Application of EPZ004777 in preparation of product for treating porcine circovirus type 2 infection

Abstract

According to the application of EPZ004777 in preparation of products for treating porcine circovirus type 2 infection, it is found for the first time that 50 [mu] g / mL of EPZ004777 has a remarkable inhibiting effect on porcine circovirus type 2 in the replication stage, and the EPZ004777 can be applied to preparation of drugs and / or feed additives for treating porcine intestinal coronavirus infection.

Description

Technical Field

[0001] The invention belongs to the field of veterinary medicines, and specifically relates to an application of EPZ004777 in the preparation of a medicine and / or feed additive for treating porcine circovirus type 2 infection. Background Art

[0002] Porcine circovirus 2 (PCV2) is a pathogen that poses a major threat to the global pig industry. The virus can significantly damage the pig's immune system, leading to an immunosuppressive state, which in turn causes a series of serious diseases, such as post-weaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS), porcine respiratory disease complex (PRDC), etc., causing huge economic losses to the health of the pig herd and the breeding industry.

[0003] In current breeding practices, farms mainly use strategies such as nutritional regulation and vaccination to enhance the immunity of pigs to fight against PCV2 invasion. However, although these measures can alleviate disease symptoms and improve the survival rate of piglets to a certain extent, they face many challenges. First, there is currently no specific treatment for PCV2, and once infected, it is difficult to cure completely. Secondly, with the continuous emergence of PCV2 variants, existing vaccines often fail to produce sufficient immune responses and cannot completely prevent the invasion and spread of the virus. Therefore, despite a series of preventive measures, it is still difficult to effectively prevent the spread of the virus and eradicate porcine circovirus disease.

[0004] In view of the above difficulties, it is particularly urgent to find new and more effective methods to prevent and control PCV2. EPZ004777, as a highly efficient H3K79 methyltransferase DOT1L selective inhibitor, has shown significant activity in the anti-tumor field. Although there are no reports on the application of EPZ004777 against porcine circovirus type 2, its unique biological properties and potential antiviral mechanisms provide us with new ideas. In view of the destructive effect of PCV2 on the pig immune system and its widespread prevalence around the world, exploring the application potential of EPZ004777 in the prevention and control of PCV2 is of great significance for developing new prevention and control strategies and reducing economic losses in the pig industry.

[0005] In summary, the present invention aims to explore the application potential of EPZ004777 in the prevention and control of porcine circovirus disease by in-depth research on the inhibitory effect of EPZ004777 on PCV2, in order to provide a more effective and reliable prevention and control method for the pig industry. Summary of the invention

[0006] Technical problem to be solved: The present invention provides an application of EPZ004777 in the preparation of a product for treating porcine circovirus type 2 infection, which is used for preparing a drug and / or feed additive for treating porcine circovirus type 2 infection.

[0007] Technical solution: Application of EPZ004777 in the preparation of products for the treatment of porcine circovirus type 2 infection.

[0008] The above products are medicines or feed additives.

[0009] The concentration of the above EPZ004777 is 50 μg / mL.

[0010] The above-mentioned medicine also includes other pharmaceutically acceptable excipients.

[0011] A pharmaceutical composition for treating porcine circovirus type 2 infection, wherein the active ingredient in the pharmaceutical composition is EPZ004777.

[0012] A feed additive for treating porcine circovirus type 2 infection, wherein the active ingredient in the additive is EPZ004777.

[0013] The above-mentioned pharmaceutical composition also includes at least one auxiliary component that enhances the antiviral effect of EPZ004777.

[0014] The feed additive also includes at least one nutrient component for improving the immunity of pigs.

[0015] The above EPZ004777 is a pure product obtained by chemical synthesis.

[0016] The above EPZ004777 is an extract obtained by a biological fermentation method.

[0017] Beneficial effects: The present invention reveals for the first time that EPZ004777 at a concentration of 50 μg / mL has a significant inhibitory effect on porcine circovirus type 2 during the replication stage. This discovery provides new ideas and methods for the treatment of porcine enteric coronavirus infections. Through further research and development, EPZ004777 is expected to be used in the preparation of therapeutic drugs for porcine enteric coronavirus infections, providing an effective disease prevention and control method for the pig farming industry. At the same time, its potential as a feed additive cannot be ignored, and it may help improve the overall health level of pigs and the benefits of farming. This innovative achievement not only enriches the technical system of pig disease prevention and control, but also provides strong support for ensuring the healthy development of animal husbandry and improving food safety and quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1The toxicity of different concentrations of EPZ004777 on PK15 cells: The cell absorbance at 450 nm was used to detect the CCK8 cell activity after PK15 cells were treated with EPZ004777 in the range of 0-300 μg / mL.

