CN115252625B

CN115252625B - Application of cyclovirobuxine D in preparation of preparation for treating African swine fever

Info

Publication number: CN115252625B
Application number: CN202210961664.9A
Authority: CN
Inventors: 赵东明; 步志高; 朱远茂; 李芳�; 孙恩成; 张振江; 华荣虹
Original assignee: Harbin Veterinary Research Institute of CAAS
Current assignee: Harbin Veterinary Research Institute of CAAS
Priority date: 2022-08-11
Filing date: 2022-08-11
Publication date: 2024-01-26
Anticipated expiration: 2042-08-11
Also published as: CN115252625A

Abstract

The invention discloses an application of cyclovirobuxine D in preparing a preparation for treating or slowing down African swine fever or inhibiting African swine fever virus proliferation. The cyclovirobuxine D can effectively inhibit African swine fever virus from adsorbing swine alveolar macrophages and also can effectively inhibit the internalization of the African swine fever virus in the swine alveolar macrophages, so that the replication of the African swine fever virus can be effectively inhibited, and a material basis of a novel active medicament and a novel pharmaceutical composition is provided for treating or slowing down African swine fever. The invention also discloses a pharmaceutical composition containing cyclovirobuxine D, which can better inhibit proliferation of African swine fever virus.

Description

Application of cyclovirobuxine D in preparation of preparation for treating African swine fever

Technical Field

The invention belongs to the field of pharmacy, and relates to application of cyclovirobuxine D in preparation of a preparation for treating African swine fever.

Background

African swine fever virus (African Swine Fever Virus, ASFV) is the only member of the African swine fever virus family (Asfarviridae), and is also the only known DNA arbovirus, and the death rate of virulent infection is up to 100%, which seriously threatens the healthy development of the pig industry in the world. The main vehicles of ASFV are pigs, wild boars and soft ticks. The virus is divided into 24 genotypes, 8 serotypes, and genes I and II are currently epidemic strains. With the popularity of ASF in China, new variant strains are continuously generated, so that the ASF prevention and control situation is more severe.

To date, no effective vaccine and antiviral drug against ASF has been developed, mainly because the viral genome is large and the structure is complex, and furthermore the viral immune escape mechanism and host protective immune mechanism are not clear. Potential antiviral drugs can be divided into two classes according to the replication mechanism of ASFV: (1) Inhibitors that act directly on ASFV by targeting viral proteins (direct acting antiviral drugs); (2) Inhibitors of cytokines that target ASFV replication (host-targeted antiviral drugs). Although various types of anti-ASFV active agents have been reported, the in vivo efficacy of these compounds has not been evaluated. There is still a lack of effective drugs for treating and preventing ASFV in clinic. Therefore, there is a need to develop anti-ASFV drug studies that can provide a material basis for ASF control.

The compounds Berbamine dihydrochloride (Berbamine (dihydrochloride), CAS: 6078-17-7) and Berbamine (Berbamine, CAS: 478-61-5) are a bisbenzylisoquinoline natural product isolated from the herb Huang Lumu. They are novel inhibitors of bcr/abl and inhibitors of NF- κb, have anti-leukemia activity, inhibit the growth of cancer cells, and induce apoptosis of human myeloma cells.

The compound Gamithromycin (CAS: 145435-72-9) is a novel macrolide antibiotic, mainly used for treating respiratory diseases of cattle.

The compound cyclovirobuxine D (CAS: 860-79-7) is an active compound extracted from Buxus lobus, and is used for treating acute myocardial ischemia.

No report is made that these 4 compounds are capable of treating or preventing ASFV infection.

Disclosure of Invention

The invention discovers that the four compounds Berbamine (Dihydrochloride), berbamine, gamithromycin and Cyclovirobuxin D can not only remarkably inhibit the replication of an ASFV-eGFP model in vitro, but also remarkably inhibit the replication of wild ASFV. Studies with different time additions of compounds have shown that they are all able to affect ASFV replication. Further studies have found that the mechanism by which these four compounds inhibit ASFV replication is different. Berbamine (Dihydrochloride) and Berbamine each achieve the effect of inhibiting ASFV replication by affecting adsorption of ASFV, cycloviridoxin D by affecting adsorption and internalization of ASFV, and Gamithromycin by affecting entry of ASFV into late endocytosis. The invention provides an application foundation for the research and development of medicines for treating or preventing ASFV.

After the different components are combined into the composition, the toxicity is low, the safety is good, and the composition can still ensure good safety. The medicines act on different stages of the normal replication cycle of the ASFV, the basic inhibition mechanism is clear, the combination at least does not have mutual antagonism in the discovered mechanism range, the synergistic superposition of the components can generate the inhibition effect on the ASFV, and the composition can better inhibit the replication of the ASFV. Under the condition of ensuring the same ASFV inhibition rate, the dosage of each medicine can be reduced compared with the dosage of each medicine used singly, thereby further reducing potential toxic and side effects. The invention discovers that any kind of combination of four compounds with novel activity has better application prospect for treating or preventing African swine fever virus infection compared with a single compound.

