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Addition of peginterferon alpha (PEG-IFN add-on) to entecavir (ETV) treatment after a short lead-in phase results in more response than ETV monotherapy in HBeAg-positive chronic hepatitis B infection (CHB). This study is the first to assess long-term efficacy of this treatment strategy. Patients who received ETV ± 24 weeks of PEG-IFN add-on in a global trial (ARES study) and completed follow-up were eligible to participate in this observational LTFU study if they had at least one combined HBeAg and HBV DNA measurement beyond week 96 of the ARES study. The primary endpoint was combined response (HBeAg loss and HBV DNA

BACKGROUND: We aimed to identify HBc amino acid differences between subgroups of chronic hepatitis B (CHB) patients.METHODS: Deep sequencing of HBc was performed in samples of 89 CHB patients (42 HBeAg positive, 47 HBeAg negative). Amino acid types were compared using Sequence Harmony to identify subgroup specific sites between HBeAg-positive and -negative patients, and between patients with combined response and non-response to peginterferon/adefovir combination therapy.RESULTS: We identified 54 positions in HBc where the frequency of appearing amino acids was significantly different between HBeAg-positive and -negative patients. In HBeAg negative patients, 22 positions in HBc were identified which differed between patients with treatment response and those with non-response. The fraction non-consensus sequence on selected positions was significantly higher in HBeAg-negative patients, and was negatively correlated with HBV DNA and HBsAg levels.CONCLUSIONS: Sequence Harmony identified a number of amino acid changes associated with HBeAg-status and response to peginterferon/adefovir combination therapy.

BACKGROUND: Antiviral treatment is currently not recommended for patients with chronic hepatitis B with a low viral load. However, they might benefit from acquiring a functional cure (hepatitis B surface antigen [HBsAg] loss with or without formation of antibodies against hepatitis B surface antigen [anti-HBs]). We assessed HBsAg loss during peg-interferon-alfa-2a (peg-IFN) and nucleotide analogue combination therapy in patients with chronic hepatitis B with a low viral load.METHODS: In this randomised controlled, open-label trial, patients were enrolled from the Academic Medical Center (AMC), Amsterdam, Netherlands. Eligible patients were HBsAg positive and hepatitis B e antigen (HBeAg) negative for more than 6 months, could be treatment naive or treatment experienced, and had alanine aminotransferase (ALT) concentrations less than 5 × upper limit of normal (ULN). Participants were randomly assigned (1:1:1) by a computerised randomisation programme (ALEA Randomisation Service) to receive peg-IFN 180 μg/week plus adefovir 10 mg/day, peg-IFN 180 μg/week plus tenofovir disoproxil fumarate 245 mg/day, or no treatment for 48 weeks. The primary endpoint was the proportion of patients with serum HBsAg loss among those who received at least one dose of study drug or had at least one study visit (modified intention-to-treat population [mITT]). All patients have finished the initial study of 72 weeks and will be observed for up to 5 years of follow-up. This study is registered with ClinicalTrials.gov, number NCT00973219.FINDINGS: Between Aug 4, 2009, and Oct 17, 2013, 167 patients were screened for enrolment, of whom 151 were randomly assigned (52 to peg-IFN plus adefovir, 51 to peg-IFN plus tenofovir, and 48 to no treatment). 46 participants in the peg-IFN plus adefovir group, 45 in the peg-IFN plus tenofovir group, and 43 in the no treatment group began treatment or observation and were included in the mITT population. At week 72, two (4%) patients in the peg-IFN plus adefovir group and two (4%) patients in the peg-IFN plus tenofovir group had achieved HBsAg loss, compared with none of the patients in the no treatment group (p=0·377). The most frequent adverse events (>30%) were fatigue, headache, fever, and myalgia, which were attributed to peg-IFN dosing. Two (4%) serious adverse events were reported in the peg-IFN plus adefovir group (admission to hospital for alcohol-related pancreatitis [week 6; n=1] and pregnancy, which was electively aborted [week 9; n=1]), three (7%) in the peg-IFN plus tenofovir group (admission to hospital after a suicide attempt during a severe depression [week 23; n=1], admission to hospital for abdominal pain [week 2; n=1], and an elective laminectomy [week 40; n=1]), and three (7%) in the no treatment group (admission to hospital for septic arthritis [week 72; n=1], endocarditis [week 5; n=1], and hyperthyroidism [week 20; n=1]).INTERPRETATION: In patients with chronic hepatitis B with a low viral load, combination treatment (peg-IFN plus adefovir and peg-IFN plus tenofovir) did not result in significant HBsAg loss compared with no treatment, which does not support the use of combination treatment in this population of patients.FUNDING: Roche, Fonds NutsOhra.

Background. Hepatitis B virus (HBV) modulates microRNA (miRNA) expression to support viral replication. The aim of this study was to identify miRNAs associated with hepatitis B e antigen (HBeAg) status and response to antiviral therapy in patients with chronic hepatitis B (CHB) , and to assess if these miRNAs are actively secreted by hepatoma cells. Methods. Plasma miRNA levels were measured by reverse-transcription quantitative polymerase chain reaction in healthy controls (n = 10) and pretreatment samples of an identification cohort (n = 24) and a confirmation cohort (n = 64) of CHB patients treated with peginterferon/nucleotide analogue combination therapy. Levels of HBV-associated miRNAs were measured in cells, extracellular vesicles, and hepatitis B surface antigen (HBsAg) particles of hepatoma cell lines. Results. HBeAg-positive patients had higher plasma levels of miR-122-5p, miR-125b-5p, miR-192-5p, miR-193b-3p, and miR- 194-5p compared to HBeAg-negative patients, and levels of these miRNAs were associated with HBV DNA and HBsAg levels. Pretreatment plasma levels of miR-301a-3p and miR-145-5p were higher in responders (combined response or HBsAg loss) compared to nonresponders. miR-192-5p, miR-193b-3p, and miR-194-5p were present in extracellular vesicles and HBsAg particles derived from hepatoma cells. Conclusions. We identified miRNAs that are associated with HBeAg status, levels of HBV DNA and HBsAg, and treatment response in CHB patients. We demonstrated that several of these miRNAs are present in extracellular vesicles and HBsAg particles secreted by hepatoma cells.

