Background & Aims New therapies for chronic hepatitis B (CHB) are urgently needed since current treatments rarely lead to cure. We evaluated whether the oral small molecule toll-like receptor (TLR7) agonist GS-9620 could induce durable anti-viral efficacy in woodchucks chronically infected with woodchuck hepatitis virus (WHV), a hepadnavirus closely related to human hepatitis B virus (HBV). Methods After evaluating the pharmacokinetics, pharmacodynamics and tolerability of oral GS-9620 in uninfected woodchucks, adult woodchucks chronically infected with WHV (n=7 per group) were dosed with GS-9620 or placebo for 4 or 8 weeks with different treatment schedules. Results GS-9620 treatment induced rapid, marked and sustained reduction in serum viral DNA (mean maximal 6.2 log10 reduction), and hepatic WHV DNA replicative intermediates, WHV cccDNA and WHV RNA, as well as loss of detectable serum WHV surface antigen (WHsAg). GS-9620 treatment also induced a sustained antibody response against WHsAg in a subset of animals. Strikingly, treatment reduced the incidence of hepatocellular carcinoma (HCC) from 71% in the placebo group to 8% in GS-9620-treated woodchucks with sustained viral load reduction. GS-9620 treatment was associated with reversible increases in serum liver enzymes and thrombocytopenia, and induced intrahepatic CD8+ T cell, NK cell, B cell and interferon (IFN) response transcriptional signatures. Conclusions The data demonstrate that short duration, finite treatment with the oral TLR7 agonist GS-9620 can induce a sustained antiviral response in the woodchuck model of CHB, and support investigation of this compound as a therapeutic approach to attain a functional cure in CHB patients.

Acute hepadnavirus infections either resolve or progress to chronicity. Factors that influence chronicity as an outcome of hepatitis B virus (HBV) infection in humans can be studied experimentally in the woodchuck model. Accordingly, several woodchuck hepatitis virus (WHV) inocula were characterized. Representative inocula had high titers of infectious virus (approximately 10 7.7 -10 9.5 woodchuck 50% infectious doses per milliliter [WID 50% /mL] by subcutaneous inoculation), with 1 WID 50% ranging between 21 and 357 physical virion particles. WHV7P1 (standard high dose, 5 ؋ 10 6 WID 50% ) produced a 72% chronicity rate (i.e., percent chronic of total infected) in neonatal woodchucks (1-3 days old). Comparable doses of WHV8P1 resulted in a lower chronicity rate in neonates (34% chronic) indicating that it represented a strain different from WHV7P1. Neonatal woodchucks were more susceptible to chronic infection by high doses of WHV7P1 (range, 65%-75% chronic) compared with 8-week-old weanlings (33% chronic) and adult woodchucks (0% chronic; i.e., all resolved). High doses of cloned wild-type viruses also induced high rates of chronicity in neonates (70%-80% chronic). Chronicity rates in neonates were decreased for low doses of WHV7P1 (500 WID 50% , 9% chronic) and for high doses of a precore WHeAg-minus mutant WHV8 clone (17% chronic). Thus, both age and viral determinants can influence chronicity as an outcome of experimental WHV infection. Standardized inocula will enable the study of mechanisms that initiate and maintain chronic hepadnavirus infection and also provide a means for developing WHV carriers for therapeutic studies. (HEPATOLOGY 2000;31:190-200.)The eastern woodchuck is naturally infected by a virus closely related to hepatitis B virus (HBV). Experimental infection of woodchucks with woodchuck hepatitis virus (WHV) has been of value in modeling virtually all aspects of HBV infection, pathogenesis, and therapy. 1,2 Development of the model has evolved to use woodchucks bred and reared under controlled environmental conditions, virus-and hostspecific assays for viral infection and disease, and standardized WHV inocula. Because titered HBV inocula have proven invaluable for developing the chimpanzee as a predictive model of HBV infection, disease, and vaccine protection, 3 titered WHV inocula were considered essential for further defining host and viral determinants that promote chronicity and disease progression to hepatocellular carcinoma (HCC) in hepadnavirus infection. In this study, we measured the infectivity of several WHV inocula and, using controlled doses of virus, showed that both animal age and viral determinants can affect chronicity as an outcome of WHV infection. These standardized inocula represent a useful resource for developing serum and tissue banks from selflimited and chronic WHV infections for studies of WHV pathogenesis, and for producing chronic carrier woodchucks at predictable frequencies. MATERIALS AND METHODSAnimals. Woodchucks were bred, maintained, and handled under contr...

