Use of bexarotene in the preparation of a medicament for anti-PRRSV or for treating porcine reproductive and respiratory syndrome

Abstract

The present invention discloses the use of bexarotene in the preparation of a medicament for anti-PRRSV or for treating porcine reproductive and respiratory syndrome (PRRS). The present invention provides a new use of the existing anti-tumor drug bexarotene, and it is found that bexarotene has an anti-PRRSV effect, can significantly inhibit the proliferation and replication of PRRSV, reduce the expression of PRRSV-N protein and the infectivity of PRRSV; at the same time, it also has a broad-spectrum anti-PRRSV effect, can inhibit a variety of PRRSV strains with different pathogenicities, especially has a significant antiviral effect against highly pathogenic HP-PRRSV; and has almost no cytotoxicity, and can be better used for anti-PRRSV infection to relieve PRRS. Since bexarotene has a small molecular weight and can easily cross the cell membrane to enter the cell to play an antiviral role, it does not need to be delivered into the cell by a carrier, which lays a foundation for the development of more new drugs against PRRSV and provides more methods for the prevention and treatment of PRRS.

Description

Technical Field

[0001] The present invention belongs to the technical field of biotechnology and pharmacy. More specifically, it relates to the use of bexarotene in the preparation of a drug for anti-PRRSV or the treatment of porcine reproductive and respiratory syndrome (PRRS). Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious porcine viral infectious disease caused by Porcine reproductive and respiratory syndrome virus (PRRSV), commonly known as "blue ear disease". PRRS was first reported in the United States in the late 1980s and is one of the pathogens that cause great harm to the world's pig farming industry. The infection of this disease in pig herds can cause fever, dyspnea, and spotted congestion or cyanosis on the skin of the limbs, and even death. After infecting sows, it can lead to abortion, causing great harm to the health of pig herds and seriously damaging the economic interests of the pig farming industry. In April 2006, highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) broke out in Jiangxi Province, China. Subsequently, the disease quickly spread to many provinces and cities across the country, with a vast epidemic area and the disease occurring in pig herds of all ages. So far, most pig-raising areas in the country are still threatened by this disease, with the incidence rate generally above 50% and the fatality rate between 20% and 100%.

[0003] PRRS is an immunosuppressive disease. PRRSV can target host immune cells, weaken the host's defense ability, and lead to an increased susceptibility to primary and secondary pathogens, which can affect the growth efficiency of pig herds and increase the incidence and mortality. To control the prevalence of PRRS in China, an inactivated vaccine was developed using the first PRRSV isolate CH-1a in China and was put into use in pig farms after being registered in 2000. In 2007, a live attenuated vaccine derived from the CH-1a isolate was also approved for registration in China. Shortly after the outbreak of atypical PRRS, China developed an inactivated vaccine using the isolated virus (JXA1). However, the effects of these vaccines in controlling PRRS are not satisfactory. In vaccinated pig farms, clinical cases related to the failure of pregnant sows to produce and the respiratory disorders of suckling piglets occur frequently and cannot provide effective clinical immune protection as expected. Generally speaking, the existing vaccines cannot effectively prevent and control PRRS. Therefore, it is particularly important to find effective anti-PRRS virus drugs, which may be a new strategy for preventing and controlling PRRS.

[0004] Due to the poor immune effect of existing commercial vaccines, the cross-protection ability of vaccines against different strains is not complete, and there is a possibility of virus virulence reversion and recombination with wild virus to form new strains during the replication process of attenuated vaccines in pigs. Currently, there is also a severe lack of drugs that can combat PRRSV. Therefore, it is of great significance to develop more safe, efficient, and broad-spectrum anti-PRRSV drugs. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects of existing PRRSV vaccines, such as poor immune effect and weak protection ability, as well as the shortage of existing anti-PRRSV drugs, and to provide the application of bexarotene in the preparation of drugs for anti-PRRSV or treating porcine reproductive and respiratory syndrome (PRRS).

[0006] The object of the present invention is to provide a new application of the old drug bexarotene in anti-PRRSV.

