CN115252625B - Application of a kind of Cyclopterygium D in the preparation of preparations for the treatment of African swine fever - Google Patents

Abstract

The invention discloses the application of Cyclopycin D in the preparation of preparations for treating or slowing down African swine fever or inhibiting the proliferation of African swine fever virus. Cyclovir Buxine D can effectively inhibit the adsorption of African swine fever virus to porcine alveolar macrophages, and can also effectively inhibit the internalization of African swine fever virus in porcine alveolar macrophages, thereby effectively inhibiting the replication of African swine fever virus and providing a therapeutic basis. Or alleviating African swine fever provides a material basis for new active drugs and pharmaceutical compositions. The invention also discloses a pharmaceutical composition containing Cyclopycin D, which can better inhibit the proliferation of African swine fever virus.

Description

Application of a kind of Cyclopterygium D in the preparation of preparations for the treatment of African swine fever

Technical field

The invention belongs to the field of pharmaceuticals and relates to the application of a kind of cyclobuxine D in the preparation of preparations for treating African swine fever.

Background technique

African Swine Fever Virus (ASFV) is the only member of the African swine fever virus family (Asfarviridae) and the only known DNA arbovirus. The mortality rate of virulent infection is as high as 100%, seriously threatening the world's pig industry. healthy development. The main vectors of ASFV are domestic pigs, wild boars and soft ticks. The virus is divided into 24 genotypes and 8 serotypes. Genotypes Ⅰ and Ⅱ are the current popular strains. With the epidemic of ASF in my country, new mutant strains continue to emerge, making the ASF prevention and control situation more severe.

So far, no effective vaccine or antiviral drug has been developed against ASF. The main reason is that the virus has a large genome and complex structure. In addition, the viral immune evasion mechanism and host protective immunity mechanism are still unclear. Based on the replication mechanism of ASFV, potential antiviral drugs can be divided into two categories: (1) inhibitors that directly act on ASFV by targeting viral proteins (direct-acting antiviral drugs); (2) targeting cells where ASFV replicates Inhibitors of factors (host-targeted antiviral drugs). Although active drugs against various types of ASFV have been reported, the in vivo efficacy of these compounds has not been evaluated. Currently, there is still a lack of effective clinical drugs to treat and prevent ASFV. Therefore, it is necessary to carry out anti-ASFV drug research, which can provide a material basis for ASF prevention and control.

The compounds Berbamine (dihydrochloride), CAS: 6078-17-7) and Berbamine (CAS: 478-61-5) are bisbenzyl compounds isolated from the Chinese herbal medicine Huanglu. Isoquinoline natural products. They are novel inhibitors of bcr/abl and inhibitors of NF-κB. They have anti-leukemia activity, can inhibit the growth of cancer cells, and induce apoptosis in human myeloma cells.

The compound Gamithromycin (CAS: 145435-72-9) is a new macrolide antibiotic mainly used to treat respiratory diseases in cattle.

The compound Cyclovirobuxin D (CAS:860-79-7) is an active compound extracted from Buxus microphylla and is used to treat acute myocardial ischemia.

There are no reports that these four compounds can treat or prevent ASFV infection.

Contents of the invention

The present invention found that four compounds, Berbamine (Dihydrochloride), Berbamine, Gamithromycin and Cyclovirobuxin D, can not only significantly inhibit the replication of ASFV-eGFP model in vitro, but also significantly inhibit the replication of wild-type ASFV. Studies by adding compounds at different times have shown that they can affect ASFV replication. Further research found that the mechanisms by which these four compounds inhibit ASFV replication are different. Berbamine (Dihydrochloride) and Berbamine each achieve the effect of inhibiting ASFV replication by affecting the adsorption of ASFV, Cyclovirobuxin D by affecting the adsorption and internalization of ASFV, and Gamithromycin by affecting the entry of ASFV into late endosomes. The present invention provides an application basis for the development of drugs for the treatment or prevention of ASFV.

After combining their different components into a composition, they have low toxicity and good safety, and the composition should still ensure good safety. These drugs act on different stages of the normal replication cycle of ASFV. The basic inhibitory mechanism is clear. When used in combination, there will be no mutual antagonism at least within the scope of the discovered mechanisms. The ingredients will synergistically superimpose the inhibitory effect on ASFV. The composition can be more Effectively inhibit ASFV replication. In order to ensure the same ASFV inhibition rate, the amount of each drug required can be reduced compared with using it alone, thereby further reducing potential toxic and side effects. The present invention discovers that any combination of four compounds with new activities has better application prospects in treating or preventing African swine fever virus infection than a single compound.

In order to solve the problems existing in the prior art, the first aspect of the present invention provides the application of an active substance in the preparation of preparations for preventing, slowing down, treating or controlling African swine fever and/or inhibiting the proliferation of African swine fever virus, The active substance is any one of cyclobuxin D, a medicinal salt of cyclobuxin D and a prodrug of cyclobuxin D, a combination of any two or a combination of three; The structural formula of Buxus D is:

In some embodiments, the active substance is the only pharmaceutically active substance.

In some embodiments, the pharmaceutically acceptable salts of cyclobuxin D include: tosylate, methanesulfonate, malate, acetate, and citric acid of gamithromycin of cyclobuxin D Salt, malonate, tartrate, succinate, lactate, benzoate, ascorbate, alpha-ketoglutarate, alpha-glycerophosphate, hydrochloride, sulfate, nitrate, Bicarbonate, carbonate, phosphate, hydrobromide and hydroiodide.

In some embodiments, the formulation is a drug.

