Application of labolol in preparation of medicine for preventing and treating feline calicivirus infection

Abstract

The invention belongs to the technical field of veterinary drugs, and particularly discloses application of laboldol serving as a medicinal ingredient in preparation of a medicine for preventing and treating FCV (Fine Calivirus) infection. According to the application disclosed by the invention, the experimental research finds that the Larburol can obviously inhibit the proliferation of FCV in cat kidney cells, and the median inhibitory concentration (IC50) of the Larburol is 1.87 mu M; in-vivo pharmacological experiments show that the laboldol significantly inhibits the replication of FCV in cats and alleviates symptoms induced by virus infection. Research results show that the laboldol can be used as a drug effect component and can be prepared into a preparation with an auxiliary agent, and the laboldol is used for preventing and treating diseases caused by FCV infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of veterinary drugs. More specifically, it relates to the application of rabdol in the preparation of drugs for preventing and treating feline calicivirus infection. Background Art

[0002] Feline calicivirus disease (FCVD) is an acute and highly contagious infectious disease caused by feline calicivirus (FCV) infecting feline animals. The main clinical symptoms of FCV infection are upper respiratory tract symptoms, oral and nasal ulcers, and fever. Acute lameness syndrome, abortion, enteritis, and severe pneumonia may also occur. Previously, a fatal systemic FCV mutant strain found in the United States and the European Union can even cause severe systemic diseases, including skin edema and ulcerative lesions, jaundice, with a mortality rate as high as 67%. FCV was first discovered in 1957. According to the European Advisory Committee on Cat Diseases, the prevalence of FCV in domestic pet cats is 10%, but in places with high cat breeding density, such as shelters, cat colonies, and catteries, up to 40% of cats are detected with FCV. FCV can show latent infection after animal infection and some animals carry the virus for life, which poses a potential hazard to cats with low antibody levels. Cats can be vaccinated with FCV vaccine to prevent FCV infection. However, due to the large number of clinical subtypes of FCV, the preventive effect of the vaccine against different subtypes of FCV infection is low, and its clinical application effectiveness is very limited.

[0003] Antiviral drugs are an effective means to prevent and control viral diseases. Chinese Patent Application CN111437285A discloses a feline calicivirus inhibitor with nitazoxanide and mizoribine as active ingredients. Through experiments, it is proved that when nitazoxanide with a concentration of 0.2 μM to 2.5 μM and mizoribine with a concentration of 0.6 μM to 20 μM are used simultaneously, the two drugs have a synergistic inhibitory effect on the replication of feline calicivirus. However, there are few research reports on drugs for preventing and treating FCVD at present, and there is still no drug with anti - FCV effect clinically. Therefore, the research and development demand for drugs against FCV infection is urgent.

[0004] Lapachol is a natural naphthoquinone compound with the chemical name 2-hydroxy-3-(3-methylbut-2-en-1-yl)naphthalene-1,4-dione, having the chemical structure of formula (I). It is widely present in various plants, including those of the Apocynaceae, Balsaminaceae, Euphorbiaceae, and Liliaceae families, and can also be chemically synthesized. Lapachol is an orally active dihydroorotate dehydrogenase (DHODH) inhibitor. By directly blocking DHODH activity, it inhibits the biosynthesis of pyrimidine nucleotides, and thus has a wide range of physiological activities such as anti-tumor and anti-parasitic effects. The replication of RNA viruses depends on the host cell to provide the raw materials required for virus synthesis, including two pyrimidine nucleotides, cytosine and uracil. Therefore, inhibiting DHODH activity can inhibit virus replication. There are many research reports on the anti-tumor effect of lapachol, but there are few research reports on its antiviral effect. There are no reports on the anti-FCV infection effect of lapachol and its application in the prevention and treatment of FCV infection, nor are there any related patent disclosures.

[0005] Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide the application of lapachol in the preparation of drugs for preventing and treating FCV infection in view of the lack of existing anti-FCV drugs and the situation of no available drugs.

[0007] The drug in the present application also contains pharmaceutically acceptable excipients; the dosage form of the drug is tablets, capsules, granules, powders, syrups, oral liquids or injections.

[0008] Lapachol in the present application has anti-FCV infection activity in vitro and in vivo.

[0009] Lapachol in the present application can inhibit the replication of FCV virus, reduce the relative expression levels of FCV p30mRNA and VP1 protein, and its effective concentration is 1.25 - 5 μM.

[0010] Furthermore, lapachol in the present application has an inhibitory effect on the replication of FCV in cats.