In order to solve the problems existing in the prior art, the first aspect of the present invention provides an application of an active substance in preparation of a preparation for preventing, slowing down, treating or controlling african swine fever and/or inhibiting african swine fever virus proliferation, wherein the active substance is cyclovirobuxine D, any one of a pharmaceutically acceptable salt of cyclovirobuxine D and a prodrug of cyclovirobuxine D, a combination of any two or a combination of three of the above; the structural formula of the cyclovirobuxine D is as follows:

in some embodiments, the active is the sole pharmaceutically active.

In some embodiments, the pharmaceutically acceptable salt of cyclovirobuxine D comprises: the tosylate, mesylate, malate, acetate, citrate, malonate, tartrate, succinate, lactate, benzoate, ascorbate, alpha-ketoglutarate, alpha-glycerophosphate, hydrochloride, sulfate, nitrate, bicarbonate, carbonate, phosphate, hydrobromide, and hydroiodide salts of gamimycin of cyclovirobuxine D.

In some embodiments, the formulation is a drug.

In some embodiments, the formulation is a feed additive.

The second aspect of the invention provides an application of an active substance in preparing a preparation for preventing, slowing down, 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, wherein the active substance is cyclovirobuxine D, any one of medicinal salts of cyclovirobuxine D and prodrugs of cyclovirobuxine D, a combination of any two or a combination of three; the structural formula of the cyclovirobuxine D is as follows:

in some embodiments, the active is the sole pharmaceutically active.

In some embodiments, the susceptible cell is a porcine alveolar macrophage.

In some embodiments, the formulation is a drug.

In some embodiments, the formulation is a feed additive.

In a third aspect the present invention provides 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 of berberine, pharmaceutical salt of berberine and prodrug of berberine, combination of any two or combination of three;

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

the structural formula of the berbamine is as follows:

the structural formula of the cyclovirobuxine D is as follows:

in some embodiments, the molar ratio of the first active to the second active is from 1:0.1 to 10 (e.g., any ratio or range between any two ratios of 1:0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

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

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

In some embodiments, 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.

In a fourth aspect, the invention provides the use of a pharmaceutical composition according to the third aspect of the invention in the manufacture of a formulation for preventing, slowing, 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.

The cyclovirobuxine D can effectively inhibit African swine fever virus from adsorbing pig alveolar macrophages, and can effectively inhibit the internalization of the African swine fever virus in susceptible cells, so that the replication of the African swine fever virus can be effectively inhibited, and a new active medicine and a material basis are provided for treating or preventing the African swine fever.

The four compounds of berbamine, berbamine hydrochloride, mi Mei added and cyclovirobuxine D have better inhibition effect on ASFV by any two, three or four drugs than by using one drug alone, and have better inhibition effect on ASFV by any three or two drugs. Therefore, any kind of combination among the four compounds has systematic synergism, and no drug compatibility reduction and effect antagonism occur.

Drawings

Figure 1 shows cytotoxicity of DMSO at different concentrations on PAMs.

Figure 2 shows cytotoxicity of PAMs by different concentrations of Ethanol.

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

FIG. 4 shows CC50 and IC50 of Gamithromycin and Cyclovirobuxin D.

FIG. 5 shows a fluorescence photograph of four drugs for ASFV-eGFP replication.

Figure 6 shows qPCR results versus WB results for the effect of four drugs on ASFV-eGFP replication.

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

Figure 8 shows WB results for four drugs affecting wild-type ASFV replication.

Figure 9 shows qPCR results versus WB results for the effect of four drug additions on ASFV-eGFP replication at different times.

Fig. 10 shows qPCR assay results for the inactivation of ASFV with 4 drugs.

FIG. 11 shows the WB assay results of 4 drugs on ASFV inactivation.

Fig. 12 shows qPCR assay results for adsorption of ASFV by 4 drugs.

Fig. 13 shows WB detection results of adsorption of ASFV by 4 drugs.

Figure 14 shows qPCR assay results for internalization of ASFV for 4 drugs.

Figure 15 shows WB detection results for the internalization of ASFV with 4 drugs.

FIG. 16 shows confocal images of gamithromycin on ASFV endocytosis at early stage of cells.

FIG. 17 shows the results of calculation of the effect of gamithromycin on ASFV endocytosis in early cellular phases.

Fig. 18 shows confocal images of gamithromycin for ASFV at late endocytosis.

FIG. 19 shows the results of calculation of the effect of gamithromycin on ASFV endocytosis during the late phase of cells.

