Application of six small molecule drugs in inhibition of canine parvovirus

A technology of canine parvovirus and medicine, applied in the field of known compounds, can solve the problems of vaccine weakening

Active Publication Date: 2020-10-30
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although inactivated or attenuated live vaccines have been widely used for prevention, the repeated emergence of mutant strains has raised concerns and concerns about the effectiveness of existing vaccines, while maternal antibodies have also been shown to weaken vaccines

Method used

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  • Application of six small molecule drugs in inhibition of canine parvovirus
  • Application of six small molecule drugs in inhibition of canine parvovirus
  • Application of six small molecule drugs in inhibition of canine parvovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1.6 Determination of inhibitory and protective effects of small molecule drugs on CPV in F81 cells

[0062] Before using F81 cells, add 4μL of small molecule drug (Closantel / Closantel Sodium / Gemcitabine HCl / Trifluridine / Gemcitabine / Cladribine) (10mM) and 4μL of control drug Cidofovir (10mM) to 156μL of maintenance medium (MM). Prepare a 250 μM drug stock solution.

[0063] Drug treatment group: F81 cells were treated with each drug stock solution. For each treatment group, 4μL of 250μM drug stock solution was added to 86μLF81 cells (25,000 cells per well), the final concentration of each drug was 10μM, after continuous treatment for 1h, 10μL of CPV (New CPV-2a strain SD6) was used to The MOI (multiplicity of infection) of 0.076 infects F81 cells treated with drugs. Cell viability was measured 40 hours after infection.

[0064] Positive control: add DMSO to F81 cells to a final concentration of 0.1% (volume fraction). Cell viability was measured 40 hours after addin...

Embodiment 2

[0069] Example 2.6 Determination of 50% effect concentration (50% antiviral efficacy, EC50) and 50% cytotoxicity concentration (50% cytotoxicity concentrations, CC50) of small molecule drugs

[0070] A dose response test was used to determine the EC50 and CC50 of the drug. details as follows:

[0071] The EC50 determination procedure is as follows: pretreat 86μL F81 cells (25,000 cells per well) with 4μL diluted drug (Closantel / Closantel Sodium / Gemcitabine HCl / Trifluridine / Gemcitabine / Cladribine) (final concentration range is 0.3125-20μM) for 1h Then, 10 μL of CPV (New CPV-2a strain SD6) (MOI=0.076) was used to infect the cells after the drug treatment.

[0072] The CC50 determination procedure is: 96 μL of F81 cells (25,000 cells per well) and 4 μL of a multiple-diluted drug (the final concentration range is 0.3125-80 μM).

[0073] Both EC50 and CC50 determinations were repeated 3 times. After 40 hours of incubation, use Cell CountingKit (TransGen Biotech, China, FC101-01) was use...

Embodiment 3

[0077] Example 3. Immunofluorescence assay (Immunofluorescence assay, IFA) detects the inhibition of the expression of virus VP2 protein by 6 small molecule drugs

[0078] Drug treatment group: 86 μL of F81 cells (25,000 cells per well) in a 96-well plate were diluted with 4 μL of drugs (Closantel / Closantel Sodium / Gemcitabine HCl / Trifluridine / Gemcitabine / Cladribine) (the final concentration of the drug was 5 μM, respectively) 10μM and 20μM) were pretreated for 1 hour, and the treated cells were infected with 10μLCPV (New CPV-2a strain SD6) (MOI=0.076). An immunofluorescence test was performed about 30 hours after infection.

[0079] Control group: Add DMSO to F81 cells in a 96-well plate to a final concentration of 0.1% (volume fraction), and perform an immunofluorescence test 30 hours later.

[0080] Immunofluorescence test:

[0081] Fixation: discard the culture medium in the 96-well plate and use 1×PBS (Gibco TM , USA, article number: 20012050) Wash 3 times, 2-3min / time. The cel...

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Abstract

The invention relates to new application of known compounds, in particular to an application of six small molecule drugs in inhibition of canine parvovirus. The invention provides the application of closantel, Closantel Sodium, gemcitabine HCl, Trifluridine, Gemcitabine and Cladribine in preparation of drugs for inhibiting replication of canine parvovirus, t active components of the medicine comprise Closantel, Closantel Sodium, Gemcitabine HCl, Trifluridine, Gemcitabine and / or Cladribine, and the structural formula of the medicine is shown in the specification. The data prove that the six small molecule drugs have very excellent inhibition effects on CPV replication, and can inhibit expression of different genotypes of CPV virus VP2 proteins.

Description

Technical field [0001] The present invention relates to new uses of known compounds, especially the use of Closantel, Closantel Sodium, Gemcitabine HCl, Trifluridine, Gemcitabine and Cladribine in inhibiting canine parvovirus. Background technique [0002] Canine parvovirus (CPV) is a single-stranded DNA virus with a simple structure. It is a member of the Parvoviridae family. It has no envelope and forms an icosahedron. The total length of the viral genome is about 5300 nt and contains 2 ORFs. The 5'end mainly encodes the early transcription regulatory proteins (NS1 and NS2), and the 3'end encodes the late transcription structural protein, namely the viral capsid protein (VP1 and VP2). VP2 protein is the main component of the capsid protein, which can bind to the transferrin receptor (TfR) on the host cell membrane to mediate parvovirus infection. CPV is one of the main pathogens of acute gastroenteritis, leukopenia and myocarditis in dogs, and it is widespread in carnivores. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/7068A61K31/277A61K31/7076A61K31/7072A61P31/20
CPCA61K31/7068A61K31/277A61K31/7076A61K31/7072A61P31/20A61K2300/00Y02A50/30
Inventor 杨兵周宏专苏霞徐福洲林路路张进齐颀
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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