A unique series of simple "unnatural" nucleosides has been discovered to inhibit hepatitis B virus (HBV) replication. Through structure-activity analysis it was found that the 3-OH group of the ␤-L-2-deoxyribose of the ␤-L-2-deoxynucleoside confers specific antihepadnavirus activity. The unsubstituted nucleosides ␤-L-2-deoxycytidine, ␤-L-thymidine, and ␤-L-2-deoxyadenosine had the most potent, selective, and specific antiviral activity against HBV replication. Human DNA polymerases (␣, ␤, and ␥) and mitochondrial function were not affected. In the woodchuck model of chronic HBV infection, viral load was reduced by as much as 10 8 genome equivalents/ml of serum and there was no drug-related toxicity. In addition, the decline in woodchuck hepatitis virus surface antigen paralleled the decrease in viral load. These investigational drugs, used alone or in combination, are expected to offer new therapeutic options for patients with chronic HBV infection.Infection with hepatitis B virus (HBV) is a major world health problem, affecting 5% of the population. More than 2 billion people have been infected with the virus, and 350 million of them are chronic carriers at risk of death from cirrhosis and liver cancer (49).Several strategies have been evaluated for the treatment of chronic HBV infection with the goal of eliminating persistent viral replication and preventing progression to chronic active hepatitis and liver failure. Currently, the only approved treatment options are alpha interferon (IFN) and lamivudine (␤-L-2Ј,3Ј-dideoxy-3Ј-thiacytidine [3TC]). Unfortunately, the rate of response to IFN is low, and drug-associated side effects are significant (24,55). Individuals who are immunosuppressed (e.g., transplant recipients or those coinfected with the human immunodeficiency virus [HIV]) rarely respond to IFN therapy (13). Lamivudine is a well-known example of the class of ␤-Lnucleoside analogs that has recently drawn attention as antiviral and anticancer agents (52). As with IFN, however, a complete antiviral response, as assessed by HBe seroconversion, is seen in only a minority of patients after 1 year of treatment (27). In addition, cessation of lamivudine therapy or development of viral resistance may lead to a marked rebound in viral replication which can be life threatening (hepatitis flare) in HIV-HBV-coinfected patients (2, 30). Lamivudine resistance is now recognized in 16 to 32% of HBV-infected patients after 1 year of treatment and in as many as 58% after 2 to 3 years (14,27,30).Since the Food and Drug Administration approved lamivudine for the treatment of HIV infection in the United States in 1996 and for HBV in 1998, intensive studies on "unnatural" L-nucleosides as agents against HIV, HBV, and herpesviruses (including Epstein-Barr virus [EBV]) and as anticancer agents have been conducted (23). Now, through an extensive structure-activity analysis, we have found that the 3Ј-OH group of the ␤-L-2Ј-deoxyribose of the ␤-L-2Ј-deoxynucleoside series confers unique specificity for anti-HBV activity. In this ...

Hepatitis B virus (HBV) is the major cause of acute and chronic hepatitis, leading to progressive development of necroinflammatory changes in the liver, which can result in cirrhosis and hepatocellular carcinoma (1, 7). Approximately 350 million people (5% of the world's population) are chronically infected with HBV, and 1 million of these patients die every year as a result of this infection (11). Although the development of an effective vaccine to prevent HBV infection has shown promising results and should lead to its eventual eradication, antiviral chemotherapy remains the only effective method to prevent the progression of the disease in chronic carriers (8). Initially, alpha interferon was used as therapy for chronic HBV infection; however, the majority of patients did not benefit, and side effects were significant in some patients (15). At present, ␤-L-2Ј,3Ј-dideoxy-3Ј-thiacytidine (lamivudine) is the only nucleoside analogue approved for use for the treatment of chronic hepatitis B; however, upon the cessation of treatment serum HBV DNA levels return to pretreatment levels. This rebound is also associated with the appearance of drug-resistant virus that is mutated at the active site of the viral reverse transcriptase (9). Therefore, the development of new antiretroviral agents active against HBV is needed.Recently, ␤-L-thymidine (L-dT) and ␤-L-2Ј-deoxycytidine (LdC) were shown to be potent and specific inhibitors of HBV replication both in vivo and in vitro (50% effective concentrations [EC 50 s], 0.19 to 0.24 M in human hepatoma 2.2.15 cells) (2). In a phase I-II clinical trial, treatment with L-dT has also been demonstrated to cause marked reductions in HBV DNA levels in chronically infected patients

The formation of 3'-amino-3'-deoxythymidine (AMT) in patients receiving 3'-azido-3'-deoxythymidine (zidovudine) and the potential role of this metabolite in zidovudine-induced toxicity was recently demonstrated by our laboratory. This study evaluated the formation of AMT versus cytochrome P450 (P450) content, cytochrome B5 (B5) content and the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase activity in human liver microsomes obtained from 24 different donors. Significant interindividual differences in total P450 content and P450 reductase activity were observed, whereas no variation was observed in B5 content. Of particular importance, metabolism of zidovudine to AMT varied widely and correlated with P450 content but not with B5 content or P450 reductase activity. The apparent values for the Michaelis-Menten constant and the maximum rate of metabolism of the reaction were 46.1 mmol/L and 3.5 nmol/min/mg microsomal protein. These large variations of AMT levels as a function of P450 suggest that major interindividual differences may be observed in the pharmacokinetics and formation of this metabolite that may affect the pharmacodynamic properties of zidovudine. Potential drug-drug interactions may occur with therapeutic agents that interact with or induce P450 (zidovudine).