Summary Background miR-122 is an important host factor for hepatitis C virus (HCV) replication. The aim of this study was to assess the safety and tolerability, pharmacokinetics, and antiviral effect of a single dose of RG-101, a hepatocyte targeted N-acetylgalactosamine conjugated oligonucleotide that antagonises miR-122, in patients with chronic HCV infection with various genotypes. Methods In this randomised, double-blind, placebo-controlled, multicentre, phase 1B study, patients were randomly assigned to RG-101 or placebo (7:1). We enrolled men and postmenopausal or hysterectomised women (aged 18–65 years) with chronic HCV genotype 1, 3, or 4 infection diagnosed at least 24 weeks before screening who were either treatment naive to or relapsed after interferon-α based therapy. Patients with co-infection (hepatitis B virus or HIV infection), evidence of decompensated liver disease, or a history of hepatocellular carcinoma were excluded. Randomisation was done by an independent, unblinded, statistician using the SAS procedure Proc Plan. The first cohort received one subcutaneous injection of 2 mg/kg RG-101 or placebo; the second cohort received one subcutaneous injection of 4 mg/kg or placebo. Patients were followed up for 8 weeks (all patients) and up to 76 weeks (patients with no viral rebound and excluding those who were randomised to the placebo group) after randomisation. The primary objective was safety and tolerability of RG-101. This trial was registered with EudraCT, number 2013-002978-49. Findings Between June 4, 2014, and Oct 27, 2014, we enrolled 32 patients with chronic HCV genotype 1 (n=16), 3 (n=10), or 4 (n=6) infections. In the first cohort, 14 patients were randomly assigned to receive 2 mg/kg RG-101 and two patients were randomly assigned to receive placebo, and in the second cohort, 14 patients were randomly assigned to receive 4 mg/kg RG-101 and two patients were randomly assigned to receive placebo. Overall, 26 of the 28 patients dosed with RG-101 reported at least one treatment-related adverse event. At week 4, the median viral load reduction from baseline was 4·42 (IQR 3·23–5·00) and 5·07 (4·19–5·35) log 10 IU/mL in patients dosed with 2 mg/kg RG-101 or 4 mg/kg RG-101. Three patients had undetectable HCV RNA levels 76 weeks after a single dose of RG-101. Viral rebound at or before week 12 was associated with the appearance of resistance associated substitutions in miR-122 binding regions in the 5′ UTR of the HCV genome. Interpretation This study showed that one administration of 2 mg/kg or 4 mg/kg RG-101, a hepatocyte targeted N-acetylgalactosamine conjugated anti-miR-122 oligonucleotide, was well tolerated and resulted in substantial viral load reduction in all treated patients within 4 weeks, and sustained virological response in three patients for 76 weeks. Funding Regulus Therapeutics, Inc.

Entecavir (ETV) is a potent inhibitor of hepatitis B viral replication, but long-term therapy may be required. We investigated whether adding on pegylated interferon (Peg-IFN) to ETV therapy enhances serological response rates. In this global investigator-initiated, open-label, multicenter, randomized trial, hepatitis B e antigen (HBeAg)-positive chronic hepatitis B (CHB) patients with compensated liver disease started on ETV monotherapy (0.5 mg/day) and were randomized in a 1:1 ratio to either Peg-IFN add-on therapy (180 µg/week) from week 24 to 48 (n = 85) or to continue ETV monotherapy (n = 90). Response was defined as HBeAg loss with HBV DNA

Background & aims The aim of this integrated analysis was to assess the efficacy of the once-daily combination of elbasvir 50 mg and grazoprevir 100 mg, with and without ribavirin in HCV genotype 4 (GT4)-infected participants enrolled in the Phase 2/3 clinical programme with elbasvir/grazoprevir. Methods Treatment-naive and treatment-experienced participants 18 years of age or older with chronic HCV GT4 infection and baseline HCV RNA ≥10 000 IU/mL were included in the analysis. The analysis population was the full analysis set (FAS; all participants who received at least 1 dose of study medication) and a total of 155 HCV GT4 participants were evaluated. The primary endpoint was sustained virologic response at week 12 (SVR12; HCV RNA less than the lower limit of quantitation at 12 weeks after the completion of study therapy). Results Overall, among GT4-infected participants treated with 12 or 16 weeks of elbasvir/grazoprevir ± ribavirin, the SVR12 efficacy rates were 96.4% (107/111) in treatment-naive participants and 88.6% (39/44) in treatment-experienced participants. The SVR12 rates were 96.0% (97/101) in treatment-naive participants treated with 12 weeks of elbasvir/grazoprevir and 100% (8/8) in treatment-experienced participants treated with 16 weeks of elbasvir/grazoprevir plus ribavirin. Efficacy was not impacted by GT4 subtype. Conclusions The regimens of 12 weeks of elbasvir/grazoprevir without ribavirin, and 16 weeks of elbasvir/grazoprevir plus ribavirin, were efficacious in HCV GT4-infected treatment-naive and treatment-experienced participants respectively. Baseline NS5A resistance-associated substitutions did not impact the efficacy of elbasvir/grazoprevir in GT4-infected participants.

Background & AimsDifferences in intrahepatic gene expression patterns may be associated with therapy response in peginterferon-treated chronic hepatitis B (CHB) patients. MethodsWe employed gene expression profiling in baseline liver biopsies of 40 CHB patients (19 HBeAg-positive; 21 HBeAg-negative) treated with peginterferon and adefovir for 48weeks, and compared expression patterns of combined responders (HBeAg loss, HBV-DNA

Background The role of natural killer (NK) cells in the process of hepatitis B virus (HBV) surface antigen (HBsAg) clearance and whether their phenotype is related to treatment outcome in patients with chronic hepatitis B are currently unknown. Methods Patients with chronic hepatitis B (HBV DNA load, >17 000 IU/mL) were treated with pegylated interferon alfa-2a and adefovir for 48 weeks. NK cell phenotype and function were analyzed in 7 responders (defined as individuals with HBsAg clearance by week 72; 3 HBV e antigen [HBeAg]-positive and 4 HBeAg-negative), 7 matched nonresponders, and 7 healthy controls. Subsequently, 34 baseline samples from HBeAg-positive patients with chronic hepatitis B were analyzed. Results During treatment, the percentage and absolute number of CD56(bright) NK cells increased significantly, whereas the percentage and absolute number of CD56(dim) NK cells decreased. At baseline, responders had a significantly lower expression of chemokine receptor CX3CR1 on CD56(bright) NK cells and inhibitory receptor NKG2A on CD56(dim) NK cells, compared with nonresponders. In addition, responders had higher CD56(bright) TRAIL expression and interferon γ production at end of treatment. These baseline differences were not found in HBeAg-positive patients who had HBeAg seroconversion without HBsAg clearance. Conclusions Combination therapy significantly influences NK cell phenotype and function. Differences between patients with chronic hepatitis B with HBsAg clearance and nonresponders suggest that NK cells play a role in the clearance of HBsAg during interferon-based combination therapy.

Tissue resident memory T cells (TRM) have been identified in various tissues, however human liver TRM to date remain unidentified. TRM can be recognized by CD69 and/or CD103 expression and may play a role in the pathology of chronic hepatitis B (CHB) and hepatitis C virus infection (CHC). Liver and paired blood mononuclear cells from 17 patients (including 4 CHB and 6 CHC patients) were isolated and CD8+ T cells were comprehensively analysed by flowcytometry, immunohistochemistry and qPCR. The majority of intrahepatic CD8+ T cells expressed CD69, a marker used to identify TRM, of which a subset co-expressed CD103. CD69 + CD8+ T cells expressed low levels of S1PR1 and KLF2 and a large proportion (>90%) was CXCR6+, resembling liver TRM in mice and liver resident NK cells in human. Cytotoxic proteins were only expressed in a small fraction of liver CD69 + CD8+ T cells in patients without viral hepatitis, however, in livers from CHB patients more CD69 + CD8+ T cells were granzyme B+. In CHC patients, less intrahepatic CD69 + CD8+ T cells were Hobit+ as compared to CHB and control patients. Intrahepatic CD69 + CD8+ T cells likely TRM which have a reduced cytolytic potential. In patients with chronic viral hepatitis TRM have a distinct phenotype.

