Entecavir (ETV) exhibits potent antiviral activity in patients chronically infected with wild-type or lamivudine (3TC)-resistant (3TC r ) hepatitis B virus (HBV). Among the patients treated in phase II ETV clinical trials, two patients for whom previous therapies had failed exhibited virologic breakthrough while on ETV.Isolates from these patients (arbitrarily designated patients A and B) were analyzed genotypically for emergent substitutions in HBV reverse transcriptase (RT) and phenotypically for reduced susceptibility in cultures and in HBV polymerase assays. After 54 weeks of 3TC therapy, patient A (AI463901-A) received 0.5 mg of ETV for 52 weeks followed by a combination of ETV and 100 mg of 3TC for 89 weeks. Viral rebound occurred at 133 weeks after ETV was started. The 3TC r RT substitutions rtV173L, rtL180M, and rtM204V were present at study entry, and the additional substitutions rtI169T and rtM250V emerged during ETV-3TC combination treatment. Reduced ETV susceptibility in vitro required the rtM250V substitution in addition to the 3TC r substitutions. For liver transplant patient B (AI463015-B), previous famciclovir, ganciclovir, foscarnet, and 3TC therapies had failed, and RT changes rtS78S/T, rtV173L, rtL180M, rtT184S, and rtM204V were present at study entry. Viral rebound occurred after 76 weeks of therapy with ETV at 1.0 mg, with the emergence of rtT184G, rtI169T, and rtS202I substitutions within the preexisting 3TC r background. Reduced susceptibility in vitro was highest when both the rtT184G and the rtS202I changes were combined with the 3TC r substitutions. In summary, infrequent ETV resistance can emerge during prolonged therapy, with selection of additional RT substitutions within a 3TC r HBV background, leading to reduced ETV susceptibility and treatment failure.Nearly 400 million people are chronically infected with hepatitis B virus (HBV) worldwide (16,19). After prolonged infections, often lasting decades, patients frequently develop severe liver disease that can lead to cirrhosis and hepatocellular carcinoma. Chronically infected patients also serve as sources of HBV transmission. There are currently three approved therapies for chronic HBV infections: interferon, lamivudine (3TC; ␤-L-2Ј,3Ј-dideoxy-3Ј-thiacytidine), and adefovir-dipivoxil, the prodrug of adefovir [ADV; 9-(2-phosphonylmethoxyethyl)adenine] (18). Interferon is administered subcutaneously and is associated with numerous adverse events, some of which can be severe, and a sustained antiviral response in only 30 to 40% of treated patients (30). 3TC treatment, administered orally, is effective in reducing viral loads but results in the frequent emergence of drug-resistant HBV due to substitutions at the Tyr-Met-Asp-Asp (YMDD) nucleotide binding site motif of viral DNA polymerase. Data from four large clinical trials revealed 3TC resistance (3TC r ) mutations in 24, 42, 53, and 70% of patients after 1, 2, 3, and 4 years of therapy, respectively (15). Treatment with ADV, recently approved by the U.S. Food and Drug Administration,...

Comprehensive monitoring of genotypic and phenotypic antiviral resistance was performed on 673 entecavir (ETV)-treated nucleoside naïve hepatitis B virus (HBV) patients. ETV reduced HBV DNA levels to undetectable by PCR (<300 copies/mL, <57 IU/mL) in 91% of hepatitis B e antigen (HBeAg)-positive and -negative patients by Week 96. Thirteen percent (n ‫؍‬ 88) of the comparator lamivudine (LVD)-treated patients experienced a virologic rebound (>1 log increase from nadir by PCR) in the first year, with 74% of these having LVD resistance (LVDr) substitutions evident. In contrast, only 3% (n ‫؍‬ 22) of ETV-treated patients exhibited virologic rebound by Week 96. Three ETV rebounds were attributable to LVDr virus present at baseline, with one having a S202G ETV resistance (ETVr) substitution emerge at Week 48. None of the other rebounding patients had emerging genotypic resistance or loss of ETV susceptibility. Genotyping all additional ETV patients with PCR-detectable HBV DNA at Weeks 48, 96, or end of dosing identified seven additional patients with LVDr substitutions, including one with simultaneous emergence of LVDr/ETVr. Generally, ETV patients with LVDr were detectable at baseline (8/10) and most subsequently achieved undetectable HBV DNA levels on ETV therapy (7/10). No other emerging substitutions identified decreased ETV susceptibility. In conclusion, ETVr emergence in ETV-treated nucleoside naïve patients over a 2-year period is rare, occurring in two patients with LVDr variants. These findings suggest that the rapid, sustained suppression of HBV replication, combined with a requirement for multiple substitutions, creates a high genetic barrier to ETVr in nucleoside naïve patients.

