740 Emergence of dual-resistance HBV mutations to adefovir-dipivoxil and lamivudine in a hepatitis B patient with combination therapy for adefovir resistance

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S A F E T Y A N D E F F I C A C Y OF V I R A M I D I N E | PLUS PEGYLATED INTERFERON A L P H A - 2 B V E R S U S RIBAVIRIN PLUS PEGYLATED INTERFERON A L P H A - 2 B IN THERAPY-NA'I'VE PATIENTS INFECTED WITH HCV: PHASE 3 RESULTS

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Y. Benhamou 1, P. Pockros2, M. Rodriguez-Torres3, S. Gordon4, M. Shiffman5, Y. Lurie 6, N. Afdhal 7, K. Lamon8, Y. Kim8, B. Murphy8.

1Hdpital Pitid-Salpdtri~re, Paris, France," :Scripps Clinic, La Jolla, CA, USA," 3Fundacion de Investigacion de Diego, Puerto Rico, USA," 4Detroit, Michigan, USA," 5Medical College of Virginia, Richmond, VA, USA," 6Tel Aviv, Israel," 7Beth Israel-Deaconess Medical Cente~ Boston, MA, USA," 8 Valeant Pharmaceuticals' Research & Development, USA Introduction: Viramidine, a liver-targeting oral pro-drug of ribavirin, does not significantly accumulate in the red blood cell (RBC). Data from a phase 2 study revealed that dosing Viramidine 600 mg BID with pegylated interferon resulted in comparable efficacy but significantly lower rates of anemia compared to ribavirin and pegylated interferon. This fixed Viramidine dose (600 mg BID) was chosen for phase 3. Methods: This multi-center, active-control, randomized, parallel group, double blind study consisted of 970 patients. Patients were randomized 2:1 to receive Viramidine 600mg BID or weight-based dosed ribavirin (1000 1200mg/day), respectively. Stratification was based on genotype, baseline viral load, and weight. Analyses assessed anemia (Hb < 10 g/dL) for superiority testing and sustained viral response (SVR) based on NGI SuperQuant assay (sensitivity <100 copies/ml) for non-inferiority testing. Results: The rate of anemia was 5% in the Viramidine cohort versus 24% in the ribavirin group (p<0.001). The study did not meet the non-inferiority efficacy endpoint on an overall intent-to-treat (ITT) basis: Viramidine 38%, ribavirin 52%, respectively. Viramidine met the noninferiority criteria in pre-determined per protocol (PP) analyses based on geographic region and weight-based dosing.

Regiona

Efficacy: Percent SVR (PP analysis) Viramidine Ribavirin Adjusted difference of proportion and 95% confidence intervals

Overall (N 637) 52% N.A.&E.U. (N 489) 51% N.A.&E.U. >75kg (N 271) 42% N.A.&E.U. ~<75kg (N 218) 62% R.O.W. (N 148) 55%

62% 56% 53% 60% 78%

0.074 (0.002,0.151) 0.029 (0.060,0.118) 0.090 (0.034,0.213) 0.049 (0.177,0.079) 0.209 (0.052,0.365)

aAbbreviations: E.U.: EuropeanUnion; N.A.: North America; R.O.W.: Rest of World.

Viramidine weight-based analysis; Patients with SVR (PP analysis) Viramidine dose ~<18 mg/kg 19 22 mg/kg ~>23mg/kg

N 323 82 16

SVR

Anemia (Hb < 10 g/dL)

47% 66% 81%

4.3% 2.4% 12.5%

Conclusions: This study confirmed the superior safety profile of Viramidine. Fixed-dose Viramidine resulted in an average dose of 15mg/kg which did not maximize the potential efficacy advantages of Viramidine compared to weight-based ribavirin. Analyses of weight-based dosing demonstrated that increasing the mg/kg dose of Viramidine improved the response without a proportionate increase in anemia. Other adverse events did not appreciably increase with higher doses of Viramidine.

