingredients of the pharmaceutical composition include a first active substance and a second active substance; The first active substance is active substance a; the second active substance is active substance b; The active substance a is triptolide; The active substance b is any one or a combination of two of vorseluoto and a pharmaceutically acceptable salt of vorseluoto; The structural formula of the triptolide is: The structural formula of the voseluoto is:

2. The pharmaceutical composition according to claim 1, wherein The molar ratio of the first active substance to the second active substance is 1:0.3-30.

3. The pharmaceutical composition according to claim 2, wherein The molar ratio of the first active substance to the second active substance is 1:2-5.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein The pharmaceutically acceptable salt of vorseluoto is selected from the group consisting of 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 vorseluoto.

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 product 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 triptolide; The structural formula of the triptolide is: The use is selected from any one of the following U1, U2 and U3 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; The influenza virus is influenza A virus, and the influenza is influenza A.

7. The use according to claim 6, characterized in that The product is selected from pharmaceuticals.

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

9. 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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