Use of coumarin derivatives and pharmaceutical compositions thereof for the preparation of a medicament against Japanese encephalitis virus

Abstract

The application discloses a coumarin derivative and application of a pharmaceutical composition thereof to preparation of an anti-Japanese encephalitis virus medicine, and relates to the technical fields of biological medicine and antiviral medicine. The application finds that dicoumarol has strong anti-Japanese encephalitis virus (JEV) activity through systematic screening of various coumarin derivatives, and can be used in combination with telzid, thereby significantly improving the inhibition of JEV replication and showing a synergistic antiviral effect. In a mouse acute encephalitis model, the compatibility of dicoumarol and nitazoxanide, the parent drug of telzid, can greatly improve the end-point survival rate of lethal infection mice, and can eliminate the sleeve-like change around the blood vessels and neuron cell necrosis, which are the signs of acute inflammation in the brain parenchyma, thereby realizing the double synergies of anti-viral replication and central nervous protection. The application discloses a new method for applying the coumarin derivative and the pharmaceutical composition thereof to preparation of an anti-JEV infection medicine.

Description

Technical Field

[0001] This invention relates to the field of biomedicine and antiviral drug technology, specifically to the application of a coumarin derivative and its pharmaceutical composition in the preparation of drugs against Japanese encephalitis virus. Background Technology

[0002] Japanese encephalitis virus (JEV), also known as Japanese encephalitis virus, belongs to the genus Flaviviridae of the family Flaviviridae. It is a highly important and dangerous mosquito-borne neurotropic virus (primarily transmitted by Culex tritaeniorhynchus mosquitoes). JEV is the pathogen that causes Japanese encephalitis (JE), which is mainly prevalent in Asia and the Western Pacific region. Clinically, JEV infection can lead to severe symptoms such as high fever, seizures, altered consciousness, meningeal irritation, and acute encephalitis. Statistics show that the mortality rate of JE is as high as 20%-30%, and as many as 30%-50% of survivors suffer from permanent neurological sequelae, such as cognitive impairment, motor paralysis, epilepsy, and speech disorders. Currently, there are no approved specific and highly effective anti-JEV drugs in clinical practice, either domestically or internationally, for patients already infected with JEV and exhibiting clinical symptoms.

[0003] Natural products and their derivatives, due to their rich structural diversity and broad biological activities, have become an important source for antiviral drug development. Among them, coumarin compounds possess various pharmacological effects, including anti-inflammatory, antioxidant, immunomodulatory, and antiviral properties. Previous studies have shown that some coumarin derivatives have inhibitory effects on certain RNA viruses, such as influenza virus. However, systematic research on the antiviral activity of different coumarin derivatives against JEV is lacking, and there are no reports of coumarin derivatives exhibiting antiviral activity against JEV.

[0004] Nitazoxanide (NTZ) is an antiparasitic drug approved by the US FDA. After oral administration, it is rapidly hydrolyzed by plasma esterases to remove acetyl groups, metabolizing into the main active product, tizoxanide (TIZ). The original NTZ drug is almost undetectable in the body [Broekhuysen J, Stockis A, LinsRL, De Graeve J, Rossignol JF. Nitazoxanide: pharmacokinetics and metabolism in man. Int J Clin Pharmacol Ther. 2000 Aug;38(8):387-94.]. In recent years, studies have confirmed that both NTZ and TIZ have broad-spectrum antiviral, anti-inflammatory, and cell metabolism and signaling pathway regulatory pharmacological activities. NTZ exerts its pharmacological effects in vivo by being metabolized into TIZ [Rossignol JF. Nitazoxanide: a first-in-class broad-spectrum antiviral agent. Antiviral Res. 2014 Oct;110:94-103.]. Since NTZ is a prodrug of TIZ, and in vitro cultured cells generally lack an in vivo metabolic enzyme activation system, in vitro cellular efficacy and mechanism studies generally directly use the active metabolite TIZ of NTZ. However, in vivo animal studies and subsequent clinical translations are more suitable for oral administration and have better bioavailability with NTZ [Guo S, Li F, Wang B, Zhao Y, Wang X, Wei H, Yu K, Hai X. Analysis of tizoxanide, active metabolite of nitazoxanide, in rat braintissue and plasma by UHPLC-MS / MS. Biomed Chromatogr. 2020 Feb;34(2):e4716.]. However, the combined anti-JEV infection effects of NTZ or TIZ with coumarin compounds have not been reported. The structural formulas of dicoumarol (DCM), nizoxinide, and tezoxinide are as follows:

[0005] Summary of the Invention

[0006] The technical problem to be solved by this invention is how to overcome the clinical bottlenecks in the prior art, such as the lack of specific and highly effective drugs for Japanese encephalitis virus infection, and the easy resistance and narrow safety range of direct-acting antiviral drugs.

[0007] The present invention solves the above-mentioned technical problems through the following technical means: This invention proposes the application of a coumarin derivative in the preparation of a drug for treating Japanese encephalitis virus, wherein the coumarin derivative is one or more of dicoumarin, 7-hydroxycoumarin, and coumarin-3-carboxylic acid.

