Application of ailanone in preparation of medicine for treating cat infectious peritonitis

Abstract

The invention discloses application of ailanone in preparation of a medicine for treating feline infectious peritonitis, and belongs to the field of biological medicine. The change of the expression quantity of the feline infectious peritonitis virus N protein is detected through indirect immunofluorescence and western blot, and the change of the quantity titer of the feline infectious peritonitis virus is detected through TCID50. Results show that ailanone has an obvious effect of resisting the feline infectious peritonitis virus, the inhibitory effect on the feline infectious peritonitis virus is enhanced along with increase of the drug concentration, and the half effective concentration is 0.09 mu M. Meanwhile, drug inhibition tests show that ailanone has no inhibition effect on feline calicivirus. The invention provides a novel therapeutic drug and strategy for treating feline infectious peritonitis.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the use of ailanthus ketone in the preparation of drugs for treating feline infectious peritonitis. Background Technology

[0002] Feline infectious peritonitis virus (FIP) is the causative virus of feline infectious peritonitis (FIP). This virus is caused by a mutation at a genetic locus in feline coronavirus, which was highly infectious before the mutation. Although theoretically the virus remains infectious, the risk of infection in other cats is low due to limited transmission routes. This disease is a common ailment in felines, but other wild felines also experience some incidence, and the mortality rate is high. The disease is typically fatal, characterized by fibrinous and granulomatous serositis, protein-rich serous effusions in body cavities, and / or granulomatous lesions (pyogenic granulomas). The pathogens of this deadly disease are feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV). Both viruses belong to the feline coronavirus (FCoV) family. FCoVs are part of the Alphacoronavirus coronavirus species and are primarily composed of nucleocapsid (N) protein, transmembrane (M) protein, envelope protein (E), and spike protein (S). FCoVs are classified into types I and II based on the amino acid sequence of their spike protein. Each of these types consists of two viruses: the FIP virus (FIPV) that causes feline infectious peritonitis (FIP) and the non-FIP feline enteric coronavirus (FECV). FIPV and FECV of the same type cannot be distinguished by antigenicity or genetic level, differing only in their pathogenicity in cats. FECV is asymptomatic in cats, but FIPV infection can induce feline infectious peritonitis (FIP). FIP is an immune-mediated and difficult-to-treat viral infection with a 100% mortality rate. Several effective antiviral drugs for the treatment of FIP have recently been reported, but these are all foreign-developed and patented drugs, and are expensive, such as GS441524 and GC376. However, their intellectual property rights belong to foreign companies, and my country lacks domestically developed drugs for treating FIP, thus urgently needing such drugs to fill the market gap.

[0003] Ailanthone, a monomeric component of traditional Chinese medicine extracted from the bark of *Ailanthus altissima*, possesses antitumor activity. It can inhibit androgen receptors and their cleavage variants, block AR-HSP90 interaction, and induce AR protein degradation. Growth inhibition effects have been demonstrated in various cancer cells. Furthermore, domestic and international literature has reported significant effects of ailanthone in inhibiting viral infections such as African swine fever. However, there are currently no reports on the effects of ailanthone on feline infectious peritonitis virus (FIP). Therefore, introducing ailanthone into the inhibition of FIP has significant practical implications. Summary of the Invention

[0004] The purpose of this invention is to provide the application of ailanthus ketone in the preparation of drugs for treating feline infectious peritonitis (FIP) to solve the problems existing in the prior art. Experiments have shown that ailanthus ketone can have a significant antiviral effect against FIP ​​virus, and the inhibitory effect on FIP virus is enhanced with increasing drug concentration. This provides a new drug and a new strategy for treating FIP.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides the use of ailanthoides in the preparation of a medicament for treating feline infectious peritonitis.

[0007] This invention also provides the use of ailanthus ketone in the preparation of medicaments that inhibit the replication of feline infectious peritonitis virus.

