CN114404438B - Use of Trifluridine in the preparation of medicines for inhibiting canine parvovirus replication - Google Patents

Abstract

The present invention relates to the novel application of known compounds, especially the application of Trifluridine in inhibiting canine parvovirus. The invention provides the use of trifluridine in preparing a medicine for inhibiting the replication of canine parvovirus, and the active ingredient of the medicine contains Trifluridine. The data prove that Trifluridine has a very excellent inhibitory effect on CPV replication, and can inhibit the expression of VP2 protein of different genotypes of CPV virus.

Description

Use of Trifluridine in the preparation of medicines for inhibiting canine parvovirus replication

This application is a divisional application of the patent application submitted on July 17, 2020 with the application number 202010689594.7 and the invention title "Application of 6 Small Molecular Drugs in Inhibiting Canine Parvovirus".

technical field

The present invention relates to the novel application of known compounds, especially the application of Trifluridine in inhibiting canine parvovirus.

Background technique

Canine parvovirus (Canine parvovirus, CPV) is a kind of single-stranded DNA virus with simple structure, which is a member of Parvoviridae. It has no envelope and forms an icosahedron. The total length of the viral genome is about 5300nt, containing two ORFs, the 5' end mainly encodes the regulatory proteins of early transcription (NS1 and NS2), and the 3' end encodes the structural proteins of late transcription, namely the viral capsid proteins (VP1 and VP2). The VP2 protein is the main component of the capsid protein, which can bind to the transferrin receptor (TfR) on the host cell membrane, thereby mediating the infection of the parvovirus. CPV is one of the main pathogens of canine acute gastroenteritis, leukopenia and myocarditis, and widely exists in carnivorous animals. Typical clinical symptoms of CPV infection include vomiting, fever, and diarrhea, especially in puppies aged 6 weeks to 6 months.

CPV virus is widely distributed in the world. After CPV-2 appeared in the late 1970s, CPV-2 and its variants have been reported in many countries on five continents. With no specific anti-CPV drugs, the only remaining treatment options, other than prophylaxis through vaccines, are supportive care and symptom-based care. CPV genome substitution rates are similar to those of RNA viruses, and the earliest CPV-2 type was replaced by three major subspecies CPV-2a, CPV-2b, and CPV-2c in just a few years. Although inactivated or attenuated live vaccines have been widely used for prevention, the frequent emergence of mutant strains has aroused concern and concern about the effectiveness of existing vaccines. At the same time, maternal antibodies have also shown a weakening effect on vaccines.

Therefore, it is urgent to develop new drugs for the prevention and treatment of CPV virus.

Contents of the invention

The inventors found that six small molecule drugs Closantel, Closantel Sodium, Gemcitabine HCl, Trifluridine, Gemcitabine and Cladribine (chemical structural formula shown in Figure 1) have inhibitory effect on the replication of canine parvovirus (CPV). The 50% cytotoxicity concentrations (CC50) and 50% antiviral efficacy (EC50) concentrations of the drugs were determined by in vitro experiments, and the indirect immunofluorescence assay (IFA) and Western blot tests were used to verify the effects of these six small molecule drugs on CPV replication in vitro.

In one aspect, the present invention provides the use of Trifluridine in the preparation of a drug for inhibiting the replication of canine parvovirus, characterized in that the active ingredient of the drug contains Trifluridine.

In some embodiments of the use of the present invention, the drug is a single active ingredient drug, and the active ingredient is Trifluridine.

In other embodiments of the use of the present invention, the drug is a compound drug, the active ingredient of which also includes Closantel, Closantel Sodium, Gemcitabine HCl, Gemcitabine and/or Cladribine. In some embodiments, other antiviral components may also be included.

In some embodiments of the use of the present invention, the medicament further comprises a pharmaceutically acceptable carrier.

In some embodiments of the use of the present invention, the dosage form of the drug is tablet, suspension or dry suspension.

In another aspect, the present invention provides a medicine for inhibiting the replication of canine parvovirus, characterized in that the active ingredient of the medicine contains Trifluridine.

In some embodiments of the medicament of the present invention, the medicament is a single active ingredient medicament, and the active ingredient is Trifluridine.

