CN114668763A - Application of etravirine in preparation of drugs for resisting infection of tick-borne encephalitis virus West Nile virus yellow fever virus and chikungunya virus - Google Patents
Application of etravirine in preparation of drugs for resisting infection of tick-borne encephalitis virus West Nile virus yellow fever virus and chikungunya virus Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- 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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- 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, and relates to application of etravirine in preparation of a medicine 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 drug is a drug for preventing or treating TBEV, WNV, YFV and CHIKV infection, wherein the drug takes etravirine as the only active component, or a drug composition containing etravirine. 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 the screened etravirine can effectively inhibit the TBEV from infecting human liver cancer cells Huh7, has low cytotoxicity, has an inhibiting effect on WNV, YFV and CHIKV, can be used as potential anti-TBEV, WNV, YFV and CHIKV drugs, and has an application prospect.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to application of etravirine in preparation of a medicine for resisting tick-borne encephalitis virus, West Nile virus, yellow fever virus and chikungunya virus infection.
Background
Etravirine (Etravirine) is a non-nucleoside reverse transcriptase inhibitor, and is inactivated by direct binding to a hydrophobic region near the active site of viral reverse transcriptase to change the conformation of enzyme protein, thereby preventing binding of new nucleotides and polymerization of double-stranded DNA, and having anti-HIV effect.
Tick-borne encephalitis virus (TBEV), also known as Tick-borne encephalitis virus, is a flavivirus genus of the Flaviviridae family. Tick-borne encephalitis (TBE) is a natural epidemic disease caused by TBEV, characterized mainly by central nervous system lesions. The main route of infection of humans with viruses is the bite by ticks carrying TBEV, and also infections due to ingestion of TBEV contaminated milk products. The distribution of TBE is obvious regional and closely related to the propagation medium. In recent years, the incidence of TBE has been on the rise in northeast china, eastern russia and northern japan, and the range of epidemic areas has been expanding. The inactivated vaccines currently used to prevent TBEV infection have low immunogenicity, short maintenance time for inducing neutralizing antibodies, and low vaccination rates for people who may be exposed outside of the forest area professional population. There is currently no TBE specific therapeutic agent.
West Nile Virus (WNV) belongs to the Flaviviridae family of Flaviviridae, is a single-stranded positive-strand RNA virus, can cause West Nile fever and West Nile virus meningoencephalitis 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. WNV disease is widely distributed in parts of Africa, the middle east, and Europe. In the summer of 1999, WNV first landed in the United states and spread rapidly in North America and Central America in the following years, leading to the greatest prevalence of arbovirus infections in countries such as the United states and Canada historically. At present, no human vaccine and therapeutic drug aiming at WNV infection exist. Although no WNV infection epidemic situation occurs in China, the possibility of introductive transmission exists.
Yellow Fever Virus (YFV) is an aedes mosquito-mediated infectious virus in the flaviviridae family, which is equivalent to ZIKV, West Nile Virus (WNV) and dengue virus (DENV), and belongs to the flaviviridae family, and uses mosquitoes as transmission vectors, and is currently mainly prevalent in tropical and subtropical regions of africa and south america. Yellow fever virus can cause yellow fever which seriously harms human health, and is manifested as jaundice, bleeding, and even multiple systems of organ failure. Although attenuated vaccines exist, the vaccine has limited coverage in endemic areas, and epidemic situations occur each year. In recent years, China has many cases of input infected people. There is currently no specific therapeutic for YFV infection.
Chikungunya fever virus (CHIKV) is a single-stranded positive-strand RNA virus, can cause Chikungunya fever, and although the lethality rate is not high, infected people lose labor force, resulting in serious social impact and economic loss. CHIKV is a mosquito-borne virus that is transmitted to humans primarily by aedes aegypti and aedes albopictus, and was first isolated in 1952-1953 in tanzania and subsequently developed in africa and asia. After 2005 CHIKV again exploded globally and sporadic incidences in france, italy and china, especially mutations in CHIKV south african type E1 protein (E1-a226V) made it spread effectively through aedes albopictus, posing a potential threat to not only tropical and subtropical areas, but also people in temperate areas where aedes albopictus is widespread. At present, no vaccine and specific therapeutic drug for CHIKV infection exist.
Disclosure of Invention
The invention aims to provide a new application of etravirine.
The invention provides application of etravirine in preparation of drugs for resisting tick-borne encephalitis virus, West Nile virus, yellow fever virus and chikungunya virus infection.
The chemical structural formula of the etravirine is as follows:
the invention relates to an application, which is characterized in that: the tick-borne encephalitis virus, West Nile virus, yellow fever virus and chikungunya virus infection resisting medicine takes etravirine as the only active component or a medicine composition containing the etravirine.