[0019] Figure 2 Effect of 50 μg / mL EPZ004777 on the replication of porcine circovirus type 2 infected PK15 cells: Fluorescence quantitative PCR (qPCR) was used to detect the expression level of PCV2 Cap gene in PK15 cells.

[0020] Figure 3 The inhibitory effect of 50 μg / mL EPZ004777 on the replication of porcine circovirus type 2: Western Blot was used to determine the effect of EPZ004777 on the expression level of PCV2 Cap protein in PK15 cells; indirect immunofluorescence was used to detect the fluorescence of PCV2 Cap protein in PK15 cells.

[0021] Figure 4 Effect of 50 μg / mL EPZ004777 on PCV2 Cap protein in PK15 cells: Indirect immunofluorescence detection of PCV2 Cap protein fluorescence in PK15 cells.

[0022] Figure 5 This is histological observation of organs of mice infected with PCV2.

[0023] Figure 6 qPCR was used to detect the effect of EPZ004777 on the viral load of mice after PCV2 infection.

[0024] Figure 7 To observe the effect of EPZ004777 treatment on PCV2 virus particles in mouse kidney tissue using electron microscopy.

[0025] Figure 8 HE staining was used to observe the effect of EPZ004777 treatment on the pathological changes of mouse kidney tissue. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0027] The materials and reagents specifically involved in the examples are as follows:

[0028] Materials: PK15 cells (ATCC, CCL-33); 8-week-old C57BL / 6 mice (Experimental Animal Center of Yangzhou University); 35-day-old three-way piglets (Jiangsu Academy of Agricultural Sciences); PCV2d-P58 virus (Jiangsu Academy of Agricultural Sciences).

[0029] Reagents: EPZ004777 (MedChemExpress); DMEM culture medium (Gibco, USA); fetal bovine serum (Gibco, USA); CCK8 kit (Nanjing Novozyme); penicillin-streptomycin mixture (Beijing Solebow Technology Co., Ltd.); 0.25% trypsin (Gibco, USA); TRIzol (Invitrogen, USA); chloroform (Yangzhou University "Precursor Chemicals" Chemical Library); DPEC water (Gibco, USA); viral genomic RNA / DNA extraction kit (TaKaRa, Takara Biotechnology (Dalian) Co., Ltd.); HI Script Q RT SuperMix for qPCR (+gDNAwiper) reverse transcription kit (Nanjing Novozyme Biotechnology Co., Ltd.); probe method qPCR reagent Premix Ex Taq TM (TaKaRa, Bao Biotechnology (Dalian) Co., Ltd.); cell lysis buffer (Shanghai Biotech Co., Ltd.); protease inhibitors (Shanghai Biotech Co., Ltd.); 5× SDS-PAGE protein loading buffer (Yisheng Biotechnology (Shanghai) Co., Ltd.); 10% protein precast gel (Shanghai Yazyme Biopharmaceutical Technology Co., Ltd.); PCV2-Cap (GeneTex, USA), PCV2-Cap protein monoclonal antibody (Abcam (Shanghai) Trading Co., Ltd.); anti-HSP90 antibody (Abcam (Shanghai) Trading Co., Ltd.); horseradish peroxidase (HRP) labeled goat anti-mouse or goat anti-rabbit secondary antibody (Nanjing Biotech Co., Ltd.); ultrasensitive ECL chemiluminescence kit (Suzhou Xinsaimei Biotechnology Co., Ltd.); 488-conjugated Goat anti-Rabbit IgG (H+L) secondary antibody (Wuhan Sanying Biotechnology Co., Ltd.); DAPI (Beijing Solebold Technology Co., Ltd.); TritonX-100 (Beijing Solebold Technology Co., Ltd.).

[0030] Example 1. Effect of EPZ004777 on PK15 cell viability

[0031] First, this example explores the effect of EPZ004777 on the viability of porcine kidney cell PK15. PK15 cells were cultured at 2×10 4 Cells were plated at a density of 100 μL / mL in a 96-well plate, and 100 μL of cell growth medium containing 10% fetal bovine serum and 1% penicillin-streptomycin was added to each well. The plates were then placed at 37°C and 5% CO. 2The cells were cultured in an incubator for 24 h, the stock solution was removed, and EPZ004777 was added at final concentrations of 0, 50, 100, 150, 200, and 300 μg / mL. After 48 h of culture in an incubator, the cells were replaced with fresh serum-free medium containing 10% cell viability detection reagent CCK8 and placed at 37°C and 5% CO. 2 Incubate for 2 hours; use a microplate reader to detect the absorbance at 450 nm and calculate the cell viability under different concentrations of EPZ004777. Figure 1 As shown, EPZ004777 has a high safety profile and exhibits no cytotoxicity at 50-300 μg / mL.