In some embodiments, the formulation is a feed additive.

The second aspect of the present invention provides an active substance prepared for preventing, slowing down, treating or controlling African swine fever virus by inhibiting the adsorption of African swine fever virus to susceptible cells and/or inhibiting the internalization of African swine fever virus in susceptible cells. Application in African swine fever and/or preparations for inhibiting the proliferation of African swine fever virus, the active substance is cyclobuxine D, any of the pharmaceutical salts of cyclobuxine D and the prodrugs of cyclobuxine D One type, any combination of two types or a combination of three types; the structural formula of the ring-dimensional box star D is:

In some embodiments, the active substance is the only pharmaceutically active substance.

In some embodiments, the pharmaceutically acceptable salts of cyclobuxin D include: tosylate, methanesulfonate, malate, acetate, and citric acid of gamithromycin of cyclobuxin D Salt, malonate, tartrate, succinate, lactate, benzoate, ascorbate, alpha-ketoglutarate, alpha-glycerophosphate, hydrochloride, sulfate, nitrate, Bicarbonate, carbonate, phosphate, hydrobromide and hydroiodide.

In some embodiments, the susceptible cells are porcine alveolar macrophages.

In some embodiments, the formulation is a drug.

In some embodiments, the formulation is a feed additive.

A third aspect of the present invention provides a pharmaceutical composition, the pharmaceutically active ingredients of the pharmaceutical composition include a first active substance and a second active substance;

The first active substance is berbamine, any one of a pharmaceutically acceptable salt of berbamine and a prodrug of berbamine, a combination of any two or a combination of three;

The second active substance is cyclobuxin D, any one of the pharmaceutical salts of cyclobuxin D and the prodrugs of cyclobuxin D, a combination of any two or a combination of three;

The structural formula of berbamine is:

The structural formula of the ring-dimensional box star D is:

In some embodiments, the molar ratio of the first active material to the second active material is 1:0.1-10 (for example, 1:0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, Any ratio among 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or the range between any two ratios).

In some embodiments, the molar ratio of the first active material to the second active material is 1:0.2-5.

In some embodiments, the molar ratio of the first active material to the second active material is 1:1.

In some embodiments, the pharmaceutically acceptable salts of berbamine include: tosylate, methanesulfonate, malate, acetate, citrate, malonate, tartrate of berbamine , succinate, lactate, benzoate, ascorbate, α-ketoglutarate, α-glycerophosphate, dihydrochloride, sulfate, nitrate, bicarbonate, carbonate, Phosphates, hydrobromides and hydroiodates.

In some embodiments, the pharmaceutically acceptable salts of cyclobuxin D include: tosylate, methanesulfonate, malate, acetate, and citric acid of gamithromycin of cyclobuxin D Salt, malonate, tartrate, succinate, lactate, benzoate, ascorbate, alpha-ketoglutarate, alpha-glycerophosphate, hydrochloride, sulfate, nitrate, Bicarbonate, carbonate, phosphate, hydrobromide and hydroiodide.

The fourth aspect of the present invention provides the use of the pharmaceutical composition described in the third aspect of the present invention in the preparation of preparations for preventing, slowing down, treating or controlling African swine fever and/or inhibiting the proliferation of African swine fever virus.

In some embodiments, the formulation is a drug.

In some embodiments, the formulation is a feed additive.

Cyclovir Buxine D can effectively inhibit the adsorption of African swine fever virus to porcine alveolar macrophages, and can effectively inhibit the internalization of African swine fever virus in susceptible cells, thereby effectively inhibiting the replication of African swine fever virus. It is a promising tool for the treatment or prevention of African swine fever virus in Africa. Swine fever provides new active drugs and material basis.

Among the four compounds: berbamine, berbamine hydrochloride, gamithromycin, and cyclibuxin D, the combination of any two, three or four compounds has a greater inhibitory effect on the ASFV virus than the use of one drug alone. Even better, the combination of any three drugs has a better inhibitory effect on the ASFV virus than the combination of any two of the three drugs, and the combination of four drugs has the best effect. It can be seen that any combination of the four compounds has a systemic synergistic effect, and no drug compatibility inefficiency or effect antagonism occurs. The combined use of the four drugs of the present invention can enhance the drug efficacy and reduce potential side effects. It also provides support for selecting medications for different infection conditions.

Description of the drawings

Figure 1 shows the cytotoxicity of different concentrations of DMSO to PAMs.

Figure 2 shows the cytotoxicity of Ethanol to PAMs at different concentrations.

Figure 3 shows Berbamine (dihydrochloride), CC50 and IC50 of Berbamine.

Figure 4 shows the CC50 and IC50 of Gamithromycin and Cyclovirobuxin D.

Figure 5 shows fluorescence photos of ASFV-eGFP replication by four drugs.

Figure 6 shows the qPCR results and WB results of the effects of four drugs on ASFV-eGFP replication.

Figure 7 shows the qPCR results of the effects of four drugs on wild-type ASFV replication.

Figure 8 shows the WB results of the effects of four drugs on wild-type ASFV replication.

Figure 9 shows the qPCR results and WB results of the effects of adding four drugs at different times on ASFV-eGFP replication.

Figure 10 shows the qPCR detection results of the inactivation effects of four drugs on ASFV.

Figure 11 shows the WB detection results of the inactivation effects of four drugs on ASFV.

Figure 12 shows the qPCR detection results of the adsorption effects of four drugs on ASFV.

Figure 13 shows the WB detection results of the adsorption effects of four drugs on ASFV.

Figure 14 shows the qPCR detection results of the internalization effects of four drugs on ASFV.