The invention relates to an application, which is characterized in that: the medicine composition containing the etravirine is a medicine composition consisting of the etravirine and one or more pharmaceutically acceptable auxiliary materials.
The invention relates to an application, which is characterized in that: the above medicine is used for preventing or treating infection of tick-borne encephalitis virus, West Nile virus, yellow fever virus and chikungunya virus.
The invention relates to an application, which is characterized in that: the content of the etravirine in the tick-borne encephalitis virus, West Nile virus, yellow fever virus and chikungunya virus infection resisting medicine is 0.1-99 wt%.
The invention relates to an application, which is characterized in that: the administration dosage form of the medicine is selected from powder, tablets, granules, capsules, solutions, emulsions and suspensions.
The invention relates to an application, which is characterized in that: the administration route of the medicine is selected from injection administration, gastrointestinal administration and skin administration.
The invention has the following functions and effects:
according to the invention, an experimental operation system of TBEV infected susceptible cells is utilized, candidate small molecule drugs capable of inhibiting TBEV infection are screened from a clinically approved drug small molecule library, etravirine is screened, which can effectively inhibit TBEV infection on human liver cancer cell Huh7, has low cytotoxicity, has an inhibiting effect on WNV, YFV and CHIKV, can be used as potential anti-TBEV, WNV, YFV and CHIKV drugs, and has an application prospect.
Drawings
FIG. 1 Effect of etravirine on protecting BHK cells against tick-borne encephalitis virus infection;
BHK cells are infected with tick-borne encephalitis virus, etravirine or DMSO solvent is added at the same time, or the BHK cells are not infected with tick-borne encephalitis virus (no virus is added), CCK8 reagent is added after 72 hours, and the light absorption value of 450nm is detected.
FIG. 2 toxicity of etravirine to Huh7 cells;
namely, etravirine and a solvent DMSO respectively treat Huh7 cells, and a CCK8 reagent is added after 72 hours to detect the light absorption value at 450 nm.
Figure 3 inhibition of tick-borne encephalitis virus by etravirine in a model of Huh7 cell infection.
FIG. 4 shows the effect of etravirine in inhibiting infection by tick-borne encephalitis virus, West Nile virus, yellow fever virus, and chikungunya virus.
Detailed Description
In order to more clearly illustrate the invention, the invention is further described below in connection with preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
Etravirine used in embodiments of the present invention may be purchased commercially.
Viruses, drugs, reagents and other materials
1. Virus: tick-borne encephalitis virus and yellow fever virus are separated and cultured in a biomedical protective research and development room of army medical university of navy, China liberation army, west nile virus and chikungunya virus are synthesized by a reverse genetics technology and cultured in an amplification way by baby hamster kidney BHK cells. All experimental procedures involving viral infection were performed at the naval military medical university P3 laboratory.
2. A compound: 978 U.S. FDA chemical drug molecule libraries were purchased from Selleck, USA.
3. Human hepatoma cell lines Huh7 and baby hamster kidney BHK cells, purchased from shanghai cell institute of chinese academy of sciences, were maintained by the biomedical protective research laboratory of the navy military medical university of chinese people's liberation army.
DMEM cell culture medium is a product of Hyclone in America, 10% of fetal bovine serum, non-essential amino acids, ampicillin and streptomycin (each 100U/ml) are added when in use, and additives of the culture medium are all products of Thermo Fisher in America.
5. Cell digest, containing 0.25% trypsin, was prepared in phosphate buffer.
The CCK8 cell activity and proliferation detection kit is a product of MedChemexpress company in the United states.
7. Mouse tick-borne encephalitis virus polyclonal antibody, West Nile virus polyclonal antibody, yellow fever virus polyclonal antibody and chikungunya fever virus polyclonal antibody are prepared by immunizing mice with formaldehyde-inactivated tick-borne encephalitis virus in the biomedical protective research laboratory of army medical university of navy, China people's liberation army.
