USE OF ADEFOVIR DIPIVOXIL AND STRUCTURAL ANALOG THEREOF FOR TREATING PSEUDORABIES VIRUS

Abstract

The present invention relates to the medical field, and in particular to the use of adefovir dipivoxil and a structural analog thereof for treating pseudorabies virus. In particular, the invention relates to the use of a compound as shown in formula (I), a stereoisomer thereof, a solvate thereof, a pharmaceutically acceptable salt thereof or a solvate of the pharmaceutically acceptable salt thereof for inhibiting the pseudorabies virus, treating pseudorabies virus infections or treating diseases related to pseudorabies virus infections.

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Claims

1. A method for treating pseudorabies virus infection or a disease related to pseudorabies virus infection, the method comprising: administering to a subject in need thereof a therapeutically effective dose of a compound of formula (I), a stereoisomer, solvate, or pharmaceutically acceptable salt of the compound, or a solvate of the pharmaceutically acceptable salt of the compound; ##STR00005## wherein R.sub.1 and R.sub.2 are each independently selected from H, C.sub.1-C.sub.4 alkyl, halo C.sub.1-C.sub.4 alkyl, —CH.sub.2OCO—(C.sub.1-C.sub.4 alkyl), and —CH.sub.2OCO-(halo C.sub.1-C.sub.4 alkyl), and wherein R.sub.1 and R.sub.2 are the same or different.

2. The method according to claim 1, wherein the pharmaceutically acceptable salt is selected from hydrochloride, aspartate, taurate, gluconate, fructonate, salicylate, and maleate.

3. The method according to claim 1, wherein the solvate is a hydrate.

4. A method for treating pseudorabies virus infection or a disease related to pseudorabies virus infection, the method comprising: administering to a subject in need thereof a pharmaceutical composition, wherein the pharmaceutical composition comprises the compound, the stereoisomer, solvate, or pharmaceutically acceptable salt of the compound, or the solvate of the pharmaceutically acceptable salt of the compound according to claim 1, and one or more pharmaceutically acceptable carriers or excipients.

5. The method according to claim 4, wherein the pharmaceutical composition further comprises a second therapeutic drug.

6. A method for treating pseudorabies virus infection or a disease related to pseudorabies virus infection, the method comprising: administering to a subject in need thereof the compound, the stereoisomer, solvate or pharmaceutically acceptable salt of the compound, or the solvate of the pharmaceutically acceptable salt of the compound according to claim 1 in combination with a second therapeutic drug.

7. A method for inhibiting pseudorabies virus, comprising: applying to the pseudorabies virus the compound, the stereoisomer, solvate, or pharmaceutically acceptable salt of the compound, or the solvate of the pharmaceutically acceptable salt of the compound according to claim 1.

8. (canceled)

9. The method according to claim 1, wherein the compound is adefovir or adefovir dipivoxil.

10. The method according to claim 1, wherein: the solvate of the compound is an adefovir dipivoxil hydrate; and/or, the solvate of the pharmaceutically acceptable salt of the compound is a hydrate of adefovir dipivoxil hydrochloride.

11. The method according to claim 1, wherein the subject is selected from porcine, bovine, canine, mouse, rabbit, sheep, and human.

12. The method according to claim 1, wherein the disease related to pseudorabies virus infection is at least one selected from the group consisting of pseudorabies disease, encephalitis, retinitis, and endophthalmitis.

13. The method according to claim 4, wherein the pharmaceutical composition is made into at least one dosage form selected from the group consisting of a tablet, a capsule, a pill, an oral liquid preparation, a granule, a powder, and an injection.

14. The method according to claim 4, wherein the pharmaceutical composition is administered orally, injected, implanted, externally applied, sprayed, or inhaled.

15. The method according to claim 5, wherein the second therapeutic drug is at least one selected from the group consisting of an interferon drug, a broad-spectrum antiviral drug, other therapeutic drug for herpes virus, and an anti-inflammatory drug.

16. The method according to claim 15, wherein: the interferon drug is selected from α interferon, β interferon, and γ interferon; the broad-spectrum antiviral drug is ribavirin; the other therapeutic drug for herpes virus is selected from acyclovir, ganciclovir, and alacyclovir; and/or the anti-inflammatory drug is selected from ibuprofen, aspirin, and indomethacin.

17. The method according to claim 6, wherein the second therapeutic drug is at least one selected from the group consisting of an interferon drug, a broad-spectrum antiviral drug, other therapeutic drug for herpes virus, and an anti-inflammatory drug.

18. The method according to claim 17, wherein: the interferon drug is selected from α interferon, β interferon, and γ interferon; the broad-spectrum antiviral drug is ribavirin; the other therapeutic drug for herpes virus is selected from acyclovir, ganciclovir, and valacyclovir; and/or the anti-inflammatory drug is selected from ibuprofen, aspirin, and indomethacin.

19. The method according to claim 6, wherein the compound, the stereoisomer, solvate, or pharmaceutically acceptable salt of the compound, or a solvate of the pharmaceutically acceptable salt of the compound and the second therapeutic drug are administered simultaneously to a subject in need of treatment.

