Application of nizoxinib and its in vivo metabolite tezoxinib in the preparation of drugs for treating feline parvovirus infection

Abstract

This invention discloses the application of nizoxinid and its in vivo metabolite tinzoxinid in the preparation of a drug for treating feline parvovirus infection, belonging to the field of novel uses of pharmaceutical compounds. The active ingredient of the drug contains nizoxinid. Based on nizoxinid and its in vivo metabolite tinzoxinid, this invention evaluates their inhibitory effect on feline parvovirus. This inhibitor can effectively inhibit the replication of feline parvovirus in vitro, and its inhibitory effect occurs during the replication phase of feline parvovirus infection. This invention is based on the ability of nizoxinid and its in vivo metabolite tinzoxinid to inhibit the replication of feline parvovirus. The corresponding drug can effectively prevent and treat feline parvovirus, and it has high safety, wide applicability, and is easy to promote and apply.

Description

Technical Field

[0001] This invention belongs to the field of new uses of pharmaceutical compounds, specifically relating to the application of nitrozonide and its in vivo metabolite tezozonide in the preparation of drugs for treating feline parvovirus infection. Background Technology

[0002] Feline parvovirus (FPV), also known as feline panleukopenia virus, is the pathogen that causes feline panleukopenia (feline distemper). First discovered by the French scholar Verge in the early 20th century, and successfully isolated and cultured by Bilin and Johnson in 1957, it opened the door to scientific understanding of the virus. As a single-stranded negative-sense DNA virus, FPV belongs to the Parvoviridae family and the Parvovirus genus, possessing extremely small viral particle size and widespread natural distribution. Its clinical features are highly typical, primarily causing high fever, vomiting, severe leukopenia, and hemorrhagic enteritis in infected cats, with extremely high morbidity and mortality rates in kittens. The virus is highly contagious and can be transmitted through direct contact or contaminated objects; the long-standing lack of specific antiviral drugs poses a challenge to prevention and control. The source of infection is infected cats and recovered carrier cats, primarily transmitted through the digestive tract, but also through the respiratory tract, vertical transmission, and indirect contact. Outbreaks are more common in winter and spring, and are more likely to occur in group-housed environments. The virus specifically targets rapidly dividing cells, leading to intestinal mucosal necrosis and bone marrow hematopoietic suppression, with a mortality rate exceeding 90% in kittens. Clinical treatment primarily involves supportive therapy, as there are no specific antiviral drugs. Therefore, there is an urgent need to develop a drug that can effectively combat feline parvovirus to inhibit or control its spread.

[0003] Nitazoxanide (NZT) is a broad-spectrum antiprotozoal, antibacterial, and antiviral drug, primarily used clinically to treat diarrhea caused by Cryptosporidium, Giardia lamblia, and Entamoeba histolytica. It was also the first drug approved by the FDA for the treatment of cryptosporidiosis. Its core mechanism of action is the inhibition of pyruvate-ferric oxidoreductase (PFOR), blocking the electron transport chain of pathogen energy metabolism, thereby killing the pathogen. After oral administration, it is rapidly hydrolyzed to tizoxanide, the main active metabolite, which also possesses anti-inflammatory and immunomodulatory potential. Recent studies have found that nitazoxanide and its metabolites also have significant antiviral activity, inhibiting the replication of various viruses, including influenza virus, norovirus, hepatitis B virus, SARS-CoV-2, and pseudorabies virus. Its antiviral mechanism is also related to cellular innate immunity mediated by the host target protein SμCLG2. In addition, nitrozonide has shown good efficacy in the treatment of canine giardiasis, with a dose of 2-4 mg / kg body weight being able to quickly clear Giardia cysts from the dog's body. However, there are currently no reports on the use of nitrozonide in the treatment of feline parvovirus infection, nor have there been any records of its application in the suppression and treatment of feline parvovirus infection. Summary of the Invention

[0004] This invention addresses the technical problems of the lack of effective treatments for feline parvovirus infection, poor treatment outcomes, and difficulty in controlling secondary infections. It provides the application of nitrozonide and its metabolite tezolinide in the preparation of drugs for treating feline parvovirus infection, expanding the application scope of nitrozonide and providing a new drug for treating feline parvovirus infection. This drug can specifically inhibit feline parvovirus replication, alleviate clinical symptoms, control secondary infections, improve the cure rate, and has high safety and low drug resistance rate.

