A pharmaceutical combination against feline infectious peritonitis virus, preparation and application thereof

Abstract

The present application relates to the technical field of antiviral drugs, and provides a drug combination for resisting feline infectious peritonitis virus, a preparation and application thereof.The drug combination comprises entrectinib, compound GS441524 and / or compound GC376.Through combination of entrectinib, compound GS441524 and compound GC376, a significantly enhanced effect of resisting feline infectious peritonitis virus (FIPV) is exhibited, and a significantly synergistic enhancement effect is obtained; when the drug combination is applied to resisting feline infectious peritonitis virus, the drug combination is not sensitive to variation of FIPV, and is not prone to drug resistance problem, and has a good clinical drug development prospect, thereby providing a new selection for drugs for treating and preventing feline infectious peritonitis.

Description

Technical Field

[0001] This invention belongs to the field of antiviral drug technology, specifically relating to a drug combination and its application in the preparation of anti-feline infectious peritonitis virus. Background Technology

[0002] Feline infectious peritonitis (FIP) is a fatal disease caused by mutant strains of feline coronavirus (FCoV). Its pathogenic mechanism involves the synergistic effect of viral subtype transformation and immunopathological response. Feline coronaviruses can be divided into type I and type II, and each type is further divided into enteric type (FECV) and lethal type (FIPV) based on pathogenicity. Approximately 90% of FCoV infections present as self-limiting feline enteric coronavirus (FeCV) infections, and only about 10% of infected individuals undergo viral mutation, transforming into the FIPV subtype. FIPV mutant strains can infect the mononuclear macrophage system, causing systemic dissemination, ultimately inducing granulomatous inflammation around blood vessels in the abdominal organs, kidneys, and central nervous system. Its core pathological mechanism is a type III hypersensitivity reaction triggered by viral antigen-antibody complex deposition, leading to increased vascular permeability and multiple organ dysfunction. Type I FCoV is more common in natural infections, but it is difficult to culture in vitro and mainly relies on macrophage proliferation; Type II FCoV is derived from the recombination of type I and canine coronavirus (CCV), easily replicates in various cell lines, and has a high viral yield. The two viral receptors also differ: type II depends on feline aminopeptidase-N (fAPN), while type I depends on feline dendritic cell-specific adhesion molecule (fDC-SIGN). Due to the complex and diverse strains of FIPV, the development of broad-spectrum antiviral drugs targeting all strains is extremely difficult, leading to feline infectious peritonitis being long considered an incurable disease. Currently, there are no officially approved drugs for the treatment of feline infectious peritonitis in China. Although the emergence of antiviral drugs such as GS-441524 (an RdRp inhibitor) and GC376 (a 3CL protease inhibitor) has brought some hope for the treatment of feline infectious peritonitis, their efficacy and potential long-term drug resistance issues still require further research and optimization. This invention arises from this. Summary of the Invention

[0003] The purpose of this invention is to provide a drug combination, formulation and application for combating feline infectious peritonitis virus.

[0004] The technical solution adopted in this invention is as follows:

[0005] The present invention provides a drug combination against feline infectious peritonitis virus, the drug combination comprising entrectinib, and compound GS441524 and / or compound GC376.

[0006] The molecular formula of entrectinib is: C 31H 34 F2N6O2;

[0007] The structural formula of entrectinib is:

[0008] .

[0009] The molecular formula of compound GS441524 is: C 12 H 13 N5O4;

[0010] The structural formula of compound GS441524 is:

[0011] .

[0012] The molecular formula of compound GC376 is: C 21 H 32 N3NaO8S;

[0013] The structural formula of compound GC376 is:

[0014] .

[0015] Theoretical Analysis: (1) Coronavirus replication depends on programmed -1 ribosome frameshift (-1 PRF), a mechanism that is highly conserved in coronaviruses but extremely rare in mammalian cells. -1 PRF is crucial for viral replication by regulating the relative expression ratio of open reading frames on viral mRNA. Studies have confirmed that inhibiting -1 PRF can effectively block coronavirus replication. (2) After the coronavirus genomic RNA enters the host cytoplasm, it can be translated simultaneously by multiple ribosomes (forming polyribosomes) to synthesize polyprotein precursors. These precursors are then mainly cleaved by the virus-encoded 3CL protease (main protease) and other proteases (such as PLpro) to produce a variety of functional proteins, including RNA-dependent RNA polymerase (RdRp).

