The current standard of care for patients with

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1 Robust Antiviral Activity of R1626, a Novel Nucleoside Analog: A Randomized, Placebo-Controlled Study in Patients with Chronic Hepatitis C Stuart K. Roberts, 1 Graham Cooksley, 2 Gregory J. Dore, 3 Richard Robson, 4 David Shaw, 5 Heather Berns, 6 George Hill, 6 Klaus Klumpp, 6 Isabel Najera, 6 and Carla Washington 6 The nucleoside analog R1479 is a potent and highly selective inhibitor of nonstructural protein 5B directed hepatitis C virus (HCV) replication in vitro. R1626, a tri-isobutyl ester prodrug of R1479, was developed to increase bioavailability and improve antiviral activity. A multicenter, observer-blinded, randomized, placebo-controlled, multiple ascending dose, phase 1b study was designed to evaluate the safety, pharmacokinetics, and antiviral activity and to potentially identify the maximum tolerated dose of R1626 in patients with chronic hepatitis C. Forty-seven treatment-naïve patients infected with HCV genotype 1 were treated with R1626 orally at doses of 500 mg, 1500 mg, 3000 mg, or 4500 mg or placebo twice daily for 14 days with 14 days of follow-up. Safety, tolerability, pharmacokinetics, and antiviral activity were assessed. Doses up to and including 3000 mg twice daily were well tolerated after 14 days of treatment. There was an increase in frequency of adverse events at the highest dose (4500 mg). Reversible mild to moderate hematological changes were observed with increasing doses. R1626 was efficiently converted to R1479, with dose-proportional pharmacokinetics observed over the entire dose range. The pharmacokinetics of R1479 were linear over the dose range evaluated. Dose-dependent and time-dependent reductions in HCV RNA were observed. Mean decreases (median; range) in viral load after 14 days of treatment with doses of 500, 1500, 3000, and 4500 mg were 0.32 (0.22; ), 1.2 (0.8; ), 2.6 (2.7; ) and 3.7 (4.1; ) log 10, respectively. No resistance to R1479 was observed after 14 days of treatment with R1626. Conclusion: These data support further studies of R1626 in combination with peginterferon alfa-2a and ribavirin for the treatment of patients with chronic HCV infection. (HEPATOLOGY 2008;48: ) Abbreviations: AE, adverse event; ALT, alanine aminotransferase; HCV, hepatitis C virus; NS5B, nonstructural protein 5B; SAE, serious adverse event. From the 1 The Alfred Hospital, Melbourne, Australia; 2 Royal Brisbane Hospital, Brisbane, Australia; 3 St. Vincents Hospital, Sydney, Australia; 4 Christchurch Clinical Studies Trust, Christchurch, New Zealand; 5 Royal Adelaide Hospital, Adelaide, Australia; and 6 Roche Palo Alto LLC, Palo Alto, CA. Received November 22, 2007; accepted March 3, Address reprint requests to: Carla Washington, Director, Clinical Pharmacology, Roche Palo Alto LLC, 3431 Hillview Ave., Palo Alto, CA carla.washington.cw1@roche.com; fax: Copyright 2008 by the American Association for the Study of Liver Diseases. Published online in Wiley InterScience ( DOI /hep Potential conflict of interest: Dr. Roberts is a consultant for, advises, and is on the speakers bureau of Roche. Dr. Dore advises, is on the speakers bureau of, and received grants from Roche and Gilead Sciences. He also advises and received grants from Bristol-Myers Squibb. Dr. Washington owns stock in Roche. Dr. Cooksley advises and is on the speakers bureau of Roche. The current standard of care for patients with chronic hepatitis C virus (HCV) infection, the combination of pegylated interferon alpha plus ribavirin for 48 weeks, results in sustained virological response in only approximately 50% of patients infected with HCV genotype 1. 1,2 Response rates are lower still in patients coinfected with human immunodeficiency virus, older patients, and those with cirrhosis. 3-5 Therefore, a considerable unmet medical need remains in this disease area, which will be addressed through the development of novel therapeutic approaches. One such approach is to target the HCV polymerase enzyme that is essential for HCV replication. The concept of polymerase inhibition to attain antiviral efficacy has been proven in viral infections such as hepatitis B virus, herpes simplex virus, and human immunodeficiency virus. Polymerase inhibitors form the largest class of approved antiviral drugs and, of these, nucleoside analogs constitute the largest chemical class. Nucleoside analogs are metabolized by cellular enzymes to the corresponding nucleoside triphosphate analogs, which competitively inhibit viral nucleic acid synthesis. 398

