The Anti-HBV Activity And Mechanism Of Novel Nucleosides And Preliminary Identification Of Its Metabolites In Rat Plasma

Objective:The morbidity and mortality of liver cirrhosis,hepatocellular carcinoma and other related diseases caused by hepatitis B virus?HBV?infection continue to increase,and HBV infection has long been a serious threat to human health with multiple transmission routes,long course and complicated pathogenesis^[1].At present,anti-hbv drugs are mainly interferon?IFN?and necleoside?acid?analogues?NAs?.Although these two drugs can inhibit HBV replication,they can not completely eliminate the covalently closed circular DNA?cccDNA?in the nucleus of hepatocytes,and long-term use will produce toxic side effects and drug resistance,so it is difficult to achieve the desired therapeutic goals^[2].Therefore,the search for safe and effective therapeutic drugs is the common goal of relevant researchers.Our research team designed some nucleoside compounds:GY001-GY005.This project aims to screen the anti-HBV activity of the five compounds,explore the anti-hbv mechanism of the compounds with better activity,and conduct the identification experiment of metabolites in rat plasma to provide data support for the non-clinical pharmacokinetics of the compounds.Methods:In this study,human hepatoma HepG2 cells,Wild-type HepG2.2.15 cells transfected with HBV gene and HepG2.2.15 cells of lamivudine-resistant HBV gene were used as screening models,and lamivudine?3TC?at a concentration of 20?M was selected as a positive drug^[3].HBsAg and HBeAg were used as indicators to compare the anti-hbv activity of these five compounds in vitro,and their effects on cell supernatant and intracellular HBV DNA copy number were detected.The best active compounds were selected to explore their anti-HBV mechanism.On this basis,the most active compound GY005 was tested for metabolites in rat plasma,which laid the foundation for the later pharmacokinetic experiments of these compounds.The study is divided into three parts,the contents of which are as follows:Part?Antihbv activity of compound GY001-GY005 in vitroThe toxicity of the compound to HepG2 cells was determined by methyl thiazolyl tetrazolium assay?MTT?,and the Concentration of cytotoxicity 50%(CC₅₀)of the cells was calculated;The enzyme-linked immunosorbent assay?ELISA?was used to detect the drug in the supernatant after 6 days.The changes of HBsAg and HBeAg were compared,and Comparison of the inhibitory effects of five compounds on the expression of HBV antigen;Real-time fluorescent Quantitative polymerase chain reaction method?Quantitative Real-time PCR,FQ-PCR?to detect compounds in cells after 6 days,compounds inside the cells and the effect of HBV DNA copy number in the cell supernatant,calculate the compounds of HepG2.2.15 cells and HepGRL1cells in supernatant and HBV DNA copy number in the cell inhibition rate,and compared with positive drug inhibition rate,determine the effect of anti-HBV in vitro.Part?Study on the mechanism of GY005 anti-HBVReverse transcription polymerase chain reaction?RT-PCR?was used to detect the effect of GY005 on the mRNA expression of HBV polymerase?Polymerase,P?gene in HepG2.2.15 cells.HepG2.2.15 cells were cultured on a 6-well cell plate,and the dosing group,positive group and blank group were set.After 6 days,cells were collected and RNA was extracted.RT-PCR was performed after reverse transcription.Part?Preliminary identification of GY005 metabolites in rat plasma.SD rats were selected,GY005 homogenized with CMC-Na,blood was collected from the eyelids after intragastric administration,plasma was separated,and its metabolites were detected by LC-MS/MS.The data were analyzed and compared with the standard products to identify metabolites.Results:1.MTT assay showed that GY001-GY005 was less toxic to HepG2 cells in vitro,and its CC₅₀ was greater than 500?M.2.The inhibition rate of HepG2.2.15 and HepGRL1 antigens by the same concentration?1?M?of GY001-GY005 was detected by ELISA.The results showed that GY001-GY005 inhibited the secretion of HBV antigen,and GY005 activity was slightly better.3.The results of FQ-PCR showed that GY001-GY005 significantly reduced the level of HBV DNA in the supernatant and cells,and the inhibition rate of the copy number was concentration-dependent.Six days after administration of HepG2.2.15cells,the activity of GY005 was more prominent.The inhibition rate of 1?M GY005on HepG2.2.15 cell supernatant and intracellular HBV DNA was 81.07%?p<0.01?and 71.85%?p<0.01?,respectively.The inhibition rate of 3TC group was about75%;The most prominent effect of HepGRL1 cells was GY005 for 6 days.The inhibition rate of HepGRL1 cell supernatant and intracellular HBV DNA by 1?M GY005 was as high as 72.97%?p<0.01?and 80.05%?p<0.01?,respectively,and the inhibition rate of 3TC group was about 20%.4.The results of RT-PCR showed that the brightness of HBV P gene mRNA bands in cells was significantly decreased after GY005 treatment compared with the blank control group and the positive control group.This indicates that GY005 can inhibit the expression of HBV P gene mRNA.5.The results of LC-MS/MS showed that the GY001,GY002 and GY003metabolites were less in the body after GY005 administration for 2 hours,while the prototype?GY005?was completely hydrolyzed.Conclusion:1.GY001-GY005 compounds are less toxic.2.GY001-GY005 compound has obvious anti-HBV effect,and the best activity is GY005.Its mechanism of action is to inhibit the expression of HBV P gene mRNA at the transcriptional level.3.GY005 is easily hydrolyzed in rats,and the hydrolyzed metabolites are GY001,GY002 and GY003.