Figure 15 shows the WB detection results of the internalization effects of four drugs on ASFV.

Figure 16 shows confocal images of ASFV in early endosomes of cells by gamithromycin.

Figure 17 shows the calculated results of the effect of gamithromycin on ASFV transport in early endosomes of cells.

Figure 18 shows confocal images of ASFV in late endosomes of cells by gamithromycin.

Figure 19 shows the calculation results of the effect of gamithromycin on endocytic trafficking of ASFV in the late phase of cells.

Figure 20 shows the TCID ₅₀ detection results of four drugs on ASFV release.

Figure 21 shows the qPCR detection results of the drugs alone and in combination.

Figure 22 shows the WB detection results of the drugs alone and in combination.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

The materials and instruments not described in the present invention are conventional materials and instruments in the art, and the operation details not described in the present invention are conventional operations in the art.

Experimental Materials

Experimental drugs: Berbamine (dihydrochloride), Berbamine, Gamithromycin and Cyclovirobuxin D (purchased from Selleck Company).

Experimental reagents: RPMI 1640 complete medium was provided by Gibco; pig serum was provided by Hyclone; CCK-8 was provided by Japan Doren Chemical Technology Co., Ltd.; PBS and dimethyl sulfoxide were provided by solarbio.

Experimental equipment: filters, pipettes, 24-well and 96-well cell culture plates are provided by Corning Company; clean operating table, carbon dioxide incubator are provided by Thermo Company of the United States; inverted optical microscope is provided by Mshot Company; enzyme-linked immunoassay detector is provided by BioTek Company .

Cells and viruses: PAMs (alveolar macrophages); ASFV wild-type strain (Pig/HLJ/2018 strain, Genebank sequence number: MK333180.1); ASFV marker strain (called ASFV-eGFP strain), in ASFV virus The expression of eGFP in the strain enables cells infected with ASFV-eGFP to emit green fluorescence, making it easier to observe the number and distribution of virus particles through fluorescence. For its preparation method, see A seven-gene-deleted African swine fever virus is safe and effective as a live attenuated ASFV-Δ6GD strain in vaccine in pigs, Sci China Life Sci, Weiye Chenet al., 2020;63(5):623-634.

Example 1: Toxicity of DMSO and Ethanol to PAMs

PAMs were added to the 96-well cell culture plate at 3×10 ⁵ cells/well, 100 μl/well, and the culture medium was RPMI1640 complete medium. Place in a 37°C 5% _CO2 incubator until the cells are completely attached. Aspirate the culture medium, and add 0.0625v/v%, 0.125v/v%, 0.25v/v%, 0.5v/v%, 1v/v%, 2v/v%, 4v/v% to the experimental wells (As) respectively. RPMI 1640 of dimethyl sulfoxide (DMSO) and 0.3125v/v%, 0.625v/v%, 1.25v/v%, 2.5v/v%, 5v/v%, 10v/v% ethanol (Ethanol) Complete medium was added to the 96-well plate at a dosage of 100 μl/well, and each concentration was repeated into 3 wells. Add RPMI 1640 complete medium to the control well (Ac) at a dosage of 100 μl/well. There are no cells in the blank well (Ab), and an equal volume of RPMI 1640 complete medium is added at a dosage of 100 μl/well. Incubate in a 37°C 5% _CO2 incubator for 48h. After 48 hours, replace with 100 μl of fresh RPMI 1640 complete medium, and add 10 μl of CCK-8 solution (tetrazolium salt solution) to each well. Incubate the culture plate in the incubator for 1-4h. Use a microplate reader to measure the absorbance at 450 nm. Calculate the cell survival rate according to the following formula: Cell survival rate % = [(OD _As - OD _Ab )/(OD _Ac - OD _Ab )] × 100%. Among them, OD _As , OD _Ab , and OD _Ac represent the OD values of the test well, blank well, and control well respectively.

The results showed that the DMSO with a concentration of 4v/v% was significantly different from the control group (p<0.0001); the DMSO with a concentration of 2v/v% and 1v/v% was significantly different from the control group (p<0.01); the concentration was There was no significant difference between 0.5v/v% DMSO and the control group (p>0.05) (Figure 1). Therefore, 0.5% DMSO has no obvious cytotoxicity to PAM and can be used as a drug-free control or a solvent for drug activity testing.

The results showed that there was a very significant difference between Ethanol at a concentration of 10v/v% and the control group (p<0.0001); there was no significant difference between Ethanol at a concentration of 5v/v% and the control group (p>0.05) (Figure 2). Therefore, 5v/v% Ethanol has no obvious cytotoxicity to PAM and can be used as a drug-free control or a solvent for drug activity testing.

Example 2: Determination of IC ₅₀ and CC ₅₀ of small molecule compounds Berbamine (dihydrochloride), Berbamine, Gamithromycin and Cyclovirobuxin D

PAMs were added to the 96-well cell culture plate at 3 × 10 ⁵ cells/well, 100 μl/well, and the culture medium was RPMI 1640 complete medium. Place in a 37°C 5% _CO2 incubator and wait until the cells are completely attached. Aspirate the culture medium, and add RPMI 1640 complete culture medium containing four compounds at different concentrations to the experimental wells (As). The specific concentrations are shown in the abscissas of each subfigure in Figure 3 and Figure 4. The value is an index of 10, in μM, in 100 μl. The amount/well was added to the 96-well plate, and each concentration was repeated into 3 wells. Add RPMI 1640 complete medium to the control well (Ac) at a dosage of 100 μl/well. There were no cells in the blank well (Ab), and only an equal volume of RPMI 1640 complete medium was added. Incubate in a 37°C 5% _CO2 incubator for 48h. After 48 hours, replace with 100 μl of fresh RPMI 1640 complete medium, and add 10 μl of CCK-8 solution to each well. Incubate the culture plate in the incubator for 1-4h. Use a microplate reader to measure the absorbance at 450 nm. Calculate the cell survival rate according to the following formula: Cell survival rate % = [(OD _As - OD _Ab )/(OD _Ac - OD _Ab )] × 100%. Among them, OD _As , OD _Ab , and OD _Ac represent the OD values of the test well, blank well, and control well respectively.

