CN116236484B - Use of Berbamine dihydrochloride in inhibiting canine parvovirus - Google Patents
Use of Berbamine dihydrochloride in inhibiting canine parvovirus Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
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- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
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Abstract
The invention relates to a novel use of a known compound, in particular to an application of Berbamine dihydrochloride in inhibiting canine parvovirus. The invention provides the use of Berbamine dihydrochloride in the manufacture of a medicament for inhibiting canine parvovirus replication, the active ingredient of which comprises Berbamine dihydrochloride. The data prove that Berbamine dihydrochloride has very excellent inhibition effect on CPV replication and can effectively inhibit the expression of canine parvovirus VP2 protein.
Description
Technical Field
The invention relates to a novel use of a known compound, in particular to an application of Berbamine dihydrochloride in inhibiting canine parvovirus.
Background
Canine parvovirus (Canine parvovirus, CPV) is one of the most common and most severe causative agents in canines. CPV is the most dangerous for puppies of 2-4 months of age, and its typical clinical manifestations include vomiting, fever, diarrhea and dehydration, with higher morbidity and mortality. CPV is a non-enveloped single-stranded, negative-strand DNA virus. CPV genomic DNA is about 5300nt in length and contains two Open Reading Frames (ORFs) encoding 2 nonstructural proteins (NS 1 and NS 2) and two structural proteins (VP 1 and VP 2), respectively. Wherein VP2 protein is the major component of capsid protein, and can bind to transferrin receptor (TfR) on host cell membrane, thereby mediating canine parvovirus infection.
CPV genome substitution rates are similar to RNA viruses, resulting in the continued emergence of new variants. Viruses have been continuously mutated from the original CPV-2 type to five subtypes CPV-2a, CPV-2b, new CPV-2a, new CPV-2b and CPV-2c, all of which are reported in China. CPV hosts are numerous and the virus is detected in the body of wolves, foxes, cats, tigers, raccoons, bears, otters, jackals, leopards, and castors. These animals play an important role in the evolution and spread of CPV. It can be seen that CPV is widely prevalent, constantly varying, and spread across multiple species hosts. Although inactivated or attenuated live vaccines have been widely used for prophylaxis, the frequent occurrence of variant strains has raised concerns and concerns about the efficacy of existing vaccines. At the same time, maternal antibodies also showed a weakening effect on the vaccine.
Therefore, development of a new drug for preventing and treating diseases caused by CPV is desired.
Disclosure of Invention
The inventors found that small molecule drug Berbamine dihydrochloride (whose chemical structural formula is shown in figure 1) has a significant inhibitory effect on Canine Parvovirus (CPV) replication. The 50% cytotoxicity (50%cytotoxicity concentrations,CC50) concentration and the 50% antiviral effect (50%antiviral efficacy,EC50) concentration of the medicine are determined through in vitro experiments, and the in vitro CPV replication inhibiting effect of Berbamine dihydrochloride is further verified through an indirect immunofluorescence test (Indirect immunofluorescence assay, IFA) and a Western blot test.
In one aspect, the invention provides the use of Berbamine dihydrochloride in the manufacture of a medicament for inhibiting canine parvovirus replication, wherein the active ingredient of the medicament comprises Berbamine dihydrochloride.
In some embodiments of the use of the invention, the medicament is a single active ingredient medicament having an active ingredient of Berbamine dihydrochloride.
In other embodiments of the use according to the invention, the medicament is a combination, the active ingredient of which comprises Berbamine dihydrochloride. The medicament may also contain other antiviral ingredients.
In some embodiments of the use of the invention, the medicament further comprises a pharmaceutically acceptable carrier.
In some embodiments of the use of the invention, the pharmaceutical is in the form of a tablet, suspension or dry suspension.
In another aspect, the invention provides a medicament for inhibiting canine parvovirus replication, wherein the active ingredient of the medicament comprises Berbamine dihydrochloride.
In some embodiments of the medicament of the invention, the medicament is a single active ingredient medicament having an active ingredient of Berbamine dihydrochloride.
