CN114917227B - Application of Ivacizumab in preparation of anti-tick-borne encephalitis virus, west nile virus, yellow fever virus and chikungunya fever virus infection medicines - Google Patents
Application of Ivacizumab in preparation of anti-tick-borne encephalitis virus, west nile virus, yellow fever virus and chikungunya fever virus infection medicines 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
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
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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, and relates to application of ivakatuo in preparation of medicines for resisting tick-borne encephalitis virus, west nile virus, yellow fever virus and chikungunya fever virus infection. According to the invention, a TBEV-infected susceptible cell experimental operation system is utilized, candidate small molecule drugs capable of inhibiting TBEV infection are screened from a clinically approved drug small molecule library, and the ivakatuo is screened out, so that TBEV can effectively inhibit human liver cancer cell Huh7 infection, has low cytotoxicity, has 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 ivakatuo in preparation of medicines for resisting tick-borne encephalitis virus, west nile virus, yellow fever virus and chikungunya fever virus infection.
Background
Ivakaftor (ivacator) is a drug (CFTR) gene (mainly G551D mutation) for treating human cystic fibrosis that modulates specific gene mutations at cystic fibrosis transmembrane conductance, accounting for 4-5 cases of cystic fibrosis. It also contains a combination of drugs in Chemical book, lumacaftor/ivaftor (trade name orkambi), which is used to treat cystic fibrosis patients with the F508del mutation in CFTR. Its main effect is increased chloride transport through enhanced channel opening probability (or gating) of CFTR proteins.
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. In recent years, TBE has developed in northeast China, eastern russian and northern japan, and the epidemic area has been expanding. Specific anti-TBEV immunoglobulins, non-specific immunoglobulins and recombinant anti-TBEV immunoglobulins have been validated or used for the prevention and treatment of TBE, but often do not achieve good results due to viral tolerance, limited effect, uncertainty in clinical effect, etc.; clinical drugs for the simultaneous treatment of diseases caused by TBEV are also extremely scarce.
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. WNV disease is widely distributed in parts of africa, middle east, europe. In the summer of 1999, WNV first entered the united states and then spread rapidly in north america and central america within a few years, resulting in the greatest prevalence of arbovirus infection historically in countries such as the united states and canada. There is currently no vaccine or therapeutic for humans with WNV infection. Although no WNV infection epidemic occurs in China so far, the possibility of inpatient transmission exists.
Yellow fever virus (Yellow fevervirus, YFV) is a virus of the genus flaviviridae, which is equivalent to the group consisting of zaka virus (ZIKV), west Nile Virus (WNV) and dengue virus (DENV), and is a mosquito-based vector of infection in insect-borne, and is currently mainly prevalent in tropical and subtropical regions of africa and south america. Yellow fever virus can cause yellow fever that is a serious hazard to human health, manifested as jaundice, bleeding, and even multiple organ failure. Although attenuated vaccines exist, the coverage rate of vaccination of vaccines in epidemic areas is limited, and epidemic situations occur every year. In recent years, there are many cases of input infectious agents in our country. There is currently no therapeutic drug against YFV infection.
Chikungunya fever virus (Chikungunya fever virus, CHIKV) is a single-stranded positive strand RNA virus that causes chikungunya fever but the infected population loses labor though the mortality is not high, resulting in serious social impact and economic loss. CHIKV is a mosquito vector virus that is transmitted to humans mainly by aedes aegypti and aedes albopictus, and was first isolated in tank sania in 1952-1953 and then outbreaked in africa and asia. After 2005 CHIKV has been exploded again several times worldwide, and has scattered input cases in france, italy and china, especially mutations (E1-a 226V) in the south africa in the east of CHIKV make it possible to spread by aedes albopictus effectively, which constitutes a potential threat not only in tropical and subtropical areas, but also to the masses in temperate areas where aedes albopictus is widely present. Currently, there is no vaccine or therapeutic against CHIKV infection.
Disclosure of Invention
The invention aims to provide a new application of ivacaine.
The invention provides application of ivatutor 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 the ivacaine is as follows:
the application provided by the invention is further characterized in that: the anti-tick-borne encephalitis virus, west nile virus, yellow fever virus and chikungunya fever virus infection medicament is a medicament composition which takes the ivacaine as the only active ingredient or contains the ivacaine.
The application provided by the invention is further characterized in that: the medicine composition containing the ivacaine refers to a medicine composition formed by the ivacaine and one or more pharmaceutically allowable auxiliary materials.
The application provided by the invention is further 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.
The application provided by the invention is further characterized in that: the content of the ivacaine in the anti-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 provided by the invention is further characterized in that: the drug is administered in the form of powder, tablet, granule, capsule, solution, emulsion, or suspension.