The results showed that compared with the control well, the CC ₅₀ of Berbamine (dihydrochloride), Berbamine, Gamithromycin and Cyclovirobuxin D were 61.85μM, 53.80μM, 132.2μM and 61.20μM respectively; the IC of Berbamine (dihydrochloride), Berbamine, Gamithromycin and Cyclovirobuxin D ₅₀ are 0.71μM, 0.87μM, 2.25μM and 0.32μM respectively. The calculated selection index (SI) of Berbamine (dihydrochloride), Berbamine, Gamithromycin and Cyclovirobuxin D were 87.11, 61.83, 58.76 and 191.3 respectively (Figure 3, Figure 4). It can be seen that these four compounds have low toxicity and good safety.

Example 3: Effects of Berbamine (dihydrochloride), Berbamine, Gamithromycin and Cyclovirobuxin D on ASFV-eGFP replication.

PAMs were cultured in a 24-well plate (1.25 × 10 ⁶ cells/well), 100 μl/well, and the culture medium was RPMI 1640 complete medium. After the cells were completely adhered, different concentrations of Berbamine (dihydrochloride), Berbamine, Gamithromycin and Cyclovirobuxin were added. D (10 μM, 5 μM and 2.5 μM, 0 μM) was incubated in RPMI 1640 complete medium for 2 h, discarded, and then added corresponding concentrations of drugs (10 μM, 5 μM and 2.5 μM, 0 μM) in RPMI 1640 complete medium and inoculated with ASFV -eGFP (MOI=0.1) was infected for 2 hours, cells were washed three times with PBS, and RPMI 1640 complete medium containing compounds at different concentrations (10 μM, 5 μM and 2.5 μM, 0 μM) was added. The treatment of blank control (Mock) was as follows: no virus or drugs were inoculated, only RPMI 1640 complete medium was added. Incubate at 37°C for 48 hours, and use a fluorescence microscope to take white light and fluorescence pictures of each well. The results are shown in Figure 5. The fluorescence picture of each well is at the top and the white light picture is at the bottom. Collect the cell supernatant and cells and perform qPCR and WB respectively.

A fluorescence microscope can take pictures under white light to observe cell viability, and fluorescence can determine ASFV infection. The white light picture shows that the cell viability is normal; the fluorescence picture shows that compared with the positive control group, as the drug concentration increases, the number of fluorescence gradually decreases.

According to the method recommended by OIE for quantitative qPCR detection of ASFV p72 gene copy number (see the document Development of a TaqMan PCR assay with internal amplification control for the detection of African swine fever virus, Donald P King et al., J Virol Methods. 2003; 107(1 ):53-61.), extract DNA from the cell supernatant for qPCR. The results are shown in the bar graph on the left side of Figure 6. The qPCR test results show that compared with the positive control group (0 μM drug group), as the drug concentration increases, the copy number of the progeny virus of ASFV p72 in the cell supernatant gradually decreases.

After lysing the cells, ASFV p54 antibody (monoclonal antibody against ASFV p54 protein, prepared by the applicant for the applicant's experiment) was used to detect the expression of ASFV p54 protein using conventional WB methods, and β-actin of PAMs was used as an internal reference in the cell. (The antibody was purchased from Proteintech Company). The results are shown in the protein electrophoresis chart on the right side of Figure 6. The WB test results showed that compared with the positive control group, as the drug concentration increased, the p54 protein band gradually became lighter.

It can be seen that these four drugs all have obvious inhibitory effects on the replication of ASFV-eGFP in a dose-dependent manner. Therefore, these four drugs have the effect of treating or slowing down the treatment of African swine fever virus infection.

Example 4: Effects of Berbamine (dihydrochloride), Berbamine, Gamithromycin and Cyclovirobuxin D on the replication of wild-type ASFV.

PAMs were cultured in 24-well plates (1.25×10 ⁶ cells/well), and the culture medium was RPMI 1640 complete medium. After the cells were completely attached, RPMI 1640 complete medium containing Berbamine (dihydrochloride), Berbamine, Gamithromycin and Cyclovirobuxin D was added. The culture medium, drug type and dosage are: 10μM Berbamine (dihydrochloride) + 0.1v/v% DMSO; 10μM Berbamine + 0.1v/v% DMSO; 10μM Gamithromycin + 0.1v/v% DMSO; 10μM Cyclovirobuxin D + 0.1v /v% Ethanol; 0.1v/v% DMSO and 0.1v/v% Ethanol, incubate for 2h, discard, then add corresponding concentration of drugs and inoculate wild-type ASFV (Pig/HLJ/2018, MOI=0.05) for 2h infection , wash the cells three times with PBS, and add RPMI1640 complete medium containing corresponding concentrations of drugs. The types and dosages of drugs added are: 10μM Berbamine (dihydrochloride) + 0.1v/v% DMSO; 10μM Berbamine + 0.1v/v% DMSO; 10μM Gamithromycin + 0.1v/v% DMSO; 10μM Cyclovirobuxin D + 0.1v/v% Ethanol; 0.1 v/v% DMSO and 0.1 v/v% Ethanol. The treatment of the blank control group (Mock) is: only add RPMI1640 complete medium. Incubate at 37°C for 48 hours, collect the cell supernatant and cells, and use the same reagents and methods as in Example 3 to conduct qPCR (results see Figure 7) and WB (results see Figure 8) detection on the samples.