[0007] The above object of the present invention is achieved by the following technical solutions:

[0008] The present invention provides a new application of the existing anti-tumor drug bexarotene in anti-PRRSV. It is found that bexarotene can significantly inhibit the replication and proliferation of PRRSV, with an EC50 of 2.946 μM. It can inhibit the expression of PRRSV-N protein and also has an obvious inhibitory effect on the recombinant virus PRRSV-TA-12Gluc, capable of reducing the virus titer. Moreover, it has an obvious anti-PRRSV effect on PRRSV strains with different pathogenicities, showing broad-spectrum characteristics. In particular, it still has a good antiviral effect against HP-PRRSV and can be used to prepare drugs for alleviating or treating porcine reproductive and respiratory syndrome, becoming a new strategy for effectively preventing and controlling PRRS. At the same time, since bexarotene is an oral drug that can be absorbed through the intestine, it can be premixed in feed for administration, and the administration method is extremely convenient. In addition, the pharmacokinetics of bexarotene indicates that the drug reaches the maximum plasma concentration approximately 2 hours after oral administration, can quickly exert its effect, laying a foundation for the development of more new anti-PRRSV drugs and providing more methods for the prevention and treatment of porcine reproductive and respiratory syndrome.

[0009] Bexarotene studied in the present invention is a high-affinity selective retinoid X receptor (RXR) agonist, belonging to an anti-tumor drug and clinically used for treating patients with cutaneous T-cell lymphoma. The chemical name of bexarotene is 4-[1-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthyl)vinyl]benzoic acid, with the molecular formula C 24 H 28 O2 and a molecular weight of 348.47800. Its structural formula is:

[0010]

[0011] Therefore, the present invention provides the use of bexarotene as an anti-PRRSV agent.

[0012] The present invention provides the use of bexarotene in the preparation of an anti-PRRSV drug.

[0013] The present invention provides the use of bexarotene in the preparation of a drug for preventing PRRSV infection.

[0014] The present invention provides the use of bexarotene in the preparation of a drug for alleviating or treating porcine reproductive and respiratory syndrome (PRRS).

[0015] Furthermore, the drug can inhibit the replication and proliferation of PRRSV.

[0016] Furthermore, the drug can inhibit the expression of the viral PRRSV-N protein.

[0017] Furthermore, the drug can reduce the virus titer.

[0018] Furthermore, the porcine reproductive and respiratory syndrome is the PRRS caused by HP-PRRSV or the PRRS caused by a viral variant.

[0019] Preferably, the viral variant is the WuH3 strain, the SD16 strain, the TA-12 strain, the JXA1 strain or the NADC30 strain.

[0020] Furthermore, the drug contains a pharmaceutically acceptable carrier or excipient.

[0021] Furthermore, the dosage form of the drug can be prepared into an injection preparation or an oral preparation, such as a powder, a capsule, a tablet, etc., but is not limited to a powder, a capsule, a tablet, and can be any dosage form acceptable in veterinary clinical practice.

[0022] Preferably, the concentration of the active ingredient of bexarotene in the drug is 1-20 μM.

[0023] More preferably, the concentration of the active ingredient of bexarotene in the drug is 2-10 μM.

[0024] The present invention has the following beneficial effects:

[0025] The present invention provides a new application of the existing anti-tumor drug bexarotene, and it is found that bexarotene has an anti-PRRSV effect, can significantly inhibit the proliferation and replication of porcine reproductive and respiratory syndrome virus (PRRSV), and reduce the infectivity of PRRSV; at the same time, it also has a broad-spectrum anti-PRRSV effect, can inhibit the expression of PRRSV-N protein of various PRRSV strains with different pathogenicities, especially has a significant antiviral effect against highly pathogenic HP-PRRSV; and it has almost no cytotoxicity, can be better used to resist PRRSV infection, and relieve porcine reproductive and respiratory syndrome. Since bexarotene has a small molecular weight and can easily penetrate the cell membrane and enter the cell to play an antiviral role, there is no need for a carrier to deliver it into the cell, which lays a foundation for the development of more new drugs against PRRSV and provides more methods for the prevention and treatment of porcine reproductive and respiratory syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the toxicity curve of bexarotene to Marc-145 cells and the median effective concentration curve of the drug.

[0027] Figure 2 It is for detecting the expression level of PRRSV-N mRNA by fluorescence quantitative PCR (RT-PCR).

[0028] Figure 3 It is the virus infection ratio of recombinant virus PRRSV-TA-12Gluc.

[0029] Figure 4 It is for detecting the expression level of PRRSV-N protein by western-blot.

[0030] Figure 5 It is the fluorescence image for detecting the expression level of PRRSV-N protein by immunofluorescence IFA (in the figure, DAPI indicates the cell nucleus, and Merge indicates the combined image of PRRSV-N protein and the cell nucleus).

