956 PHARMACOKINETICS, TOLERABILITY AND EFFECT OF FOOD ON HCV POLYMERASE INHIBITOR ABT-333 FOLLOWING SINGLE ASCENDING DOSES IN HEALTHY ADULT VOLUNTEERS

05h: VIRAL HEPATITIS − h) HEPATITIS C − CLINICAL (NEW COMPOUNDS, RESISTANCE) respective EC50 values together with G418 (400 mg/ml) to select drugresistant replicon clones. The NS5B polymerase coding regions of resistant replicons were sequenced, and single-mutant replicons were constructed by site-directed mutagenesis. Results: The most frequently observed resistance mutations selected by ABT-333 and ABT-072 were C316Y, M414T, Y448H/C, or S556G. Phenotypic characterization of the resistant variants showed a loss susceptibility to either ABT-333 or ABT-072 ranging from less than ten to more than one thousand-fold. Most resistant mutants, however, have significantly lower replication capacity than wild type. Combinations of ABT-333 or ABT-072 with interferon in both short-term and long-term replicon assays exhibit additive to synergistic antiviral interactions. Conclusions: ABT-333 and ABT-072 are highly potent non-nucleoside inhibitors of HCV polymerase. Replicon resistance selection studies with these compounds have revealed mutant variants with reduced susceptibility. Combination treatments of ABT-333 or ABT-072 with IFN show additive to synergistic interactions in HCV replicon, suggesting therapeutic utility of these combinations in vivo. 954 BI 201335, A POTENT HCV NS3 PROTEASE INHIBITOR, IN TREATMENT-NAIVE AND -EXPERIENCED CHRONIC HCV GENOTYPE-1 INFECTION: GENOTYPIC AND PHENOTYPIC ANALYSIS OF THE NS3 PROTEASE DOMAIN G. Kukolj1 , Y. Benhamou2 , M.P. Manns3 , M. Bourli`ere4 , S. Pol5 , M. Schuchmann6 , M. Cartier1 , D. Huang7 , L. Lagac´e1 , G. Steinmann8 , J.O. Stern7 . 1 Biological Sciences, Boehringer Ingelheim (Canada) Ltd. R&D, Laval, Quebec, Canada; 2 Service H´epato-Gastroent´erologie, Batiment des Cliniques M´edicales, Hopital Pitie Salpetriere, Paris, France; 3 Zentrum Innere Medizin, Abteilung f¨ur Gastroenterologie, Hepatologie und Endokrinologie, Medizinische Hochschule Hannover, Hannover, Germany; 4 Service d’H´epato-Gastro-Ent´erologie, Hopital Saint Joseph, Marseille, 5 Pˆole M´edico Chirurgical d’H´epato-GastroEnt´erologie, Unit´e d’H´epatologie, Hopital Cochin, Paris, France; 6 I. Med. Klinik und Poliklinik, Klinikum der Johannes Gutenberg, Universit¨at Mainz, Mainz, Germany; 7 Virology, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT, USA; 8 Clinical Research, Virology, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany E-mail: [email protected] Background: BI 201335, a potent and specific HCV NS3/4A protease inhibitor, has been studied with chronic genotype (GT) 1 HCV infection for 14-days monotherapy in treatment-naive patients followed by combination with PegIFN/RBV for an additional 14 days; and in treatment-experienced patients for 28 days as combination therapy with PegIFN/RBV. All dose groups achieved median viral load reductions of 3 log10 . On-treatment viral rebound was observed in most patients receiving monotherapy, but in only 3/19 treatment-experienced patients receiving triple combination therapy with lower dosages of BI 201335. Genotypic and phenotypic characterization of viral isolates from this study has been performed. Methods: HCV RNA was collected and extracted from 53 patients at baseline, days 14 and 28, and during follow-up. Population sequencing was performed on the complete NS3/4A region. Clonal sequencing permitted the quantification and identification of major and minor variants (LLOQ: 5%) in the NS3 protease domain. Chimeric HCV sub-genomic replicons were constructed to determine BI 201335 EC50 values against patientderived protease sequences. Results: Population sequencing of baseline isolates revealed one encoding an NS3 V/I170T substitution that conferred an 8-fold reduction in BI 201335 EC50 relative to the subtype reference. Mean EC50 values for the other GT 1a (10±8 nM) or 1b (9±4 nM) baseline-derived samples were within three-fold of the reference values. The predominant GT 1a resistance mutations in on-treatment viral rebound samples encoded an R155K substitution, whereas GT 1b viruses mainly encoded changes at D168, with valine as a predominant substitution. R155K variants conferred reductions in sensitivity to BI 201335 with EC50 values of 1.8−6.5 mM, whereas the EC50 s for D168V variants were 3.6 −15 mM. Rapid changes