In some other embodiments of the medicament of the present invention, the medicament is a compound medicine, the active ingredients of which further include Closantel, Closantel Sodium, Gemcitabine HCl, Gemcitabine and/or Cladribine. 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 liquid or solid carrier component.

In some embodiments of the medicament of the present invention, the dosage form of the medicament is tablet, suspension or dry suspension.

Where permitted by law, the present invention claims a method for treating canine parvovirus infection, which is characterized in that it comprises administering an effective dose of any of the above-mentioned drugs to the infected individual, and after a predetermined time of on-time administration, the level of canine parvovirus in the serum of the infected individual is significantly reduced or reduced to an undetectable level.

Closantel Sodium, Chinese name: Closantel Sodium, CAS number: 61438-64-0, molecular formula: C ₂₂ H ₁₃ Cl ₂ I ₂ N ₂ O ₂ .Na, its structural formula is shown in Figure 1A, is a Gram-positive antibacterial activity inhibitor.

Closantel, Chinese name: Closantel, CAS number: 57808-65-8, molecular formula: C ₂₂ H ₁₄ Cl ₂ I ₂ N ₂ O ₂ , its structural formula is shown in Figure 1B, is a Gram-positive antibacterial activity inhibitor.

Gemcitabine HCl, Chinese name: gemcitabine hydrochloride, CAS number: 122111-03-9, molecular formula: C ₉ H ₁₁ F ₂ N ₃ O ₄ .HCl, its structural formula 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 ₉ H ₁₁ F ₂ N ₃ O ₄ , its structural formula is shown in Figure 1E, is a DNA and nucleic acid synthesis inhibitor.

Cladribine, Chinese name: cladribine, CAS number: 4291-63-8, molecular formula: C ₁₀ H ₁₂ ClN ₅ O ₃ , its structural formula is shown in Figure 1D, is an adenosine deaminase inhibitor.

Trifluridine, Chinese name: trifluorothymidine, CAS number: 70-00-8, molecular formula: C ₁₀ H ₁₁ F ₃ N ₂ O ₅ , its structural formula is shown in Figure 1F, is an anti-herpes virus drug.

The inventors found that Closantel, Closantel Sodium, Gemcitabine HCl, Trifluridine, Gemcitabine and Cladribine could inhibit the replication of CPV strains, and measured their protective effects on CPV inhibition in F81 cells. The results showed that the six small molecule drugs could well protect cells after CPV infection.如图2所示，当药物终浓度为10μM时，Closantel，Closantel Sodium，Gemcitabine HCl，Trifluridine，Gemcitabine和Cladribine对F81细胞的抑制保护率分别为80.64±7.87，69.76±6.06，64.18±0.97，57.11±5.45，56.1±2.09和50.92±1.58％，明显高于对照药物Cidofovir(中文名：西多福韦，CAS号：113852-37-2，分子式：C ₈ H ₁₄ N ₃ O ₆ P，广谱抗DNA病毒药物)的-1.28±1.03％的保护率。

The 50% effect concentration (EC50) and 50% cytotoxicity concentration (CC50) of Closantel, Closantel Sodium, Gemcitabine HCl, Trifluridine, Gemcitabine and Cladribine on CPV strain SD6 (New CPV-2a) were further measured. Six small molecule drugs inhibited the EC50 of strain SD6 on F81 cell infection, and the CC50 results are shown in Figure 3 and Figure 4.

The inventors further carried out immunofluorescence assay (IFA), and the results showed that when the final drug concentration was 5 μM, the fluorescent signal for detecting VP2 expression was lower than that of the control group without drug addition, and when the final drug concentration was 10 μM or 20 μM, almost no fluorescent signal could be detected, which proved that the six small molecule drugs could successfully inhibit the replication of CPV in F81 cells (Figure 5).

In addition, the results of Western blot showed that the six small molecule drugs could inhibit the expression of VP2 proteins of different genotypes of viruses, and with the increase of drug concentration, the expression of VP2 proteins of different genotypes gradually decreased, which further verified the effect of the drugs on inhibiting CPV replication (Figure 6).

In summary, the present invention has screened out effective anti-canine parvovirus (CPV) drug candidates Closantel, ClosantelSodium, Gemcitabine HCl, Trifluridine, Gemcitabine and Cladribine, because it is a mature clinical drug in use, it will provide a reference for clinical medication, and it will be developed as an anti-CPV drug, which has a great application prospect.