In other embodiments of the medicament of the present invention, the medicament is a compound medicament, the active ingredient of which comprises Berbamine dihydrochloride. The medicament may also contain other antiviral ingredients.
In some embodiments of the medicament of the invention, the medicament further comprises a pharmaceutically acceptable carrier. The carrier may be liquid or solid.
In some embodiments of the medicament of the invention, the medicament is in the form of a tablet, suspension or dry suspension.
The present invention claims, where applicable by law, a method of treating canine parvovirus infection comprising administering to an infected individual an effective dose Berbamine dihydrochloride, and after a predetermined time of administration, causing the canine parvovirus level in the serum of the infected individual to be significantly reduced or to an undetectable level.
Berbamine dihydrochloride described herein is a novel inhibitor of bcr/ab1, wherein the text is named: berbamine hydrochloride, CAS number: 6078-17-7, the molecular formula is: c 37H40N2O6, 2HCl, the structural formula is:
The inventors found that the small molecule drug Berbamine dihydrochloride can effectively inhibit CPV replication, and tested the inhibition effect on CPV in F81 cells, and the result shows that Berbamine dihydrochloride can well protect F81 cells after CPV infection. As shown in FIG. 2, the F81 cell protection rate (71.23.+ -. 6.1%) of Berbamine dihydrochloride was significantly higher than that of the control drug Cidofovir (0.86.+ -. 8.44%) when the drug concentration was 10. Mu.M. Cidofovir is a broad-spectrum anti-DNA virus drug, wherein the name is: cidofovir, CAS number: 113852-37-2, the molecular formula is: c 8H14N3O6 P.
Further, it was determined that Berbamine dihydrochloride inhibited the 50% effector concentration (EC 50) of CPV strain SD6 (New CPV-2 a) on F81 cells was 8.04 μm, and that Berbamine dihydrochloride had a 50% cytotoxic concentration (CC 50) on F81 cells of 17.66 μm (fig. 3).
Further, the inhibition of canine parvovirus VP2 protein expression by Berbamine dihydrochloride was examined by immunofluorescence assay (IFA). As shown in FIG. 4, the fluorescence signal of canine parvovirus VP2 protein decreased with increasing Berbamine dihydrochloride concentration. When Berbamine dihydrochloride final concentration was 5 μm, the fluorescence signal was reduced in the drug-treated group compared to the control group; the drug treated group showed a significant decrease in fluorescence signal compared to the control group at Berbamine dihydrochloride final concentrations of 10 μm and 15 μm. Thus, berbamine dihydrochloride was shown to be effective in inhibiting CPV replication in F81 cells.
Further, the inhibition of Berbamine dihydrochloride on canine parvovirus VP2 protein expression was examined by Western blot. The results showed that the expression level of canine parvovirus VP2 protein gradually decreased with increasing Berbamine dihydrochloride concentration, further verifying the effect of the drug in inhibiting CPV replication (FIG. 5).
In summary, the present invention provides candidate drug Berbamine dihydrochloride effective against Canine Parvovirus (CPV), which provides a reference for clinical use. Berbamine dihydrochloride is developed into an anti-CPV drug, and has good application prospect.
Drawings
FIG. 1 is a structural formula Berbamine dihydrochloride.
FIG. 2 shows experimental results of in vitro inhibition of CPV replication by small molecule drugs. The abscissa is the drug name and the ordinate is the percent (%) CPE inhibition of the drug on CPV in F81 cells. The percentage CPE inhibition of Berbaminedihydrochloride on CPV in F81 cells was 71.23 ±6.1%. The control drug Cidofovir had a CPE inhibition percentage of 0.86.+ -. 8.44% for CPV in F81 cells.
FIG. 3 shows the results of a 50% effector concentration (EC 50) of Berbaminedihydrochloride for inhibiting infection of F81 cells by CPV strain SD6 (NewCPV-2 a) and a 50% cytotoxicity concentration (CC 50) of Berbaminedihydrochloride on F81 cells.