[0005] To achieve the above objectives, this application provides the use of nizoxinid and its in vivo metabolite tezoxinid in the preparation of a drug for treating feline parvovirus infection, wherein the active ingredient of the drug contains nizoxinid.

[0006] Furthermore, the active ingredient also includes tinizonide, the in vivo metabolite corresponding to nizozonide.

[0007] Furthermore, the drug is a single active ingredient drug, whose active ingredient contains only nitrozonide.

[0008] Furthermore, the drug is a compound drug, and its active ingredients include nitrozonidine and other antitoxic components.

[0009] Furthermore, the other antiviral components are at least one of antiviral drugs, antibacterial drugs, immunomodulators, and fluid replacement.

[0010] Furthermore, the drug also contains a pharmaceutically acceptable carrier.

[0011] Furthermore, the dosage form of the drug is tablets, suspensions, or dry suspensions.

[0012] The present invention has the following advantages over the prior art: This invention evaluates the inhibitory effect of nitrozonide and its in vivo metabolite tinozonide on feline parvovirus (FPV). This inhibitor effectively suppresses FPV replication in vitro, with the inhibitory effect occurring during the replication phase of FPV infection. This invention is based on the ability of nitrozonide and its in vivo metabolite tinozonide to inhibit FPV replication; the corresponding drugs contained within them effectively prevent and treat FPV, and exhibit high safety, broad applicability, and ease of widespread application. Attached Figure Description

[0013] Figure 1 This is a graph showing the toxicity results of different concentrations of nitrozonidine on CRFK cells.

[0014] Figure 2 This is a graph showing the toxicity results of different concentrations of tezolid on CRFK cells.

[0015] Figure 3 This is an indirect immunofluorescence image showing the inhibitory effect of different concentrations of nitrozonide on FPV.

[0016] Figure 4 This is an indirect immunofluorescence OD analysis graph showing the inhibitory effect of different concentrations of nitrozonide on FPV.

[0017] Figure 5 This is an indirect immunofluorescence image showing the inhibitory effect of different concentrations of tezolid on FPV.

[0018] Figure 6 This is an indirect immunofluorescence OD analysis graph showing the inhibitory effect of different concentrations of tezolid on FPV.

[0019] Figure 7 This is a diagram illustrating the time intervals for adding nitrozonide and tezolid.

[0020] Figure 8 This is an indirect immunofluorescence image showing the inhibitory effect of adding nitrozonidine at different times on FPV.

[0021] Figure 9 This is an indirect immunofluorescence image showing the inhibitory effect of adding tezolid at different times on FPV. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of the embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0023] In the embodiments, it should be noted that any processes not specifically described below are those that can be implemented or understood by those skilled in the art by referring to existing technology. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0024] Experimental materials: 1. Drug Sources: Nitazoxanide (NTZ) was purchased from Shanghai Titan Technology Co., Ltd., product number 65650A. Tizoxanide (TIZ) was purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number T91166.

[0025] 2. Biochemical reagents: DMEM, PBS, and FBS were purchased from Gibco. DMSO was purchased from Solarbio. CCK8 was purchased from Shanghai Titan Technology Co., Ltd. Anti-feline parvovirus IgG (primary antibody) was purchased from Guangzhou Qianxun Biotechnology Co., Ltd., and Cy3-labeled goat anti-mouse IgG (secondary antibody) was purchased from Beyotime.