[0008] Preferably, the effective concentration of the ailanthus ketone for inhibiting the replication of feline infectious peritonitis virus is ≥0.625 μM.

[0009] The present invention also provides a medicament for treating feline infectious peritonitis, wherein the sole active ingredient of the medicament is ailanthoone, and the medicament further comprises pharmaceutically acceptable excipients.

[0010] Preferably, the dosage form of the drug includes powder, tablet, granule, capsule, solution, emulsion and suspension.

[0011] The present invention discloses the following technical effects:

[0012] This invention detects changes in the expression level of feline infectious peritonitis virus (FIP) N protein, TCID, using indirect immunofluorescence and Western blotting. 50 The titer of feline infectious peritonitis virus (FIP) was measured. Results showed that ailanthus ketone had a significant antiviral effect against FIP, with the inhibitory effect increasing with increasing drug concentration, reaching a half-maximal effective concentration (IC50) of 0.09 μM. However, drug inhibition assays indicated that ailanthus ketone had no inhibitory effect on feline calicivirus. This invention provides a novel therapeutic drug and strategy for treating feline infectious peritonitis. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1 The image shows the inhibitory effect of ailanthus ketone on feline infectious peritonitis virus (FIPV) at the cellular level; where A is the cell control, B is the image of lesions after 48 hours of FIPV infection with CRFK, and C is the image of cell infection after adding ailanthus ketone to infected cells.

[0015] Figure 2 The image shows the inhibitory effect of ailanthus ketone on feline calicivirus (FCV) at the cellular level; where A is the cell control, B is the image of lesions after 24 hours of feline calicivirus infection of CRFK, and C is the image of cell infection after ailanthus ketone was added to infected cells.

[0016] Figure 3 CC of the cytotoxic effects of ailanthus ketone 50 The determination;

[0017] Figure 4 TCID of ailanthoides at different stages of feline infectious peritonitis virus (CRFK) infection 50 Measurement;

[0018] Figure 5 Indirect immunofluorescence assay for the antiviral activity of ailanthus ketone;

[0019] Figure 6 EC for antiviral activity of ailanthus ketone 50 The determination;

[0020] Figure 7 The results of Western blotting identification of the antiviral activity of ailanthus ketone are presented. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] The virus used in the following examples is the feline infectious peritonitis S protein replacement strain rQS-79. The construction method is described in the reference "Wang Gang et al. Establishment of Full-length cDNA Clones and an Efficient Oral Infection Model for Feline Coronavirus in Cats.[J]. Journal of virology, 2021, : JVI0074521-JVI0074521; the feline kidney cells (CRFK) were purchased from ATCC.

[0027] Ailanthone, its chemical formula is C 20 H 24 O7, with a molecular weight of 376.40 and CAS number 981-15-7. The ailanthus ketone involved in the embodiments of this invention was purchased from MedChemExpress (MCE).

[0028] Example 1: Verification of the inhibitory effect of ailanthus ketone on feline infectious peritonitis virus.

[0029] 1) Take CRFK cells in good growth condition, digest and passage them, and adjust the cell density to 1×10⁶ cells / year using cell growth medium (DMEM containing 10% fetal bovine serum). 5 Cells / mL were seeded into 96-well plates, 100 μL per well, and incubated at 37℃ in a 5% CO2 incubator for 14 h.

[0030] 2) After 16 h, remove the 96-well plate, discard the culture medium in the wells, and wash three times with sterile PBS;

[0031] 3) Add ailanthus ketone at a final drug concentration of 10 μM; add feline infectious peritonitis virus at a viral titer of 0.01 MOI; and use 100 μL of cell maintenance medium (containing DMEM with a volume percentage of 2% fetal bovine serum) as a control.

[0032] 4) After 24 hours, observe the cellular pathological changes under a microscope and take photos for recording.