Woodchucks were used to study the antiviral activity and toxicity of fialuridine (FIAU; 1,-2Јdeoxy-2Јfluoro-1-␤-Darabinofuranosyl-5-iodo-uracil). In an initial experiment, groups of six chronic woodchuck hepatitis virus (WHV) carrier woodchucks received daily doses of FIAU by intraperitoneal injection for 4 weeks. At 0.3 mg/kg/d, the antiviral effect was equivocal, but at 1.5 mg/kg/d, FIAU had significant antiviral activity. No evidence of drug toxicity was observed during the 4-week period of treatment or during posttreatment follow-up. In a second experiment, groups of nine WHV carriers or uninfected woodchucks were given 1.5 mg/kg/d of FIAU orally for 12 weeks, and the results compared with placebo-treated controls. After 4 weeks, the serum WHV-DNA concentration in the FIAUtreated carrier group was two to three logs lower than that in the placebo-treated group. After 12 weeks of FIAU treatment, serum WHV DNA was not detectable by conventional dot-blot analysis, hepatic WHV-DNA replicative intermediates (RI) had decreased 100-fold, and hepatic expression of WHV core antigen was remarkably decreased. No evidence of toxicity was observed after 4 weeks, but, after 6 to 7 weeks, food intake decreased and, after 8 weeks, the mean body weights of woodchucks treated with FIAU were significantly lower than controls. Anorexia, weight loss, muscle wasting, and lethargy became progressively severe, and all FIAU-treated woodchucks died or were euthanized 78 to 111 days after treatment began. Hepatic insufficiency (hyperbilirubinemia, decreased serum fibrinogen, elevated prothrombin time), lactic acidosis, and hepatic steatosis were characteristic findings in the final stages of FIAU toxicity in woodchucks. The syndrome of delayed toxicity in woodchucks was similar to that observed previously in humans treated with FIAU, suggesting that the woodchuck should be valuable in future investigations of the molecular mechanisms of FIAU toxicity in vivo and for preclinical toxicological evaluation of other nucleoside analogs before use in patients. (HEPATOLOGY 1998;28:179-191.)

L-FMAU has markedly less toxicity than its D-enantiomer, D-FMAU, and L-FMAU does not induce an increase in lactic acid production in the human hepatoblastoma cell line, HepG2, as observed following exposure to D-FMAU and the related nucleoside analogue, D-FIAU (fialuridine). [1][2][3][5][6][7][8][11][12][13][14][15] Fialuridine demonstrated potent anti-HBV activity in clinical trials for chronic HBV infection, but also induced severe delayed toxicity associated with lactic acidosis and hepatic failure that resulted in the death of several patients. 13,14 D-FMAU, D-FIAU, and D-FEAU have been shown to be potent inhibitors of woodchuck hepatitis virus (WHV) replication in chronically infected woodchucks, but all of these nucleoside analogues also demonstrated severe toxicity at daily doses greater than 2 mg/kg body weight. 11,12,15 WHV and its natural host, the Eastern woodchuck (M. monax), constitute a useful model of HBV-induced disease, including hepatocellular carcinoma (HCC). 16,17 A variety of published studies from several laboratories have reported the use of chronic WHV infection in woodchucks to investigate potential antiviral therapies for chronic HBV infection. 11,15,[18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] 26,31,32,34,35 ribavirin, 34 thymosin ␣1, 20 vidarabine (ara-A), 21,34 zidovudine (AZT), 34 combination therapy with either lamivudine/interferon alfa 35 or lamivudine/famciclovir 32 ) have shown similar relative antiviral activities against WHV and similar toxicity profiles in chronically infected woodchucks. These studies demonstrate that the WHV/woodchuck model of chronic HBV infection can be considered to be a predictive model for new, potential therapeutic applications against HBV infection in humans, especially nucleosides.