FIG. 4 shows the result of Berbamine dihydrochloride immunofluorescence assay for inhibiting expression of canine parvovirus VP2 protein. The upper panel is the detection result of the Control group, wherein Control FITC is the FITC fluorescence signal (showing virus number) of the Control group, control DAPI is the DAPI fluorescence signal (showing cell number) of the Control group, and Control Merge is the combined graph of the FITC fluorescence signal and the DAPI fluorescence signal of the Control group. The lower panel is the result of the Berbamine dihydrochloride treatment group and is a combined plot of FITC fluorescent signal and DAPI fluorescent signal, where 5. Mu.M, 10. Mu.M, and 15. Mu.M represent the final concentration of Berbamine dihydrochloride in the assay system.
FIG. 5 shows the result of Westernblot analysis of Berbaminedihydrochloride for inhibiting expression of VP2 protein of canine parvovirus. The upper panel shows the detection result of canine parvovirus VP2 protein, and the lower panel shows the detection result of beta-actin. VP2 is the target protein detected, and beta-actin is used as an internal reference. DMSO represents control group; 5, 10, 20 represent drug-treated groups with Berbaminedihydrochloride final concentrations of 5. Mu.M, 10. Mu.M and 20. Mu.M, respectively.
Detailed Description
The present invention will be described in detail with reference to the following examples, which are to be understood as merely illustrative and explanatory of the invention, and are not in any way limiting to the scope of the invention.
The main reagents used in the following examples
Berbaminedihydrochloride: purchased from seleck (USA), drug parameters: s3609. Berbaminedihydrochloride A medicinal solution was prepared using DMSO as a solvent in the following examples.
Cidofovir: purchased from seleck (USA), drug parameters: s1516. Cidofovir A medicinal solution was prepared using DMSO as a solvent in the following examples.
DMSO (dimethyl sulfoxide): CAS number 67-68-5, sigma-Aldrich, D2650.
CellTiterAQueousOneSolutionCellProliferationAssay (MTS): purchased from PromegaBiotech, USA, catalog number: G3580.
PBS: purchased from Gibco TM, USA, cat: 20012050.
Mouse anti-VP 2 monoclonal antibody: purchased from INGENASA, spanish, cat No.: m.15.cpv.i5f8.
FITC-labeled goat anti-mouse IgG (h+l) secondary antibody: purchased from ThermoScientific, USA, cat No.: A16079.
4, 6-Diamidino-2-phenylindole Dihydrochloride (DAPI): CAS number 28718-90-3, available from ThermoScientific, D3571.
Mammalian total protein extraction kitMammalianTotalProteinExtractionKit): purchased from TransGenBiotech, china, catalog number: DE101-01.
20 XTBS-Tween 20: purchased from Thermo Scientific, cat No. 28360.
Beta-actin monoclonal antibody (AC-15): purchased from Thermo Scientific, USA, cat: MA1-91399.
HRP-labeled goat anti-mouse IgG: purchased from Thermo Scientific, USA, cat: 31430.
SuperSignal TM West Pico PLUS chemiluminescent substrate detection kit, available from Thermo Scientific, USA, cat: 34579.
Cells and viruses used in the examples below
F81 cells were purchased from ATCC (AMERICAN TYPE Culture Collection, american type culture Collection, also known as American type culture Collection). The F81 cells are cat kidney cells, and are engineering cell lines transformed from cat kidney primary cells through passage cloning screening.
New CPV-2a strain SD6 is isolated and stored by Beijing city key laboratory of livestock epidemic prevention and control technology of livestock and poultry veterinary institute of the national academy of sciences of agriculture and forestry in Beijing city. This strain is described in non-patent document "Hongzhuan Zhou,Xia Su,Lulu Lin,JinZhang,Qi Qi,Fangfang Guo,Fuzhou Xu,Bing Yang.Inhibitory Effects ofAntiviralDrugCandidates on Canine Parvovirus in F81Cells.Viruses.2019Aug;11(8)：742.doi：10.3390/v11080742.PMID：31412574.". The homology of the VP2 protein coding gene of the New CPV-2a strain SD6 with the nucleotide sequences of the VP2 protein coding genes corresponding to BJ14-7 (New CPV-2a strain, genBank: KT162031, PMID: 26692471) and BJ14-1 (New CPV-2b strain, genBank: KT162022, PMID: 26692471) is up to 99.9% and 99.3%. Applicant states that: new CPV-2a strain SD6 can be provided to the public for research purposes within twenty years from the date of filing.
