Use of clofazimine in the preparation of a medicament for the prevention and / or treatment of porcine epidemic diarrhea virus infection

Abstract

The application discloses application of clofazimine in preparation of a medicine for preventing and / or treating porcine epidemic diarrhea virus (PEDV) infection. The clofazimine is safe and has small toxic and side effects against the PEDV, provides a new direction for research and development of the medicine for preventing and / or treating the PEDV infection, and has important research significance and application significance for research and development of the medicine for preventing and / or treating the PEDV infection. The clofazimine presents a dose-dependent mode to inhibit replication of the PEDV, and provides a scientific and reliable theoretical basis for clinical treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the use of clofazimine in the preparation of drugs for the prevention and / or treatment of porcine epidemic diarrhea virus infection. Background Technology

[0002] Porcine epidemic diarrhea (PED) is an acute, highly contagious intestinal infectious disease caused by porcine epidemic diarrhea virus (PEDV). PEDV belongs to the genus *Alphacoronavirus* of the family Coronaviridae. Infected piglets exhibit vomiting, watery diarrhea, dehydration, and even death, causing significant economic losses to the pig farming industry. Currently, vaccination remains the primary means of preventing PED, but its protective efficacy is limited. Given that there are currently no approved drugs specifically for treating PEDV, there is a need to develop a drug that can effectively combat PEDV.

[0003] Clofazimine (CFZ), also known as chlorpheniramine, is a drug that inhibits the activity of Mycobacterium tuberculosis and Mycobacterium leprae. Its mechanism of action against Mycobacterium leprae involves interfering with nucleic acid metabolism and inhibiting bacterial protein synthesis. Its action is slower than that of dapsone and it is currently mainly used to treat infectious diseases caused by Mycobacterium leprae resistant to dapsone. Due to its effective treatment of Mycobacterium leprae infections, it was recommended by the WHO in 1981 as part of a combination therapy regimen for leprosy. CFZ significantly interferes with both the energy and nucleic acid metabolism of Mycobacterium tuberculosis. Because CFZ's target and molecular mechanism of action in treating multidrug-resistant and extensively drug-resistant tuberculosis differ from other drugs, it has become an important component of treatment regimens for drug-resistant tuberculosis. Summary of the Invention

[0004] This invention provides the application of clofazimine in the preparation of drugs for the prevention and / or treatment of porcine epidemic diarrhea virus infection, which has important research and application significance for the research and development of drugs for the prevention and / or treatment of porcine epidemic diarrhea virus infection.

[0005] This invention provides the use of clofazimine in the preparation of medicaments for the prevention and / or treatment of porcine epidemic diarrhea virus infection.

[0006] In one embodiment of the invention, clofazimine inhibits the replication of porcine epidemic diarrhea virus in a dose-dependent manner.

[0007] In one embodiment of the present invention, clofazimine inhibits the invasion and replication processes of porcine epidemic diarrhea virus.

[0008] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0009] 1. Clofazimine is effective against swine epidemic diarrhea virus (PEDV). It is safe and has few side effects, providing a new direction for the research and development of drugs for the prevention and / or treatment of PEDV infection. It has important research and application significance for the research and development of drugs for the prevention and / or treatment of PEDV infection.

[0010] 2. Clofazimine inhibits the replication of porcine epidemic diarrhea virus in a dose-dependent manner, providing a scientific and reliable theoretical basis for clinical treatment.

[0011] 3. Clofazimine has a mature preparation process, and therefore has a promising application prospect in the preparation of drugs for the prevention and / or treatment of porcine epidemic diarrhea virus infection.

[0012] Of course, any product implementing this invention does not necessarily need to achieve all of the advantages described above at the same time.

[0013] Instruction manual illustrations

[0014] Figure 1 The cell survival rate curves of Vero cells on different concentrations of chlorfazimine;

[0015] Figure 2 Figure 1 shows the results of detecting the mRNA level of PEDV virus N protein in Vero cells by different concentrations of clofazimine.

[0016] Figure 3 Figure 1 shows the protein level detection results of different concentrations of clofazimine inhibiting the N protein of PEDV virus on Vero cells.

[0017] Figure 4 Figure 1 shows the results of detecting the inhibition of PEDV viral titers by different concentrations of clofazimine on Vero cells.

[0018] Figure 5 Figure 1 shows the results of IFA assay for the inhibition of PEDV virus by different concentrations of clofazimine on Vero cells.

