Use of resatorvid in the preparation of a medicine for resisting swine encephalomyelitis virus infection

Abstract

This invention relates to the application of ressatopic vir in the preparation of drugs against porcine hemagglutinating encephalomyelitis virus (PCEV) infection, belonging to the field of veterinary drug technology. It solves the technical problem that there are currently no specific therapeutic drugs for PCEV in the prior art. Specifically, this invention was studied through in vitro and in vivo mouse models of PCEV infection. The results showed that ressatopic vir can directly inhibit the replication of PCEV at the gene and protein levels both in vitro and in vivo, reducing viral titers. This demonstrates that ressatopic vir has a significant anti-PCEV effect and can be used in the development of new drugs against PCEV infection, and is of great significance for the identification of drug targets.

Description

Technical Field

[0001] This invention belongs to the field of veterinary drug technology, specifically relating to the application of Resapride in the preparation of a drug for treating porcine hemagglutinating encephalomyelitis virus infection. Background Technology

[0002] Resaptovir (TAK-242) is a small-molecule selective inhibitor of TLR4 signaling with anti-inflammatory activity. TAK-242 directly binds to the Cys747 residue in the intracellular region of TLR4, disrupting the interaction between TLR4 and its adaptor molecules MYD88 and TRIF, thereby inhibiting TLR4 signaling and blocking the production of NO, TNF-α, and IL-6 induced by pathogens.

[0003] Porcine hemagglutinating encephalomyelitis virus (PHEV) belongs to the order Nidovirales, family Coronaviridae, and genus Coronavirus. It is a single-stranded positive-sense RNA virus, 70–130 nm in diameter, with an envelope containing 20–30 nm petal-like projections. Coronaviruses are widespread in nature. Since the outbreak of Severe Acute Respiratory Syndrome (SARS), the frequency and severity of new coronavirus diseases have been increasing. PHEV, along with SARS-CoV, MERS-CoV, and SARS-CoV-2, belongs to the genus β-coronavirus. It was the first coronavirus discovered to infect pigs and is the only known neurotropic coronavirus capable of causing severe central nervous system infection in pigs. Porcine hemagglutinating encephalomyelitis caused by PHEV infection is an acute, highly contagious disease. Based on clinical manifestations, it can be classified into encephalomyelitis type, vomiting-wasting type, and influenza type. These three types can coexist in the same pig herd or occur in different pig herds or regions. Currently, there are no specific drugs or vaccines for this disease, so there is an urgent need to find new anti-PHEV drugs to provide technical reserves for guiding comprehensive epidemic prevention and control in clinical practice. Summary of the Invention

[0004] The purpose of this invention is to provide an application of ressatopic valerate in the preparation of a drug for treating swine hemagglutinating encephalomyelitis virus infection. This invention is the first to discover that ressatopic valerate has an anti-PHEV infection effect.

[0005] The technical solution adopted by the present invention to achieve the above objectives is as follows.

[0006] This invention provides the use of Resapride in the preparation of drugs for treating porcine hemagglutinating encephalomyelitis virus infection, wherein the use is one or both of the following (a1) and (a2):

[0007] (a1) The use of ressatopic vir in the preparation of drugs for the treatment of PHEV infection;

[0008] (a2) Application of Resaptovir in the preparation of drugs for the prevention of PHEV infection.

[0009] Preferably, the anti-PHEV infection drug is a pharmaceutical composition containing resadorvi as the sole active ingredient.

[0010] More preferably, the pharmaceutical composition refers to a pharmaceutical composition consisting of ressatopic valerate and one or more pharmaceutically permissible excipients.

[0011] Preferably, the dosage form of the anti-PHEV infection drug is powder, granules, capsules or solution.

[0012] Preferably, the route of administration of the anti-PHEV infection drug is intravenous injection, intraperitoneal injection, oral administration, nebulized inhalation, or intracerebral administration.