Description of drawings

Figure 1. Structural formulas of the small molecule drugs Closantel, Closantel Sodium, Gemcitabine HCl, Trifluridine, Gemcitabine and Cladribine.

Figure 2. Experimental results of small molecule drugs inhibiting CPV replication in vitro; the abscissa is the name of the drug, and the ordinate is the percentage of CPE inhibition. Closantel, Closantel Sodium, Gemcitabine HCl, Trifluridine, Gemcitabine and Cladribine inhibited percentages of F81 cells 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 inhibition percentage of control drug Cidofovir on F81 cells was -1.28±1.03%.

Figure 3. CC50 and EC50 assay results of small molecule drugs on F81 cells; A, B, and C are the CC50 and EC50 assay results of Closantel Sodium, Closantel and Gemcitabine HCl, respectively.

Figure 4. CC50 and EC50 assay results of small molecule drugs on F81 cells; D, E, and F respectively CC50 and EC50 assay results of Cladribine, Gemcitabine and Trifluridine.

Figure 5. Immunofluorescence detection results of small molecule drugs inhibiting viral VP2 protein expression. The control FITC is the FITC fluorescence signal of the control group (displaying the number of viruses), the control DAPI is the DAPI fluorescence signal of the control group (displaying the number of cells), and the control combination is the merged figure of the FITC fluorescence signal and the DAPI fluorescence signal of the control group. The pictures of Closantel Sodium, Closantel, Gemcitabine HCl, Cladribine, Gemcitabine and Trifluridine are merged pictures of FITC fluorescence signal and DAPI fluorescence signal. 5 μM, 10 μM and 20 μM represent the final concentration of the drug in the experimental system.

Figure 6. Results of inhibition experiments (Western blot experiments) of small molecule drugs on different genotypes of CPV strains. DMSO represents the control group, 5, 10, and 20 represent the treatment groups with final drug concentrations of 5 μM, 10 μM and 20 μM, respectively. VP2 is the target protein for detection, and β-actin is used as an internal reference.

Detailed ways

The present invention will be described in detail below in conjunction with the examples. It should be understood that the following examples are only used as explanations and descriptions 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), 100 U/ml penicillin, 100 μg/ml streptomycin.

Maintenance medium (MM): DMEM (Gibco, USA) plus 2% fetal bovine serum (Gibco, USA) and 100 U/ml penicillin and 100 μg/ml streptomycin.

Closantel: purchased from Selleck (USA), drug parameter: S4106 (concentration: 10 mM).

Closantel Sodium: purchased from Selleck (USA), drug parameter: S4105 (concentration: 10 mM).

Gemcitabine HCl: purchased from Selleck (USA), drug parameter: S1149 (concentration: 10 mM).

Trifluridine: purchased from Selleck (USA), drug parameter: S1778 (concentration: 10 mM).

Gemcitabine: purchased from Selleck (USA), drug parameter: S1714 (concentration: 10 mM).

Cladribine: purchased from Selleck (USA), drug parameter: S1199 (concentration: 10 mM).

Cidofovir: purchased from Selleck (USA), drug parameter: S1516 (concentration: 10 mM).

Anti-VP2 monoclonal antibody was purchased from INGENASA, Spain, product number: M.15.CPV.I5F8.

FITC-labeled goat anti-mouse IgG (H+L) secondary antibody (1:200 dilution) was purchased from Thermo Scientific, USA, catalog number: A16079.

β-actin monoclonal antibody (AC-15), purchased from Thermo Scientific, USA, catalog number: MA1-91399.

HRP-labeled goat anti-mouse IgG was purchased from Thermo Scientific, USA, catalog number: 31430.

Cell Counting Kit, purchased from TransGen Biotech, China, catalog number: FC101-01.

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 was purchased from ThermoScientific, USA, catalog number: 34579.

DMSO (dimethylsulfoxide): CAS No. 67-68-5, Sigma-Aldrich, D2650.

4,6-diamidino-2-phenylindole dihydrochloride (DAPI): CAS No. 28718-90-3, Thermo Scientific, D3571.