[0019] Figure 6 Figure 1 shows the results of CPE detection of PEDV virus on Vero cells at different concentrations of clofazimine.

[0020] Figure 7 The graph shows the protein level results of different concentrations of clofazimine inhibiting PEDV replication in LLC-PK1 cells;

[0021] Figure 8 The figure shows the results of chlorfazimine detection of PEDV N protein at the mRNA level at different stages. Detailed Implementation

[0022] In this document, the range expressed as "from one value to another" is a concise way of representing a range to avoid listing all the values ​​in that range in the specification. Therefore, the description of a particular range of values ​​covers any value within that range as well as the smaller range of values ​​defined by that value, just as if the arbitrary value and the smaller range of values ​​were explicitly stated in the specification.

[0023] The term “treatment” as used in this article includes the containment, mitigation, termination, reduction, improvement, or reversal of the progression or severity of existing symptoms, conditions, illnesses, or diseases, and treatment may be applied for prevention or therapeutic purposes.

[0024] Previous studies on anti-PEDV drugs have shown that usnic acid exhibits significant toxicity to Vero cells at concentrations above 20 μM, with a minimum inhibitory concentration (MIC) of 2.5 μM. Lysimachia christinae extract at concentrations below 1 mg / mL is not toxic to Vero cells, but exhibits strong inhibitory activity against PEDV at concentrations above 10 μg / mL. Aloe vera extract has a maximum safe concentration of 16 mg / mL for Vero cells, at which concentration 16 mg / mL completely inhibits PEDV proliferation in both Vero and IPEC-J2 cells. In this invention, CFZ exhibits PEDV inhibitory activity, and CC on Vero cells... 50 When the concentration of CFZ is >300μM and 0.625μM or higher, it can effectively inhibit the proliferation of PEDV in Vero cells and LLC-PK1 cells, acting on both the viral invasion and replication stages, and exerting good anti-PEDV activity.

[0025] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Improvements and adjustments made by those skilled in the art based on the present invention in practical applications still fall within the scope of protection of the present invention.

[0026] Experimental materials:

[0027] Chlorfazimine was purchased from Shanghai E. En Chemical Technology Co., Ltd.

[0028] Dimethyl sulfoxide was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.

[0029] Vero cells are preserved in the Shanghai Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai Jiao Tong University.

[0030] LLC-PK1 cells were provided by the Shanghai Veterinary Research Institute of the Chinese Academy of Agricultural Sciences.

[0031] The CCK8 test kit was purchased from Shanghai Taoshu Biotechnology Co., Ltd.

[0032] Solution preparation: Dissolve clofazimine (CFZ) powder in dimethyl sulfoxide (DMSO) solution to a final concentration of 20 mM, then filter through a 0.22 μm sterile filter to prepare CFZ stock solution, and store at -80℃ for later use.

[0033] Example 1

[0034] The toxicity test of clofazimine on Vero cells was conducted as follows:

[0035] Vero cells were seeded in 96-well cell culture plates and cultured at 37°C with 5% CO2. After the cells grew into a monolayer, they were treated with CFZ at concentrations of 0.16 μM, 0.31 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM, 160 μM, and 320 μM. Three replicates were set up for each concentration of the drug, and the cells were incubated at 37°C for 24 h. A DMSO negative control and a cell-free well containing only culture medium were also set up. Cell viability was detected using a CCK8 assay kit. The cells were incubated at 37°C with 5% CO2 for 2–3 h, and the absorbance at 450 nm was measured using an INESA-722N spectrophotometer.

[0036] Test results as follows Figure 1 As shown, in the cytotoxicity experiment of clofazimine against Vero cells, the average survival rate of Vero cells fluctuated around 100% at concentrations ranging from 0.16 to 320 μM. Furthermore, by plotting cell survival curves at different concentrations, the formula (A) was used to... (加药) -A (培养基) ) / (A (对照组) -A (培养基) )×100% of which (A (加药) The absorbance of the experimental wells (containing cells, culture medium, CCK-8 solution, and drug solution), A (对照组) The absorbance of the control well (containing cells, culture medium, and CCK-8 solution, but without drugs) is A. (培养基) Calculate the CLC50 of clofazimine on Vero cells based on the absorbance of the blank wells (containing culture medium and CCK-8 solution, but excluding cells and drugs). 50 >300μM.