[0008] Preferably, the coumarin derivative can inhibit the replication of Japanese encephalitis virus and reduce viral protein expression and viral RNA levels.

[0009] Dicoumarol: CAS No. 66-76-2.

[0010] 7-Hydroxycoumarin: CAS No. 93-35-6.

[0011] Coumarin-3-carboxylic acid: CAS number 531-81-7.

[0012] Preferably, the drug uses a coumarin derivative as the active pharmaceutical ingredient and is formulated into any pharmaceutically acceptable dosage form.

[0013] Preferably, the dosage form is a tablet, capsule, granule, oral preparation, or injection.

[0014] The present invention also provides a pharmaceutical composition wherein the active ingredient in the pharmaceutical composition comprises: (1) Dicoumarin; and (2) Nitrozonide or tezolid.

[0015] Preferably, the pharmaceutical composition is formulated into any pharmaceutically acceptable dosage form using dicumarol, nitrozonide, or tezolinide as the active pharmaceutical ingredient.

[0016] Preferably, the dosage form is a tablet, capsule, granule, oral preparation, or injection.

[0017] Preferably, the molar ratio of the coumarin, nitrozonide, or tezolidinide is 1:(2~4), and more preferably 1:3.

[0018] The present invention also proposes the use of the above-mentioned pharmaceutical composition in the preparation of a drug for treating Japanese encephalitis virus infection.

[0019] Preferably, the efficacy of the drug specifically includes: improving the survival rate of JEV-infected hosts, slowing down weight loss, and reducing brain tissue pathological damage caused by central nervous system inflammation.

[0020] Preferably, the in vivo dosage is 10-50 mg / kg of dicumarol and 80-120 mg / kg of nitrozanide; more preferably, it is 30 mg / kg of dicumarol and 100 mg / kg of nitrozanide.

[0021] The present invention also proposes the use of the above-mentioned pharmaceutical composition in the preparation of drugs for the prevention and treatment of epidemic encephalitis B caused by Japanese encephalitis virus infection.

[0022] The beneficial effects of this invention are as follows: (1) The core natural products are targeted more precisely, and the antiviral efficacy has a specific advantage: This invention, through systematic structural difference screening, confirms that not all coumarin derivatives possess anti-JEV activity. 7-HC, DCM, and C3AC exhibit excellent anti-JEV replication activity and can serve as candidate lead compounds for the preparation of synergistic anti-JEV drugs in combination with tinizonide. Among them, the bicyclic dimer structure—dicoumarin (DCM)—exhibits the strongest antiviral activity, with an in vitro half-maximal inhibitory concentration (IC50) for inhibiting JEV replication (EC50). 50 The concentration reached 5.714 μM, demonstrating significantly better efficacy than 7-HC (EC). 50 = 36.13μM) and C3AC (EC 50 Monocyclic coumarin derivatives (e.g., 23.82 μM) have excellent prospects for single-drug development.

[0023] (2) Specific synergistic compatibility characteristics were discovered: Experiments have shown that when common monocyclic coumarins (such as 7-HC and C3AC) are used in combination with the broad-spectrum antiviral drug tinizonide, no additive or enhancing effect is produced at either the protein or mRNA level. However, this invention, by combining dicumarol with tinizonide, exhibits a significant synergistic antiviral effect, significantly reducing the expression level and mRNA replication level of JEV NS3 protein, and suppressing JEV NS3 mRNA transcription and protein translation depth to near baseline levels, providing a new pathway to overcome the bottleneck of single-drug efficacy in clinical practice.

[0024] (3) Significant protective effect in vivo and reversal effect on central nervous system pathology: In a fatal acute encephalitis infection model in BALB / c mice, single-drug therapy with NTZ or DCM only maintained a endpoint survival rate of approximately 30%. However, the DCM+NTZ combination therapy of this invention significantly increased the survival rate to approximately 50%, and also significantly delayed death and stabilized the body weight of affected mice. Simultaneously, the composition of this invention can extremely significantly reduce JEV-induced perivascular cuff-like changes, abnormal microglial cell proliferation, and neuronal necrosis and degeneration, achieving pathological repair and neuroprotection of damaged brain tissue, and possessing extremely high clinical pharmaceutical translation and industrial application value.

[0025] This invention provides a novel application of a pharmaceutical composition consisting of dicoumarol (DCM) and nitazoxanide (NTZ) in the preparation of drugs against Japanese encephalitis virus (JEV). Since nitazoxanide is rapidly metabolized in vivo to the active metabolite tizoxanide (TIZ), the active ingredient exerting its pharmacological effect in the pharmaceutical composition of dicoumarol and nitazoxanide is tizoxanide (TIZ). Based on the pharmacokinetic characteristics of NTZ's rapid in vivo metabolism and activation to generate the active metabolite TIZ, the in vitro cellular and molecular mechanism studies of this invention were conducted using the active metabolite TIZ, while the in vivo animal protection and administration utilized the clinically available orally administered prodrug NTZ. This invention discloses that the pharmaceutical composition of dicoumarol and nitazoxanide not only efficiently inhibits JEV replication at the molecular and cellular levels, but also exhibits a synergistic protective effect in animals, significantly improving survival rates and reversing pathological damage to central nervous system tissues.