The media used in the following examples
Maintenance Medium (MM): DMEM (Gibco, USA) containing 2% foetal calf serum (Gibco, USA), 100U/ml penicillin and 100. Mu.g/ml streptomycin.
Unless otherwise indicated, all reagents used in the examples below are conventional in the art and are commercially available or formulated according to conventional methods in the art, and are of laboratory grade. Unless otherwise indicated, the experimental methods and experimental conditions used in the following examples are those conventional in the art, and reference may be made to the relevant experimental manuals, well-known documents or manufacturer's instructions. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Example 1.Berbamine dihydrochloride determination of the inhibition of CPV in F81 cells
F81 cells were purchased prior to use. mu.L of 10mM Berbamine dihydrochloride (U.S. Selleck, S3609) and 4. Mu.L of 10mM control drug Cidofovir (U.S. Selleck, S1516) were each added to 156. Mu.L of Maintenance Medium (MM) to prepare 250. Mu.M drug stock solutions.
Drug treatment group: f81 cells were treated with drug stock solutions Berbamine dihydrochloride and Cidofovir, respectively. The method comprises the following steps: after adding 4. Mu.L of 250. Mu.M stock solution of drug to 86. Mu. L F81 cells (25,000 cells per well) and continuing the treatment for 1 hour, the drug-treated F81 cells were infected with 10. Mu.L of CPV (New CPV-2a type strain SD 6) at MOI (multiplicity of infection ) of 0.076, and cell viability was examined 40 hours after infection. The final concentration of drug in the test system was 10. Mu.M.
Positive control: DMSO (Sigma-Aldrich, D2650) was added to F81 cells to a final concentration of 0.1% (volume fraction). Cell viability was measured 40 hours after DMSO addition.
Negative control: to F81 cells, DMSO (Sigma-Aldrich, D2650) was added to a final concentration of 0.1% (volume fraction), and after 1h F81 cells were infected with CPV (New CPV-2a strain SD 6) at an MOI of 0.076. Cell viability was measured 40 hours after infection.
By CellTiterAQueous One Solution Cell Proliferation Assay (MTS) (Promega Biotech, USA, G3580) cell viability was measured according to the kit instructions. Wells without CPV infection were used as positive (cell) control wells for 100% inhibition protection, and wells without any drug added to infect CPV (New CPV-2a strain SD 6) were used as negative (virus) control wells for 0% inhibition protection. Percent inhibition was calculated using the following formula: percent CPE inhibition (%) = (OD 450 of drug treated group-OD 450 of negative control)/(OD 450 of positive control-OD 450 of negative control) ×100. The test was repeated 3 times.
The experimental results are shown in fig. 2:
the inhibition protection test result shows that Berbamine dihydrochloride can well protect cells after CPV infection. According to the experimental definition, wells without CPV infection served as 100% inhibition protection positive control, while wells infected with CPV (without any drug added) served as 0% inhibition protection negative control. The inhibition protection rate (i.e. percent CPE inhibition) of Berbamine dihydrochloride was 71.23 ±6.1% as calculated by the formula, whereas the inhibition protection rate (i.e. percent CPE inhibition) of the broad-spectrum anti-DNA virus drug Cidofovir was only 0.86±8.44%, the difference between them being significant (P < 0.05).
Example 2 determination of 50% Effect concentration (EC 50) and 50% cytotoxicity concentration (CC 50) of Berbaminedihydrochloride in inhibiting CPV infected cells
The 50% effector concentration (50% anti-viralefficacy, EC 50) and 50% cytotoxic concentration (50%cytotoxicity concentrations,CC50) of Berbamine dihydrochloride to F81 cells was determined by dose-response assay Berbamine dihydrochloride to inhibit infection of F81 cells with CPV strain SD6 (NewCPV-2 a).
