Use of verteporfin in the preparation of a medicine for resisting infection of bandavirus dabie

Abstract

The present application relates to the technical field of medicine, and relates to application of verteporfin in preparation of medicine for resisting bandavirus infection. Verteporfin is an autophagy inhibitor, can inhibit autophagosome formation by directly targeting and modifying p62, and is mainly used for treating senile macular degeneration treated by photodynamic therapy. The present application reports the application of verteporfin in the medicine for resisting bandavirus infection, which is reported for the first time in the world.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of verteporfin in preparation of a medicine for resisting Dabie bandavirus infection. BACKGROUND

[0002] Verteporfin is a benzoporphyrin derivative, also known as a phenyl chlorin derivative monobasic acid. The chemical name is 13-methyl trans-(±)-18-vinyl-4,4a-dihydro-3,4-bis(methoxycarbonyl)-4a,8,14,19-tetramethyl-23H,25H-benzoporphyrin-9,13-dipropyl ester. Verteporfin is an autophagy inhibitor, which can inhibit autophagosome formation by directly targeting and modifying p62 (a scaffold and adaptor protein that can bind polyubiquitinated proteins to be degraded and LC3 on the autophagosome membrane), and is mainly used for treating age-related macular degeneration in photodynamic therapy. In addition, verteporfin can inhibit the expression levels of Yes-associated protein (YAP) and its target genes cysteine-rich protein 61 (CYR61) and connective tissue growth factor (CTGF), thereby playing an anticancer role. Studies have shown that verteporfin has inhibitory effect on various cancer cells. Therefore, verteporfin has high application value.

[0003] Dabie bandavirus (DBV) is a new bunyavirus, which was first discovered in China in 2009. The fever with thrombocytopenia syndrome (SFTS) caused by the virus belongs to an acute infectious disease, and the mortality rate is as high as 30%. In recent years, the transmission range of the virus has gradually expanded, which has brought great threat to human life and health. The World Health Organization has listed the Dabie bandavirus as a priority pathogen, and SFTS has been listed as one of the top ten infectious diseases by the World Health Organization. So far, there is no specific and effective vaccine and treatment drug, and the number of SFTS death cases is increasing year by year. Therefore, it is urgent to develop safe and efficient anti-Dabie bandavirus drugs. SUMMARY

[0004] The application aims to provide a new use of verteporfin.

[0005] The application provides application of verteporfin in preparation of a medicine for resisting Dabie bandavirus infection.

[0006] The chemical structural formula of the verteporfin is as follows:

[0007]

[0008] The application utilizes an experimental operation system of Dabie bandavirus infection of susceptible cells, screens candidate small molecule drugs capable of inhibiting Dabie bandavirus infection from a clinically approved small molecule drug library, and screens out that verteporfin can effectively inhibit Dabie bandavirus infection and has smaller cytotoxicity, and can be used as a potential anti-Dabie bandavirus drug and has application prospects.

[0009] The application has the characteristics that the dosage form of the drug is a gastrointestinal administration dosage form.

[0010] The application has the characteristics that the administration dosage form of the drug is selected from powders, tablets, granules, capsules, solutions, emulsions, suspensions.

[0011] The application has the characteristics that the dosage form of the drug is a non-gastrointestinal administration route dosage form.

[0012] The application has the characteristics that the administration route dosage form of the drug is selected from injection administration dosage forms, respiratory tract administration dosage forms, nose drops, skin administration dosage forms, mucous membrane administration dosage forms or cavity administration dosage forms. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 . Effect of verteporfin on protecting Huh7 cells from Dabie bandavirus infection

[0014] That is, Huh7 cells are infected with Dabie bandavirus, and verteporfin or solvent DMSO is added at the same time, and 24 hours later, the infection of the virus on the cells is detected by immunofluorescence technology, and the inhibition rate of the compound on the Dabie bandavirus infection at a concentration of 10 μM is calculated according to the number of positive clones in each well.

[0015] Figure 2 . Toxicity of verteporfin on Huh7 cells

[0016] That is, Huh7 cells are treated with different concentrations of verteporfin and solvent DMSO, respectively, and 24 hours later, CCK8 reagent is added, and the absorbance at 450 nm is detected.

