CN111012778A - Application of Nitazoxanide in inhibiting canine parvovirus - Google Patents

Abstract

The invention discloses application of Nitazoxanide in inhibiting canine parvovirus, and relates to new application of a known medicament. The data prove that the small molecule drug Nitazoxanide has excellent inhibition effect on CPV replication and can inhibit the expression of VP2 protein of different genotypes.

Description

Application of Nitazoxanide in inhibiting canine parvovirus

Technical Field

The invention relates to a new application of a compound, in particular to an application of Nitazoxanide in inhibiting canine parvovirus.

Background

Canine Parvovirus (CPV), a member of the family parvoviridae, is a non-enveloped, single-stranded DNA virus. 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 at 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 agents for the prevention and treatment of CPV virus is urgently needed.

Disclosure of Invention

The inventor of the invention unexpectedly finds that the small molecule drug Nitazoxanide shown as the formula I has very excellent inhibition effect on CPV replication. The concentration of the drug in 50% cytotoxicity (CC 50) and 50% antiviral effect (EC 50) is determined by in vitro experiments, and the effect of the drug in inhibiting CPV replication in vitro is further verified by using Indirect immunofluorescence assay (IFA) and Western blot.

The technical scheme of the invention is as follows:

the invention provides application of Nitazoxanide in preparing a medicament for inhibiting canine parvovirus replication, which is characterized in that the active ingredient of the medicament comprises Nitazoxanide.

Preferably, the medicament is a single active ingredient medicament, the active ingredient of which comprises Nitazoxanide only.

Preferably, the medicament is a compound medicament, and the active ingredients of the compound medicament comprise Nitazoxanide and other antiviral ingredients.

Preferably, the medicament further comprises a pharmaceutically acceptable carrier.

Preferably, the pharmaceutical is in the form of a tablet, suspension or dry suspension.

In another aspect of the present invention, there is provided a medicament for inhibiting canine parvovirus replication, wherein the active ingredient of the medicament comprises nitizoxanide.

Preferably, the medicament is a single active ingredient medicament, the active ingredient of which comprises Nitazoxanide only.

Preferably, the medicament is a compound medicament, and the active ingredients of the compound medicament comprise Nitazoxanide and other antiviral ingredients.

Preferably, the medicament further comprises a pharmaceutically acceptable carrier component, either liquid or solid.

Preferably, 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 the level of canine parvovirus in the serum of said infected individual is significantly reduced or reduced to undetectable levels after a predetermined period of time on schedule.

Nitazoxanide (Nitazoxanide) has previously been reported for the treatment of cryptosporidiosis, Giardia and other parasitic infections, and has the chemical formula o [ N- (5-nitrothiazol-2-yl) carbamoyl]Phenol acetate of the formula C₁₂H₉N₃O₅S, having the following structural formula I:

the inventor finds that Nitazoxanide can inhibit the replication of a CPV strain, and measures the CPV inhibition protection effect of Nitazoxanide on F81 cells, and the result shows that Nitazoxanide can well protect cells after CPV infection, and as shown in figure 1, the Nitazoxanide inhibition protection rate is as high as 103 +/-3.46% at 10 mu M, which is obviously higher than the protection rate of a control Cidofovir of 0.11 +/-0.70%.

Further, the 50% effective concentration (50% antiviral efficacy, EC50) and 50% cytotoxic concentration (50% cytoxicity concentrations, CC50) of Nitazoxanide against CPV strain IS1(New CPV-2a) were determined, and the results showed that Nitazoxanide inhibited the infection of F81 cells by strain IS1 with EC50, CC50 and SI values of 2.02. mu.M, 26.05. mu.M and 12.90, respectively, and the results are shown in FIG. 2.

The inventor further performs an immunofluorescence IFA test, and the results of the immunofluorescence test show that the fluorescence signal for detecting the expression of VP2 is obviously lower than that of an untreated control group at the concentration of 5 mu M, and the fluorescence signal is hardly detected at the concentrations of 10 mu M and 20 mu M, so that the Nitazoxanide can successfully inhibit the replication of CPV in F81 cells.

In addition, Western blot results show that Nitazoxanide can inhibit the expression of VP2 protein of different genotypes of viruses, and the expression quantity of VP2 protein of different genotypes is gradually reduced with the increase of the concentration of the drug, so that the effect of the drug in inhibiting CPV replication is further verified.

In conclusion, the invention screens out the candidate drug Nitazoxanide which can effectively resist CPV, and because the Nitazoxanide is a mature clinical drug in use, the Nitazoxanide provides reference for clinical medication, and is developed into the CPV-resistant drug, so that the invention has a great application prospect.