2 HEPATOLOGY, Vol. 48, No. 2, 2008 ROBERTS ET AL. 399 R1479 was discovered as a potent and specific inhibitor of nonstructural protein 5B (NS5B) directed HCV replication in cell culture. 6 The corresponding nucleotide triphosphate is a competitive inhibitor of HCV polymerase and inhibits RNA chain extension after incorporation into nascent HCV RNA. 6 Nonclinical pharmacological assessment of R1479 supported its promise as an effective and selective antiviral drug for the treatment of HCV infection. R1626, a tri-isobutyl ester prodrug of R1479, was therefore developed to improve oral bioavailability of R1479 and thus optimally deliver R1479 to achieve maximal antiviral activity in vivo. R1626 is rapidly converted to R1479, predominately by esterases, in the epithelial layer of the gastrointestinal tract. Animal studies have demonstrated that R1626 has 10-fold greater permeability, providing significantly greater exposure to R1479 than dosing with the parent drug itself. The majority of administered R1626 is excreted as R1479 in the urine. Based on in vitro data, R1479 is not a substrate, inhibitor, or inducer of cytochrome P450 (CYP450) enzymes (Roche, data on file). Preliminary data in healthy volunteers suggest that R1626 is well tolerated as single doses up to 12,000 mg, with no reported serious adverse events. 7 The primary objective of this phase 1b study was to perform an initial assessment of the safety, tolerability, pharmacokinetics, and antiviral activity, and to potentially identify the maximum tolerated dose after administration of multiple ascending doses of R1626 in treatment-naïve patients infected with HCV genotype 1. Patients and Methods Patients The study was conducted in accordance with good clinical practice, the Declaration of Helsinki, and appropriate requirements with regard to independent ethics committee review, informed consent, and regulations pertaining to the protection of subjects in research studies. All participating subjects signed an informed consent form before receiving the study drug. Eligible patients were females of nonchildbearing potential and men aged between 18 and 60 years who were treatment-naïve and had chronic liver disease consistent with chronic hepatitis C genotype 1 infection (defined as persistence of HCV infection diagnosed by the detection of HCV RNA in the blood for at least 6 months from initial detection). All patients had serological evidence of HCV infection by an anti-hcv antibody test, at least 1 measurement of serum HCV RNA of 10 5 IU/mL or greater (Roche COBAS Amplicor Monitor version 2.0; Roche Molecular Systems Inc., Branchburg, NJ), alanine aminotransferase (ALT) 5 times the upper limit of normal or less at screening, and a liver biopsy showing Metavir fibrosis stage of 2 or less. Liver biopsies had to be obtained within 36 months before screening, or during the screening period if not performed previously. Patients who had been previously treated with any interferon treatment, in combination or without ribavirin, or any other systemic antiviral therapy or investigational drug for HCV were excluded from the study. Other exclusion criteria included infection with hepatitis A or B viruses, or human immunodeficiency virus, a history or evidence of other medical conditions associated with chronic liver disease, renal disease, or immunologically mediated disease, or a history of severe psychiatric disease. All patients had a complete medical and medication history taken during screening and underwent a physical examination including hematology, chemistry, urinalysis, and electrocardiogram. Study Design and Procedures This multicenter, observer-blinded, randomized, placebo-controlled, multiple ascending dose study was conducted in Australia and New Zealand. Screening was conducted between April 22, 2005 and May 24, 2006; the last patient observation took place on July 18, A total of 48 patients were enrolled into the study and, after confirmation of eligibility, were randomized to treatment with R1626 orally at doses of 500 mg, 1500 mg, 3000 mg, or 4500 mg or matched placebo twice daily for 14 days. All medicines and placebos were provided by F. Hoffmann-La Roche (Basel, Switzerland). The allocation sequence was generated by Roche, and an interactive voice response system was used to implement random allocation sequence when