FIG. 20 shows TCID effect of four drugs on ASFV release ₅₀ And (5) detecting a result.

Figure 21 shows qPCR detection results for both single and combined drug administration.

Fig. 22 shows WB detection results for both individual and combined administration.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the embodiments of the present invention will be described in further detail with reference to the accompanying drawings.

Materials and apparatuses not described in the present invention are conventional materials and apparatuses in the art, and details of operation not described in the present invention are conventional operations in the art.

Experimental materials

Experimental medicine: berbamine (dihydrochloride), berbamine, gamithromycin and Cyclovirobuxin D (available from Selleck Corp.).

Experimental reagent: RPMI1640 complete medium was supplied by Gibco corporation; pig serum is supplied by Hyclone company; CCK-8 is provided by Japan Tonic chemical Co., ltd; PBS and dimethyl sulfoxide were supplied by the company sorabio.

Experimental equipment: filters, pipettes, 24-well and 96-well cell culture plates were supplied by corning corporation; clean bench, carbon dioxide incubator, available from Thermo company, usa; the inverted optical microscope is provided for Mshot company; the ELISA was supplied by BioTek.

Cell and virus: PAMs (alveolar macrophages); ASFV wild-type strain (Pic/HLJ/2018 strain, genebank accession number: MK 333180.1); ASFV marker strains (called ASFV-eGFP strains), in which eGFP is expressed, enable cells infected with ASFV-eGFP to fluoresce green, facilitating the observation of the number and distribution of virions by fluorescence, see A seven-gene-deleted African swine fever virus is safe and effective as a live attenuated vaccine in pigs, sci China Life Sci, weiye Chen et al 2020;63 (5) ASFV-delta 6GD strain in 623-634.

Example 1: toxicity of DMSO and Ethanol to PAMs

PAMs are used in 3X 10 ⁵ The individual cells/well were added to 96 well cell culture plates at 100 μl/well and the medium was RPMI1640 complete medium. Placing at 37 ℃ 5% CO ₂ And (5) culturing the cells in the incubator after the cells are completely adhered. The culture broth was aspirated, and the experimental wells (As) were each filled with 0.0625v/v%,0.125v/v%,0.25v/v%,0.5v/v%,1v/v%,2v/v%,4v/v% dimethyl sulfoxide (DMSO) and 0.3125v/v%,0.625v/v%,1.25v/v%,2.5v/v%,5v/v%,10v/v% Ethanol (Ethanol) in RPMI1640 complete medium, 100. Mu.l/well was filled into 96-well plates, and 3 wells were repeated for each concentration. Control wells (Ac) were added to RPMI1640 complete medium at 100 μl/well. Blank wells (abs) were free of cells and an equal volume of RPMI1640 complete medium was added at 100 μl/well. 37 ℃ 5% CO ₂ Incubators were incubated for 48h. After 48h, 100. Mu.l of fresh RPMI1640 complete medium was changed and 10. Mu.l of CCK-8 solution (tetrazolium salt solution) was added to each well. The plates were incubated in an incubator for 1-4h. The absorbance at 450nm was measured with a microplate reader. Cell viability was determined according to the following formula: cell viability% = [ (OD) _As -OD _Ab )/(OD _Ac -OD _Ab )]X 100%. Wherein OD _As 、OD _Ab 、OD _Ac OD values are represented for test wells, blank wells, control wells, respectively.

The results showed that DMSO at 4v/v% was very different compared to the control group (p < 0.0001); DMSO at concentrations of 2v/v% and 1v/v% were significantly different compared to the control group (p < 0.01); DMSO at 0.5v/v% was not significantly different compared to the control (p > 0.05) (fig. 1). Therefore, 0.5% DMSO has no significant cytotoxicity to PAM and can be used as a drug-free control or solvent for drugs for pharmaceutical activity testing.

The results showed that the concentration of 10v/v% of Ethanol was very different compared to the control group (p < 0.0001); ethanol at a concentration of 5v/v% was not significantly different from the control (p > 0.05) (FIG. 2). Therefore, 5v/v% of Ethanol has no obvious cytotoxicity to PAM, and can be used as a drug-free control or a solvent of a drug for testing the activity of the drug.