Background and aim: Precore (PC) and basal core promoter (BCP) mutations are associated with responses to interferon-based treatment in HBeAg-positive chronic hepatitis B (CHB) patients. Here, we identify viral minority variants in these regions and assess association with response to peginterferon-alfa (Peg-IFN) and adefovir combination therapy. Patients and methods: Ultra-deep pyrosequencing analysis of the BCP and PC region was performed for 89 CHB patients (42 HBeAg-positive; 47 HBeAg-negative), at baseline and during treatment. Specifically, associations of individual positions with the HBeAg-negative phenotype were studied, as well as the association of the most prevalent mutations with combined response in HBeAg-positive and negative patients at week 72 (HBeAg negativity, HBV-DNA

Interferon (IFN) negatively impacts patients' well-being and patient-reported outcomes (PROs). Our aim was to assess PROs during treatment with an IFN-free regimen [sofosbuvir (SOF)+ribavirin (RBV)]. Four PRO questionnaires [Short Form-36 (SF-36), Chronic Liver Disease Questionnaire-HCV (CLDQ-HCV), Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-F), Work Productivity and Activity Index: Specific Health Problem (WPAI:SHP)] were administered at baseline, end-of-treatment and post-treatment to 334 HCV genotype 2 and 3 patients (naïve or treatment-experienced) enrolled in the VALENCE study. Of these, 250 genotype 3 patients were treated for 24 weeks while 73 genotype 2 and 11 genotype 3 patients received 12 weeks of treatment. Baseline PRO scores were similar between the two arms of the study. Throughout and after treatment, patients receiving 12 or 24 weeks had similar FACIT-F, CLDQ-HCV, SF-36 and WPAI:SHP scores (all p>0.05). Compared to their own baseline scores, patients receiving SOF+RBV experienced modest declines in some aspects of SF-36, CLDQ-HCV, fatigue and WPAI:SHP scores (p = 0.04 to 0.05). In patients achieving SVR-12 (regardless of the regimen), significant improvements were noted in general health (p = 0.0004), CLDQ-HCV (p

Background&aims With the introduction of DAA's, the majority of treated chronic hepatitis C patients (CHC) achieve a viral cure. The exact mechanisms by which the virus is cleared after successful therapy, is still unknown. The aim was to assess the role of the immune system and miRNA levels in acquiring a sustained virological response after DAA treatment in CHC patients with and without prior RG-101 (anti-miR-122) dosing. Methods In this multicenter, investigator-initiated study, 29 patients with hepatitis C virus (HCV) genotype 1 (n = 11), 3 (n = 17), or 4 (n = 1) infection were treated with sofosbuvir and daclatasvir ± ribavirin. 18 patients were previously treated with RG-101. IP-10 levels were measured by ELISA. Ex vivo HCV-specific T cell responses were quantified in IFN-γ-ELISpot assays. Plasma levels of miR-122 were measured by qPCR. Results All patients had an SVR12. IP-10 levels rapidly declined during treatment, but were still elevated 24 weeks after treatment as compared to healthy controls (median 53.82 and 39.4 pg/mL, p = 0.02). Functional IFN-γ HCV-specific T cell responses did not change by week 12 of follow-up (77.5 versus 125 SFU/106 PBMC, p = 0.46). At follow-up week 12, there was no difference in plasma miR-122 levels between healthy controls and patients with and without prior RG-101 dosing. Conclusions Our data shows that successful treatment of CHC patients with and without prior RG-101 dosing results in reduction of broad immune activation, and normalisation of miR-122 levels (EudraCT: 2014-002808-25). Trial registration EudraCT: 2014-002808-25.

Background Acute hepatitis B virus infection in adults is generally self-limiting but may lead to chronicity in a minority of patients. Methods We included 9 patients with acute hepatitis B virus (HBV) infection and collected longitudinal follow-up samples. Natural killer (NK) cell characteristics were analyzed by flowcytometry. HBV-specific T-cell function was analyzed by in vitro stimulation with HBV peptide pools and intracellular cytokine staining. Results Median baseline HBV DNA load was 5.12 log IU/mL, and median ALT was 2652 U/mL. Of 9 patients, 8 cleared HBsAg within 6 months whereas 1 patient became chronically infected. Early time points after infection showed increased CD56bright NK cells and an increased proportion of cells expressing activation markers. Most of these had normalized at week 24, while the proportion of TRAIL-positive CD56bright NK cells remained high in the chronically infected patient. In patients who cleared HBV, functional HBV-specific CD8+ and CD4+ responses could be observed, whereas in the patient who developed chronic infection, only low HBV-specific T-cell responses were observed. Conclusions NK cells are activated early in the course of acute HBV infection. Broad and multispecific T-cell responses are observed in patients who clear acute HBV infection, but not in a patient who became chronically infected.

BACKGROUND The stability and propagation of hepatitis C virus (HCV) is dependent on a functional interaction between the HCV genome and liver-expressed microRNA-122 (miR-122). Miravirsen is a locked nucleic acid-modified DNA phosphorothioate antisense oligonucleotide that sequesters mature miR-122 in a highly stable heteroduplex, thereby inhibiting its function. METHODS In this phase 2a study at seven international sites, we evaluated the safety and efficacy of miravirsen in 36 patients with chronic HCV genotype 1 infection. The patients were randomly assigned to receive five weekly subcutaneous injections of miravirsen at doses of 3 mg, 5 mg, or 7 mg per kilogram of body weight or placebo over a 29-day period. They were followed until 18 weeks after randomization. RESULTS Miravirsen resulted in a dose-dependent reduction in HCV RNA levels that endured beyond the end of active therapy. In the miravirsen groups, the mean maximum reduction in HCV RNA level (log(10) IU per milliliter) from baseline was 1.2 (P = 0.01) for patients receiving 3 mg per kilogram, 2.9 (P = 0.003) for those receiving 5 mg per kilogram, and 3.0 (P = 0.002) for those receiving 7 mg per kilogram, as compared with a reduction of 0.4 in the placebo group. During 14 weeks of follow-up after treatment, HCV RNA was not detected in one patient in the 5-mg group and in four patients in the 7-mg group. We observed no dose-limiting adverse events and no escape mutations in the miR-122 binding sites of the HCV genome. CONCLUSIONS The use of miravirsen in patients with chronic HCV genotype 1 infection showed prolonged dose-dependent reductions in HCV RNA levels without evidence of viral resistance. (Funded by Santaris Pharma; ClinicalTrials.gov number, NCT01200420.)

Background In this study, we aimed to identify baseline predictors of response in chronic hepatitis B patients treated with a combination of pegylated interferon (PEG-IFN)-α2a and adefovir. Methods We treated 92 chronic hepatitis B patients (44 hepatitis B e antigen [HBeAg]-positive and 48 HBeAg-negative) with HBV DNA>100,000 copies/ml (>17,182 IU/ ml) with PEG-IFN and adefovir for 48 weeks and followed them up for 2 years. Baseline markers for HBeAg loss, combined response (HBeAg negativity, HBV DNA levels ≤2,000 IU/ml and alanine aminotransferase [ALT] normalization) and hepatitis B surface antigen (HBsAg) loss were evaluated. Results Two years after the end of treatment, rates of HBeAg loss and HBsAg loss in HBeAg-positive patients were 18/44 (41%) and 5/44 (11%), respectively. In HBeAg-negative patients, rates of combined response and HBsAg loss were 12/48 (25%) and 8/48 (17%), respectively. HBeAg-negative patients with HBsAg loss had lower baseline HBsAg levels than those without HBsAg loss (mean HBsAg 2.35 versus 3.55 log10 IU/ml; PConclusions With combination therapy of PEG-IFN and adefovir for 48 weeks, a high rate of HBsAg loss was observed in both HBeAg-positive (11%) and HBeAg-negative (17%) patients 2 years after treatment ended. In HBeAg-negative patients, a low baseline HBsAg level was a strong predictor for HBsAg loss.