[0033] The results are as follows Figure 1 As shown, under the microscope, normal CRFK cells exhibit good growth, intact morphology, and clear boundaries. However, CRFK cells infected with feline infectious peritonitis virus (FIPV) showed typical "membrane fusion" lesions one day later, with visible nucleus aggregation and very pronounced lesions. Simultaneous treatment of cells with ailanthus and FIPV significantly reduced membrane fusion and improved cell growth.

[0034] Example 2: Verification of the inhibitory effect of ailanthus ketone on feline calicivirus

[0035] 1) Take CRFK cells in good growth condition, digest and passage them, and adjust the cell density to 1×10⁶ cells / year using cell growth medium. 5 Cells / mL were seeded into 96-well plates, 100 μL per well, and incubated at 37℃ in a 5% CO2 incubator for 14 h.

[0036] 2) After 16 h, remove the 96-well plate, discard the culture medium in the wells, and wash three times with sterile PBS;

[0037] 3) Add ailanthus ketone at a final drug concentration of 10 μM; add feline calicivirus at a viral titer of 0.1 MOI; and add 100 μL of cell maintenance medium in a single column as a control.

[0038] 4) After 24 hours, observe the cellular pathological changes under a microscope and take photos for recording.

[0039] The results are as follows Figure 2 As shown, under the microscope, normal CRFK cells exhibit good growth, intact morphology, and clear boundaries. However, CRFK cells infected with feline calicivirus (FCV) showed typical "cell rounding and detachment" lesions one day later, with very obvious lesions. Treatment of cells with both ailanthus ketone and feline calicivirus did not improve the cytopathic effect of rounding and detachment.

[0040] Example 3: Detection of cell viability by ailanthoides

[0041] 1) Take CRFK cells in good growth condition, digest and passage them, and adjust the cell density to 1×10⁶ cells / year using cell growth medium. 5 Cells / mL were seeded into 96-well plates, 100 μL per well, and incubated at 37℃ in a 5% CO2 incubator for 14 h.

[0042] 2) After 16 h, remove the 96-well plate, discard the culture medium in the wells, and wash three times with sterile PBS;

[0043] 3) Dilute ailanthoone with cell maintenance medium to 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM and 1.25 μM, and then add it to CRFK cells in sequence, with 100 μL of cell maintenance medium added in a column as a control.

[0044] 4) After 72 hours, cell viability was assessed using a CCK-8 assay kit. Cell viability measurement reflects the toxic effects of ailanthophyll on cells. The cell viability (%) was calculated using the formula: (Average luminescence value of cells treated with the compound / Average luminescence value of the control group) × 100%.

[0045] like Figure 3 As shown, ailanthus ketone has relatively low toxicity to CRFK cells, and its CC... 50 It is 15.72 μM.

[0046] Simultaneously, CRFK cells infected with feline calicivirus (FCV) were inoculated using the same method, and cell viability was assessed using a CCK-8 assay kit. Results are as follows: Figure 2 As shown, under the microscope, normal CRFK cells exhibited good growth, intact morphology, and clear boundaries. However, CRFK cells infected with feline calicivirus (FCV) showed typical "cell rounding and detachment" lesions after 2 days, with very obvious lesions. When cells were treated simultaneously with ailanthus and FCV, the cell rounding and detachment were essentially the same as in the virus control group. This indicates that ailanthus cannot inhibit FCV infection.

[0047] Example 4: Antiviral activity of ailanthus ketone (TCID) 50 Measurement

[0048] 1) Digest and passage CRFK cells, and adjust the cell density to 8 × 10⁶ cells / year using cell growth medium. 5 / mL, inoculated into 12-well plates, 1mL / well, and incubated at 37℃ in a 5% CO2 incubator for 14 h;

[0049] 2) Take out a 12-well plate, add feline infectious peritonitis virus at a viral titer of 0.01 MOI, gently shake the plate to mix, and set up a virus control and a cell control in each 12-well plate. At the same time, add 5 μM ailanthus ketone 1 hour before virus infection, at infection (when virus and drug are added simultaneously), and 1 hour after infection, and incubate at 37°C in a 5% CO2 incubator.