[0032] Example 2. This example verifies that EPZ004777 can play an anti-PCV2 infection role in PK15 cells

[0033] Porcine kidney cells (PK15, ATCC, CCL-33) were cultured at 2.0×10 5 The cells were seeded into 6-well plates at a density of 100 μg / well and placed in a DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin mixture at 37°C and 5% CO. 2 When the cells grow to 70% confluence in an incubator, PK15 cells are inoculated with PCV2 (MOI = 1) and placed in a 37°C, 5% CO 2 After 2 h in the incubator, the cells were washed three times with PBS to remove uninfected viruses, 1 mL of serum-free DMEM cell culture medium was added, and an equal volume of EPZ004777 (final concentration of 50 μg / mL) was added to the cells and placed at 37°C, 5% CO 2 The PCV2 virus genomic DNA was extracted using the TaKaRaMiniBEST Viral RNA / DNA Extraction Kit Ver and the Premix Ex Taq TM The probe method fluorescence quantitative kit was used to detect the number of PCV2 virus copies. Figure 2 As shown, porcine kidney cells (PK15, ATCC, CCL-33) were cultured at 2.0×10 5 The cells were seeded into 6-well plates at a density of 100 μg / well and placed in a DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin mixture at 37°C and 5% CO. 2 When the cells grow to 70% confluence in an incubator, PK15 cells are inoculated with PCV2 (MOI = 1) and placed in a 37°C, 5% CO 2After 2 h in the incubator, the cells were washed three times with PBS to remove uninfected viruses, 1 mL of serum-free DMEM cell culture medium was added, and an equal volume of EPZ004777 (final concentration of 50 μg / mL) was added to the cells and placed at 37°C, 5% CO 2 After culturing in the incubator for 48 hours, the cell samples were collected with protein lysis buffer containing protease inhibitors, and centrifuged at 4°C, 12000rpm for 15 minutes to obtain the supernatant protein of the cell lysate. The protein concentration in the protein sample was determined by the BCA method, and the target protein loading amount was adjusted according to the concentration value. 5× non-denaturing protein loading buffer was added, and the sample was boiled in boiling water for 10 minutes and then briefly centrifuged; the protein loading amount was adjusted with the same content of HSP90 as the standard, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed; the sample was transferred at a constant voltage of 110V. The protein samples were transferred to a 0.22 μm polyvinylidene fluoride (PVDF) membrane after 1 hour of incubation at room temperature with TBST buffer containing 5% (v / v) skim milk powder for 1 hour; mouse anti-PCV2-Cap and mouse anti-HSP90 were used as primary antibodies, respectively, and incubated at 4°C overnight; TBST was washed 3 times, 10 minutes each time, and then horseradish peroxidase (HRP)-labeled goat anti-mouse secondary antibody was added and incubated at room temperature for 1 hour; TBST was washed 3 times, and the protein levels of PCV2-Cap and HSP90 were visualized by Western blot using an ultrasensitive ECL chemiluminescence kit. The results are shown in Figure 3 As shown, EPZ004777 can significantly inhibit the expression of PCV2-Cap protein.

[0034] Porcine kidney cells (PK15, ATCC, CCL-33) were cultured at 2.0×10 5 The cells were seeded into 6-well plates at a density of 100 μg / well and placed in a DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin mixture at 37°C and 5% CO. 2 When the cells grow to 70% confluence in an incubator, PK15 cells are inoculated with PCV2 (MOI = 1) and placed in a 37°C, 5% CO 2 After 2 h in the incubator, the cells were washed three times with PBS to remove uninfected viruses, 1 mL of serum-free DMEM cell culture medium was added, and an equal volume of EPZ004777 (final concentration of 50 μg / mL) was added to the cells and placed at 37°C, 5% CO 2After 48 hours of incubation in the incubator, indirect immunofluorescence experiments were performed: 4% paraformaldehyde was used to fix the cells with different treatments for 30 minutes, and then washed three times with PBS; 0.5% TritonX-100 was added to permeabilize the membrane for 15 minutes to increase the permeability of the antibody to the cell membrane; then washed three times with PBS; 3% BCA was added to block for 2 hours, and washed twice with PBS; rabbit anti-PCV2-Cap protein primary antibody (1:100) was added, and incubated at 4°C overnight. Washed three times with PBS; added 488-conjugated Goat anti-Rabbit IgG (H+L) (1:200) secondary antibody was incubated at room temperature for 1 hour. Washed three times with PBS; 1μg / mL DAPI was added to stain the cell nucleus, and washed three times with PBS to remove the excess DAPI. Observed under a fluorescence microscope.

[0035] The results are as follows Figure 4 As shown, the specific fluorescence of the red PCV2-Cap protein in cells was significantly reduced after EPZ004777 treatment, and the blue DAPI represents the cell nucleus.