[0013] Preferably, in cells, the concentration of ressatopic valerate in the anti-PHEV infection drug is 1 μM to 20 μM.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] The application of resadorvir in the preparation of anti-PHEV infection drugs of the present invention was studied using an in vitro PHEV-infected cell model. Results showed that resadorvir at concentrations of 1 μM to 20 μM could inhibit viral proliferation in cells, with effects observed at the viral titer, gene, and protein levels. Furthermore, resadorvir exhibited a significant inhibitory effect on PHEV without cytotoxicity and had a high selectivity index. This indicates that resadorvir can be used for the development of new anti-PHEV drugs and is of significant importance for the identification of drug targets. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This diagram illustrates the effect of different concentrations of resatope solution on the viability of mouse neuroblastoma cells (N2a) in Example 1 of this invention. The horizontal axis represents the concentration of resatope, and the left vertical axis represents the effect of resatope on cell viability.

[0018] Figure 2This refers to the inhibitory effect of 0.01 μM to 20 μM Resapride solution on PHEV-infected cells in vitro for 4 hours at the mRNA level, as shown in Example 1 of this invention.

[0019] Figure 3 This refers to the inhibitory effect of 0.01 μM to 20 μM Resapride solution on PHEV-infected cells in vitro for 24 hours at the mRNA level, as shown in Example 1 of this invention.

[0020] Figure 4 In the diagram, A represents the inhibitory effect of 0.01 μM to 20 μM Resatopivir solution on PHEV-infected cells in vitro for 4 hours at the protein level in Example 1 of this invention; B is a graph showing the PHEV protein level analysis.

[0021] Figure 5 In the diagram, A represents the inhibitory effect of 0.01 μM to 20 μM Resapride solution on PHEV-infected cells in vitro for 24 hours at the protein level in Example 1 of this invention; B is a graph showing the PHEV protein level analysis.

[0022] Figure 6 This invention illustrates the inhibitory effect of 20 μM Resapride solution on PHEV at the cellular level in Example 1 of this invention.

[0023] Figure 7 The curves showing the changes in body weight of mice in different groups in Example 2 of this invention are shown.

[0024] Figure 8 The survival curves of mice in different groups in Example 2 of this invention are shown.

[0025] Figure 9 This refers to the inhibitory effect of Resapride solution on PHEV in vivo at the protein level in Example 2 of the present invention.

[0026] Figure 10 This refers to the inhibitory effect of Resapride solution on PHEV in vivo at the protein level in Example 2 of the present invention. Detailed Implementation

[0027] To further understand the present invention, preferred embodiments of the present invention are described below. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims of the present invention.

[0028] The application of Resapordil in the preparation of drugs for treating porcine hemagglutinating encephalomyelitis virus infection according to the present invention can be one or both of (a1) and (a2):

[0029] (a1) The use of ressatopic vir in the preparation of drugs for the treatment of PHEV infection;

[0030] (a2) Application of Resaptovir in the preparation of drugs for the prevention of PHEV infection.

[0031] In the above technical solution, the anti-PHEV infection drug is a pharmaceutical composition containing Resapordil as the sole active ingredient. The pharmaceutical composition refers to a pharmaceutical composition consisting of Resapordil and one or more pharmaceutically permissible excipients.

[0032] In the above technical solutions, the dosage form of the anti-PHEV infection drug is powder, granules, capsules, or solution. It should be noted that the dosage form of the anti-PHEV infection drug is not limited to these; other dosage forms known to those skilled in the art are also applicable to this invention.

[0033] In the above technical solutions, the administration routes of the anti-PHEV infection drugs are intravenous injection, intraperitoneal injection, oral administration, nebulized inhalation, or intracerebral administration. It should be noted that the administration routes of the anti-PHEV infection drugs are not limited to these, and other administration routes known to those skilled in the art are also applicable to this invention.

[0034] In the above technical solutions, preferably, the concentration of ressatopic valerate in the anti-PHEV infection drug is 1 μM to 20 μM in cells.

[0035] In this invention, Resatorvaline is a prior art technology, and its chemical structural formula is as follows:

[0036]

[0037] It can be obtained through commercial purchase.

[0038] The terminology used in this invention generally has the meaning as understood by one of ordinary skill in the art, unless otherwise stated.

[0039] The present invention will be further illustrated below with reference to the embodiments.

[0040] In the following embodiments, various processes and methods not described in detail are conventional methods known in the art. Unless otherwise specified, the materials, reagents, apparatus, instruments, equipment, etc., used in the following embodiments are commercially available.