MicroRNA-122 is an important host factor for the hepatitis C virus (HCV). Treatment with RG-101, an N-acetylgalactosamine-conjugated anti-microRNA-122 oligonucleotide, resulted in a significant viral load reduction in patients with chronic HCV infection. Here, we analyzed the effects of RG-101 therapy on antiviral immunity. Thirty-two chronic HCV patients infected with HCV genotypes 1, 3, and 4 received a single subcutaneous administration of RG-101 at 2 mg/kg (n = 14) or 4 mg/kg (n = 14) or received a placebo (n = 2/dosing group). Plasma and peripheral blood mononuclear cells were collected at multiple time points, and comprehensive immunological analyses were performed. Following RG-101 administration, HCV RNA declined in all patients (mean decline at week 2, 3.27 log10 IU/mL). At week 8 HCV RNA was undetectable in 15/28 patients. Plasma interferon-γ-induced protein 10 (IP-10) levels declined significantly upon dosing with RG-101. Furthermore, the frequency of natural killer (NK) cells increased, the proportion of NK cells expressing activating receptors normalized, and NK cell interferon-γ production decreased after RG-101 dosing. Functional HCV-specific interferon-γ T-cell responses did not significantly change in patients who had undetectable HCV RNA levels by week 8 post-RG-101 injection. No increase in the magnitude of HCV-specific T-cell responses was observed at later time points, including 3 patients who were HCV RNA-negative 76 weeks postdosing.Dosing with RG-101 is associated with a restoration of NK-cell proportions and a decrease of NK cells expressing activation receptors; however, the magnitude and functionality of ex vivo HCV-specific T-cell responses did not increase following RG-101 injection, suggesting that NK cells, but not HCV adaptive immunity, may contribute to HCV viral control following RG-101 therapy. (Hepatology 2017;66:57-68).

Background The addition of direct-acting antivirals to pegylated interferon-α plus ribavirin for the treatment of chronic HCV infection can result in an increased sustained viral response rate and may permit reduction in treatment duration. IDX320 is a potent non-covalent macrocyclic inhibitor of the HCV NS3/4A protease. Methods This was a randomized double-blind placebo-controlled single- and multiple-dose study to assess the safety, tolerability, antiviral activity and pharmacokinetics of IDX320 in healthy volunteers (HV) and patients with chronic HCV genotype 1 infection. HV ( n=48) received single or multiple ascending doses of IDX320. Two HCV-infected patients received a single dose of 200 mg IDX320. Dosages for other HCV-infected patients were as follows: placebo, 50, 100, 200 or 400 mg of IDX320 orally once daily for 3 days ( n=30) or placebo/200 mg of IDX320 twice-daily for 3 days ( n=8). Results In total, 48 HV and 40 HCV-infected patients were enrolled and all completed the study. There were no serious adverse events. The majority of adverse events were of mild or moderate intensity. Pharmacokinetics supported a once-daily dosing regimen. A rapid decline in plasma HCV RNA was observed in all patients. In the multiple-dose study, mean HCV RNA reductions were 2.6, 3.1, 3.1, 3.3 and 3.8 log10 IU/ml after 3 days in the IDX320 50, 100, 200, 400 mg once-daily and 200 mg twice-daily treatment groups, respectively. This compared to a mean HCV RNA reduction of 0.04 log10 in the placebo group. Conclusions Once-daily IDX320 dosing demonstrated potent dose-dependent antiviral activity in treatment-naive HCV genotype-1-infected patients.

Background PHX1766 is a novel HCV NS3/4 protease inhibitor with robust potency and high selectivity in replicon studies (50% maximal effective concentration 8 nM). Two clinical trials investigated the safety, tolerability, pharmacokinetics and antiviral activity of PHX1766 in healthy volunteers (HV) and chronic hepatitis C patients, by use of a dose-adaptive overlapping clinical trial design. Methods Two randomized, double-blind, placebo-controlled clinical trials were conducted. Single doses of PHX1766 or placebo were administered to 25 HV and six HCV genotype 1-infected patients (50 mg once daily –1,000 mg once daily, 250 mg twice daily and 100 mg of a new formulation of PHX1766 once daily). Multiple doses of PHX1766 or placebo were administered to 32 HV and seven HCV genotype 1-infected patients (50 mg once daily –800 mg twice daily). Results Oral administration of PHX1766 was safe and well tolerated at all dose levels with rapid absorption (time at which concentration maximum is reached of 1–4 h) and with mean terminal half-lives of 4–23 h. Multiple doses of PHX1766 800 mg twice daily in HCV patients produced an area under the plasma concentration–time curve from time of drug administration to the last time point with a measurable concentration after dosing accumulation ratio of 2.3. The mean maximal observed HCV RNA decline was 0.6 log10 IU/ml in the first 24 h in the single-dose protocol and 1.5 log10 IU/ml after 6 days of PHX1766 dosing. Conclusions An overlapping, dose-adaptive single-dose and multiple-dose escalating design in HV and HCV-infected patients proved to be highly efficient in identifying a therapeutic dose. Although in vitro replicon studies indicated a robust HCV RNA viral decline of PHX1766, the study in HCV patients demonstrated only modest viral load reduction.

Background and Aim: We investigated whether intrahepatic markers could predict response in chronic hepatitis B virus (HBV) patients treated with peg-interferon and adefovir for 48 weeks. Methods: Intrahepatic covalently closed circular DNA (cccDNA), total intrahepatic HBV DNA and the proportion of hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) positive hepatocytes in 16 hepatitis B e antigen (HBeAg) positive and 24 HBeAg negative patients were measured at baseline and at end of treatment. Results: Baseline intrahepatic markers were not associated with sustained virological response (SVR) defined as HBV DNA

Aliment Pharmacol Ther 2011; 34: 443–453 Summary Background The ANA773 is an oral prodrug of a small-molecule toll-like receptor (TLR)7 agonist. Preclinical and healthy volunteer clinical studies with ANA773 have demonstrated induction of endogenous interferon-α (IFN-α) of multiple subtypes, which supports the potential utility in the treatment of chronic hepatitis C virus (HCV) infection. Aim To examine safety, tolerability, pharmacodynamics, pharmacokinetics and anti-viral activity of ANA773. Methods The ANA773 was investigated in a double-blind, placebo-controlled study in 34 patients chronically infected with HCV of any genotype. Patients were treatment-naive or had relapsed following previous interferon-based treatment. This dose escalation study was composed of four dose groups (800, 1200, 1600 and 2000 mg). In each group, six to eight patients received ANA773 and two received placebo. Patients were dosed with ANA773 every-other-day for either 28 days (800, 1200 or 1600 mg) or 10 days (2000 mg). Results Mild to moderate adverse events were reported, with an increase in frequency and intensity with increasing dose. No serious AEs were reported and there were no early discontinuations. There were dose-related increases in various markers of IFN-α response. The mean maximum change in serum HCV RNA level from baseline was −0.34, −0.29, −0.40, −0.97 and −1.26 log10 in the placebo, 800, 1200, 1600 and 2000 mg cohorts, respectively. At the 2000 mg dose, ANA773 significantly (P = 0.037) reduced serum HCV RNA levels (range: 0.14 to −3.10 log10). Conclusion The ANA773 was generally well tolerated and resulted in a dose-related IFN-dependent response leading to a significant decrease in serum HCV RNA levels in the 2000 mg dose group.