[0016] The drug combination in this application employs entrectinib (a ribosomal frameshift inhibitor) in combination with compound GC376 (a 3CL protease inhibitor) and / or compound GS441524 (an RdRp inhibitor). Entrectinib reduces viral protein synthesis during the early stages of viral translation by inhibiting -1 PRF, thereby decreasing the abundance of key enzymes required for subsequent viral replication (such as 3CL protease and RdRp). This results in a synergistic enhancement effect with antiviral drugs that directly target these enzymes (compounds GC376 and GS441524) (see [link to relevant documentation]). Figure 1(As shown). That is, the drug combination of this application can improve the drug's efficiency in inhibiting the virus by reducing the number of target proteins. Furthermore, this drug combination works through a highly conserved -1 PRF mechanism, is insensitive to viral gene mutations, and can better avoid drug resistance problems.

[0017] Preferably, when the drug combination includes entrectinib and compound GS441524, the molar ratio of entrectinib to compound GS441524 is 3:1 to 1:10;

[0018] When the drug combination includes entrectinib and compound GC376, the molar ratio of entrectinib to compound GC376 is 3:1 to 1:10.

[0019] This invention provides an antiviral formulation for feline infectious peritonitis virus, comprising any of the drug combinations described above.

[0020] Preferably, the formulation is an oral formulation or an injectable formulation.

[0021] This invention provides the use of any of the above-described drug combinations in the preparation of drugs against feline infectious peritonitis virus.

[0022] Preferably, it is at least one of the following:

[0023] To prepare products that inhibit the replication and proliferation of feline infectious peritonitis virus;

[0024] Prepare products for the treatment and / or prevention of diseases caused by infection with the feline infectious peritonitis virus.

[0025] Preferably, the feline infectious peritonitis virus includes FIPV 79-1146.

[0026] Preferably, the disease caused by the feline infectious peritonitis virus infection is feline infectious peritonitis.

[0027] Preferably, the anti-feline infectious peritonitis virus drug inhibits the replication and proliferation of feline infectious peritonitis virus in CRFK cells.

[0028] Preferably, the concentration of entrectinib is 30-1000 nM, and the concentration of compound GS441524 or compound GC376 is 100-1000 nM.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] 1. This application reveals that the combination of entrectinib with compound GS441524 and with compound GC376 significantly enhances antiviral efficacy, exhibiting a synergistic effect in inhibiting viruses, particularly demonstrating a significantly enhanced effect against feline infectious peritonitis virus (FIPV). Therefore, the drug combinations of this application can be used to treat and prevent diseases caused by FIPV infection, to prepare anti-FIPV drugs, and to prepare drugs for treating and preventing diseases caused by FIPV infection. This drug combination shows good anti-FIPV efficacy, is insensitive to FIPV mutations, and is unlikely to develop drug resistance, demonstrating promising application prospects.

[0031] 2. Entrectinib exhibits a very strong promoting effect on compounds GS441524 and GC376; compounds GS441524 and GC376 also exhibit a promoting effect on entrectinib, but the promoting effect is relatively weak. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 This is a schematic diagram of the target sites of the drug combination.

[0034] Figure 2 The inhibition rate matrix of FIPV by the combination of entrectinib and compound GS441524.

[0035] Figure 3 The left and right plots show the changes in inhibition rate of FIPV when entrectinib is used in combination with compound GS441524.

[0036] Figure 4 The curves showing the growth rate of the inhibition rate of the promoting effect of entrectinib on compound GS441524 are shown in (a) and (b) respectively.

[0037] Figure 5 Experimental results showing the inhibitory effects of entrectinib and / or compound GS441524 on FIPV-induced CPE in CRFK cells. Among them, (a) is a schematic diagram of the time distribution of CRFK cells after FIPV virus treatment and drug treatment; (b) is a cell CPE image of each group at two magnifications; and (c) is a bar chart of viral titer for each group.

[0038] Figure 6 The inhibition rate matrix of FIPV by the combination of entrectinib and compound GC376.

[0039] Figure 7 The left and right plots show the changes in inhibition rate of FIPV when entrectinib is used in combination with compound GC376.

[0040] Figure 8 The curves showing the growth rate of the inhibition rate of entrectinib on the promoting effect of compound GC376 are shown in (a) and (b) respectively.