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M A T H E M A T I C A L M O D E L I N G OF S U B G E N O M I C HEPATITIS C VIRAL REPLICATION IN HUH-7 CELLS

H. Dahari 1, R.M. Ribeiro 1, C.M. Rice 2, A.S. Perelson 1. 1Theoretical Biology and Biophysics, MS-K710, Los Alamos National Laboratory, NM, USA," 2Center for the study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY, USA

Background and Aims: Cell-based hepatitis C virus (HCV) replicon systems have provided a means for understanding HCV replication mechanisms and for testing new antiviral agents. Based on quantitative data of subgenomic HCV replication in Huh-7 cells, we sought to gain a better understanding of its dynamics from transfection to steady state using a mathematical model. Methods: We developed a mathematical model of HCV replication assuming that the strategy of replication involves double-stranded RNA templates, and that the translation of the HCV polyprotein occurs in the cytoplasm, while HCV replication occurs in a vesicular membranous structure (VMS). We tested the robustness of our model results by simulating one thousand random combinations of parameter values. Results: According to our model we predict the following: (i) about 6 • 103 ribosomes are involved in generating millions of HCV NS5B-polymerase molecules in a Huh-7 cell, (ii) the observed 10:1 asymmetry of plus- to minus-strand RNA levels can be explained by a higher affinity (200-fold) interaction of HCV NS5B-polymerase complexes with the minus-strand RNA over the plus-strand RNA in order to initiate replication, (iii) the latter higher affinity (>200-fold) leads to ~6:1 plus-to-minus strand ratio in VMS, in agreement with recent experimental results, and (iv) transfection of higher numbers of replication competent plus-strand RNA leads to faster attainment of the RNA steady-state, but does not change its magnitude. Conclusions: Our results shed light on the intracellular dynamics of subgenomic HCV RNA replication from transfection to steady state within Huh-7 cells. Fully permissive HCV replication systems have been developed and the model presented here is a first step towards building a comprehensive model for intracellular HCV replication. Moreover, the model can serve as an important tool in understanding HCV replication mechanisms, and may prove useful in designing and evaluating new antivirals against HCV.

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E M E R G E N C E OF DUAL-RESISTANCE H B V MUTATIONS TO ADEFOVIR-DIPIVOXIL A N D L A M I V U D I N E IN A HEPATITIS B PATIENT WITH C O M B I N A T I O N T H E R A P Y F O R A D E F O V I R RESISTANCE

S.Y. Kwon 1, C.H. Lee 1, W.H. Choi 1, J.E. Yeon2, K.S. Byun 2. 1Dept. Internal Medicine, Konkuk Univ. Hospital, Seoul, South Korea," :Dept. Internal Medicine, Korea Univ. Guro Hospital, Seoul, South Korea

Backgrounds: Adefovir dipivoxil (ADV) is used successfully for treatment of patients with lamivudine (LMV)-resistant hepatitis B. ADV-resistant mutation is not uncommon in LMV-resistant hepatitis B. The emergence of multiple drug resistant HBV is a major clinical concern in longitudinal sequential monotherapy or combination therapy. We report a case of dual mutation of YMDD and rtA181 during ADV LMV combination therapy for ADV resistance. Case: Patient was a 58-year-old Korean man with eAg-positive hepatitis B cirrhosis. He had been previously treated with LMV for 23 months, developed LMV resistance (rtM204I/V, rtL180M) despite good compliance. ADV was started, and the viral load dropped rapidly with restoration of YMDD wild type. After 16 months of ADV therapy, ADV resistance

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(rtA181V) resulted in viral breakthrough. LMV was added to ADV, however, anti-viral response was suboptimal. Resistance mutation to both ADV and LMV developed with hepatitis B flare-up. ADV was replaced by tenofovir, resulting in a significant drop in viral load. Conclusion: The multiple drug resistant mutation will be a troublesome question in the anti-viral therapy for hepatitis B. Combination treatment with different cross-resistant profile or switch to a new potent anti-viral agent should be needed for the multiple-drug resistant cases.

