CN115006386B - Application of nifuruzide in the preparation of drugs against tick-borne encephalitis virus, West Nile virus, yellow fever virus and chikungunya virus infection - Google Patents

Abstract

The present invention relates to the field of medical technology, and relates to the use of nifuruzide in preparing anti-tick-borne encephalitis virus (TBEV), West Nile virus (WNV), yellow fever virus (YFV) and chikungunya virus (CHIKV) infection drugs. applications in. The anti-TBEV, WNV, YFV and CHIKV infection medicine is a medicine with nifuruzide as the only active ingredient, or a pharmaceutical composition containing nifuruzide, for preventing or treating TBEV, WNV, YFV and CHIKV infection. Using the experimental operation system of TBEV-susceptible cells, we screened small molecule drugs that can inhibit infection from the clinical drug small molecule library, and found that nifuruzide can effectively inhibit TBEV infection of human liver cancer cell Huh7 with low cytotoxicity, and It has inhibitory effects on WNV, YFV, and CHIKV, and can be used as a potential anti-TBEV, WNV, YFV, and CHIKV drug, and has application prospects.

Description

Nifurazide in preparation against tick-borne encephalitis virus, West Nile virus, yellow fever virus and chitosan Application in drugs for Kenya fever virus infection

Technical field

The invention belongs to the field of medical technology, and specifically relates to the application of nifuruzide in the preparation of medicines against tick-borne encephalitis virus, West Nile virus, yellow fever virus and chikungunya virus infection.

Background technique

Nifuroxazide (NFX) is an oral nitrofuran antibiotic, mainly used to prevent and treat bacillary dysentery and enteritis. In recent years, studies have found that nifuruzide has potential anti-tumor effects and has anti-tumor cell proliferation and metastasis effects on liver cancer, breast cancer, multiple myeloma, and melanoma. An important target of nifuruzide's anti-tumor effect is signal transducer and activator of transcription 3 (STAT3). NFX inhibits the phosphorylation of STAT3 by inhibiting the JAK family kinases JAK2 and Tyk2, thereby inhibiting STAT3 activity.

Tick-borne encephalitis virus (TBEV), also known as forest encephalitis virus, belongs to the genus Flavivirus of the family Flaviviridae. Tick-borne encephalitis (TBE) is a natural focal disease caused by TBEV and mainly characterized by central nervous system lesions. The main way humans are infected with the virus is through bites from ticks carrying TBEV, and they can also be infected by ingesting TBEV-contaminated dairy products. The distribution of TBE has obvious regional characteristics and is closely related to the communication media. There are currently no specific treatments for TBE.

West Nile virus (WNV) belongs to the genus Flaviviridae of the family Flaviviridae. It is a single-stranded positive-sense RNA virus that can cause West Nile fever and West Nile virus meningoencephalitis in humans and animals. Culex mosquitoes are the main virus. Birds are the intermediate hosts of the virus, while humans and horses are the terminal hosts. There is currently no human vaccine or treatment for WNV infection.

Yellow fever virus (YFV) is an arbovirus vectored by Aedes mosquitoes. It belongs to the genus Flaviviridae of the family Flaviviridae along with Zika virus (ZIKV), West Nile virus (WNV) and dengue virus (DENV). Mosquitoes are the vector of transmission. Yellow fever virus can cause yellow fever, which seriously endangers human health, manifesting as jaundice, bleeding, and even multi-system organ failure. There is currently no specific treatment for YFV infection.

Chikungunya fever virus (CHIKV) belongs to the genus Alphavirus in the family Membranoviridae. It is a single-stranded positive-sense RNA virus that can cause chikungunya fever. Although the fatality rate is not high, the number of infected people is very high. labor force, resulting in serious social impact and economic losses. CHIKV is a mosquito-borne virus primarily transmitted to humans by Aedes aegypti and Aedes albopictus mosquitoes. There is currently no vaccine or specific treatment for CHIKV infection.

Contents of the invention

The present invention aims to provide a new use of nifuruzide.

The invention provides the application of nifuruzide in the preparation of medicines against tick-borne encephalitis virus, West Nile virus, yellow fever virus and chikungunya virus infection.

The chemical structural formula of nifuruzide is as follows:

The application of the present invention is characterized in that: the above-mentioned anti-tick-borne encephalitis virus, West Nile virus, yellow fever virus and chikungunya virus infection medicine uses nifuruzide as the only active ingredient, or contains nitrile Pharmaceutical compositions of fruzide.

