CN114306364B - Application of gemcitabine hydrochloride and gemcitabine in inhibiting canine parvovirus - Google Patents
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Abstract
The invention relates to a new application of a known compound, in particular to application of Gemcitabine HCl and Gemcitabine in inhibiting canine parvovirus. The invention provides application of Gemcitabine HCl and Gemcitabine in preparing a medicament for inhibiting canine parvovirus replication, wherein active ingredients of the medicament comprise Gemcitabine HCl and/or Gemcitabine. The data prove that Gemcitabine HCl and Gemcitabine have very excellent inhibition effect on CPV replication, and can inhibit the expression of VP2 proteins of CPV viruses with different genotypes.
Description
The application is a divisional application of patent application with the application number of 202010689594.7 and the invention name of 'application of 6 small molecule drugs in inhibiting canine parvovirus' submitted by month 07 in 2020.
Technical Field
The invention relates to a new application of a known compound, in particular to application of Gemcitabine HCl and Gemcitabine in inhibiting canine parvovirus.
Background
Canine parvovirus (Canine parvovirus, CPV) is a simple single stranded DNA virus that is a member of the parvoviridae family, non-enveloped, icosahedral. The viral genome is about 5300nt in length, contains 2 ORFs, and encodes mainly early transcribed regulatory proteins (NS 1 and NS 2) at the 5 'end and late transcribed structural proteins (VP 1 and VP 2) at the 3' end. VP2 protein is the major component of capsid proteins, and can bind to transferrin receptor (TfR) on the host cell membrane, thereby mediating infection by parvovirus. CPV is one of the major etiologies in canine acute gastroenteritis, leukopenia and myocarditis, and is widely found in predators. Typical clinical symptoms of infection with CPV include vomiting, fever, diarrhea, especially puppies of 6 weeks to 6 months susceptibility.
CPV is widely distributed worldwide, and after the appearance of CPV-2 in the late 70 s of the 20 th century, CPV-2 and its variants have been reported in various countries in five continents. Since there is no specific anti-CPV drug, the only treatment options left are supportive care and symptom-based care, except through vaccine prophylaxis. CPV genome substitution rates are similar to RNA viruses, with the earliest CPV-2 type being replaced by three major subspecies CPV-2a, CPV-2b and CPV-2c for only a few years. 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 effectiveness of existing vaccines, while maternal antibodies have also been shown to impair the vaccine.
Therefore, development of a new drug for preventing and treating CPV virus is desired.
Disclosure of Invention
The inventors found that 6 small molecule drugs, closal Sodium, gemcitabine HCl, trifluradine, gemcitabine and Cladribine (chemical formula shown in FIG. 1), have inhibitory effects on Canine Parvovirus (CPV) replication. The concentration of 50% cytotoxicity (50%cytotoxicity concentrations,CC50) and 50% antiviral effect (50%antiviral efficacy,EC50) of the drug is determined by in vitro experiments, and the effect of the 6 small molecule drugs on inhibiting CPV replication in vitro is further verified by using an indirect immunofluorescence assay (Indirect immunofluorescence assay, IFA) and a Western blot assay.
In one aspect, the invention provides the use of Gemcitabine HCl and Gemcitabine in the preparation of a medicament for inhibiting canine parvovirus replication, wherein the active ingredients of the medicament comprise Gemcitabine HCl and/or Gemcitabine.
In some embodiments of the use of the invention, the medicament is a single active ingredient medicament, the active ingredient of which is Gemcitabine HCl or Gemcitabine.
In other embodiments of the use of the invention, the medicament is a combination, the active ingredient of which further comprises Closal, closal Sodium, trifluradine, and/or 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 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, which is characterized in that the active ingredient of the medicament comprises Gemcitabine HCl and/or Gemcitabine.
In some embodiments of the medicament of the invention, the medicament is a single active ingredient medicament, the active ingredient of which is Gemcitabine HCl or Gemcitabine.
In other embodiments of the medicament of the invention, the medicament is a combination medicament, the active ingredient of which further comprises Closal, closal Sodium, trifluradine, and/or Cladridine. In some embodiments, other antiviral components may also be included.
In some embodiments of the medicament of the invention, the medicament further comprises a pharmaceutically acceptable liquid or solid carrier component.
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 of any of the above agents, wherein upon time of administration for a predetermined period, canine parvovirus levels in the serum of said infected individual are significantly reduced or reduced to undetectable levels.