The EC50 determination steps are: 86 μ L F81 cells (25,000 cells per well) were pre-treated with Berbamine dihydrochloride diluted at a 4 μl fold ratio (final concentration range of 0.625-10 μM) for 1h, then infected with Berbaminedihydrochloride treated F81 cells with 10 μl CPV (New CPV-2a strain SD 6) at a MOI (multiplicity of infection) of 0.076.
The CC50 measurement steps are as follows: 96 μ L F81 cells (25,000 cells per well) were mixed with 4 μl of diluted Berbamine dihydrochloride (final concentration range 2.5-80 μΜ).
Both the EC50 assay and the CC50 assay were performed in 3 replicates. After incubation for 40 hours, cellTiter was usedAQueous One Solution Cell Proliferation Assay (MTS) (Promega Biotech, USA, G3580) was assayed for cell viability according to the kit instructions to determine the inhibition efficiency of Berbamine dihydrochloride against CPV infection and the toxicity of Berbamine dihydrochloride against F81 cells, and EC50 and CC50 values were calculated by the dose-response nonlinear regression analysis method in GRAPHPAD PRISM software.
The experimental results are shown in fig. 3:
berbamine dihydrochloride 50% antiviral effect concentration (EC 50) of 50% inhibiting infection of F81 cells with New CPV-2a type strain SD6 was 8.04. Mu.M.
Berbamine dihydrochloride 50% cytotoxic concentration (CC 50) on F81 cells was 17.66. Mu.M.
EXAMPLE 3 detection of Berbamine dihydrochloride inhibition of canine parvovirus VP2 protein expression by immunofluorescence assay (Immunofluorescenceassay, IFA)
Drug treatment group: 86. Mu.L of F81 cells (25,000 cells per well) in 96-well plates were pre-treated with Berbaminedihydrochloride (final concentrations of 5. Mu.M, 10. Mu.M and 15. Mu.M, respectively) diluted by a 4. Mu.L-fold ratio for 1 hour, and the treated cells were infected with 10. Mu.L of CPV (New CPV-2a strain SD 6) (MOI=0.076) and immunofluorescence assay was performed 30 hours after infection.
Control group: to F81 cells in 96-well plates, DMSO (Sigma-Aldrich, D2650) was added to a final concentration of 0.1% (volume fraction), and after 1h the cells were infected with CPV (New CPV-2a strain SD 6) (MOI=0.076), and immunofluorescence experiments were performed 30 hours after infection.
The immunofluorescence assay was performed as follows:
Fixing: the culture medium in the 96-well plate was discarded, and washed 3 times with 1 XPBS (Gibco TM, USA, cat# 20012050) for 2-3 min/time. Cells were fixed with 80% acetone (4:1 acetone to water volume ratio), 100. Mu.L of 80% acetone was added to each well and incubated at room temperature for 10min. Washing with 1 XPBS for 3 times and 2-3 min/time.
Closing: 3% BSA (3 g BSA per 100mL 1 XPBS) was added at room temperature and blocked for 1 hour at room temperature. Washing with 1 XPBS for 10min.
An antibody: incubation with mouse anti-VP 2 monoclonal antibody (INGENASA, spain, M.15.CPV. I5F 8) (diluted 1:100 in 1 XPBS) for 40 min. The antibody solution was discarded, and washed 3 times with 1 XPBS for 10 min/time.
And (2) secondary antibody: incubation with FITC-labeled goat anti-mouse IgG (H+L) secondary antibody (Invitrogen, USA, A16079) (diluted 1:200 in 1 XPBS) for 40min. The antibody solution was discarded, and washed 3 times with 1 XPBS for 10 min/time.
Dyeing: dyeing with 4, 6-diamidino-2-phenylindole dihydrochloride (DAPI, 1. Mu.g/ml) and reacting for 10min at room temperature in the absence of light. The staining solution was discarded, washed 3 times with 1 XPBS for 10 min/time.
Cells were examined with an Axio observer.z1 inverted fluorescence microscope (Zeiss, germany) at 10 x magnification and analyzed by Image J Image processing software.