The application involved in the present invention is characterized in that: the above-mentioned pharmaceutical composition containing nifuruzide refers to a pharmaceutical composition composed of nifuruzide and one or more pharmaceutically acceptable excipients.

The application of the present invention is characterized in that the above-mentioned medicine is used to prevent or treat infections by tick-borne encephalitis virus, West Nile virus, yellow fever virus and chikungunya virus.

The application involved in the present invention is characterized in that: the content of nifuruzide in the above-mentioned medicine against tick-borne encephalitis virus, West Nile virus, yellow fever virus and chikungunya virus infection is 0.1 to 99wt%.

The application involved in the present invention is characterized in that the dosage form of the above-mentioned medicine is selected from the group consisting of powder, tablet, granule, capsule, solution, emulsion and suspension.

The application involved in the present invention is characterized in that the dosage form of the above-mentioned drug administration route is selected from the group consisting of injection dosage form, gastrointestinal dosage form, and skin dosage form.

Functions and effects of the present invention:

The present invention uses an experimental operation system for TBEV to infect susceptible cells, and screens candidate small molecule drugs that can inhibit TBEV infection from a clinically approved drug small molecule library. It is screened out that nifuruzide can effectively inhibit TBEV infection of human liver cancer cell Huh7. It has low cytotoxicity and has inhibitory effects on WNV, YFV, and CHIKV. It can be used as a potential anti-TBEV, WNV, YFV, and CHIKV drug and has application prospects.

Description of the drawings

Figure 1. The effect of nifuruzide in protecting BHK cells against tick-borne encephalitis virus infection;

That is, BHK cells were infected with tick-borne encephalitis virus, and nifuruzide or the solvent DMSO was added at the same time, or they were not infected with tick-borne encephalitis virus (no virus was added). After 72 hours, CCK8 reagent was added to detect the absorbance value at 450nm.

Figure 2. Toxicity of nifuruzide to Huh7 cells;

That is, Huh7 cells were treated with nifuruzide and the solvent DMSO respectively. After 72 hours, CCK8 reagent was added to detect the absorbance value at 450nm.

Figure 3. Inhibitory effect of nifuruzide on tick-borne encephalitis virus in Huh7 cell infection model.

Figure 4. The effect of nifuruzide in inhibiting tick-borne encephalitis virus, West Nile virus, yellow fever virus, and chikungunya virus infection.

Detailed ways

In order to illustrate the present invention more clearly, the present invention will be further described below with reference to preferred embodiments. Those skilled in the art should understand that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of the present invention.

Nifurozide used in the embodiments of the present invention can be purchased commercially.

1. Viruses, drugs, reagents and other materials

1. Viruses: Tick-borne encephalitis virus and yellow fever virus were isolated, amplified and cultured by the Biomedical Protection Teaching and Research Office of the Naval Medical University of the Chinese People's Liberation Army. West Nile virus and chikungunya virus were synthesized using reverse genetics technology and were produced by Expansion and culture of baby hamster kidney BHK cells. All experimental operations involving viral infection were performed in the P3 laboratory of the Naval Medical University.

2. Compounds: 978 U.S. FDA chemical drug molecule libraries, purchased from Selleck Company in the United States.

3. Human liver cancer cell line Huh7 and baby hamster kidney BHK cells were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences and preserved by the Biomedical Protection Teaching and Research Office of the Naval Medical University of the Chinese People's Liberation Army.

4. The DMEM cell culture medium is a product of Hyclone Company of the United States. When used, 10% fetal bovine serum, non-essential amino acids, ampicillin and streptomycin (100 U/ml each) are added. The culture medium additives are all products of Thermo Fisher Company of the United States.

5. Cell digestion solution, containing 0.25% trypsin, prepared with phosphate buffer saline.

6. CCK8 cell activity and proliferation detection kit is a product of MedChemExpress Company of the United States.

7. Mouse tick-borne encephalitis virus polyclonal antibodies, West Nile virus polyclonal antibodies, yellow fever virus polyclonal antibodies, and chikungunya virus polyclonal antibodies were obtained from the Biomedical Protection Teaching and Research Office of the Naval Medical University of the Chinese People's Liberation Army using formaldehyde. Prepared by immunizing mice with inactivated tick-borne encephalitis virus.