Background Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) may become an important predictor for treatment outcome or long-term follow-up. Aim To detect cccDNA in formalin-fixed, paraffin embedded (FFPE) and to compare with cryo-preserved liver tissue. Methods Biopsies of 56 chronic hepatitis B patients were collected. Cryo-preserved and FFPE liver biopsies were available from 37 out of 56 patients. Paraffin was extracted with 1 ml xylene, followed by 100% alcohol and acetone. For the detection of cccDNA, selective primers were used. For quantification of hepatocytes a commercial Taqman beta-actin control kit was used. Results The cccDNA was detected in 80% of FFPE and in 100% of cryo-preserved liver specimens. Recovery of hepatocytes and cccDNA was approximately a 100-fold lower in FFPE liver tissue, but intrahepatic cccDNA levels were comparable. In FFPE and cryo-preserved liver tissue, intrahepatic cccDNA levels correlated strongly with HBV DNA, hepatitis B e antigen (HbeAg), and plasma cccDNA levels. HbeAg positive chronic hepatitis B patients had significantly higher intrahepatic cccDNA levels compared with HBeAg negative patients (P 3; HBV DNA > 20 000 IU/ml) and inactive hepatitis (histological activity index scorer

Hepatitis C virus (HCV) genotype 4 (HCV-4) infection is considered to be difficult to treat and has become increasingly prevalent in European countries, including The Netherlands. Using a molecular epidemiological approach, the present study investigates the genetic diversity and evolutionary origin of HCV-4 in Amsterdam, The Netherlands. Phylogenetic analysis of the NS5B sequences (668 bp) obtained from 133 patients newly diagnosed with HCV-4 infection over the period from 1999 to 2008 revealed eight distinct HCV-4 subtypes; the majority of HCV-4 isolates were of subtypes 4d (57%) and 4a (37%). Three distinct monophyletic clusters were identified, with each one having a specific epidemiological profile: (i) Egyptian immigrants infected with HCV-4a ( n = 46), (ii) Dutch patients with a history of injecting drug use infected with HCV-4d ( n = 44), and (iii) Dutch human immunodeficiency virus (HIV)-positive men who have sex with men (MSM) infected with HCV-4d ( n = 26). Subsequent molecular clock analyses confirmed that the emergence of HCV-4 within these three risk groups coincided with (i) the parenteral antischistosomal therapy campaigns in Egypt (1920 to 1960), (ii) the popularity of injecting drug use in The Netherlands (1960 to 1990), and (iii) the rise in high-risk sexual behavior among MSM after the introduction of highly active antiretroviral therapy (1996 onwards). Our data show that in addition to the influx of HCV-4 strains from countries where HCV-4 is endemic, the local spread of HCV-4d affecting injecting drug users and, in recent years, especially HIV-positive MSM will further increase the relative proportion of HCV-4-infected patients in The Netherlands. HCV-4-specific agents are drastically needed to improve treatment response rates and decrease the future burden of HCV-4-related disease.

Background Telaprevir (TVR) is a hepatitis C virus (HCV) NS3.4A protease inhibitor that has exhibited antiviral activity in patients with HCV genotype 1 infection. The viral dynamics in patients dosed with TVR were compared with those reported for patients treated with interferon (IFN). Methods The dynamics of wild-type HCV genotype 1 in patients dosed with TVR monotherapy ( n=36) and TVR plus pegylated interferon (PEG-IFN)-α2a ( n=8) were quantified using a biphasic viral dynamic model. Results Patients dosed with either TVR monotherapy or TVR plus PEG-IFN-α2a had median first and second phase decreases of 12 per day and 1.1 per day, respectively. The second phase decrease was approximately 10-fold higher than reported values for IFN-based treatments ( P10. In patients dosed with TVR mono-therapy, increased TVR dosage of the same schedule was related to better blockage. Conclusions These results suggested that TVR-based regimens for chronic HCV infection will lead to an early and more rapid viral decrease that could potentially result in higher sustained viral response rates as well as offer the potential for a reduced duration of treatment.

Chronic hepatitis B virus (HBV) infection is characterized by functional impairment of HBV-specific T cells. Understanding the mechanisms behind T cell dysfunction and restoration is important for the development of optimal treatment strategies. In this study we have first analysed the phenotype and function of HBV-specific T cells in patients with low viral load (HBV DNA 17,182IU/ml) treated with peginterferon/adefovir combination therapy who had various treatment outcomes. HBV-specific T cells could be detected directly ex vivo in 7/22 patients with low viral load. These showed an early differentiated memory phenotype with reduced ability to produce IL-2 and cytotoxic molecules such as granzyme B and perforin, but with strong proliferative potential. In a cohort of 28 chronic hepatitis B patients with high viral load treated with peginterferon and adefovir, HBV-specific T cells could not be detected directly ex vivo. However, HBV-specific T cells could be selectively expanded in vitro in patients with therapy-induced HBsAg clearance (HBsAg loss n=7), but not in patients without HBsAg clearance (n=21). Further analysis of HBV-specific T cell function with peptide pools showed broad and efficient antiviral responses after therapy. Our results show that peginterferon based combination therapy can induce HBV-specific T cell restoration. These findings may help to develop novel therapeutic strategies to reconstitute antiviral functions and enhance viral clearance

Telaprevir (VX-950), an inhibitor of the hepatitis C virus (HCV) NS3/4A protease, substantially decreased plasma HCV RNA levels in a prior clinical study. The present study evaluated viral kinetics and safety during dosing with telaprevir alone and in combination with peginterferon alfa-2a for 14 days. Previously untreated patients with genotype 1 hepatitis C were randomized to receive placebo and peginterferon alfa-2a (n = 4); telaprevir (n = 8); or telaprevir and peginterferon alfa-2a (n = 8). Telaprevir was given as 750 mg oral doses every 8 hours; peginterferon alfa-2a was given as weekly 180 μg subcutaneous injections. The median change in HCV RNA from baseline to day 15 was −1.09 log10 (range, −2.08 to −0.46) in the placebo and peginterferon alfa-2a group; −3.99 log10 (range, −5.28 to −1.26) in the telaprevir group, and −5.49 log10 (range, −6.54 to −4.30) in the telaprevir and peginterferon alfa-2a group. Day 15 HCV RNA levels were undetectable in 4 patients who received telaprevir and peginterferon alfa-2a and in 1 patient who received telaprevir alone. No viral breakthrough occurred in patients who received telaprevir and peginterferon alfa-2a. The majority of adverse events were mild. There were no serious adverse events or premature discontinuations. Twelve weeks after starting off-study standard therapy, HCV RNA was undetectable in all 8 patients in the telaprevir and peginterferon alfa-2a group, 5 patients in the telaprevir group, and 1 patient in the placebo and peginterferon alfa-2a group. Conclusion: This study confirmed the substantial antiviral effects of telaprevir and showed an increased antiviral effect of telaprevir combined with peginterferon alfa-2a. (HEPATOLOGY 2007;46:640–648.)

