Application of carboplatin in preparation of medicine for preventing and / or treating negative-chain RNA virus infection
By using carboplatin to inhibit the replication of ruminant virus in Vero cells, the problem of lack of effective treatment of ruminant virus in the prior art was solved, and the effect of significantly reducing viral titer and N protein expression was achieved, providing a new method to prevent and treat negative-strand RNA virus infection.
Patent Information
- Application Number
- CN202311466358.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-11-07
- Publication Date
- 2025-05-09
AI Technical Summary
The prior art lacks effective treatments to deal with diseases caused by PPRV, and there are problems with the stability and safety of commonly used vaccines.
Using carboplatin as a drug, by inhibiting the replication of ruminant virus in Vero cells, significantly reducing viral titer and N protein expression, thus providing a new method to prevent and treat negative-strand RNA virus infection.
Carboplatin has no significant effect on cell survival, but it can significantly inhibit the replication of PPRV in Vero cells and N protein expression, and has broad application prospects.
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Figure CN119950515A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and specifically relates to the application of carboplatin in preparing a drug for preventing and / or treating negative-strand RNA virus infection. Background Art
[0002] Negative-strand RNA viruses are an important class of pathogens with a wide range of hosts. Their RNA cannot directly function as mRNA. They must first synthesize a complementary strand (positive strand) as mRNA, then translate protein molecules, and then undergo nucleic acid replication to become a template for synthesizing progeny viral RNA. So far, negative-strand RNA viruses are divided into at least eight virus families and four unclassified genera, namely: Bornaviridae, Filoviridae, Paramyxoviridae, Rhabdovieidae, Arenaviridae, Bunyaviridae, Ophioviridae, OrLbomyxoviridae, Deltavirus, Ernavirus, Tenuivirus, and Varicosavirus.
[0003] Peste des petits ruminants virus (PPRV) belongs to the Morbillivirus genus of the Paramyxoviridae family. Other members of the same genus include Canine distemper virus (CDV), Cetacean morbillivirus virus (CeMV), Phocine distemper virus (PDV), Rinderpest virus (RPV) and Measles virus (MV). PPRV is an enveloped single-stranded negative-sense RNA virus with only one serotype. It causes an acute, febrile, highly contagious disease of small ruminants such as goats and sheep - Peste des petits ruminants (PPR), commonly known as sheep plague, also known as pseudo-rinderpest of small ruminants, pneumococcal enteritis, and stomatitis-pneumococcal enteritis syndrome. The morbidity rate is as high as 90% to 100%, and the mortality rate is 50% to 100%. Clinically, small ruminants mainly show symptoms such as high fever, stomatitis, pneumonia, diarrhea, etc. PPR often occurs during the transition of seasons and in winter and spring. It has the characteristics of short incubation period, fast transmission speed, high morbidity and high mortality. It is a type of animal epidemic in my country, which has caused serious economic losses to my country's sheep industry and also restricted the development of the sheep industry.
[0004] At present, there is no effective treatment for PPR. The outbreak is mainly prevented by comprehensive prevention and control measures such as killing at epidemic sites, isolating and restricting animal movement, and emergency vaccination. The main measure for preventing and controlling PPR is vaccination. Researchers have developed four PPRV homologous attenuated vaccines: Nigeria 75 / 1, Sungri / 96, Arasur / 87, and Coimbatore / 97. Among them, Nigeria 75 / 1 and Sungri / 96 are the most widely used, which can produce good protective immunity against four lineages of PPRV. The prevalent small ruminant virus lineage in my country is IV lineage. The use of Nigeria 75 / 1 attenuated vaccine may increase the probability of virus mutation and recombination, and even the risk of enhanced virulence. In addition, because PPRV is not heat-resistant, the stability of Nigeria 75 / 1 vaccine is poor, which is not conducive to transportation and use at the grassroots level.
[0005] Carboplatin is a non-specific drug for the cell cycle. It mainly acts on the N7 and O6 atoms of guanine in DNA, causing cross-links between and within DNA chains, destroying DNA molecules, and interfering with DNA synthesis. It is a broad-spectrum anti-tumor drug. It is mainly used for small cell lung cancer, ovarian cancer, testicular tumors, head and neck squamous cell carcinoma, etc.; it can be used for non-small cell lung cancer, bladder cancer, cervical cancer, pleural mesothelioma, melanoma and endometrial cancer, etc. At present, there is no literature that publicly discloses that carboplatin has the effect of inhibiting the replication of negative-strand RNA viruses.