To evaluate the effects of these 4 drugs on wild-type ASFV replication, qPCR and WB analysis were performed. The qPCR results showed that after adding these four drugs, the copy number of ASFV p72 in the supernatant was significantly lower than that of the positive control group (0.1v/v% DMSO and 0.1v/v% Ethanol), indicating that they can inhibit wild-type ASFV of copy. WB results showed that after adding these four drugs, the expression of ASFV p54 protein was significantly lower than that of the positive lighting group. Therefore, the four drugs can also significantly inhibit the replication of wild-type ASFV (Figure 7, Figure 8). It can be seen that these four drugs have the effect of treating or slowing down the treatment of African swine fever virus infection.

Example 5: Effects of Berbamine (dihydrochloride), Berbamine, Gamithromycin and Cyclovirobuxin D on the replication phase of ASFV

In order to study the effects of Berbamine (dihydrochloride), Berbamine, Gamithromycin and Cyclovirobuxin D on different stages of ASFV replication, PAMs were cultured on 24-well plates (1.25x10 ⁶ /well), and the culture medium was RPMI 1640 complete medium. After the cells were attached After the wall, ASFV-eGFP (MOI=0.5) was inoculated, and 4 drugs were added at -2, 0, 2, 4, 8 and 16 hours of the inoculation time. The type and dosage of drugs were: 10 μM Berbamine (dihydrochloride) + 0.1 v/v% DMSO; 10 μM Berbamine + 0.1 v/v% DMSO; 10 μM Gamithromycin + 0.1 v/v% DMSO; 10 μM Cyclovirobuxin D + 0.1 v/v% Ethanol; 0.1 v/v% DMSO and 0.1 v/v% Ethanol. Berbamine (dihydrochloride), Berbamine, Gamithromycin were mixed with 0.1v/v% DMSO positive control and drug-free blank control (Mock); Cyclovirobuxin D was mixed with 0.1v/v% Ethanol positive control and drug-free blank control (Mock). In order to strictly follow the ASFV replication cycle, samples were collected 24 hours after infection, and qPCR and WB assays were performed on each sample using the same reagents and methods as in Example 3.

In order to analyze the effects of these four drugs on different stages of ASFV replication, the ASFV p72 gene copy numbers of the four drugs were measured at -2, 0, 2, 4, 6, 8 and 16 h time points before and after ASFV infection of PAMs. The addition of these drugs significantly inhibited ASFV replication compared with the positive control, especially in the early stages of ASFV replication. The qPCR results showed that after adding Berbamine (dihydrochloride), the CT values of Berbamine and Cyclovirobuxin D were significantly higher than those of the positive control group from -2h to 4h, indicating that the three drugs had the most obvious inhibitory effect on the early stage of viral replication. The results of WB also showed that the above three drugs had better inhibitory effects in the early stages of replication. When adding Gamithromycin, both qPCR and WB results showed that Gamithromycin could significantly inhibit virus replication when added from -2h to 8h, indicating that it has the effect of inhibiting ASFV replication in both the early and middle stages of virus replication. Therefore, Berbamine (dihydrochloride), Berbamine and Cyclovirobuxin D can inhibit early ASFV replication, while Gamithromycin can inhibit early and mid-stage ASFV replication (Figure 9).

Example 6: Direct inactivation of ASFV by Berbamine (dihydrochloride), Berbamine, Gamithromycin and Cyclovirobuxin D.

To evaluate whether these four drugs can directly kill ASFV, PAMs were spread in a 24-well plate (1.25x10 ⁶ /well). The culture medium was RPMI 1640 complete medium, 500μl/well, and waited for the cells to completely adhere to the wall. ASFV-eGFP (MOI=1) and the corresponding drugs (2μM Berbamine (dihydrochloride) + 0.02v/v% DMSO; 2μM Berbamine + 0.02v/v% DMSO; 2μM Gamithromycin + 0.02v/v% DMSO; 2μM Cyclovirobuxin D +0.02v/v% Ethanol; 0.1v/v% DMSO and 0.02v/v% Ethanol), mix and incubate at 37°C for 1 hour. The treatment of blank control (Mock) is: only add RPMI 1640 complete medium. The mixture was then diluted 20-fold to eliminate the potential effects of these drugs on ASFV-eGFP infection. Then, the above virus-containing mixture was added to the adherent cells and cultured at 37°C for 2 hours. After discarding the supernatant, the cells were washed three times with PBS, and RPMI 1640 complete medium containing 20 v/v% porcine serum was added. Collect the cell supernatant and cells after 48 hours, use the same reagents and methods as in Example 3 for each sample, and use qPCR and WB to detect the copy number of the ASFV p72 gene in the supernatant (see Figure 10 for the results) and the expression level of p54 protein ( The results are shown in Figure 11). The results showed that there was no significant difference compared with the control group, and none of the four drugs could directly kill the virus.

Example 7: Inhibitory effects of Berbamine (dihydrochloride), Berbamine, Gamithromycin and Cyclovirobuxin D on ASFV-adsorbed cells.