Closantel sound, chinese name: cyanoiodisalate sodium, CAS number: 61438-64-0, molecular formula: c (C) 22 H 13 Cl 2 I 2 N 2 O 2 Na, the structural formula of which is shown in figure 1A, is a gram-positive antibacterial activity inhibitor.
Closantel, chinese name: cyanoioxazin, CAS number: 57808-65-8, molecular formula: c (C) 22 H 14 Cl 2 I 2 N 2 O 2 The structural formula is shown in figure 1B, and is a gram-positive antibacterial activity inhibitor.
Gemcitabine HCl, chinese name: gemcitabine hydrochloride, CAS no: 122111-03-9, molecular formula: c (C) 9 H 11 F 2 N 3 O 4 HCl, the structural formula of which is shown in figure 1C, is a DNA and nucleic acid synthesis inhibitor.
Gemcitabine, chinese name: gemcitabine, CAS number: 95058-81-4, molecular formula: c (C) 9 H 11 F 2 N 3 O 4 The structural formula is shown in figure 1E, and is a DNA and nucleic acid synthesis inhibitor.
Cladribine, chinese name: cladribine, CAS No.: 4291-63-8, molecular formula: c (C) 10 H 12 ClN 5 O 3 The structural formula is shown in figure 1D, and is an adenosine deaminase inhibitor.
Trifluridine, chinese name: trifluoro thymidine, CAS no: 70-00-8, molecular formula: c (C) 10 H 11 F 3 N 2 O 5 The structural formula is shown in figure 1F, and is an anti-herpesvirus drug.
The inventor finds that Closal, closal Sodium, gemcitabine HCl, trifluradine, gemcitabine and Cladribine can inhibit the replication of CPV strains, and determines the CPV inhibition and protection effect on F81 cells, and the result shows that 6 small molecule drugs can well protect cells after CPV infection. As shown in FIG. 2, when the final concentration of the drug was 10. Mu.M, the inhibition protection rates of Closal, closal Sodium, gemcitabine HCl, trifluradine, gemcitabine and Cladribine on F81 cells were 80.64.+ -. 7.87, 69.76.+ -. 6.06, 64.18.+ -. 0.97, 57.11.+ -. 5, respectively.45 56.1+ -2.09 and 50.92+ -1.58% higher than that of the control drug Cidofovir (Chinese name: cidofovir, CAS number: 113852-37-2, molecular formula: C) 8 H 14 N 3 O 6 P, broad-spectrum anti-DNA virus drug) 1.28.+ -. 1.03%.
Further, the 50% effector concentration (EC 50) and 50% cytotoxic concentration (CC 50) of Closal, closal Sodium, gemcitabine HCl, triflurabine, gemcitabine and Cladribine on CPV strain SD6 (New CPV-2 a) were determined, and the EC50 of F81 cell infection by 6 small molecule drug-inhibiting strains SD6 was determined, and the CC50 results are shown in FIGS. 3 and 4.
The inventors further conducted immunofluorescence assay (IFA), and as a result, it was shown that the fluorescence signal of VP2 expression was detected at a drug final concentration of 5 μm, i.e., lower than that of the control group without drug, and that almost no fluorescence signal was detected at a drug final concentration of 10 μm or 20 μm, demonstrating that 6 small molecule drugs were able to successfully inhibit CPV replication in F81 cells (fig. 5).
In addition, western blot results show that 6 small molecule drugs can inhibit the expression of VP2 proteins of viruses with different genotypes, and the expression quantity of VP2 proteins with different genotypes is gradually reduced along with the increase of the concentration of the drugs, so that the effect of the drugs on inhibiting CPV replication is further verified (figure 6).
In conclusion, candidate drugs Closal, closal Sodium, gemcitabine HCl, trifluradine, gemcitabine and Cladribine which are effective against Canine Parvovirus (CPV) are screened out, and as the candidate drugs are mature clinical drugs, reference is provided for clinical medication, and the candidate drugs are developed into anti-CPV drugs, so that the preparation method has a wide application prospect.
Drawings
FIG. 1 shows the structural formulae of the small molecule drugs Closal, closal Sodium, gemcitabine HCl, trifluradine, gemcitabine and Cladribine.
FIG. 2. 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. The percentage of inhibition of F81 cells by Closalte, closalte Sodium, gemcitabine HCl, trifluradine, gemcitabine and Cladribine was 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 inhibition percentage of the control drug Cidofovir on F81 cells is-1.28+/-1.03%.
