CN115006386B - Application of nifurazite in preparation of medicines for resisting tick-borne encephalitis virus west nile virus yellow fever virus and chikungunya fever virus infection - Google Patents
Application of nifurazite in preparation of medicines for resisting tick-borne encephalitis virus west nile virus yellow fever virus and chikungunya fever virus infection Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
- A61K31/345—Nitrofurans
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention relates to the technical field of medicines, in particular to application of nifurazite in preparing medicines for resisting tick-borne encephalitis virus (TBEV), west Nile Virus (WNV), yellow Fever Virus (YFV) and chikungunya fever virus (CHIKV) infection. The anti TBEV, WNV, YFV and CHIKV infection medicines are medicines for preventing or treating TBEV, WNV, YFV and CHIKV infection by taking nifurazite as the only active ingredient or a medicine composition containing nifurazite. By utilizing an experimental operation system of TBEV susceptible cells, small-molecule drugs capable of inhibiting infection are screened from a clinical drug small-molecule library, and nifurazimine can effectively inhibit TBEV from infecting human liver cancer cells Huh7, has low cytotoxicity and inhibition effect on WNV, YFV, CHIKV, can be used as potential anti-TBEV, WNV, YFV, CHIKV drugs, and has application prospects.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to application of nifurazite in preparation of medicines for resisting tick-borne encephalitis virus, west nile virus, yellow fever virus and chikungunya fever virus infection.
Background
Nifurazide (NFX) is an oral nitrofuran antibiotic and is mainly used for preventing and treating bacillary dysentery and enteritis. In recent years, the research discovers that nifurazimine has potential anti-tumor effect and anti-tumor cell proliferation and metastasis effect on liver cancer, breast cancer, multiple myeloma and melanoma. An important target for nifurazite to exert an anti-tumor effect is signal transduction and transcriptional activator 3 (Signal transducer and activator oftranscription, STAT 3), NFX inhibits phosphorylation of STAT3 by inhibiting JAK family kinases JAK2 and Tyk2, thereby inhibiting STAT3 activity.
Tick borne encephalitis virus (Tick-borne encephalitis virus, TBEV), also known as forest encephalitis virus, is a flaviviridae genus of flaviviridae. Tick Borne Encephalitis (TBE) is a natural epidemic disease caused by TBEV and characterized mainly by central nervous system lesions. The main route of human infection with viruses is by biting with ticks carrying TBEVs, and also by ingestion of TBEV-contaminated milk products. The distribution of TBE has obvious locality and is closely related to the propagation medium. No TBE specific therapeutic drug exists at present.
West Nile Virus (WNV) belongs to the genus Flaviviridae, a single-stranded positive strand RNA virus that causes West Nile fever and West Nile virus meningoepithymen encephalitis in humans and animals, culex is the main transmission medium, birds are the intermediate hosts of the virus, and humans and horses are the end hosts. There is currently no vaccine or therapeutic for humans with WNV infection.
Yellow fever virus (Yellow fevervirus, YFV) is an insect-borne virus mediated by aedes, and belongs to the flaviviridae genus, such as the species zaka virus (ZIKV), west Nile Virus (WNV), and dengue virus (DENV), all of which are mosquito-mediated. Yellow fever virus can cause yellow fever that is a serious hazard to human health, manifested as jaundice, bleeding, and even multiple organ failure. There is currently no specific therapeutic drug against YFV infection.
Chikungunya fever virus (Chikungunya fevervirus, CHIKV) belongs to the genus chikungunya of the family of the membrane viruses, is a single-stranded positive strand RNA virus, can cause chikungunya fever, and has low mortality but causes serious social and economic losses due to loss of labor force of infected people. CHIKV is a mosquito vector virus that is transmitted to humans mainly by aedes aegypti and aedes albopictus. Currently, there is no vaccine or specific therapeutic against CHIKV infection.
Disclosure of Invention
the invention aims to provide a new application of nifurazite.
the invention provides an application of nifurazite in preparing medicines for resisting tick-borne encephalitis virus, west nile virus, yellow fever virus and chikungunya fever virus infection.
the chemical structural formula of nifurazite is as follows:
The application related to the invention is characterized in that: the tick-borne encephalitis virus, west nile virus, yellow fever virus and chikungunya fever virus infection medicament is a medicament composition which takes nifurazite as the only active ingredient or contains nifurazite.