Example 2: berbamine (dihydrochloride) Berbamine, gamithromycin and Cycloviridoxixin D small molecule Compound IC ₅₀ And CC ₅₀ Is (are) determined by

PAMs are used in 3X 10 ⁵ Each well was added to a 96-well cell culture plate at 100. Mu.l/well in RPMI1640 complete medium. Placing in 37 ℃ 5% CO ₂ Incubator, after the cells are completely adhered. The culture medium was aspirated, and experimental wells (As) were each filled with RPMI1640 complete medium containing four compounds at different concentrations, see the abscissa of each plot of FIGS. 3 and 4, with a 10 index, in. Mu.M, and each 100. Mu.l/well was filled into 96-well plates, and 3 wells were repeated for each concentration. Control wells (Ac) were added to RPMI1640 complete medium at 100 μl/well. Blank wells (abs) were free of cells and only an equal volume of RPMI1640 complete medium was added. 37 ℃ 5% CO ₂ Incubators were incubated for 48h. After 48h, 100. Mu.l of fresh RPMI1640 complete medium was changed and 10. Mu.l of CCK-8 solution was added to each well. The plates were incubated in an incubator for 1-4h. The absorbance at 450nm was measured with a microplate reader. Cell viability was determined according to the following formula: cell viability% = [ (OD) _As -OD _Ab )/(OD _Ac -OD _Ab )]X 100%. Wherein OD _As 、OD _Ab 、OD _Ac OD values are represented for test wells, blank wells, control wells, respectively.

The results showed CC of Berbamine (dihydrochloride), berbamine, gamithromycin and Cyclovirobuxin D compared to control wells ₅₀ 61.85. Mu.M, 53.80. Mu.M, 132.2. Mu.M and respectively61.20. Mu.M; berbamine (dihydrochloride) IC of Berbamine, gamithromycin and Cyclovirobuxin D ₅₀ 0.71. Mu.M, 0.87. Mu.M, 2.25. Mu.M and 0.32. Mu.M, respectively. The Selection Indices (SI) of Berbamine (dihydrochloride), berbamine, gamithromycin and Cycloviridoxin D were calculated to be 87.11, 61.83, 58.76 and 191.3, respectively (FIGS. 3, 4). Therefore, the four compounds have low toxicity and good safety.

Example 3: berbamine (dihydrochloride) effects of Berbamine, gamithromycin and Cycloviridoxin D drugs on ASFV-eGFP replication.

PAMs were cultured in 24-well plates (1.25X10) ⁶ Cells/well), 100 μl/well, of RPMI1640 complete medium, after cells were completely attached, RPMI1640 complete medium containing different concentrations of Berbamine (dihydrochloride), berbamine, gamithromycin and Cyclovirobuxin D (10 μΜ,5 μΜ and 2.5 μΜ,0 μΜ) was added, incubated for 2h, and after discarding, RPMI1640 complete medium containing the corresponding concentrations of the drug (10 μΜ,5 μΜ and 2.5 μΜ,0 μΜ) and RPMI1640 complete medium containing the compound of the corresponding concentration of drug (10 μΜ,5 μΜ and 2.5 μΜ,0 μΜ) were added and ASFV-eGFP (moi=0.1) was inoculated for 2h, cells were washed 3 times with PBS, and RPMI1640 complete medium containing the different concentrations of the compound (10 μΜ,5 μΜ and 2.5 μΜ,0 μΜ) was added. The treatment of the blank (Mock) was: no virus inoculation and drug addition only RPMI1640 complete medium. Incubated at 37℃for 48h, and white light and fluorescence pictures were taken for each well with a fluorescence microscope, with the results shown in FIG. 5, with the fluorescence picture for each well on top and the white light picture on the bottom. The cell supernatants and cells were collected and qPCR and WB were performed, respectively.

The fluorescent microscope can observe the cell activity condition by taking pictures under white light, and the ASFV infection condition can be determined by fluorescence. The white light picture can see that the cell activity is normal; the fluorescence photograph shows that the number of fluorescence gradually decreases with increasing drug concentration compared to the positive control group.

The quantitative qPCR method for detecting the copy number of ASFV P72 gene was recommended by OIE (see 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), and the DNA from the cell supernatant was extracted for qPCR. Referring to the left bar chart of fig. 6, qPCR test results showed that the progeny virus copy number of ASFV p72 in the cell supernatant gradually decreased with increasing drug concentration, as compared to the positive control group (0 μm drug group).

After cell lysis, the expression level of ASFV p54 protein was detected by a conventional WB method using ASFV p54 antibody (monoclonal antibody against ASFV p54 protein, prepared by the applicant's own experiments), while setting PAMs beta-actin as an intracellular reference (antibodies purchased from proteontech), as a result of which see fig. 6, right-side protein electrophoresis chart. The WB test results showed that the p54 protein band gradually faded with increasing drug concentration compared to the positive control group.

Therefore, all the 4 medicines have obvious inhibiting effect on the replication of ASFV-eGFP and are dose-dependent. Therefore, the 4 medicines have the effect of treating or slowing down the treatment of African swine fever virus infection.

Example 4: berbamine (dihydrochloride) effects of Berbamine, gamithromycin and Cycloviridoxin D drugs on wild-type ASFV replication.

