Agonistic Activation of Cytosolic DNA Sensing Receptors in Woodchuck Hepatocyte Cultures and Liver for Inducing Antiviral Effects.

Immune modulation for the treatment of chronic hepatitis B (CHB) has gained more traction in recent years, with an increasing number of compounds designed for targeting different host pattern recognition receptors (PRRs). These agonistic molecules activate the receptor signaling pathway and trigger an innate immune response that will eventually shape the adaptive immunity for control of chronic infection with hepatitis B virus (HBV). While definitive recognition of HBV nucleic acids by PRRs during viral infection still needs to be elucidated, several viral RNA sensing receptors, including toll-like receptors 7/8/9 and retinoic acid inducible gene-I-like receptors, are explored preclinically and clinically as possible anti-HBV targets. The antiviral potential of viral DNA sensing receptors is less investigated. In the present study, treatment of primary woodchuck hepatocytes generated from animals with CHB with HSV-60 or poly(dA:dT) agonists resulted in increased expression of interferon-gamma inducible protein 16 (IFI16) or Z-DNA-binding protein 1 (ZBP1/DAI) and absent in melanoma 2 (AIM2) receptors and their respective adaptor molecules and effector cytokines. Cytosolic DNA sensing receptor pathway activation correlated with a decline in woodchuck hepatitis virus (WHV) replication and secretion in these cells. Combination treatment with HSV-60 and poly(dA:dT) achieved a superior antiviral effect over monotreatment with either agonist that was associated with an increased expression of effector cytokines. The antiviral effect, however, could not be enhanced further by providing additional type-I interferons (IFNs) exogenously, indicating a saturated level of effector cytokines produced by these receptors following agonism. In WHV-uninfected woodchucks, a single poly(dA:dT) dose administered via liver-targeted delivery was well-tolerated and induced the intrahepatic expression of ZBP1/DAI and AIM2 receptors and their effector cytokines, IFN-ß and interleukins 1ß and 18. Receptor agonism also resulted in increased IFN-γ secretion of peripheral blood cells. Altogether, the effect on WHV replication and secretion following in vitro activation of IFI16, ZBP1/DAI, and AIM2 receptor pathways suggested an antiviral benefit of targeting more than one cytosolic DNA receptor. In addition, the in vivo activation of ZBP1/DAI and AIM2 receptor pathways in liver indicated the feasibility of the agonist delivery approach for future evaluation of therapeutic efficacy against HBV in woodchucks with CHB.

Suppression of lamivudine-resistant B-domain mutants by adefovir dipivoxil in the woodchuck hepatitis virus model.

Adult woodchucks (Marmota monax) chronically infected with woodchuck hepatitis virus (WHV) were treated orally with lamivudine (15 mg/kg per day) for 57 weeks. After 20 weeks of treatment a 2-3 log reduction in serum WHV DNA was detected. Serum titers of WHV then increased gradually, in the presence of lamivudine treatment, reaching pre-treatment values by week 40. Viral recrudescence was associated with development of mutations in the B domain of the WHV polymerase gene. Mutations observed in the highly conserved FLLA motif of the B domain were L564V, L565M, and A566T, with A566T being the most frequently observed. Beginning on week 57 of lamivudine treatment, one group (n = 3) was treated orally with adefovir dipivoxil at a dose of 15 mg/kg per day plus lamivudine, and a second group (n = 3) was treated with H2O placebo plus lamivudine. In woodchucks treated with adefovir dipivoxil, two had the A566T mutation, and one had both A566T and L565V. In the group maintained on lamivudine monotherapy, A566T alone was present in one animal, another carried both A566T and L565V, and in the third, no B-domain mutations were detected. There was a 4.5 log reduction in serum WHV DNA after 12 weeks of treatment with the adefovir/lamivudine combination, while in the lamivudine monotherapy controls, WHV DNA decreased by only 0.83 log (P > 0.001). A slight recurrence in serum titers of WHV DNA was observed one week after withdrawal of adefovir treatment but no further increase in viral load was observed during the remainder of the 12-week post-treatment follow-up period. The results demonstrate that supplemental adefovir dipivoxil treatment is effective in suppressing replication of lamivudine-resistant B-domain mutants in the woodchuck model of hepatitis B virus infection.

Korean medicinal plant extracts exhibit antiviral potency against viral hepatitis.

OBJECTIVES: Investigation of natural ethnopharmacologic extracts exhibiting antiviral potential may lead to the discovery of new therapeutics for the treatment of chronic viral hepatitis infections. Traditional Korean medicinal herbs have been identified that exhibit potency against hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. Research on the antiviral potential of naturally derived extracts is facilitated through the use of appropriate animal and liver cell culture models for these hepatotrophic pathogens. Objectives of this study were to demonstrate antiviral activity of an aqueous extract of herbal formulation KYH-1 in surrogate in vitro assays for HBV and HCV and identify mechanisms of action. METHODS: Antiviral potency of KYH-1 was measured in tissue culture systems that support replication of the woodchuck hepatitis virus (WHV), and the bovine viral diarrhea virus (BVDV). These assays serve as surrogate models for HBV and HCV, respectively. A recombinant HBV polymerase gene expression assay was used to define a molecular target. RESULTS: KYH-1 exhibited potent antiviral activity against WHV and to a lesser extent against BVDV. KYH-1 and its constituent components inhibited HBV polymerase priming in vitro. Additionally, KYH-1 suppressed HBV replication in a human hepatoblastoma cell line. CONCLUSION: Evaluation of naturally derived products for antiviral activity against HBV and HCV in standardized surrogate assays provides a scientific basis for potential use as complementary or alternative medicines. This study provides significant results justifying preclinical evaluation of KYH-1 as an antiviral therapy for HBV infections.

