CN111840308B - Application of 6 small-molecule drugs in inhibition of canine parvovirus - Google Patents
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Abstract
The invention relates to a new application of a known compound, in particular to an application of 6 small molecule drugs in inhibiting canine parvovirus. The invention provides application of Closantel, Closantel Sodium, Gemcitabine HCl, Trifluridine, Gemcitabine and Cladribine in preparing a medicament for inhibiting canine parvovirus replication, wherein the active ingredients of the medicament comprise Closantel, Closantel Sodium, Gemcitabine HCl, Trifluridine, Gemcitabine and/or Cladribine. The data prove that the 6 small molecular drugs have very excellent inhibition effect on CPV replication and can inhibit the expression of CPV VP2 protein of different genotypes.
Description
Technical Field
The invention relates to a new application of a known compound, in particular to an application of Closantel, Closantel Sodium, Gemcitabine HCl, Trifluridine, Gemcitabine and Cladribine in inhibiting canine parvovirus.
Background
Canine Parvovirus (CPV) is a single-stranded DNA virus with a simple structure, is a member of the family parvoviridae, is non-enveloped and is icosahedral. The viral genome is about 5300nt in length, and contains 2 ORFs, the 5 'end mainly encodes early-transcribed regulatory proteins (NS1 and NS2), and the 3' end encodes late-transcribed structural proteins, namely viral capsid proteins (VP1 and VP 2). The VP2 protein is the major component of capsid protein, and can bind to transferrin receptor (TfR) on host cell membrane, thereby mediating parvovirus infection. CPV is one of the major causes of acute gastroenteritis, leukopenia and myocarditis in dogs and is widely found in carnivores. Typical clinical symptoms of CPV infection include vomiting, fever, diarrhea, and susceptibility to puppies particularly 6 weeks to 6 months.
CPV virus is widely distributed worldwide, and after the emergence of CPV-2 in the late 70 s of the 20 th century, CPV-2 and its variants have been reported in several countries in five continents. Since there are no specific anti-CPV drugs, the only treatment options left are supportive therapy and symptom-based care, except for prevention by vaccine. CPV genome replacement rates are similar to RNA viruses, and only for a few years the earliest CPV-2 type was replaced by three major subspecies, CPV-2a, CPV-2b and CPV-2 c. Although inactivated or live-attenuated vaccines have been widely used for prophylaxis, the frequent emergence of variant strains has raised concerns and concerns about the efficacy of existing vaccines, and at the same time, maternal antibodies have also been shown to impair the vaccine.
Therefore, the development of new drugs for preventing and treating CPV virus is urgently needed.
Disclosure of Invention
The inventor finds that 6 small-molecule drugs Closantel, Closantel Sodium, Gemcitabine HCl, Trifluridine, Gemcitabine and Cladribine (chemical structural formula is shown in figure 1) have inhibition effect on Canine Parvovirus (CPV) replication. The concentration of 50% cytotoxicity (CC50) and 50% antiviral effect (EC50) of the drug was determined by in vitro experiments, and the effect of these 6 small molecule drugs in inhibiting CPV replication in vitro was further verified by Indirect immunofluorescence assay (IFA) and Western blot assay.
In one aspect, the present invention provides the use of Closantel, Closantel Sodium, Gemcitabine HCl, Trifluridine, Gemcitabine and Cladribine for the preparation of a medicament for inhibiting canine parvovirus replication, wherein the active ingredient of the medicament comprises Closantel, Closantel Sodium, Gemcitabine HCl, Trifluridine, Gemcitabine and/or Cladribine.
In some embodiments of the use of the invention, the medicament is a single active ingredient medicament having one of Closantel, Closantel Sodium, Gemcitabine HCl, Trifluridine, Gemcitabine and Cladribine as an active ingredient.