[0041] Figure 9 The experimental results show the inhibitory effects of entrectinib and / or compound GC376 on FIPV-induced CRFK cell cytotoxicity (CPE). (a) is a schematic diagram of the time distribution of CRFK cells after FIPV and drug treatment; (b) are images of cell CPE in each group at two magnifications; and (c) is a bar chart of viral titers in each group. Detailed Implementation

[0042] The present invention will be described in detail below through embodiments and test examples. The examples given are for the purpose of better illustrating the content and advantages of the present invention, but should not be construed as limiting the scope of the invention to the examples provided. Non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0043] Example 1

[0044] The inhibitory effect of entrectinib in combination with compound GS441524 on FIPV (immunofluorescence assay)

[0045] 1. Experimental Methods

[0046] 1.1 Grouping scheme for the combined use of entrectinib and compound GS441524

[0047] All the following experiments were conducted in a P2 laboratory, and all operations complied with biosafety management regulations.

[0048] CRFK (feline kidney) cells and FIPV virus (FIPV79-1146) were provided and preserved by the Laboratory of Human Diseases and Immunotherapy, West China Hospital, Sichuan University. Other reagents and materials, unless otherwise specified, were sourced from commercial channels.

[0049] FIPV virus was cultured in CRFK cells. Two hours after infection, the culture medium was replaced with normal medium, and different concentrations of the combined drug (entrectinib and compound GS441524) were added using a checkerboard dosing method. Specifically, the concentrations of entrectinib, from lowest to highest, were: 0, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, and 1 μM. The concentrations of compound GS441524, from highest to lowest, were: 1, 0.3, 0.1, 0.03, 0.01, 0.003, 0.001, and 0 μM. The checkerboard dosing design for entrectinib and compound GS441524 is shown in Table 1.

[0050] Table 1. Checkerboard dosing design for entrectinib and compound GS441524 (expressed as: concentration of entrectinib / concentration of compound GS441524)

[0051]

[0052] 1.2 Immunofluorescence

[0053] (1) Drug preparation

[0054] Entrectinib: Prepared as a 10 mM stock solution using DMSO. GS441524: Prepared as a 10 mM stock solution using sterile water and hydrochloric acid. All stock solutions were serially diluted with complete cell culture medium before the experiment. In the combination therapy group, the drugs were mixed according to the pre-defined ratio.

[0055] (2) Cell infection and drug treatment (96-well plate immunofluorescence assay)

[0056] Cells in the logarithmic growth phase were spaced at 1 × 10⁶ cells per well. 4 Cells were seeded at a density of [number] cells / well in 96-well cell culture plates and incubated overnight at 37°C with 5% CO2 until approximately 80% confluence. The old culture medium was discarded, and for some experimental groups, fresh culture medium containing different concentrations of single or combined drugs was added to pretreat the cells for 2 hours. The virus control group and each drug-treated group were given FIPV virus solution diluted to an appropriate MOI (e.g., 0.1) with maintenance medium (containing 2% fetal bovine serum). The cell control group was given an equal volume of virus-free maintenance medium. The culture plates were incubated at 37°C for 2 hours to allow for complete virus adsorption. After adsorption, the virus solution was discarded, and the cells were gently washed once with pre-warmed PBS (phosphate-buffered saline) to remove any unadsorbed virus. Maintenance medium containing the appropriate concentration of drug (single or combined) was added to each well. Eight replicates were set for each drug concentration. The culture plates were returned to the incubator and cultured for another 48 hours.

[0057] (3) Immunofluorescence staining

[0058] After culture, the culture medium was aspirated, and 100 μL of 4% paraformaldehyde was added to each well for fixation at room temperature for 15 minutes. The cells were washed three times with PBS. 100 μL of PBS containing 0.1% Triton X-100 was added, and the cells were permeated at room temperature for 10 minutes. The cells were washed three times with PBS. 100 μL of PBS containing 5% bovine serum albumin (BSA) was added to each well for blocking at room temperature for 1 hour. The blocking solution was discarded, and anti-FIPV spike protein (S) specific primary antibody diluted with PBS containing 1% BSA was added to each well, and the cells were incubated overnight at 4°C. The next day, the cells were washed three times with PBS for 5 minutes each time. Fluorescently labeled secondary antibody (Alexa Fluor® 555) was added to each well, and the cells were incubated at room temperature in the dark for 1 hour. After washing three times with PBS, anti-fluorescence quenching mounting medium containing Hochest-33342 (a DNA-specific dye) was added to each well to stain the cell nuclei. Images of multiple fields of view per well were automatically acquired using a fluorescence microscope or a high-content imaging system. The proportion of fluorescent positive cells or the average fluorescence intensity in each field of view is quantified using analytical software to assess viral protein expression levels.