Entecavir (ETV) is a deoxyguanosine analog approved for use for the treatment of chronic infection with wild-type and lamivudine-resistant (LVDr) hepatitis B virus (HBV).In LVD-refractory patients, 1.0 mg ETV suppressed HBV DNA levels to below the level of detection by PCR (<300 copies/ml) in 21% and 34% of patients by Weeks 48 and 96, respectively. Prior studies showed that virologic rebound due to ETV resistance (ETVr) required preexisting LVDr HBV reverse transcriptase substitutions M204V and L180M plus additional changes at T184, S202, or M250. To monitor for resistance, available isolates from 192 ETV-treated patients were sequenced, with phenotyping performed for all isolates with all emerging substitutions, in addition to isolates from all patients experiencing virologic rebounds. The T184, S202, or M250 substitution was found in LVDr HBV at baseline in 6% of patients and emerged in isolates from another 11/187 (6%) and 12/151 (8%) ETV-treated patients by Weeks 48 and 96, respectively. However, use of a more sensitive PCR assay detected many of the emerging changes at baseline, suggesting that they originated during LVD therapy. Only a subset of the changes in ETVr isolates altered their susceptibilities, and virtually all isolates were significantly replication impaired in vitro. Consequently, only 2/187 (1%) patients experienced ETVr rebounds in year 1, with an additional 14/151 (9%) patients experiencing ETVr rebounds in year 2. Isolates from all 16 patients with rebounds were LVDr and harbored the T184 and/or S202 change. Seventeen other novel substitutions emerged during ETV therapy, but none reduced the susceptibility to ETV or resulted in a rebound. In summary, ETV was effective in LVD-refractory patients, with resistant sequences arising from a subset of patients harboring preexisting LVDr/ETVr variants and with approximately half of the patients experiencing a virologic rebound.More than 350 million people worldwide are chronically infected with hepatitis B virus (HBV) (32); and many will ultimately develop severe liver disease, including cirrhosis, hepatocellular carcinoma, and liver failure. Significant improvements in patient outcomes have been realized since the use of antiviral therapy for HBV. Due to the poor efficacies of these therapies and the emergence of viral resistance, however, additional therapies are needed (16). Prior to 2005, HBV therapies included parenteral regimens containing interferon alfa and the oral nucleoside/nucleotide analogs lamivudine (LVD) and adefovir dipivoxil (ADV). However, interferon alfa shows poor response rates and poor sustained efficacy (ϳ30 to 40% [reviewed in reference 18]), has low tolerability, and is contraindicated in patients with decompensated liver disease. LVD and ADV are associated with the development of viral resistance. LVD resistance (LVDr) is reported to occur in 24% of patients treated for 1 year, and this rate increases to 70% after 4 years (19). The rate of ADV resistance (ADVr) in nucleoside-naïve HBeAg-negative HBV patients has been r...

Entecavir (ETV; Baraclude) is a novel deoxyguanosine analog with activity against hepatitis B virus (HBV).ETV differs from the other nucleoside/tide reverse transcriptase inhibitors approved for HBV therapy, lamivudine (LVD) and adefovir (ADV), in several ways: ETV is >100-fold more potent against HBV in culture and, at concentrations below 1 M, displays no significant activity against human immunodeficiency virus (HIV). Additionally, while LVD and ADV are obligate DNA chain terminators, ETV halts HBV DNA elongation after incorporating a few additional bases. Three-dimensional homology models of the catalytic center of the HBV reverse transcriptase (RT)-DNA-deoxynucleoside triphosphate (dNTP) complex, based on the HIV RT-DNA structure, were used with in vitro enzyme kinetic studies to examine the mechanism of action of ETV against HBV RT. A novel hydrophobic pocket in the rear of the RT dNTP binding site that accommodates the exocyclic alkene moiety of ETV was predicted, establishing a basis for the superior potency observed experimentally. HBV DNA chain termination by ETV was accomplished through disfavored energy requirements as well as steric constraints during subsequent nucleotide addition. Validation of the model was accomplished through modeling of LVD resistance substitutions, which caused an eightfold decrease in ETV susceptibility and were predicted to reduce, but not eliminate, the ETV-binding pocket, in agreement with experimental observations. ADV resistance changes did not affect the ETV docking model, also agreeing with experimental results. Overall, these studies explain the potency, mechanism, and cross-resistance profile of ETV against HBV and account for the successful treatment of naive and LVD-or ADV-experienced chronic HBV patients.More than 350 million people worldwide are chronically infected with hepatitis B virus (HBV), and a significant proportion of them will ultimately develop severe liver disease, including cirrhosis, hepatocellular carcinoma, and other severe complications (42). Entecavir (ETV; formerly called BMS-200475), lamivudine (LVD or 3TC; ␤-L-2Ј,3Ј-dideoxy-3Ј-thiacytidine), adefovir-dipivoxil prodrug [ADV or PMEA; 9-(2-phosphonylmethoxyethyl) adenine], and most recently, telbivudine (LdT) are oral HBV nucleoside/ tide reverse transcriptase inhibitors (NRTIs) approved for the treatment of chronic HBV infection. Upon entry into the cell, all of these inhibitors require subsequent phosphorylation by cellular enzymes to generate their active moieties. The combination of intrinsic potency, exposure level, efficiency of intracellular phosphorylation, and genetic barriers to resistance all contribute to the initial and long-term efficacy of these molecules.NRTIs inhibit the only known enzymatic target of HBV, the viral polymerase (Pol), which is characterized by several unique biological features (reviewed in reference 39). The mRNA encoding Pol serves as the template for synthesis of genomic virion DNA through reverse transcriptase (RT) activity. The RNA template is the mRNA that...

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