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SAFETY, TOLERABILITY AND ANTIVIRAL ACTIVITY OF PRADEFOVIR MESYLATE IN PATIENTS WITH CHRONIC HEPATITIS B VIRUS INFECTION: 48-WEEK ANALYSIS OF A PHASE 2 STUDY

K.S. Lee 1, S.G. Lim2, W.L. Chuang 3, S.G. Hwang 4, M. Cho 5, M.Y. Lai 6, Y.C. Chao 7, T.T. Chang 8, K.H. Han 9, C.M. Lee 1~ S.H. U m 11 , J.E. Yeon 12, S.S. Yang 13, E.K. Teo 14, C.Y. Peng 15, H.H. Lin 16, S.S. Yang 17, T.I. Huo 18, T. Nguyen 19, T.Y. Chen 2~ K.Q. Hu 21 , Y. Xu 22, J.Z. Sullivan-Bdlyai22. gYonsei University College of Medicine, Seoul,

South Korea," 2National University Hospital, Singapore," 3Kaohsiung Medical University Hospital, Kaoshiung, Taiwan," 4Bundang CHA Hospital, Kyunggi-do, South Korea," 5Pusan National University Hospital, Pusan, South Korea," 6National Taiwan University Hospital, Taipei, Taiwan," 7Tri-Service General Hospital, Taipei, Taiwan," 8National Cheng Kung University Hospital, Tainan, Taiwan," 9Yonsei University, Seoul, South Korea," l~ Chang-Gung Memorial Hospital, Kaoshiung Hsien, Taiwan," 11Korea University Medical Center (Anam), Seoul, South Korea," g2Korea University Medical Center (Kuro), Seoul, South Korea," 13Cathay General Hospital, Taipei, Taiwan," 14Changi General Hospital, Singapore," 15China Medical University Hospital, Taichung, Taiwan,"16Tzu Chi General Hospital, Hualien, Taiwan," 17Taiehung Veterans General Hospital, Taiehung, Taiwan," 18Taipei Veterans General Hospital, Taipei, Taiwan," 19Research & Education, Inc., San Diego, CA, USA," 2~ Shan Medical University Hospital, Taiehung, Taiwan," 21University of California Irvine, Orange, CA, USA," 22Valeant Researeh & Development, Costa Mesa, CA, USA Background: Pradefovir (PDV) is a liver-targeted prodrug of PMEA with potentially improved efficacy and less nephrotoxicity than adefovir dipivoxil (ADV). Aims: To evaluate the safety and efficacy of PDV in chronic HBV infection. Methods: Randomized, open-label, parallel-group, multicenter study comparing ADV 10mg/d and PDV 5, 10, 20, and 30mg/d for 48 weeks. Inclusion/exclusion criteria included HBV DNA > 5 • 105 c/mL; alanine aminotransferase (ALT) 1.2 10 • ULN; no prior ADV. HBV DNA was measured by COBAS Amplicor| (LLQ 169 copies/mL). Results: 244 patients were randomized. 242 received >1 dose (ITT). Patients were 100% Asian, 83% male with an average age of 38 years. 70% were HBeAg positive. 0.4% were infected with genotype A, 32.6% with genotype B, and 66.9% with genotype C. Mean baseline HBV DNA was 7.9 8.21og10 c/mL. Median baseline ALT was 2.0 2.8 • ULN. The majority of patients had previously received IFN or antivirals. Reductions from baseline in HBV DNA were 4.09• 4.84• 4.89• and 5.54• (mean• logl0 copies/mL) for the PDV 5 m g (n 47), 10mg (n 49), 2 0 m g (n 48) and 3 0 m g (n 48) groups, respectively, vs 4.19• for the ADV 10mg (n 50) group. ANCOVA model indicated that P 0.83, 0.007, 0.007, and <0.001 versus ADV for the PDV 5, 10, 20 and 30 mg groups, respectively. The proportions of patients with HBV D N A < 4 0 0 c / m L were 45%, 63%, 56% and 71% for the PDV 5mg, 10mg, 2 0 m g and 30mg groups, respectively, and 36% for the ADV 10 mg group. Diarrhea, dyspepsia, nasopharyngitis, upper respiratory tract infection and headache were the most frequently reported adverse events (AEs) by PDV 3 0 m g patients. Most AEs were mild. AEs and laboratory safety values were comparable to the ADV 10mg group.

