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.Despite the existence of efficient vaccines, hepatitis B virus (HBV) infection remains a major public health problem worldwide with 400 million chronic carriers, who are exposed to a risk of developing liver cirrhosis and hepatocellular carcinoma (24). To date, alpha interferon and lamivudine (2Ј,3Ј-dideoxy-3Ј-thiacytidine, or 3TC) are the only approved drugs for chronic HBV infection. Alpha interferon therapy is only partially effective and is frequently limited by adverse effects (16). 3TC, a cytidine analog, is a very efficient inhibitor of HBV replication (23). Although 3TC efficiently inhibits HBV replication, the slow kinetics of viral elimination during 3TC therapy (33) and the spontaneous viral genome variability lead to the emergence of drug-resistant mutants which carry mutations affecting the reverse transcriptase (RT) domain (30,32,31,36). Approximately 50% of treated patients develop viral resistance after 3 years of treatment with 3TC (26). Resistance to nucleoside analogs is associated with substitutions in the nucleic acid sequence of the polymerase gene causing changes in the amino acid sequence of the HBV RT, notably in the YMDD motif within the catalytic site. The most common polymerase variant is the rtL180M-plus-M204V change (according to the r...

A newly developed in vitro model of the human epithelial airway barrier to study the toxic potential of nanoparticles

“…This inhibitory activity closely resembles that obtained using the DHBV polymerase in the rabbit reticulocyte lysate (Seigneres, 2003). It is clear that the exact determination of the IC 50 measured in our work requires further investigation, because the assay was based on the presence of the endogenous deoxynucleotides for the reverse transcription activity.…”

“…Several animal studies have been performed in the past for the screening of new antiviral molecules that inhibit the hepadnavirus replication, either in vivo with studies involving the sacrifice of the experimental animals (Addison et al, 2002;Cao and tavis, 2006;Cavanaugh et al, 1997;Colledge et al, 1997;Cullen et al, 1997;Fourel et al, 1994;Genovesi et al, 2000;Hafkemeyer et al, 1996;Howe et al, 1996;Lin et al, 1998;Lofgren et al, 1996;Luscombe et al, 1996;Nicoll et al, 2000;Nicoll et al, 1998;Offensperger et al, 1993a;Rahn et al, 1997;Rajagopalan et al, 1996;Seigneres et al, 2000;Severini et al, 1995;Tomita et al, 2000;Urban et al, 2001;Witcher et al, 1997) or in vivo with additional in vitro studies using the rabbit reticulocyte lysate system (Aguesse-Germon et al, 1998;Doong et al, 1991;Guo et al, 2007;Jacquard et al, 2006;Le Guerhier et al, 2001;Offensperger et al, 1993b;Robaczewska et al, 2005;Seifer et al, 1998;Seigneres et al, 2001;Seigneres et al, 2003;Shaw et al, 1996;Zoulim et al, 1996) (Tab. 1).…”

Mechanism of viral persistence and resistance to nucleoside and nucleotide analogs in chronic Hepatitis B virus infection

“…The combination of such compounds was found to be either additive and more rarely synergistic in polymerase assays (reverse transcriptase activity) (Seigneres et al, 2003) as well as in tissue culture experiments (viral DNA synthesis) or in chronically infected woodchucks (Colledge et al, 2000;Colledge et al, 1997;Korba, 1996;Korba et al, 2000;Seigneres et al, 2003).…”

“…Although some of the combinations based on the inhibition of all three steps of viral genome replication inhibited more potently viral DNA synthesis than the single drugs, none were able to completely prevent the initial formation of viral cccDNA following de novo infection of hepatocytes, or to clear cccDNA once chronic infection of hepatocytes has been established in tissue culture (Seigneres et al, 2003). However, such combinations by inhibiting more potently viral DNA synthesis may delay the onset of viral resistance by limiting the chance of a given mutation to occur.…”

Effect of a Combination of Clevudine and Emtricitabine with Adenovirus-Mediated Delivery of Gamma Interferon in the Woodchuck Model of Hepatitis B Virus Infection

“…However, clearance of viral cccDNA was never observed with either FTC or L-FMAU (8,29,43). Furthermore, the combination of L-FMAU and FTC (L-FMAU/ FTC) was shown to be additive in vitro in tissue culture and in vivo in the DHBV model (33). Both compounds are currently under evaluation in human clinical trials (phase II for L-FMAU and phase III for FTC) for their efficacy in the treatment of chronic hepatitis B (11).…”