Combine PAMs (1.25x10 ⁶ /well) in a 24-well cell culture plate (the culture medium is RPMI 1640 complete medium) with ASFV-eGFP (MOI=0.1) and 10 μM Berbamine (dihydrochloride) + 0.1v/v% DMSO respectively; 10 μM Berbamine + 0.1 v/v% DMSO; 10 μM Gamithromycin + 0.1 v/v% DMSO; 10 μM Cyclovirobuxin D + 0.1 v/v% Ethanol. Incubate at 4°C for 1 hour to allow virus to adsorb to cells but prevent virus internalization. Use Buffer (0.1v/v% DMSO and 0.1v/v% Ethanol) as the positive control, and Mock as the blank well control (no virus or drugs added). Discard the supernatant and wash with 4°C pre-cooled PBS. Cells were cultured 3 times to remove unbound virus, and RPMI 1640 complete medium was added, 500 μl/well. The cell culture plate was then transferred to 37°C and incubated for 48 h. Collect cell supernatant and cells. The same reagents and methods as in Example 3 were used to perform qPCR (see Figure 12 for results) and WB (see Figure 13 for results) on each sample. The copy number of ASFV p72 in the supernatant was detected by qPCR, and the expression level of viral p54 protein in cells was detected by WB. The results showed that Berbamine (dihydrochloride), Berbamine and Cyclovirobuxin D had an effect on the adsorption of ASFV, while Gamithromycin had no obvious effect on the adsorption of ASFV (Figure 12, Figure 13).

Example 8: Inhibitory effects of Berbamine (dihydrochloride), Berbamine, Gamithromycin and Cyclovirobuxin D on the internalization of ASFV in cells.

PAMs were cultured with ASFV-eGFP (MOI=0.1) for 1 h at 4°C in RPMI 1640 complete medium, 500 μl/well, and then the cells were washed three times with pre-cooled PBS at 4°C. Add these four drugs into the wells of the cell culture plate, the four drugs are 10 μM Berbamine (dihydrochloride) + 0.1 v/v% DMSO; 10 μM Berbamine + 0.1 v/v% DMSO; 10 μM Gamithromycin + 0.1 v/v% DMSO; 10 μM Cyclovirobuxin D+0.1v/v%Ethanol. Use Buffer (0.1v/v% DMSO and 0.1v/v% Ethanol) as the positive control, Mock as the blank well control (no added viruses and drugs), 500 μl/well, and the culture medium is RPMI 1640 complete medium. The cell culture plate was then switched to 37°C. After 1 h, wash three times with PBS to remove compounds, and then add fresh RPMI 1640 complete medium. The time point of moving the cells to 37°C is 0 h, collecting the cell supernatant and cells at 48 h, and using the same reagents and methods as in Example 3 to detect the virus by qPCR (results are shown in Figure 14) and WB (results are shown in Figure 15). replication and protein expression.

Both qPCR and WB results showed that Cyclovirobuxin D affects the internalization of ASFV, while Berbamine (dihydrochloride), Berbamine and Gamithromycin do not affect the internalization of ASFV (Figure 14, Figure 15).

Example 9: Effect of Gamithromycin on ASFV in early endosomes of cells.

PAMs were cultured in confocal small dishes (1.2x10 ⁶ /dish). The culture medium was RPMI 1640 complete medium, 1mL/dish. ASFV with a dose of MOI=5 was inoculated into the PAMs and incubated at 4°C for 1 hour. Then the sample group Add 10 μM matithromycin + 0.1v/v% DMSO, add the same amount of 1640 complete medium + 0.1v/v% DMSO to the positive control group, and add Mock (without virus and drugs) as the negative control, and incubate at 37°C for 15min and 30min respectively. , 45min and 60min after sampling. When sampling, rinse once with cold PBS, add 4% paraformaldehyde to fix the cells at room temperature for 30 minutes, rinse 3 times with PBS, add 1 mL of 0.25% TritonX100 and let stand for 15 minutes to permeate the membrane, then wash 3 times with PBS, and add 0.5% BSA The solution was blocked at room temperature for 1 h. The primary antibodies were rabbit-derived anti-ASFV P72 protein polyclonal antibody serum (prepared by the applicant's own laboratory) and mouse-derived Rab5 monoclonal antibody (purchased from proteintech company), using 0.5% BSA solution at 1:500 and 1 respectively. :200 multiple dilution and incubate at 4°C overnight. Wash 3 times with PBS, add TRITC-goat anti-Rabbit IgG (H+L) (purchased from Boaolong Company) and FITC-goat anti-Mouse IgG (H+L) (purchased from Rdbio Company) as secondary antibodies. Incubate at room temperature for 1 hour in the dark, and rinse 3 times with PBS in the dark. Finally, add Hochest nuclear dye (purchased from Thermo Company) at room temperature for 15 minutes in the dark, rinse 3 times with PBS in the dark, observe and save the photos with a Leica LSM800 laser confocal microscope (see Figure 16 for fluorescence photo results), and analyze the intracellular contents with ZEN software Pearson coefficient of colocalization (see Figure 17 for results).

After adsorption and internalization on the cell surface, ASFV enters the cell and is transported from the cytoplasm to the nucleus through the endosomal transport pathway. The endosomes involved in this transport pathway can be specifically divided into early endosomes and late endosomal transport. , each can be distinguished by different molecular markers. For example, early endosome Rab5 is its specific marker. Previous experiments have shown that the drug Gamithromycin neither affects the adsorption nor internalization of ASFV, so it is speculated that this drug affects the early endocytic transport process of ASFV in cells. Through confocal experiments, the early endosome transport of ASFV was tracked using the early endosome marker molecules Rab5 and ASFVP72 proteins. It can be seen from the present invention that there is no significant difference between the sample treatment group and the positive control group, indicating that ASFV enters the next stage after being transported by early endosomes. It was also shown that Gamithromycin drug basically did not affect the early endocytic transport process of ASFV in cells (Fig. 16, Fig. 17).

