Application of sofosbuvir in preparation of medicines for preventing and treating coronavirus
A coronavirus and drug technology, applied in the direction of antiviral agents, drug combinations, pharmaceutical formulations, etc., can solve problems such as the in vivo efficacy is still under observation, and there are no antiviral drugs and vaccines.
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Embodiment 1
[0030] Example 1 Virus Amplification
[0031] African green monkey kidney cells (VeroE6) were divided into 3×10 5Each well was inoculated into a 96-well plate, placed in minimum Eagle’s medium (MEM; GibcoInvitrogen) containing 10% fetal bovine serum (FBS; GibcoInvitrogen) at 37°C, 5% CO 2 Cultivate until the monolayer grows. Inoculate a 100-fold dilution of the clinical isolates of the novel coronavirus pneumonia into a 96-well plate full of monolayer cells, and place at 37°C, 5% CO 2 Culture for two days (including the normal control group).
[0032] Two days later, the lesion degree reached more than 75%. Put it in a -80°C ultra-low temperature refrigerator, freeze and thaw once repeatedly, collect the virus liquid amplified by the cells, centrifuge at 3000r / min for 30 minutes, remove the sediment, put it in a small tube and put it in a -80°C ultra-low temperature freezer Long-term preservation.
Embodiment 2
[0033] Embodiment 2 sofosbuvir drug toxicity evaluation
[0034] After the sofosbuvir powder is dissolved in DMSO, add culture medium to dilute to 20mg / mL, the final concentration of DMSO is 1%, filter through a 0.22μm filter membrane and store at 4°C; after filtering, store at 4°C. Press about 2.5×10 per hole 4 Cells were inoculated into 96-well plates. After 24-48 hours, when the cells had grown into a monolayer, the culture medium was discarded, and 100 μL / well of drugs of different dilutions were added, and 100 μL / well of MEM was added to the control wells of normal cells, and kept at 37°C in 5% CO. 2 Continue culturing for 2 to 5 days, add 20 μL of CCK8 solution (5 mg / mL) to each well, and place at 37°C in 5% CO 2 Continue to incubate for 4 hours in the incubator. Discard the culture supernatant, add 100 μL dimethyl sulfoxide (DMSO) to each well, and shake at low speed for 10 minutes to fully dissolve the crystals. Select a wavelength of 490nm, and measure the light ab...
Embodiment 3
[0035] Example 3 Sofosbuvir anti-new coronary pneumonia novel coronavirus efficacy evaluation
[0036] To evaluate the antiviral efficacy of the drug, VeroE6 cells were grown at a density of 5×10 4 Cells / well were cultured overnight in a 48-well cell culture dish. Add virus (MOI 0.05) to infect for 2 hours. Then add 2-fold gradient dilution of the drug, each concentration set 4 duplicate wells, with 200 μM as the initial concentration of the drug, at 34 ° C, 5% CO 2 Incubate for 2 days in the incubator. Record the cytopathic effect (Cytopathogenic Effect, CPE. The occurrence of CPE in the cells is recorded according to the 6th grade standard. After recording the CPE, stain with the CCK8 method to determine the OD value. Use the Reed-Muench method to calculate the half effective concentration of the drug (EC 50 ) was 1.12 μM. It can be seen from the experimental results that the antiviral half effective concentration of sodium foscarnet is low and the antiviral effect is go...
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