20. The method according to claim 6, wherein the compound, the stereoisomer, solvate, or pharmaceutically acceptable salt of the compound, or a solvate of the pharmaceutically acceptable salt of the compound and the second therapeutic drug are administered separately to a subject in need of treatment.

21. The method according to claim 6, wherein the compound, the stereoisomer, solvate, or pharmaceutically acceptable salt of the compound, or a solvate of the pharmaceutically acceptable salt of the compound and the second therapeutic drug are in the same preparation unit.

22. The method according to claim 6, wherein the compound, the stereoisomer, solvate, or pharmaceutically acceptable salt of the compound, or a solvate of the pharmaceutically acceptable salt of the compound and the second therapeutic drug are in different preparation units.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0053] FIG. 1 shows the inhibitory effect of adefovir dipivoxil on pseudorabies virus replication in Example 1.

[0054] FIG. 2 shows the results of EC.sub.50 assay on the inhibition of adefovir dipivoxil against pseudorabies virus in Example 1.

[0055] FIG. 3 shows that adefovir dipivoxil has no inhibitory activity against EB virus in Example 1.

[0056] FIG. 4 shows the inhibitory effect of adefovir dipivoxil on the release of pseudorabies virus from cells in Example 2.

DETAILED DESCRIPTION OF EMBODIMENTS

[0057] The technical solutions in the embodiments of the invention will be described clearly and completely below in connection with the drawings in the embodiments of the invention, and it will be apparent that the embodiments described here are merely a part, not all of the embodiments of the invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the invention fall within the scope of the invention.

[0058] In the following embodiments, the used test materials and their sources are as follows:

[0059] Adefovir dipivoxil was purchased from MCE; high-glucose DMEM medium, fetal bovine serum FBS (Gibco), and 96-well plate (Nunc brand) were purchased from Thermo Company.

[0060] In the following Examples 1 to 2, adefovir dipivoxil was diluted with culture medium into a stock solution with a concentration of 10 mmol/L, and then diluted with culture medium to use concentrations of different gradients during use.

[0061] The cell line selected in the following examples is: porcine kidney cell PK-15 (culture medium is high-glucose DMEM medium+10% FBS, purchased from ATCC® CCL-33).

EXAMPLE 1

Inhibitory Effect of Adefovir Dipivoxil on Pseudorabies Virus Replication

[0062] The experimental method was immunofluorescence method. The principle of this method is that according to the principle of antigen-antibody reaction, a known antigen or antibody is first labeled with a fluorescent group, and then the fluorescent antibody (or antigen) is used as a probe to check the corresponding antigen (or antibody) in cells or tissues. Fluorescence microscopy can be used to visualize the cells or tissues where the fluorescence is located, to determine the nature and localization of antigens or antibodies, and to determine the amount using quantitative techniques such as flow cytometry.

[0063] The specific experimental steps were as follows: the cells with a density of about 90% to 95% were digested with preheated trypsin and then re-suspended in a complete medium and pipetted sufficiently to obtain a single cell suspension and then counting was carried out. A 12-well plate for cell culture was prepared, a few drops of culture medium were first added in each well on the position where the slides were to be placed according to the size of the slides, and then the slides were placed on the droplets and pressed tightly so that the slides and the culture dish could be attached together by the tension of the medium to prevent the slides from floating when the cell suspension was added, and a double-layered cell patch would not be caused. The cells at an appropriate density were seeded into the 12-well plate. 24 hours or 48 hours later, according to the speed of cell growth, the cell density was observed and determined. After the12-well plate was full of cells, different concentrations of adefovir dipivoxil and 100 pfu of pseudorabies virus were then added in sequence, and immunofluorescence detection was then carried out 24 h after infection. During cell fixation, immunofluorescence and medium removal, PBS was generally added to wash the cells 3 times, 5 min each time. 1 ml of 4% paraformaldehyde was added to the wells for cell fixation, and the cells were fixed at room temperature for 20 min. The paraformaldehyde was removed by absorption, and rinsing with PBS was carried out three times, 5 min each time. 0.5% Triton X-100 (prepared in PBS) was added to the wells for permeabilization at room temperature for 20 min in order to permeabilize cells. The Triton X-100 was then removed and rinsing with PBS was carried out three times, 5 min each time. Blocking with 10% goat serum homologous to the secondary antibody (prepared in PBS) or with 5% BSA was carried out for 2 h (the selected blocking solution was the same as the antibody dilution used in the subsequent operation). Rinsing with PBS was not required after the blocking. The blocking solution was removed by absorption and a sufficient amount of the primary antibody with the appropriate concentration was added dropwise to each well (the antibody was used at a recommended concentration according to the operating instruction of the antibody at the first time, and the appropriate concentration of the antibody could be explored for subsequent stages of the experiment), and incubation was then carried out overnight in a humidified box at 4° C. The primary antibody was removed by absorption and rinsing with PBS was carried out three times, 5 min each time. A sufficient amount of the secondary antibody with an appropriate concentration was added dropwise at 37° C. to the well, and incubation was then carried out for 1 h at room temperature in the dark. To be noted that the operation should be carried out in a dark place because the secondary antibody is labeled with fluorescein. The secondary antibody was removed by absorption and rinsing with PBS was carried out three times, 5 min each time. DAPI was added dropwise to the glass slide, or Hoechst was used to counterstain the nuclei, usually with blue fluorescence; incubation was then carried out in the dark for 5 to 10 min. Cells were gently rinsed three times with PBS, 5 min each time, to remove excess DAPI. To take the cell slide, because the cell slide was tightly connected with the bottom of the petri dish and the tension was large, a needle tip of a syringe can be bent to the back to make a small hook, so that the cell slide can be gently hooked up and taken out with small tweezers. The liquid on the cell slide was dried with absorbent paper, the cell slide was then blocked with a blocking solution containing an anti-fluorescence quencher (to be noted that the cell slide should be reversely attached to a poly-lysine slide). Then, observation and image acquisition were carried out under a fluorescence microscope. To be noted that an excitation light source corresponding to the antibody is selected.