[0041] Example 1

[0042] Resalitopivir in vitro anti-PHEV assay

[0043] 1.1 Dissolution and storage of Resapride

[0044] Weigh 10 mg of Resaporvec (Selleck, S745501) and add it to 552.8 μL of DMSO solution to prepare a 50 mM Resaporvec solution. Store at -80°C.

[0045] 1.2 Culture of mouse neuroblastoma-producing cells (N2a cells)

[0046] Cells were removed from liquid nitrogen, rapidly dissolved at 37°C, resuspended in DMEM containing 8 wt% fetal bovine serum, and cultured in DMEM containing 8 wt% fetal bovine serum.

[0047] 1.3 Determination of Resatorvaline cytotoxicity concentration

[0048] N2a cells were seeded into 96-well plates, 100 μL of cell suspension per well, and cultured in a 37°C, 5% CO2 cell culture incubator. When the cells reached 70%–80% confluence, resapride was prepared into DMEM solutions of different concentrations (10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, and 80 μM) and added to the 96-well plates by medium exchange. A blank control group was also included. After 24 h of incubation, medium containing 8 wt% CCK-8 was prepared and added by medium exchange. Color development was performed after 24 h of incubation, and the absorbance at 450 nm was measured. Cell viability was calculated.

[0049] The calculation formula is as follows:

[0050] Cell viability = [(experimental wells - blank wells) / (control wells - blank wells)] × 100%

[0051] Experimental results are as follows Figure 1 As shown, Resapride concentrations of 0.01 μM to 20 μM had no significant effect on cell viability. Cell viability was significantly reduced at 30 μM.

[0052] 1.4 Inhibitory effect of Resapride on PHEV infection

[0053] The inhibitory effect of Resapride solutions with concentrations ranging from 0.01 μM to 20 μM on PHEV will be explored. The experiment will verify the effect at the mRNA level, protein expression level, and cellular level.

[0054] 1.4.1 RT-PCR detection method

[0055] N2a cells were seeded in 12-well plates. When the cells reached 70%–80% confluence, they were inoculated with PHEV at MOI 2 and MOI 1, respectively. After 1 hour, one well was replaced with DMEM solution containing 2 wt% fetal bovine serum and the highest drug concentration DMSO as the DMSO control group. The other seven wells were replaced with DMEM solution containing 0.01 μM, 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, and 20 μM resatoridone, respectively, and DMEM solution containing 2 wt% fetal bovine serum as the drug groups. Each well was in triplicate. After incubation for 4 hours and 24 hours, total RNA was extracted from adherent cells, and the mRNA expression level of PHEVN in the cells was detected.

[0056] Method for extracting total RNA from cells (RNA extraction and reverse transcription): After incubation, collect the supernatant and wash three times with PBS. Add 1 mL of RNAiso plus to each well. After complete cell lysis, collect the lysis buffer into a 1.5 mL centrifuge tube. Add 200 μL of chloroform to each sample tube and shake vigorously, then incubate on ice for 10 min. After incubation, centrifuge at 4°C and 12000 rpm for 15 min. After centrifugation, aspirate 400 μL of the supernatant to a 1.5 mL centrifuge tube and add 400 μL of isopropanol. Mix well and incubate at -20°C for 30 min. After incubation, centrifuge at 4°C and 12000 rpm for 10 min, then discard the isopropanol. Add 1 mL of 75% ethanol to each sample tube, mix well, centrifuge at 4°C and 12000 rpm for 5 min, then discard the ethanol and incubate for 3 min. Add 20 μL of RNase-free, dehydrogenase-free (DEPC) solution to each tube, mix thoroughly by pipetting to obtain RNA solution, and determine the sample concentration. Reverse transcription is performed according to the sample concentration, with a total volume of 40 μL, including 4 μL Oligo(dT)18 primer, 8 μL 5×ReverseTranscriptate M-MLV Buffer, 8 μL 2.5 mM dNTP, 1 μL RRI, 1 μL M-MLV-RT, 4 μg RNA, and the remainder is made up with DEPC solution. Incubate at 42℃ for 1 h, then at 70℃ for 10 min to obtain cDNA. Real-time fluorescent PCR is performed using the reverse-transcribed cDNA as a template. The cDNA amplification reaction volume is 20 μL: 10 μL SYBR GreenMix, 1 μL forward primer, 1 μL reverse primer, 6 μL water, and 2 μL cDNA template.