BACKGROUND Treatment of patients with chronic hepatitis B (CHB) with nucleos(t)ide analogues (NAs) suppresses hepatitis B virus (HBV) DNA production but does not affect the synthesis of the RNA pregenome or HBV messenger RNA. Whether HBV RNA-containing particles continue to be secreted into the bloodstream remains controversial. METHODS We developed a sensitive polymerase chain reaction (PCR) assay to quantify the HBV RNA load in a supernatant of NA-treated HepG2-2.2.15 cells and in plasma specimens from 20 patients with CHB who were receiving NA therapy and 86 patients treated with pegylated interferon alfa (Peg-IFN) and adefovir. RESULTS Treatment of HepG2-2.2.15 cells with NAs for 9 days reduced HBV DNA levels (by 1.98 log10 copies/mL), whereas HBV RNA levels increased (by 0.47 log10 copies/mL; P < .05). During long-term NA treatment of patients with CHB, HBV RNA levels remained higher than HBV DNA levels. Peg-IFN-based treatment induced a stronger decrease in the HBV RNA load than NA monotherapy, and this decline was more pronounced in responders than in nonresponders. In HBV e antigen-negative patients, a lower baseline plasma HBV RNA level was independently associated with response to Peg-IFN and adefovir (odds ratio, 0.44; P = .019). Immunoprecipitation with HBV core antigen-specific antibodies after removal of the HBV surface antigen envelope demonstrated the association of plasma HBV RNA with virions. CONCLUSIONS HBV RNA is present in virions in plasma specimens from patients with CHB. HBV RNA levels vary significantly from those of established viral markers during antiviral treatment, which highlights its potential as an independent marker in the evaluation of patients with CHB.

Background and Aims HBsAg loss is a desired, but rare, treatment-induced clinical endpoint in chronic hepatitis B (CHB). Few studies have evaluated viral factors contributing to HBsAg loss. Methods This study evaluated baseline interpatient sequence diversity across the HBV genome in tenofovir disoproxil fumarate-treated patients who lost HBsAg and compared it to that of control patients with high HBsAg levels throughout therapy. Twenty-one HBeAg+ patients (14 genotype (GT) A and 7 GT D) who achieved HBsAg loss and 27 controls (17 GT A and 10 GT D), were analyzed. Population sequencing was performed on baseline samples and pairwise genetic distances were calculated for 17 overlapping regions across the HBV genome as a measure of interpatient viral diversity. Results Overall, viral diversity was up to 10-fold higher across GT D patients compared to GT A patients throughout the HBV genome. Within the pol/RT and HBs genes, interpatient viral diversity was significantly lower among HBsAg loss patients for both GT A and D, with the difference driven largely by a reduction in diversity in the small S gene. Conversely, interpatient viral diversity was generally higher in HBsAg loss patients across the HBx gene regulatory elements and precore region. Conclusion In HBsAg loss patients, less interpatient viral diversity was observed within structural-coding regions while specific regions across the HBx and precore genes encoding nonstructural regulatory elements generally displayed higher interpatient viral diversity. These distinct patterns may reflect different responses to adaptive pressure for HBV genomic structural and nonstructural elements.

MicroRNAs regulate gene expression by binding to the 3‘-untranslated region (UTR) of target messenger RNAs (mRNAs). The importance of microRNAs has been shown for several liver diseases, for example, viral hepatitis. Micro-RNA-122 is highly abundant in the liver and is involved in the regulation of lipid metabolism. MicroRNA-122 is also an important host factor for the HCV and promotes HCV replication. In contrast to HCV, microRNA-122 inhibits replication of the HBV. MicroRNA-122 acts as a tumour suppressor and reduced levels of microRNA-122 are associated with hepatocellular carcinoma. MicroRNAs other than microRNA-122 have been linked to viral hepatitis, fibrosis and inflammation. In this review, we discuss function and clinical implications of microRNA-122 and other microRNAs in liver diseases, especially viral hepatitis.

Background We studied whether hepatitis B surface antigen (HBsAg)/anti-HBs immune complex levels in chronic hepatitis B (CHB) patients receiving antiviral therapy could be used as a response marker at baseline (BL) or early during treatment to predict treatment outcome. Methods An experimental array-based assay (immunological multi-parameter chip technology [IMPACT]; Roche Diagnostics, Penzberg, Germany) served to determine HBsAg, anti-HBs and complex levels. We tested a panel of serum samples of 40 hepatitis B e antigen (HBeAg)-positive and 44 HBeAg-negative patients who received pegylated interferon and adefovir for 48 weeks. Results HBsAg loss occurred in 4 of 40 HBeAg-positive and 4 of 44 HBeAg-negative patients. A total of 14 of 40 HBeAg-positive patients lost HBeAg and 12 of them formed anti-HBe. At BL, complexes were present in 83 (99%) patients, whereas free anti-HBs levels were detectable in 5 patients. Complex levels at BL and week 12 were higher in HBeAg-positive patients with HBeAg loss, compared to patients who retained HBeAg ( P=0.002 and P=0.005, respectively). Receiver operating characteristic analysis for HBeAg loss in HBeAg-positive patients at BL and week 12 showed area-under-the-curve values of 0.79 ( P=0.002) and 0.82 ( P=0.003) for complex levels. We found no correlation in either HBeAg-positive or -negative patients between complex levels and HBsAg loss. Conclusions We demonstrated for the first time that before and during treatment HBsAg/anti-HBs immune complex levels can predict HBeAg loss in HBeAg-positive CHB patients treated with pegylated interferon and adefovir. Complexes were present in almost all patients at BL and were higher in patients who lost HBeAg. In conclusion, determining HBsAg/anti-HBs immune complex levels before and early during treatment could aid in selecting CHB patients with an optimal chance to achieve HBeAg loss.

Background and aims: MicroRNA-122 (miR-122) is an important host factor for hepatitis C virus (HCV) and promotes HCV RNA accumulation. Decreased intra-hepatic levels of miR-122 were observed in patients with hepatocellular carcinoma, suggesting a potential role of miR-122 in the development of HCC. Miravirsen targets miR-122 and resulted in a dose dependent and prolonged decrease of HCV RNA levels in chronic hepatitis C patients. The aim of this study was to establish the sustained virological response rate to peginterferon (P) and ribavirin (R) following miravirsen dosing and to assess long-term safety in patients treated with miravirsen. Methods: In this multicenter, retrospective follow-up study we included 36 treatment naive patients with chronic hepatitis C genotype 1 who received five weekly subcutaneous injections with miravirsen or placebo over a 29-day period in a phase 2a study. Patients were offered PR therapy 3 weeks (3 mg/kg group) or 6 weeks (5 or 7 mg/kg group) after completion of miravirsen or placebo dosing. Results: PR therapy was started in 14/36 patients of whom 12 had received miravirsen. SVR was achieved in 7/12 patients previously dosed with miravirsen. All patients dosed with 7 mg/kg miravirsen who were subsequently treated with PR achieved SVR. One patient had a prolonged undetectable HCV RNA period from week 14 to week 29 after baseline without subsequent antiviral therapy and relapsed thereafter. None of the patients treated with anti-miR-122 developed HCC or other liver-related complications. Conclusion: No long-term safety issues were observed among 27 miravirsen-treated patients. Targeting miR-122 may be an effective and safe treatment strategy for HCV infection and should be investigated in larger clinical trials. (C) 2014 Elsevier B.V. All rights reserved.