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in mutant prevalence at the end of dosing suggest that D168V variants are significantly less fit than R155K variants. Conclusion: HCV NS3 variants that confer resistance to BI 201335 were selected during treatment; these variants do not alter the sensitivity to PegIFN/RBV as the majority of treatment naive patients with resistant virus subsequently displayed anti-viral responses during combination therapy. 955 PRECLINICAL POTENCY, PHARMACOKINETIC AND ADME CHARACTERIZATION OF ABT-333, A NOVEL NON-NUCLEOSIDE HCV POLYMERASE INHIBITOR C. Maring, R. Wagner, D. Hutchinson, C. Flentge, W. Kati, G. Koev, Y. Liu, D. Beno, J. Shen, Y.Y. Lau, Y. Gao, J. Fischer, S. Vaidyanathan, B.H. Lim, J. Beyer, R. Mondal, A. Molla. Global Pharmaceutical Research & Development, Abbott, Abbott Park, Illinois, USA E-mail: [email protected] Background and Aims: ABT-333 is a novel non-nucleoside HCV polymerase inhibitor with potent activity against genotype 1a and 1b HCV replicons. This compound is currently being evaluated for safety, pharmacokinetics and antiviral activity in chronically infected HCV patients. The objective of this report will be to disclose the potency, pharmacokinetic and ADME profile of ABT-333 in preclinical model systems. Methods: The polymerase inhibition profile and selectivity of ABT-333 was determined with a panel of HCV NS5B and human polymerase enzymes. Its HCV replicon potency was assessed with and without 40% human plasma. ADME studies with 3H-ABT-333 were conducted in vitro with human microsomes and hepatocytes and in vivo in rats. ABT-333 plasma pharmacokinetics in multiple species was determined after IV and oral dosing, and its liver levels at 12 h after oral dosing. Results: ABT-333 exhibits highly potent (IC50 < 10 nM) and selective activity for genotype 1a and 1b HCV polymerases. The EC50 s for ABT333 in genotype 1 replicons range from 2 to 7 nM and its potency is attenuated by 10- to 14-fold in 40% human plasma. ABT-333 has high Caco-2 permeability and good in vitro stability in human microsomes. ABT-333 does not inhibit CYP enzymes or induce CYP3A4 expression in hepatocytes. Metabolite profiles in hepatocytes indicate a primary oxidative pathway followed by subsequent conjugation primarily as a glucuronide. Subsequent rat ADME studies with ABT-333 confirmed this in vitro metabolism profile and showed that it is eliminated primarily by biliary excretion. ABT-333 has oral bioavailability in rat and dog and is highly distributed to liver producing 12-hour drug levels at significant multiples over its EC50 . Conclusion: ABT-333 is a potent non-nucleoside inhibitor of genotype 1a and 1b HCV polymerase with oral bioavailability in rat and dog. The compound exhibits a preclinical ADME profile of high permeability, in vitro metabolic stability and low potential for drug interactions that predicts favorable pharmacokinetics in humans. 956 PHARMACOKINETICS, TOLERABILITY AND EFFECT OF FOOD ON HCV POLYMERASE INHIBITOR ABT-333 FOLLOWING SINGLE ASCENDING DOSES IN HEALTHY ADULT VOLUNTEERS R. Menon1 , D. Cohen2 , A. Nada1 , E. Olson Dumas1 , Y.-L. Chiu1 , T. Podsadecki2 , W. Awni1 , B. Bernstein2 , C. Klein1 . 1 Clinical Pharmacology and Pharmacometrics, 2 Global Pharmaceutical Research and Development, Abbott Laboratories Inc, North Chicago, Illinois, USA E-mail: [email protected] Background and Aims: ABT-333 is a novel non-nucleoside NS5B polymerase inhibitor being developed for the treatment of HCV genotype 1 infection. The objectives of this study were to evaluate the safety, tolerability and pharmacokinetics (PK) of escalating, single, oral doses of ABT-333 and to determine the effect of food on the PK of ABT-333 in healthy subjects. Methods: The double-blind, placebo-controlled, single-ascending dose (10 to 1200 mg) portion of this study was conducted in 9 sequential groups. In