the number of sites increased from 3 to 5. Investigators, patients, and individuals involved in data collection were all blinded to group assignment. Patients were treated for 14 days and then followed for an additional 14 days. A single patient in the 3000-mg placebo group withdrew before the study start. Safety Assessments. Safety and tolerability were monitored at regular intervals from predose through to a follow-up visit 14 days after completion of dosing. Safety assessments included physical examinations, vital signs, electrocardiograms, clinical laboratory assessments, incidence of clinical adverse events, concomitant medications, laboratory assessments, and laboratory abnormalities. Any clinically significant abnormal laboratory test results were followed until resolved or stabilized. Adverse events were recorded and assessed for severity and possible relationship to study drug at each study visit. Pharmacokinetics. Blood samples for pharmacokinetic determination of plasma concentrations of R1479, R1626, and its metabolites were collected at the time of

3 400 ROBERTS ET AL. HEPATOLOGY, August 2008 the morning dose as follows: on days 1 and 7 at predose and at 1, 2, 4, 6, 8, 10, and 12 hours postdose; on day 14 predose and at 1, 2, 4, 6, 8, 10, 12, 16, 24, 48 and 72 hours postdose. Plasma samples were collected before dosing on days 2, 5, and 10 for evaluation of trough concentrations. Urine was also collected at 0 to 6 hour and 6 to 12 hour intervals to assess R1479 concentrations. Pharmacokinetics parameters were calculated using noncompartmental methods (WinNonlin, version 5.0 Pharsight Corp., Mountain View, CA). Efficacy Analyses. Antiviral activity was assessed by change from baseline in serum HCV RNA level. Qualitative HCV RNA tests (Roche COBAS Amplicor HCV Test, version 2.0; Roche Molecular Systems Inc., Branchburg, NJ) were performed at the screening visit. Quantitative tests (Roche COBAS Amplicor HCV Monitor test, version 2.0) were also performed on all positive qualitative tests at this visit. Quantitative tests were performed at subsequent study visits. The limit of HCV RNA quantification was 600 IU/mL. Additional qualitative HCV RNA measurements were performed on all samples below the limit of quantification. The limit of detection was 50 IU/mL. Serum samples for HCV RNA assessment were obtained during screening (days 14 and &,minus1) at a time corresponding to the planned dosing time; predose, and at 4 and 12 hours after the morning dose on day 1, predose on treatment days 2, 5, 7, 12, and 14, and posttreatment on days 15, 21, and 28. Similarly, serum ALT activity was also measured in the samples used for HCV RNA analysis. Resistance Monitoring. The analysis of resistance in this study was based on the definition of drug resistance as a new ability of a virus to replicate in the presence of a drug that once inhibited its replication. This new ability of the virus to replicate in the presence of the drug will be reflected in an increase in plasma viral load. Therefore, samples were selected from patients that showed an initial sensitivity to the drug followed by a viral load rebound while on treatment. Monitoring for the development of resistance to R1479 was performed in patients experiencing virological rebound, defined as a 0.5 log 10 IU/mL or more decrease in viral load followed by a subsequent ontreatment increase of 0.5 log 10 IU/mL or greater above nadir. In addition, samples were selected from patients that did not respond to treatment ( 0.5 log IU/mL viral load reduction at any time on treatment), because these patients may have carried a virus naturally resistant to inhibition by the drug. The sensitivity of the clinical isolates to R1479 was determined using the HCV NS5B phenotypic assay as previously described. 