As a result, as shown in FIG. 4, the fluorescence signal of canine parvovirus VP2 protein was strong in the control group that was not treated with the drug, indicating that there was a large amount of canine parvovirus VP2 protein expressed (FIG. 4, control). In the drug-treated group, the fluorescence signal of canine parvovirus VP2 protein gradually decreased with increasing Berbamine dihydrochloride concentration. When Berbamine dihydrochloride final concentration was 5uM, the fluorescence signal was reduced in the drug-treated group compared to the control group; at Berbamine dihydrochloride final concentrations of 10 μm and 15 μm, the fluorescence signal was significantly reduced in the drug-treated group compared to the control group (fig. 4,Berbamine dihydrochloride). Thus, berbamine dihydrochloride was shown to be able to successfully inhibit CPV replication in F81 cells.
Example 4 detection of Berbamine dihydrochloride inhibition of canine parvovirus VP2 protein expression by Western blot assay
Drug treatment group: f81 cells were seeded at 7.5×10 5 cells per well in 6-well plates and pre-treated with different concentrations of Berbamine dihydrochloride (final concentrations of 5 μm,10 μm and 20 μm, respectively) for 1 hour, cells after Berbamine dihydrochloride treatment at each concentration were infected with CPV (New CPV-2a strain SD 6) (moi=0.076), and after 40 hours incubation total cell proteins were extracted and subjected to Westernblot assay.
Control group: DMSO (Sigma-Aldrich, D2650) was added to F81 cells to a final concentration of 0.1% (volume fraction), cells were infected with CPV (New CPV-2a type strain SD 6) at a MOI of 0.076 after 1h, and after 40 hours of incubation the total cell proteins were extracted and Western blot assays were performed.
Protein extraction and Western blot test:
Cells were collected and lysed using a mammalian total protein extraction kit ProteinExt (TransGen Biotech, china, DE1o 1-01) according to the kit instructions. Equal amounts of cell lysates were transferred to PVDF membrane (Millipore, USA) after SDS-PAGE proteins. The transferred PVDF film was blocked with TBS-Tween20 containing 5% skimmed milk (5 g skimmed milk powder per 100mL TBS-Tween 20) at room temperature for l hours. Mouse anti-VP 2 monoclonal antibody (INGENASA, spain, M.15.CPV. I5F 8) (diluted 1:800 with TBS-Tween 20) and beta-actin monoclonal antibody (AC-15) (Thermo Scientific, USA, MA 1-91399) (diluted 1:4000 with TBS-Tween 20) were used, respectively, for overnight incubation at 4 ℃. TBS-Tween20 was washed 3 times, 10 min/time. HRP-labeled goat anti-mouse IgG (Thermo Scientific, USA, cat# 31430) (diluted 1:6000 with TBS-Tween 20) was used for incubation at 37℃for 1 hour. TBS-Tween20 was washed 3 times, 10 min/time. The strips were detected using a SuperSignal TM West Pico PLUS chemiluminescent substrate detection kit (Thermo Scientific, USA, 34579) according to the kit instructions and imaged by a chemiluminescent device (Protein simple, USA).
As shown in FIG. 5, the Western blot results show that Berbamine dihydrochloride can inhibit VP2 protein expression of New CPV-2a strain SD6, and the difference is obvious compared with a control group without adding a drug. And the expression level of the VP2 protein of the canine parvovirus gradually decreases along with the increase of Berbamine dihydrochloride concentration. At Berbamine dihydrochloride final concentrations of 10 μm and 20 μm, the bands were weak, demonstrating that Berbamine dihydrochloride was able to well inhibit the replication of New CPV-2a strain SD6 (FIG. 5).
Claims (5)
1. Use of berbamine hydrochloride for the manufacture of a medicament for inhibiting canine parvovirus replication, characterized in that the active ingredient of the medicament comprises berbamine hydrochloride.
2. The use according to claim 1, wherein the medicament is a single active ingredient medicament, the active ingredient of which is berbamine hydrochloride.
3. The use according to claim 1, wherein the medicament is a compound medicament, the active ingredient of which comprises berbamine hydrochloride.
4. The use according to any one of claims 1-3, wherein the medicament further comprises a pharmaceutically acceptable carrier.
5. The use according to claim 4, wherein the pharmaceutical dosage form is a tablet, suspension or dry suspension.
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