[0064] The inhibitory effect of adefovir dipivoxil on pseudorabies virus replication is shown in FIG. 1. The concentrations of adefovir dipivoxil used were 5 μmol/L, 2.5 μmol/L, 1.25 μmol/L, 0.625 μmol/L, 0.313 μmol/L, 0.156 μmol/L, and 0.078 μmol/L. As can be seen from FIG. 1, the adefovir dipivoxil of a high concentration can significantly inhibit the replication and proliferation of pseudorabies virus, confirming that adefovir dipivoxil has a significant proliferation inhibitory effect on pseudorabies virus.

[0065] The results of EC.sub.50 assay on the inhibition of adefovir dipivoxil against pseudorabies virus are shown in FIG. 2. The concentrations of adefovir dipivoxil used were 5 μmol/L, 2.5 μmol/L, 1.25 μmol/L, 0.625 μmol/L, 0.313 μmol/L, 0.156 μmol/L, and 0.078 μmol/L. As can be seen from FIG. 2, the EC.sub.50 of adefovir dipivoxil for the inhibition against pseudorabies virus proliferation reaches nanomolar level, confirming that adefovir dipivoxil has a good inhibitory effect on pseudorabies virus.

[0066] As a comparison, the inhibitory activity of adefovir dipivoxil against EB virus replication was tested, and the results are shown in FIG. 3. The concentrations of adefovir dipivoxil used were 5 μmol/L, 2.5 μmol/L, 1.25 μmol/L, 0.625 μmol/L, 0.313 μmol/L, 0.156 μmol/L, and 0.078 μmol/L. As can be seen from FIG. 3, adefovir dipivoxil has no inhibitory activity against EB virus which is also a herpes virus.

EXAMPLE 2

Inhibitory Effect of Adefovir Dipivoxil on the Release of Pseudorabies Virus from Cells

[0067] The method used was virus plaque titration. According to the principle of this method, virus solution of each dilution is inoculated into a monolayer cell culture environment, after 2 hours of adsorption, the monolayer cells are covered with agarose, and the virus infects the cells and proliferates in the cells, causing the cells to rupture and die. Due to the limitations of the solid medium, the released virus can only spread around from the initially infected cells. After several proliferation cycles, a localized diseased cell area, i.e., the viral plaque, is formed. After staining with a dye, the living cells are red, while the cells in the plaque area are not stained, forming an unstained area.

[0068] The corresponding virus host cells were prepared and then digested evenly, and next the cell concentration was adjusted. The host cells were inoculated in a six-well plate at an appropriate concentration. After the cells grew into a monolayer, the culture medium was removed by absorption. The virus solution was prepared and diluted at a ten-fold gradient to obtain five solutions of virus with different concentrations. An appropriate amount of virus solution was added dropwise to each well and then absorbed for 2 h at 37° C., and extra virus solution was removed. 2% agarose solution with a low melting point was prepared and put in a water bath with a temperature of 40° C. to 50° C. for later use. The above agarose and 2× cell maintenance solution were mixed at a ratio of 1:1. The mixture was then added to the wells in a dose of 2 mL/well, and then cooled and solidified into a covering layer. The culture plate was placed upside down in a 37° C. carbon dioxide incubator. 48 hours later, the cells were fixed with 10% formaldehyde for 30 min. The covering gel was then removed, and staining with 1% crystal violet was carried out for 15 min. After the dye solution was removed, the viral plaques were counted.

[0069] Different concentrations of adefovir dipivoxil and 100 pfu of pseudorabies virus were added to PK15 cells. After 24 hours of cultivation, the culture supernatant of PK15 cells was collected for virus plaque titration test. The results are shown in FIG. 4. The drug concentrations used were 5 μmol/L, 2.5 μmol/L, 1.25 μmol/L, 0.625 μmol/L, 0.313 μmol/L, 0.156 μmol/L, and 0.078 μmol/L. As can be seen from FIG. 4, adefovir dipivoxil can significantly inhibit the release of pseudorabies virus from cells.

[0070] The embodiments mentioned above are merely preferred embodiments of the invention and not intended to limit the invention. Any of modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the invention shall be covered in the protection scope of the invention.