Inhibition of human hepatitis B virus (HBV) by a novel non-nucleosidic compound in a transgenic mouse model

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Abstract

BAY 41-4109 is a member of a class of heteroaryl-pyrimidines that was recently identified as potent inhibitors of human hepatitis B virus (HBV) replication. We have investigated the antiviral activity of BAY 41-4109 (methyl (R)-4-(2-chloro-4-fluorophenyl)-2-(3,5-difluoro-2-pyridinyl)-6-methyl-1,4-dihydro-pyrimidine-5-carboxylate) in HBV-transgenic mice (Tg [HBV1.3 fsX3′5′]). Bay 41-4109 was administered per os using different schedules (b.i.d. or t.i.d. for up to 28 days) and dosages ranging from 3 to 30 mg/kg. The compound reduced viral DNA in the liver and in the plasma dose-dependently with efficacy comparable to 3TC. In contrast to 3TC-treated mice, we found a reduction of cytoplasmic hepatitis B virus core antigen (HBcAg) in liver sections of BAY 41-4109-treated mice, which indicated a different mode of action. Pharmacokinetic studies in mice have shown rapid absorption, a bioavailability of 30% and dose-proportional plasma concentrations. We conclude that BAY 41-4109 is a new anti-HBV drug candidate.

Introduction

Over 250 million people worldwide are persistently infected with human hepatitis B virus (HBV). As a direct consequence of chronic HBV infection, these people have an increased risk of developing liver cancer. Hepatitis B vaccines have been available for a long time, but HBV infection remains a global health problem, responsible for about 1.2 million deaths annually. New efficacious anti-HBV drugs have been developed recently.

The efficacy of interferon (IFN) is partial and of limited duration, with less than 30% of the chronic carriers being treated with IFN responding to treatment. In addition, approximately 50% of those patients who respond to IFN therapy experience recurrence of viremia after cessation of the treatment (Fattovich et al., 1988, Thomas, 1998). The many side effects and lack of universal applicability of IFN have led to the investigation of other compounds. Nucleoside analogues such as Lamivudine [3TC, (−)-β-l-2′,3′-dideoxy-3′-thiacytidine] or Adefovir [9-(2-phosphonylmethoxyethyl)adenine] are now used or are in trials to establish a role in first line therapy (Poordad and Gish, 1999, DeClercq, 2001). However, despite the fact that anti-HBV drugs are capable of reducing viral loads very rapidly, the initial response is followed by a slow elimination of residual virus. In addition, the emergence of drug resistance during the slower phase of HBV elimination occurs frequently in 3TC-treated patients and limits a long termed treatment of HBV infection. Therefore, new and effective anti-HBV drugs are highly desired.

Here, we report that BAY 41-4109 (methyl (R)-4-(2-chloro-4-fluorophenyl)-2-(3,5-difluoro-2-pyridinyl)-6-methyl-1,4-dihydro-pyrimidine-5-carboxylate) is active in a transgenic mouse model of HBV infection.

Section snippets

HepG 2.2.15 cell assay for inhibition of HBV replication

Compounds were tested for their ability to inhibit HBV replication as described previously (Korba and Gerin, 1992). Briefly, HepG2.2.15 (Sells et al., 1987) cells were plated in 96 well plates and incubated together with dilutions of the compound at 37 °C and 5% CO2. Medium was changed at day 4. Eight days after exposure to the compounds began, the supernatants were collected, the cells lysed and assayed for the presence of HBV-DNA by dot-blot hybridization and LumiImager quantification (Roche

BAY 41-4109 reduces HBV-specific DNA in HBV-transgenic mice

In experiments using HepG2.2.15 cells (Sells et al., 1987) the inhibitory concentration 50 (IC50) of BAY 41-4109 (Fig. 1) against HBV was estimated to be 53 nM, the IC50 of 3TC against HBV was estimated to be 220 nM.

The pharmacokinetic data showed that even low dosages are sufficient to exceed in vitro IC50 levels in plasma (see below and Table 1). However, since the compound displayed a relatively short half-life of 2 h in mice, we decided to administer the compound twice a day (b.i.d.).

We

Discussion

The purpose of our study was the characterization of a novel class of heteroaryl-pyrimidines in a relevant animal model of HBV replication. Several animal models have been described. Whereas the duck hepatitis B virus (DHBV) (Mason et al., 1980) has been the hepadnavirus of choice for molecular studies of hepadnaviruses, the woodchuck—woodchuck hepatitis B virus (WHV) (Summers et al., 1978) model provides the best studied in vivo system for investigating the pathogenesis of virus-caused

Acknowledgments

We thank Uwe Reimann, Holger Dethlefsen and Diana Guntermann for excellent technical support.

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