[0059] The formula for calculating the virus inhibition rate is:

[0060] Inhibition rate (%) = [1 - (fluorescence signal in the drug-treated group - fluorescence signal in the cell control group) / (fluorescence signal in the virus control group - fluorescence signal in the cell control group)] × 100%

[0061] 1.3 Data Analysis

[0062] The fluorescence values ​​were compiled into an Excel spreadsheet and uploaded to the SynergyFinder software. The mode of action was Inhibition, and the algorithm mode was ZIP (Zero Interaction Potency).

[0063] 2. Experimental Results

[0064] 2.1 Synergistic effect analysis results of Entrectinib and GS441524

[0065] The combination drug (i.e., the drug combination) was Entrectinib + GS441524. The synergistic effect analysis results of this drug combination (Entrectinib and GS441524) are shown in Table 2.

[0066] Table 2. Synergistic effect analysis results of Entrectinib + GS441524 combination.

[0067]

[0068] The zero-interaction synergy score (ZIP) for the combination of Entrectinib and GS441524 was 7.45, which is greater than 2, and the most synergistic area score was 34.33. This indicates that the combination of Entrectinib and GS441524 significantly enhances the efficacy of the drugs and has a significant synergistic effect.

[0069] 2.2 Inhibition rate of FIPV by the combined use of Entrectinib and GS441524

[0070] Figure 2 This is a dose-response matrix (DRC) plot showing the inhibition rate of FIPV (fibrillated fibroblast growth factor) by the combination of entrectinib and compound GS441524. ZIP model analysis was used to obtain the FIPV inhibition rate (inh. %) corresponding to each dose concentration. Figure 2 The changes in FIPV inhibition rate under different ratios of Entrectinib + GS441524 combination concentrations are shown. The darker the color (red), the higher the inhibition rate of the combination.

[0071] Figure 3 Planar and three-dimensional plots showing the changes in inhibition rate of FIPV when entrectinib is used in combination with compound GS441524. Figure 3 The left side of the middle section shows a plot of the inhibition rate of Entrectinib and GS441524 against FIPV as the concentration increases. Figure 3 The right side of the image shows a 3D plot illustrating the changes in the inhibition rate of FIPV under different ratios of Entrectinib and GS441524. The zero-interaction potency synergy score (ZIPSynergy score) for the combination of Entrectinib and GS441524 is 7.446.

[0072] Figure 4 The graphs show the growth rate of the inhibition rate of entrectinib on the promoting effect of compound GS441524 (a) and the growth rate of the inhibition rate of GS441524 on the promoting effect of entrectinib (b). The horizontal axis represents drug concentration (nM), and the vertical axis represents the inhibition rate (%).

[0073] According to Table 2, Figure 2 , Figure 3As shown, when using compound GS441524 alone, the inhibition rate increased from approximately 0% to 47.34% as the concentration increased from 1 nM to 1000 nM. The maximum single-drug inhibition rate of GS441524 was less than 50%, indicating that the efficacy of GS441524 alone was limited. When using entrectinib alone, the inhibition rate increased from approximately 0% to 65.34% as the concentration increased from 1 nM to 1000 nM. The maximum single-drug inhibition rate of entrectinib was 65.34%, indicating that the efficacy of entrectinib alone was moderate. However, based on the results of multiple combinations of entrectinib and GS441524, it can be seen that the combination of entrectinib and GS441524 exhibits a significant synergistic enhancement effect. For example, the combination of Entrectinib 100 nM and GS441524 300 nM achieved an inhibition rate of 83.81%, while the inhibition rate of Entrectinib alone at 100 nM was only 11.7%, and the inhibition rate of GS441524 alone at 300 nM was only 22.49%, with the sum of the two being 34.19%, far lower than 83.81%; there are also many other similar highly synergistic combinations (see...). Figure 2 and Figure 3 (As shown). This confirms that the combination of entrectinib and compound GS441524 has a clear and strong synergistic anti-FIPV effect.