[0057] PHEV forward primer: 5′-TCTGGGAATCCTGACGAG-3′;

[0058] PHEV reverse primer: 5′-AGGCGCTGCAACACTTAC-3′;

[0059] GAPDH forward primer: 5′-CTCAACTACATGGTCTACATGTTC-3′;

[0060] GAPDH reverse primer: 5′-ATTTGATGTTAGTGGGGTCTCGCTC-3′;

[0061] The testing procedure was as follows: 95℃ for 2 min; 94℃ for 15 s; 60℃ for 15 s, 40 cycles; 72℃ for 10 min.

[0062] Using the housekeeping gene GAPDH as a control, 2 ﹣△△CT Analyze the changes in PHEV mRNA transcription levels.

[0063] The test results are as follows Figure 2 , Figure 3 As shown, ressatope at concentrations of 1 μM to 20 μM significantly inhibited the replication of PHEV.

[0064] 1.4.2 Western blotting detection method

[0065] N2a cells were seeded in 12-well plates. When the cells reached 70-80% confluence, they were treated with 7 different concentrations of PHEV. After incubation, intracellular proteins were extracted, and the protein expression level of PHEV in the cells was detected.

[0066] Method for extracting intracellular proteins: After incubation, remove the 12-well plate from the cell culture incubator and wash three times with PBS. Discard the PBS, add 1 mL of PBS, scrape the cells with a cell scraper, and transfer them to 1.5 mL centrifuge tubes. Centrifuge at 12,000 rpm for 10 min at 4°C. Discard the supernatant, add 200 μL of RIPA protein lysis buffer (100:1 volume ratio) and protein inhibitor (PMSF) to each tube, mix by pipetting, and lyse on ice for 30 min. Centrifuge at 12,000 rpm for 10 min at 4°C, collect 180 μL of the supernatant, add 45 μL of 5X loading buffer, and boil in water for 10 min. Subsequently, separate the protein samples using SDS-PAGE, transfer them to polyvinylidene fluoride (PVDF) membranes, block with 5 wt% skim milk powder, incubate with primary and secondary antibodies sequentially, and finally develop using chemiluminescent substrate (ECL). The primary antibody used was a self-made antibody against PHEV, and the secondary antibody used was a horseradish peroxidase-labeled goat anti-rabbit antibody at a concentration of 1:10000.

[0067] The test results are as follows Figure 4 , Figure 5 As shown, ressatope at concentrations of 1 μM to 20 μM significantly inhibited the replication of PHEV.

[0068] 1.4.3 Indirect Immunofluorescence Assay Method

[0069] N2a cells were seeded in 12-well cell slides. When the cells reached 60%–70% confluency, they were inoculated with PHEV at an MOI of 1 for 1 hour before being drug-treated at a concentration of 20 μM. Cells were fixed with 4% paraformaldehyde solution at 0, 4, 8, 12, 24, and 48 hours after drug treatment, and the PHEV content at the cellular level was measured.

[0070] Indirect immunofluorescence assay procedure: Discard the nutrient solution in the 12-well plate, fix with 4 wt% paraformaldehyde preheated to 37°C at room temperature for 10 min, then discard the solution. Fix with methanol precooled to -40°C at 4°C for 10 min, then discard the solution. Wash three times with PBS, permeabilize with 0.5 wt% Triton X-100 at room temperature for 5 min, wash three times with PBS, block with 5 wt% skim milk powder at 37°C for 1 h, wash three times with PBS, remove the slide, label it, and place it in a dark box to incubate with the self-made PHEV-N protein antibody overnight at 4°C. Wash with PBS for 15 min, incubate with goat anti-rabbit red fluorescent antibody in the dark at 37°C for 1 h, wash with PBS for 15 min, mount with anti-fluorescence quenching mounting medium, observe and photograph using a fluorescence microscope.

[0071] The test results are as follows Figure 6 As shown, Resatorvaline significantly affected PHEV replication at different time points.

