Kevin A. Pokornowski

Long‐term monitoring shows hepatitis B virus resistance to entecavir in nucleoside‐naïve patients is rare through 5 years of therapy

Patients with chronic hepatitis B virus (HBV) infection who develop antiviral resistance lose benefits of therapy and may be predisposed to further resistance. Entecavir (ETV) resistance (ETVr) results from HBV reverse transcriptase substitutions at positions T184, S202, or M250, which emerge in the presence of lamivudine (LVD) resistance substitutions M204I/V ± L180M. Here, we summarize results from comprehensive resistance monitoring of patients with HBV who were continuously treated with ETV for up to 5 years. Monitoring included genotypic analysis of isolates from all patients at baseline and when HBV DNA was detectable by polymerase chain reaction (≥300 copies/mL) from Years 1 through 5. In addition, genotyping was performed on isolates from patients experiencing virologic breakthrough (≥1 log10 rise in HBV DNA). In vitro phenotypic ETV susceptibility was determined for virologic breakthrough isolates, and for HBV containing novel substitutions emerging during treatment. The results over 5 years of therapy showed that in nucleoside‐naïve patients, the cumulative probability of genotypic ETVr and genotypic ETVr associated with virologic breakthrough was 1.2% and 0.8%, respectively. In contrast, a reduced barrier to resistance was observed in LVD‐refractory patients, as the LVD resistance substitutions, a partial requirement for ETVr, preexist, resulting in a 5‐year cumulative probability of genotypic ETVr and genotypic ETVr associated with breakthrough of 51% and 43%, respectively. Importantly, only four patients who achieved <300 copies/mL HBV DNA subsequently developed ETVr. Conclusion: Long‐term monitoring showed low rates of resistance in nucleoside‐naïve patients during 5 years of ETV therapy, corresponding with potent viral suppression and a high genetic barrier to resistance. These findings support ETV as a primary therapy that enables prolonged treatment with potent viral suppression and minimal resistance. (HEPATOLOGY 2009.)

Clinical Emergence of Entecavir-Resistant Hepatitis B Virus Requires Additional Substitutions in Virus Already Resistant to Lamivudine

ABSTRACT Entecavir (ETV) exhibits potent antiviral activity in patients chronically infected with wild-type or lamivudine (3TC)-resistant (3TCr) 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 3TCr 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 3TCr 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 3TCr background. Reduced susceptibility in vitro was highest when both the rtT184G and the rtS202I changes were combined with the 3TCr substitutions. In summary, infrequent ETV resistance can emerge during prolonged therapy, with selection of additional RT substitutions within a 3TCr HBV background, leading to reduced ETV susceptibility and treatment failure.

Entecavir resistance is rare in nucleoside naïve patients with hepatitis B

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. (HEPATOLOGY 2006;44:1656–1665.)

Two-Year Assessment of Entecavir Resistance in Lamivudine-Refractory Hepatitis B Virus Patients Reveals Different Clinical Outcomes Depending on the Resistance Substitutions Present

ABSTRACT 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.

Comprehensive evaluation of hepatitis B virus reverse transcriptase substitutions associated with entecavir resistance