The application related to the invention is characterized in that: the pharmaceutical composition containing nifurazite refers to a pharmaceutical composition composed of nifurazite and one or more pharmaceutically acceptable auxiliary materials.
The application related to the invention is characterized in that: the medicine can be used for preventing or treating tick-borne encephalitis virus, west nile virus, yellow fever virus and chikungunya fever virus infection.
The application related to the invention is characterized in that: the content of nifurazide in the tick-borne encephalitis virus, the west nile virus, the yellow fever virus and the chikungunya fever virus infection medicament is 0.1-99 wt%.
the application related to the invention is characterized in that: the medicine is selected from powder, tablet, granule, capsule, solution, emulsion, and suspension.
The application related to the invention is characterized in that: the administration route of the above drugs is selected from injection administration, gastrointestinal tract administration, and skin administration.
The invention has the following functions and effects:
According to the invention, a candidate small molecule drug capable of inhibiting TBEV infection is screened from a clinically approved drug small molecule library by utilizing an experimental operation system of TBEV infected susceptible cells, and nifurazidine is screened out, so that TBEV can be effectively inhibited from infecting human liver cancer cells Huh7, has low cytotoxicity, has inhibition effect on WNV, YFV, CHIKV, can be used as a potential anti-TBEV, WNV, YFV, CHIKV drug, and has application prospect.
Drawings
FIG. 1. The effect of nifurazidine on protecting BHK cells against tick-borne encephalitis virus infection;
I.e., BHK cells were infected with tick-borne encephalitis virus, with the addition of nifurazite or solvent DMSO, or without tick-borne encephalitis virus (no virus added), and after 72 hours, CCK8 reagent was added to detect absorbance at 450 nm.
FIG. 2 toxicity of nifurazidine on Huh7 cells;
i.e. nifurazit and solvent DMSO were used to treat Huh7 cells separately, and after 72 hours CCK8 reagent was added to detect absorbance at 450 nm.
FIG. 3 inhibition of tick-borne encephalitis virus by nifurazidine in a model of Huh7 cell infection.
figure 4 effects of nifurazite on inhibition of tick-borne encephalitis virus, west nile virus, yellow fever virus, chikungunya virus infection.
Detailed Description
In order to more clearly illustrate the present invention, the present invention will be further described with reference to preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and that this invention is not limited to the details given herein.
nifurazimine used in the embodiments of the present invention may be purchased commercially.
1. Viruses, drugs, agents, and other materials
1. Virus: tick-borne encephalitis virus and yellow fever virus are separated and amplified and cultured by a biomedical protection teaching and research room of the naval university of Chinese people's liberation army, west Nile virus and chikungunya fever virus are synthesized by reverse genetics technology, and are amplified and cultured by baby hamster kidney BHK cells. All experimental procedures involving viral infection were performed in the navy university of medical university P3 laboratory.
2. A compound: 978 U.S. FDA chemical drug molecular libraries were purchased from Selleck, inc.
3. Human liver cancer cell lines Huh7 and baby hamster kidney BHK cells are purchased from Shanghai cell institute of China academy of sciences and stored by biomedical protection textroom of the navy university of the liberation army of Chinese people.
The DMEM cell culture fluid is a product of Hyclone company in the United states, 10% of fetal calf serum, non-essential amino acids, ampicillin and streptomycin (100U/ml each) are added when the DMEM cell culture fluid is used, and the culture fluid additives are all products of Thermo Fisher company in the United states.
5. cell digests, containing 0.25% trypsin, were prepared with phosphate buffer.
The CCK8 cell activity and proliferation detection kit is manufactured by MedChemexpress company of America.
7. the mouse tick-borne encephalitis virus polyclonal antibody, the West Nile virus polyclonal antibody, the yellow fever virus polyclonal antibody and the chikungunya fever virus polyclonal antibody are prepared by immunizing mice with formaldehyde-inactivated tick-borne encephalitis virus in a biomedical protection teaching laboratory of the university of army of navy of the Chinese people liberation army.
