Untying relaxed circular DNA of hepatitis B virus by polymerase reaction provides a new option for accurate quantification and visualization of covalently closed circular DNA.

Hepatitis B virus (HBV) is a small hepatotropic DNA virus that replicates via an RNA intermediate. After entry, the virus capsid carries relaxed circular DNA (rcDNA) into the nucleus where the viral genome is converted into covalently closed circular DNA (cccDNA), which serves as the template for all viral transcripts. To monitor cccDNA levels, preprocessing methods to eliminate rcDNA have emerged for quantitative PCR, although Southern blotting is still the only method to discriminate cccDNA from other DNA intermediates. In this study, we have established a robust method for untying mature rcDNA into double stranded linear DNA using specific polymerases. Untying rcDNA provides not only an alternative method for cccDNA quantification but also a sensitive method for visualizing cccDNA. We combined this method with plasmid-safe DNase and T5 exonuclease preprocessing and revealed that accurate quantification requires cccDNA digestion by a restriction enzyme because heat stability of cccDNA increases after T5 exonuclease treatment. In digital PCR using duplex TaqMan probes, fewer than 1000 copies of cccDNA were successfully visualized as double positive spots that were distinct from single positives derived from untied rcDNA. This method was further applied to the infection model of primary hepatocytes treated with nucleoside analogues and a core protein allosteric modulator to monitor cccDNA levels. Relative quantification of cccDNA by human genome copy demonstrated the possibility of precise evaluation of cccDNA level per nucleus. These results clearly indicate that the sequential reaction from untying rcDNA is useful to investigate cccDNA fates in a small fraction of nuclei.

Efficacy and safety of telaprevir with natural human interferon-ß and ribavirin in Japanese chronic hepatitis C patients with depression.

AIM: To assess the efficacy and safety of telaprevir (TVR) when used in combination with natural human interferon-ß (IFN-ß) and ribavirin (RBV) for genotype 1 patients with depression compared to IFN-ß/RBV therapy in Japan. We also examined the efficacy of the TVR/IFN-ß/RBV therapy in treatment failure genotype 2 patients with depression. METHODS: For the genotype 1 patients, 30 patients received TVR (750 mg every 8 h) for 12 weeks combined with IFN-ß and RBV for 24 weeks (Group A), and 30 received IFN-ß and RBV for 48 weeks (Group B). For the genotype 2 patients, 14 patients were dosed only with the TVR-based regimen. RESULTS: The sustained virologic response (SVR) rates for Group A and Group B were 63.3% and 20.0%, respectively (P = 0.001, likelihood ratio test). The SVR rate for genotype 2 patients previously treated with pegylated IFN and/or RBV was 71.4%. No patient dropped out due to exacerbation of depression. The trend of platelet counts after the drugs were given was similar in the TVR/IFN-ß/RBV therapy group and the IFN-ß/RBV therapy group. Common resistance-associated variants of TVR were identified in 4 of the 13 patients who did not achieve SVR. CONCLUSION: This study showed that an addition of TVR to IFN-ß/RBV therapy raised SVR in previously treated and untreated genotype 1 patients and previously treated genotype 2 patients with chronic hepatitis C and depression.

Efficacy and safety of telaprevir with pegylated interferon α-2a and ribavirin in Japanese patients.

AIM: To assess the efficacy and safety of telaprevir (TVR) in combination with pegylated interferon α-2a (PEG-IFNα-2a) and ribavirin (RBV) for treatment-naïve patients and relapsed patients compared to previous TVR-based triple therapy in Japan. METHODS: The study group included 35 treatment-naïve (median age, 55 years) and 19 relapsed (median age, 55 years) patients with genotype 1 hepatitis C virus infection. Patients received TVR (750 mg every 8 h) for 12 weeks, in combination with PEG-IFNα-2a and RBV. RESULTS: The sustained virological response (SVR24 ) rates for naïve and relapsed patients were 85.7% (30/35) and 94.7% (18/19), respectively. The discontinuation rate of all study drugs due to adverse events was 5.6% (3/54). Among the 54 patients, grade 3 skin disorders and grade 3 anemia (<8.0 g/dL) were reported in 2 (3.7%) and 6 patients (11.1%), respectively. Although the overall safety profiles were similar for the TVR/PEG-IFNα-2a/RBV and TVR/PEG-IFNα-2b/RBV regimens (previous study), the proportion of patients discontinuing all study drugs due to adverse events was lower in the patients treated with the TVR/PEG-IFNα-2a/RBV regimen (3/54, 5.6%) than TVR/PEG-IFNα-2b/RBV regimen (44/267, 16.5%). CONCLUSION: Telaprevir in combination with PEG-IFNα-2a/RBV provided a high sustained virological response rate for the treatment of genotype 1 hepatitis C virus in both treatment-naïve and relapsed patients in Japan. Telaprevir-based therapy may provide a useful treatment option for patients who are difficult to treat due to NS5A (Y93, L31) and NS3/4A (D168) variants.