[0050] 3) After about 48 hours, 70% of the virus control showed lesions. The 12-well plate was then transferred to an ultra-low temperature freezer at -80°C for one freeze-thaw cycle.

[0051] 4) Collect the liquid from each well into an EP tube, centrifuge at 4000 rpm for 10 min, and then collect the supernatant to determine the toxicity.

[0052] 5) Prepare cells in good growth condition, digest them with trypsin after they reach about 80% growth, and then plate them into 96-well plates;

[0053] 6) When the cells in the 96-well plate reach approximately 80% confluence, serially dilute the virus 10-fold in sterile 2 mL EP tubes (use 10 mL for the first time). -1 Up to 10 -12 Each well contains 100 μL of virus suspension, with 8 wells for each dilution, and control wells are also provided.

[0054] 7) The operation must be carried out on ice to ensure the stability of the virus titer, and the instrument must be shaken evenly to prevent the virus from agglomerating, which would lead to a large deviation in the measured virus titer.

[0055] 8) Smoothly transfer the 96 plates to a cell culture incubator containing 5% CO2 at 37°C;

[0056] 9) Observe the results after 48 hours, remove the 96-well plate and examine the cytopathic effects under a microscope, and record the results;

[0057] 10) Calculate the virus TCID using the Reed and Muench methods. 50 .

[0058] like Figure 4 As shown, ailanthus ketone mainly exerts its effect after the virus is adsorbed; adding it 1 hour after virus adsorption can completely inhibit viral replication.

[0059] Example 5: Indirect immunofluorescence (IFA) detection of the viral activity of ailanthus ketone

[0060] 1) Take CRFK cells in good growth condition, digest and passage them, and adjust the cell density to 1×10⁶ cells / year using cell growth medium. 5 / mL, inoculated into 100 μL / well of a 96-well plate, and incubated at 37℃ in a 5% CO2 incubator for 12 h;

[0061] 2) After 12 h, the cells grew into a monolayer. The 96-well plate was washed twice with serum-free DMEM medium, replaced with cell maintenance medium, and ailanthus ketone was added for 2-fold serial dilution. Then, feline infectious peritonitis virus solution with 0.01 MOI was added to make the final concentrations of ailanthus ketone 5 μM, 2.5 μM, 1.25 μM, 0.625 μM and 0.3125 μM. At the same time, a virus control group and a cell control group were set up.

[0062] 3) 24 hours after infection, discard the culture medium in the well, wash twice with PBS, and then add 4% paraformaldehyde and incubate at room temperature for 30 minutes.

[0063] 4) Discard the waste liquid, wash twice with PBS, add blocking buffer containing 1% BSA in PBST, and let stand at 37°C for 1 h.

[0064] 5) After washing twice with PBS, add the primary antibody (FIPV N protein rabbit polyclonal antibody) diluted 1:1000 and incubate at 37°C for 1 h.

[0065] 6) After washing 4 times with PBST, add goat anti-rabbit fluorescent secondary antibody at a ratio of 1:2000 and incubate at 37°C in the dark for 1 h.

[0066] 7) Discard the secondary antibody, wash 4 times with PBS, and observe and photograph under a fluorescence microscope.

[0067] like Figure 5 As shown, feline infectious peritonitis virus (FIPV) was virtually undetectable at 2.5 μM with ailanthus. As the drug concentration decreased, the detected FIPV levels increased continuously, reaching a significant amount at 0.625 μM in the cells.

[0068] The half-maximal effective concentration (EC50) reflects the inhibitory effect of ailanthus ketone on the virus; that is, the drug concentration corresponding to 50% of the maximum biological effect (virus inhibition). Results are as follows... Figure 6 As shown in the EC50 curve, ailanthus has a very good therapeutic effect on feline infectious peritonitis virus (FIP). 50 The specific value is 0.09 μM.