8. Alexa Fluor 488-labeled anti-mouse IgG is a product of Thermo Fisher Company of the United States.

2. Experimental methods:

(1) Screening anti-tick-borne encephalitis virus drugs from the FDA drug small molecule library containing 978 small molecule compounds

Use complete DMEM culture medium to subculture baby hamster kidney BHK cells in a T75 cell culture flask, inoculate them on a 96-well plate, 10,000 cells per well, and culture for 12 hours in 100 μL of DMEM culture medium. Then, 50 μL of complete DMEM culture medium containing 1000 PFU (plaque forming units) of tick-borne encephalitis virus was added to each well; at the same time, 50 μL of complete DMEM culture medium containing FDA small molecule chemical drugs was added. The final concentration of the drug was 10 μM. Each concentration was repeated 3 times. Wells, add an equal volume of solvent DMSO as a control without adding drugs. Place it in a 37°C, 5% _CO2 incubator for culture. After 72 hours, it can be seen under the microscope that all cells in the DMSO-added wells have become round or fallen off. Add 10 μL of CCK8 cell activity and proliferation detection reagent to each well and place it in a 37°C, 5% CO ₂ incubator. After 30 minutes, use a multifunctional microplate reader to detect the absorbance value of each well at a wavelength of 450 nm to calculate the protection of cells by the drug. Rate = (absorbance value at 450nm of cells with drug added - absorbance value at 450nm of cells in DMSO wells)/absorbance value at 450nm of cells without virus and DMSO added * 100%. The protection of cells infected by tick-borne encephalitis virus at a concentration of 10 μM for each drug was obtained. Rate. The results showed that nifuruzide had a significant protective effect on cells. The 450nm absorbance values of the two antibiotics and DSMO-treated cells are shown in Figure 1. The calculated cell protection rates of nifuruzide were 89.7% respectively.

(2) Toxicity of nifuruzide to cells

The cultured human liver cancer cell line Huh7 and baby hamster kidney BHK cells were seeded in a 96-well plate, with 10,000 cells per well and 100 μL of culture medium. After 12 hours, the original culture medium was aspirated, and a concentration gradient dilution of nitrate was added to each well. 100 μL of fruzide's complete DMEM culture medium. The final concentrations of nifuruzide were 2.5, 5, 10, 20, 40 and 80 μM. Each concentration was repeated in 3 wells. The solvent DMSO content of 80 μM drug was used as a control without adding drug. . Place it in a 37°C, 5% _CO2 incubator for culture. After 48 hours, add 10 μL of CCK8 cell activity and proliferation detection reagent to each well and place it in a 37°C, 5% CO ₂ incubator. After 30 minutes, use a multifunctional microplate reader to detect the absorbance value of each well at a wavelength of 450 nm. According to The difference in absorbance value at 450nm between wells treated with drugs at different concentrations and wells treated with solvents was used to evaluate the cytotoxicity of the drug.

The results showed that when the concentration was equal to or lower than 80 μM, there was no significant difference between the two types of cells treated with nifuruzide and the cells treated with DMSO solvent. Figure 2 shows the absorbance at 450 nm of Huh7 cells treated with nifuruzide and cells treated with DMSO solvent at 80 μM.

(3) Inhibitory effect of nifuruzide on tick-borne encephalitis virus in cell infection model

The subcultured human liver cancer cell line Huh7 was inoculated into a 96-well plate, with 10,000 cells per well and 100 μL of culture medium, and cultured for 12 hours. Then, 50 μL of complete DMEM culture medium containing 1000 PFU of tick-borne encephalitis virus was added to each well; at the same time, 50 μL of complete DMEM culture medium containing nifuruzide was added. The final concentration of the drug was 5 μM. Repeat 3 wells for each concentration to add an equal volume of solvent. DMSO was used as a control without adding drugs and was cultured in a 37°C, 5% _CO2 incubator.

After 20 hours, use immunofluorescence technology to detect the infection of the cells by the virus. The specific operations are as follows: Aspirate the culture medium in the culture plate, add 100 μL methanol to each well, put the culture plate in a -20°C refrigerator, and take out the culture after 20 minutes. plate, aspirate the methanol, wash each well once with phosphate buffer saline (PBS), then add 100 μL of PBS containing 3% bovine serum albumin (BSA) (hereinafter referred to as 3% BSA-PBS), and place it on a horizontal shaker , shake slowly at room temperature for 1 hour, aspirate the 3% BSA-PBS in the culture plate, add 100 μL of 1% BSA-PBS containing anti-tick-borne encephalitis virus polyclonal antibody to each well (500-fold dilution of the antibody), and shake slowly at room temperature for 1 hour hour, aspirate the anti-tick-borne encephalitis virus polyclonal antibody working solution in the culture plate, wash each well three times with PBS, and then add 100 μL of 1% BSA-PBS containing Alexa Fluor488-labeled anti-mouse IgG ( Fluorescein antibody 1500 times dilution), shake slowly at room temperature for 1 hour in the dark, and aspirate the fluorescein antibody working solution in the culture plate. Add 100 μL of DAPI nuclear staining solution to each well, shake slowly at room temperature for 10 minutes in the dark, and aspirate the fluorescein antibody working solution in the culture plate. DAPI cell nuclear staining solution, wash each well three times with PBS, and use a cell imaging and analysis system (BioTek Cytation 5Imaging Reader) to take pictures of the fluorescence distribution of cells in each well. Take pictures of four fields of view in each well, analyze and calculate the percentage of green fluorescence-positive cells, that is, the virus infection rate.