[0017] Figure 3 . Inhibition activity of different concentrations of verteporfin on Dabie bandavirus in a Huh7 cell infection model

[0018] That is, Huh7 cells are infected with Dabie bandavirus, and verteporfin or solvent DMSO is added at the same time, and 24 hours later, the infection of the virus on the cells is detected by immunofluorescence technology, and the inhibition rate of the compound on the Dabie bandavirus infection at different concentrations is calculated according to the number of positive clones in each well. DETAILED DESCRIPTION

[0019] In order to make the present application clearer, the present application will be further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the specific descriptions below are illustrative rather than limiting and should not limit the scope of protection of the present application.

[0020] The verteprofin used in the embodiments of the present application can be obtained by market purchase.

[0021] I. Viruses, drugs, reagents and other materials

[0022] 1. Virus: Dabie bandavirus, isolated from peripheral blood samples of infected persons by the Eastern Regional Center for Disease Control using human laryngeal epithelial carcinoma cell line Hep2 cells.

[0023] 2. Compound: 2580 FDA chemical drug molecules in the United States, purchased from Selleck Company in the United States.

[0024] 3. Human hepatoma cell line Huh7 cells, purchased from Shanghai Cell Institute of Chinese Academy of Sciences, and stored in the Biomedical Defense Teaching and Research Office of the Navy Medical University of the People's Liberation Army.

[0025] 4. DMEM cell culture medium is a product of Hyclone Company in the United States, and 10% fetal bovine serum, non-essential amino acids, ampicillin and streptomycin (100 U / ml each) are added when used. The culture medium additives are all products of Thermo Fisher Company in the United States.

[0026] 5. Cell digestion solution contains 0.25% trypsin, prepared with phosphate buffer.

[0027] 6. CCK8 cell activity and proliferation detection kit is a product of Dojindo Company in Japan.

[0028] 7. Mouse anti-Dabie bandavirus monoclonal antibody prepared by the Eastern Regional Center for Disease Control.

[0029] 8. Fluorescein Alexa Fluor 488-labeled anti-mouse IgG is a product of Thermo Fisher Company in the United States.

[0030] II. Experimental methods

[0031] (I) Screening of Dabie bandavirus drugs from FDA drug small molecule library containing 2580 small molecule compounds

[0032] Huh7 cells were subcultured in T25 cell culture flasks with complete DMEM medium, seeded in 96-well plates at 50,000 cells per well in 100 μL of DMEM medium, and incubated for 12 h. Then 50 μL of complete DMEM medium containing Dabie bandavirus was added to each well at a MOI (multiplicity of infection) of 1, and 50 μL of complete DMEM medium containing an FDA small-molecule chemical drug was added to each well at a final concentration of 10 μM, with 3 wells for each concentration, and an equal-concentration solvent DMSO as a control without the drug. The plates were incubated in a 37 °C, 5% CO2incubator. After 24 h, the infection of the cells by the virus was detected by immunofluorescence, as follows. The culture medium was removed from the plates, 100 μL of methanol was added to each well, and the plates were placed in a -20 °C refrigerator for 30 min. Then the methanol was removed from the plates, and each well was washed once with phosphate-buffered saline (PBS). Then 100 μL of 3% bovine serum albumin (BSA)-containing PBS (hereinafter referred to as 3% BSA-PBS) was added to each well, and the plates were slowly shaken on a horizontal shaker at room temperature for 2 h. Then 100 μL of 1% BSA-PBS containing an anti-Dabie bandavirus monoclonal antibody (antibody diluted 1:3000) was added to each well, and the plates were slowly shaken at room temperature for 1 h. Then the anti-Dabie bandavirus monoclonal antibody working solution was removed from the plates, and each well was washed 3 times with PBS. Then 100 μL of 1% BSA-PBS containing a fluorescein Alexa Fluor 488-labeled anti-mouse IgG (fluorescein antibody diluted 1:2000) was added to each well, and the plates were slowly shaken at room temperature in the dark for 2 h. Then the fluorescein antibody working solution was removed from the plates, and 100 μL of DAPI cell nucleus staining solution was added to each well, and the plates were slowly shaken at room temperature in the dark for 10 min. Then the DAPI cell nucleus staining solution was removed from the plates, and each well was washed 3 times with PBS. The fluorescence distribution of the cells in each well was photographed using a cell imaging and analysis system (BioTek Cytation 5 Imaging Reader). The inhibition rate of each compound on the infection of Dabie bandavirus at a concentration of 10 μM was calculated according to the number of positive clones in each well, as follows: % inhibition = [(number of positive clones in the DMSO well at the same concentration - number of positive clones in the drug-containing well) / number of positive clones in the DMSO well at the same concentration] x 100. As shown in Table 1, at 10 μM, verterporfin completely inhibited the infection of Dabie bandavirus. Figure 1 As shown in Table 1, at 10 μM, verterporfin completely inhibited the infection of Dabie bandavirus.