In other embodiments of the use of the present invention, the medicament is a combination medicament comprising as active ingredients two or more of Closantel, Closantel Sodium, Gemcitabine HCl, Trifluridine, Gemcitabine and Cladridine. In some embodiments, other antiviral components may also be included.
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 present invention, the pharmaceutical is in the form of a tablet, suspension or dry suspension.
In another aspect, the present invention provides a medicament for inhibiting canine parvovirus replication, wherein the active ingredient of the medicament comprises Closantel, Closantel Sodium, Gemcitabine HCl, trifluraline, Gemcitabine and/or Cladribine.
In some embodiments of the agents of the present invention, the agent is a single active ingredient agent having one of Closantel, Closantel Sodium, Gemcitabine HCl, Trifluridine, Gemcitabine and Cladribine as an active ingredient.
In other embodiments of the pharmaceutical of the present invention, the pharmaceutical is a combination drug, and the active ingredient comprises two or more of Closantel, Closantel Sodium, Gemcitabine HCl, Trifluridine, Gemcitabine and Cladridine. In some embodiments, other antiviral components may also be included.
In some embodiments of the medicament of the present invention, the medicament further comprises a pharmaceutically acceptable carrier component that is liquid or solid.
In some embodiments of the pharmaceutical of the present invention, the pharmaceutical is in the form of a tablet, suspension, or dry suspension.
The present invention claims, where permitted by law, a method of treating canine parvovirus infection, comprising administering to an infected individual an effective amount of any of the foregoing agents, wherein after a predetermined period of time, the level of canine parvovirus in the serum of said infected individual is significantly reduced or reduced to an undetectable level.
Closantel Sodium, chinese name: closantel sodium, CAS number: 61438-64-0, formula: c22H13Cl2I2N2O2Na, having the formula shown in FIG. 1A, is a gram-positive inhibitor of antibacterial activity.
Closantel, chinese name: closantel, CAS number: 57808-65-8, molecular formula: c22H14Cl2I2N2O2The structural formula is shown in figure 1B, and the compound is a gram-positive antibacterial activity inhibitor.
Gemcitabine HCl, chinese name: gemcitabine hydrochloride, CAS number: 122111-03-9, formula: c9H11F2N3O4HCl, having the structural formula shown in fig. 1C, is an inhibitor of DNA and nucleic acid synthesis.
Gemcitabine, chinese name: gemcitabine, CAS number: 95058-81-4, molecular formula: c9H11F2N3O4The structural formula is shown in figure 1E, and is a DNA and nucleic acid synthesis inhibitor.
Cladripine, chinese name: cladribine, CAS number: 4291-63-8, molecular formula: c10H12ClN5O3The structural formula is shown in figure 1D, and the adenosine deaminase inhibitor is provided.
Trifluridine, chinese name: trifluorothymidine, CAS No.: 70-00-8, molecular formula: c10H11F3N2O5The structural formula is shown in figure 1F, and is an anti-herpes virus drug.
The inventor finds that Closantel, Closantel Sodium, Gemcitabine HCl, Trifluridine, Gemcitabine and Cladribine can inhibit the replication of a CPV strain, and determines the CPV inhibition protection effect on F81 cells, and the result shows that 6 small-molecule drugs can well protect the cells after CPV infection. As shown in figure 2, when the final concentration of the drug is 10 μ M, the inhibition protection rates of F81 cells by Closantel, Closantel Sodium, Gemcitabine HCl, Trifluridine, Gemcitabine and Cladribine are 80.64 + -7.87, 69.76 + -6.06, 64.18 + -0.97, 57.11 + -5.45, 56.1 + -2.09 and 50.92 + -1.58 respectively, which are obviously higher than those of a control drug Cidofovir (Chinese name: Cidofovir, CAS number: 113852-37-2, molecular formula: C: 37-2)8H14N3O6P, broad spectrum anti-DNA virus drug) of-1.28 ± 1.03%.