8 Briefly, HCV RNA was extracted from patients serum and the NS5B coding region was amplified and cloned into replicon vectors. In vitro transcribed replicon RNA containing the NS5B region from the clinical isolates was transfected into Huh-7 cells. Compounds (or medium as a control) were added 24 hours posttransfection in three-fold dilutions at a final dimethylsulfoxide concentration of 1%. Firefly luciferase reporter signal was read 72 hours after addition of compounds using the Luciferase Assay System (Promega Corporation, Madison, WI). The median inhibitory concentration values were assessed as the compound concentration at which a 50% reduction in the levels of firefly luciferase reporter was observed as compared with control samples in the absence of compound. Dose response curves and median inhibitory concentration values were generated by using the XLfit3 program (ID Business Solutions, Ltd., Surrey, UK). The entire region of the HCV genome encoding NS5B protein, the target of R1479 inhibitory activity, was sequenced using primers covering both DNA strands. Sequencing reactions were performed using an ABI 3730 xl DNA Analyzer and chromatograms analyzed using Sequencher (Gene Codes Corporation, Ann Arbor, MI) and VNTI (InforMax Inc., MD). Results Patient Characteristics A total of 47 patients were randomized and received treatment 35 active drug and 12 placebo, as follows: 500 mg twice daily (9 active and 2 placebo); 1500 mg twice daily (9 active and 3 placebo); 3000 mg twice daily (8 active and 4 placebo); 4500 mg twice daily (9 active and 3 placebo). Demographic data and baseline HCV RNA levels were similar across the groups (Table 1). Forty-six patients completed treatment and 1 patient withdrew from the study prematurely. Pharmacokinetic Results After oral administration, R1626 was efficiently converted to R1479 with near dose proportional pharmacokinetics observed over the entire dose range (Fig. 1). Linear pharmacokinetics were observed for all doses evaluated C min [minimum or trough concentration], C max [maximum concentration], and area under the curve). After 14 days of treatment with R1626, mean trough concentrations (C min ) ranged from 0.86 to 5.98 g/ml, and mean maximum concentrations ranged from 3.7 to 25.1 g/ml. The mean elimination half-life ranged from 20 to 25 hours. The effective half-life of R1479 ranged from 4.62 to 5.87 hours, compatible with a twice-daily dosing regimen mean. R1479 was the major component excreted in urine; negligible concentrations ( 1%) of R1626 were recovered. Approximately 57% to 71% of

4 HEPATOLOGY, Vol. 48, No. 2, 2008 ROBERTS ET AL. 401 Table 1. Summary of Demographic and Baseline Characteristics R1626 Placebo 500 mg Twice Daily 1500 mg Twice Daily 3000 mg Twice Daily 4500 mg Twice Daily N Mean age, years (range) 43.0 (26 58) 35.9 (20 51) 42.7 (28 60) 44.6 (27 52) 46.9 (31 57) Sex (M/F) 10/2 7/2 9/0 7/1 9/0 Body mass index (kg/m 2 ), Mean SD Ethnicity White Other HCV RNA (log 10 IU/mL) Mean Median Range ALT (IU/L) Mean Range ALT, alanine aminotransferase; HCV, hepatitis C virus administered R1626 was recovered as R1479 over 12 hours after the last dose of R1626 on day 14. Assessment of Antiviral Activity Reduction in HCV RNA Level. Time-related and dose-related decreases in serum HCV RNA were observed, with maximum mean (median; range) decreases of 0.32 (0.22; ), 1.2 (0.8; ), 2.6 (2.7; ), and 3.7 (4.1; ) log 10 at day 14 for the 500-mg, 1500-mg, 3000-mg, and 4500-mg dose groups, respectively (Fig. 2). In contrast, the HCV RNA levels in patients in the placebo arm showed very little change from baseline during the dosing period. All patients in the 4500-mg group and 63% of patients in the 3000-mg group had a mean decrease in HCV RNA from baseline of at least 2 log 10 (Table 2). In the 3000-mg twice daily R1626 group, 2 of 8 patients (25%) were below the limit of quantification (600 IU/mL) by day 14. At a dose of 4500 mg twice daily, 8 of 9 patients (88.8%) had viral levels that fell below quantitative levels, and 5 of 9 patients were polymerase chain reaction negative ( 50 IU/mL) after 14 days of treatment. HCV RNA levels returned toward baseline values after discontinuation of R1626. In correlating decreases in viral load to exposure, higher exposures were generally associated with greater decreases in viral load (Fig. 3). A detailed analysis of the virological response according to dose is given in Table 2. The HCV RNA profiles of individual patients during and after treatment with R1626 (1500-mg, 3000-mg, or 4500-mg dose) are shown in Fig. 4. ALT Response. Time-related and dose-related decreases were observed in changes in mean ALT from baseline (Fig. 5). Decreases ranged from 28 IU/mL (38%) in the 500-mg twice daily group to 67 IU/mL (72%) in the 3000-mg twice daily group. Five of 8 patients who received R1626 in the 3000-mg group and 7 of 9 patients in the 4500-mg group demonstrated on-treatment normal- Fig. 1. Linear mean plasma concentration of R1479 ( g/ml) versus time profile following administration of R1626 (day 14). Fig. 2. Mean reduction in HCV RNA levels (log 10 ) from baseline.

