Effects of pyrimidine and purine analog combinations in the duck hepatitis B virus infection model

Abstract

To design new strategies of antiviral therapy for chronic hepatitis B, we have evaluated the antiviral activity of the combination of amdoxovir (DAPD), emtricitabine [(-)FTC], and clevudine (L-FMAU) in the duck hepatitis B virus (DHBV) model. Using their triphosphate (TP) derivatives in a cell-free system expressing a wild-type active DHBV reverse transcriptase (RT), the three dual combinations exhibited a greater additive inhibitory effect on viral minus-strand DNA synthesis than the single drugs, according to the Bliss independence model. Both dual combinations with DAPD TP were the most efficient while the triple combination increased the inhibitory effect on the DHBV RT activity in comparison with the dual association, however, without additive effect. Postinoculation treatment of experimentally infected primary duck hepatocytes showed that dual and triple combinations potently inhibited viral DNA synthesis during treatment but did not inhibit the reinitiation of viral DNA synthesis after treatment cessation. Preinoculation treatment with the same combinations exhibited antiviral effects on intracellular viral DNA replication, but it was unable to prevent the initial covalently closed circular DNA (cccDNA) formation. Short-term in vivo treatment in acutely infected ducklings showed that the dual combinations were more-potent inhibitors of virus production than the single treatments, with the L-FMAU and FTC combination being the most potent. A longer administration of L-FMAU and FTC for 4 weeks efficiently suppressed viremia and viral replication. However, no viral clearance from the liver was observed, suggesting that the enhanced antiviral effect of this combination was not sufficient for cccDNA suppression and HBV eradication from infected cells.

FIG. 1.

Qualitative analysis of the inhibitory activity of dual and triple combinations with DAPD TP, FTC TP and l-FMAU TP on wild-type RT activity. The result of one typical experiment for one of the tested combinations with two radiolabeled deoxynucleoside TPs is presented. Autoradiograms of electrophoresis show the inhibition of viral minus-strand DNA synthesis by a single drug or a dual combination. Drug concentrations are indicated at the tops of the autoradiograms.

FIG. 2.

Inhibitory effect of dual and triple combinations on intracellular DHBV DNA synthesis during drug administration and after treatment cessation in a postinoculation (A) or preinoculation (B) administration. Autoradiograms of Southern blot hybridizations of intracellular viral DNA are shown. The low concentrations of DAPD, FTC, and l-FMAU are 1, 0.5, and 0.1 μM, respectively; the high concentrations of DAPD, FTC, and l-FMAU are 2, 1, and 0.5 μM, respectively. Replication intermediates are represented as single strand (SS), linear (L), and relaxed circular (RC). Lanes: 1, no treatment; 2 and 9, DAPD; 3 and 10, FTC; 4 and 11, l-FMAU; 5 and 12, DAPD and l-FMAU; 6 and 13, DAPD and FTC; 7 and 14, FTC and l-FMAU; 8 and 15, DAPD, FTC, and l-FMAU.

FIG. 3.

Dual and triple combination therapy decreased viremia in chronically infected ducks. Experimental protocols of the two short-term studies (studies 1 [A] and 2 [B]) and the long-term study (study 3 [C]) are described in detail in Materials and Methods and in Tables 3 and 4. Viremia (in picograms/milliliter) was monitored by DHBV DNA quantification by using a dot blot assay and/or real-time PCR. The mean viremia levels for each group from studies 1, 2, and 3 are plotted on the graphs shown in panels A, B, and C, respectively; the first two are displayed on a log y axis. The black bar indicates the antiviral treatment period. The limit of detection of DHBV DNA was 200 pg/ml for dot blot analysis and 100 copies/ml for the real-time PCR assay. In studies 1 and 3, all ducks were sacrificed at the end of study while in study 2 only half of the ducks were sacrificed at the end of treatment.