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Poster Session − Saturday, April 25

each dose group, healthy adult subjects were randomized to receive oral doses of either ABT-333 (8) or Placebo (2) under nonfasting conditions. The single-dose, open-label, food-effect portion of this study dosed 8 healthy adult subjects with 100 mg ABT-333 under fasting and nonfasting conditions in a 2-period crossover design. Plasma samples were collected for up to 72 hours after dosing for pharmacokinetic analyses. Safety and tolerability were monitored throughout the study. Results: Maximum plasma concentrations (Cmax ) of ABT-333 occurred at approximately 3 hours post-dose. The mean half-life (t1/ 2 ) was 5 to 8 hours. The Cmax and area under the plasma concentration-time curve (AUC) increased proportionally across all treatment groups and mean values ranged from 31 ng/mL to 3053 ng/mL (Cmax ) and 317 to 23939 ng.hr/mL (AUC). Food did not impact the Cmax and AUC of ABT333. Single doses of ABT-333 up to the maximum dose tested, 1200 mg, were well tolerated with no dose limiting toxicity identified. There were no clinically significant changes from baseline in laboratory values, vital signs, or ECG recordings. Conclusions: The PK of ABT-333 increased in a dose proportional fashion. Food did not impact the Cmax and AUC of ABT-333. ABT-333 was well tolerated up to the maximum dose tested, 1200 mg. 957 PHARMACOKINETICS, SAFETY AND TOLERABILITY OF THE HCV POLYMERASE INHIBITOR ABT-333 FOLLOWING MULTIPLE ASCENDING DOSES AND EFFECT OF CO-ADMINISTRATION OF KETOCONAZOLE IN HEALTHY SUBJECTS R. Menon1 , D. Cohen2 , A. Nada1 , Y.-L. Chiu1 , T. Podsadecki1 , W. Awni1 , B. Bernstein2 , C. Klein1 . 1 Clinical Pharmacology and Pharmacometrics, 2 Global Pharmaceutical Research and Development, Abbott Laboratories Inc, North Chicago, Illinois, USA E-mail: [email protected] Background and Aims: ABT-333 is a novel non-nucleoside NS5B polymerase inhibitor being developed for the treatment of HCV genotype 1 infection. This study evaluated the safety, tolerability and pharmacokinetics of multiple oral doses of ABT-333 and assessed the effect of a potent CYP3A inhibitor on the pharmacokinetics of ABT-333 in healthy subjects. Methods: Healthy adult subjects received ABT-333 200 and 400 mg BID for 10 days in a double-blind, placebo-controlled fashion in sequential groups. In each dose group, subjects were randomized to receive oral doses of either ABT-333 (8) or Placebo (4) under nonfasting conditions. Serial plasma samples were collected on Day 1 and Days 10 to 13. Plasma samples were also collected prior to morning dose on days 2, 3, 5, 7, 8 and 9. Safety and tolerability were monitored throughout the study. In the 200 mg dose group, on day 11, an additional morning dose of ABT-333 was co-administered with 400 mg ketoconazole. Results: The mean maximum plasma concentration (Cmax ) and area under the concentration-time curve (AUC12 ) for the 200 mg dose group on Day 10 were 405 ng/mL and 2807 ng*hr/mL, respectively. The dose-normalized maximum plasma concentration and area under the concentration-time curve were comparable for the 200 and 400 mg BID dose groups. Minimal accumulation between Day 1 and 10 was noted with multiple dose administration. The Cmax and AUC values for evening dose and morning dose on Day 10 were comparable indicating no significant diurnal variation in the pharmacokinetics of ABT-333. Ketoconazole codosing resulted in a modest increase in ABT-333 Cmax (53%) and AUC (64%). ABT-333 was well tolerated with few adverse events and no clinically significant dose-related changes from baseline in laboratory values, vital signs, or ECG recordings. Conclusions: The pharmacokinetics of ABT-333 were dose proportional following 10 days of administration. ABT-333 showed minimal accumulation with no diurnal variation. As the fold increase in ABT-333 exposure in the presence of ketoconazole was between 1.25 and 2, based on FDA guidance, ABT-333 is not a “sensitive substrate” of CYP3A. ABT-333 was well tolerated following multiple dosing for 10 days.