Rapid emergence of telaprevir resistant hepatitis C virus strain from wildtype clone in vivo.

UNLABELLED: Telaprevir is a potent inhibitor of hepatitis C virus (HCV) NS3-4A protease. However, the emergence of drug-resistant strains during therapy is a serious problem, and the susceptibility of resistant strains to interferon (IFN), as well as the details of the emergence of mutant strains in vivo, is not known. We previously established an infectious model of HCV using human hepatocyte chimeric mice. Using this system we investigated the biological properties and mode of emergence of mutants by ultra-deep sequencing technology. Chimeric mice were injected with serum samples obtained from a patient who had developed viral breakthrough during telaprevir monotherapy with strong selection for resistance mutations (A156F [92.6%]). Mice infected with the resistant strain (A156F [99.9%]) developed only low-level viremia and the virus was successfully eliminated with interferon therapy. As observed in patients, telaprevir monotherapy in viremic mice resulted in breakthrough, with selection for mutations that confer resistance to telaprevir (e.g., a high frequency of V36A [52.2%]). Mice were injected intrahepatically with HCV genotype 1b clone KT-9 with or without an introduced resistance mutation, A156S, in the NS3 region, and treated with telaprevir. Mice infected with the A156S strain developed lower-level viremia compared to the wildtype strain but showed strong resistance to telaprevir treatment. Although mice injected with wildtype HCV showed a rapid decline in viremia at the beginning of therapy, a high frequency (11%) of telaprevir-resistant NS3 V36A variants emerged 2 weeks after the start of treatment. CONCLUSION: Using deep sequencing technology and a genetically engineered HCV infection system, we showed that the rapid emergence of telaprevir-resistant HCV was induced by mutation from the wildtype strain of HCV in vivo.

Antiviral effects of peginterferon alpha-2b and ribavirin following 24-week monotherapy of telaprevir in Japanese hepatitis C patients.

BACKGROUND/AIMS: Anemia is commonly observed as a side effect in a treatment with protease inhibitors combined with peginterferon alpha and ribavirin for hepatitis C virus infection. This study assessed the safety, tolerability, viral kinetics, and selection of variants in telaprevir monotherapy for 24 weeks, and outcomes of the off-study treatment with peginterferon alpha-2b and ribavirin among Japanese female patients at a median age of 54 years who were difficult to treat with the standard therapy (peginterferon alpha-2b and ribavirin) alone in Japan. METHODS: Four treatment-naïve patients with chronic hepatitis C virus subtype 1b infection received telaprevir (750 mg every 8 h) alone for 24 weeks. All patients then started the off-study treatment with peginterferon alpha-2b and ribavirin. Safety, tolerability, hepatitis C virus RNA levels, and emergence of telaprevir-resistant variants were monitored. RESULTS: During the 24 weeks of telaprevir monotherapy, there was no discontinuation due to adverse events, but 2 patients stopped the intake at weeks 6 and 15 because of viral breakthrough. Emergence of telaprevir-resistant variants was observed in 3 patients who showed viral breakthrough. These variants were eliminated by the off-study treatment, and sustained virological response was achieved in all patients. CONCLUSIONS: Anemia was manageable by carefully adjusting the ribavirin dosage in the standard therapy that followed telaprevir monotherapy. This sequential regimen seems to be safer and more tolerable than the triple combination of telaprevir, peginterferon alpha, and ribavirin, especially among elderly females with low baseline hemoglobin.

Elimination of hepatitis C virus by short term NS3-4A and NS5B inhibitor combination therapy in human hepatocyte chimeric mice.