Drawings

FIG. 1 Nitazoxanide is capable of inhibiting CPV replication in vitro,

FIG. 2 Nitazoxanide EC50(A) and CC50(B) in F81 cells,

FIG. 3 shows that the immunofluorescence method detects the Nitazoxanide inhibits the expression of virus VP2 protein,

FIG. 4 Western blot to detect VP2 expression of F81 cells after treatment with Nitazoxanide of different concentrations and infection with different types of IS1 and IS 2.

Detailed Description

Experimental reagent

Growth Medium (GM): DMEM (Gibco, USA) plus 10% fetal bovine serum, 100U/ml penicillin, 100. mu.g/ml streptomycin.

Maintenance Medium (MM): DMEM plus 2% fetal calf serum and 100U/ml penicillin and 100. mu.g/ml streptomycin.

Nitazoxanide: from seleck (usa), drug parameters: s1627 (concentration: 10 mM).

Cidofovir: from seleck (usa), drug parameters: s1516 (concentration: 10 mM).

anti-VP 2 monoclonal antibody, purchased from INGENASA, spain, m.15.cpv.i5 f8.

FITC-labeled goat anti-mouse IgG (H + L) secondary antibody (1: 200 dilution) (Thermo Scientific, USA), A16079

β -actin (AC-15), available from Thermo Scientific, USA, MA 1-91399.

Trans

Cell Counting Kit，TransGen Biotech，China，FC101-01。

SuperSignal^TMWest Pico PLUS chemiluminescent substrate detection kit, Thermo Scientific, USA, 34579.

Cells and viruses

F81 cells were purchased from ATCC and cultured in Growth Medium (GM) consisting of DMEM (Gibco, USA), 10% fetal bovine serum and 100U/ml penicillin and 100. mu.g/ml streptomycin. Maintenance Medium (MM) consisted of DMEM, 2% fetal bovine serum and 100U/ml penicillin and 100. mu.g/ml streptomycin.

The New CPV-2a type strain IS1 and the New CPV-2b strain IS2 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 of agriculture and forestry, academy of sciences in Beijing City. The nucleotide sequence homology of the VP2 protein coding gene and the corresponding VP2 protein coding gene of BJ14-7(KT162031, New CPV-2a) and BJ14-1(KT162022, New CPV-2b) is as high as 99.9% and 99.3%.

The applicants claim that it IS possible to provide the public with experiments for research purposes with strain IS1 of the New CPV-2a type and strain IS2 of the New CPV-2b type within two decades from the filing date.

Example 1 measurement of protective Effect of Nitazoxanide on CPV inhibition in F81 cells

F81 cells before use, 4. mu.L of Nitazoxanide (10mM) and Cidofovir (10mM) drugs were transferred to a new 96-well plate and diluted into wells containing 16. mu.L of maintenance medium to prepare 250. mu.M drug stock solutions.

86 μ L F81 cells (25,000 cells per well) were treated with 4 μ L of the above diluted drug stock solution at a final concentration of 10 μ M for 1h each drug.

F81 cells were infected with 10 μ L of CPV at an MOI of 0.012 after drug treatment. F81 cells containing 0.1% DMSO and cells containing IS1(New CPV-2 a)/0.1% DMSO mixtures were used as positive or negative controls, respectively. Trans was used 48 hours after infection

Cell Counting Kit (TransGen Biotech, China) examined Cell viability. Wells not infected with CPV served as positive (cell) control wells with 100% inhibition protection, and wells infected with IS1(New CPV-2a)Negative (viral) 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 experimental results are as follows:

the inhibition protection test result shows that the Nitazoxanide can well protect cells infected by CPV. According to the experimental definition, the holes without CPV infection are used as a positive control of 100% inhibition protection, and the holes infected with CPV (without any drug) are used as a negative control of 0% inhibition protection, and the Nitazoxanide inhibition protection rate (namely CPE inhibition percentage) is as high as 103 +/-3.46%, while the broad-spectrum anti-DNA virus drug Cidofovir has the inhibition protection rate (namely CPE inhibition percentage) of only 0.11 +/-0.70%, and the difference is very significant (P <0.0001), and the result is shown in FIG. 1.

Example 2 determination of the Nitazoxanide 50% Effect concentration (50% antiviral efficacy, EC50) and 50% cytotoxic concentration (50% cytotoxic concentrations, CC50)

The EC50 and CC50 of the drugs were determined using a dose response assay. Specifically, as follows, the following description will be given,

the EC50 determination procedure was: 86 μ L F81 cells (25,000 cells per well) were pretreated for 1h with 4 μ L of drug diluted in multiples (final concentration range 0.3125-20 μ M) and then infected with 10 μ L of CPV (MOI ═ 0.012).

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).

EC50 and CC50s assays were performed in 3 replicates. After 48 hours incubation, with Trans

Cell viability was measured with CellCounting Kit (transcgen Biotech, China) to determine the inhibition efficiency of nitizoxanide against CPV infection and the toxicity of nitizoxanide against F81 cells, and EC50 and CC50 values were calculated by dose-response nonlinear regression analysis in GraphPad Prism software.