[0069] Example 6: Western Blot Determination of Viral Activity of Ailanthone

[0070] 1) Take cells treated with different concentrations of drugs, discard the cell culture medium, wash the cells once with pre-cooled PBS, aspirate the PBS, resuspend the cells in 1 mL (6-well plate) of PBS, and put them into 2 mL EP tubes.

[0071] 2) Centrifuge at 4℃ and 4000 r / min for 5 min, discard the supernatant, add 120 μL (6-well plate) of cell lysis buffer, resuspend, and rotate at 4℃ for 25 min.

[0072] 3) Centrifuge at 12000 r / min for 20 min at 4℃, collect the supernatant, add 5× loading buffer, boil in boiling water for 10 min, then freeze for 10 min, and store at 4℃.

[0073] 4) Prepare a 12% separating gel, mix it well, add it to a glass plate, add a layer of deionized water on the separating gel, and place it in a fume hood at room temperature for about 30 minutes.

[0074] 5) After the separating gel has fully polymerized, add the prepared 5% concentrate onto the separating gel, insert the comb, and place it in a fume hood at room temperature for about 30 minutes.

[0075] 6) Place the prepared polyacrylamide gel in a vertical electrophoresis tank, add an appropriate amount of 1×SDS-PAGE electrophoresis buffer to the middle tank and the outer tank, and add an appropriate volume of the prepared protein sample and protein marker to the sample well.

[0076] 7) Connect to a power supply of 80V for 30 minutes, then perform electrophoresis at 120V for 1 hour and 20 minutes;

[0077] 8) Transfer: After SDS-PAGE electrophoresis, based on the molecular weight of the target protein, and using the protein marker as a control, cut the gel and trim appropriate sizes of filter paper and PVDF membrane. Place the cut filter paper in the transfer buffer, soak the cut PVDF membrane in methanol, then soak it in pure water and the transfer buffer, and finally place it in the transfer tank and transfer at 120V for 60 min.

[0078] 9) Blocking: Gently transfer the membrane into a clean petri dish using tweezers, add an appropriate amount of blocking solution (TBST in 5% skim milk), and incubate at room temperature on a shaker for 2 hours;

[0079] 10) Primary antibody incubation: Rinse the membrane with TBST, place it in a petri dish containing pre-diluted primary antibody, and incubate on a shaker at room temperature for 2.5 h;

[0080] 11) Secondary antibody incubation: Wash the membrane three times with TBST for 15 min each time, then place the membrane in a petri dish containing pre-diluted secondary antibody and incubate on a shaker at room temperature for 2 h. After incubation, wash the membrane six times with TBST for 5 min each time.

[0081] 12) Color development: Mix equal volumes of solution A and solution B, and develop the color in a colorimetric instrument;

[0082] 13) After color development and photographing, wash the membrane six times with TBST, 5 min each time. Incubate GAPDH primary antibody and goat anti-rabbit secondary antibody in the same way, and photograph and save after color development.

[0083] like Figure 7 As shown, Western blotting results indicated that feline infectious peritonitis virus (FIP) N protein was virtually undetectable at 0.625 μM with ailanthus. As the drug concentration decreased, the detected FIP level increased, reaching a significant amount at 0.3125 μM.

[0084] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Use of jatrophane in the preparation of a medicament for treating feline infectious peritonitis.

2. Use of jatrophane in the preparation of a medicament for inhibiting the replication of feline infectious peritonitis virus.

3. Use according to claim 2, wherein the compound is ###0002### The effective concentration of the jatrophane for inhibiting the replication of feline infectious peritonitis virus is ≥0.625 μM.

4. A medicament for the treatment of feline infectious peritonitis, characterized in that, The only active ingredient of the medicament is jatrophane, and the medicament further comprises a pharmaceutically acceptable adjuvant.

5. The medicament according to claim 3, wherein The dosage form of the medicament comprises powders, tablets, granules, capsules, solutions, emulsions and suspensions.

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