Example 10: Effect of Gamithromycin on ASFV in late endosomes.

The marker molecule for late endosomes is LBPA, so the primary anti-mouse Rab5 monoclonal antibody in Example 9 was replaced with a murine LBPA monoclonal antibody (purchased from Merck-Millipore). In other aspects, the same reagent method as in Example 9 was used to conduct laser confocal experiments on each sample at 60 min, 90 min, 120 min and 150 min respectively (see Figures 18 and 19 for the results).

As the endosomal trafficking pathway proceeds, ASFV undergoes transport in early endosomes and subsequently enters late endosomes. Through laser confocal experiments, the present invention uses the specific marker LBPA of late endosomes to track and observe the late endosome transport stage during ASFV infection. The present invention found that there were significant differences between the sample treatment group adding Gamithromycin drug and the positive control group at 60min, 90min, 120min and 150min, indicating that the transport stage of ASFV into late endosomes was affected. It was also shown that the drug Gamithromycin affects the endosome transport process of ASFV in late cellular stages (Fig. 18, Fig. 19).

Example 11: Effects of Berbamine (dihydrochloride), Berbamine, Gamithromycin and Cyclovirobuxin D on the release of ASFV in cells.

PAMs were cultured in a 24-well cell culture plate. The culture medium was RPMI 1640 complete medium, 500 μl/well. ASFV-eGFP (MOI=0.3) was incubated with PAMs at 37°C for 2 h. Washed three times with PBS, fresh RPMI 1640 was added. culture medium. 16 hours after infection, ASFV was treated with 10 μM Berbamine (dihydrochloride) + 0.1 v/v% DMSO; 10 μM Berbamine + 0.1 v/v% DMSO; 10 μM Gamithromycin + 0.1 v/v% DMSO; 10 μM Cyclovirobuxin D + 0.1 v/v% Ethanol Infect the cells for 1 hour, use 0.1v/v% DMSO and Ethanol as controls, and Mock as the blank well control (no virus or drugs are added), then wash with PBS three times, and add fresh RPMI1640 complete medium. When the first cycle of ASFV is completed and new viral particles are released, 24 hours after infection, cultures are collected and ASFV titers are measured by TCID ₅₀ .

The results showed that the virus titers after the action of Berbamine (dihydrochloride), Berbamine, Gamithromycin and Cyclovirobuxin D were not significantly different from the positive control group. This shows that these drugs do not affect the release of ASFV in cells (see Figure 20 for the results).

Example 12: Effects of 4 drug combinations on ASFV replication

PAMs were cultured in 24-well plates (1.25 × 10 ⁶ cells/well), and the culture medium was RPMI 1640 complete medium. After the cells were completely adhered, add Berbamine (dihydrochloride) (code: BD), Berbamine (code: B) , RPMI 1640 complete culture medium of Gamithromycin (code: G) and Cyclovirobuxin D (code: CD). The drug types and dosages are: 2.5μM Berbamine (dihydrochloride); 2.5μM Berbamine; 2.5μM Gamithromycin; 2.5μM Cyclovirobuxin D; The combination of 2.5μM Berbamine (dihydrochloride) and 2.5μM Cyclovirobuxin D; the combination of 2.5μM Berbamine and 2.5μM Cyclovirobuxin D; the combination of 2.5μM Gamithromycin and 2.5μM Cyclovirobuxin D; the combination of 2.5μM Berbamine (dihydrochloride) and 2.5μM Gamithromycin; 2.5μM Berbamine Combination with 2.5 μM Gamithromycin; Combination of 2.5 μM Berbamine (dihydrochloride) and 2.5 μM Berbamine; Combination of 2.5 μM Berbamine (dihydrochloride), 2.5 μM Berbamine and 2.5 μM Gamithromycin; Combination of 2.5 μM Berbamine, 2.5 μM Cyclovirobuxin D and 2.5 μM Gamithromycin ; Combination of 2.5μM Berbamine (dihydrochloride), 2.5μM Cyclovirobuxin D and 2.5μM Gamithromycin; Combination of 2.5μM Berbamine (dihydrochloride), 2.5μM Cyclovirobuxin D and 2.5μM Berbamine; 2.5μM Berbamine (dihydrochloride), 2.5μM Cyclovirobuxin D, 2.5μM A combination of Berbamine and 2.5 μM amithromycin; at the same time, 0.1v/v% DMSO and 0.1v/v% Ethanol were added to the above solution and positive control. The Mock was a negative control (without adding drugs and viruses) and was incubated for 2 hours. After discarding, the corresponding Concentration of the drug and inoculation with ASFV-eGFP (MOI = 0.2) for 2 h, the cells were washed three times with PBS, and then RPMI 1640 complete medium containing the same drug at the corresponding concentration was added to the drug wells again. Incubate at 37°C for 48 hours, collect the cell supernatant and cells, and use the same reagents and methods as in Example 3 to perform qPCR (results see Figure 21 and Table 1) and WB (results see Figure 22) detection on the samples. In Table 1, the drugs are shown in the first column, the p72 gene copy numbers corresponding to each drug and treatment are shown in column 2, and the comparison of the p72 gene copy numbers corresponding to each drug and other drug treatments are shown in columns 3-6, *** means significant difference, NA means no comparison.

