Application of difluoromethyl ornithine in preparation of medicine for preventing and / or treating diseases caused by porcine circovirus type 2 infection
By using difluoromethylornithine to inhibit the replication of porcine cyclovirus type 2 (PCV2) and the expression of Cap protein, the problem of difficulty in effectively inhibiting PCV2 infection in the prior art was solved, and the effect of significantly reducing viral load and inhibiting viral replication was achieved.
Patent Information
- Application Number
- CN202510493473.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-18
- Publication Date
- 2025-07-01
AI Technical Summary
The prior art is difficult to effectively inhibit pig circovirus type 2 (PCV2) infection, resulting in decreased immunity and the risk of secondary infection.
Difluoromethylornithine is used to reduce viral load by inhibiting the replication of PCV2 or the expression of Cap protein, and to prepare drugs to prevent and treat PCV2 infection.
Significantly reduce the PCV2 viral load in mice, reduce viral titer, inhibit viral replication, improve immunity, and reduce the risk of secondary infection.
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Figure CN120227367A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and specifically relates to the use of difluoromethylornithine in the preparation of a medicament for preventing and / or treating diseases caused by porcine circovirus type 2 infection. Background Art
[0002] Porcine circovirus type 2 (PCV2) belongs to the genus Circovirus in the family Circoviridae. It has an icosahedral structure, no envelope, and is a single-stranded circular DNA virus with a genome length of approximately 1.76 kb. PCV2 has strong infectivity and is susceptible to pig herds of different ages. It often causes porcine circovirus-associated diseases (PCVAD), including porcine dermatitis and nephropathy syndrome (PDNS), postweaning multisystemic wasting syndrome (PMWS), sow reproductive failure disorder (PRFD), and porcine respiratory disease complex (PRDC), etc. When a pig herd is infected with PCV2 alone, it usually causes mild clinical symptoms and does not cause extensive tissue damage, but it will lead to a significant decline in immunity, thereby increasing the susceptibility to other diseases and causing secondary infections. Currently, vaccination is the most effective measure to resist PCV2. However, research on aspects such as host immunity and pathogenic mechanisms caused by pathogen variation still needs to be broken through. Therefore, the screening and research of drugs for inhibiting porcine circovirus type 2 infection are crucial. Summary of the Invention
[0003] In view of the deficiencies of the prior art, the present invention provides the use of difluoromethylornithine in the preparation of a medicament for preventing and / or treating diseases caused by porcine circovirus type 2 infection. The present invention discovers that difluoromethylornithine can inhibit porcine circovirus type 2 from infecting host cells and mice, can reduce the virus titer of porcine circovirus type 2, inhibit the expression of the Cap protein of porcine circovirus type 2, and significantly reduce the viral load in mice. It can be used to prepare a medicament for resisting porcine circovirus type 2 infection and for inhibiting virus replication.
[0004] The technical solution provided by the present invention is as follows:
[0005] The present invention provides the use of difluoromethylornithine in the preparation of a medicament for preventing and / or treating diseases caused by porcine circovirus type 2 infection.
[0006] Furthermore, the difluoromethylornithine treats diseases caused by porcine circovirus type 2 infection by inhibiting the replication of porcine circovirus type 2 or the expression of the Cap protein.
[0007] Furthermore, the difluoromethylornithine can reduce the viral load of porcine circovirus type 2 in the spleen, lung, and inguinal lymph nodes.
[0008] Furthermore, the difluoromethylornithine inhibits the replication of porcine circovirus type 2 in cells in a dose-dependent manner.
[0009] Furthermore, the difluoromethylornithine can inhibit the weight loss caused by porcine circovirus type 2 infection.
[0010] Furthermore, the dosage of the difluoromethylornithine is 10 - 200 µM.
[0011] The present invention also provides a pharmaceutical preparation, which comprises the above-mentioned difluoromethylornithine and pharmaceutically acceptable excipients.
[0012] Furthermore, the pharmaceutical preparation further includes one or more pharmaceutically acceptable carriers.