Antiviral efficacy and pharmacokinetics of oral adefovir dipivoxil in chronically woodchuck hepatitis virus-infected woodchucks.

The antiviral efficacy of orally administered adefovir dipivoxil was evaluated in an 18-week study (12 weeks of treatment and 6 weeks of recovery) conducted with woodchucks chronically infected with woodchuck hepatitis virus (WHV). Adefovir dipivoxil is a prodrug of adefovir designed to enhance its oral bioavailability. Following administration of 15 mg of adefovir dipivoxil per kg of body weight in four WHV-infected animals, the mean maximum concentration of adefovir in serum was 0.462 microg/ml, with an elimination half-life of 10.2 h, and the oral bioavailability of adefovir was estimated to be 22.9% (+/-11.2%). To study antiviral efficacy, the animals were divided into three groups. There were six animals each in a high-dose group (15 mg/kg/day) and a low-dose group (5 mg/kg/day). A vehicle control group consisted of five animals because WHV DNA was detectable only by PCR at the time of the study in one of the original six animals. Efficacy was evaluated by determining the levels of WHV DNA in serum. The geometric mean WHV DNA level for the high-dose group diminished by >40-fold (>1.6 log(10)) after 2 weeks of treatment and >300-fold (>2.5 log(10)) at 12 weeks. There was a >10-fold reduction in five of six low-dose animals by 2 weeks, but levels were unchanged in one animal. By 12 weeks of treatment there was a >45-fold (>1.6 log(10)) reduction of WHV DNA levels, and serum WHV DNA levels were below the limit of quantification in three of six animals. Viral DNA levels returned to pretreatment levels during the 6-week recovery period. There were no clinically significant changes in body weight, hematology, or serum chemistry values, including bicarbonate or lactate, in any of the treated animals. No histologic evidence of liver injury was apparent in the biopsies. Under the conditions of this study, adefovir dipivoxil was an effective antihepadnaviral agent.

Treatment of chronic hepadnavirus infection in a woodchuck animal model with an inhibitor of protein folding and trafficking.

A novel strategy for anti-viral intervention of hepatitis B virus (HBV) through the disruption of the proper folding and transport of the hepadnavirus glycoproteins is described. Laboratory reared woodchucks chronically infected with woodchuck hepatitis virus (WHV) were treated with N-nonyl-deoxynojirimycin (N-nonyl-DNJ), an inhibitor of the endoplasmic reticulum (ER) alpha-glucosidases. The woodchucks experienced significant dose dependent decreases in enveloped WHV, resulting in undetectable amounts in some cases. The reduction in viremia correlated with the levels of hyperglucosylated glycan in the serum of treated animals. This correlation supports the mechanism of action associated with the drug and highlights the extreme sensitivity of the virus to this type of glycan inhibitor. At N-nonyl-DNJ concentrations that prevented WHV secretion, the glycosylation of most serum glycoproteins appeared unaffected, suggesting great selectivity for this class of therapeutics. Indeed, this may account for the low toxicity of the compound over the treatment period. We provide the first evidence that glucosidase inhibitors can be used in vivo to alter specific steps in the N-linked glycosylation pathway and that this inhibition has anti-viral effects.

Preclinical investigation of L-FMAU as an anti-hepatitis B virus agent.

Preclinical aspects of a potent anti-hepatitis B virus (HBV) L-nucleoside, 1-(2-fluoro-5-methyl-beta-L-arabino-furanosyl)uracil (L-FMAU) are described. L-FMAU was prepared from L-ribose derivatives via either L-xylose or L-arabinose. L-FMAU shows potent antiviral activity against hepatitis B virus (EC50 5.0 microM in H1 cells) with high selectivity in vitro. L-FMAU is not incorporated into mitochondrial DNA and no significant lactic acid production was observed in vitro. L-FMAU is phosphorylated by thymidine kinase as well as deoxycytidine kinase, ultimately to the triphosphate, which inhibits HBV DNA polymerase as the mechanism of antiviral action. Preliminary in vivo toxological studies suggest no apparent toxicity for 30 days at 50 mg/kg/day in mice and for 3 months in woodchucks (10 mg/kg/day). L-FMAU also has respectable bioavailability in rats. L-FMAU shows potent anti-HBV activity in vivo against woodchuck hepatitis virus in chronically infected woodchucks and there is no significant virus rebound after cessation of the drug treatment.