BACKGROUND & AIMS: The current treatment regimen for chronic hepatitis C virus (HCV) infection is peg-interferon plus ribavirin combination therapy. The majority of developing therapeutic strategies also contain peg-interferon with or without ribavirin. However, interferon is expensive and sometimes intolerable for some patients because of severe side effects. METHODS: Using human hepatocyte chimeric mice, we examined whether a short term combination therapy with the HCV NS3-4A protease inhibitor telaprevir and the RNA polymerase inhibitor MK-0608 with or without interferon eradicates the HCV from infected mice. The effect of telaprevir and MK-0608 combination therapy was examined using subgenomic HCV replicon cells. RESULTS: Combination therapy with the two drugs enhanced inhibition of HCV replication compared with either drug alone. In in vivo experiments, early emergence of drug resistance was seen in mice treated with either telaprevir or MK-0608 alone. However, emergence was prevented by the combination of these drugs. Mice treated with a triple combination therapy of telaprevir, MK-0608, and interferon became negative for HCV RNA soon after commencement of the therapy, and HCV RNA was not detected in serum of these mice 12 weeks after cessation of the therapy. Furthermore, all mice treated with a high dose telaprevir and MK-0608 combination therapy for 4 weeks became negative for HCV RNA 1 week after the beginning of the therapy and remained negative after 18 weeks. CONCLUSIONS: Eradication of HCV from mice with only 4 weeks of therapy without interferon points the way to future combination therapies for chronic hepatitis C patients.

Practical evaluation of a mouse with chimeric human liver model for hepatitis C virus infection using an NS3-4A protease inhibitor.

A small-animal model for hepatitis C virus (HCV) infection was developed using severe combined immunodeficiency (SCID) mice encoding homozygous urokinase-type plasminogen activator (uPA) transplanted with human hepatocytes. Currently, limited information is available concerning the HCV clearance rate in the SCID mouse model and the virion production rate in engrafted hepatocytes. In this study, several cohorts of uPA(+/+)/SCID(+/+) mice with nearly half of their livers repopulated by human hepatocytes were infected with HCV genotype 1b and used to evaluate HCV dynamics by pharmacokinetic and pharmacodynamic analyses of a specific NS3-4A protease inhibitor (telaprevir). A dose-dependent reduction in serum HCV RNA was observed. At telaprevir exposure equivalent to that in clinical studies, rapid turnover of serum HCV was also observed in this mouse model and the estimated slopes of virus decline were 0.11-0.17 log(10) h(-1). During the initial phase of treatment, the log(10) reduction level of HCV RNA was dependent on the drug concentration, which was about fourfold higher in the liver than in plasma. HCV RNA levels in the liver relative to human endogenous gene expression were correlated with serum HCV RNA levels at the end of treatment for up to 10 days. A mathematical model analysis of viral kinetics suggested that 1 g of the chimeric human liver could produce at least 10(8) virions per day, and this may be comparable to HCV production in the human liver.

Antiviral activities of MCC-478, a novel and specific inhibitor of hepatitis B virus.

MCC-478 is a newly synthesized 2-amino-6-arylthio-9-phosphonomethoxyethylpurine bis(2,2,2-trifluoroethyl) ester derivative. MCC-478 showed a substantially higher (ca. 80-fold) anti-hepatitis B virus (HBV) activity than that of lamivudine, despite no significant anti-human immunodeficiency virus activity. Since the bis(2,2,2-trifluoroethyl) ester group was used to improve the oral bioavailability of the phosphonomethoxyethylpurine derivatives, two monoester derivatives and one phosphonic acid derivative were also evaluated. It was suggested that these hydrolyzed derivatives, which appeared in animals given MCC-478, have enough anti-HBV activity to contribute to efficacy in vivo. Furthermore, no apparent cytotoxic effects or reductions of mitochondrial DNA content by MCC-478 and its derivatives were observed. These results indicated that MCC-478 may be a new promising anti-HBV agent.

2-Amino-6-arylthio-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl) esters as novel HBV-specific antiviral reagents.

Novel 2-amino-6-arylthio-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl) esters were synthesized and evaluated for antihepatitis B virus (HBV) activity in vitro using HB611, HuH-6 cell line, stably transfected with the HBV genome. Among the compounds synthesized, 2-amino-6-phenylthio-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester (8), 2-amino-6-(4-methoxyphenylthio)-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester (16), 2-amino-6-(3-methoxyphenylthio)-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester (17), and 2-amino-6-(2-methoxyphenylthio)-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester (18) showed considerably high anti-HBV activity, as represented by IC(50) values of 0.05, 0.03, 0.04, and 0.08 microM, respectively, and exhibited low cytotoxicity, as represented by CC(50) values of more than 1000 microM. It was suggested that these compounds did not have anti-HIV activity, and compound 8 showed only weak anti-HSV-1 activity. An antiviral agent, 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA), which was used as a control in the present study, showed moderate anti-HBV activity, as represented by an IC(50) value of 0.2 microM. Furthermore, compound 16 was administered orally to mice at a dose of 100 mg/kg in order to examine its gastrointestinal absorbability. Consequently, the main active metabolite was observed in mouse plasma, with especially high concentrations in the liver.