[0026] This invention develops a novel, highly effective, and low-toxicity coumarin drug targeting JEV and explores its application in combination with TIZ for antiviral treatment, which has significant research value and clinical application prospects.

[0027] Of course, implementing any product or method of the present invention does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0028] Figure 1 The chemical structural formulas of dicumarol, nitrozonide, and tezolidinide are shown. Figure 2 This is a schematic diagram illustrating the cytotoxicity of different coumarin derivatives (4-HC, 7-HC, DCM, C3AC, DEAC) to host cells in Example 1 of the present invention; wherein (A) 4-hydroxycoumarin (4-HC), (B) 7-hydroxycoumarin (7-HC), (C) dicoumarin (DCM), (D) coumarin-3-carboxylic acid (C3AC), and (E) 7-(diethylamino)coumarin (DEAC); Figure 3 This is a graph showing the effect of different coumarin derivatives (4-HC, 7-HC, DCM, C3AC, DEAC) on the mRNA expression level of the NS3 gene in JEV-infected cells in Example 1 of the present invention. Figure 4 The images show the Western blot detection and quantitative analysis of the expression level of NS3 protein in JEV-infected cells by different coumarin derivatives (4-HC, 7-HC, DCM, C3AC, DEAC) in Example 1 of this invention; where (A) is the Western blot detection image and (B) is the quantitative analysis image. Figure 5 The half-maximal inhibitory concentration (EC50) of different coumarin derivatives (7-HC, C3AC, DCM) against Japanese encephalitis virus in Example 2 of this invention. 50 Figure 1; where (A) 7-hydroxycoumarin (7-HC), (B) coumarin-3-carboxylic acid (C3AC), and (C) dicoumarin (DCM); Figure 6 The images show Western blot detection and quantitative analysis of JEV NS3 protein expression by 7-hydroxycoumarin (7-HC) and tezolinone (TIZ) alone and in combination, as described in Example 3 of this invention; where (A) is the Western blot detection image and (B) is the quantitative analysis image. Figure 7 This is a graph showing the effect of 7-hydroxycoumarin (7-HC) and tezolinone (TIZ) alone and in combination on the expression level of JEV NS3 mRNA in Example 3 of the present invention. Figure 8 The images show Western blot detection and quantitative analysis of JEV NS3 protein expression using coumarin-3-carboxylic acid (C3AC) and tezolinide (TIZ) alone and in combination, as described in Example 3 of this invention; where (A) is the Western blot detection image and (B) is the quantitative analysis image. Figure 9 This is a graph showing the effect of coumarin-3-carboxylic acid (C3AC) and tezolinone (TIZ) alone and in combination on the expression level of JEV NS3 mRNA in Example 3 of the present invention. Figure 10 The images show Western blot detection and quantitative analysis of JEV NS3 protein expression using dicumarol (DCM) and tezolinone (TIZ) alone and in combination, as described in Example 3 of this invention; where (A) is the Western blot detection image and (B) is the quantitative analysis image. Figure 11 This is a graph showing the effect of dicoumarin (DCM) and tezolinone (TIZ) alone and in combination on the expression level of JEV NS3 mRNA in Example 3 of the present invention. Figure 12This is a schematic diagram illustrating the dose-effect synergistic verification of dicumarol (DCM) and tezolinone (TIZ) in Example 4 of the present invention; wherein (A) is a Western blot diagram of the effect of different concentrations of DCM and TIZ alone and in combination on the expression of JEV NS3 protein, (B) is a quantitative analysis diagram of the relative expression level of JEV NS3 protein when different concentrations of DCM and TIZ are used alone and in combination, and (C) is a diagram of the effect of different concentrations of DCM and TIZ alone and in combination on the expression level of JEV NS3 mRNA; Figure 13 This is a schematic diagram illustrating the in vivo protective effect and brain tissue pathological influence of dicumarol (DCM) and nitrozonidine (NTZ) alone and in combination on mice infected with Japanese encephalitis virus in Example 5 of the present invention; wherein (A) is a schematic diagram of the in vivo experimental drug administration and sample collection time of the BALB / c mouse JEV infection model, (B) is a survival curve analysis diagram of mice in each group within 12 days after infection, (C) is a curve of weight change of mice in each group within 9 days after infection, and (D) is a pathological morphological observation diagram of brain tissue of mice in each group (HE staining, top: gross brain tissue section; bottom: high magnification brain tissue pathological details). Note: This indicates that the p-value is less than 0.05. This indicates that the p-value is less than 0.01. ns indicates that the P-value is less than 0.001, and ns indicates that the P-value is greater than or equal to 0.05. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art.

[0030] Unless otherwise specified, the test materials and reagents used in the following examples are commercially available or prepared by known methods.

[0031] Unless otherwise specified, all techniques or conditions described in the embodiments can be performed in accordance with the techniques or conditions described in the literature in this field or in the product manual. Unless otherwise specified, the quantitative experiments in the following embodiments are all repeated three times or more, and the results are averaged.