Virologic resistance emerging during entecavir (ETV) therapy for hepatitis B virus (HBV) requires three substitutions in the viral reverse transcriptase (RT), signifying a high barrier to resistance. Two of these substitutions are associated with lamivudine resistance (LVDr) in the tyrosine‐methionine‐aspartate‐aspartate (YMDD) motif (rtM204V and rtL180M), whereas the other occurs at one or more positions specifically associated with ETV resistance (ETVr): rtT184, rtS202, or rtM250. Although a variety of substitutions at these primary ETVr positions arise during ETV therapy, only a subset give rise to clinical virologic breakthrough. To determine the phenotypic impact of observed clinical and potential new ETVr substitutions, a comprehensive panel of clones containing every possible amino acid at the three primary ETVr positions in LVDr HBV was constructed and analyzed in vitro. A range of replication capacities was observed for the panel, but none of the mutations rescued replication of the LVDr mutant to the wild‐type level. More clones with residue rtS202 substitutions were severely impaired than those at rtT184 or rtM250. A wide variety of ETV susceptibilities was observed, ranging from approximately eight‐fold (no increase over the LVDr parent) to greater than 400‐fold over the wild‐type. A correlation was identified between clinically observed substitutions and those displaying higher in vitro replication and resistance, especially those from virologic breakthrough patients. Conclusion: The high number of tolerated and resistant ETVr substitutions is consistent with models predicting that the mechanism for ETVr is through enhancement of LVDr changes in the RT deoxyribonucleotide triphosphate (dNTP)‐binding pocket. (HEPATOLOGY 2008.)

A Novel Small Molecule Inhibitor of Hepatitis C Virus Entry

Small molecule inhibitors of hepatitis C virus (HCV) are being developed to complement or replace treatments with pegylated interferons and ribavirin, which have poor response rates and significant side effects. Resistance to these inhibitors emerges rapidly in the clinic, suggesting that successful therapy will involve combination therapy with multiple inhibitors of different targets. The entry process of HCV into hepatocytes represents another series of potential targets for therapeutic intervention, involving viral structural proteins that have not been extensively explored due to experimental limitations. To discover HCV entry inhibitors, we utilized HCV pseudoparticles (HCVpp) incorporating E1-E2 envelope proteins from a genotype 1b clinical isolate. Screening of a small molecule library identified a potent HCV-specific triazine inhibitor, EI-1. A series of HCVpp with E1-E2 sequences from various HCV isolates was used to show activity against all genotype 1a and 1b HCVpp tested, with median EC50 values of 0.134 and 0.027 µM, respectively. Time-of-addition experiments demonstrated a block in HCVpp entry, downstream of initial attachment to the cell surface, and prior to or concomitant with bafilomycin inhibition of endosomal acidification. EI-1 was equally active against cell-culture adapted HCV (HCVcc), blocking both cell-free entry and cell-to-cell transmission of virus. HCVcc with high-level resistance to EI-1 was selected by sequential passage in the presence of inhibitor, and resistance was shown to be conferred by changes to residue 719 in the carboxy-terminal transmembrane anchor region of E2, implicating this envelope protein in EI-1 susceptibility. Combinations of EI-1 with interferon, or inhibitors of NS3 or NS5A, resulted in additive to synergistic activity. These results suggest that inhibitors of HCV entry could be added to replication inhibitors and interferons already in development.

Hepatitis B virus quasispecies susceptibility to entecavir confirms the relationship between genotypic resistance and patient virologic response.

BACKGROUND/AIMS The efficacy of anti-viral therapy for chronic hepatitis B virus (HBV) is lost upon the emergence of resistant virus. Using >500 patient HBV isolates from several entecavir clinical trials, we show that phenotypic susceptibility correlates with genotypic resistance and patient virologic responses. METHODS The full-length HBV or reverse transcriptase gene was amplified from patient sera, sequenced, and cloned into an HBV expression vector. Entecavir susceptibilities of individual virus clones and patient quasispecies populations were analyzed in conjunction with the sequenced resistance genotype and the patients virologic response. RESULTS Entecavir susceptibility decreased approximately 8-fold for isolates with various constellations of lamivudine resistance substitutions. The spectrum of additional substitutions that emerged during therapy at residues rtT184, rtS202, or rtM250 displayed varying levels of entecavir susceptibility according to the specific resistance substitutions and the proportion of resistant variants in the quasispecies. Phenotypic analyses of samples associated with virologic breakthrough confirmed the role of these residue changes in entecavir resistance. Additional longitudinal phenotypic analyses showed that decreased susceptibility correlated with both genotypic resistance and increased circulating HBV DNA. CONCLUSIONS HBV phenotypic analysis provides additional insight as part of a resistance monitoring program that includes genotypic analysis and quantification of circulating virus.