8. Fluorescein Alexa Fluor 488-labeled anti-mouse IgG is a product of Thermo Fisher, inc. of America.
2. The experimental method comprises the following steps:
Screening anti-tick borne encephalitis virus drugs from FDA drug small molecule library containing 978 small molecule compounds
Baby hamster kidney BHK cells were subcultured in T75 cell culture flasks with complete DMEM medium, inoculated in 96-well plates with 10000 cells per well, 100. Mu.L of DMEM medium, and cultured for 12 hours. Subsequently 50. Mu.L of complete DMEM medium containing 1000PFU (plaque forming units) tick-borne encephalitis virus was added to each well; simultaneously, 50 mu L of complete DMEM culture solution containing FDA small molecule chemical drugs is added, the final concentration of the drugs is 10 mu M, 3 holes are repeated for each concentration, and an equal volume of solvent DMSO is added to serve as a control without adding the drugs. Placing at 37deg.C and 5% CO2Culturing in incubator. After 72 hours, all cells in the DMSO-added wells were seen to round or shed under the microscope. 10 mu L of CCK8 cell activity and proliferation detection reagent is added into each hole, and the mixture is placed at 37 ℃ and 5% CO2And detecting the light absorption value of each hole at the wavelength of 450nm by using a multifunctional enzyme-labeled instrument after 30 minutes in the incubator, and calculating the protection rate of the medicines to cells= (the light absorption value of 450nm of the medicine-the light absorption value of 450nm of the DMSO hole cells)/the light absorption value of 450nm of the cells without adding viruses and DMSO (the concentration of 10 mu M) to obtain the protection rate of each medicine to tick-borne encephalitis virus infected cells. The results show that nifurazidine has remarkable protection effect on cells, the light absorption value of two antibiotics and DSMO treated cells at 450nm is shown in figure 1, and the calculated cytoprotection rates of nifurazidine are 89.7% respectively.
Toxicity of nifurazide to cells
Cultured human liver cancer cell lines Huh7 and baby hamster kidney BHK cells are respectively inoculated in a 96-well plate, 10000 cells are respectively arranged in each well, 100 mu L of culture solution is absorbed after 12 hours, 100 mu L of complete DMEM culture solution with concentration gradient diluted nifurazimine is added in each well, the final concentration of nifurazimine is respectively 2.5, 5, 10, 20, 40 and 80 mu M, 3 wells are repeated for each concentration, and the solvent DMSO content of 80 mu M of the drug is used as a control without adding the drug. Placing at 37deg.C and 5% CO2Culturing in incubator. After 48 hours, 10. Mu.L of CCK8 cell activity and proliferation detection reagent was added to each well, and the mixture was placed at 37℃in 5% CO2In the incubator, detecting the light absorption value of each hole to 450nm wavelength by using a multifunctional enzyme-labeled instrument after 30 minutes, and evaluating the cytotoxicity of the medicine according to the difference of the light absorption values of the medicine treatment holes with different concentrations and the solvent holes.
The results showed that at concentrations equal to or below 80 μm, there was no significant difference between nifurazidine treated cells and DMSO solvent treated cells. FIG. 2 shows the absorbance at 80. Mu.M of nifurazit-treated Huh7 cells versus DMSO-solvent-treated cells at 450 nm.
Inhibition of tick-borne encephalitis virus by nifurazidine in cell infection model
the subcultured human liver cancer cell line Huh7 was inoculated into 96-well plates with 10000 cells per well, 100. Mu.L of the culture medium, and cultured for 12 hours. Subsequently 50. Mu.L of complete DMEM medium containing 1000PFU tick-borne encephalitis virus was added to each well; simultaneously adding 50 μl of complete DMEM culture solution containing nifurazite, 5 μm final concentration of the drug, repeating 3 holes for each concentration, adding equal volume of solvent DMSO as control without drug, and placing at 37deg.C and 5% CO2Culturing in incubator.