[0011] Preferably, the oral administration dose of lapachol in the present application is 5 mg / kg, administered once a day for 7 consecutive days, which can significantly inhibit the replication of FCV in cats and relieve symptoms such as oral ulcers induced by virus infection.

[0012] The present invention has the following beneficial effects: Lapachol described in the present invention significantly inhibits the proliferation of FCV in feline kidney cells, significantly reduces the replication of FCV in cats, and relieves the disease symptoms induced by FCV infection. In addition, the lapachol has advantages such as a wide source and good availability, and is a candidate drug with application prospects for the preparation of anti-feline calicivirus infection drugs. Brief Description of the Drawings

[0013] The accompanying drawings here are incorporated into the description and form a part of this description, showing embodiments consistent with the present invention, and are used together with the description to explain the principles of the present invention.

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or in the prior art, the accompanying drawings required for use in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0015] In the accompanying drawings:

[0016] 1. Figure 1 Is the measurement result of the half cytotoxic concentration (CC 50 ) of arabinol on feline kidney cells and the half inhibitory concentration (IC 50 ) of arabinol on FCV in feline kidney cells in Example 1.

[0017] 2. Figure 2 Is the statistical chart of experimental data on the inhibitory effect of arabinol at different concentrations on the synthesis of FCV VP1 protein in feline kidney cells analyzed by immunofluorescence method in Example 2.

[0018] 3. Figure 3 Is the statistical chart of experimental data on the inhibitory effect of arabinol at different concentrations on the relative expression level of FCV p30mRNA in feline kidney cells analyzed by RT-qPCR in Example 3.

[0019] 4. Figure 4 Is the statistical chart of experimental data on the inhibitory effect of arabinol on the replication of FCV-infected feline virus analyzed by end-point dilution method in Example 4. Detailed implementation manners

[0020] The following further illustrates the present invention in conjunction with the description of the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0021] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0022] Example 1: Determination of the half cytotoxic concentration (CC 50 ) of arabinol on feline kidney cells and the half inhibitory concentration (IC 50 ) of arabinol on FCV in feline kidney cells

[0023] (1) Determination of the half cytotoxic concentration (CC 50 ) of arabinol on feline kidney cells

[0024] Inoculate CRFK cells with a density of approximately 1.5×10 5 / mL into a 96-well plate, 100 μL per well. After they grow to a monolayer, wash twice with PBS. Dilute the compound serially with DMEM maintenance medium containing 2% FBS. Set up 9 concentration gradients of laberol treatment groups from 1.88 to 480 μM, a solvent control group containing 8‰ DMSO, and a blank control group, 100 μL per well. After culturing in a 37 °C, 5% CO 2 incubator for 24 h, discard the supernatant, add 0.5 mg / mL MTT solution, 100 μL per well, and incubate in the dark at 37 °C. After 4 h, terminate the incubation, discard the supernatant, add 150 μL DMSO to each well, and oscillate with a low-speed shaker for 10 min to fully dissolve the formazan crystals. Detect the OD value at a wavelength of 570 nm with a full-wavelength microplate reader and calculate the cell survival rate.

[0025] The formula for calculating the cell survival rate is as follows:

[0026]

[0027] The results are as Figure 1 shown. The half cytotoxic concentration (CC 50 ) of laberol against feline kidney cells is 1266 μM.

[0028] (2) Determination of the half inhibitory concentration (IC 50 ) of laberol against FCV in feline kidney cells

[0029] Seed CRFK cells in a 96-well plate. After they grow to a monolayer, wash twice with PBS. Dilute the virus with DMEM maintenance medium containing 2% FBS to a virus solution of 100 TCID s0 . Set up a blank control group and incubate in a 37 °C, 5% CO 2 incubator for 1 h. Set up a solvent control group and different concentration laberol treatment groups, 100 μL per well. After culturing for 24 h, discard the supernatant, terminate the culture, and incubate with 4% paraformaldehyde at room temperature for fixation, 150 μL per well. After 15 min, gently wash three times with PBS, add 50 μL of 0.3% Triton-X100 solution to each well, and incubate at room temperature; after 10 min, gently wash three times with PBS, add 100 μL of 2% BSA solution to each well, and incubate at 37 °C; after 1 h, gently wash three times with PBS, add 50 μL of Anti-FCV Antibody (diluted 1:1000) to each well, and incubate overnight at 4 °C; wash three times with PBS, 5 min each time, and add goat Anti-Mouse lgG H&L to each well in the dark (1:1000 dilution) 50 μL, incubated at 37 °C; after 1 h, washed three times with PBS, 5 min each time, added 100 μL of DAPI (300 nM) solution to each well in the dark, and incubated at room temperature. After 5 min, washed three times with PBS, 5 min each time, observed and photographed under an inverted fluorescence microscope. The fluorescence density (blue fluorescence and green fluorescence) of each well was digitally processed using Image J software, and the IC 50 value was determined, and calculated using the nonlinear regression function with GraphPad Prism 8.0 software. The calculation formula is as follows:

[0030]

[0031] The results were as Figure 1 , and the half-maximal inhibitory concentration (IC 50 ) of arabinitol against FCV in feline kidney cells was 1.87 μM.

[0032] (3) Selectivity index (SI) of arabinitol against FCV in feline kidney cells

[0033] According to the half-maximal cytotoxic concentration (CC 50 ) of arabinitol on feline kidney cells and the half-maximal inhibitory concentration (IC 50 ) of arabinitol against FCV in feline kidney cells, the selectivity index (SI) was calculated. The calculation formula was: SI = CC 50 / IC 50 . The results showed that arabinitol of the present invention had significant antiviral activity against FCV at non-cytotoxic concentrations, with an SI of 677 and high safety.

[0034] Example 2: Analysis of the inhibitory effect of different concentrations of arabinitol on the synthesis of FCV VP1 protein in feline kidney cells by immunofluorescence assay

[0035] CRFK cells were seeded in 96-well plates. After the monolayer was confluent, washed twice with PBS, and the virus was diluted into a virus solution of 100 TCID s0 using DMEM maintenance medium containing 2% FBS. A blank control group was set up and incubated in a 37 °C, 5% CO 2 incubator for 1 h. A solvent control group and different concentrations of arabinitol treatment groups were set up, 100 μL per well. After 24 h of culture, the supernatant was discarded, the culture was terminated, fixed by incubation with 4% paraformaldehyde at room temperature, and the IFA test was performed according to the method of Example 1. Observed under a fluorescence microscope, green fluorescence represents FCV VP1 protein.

[0036] The experimental results are shown in Figure 2, among which, compared with the virus-infected control group, "***" indicates extremely significant differences (P<0.001), and ns indicates no significant differences. As can be seen from the figure, the drug labetol described in the present invention has a significant inhibitory effect on the synthesis of FCV VP1 protein in CRFK cells within the concentration range of 2.5-5 μM, and shows a good dose-dependent relationship, indicating that labetol has a significant inhibitory effect on the synthesis of FCV VP1 protein at non-cytotoxic concentrations.

[0037] Example 3: RT-qPCR analysis of the inhibitory effect of different concentrations of labetol on the relative expression level of FCV p30 mRNA in feline kidney cells

[0038] Seed CRFK cells in a 12-well plate. After the monolayer is confluent, wash twice with PBS. Dilute the virus into a virus solution with 100 TCID 50 using DMEM maintenance medium containing 2% FBS. Set up a blank control group and incubate at 37°C and 5% CO 2 in an incubator for 1 h. Set up a solvent control group and treatment groups with different concentrations of labetol, 100 μL per well. After culturing for 24 h, terminate the culture. Place the cell plate at -80°C and 4°C and freeze-thaw three times repeatedly to fully release the virus into the supernatant. Extract total RNA using a total RNA extraction kit, reverse transcribe the RNA into cDNA using a cDNA first-strand synthesis kit, and store the product at 4°C for temporary use or at -20°C for long-term storage. Using the SYBR Green dye method, with SYBR Green I as the fluorescent dye, the first-strand cDNA obtained by reverse transcription as the template, and GAPDH as the internal reference gene, perform fluorescence quantitative PCR using primers FCV-p30-F and FCV-p30-R to detect the relative expression level of FCV p30 mRNA.

[0039] Among them, the sequences of the specific primers are as follows:

[0040] FCV-p30-F: 5'-AGCAGGACAGTTTCAAGCCATCA-3’

[0041] FCV-p30-R: 5'-ATCCATCCAGTGTCGCAACATAGC-3’

[0042] GAPDH-F: 5'-TGGAAAGCCCATCACCATC-3’

[0043] GAPDH-R: 5'-ACTCCACAACATACTCAGCACCA-3’

[0044] The test results are as Figure 3As shown, the ordinate represents the relative expression of FCV p30mRNA, and compared with the virus infection control group, "***" indicates a very significant difference (P<0.001), and ns indicates no significant difference. As can be seen from the figure, compared with the virus control group, the rabbis alcohol of the present invention has a significant inhibitory effect on the FCV p30mRNA level in cat kidney cells within a concentration range of 2.5 to 5 μM, and presents a significant dose-dependent relationship, indicating that rabbis alcohol has an inhibitory effect on the replication of FCV in cat kidney cells.