Further, 50% effector concentration (EC50) and 50% cytotoxic concentration (CC50) of Closantel, Closantel Sodium, Gemcitabine HCl, Trifluridine, Gemcitabine and Cladribine against CPV strain SD6(New CPV-2a) were determined, and the results of 6 small molecule drugs inhibiting EC50 infection of F81 cells by strain SD6 and CC50 are shown in FIG. 3 and FIG. 4.
The inventors further performed an immunofluorescence assay (IFA), and the results showed that the fluorescence signal expressed by VP2 was lower than that of the control group without drug when the final drug concentration was 5. mu.M, and almost no fluorescence signal was detected when the final drug concentration was 10. mu.M or 20. mu.M, demonstrating that 6 small molecule drugs can successfully inhibit CPV replication in F81 cells (FIG. 5).
In addition, Western blot results show that the 6 small molecule drugs can inhibit the expression of VP2 proteins of different genotypes of viruses, and the expression levels of VP2 proteins of different genotypes are gradually reduced with the increase of the drug concentration, so that the effect of the drug in inhibiting CPV replication is further verified (FIG. 6).
In conclusion, the candidate drugs Closantel, Closantel Sodium, Gemcitabine HCl, Trifluridine, Gemcitabine and Cladribine which can effectively resist Canine Parvovirus (CPV) are screened out, and the candidate drugs are mature clinical drugs, so that the reference is provided for clinical medication, and the candidate drugs are developed into the CPV-resistant drugs, and have a great application prospect.
Drawings
FIG. 1 shows the structural formulas of the small molecule drugs Closantel, Closantel Sodium, Gemcitabine HCl, Trifluridine, Gemcitabine and Cladridine.
FIG. 2 shows the experimental results of the in vitro inhibition of CPV replication by small molecule drugs; the abscissa is the drug name and the ordinate is the percentage of CPE inhibition. The percent inhibition of F81 cells by Closantel, Closantel Sodium, Gemcitabine HCl, Trifluridine, Gemcitabine and Cladribine were 80.64 + -7.87, 69.76 + -6.06, 64.18 + -0.97, 57.11 + -5.45, 56.1 + -2.09 and 50.92 + -1.58%, respectively. The percent inhibition of F81 cells by the control drug Cidofovir was-1.28. + -. 1.03%.
FIG. 3 results of CC50 and EC50 assays of small molecule drugs on F81 cells; a, B, C are the results of CC50 and EC50 assays for Closantel Sodium, Closantel and Gemcitabine HCl, respectively.
FIG. 4. results of CC50 and EC50 assays of small molecule drugs on F81 cells; the results of CC50 and EC50 assays for D, E and F respectively, Cladribine, Gemcitabine and Trifluridine.
FIG. 5 shows the results of immunofluorescence assay for the inhibition of viral VP2 protein expression by small molecule drugs. 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 pool is a pool plot of FITC fluorescence signal and DAPI fluorescence signal of the control group. The pictures of Closantel Sodium, Closantel, Gemcitabine HCl, Cladribine, Gemcitabine and Trifluridine are all merged pictures of FITC fluorescence signal and DAPI fluorescence signal. 5 μ M, 10 μ M and 20 μ M represent the final concentration of drug in the experimental system.
FIG. 6 shows the inhibition experiment results of small molecule drugs on different genotype CPV strains (Western blot experiment). DMSO represents the control group, and 5, 10, 20 represent the treatment groups with final drug concentrations of 5. mu.M, 10. mu.M and 20. mu.M, respectively. VP2 is the target protein for detection, and beta-actin is used as an internal reference.
Detailed Description
The present invention is described in detail below with reference to examples, it being understood that the following examples are only illustrative and illustrative of the present invention and do not limit the scope of the present invention in any way.
Experimental reagent
Growth Medium (GM): DMEM (Gibco, USA) plus 10% fetal bovine serum (Gibco, USA), 100U/ml penicillin, 100. mu.g/ml streptomycin.