Table 1. Significance analysis of ASFV p72 gene copy number differences by qPCR

In order to evaluate the impact of the combined use of these 4 drugs on ASFV replication, qPCR and WB analysis were performed. The qPCR results showed that after adding any two, three or four combinations of these four drugs, the copy number of ASFV p72 in the cell supernatant was significantly lower than when the corresponding drugs were used alone. control group, indicating that they can inhibit the replication of ASFV. WB results showed that after adding any two, three or four combinations of these four drugs, compared with the corresponding drug alone, the ASFV p54 protein expression was significantly lower than the corresponding drug alone in the control group. Therefore, the combination of two, three and four groups of four drugs: berbamine, berbamine hydrochloride, gamithromycin and cyclitin D can also significantly inhibit the replication of ASFV, showing that among these four drugs The synergistic effect between them (Figure 21 and Figure 22). It can be seen that the combined use of berbamine, berbamine hydrochloride, gamithromycin and cyclobuxine D has the effect of treating or slowing down the treatment of African swine fever virus infection.

It is known from common technical knowledge that the present invention can be implemented by other embodiments without departing from its spirit or essential characteristics. Therefore, the above-disclosed embodiments are in all respects illustrative and not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are included in the present invention.

Claims (20)

1. Use of an active substance in the preparation of a formulation for slowing, treating or controlling african swine fever and/or inhibiting african swine fever virus proliferation, the active substance being a combination of either or both of cyclovirobuxine D and a pharmaceutically acceptable salt of cyclovirobuxine D; the structural formula of the cyclovirobuxine D is as follows:

2. the use according to claim 1, wherein the active substance is the sole pharmaceutically active substance.

3. The use of claim 1, wherein the pharmaceutically acceptable salt of cyclovirobuxine D comprises: toluene sulfonate, methane sulfonate, malate, acetate, citrate, malonate, tartrate, succinate, lactate, benzoate, ascorbate, alpha-ketoglutarate, alpha-glycerophosphate, hydrochloride, sulfate, nitrate, bicarbonate, carbonate, phosphate, hydrobromide, and hydroiodide of cyclovirobuxine D.

4. The use according to claim 1, wherein the formulation is a medicament.

5. The use according to claim 1, wherein the formulation is a feed additive.

6. Use of an active substance, which is a combination of either or both of cyclovirobuxine D and a pharmaceutically acceptable salt of cyclovirobuxine D, for the preparation of a formulation for slowing, treating or controlling african swine fever and/or inhibiting proliferation of african swine fever virus by inhibiting adsorption of african swine fever virus to susceptible cells and/or inhibiting internalization of african swine fever virus in susceptible cells; the structural formula of the cyclovirobuxine D is as follows:

7. the use according to claim 6, wherein the active substance is the sole pharmaceutically active substance.

8. The use of claim 6, wherein the pharmaceutically acceptable salt of cyclovirobuxine D comprises: toluene sulfonate, methane sulfonate, malate, acetate, citrate, malonate, tartrate, succinate, lactate, benzoate, ascorbate, alpha-ketoglutarate, alpha-glycerophosphate, hydrochloride, sulfate, nitrate, bicarbonate, carbonate, phosphate, hydrobromide, and hydroiodide of cyclovirobuxine D.

9. The use of claim 6, wherein the susceptible cell is a porcine alveolar macrophage.

10. The use of claim 6, wherein the formulation is a medicament.

11. The use according to claim 6, wherein the formulation is a feed additive.

12. A pharmaceutical composition, the pharmaceutically active ingredient of which comprises a first active substance and a second active substance;

the first active substance is any one or the combination of two of berberine and pharmaceutical salts of berberine;

the second active substance is any one or the combination of two of cyclovirobuxine D and medicinal salt of cyclovirobuxine D;

the structural formula of the berbamine is as follows:

the structural formula of the cyclovirobuxine D is as follows:

13. the pharmaceutical composition according to claim 12, wherein the molar ratio of the first active substance to the second active substance is from 1:0.1 to 10.

14. The pharmaceutical composition according to claim 13, wherein the molar ratio of the first active substance to the second active substance is 1:0.2-5.

15. The pharmaceutical composition according to claim 14, wherein the molar ratio of the first active substance to the second active substance is 1:1.

16. The pharmaceutical composition of claim 12, wherein the pharmaceutically acceptable salt of berbamine comprises: toluene sulfonate, methane sulfonate, malate, acetate, citrate, malonate, tartrate, succinate, lactate, benzoate, ascorbate, alpha-ketoglutarate, alpha-glycerophosphate, dihydrochloride, sulfate, nitrate, bicarbonate, carbonate, phosphate, hydrobromide, and hydroiodide of berbamine.

17. The pharmaceutical composition of claim 12, wherein the pharmaceutically acceptable salt of cyclovirobuxine D comprises: toluene sulfonate, methane sulfonate, malate, acetate, citrate, malonate, tartrate, succinate, lactate, benzoate, ascorbate, alpha-ketoglutarate, alpha-glycerophosphate, hydrochloride, sulfate, nitrate, bicarbonate, carbonate, phosphate, hydrobromide, and hydroiodide of cyclovirobuxine D.

18. Use of a pharmaceutical composition according to any one of claims 12 to 17 for the preparation of a formulation for slowing, treating or controlling african swine fever and/or inhibiting african swine fever virus proliferation.

19. The use of claim 18, wherein the formulation is a medicament.

20. The use according to claim 18, wherein the formulation is a feed additive.