[0032] Example 1: Preliminary screening of cytotoxic and anti-Japanese encephalitis virus (JEV) activity of coumarin derivatives 1. Experimental Materials 1.1 Cells and Virus Strains The Japanese encephalitis virus strain NJ2008 was donated by the laboratory of Qiu Yafeng at the Shanghai Veterinary Research Institute.

[0033] 1.2 Main Reagents and Compounds 4-Hydroxycoumarin (4-HC), 7-hydroxycoumarin (7-HC), dicoumarin (DCM), coumarin-3-carboxylic acid (C3AC), and 7-(diethylamino)coumarin (DEAC) were all purchased from Aladdin Biotech. Tizonide (TIZ, CAS No.: 173903-47-4) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., with a purity greater than 98%. The CCK-8 cell viability assay kit was purchased from Suzhou Xinsaimei Biotechnology Co., Ltd.; the RNA extraction kit, reverse transcription kit, and qPCR fluorescent staining reagent were all purchased from Beijing TransGen Biotech Co., Ltd.; JEV NS3 rabbit anti-polyclonal antibody, GAPDH internal control antibody, and HRP-labeled goat anti-rabbit IgG secondary antibody were all purchased from Wuhan Sanying Biotech Co., Ltd.

[0034] 4-Hydroxycoumarin: CAS No. 1076-38-6.

[0035] 7-(diethylamino)coumarin: CAS No. 20571-42-0.

[0036] 2. Experimental Methods 2.1 Cell Culture: Cultured at 37°C in a 5% CO2 incubator. Used DMEM medium containing 10% FBS, 100 U / mL penicillin, and streptomycin. Cells were passaged at 90% confluence at a ratio of 1 / 3 to 1 / 4.

[0037] 2.2 Cytotoxicity assay (CCK-8 assay): BHK21 cells were administered at 8 × 10⁸ 3Cells were seeded per well (100 μl) in a 96-well cell culture plate and allowed to adhere. The five coumarin derivatives were then seeded in DMEM (Dur+2% serum) at nine different concentrations: 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, and 100 μM, with three replicates for each concentration. After 24 hours of culture, the culture supernatant was discarded, and 100 μl of fresh DMEM medium containing 10% CCK-8 reagent was added to each well. The cells were then incubated for another hour, and the absorbance at 450 nm was measured using a microplate reader to calculate cell viability.

[0038] 2.3 Virus culture: BHK21 cells were seeded into culture containers and cultured in complete medium containing serum until cell confluence reached 70%–90%. After discarding the medium, the cells were washed once with serum-free medium or cell maintenance medium. Virus (Japanese encephalitis virus NJ2008 strain) was inoculated into the cell culture system at a set multiplicity of infection (MOI) of 0.1. Two hours after virus adsorption, medium containing 2% serum was added and the cells were cultured until the medium turned orange-yellow, at which point the virus was harvested.

[0039] 2.4 Initial screening of compounds BHK21 cells were seeded in 12-well plates (2 × 10⁻⁶ cells per well). 5 Cells per well were cultured for 24 hours. The culture medium was discarded, and the cells were washed twice with phosphate-buffered saline (PBS). Serum-free medium containing Japanese encephalitis virus (JEV) virus solution (MOI = 0.1) was added, and the cells were incubated at 37°C for 2 hours for adsorption. The virus solution was removed, and unadsorbed virus was washed away with phosphate-buffered saline (PBS). Then, maintenance solutions containing non-toxic concentrations (e.g., 5 μM or 50 μM) of the aforementioned five coumarin derivatives were added. A normal cell control group and a JEV-infected group were also established. Cells were cultured for another 24 hours, and cell samples were collected.

[0040] 2.5 Real-time quantitative PCR (RT-qPCR) Total RNA was extracted from cells in each group using the TRIzol method, and 1 μg of total RNA was reverse transcribed into cDNA using a reverse transcription kit. Using the cDNA as a template, quantitative PCR amplification of the JEV replication marker NS3 gene was performed using specific primers.

[0041] The primer sequences for RT-qPCR amplification are as follows: Specific amplification gene-NS3 primers, NS3-F:5'- GGAGAAGGCAGAGTCATC-3' (SEQ ID NO.1) NS3-R:5'- GGCTAGGTTACCGTCATC-3' (SEQ ID NO.2) Specific gene amplification - GAPDH primers, GAPDH -F:5'- GAAGGTGGTGAAGCAGGCATCT-3' (SEQ ID NO.3) GAPDH -R:5'-CGGCATCGAAGGTGGAAGAGTG-3' (SEQ ID NO.4) Use 2 -△△Ct The relative expression levels of NS3 mRNA in each group were calculated using the method.

[0042] 2.6 Western Blot Total protein was extracted from cells in each group using RIPA lysis buffer containing a recombinant protease inhibitor, and protein concentration was determined by the BCA method. 30 μg of protein sample was subjected to 10% SDS-PAGE electrophoresis and then transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 2 hours, and then JEV NS3 antibody (1:10000 dilution) and GAPDH antibody (1:30000 dilution) were added, followed by overnight incubation at 4°C. The next day, the membrane was washed three times with TBST, and then incubated with HRP-labeled goat anti-rabbit IgG secondary antibody (1:3000 dilution) at room temperature for 1 hour. After washing, the membrane was developed using ECL chemiluminescence imaging, and the bands were quantitatively analyzed using ImageJ software based on their grayscale values.