958 NO EVIDENCE OF R7128 DRUG RESISTANCE AFTER UP TO 4 WEEKS TREATMENT OF GT 1, 2 AND 3 HEPATITIS C VIRUS INFECTED INDIVIDUALS S. Le Pogam1 , A. Seshaadri1 , A. Kosaka1 , S. Hu1 , A. Beard2 , S. Julian1 , C. Nick1 , I. Najera1 . 1 Roche Palo Alto LLC, Palo Alto, CA, 2 Pharmasset, NJ, USA E-mail: [email protected] Background and Aims: R7128, a prodrug of PSI-6130, has demonstrated antiviral efficacy after 2 weeks’ monotherapy in treatment-experienced HCV GT-1-infected patients and after 4 weeks therapy in combination with pegylated interferon alpha 2a and ribavirin in GT-1 infected treatmentnaive patients and in GT-2/3 infected treatment-experienced patients. The aim of the study was to: i. monitor the emergence of drug resistance to R7128 in vivo, ii. determine whether the NS5B S282T substitution exists in the patient’s quasispecies at low frequency (clonal analysis), iii. determine the frequency of S282T in a patient’s quasispecies necessary to observe a decrease in PSI-6130 sensitivity. Methods: Viral resistance monitoring was performed by sequence (population and clonal studies) and phenotypic analysis of the NS5B gene from isolates at baseline and at on-treatment time points. The S282T mutation was introduced into NS5B clinical isolates at different ratios of wild-type/ S282T replicons to determine their effect on PSI-6130-sensitivity. Results: Population sequence analysis of the NS5B region revealed no evidence of the S282T resistance mutation. NS5B quasispecies sequence information from 42 GT-1 infected patients (3400 NS5B sequences from untreated patients and 800 from patients undergoing R7128 treatment) showed no S282T variants. All NS5B isolates from on-treatment time points were sensitive to inhibition by PSI-6130. In vitro studies showed that NS5B S282T confers a low replication capacity and a low level of resistance regardless of the genetic background it is engineered into. Mutant S282T needs to be present at a high proportion in the viral population in order to observe a decrease in PSI-6130 sensitivity. Conclusions: No evidence for the development of viral resistance to R7128 after 2 weeks or 4 weeks of therapy was observed. No S282T variants were found in any treatment-naive or R7128 treated patients at population or quasispecies level, confirming clinically the high genetic barrier of this nucleoside polymerase inhibitor in short-term studies. Given the low replication capacity of the S282T in vitro engineered mutant, and the low level of phenotypic resistance it confers, it is likely that S282T variants must become predominant in the viral population to observe a clinically-relevant decrease in PSI-6130 sensitivity. 959 SUBTYPE SPECIFIC RESISTANCE PROFILES AFTER SELECTION OF HCV GENOTYPE 1A OR GENOTYPE 1B REPLICONS WITH TELAPREVIR OR HCV-796 M. McCown, S. Rajyaguru, N. Cammack, I. Najera. Roche Palo Alto LLC, Palo Alto, CA, USA E-mail: [email protected] Background and Aims: Resistant variants have been described in HCV infected patients treated with the protease inhibitor Telaprevir or with the non-nucleoside polymerase inhibitor HCV-796. Interestingly, the resistance substitutions identified in Telaprevir treated patients varied depending on the HCV subtype. In this study, we determined the kinetics of resistance selection to Telaprevir and HCV-796 and their resistance profiles using the HCV replicon system. Methods: GT-1a and GT-1b replicon cells were treated at 15X the EC50 with either Telaprevir or HCV-796 and the emergence of resistance was monitored over a three week period. At the indicated time-points, the cellular RNA was extracted and either the NS3 protease (for Telaprevir) or the NS5B (for HCV-796) regions were amplified and sequenced. Results: Treatment of GT-1b replicon cells with Telaprevir selected substitutions at NS3 position 156 whereas in GT-1a replicon cells a substitution at position 155, in addition to 156, was identified. This result is consistent