[0074] according to Figure 4 As shown in (a), by observing the inflection point, it can be seen that 30 nM Entrectinib can significantly enhance the inhibitory effect of compound GS441524 on FIPV; from Figure 4 As can be seen in (b), the inflection point of the inhibition rate of Entrectinib on FIPV occurred in the group where the concentration of compound GS441524 was 100 nM, which indicates that the promoting effect of compound GS441524 on Entrectinib was relatively weak, while the promoting effect of Entrectinib on compound GS441524 was very strong.

[0075] Example 2

[0076] In vitro antiviral efficacy assay of entrectinib in combination with compound GS441524

[0077] 1. Experimental Methods

[0078] TCID 50 Method for determining viral titer

[0079] Following the same procedure as steps 1.2(1) and (2) in Example 1, cells in the drug-treated experimental group were cultured (48 h) in combination with entrectinib (0.5 μM) and GS441524 (0.5 μM), and the supernatant of each drug-treated experimental group was collected in parallel. The collected supernatant was serially diluted 10-fold with maintenance medium (e.g., 10... -1 Up to 10 -6 CRFK cells were seeded in 96-well plates and cultured overnight. Old culture medium was discarded, and each dilution was seeded in 8 replicates (100 μL per well). Cell control wells were also included. Cells were incubated at 37°C for 5–7 days, and cytopathic effect (CPE) was observed daily under a microscope. The number of wells showing CPE at each dilution was recorded. The median tissue culture infection dose (TCID) of the supernatant in each treatment group was calculated using the Spearman-Kärber method. 50 / mL).

[0080] 2. Experimental Results

[0081] See the experimental results. Figure 5 As shown, Figure 5 Experimental results on the inhibitory effects of entrectinib and / or compound GS441524 on FIPV-induced CRFK cell CPE. Figure 5 (a) is a schematic diagram showing the time distribution of CRFK cells after treatment with FIPV virus and drugs (GS441524 and / or Entrection). Figure 5 In the middle (b), from left to right, are cell photographs of the uninfected FIPV cell control group, the FIPV-infected virus control group with added DMSO, the GS441524 0.5μM administration group, the Entrectinib 0.5μM administration group, the GS441524 1μM administration group, the Entrectinib 1μM administration group, and the Entrectinib 0.5μM + GS441524 0.5μM administration group. Figure 5 In the middle (c), the calculated viral titer is shown for each group.

[0082] according to Figure 5As shown in (b), the uninfected CRFK cells in the cell control group were in good condition with little apoptosis; the cells in the virus control group after the addition of FIPV showed a large number of cell peculiarities (CPEs); neither Entrectinib 0.5μM nor GS441524 0.5μM alone could effectively inhibit viral amplification, and a large number of CPEs were present in the CRFK cells; neither Entrectinib 1μM nor GS441524 1μM alone could effectively inhibit viral amplification, and a large number of CPEs were still present in the CRFK cells, with inhibition rates of 65.34% and 47.34% respectively when used alone; however, when Entrectinib 0.5μM + GS441524 0.5μM (total 1μM) were used in combination, CPEs were significantly reduced, the cell condition was close to that of the uninfected cell control group, and the inhibition rate was close to 100%. Figure 5 The TCID50 results in (c) showed that the viral load in the combination group was significantly lower than that in the viral control group (DMSO) and the single-drug group. This indicates that the combination of Entrectinib 0.5 μM and GS441524 0.5 μM has a highly significant synergistic effect in inhibiting viral amplification and suppressing CPE.

[0083] Example 3

[0084] The inhibitory effect of entrectinib combined with compound GC376 on FIPV (immunofluorescence assay)

[0085] 1. Experimental Methods

[0086] The experimental method in this embodiment is basically the same as that in "1. Experimental Method" of Example 1, except that compound GS441524 is replaced with compound GC376. Further details will not be provided here.

[0087] 2. Experimental Results

[0088] 2.1 Results of synergistic effect analysis of Entrectinib and GC376

[0089] The combination drug (i.e., the drug combination) was Entrectinib + GC376. The synergistic effect analysis results of this drug combination (Entrectinib and GC376) are shown in Table 3.

[0090] Table 3. Synergistic effect analysis results of Entrectinib + GC376 combination

[0091]

[0092] The zero-interaction synergy score (ZIP Synergy score) for the combination of Entrectinib and GC376 was 8.58, which is greater than 2, and the most synergistic area score was 39.30, indicating that the combination of Entrectinib and GC376 significantly enhanced the efficacy of the drugs and had a significant synergistic effect.