PAMs were cultured in 24-well plates (1.25X10) ⁶ Cell/well), the culture solution is RPMI1640 complete culture medium, after the cells are completely adhered, the RPMI1640 complete culture medium containing Berbamine (dihydrochloride), berbamine, gamithromycin and Cyclovirobuxin D drugs is added, and the adding types and the adding amounts are as follows: 10. Mu.M Berbamine (dihydrochloride) +0.1v/v% DMSO; 10. Mu.M Berbamine+0.1v/v% DMSO; 10. Mu.M gamithromycin+0.1v/v% DMSO;10 mu M Cyclovirobuxin D +0.1v/v% Ethanol;0.1v/v% DMSO and 0.1v/v% Ethanol, incubated for 2h, discarded, added with the corresponding concentration of drug and inoculated with wild-type ASFV (Pig/HLJ/2018, moi=0.05) for 2h, washed 3 times with PBS, and added with RPMI1640 complete medium containing the corresponding concentration of drug. The types and the dosage of the medicine are respectively as follows: 10. Mu.M Berbamine (dihydrochloride) +0.1v/v% DMSO; 10. Mu.M Berbamine+0.1v/v% DMSO; 10. Mu.M gamithromycin+0.1v/v% DMSO;10 mu M Cyclovirobuxin D +0.1v/v% Ethanol;0.1v/v% DMSO and 0.1v/v% EtOH. The treatment of the blank (Mock) group was: only RPMI1640 complete medium was added. Culturing at 37℃for 48h, collecting cell supernatants and cells, and performing qPCR (see FIG. 7 for results) and WB (see FIG. 8 for results) detection on the samples using the same reagents and methods as in example 3, respectively.

To evaluate the effect of these 4 drugs on wild-type ASFV replication, qPCR and WB analysis was performed. qPCR results showed that the copy number of ASFV p72 in the supernatant was significantly lower than the positive control (0.1 v/v% DMSO and 0.1v/v% Ethanol) after addition of these 4 drugs, indicating that they could inhibit replication of wild-type ASFV. WB results showed that ASFV p54 protein expression was significantly lower than in positive versus illuminated groups after addition of these 4 drugs. Thus, 4 drugs were also able to significantly inhibit replication of wild-type ASFV (fig. 7, 8). Thus, the 4 medicines have the effect of treating or slowing down the treatment of African swine fever virus infection.

Example 5: berbamine (dihydrochloride) Effect of Berbamine, gamithromycin and Cycloviridoxin D drugs on ASFV replication stage

To study Berbamine (dihydrochloride) the effects of Berbamine, gamithromycin and cyclopirobuxin D drugs on different stages of ASFV replication, PAMs were cultured on 24-well plates (1.25 x10 ⁶ Hole), the culture solution is RPMI1640 complete culture medium, after cells are attached, ASFV-eGFP (MOI=0.5) is inoculated, 4 medicines are respectively added in-2,0,2,4,8 and 16 hours of inoculation time, and the adding types and the using amounts are as follows: 10. Mu.M Berbamine (dihydrochloride) +0.1v/v% DMSO; 10. Mu.M Berbamine+0.1v/v% DMSO; 10. Mu.M gamithromycin+0.1v/v% DMSO;10 mu M Cyclovirobuxin D +0.1v/v% Ethanol;0.1v/v% DMSO and 0.1v/v% EtOH. Berbamine (dihydrochloride) Berbamine, gamithromycin was formulated with 0.1v/v% DMSO positive control and no drug blank (Mock); cyclovirobuxin D was formulated with 0.1v/v% Ethanol positive control and no drug blank (Mock). To strictly follow the ASFV replication cycle, samples were collected 24h post-infection and qPCR and WB assays were performed on each sample using the same reagents and methods as in example 3.

To analyze the effect of these 4 drugs on different stages of ASFV replication, the ASFV p72 gene copy numbers of the 4 drugs at-2, 0,2,4, 6, 8 and 16h time points, respectively, were determined before and after infection with PAMs. The addition of these drugs significantly inhibited ASFV replication compared to the positive control, particularly at the early stages of ASFV replication. qPCR results show that CT values of the added Berbamine (dihydrochloride) Berbamine and Cycloviridoxin D are obviously higher than those of a positive control group in-2 to 4 hours, and the inhibition effect of 3 medicaments on the early stage of virus replication is obvious. The results of WB also indicate that the above 3 drugs have better inhibitory effect in the early stages of replication. The results of qPCR and WB show that Gamithromycin can obviously inhibit viral replication in the period of-2 to 8 hours, and the Gamithromycin has the effect of inhibiting ASFV replication in the early and middle stages of viral replication. Thus, berbamine (dihydrochloride) Berbamine and Cyclospiraxin D were able to inhibit early and mid-phase replication of ASFV, while Gamithromycin was able to inhibit early and mid-phase replication of ASFV (FIG. 9).