[0031] Figure 6 It is the result image of plaque assay for detecting virus titer.

[0032] Figure 7 It is the expression level of PRRSV-N protein of different PRRSV strains. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0033] The following further illustrates the present invention in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

[0034] Unless otherwise specified, the reagents and materials used in the following embodiments are all commercially available.

[0035] The cells and PRRSV strains used in the following examples were all sourced and stored in the laboratory of the research group of this invention; bexarotene was provided by MedChemExpress (MCE) company, CAS No. 153559-49-0.

[0036] For the statistical analysis of the following examples, all experiments were independently repeated at least 3 times, and the results were expressed as the mean and standard error. One-way ANOVA and T-tests were used for analysis; all statistical analyses were performed using P<0.05 as the test criterion for significant statistical differences, and the analysis software was SPSS16.0 and GraphPad Prism5.

[0037] Example 1 Cytotoxicity of Bexarotene and Half Maximal Effective Concentration of the Drug

[0038] 1. Detection of Drug Cytotoxicity

[0039] The CCK-8 kit (purchased from Nanjing Novozymes Biotechnology Co., Ltd.) was used for the detection of drug cytotoxicity. Marc-145 cells (or PAMs cells) were cultured in DMEM medium containing 10% fetal bovine serum for about 18 h until the cell density reached 60-70%. The culture medium was discarded, and nutrient solutions containing serially diluted bexarotene (diluted with DMEM medium, and the dilution concentrations were 2.5, 5.0, 10.0, 20.0 μM respectively) were added and allowed to act for 36 h. At the same time, a negative control group and a blank control group were set. Then, CCK-8 Solution was added and the cells were cultured for another 2 h; the absorbance at 450 nm was measured using a multifunctional microplate reader, and a cytotoxicity graph of bexarotene was plotted. Taking the cell viability of the negative control group as 100%, the percentage of cell survival = [(absorbance of cells treated with serially diluted bexarotene - absorbance of the blank control group) / (absorbance of the negative control group - absorbance of the blank control group)] × 100%, which was the relative cell viability of bexarotene at different concentrations.

[0040] The cytotoxicity of bexarotene is as Figure 1 shown. The CC50 curve of the cells in the figure shows that when the concentration of bexarotene reaches 20.0 μM, it still does not show cytotoxicity, indicating that bexarotene has extremely low toxicity to cells.

[0041] 2. Half Maximal Effective Concentration of the Drug

[0042] Marc-145 cells (or PAMs cells) were cultured in DMEM medium containing 10% fetal bovine serum for about 18 h until the cell density reached 70-75%. Then the culture medium was discarded and replaced with DMEM medium containing 2% fetal bovine serum and a serial dilution of bexarotene (diluted with DMEM medium at concentrations of 2.5, 5.0, 10.0, 20.0 μM). HP-PRRSV was inoculated at a multiplicity of infection (MOI) of 0.5. A control group without bexarotene and a negative control group were set up. Then the cells were cultured in an incubator at 37 °C and 5% CO2. After 36 h, the cells were washed three times with PBS and fixed and permeabilized with ice-cold methanol. Subsequently, the expression level of PRRSV-N protein was detected by immunofluorescence (IFA). Taking the PRRSV-N fluorescence intensity of the control group without bexarotene as 100%, the effective concentration was calculated according to the formula: effective concentration = (PRRSV-N fluorescence intensity of the experimental groups with different concentrations of bexarotene / PRRSV-N fluorescence intensity of the control group without bexarotene). A drug effective concentration curve was plotted and the half-maximal effective concentration (EC50) of the drug was calculated.

[0043] The results of the half-maximal effective concentration of bexarotene were as Figure 1 shown. According to the EC50 of the drug semi-effective concentration curve, the half-maximal effective concentration of bexarotene was 2.946 μM, indicating that bexarotene had a good anti-PRRSV effect.

[0044] Example 2 Antiviral assay of bexarotene

[0045] 1. Detection by fluorescence quantitative PCR (RT-PCR)

[0046] Marc-145 cells (or PAMs cells) were cultured in DMEM medium containing 10% fetal bovine serum for about 18 h until the cell density reached 70-75%. Then the culture medium was discarded and replaced with DMEM medium containing 2% fetal bovine serum and a serial dilution of bexarotene (diluted with DMEM medium at concentrations of 5.0, 7.5, 10.0 μM). HP-PRRSV was inoculated at a multiplicity of infection (MOI) of 0.5. A control group without bexarotene was set up. Then the cells were cultured in an incubator at 37 °C and 5% CO2. After 36 h, the cells were washed three times with PBS, then digested with 0.25% trypsin, and the RNA in the cells was extracted. After measuring the RNA concentration, quantitative reverse transcription was performed, and finally the expression level of PRRSV-N mRNA was detected by fluorescence quantitative PCR.