Maintenance Medium (MM): DMEM (Gibco, USA) plus 2% fetal bovine serum (Gibco, USA) and 100U/ml penicillin and 100. mu.g/ml streptomycin.
Closantel: from seleck (usa), drug parameters: s4106 (concentration: 10 mM).
Closantel Sodium: from seleck (usa), drug parameters: s4105 (concentration: 10 mM).
Gemcitabine HCl: from seleck (usa), drug parameters: s1149 (concentration: 10 mM).
Trifluridine: from seleck (usa), drug parameters: s1778 (concentration: 10 mM).
Gemcitabine: from seleck (usa), drug parameters: s1714 (concentration: 10 mM).
Cladripine: from seleck (usa), drug parameters: s1199 (concentration: 10 mM).
Cidofovir: from seleck (usa), drug parameters: s1516 (concentration: 10 mM).
anti-VP 2 monoclonal antibody, purchased from INGENASA, spain, cat No.: M.15.CPV.I5F8.
Secondary FITC-labeled goat anti-mouse IgG (H + L) antibody (1: 200 dilution) was purchased from Thermo Scientific, USA, cat #: A16079.
beta-actin monoclonal antibody (AC-15), purchased from Thermo Scientific, USA, cat #: MA 1-91399.
HRP-labeled goat anti-mouse IgG was purchased from Thermo Scientific, USA, cat #: 31430.
Extraction kit for total mammalian protein (
Mammalian Total Protein Extraction Kit), transcgen Biotech, China, catalog No.: DE 101-01.
SuperSignal TMWest Pico PLUS chemiluminescent substrate detection kit, purchased from Thermo Scientific, USA, cat #: 34579.
DMSO (dimethyl sulfoxide): CAS number 67-68-5, Sigma-Aldrich, D2650.
4, 6-diamidino-2-phenylindole Dihydrochloride (DAPI): CAS number 28718-90-3, Thermo Scientific, D3571.
20 × TBS-Tween 20: purchased from Thermo Scientific, cat # 28360.
PBS from GibcoTMUSA, cargo number: 20012050.
cells and viruses
F81 cells were purchased from ATCC (American type culture Collection, also known as American type culture Collection). F81 cells were cultured in Growth Medium (GM) consisting of DMEM (Gibco, USA), 10% fetal bovine serum (Gibco, USA) and 100U/ml penicillin and 100. mu.g/ml streptomycin. Maintenance Medium (MM) consisted of DMEM (Gibco, USA), 2% fetal bovine serum (Gibco, USA) and 100U/ml penicillin and 100. mu.g/ml streptomycin.
The New CPV-2a type strain SD6 and the New CPV-2b type strain SD3 are separated and stored in a key laboratory in Beijing City in the prevention and control technology of the livestock and poultry epidemic disease of the institute of livestock and veterinary, academy of agriculture and forestry, Beijing City. The nucleotide sequence homology of VP2 protein coding genes of the strain SD6 and the strain SD3 with corresponding VP2 protein coding genes of BJ14-7(GenBank: KT162031, New CPV-2a) and BJ14-1(GenBank: KT162022, New CPV-2b) is as high as 99.9% and 99.3%. Non-patent documents describing strain SD6 and strain SD3 are: hongzhuan Zhou, Xia Su, Lulu Lin, Jin Zhuang, Qi Qi, Fangfang Guo, Fuzhou Xu, Bing Yang. inhibition Effects of anti Drug drugs on cancer Parvovirus in F81 cells. viruses.2019 Aug; 742: doi 10.3390/v11080742: PMID 31412574.
The applicants state that within twenty years from the filing date, New CPV-2a strain SD6 and New CPV-2b strain SD3 can be provided to the public for research purposes.
