Application of macrolide derivative in preparation of anti-poxviridae virus medicine

Abstract

The invention discloses an application of macrolide derivatives in preparation of drugs for resisting poxviridae viruses. It is found that the macrolide derivative T8 can reduce the replication level of LSDV, and the derivative is low in toxicity, has no toxic or side effect on MDBK cells, can serve as an inhibitor for efficiently resisting bovine nodular skin disease viruses, provides a powerful theoretical basis and a practical basis for further research and development of antiviral drugs, and has good application prospects. Wide application prospects are realized in the fields of breeding industry and biological medicines.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to an application of a macrolide derivative T8 in preparation of an antiviral drug. BACKGROUND

[0002] CN117304241A discloses a macrolide compound, the structure of which is shown as T8. The compound can be used for treating pathogen infection, especially treating animal pathogen infection, and has significant effect, but no other use has been found at present.

[0003]

[0004] Lumpy skin disease (LSD) is caused by lumpy skin disease virus (LSDV) of capripoxvirus genus, and the virus has the characteristics of long environmental survival period and complex transmission route, and has become a major threat to the development of global animal husbandry, but there is no specific drug for treating LSDV at present.

[0005] Antiviral drugs refer to drugs for preventing and treating animal viral diseases. These drugs can block the spread of viruses in the animal body by inhibiting key enzymes in the viral replication process or blocking the interaction between viruses and host cells. In the entire replication cycle of viruses, any link can be a target for inhibitors. The research and application of veterinary antiviral drugs are of great significance for maintaining animal health and ensuring animal husbandry production. Antiviral drugs can provide "chemical protection" before the production of vaccines and antibodies, which is particularly important for newborn calves, immunosuppressed individuals and newly emerging variants.

[0006] At present, there is no anti-LSDV drug approved, so it is still an important task for drug workers to develop a new generation of antiviral drugs with high efficiency, low toxicity and clear mechanism of action. SUMMARY

[0007] In a first aspect, the present application first finds a new application of a macrolide compound T8 or a pharmaceutically acceptable salt thereof in preparation of an antichthonous poxvirus drug.

[0008] The structural formula of the macrolide derivative T8 is as follows: .

[0009] The poxvirus includes orthopoxvirus genus and capripoxvirus genus.

[0010] The orthopoxvirus genus includes vaccinia virus.

[0011] The capripoxvirus genus includes lumpy skin disease virus.

[0012] The macrolide derivative T8 is used at concentrations ranging from 12.5 to 6400 ppm. Specifically, concentrations include 6400 ppm, 800 ppm, 100 ppm, and 12.5 ppm.

[0013] The drug is a compound drug with macrolide derivative T8 as the sole active ingredient or with other antiviral active ingredients from the Poxviridae family.

[0014] The drug also includes pharmaceutically acceptable carriers, excipients, or additives; the carriers or excipients are selected from one or more of diluents, binders, adsorbents, fillers, and disintegrants; the additives are selected from one or more of stabilizers, bactericides, buffers, isotonic agents, chelating agents, pH control agents, and surfactants.

[0015] The dosage forms of the drug are tablets, capsules, oral liquids, lozenges, granules, powders, pills, powders, ointments, elixirs, suspensions, powders, and solutions.

[0016] Secondly, the present invention also provides a method for treating bovine nodular dermatitis in a subject, comprising administering to the subject an effective amount of a macrolide derivative T8, or a tautomer, cis or trans isomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, or the pharmaceutical composition thereof.

[0017] Thirdly, the present invention also provides a veterinary drug composition for treating bovine nodular dermatitis, characterized in that it comprises the macrolide derivative T8.

[0018] Fourthly, the present invention also provides a pharmaceutical preparation for treating bovine nodular dermatitis, comprising the macrolide derivative T8.

[0019] The dosage form of the pharmaceutical preparation may be tablets, capsules, oral liquids, lozenges, granules, powders, pills, powders, ointments, elixirs, suspensions, powders, or solutions.

[0020] Compared with the prior art, the beneficial effects achieved by the present invention are: This invention marks the first discovery that the macrolide derivative T8 possesses broad-spectrum and highly effective antiviral activity, inhibiting the replication of LSDV (Likely a type of goatpox virus). This provides a new and safe technical approach for developing safe and reliable antiviral drugs against poxvirus infections. Experimental results show that when compound T8 is used at concentrations of 12.5-6400 ppm, it can inhibit the replication of bovine nodular dermatitis virus (LSDV), and the inhibitory effect on LSDV is dose-dependent. Furthermore, T8 exhibits low cytotoxicity and low C2C. 50With a value >6400ppm, compound T8 can be used as a highly effective macrolide inhibitor against LSDV, and has broad application prospects in the fields of aquaculture and biomedicine. Attached Figure Description

[0021] Figure 1 The figure shows the titer results of T8 inhibiting LSDV virus in a dose-dependent manner.