After 20 hours, the infection of the cells by the virus is detected by immunofluorescence technique, and the specific operation is as follows: the culture medium in the culture plate was aspirated, 100. Mu.L of methanol was added to each well, the culture plate was placed in a-20deg.C refrigerator, after 20 minutes, the culture plate was removed, the methanol was aspirated, each well was washed with Phosphate Buffer (PBS) once, then 100. Mu.L of PBS containing 3% Bovine Serum Albumin (BSA) (hereinafter abbreviated as 3% BSA-PBS) was added, the plate was placed on a horizontal shaking table, the plate was slowly shaken at room temperature for 1 hour, 100. Mu.L of 1% BSA-PBS containing anti-tick-encephalitis virus polyclonal antibody was added to each well, the plate was slowly shaken at room temperature for 1 hour, each well was washed 3 times with PBS, then 100. Mu.L of 1% BSA-PBS containing fluorescein Alexa Fluor 488-labeled anti-mouse IgG was added (1500 times with fluorescein antibody), the plate was slowly shaken at room temperature for 1 hour, the plate was blotted with fluorescein antibody working solution, 100. Mu.L of DAP nuclear staining solution was added to each well, the plate was slowly shaken at room temperature for 10 minutes, and the plate was subjected to image analysis by a fluorescent Imaging system for 5 times of fluorescent cell staining by shaking, and the plate was subjected to image analysis by using the image Reader per well. Four fields were photographed per well and the percentage of green fluorescent positive cells, i.e. the viral infection rate, was analyzed and calculated.
The results are shown in figure 3, which shows a significant decrease in the rate of nifurazidine-treated virus infection compared to DMSO solvent-treated wells as controls. The results demonstrate that nifurazidine significantly inhibited infection of Huh7 cells with tick-borne encephalitis virus at a concentration of 5 μm.
Quantitative determination of the infection Activity of nifurazite in inhibiting tick-borne encephalitis virus, west Nile virus, yellow fever virus and chikungunya fever virus
In order to observe whether nifurazite has anti-inhibitory activity on tick-borne encephalitis virus, west nile virus, yellow fever virus and chikungunya fever virus, we further quantitatively detected the activity of nifurazite against tick-borne encephalitis virus, west nile virus, yellow fever virus and chikungunya fever virus, namely by detecting the antiviral activity at different concentrations, the half inhibitory concentration (IC 50) of the drug on the virus is calculated. The drug was set up with four concentration gradients of 0.2, 1,5, 25 μm in serial five dilutions. The detection method was the same as that described in (III) except that the concentration gradient was set for the drug treatment. After virus infected cells are combined with a virus antibody and a fluorescent antibody and cell nuclei are stained, cell imaging and analysis systems are used for taking pictures of the cells, four visual fields are taken for each hole, the percentage of green fluorescent positive cells, namely the virus infection rate, is analyzed and calculated, and the IC50 of nifurazite on tick-borne encephalitis virus, west Nile virus, yellow fever virus and chikungunya fever virus is calculated according to the positive cell percentage of each concentration gradient drug treatment hole; tick-borne encephalitis virus IC50: 3.56. Mu.M; west nile virus IC50: 4.13. Mu.M; yellow fever virus IC50: 4.32. Mu.M; chikungunya virus IC50: 5.08. Mu.M.
The in vitro and in vivo experimental results show that nifurazite has remarkable tick-borne encephalitis virus, west nile virus, yellow fever virus and chikungunya fever virus infection activity, and can be used for preparing medicines for resisting tick-borne encephalitis virus, west nile virus, yellow fever virus and chikungunya fever virus infection.
The foregoing has shown and described the principal features of the invention and the advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
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.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2008127291A2 (en) * | 2006-10-10 | 2008-10-23 | Los Alamos National Security, Llc | Advanced drug development and manufacturing |
| CN107723304A (en) * | 2016-08-10 | 2018-02-23 | 中国科学院上海生命科学研究院 | Applications of the PRKAR2A in inflammatory resolution |
| WO2021189444A1 (en) * | 2020-03-24 | 2021-09-30 | 中国人民解放军海军军医大学 | Use of rifamycin antibiotics in preparation of drugs against yellow fever virus infections |
| WO2021196654A1 (en) * | 2020-03-31 | 2021-10-07 | 武汉大学 | Use of glycosyl polyether compound in preparation of anti-rna virus drugs |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008127291A2 (en) * | 2006-10-10 | 2008-10-23 | Los Alamos National Security, Llc | Advanced drug development and manufacturing |
| CN107723304A (en) * | 2016-08-10 | 2018-02-23 | 中国科学院上海生命科学研究院 | Applications of the PRKAR2A in inflammatory resolution |
| WO2021189444A1 (en) * | 2020-03-24 | 2021-09-30 | 中国人民解放军海军军医大学 | Use of rifamycin antibiotics in preparation of drugs against yellow fever virus infections |
| WO2021196654A1 (en) * | 2020-03-31 | 2021-10-07 | 武汉大学 | Use of glycosyl polyether compound in preparation of anti-rna virus drugs |
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