[0026] Example 1: To determine the cytotoxicity of nizoxinib and tezoxinib, the cytotoxicity of nizoxinib and tezoxinib was detected using the CCK8 assay kit. The specific procedures were as follows: Nizoxinib and tezoxinib were dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 50 mmol / L, and then dissolved in DMEM containing 2% FBS to different concentrations. After CRFK cells were cultured into a monolayer in 96-well cell culture plates, the 10% FBS DMEM culture medium was discarded. 100 μL of nizoxinib or tezoxinib solution containing different concentrations (256, 128, 64, 32, 16, 8, 4 μmol / L) was added to each well. The plates were incubated at 37°C in a 5% CO2 incubator. After 48 h, the culture was terminated, the supernatant was discarded, and 100 μL of 2% FBS DMEM culture medium was added to each well. CCK-8 solution was added to each cell well at a density of 10 μL. After incubation at 37°C and 5% CO2 for 3 h, the OD value of each well was measured at 450 nm using a microplate reader to calculate cell viability. GraphPad Prism 8.0 software was used to calculate the half-maximal cytotoxic concentration (CC50) of the two active pharmaceutical ingredients on CRFK cells.

[0027] From the appendix Figure 1 It can be seen that when the concentration of nitrozonidine is not higher than 380.8 μmol / L, the viability of normal CRFK cells can be maintained above 50%. (From the attached...) Figure 2 It can be seen that when the concentration of tezolid is not higher than 208.3 μmol / L, the viability of normal CRFK cells can be guaranteed to be above 50%.

[0028] Example 2: Antiviral activity of nitrozonide and tezolid against feline parvovirus: 100 μL of CRFK cells were seeded into 96-well plates and cultured overnight in an incubator; when the cell confluence in each well reached approximately 60%, the old culture medium was discarded, and the cells were washed twice with sterile PBS buffer; 100 µL of diluted virus solution (100 TCID50) was added to each well. 50 Incubate the cells in an incubator for 1 hour; wash twice with sterile PBS buffer to remove unadsorbed virus particles, then add 100 µL of nitrozonide or tezolinide at a series of concentrations to each well, and continue incubation for 48 hours. Observe CPE, and then perform indirect immunofluorescence detection as follows: Fixation: Wash twice with PBS buffer, add 4% paraformaldehyde (50 µL per well), and let stand at room temperature for 15-20 min; Permeabilization: Wash twice with PBS buffer, add 0.3% Triton solution diluted with PBS buffer (50 µL per well), and let stand at room temperature for 10 min; Blocking: Wash twice with PBS buffer, add 5% BSA solution prepared with PBS buffer (50 µL per well), and incubate at 37°C for 1 h; Primary antibody: Wash twice with PBS buffer, add 100 µL of primary antibody solution diluted with PBS buffer (1:500) to each well, and incubate overnight at 4°C; recover the primary antibody solution and wash three times with PBS buffer; Secondary antibody: Protect from light, add 50 µL of secondary antibody solution diluted with PBS buffer (1:500) to each well, and let stand for 1 h; wash 3 times with PBS buffer; DAPI: Protect from light. Add 50 µL of DAPI (3 µM) diluted with PBS buffer to each well, let stand at room temperature for 10 min, and wash twice with PBS buffer. Recording: Results were observed and photographed using a fluorescence inverted microscope. Grayscale analysis was performed on the results using ImageJ software. Finally, the results were analyzed and plotted using SPSS 18.0 and Graphpad Prism 8 software.

[0029] From the appendix Figure 3 It was found that the addition of 5 μmol / L nitrozonide significantly inhibited the proliferation of feline parvovirus in CRFK cells. Further analysis by appendix... Figure 4 It was found that when the EC50 of nitrozonidine was 1.66 μmol / L, and the concentration of nitrozonidine was greater than 1.66 μmol / L, it significantly reduced the synthesis of VP2 protein of feline parvovirus, showing a dose-dependent decreasing trend. This indicates that nitrozonidine can inhibit the replication of feline parvovirus within a certain concentration range. (See attached...) Figure 5 It was found that the addition of 5 μmol / L tezolinide significantly inhibited the proliferation of feline parvovirus in CRFK cells. Further analysis by appendices... Figure 6 It was found that when the EC50 of tinizonide was 1.73 μmol / L, and the concentration of tinizonide was greater than 1.73 μmol / L, it significantly reduced the synthesis of VP2 protein of feline parvovirus, and the results showed a dose-dependent decreasing trend. This indicates that tinizonide can inhibit the replication of feline parvovirus within a certain concentration range.