Unless otherwise specified, the reagents used in the following examples are conventional in the art, and are either commercially available or formulated according to methods conventional in the art, and may be of laboratory pure grade. Unless otherwise specified, the experimental methods and experimental conditions used in the following examples are all conventional in the art, and reference may be made to relevant experimental manuals, well-known literature, 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 1.6 determination of the protective Effect of Small molecule drugs on CPV inhibition in F81 cells
F81 cells before use, 4. mu.L of small molecule drug (Closantel/Closantel Sodium/Gemcitabine HCl/Trifluridine/Gemcitabine/Cladribine) (10mM) and 4. mu.L of control drug Cidofovir (10mM) were added to 156. mu.L of Maintenance Medium (MM), respectively, to prepare 250. mu.M drug stock solutions.
Drug treatment group: f81 cells were treated separately with each drug stock solution. To 86 μ L F81 cells (25,000 cells per well) was added 4 μ L of 250 μ M drug stock solution at a final concentration of 10 μ M of each drug, and after 1h of treatment, the drug-treated F81 cells were infected with 10 μ L of CPV (New CPV-2a type strain SD6) at an MOI (multiplicity of infection) of 0.076. Cell viability was measured 40 hours after infection.
Positive control: DMSO 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: DMSO was added to F81 cells to a final concentration of 0.1% (volume fraction), and F81 cells were infected with CPV (New CPV-2a type strain SD6) at an MOI of 0.076 after 1 h. Cell viability was measured 40 hours after infection.
By using
Cell Counting Kit (TransGen Biotech, China, FC101-01) Cell viability was determined according to the Kit instructions. Wells without CPV infection served as positive (cell) control wells with 100% inhibition protection, and wells infected with CPV (New CPV-type 2a strain SD6) served as negative (virus) control wells with 0% inhibition protection. Percent inhibition was calculated using the following formula: percent CPE inhibition (OD 450 of drug-treated cells-OD 450 of negative control)/(OD 450 of positive control-OD 450 of negative control) 100, the experiment was repeated 3 times.
The results are shown in FIG. 2:
the inhibition protection test result shows that the 6 kinds of small molecule drugs can well protect cells infected by CPV. The wells without CPV infection were defined experimentally as a positive control for 100% inhibition protection, and the wells infected with CPV (without any drug) were defined as a negative control for 0% inhibition protection, and the inhibition protection rates (i.e., percentage of CPE inhibition) of Closantel, Closantel Sodium, Gemcitabine HCl, Trifluridine, Gemcitabine and Cladribine were 80.64 + -7.87, 69.76 + -6.06, 64.18 + -0.97, 57.11 + -5.45, 56.1 + -2.09 and 50.92 + -1.58%, respectively, while the inhibition protection rate (i.e., percentage of CPE inhibition) of the broad spectrum anti-DNA virus drug Cidofovir was only-1.28 + -1.03%, with a very significant difference (P <0.0001), as shown in FIG. 2.
Example 2.6 determination of 50% Effect concentration (50% antiviral efficacy, EC50) and 50% cytotoxic concentration (50% cytoxicity concentrations, CC50) of Small molecule drugs
The EC50 and CC50 of the drugs were determined using a dose response assay. The method comprises the following specific steps:
the EC50 determination procedure was: 86 μ L F81 cells (25,000 cells per well) were pretreated with 4 μ L of drug diluted at a fold ratio (Closantel/Closantel Sodium/Gemcitabine HCl/Trifluridine/Gemcitabine/Cladribine) (final concentration range 0.3125-20 μ M) for 1h, and then drug-treated cells were infected with 10 μ L of CPV (New CPV-2a strain SD6) (MOI ═ 0.076).
The CC50 determination steps are as follows: 96 μ L F81 cells (25,000 cells per well) were mixed with 4 μ L of drug diluted in multiples (final concentration range 0.3125-80 μ M).