[0013] Furthermore, the pharmaceutically acceptable carriers include: excipients, fillers, binders, wetting agents, disintegrants, diluents and / or surfactants.
[0014] Furthermore, the dosage form of the pharmaceutical preparation is any one or more of tablets, capsules, oral liquids, granules, pills or injections.
[0015] Beneficial effects
[0016] The present invention for the first time confirms that difluoromethylornithine can inhibit porcine circovirus type 2 from infecting host cells and mice, can reduce the virus titer of porcine circovirus type 2 and the expression of Cap protein, significantly reduce the viral load in mice, and can be used for preparing drugs against porcine circovirus type 2 infection to inhibit virus replication. Description of the drawings
[0017] Figure 1 It is the viability of PK-15 cells treated with difluoromethylornithine at different concentrations.
[0018] Figure 2 It is a schematic diagram of the western blot detection results of viral Cap protein in PCV2-infected PK-15 cells under different treatments.
[0019] Figure 3 It is a schematic diagram of the detection results of the viral copy number in PCV2-infected PK-15 cells under different treatments.
[0020] Figure 4 It is a schematic diagram of the detection results of the virus titer in PCV2-infected PK-15 cells under different treatments.
[0021] Figure 5 It is a schematic diagram of the change of the mouse body weight curve.
[0022] Figure 6Schematic diagram of the results of difluoromethylornithine inhibiting the viral load in the lungs of mice.
[0023] Figure 7 Schematic diagram of the results of difluoromethylornithine inhibiting the viral load in the spleen of mice.
[0024] Figure 8 Schematic diagram of the results of difluoromethylornithine inhibiting the viral load in the inguinal lymph nodes of mice. Detailed implementation manners
[0025] The present invention will be further elaborated in detail below in conjunction with the accompanying drawings of the specification and specific embodiments. The embodiments are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.
[0026] Example 1 Effect of the drug on cell viability
[0027] I. Experimental method
[0028] (1) Cell culture
[0029] Inoculate porcine kidney cell line PK-15 cells into a culture plate and culture them with Dulbecco's modified Eagle's medium (DMEM; Gibco, Waltham, MA, USA) containing 10% fetal bovine serum (S711-001S; LONSERA, Ltd, China). The culture conditions are: 37 °C, 5% CO2.
[0030] (2) Drug preparation
[0031] Dissolve difluoromethylornithine (CAS No.: 70052-12-9) with 100% DMSO (purchased from Solarbio) to obtain difluoromethylornithine stock solutions with concentrations of 10 mM, 50 mM, 100 mM and 200 mM.
[0032] (3) Drug addition treatment
[0033] After the PK-15 cells are passaged and cultured for 24 h until the cell density reaches 80%, discard the old medium, wash 3 times with 1×PBS, add Dulbecco's modified Eagle's medium (DMEM; Gibco, Waltham, MA, USA) for maintenance culture and different concentrations of difluoromethylornithine stock solutions. The final concentrations of difluoromethylornithine are 10 μM, 50 μM, 100 μM and 200 μM respectively, and incubate at 5% CO2 for 48 h. Use DMSO as a control.
[0034] (4) Cell viability assay
[0035] Incubate PK-15 cells treated with different concentrations of the drug with CCK-8 solution. Add 10 μL of CCK-8 solution to each well of the cell plate, incubate the culture plate in the incubator for 2 hours, and gently mix it on the shaker before reading the plate. Then, read the absorbance at 450 nm using a microplate reader and calculate the cell viability.
[0036] II. Experimental results
[0037] As Figure 1 shown, difluoromethylornithine has low toxicity. Even when the dosage reaches 200 μM, the cell viability can still reach more than 90%, indicating low cytotoxicity and good safety.
[0038] Example 2: Difluoromethylornithine inhibits PCV2 replication
[0039] I. Experimental methods
[0040] (1) Cell culture
[0041] The method is the same as that of "Cell culture" in Example 1.