5 402 ROBERTS ET AL. HEPATOLOGY, August 2008 Table 2. Maximum Reduction in the HCV RNA Level from Baseline to Day 14 According to Dose R1626 dose (mg bid) N Magnitude of Viral Load Reduction on Day 14 (log 10 IU/mL) >1 log 10 >2 log 10 >3 log 10 >4 log 10 Mean Median Range Rebound [n] Nonresponse [n] (11%) 2 (22%) (38%) 2 (25%) 3 (38%) (11%) 3 (33%) 5 (56%) ization of ALT levels. Similar decreases were observed in aspartate aminotransferase levels. Resistance Evaluation The evaluation of resistance selection was based on the definition of drug resistance as the renewed ability of a virus to replicate in the presence of a drug that once inhibited its replication. After an initial viral load reduction attributable to the antiviral effect, this capacity of the virus to replicate in the presence of the drug will result in an increase in plasma HCV RNA level. The selection of drug-resistant virus is therefore associated with a viral rebound profile. The viral load profiles of all study participants were assessed for a viral rebound phenotype defined as a viral load reduction 0.5 log 10 IU/mL or greater followed by a 0.5 log 10 or greater increase in HCV RNA level from nadir during treatment with R1626. Among all study participants from all dose groups, only 3 patients showed a viral rebound phenotype. These 3 patients all received the 500 mg twice daily dose. Pretreatment and end-of-treatment samples from these 3 patients were assessed for potential selection of resistance by phenotypic and sequencing characterization. Sequence analysis of the entire NS5B coding region showed no common amino acid changes across the 3 patients. Phenotypic characterization of these clinical isolates showed no change in susceptibility to R1479, with a 0.7- Fig. 3. Decrease in HCV RNA viral concentration versus R1479 exposure on last day of dosing (day 14). fold to 1.4-fold shift in median inhibitory concentration at day 14 compared with baseline (Table 3); these values are well within the 2.6-fold variability of the assay compared with baseline sample and compared with reference controls (HCV genotype 1a H77 and genotype 1b Con reference strains). 8 Therefore, there was no evidence for selection of resistance in this study after 14 days of monotherapy with R1626. Three other patients from the 500-mg dose group showed no response to the drug throughout the treatment period. To study whether the lack of response was a result of reduced sensitivity of these patients to R1479 because of the presence of R1479 resistance mutation (S96T) or any other amino acid substitution, samples from these patients at baseline, at the end of treatment (day 14), as well as during the follow-up period (days 21 and 28) were selected for further analyses. Sequence studies showed no S96T amino acid substitution in the NS5B coding region in the consensus sequence of any of the 3 patients at any of the time points. The phenotypic assay showed that all baseline samples were sensitive to R1479, as were the samples analyzed at each subsequent time point (Table 3). To investigate whether the lack of virological response in the 3 patients was attributable to the presence of R1479 resistance mutation (S96T or S96T/N142T) at low level in the virus quasispecies of baseline samples, clonal sequence analysis of between 96 and 106 NS5B polymerase molecular clones for each sample spanning the NS5B coding region was performed. This revealed the absence of preexisting amino acid substitutions among the quasispecies of these patients that could be responsible for resistance to R1479 at a low level. Safety Results The summary of adverse events is given in Table 4. Most adverse events (approximately 67%) were rated as mild by the investigator. Approximately 25% to 33% of the adverse events were described as moderate by the investigator in all but the 4500-mg dose group, whereas half were rated as moderate in intensity. Seven patients reported a total of 13 events that were considered severe; of these, headache was the most commonly reported. Other severe adverse events included dizziness, vomiting, nau-

6 HEPATOLOGY, Vol. 48, No. 2, 2008 ROBERTS ET AL. 403 Fig. 4. Reduction in HCV RNA levels (log 10 ) in individual patients during and after dosing with R1626 (1500 mg, 3000 mg or 4500 mg) or placebo. sea, retching, back pain, and neck pain. No patient was withdrawn from the study because of an adverse event. Gastrointestinal events accounted for a significant proportion of adverse events in the 3000-mg and 4500-mg groups, with nausea and diarrhea being the most common adverse events in both groups. Two patients in the 4500-mg group each missed 1 dose of the study drug because of an adverse event; 1 patient missed the day 5 dose because of vomiting but continued with the study schedule the following day; 1 patient missed the day 14 dose because of severe nausea. In the 3000-mg group, of the 33 adverse events reported, 12 were gastrointestinal events reported by 6 patients, including 3 with mild or moderate nausea and 2 with mild or moderate diarrhea. Two patients had underlying conditions (gastroesophageal reflux disease and irritable bowel syndrome) that may have influenced these findings. One patient missed 1 dose (on day 5) because of vomiting. There was 1 serious adverse event (metastatic gastric cancer) reported in this group. Based on the temporal relationship of the event and the study drug, and the advanced stage of the gastric cancer at diagnosis, this was considered unlikely to be related to R1626 administration. With the exception of hematological values and liver enzyme levels, results for clinical laboratory tests (serum chemistry and urinalysis) were unremarkable and similar across the treatment groups (data not shown). Time-related and dose-related mild-to-moderate hematological Fig. 5. Reduction in ALT levels (U/L) in individual patients during and after dosing with R1626 (500 mg, 1500 mg, 3000 mg, or 4500 mg) or placebo.