[0043] 3. Experimental Results 3.1 Cytotoxicity The results are as follows Figure 2 As shown in Figures A, B, D, and E, 4-hydroxycoumarin (4-HC), 7-hydroxycoumarin (7-HC), coumarin-3-carboxylic acid (C3AC), and 7-(diethylamino)coumarin (DEAC) showed no cytotoxicity at concentrations up to 100 μM. In this example, the dosage of coumarin compounds used in the cellular antiviral experiment was 50 μM, which is well within the safe and non-toxic concentration range. The results are as follows... Figure 2 As shown in Figure C, dicumarol exhibits no cytotoxicity against BHK21 cells at concentrations below 5.0 μM. In this embodiment, the dose of dicumarol used in the cellular-level antiviral experiment was 5.0 μM, which is well within the safe and non-toxic concentration range.

[0044] 3.2 Antiviral effect Real-time quantitative PCR results showed that 7-HC, DCM, and C3AC significantly inhibited the relative expression of JEV NS3 mRNA. Figure 3 Consistent with the transcriptional results above, Western blot analysis confirmed that treatment with these coumarin derivatives significantly downregulated the expression levels of JEV NS3 viral proteins in cells. Figure 4 (A). Quantitative analysis of the band grayscale further confirmed that 7-HC, DCM, and C3AC all had statistically significant inhibitory effects on NS3 protein expression (P<0.01). Figure 4 (B) In summary, the above experimental results indicate that 7-HC, DCM, and C3AC possess excellent anti-JEV replication activity and can serve as candidate lead compounds for the preparation of synergistic anti-JEV drugs in combination with tezolinide.

[0045] Example 2: EC of coumarin derivatives 50 Measurement 1. Experimental Materials 1.1 Cells and Virus Strains The Japanese encephalitis virus strain NJ2008 was donated by the laboratory of Qiu Yafeng at the Shanghai Veterinary Research Institute.

[0046] 1.2 Main Reagents and Compounds 7-Hydroxycoumarin (7-HC), discoumarin (DCM), and coumarin-3-carboxylic acid (C3AC) were purchased from Aladdin. The CCK-8 cell viability assay kit was purchased from SinSmithKline.

[0047] 2. Experimental Methods 2.1 Cell Culture: Cultured at 37°C in a 5% CO2 incubator. Used DMEM medium containing 10% FBS, 100 U / mL penicillin, and streptomycin. Cells were passaged at 90% confluence at a ratio of 1 / 3 to 1 / 4.

[0048] 2.2 EC 50 Detection (CCK-8 method): BHK21 cells were seeded in 96-well plates and then seeded with JEVs (MOI = 0.1). After 2 hours of adsorption, serially diluted 7-HC, C3AC, and DCM solutions were added (concentration gradients of 0.1, 1, 10, and 100 μM, etc.), and the cells were cultured for another 24 hours. Cell viability was assessed to determine the viral inhibition rate. Using GraphPad Prism software, a nonlinear regression model was used to fit the dose-response curve with the logarithm of compound concentration as the X-axis and the viral inhibition rate (%) as the Y-axis, and the half-maximal inhibitory concentration (IC50) of each compound was accurately calculated. 50 ).

[0049] 3. Experimental Results The fitted curve and quantitative data are as follows: like Figure 5 As shown in Figure A, the EC50 for 7-HC inhibition of JEV is 36.13 μM. Figure 5 As shown in Figure B, C3AC inhibits the EC of JEV. 50 It is 23.82 μM. For example... Figure 5 As shown in Figure C, DCM inhibits the EC of JEV. 50 The concentration was only 5.714 μM. Among all the effective coumarin compounds screened, dicoumarin exhibited an extremely low half-maximal inhibitory concentration (IC50) and the strongest anti-JEV replication activity, with molecular efficacy significantly superior to other monocyclic coumarin derivatives.

[0050] Example 3: Synergistic antiviral effect of dicumarol and tezolinone 1. Experimental Materials 1.1 Cells and Virus Strains The Japanese encephalitis virus strain NJ2008 was donated by the laboratory of Qiu Yafeng at the Shanghai Veterinary Research Institute.

[0051] 1.2 Main Reagents and Compounds 7-Hydroxycoumarin (7-HC), dicoumarin (DCM), and coumarin-3-carboxylic acid (C3AC) were all purchased from Aladdin Biotech. Tizonide (TIZ, CAS No.: 173903-47-4) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., with a purity greater than 98%. RNA extraction kit, reverse transcription kit, and qPCR fluorescent staining reagent were all purchased from TransGen Biotech. JEVNS3 rabbit anti-polyclonal antibody, GAPDH internal control antibody, and HRP-labeled goat anti-rabbit IgG secondary antibody were all purchased from Wuhan Sanying Biotech.

