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Application of calcein and oseltamivir in drug for preventing and treating H7N9 influenza

A calcein, H7N9 technology, applied in the direction of medical preparations containing active ingredients, pharmaceutical formulations, organic active ingredients, etc., can solve the problem of no anti-influenza virus and other issues

Active Publication Date: 2016-11-09
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are no reports of its anti-influenza virus efficacy

Method used

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  • Application of calcein and oseltamivir in drug for preventing and treating H7N9 influenza
  • Application of calcein and oseltamivir in drug for preventing and treating H7N9 influenza
  • Application of calcein and oseltamivir in drug for preventing and treating H7N9 influenza

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] H7N9 wild-type or mutant neuraminidase solution was prepared with pH 6.5 MES buffer (32.5mM), a certain amount of neuraminidase was added to the reaction buffer to make the total volume 40 μL, and then 10 μL of different concentration of calcein or oseltamivir carboxylate. Place in a 37°C incubator for 30 minutes, then add 50 μL of 20 μM MU-NANA substrate buffer solution, the final reaction system is 100 μL, the reaction solution is placed in a black microplate plate, and detected on a microplate reader. The excitation wavelength is 360 nm, and the emission wavelength is 360 nm. The wavelength is 450nm, and the detection time is 8min. The change of the detected fluorescence intensity represents the activity of the enzyme to degrade the substrate. The test results are: IC of oseltamivir carboxylate on H7N9 wild-type and mutant neuraminidase 50 1.75±0.12nmol / L and 26.52±1.06μmol / L, respectively; the IC of calcein on H7N9 wild-type and mutant 50 They were 12.68±2.26μm / L...

Embodiment 2

[0016] According to the determined IC of calcein and oseltamivir carboxylate on H7N9 wild-type and mutant neuraminidase 50 To determine the adding concentration of calcein and oseltamivir carboxylate. The concentrations of calcein and oseltamivir carboxylate were chosen to be 0.25, 0.5, 1, 2 and 4 times the IC of H7N9 wild-type and mutant neuraminidase, respectively 50 concentration. And the ratio of the concentration of calcein added to the concentration of oseltamivir carboxylate is maintained consistent, its IC 50 value ratio. First, add 30 μL of diluted H7N9 wild-type or mutant neuraminidase solution to a 96-well ELISA plate, and then add 10 μl of calcein of different concentrations and 10 μL of corresponding concentrations of oseltamivir carboxylate to each well , each concentration was repeated 4 times. After incubating at 37°C for 30 min, 50 μl of 20 μM NA-specific substrate MU-NANA was added to detect the fluorescence intensity of H7N9 wild-type and mutant neuramin...

Embodiment 3

[0021] In order to detect the effect of the mixture of calcein and oseltamivir carboxylate on MDCK cells against H7N9 wild-type and mutant influenza viruses, MDCK cells in logarithmic growth phase were selected. Spread 96-well plates, 3000 / well, 37°C, 5% CO 2 Cultivate in an incubator for 24 hours until the confluence of monolayer cells reaches 50-60%. The next day, the culture medium was discarded and washed twice with serum-free DMEM to remove residual serum. Then use 100 μL of H7N9 wild-type or mutant influenza virus allantoic fluid (100-fold concentration of TCID 50 concentration) and 100 μl of mixtures of calcein and oseltamivir carboxylate at different concentrations were mixed for 2 hours at 37°C. IC50 concentrations of 0.25, 0.5, 1, 2 and 4 times, the ratio of the concentration of calcein added to the concentration of oseltamivir carboxylate remained consistent, and its IC 50 value ratio. After that, the incubation mixture was added to the 96 wells where the cells ...

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Abstract

Provided is application of calcein and oseltamivir in a drug for preventing and treating H7N9 influenza. According to the technical scheme, the method comprises the steps that an H7N9 wild-type or mutant-type influenza virus neuraminidase solution and a calcein and oseltamivir mixed solution are subjected to incubation for 30 min, and a substrate is added for detection; it is shown through experimental results that combined usage of the two drugs has inhibiting effects on activity of both H7N9 wild-type and mutant-type influenza virus neuraminidase, the anti-influenza effect of combined usage of calcein and oseltamivir at a cellular level is detected, and it is found that combined usage of calcein and oseltamivir can have a treatment effect on cells infected with H7N9 wild-type and mutant-type influenza viruses; finally, the influence of combined usage of calcein and oseltamivir on a mouse influenza model is detected, reduction of weight of a mouse with influenza can be obviously relieved, the mouse gradually restores to the healthy level, and the lung index of the mouse with the influenza can be significantly decreased.

Description

technical field [0001] The invention specifically relates to the application of the combination of calcein and oseltamivir in the medicine for preventing and treating H7N9 influenza virus. Background technique [0002] Influenza is an acute viral respiratory infectious disease caused by influenza virus. It is highly contagious and spreads rapidly. It brings serious threats to global human health and huge economic losses every year. It is still beyond the control of human beings. Worldwide infectious disease. The randomness of influenza outbreaks is not regular at all, and it is impossible to predict the next virus outbreak. So far, it is impossible to effectively prevent the occurrence of unknown influenza through vaccines, and anti-influenza virus drugs have become the most effective means to treat the development of influenza. In recent years, significant progress has been made in targeted research on inhibitor drugs, mainly including matrix protein 2 (M2) ion channel in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/365A61K31/215A61P31/16
CPCA61K31/215A61K31/365A61K2300/00
Inventor 王烨房学迅于淼李源博张金瑞李响于大海
Owner JILIN UNIV
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