Example 6: berbamine (dihydrochloride) direct inactivation of ASFV by Berbamine, gamithromycin and Cycloviridoxin D drugs

To evaluate whether these 4 drugs can directly kill ASFV, PAMs were plated in 24-well plates (1.25x10 ⁶ Well), the culture medium was RPMI1640 complete medium, 500. Mu.l/well, and cells were allowed to stand for complete attachment. ASFV-eGFP (MOI=1) and the corresponding drugs (2. Mu.M Berboxamine) +0.02v/v% DMSO; 2. Mu.M Berboxamine+0.02 v/v% DMSO; 2. Mu.M gamithromycin+0.02v/v% DMSO; 2. Mu. M Cyclovirobuxin D +0.02v/v% EtOH; 0.1v/v% DMSO and 0.02v/v% EtOH) were mixed and incubated for 1h at 37 ℃. The treatment of the blank (Mock) was: only RPMI1640 complete medium was added. The mixture was then diluted 20-fold to eliminate the potential effect of these drugs on ASFV-eGFP infection. Then, the above virus-containing mixture was added to the adherent cells, and cultured at 37℃for 2 hours. After discarding the supernatant, cells were washed 3 times with PBS and RPMI1640 complete medium containing 20v/v% porcine serum was added. Cell supernatants and cells were collected after 48 hours, and the copy number of ASFV p72 gene and the expression amount of p54 protein in the supernatants (see FIG. 10 for results) were examined by qPCR and WB for each sample using the same reagents and method as in example 3 (see FIG. 11 for results). The results showed that there was no significant difference compared to the control, and none of the 4 drugs could directly kill the virus.

Example 7: berbamine (dihydrochloride) inhibition of ASFV-adsorbing cells by Berbamine, gamithromycin and Cycloviridoxin D drugs.

PAMs in 24 well cell culture plates (1.25x10) ⁶ Well) (RPMI 1640 complete medium) with ASFV-eGFP (moi=0.1) and 10 μm Berbamine) +0.1v/v% DMSO, respectively; 10. Mu.M Berbamine+0.1v/v% DMSO; 10. Mu.M gamithromycin+0.1v/v% DMSO;10 mu M Cyclovirobuxin D +0.1v/v% EtOH, incubated at 4℃for 1h to allow virus to adsorb to cells but prevent virus internalization. Buffer (both 0.1v/v% DMSO and 0.1v/v% EtOH) was used as positive control, mock as blank well control (no virus and drug added), the supernatant was discarded, the cells were washed 3 times with PBS pre-chilled at 4deg.C to remove unbound virus, and RPMI1640 complete medium was added at 500 μl/well. The cell culture plates were then transferred to 37℃and incubated for 48h. Cell supernatants and cells were collected. qPCR (see fig. 12 for results) and WB (see fig. 13 for results) were performed on each sample using the same reagents and methods as in example 3, respectively. The supernatant was assayed for ASFV p72 copy number by qPCR and the cells were assayed for viral p54 protein expression level by WB. The results showed that Berbamine (dihydrochloride), berbamine and Cyclovirobuxin D had an effect on ASFV adsorption, whereas Gamithromycin had no significant effect on ASFV adsorption (fig. 12, fig. 13).

Example 8: berbamine (dihydrochloride) inhibition of ASFV internalization by Berbamine, gamithromycin and Cycloviridoxin D drugs.

PAMs were incubated with ASFV-eGFP (moi=0.1) at 4 ℃ for 1h in RPMI1640 complete medium, 500 μl/well, and cells were washed 3 times with PBS pre-chilled at 4 ℃. These 4 drugs were added to the wells of the cell culture plate, and each of the four drugs was 10. Mu.M Berbamine (dihydrochloride) +0.1v/v% DMSO; 10. Mu.M Berbamine+0.1v/v% DMSO; 10. Mu.M gamithromycin+0.1v/v% DMSO;10 mu M Cyclovirobuxin D +0.1v/v% Ethanol. Buffer (0.1 v/v% DMSO and 0.1v/v% EtOH) was used as positive control, mock as blank well control (no virus or drug added), 500 μl/well, and RPMI1640 complete medium. The cell culture plates were then switched to 37 ℃. After 1h, the compound was removed by washing 3 times with PBS, and then fresh RPMI1640 complete medium was added. Cells were removed to 37℃at time point of 0h, cell supernatants and cells were collected at 48h, and replication and protein expression of viruses were determined by qPCR (see FIG. 14 for results) and WB (see FIG. 15 for results) using the same reagents and methods as in example 3, respectively.

Both qPCR and WB results showed that Cyclovirobuxin D affected the internalization of ASFV, whereas Berbamine (dihydrochloride), berbamine and Gamithromycin did not (fig. 14, fig. 15).