8. Fluorescein Alexa Fluor 488-labeled anti-mouse IgG was a product of Thermo Fisher, USA.
II, an experimental method:
screening tick-borne encephalitis virus resisting medicines from FDA medicine micromolecule library containing 978 micromolecule compounds
Baby hamster kidney BHK cells were subcultured in a T75 cell culture flask using complete DMEM medium, inoculated into a 96-well plate at 10000 cells per well in 100. mu.L of DMEM medium, and cultured for 12 hours. Subsequently 50 μ L of complete DMEM medium containing 1000PFU (plaque forming unit) tick-borne encephalitis virus was added to each well; at the same time, 50 μ L of complete DMEM medium containing FDA small molecule chemical drug was added, with a final drug concentration of 10 μ M, and 3 wells were repeated at each concentration, with the addition of an equal volume of solvent DMSO as a control without drug. Placing at 37 ℃ and 5% CO2Culturing in an incubator. After 72 hours, all cells in the DMSO wells were microscopically rounded or exfoliated. Adding CCK8 cell activity and proliferation detection reagent 10 μ L into each well, placing at 37 deg.C and 5% CO2In an incubator, after 30 minutes, an absorbance value of each well at the wavelength of 450nm is detected by a multifunctional microplate reader, and the protection rate of the drug to cells (the absorbance value of the drug added to the cells at 450 nm-the absorbance value of the cells at the DMSO wells at 450 nm)/the absorbance value of the cells without the added virus and DMSO at 450nm is calculated as 100 percent, so that the protection rate of each drug to tick-borne encephalitis virus infected cells at the concentration of 10 mu M is obtained. The results show that etravirine has effects on cellsThe protection effect is remarkable, the 450nm light absorption values of the two antibiotics and the DSMO treated cells are shown in figure 1, and the cell protection rates of the etravirine obtained through calculation are 91.1% respectively.
Toxicity of (di) etravirine to cells
The cultured human hepatoma cell line Huh7 and baby hamster kidney BHK cells were inoculated into a 96-well plate with 10000 cells per well and 100. mu.L of culture solution, 12 hours later, the original culture solution was aspirated off, 100. mu.L of complete DMEM culture solution of etravirine was added to each well at a concentration gradient dilution, the final concentration of etravirine was 2.5, 5, 10, 20, 40 and 80. mu.M, 3 wells were repeated at each concentration, and the content of DMSO as a solvent of 80. mu.M drug was used as a control without drug. Placing at 37 ℃ and 5% CO2Culturing in an incubator. After 48 hours, 10. mu.L of CCK8 cell activity and proliferation detection reagent was added to each well, and the mixture was incubated at 37 ℃ with 5% CO2And (3) in the incubator, detecting the light absorption value of each hole at the wavelength of 450nm by using a multifunctional microplate reader after 30 minutes, and evaluating the cytotoxicity of the medicament according to the difference of the light absorption values of 450nm of the medicament treatment holes and the solvent holes with different concentrations.
The results show that there was no significant difference between the etravirine-treated and DMSO solvent-treated cells when the concentration was equal to or lower than 80 μ M. FIG. 2 shows the 450nm absorbance of etravirine-treated Huh7 cells with DMSO solvent-treated cells at 80 μ M.
Inhibition of tick-borne encephalitis virus by etravirine in a model of cell infection
The subcultured human hepatoma cell line Huh7 was inoculated into a 96-well plate at 10000 cells per well in 100 μ L of culture medium and cultured for 12 hours. Subsequently 50 μ L of complete DMEM medium containing 1000PFU tick-borne encephalitis virus was added per well; simultaneously adding 50 μ L of complete DMEM culture solution containing etravirine, with final drug concentration of 5 μ M, repeating for 3 wells at each concentration, adding equal volume of solvent DMSO as control without drug, placing at 37 deg.C and 5% CO2Culturing in an incubator.
After 20 hours, detecting the infection of the cells by the virus by using an immunofluorescence technique, and specifically performing the following steps: removing the culture medium from the culture plate by pipetting, adding 100. mu.L of methanol per well, placing the culture plate in a refrigerator at-20 deg.C for 20 minutes, taking out the culture plate, pipetting methanol, washing the wells with Phosphate Buffered Saline (PBS) once per well, adding 100. mu.L of PBS containing 3% Bovine Serum Albumin (BSA) (hereinafter referred to as 3% BSA-PBS), placing the plate on a horizontal shaker, slowly shaking at room temperature for 1 hour, pipetting 3% BSA-PBS in the culture plate, adding 100. mu.L of 1% BSA-PBS containing polyclonal antibody against tick-borne encephalitis virus (antibody 500-fold dilution) per well, slowly shaking at room temperature for 1 hour, pipetting the working solution of polyclonal antibody against tick-borne encephalitis virus from the culture plate, washing 3 times per well with PBS, adding 100. mu.L of 1% BSA-PBS containing Alexa Fluor 488-labeled anti-mouse IgG (fluorescein antibody 1500-fold dilution), the cells were gently shaken at room temperature for 1 hour in the dark, the fluorescein antibody working solution in the culture plate was aspirated, 100. mu.L of DAPI cell nucleus staining solution was added to each well, the DAPI cell nucleus staining solution in the culture plate was aspirated for 10 minutes in the dark, each well was washed with PBS 3 times, and the fluorescence distribution of the cells in each well was photographed using a cell Imaging and analysis system (BioTek staining 5 Imaging Reader). Four fields of view are photographed per well, and the percentage of green fluorescence positive cells, i.e., the rate of virus infection, is analyzed and calculated.