7 404 ROBERTS ET AL. HEPATOLOGY, August 2008 Patient Table 3. Phenotypic Characterization of Patients with Virological Rebound or Nonresponse Baseline R1479 IC 50 ( M) Day 14 R1479 IC 50 ( M) R1479 IC 50 Fold Shift From Baseline Patients 1, 2, and 3 had viral rebound, which was defined as: viral load reduction 0.5 log 10 IU/mL followed by a 0.5 log 10 increase in the HCV RNA level from the nadir; patients 4, 5, and 6 were nonresponders (all in 500-mg dose arm). changes (decreases in hematocrit, hemoglobin, red blood cells, reticulocytes, white blood cells, neutrophils, monocytes, and platelets) were observed (Fig. 6A-D). In most cases, the severity of these abnormalities did not exceed grade 1 (mild adverse event). Four patients had grade 2 abnormalities (low white blood cell counts in 2; low neutrophil counts in 1; low white blood cell and low neutrophil counts in 1). Patients in the 4500-mg group showed the greatest number of hematological abnormalities. At day 14, reductions in hemoglobin level from baseline were 1.0, 1.3, 1.6, and 1.9 g/dl for the placebo, 1500-mg, 3000-mg, and 4500-mg groups, respectively. A drop of approximately 1 g/dl in hemoglobin level would be anticipated because of the need to draw an approximately 500-mL blood sample as per the study protocol. By day 28 (14 days after the last dose), hematological parameters for most patients were returning toward baseline levels. One patient in the highest dose group (4500 mg) required an additional 5 weeks to return to baseline values. Nine patients (3 in each of the 1500-mg, 3000-mg, and 4500-mg dose groups) had at least 1 elevated total bilirubin value ( 17 M) during the study; however, 7 of these patients had total bilirubin values greater than 17 M before R1626 dosing (day 1). The elevations did not appear to be dose related. The highest total bilirubin value observed was 34.9 M at day 14 in a patient in the 4500-mg group. In 7 of 9 patients, values returned to within the normal range ( 17 M) by day 28 (2 weeks after the completion of dosing), including the patient with the highest total bilirubin value. The 2 patients whose total bilirubin values were still elevated at day 28 (both in the 1500-mg group) had elevated bilirubin values before administration of R1626. The baseline and onstudy direct bilirubin values for these patients were within the normal range (0-7.0 M). No patients had clinically significant changes in vital signs or electrocardiograms. Discussion Nucleoside analogs targeting the HCV polymerase enzyme are a novel class of drug that hold promise for improving treatment efficacy in patients chronically infected with HCV. Based on potent in vitro data, 6 R1626, a triisobutyl ester prodrug of R1479, was developed to increase the oral bioavailability of R1479 and so improve antiviral activity in vivo. The current study, designed to evaluate the safety, tolerability, pharmacokinetics, and antiviral activity of R1626 administered for 14 days to treatment-naïve patients chronically infected with HCV genotype 1, showed the agent to be generally well tolerated up to 3000 mg, with increasing adverse events at the highest dose (4500 mg). Hematological changes (including hemoglobin, hematocrit, reticulocytes, and red and white blood cells) were observed with increasing doses; however, these tended to be mild to moderate and reversible, recovering to baseline levels by day 28. The most frequent and marked changes were observed with the 4500-mg dose. The cause of these hematological effects is currently unclear; there is no evidence of hemolytic effects of R1479 in vitro. 9 Gastrointestinal disturbances, in particular nausea and diarrhea, were most pronounced in the 4500-mg dose group, and, as a result, this dose would not be considered for use in future clinical trials with R1626. The data from this study demonstrate that R1626 is capable of significantly reducing HCV RNA in genotype 1 infected patients when used as monotherapy. Timerelated, dose-related, and exposure-related decreases were observed after multiple doses of R1626, with declines in viral load being seen as early as 4 hours after the first dose. Table 4. Summary of AEs Placebo 500 mg bid 1500 mg bid R mg bid 4500 mg bid N Total number of AEs AEs reported by 2 patients Headache Dizziness Altered taste Lethargy Nausea Diarrhea Flatulence Vomiting Abdominal pain Rash SAEs reported AE, adverse event; SAE, serious adverse event.