Example 9: effects of Gamithromycin drug on early endocytosis of ASFV.

PAMs were found in confocal cuvettes (1.2x10) ⁶ Dish) was incubated with RPMI1640 complete medium, 1 mL/dish, ASFV at moi=5 was inoculated into PAMs, incubated at 4 ℃ for 1h, then 10 μm gamithromycin+0.1v/v% DMSO was added to the sample group, equal amount of 1640 complete medium+0.1v/v% DMSO was added to the positive control group, negative control was Mock (no virus and drug added), and incubation was performed at 37 ℃ for 15min, 30min, 45min and 60min, respectively, followed by sampling. The sample was washed once with PBS which was cooled, cells were fixed at room temperature for 30min with 4% paraformaldehyde, washed 3 times with PBS, allowed to stand for 15min for membrane permeation with 0.25%TritonX100 1mL, washed 3 times with PBS, and blocked at room temperature for 1h with 0.5% BSA solution. The antibodies were rabbit anti-ASFV P72 polyclonal serum (prepared by the applicant's own laboratory) and murine Rab5 monoclonal (purchased from proteontech) diluted with 0.5% bsa solution at 1:500 and 1:200 fold, respectively, and incubated overnight at 4 ℃. Rinse 3 times with PBS, add TRITC-coat anti-Rabbit IgG (H+L) (from Boolong Co.) and FITC-coat anti-Mouse IgG (H+L) (from Rdbio Co.) as secondary antibodies, incubate 1H at room temperature in the dark, rinse 3 times with PBS in the dark. Finally, hochest nuclear dye (available from Thermo Inc.) was added and washed 3 times in PBS in the absence of light for 15min at room temperature, photographs were observed and saved using a Leica LSM800 laser confocal microscope (see FIG. 16 for fluorescence photograph results), and the intracellular co-localized Pearson coefficients were analyzed by ZEN software (see FIG. 17 for results).

ASFV is absorbed and internalized on the cell surface, enters the cell, is transported to the cell nucleus in cytoplasm through an endosomal transport path, and endosomes participating in the transport path can be specifically divided into an early endosome transport part and a late endosome transport part, and can be respectively distinguished by different molecular markers, for example, the early endosome Rab5 is a specific marker. Previous experiments demonstrated that Gamithromycin drug neither affects the adsorption nor internalization of ASFV, so it was hypothesized that this drug affects the early endocytosis transport process of ASFV. Early endocytosis transport of ASFV was tracked by confocal experiments using the target early endocytosis marker molecule Rab5 and ASFV P72 protein. The invention can show that the sample treatment group and the positive control group have no significant difference, which indicates that ASFV enters the next stage through early endocytosis. It was also shown that Gamithromycin drug did not substantially affect the early endocytosis transport process of ASFV in cells (fig. 16, fig. 17).

Example 10: effects of Gamithromycin drug on ASFV late endocytosis.

The marker molecule for late endocytosis was LBPA, so the primary murine Rab5 monoclonal antibody of example 9 was replaced with murine LBPA monoclonal antibody (available from Merck-Millipore). Otherwise, laser confocal tests were performed on each sample at 60min, 90min, 120min and 150min using the same method and reagent as in example 9 (see fig. 18 and 19 for the results).

As the endosomal transport pathway proceeds, ASFV undergoes transport in early endocytosis and then enters late endocytosis. The invention uses the specific marker LBPA of the late endocytosis to track and observe the late endosomal transport stage in the ASFV infection process through a laser confocal test. The invention discovers that the sample treatment group added with the Gamithromycin medicament and the positive control group have significant differences in 60min, 90min, 120min and 150min, which indicates that ASFV is influenced in the transportation stage into late endocytosis. Gamithrombin drug was also shown to affect ASFV transport during late endocytosis of cells (FIG. 18, FIG. 19).

Example 11: berbamine (dihydrochloride) effects of Berbamine, gamithromycin and Cycloviridoxin D drugs on ASFV release in cells.

PAMs were cultured in 24-well cell culture plates in RPMI1640 complete medium at 500 μl/well, ASFV-eGFP (moi=0.3) incubated with PAMs at 37 ℃ for 2h, washed 3 times with pbs, and fresh RPMI1640 complete medium was added. 16h after infection, 10. Mu.M Berberine (dihydrochloride) +0.1v/v% DMSO is used; 10. Mu.M Berbamine+0.1v/v% DMSO; 10. Mu.M gamithromycin+0.1v/v% DMSO; ASFV infected cells were treated with 10. Mu. M Cyclovirobuxin D +0.1v/v% EtOH for 1h, with 0.1v/v% DMSO and EtOH as controls, mock as blank well control (no virus and drug added), then washed 3 times with PBS, and fresh RPMI1640 complete medium was added. When the first cycle of ASFV is completed and new viral particles are released, 24 hours after infection, cultures are harvested and passed through TCID ₅₀ ASFV titer was measured.