[0006] The inventors unexpectedly discovered during the research process that carboplatin has the effect of inhibiting the replication of negative-strand RNA viruses, and the negative-strand RNA virus is peste des petits ruminants virus. The carboplatin has no significant effect on cell survival rate, and the virus titer in Vero cells treated with carboplatin is significantly reduced; it can significantly inhibit the replication of PPRV in Vero cells, and the inhibitory effect is most significant when the carboplatin concentration is 100 μM; and the N protein level in Vero cells treated with carboplatin (100 μM) is significantly reduced, which has broad application prospects. Summary of the invention
[0007] In view of the above technical problems, the primary purpose of the present invention is to provide the use of carboplatin and its pharmaceutically acceptable salts in the preparation of drugs for preventing negative-strand RNA virus infection.
[0008] Preferably, the negative-strand RNA virus is Peste des Petits Ruminants virus.
[0009] The second object of the present invention is to provide the use of carboplatin and its pharmaceutically acceptable salts in the preparation of drugs for treating negative-strand RNA virus infections.
[0010] Preferably, the negative-strand RNA virus is Peste des Petits Ruminants virus.
[0011] Preferably, the carboplatin is added with a pharmaceutically acceptable carrier and / or excipient to prepare any pharmaceutically acceptable dosage form.
[0012] Preferably, the dosage form includes any one of tablets, sprays, granules, capsules, oral solutions and injections.
[0013] The third object of the present invention is to provide the use of carboplatin and its pharmaceutically acceptable salts in the preparation of negative-strand RNA virus vaccine adjuvants.
[0014] Preferably, the negative-strand RNA virus is Peste des Petits Ruminants virus.
[0015] Preferably, the carboplatin is added with a pharmaceutically acceptable carrier and / or excipient to prepare any pharmaceutically acceptable dosage form.
[0016] Preferably, the dosage form includes any one of tablets, sprays, granules, capsules, oral solutions and injections.
[0017] The beneficial effects of the present invention are as follows: the present invention provides the use of carboplatin and a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and / or treating negative-strand RNA virus infection, and experimental results show that the carboplatin has no significant effect on cell survival rate, and the virus titer in Vero cells treated with carboplatin is significantly reduced; it can significantly inhibit the replication of PPRV in Vero cells, and the inhibitory effect is most significant when the carboplatin concentration is 100 μM; and the N protein level in Vero cells treated with carboplatin (100 μM) is significantly reduced, and the present invention has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 Effects of different concentrations of carboplatin on the activity of Vero cells
[0019] Figure 2 100 μM carboplatin significantly inhibited the viral titer of PPRV in Vero cells
[0020] Figure 3 Carboplatin significantly inhibits the replication of PPRV in Vero cells
[0021] Figure 4 100 μM carboplatin significantly inhibited the expression of PPRVN protein DETAILED DESCRIPTION
[0022] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, but not for limiting the scope of the present invention.
[0023] The experimental methods in the following examples are conventional methods unless otherwise specified; the experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified.
[0024] In the following examples, the Chinese aliases of carboplatin are carbon platinum, carboplatin, platinum ercar, cis-diaminocyclobutane platinum, cis-diaminocyclobutane carboxylic acid platinum; the foreign names are Carboplatin, Paraplatin, Carboplatin, Ercar, all of which represent the same drug.
[0025] Example 1: Effect of carboplatin in inhibiting peste des petits ruminants virus
[0026] 1. Experimental Materials
[0027] 1.1 Reagents and Antibodies
[0028] Mouse anti-β-tubulin monoclonal antibody (66240) was purchased from proteintech; carboplatin (S1215) was purchased from Selleck; mouse anti-N monoclonal antibody was prepared by the laboratory; the strain used was PPRV Nigeria 75 / 1 vaccine strain.
[0029] 1.2 Cells and culture
[0030] Vero cells were purchased from ATCC (CCL-81) and cultured in 100 mL L -1 Fetal bovine serum (Gibco, C20012500BT), 100 U·mL -1 Penicillin and 100 μg mL -1 The cells were cultured in DMEM medium (Gibco, C11330500BT) containing streptomycin at 37°C, 50 mL / L -1 CO 2 of moist conditions.