[0042] (2) Virus inoculation
[0043] After the cell density reaches 80%, infect PK-15 cells with PCV2 (PCV2 strain BJW, GenBank: AY847748.1) at a multiplicity of infection (MOI) of 1.0. The specific operation is as follows: Infect at 37°C and 5% CO2 for 1 h; discard the old culture medium, wash the cells 2 times with phosphate-buffered saline (PBS); add Dulbecco's modified Eagle's medium (DMEM; Gibco, Waltham, MA, USA) containing 5% fetal bovine serum (S711-001S; LONSERA, Ltd, China), and continue to culture at 37°C and 5% CO2 for 24 h, 48 h, and 72 h.
[0044] (3) Drug addition treatment
[0045] During virus infection, perform drug addition treatment on PK-15 cells.
[0046] Difluoromethylornithine with final concentrations of 10 μM, 50 μM, 100 μM, and 200 μM was added to the cell culture medium respectively, and a control group with DMSO added was set up. Then the cells were placed in a 37°C, 5% CO2 cell culture incubator and cultured for another 48 h.
[0047] (4)Detection by Western blot
[0048] PK-15 cells were lysed with RIPA lysis buffer containing 1% protease inhibitor on ice for 30 min. The lysed cell mixture was added to a 1.5 mL centrifuge tube. Using a cell disruptor at 70 W, working for 5 s, with a 5 s interval, repeated three times; then centrifuged at 4°C, 12,000 rpm for 15 min, and the supernatant was collected into a new centrifuge tube to obtain the protein sample of the cells.
[0049] The total protein concentration of the protein sample of the cells was detected using a BCA protein concentration assay kit. 6× Loading Buffer protein loading buffer was added, mixed well, and then placed in a boiling sample device at 100°C for 10 min to obtain the protein loading solution.
[0050] The expression of viral Cap protein in the protein loading solution was detected by SDS-PAGE gel electrophoresis.
[0051] The electrophoresis conditions were: concentrating at 80 V for 30 min and separating at 120 V for 60 min. After electrophoresis, the gel was placed in a transfer system to transfer the protein bands to the NC membrane.
[0052] After the transfer, the NC membrane was placed in 5% skim milk and slowly blocked on a shaker at room temperature for 2 h; the blocking solution was discarded, and the NC membrane was washed 3 times with PBS to remove 5% skim milk, 5 min each time; the NC membrane was incubated with PCV2 Cap mouse monoclonal antibody (prepared and stored in our laboratory) on a shaker at 4°C overnight; the NC membrane was washed 3 times with PBS to remove the Cap mouse monoclonal antibody, 5 min each time; horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (04-18-06, purchased from KPL) was added and incubated on a shaker at room temperature for 1 h; the NC membrane was washed 3 times with PBS to remove HRP-labeled goat anti-mouse IgG, 5 min each time.
[0053] (5)Detection of viral genome copy number by real-time quantitative polymerase chain reaction (RT-qPCR) Use the EasyPure® Viral DNA / RNA Kit to extract viral DNA, and perform fluorescence quantitative PCR detection using Taq Pro Universal SYBR qPCR Master Mix.
[0054] The gene-specific primers used for fluorescence quantitative PCR are shown in Table 1, the reaction system is shown in Table 2, and the reaction program is shown in Table 3.
[0055] Table 1 Information of primers used for fluorescence quantitative PCR
[0056]
[0057] Table 2 Reaction system for detecting viral load by fluorescence quantitative PCR
[0058]
[0059] Table 3 Fluorescence quantitative PCR reaction program
[0060]
[0061] (6)PCV2 TCID 50 Determination
[0062] The collected virus solution to be tested that has been repeatedly frozen and thawed three times is serially diluted 10-fold in 1.5 mL Eppendorf tubes with DMEM medium containing 2% FBS, i.e., 10 -1 ~10 -8 . Sequentially inoculate the virus solution of each dilution into a 96-well plate pre-seeded with PK-15 cells at a cell density of 50 - 60%. Add the diluted virus solution, and simultaneously set normal cells without virus inoculation as the negative control, with each group repeated three times. Then place the 96-well plate in a cell culture incubator at 37 °C and 5% CO2 and continue to culture for 48 h. After collecting the cell samples, fix them with paraformaldehyde at room temperature for 30 min. After fixation, add 5% skim milk and place it on a shaker for slow blocking for 2 h; discard the blocking solution, wash 3 times with PBS to remove 5% skim milk, 5 min each time; add PCV2 Cap mouse monoclonal antibody to the 96-well plate and place it on a shaker, and incubate overnight at 4 °C; wash 3 times with PBS to remove Cap mouse monoclonal antibody, 5 min each time; add the corresponding fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG, and incubate on a shaker at room temperature for 1 h; wash 3 times with PBS to remove FITC-labeled goat anti-mouse IgG, 5 min each time. Subsequently, add PBS and observe under a fluorescence microscope, and finally calculate the virus titer by the Karber method.