20×TBS-Tween 20: purchased from Thermo Scientific, Cat. No. 28360.

PBS was purchased from Gibco ^™ , USA, catalog number: 20012050.

cells and viruses

F81 cells were purchased from ATCC (American 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 100 U/ml penicillin and 100 μg/ml streptomycin. Maintenance medium (MM) consisted of DMEM (Gibco, USA), 2% fetal bovine serum (Gibco, USA) and 100 U/ml penicillin and 100 μg/ml streptomycin.

New CPV-2a strain SD6 and New CPV-2b strain SD3 were isolated and preserved by the Beijing Key Laboratory of Animal Disease Prevention and Control, Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agriculture and Forestry Sciences. The nucleotide sequence homology of the VP2 protein coding genes of strain SD6 and strain SD3 with the corresponding VP2 protein coding genes of BJ14-7 (GenBank: KT162031, New CPV-2a) and BJ14-1 (GenBank: KT162022, New CPV-2b) was as high as 99.9% and 99.3%. The non-patent literature that records strain SD6 and strain SD3 is: Hongzhuan Zhou, Xia Su, Lulu Lin, Jin Zhang, Qi Qi, Fangfang Guo, Fuzhou Xu, Bing Yang. Inhibitory Effects of Antiviral Drug Candidates on Canine Parvovirus in F81 Cells. Viruses. 2019Aug; 11(8):742.do i:10.3390/v11080742.PMID:31412574.

The applicant declares that within 20 years from the filing date, the New CPV-2a strain SD6 and the New CPV-2b strain SD3 can be provided to the public for experiments for research purposes.

Unless otherwise specified, the reagents used in the following examples are all conventional reagents in this field, which are commercially available or prepared according to conventional methods in this field, and the specifications are laboratory pure grade. Unless otherwise specified, the experimental methods and experimental conditions used in the following examples are conventional experimental methods and experimental conditions in the art, and reference can be made to relevant experimental manuals, 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. Determination of the protective effect of 6 small molecule drugs on CPV inhibition in F81 cells

Before the F81 cells were purchased and used, 4 μL of the small molecule drug (Closantel/Closantel Sodium/Gemcitabine HCl/Trifuridine/Gemcitabine/Cladribine) (10 mM) and 4 μL of the control drug Cidofovir (10 mM) were added to 156 μL of the maintenance medium (MM) to prepare a 250 μM drug stock solution.

Drug treatment group: each drug stock solution was used to treat the F81 cells respectively. For each treatment group, 4 μL of 250 μM drug stock solution was added to 86 μL of F81 cells (25,000 cells per well), and the final concentration of each drug was 10 μM. After continuous treatment for 1 h, 10 μL of CPV (New CPV-2a strain SD6) was used to infect the drug-treated F81 cells at an MOI (multiplicity of infection, multiplicity of infection) of 0.076. Cell viability was assayed 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 the addition of DMSO.

Negative control: DMSO was added to F81 cells to a final concentration of 0.1% (volume fraction), and F81 cells were infected with CPV (NewCPV-2a strain SD6) at an MOI of 0.076 after 1 h. Cell viability was assayed 40 hours after infection.

use Cell Counting Kit (TransGen Biotech, China, FC101-01) was used to detect cell viability according to the instructions of the kit. Wells without CPV infection were used as positive (cell) control wells for 100% inhibitory protection, and wells infected with CPV (New CPV-2a strain SD6) were used as negative (virus) control wells for 0% inhibitory protection. The inhibition percentage was calculated using the following formula: CPE inhibition percentage=(OD450 of drug-treated cells-OD450 of negative control)/(OD450 of positive control-OD450 of negative control)*100, and the experiment was repeated 3 times.

The result is shown in Figure 2:

The results of the inhibition protection test showed that the 6 small molecule drugs could well protect the cells after CPV infection. According to the test definition, wells without CPV infection are used as 100% inhibition protection positive control, while wells infected with CPV (without any drugs) are used as 0% inhibition protection negative control. After formula calculation, the inhibition protection rates (i.e. CPE inhibition percentage) of Closantel, Closantel Sodium, GemcitabineHCl, Trifluridine, Gemcitabine and Cladribine are 80.64 ± 7.87, 69.76 ± 6.06, 64.1 respectively 8±0.97, 57.11±5.45, 56.1±2.09 and 50.92±1.58%, while the inhibitory protection rate (i.e. CPE inhibition percentage) of the broad-spectrum anti-DNA virus drug Cidofovir was only -1.28±1.03%, the difference between the two was extremely significant (P<0.0001), as shown in Figure 2.