As a result:

the concentration of the anti-viral effect of the inhibitor strain of Nitazoxanide IS1 on F81 cell infection was 2.02. mu.M, while the concentration of the toxicity of Nitazoxanide on F81 cell was 26.05. mu.M, and the results are shown in FIG. 2. The selection coefficient (SI) is CC50/EC 50-12.90.

Example 3 Immunofluorescence assay (IFA)

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 diluted in multiples (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 (MOI ═ 0.012). Approximately 30 hours after infection, cells were fixed with 80% acetone and then treated with 1: 100 dilution of mouse anti-VP 2 monoclonal antibody (INGENASA, Spain) was incubated for 40 min, washed and then incubated with a secondary FITC-labeled goat anti-mouse IgG (H + L) antibody (1: 200 dilution) (Invitrogen, USA). Finally, staining was performed with 4, 6-diamidino-2-phenylindole (DAPI). After washing, cells were examined at 20x magnification with an In CellAnalyzer 2500HS instrument (Operetta, PerkinElmer, USA) and analyzed by ImageJ.

As can be seen from the results, the control group to which no drug was added was detected using a monoclonal antibody against VP2, and a large amount of VP2 protein was expressed in the visual field. In the group to which the Nitazoxanide drug was added, as the concentration of Nitazoxanide increased, the detectable VP2 protein signal decreased, and the difference was significant at 5. mu.M compared to the control, and almost no fluorescence signal was detected at 10. mu.M and 20. mu.M. Nitazoxanide was shown to be successful in inhibiting CPV replication in F81 cells (FIG. 3).

Example 4 inhibition experiment of Nitazoxanide against different genotype CPV strains

F81 cells were plated at 7.5X 10 per well⁵Cells were seeded in 6-well plates and pre-treated with different concentrations of drugs (final concentrations of 5 μ M, 10 μ M and 20 μ M, respectively) for 1 hour, and the treated cells were infected (MOI 0.012) with different genotype CPV strains IS1(New CPV-2a) and IS2(New CPV-2b), and cells that were not drug-treated were used as controls.

After 48 hours incubation, cells were collected and used to extract the kit ProteinExt (Trans) from total mammalian proteinGenBiotech, China) equal amounts of cell lysates were transferred to PVDF membrane (Millipore, USA) after protein electrophoresis on SDS-PAGE, blocked for 1 hour at room temperature with TBS-Tween 20 containing 5% skim milk, diluted with anti-VP 2 monoclonal antibodies (INGENASA, Spanish, M.15.CPV.I5F8) and β -actin (AC-15) (Thermo Scientific, USA, MA1-91399), respectively, at 1: 800 and 1: 4000 and incubated overnight at 4 ℃, washed, incubated with HRP-labeled goat anti-mouse IgG for 1 hour at 37 ℃ and bands with SuperSignal^TMWest Pico PLUS chemiluminescent substrate detection kit (Thermo Scientific, USA, 34579) and imaged by a chemiluminescent device (Protein simple, USA).

Western blot results show that Nitazoxanide can inhibit the expression of VP2 proteins of different genotypes of IS1(New CPV-2a) and IS2(New CPV-2b), and the difference IS obvious compared with a control without adding drugs (adding 0.1% DMSO). And with the increase of the drug concentration, the expression level of 2 genotype VP2 proteins is gradually reduced, and the bands are weaker at the concentrations of 10 mu M and 20 mu M, which proves that the Nitazoxanide can well inhibit the replication of the virus for different genotype strains (FIG. 4).

Claims (10)

1. Use of Nitazoxanide for the preparation of a medicament for inhibiting canine parvovirus replication, wherein the active ingredient of said medicament comprises Nitazoxanide.
2. 2. Use according to claim 1, wherein the medicament is a single active ingredient medicament comprising as active ingredient only Nitazoxanide.
3. 3. The use according to claim 1, wherein the medicament is a combination medicament, the active ingredients of which comprise Nitazoxanide and other antiviral ingredients.
4. 4. The use according to any one of claims 1 to 3, wherein the medicament further comprises a pharmaceutically acceptable carrier.
5. 5. Use according to claim 4, wherein the medicament is in the form of a tablet, suspension or dry suspension.
6. 6. A medicament for inhibiting canine parvovirus, wherein the active ingredient of the medicament comprises nitizoxanide.
7. 7. The medicament according to claim 6, wherein the medicament is a single active ingredient medicament comprising Nitazoxanide alone as an active ingredient.
8. 8. The drug according to claim 6, which is a combination drug comprising Nitazoxanide and other antiviral ingredients as active ingredients.
9. 9. The medicament according to any one of claims 6 to 8, further comprising a pharmaceutically acceptable liquid or solid carrier component.
10. 10. The medicament of claim 9, wherein the medicament is in the form of a tablet, suspension or dry suspension.

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