[0052] 2. Experimental Methods 2.1 Cell Culture: Cultured at 37°C in a 5% CO2 incubator. Used DMEM medium containing 10% FBS, 100 U / mL penicillin, and streptomycin. Cells were passaged at 90% confluence at a ratio of 1 / 3 to 1 / 4.

[0053] 2.2 Virus culture: BHK21 cells were seeded into culture vessels and cultured in complete medium containing serum until cell confluence reached 70%–90%. After discarding the medium, the cells were washed once with serum-free medium or maintenance medium. The virus was inoculated into the cell culture system at the set multiple of infection (MOI) of 0.1. Two hours after virus adsorption, medium containing 2% serum was added and cultured until the medium turned orange-yellow, at which point the virus was harvested.

[0054] 2.3 Combined treatment with compounds: To explore host-targeted multi-target combination therapy synergistic strategies, this embodiment evaluated the biological effects of candidate compounds combined with the broad-spectrum antiviral drug tinizonide. Adherent cells were divided into: (1) Control group, (2) JEV infection group, (3) derivative monotherapy group (7-HC or C3AC or DCM alone), (4) TIZ monotherapy group, and (5) combination therapy group (7-HC or C3AC or DCM + TIZ).

[0055] After inoculating and washing the cells with the virus, maintenance solutions containing the corresponding single or combined drugs were added according to the above groupings. The concentrations of each component in the maintenance solution were controlled within their respective toxicity safety thresholds. After 24 hours of incubation, cells were simultaneously collected for Western blotting quantification of proteins and RT-qPCR gene transcription analysis.

[0056] 2.4 Real-time quantitative PCR (RT-qPCR) Total RNA was extracted from cells in each group using the TRIzol method, and 1 μg of total RNA was reverse transcribed into cDNA using a reverse transcription kit. Using the cDNA as a template, quantitative PCR amplification of the JEV replication marker NS3 gene was performed using specific primers.

[0057] The primer sequences for RT-qPCR amplification are as follows: Specific amplification gene-NS3 primers, NS3-F:5'- GGAGAAGGCAGAGTCATC-3' (SEQ ID NO.5) NS3-R:5'- GGCTAGGTTACCGTCATC-3' (SEQ ID NO.6) Specific gene amplification - GAPDH primers, GAPDH -F:5'- GAAGGTGGTGAAGCAGGCATCT-3' (SEQ ID NO.7) GAPDH -R:5'-CGGCATCGAAGGTGGAAGAGTG-3' (SEQ ID NO.8) Use 2 -△△Ct The relative expression levels of NS3 mRNA in each group were calculated using the method.

[0058] 2.5 Western Blot Total protein was extracted from cells in each group using RIPA lysis buffer containing a recombinant protease inhibitor, and protein concentration was determined by the BCA method. 30 μg of protein sample was subjected to 10% SDS-PAGE electrophoresis and then transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 2 hours, and then JEV NS3 antibody (1:10000 dilution) and GAPDH antibody (1:30000 dilution) were added, followed by incubation overnight at 4°C. The next day, the membrane was washed three times with TBST, and then incubated with HRP-labeled secondary antibody (1:3000 dilution) at room temperature for 1 hour. After washing, the membrane was developed using ECL chemiluminescence imaging, and the bands were quantitatively analyzed using ImageJ software based on their grayscale values.

[0059] 3. Experimental Results 3.1 Effects of combined drug therapy on JEV NS3 protein translation levels: Treatment with 7-HC, C3AC, or DCM alone significantly reduced the expression level of JEV NS3 protein. Figure 6 China A Figure 8 China A Figure 10 (A). Notably, compared to the individual monotherapy groups, the combination of dicumarol and tezolinone (DCM+TIZ) significantly enhanced the inhibitory effect on NS3 protein expression; in contrast, the combination of 7-HC or C3AC with TIZ failed to produce any additional enhancement. Figure 6 B, Figure 8 B, Figure 10 (See section B). This result indicates that common monocyclic coumarin derivatives (7-HC, C3AC) cannot produce a compatibility advantage with TIZ, while the bicyclic dimer coumarin (DCM) targeted in this invention exhibits a specific synergistic effect of protein translation inhibition when used in combination with TIZ.

[0060] 3.2 Effects of combined drug therapy on JEV NS3 mRNA transcription levels: Highly consistent experimental results were also observed at the gene transcription level. Real-time quantitative PCR analysis showed that single treatment with the above three compounds significantly downregulated the expression level of NS3 mRNA. Figure 7 , Figure 9 , Figure 11However, when administered in combination, the DCM+TIZ combination group showed enhanced inhibition of JEV NS3 mRNA compared to the DCM monotherapy group. Figure 11 While the other two groups of compounds showed no significant difference at the transcriptional level when used in combination, the results were as follows:

[0061] This invention, through extremely rigorous and systematic multi-level screening and dose-effect correlation experiments, has for the first time clearly demonstrated that not all coumarin molecules can produce compatibility advantages with the broad-spectrum antiviral drug tinizonide (TIZ). This combination can achieve complete clearance of Japanese encephalitis virus in the central nervous system and host cells while significantly reducing the dosage of single drugs and the risk of cytotoxicity. It has extremely high clinical translational value in the preparation of novel, safe, and highly effective multi-target anti-Japanese encephalitis drug compositions.