Both EC50 and CC50 assays were performed in 3 replicates. After 40 hours of incubation, the cells were incubated with
Cell Counting Kit (TransGen Biotech, China, FC101-01) determines Cell viability according to the Kit using instructions, further determines the inhibition efficiency of 6 small molecule drugs on CPV infection and the toxicity of each drug on F81 cells, and calculates EC50 and CC50 values by a dose response nonlinear regression analysis method in GraphPad Prism software.
The results are shown in FIGS. 3 and 4:
the median antiviral effect concentrations (EC50) of Closantel, Closantel Sodium, Gemcitabine HCl, Trifluridine, Gemcitabine and Cladribine inhibitory strain SD6(New CPV-2a) on F81 cell infection were 7.77. mu.M, 6.01. mu.M, 0.68. mu.M, 9.35. mu.M, 0.62. mu.M and 0.32. mu.M, respectively.
The half-toxic concentrations of Closantel, Closantel Sodium, Gemcitabine HCl, Trifluridine, Gemcitabine and Cladribine to F81 cells (CC50) were 13.69. mu.M, 19.08. mu.M, 141.6. mu.M, > 160. mu.M, 40.03. mu.M and 40.21. mu.M, respectively.
Example 3 immunofluorescent assay (IFA) to detect inhibition of viral VP2 protein expression by 6 small molecule drugs
Drug treatment group: 86 μ L (25,000 cells per well) of F81 cells in a 96-well plate were pretreated for 1 hour with 4 μ L of drug (Closantel/Closantel Sodium/Gemcitabine HCl/Trifluridine/Gemcitabine/Cladribine) diluted at a fold ratio (drug final concentrations of 5 μ M, 10 μ M and 20 μ M, respectively), and the treated cells were infected with 10 μ L of CPV (New CPV-2a type SD6) (MOI strain 0.076). Immunofluorescence assays were performed approximately 30 hours after infection.
Control group: DMSO was added to F81 cells in a 96-well plate to a final concentration of 0.1% (volume fraction), and immunofluorescence assay was performed after 30 hours.
Immunofluorescence assay:
fixing: discard the culture medium in 96-well plate and use 1 XPBS (Gibco)TMUSA, cargo number: 20012050) for 3 times, 2-3 min/time. Cells were fixed with 80% acetone (acetone to water volume ratio 4:1), 100. mu.L of 80% acetone was added to each well and incubated at room temperature for 10 min. Washing with 1 × PBS 3 times, 2-3 min/time.
And (3) sealing: blocking was performed by adding 3% BSA (3 g BSA in 100mL 1 XPBS) at room temperature for 1 hour at room temperature. Wash with 1 × PBS for 10 min.
A first antibody: the cells were incubated with mouse anti-VP 2 monoclonal antibody (INGENASA, Spain, M.15.CPV.I5F8) (1: 100 dilution, 1 × PBS) for 40 min. Antibody liquid was discarded and washed 3 times with 1 × PBS for 10 min/time.
Secondary antibody: the cells were incubated with FITC-labeled secondary goat anti-mouse IgG (H + L) (Invitrogen, USA, A16079) (1: 200 dilution, 1 XPBS) for 40 min. Antibody liquid was discarded and washed 3 times with 1 × PBS for 10 min/time.
Dyeing: stain with 4, 6-diamidino-2-phenylindole dihydrochloride (DAPI, 1. mu.g/ml) and react for 10min at room temperature in the dark. Discard the staining solution, wash 3 times with 1 × PBS, 10 min/time.
Cells were examined at 20x magnification using the In Cell Analyzer 2500HS Cell imaging analysis system (Operetta, PerkinElmer, USA) and analyzed by Image J Image processing software.
As shown in FIG. 5, the control group without any drug was detected by using a monoclonal antibody against VP2, and a large amount of VP2 protein was expressed in the visual field (control FITC in FIG. 5). In the treatment group to which a small molecule drug (Closantel/Closantel Sodium/Gemcitabine HCl/Trifluridine/Gemcitabine/Cladribine) was added, a decrease in VP2 protein signal was detected with an increase in drug concentration, a difference was shown in comparison with the control group at a final drug concentration of 5. mu.M, and fluorescence signals were gradually detected at final drug concentrations of 10. mu.M and 20. mu.M. It was demonstrated that 6 small molecule drugs were able to successfully inhibit CPV replication in F81 cells (fig. 5).
