Search

A novel codrug, α-DDB-FNCG, was synthesized through coupling of α-biphenyl dimethyl dicarboxylate (α-DDB) and the nucleoside analogue FNCG, via an ester bond. The anti-HBV activity and hepatoprotective effects of this compound were investigated both in vitro and in vivo. In HBV-transfected HepG2.2.15 cell line, the secretion of HBsAg and HBeAg as well as the levels of extracellular and intracellular viral DNA were determined by ELISA and real-time fluorescent quantitative Polymerase Chain Reaction (FQ-PCR), respectively. In DHBV-infected ducks, the viral DNA levels in serum and liver were determined by FQ-PCR. In addition, the levels of alanine transaminase (ALT) and aspartate aminotransferase (AST) in both serum and liver were also examined. The improvement of ducks' livers was evaluated by histopathological analysis. It has been demonstrated that α-DDB-FNCG could suppress the levels of HBV antigens and viral DNA in a time- and dose-dependent manner in the HepG2.2.15 cell line. Furthermore, this codrug could also significantly inhibit the viral DNA replication and reduce the ALT and AST levels in both serum and liver of DHBV-infected ducks, with improved hepatocellular architecture in drug-treated ducks. In short, these results suggest that α-DDB-FNCG could be a promising candidate for further development of new anti-HBV agents with hepatoprotective effects.

Ethnopharmacological Relevance: Hydrocotyle sibthorpioides (Apiaceae) have been used as a folk remedy for the treatment of fever, edema, detoxication, throat pain, psoriasis and hepatitis B virus infections in China. The aim of this study is to isolate and identify an anti-HBV compound from this herb., Materials and Methods: A compound (saponin) was isolated from the active ethanol extract using bioassay-guided screening. The structure of the saponin was elucidated by spectroscopic methods and compared with published data. The anti-HBV activity of the saponin was evaluated by detecting the levels of HBV antigens, extracellular HBV DNA, nuclear covalent closed circular DNA (cccDNA) and five HBV promoters in HepG2.2.15 cells. In addition, the levels of serum HBsAg/HBeAg, DHBV DNA, ALT/AST and hepatic pathological changes were analyzed in DHBV-infected ducks., Results: The chemical analysis indicated that the saponin isolated from Hydrocotyle sibthorpioides is asiaticoside. The pharmacodynamics experimental studies showed that asiaticoside effectively suppressed the levels of HBsAg/HBeAg, extracellular HBV DNA and intracellular cccDNA in a dose-dependent manner. Furthermore, experiments demonstrated that asiaticoside markedly reduced viral DNA transcription and replication by inhibiting the activities of core, s1, s2, and X gene promoters. In addition, asiaticoside markedly reduced DHBV replication without any obvious signs of toxicity. The levels of serum DHBV DNA, HBsAg/HBeAg were increased 3 days after drug withdrawal, but the levels rebounded slightly in the asiaticoside treatment groups compared with the 3TC treatment group. Moreover, analysis of the serum ALT/AST levels and the liver pathological changes indicated that asiaticoside could alleviate liver damage., Conclusions: Our results show that asiaticoside could efficiently inhibit HBV replication both in vitro and in vivo, and asiaticoside may be a major bioactive ingredient in Hydrocotyle sibthorpioides., (© 2013 Published by Elsevier Ireland Ltd.)