The results showed that Berbamine (dihydrochloride), berbamine, gamithromycin and Cyclovirobuxin D showed no significant difference in viral titers after the action from the positive control group, and thus it was seen that these drugs did not affect ASFV release in cells (see fig. 20 for results).

Example 12: effect of 4 drug combinations on ASFV replication

PAMs were cultured in 24-well plates (1.25X10) ⁶ Cell/well), the culture medium was RPMI1640 complete medium, and after the cells were completely attached, a culture medium containing Berbamine (dihydrochloride) (code: BD), berbamine (code: b) Gamithromycin (code: g) And cyclitobiuxin D (code: CD) RPMI1640 complete medium of the drug, the type and amount of the drug added are respectively: 2.5. Mu.M Berbamine (dihydride); 2.5. Mu.M Berbamine; 2.5. Mu.M Gamithromycin;2.5 μ M Cyclovirobuxin D; 2.5. Mu.M Berbamine (dihydrochlodide) and 2.5. Mu. M Cyclovirobuxin D; a combination of 2.5 μm Berbamine and 2.5 μ M Cyclovirobuxin D; 2.5. Mu.M Gamithromycin in combination with 2.5. Mu. M Cyclovirobuxin D; 2.5. Mu.M Berbamine (dihydrochloride) and 2.5. Mu.M Gamithromycin; a combination of 2.5 μm Berbamine and 2.5 μm Gamithromycin; 2.5. Mu.M Berbamine (dihydrochlodide) and 2.5. Mu.M Berbamine; a combination of 2.5. Mu.M Berbamine (dihydrochloride), 2.5. Mu.M Berbamine, and 2.5. Mu.M Gamithromycin; 2.5. Mu.M Berbamine, 2.5. Mu.M CyclovirA combination of uxin D and 2.5 μm Gamithromycin; a combination of 2.5 μm Berbamine (dihydrochloride), 2.5 μm M Cyclovirobuxin D and 2.5 μm Gamithromycin; 2.5. Mu.M Berbamine (dihydrochloride), 2.5. Mu. M Cyclovirobuxin D, and 2.5. Mu.M Berbamine; 2.5. Mu.M Berbamine (dihydrochloride), 2.5. Mu. M Cyclovirobuxin D, 2.5. Mu.M Berbamine, and 2.5. Mu.M Gamithromycin; meanwhile, 0.1v/v% DMSO and 0.1v/v% Ethanol are added into the solution and the positive control, the Mock is taken as a negative control (no drug and virus are added), the negative control is incubated for 2 hours, the negative control is discarded, the corresponding concentration of drug and ASFV-eGFP (MOI=0.2) are inoculated for 2 hours, the cells are washed for 3 times by PBS, and RPMI1640 complete culture medium containing the same drug with the corresponding concentration is respectively added into the drug adding holes. After culturing at 37℃for 48 hours, cell supernatants and cells were collected, and qPCR (see FIG. 21 and Table 1 for results) and WB (see FIG. 22) were performed on the samples using the same reagents and methods as in example 3, respectively. In table 1, the drugs are shown in the first column, the p72 gene copy numbers for each drug and treatment are shown in column 2, the comparison of the p72 gene copy numbers for each drug and other drug treatments are shown in columns 3-6, the differences are significant, and NA represents no comparison.

TABLE 1 significance analysis of ASFV p72 Gene copy number differences by qPCR

To evaluate the effect of the combination of these 4 drugs on ASFV replication, qPCR and WB analysis was performed. qPCR results showed that the copy number of ASFV p72 in the cell supernatant was significantly lower than the corresponding drug alone control group after addition of any two, three and four combinations of these 4 drugs, indicating that they could inhibit replication of ASFV. WB results showed that after addition of any two, three and four combinations of these 4 drugs, ASFV p54 protein expression was significantly lower than for the control group with the corresponding drug alone. Therefore, the combination of two, three and four groups of the 4 drugs of berbamine, berbamine hydrochloride, and Mi Mei plus cyclovirobuxine D can also significantly inhibit the replication of ASFV, and exhibit a synergistic effect between the 4 drugs (FIGS. 21 and 22). Therefore, the berberine hydrochloride, the Mi Mei element and the cyclovirobuxine D medicine are used in combination to treat or slow down the treatment of the African swine fever virus infection.

It will be appreciated by those skilled in the art that the present invention can be carried out in other embodiments without departing from the spirit or essential characteristics thereof. Accordingly, the above disclosed embodiments are illustrative in all respects, and not exclusive. All changes that come within the scope of the invention or equivalents thereto are intended to be embraced therein.

Claims

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.