Example 2. Determination of 50% effect concentration (50% antiviral efficacy, EC50) and 50% cytotoxicity concentration (50% cytotoxicity concentrations, CC50) of 6 kinds of small molecule drugs

The EC50 and CC50 of the drugs were determined by dose-response assay. details as follows:

The EC50 determination procedure is as follows: 86 μL of F81 cells (25,000 cells per well) were pretreated with 4 μL of doubly diluted drug (Closantel/Closantel Sodium/Gemcitabine HCl/Trifluridine/Gemcitabine/Cladribine) (final concentration range 0.3125-20 μM) for 1 h, and then 10 μL of CPV (New CPV-2a strain SD6 ) (MOI=0.076) to infect the drug-treated cells.

The CC50 determination procedure is as follows: 96 μL of F81 cells (25,000 cells per well) are mixed with 4 μL of doubly diluted drug (final concentration range is 0.3125-80 μM).

Both EC50 and CC50 determinations were performed in triplicate. After 40 hours of incubation, the Cell Counting Kit (TransGen Biotech, China, FC101-01) was used to measure cell viability according to the kit instruction manual, and then to determine the inhibitory efficiency of 6 small molecule drugs on CPV infection and their respective toxicity to F81 cells, and the EC50 and CC50 values were calculated by the dose-response nonlinear regression analysis method in GraphPad Prism software.

The results are shown in Figure 3 and Figure 4:

Closantel, Closantel Sodium, Gemcitabine HCl, Trifluridine, Gemcitabine and Cladribine inhibited strain SD6 (New CPV-2a) on the infection of F81 cells with a median antiviral effect concentration (EC50) of 7.77 μM, 6.01 μM, 0.68 μM, 9.35 μM, 0.62 μM and 0.32 μM, respectively.

Closantel, Closantel Sodium, Gemcitabine HCl, Trifluridine, Gemcitabine, and Cladribine had a median toxic concentration (CC50) of 13.69 μM, 19.08 μM, 141.6 μM, >160 μM, 40.03 μM and 40.21 μM on F81 cells, respectively.

Example 3. Immunofluorescence assay (IFA) detection of inhibition of viral VP2 protein expression by six small molecule drugs

Drug treatment group: 86 μL of F81 cells in a 96-well plate (25,000 cells per well) were pretreated with 4 μL of doubly diluted drugs (Closantel/Closantel Sodium/Gemcitabine HCl/Trifluridine/Gemcitabine/Cladribine) (the final drug concentrations were 5 μM, 10 μM and 20 μM, respectively) for 1 hour, and the treated cells were treated with 10 μL of LCPV (New CPV -2a strain SD6) (MOI=0.076) infection. 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 an immunofluorescence test was performed 30 hours later.

Immunofluorescence test:

Fixation: Discard the culture medium in the 96-well plate, and wash 3 times with 1×PBS (Gibco ^TM , USA, catalog number: 20012050), 2-3 min each time. Cells were fixed with 80% acetone (the volume ratio of acetone and water was 4:1), 100 μL of 80% acetone was added to each well, and incubated at room temperature for 10 min. Wash 3 times with 1×PBS, 2-3min/time.

Blocking: add 3% BSA (3 g BSA to 100 mL 1×PBS) at room temperature for blocking, room temperature for 1 hour. Wash with 1×PBS for 10 minutes.

Primary antibody: Incubate with mouse anti-VP2 monoclonal antibody (INGENASA, Spain, M.15.CPV.I5F8) (1:100 dilution, diluted with 1×PBS) for 40 minutes. Discard the antibody solution and wash 3 times with 1×PBS, 10min each time.

Secondary antibody: Incubate with FITC-labeled goat anti-mouse IgG (H+L) secondary antibody (Invitrogen, USA, A16079) (diluted 1:200, diluted with 1×PBS) for 40 minutes. Discard the antibody solution and wash 3 times with 1×PBS, 10min each time.