[0037] Example 2

[0038] The application of CFZ in combating porcine epidemic diarrhea virus (PEDV) shows that CFZ inhibits PEDV replication in a dose-dependent manner.

[0039] Vero and LLC-PK1 cells were passaged into 6-well plates. After the cells reached a confluent monolayer, the culture medium was discarded, and the cells were washed twice with PBS. 1 mL of viral infection medium (MOI = 0.1) was added to each well, and the plates were incubated at 37°C for 1 hour. This ensured the virus completed its adsorption and invasion process. The viral solution was then discarded, and the cells were washed twice with PBS. The plates were then replaced with infection medium and cultured for another hour. Simultaneously, different concentrations of CFZ (CFZ diluted with DMSO to 0.16 μM, 0.31 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, 15 μM, and 20 μM) were added to each well. A DMSO negative control group was also included. The plates were then incubated at 37°C for another 10 hours.

[0040] Viral infection status was determined by observing cytopathic effects, IFA, viral titer, viral N protein expression level, and N protein mRNA level.

[0041] Test results as follows Figures 2-7 As shown, after CFZ treatment, Vero cells exhibited significantly lower viral N protein expression levels, N protein mRNA levels, and progeny virus titers compared to the DMSO control group. CPE results showed a significant reduction in cytopathic effects in the CFZ-treated group. Indirect immunofluorescence results indicated a significant dose-dependent decrease in green fluorescence in the CFZ-treated group. LLC-PK1 cells also showed a significant dose-dependent decrease in viral N protein expression after CFZ treatment. Figures 2-7 It can be seen that CFZ can significantly inhibit PEDV activity.

[0042] Example 3:

[0043] The application of CFZ in combating porcine epidemic diarrhea virus: CFZ plays an antiviral role in the virus's invasion and replication process.

[0044] To analyze the inhibitory effect of CFZ on PEDV infection at which stage of cell infection, Vero cells were seeded into 6-well plates. After the cells grew into a monolayer, the culture medium was discarded, and the cells were washed twice with PBS. CFZ at a concentration of 20 μM was added at 16 h before infection, during infection, from during infection to 1 h after infection, from during infection to 10 h after infection, 1 h after infection, 2 h after infection, and 5 h after infection to infect cells with PEDV at a concentration of 0.1 MOI. Cells were then incubated at 37°C in a 5% CO2 incubator for 10 hours after inoculation (one viral life cycle). A DMSO negative control was also included. Cells were harvested 10 h after inoculation, and cellular RNA was extracted and quantitatively analyzed using fluorescence to detect the expression level of viral N protein mRNA.

[0045] Test results as follows Figure 8As shown, when CFZ was added 16 hours before infection, during infection to 1 hour after infection, during infection to 10 hours after infection, 1 hour after infection, 2 hours after infection, and 5 hours after infection, the mRNA level of viral N protein was lower than that in the control group. Treatment with CFZ during inoculation strongly inhibited PEDV infection, indicating that CFZ can inhibit viral entry. CFZ also blocked PEDV infection after entry, as reduced viral replication was observed when CFZ was added 5 hours after infection, indicating that CFZ plays a role in the viral adsorption, invasion, and replication phases.

[0046] The above description discloses only preferred embodiments of the present invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to make good use of the invention. The present invention is limited only by the claims and their full scope and equivalents.

[0047] Under the guidance of the present invention and the above embodiments, those skilled in the art will readily foresee that all the raw materials or their equivalents, processing methods or their equivalents listed or exemplified in the present invention can achieve the present invention, and that the upper and lower limits and range values ​​of the parameters of each raw material and processing method can also achieve the present invention. Examples are not listed one by one here.

Claims

1. Use of clofazimine for the manufacture of a medicament for the prevention and / or treatment of porcine epidemic diarrhea virus infection.

2. The use of clofazimine according to claim 1 in the preparation of a medicament for the prevention and / or treatment of porcine epidemic diarrhea virus infection, characterized in that, Clofazimine inhibits the entry process and the replication process of porcine epidemic diarrhea virus.

3. Use of clofazimine according to claim 1 for the manufacture of a medicament for the prevention and / or treatment of porcine epidemic diarrhea virus infection, characterized in that, Clofazimine inhibits the replication of porcine epidemic diarrhea virus in a dose-dependent manner.

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