[0063] II. Experimental results
[0064] 1. Western blot detection results
[0065] The results are as Figure 2 shown. During the culture of PK-15 cells, after adding difluoromethylornithine, the expression level of PCV2 Cap protein was significantly reduced and showed a dose-dependent manner. It indicates that difluoromethylornithine significantly inhibits the expression of PCV2 Cap protein and inhibits the replication of PCV2.
[0066] 2. Fluorescent quantitative PCR detection results
[0067] The results are as Figure 3 shown. During the culture of PK-15 cells, after adding difluoromethylornithine, the genomic copy number of PCV2 was significantly reduced and showed a dose-dependent manner. It indicates that difluoromethylornithine significantly inhibits the replication of PCV2.
[0068] 3. TCID 50 detection results
[0069] The results are as Figure 4 shown. During the culture of PK-15 cells, after adding difluoromethylornithine, the virus titer of PCV2 was significantly reduced and showed a dose-dependent manner. It indicates that difluoromethylornithine significantly inhibits the replication of PCV2.
[0070] Example 3: Difluoromethylornithine inhibits the replication of PCV2 in BALB / c mice
[0071] I. Experimental method
[0072] (1) Experimental animals and grouping
[0073] The experimental animals were female BALB / c mice, 5 - 6 weeks old, with normal health status. They were randomly divided into four groups, with 8 mice in each group. The grouping and treatment were as follows:
[0074] The first group (negative control group): 8 BALB / c mice were intraperitoneally injected with 100 μL of DMEM medium respectively. The negative control group was not inoculated with the virus and not treated with drugs.
[0075] The second group (single drug treatment group): 8 BALB / c mice were intraperitoneally injected with 100 μL of DMEM medium respectively, and at the same time given 2% difluoromethylornithine aqueous solution, continuously treated for 28 days, and not inoculated with the virus.
[0076] The third group (infection group): 8 BALB / c mice were intraperitoneally injected with 100 μL of pre-prepared PCV2 virus solution, and not treated with drugs.
[0077] Group 4 (drug treatment group): 8 BALB / c mice were inoculated with 100 µL of PCV2 virus solution by intraperitoneal injection, and at the same time, 2% difluoromethylornithine aqueous solution was administered for 28 consecutive days.
[0078] Record the original body weights of the mice in each group before treatment. After treatment, weigh the mice every day within a fixed time range. On the 7th, 14th, 21st, and 28th days after treatment, randomly select 2 mice from each group, sacrifice them by cervical dislocation after dissection, collect the lungs, spleens, and inguinal lymph nodes, weigh the obtained organs, and store them at -80 °C for later use.