Background & Aims Achievement of HBsAg loss remains the hallmark of chronic hepatitis B treatment. In order to identify host factors contributing to treatment-induced HBsAg loss, we performed a genome-wide screen of single nucleotide polymorphisms (SNPs) and studied its immunological consequence. Methods Chronic hepatitis B patients (40 HBeAg-positive and 44 HBeAg-negative) treated with peginterferon alfa-2a and adefovir were genotyped for 999,091 SNPs, which were associated with HBsAg loss at week 96 (n=9). Plasma carnitine levels were measured by tandem-mass spectrometry, and the effect of carnitine on the proliferative capacity of hepatitis B virus (HBV)-specific and non-specific CD8 T cells was studied in vitro . Results One polymorphism, rs12356193 located in the SLC16A9 gene, was genome-wide significantly associated with HBsAg loss at week 96 ( p =1.84×10 −8 ). The previously reported association of rs12356193 with lower carnitine levels was confirmed in our cohort, and baseline carnitine levels were lower in patients with HBsAg loss compared to patients with HBsAg persistence ( p =0.02). Furthermore, we demonstrated that carnitine suppressed HBV-specific CD8 T cell proliferation. Conclusions In chronic hepatitis B patients treated with peginterferon and adefovir, we identified strong associations of SLC16A9 gene variation and carnitine levels with HBsAg loss. Our results further suggest that a lower baseline plasma carnitine level increases the proliferative capacity of CD8 T cells, making patients more susceptible to the immunological effect of this treatment. These novel findings may provide new insight into factors involved in treatment-induced HBsAg loss, and play a role in the prediction of treatment outcome.

Chronic systemic 'latent' viral infections such as Cytomegalovirus infection (CMV) are known to leave a fingerprint in the total T-cell population. We investigated whether chronic infections with a 'persistent' viremia, such as chronic hepatitis B and C (CHB, CHC), characterized by local organ-specific inflammation, also impact the total peripheral T-cell population or other virus specific T-cells that do not target hepatitis viruses. No phenotypic or functional differences were found between CD8(+) T-cells or CMV- or Epstein-Barr virus specific T-cells in viral hepatitis and healthy controls (HC). However, expression of chemokine-receptor CXCR3 was significantly higher on total peripheral CD8(+) T-cells of CHB or CHC patients compared to HC (p

Worldwide approximately 130-210 million people suffer from chronic hepatitis C. Adequate antiviral therapy reduces morbidity and mortality caused by chronic hepatitis C and prevents further spread of the hepatitis C-virus (HCV). The current standard treatment of chronic hepatitis C, consisting of the combination of pegylated interferon-α (peginterferon) and ribavirin, lasts 24-48 weeks, and is accompanied by significant side effects and has a suboptimal chance of success. Protease inhibitors, which have recently been registered, belong to a new class of medicines which directly affect the life cycle of HCV. Protease inhibitors, in combination with peginterferon and ribavirin, provide almost double the chance of curing in patients with HCV genotype 1. Treatment duration can be shortened in a considerable proportion of these patients. Since treatment with protease inhibitors can lead to resistant virus strains and this therapy leads to additional side effects, the complexity of treatment will increase.

Background & Aims Telaprevir, a hepatitis C virus NS3/4A protease inhibitor has significantly improved sustained viral response rates when given in combination with pegylated interferon alfa-2a and ribavirin, compared with current standard of care in hepatitis C virus genotype 1 infected patients. In patients with a failed sustained response, the emergence of drug-resistant variants during treatment has been reported. It is unclear to what extent these variants persist in untreated patients. The aim of this study was to assess using ultra-deep pyrosequencing, whether after 4 years follow-up, the frequency of resistant variants is increased compared to pre-treatment frequencies following 14 days of telaprevir treatment. Methods Fifteen patients from 2 previous telaprevir phase 1 clinical studies (VX04-950-101 and VX05-950-103) were included. These patients all received telaprevir monotherapy for 14 days, and 2 patients subsequently received standard of care. Variants at previously well-characterized NS3 protease positions V36, T54, R155 and A156 were assessed at baseline and after a follow-up of 4±1.2 years by ultra-deep pyrosequencing. The prevalence of resistant variants at follow-up was compared to baseline. Results Resistance associated mutations were detectable at low frequency at baseline. In general, prevalence of resistance mutations at follow-up was not increased compared to baseline. Only one patient had a small, but statistically significant, increase in the number of V36M and T54S variants 4 years after telaprevir-dosing. Conclusion In patients treated for 14 days with telaprevir monotherapy, ultra-deep pyrosequencing indicates that long-term persistence of resistant variants is rare.

Background ANA773, an oral prodrug of a small-molecule Toll-like receptor (TLR)7 agonist, induces a dose-related decrease in serum HCV RNA levels in chronic hepatitis C patients. Methods The prodrug ANA773 was administered to healthy individuals and chronic hepatitis C patients. At different time points during the course of treatment, modulation of the phenotype and function of peripheral leukocytes were evaluated to determine the role of distinct immune cells on the clinical outcome of therapy. Results Early after administration of the TLR7 agonist, a mild transient reduction of the number of lymphocytes was observed in both healthy individuals and chronic hepatitis C patients. Moreover, repeated administration of ANA773 resulted in transiently reduced numbers of myeloid and plasmacytoid dendritic cells (DC) in blood. Interestingly, reduced plasmacytoid DC numbers as well as increased serum interferon (IFN)-α and IFN-γ inducible protein (IP)-10 levels were observed only in virological responders (≥1 log10 IU/ml reduction of HCV RNA levels upon ANA773 treatment), but were absent in virological non-responders. In vitro stimulation of peripheral blood mononuclear cells from virological responders showed a high frequency of IFN-α-producing plasmacytoid DC upon stimulation in vitro with ANA773, whereas no IFN-α was induced in non-responders. Conclusions These findings indicate that the viral load decline in chronic hepatitis C patients treated with the TLR7 agonist ANA773 is likely due to intrinsic differences in the induction of endogenous IFNs and IFN-stimulated gene products (IFN-α and IP-10) upon TLR7 ligation.

In the TMC435-C101 study, 6 patients infected with hepatitis C virus genotype 1 were treated with the protease inhibitor TMC435 (200 mg once daily) as monotherapy for 5 days. Approximately 1.5 years later, 5 of these patients were re-treated with TMC435 (200 mg once daily) plus pegylated interferon alfa-2a and ribavirin (PegIFN alpha-2a and RBV) for 4 weeks, followed by PegIFN alpha-2a and RBV until week 48 (in the Optimal Protease inhibitor Enhancement of Response to therApy [OPERA-1] study). TMC435-resistant variants, which emerged in all 5 patients during the TMC435-C101 study, were no longer detected at the beginning of the OPERA-1 study based on virus population sequencing. During the OPERA-1 study, 3 patients had a sustained virologic response; deep sequencing indicated low-level persistence of resistant variants in the remaining 2 patients, which might have affected their response to re-treatment. Clinical trials.gov Number, NCT00561353

Background Standard treatment of chronic hepatitis C with pegylated interferon and ribavirin is associated with suboptimal virological response rates and substantial side effects. This study describes the in vitro and in vivo development of JTK- 652, a novel pyrrolopyridazin-derived HCV infection inhibitor. Methods JTK-652 was evaluated in multiple cell lines using an in vitro HCV infection model consisting of HCV pseudotype vesicular stomatitis virus bearing HCV E1/E2 envelope proteins. Safety, tolerability, pharmacokinetics and efficacy of JTK-652 were tested in a randomized double- blind and placebo-controlled study in healthy male volunteers ( n=36) and chronic hepatitis C patients. A total of 10 HCV genotype-1-infected patients (treatment-naive [ n=2] and treatment-experienced [ n=8]) with HCV RNA>1x105 IU/ml received an oral dose of 100 mg JTK-652 three times daily or placebo (8:2 ratio) for 4 weeks. Results JTK-652 showed potent inhibitory activity against HCV genotype 1a and 1b pseudotype viruses bearing HCV E1/E2 envelope proteins in HepG2 cells and in human primary hepatocytes. No significant clinical laboratory, vital sign, ECG or physical examination abnormalities were observed during the Phase I trial. JTK-652 was found to be well tolerated. No significant changes in HCV RNA levels compared with baseline were observed at the end of treatment. Conclusions Although results from the preclinical studies indicated that JTK-652 has well-established antiviral properties and a Phase I clinical trial has showed that JTK-652 was safe and well tolerated at a 100 mg three times daily dose level, plasma HCV RNA levels in chronically HCV- infected patients did not decrease during 28 days of dosing at a 100 mg three times daily dose level.