8 HEPATOLOGY, Vol. 48, No. 2, 2008 ROBERTS ET AL. 405 Fig. 6. Mean changes in hematological parameters from baseline to day 28. The maximum decrease in HCV RNA levels was noted on the last day of dosing, and HCV RNA profiles showed a consistent reduction in viral load across individual patients at the 3000-mg and 4500-mg doses. The decreases in HCV RNA from baseline observed with R1626 indicates potent antiviral activity and lack of viral load rebound in the significant majority of patients after 14 days of monotherapy Hepatitis C virus is typically found as a genetically heterogeneous, monophyletic population of viral variants (quasispecies), the composition of which changes rapidly as escape variants that evade cellular and humoral immune responses are selected. This high genetic heterogeneity, together with the high mutation rate and short generation time of HCV, represents an opportunity for the virus to evade antiviral treatment. Longer periods of monotherapy with R1626 may be required to select for replication-competent, R1626-resistant HCV variants. Viral rebound during treatment was observed in 3 patients, all of whom received the lowest dose of 500 mg. The rebound appears to be attributable to inadequate drug levels, because there was no evidence of viral resistance from phenotypic and sequencing analysis. Nonresponse was observed in 3 other patients of the lowest-dose group (500 mg). Also, the nonresponse appeared to be attributable to inadequate drug levels, because there was no evidence of viral resistance from phenotypic and sequencing analysis including clonal analysis of the baseline samples. The lack of resistance observed in this study may reflect the potency of R1626 as an antiviral agent, with the low fitness (4%-5% compared with wild-type) shown in vitro by R1479-resistant NS5B mutants S96T and S96T/N142T contributing to a high barrier to resistance development. 13 The lack of selection of resistance to R1479 (despite the inadequate drug levels) is in contrast to the observations to date with other antivirals in development. Viral resistance according to phenotyping and sequencing analysis emerged during 14-day treatment with VX-950, an inhibitor of the HCV NS3 4A protease, 14 and antiviral resistance was also selected within the first week of monotherapy with the nonnucleoside polymerase inhibitor HCV

9 406 ROBERTS ET AL. HEPATOLOGY, August 2008 In conclusion, 14 days of treatment with R1626 was generally well tolerated up to a dose of 3000 mg twice daily and resulted in robust time-dependent, dose-dependent, and exposure-dependent decreases in HCV viral load. There was no evidence of resistance development during 14 days of treatment. Given the promising profile, further studies are warranted to determine the efficacy and safety of R1626 in combination with peginterferon alfa-2a plus ribavirin, the current standard of care, in patients with chronic HCV infection. Acknowledgment: The authors thank all the clinical study investigators and coordinators involved in the trial, Michael Brandl (formulation development), Scott Fettner and Yaping Tu (pharmacokinetic analysis), David Ipe (statistical analysis), Marie Mannino (safety monitoring), Sophie Le Pogam, Alan Kosaka, and Sharon Jiang (phenotypic and sequencing characterization of samples for monitoring of resistance), the R1626 Study Management Team, the R1626 Research Development Team, and the Life Cycle Team. Karen Searle (a medical writer) provided editorial assistance. References 1. Fried MW, Shiffman ML, Reddy KR, Smith C, Marinos G, Goncales FL Jr, et al. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med 2002;347: Manns MP, McHutchison JG, Gordon SC, Rustgi VK, Shiffman M, Reindollar R, et al. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial. Lancet 2001;358: Torriani FJ, Rodriguez-Torres M, Rockstroh JK, Lissen E, Gonzalez-Garcia J, Lazzarin A, et al. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection in HIV-infected patients. N Engl J Med 2004; 351: Chung RT, Andersen J, Volberding P, Robbins GK, Liu T, Sherman KE, et al. Peginterferon alfa-2a plus ribavirin versus interferon alfa-2a plus ribavirin for chronic hepatitis C in HIV-coinfected persons. N Engl J Med 2004;351: Pawlotsky JM. Treating hepatitis C in difficult-to-treat patients. N Engl J Med 2004;351: Klumpp K, Leveque V, Le Pogam S, Ma H, Jiang WR, Kang H, et al. The novel nucleoside analogue R1479 (4 -azidocytidine) is a potent inhibitor of NS5B-dependent RNA synthesis and hepatitis C virus replication in cell culture. J Biol Chem 2006;281: Robson R, Berns H, Brandl M, Fettner S, Haznedar J, Hill G, et al. Safety, tolerability and pharmacokinetics of R1626, a novel nucleoside analog targeting HCV polymerase: results from a phase 1 single dose escalation trial in healthy subjects [Abstract]. Clin Pharmacol Ther 2007;81(Suppl 1):S Le Pogam S, Seshaadri A, Kosaka A, Chiu S, Kang H, Hu S, et al. Existence of hepatitis C virus NS5B variants naturally resistant to non nucleosides, but not to nucleosides, polymerase inhibitors among untreated patients. J Antimicrobial Chemotherapy, March 20, E-pub ahead of print. 9. Jiang WR, Symons J, Cammack N, Klumpp K. R1479, a novel nucleoside analog and potent inhibitor of HCV replication, does not affect ribavirininduced hemolysis and erythrocyte fragility in vitro. J Hepatol 2007; 46(Suppl 1):S Godofsky E, Afdhal N, Rustgi V, Shick L, Duncan L, Zhou X-J, et al. First clinical results for a novel antiviral treatment for hepatitis C: a phase I/II dose escalation trial assessing tolerance, pharmacokinetics and antiviral activity of NM283 [Abstract 96]. J Hepatol 2004;40(Suppl 1): Afdhal N, Godofsky E, Dienstag J, Rustgi V, Schick L, McEniry D, et al. Final phase I/II trial results for NM283, a new polymerase inhibitor for hepatitis C: antiviral efficacy and tolerance in patients with HCV-1 infection, including previous interferon failures [Abstract LB-03]. HEPATOL- OGY 2004;40(Suppl 1):726A. 12. Chandra P, Raible D, Harper D, Speth J, Villano S, Bichier G. Antiviral activity of the non-nucleoside polymerase inhibitor, HCV-796, in patients with chronic hepatitis C virus: preliminary results from a randomized, double-blind, placebo-controlled, ascending multiple dose study [Abstract 1]. Gastroenterology 2006;130(Suppl 2):A Le Pogam S, Jiang W, Leveque V, Rajyaguru S, Ma H, Kang H, et al. In vitro selected Con1 subgenomic replicons resistant to 2 -C-methyl-cytidine or to R1479 show lack of cross resistance. Virology 2006;351: Reesink HW, Zeuzem S, Weegink CJ, Forestier N, Van Vliet A, Van De Wetering De RooiJ J, et al. Rapid decline of viral RNA in hepatitis C patients treated with VX-950: a phase Ib, placebo-controlled, randomized study. Gastroenterology 2006;131: Villano S, Howe A, Raible D, Harper D, Speth J, Bichier G. Analysis of HCV NS5B genetic variants following monotherapy with HCV-796, a non-nucleoside polymerase inhibitor, in treatment naive HCV-infected patients [Abstract 1127]. HEPATOLOGY 2006;44(Suppl 1):607A-608A.