[0047] The detection results were as Figure 2 shown, indicating that bexarotene had a significant inhibitory effect on the expression of PRRSV-N mRNA. Moreover, as the concentration of bexarotene increased, the inhibitory effect became more obvious, showing a dose-dependent relationship, indicating that bexarotene could inhibit the expression of PRRSV-N mRNA.

[0048] 2. Detection of the expression level of the recombinant virus Gaussia luciferase protein

[0049] Culture Marc-145 cells (or PAMs cells) with DMEM medium containing 10% fetal bovine serum for about 18 h until the cell density reaches 70-75%. Discard the medium and replace it with DMEM medium containing 2% fetal bovine serum with a serial dilution of bexarotene (the dilution concentrations are: 5.0, 7.5, 10.0 μM). Inoculate the PRRSV-TA-12Gluc recombinant virus (the strain was donated by the research group of Professor Li Yanhua from Yangzhou University) at a multiplicity of infection MOI = 0.5, and set a control group without bexarotene. Then culture in an incubator at 37 °C and 5% CO2. After 36 h, collect the cell supernatant into an enzyme-linked immunosorbent assay (ELISA) plate, add the coelenterazine reaction substrate, and then use a multifunctional microplate reader to detect the absorbance at 480 nm. Take the absorbance of the control group without bexarotene as 100%. After treating MARC-145 cells infected with TA-12 recombinant PRRSV expressing Gaussia luciferase (Gluc) gene with bexarotene at different concentrations, detect the expression level of Gluc protein, calculate the expression amount of Gaussia luciferase protein and convert it into the virus infection ratio.

[0050] The results are as Figure 3 shown, indicating that bexarotene at different concentrations can significantly inhibit the expression of the Gaussia luciferase protein of the recombinant virus PRRSV-TA-12Gluc, suggesting that bexarotene has an obvious inhibitory effect on the recombinant virus PRRSV-TA-12Gluc.

[0051] 3. Detection by Western blot

[0052] Culture Marc-145 cells (or PAMs cells) with DMEM medium containing 10% fetal bovine serum for about 18 h until the cell density reaches 70-75%. Discard the medium and replace it with DMEM medium containing 2% fetal bovine serum with a serial dilution of bexarotene (diluted with DMEM medium, the dilution concentrations are: 5.0, 7.5, 10.0 μM). Inoculate HP-PRRSV at a multiplicity of infection MOI = 0.5, and set a control group without bexarotene and a blank control group. Then culture in an incubator at 37 °C and 5% CO2. After 36 h, wash the cells 3 times with PBS, then digest the cells with 0.25% trypsin, measure the protein concentration after cell lysis, and detect the expression level of PRRSV-N protein by Western blot.

[0053] The detection results are as Figure 4As shown, bexarotene inhibited the expression of PRRSV-N protein, and when the concentration of bexarotene reached 7.5 μM, PRRSV-N protein was hardly detectable, indicating that bexarotene has excellent anti-PRRSV ability.

[0054] 4. Immunofluorescence (IFA) assay

[0055] Marc-145 cells (or PAMs cells) were cultured in DMEM medium containing 10% fetal bovine serum for about 18 h until the cell density reached 70 - 75%. The culture medium was discarded and replaced with DMEM medium containing 2% fetal bovine serum with 10.0 μM bexarotene. HP-PRRSV was inoculated at a multiplicity of infection (MOI) of 0.5, and a control group without bexarotene and a blank control group were set up. Then, the cells were cultured in an incubator at 37 °C and 5% CO2. After 36 h, the cells were washed 3 times with PBS, fixed and permeabilized with ice-cold methanol, and the expression level of PRRSV-N protein was detected by immunofluorescence (IFA).

[0056] The detection results are as Figure 5 shown. Compared with the control group without bexarotene, the cells treated with bexarotene were hardly infected with PRRSV, and the proliferation of PRRSV was inhibited, indicating that bexarotene has anti-PRRSV infection activity.