[0093] 2.2 Inhibition rate of FIPV by the combination of Entrectinib and GC376

[0094] Figure 6 This is a dose-response matrix (DRC) showing the inhibition rate of FIPV (fibrillated fibroblast growth factor) in combination with entrectinib and compound GC376. The inhibition rate of FIPV at each dose concentration was obtained through ZIP model analysis. Figure 6 The changes in FIPV inhibition rate under different ratios of Entrectinib + GC376 combination are shown. The darker the color (red), the higher the inhibition rate of the combination.

[0095] Figure 7 Planar and three-dimensional plots showing the changes in inhibition rate of FIPV when entrectinib is used in combination with compound GC376. Figure 7 The left side of the middle section shows a plot showing the change in the inhibition rate of Entrectinib and GC376 on FIPV as the concentration increases. Figure 7 The right side of the image shows a 3D plot illustrating the changes in the inhibition rate of FIPV under different ratios of Entrectinib and GC376. The zero-interaction power synergy score (ZIP Synergy score) for the combination of Entrectinib and GC376 is 8.578.

[0096] Figure 8 The graphs show the growth rate of the inhibition rate of entrectinib's promoting effect on compound GC376 (a) and the growth rate of the inhibition rate of compound GC376's promoting effect on entrectinib (b). The horizontal axis represents drug concentration (nM), and the vertical axis represents inhibition rate (%).

[0097] According to Table 3, Figure 6 , Figure 7As shown, when using compound GC376 alone, the inhibition rate increased from approximately 0% to 55.13% as the concentration increased from 1 nM to 1000 nM, with the maximum single-drug inhibition rate of GC376 being 55.13%, indicating that the efficacy of GC376 alone is relatively limited. When using entrectinib alone, the inhibition rate increased from approximately 0% to 66.34% as the concentration increased from 1 nM to 1000 nM, with the maximum single-drug inhibition rate of entrectinib being 66.34%, indicating that the efficacy of entrectinib alone is moderate. However, based on the results of multiple combinations of entrectinib and GC376, it can be seen that the combination of entrectinib and GC376 exhibits a significant synergistic enhancement effect. For example, the inhibition rate of Entrectinib 100 nM + GC376 300 nM reached 95.49%, while the inhibition rate of Entrectinib alone at 100 nM was only 10.7%, and the inhibition rate of GC376 alone at 300 nM was only 22.27%, with the sum of the two being 32.97%, far lower than 95.49%; there are also many other similar highly synergistic combinations (see...). Figure 6 and Figure 7 (As shown). Therefore, it can be determined that the combination of entrectinib and compound GC376 has a clear and strong synergistic anti-FIPV effect.

[0098] according to Figure 8 As shown in (a), by observing the inflection point, it can be seen that 30 nM Entrectinib can significantly enhance the inhibitory effect of compound GC376 on FIPV; from Figure 8 As can be seen in (b), the inflection point of the inhibition rate of Entrectinib against FIPV occurred in the group where compound GC376 was 100 nM, which indicates that the promoting effect of compound GC376 on Entrectinib was relatively weak, while the promoting effect of Entrectinib on compound GC376 was very strong.

[0099] Example 4

[0100] In vitro antiviral efficacy assay of entrectinib in combination with compound GC376

[0101] 1. Experimental Methods

[0102] TCID 50 Method for determining viral titer

[0103] Following the same procedure as steps 1.2(1) and (2) in Example 1, cells in the drug-treated experimental group were cultured for 48 h with a combination of entrectinib (0.5 μM) and GC376 (0.5 μM). Supernatants from each drug-treated experimental group were collected in parallel. The collected supernatants were serially diluted 10-fold with maintenance medium (e.g., 10...). -1 Up to 10-6 CRFK cells were seeded in 96-well plates and cultured overnight. Old culture medium was discarded, and each dilution was seeded in 8 replicates (100 μL per well). Cell control wells were also included. Cells were incubated at 37°C for 5–7 days, and cytopathic effect (CPE) was observed daily under a microscope. The number of wells showing CPE at each dilution was recorded. The median tissue culture infection dose (TCID) of the supernatant in each treatment group was calculated using the Spearman-Kärber method. 50 / mL).