FIG. 3 CC50 and EC50 measurements on F81 cells with small molecule drugs; a, B, and C are the CC50 and EC50 measurements of Closal Sodium, closal and Gemcitabine HCl, respectively.
FIG. 4 CC50 and EC50 measurements on F81 cells with small molecule drugs; CC50 and EC50 determinations of D, E, F, cladribine, gemcitabine and trifluradine, respectively.
FIG. 5 immunofluorescence assay of small molecule drug inhibiting expression of viral VP2 protein. Control FITC is FITC fluorescence signal (showing virus number) of control group, control DAPI is DAPI fluorescence signal (showing cell number) of control group, control combination is a combined plot of FITC fluorescence signal and DAPI fluorescence signal of control group. The pictures of Closal Sodium, closal, gemcitabine HCl, cladribine, gemcitabine and Triflunidine are all combined plots of FITC fluorescence signal and DAPI fluorescence signal. 5. Mu.M, 10. Mu.M and 20. Mu.M represent the final concentration of drug in the experimental system.
FIG. 6 shows the results of experiments (Western blot experiments) of small molecule drugs on CPV strains of different genotypes. DMSO represents control groups, 5, 10, 20 represent treatment groups with final drug concentrations of 5 μm,10 μm and 20 μm, respectively. VP2 is the target protein detected, and beta-actin is used as an internal reference.
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.
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: purchased from seleck (USA), drug parameters: s4106 (concentration: 10 mM).
Closantel Sodium: purchased from seleck (USA), drug parameters: s4105 (concentration: 10 mM).
Gemcitabine HCl: purchased from seleck (USA), drug parameters: s1149 (concentration: 10 mM).
Trifluradine: purchased from seleck (USA), drug parameters: s1778 (concentration: 10 mM).
Gemcitabine: purchased from seleck (USA), drug parameters: s1714 (concentration: 10 mM).
Cladribine: purchased from seleck (USA), drug parameters: s1199 (concentration: 10 mM).
Cidofovir: purchased from seleck (USA), drug parameters: s1516 (concentration: 10 mM).
anti-VP 2 monoclonal antibodies, available from engenasa, spain, cat: m.15.cpv.i5f8.
FITC-labeled goat anti-mouse IgG (h+l) secondary antibodies (1:200 dilution) were purchased from Thermo Scientific, USA, cat: A16079.
beta-actin monoclonal antibody (AC-15), available from Thermo Scientific, USA, cat: MA1-91399.
HRP-labeled goat anti-mouse IgG was purchased from Thermo Scientific, USA, cat: 31430.
Mammalian total protein extraction kit
Mammalian Total Protein Extraction Kit), transGen Biotech, china, catalog number: DE101-01.
SuperSignal TM West Pico PLUS chemiluminescent substrate detection kit, available 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 XTBS-Tween 20: purchased from Thermo Scientific, cat No. 28360.
PBS was purchased from Gibco TM USA, cat No.: 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. The Maintenance Medium (MM) consists of DMEM (Gibco, USA), 2% fetal bovine serum (Gibco, USA) and 100U/ml penicillin and 100. Mu.g/ml streptomycin.
New CPV-2a type strain SD6 and New CPV-2b type strain SD3 are isolated and stored by Beijing city key laboratory of livestock epidemic prevention and control technology of livestock and poultry and veterinary institute of the national academy of sciences of agriculture and forestry in Beijing city. The homology of VP2 protein coding genes of the strain SD6 and the strain SD3 with the nucleotide sequences of corresponding VP2 protein coding genes of BJ14-7 (GenBank: KT162031, new CPV-2 a) and BJ14-1 (GenBank: KT162022, new CPV-2 b) is up to 99.9% and 99.3%. The non-patent documents describing strain SD6 and strain SD3 are: hongzhuan Zhou, xia Su, lu Lin, jin Zhang, qi, fangfang Guo, fuzhou Xu, bing yang. Inhibit Effects of Antiviral Drug Candidates on Canine Parvovirus in F cells, viruses 2019aug;11 (8) 742.doi:10.3390/v11080742.PMID 31412574.
The applicant stated that New CPV-2a strain SD6 and New CPV-2b strain SD3 could be provided to the public for research purposes within twenty years from the date of application.