[0079] (2)Determination of PCV2 copy number in tissues
[0080] Extraction of viral DNA from tissues: Take the lungs, spleens, and inguinal lymph nodes out of the -80 °C refrigerator, thaw them at room temperature and place them in grinding tubes respectively. Pipette 300 µL of PBS into the grinding tubes, grind them using a grinder. After grinding evenly, place them in the -80 °C refrigerator, freeze-thaw three times, centrifuge at 12,000 rpm for 10 min, and take the supernatant and place it in a new EP tube. Pipette 20 µL of proteinase K into the EP tube containing 200 µL of the solution to be tested, then add 200 µL of BB5, vortex for 15 s, and incubate at 56 °C for 15 min. After incubation, add 250 µL of absolute ethanol, vortex for 15 s, and let it stand at room temperature for 15 min. Transfer the standing mixed solution to a centrifugal column, centrifuge at 12,000 rpm at room temperature for 1 min, and repeat twice. Add 500 µL of WB5 to the centrifugal column, centrifuge at 12,000 rpm at room temperature for 1 min, and repeat twice. Subsequently, centrifuge at 12,000 rpm at room temperature for 1 min, and retain the centrifugal column. Place the centrifugal column in a 1.5 mL EP tube, add 30 µL of RNAse-free Water, let it stand at room temperature for 1 min. After standing, centrifuge at 12,000 rpm at room temperature for 1 min, and store the eluted DNA in the EP tube at -20 °C.
[0081] The fluorescence quantitative PCR detection is the same as the method of "Real-time quantitative polymerase chain reaction (RT-qPCR) for detecting the copy number of viral genomes" in Example 2.
[0082] II. Experimental results
[0083] 1. Changes in mouse body weight
[0084] The results are as Figure 5 shown. During the entire experimental process, record the body weights of the mice every day. Compared with the infection group, the drug treatment group has no effect on the body weights of the mice.
[0085] 2. Detection results of PCV2 copy number in mouse lungs
[0086] The results are as follows Figure 6 shown. In BALB / c mice, compared with the infected group, the genomic copy number of PCV2 in the lungs of the drug-treated group was significantly reduced. This indicates that difluoromethylornithine significantly inhibits the replication of PCV2 in the lungs of mice.
[0087] 3. Detection results of PCV2 copy number in mouse spleens
[0088] The results are as follows Figure 7 shown. In BALB / c mice, compared with the infected group, the genomic copy number of PCV2 in the spleens of the drug-treated group was significantly reduced. This indicates that difluoromethylornithine significantly inhibits the replication of PCV2 in the spleens of mice.
[0089] 4. Detection results of PCV2 copy number in mouse inguinal lymph nodes
[0090] The results are as follows Figure 8 shown. In BALB / c mice, compared with the infected group, the genomic copy number of PCV2 in the inguinal lymph nodes of the drug-treated group was significantly reduced. This indicates that difluoromethylornithine significantly inhibits the replication of PCV2 in the inguinal lymph nodes of mice.
[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For those of ordinary skill in the art, based on the above description and ideas, other different forms of changes or modifications can be made. It is not necessary and impossible to enumerate all the implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.
Claims
1. Use of difluoromethylornithine in the preparation of drugs for preventing and / or treating diseases caused by porcine circovirus type 2 infection.
2. The use according to claim 1, characterized in that: The difluoromethylornithine can treat diseases caused by porcine circovirus type 2 infection by inhibiting porcine circovirus type 2 replication or the expression of Cap protein.
3. The use according to claim 1, characterized in that: The difluoromethylornithine can reduce the viral load of porcine circovirus type 2 in the spleen, lungs and inguinal lymph nodes.
4. The use according to claim 1, characterized in that: The difluoromethylornithine inhibits the replication of porcine circovirus type 2 in cells in a dose-dependent manner.
5. The use according to claim 1, characterized in that: The difluoromethylornithine can inhibit the weight loss caused by porcine circovirus type 2 infection.
6. The use according to claim 1, characterized in that: The dosage of the difluoromethylornithine is 10-200 μM.
7. A pharmaceutical preparation, characterized in that The invention comprises the difluoromethylornithine as claimed in claim 1, and pharmaceutically acceptable excipients.
8. The pharmaceutical preparation according to claim 7, characterized in that The medicament further comprises one or more pharmaceutically acceptable carriers.
9. The pharmaceutical preparation according to claim 7, characterized in that The pharmaceutically acceptable carrier includes: an excipient, a filler, a binder, a wetting agent, a disintegrant, a diluent and / or a surfactant.
10. The pharmaceutical preparation according to any one of claims 7 to 9, characterized in that The dosage form of the pharmaceutical preparation is any one or more of tablets, capsules, oral liquids, granules, pills or injections.