[0022] Figure 2 The graph shows the toxicity of different concentrations of T8 on MDBK cells. Detailed Implementation

[0023] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0025] Unless otherwise specified, all reagents, materials, instruments, etc. used in the following examples are commercially available.

[0026] The experimental methods described below are standard operating procedures in molecular biology, cell biology, or virology, which researchers in the field can easily understand and perform.

[0027] Routine tests in the examples: 1. Cell preparation When passaged MDBK cells, DMEM culture medium containing 10% fetal bovine serum and 1% penicillin and streptomycin was used, and DMEM culture medium containing 2.5% fetal bovine serum and 1% penicillin and streptomycin was used as the maintenance medium.

[0028] 2. Virus culture LSDV virus culture: Culture approximately 10 flasks of T75 MDBK cells, inoculate with viral stock at 0.01 MOI, scrape off cells 72 hours after infection, transfer to 50 mL centrifuge tubes, centrifuge at 2500 rpm for 10 min, and discard the supernatant. Resuspend in 1.5 mL of 10 mM Tris-Cl per T75 flask and place on ice. Lyse the cell suspension by homogenizing 40 times in a glass homogenizer with a tight pestle, transfer to 50 mL tubes, and centrifuge at 300 g, 4 °C for 5 min, saving the supernatant; sonicate the combined supernatant (lysate) on ice for 90 seconds; separate the sonicated lysate into layers on a 36% sucrose pad in a sterile centrifuge tube, with a maximum tube volume of 38.5 mL, and fill the tube to the top 2-3 mm. Make up the volume with 10 mM Tris-Cl (pH 9.0); centrifuge at 33000g for 80 minutes, aspirate and discard the supernatant, resuspend the virus particles in 1 mL of 1 mM Tris-Cl (pH 9.0), and operate on ice. Store at -80°C.

[0029] 3. Evaluation of the antiviral activity of the test compounds 1) Trypsin digestion of fused monolayer MDBK cells; 2) In a 24-well plate, lay 1×10⁻⁶ molten metal in each well. 5 One MDBK cell was incubated overnight at 37°C in a 5% CO2 incubator. 3) Infect the above cells with LSDV-GFP virus at MOI=0.01, and add appropriate concentrations (6400ppm, 800ppm, 100ppm, 12.5ppm) of the test compound. Replace the maintenance medium containing the test compound after 2 hours.

[0030] 4) Collect virus-containing cells after culturing for 72 hours.

[0031] 4. Fluorescent spot test to determine virus titer 1) Trypsin digestion of fused monolayer MDBK cells; 2) In a 24-well plate, lay 1×10⁻⁶ molten metal in each well. 5 One MDBK cell was incubated overnight at 37°C in a 5% CO2 incubator. 3) The collected cells were repeatedly frozen and thawed three times in advance to release the virus. Eight 10-fold serial dilutions of the virus were prepared in DMEM medium (2.5% fetal bovine serum, 1% penicillin and streptomycin). 4) Remove the culture medium from the 24-well plate and use 200 μL of 10 -2 10 -3 10 -4 10 -5 10 -6Cells in the wells were infected with the virus dilution (three replicates per dilution), and the inoculum was discarded after 2 hours in a CO2 incubator.

[0032] 5) After 2 hours, change the medium to 0.75% w / v carboxymethyl cellulose. Place the 24-well plate in a 37°C, 5% CO2 incubator and incubate for 72 hours to allow the virus to fully infect the cells.

[0033] 6) After 72 hours of incubation, observe the fluorescent patches in the wells under a 4x fluorescence microscope. Select an appropriate dilution for the number of fluorescent patches (between 20 and 100 patches is recommended) to ensure accurate counting.

[0034] 7) PFU / mL = number of fluorescent patches × virus dilution factor ÷ inoculation volume (mL).

[0035] 5. Detection of cytotoxicity (CCK8 assay) 1) Collect cells in the logarithmic growth phase, adjust the cell suspension concentration, and divide them into 96-well plates, 180 μL per well, 3000-10000 cells / well.

[0036] 2) Incubate at 37℃ in a 5% CO2 incubator to allow the cells to adhere to the wall and incubate for 24 hours.

[0037] 3) Add appropriate concentrations (6400ppm, 1600ppm, 400ppm, 100ppm, 25ppm, 6.4ppm, 1.6ppm, 0.4ppm) of the test compound and continue culturing for 72h.