Patients with genotype 1 hepatitis C virus (HCV) who do not have a sustained response to therapy with peginterferon alfa and ribavirin have a low likelihood of success with retreatment. We randomly assigned patients with HCV genotype 1 who had not had a sustained virologic response after peginterferon alfa-ribavirin therapy to one of four treatment groups: 115 patients to the T12PR24 group, receiving telaprevir (1125-mg loading dose, then 750 mg every 8 hours) for 12 weeks and peginterferon alfa-2a (180 microg per week) and ribavirin (1000 or 1200 mg per day, according to body weight) for 24 weeks; 113 patients to the T24PR48 group, receiving telaprevir for 24 weeks and peginterferon alfa-2a and ribavirin for 48 weeks (at the same doses as in the T12PR24 group); 111 patients to the T24P24 group, receiving telaprevir and peginterferon alfa-2a for 24 weeks (at the same doses as in the T12PR24 group); and 114 patients to the PR48 (or control) group, receiving peginterferon alfa-2a and ribavirin for 48 weeks (at the same doses as in the T12PR24 group). The primary end point was sustained virologic response (undetectable HCV RNA levels 24 weeks after the last dose of study drugs). The rates of sustained virologic response in the three telaprevir groups--51% in the T12PR24 group, 53% in the T24PR48 group, and 24% in the T24P24 group--were significantly higher than the rate in the control group (14%; P

Telaprevir (TVR) is a hepatitis C virus (HCV) NS3.4A protease inhibitor that has exhibited antiviral activity in patients with HCV genotype 1 infection. The viral dynamics in patients dosed with TVR were compared with those reported for patients treated with interferon (IFN).The dynamics of wild-type HCV genotype 1 in patients dosed with TVR monotherapy (n=36) and TVR plus pegylated interferon (PEG-IFN)-alpha2a (n=8) were quantified using a biphasic viral dynamic model.Patients dosed with either TVR monotherapy or TVR plus PEG-IFN-alpha2a had median first and second phase decreases of 12 per day and 1.1 per day, respectively. The second phase decrease was approximately 10-fold higher than reported values for IFN-based treatments (P0.0001). Patients dosed with TVR plus PEG-IFN-alpha2a had a median remaining viral production after blockage (1-epsilon) of -2.37 log(10). In patients dosed with TVR monotherapy, increased TVR dosage of the same schedule was related to better blockage.These results suggested that TVR-based regimens for chronic HCV infection will lead to an early and more rapid viral decrease that could potentially result in higher sustained viral response rates as well as offer the potential for a reduced duration of treatment.

Chronic hepatitis B and C affect approximately 500 million people in the world, with substantial disease burden including liver cirrhosis and hepatocellular carcinoma. For chronic hepatitis B, two treatment strategies are currently available, both with suboptimal response and significant side effects. Promising new drugs are approaching the stage of approval; however, these agents still need further development to control this disease. Based on the understanding of the hepatitis C virus life cycle, new treatment developments for chronic hepatitis C tend to succeed rapidly; therefore, it is only a matter of time before new therapies emerge. This review summarizes the most important new agents available for treatment of chronic hepatitis B and C.

Although blood component transfusion is currently regarded as safe, adverse events may occur in recipients of these products. Among those, blood borne viral, bacterial and parasitic infections are best known. For detection of bacterial contamination in platelet concentrates various methods are available or under investigation. One of these methods, real-time polymerase chain reaction (PCR) with particular focus on real-time 16S rDNA detection, will be discussed in this review.

Telaprevir (VX-950) is an orally active, specifically targeted antiviral therapy for hepatitis C virus (HCV) that has been shown to profoundly reduce plasma HCV RNA in genotype 1 patients. Using a highly sensitive sequencing assay that detects minor populations of viral variants (≥5%), mutations were identified that conferred low-level (V36M/A, T54A, or R155K/T) or high-level (A156V/T and 36/155) resistance to telaprevir in vitro. We report a detailed kinetic analysis of these variants in 16 patients given telaprevir or telaprevir + pegylated interferon–alpha-2a (PEG-IFN–alpha-2a) for 14 days. In 4 patients who had a viral rebound on telaprevir alone, the R155K/T and A156V/T variants were detected during the initial steep decline in HCV RNA. During the rebound phase, the R155K/T and A156V/T variants were replaced by V36(M/A)/R155(K/T) double mutant variants. In the remaining 12 patients given telaprevir alone or with telaprevir/PEG-IFN–alpha-2a, the A156V/T variant was detected in some patients, but viral levels continued to decline in all patients. Conclusion: These studies suggest that the initial antiviral response to telaprevir is due to a sharp reduction in wild-type virus, which uncovers pre-existing telaprevir-resistant variants. In patients given telaprevir alone, viral rebound can result from the selection of variants with greater fitness. However, the combination of telaprevir and PEG-IFN–alpha-2a inhibited both wild-type and resistant variants. In the present study, every patient who began PEG-IFN–alpha-2a and ribavirin after the 14-day dosing period had undetectable HCV RNA levels at 24 weeks, indicating that telaprevir-resistant variants are sensitive to PEG-IFN–alpha-2a and ribavirin. (HEPATOLOGY 2007.)

BACKGROUND & AIMS: VX-950 specifically inhibits the NS3.4A protease of hepatitis C and has antiviral activity in vitro. This phase I, placebo-controlled, double-blind study evaluated the antiviral activity, pharmacokinetics, and safety of VX-950 in patients with chronic hepatitis C (CHC). METHODS: Thirty-four patients with genotype 1 CHC were randomized to receive placebo or VX-950 at doses of 450 mg or 750 mg every 8 hours or 1250 mg every 12 hours for 14 days. Of the 34 participants, 27 (79%) had failed prior treatment. Patients were monitored for safety and tolerability of VX-950. Plasma VX-950 concentrations and HCV RNA levels were measured. RESULTS: VX-950 was well tolerated and had substantial antiviral effects: viral loads dropped > or =2 log(10) in all 28 patients treated with VX-950 and > or =3 log(10) in 26 (93%) of the 28 patients. In the 750-mg-dose group, which had the highest trough plasma drug concentrations, the median reduction of HCV RNA was 4.4 log(10) after 14 days. In the 450-mg and 1250-mg groups, the maximal effect was seen between days 3 and 7 of dosing, and median HCV RNA increased between days 7 and 14; median reductions at day 14 were 2.4 log(10) and 2.2 log(10), respectively. Median alanine aminotransferase levels decreased during dosing in all VX-950 groups. CONCLUSIONS: VX-950 was well tolerated and demonstrated substantial antiviral activity. Some patients had viral breakthrough during dosing, related to selection of variants with decreased sensitivity to VX-950. The results support further studies of VX-950 in patients with CHC