As a result, as shown in fig. 3, the infection rate of etravirine-treated virus was significantly lower than that of DMSO-solvent-treated wells as a control. The results demonstrate that etravirine significantly inhibits infection of Huh7 cells by tick-borne encephalitis virus at a concentration of 5 μ M.
(IV) quantitative determination of Equvirin inhibition Activity against infection by tick-borne encephalitis virus, West Nile virus, yellow fever virus, chikungunya virus
In order to observe whether etravirine has inhibitory activity against tick-borne encephalitis virus, west nile virus, yellow fever virus and chikungunya virus, quantitative detection of the activity against tick-borne encephalitis virus, west nile virus, yellow fever virus and chikungunya virus is further carried out on etravirine, namely, half inhibitory concentration (IC50) of a drug to the virus is calculated by detecting antiviral activity at different concentrations. Four concentration gradients of 0.2, 1, 5, 25 μ M were set for the drug at five consecutive dilutions. The detection method was the same as that described in (iii) except that the drug treatment was set to a concentration gradient. After virus-infected cells were stained with virus antibody and fluorescent antibody, a cell image was taken with a cell imaging and analysis system, four fields were taken per well, and the percentage of green fluorescence positive cells, i.e., the virus infection rate, was analyzed and calculated (fig. 4). Calculating the IC50 of etravirine for tick-borne encephalitis virus, West Nile virus, yellow fever virus and chikungunya fever virus according to the positive cell percentage of each concentration gradient drug treatment hole; tick-borne encephalitis virus IC 50: 2.17 μ M; west nile virus IC 50: 4.76 μ M; yellow fever virus IC 50: 4.59 μ M; chikungunya virus IC 50: 5.56. mu.M.
The in vitro and in vivo experimental results show that etravirine has significant activity of infection by tick-borne encephalitis virus, west nile virus, yellow fever virus and chikungunya virus, can be used for preparing a medicament for resisting tick-borne encephalitis virus infection, and is used for preventing and treating tick-borne encephalitis virus infection.
The principal features of the invention and advantages of the invention have been shown and described above. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (7)
1. Use of etravirine in the manufacture of a medicament for the treatment of tick-borne encephalitis virus, West Nile virus, yellow fever virus and chikungunya virus infection.
2. Use according to claim 1, characterized in that: the tick-borne encephalitis virus, West Nile virus, yellow fever virus and chikungunya virus infection resisting medicine takes etravirine as the only active component, or a medicine composition containing the etravirine.
3. Use according to claim 2, characterized in that: the medicine composition containing the etravirine is a medicine composition formed by the etravirine and one or more pharmaceutically-allowable auxiliary materials.
4. Use according to any one of claims 1 to 3, characterized in that: the medicament is used for preventing or treating infection of tick-borne encephalitis virus, West Nile virus, yellow fever virus and chikungunya virus.
5. Use according to any one of claims 1 to 4, characterized in that: the content of the etravirine in the tick-borne encephalitis virus, West Nile virus, yellow fever virus and chikungunya virus infection resisting medicine is 0.1-99 wt%.
6. Use according to any one of claims 1 to 4, characterized in that: the administration dosage form of the medicine is selected from powder, tablets, granules, capsules, solutions, emulsions and suspensions.
7. Use according to any one of claims 1 to 4, characterized in that: the administration route of the drug is selected from injection administration, gastrointestinal administration and skin administration.
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| US20210128554A1 (en) * | 2016-12-23 | 2021-05-06 | Temple University - Of The Commonwealth System Of Higher Education | Anti-flaviviridae activity of anti-retroviral non-nucleoside (nnrtis) and nucleoside reverse transcriptase inhibitors (nnrtis) |
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| US20160213647A1 (en) * | 2015-01-28 | 2016-07-28 | Arno Therapeutics, Inc. | Compositions and methods for inhibiting viral infection |
| US20210128554A1 (en) * | 2016-12-23 | 2021-05-06 | Temple University - Of The Commonwealth System Of Higher Education | Anti-flaviviridae activity of anti-retroviral non-nucleoside (nnrtis) and nucleoside reverse transcriptase inhibitors (nnrtis) |
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| CN117462552A (en) * | 2023-12-27 | 2024-01-30 | 深圳国家感染性疾病临床医学研究中心 | Application of itravirin and/or derivatives thereof in preparation of antituberculosis drugs |
| CN117462552B (en) * | 2023-12-27 | 2024-05-17 | 深圳国家感染性疾病临床医学研究中心 | Application of itravirin and/or derivatives thereof in preparation of antituberculosis drugs |
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