The results are shown in Figure 3. Compared with the wells treated with DMSO solvent as a control, the virus infection rate of nifuruzide treatment was significantly reduced. The results demonstrated that at a concentration of 5 μM, nifuruzide could significantly inhibit the infection of Huh7 cells by tick-borne encephalitis virus.

(4) Quantitative determination of the activity of nifuruzide in inhibiting tick-borne encephalitis virus, West Nile virus, yellow fever virus, and chikungunya virus infection

In order to observe whether nifuruzide has anti-inhibitory activity against tick-borne encephalitis virus, West Nile virus, yellow fever virus, and chikungunya virus, we further tested nifuruzide against tick-borne encephalitis virus, tick-borne encephalitis virus, and chikungunya virus. Quantitative detection of the activity of West Nile virus, yellow fever virus, and chikungunya virus, that is, by detecting the antiviral activity at different concentrations, the half inhibitory concentration (IC50) of the drug against the virus is calculated. Four concentration gradients of 0.2, 1, 5, and 25 μM were set for the drug with five consecutive dilutions. The detection method is the same as described in (3) except that the concentration gradient is set for drug treatment. After the virus-infected cells are combined with viral antibodies and fluorescent antibodies and the nuclei are stained, the cells are photographed using a cell imaging and analysis system. Four fields of view are photographed in each well, and the percentage of green fluorescence-positive cells, that is, the virus infection rate, is analyzed and calculated. The percentage of positive cells in the wells treated with concentration gradient drugs was used to calculate the IC50 of nifuruzide against tick-borne encephalitis virus, West Nile virus, yellow fever virus, and chikungunya virus; IC50 for tick-borne encephalitis virus: 3.56 μM ; West Nile virus IC50: 4.13μM; Yellow fever virus IC50: 4.32μM; Chikungunya virus IC50: 5.08μM.

The above-mentioned in vitro and in vivo experimental results show that nifuruzide has significant activity against tick-borne encephalitis virus, West Nile virus, yellow fever virus, and chikungunya virus infection, and can be used to prepare anti-tick-borne encephalitis virus , West Nile virus, yellow fever virus, chikungunya virus infection drugs.

The main features of the invention and the advantages of the invention have been shown and described above. Those skilled in the industry should understand that the present invention is not limited by the above embodiments. The above embodiments and descriptions only illustrate the principles of the present invention. The present invention may have various modifications without departing from the spirit and scope of the present invention. These changes and improvements all fall within the scope of the claimed invention. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims (7)

1. Application of nifurazite in preparing medicines for resisting tick-borne encephalitis virus, west nile virus, yellow fever virus and chikungunya fever virus infection is provided.

2. The use according to claim 1, characterized in that: the tick-borne encephalitis virus, west nile virus, yellow fever virus and chikungunya fever virus infection resistant medicament is a medicament composition which takes nifurazite as the only active ingredient or contains nifurazite.

3. The use according to claim 2, characterized in that: the pharmaceutical composition containing nifurazite refers to a pharmaceutical composition composed of nifurazite and one or more pharmaceutically acceptable auxiliary materials.

4. A use according to any one of claims 1-3, characterized in that: the medicament is used for preventing or treating tick-borne encephalitis virus, west nile virus, yellow fever virus and chikungunya fever virus infection.

5. A use according to any one of claims 1-3, characterized in that: the content of nifurazide in the tick-borne encephalitis virus, west nile virus, yellow fever virus and chikungunya fever virus infection resistant medicament is 0.1-99 wt%.

6. A use according to any one of claims 1-3, characterized in that: the drug is administered in the form of powder, tablet, granule, capsule, solution, emulsion, or suspension.

7. A use according to any one of claims 1-3, characterized in that: the administration route of the medicine is selected from injection administration form, gastrointestinal tract administration form and skin administration form.