[0031] 2. Experimental Methods
[0032] 2.1CCK-8 detection
[0033] Add 100 μL of Vero cell suspension to a 96-well plate and incubate the plate at 37°C with 5% CO. 2 The cells were cultured in a humidified environment for 24 h. 10 μL of carboplatin at different concentrations was added to each well of the culture plate, and the culture plate was incubated in a cell culture incubator for 24 h (37°C, 5% CO 2 Humid environment). Add 10 μL CCK-8 solution to each well of the culture plate (be careful not to generate bubbles in the wells to affect the OD value reading), and incubate the culture plate in a cell culture incubator for 4 hours. Measure the absorbance at 450 nm using a microplate reader (PerkiEelmer, HH3500).
[0034] 2.2 Drug treatment and viral infection
[0035] 2 mL of Vero cell suspension was added to a 6-well plate, and different concentrations of carboplatin were added. An equal volume of PBS was added to the control group. The culture plate was incubated at 37°C and 5% CO. 2 Culture in a humid environment for 24 hours. After 24 hours, PPRV (MOI = 1) was used to infect the pretreated cells. The control group was added with an equal amount of PBS. The culture plate was placed at 37°C for 1 hour for adsorption. The cells were washed with PBS, the unadsorbed virus was discarded, and DMEM medium containing 2% FBS was added, and the corresponding concentration of carboplatin was added. Incubate at 37°C, 5% CO 2 After continuing to culture in a humid environment for 48 h, the cells were collected and the total RNA and total protein were extracted.
[0036] 2.3RT-qPCR
[0037] At predetermined time points (48 hpi), cells were collected and analyzed using Thermo GeneJET TM Total RNA was extracted from cells using RNA purification kit (K0732). First-strand cDNA was synthesized using ReverTraAce qPCR RT Master Mix with gDNA Remover (TOYOBO121800) reverse transcription kit. TM SYBR TM Real-time fluorescence quantitative PCR was performed using Green Master Mix (Applied Biosystems A25742). Primers were synthesized by Shanghai Bioengineering, and the primer sequences are as follows:
[0038] PPRV forward primer: 5′-AGAGTTCAATATGTTRTTAGCCTCCAT-3′
[0039] PPRV reverse primer: 5′-TTCCCCARTCACTCTYCTTTGT-3′
[0040] GAPDH forward primer: 5′-GCTGATCCCAACATTCGCTT-3′
[0041] GAPDH reverse primer: 5′-GTGTCCGGCAGAAGAGTTTG-3′
[0042] 2.4Western blot
[0043] (1) Sample preparation: 48 hours after PPRV infection, remove the cell culture plate from the incubator and place it on ice. Wash the cells with pre-cooled PBS, add 300 μL of RIPA lysis buffer to each well, place on ice for 2 minutes, and collect the cells with a cell scraper. Ultrasonicate for 1 minute, centrifuge at 16,000 g, 4°C for 10 minutes. Carefully aspirate the supernatant, add an equal amount of 2×SDS Sample Buffer, and mix by pipetting. Heat and denature at 95°C for 5 minutes in a thermostat.
[0044] (2) Prepare 10% SDS-PAGE gel using TGX Stain-Free Acrylamide Gel Preparation Kit (Bio-Rad, 1610183):
[0045] Table 1 Gel preparation volume
[0046]
[0047] (3) SDS-PAGE electrophoresis: add an equal amount (10 μl) of protein sample to each well and run at a constant voltage of 80 V for 30 min; or at a constant voltage of 120 V for 90 min.
[0048] (4) Transfer: From negative electrode to positive electrode, the order is: sponge - filter paper - gel - PVDF membrane (activated with methanol) - filter paper - sponge. Constant current 200 mA, 2 h.
[0049] (5) Blocking: The PVDF membrane was blocked in 5% skimmed milk powder on a shaker at room temperature for 2 h.
[0050] (6) Incubation with primary antibody: Place the blocked PVDF membrane (Immobilon, IPVH00010) in the corresponding primary antibody dilution solution and incubate overnight at 4°C; on the next day, wash the membrane three times with 1× TBST buffer, each time for 10 min.
