Application of n-formyl-3,4-methylenedioxybenzyl-γ-butyrolactone in the preparation of anti-hepatitis B virus medicine

A hepatitis B virus and drug technology, applied in the field of medicine, can solve the problem that there is no report on the inhibitory effect of hepatitis B virus

Active Publication Date: 2018-11-23
康珞生物科技(武汉)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] There is no report on the direct inhibitory effect of KNK437 on HBV

Method used

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  • Application of n-formyl-3,4-methylenedioxybenzyl-γ-butyrolactone in the preparation of anti-hepatitis B virus medicine
  • Application of n-formyl-3,4-methylenedioxybenzyl-γ-butyrolactone in the preparation of anti-hepatitis B virus medicine
  • Application of n-formyl-3,4-methylenedioxybenzyl-γ-butyrolactone in the preparation of anti-hepatitis B virus medicine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] [Example 1] Cytotoxicity of KNK437 on HepG2.215 and Huh7 cell lines

[0030] HepG2.2.15 and HuH7 cells were treated with 10 4 The amount of cells / well was seeded in a 96-well plate, and after 12 hours of culture, different concentrations of KNK437 were added for stimulation. The concentrations of KNK437 were: 0 μM, 0.1 μM, 1 μM, 10 μM, 20 μM, 50 μM, 100 μM, 500 μM; treatment time 2 days, 4 days, and 6 days, respectively. In the final test, remove the supernatant, wash three times with PBS, and then add fresh serum-free medium containing 10% CCK-8 solution, and continue to incubate for 2 hours. Microplate reader to measure OD of each well 490 absorbance value. The absorbance value of the unmedicated control was taken as the relative value 1 of the cell growth rate, and the absorbance values ​​of other wells were compared with it to obtain the relative value of the respective cell growth rate.

[0031] Experimental results ( figure 1 ), it can be seen that KNK437 has ...

Embodiment 2

[0033] [Example 2] Detection of HBsAg and HBeAg in the supernatant of three liver cancer cell lines treated with KNK437

[0034] (1) Add 1 μM, 10 μM and 20 μM KNK437 to HepG2.2.15 and Huh7 cells transfected with 1.1 / 1.3×HBV plasmid, and 30 nM entecavir (ETV) as an anti-HBV positive control drug for 6 days, every two days The medium was changed, and the cell supernatant was stored at -40°C after 6 days of collection;

[0035] (2) 20 μM KNK437 was added to HepG2.2.15 and Huh7 cell culture medium transfected with 1.1 / 1.3×HBV plasmid, and the cells were treated for 2 days, 4 days, 6 days, and the positive control 30nM ETV for 6 days. Change the fresh culture medium every two days, and collect the cell supernatant at -40°C for use;

[0036] (3) The cell supernatants collected in (1) and (2) were thawed and centrifuged at 10,000 g for 5 minutes to remove cell residues, and then the supernatants were preheated in a 37°C incubator for 30 minutes;

[0037] (4) Take 500 μl of each sam...

Embodiment 3

[0046] [Example 3] Detection of viral mRNA levels in three liver cancer cell lines treated with KNK437

[0047] (1) 1μM, 10μM and 20μM KNK437, and 30nM entecavir (ETV) were added to HepG2.2.15 inoculated in six-well plate and Huh7 cell culture medium transfected with 1.1 / 1.3×HBV plasmid as anti-HBV positive control After 6 days of drug treatment, the medium was changed every two days. After 6 days, the corresponding RNAiso Plus lysis solution was added to each well of the cells. Generally, 1 ml of RNAiso Plus lysis solution was added to each well of a six-well cell culture dish. Place at room temperature for 5 minutes to fully lyse the cells, blow the cells evenly, and then collect them into a 1.5ml RNase-free centrifuge tube;

[0048] (2) 20 μM KNK437 was added to HepG2.2.15 inoculated in six-well plate and Huh7 cell culture medium transfected with 1.1 / 1.3xHBV plasmid, and the cells were treated for 2 days, 4 days, 6 days, and positive control 30nM ETV was treated for 6 days...

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Abstract

The invention discloses the functions in resisting hepatitis B virus (HBV) of N-formoxyl-3,4-methylenedioxy-benzyl-gamma-butyrolactone (KNK437). The applications comprise the following specific analysis steps: processing three cell lines by KNK437 at different concentrations for certain time or processing three cell lines by KNK437 at specific concentration for different time, then respectively collecting the cell culture supernatant, total RNA in cells and DNA in cells, and carrying out detection sequentially by adopting the euzymelinked immunosorbent assay and the fluorogenic quantitative PCR, wherein the result shows that KNK437 can obviously reduce the secretion of hepatitis B associated antigens, and inhibit the transcription and copying of hepatitis B viruses in the cells, namely, the medicine has extremely great clinical application prospects in the hepatitis B treatment, the action targets are different from the action targets of the traditional HBV-resisting medicines, and therefore, KNK437 is expected to overcome the defects of large side effects, low response rate and easy formation of drug-resistant strains existing in nucleotide analog medicines.

Description

technical field [0001] The invention belongs to the technical field of medicine, and relates to a new medicinal use of N-formyl-3,4-methylenedioxybenzyl-γ-butyrolactone (KNK437), in particular to the preparation of an anti-hepatitis B virus medicine. applications in . Background technique [0002] Hepatitis B virus (HBV) is a small-molecule DNA virus that can replicate by reverse transcription, which can infect the host and cause hepatitis B (Hepatitis B) disease. HBV infection is a worldwide public health problem. About 2 billion people in the world have been infected with HBV, of which about 350 million developed chronic hepatitis B. Chronic infection, however, can lead to liver fibrosis, cirrhosis, and liver cancer. Since 1992, my country has implemented the policy of free vaccination of newborns. After vaccination, the carrier rate of children under 5 years old has dropped to 0.96%. Vaccination is a very effective way to prevent hepatitis B, but its protection rate can...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K31/4025A61P31/20
CPCA61K31/4025
Inventor 胡康洪黄亚运程智逵孙鸽魏艳红
Owner 康珞生物科技(武汉)有限公司
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