[0062] Example 4: Validation of the dose-effect synergistic effect of dicumarol and tezolinide 1. Experimental Materials 1.1 Cells and Virus Strains The Japanese encephalitis virus strain NJ2008 was donated by the laboratory of Qiu Yafeng at the Shanghai Veterinary Research Institute.

[0063] 1.2 Main Reagents and Compounds Dicoumarol was purchased from Aladdin Biotech. Tizoxin (TIZ, CAS No.: 173903-47-4) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., with a purity greater than 98%. RNA extraction kit, reverse transcription kit, and qPCR fluorescent staining reagent were all purchased from TransGen Biotech. JEV NS3 rabbit anti-polyclonal antibody, GAPDH internal control antibody, and HRP-labeled goat anti-rabbit IgG secondary antibody were all purchased from Wuhan Sanying Biotech.

[0064] 2. Experimental Methods Adherent cells were seeded with JEV (MOI = 0.1), and unadsorbed virus was washed away after 2 hours of adsorption. The experimental groups were further subdivided into different drug concentrations and ratios: JEV pure infection model group; DCM single-drug group; TIZ single-drug group; Combination 1 (5 μM DCM + 10 μM TIZ, molar ratio 1:2); Combination 2 (5 μM DCM + 15 μM TIZ, molar ratio 1:3); Combination 3 (3.75 μM DCM + 15 μM TIZ, molar ratio 1:4). After incubation in maintenance solution for 24 hours, cells from each group were collected, and the expression abundance of JEV NS3 was determined using Western blotting and RT-qPCR.

[0065] 3. Experimental Results 3.1 Effects of different ratios of combined drug administration on the translation level of JEV NS3 protein: Compared with the JEV pure infection model group, the expression of NS3 protein was only partially attenuated by DCM or TIZ alone (p<0.05). However, when DCM and TIZ were administered in combination, as the concentration of TIZ in the combination system increased from 10 μM to 15 μM, the expression of NS3 protein decreased in a concentration-dependent manner while DCM was maintained at 5 μM, and the statistically highest inhibition reduction was achieved in the 5 μM DCM + 15 μM TIZ combination group (p<0.001). If DCM was reduced back to 3.75 μM and combined with 15 μM TIZ, the expression of NS3 protein slightly recovered, but was still significantly better than the single-drug group (p<0.01). Figure 12 (A, B)

[0066] 3.2 Effects of different ratios of combined drug administration on JEV NS3 mRNA transcription levels: RT-qPCR quantification results showed completely consistent characteristics. Figure 12 As shown in Figure C, the inhibitory efficiency of the single-drug group was limited. In the combination-drug group, the 5 μM DCM + 15 μM TIZ group reduced the relative transcriptional level of JEV NS3 mRNA to the lowest baseline level in the entire group.

[0067] The synergistic inhibitory effect of dicumarol and tezolinone against Japanese encephalitis virus exhibits a clear concentration-dependent relationship. Experiments show that controlling the molar ratio of the two at 1:3 significantly enhances their in vitro antiviral activity, thus providing ample technical support for the precise determination of the active ingredient dosage in subsequent formulations.

[0068] Example 5: Synergistic anti-JEV protective effect of dicumarol and nitrozonide in vivo 1. Experimental Materials 1.1 Mice Four-week-old female BALB / c mice were purchased from Suzhou Spaford Company.

[0069] 1.2 Main Reagents and Compounds Dicoumarol was purchased from Aladdin Pharmaceutical Co., Ltd. Nitrozonide (NTZ) was provided by Jiangsu Qihui Pharmaceutical Technology Co., Ltd., with a purity greater than 99%. Hematoxylin-eosin was purchased from Beyotime Pharmaceutical Co., Ltd. JEV NS3 rabbit anti-polyclonal antibody, GAPDH internal control antibody, and HRP-labeled goat anti-rabbit IgG secondary antibody were all purchased from Wuhan Sanying Pharmaceutical Co., Ltd.

[0070] 2. Experimental Methods 2.1 Animal grouping and modeling: Healthy BALB / c mice aged 4 weeks were randomly divided into 5 groups: (1) Mock control group, (2) JEV pure infection model group, (3) NTZ monotherapy group (100 mg / kg), (4) DCM monotherapy group (30 mg / kg), and (5) NTZ+DCM combination therapy group. Except for the Mock group, all other groups of mice were injected intraperitoneally with 1×10 6 Modeling with JEV virus fluid from PFU (Phenylephrine-induced cytokine) Figure 13 (A)

[0071] 2.2 Dosing regimen: Following viral inoculation, mice in the NTZ monotherapy and combination therapy groups were administered 100 mg / kg of NTZ daily via gavage; mice in the DCM monotherapy and combination therapy groups were administered 30 mg / kg of DCM daily via intraperitoneal injection. Administration continued for 5 days.