Exposure to 10 &M L-FddCMP-bisSATE led to formation of intracellular L-FddCTP levels of 410.1(+/-) +/- 46.2 and 242.1 +/- 13.2 pmol/10(6) cells in unstimulated and PHAstimulated PBM cells, respectively; whereas, exposure of cells to the parent nucleoside, L-FddC, generated 5-10-fold less L-FddCTP. In Hep-G2 cells and EGF/HGF stimulated and unstimulated primary cultured hepatocytes, the active metabolite reached 113 +/- 29, 23.9 +/- 15.6, and 20.6 +/- 10.5 pmol/10(6) cells. Three other metabolites, L-FddCMP-monoSATE, L-FddCMP-SH, and M I, were detected intracellularly and extracellularly in all cell types examined. Intravenous administered dose of 3 mg/kg L-FddCMP-bisSATE to rhesus monkeys resulted in plasma concentration levels of 2.06 +/- 1.00 and 0.39 +/- 0.15 &M of L-FddCMP-monoSATE and L-FddC, respectively, while the prodrug was completely cleared metabolically within 15 min. Following oral administration of an equivalent dose, the absolute oral bioavailability of L-FddC derived from L-FddCMP-bisSATE administration was 65%.

.28).Recent findings have indicated that several ␤-L-nucleoside analogs exhibit high and potent antiviral activity against both human immunodeficiency virus (HIV) (12) and hepatitis B virus (HBV) (2, 3, 5, 9, 13) replication accompanied by low host cellular toxicity when compared to their respective natural ␤-D-counterparts (15). These findings prompted a search for novel ␤-L-nucleoside derivatives which could have synergistic activities and/or do not exhibit cross-resistance with currently available chemotherapeutic nucleoside analogs so that they could be used in combination therapy regimens. We have found that ␤-L-dideoxyadenosine (ddA)-5Ј-triphosphate (␤-LddATP) is a potent inhibitor of both HIV type 1 reverse transcriptase and woodchuck hepatitis virus DNA polymerase (A. Faraj, L. Placidi, C. Perigaud, E. Cretton-Scott, G. Gosselin, L. T. Martin, C. Pierra, R. F. Schinazi, J. L. Imbach, and J. P. Sommadossi, Prog. Abstr. 13th Int. Round Table Nucleosides Nucleotides Biol. Appl., abstr. 135, 1998). However, ␤-L-ddA per se has a limited anti-HBV activity (50% effective concentration [EC 50 ], 5 to 6 M) in HBV DNA-transfected human hepatoblastoma-derived HepG2 cells (2.2.15 cells) and no anti-HIV activity (EC 50 , Ͼ100 M) in peripheral blood mononuclear cells (PBMC) (1,4,8,10). Previous studies showed that ␤-L-ddA is phosphorylated by 2Ј-deoxycytidine kinase (EC 2.7.1.74) with a high K m of 220 M (6). In addition, ␤-L-ddA is not a substrate for the catabolic enzyme purine nucleoside phosphorylase (EC 2.4.2.1) (11). The purpose of the present study was to investigate the intracellular metabolism of ␤-LddA in HepG2 cells, PBMC, and primary cultured hepatocytes in order to understand its limited in vitro antiviral activity. MATERIALS AND METHODS ␤-L-ddA was synthesized as previously described (10). [2Ј,3Ј,8-3 H]␤-L-ddA (18.2 mCi/mmol) was obtained by tritium reduction of ␤-L-2Ј,3Ј-didehydro-2Ј,3Ј-dideoxyadenosine (Moravek Biochemical). [2Ј,3Ј,8-3 H]␤-L-ddA was prepared by heterogeneous catalytic exchange with tritium gas in the presence of palladium catalyst and was Ͼ96% pure as ascertained by the high-performance liquid chromatography (HPLC) method described below. The presence of the tritium in both the base and L-dideoxyribose allowed us to follow the metabolism of this molecule.Cell culture conditions and preparation of samples. HepG2 cells were grown in 225-cm 2 tissue culture flasks in minimal essential medium with nonessential amino acids supplemented with 10% heat-inactivated dialyzed fetal bovine serum (FBS), 1% sodium pyruvate, and 1% penicillin-streptomycin. The medium was changed every 3 days, and the cells were subcultured once a week. After detachment of the adherent monolayer with a 10-min exposure to 30 ml of trypsin-EDTA and three consecutive washes with medium, confluent HepG2 cells (2 ϫ 10 6 /ml) were resuspended in a final volume of 10 ml of medium per time period and exposed to 10 M [ 3 H]␤-L-ddA (1,000 dpm/pmol). The cells were maintained at 37°C under a 5% CO 2 atmosphere for specified ti...