One patient taking PDV 10 mg reported a serious, treatment-related AE; hepatoma. Conclusion: The 48-week analysis demonstrates that PDV is safe, well tolerated, and PDV 10, 20, or 30 mg has significantly greater anti-HBV activity than ADV 10 mg.

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I~-CATENIN PATHWAY CONTRIBUTES TO A B E R R A N T DNP73 EXPRESSION IN HUMAN HCC

E. Palescandolo 1'2'3, V. Schinzari 1,2, S. Vossio 1'2, A. Rossini4, E. Cariani 4, M. Levrero 1'2'3. 1Fondazione A Cesalpino and Dept of

Internal Medicine, University of Rome "La Sapienza", Rome, Italy," 2Dept. Molecular Oncogenesis Laboratory, Regina Elena Cancer Cente~ Rome, Italy," 3Rome Oncogenomic Cente~ Regina Elena Cancer Institute, Rome, Italy," 4Dept of Medicine and Laboratory, A. (2 Spedali Civili, Brescia, Italy At least four pathways (i.e. pRb, p53, TGF-[J and [J-catenin) that regulate cell proliferation and cell death are affected in HCCs. The wnt/[J-catenin pathway is a common target for HCV and HBV in HCCs, also independently from axinl/[J-catenin genes mutations: (a) the Frizzled 7 receptor, that regulates wt [J-catenin stabilisation/activation, is overexpressed in >90% of virus-related HCCs; (b) HCV core and NS5A and HBV HBx proteins induce [J-catenin accumulation. In regions where exposure to aflatoxin is low p53 mutations are rarely observed (<4% of HCCs) whereas the p53 paralog and tumor suppressor p73 is deregulated, p73 gene give rise to multiple protein isoforms due to both alternative P1 and P2 promoters utilization and alternative mRNA splicing. TAp73s proteins mimic p53 function whereas DNp73 isoforms do not activate transcription but instead have oncogenic effects by acting as dominant negative inhibitors of both TAp73 isoforms and p53. We and others have reported that DNp73 accumulates progressively in chronic hepatitis, cirrhosis and HCC and confers to HCC cells a chemoresistant phenotype. Here we show that exogenously expressed wt or tumor-derived (Ser33Tyr mutant which cannot be phosphorylated by GSK-3~ and targeted to proteolysis by the APC/axinl complex) ~-catenin activate both the P2p73 promoter in transient luciferase reporter experiments and endogenous DNp73 expression, as detected by real time PCR quantification of DNp73 mRNAs. The P2p73 promoter (2627/+76) contains activating p53, NF-KB, AP1 and ~-catenin/TCF binding sites as well as ISRE and RARE elements. By combining transactivation assays and chromatin immunoprecipitation analysis we found that the P2p73 promoter is regulated in hepatocytes by the coordinated activity of ~-catenin, NF-KB/p65, p53 and TAp73 and that [J-catenin cooperates with p53 and p73 in the activation of DNp73 expression. Abrogation of [J-catenin expression by specific siRNA in Huh-6 cells (carrying a G34V [J-catenin mutation and wt p53 alleles) led to decreased expression of DNp73 and strong potentiation of apoptosis in response to DNA damaging agents. Altogether our findings link [J-catenin overexpression with p53 and TAp73 functional inactivation that is mediated by DNp73 overexpression and does not require the selection of p53 gene inactivating mutations.