To examine the effect of Gankang Suppository on duck hepatitis B virus (DHBV), the serum biochemistry and hepatic histology in an animal model of DHBV infection, a model of DHBV infection was established by infecting 1-day-old Yingtaogu ducklings with DHBV-positive serum. The successful model was confirmed by PCR assay and 48 ducklings infected with DHBV were randomly divided into 3 groups: a Gankang Suppository treatment group, an acyclovir (ACV) group and a DHBV model group (control), with each group having 16 animals. All the animals were given the medicines for 4 weeks in a row. The serum of the animals was taken 14 and 28 days after the medication and 7 days after drug discontinuation. Real-time PCR was performed to detect the copy numbers of DHBV DNA in the serum. ALT and AST were dynamically monitored. The ducklings were sacrificed on the 7th day after the discontinuation of the treatment and livers were harvested and examined for inflammation and degeneration of liver cells by using HE staining. The results showed that on day 14, 28 after the treatment and day 7 after the withdrawal, the logarithmic values (log) of DHBV DNA copy numbers in ducklings of Gankang Suppository treatment group were significantly lower than that before the treatment (P=0.0092, P=0.0070, P=0.0080, respectively). Compared with DHBV model control group, the ALT level was significantly decreased (P=0.0020, P=0.0019, respectively) on day 28 after the treatment and on day 7 after the withdrawal. The AST level was also reduced on day 14 after the treatment (P=0.0298). Compared with the ACV control group, the level of ALT was lower on day 7 after the withdrawal (P=0.0016). Histologically, the hepatocyte swelling, vacuolous degeneration and acidophilic degeneration in Gankang Suppository treatment group were alleviated 7 days after the withdrawal as compared with model control group (P=0.0282, P=0.0084, P=0.0195, respectively). It is concluded that Gankang Suppository can effectively suppress DHBV replication, reduce the levels of serum ALT and AST and improve hepatic histology.

The antiviral activity and ability of mismatched double-stranded RNA (m-dsRNA), r(I)n.r(C12-U)n, to induce interferon (IFN) were evaluated in ducks chronically infected with duck hepatitis B virus (DHBV). When m-dsRNA was administered intravenously at a single dose of 5 mg/kg, serum DHBV DNA concentrations decreased significantly for 3 days (P < 0.002). However, the DHBV DNA concentrations returned to the pretreatment levels 4 days after treatment. Inhibition of DHBV DNA replication in the liver was also observed 2 days after treatment. Serum IFN activity peaked 3 hours after administration of m-dsRNA, then rapidly declined. 2'-5' Oligo-adenylate synthetase (2'-5'AS) activity increased gradually after treatment and remained elevated for at least 48 hours. In ducks receiving m-dsRNA once daily for 7 consecutive days, serum DHBV DNA concentrations on the last day of treatment were decreased by 76 +/- 12% (P < 0.05) in ducks that received 0.2 mg of m-dsRNA per kg and by 65 +/- 12% (P < 0.05) in ducks that received 1 mg of m-dsRNA per kg. This decrease persisted for at least 2 weeks after the cessation of treatment in all ducks. These results suggest that m-dsRNA effectively inhibits DHBV replication in vivo, and that IFN induction and stimulation of 2'-5'AS activity contribute to the inhibition of DHBV replication by m-dsRNA.

We investigated effects of various DNAs on duck hepatitis B virus replication in vivo. One-day-old ducks were infected intravenously with DHBV. Various DNAs were then injected intravenously, and duck hepatitis B virus levels were followed for up to 20 days after the inoculation. When M13 bacteriophage DNA (M13 DNA), heat-denatured Escherichia coli DNA or phi X 174 phage DNA was injected intravenously at a dose of 2.45 mg/kg body wt daily for 10 days, a significant decrease of serum duck hepatitis B virus DNA was detected within 10 days. The efficacy was twice that reported with antisense DNA on a weight basis and far more than that reported on a molar basis. M13 DNA was superior, on the basis of effective dose, to acyclovir as an anti-duck hepatitis B virus agent. On treatment with M13 DNA, serum 2-5 A synthetase level was increased five to six times, suggesting that the antiviral effect of M13 DNA is at least partly due to induction of endogenous interferon, which in turn induces 2-5 A synthetase. No significant inhibitory effect on replication of duck hepatitis B virus was demonstrated by DNAs obtained from herring testes, herring sperm, salmon testes, human placenta or calf thymus. On discontinuation of M13 DNA injection on day 10, duck hepatitis B virus reappeared in the serum at later time points. Digestion of M13 DNA with S1 nuclease resulted in marked reduction of antiviral activity. These results show that M13 DNA, not its digested product, has potent antiviral activity.