The application provided by the invention is further characterized in that: the administration route of the medicine is selected from injection administration form, gastrointestinal tract administration form, respiratory tract administration form, nasal drop, skin administration form, mucous membrane administration form or cavity tract administration form.
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 the screened ivakatuo 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 a potential anti-TBEV, WNV, YFV, CHIKV drug, and has an application prospect.
Drawings
FIG. 1. Effect of Ivacizumab to protect BHK cells against tick-borne encephalitis virus infection;
i.e., BHK cells were infected with tick-borne encephalitis virus while either Ivaccator solvent DMSO was added, or not (no virus added), and after 72 hours CCK8 reagent was added to detect absorbance at 450 nm.
FIG. 2 toxicity of Ivacizumab to Huh7 cells;
i.e. the Huh7 cells were treated with the Ivacizumab and solvent DMSO, respectively, and after 72 hours, CCK8 reagent was added to detect the absorbance at 450 nm.
Figure 3 inhibition of tick-borne encephalitis virus by ivatutor in a model of Huh7 cell infection.
Figure 4. Effect of ivacaine in inhibiting 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.
The ivacaine used in the embodiment of the invention can be purchased in a commercial manner.
1. Viruses, drugs, agents, and other materials
1. Virus: tick-borne encephalitis virus and yellow fever virus are isolated 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 virus in a biomedical protection teaching laboratory of the university of naval of the liberation army of Chinese people.
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% CO 2 Culturing 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% CO 2 And 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 the ivacaine has remarkable protective effect on cells, the two antibiotics and the DSMO treated cells have the light absorption value of 450nm as shown in figure 1, and the calculated cell protection rates of the ivacaine are 92.2% respectively.
Toxicity of Ivacizumab to cells
The cultured human liver cancer cell line Huh7 and baby hamster kidney BHK cells are respectively inoculated in a 96-well plate10000 cells per well, 100 μl of culture solution, after 12 hours, the original culture solution was aspirated, 100 μl of complete DMEM culture solution of concentration gradient diluted ivakapton was added per well, the final concentration of ivakapton was 2.5, 5, 10, 20, 40 and 80 μΜ,3 wells were repeated for each concentration, and the solvent DMSO content of 80 μΜ drug was used as a non-drug control. Placing at 37deg.C and 5% CO 2 Culturing 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% CO 2 In 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 show that at concentrations equal to or below 80 μm, there is no significant difference between both cells treated with ivakatutor and cells treated with DMSO solvent. FIG. 2 shows the absorbance at 80. Mu.M of the Huh7 cells treated with ivakaptol versus 450nm of the DMSO-solvent-treated cells.
Inhibition of tick-borne encephalitis virus by Ivacizumab 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 ivakaptone, 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% CO 2 Culturing 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 AlexaFluor 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 DAPI 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 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 reduction in the infection rate of the ivakatutor-treated virus compared to DMSO solvent-treated wells as controls. The results demonstrate that at a concentration of 5 μm, ivakapton significantly inhibited infection of Huh7 cells by tick-borne encephalitis virus.
(IV) quantitative determination of Ivaccinal to inhibit tick-borne encephalitis virus, west Nile virus, yellow fever virus, chikungunya virus infection Activity
In order to observe whether the Ivacizumab has anti-inhibitory activity on tick-borne encephalitis virus, west nile virus, yellow fever virus and chikungunya fever virus, the Ivacizumab virus activity is quantitatively detected, namely the half inhibitory concentration (IC 50) of the drug on the virus is calculated by detecting the antiviral activity at different concentrations. 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 were combined with the virus antibody and fluorescent antibody and the nuclei were stained, cell photographing was performed with a cell imaging and analysis system, four fields were photographed per well, and the percentage of green fluorescent positive cells, i.e., the virus infection rate, was analyzed and calculated (fig. 4). Calculating the IC50 of the Ivacizumab to 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 IC50: 3.17. Mu.M; west nile virus IC50: 4.75. Mu.M; yellow fever virus IC50: 1.22. Mu.M; chikungunya virus IC50: 6.43. Mu.M.
The in vitro and in vivo experimental results show that the ivatutor has obvious 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 Ivacizumab in preparing medicine 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 anti-tick-borne encephalitis virus, west nile virus, yellow fever virus and chikungunya fever virus infection medicament is a medicament composition which takes the ivacaine as the only active ingredient or contains the ivacaine.
3. The use according to claim 2, characterized in that: the medicine composition containing the ivacaine refers to a medicine composition formed by the ivacaine and one or more pharmaceutically allowable 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 the ivacaine in the anti-tick-borne encephalitis virus, the west nile virus, the yellow fever virus and the chikungunya fever virus infection 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, respiratory tract administration form, nasal drop, skin administration form, mucous membrane administration form or cavity tract administration form.
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