[0030] Example 3: Effect of adding nitrozonide or tezolid at different times on FPV virus replication: 100 μL of CRFK cells were seeded in 96-well plates and cultured overnight in an incubator; when the cell confluence in each well was about 60%, the old culture medium was discarded, and the cells were washed twice with sterile PBS buffer. 100 μL of nitrozonide containing 100 TCID3 was then added to the plate. 50 Feline parvovirus was added to a 96-well cell culture plate containing a monolayer of CRFK cells (repeat to 3 wells) and mixed with a solution containing 100 TCID50. 50Feline parvovirus and DMEM containing 0.4% DMSO were used as a mimicry treatment. To determine whether the antiviral effects of the two drugs in CRFK cells were time-dependent, see attached... Figure 7 As shown in the time-added diagram, nitrozonide or tezolinide at a final concentration of 5 μmol / L was used at -2 h, 2 h, 4 h, and 6 h after feline parvovirus infection. After incubation at 37°C and 5% CO2 in a carbon dioxide incubator for 48 h, indirect immunofluorescence detection was performed, following the specific steps in Example 2.

[0031] From the appendix Figure 8 and attached Figure 9 Compared with the DMSO group, the expression levels of feline parvovirus VP2 protein were significantly reduced in the nitrozonide and tinzolide treatment groups at -2 h, 2 h, 4 h, and 6 h post-infection, indicating that nitrozonide and tinzolide (5 μmol / L) still had an antiviral effect against feline parvovirus 6 h after infection. This suggests that nitrozonide and tinzolide can play a role in the replication phase of feline parvovirus infection.

[0032] As demonstrated in Examples 1-3, nizoxinid and its metabolite tinizonide significantly inhibit feline parvovirus (FPV) replication, and this inhibition occurs during the FPV replication phase. Nizoxinid concentrations greater than 1.66 µM significantly inhibited FPV replication, and tinizonide concentrations greater than 1.73 µM significantly inhibited FPV replication. Compared to the simulation group, when the concentration of nizoxinid and its metabolite tinizonide reached 5 µM, the inhibition of FPV in CRFK cells reached approximately 90%, demonstrating a significant effect. Furthermore, analysis of the results of experiments on FPV replication with different additions of nizoxinid or tinizonide at different time points showed that the inhibition of FPV primarily occurred during the replication phase.

[0033] It should be noted that, in the description of this invention, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified.

[0034] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention are included within the scope of protection of the present invention.

Claims

1. The use of nitrozonide and its in vivo metabolite tezolidinide in the preparation of a drug for treating feline parvovirus infection, characterized in that, The active ingredient in the drug contains nitrozonidine.

2. The application of nitrozonide and its in vivo metabolite tezolid according to claim 1 in the preparation of a drug for treating feline parvovirus infection, characterized in that, The active ingredient also includes tinazole, the in vivo metabolite of nitrozonide.

3. The application of nitrozonide and its in vivo metabolite tezolid according to claim 1 in the preparation of a drug for treating feline parvovirus infection, characterized in that, The drug is a single active ingredient drug, and its active ingredient contains only nitrozonide.

4. The application of nitrozonide and its in vivo metabolite tezolid according to claim 1 in the preparation of a drug for treating feline parvovirus infection, characterized in that, The drug is a compound drug, and its active ingredients include nitrozonidine and other antitoxic components.

5. The application of nizoxinid and its in vivo metabolite tezoxinid according to claim 4 in the preparation of a drug for treating feline parvovirus infection, characterized in that, The other antiviral components are at least one of antiviral drugs, antibacterial drugs, immunomodulators, and fluid replacement.

6. The use of nitrozonide and its in vivo metabolite tezolid according to any one of claims 1-5 in the preparation of a drug for treating feline parvovirus infection, characterized in that, The drug also contains a pharmaceutically acceptable carrier.

7. The application of nitrozonide and its in vivo metabolite tezolid according to claim 6 in the preparation of a drug for treating feline parvovirus infection, characterized in that, The dosage form of the drug is tablets, suspensions, or dry suspensions.

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