Example 4.6 experiments on inhibition of CPV strains of different genotypes by small molecule drugs
Drug treatment group: f81 cells were plated at 7.5X 10 per well5The cells were seeded in a 6-well plate and pretreated with different concentrations of drugs (Closantel/Closantel Sodium/Gemcitabine HCl/Trifluridine/Gemcitabine/Cladridine) (drug final concentrations of 5. mu.M, 10. mu.M and 20. mu.M, respectively), each drug-treated cell was infected with CPV of different genotypes (New CPV-2a type strain SD6 and New CPV-2b type strain SD3) (MOI 0.076), and total cell proteins were extracted and subjected to Western blot assay after 40 hours of incubation.
Control group: DMSO was added to F81 cells to a final concentration of 0.1% (volume fraction), and after 40 hours of incubation, total cell protein was extracted and subjected to Western blot assay.
Protein extraction and Western blot assay:
cells were collected and lysed using the mammalian total protein extraction kit ProteinExt (TransGen Biotech, China, DE101-01) according to the kit instructions. Equal amounts of cell lysate were electrophoresed through SDS-PAGE proteins and transferred to PVDF membrane (Millipore, USA). After blocking with 5% skim milk in TBS-Tween20 (100mL TBS-Tween20 with 5g skim milk powder added) for 1 hour at room temperature, anti-VP 2 monoclonal antibody (INGENASA, Spain, M.15.CPV.I5F8) and β -actin monoclonal antibody (AC-15) (Thermo Scientific, USA, MA1-91399) were used as follows, respectively, 1: 800 and 1: 4000 dilutions (diluted with TBS-Tween 20) and incubation at 4 ℃ overnight. TBS-Tween20 was washed 3 times for 10 min/time. HRP-labeled goat anti-mouse IgG (Thermo Scientific, USA, cat # 31430) was used (diluted 1: 6000 with TBS-Tween 20) and incubated at 37 ℃ for 1 hour. TBS-Tween20 was washed 3 times, 10 min/time. Use of SuperSignalTMWest Pico PLUS chemiluminescent substrate detection kit (Thermo Scientific, USA, 34579) strips were detected according to the kit instructions and imaged by a chemiluminescent device (Protein simple, USA).
The Western blot result is shown in FIG. 6, the 6 small molecular drugs can inhibit the expression of VP2 proteins of two different genotypes of CPV viruses, namely a strain SD6(New CPV-2a) and a strain SD3(New CPV-2b), and the difference is obvious compared with a control group without drugs. And with the increase of the drug concentration, the VP2 protein expression levels of the two genotypes of virus are gradually reduced, and the bands are weaker at the final drug concentrations of 10 mu M and 20 mu M, which proves that the 6 small molecular drugs can well inhibit the virus replication for CPV strains of different genotypes (FIG. 6).
Claims (4)
1. The application of closantel and closantel sodium in preparing the medicine for inhibiting the canine parvovirus replication is characterized in that the active ingredients of the medicine comprise the closantel and/or the closantel sodium, and the structural formula of the medicine is as follows:
2. the use according to claim 1, wherein the medicament is a single active ingredient medicament, the active ingredient of which is closantel or sodium closantel.
3. Use according to claim 1 or 2, wherein the medicament further comprises a pharmaceutically acceptable carrier.
4. Use according to claim 3, wherein the medicament is in the form of a tablet, suspension or dry suspension.
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| CN202210110277.4A CN114306364B (en) | 2019-07-18 | 2020-07-17 | Application of gemcitabine hydrochloride and gemcitabine in inhibiting canine parvovirus |
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