[0033] (ii) Toxicity of verterporfin to cells

[0034] Cultured human hepatocellular carcinoma cell line Huh7 was seeded into 96-well plates at a density of 50,000 cells per well, with 100 μL of culture medium per well. After 12 h, the original culture medium was aspirated, and 100 μL of complete DMEM culture medium containing serially diluted verteporfen was added to each well. Verteporfen final concentrations were 0.0064, 0.032, 0.16, 0.8, 4, and 20 μM (9 gradients), with each concentration repeated in triplicate. An equal concentration of DMSO served as a control without drug treatment. The plates were incubated at 37°C in a 5% CO2 incubator. After 24 h, 10 μL of CCK8 cell viability and proliferation assay reagent was added to each well, and the plates were incubated at 37°C in a 5% CO2 incubator. After 30 min, the absorbance at 450 nm was measured using a multi-mode microplate reader. The cytotoxicity of the drug was evaluated based on the difference in absorbance at 450 nm between wells treated with different drug concentrations and those treated with solvent. Figure 2 The results showed that verteporfen had low cytotoxicity; when the concentration was equal to or below 20 μM, there was no significant difference between the two cell types treated with verteporfen and those treated with DMSO solvent.

[0035] (III) Inhibitory effect of verteporfen on Dabiebanda virus in a cell infection model

[0036] Human hepatocellular carcinoma cell line Huh7, passaged and cultured, was seeded into 96-well plates at 50,000 cells per well with 100 μL of culture medium. After 12 h of culture, the original culture medium was aspirated, and then 50 μL of complete DMEM culture medium containing Dabiebanda virus was added to each well (MOI: multiplicity of infection) of 1. Simultaneously, 50 μL of complete DMEM culture medium containing verteporfen was added to each well at six concentration gradients: 0.0064, 0.032, 0.16, 0.8, 4, and 20 μM. Each concentration was repeated in triplicate. DMSO was used as a control without the drug. The plates were incubated at 37°C in a 5% CO2 incubator. After 24 h, the viral infection status of the cells was detected using immunofluorescence, following the same procedure as described in section (I). The inhibition rate of the compound against Dabie Bandar virus infection at different concentrations was calculated based on the number of positive clones in each well. Inhibition rate % = [(Number of positive clones in wells with the same concentration of DMSO - Number of positive clones in wells with added drug) / Number of positive clones in wells with the same concentration of DMSO] × 100. Results are as follows... Figure 3 As shown, verteporfen significantly inhibited Dabiebanda virus infection of Huh7 cells.

[0037] The above experimental results indicate that verteporfen has significant anti-Dabiebanda virus activity and can be used to prepare drugs against Dabiebanda virus infection for the prevention and treatment of Dabiebanda virus infection.

[0038] The foregoing generally describes the main features of the application and advantages of the application. It should be understood that the application is not limited in scope to the above embodiments and that the application can be practiced with various modifications and alterations without departing from the spirit and scope of the application. The application is limited only by the claims that follow and equivalents thereof.

Claims

1. Use of verteporfin for the preparation of a medicament against infection by the bandavirus of the Great Basin, characterized in that: The anti-Giardia lamblia infection drug is veriteporfin as the only active ingredient.

2. Use according to claim 1, characterized in that: The drug comprises veriteporfin and pharmaceutically acceptable one or more auxiliary materials.

3. Use according to any one of claims 1-2, characterized in that: The drug is used for treating Giardia lamblia infection.

4. Use according to claim 1, characterized in that: The drug administration form is selected from powders, tablets, granules, capsules, solutions, emulsions, suspensions.

5. The use according to claim 1, characterized in that: The drug administration route is selected from injection administration, gastrointestinal administration, and skin administration.

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