Ultrasensitive Genotypic Detection of Antiviral Resistance in Hepatitis B Virus Clinical Isolates

ABSTRACT Amino acid substitutions that confer reduced susceptibility to antivirals arise spontaneously through error-prone viral polymerases and are selected as a result of antiviral therapy. Resistance substitutions first emerge in a fraction of the circulating virus population, below the limit of detection by nucleotide sequencing of either the population or limited sets of cloned isolates. These variants can expand under drug pressure to dominate the circulating virus population. To enhance detection of these viruses in clinical samples, we established a highly sensitive quantitative, real-time allele-specific PCR assay for hepatitis B virus (HBV) DNA. Sensitivity was accomplished using a high-fidelity DNA polymerase and oligonucleotide primers containing locked nucleic acid bases. Quantitative measurement of resistant and wild-type variants was accomplished using sequence-matched standards. Detection methodology that was not reliant on hybridization probes, and assay modifications, minimized the effect of patient-specific sequence polymorphisms. The method was validated using samples from patients chronically infected with HBV through parallel sequencing of large numbers of cloned isolates. Viruses with resistance to lamivudine and other l-nucleoside analogs and entecavir, involving 17 different nucleotide substitutions, were reliably detected at levels at or below 0.1% of the total population. The method worked across HBV genotypes. Longitudinal analysis of patient samples showed earlier emergence of resistance on therapy than was seen with sequencing methodologies, including some cases of resistance that existed prior to treatment. In summary, we established and validated an ultrasensitive method for measuring resistant HBV variants in clinical specimens, which enabled earlier, quantitative measurement of resistance to therapy.

High-Throughput Screening and Rapid Inhibitor Triage Using an Infectious Chimeric Hepatitis C Virus

The recent development of a Hepatitis C virus (HCV) infectious virus cell culture model system has facilitated the development of whole-virus screening assays which can be used to interrogate the entire virus life cycle. Here, we describe the development of an HCV growth assay capable of identifying inhibitors against all stages of the virus life cycle with assay throughput suitable for rapid screening of large-scale chemical libraries. Novel features include, 1) the use of an efficiently-spreading, full-length, intergenotypic chimeric reporter virus with genotype 1 structural proteins, 2) a homogenous assay format compatible with miniaturization and automated liquid-handling, and 3) flexible assay end-points using either chemiluminescence (high-throughput screening) or Cellomics ArrayScan™ technology (high-content screening). The assay was validated using known HCV antivirals and through a large-scale, high-throughput screening campaign that identified novel and selective entry, replication and late-stage inhibitors. Selection and characterization of resistant viruses provided information regarding inhibitor target and mechanism. Leveraging results from this robust whole-virus assay represents a critical first step towards identifying inhibitors of novel targets to broaden the spectrum of antivirals for the treatment of HCV.

W1805 Entecavir Maintains a High Genetic Barrier to HBV Resistance Through 6 Years in NaïVE Patients

• Resistance is rare through 6 years – 1.2% cumulative probability of genotypic resistance. LVD-Refractory: • The 6 year cumulative probability of genotypic resistance is 57%. – 74/187 (40%) patients achieved HBV DNA < 300 c/mL while on treatment with ETV; of those 74 patients, only 5 (7%) subsequently developed genotypic ETV resistance. Entecavir Maintains a High Genetic Barrier to HBV Resistance Through 6 Years in Naïve Patients D.J. Tenney1, K.A. Pokorowski1, R.E. Rose1, C.J. Baldick1, B.J. Eggers1, J. Fang1, M.J. Wichroski1, U. Diva1, D. Xu1, R.B. Wilber1, H. Brett-Smith1 & U.H. Iloeje1 1Research and Development, Bristol-Myers Squibb Company, United States