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 laboratory 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 1.6 determination of the inhibitory protective Effect of small molecule drugs on CPV in F81 cells
F81 cell purchase before use, 4. Mu.L of small molecule drug (Closal/Closal Sodium/Gemcitabine HCl/Triflunidine/Gemcitabine/Cladribine) (10 mM) and 4. Mu.L of control drug Cidofovir (10 mM) 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 mu L F81 cells (25,000 cells per well) per treatment group was added 4. Mu.L of 250. Mu.M stock solution of each drug at a final concentration of 10. Mu.M, and after 1 hour of continuous treatment, F81 cells treated with the drug were infected with 10. Mu.L of CPV (New CPV-2a type strain SD 6) at a MOI (multiplicity of infection, 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: to F81 cells, DMSO was added to a final concentration of 0.1% (volume fraction), and after 1h the 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 using
Cell Counting Kit (TransGen Biotech, china, FC 101-01) cell viability was measured according to the kit instructions. Wells without CPV infection served as positive (cell) control wells with 100% inhibition protection, wells infected with CPV (New CPV-2a strain SD 6) served as negative (virus) control wells with 0% inhibition protection. Percent inhibition was calculated using the following formula: CPE inhibition percentage= (OD 450 of drug treated cells-OD 450 of negative control)/(OD 450 of positive control-OD 450 of negative control) 100, the test was repeated 3 times.
The results are shown in FIG. 2:
the inhibition and protection test results show that 6 small molecule drugs can well protect cells after CPV infection. Wells without CPV infection were defined as 100% inhibition protection positive control and wells infected with CPV (without any drug added) as 0% inhibition protection negative control, calculated by the formula, the inhibition protection rates (i.e. CPE inhibition percentages) for closal, closal Sodium, gemcitabine HCl, trifluradine, 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, whereas the inhibition protection rates (i.e. CPE inhibition percentages) for the broad-spectrum anti-DNA virus drug Cidofovir were only-1.28±1.03%, the two very significant (P < 0.0001), as shown in fig. 2.
EXAMPLE 2.6 determination of 50% Effect concentration (50%antiviral efficacy,EC50) and 50% cytotoxicity concentration (50%cytotoxicity concentrations,CC50) of Small molecule drug
The EC50 and CC50 of the drug were determined using a dose response assay. The method comprises the following steps:
the EC50 determination steps are: 86 mu L F cells (25,000 cells per well) were pretreated with 4. Mu.L of drug diluted at a double ratio (Closal/Closal Sodium/Gemcitabine HCl/triflurabine/Clamcitabine/Cladribine) (final concentration range of 0.3125-20. Mu.M) for 1h, and then the drug-treated cells were infected with 10. Mu.L of CPV (New CPV-2a type strain SD 6) (MOI=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 the drug diluted at a final concentration ranging from 0.3125-80 μΜ.
Both EC50 and CC50 assays were performed in 3 replicates. After incubation for 40 hours, using
Cell Counting Kit (TransGen Biotech, china, FC 101-01) cell viability was determined according to the kit instructions, and further the inhibition efficiency of the 6 small molecule drugs against CPV infection and their respective toxicity to F81 cells was determined, and EC50 and CC50 values were calculated by the dose-response nonlinear regression analysis method in GraphPad Prism software.
The results are shown in fig. 3 and 4:
the half antiviral effect concentrations (EC 50) of Closalte, closal Sodium, gemcitabine HCl, trifluradine, gemcitabine and Cladribine inhibiting strains SD6 (New CPV-2 a) 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.
Closal, closal Sodium, gemcitabine HCl, trifluradine, gemcitabine and Cladribine have F81 cell half-toxicity concentrations (CC 50) of 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 immunofluorescence assay (Immunofluorescence assay, IFA) to detect inhibition of viral VP2 protein expression by 6 small molecule drugs
Drug treatment group: 86. Mu.L of F81 cells (25,000 cells per well) in 96-well plates were pretreated with 4. Mu.L of a drug diluted at a doubling ratio (Closal/Closal Sodium/Gemcitabine HCl/triflurabine/Gemcitabine/Cladribine) (final drug concentrations of 5. Mu.M, 10. Mu.M and 20. Mu.M, respectively) for 1 hour, and the treated cells were infected with 10. Mu.L of CPV (New CPV-2a type strain SD 6) (MOI=0.076). Immunofluorescence assays were performed about 30 hours after infection.
Control group: DMSO was added to F81 cells in 96-well plates to a final concentration of 0.1% (volume fraction) and immunofluorescence assay was performed after 30 hours.