[0045] Example 4: Endpoint dilution analysis of the inhibitory effect of rabosol on viral replication in FCV-infected cats

[0046] 16 60-day-old kittens were randomly divided into 4 groups, namely, FCV virus control group, low-dose Labosol treatment group, high-dose Labosol treatment group and blank (unchallenged) control group, 4 in each group, and each group of cats was housed in separate cages. The FCV challenge group and the Labosol treatment group were challenged by nasal drops, and the negative control group was nasal drops with an equal amount of DMEM. On the 0th day of the challenge, the treatment groups were treated with low-dose 2.5 mg / kg and high-dose 5 mg / kg Labosol, respectively. The drug was filled into No. 3 capsules with excipients and administered orally once a day for 7 consecutive days. On the 7th day after infection, oral and nasal swabs were taken and placed in a centrifuge tube containing 650 μL DMEM culture medium (containing double antibodies: penicillin 100 U / mL and streptomycin 0.1 mg / mL), vortexed thoroughly to mix, and centrifuged at 3000 rpm for 5 min to collect all the attachments on the oral and nasal swabs to the bottom of the centrifuge tube. The supernatant was extracted and filtered with a 2.5 μM pore membrane filter for virus titer detection. The remaining sample liquid was placed in a -20°C refrigerator for use.

[0047] CRFK cells were plated in a 96-well plate, and after the monolayer was fully grown, the cells were washed twice with PBS. The collected samples were diluted 10-fold with DMEM containing 2% FBS and added to the cell plate. Each group had 4 replicate wells, with 100 μL per well. The plates were placed at 37°C and 5% CO. 2 Incubate in a constant temperature incubator. After 24 hours, observe the cytopathic effects of different sample groups, calculate the virus titer, and record and analyze it.

[0048] The test results are as follows Figure 4As shown, the abscissa represents different treatment groups, and the ordinate represents the virus titer. In the figure, compared with the virus challenge group, "**" indicates a significant difference (P < 0.01), and ns indicates no significant difference. It can be seen from the figure that compared with the virus challenge control group, labeolol at an oral dose of 5 mg / kg significantly reduced the FCV virus titer in the oral and nasal secretions of infected kittens, indicating that labeolol has a significant inhibitory effect on the replication of FCV virus in cats. The macroscopic observation results showed that there were ulcerous patches in the oral cavity of FCV-infected cats, and their activities and food intake decreased starting from the 3rd day after infection. The ulcerous patches in the labeolol treatment group were significantly reduced, and the activities and food intake were better than those in the virus control group. Among them, the curative effect of the 5 mg / kg labeolol group was better than that of the 2.5 mg / kg group.

[0049] It can be understood that the above embodiments only represent the preferred embodiments of the present invention, and the description thereof is relatively specific and detailed, but it should not be construed as a limitation to the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, the above technical features can be freely combined, and several deformations and improvements can also be made, which all belong to the protection scope of the present invention. Therefore, all equivalent transformations and modifications made to the scope of the claims of the present invention shall fall within the scope covered by the claims of the present invention.

Claims

1. Use of rabic alcohol in the preparation of a drug for preventing and treating feline calicivirus infection, wherein the rabic alcohol has a structure of formula (I), 2. The use of Labcohol according to claim 1 in the preparation of a drug for preventing and treating feline calicivirus infection, characterized in that: The labechol has inhibitory activity against feline calicivirus replication.

3. The use of Labcohol according to claim 2 in the preparation of a drug for preventing and treating feline calicivirus infection, characterized in that The effective inhibitory concentration of rabony alcohol on feline calicivirus replication in cat kidney cells is 1.25-5 μM.

4. The use of Labcohol according to claim 1 in the preparation of a drug for preventing and treating feline calicivirus infection, characterized in that: The labechol has an inhibitory effect on the replication of feline calicivirus in the body and alleviates the disease symptoms induced by viral infection.

5. The use of Labcohol according to claim 1 in preparing a drug for preventing and treating feline calicivirus infection, characterized in that: The medicine also contains pharmaceutically acceptable excipients; the dosage form of the medicine is tablets, capsules, granules, powders, syrups, oral solutions or injections.

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