[0036] Example 3. This example successfully established a PCV2 infection mouse model

[0037] PCV2 was intraperitoneally inoculated into 6-8 week old C57BL / 6 mice. The body weight of the challenged mice was measured on the 7th, 14th, 21st and 28th day after challenge. PCV2 viral genomic DNA was extracted using TaKaRa MiniBEST Viral RNA / DNA Extraction Kit Ver and then purified using Premix Ex Taq TM The viral load in serum and organs (heart, liver, spleen, lung, kidney, and brain) was detected by probe-based fluorescence quantitative kit. Lung, spleen, and kidney tissues were collected on days 7, 14, 21, and 28 after infection for pathological examination by HE staining.

[0038] The results are shown in Table 1. The weekly body weight changes showed that the weight gain rate of PCV2-challenged mice from 1 to 14 days after challenge was significantly lower than that of the control group, decreasing from 10.85% to 6.79%.

[0039] Table 1 Changes in weight gain of mice challenged with PCV2

[0040]

[0041] The results are shown in Table 2. PCV2-P58 strain was intraperitoneally inoculated into 8-week-old C57BL / 6 (10 7 TCID50 / mL, 1mL / mouse) During the 28-day observation period, the highest viral load in the serum after challenge was on the 7th day, with a copy number of up to 10 5The highest viral load in organs occurred on day 21 after infection, with the highest copy numbers in spleen, liver, and lung reaching 10 10 The amount in the kidney and heart is the lowest, which is 10 5 .

[0042] Table 2 Virus load in serum and organs of PCV2-infected mice

[0043]

[0044] The results are as follows Figure 5 The pathological examination showed that some of the challenged mice developed interstitial pneumonia, and inflammatory cell infiltration was observed in the spleen and kidneys.

[0045] Example 4. This example verifies that EPZ004777 can reduce PCV2 infection in mice

[0046] Since this study has determined in the early preparation stage that 40 mg / kg is the maximum safe dose of EPZ004777 in C57BL / 6 mice, the added dose of the drug is 40 mg / kg. The 12 SPF-level C57BL / 6 male mice used in the experiment were all 6-8 weeks old. In order to complete the comparative analysis, they were divided into two groups of equal number according to different body weights, one group was the PCV2 group, and the other group was the PCV2+EPZ004777 group (40 mg / kg). During the entire experimental period, all these male mice were raised at the Experimental Animal Center of Yangzhou University (Yangzhou, Jiangsu). After completing 7 days of adaptive feeding, an equal volume of normal saline was injected into the peritoneal cavity of the blank control group mice, and each male mouse in the experimental group was injected with PCV2 virus (10 7 TCID50 / mL, 1mL / head), starting from the 5th day, each male mouse in the experimental group was intraperitoneally injected with the corresponding drug for 5 consecutive days. On the 11th day (6th day of medication), each male mouse in the experimental group was autopsied, and kidney samples were collected according to the experimental process, and all DNA and total protein in their kidneys were extracted, and then qPCR determination and electron microscopy were performed to observe the PCV2 virus load, and sections were prepared according to the experimental purpose to observe the pathological histological changes.

[0047] The results are as follows Figure 6 As shown, the PCV2 viral load in the kidney tissue of mice treated with EPZ004777 was significantly lower than that in the PCV2 group (P<0.01).

[0048] The results are as follows Figure 7 As shown, electron microscopy revealed that obvious PCV2 virus particles were found in the kidney tissues of mice in the PCV2 group, while no virus particles were found in the EPZ004777-treated group.

[0049] The results are as follows Figure 8 As shown in the figure, histopathological sections showed that the glomeruli in the renal cortex of mice in the EPZ004777 treatment group were evenly distributed, and there was no obvious interstitial proliferation. However, a small amount of vascular congestion and inflammatory cell infiltration occurred in the PCV2 group.

[0050] The above examples are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent transformation or modification made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of EPZ004777 in the preparation of products for the treatment of porcine circovirus type 2 infection.

2. The use according to claim 1, characterized in that: The product is a medicine or a feed additive.

3. The use according to claim 1, characterized in that: The concentration of EPZ004777 is 50 μg / mL.

4. The use according to claim 2, characterized in that: The medicine also includes other pharmaceutically acceptable excipients.

5. A pharmaceutical composition for treating porcine circovirus type 2 infection, characterized in that: The active ingredient in the pharmaceutical composition is EPZ004777.

6. A feed additive for treating porcine circovirus type 2 infection, characterized in that: The active ingredient in the additive is EPZ004777.

7. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition also includes at least one auxiliary ingredient that enhances the antiviral effect of EPZ004777.

8. The feed additive according to claim 6, characterized in that The feed additive also includes at least one nutrient component for improving the immunity of pigs.

9. The use according to any one of claims 1 to 5, characterized in that: The EPZ004777 is a pure product obtained by chemical synthesis.

10. The use according to any one of claims 1 to 5, characterized in that: The EPZ004777 is an extract prepared by a biological fermentation method.

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