[0057] 5. Plaque assay (PFU)

[0058] Marc-145 cells (or PAMs cells) were cultured in DMEM medium containing 10% fetal bovine serum for about 18 h until the cell density reached 70 - 75%. The culture medium was discarded and replaced with DMEM medium containing 2% fetal bovine serum with 10.0 μM bexarotene. HP-PRRSV was inoculated at a multiplicity of infection (MOI) of 0.5, and a control group without bexarotene and a blank control group were set up. Then, the cells were cultured in an incubator at 37 °C and 5% CO2. After 36 h, the cell supernatant was collected for plaque assay to detect the virus titer.

[0059] The detection results are as Figure 6 shown. Compared with the control group without bexarotene, the number of plaques was significantly reduced, indicating that bexarotene can significantly inhibit PRRSV replication, and the titer of HP-PRRSV was significantly reduced after treatment with bexarotene, indicating that bexarotene has significant antiviral activity.

[0060] Example 3 Broad-spectrum anti-PRRSV effect of bexarotene

[0061] Marc-145 cells (or PAMs cells) were cultured in DMEM medium containing 10% fetal bovine serum for about 18 h until the cell density reached 70-75%. The culture medium was discarded and replaced with DMEM medium containing 2% fetal bovine serum and a serial dilution of bexarotene (diluted with DMEM medium, with dilution concentrations of 5.0, 7.5, and 10.0 μM). Different pathogenic PRRSV strains (WuH3 strain, SD16 strain, TA-12 strain, JXA1 strain, NADC30 strain) were inoculated at a multiplicity of infection (MOI) of 0.5. A control group without bexarotene and a blank control group were set up. Then, the cells were cultured in an incubator at 37°C and 5% CO2. After 36 h, the cells were washed 3 times with PBS, and then digested with 0.25% trypsin. After lysing the cells, the protein concentration was measured, and the expression level of PRRSV-N protein was detected by Western blot.

[0062] The detection results are as Figure 7 shown, indicating that bexarotene has a significant anti-PRRSV effect on different pathogenic PRRSV strains and shows a dose-dependent relationship. At 10 μM, the expression of PRRSV-N protein was hardly detectable. The above results suggest that bexarotene has a broad-spectrum anti-PRRSV effect.

[0063] In summary, the present invention provides a new application of the old drug bexarotene for treating cutaneous T-cell lymphoma, and it is found that bexarotene has an anti-PRRSV effect, can significantly inhibit the proliferation and replication of porcine reproductive and respiratory syndrome virus (PRRSV), and reduce the infectivity of PRRSV. At the same time, it also has a broad-spectrum anti-PRRSV effect, can inhibit the expression of PRRSV-N protein of multiple different pathogenic PRRSV strains, especially has a significant antiviral effect against highly pathogenic HP-PRRSV. And it has almost no cytotoxicity, can be better used to resist PRRSV infection and relieve porcine reproductive and respiratory syndrome. Because bexarotene has a small molecular weight and can easily penetrate the cell membrane into the cell to play an antiviral role, there is no need for a carrier to deliver it into the cell, which lays a foundation for the development of more new drugs against PRRSV and provides more methods for the prevention and treatment of porcine reproductive and respiratory syndrome.

[0064] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. The use of bexarotene in an anti-PRRSV preparation, characterized in that: The structural formula of bexarotene is:

2. The use of bexarotene in the preparation of an anti-PRRSV drug, characterized in that: The structural formula of bexarotene is:

3. The use of bexarotene in the preparation of a medicament for preventing PRRSV infection, characterized in that: The structural formula of bexarotene is:

4. Use of bexarotene in the preparation of a drug for alleviating or treating porcine blue ear disease, characterized in that: The structural formula of bexarotene is:

5. The use according to any one of claims 1 to 4, characterized in that: The drug can inhibit the replication and proliferation of PRRSV.

6. The use according to any one of claims 1 to 4, characterized in that: The drug can inhibit the expression of viral PRRSV-N protein.

7. The use according to any one of claims 1 to 4, characterized in that: The drug is able to reduce viral titers.

8. The use according to claim 4, characterized in that: The porcine blue ear disease is blue ear disease caused by HP-PRRSV or blue ear disease caused by virus variants.

9. The use according to claim 8, characterized in that: The virus variants are WuH3, SD16, TA-12, JXA1 or NADC30.

10. The use according to any one of claims 1 to 4, characterized in that: The medicine contains a pharmaceutically acceptable carrier or excipient.

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