Staining: stain with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI, 1 μg/ml), and react at room temperature for 10 minutes in the dark. Discard the staining solution, wash 3 times with 1×PBS, 10min each time.

Cells were examined at 20x magnification with an In Cell Analyzer 2500HS cell imaging analysis system (Operetta, PerkinElmer, USA), and analyzed by Image J image processing software.

The results are shown in FIG. 5 . The control group without any drug was detected by anti-VP2 monoclonal antibody, and there was a large amount of VP2 protein expression in the field of view (the control FITC in FIG. 5 ). In the treatment group with small molecule drugs (Closantel/ClosantelSodium/Gemcitabine HCl/Trifuridine/Gemcitabine/Cladribine), as the concentration of the drug increased, the signal of VP2 protein decreased. When the final concentration of the drug was 5 μM, it showed a difference compared with the control group. When the final concentration of the drug was 10 μM and 20 μM, the detected fluorescence signal gradually decreased. It was proved that six small molecule drugs could successfully inhibit the replication of CPV in F81 cells (Fig. 5).

Example 4. Inhibition experiments of 6 small molecule drugs on different genotypes of CPV strains

Drug treatment group: F81 cells were seeded in ⁶ -well plates at 7.5×105 cells per well, and pretreated with different concentrations of drugs (Closantel/Closantel Sodium/Gemcitabine HCl/Trifluridine/Gemcitabine/Cladribine) (the final drug concentrations were 5 μM, 10 μM and 20 μM, respectively) for 1 hour. Type 2a strain SD6 and New CPV-2b type strain SD3) were infected (MOI=0.076), and after incubation for 40 hours, total cell protein was extracted and subjected to Western blot test.

Control group: DMSO was added to F81 cells to a final concentration of 0.1% (volume fraction), and after incubation for 40 hours, total cell protein was extracted and subjected to Western blot test.

Protein extraction and Western blot test:

Cells were collected and lysed using the mammalian total protein extraction kit ProteinExt (TransGen Biotech, China, DE101-01) according to the kit instructions. The same amount of cell lysate was transferred to PVDF membrane (Millipore, USA) after SDS-PAGE protein electrophoresis. After blocking with TBS-Tween 20 containing 5% skimmed milk (100mL TBS-Tween 20 with 5g skimmed milk powder) at room temperature for 1 hour, use anti-VP2 monoclonal antibody (INGENASA, Spain, M.15.CPV.I5F8) and β-actin monoclonal antibody (AC-15) (Thermo Scientific, USA, MA1-91399), respectively, according to 1:800 and 1 : 4000 dilution (diluted with TBS-Tween 20), and incubated overnight at 4°C. TBS-Tween 20 wash 3 times, 10min/time. HRP-labeled goat anti-mouse IgG (Thermo Scientific, USA, catalog number: 31430) (diluted with TBS-Tween 20 according to 1:6000) was used to incubate at 37° C. for 1 hour. TBS-Tween 20 wash 3 times, 10min/time. SuperSignal ^TM West Pico PLUS Chemiluminescent Substrate Detection Kit (Thermo Scientific, USA, 34579) was used to detect the bands according to the instructions of the kit, and imaged by a chemiluminescent device (Protein simple, USA).

The results of Western blot are shown in Figure 6. All the six small molecule drugs can inhibit the expression of VP2 protein of two different genotypes of CPV viruses, strain SD6 (New CPV-2a) and strain SD3 (New CPV-2b). Compared with the control group without drugs, the difference is obvious. And with the increase of drug concentration, the expression of VP2 protein of the two genotypes gradually decreased, and the bands were weaker when the final concentration of the drug was 10 μM and 20 μM, which proved that the above six small molecule drugs could well inhibit the replication of the virus against CPV strains of different genotypes (Figure 6).

Claims (3)

1. Trifluorothymidine is a single active ingredient in the preparation of medicines that inhibit canine parvovirus replication, characterized in that the structural formula of said trifluorothymidine is as follows: . 2. The use according to claim 1, characterized in that the medicament further comprises a pharmaceutically acceptable carrier. 3. The use according to claim 2, characterized in that the dosage form of the medicine is tablet, suspension or dry suspension.