Immunofluorescence assay:
fixing: the culture medium in the 96-well plate was discarded, and 1 XPBS (Gibco) TM USA, cat No.: 20012050 3 times, 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: the mixture was blocked by adding 3% BSA (100 mL 1 XPBS in 3g BSA) at room temperature for 1 hour. Washing with 1 XPBS for 10min.
An antibody: incubation with mouse anti-VP 2 monoclonal antibody (INGENASA, spain, M.15.CPV. I5F8) (1:100 dilution, 1 XPBS dilution) 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) (1:200 dilution, 1 XPBS dilution) for 40 min. 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 In Cell Analyzer 2500 HS Cell imaging analysis system (operatta, perkinElmer, USA) at 20x magnification and analyzed by Image J Image processing software.
As a result, as shown in FIG. 5, the control group to which no drug was added was detected by using the anti-VP 2 monoclonal antibody, and a large amount of VP2 protein was expressed in the visual field (control FITC of FIG. 5). In the treatment group to which small molecule drug (Closal/Closal Sodium/Gemcitabine HCl/Triflunidine/Gemcitabine/Cladribine) was added, a decrease in VP2 protein signal was detected with an increase in drug concentration, and a difference was shown at a drug final concentration of 5. Mu.M compared with the control group, and the detected fluorescence signal was gradually decreased at drug final 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 of inhibition of small molecule drugs on CPV strains of different genotypes
Drug treatment group: f81 cells were plated at 7.5X10 cells per well 5 Individual cells were seeded in 6-well plates and pre-treated with different concentrations of drug (Closantel/Closantel Sodium/Gemcitabine HCl/trifluradine/Gemcitabine/cladridine) (drug final concentrations of 5 μm,10 μm and 20 μm, respectively) for 1 hour, cells treated with each drug were infected with CPV of different genotypes (New CPV-2a strain SD6 and New CPV-2b strain SD 3), respectively (moi=0.076), and after 40 hours the total protein of the cells was extracted and subjected to Western blot assay.
Control group: DMSO was added to F81 cells to a final concentration of 0.1% (volume fraction), and after 40 hours incubation the total cell proteins were extracted and Western blot tested.
Protein extraction and Western blot test:
cells were collected and lysed using a mammalian total protein extraction kit ProteinExt (TransGen Biotech, china, DE 101-01) according to the kit instructions. Equal amount of fineCell lysates were transferred to PVDF membrane (Millipore, USA) after SDS-PAGE proteins. After blocking with TBS-Tween20 containing 5% skim milk (5 g of skim milk powder added to 100mL TBS-Tween 20) at room temperature for 1 hour, anti-VP 2 monoclonal antibody (INGENASA, spain, M.15.CPV.I5F8) and beta-actin monoclonal antibody (AC-15) (Thermo Scientific, USA, MA 1-91399) were used at 1:800 and 1:4000 dilution (with TBS-Tween 20) and incubation overnight 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. Using SuperSignal TM West Pico PLUS chemiluminescent substrate detection kit (Thermo Scientific, USA, 34579) detects bands according to kit instructions and images by a chemiluminescent device (Protein simple, USA).
As shown in FIG. 6, the Western blot results show that 6 small molecule drugs can inhibit VP2 protein expression of two different genotypes of CPV viruses, namely a strain SD6 (New CPV-2 a) and a strain SD3 (New CPV-2 b), and the difference is obvious compared with a control group without the drugs. And the VP2 protein expression amount of the two types of genotype viruses is gradually reduced along with the increase of the drug concentration, and the bands are weaker when the final drug concentration is 10 mu M and 20 mu M, so that the CPV strains with different genotypes can be well inhibited by the 6 types of micromolecule drugs (figure 6).
Claims (4)
1. Use of gemcitabine hydrochloride and gemcitabine in the manufacture of a medicament for inhibiting canine parvovirus replication, wherein the active ingredient of the medicament comprises gemcitabine hydrochloride and/or gemcitabine, having the chemical structural formula:
2. the use according to claim 1, wherein the medicament is a single active ingredient medicament, the active ingredient of which is gemcitabine hydrochloride or gemcitabine.
3. The use according to claim 1 or 2, wherein the medicament further comprises a pharmaceutically acceptable carrier.
4. The use according to claim 3, wherein the pharmaceutical dosage form is a tablet, suspension or dry suspension.
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