Hepatitis B virus
(HBV), a small DNA virus that replicates through an RNA intermediate, is a leading cause of chronic hepatitis. The World Health Organization estimated in 1996 that 350 million people were persistently infected with the virus worldwide (8
). Despite the availability of an effective vaccine against HBV, the prevalence of chronic infection has not significantly decreased (2
). Persons with chronic hepatitis B not only suffer the wide range of symptoms associated with hepatitis but are at significant risk for the development of cirrhosis and/or primary hepatocellular carcinoma. Chronic carriers of HBV, moreover, constitute a reservoir for new infections.
Therapy currently consists of treatment with alpha interferon, which is associated with several undesirable side effects and variable, sometimes low, response rates (3
), and treatment with lamivudine (3TC), a pyrimidine dideoxynucleoside (reviewed in reference 5
). While effective in reducing viral load, 3TC treatment leads to resistance in both immunocompromised and immunocompetent patients with chronic HBV infections who receive the compound for extended periods (1
). Consequently, there is an urgent need for new anti-HBV agents that are both safe and effective.
A number of compounds, most of which are nucleoside analogs that inhibit HBV polymerase and thereby interfere with replication, are currently under investigation for use in the chemotherapy of chronic HBV infection (for reviews, see references 3
, and 19
). One of the most promising novel agents is entecavir (ETV; formerly BMS-200475), a guanosine analog that displays potent and selective inhibition of HBV. In HBV-producing HepG2 2.2.15 hepatoblastoma cell cultures, ETV exhibited potency in the nanomolar range, with a 50% effective concentration (EC50
) of 0.00375 μM (7
). ETV was also shown to be selective, since it had only modest activity against a panel of six unrelated RNA and DNA viruses (EC50
s ranged from 10 to 80 μM) (7
). Moreover, the concentration of ETV needed to cause 50% cytotoxicity (CC50
) in HepG2 2.2.15 cell cultures was 30 μM, yielding a favorable selectivity index (CC50
) of 8,000. Most importantly, ETV had no appreciable adverse effect on the mitochondrial DNA of proliferating HepG2 cells (7
Studies of the mechanisms of action (14
) confirmed that ETV triphosphate directly inhibits hepadnaviral polymerases and effectively suppresses the priming and elongation steps of HBV replication. To be effective antivirals, nucleoside analogs must be efficiently converted to their active triphosphate form. Phosphorylation studies examining ETV in both HepG2 and HBV-transfected HepG2 2.2.15 hepatoblastoma cells showed that ETV is readily phosphorylated by cellular enzymes to its triphosphate form with little accumulation of the intermediate mono- and diphosphate forms of ETV (18
). In addition, the intracellular half-life was determined to be relatively long (15 h) (18
Previous in vivo studies utilizing oral treatment of woodchucks (Marmota monax
) chronically infected with Woodchuck hepatitis virus
(WHV) have demonstrated the potency and effectiveness of ETV in reducing viral DNA concentrations to undetectable levels after daily administration of 0.02, 0.1, or 0.5 mg/kg of body weight for a period of 3 months (4
). ETV was well tolerated, with no evidence of toxicity during the treatment or the 3-month posttreatment follow-up period.
The Duck hepatitis B virus
(DHBV)-infected Pekin duck (Anas platyrhynchos
) is also widely accepted as a model for evaluating new agents directed against HBV. The replication cycles of DHBV, WHV, and HBV are essentially the same (for reviews, see references 10
). The purpose of the present study was to evaluate the ability of ETV to inhibit hepadnavirus replication in this alternative HBV animal model by measuring levels of DHBV DNA in infected primary duck hepatocyte cultures and in DHBV-infected Pekin ducklings. In the in vivo evaluation, ducklings received ETV orally in three different dosages, a control group received the vehicle (distilled water) as a placebo, and a reference group received 3TC in a single dosage.