[0038] 4) Carefully aspirate the supernatant, add 90 μL of fresh culture medium, then add 10 μL of CCK8 solution, and continue culturing for 2-4 hours.

[0039] 5) Measure the absorbance of each well at 450 nm using an ELISA reader.

[0040] 6) Simultaneously set up zeroing wells (culture medium, CCK8 solution) and control wells (cells, drug dissolution medium of the same concentration, culture medium, CCK8 solution), with 8 replicates for each group.

[0041] 7) Use Graphpad Prism software to plot the dose-response curve and calculate the median toxicity concentration (CC). 50 value.

[0042] Example 1: Effect of macrolide derivative T3 on LSDV-GFP viral replication 1) MDBK cells were infected with LSDV-GFP strain at 0.01 MOI. Different doses of macrolide derivative compound T8 (6400ppm, 800ppm, 100ppm, 12.5ppm) (prepared with DMSO, the same below) were added at the same time during viral infection. A DMSO control was also set up. After incubation for 2 hours, the supernatant was discarded, the cells were washed twice with PBS, and cell maintenance medium was added. The cells were cultured in an incubator for 72 hours. The samples were collected and titrated on MDBK cells. The titer was determined by fluorescent plaque counting method.

[0043] like Figure 1 As shown, the results indicate that the viral titer was significantly reduced after the addition of macrolide derivative compound T8 compared with the DMSO control group, indicating that compound T8 at concentrations of 12.5 ppm to 6400 ppm can inhibit LSDV-GFP replication in a dose-dependent manner.

[0044] Example 2: Detection of the cytotoxicity of macrolide derivative T8 on MDBK cells Collect cells in the logarithmic growth phase, adjust the cell suspension concentration, and aliquot 180 μL into 96-well plates (3000-10000 cells / well). Incubate at 37°C in a 5% CO2 incubator until cell adhesion is achieved, and culture for 24 h. Add appropriate concentrations of the test compound T8 (6400 ppm, 1600 ppm, 400 ppm, 100 ppm, 25 ppm, 1.6 ppm, 0.4 ppm) and continue culturing for 72 h. Carefully aspirate the supernatant, add 90 μL of fresh culture medium, and then add 10 μL of CCK8 solution, and continue culturing for 4 h. Measure the absorbance of each well at 450 nm using a microplate reader. Simultaneously set up zero wells (culture medium, CCK8 solution) and control wells (cells, drug solution of the same concentration, culture medium, CCK8 solution), with 8 replicates per group. Plot the dose-response curve using Graphpad Prism software and calculate the half-maximal toxicity concentration (MCC) of CCK8. 50 value.

[0045] The results are as follows Figure 2 As shown, even at the highest tested concentration of 6400 ppm, cell viability was greater than 80%, comparable to the DMSO control group. No significant decrease in cell viability was observed across concentrations ranging from 0.4 ppm to 6400 ppm, indicating that the compound did not exhibit significant toxicity within this concentration range. Therefore, it can be inferred that CC... 50 Value > 6400ppm.

[0046] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Application of macrolide derivative T8 or its pharmaceutical salt in the preparation of antiviral drugs for poxviridae; The structural formula of the macrocyclic lactone derivative T8 is as follows: 。 2. The application according to claim 1, characterized in that, The poxviruses mentioned include the genera orthopoxvirus and goatpoxvirus.

3. The application according to claim 2, characterized in that, The orthopoxvirus genus includes vaccinia virus.

4. The application according to claim 2, characterized in that, The goatpoxvirus genus includes bovine nodular dermatovirus.

5. The application according to any one of claims 1-4, characterized in that, The macrolide derivative T8 is used at a concentration of 12.5-6400 ppm.

6. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable carriers, excipients, or additives; the carriers or excipients are selected from one or more of diluents, binders, adsorbents, fillers, and disintegrants; the additives are selected from one or more of stabilizers, bactericides, buffers, isotonic agents, chelating agents, pH control agents, and surfactants.

7. The application according to claim 1, characterized in that, The dosage forms of the drug are tablets, capsules, oral liquids, lozenges, granules, powders, pills, powders, ointments, elixirs, suspensions, powders, and solutions.

8. A method for treating bovine nodular dermatitis in subjects, characterized in that, This includes administering to a subject an effective amount of a macrolide derivative T8, or a tautomer, cis or trans isomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, or the pharmaceutical composition thereof.

9. A veterinary drug composition for treating nodular skin disease in cattle, characterized in that, This includes the macrocyclic lactone derivative T8.

10. A pharmaceutical preparation for treating bovine nodular dermatitis, characterized in that, This includes the macrocyclic lactone derivative T8.

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