[0051] (7) Incubation with secondary antibody: Add 2 μL of the corresponding secondary antibody to 15 mL of 5% skim milk powder, shake well, and incubate the membrane in the secondary antibody dilution on a shaker at room temperature for 1 h; then wash the membrane three times with 1× TBST buffer, each time for 10 min.
[0052] (8) Exposure: ECL chemiluminescent solution (Affinity, KF8001) was dropped onto the membrane for development, and the results were observed using an automatic chemiluminescent gel imaging analysis system (BLT Photon Technology, GV60S10217303).
[0053] 2.5TCID 50 Determination
[0054] When the Vero cells in the 96-well plate adhered to the wall and grew to 80% to 85%, the cells were washed with PBS, and the collected virus stock solution infected for 48 hours was diluted 10 times with serum-free DMEM medium, for a total of 8 dilutions (10 -1 -10 -8 ), each dilution was repeated in 8 wells, 100 μL / well, and 100 μL serum-free DMEM medium was added to each well of the control group at 37°C and 5% CO 2 Incubate in the incubator for 1 hour, remove the culture plate, aspirate the virus solution (absorb from low concentration to high concentration to avoid cross-well), add 200 μL of DMEM medium containing 2% FBS to each well, and place the culture plate at 37°C and 5% CO 2 After 7 days, the cytopathic effect (CPE) was observed and the virus titer was calculated by the Reed-Muench method and expressed as the half tissue culture infectious dose (TCID 50 ).
[0055] 3. Results
[0056] (1) After Vero cells were treated with different concentrations of carboplatin for 24 h, the results of cell viability assay using CCK-8 were as follows: Figure 1 As shown, compared with the control group, treatment of cells with 20, 40, 60, 80, and 100 μM carboplatin had no significant effect on cell survival.
[0057] (2) Determination of TCID of virus solution 50 The results are as follows Figure 2 As shown, the viral titer was significantly reduced in Vero cells treated with carboplatin (100 μM) compared to untreated controls.
[0058] (3) The results of RT-qPCR detection of PPRV mRNA levels are as follows Figure 3 As shown, carboplatin can significantly inhibit the replication of PPRV in Vero cells in the concentration range of 40 μM to 100 μM, and the inhibitory effect is most significant when the carboplatin concentration is 100 μM.
[0059] (4) The results of western blot detection of PPRVN protein expression are as follows Figure 4 As shown, the level of N protein was significantly decreased in Vero cells treated with carboplatin (100 μM).
[0060] In summary, the present invention provides the use of carboplatin and its pharmaceutically acceptable salts in the preparation of drugs for preventing and / or treating negative-strand RNA virus infections. Experimental results show that the carboplatin has no significant effect on cell survival rate, and the virus titer in Vero cells treated with carboplatin is significantly reduced; it can significantly inhibit the replication of PPRV in Vero cells, and the inhibitory effect is most significant when the carboplatin concentration is 100 μM; and the N protein level in Vero cells treated with carboplatin (100 μM) is significantly reduced, which has broad application prospects.
Claims
1. Use of carboplatin and its pharmaceutically acceptable salts in the preparation of drugs for preventing negative-strand RNA virus infection.
2. The use according to claim 1, characterized in that The negative-strand RNA virus is peste des petits ruminants virus.
3. Use of carboplatin and its pharmaceutically acceptable salts in the preparation of drugs for treating negative-strand RNA virus infections.
4. The use according to claim 3, characterized in that The negative-strand RNA virus is peste des petits ruminants virus.
5. The use according to any one of claims 1 to 4, characterized in that: The carboplatin is added with pharmaceutically acceptable carriers and / or excipients to prepare any pharmaceutically acceptable dosage form.
6. The use according to claim 5, characterized in that The dosage form includes any one of tablets, sprays, granules, capsules, oral liquids and injections.
7. Use of carboplatin and its pharmaceutically acceptable salts in the preparation of negative-strand RNA virus vaccine adjuvants.
8. The use according to claim 7, characterized in that The negative-strand RNA virus is peste des petits ruminants virus.
9. The use according to any one of claims 7 to 8, characterized in that: The carboplatin is added with a pharmaceutically acceptable carrier and / or excipient to prepare any pharmaceutically acceptable dosage form.
10. The use according to claim 9, characterized in that The dosage form includes any one of tablets, sprays, granules, capsules, oral liquids and injections.