[0072] Example 2

[0073] Resalitopivir in vivo anti-PHEV assay

[0074] 2.1 Preparation, administration and dosage of Resaporvec solution: The Resaporvec powder from Example 1 was dissolved sequentially in DMSO, PEG300, Tween 80 and ddH2O solvents, with the final proportions of each solvent being 5%, 40%, 5%, and 50%, respectively. Each mouse was intraperitoneally injected with 3 mg / kg / day.

[0075] 2.2 Experimental Grouping

[0076] Three-week-old male BALB / c mice were divided into four groups: a blank control group (MOCK) of 3 mice, a drug-treated group (TAK-242) of 3 mice, a virus-treated group (PHEV) of 6 mice, and a virus-treated and drug-treated group (PHEV+TAK-242) of 6 mice.

[0077] 2.3 Detection Methods

[0078] RT-PCR detection method: Add 1 mL of RNAiso plus to 0.1 g of brain tissue, and perform the remaining operations as in 1.4.1.

[0079] Western boltting detection method: 0.1g brain tissue was added to 1mL of protein lysis buffer, and the rest of the operation was the same as in 1.4.2.

[0080] The average daily body weight changes of mice in different groups are as follows: Figure 7 As shown, the average daily weight curves of mice in the MOCK group and TAK-242 group generally showed a continuous upward trend, while the average daily weight curves of mice in the PHEV group and PHEV+TAK-242 group both showed a trend of first increasing and then decreasing. However, the weight of mice in the PHEV+TAK-242 group increased faster than that in the PHEV group, and the weight loss was smaller.

[0081] Survival rates of mice in different groups, such as Figure 8 As shown, no mice in the MOCK group or TAK-242 group died, while the survival time of mice in the PHEV+TAK-242 group was significantly longer than that of the PHEV group.

[0082] Changes in PHEV mRNA and protein expression levels in the PHEV group and the PHEV+TAK-242 group are as follows: Figure 9 , Figure 10 As shown, compared with the virus-infected group, the expression levels of PHEV mRNA and protein in the drug-infected group were significantly reduced, indicating that in vivo experiments, ressatopic virgin can significantly inhibit PHEV replication and exert a good preventive and therapeutic effect.

[0083] Obviously, the above embodiments are merely examples for clear illustration and are not intended to limit the scope of the embodiments. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all embodiments here. However, obvious variations or modifications derived therefrom are still within the protection scope of this invention.

Claims

1. The use of Resatinovir in the preparation of a drug for the treatment of infection with the porcine encephalomyelitis virus, characterized in that, The application is one or both of the following (a1) and (a2): (a1) The use of ressatopic in the preparation of drugs for the treatment of porcine hemagglutinating encephalomyelitis virus infection; (a2) Application of Resaptovir in the preparation of drugs for the prevention of swine hemagglutination encephalomyelitis virus infection.

2. Use of resatorvid according to claim 1 for the preparation of a medicament against infection with the porcine haemagglutinating encephalomyelitis virus, characterized in that, The drug for treating swine hemagglutination encephalomyelitis virus infection is a pharmaceutical composition containing ressatopicil as the sole active ingredient.

3. Use of resatorvid according to claim 2 for the preparation of a medicament against infection with the porcine haemagglutinating encephalomyelitis virus, characterized in that, The pharmaceutical composition refers to a pharmaceutical composition consisting of Resaptor and one or more pharmaceutically permissible excipients.

4. The use of Resapordil according to claim 1 in the preparation of a drug for treating porcine hemagglutinating encephalomyelitis virus infection, characterized in that, The dosage form of the drug for treating swine hemagglutinating encephalomyelitis virus infection is powder, granules, capsules, or solution.

5. The use of Resapordil according to claim 1 in the preparation of a drug for treating porcine hemagglutinating encephalomyelitis virus infection, characterized in that, The administration routes of the anti-swine hemagglutination encephalomyelitis virus infection drug are intravenous injection, intraperitoneal injection, oral administration, nebulized inhalation, or intracerebral administration.

6. The use of Resapordil according to claim 1 in the preparation of a drug for treating porcine hemagglutinating encephalomyelitis virus infection, characterized in that, In cells, the concentration of ressatopic valerate in the anti-porcine hemagglutinating encephalomyelitis virus infection drug is 1 μM to 20 μM.

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