[0104] 2. Experimental Results

[0105] See the experimental results. Figure 9 As shown, Figure 9 Experimental results on the inhibitory effects of entrectinib and / or compound GC376 on FIPV-induced CRFK cell CPE. Figure 9 (a) is a schematic diagram showing the time distribution of CRFK cells after treatment with FIPV virus and drugs (GC376 and / or Entrection). Figure 9 In the middle (b), from left to right, are cell photographs of the uninfected FIPV cell control group, the virus control group infected with FIPV and supplemented with DMSO, the GC376 0.5μM administration group, the Entrectinib 0.5μM administration group, the GC376 1μM administration group, the Entrectinib 1μM administration group, and the Entrectinib 0.5μM + GC376 0.5μM administration group. Figure 9 In the middle (c), the calculated viral titer is shown for each group.

[0106] according to Figure 9 As shown in (b), the uninfected CRFK cells in the cell control group were in good condition with little apoptosis; the cells in the virus control group that were added with FIPV showed a large number of CPEs.

[0107] Neither Entrectinib 0.5μM nor GC376 0.5μM alone effectively inhibited viral amplification, resulting in a large number of cell-borne pathogens (CPEs) in CRFK cells. Similarly, neither Entrectinib 1μM nor GC376 1μM alone significantly inhibited viral amplification, with CPEs still present in CRFK cells. The inhibition rates of Entrectinib 1μM and GC376 1μM alone were 66.34% and 55.13%, respectively. However, the combined use of Entrectinib 0.5μM and GC376 0.5μM (total 1μM) significantly reduced CPEs, resulting in cell states close to those of the uninfected control group, with an inhibition rate approaching 100%. Figure 9The TCID50 results in (c) showed that the viral load in the combination group was significantly lower than that in the viral control group (DMSO) and the single-drug group. This demonstrates that the combination of Entrectinib 0.5 μM and GC376 0.5 μM has a highly significant synergistic effect in inhibiting viral amplification and CPE.

[0108] In summary, the drug combination and formulation provided by this invention, when used in combination with Entrectinib or compound GS441524 or compound GC376, significantly enhances antiviral efficacy and exhibits a synergistic effect in inhibiting viruses, particularly demonstrating a significantly enhanced effect against feline infectious peritonitis virus (FIPV). The drug combination of this invention can be used to treat and prevent diseases caused by FIPV infection, to prepare anti-FIPV drugs, and to prepare drugs for treating and preventing diseases caused by FIPV infection. This drug combination shows good anti-FIPV efficacy, is insensitive to FIPV mutations, and is unlikely to develop drug resistance, demonstrating promising application prospects.

Claims

1. The application of a drug combination against feline infectious peritonitis virus (FIPV) in the preparation of an anti-FIPV drug, characterized in that, The drug combination includes entrectinib, and either compound GS441524 or compound GC376. When the drug combination includes entrectinib and compound GS441524, the molar ratio of entrectinib to compound GS441524 is 3:1 to 1:

10. When the drug combination includes entrectinib and compound GC376, the molar ratio of entrectinib to compound GC376 is 3:1 to 1:

10.

2. The use of the drug combination according to claim 1 in the preparation of a drug against feline infectious peritonitis virus, characterized in that, The drug for treating feline infectious peritonitis virus is available in oral or injectable formulations.

3. The use of the drug combination according to claim 1 in the preparation of a drug against feline infectious peritonitis virus, characterized in that, The feline infectious peritonitis virus mentioned is FIPV 79-1146.

4. The use of the drug combination according to claim 1 in the preparation of a drug against feline infectious peritonitis virus, characterized in that, The concentration of entrectinib is 30~1000 nM, and the concentration of compound GS441524 or compound GC376 is 100~1000 nM.

5. The use of a pharmaceutical combination against feline infectious peritonitis virus (FIPV) in the preparation of a medicament for treating and / or preventing diseases caused by FIPV infection, characterized in that, The drug combination includes entrectinib, and either compound GS441524 or compound GC376. When the drug combination includes entrectinib and compound GS441524, the molar ratio of entrectinib to compound GS441524 is 3:1 to 1:

10. When the drug combination includes entrectinib and compound GC376, the molar ratio of entrectinib to compound GC376 is 3:1 to 1:

10. The disease caused by the feline infectious peritonitis virus infection is feline infectious peritonitis.

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