[0072] 2.3 Clinical indicator monitoring: The weight of mice in each group was measured daily, and their clinical manifestations (such as hair standing on end, arched back, paralysis, etc.) were closely observed. The mortality of mice in each group over 12 days was recorded, and survival rate curves were plotted.

[0073] 2.4 Pathological observation of brain tissue: On day 5 post-infection, mice from each group were collected, perfused and fixed, and brain tissue was harvested. After fixation, dehydration, paraffin embedding, and sectioning, standard hematoxylin-eosin (HE) staining was performed, and the pathological damage and inflammatory cell infiltration in each neuroanatomical region were observed under an optical microscope.

[0074] 3. Experimental Results 3.1 In vivo survival rate protection effect: The survival rate of mice in the normal control group was 100%. Mice in the JEV pure infection model group began to die on day 5 after viral inoculation, with the survival rate dropping to 0% by day 8, demonstrating extremely high lethality. After treatment with NTZ or DCM monotherapy, the endpoint survival rate of mice was only maintained at approximately 30%. However, the endpoint survival rate of mice in the NTZ+DCM combination therapy group was significantly increased to approximately 50%, and the onset and death time of mice was significantly delayed, demonstrating a strong synergistic protective barrier in vivo. Figure 13 (B)

[0075] 3.2 Monitoring of weight changes: Following JEV infection, mice in the pure model group experienced reduced food intake and a severe, irreversible dose-dependent weight loss starting from day 4 after viral inoculation due to encephalitis symptoms. NTZ or DCM monotherapy groups mitigated the weight loss to some extent. However, the NTZ+DCM combination therapy group showed the most moderate weight loss throughout the entire disease monitoring period, significantly approaching that of normal mice, confirming that this combination effectively improves the quality of life in affected mice. Figure 13 (C)

[0076] 3.3 Improvement of brain tissue histopathological damage: Control (Normal Control Group): The brain parenchyma structure was intact, neurons were neatly arranged, and no vascular cuffs or inflammatory cell infiltration were observed. JEV (Pure Infection Model Group): The brain tissue showed typical acute severe encephalitis lesions, with large areas of neuronal degeneration and necrosis, reticular vacuolation of neural structures (as shown by yellow arrows), accompanied by extremely significant perivascular cuff-like changes (as shown by red arrows) and a large number of inflammatory microglia infiltrations (as shown by black / blue arrows). NTZ and DCM Single Drug Groups: Although the pathological damage and vascular cuff phenomenon in the brain were partially alleviated, obvious inflammatory infiltration and neuronal degeneration were still observed locally (as shown by yellow and black arrows). NTZ+DCM Combined Drug Group: The pathological manifestations of mouse brain tissue were fundamentally and comprehensively reversed and significantly improved. The morphology of neurons in the brain parenchyma basically returned to normal, with no obvious neuronal necrosis, and the perivascular inflammatory cell cuffs and microglial cell proliferation and infiltration were significantly reduced or basically disappeared. Figure 13 (D).

[0077] In vivo experimental results strongly confirm that dicumarol (DCM) and nitrozonidine (NTZ) possess excellent synergistic antiviral and neuroprotective activities in mammals. This drug combination significantly improved the survival rate and stabilized the body weight of mice lethally infected with JEV, and further blocked JEV-induced neuropathological damage and reduced the inflammatory flare-up of the central nervous system. This provides extremely reliable and robust clinical translational evidence for the preparation of drug compositions for the treatment of Japanese encephalitis or acute encephalitis caused by flaviviruses.

[0078] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of a coumarin derivative in the preparation of a drug against Japanese encephalitis virus, characterized in that, The coumarin derivative is one or more of dicoumarin, 7-hydroxycoumarin, and coumarin-3-carboxylic acid.

2. The application according to claim 1, characterized in that, The coumarin derivatives can inhibit the replication of Japanese encephalitis virus and reduce viral protein expression and viral RNA levels.

3. The application according to claim 1, characterized in that, The coumarin derivative is dicoumarin.

4. The application according to claim 1, characterized in that, The drug is formulated with coumarin derivatives as the active pharmaceutical ingredient and can be prepared in any pharmaceutically acceptable dosage form.

5. A pharmaceutical composition, characterized in that, The active ingredient in the pharmaceutical composition includes (1) dicumarol. (2) Nitrozonide or Tezoxinide.

6. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition is formulated in any pharmaceutically acceptable dosage form using dicumarol, nitrozonide, or tezolinide as the active pharmaceutical ingredient.

7. The pharmaceutical composition according to claim 5, characterized in that, The molar ratio of the coumarin, nitrozonide, or tezolidinide is 1:(2~4).

8. Use of a pharmaceutical composition as described in any one of claims 5-7 in the preparation of a medicament for treating Japanese encephalitis virus infection.

9. The application according to claim 8, characterized in that, The specific effects of the drug include: improving the survival rate of JEV-infected hosts, slowing down weight loss, and reducing brain tissue pathological damage caused by central nervous system inflammation.

10. Use of a pharmaceutical composition as described in any one of claims 5-7 in the preparation of a drug for the prevention and treatment of epidemic Japanese encephalitis caused by Japanese encephalitis virus infection.

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