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Once Daily Sofosbuvir/Ledipasvir Fixed Dose Combination with or without Ribavirin: the ELECTRON trial

Edward J. Gane1, Catherine A. Stedman2, Robert H. Hyland3, Xiao Ding3, Evguenio S. Svarovskoia3, Phil S. Pang3, William T. Symonds3
1Auckland Clinical Studies, Auckland, New Zealand; 2Christchurch Clinical Studies Trust, Christchurch, New Zealand; 3Gilead Science, Inc, Foster City, CA

Background: In previous Phase 2 studies, the addition of an NS5A inhibitor (either l edipasvir (LDV) or daclatasvir) to the combination of the HCV nucleotide analog sofosbuvir (SOF) plus ribavirin (RBV) for 12 weeks resulted in high rates of sustained viral response (SVR) in non-cirrhotic patients infected with HCV genotype 1 (GT-1). In the ELECTRON Phase 2 study, we evaluated the safety and efficacy of a fixed-dose combination (FDC) tablet of SOF plus LDV in additional patient populations, including those with cirrhosis and with non-GT-1 infection. We also evaluated the need for RBV and for 12 weeks' duration. Methods: We enrolled 4 arms: prior null-responder HCV GT-1 patients with compensated cirrhosis were randomized to receive open-label SOF/LDV FDC with or without RBV for 12 weeks; treatment-naīve HCV GT-1 patients without cirrhosis received SOF/LDV FDC plus RBV for 6 weeks; and treatment-naϊve HCV non-GT-1 patients without cirrhosis were assigned to receive SOF/LDV FDC plus RBV for 12 weeks. Results: 54 patients were enrolled. Of the GT-1 prior null-responder group, 79% were genotype 1a, and 32% were IL28B CC. Of the treatment-naϊve GT-1 group, 84% were genotype 1a, and 20% were IL28B CC. Of the non-GT-1 group 20% were GT-2, and 80% GT-3.Efficacy results are tabulated. SOF/LDV FDC with or without RBV was generally well tolerated; There were no SAEs or treatment discontinuations. Adverse events were generally mild, and Grade 3/4 laboratory abnormalities were infrequent and consistent with the safety profile of RBV. No toxicity attributable to SOF/LDV FDC was identified. Conclusions: SOF/LDV FDC elicited rapid decline in HCV RNA in all patient populations with no viral breakthrough observed. In treatment-naīve GT-1 patients without cirrhosis, reduction in duration from 12 to 6 weeks increased the rate of relapse. In the prior null responder GT-1 patients with cirrhosis, the addition of RBV to SOF/LDV FDC decreased the rate of relapse, suggesting that either RBV or a third DAA may be useful in this difficult-to-treat patient population. Promising SVR rates achieved in genotype 2 or 3 patients support further evaluation of SOF/LDV FDC in patients with HCV GT-2 or GT-3.

Randomized GT 1 Prior Null Responders (Cirrhotics)GT 1 Treatment Nave (Non-Cirrhotics)GT 2/3 Treatment Nave (Non-Cirrhotics)
SOF/LDV FDC x 12 weeks (n=10)SOF/LDV FDC+RBV x 12 weeks (n=9)SOF/LDV FDC+RBV x 6 weeks (n=25)SOF/LDV FDC x 12 weeks (n=10)
  1. *One subject was lost fo follow up without post-treatment data.

RVR80%66%100%100%
EOTR100%100%100%100%
SVR480%89%*88%80%
SVR 12PendingPending68%80%

Disclosures:

Edward J. Gane - Advisory Committees or Review Panels: Roche, AbbVie, Novartis, Tibotec, Gilead Sciences, Janssen Cilag, Vertex, Achillion; Speaking and Teaching: Novartis, Gilead Sciences, Roche

Catherine A. Stedman - Advisory Committees or Review Panels: Jansen; Speaking and Teaching: Gilead

Robert H. Hyland - Employment: Gilead Sciences, Inc Xiao Ding - Employment: Gilead Sciences

Evguenia S. Svarovskaia - Employment: Gilead Sciences Inc.; Stock Shareholder: Gilead Sciences Inc.

Phil S. Pang - Employment: Gilead Sciences William T. Symonds - Employment: Gilead

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The Combination of Alisporivir with an NS5A Inhibitor Provides Additive to Synergistic Antiviral Activity, no Cross-Resistance and a High Barrier to HCV Resistance

Udayan Chatterji1, Kelly A. Wong2, Weidong Zhong2, Clifford Brass3, Nikolai V. Naoumov4, Philippe Gallay 1
1TheScripps Research Institute, La Jolla, CA; 2Navartis Institutes for BioMedical Research, Emeryville, CA; 3Novartis Pharmaceuticals Corporation, East Hanover, NJ; 4Novartis Pharma AG, Basel, Switzerland

Two principal groups of HCV antivirals exist: direct-acting antivirals (DAAs) and host-targeting antivirals (HTAs). DAAs target viral proteins e. g. NS3 protease, NS5B polymerase or NS5A, while HTAs target host proteins critical for HCV replication. Both groups of antivirals were shown to profoundly reduce HCV replication in vitro and in patients with hepatitis C. In this study, we investigated whether particular DAAs exhibit additive, synergistic or antagonistic effects when combined with an HTA - Alisporivir (ALV), which targets cyclophilin A (CypA). We used different HCV genotypes established as stable luciferase reporter replicon cell lines to test the effect of ALV in combination with different classes of DAAs: NS3, NS5A and NS5B inhibitors. Compounds were tested at concentrations near EC50 values calculated in reporter cell lines specific to each genotype. Cell viability and reporter assays were carried out in five replicates for each drug combination and the normalized data were used to analyze the synergy between different combinations using MacSynergyII. In contrast to the DAAs tested, ALV was found to exert potent antiviral activity against all HCV genotypes tested (EC50 of 8, 16, 17 and 20 nM for genotypes 1a, 1b, 2a and 3a, respectively). The combinations of ALV with DAAs exerted mostly additive effects against genotypes 1a and 1b (synergy values of ∼ 0). Importantly, combining ALV with certain DAA classes exerted synergistic antiviral effect on genotypes 2a and 3a. The most profound synergistic effect was observed with the combination of ALV with NS5A inhibitors (NS5Ai) (synergy values of 15 to 68). This is consistent with the finding that ALV blocks the contact between CypA and domain II of NS5A, whereas NS5Ai target domain I of NS5A. Thus, the synergistic effects of the combination of ALV and NS5Ai may arise from the mechanism of action of these two classes of antivirals targeting distinct loci of NS5A. Importantly, we did not observe any cross-resistance between ALV and NS5Ai. Because NS5Ai and ALV exhibit a low and a high barrier to HCV resistance, respectively, we examined HCV resistance to a combination of the drugs. No resistant mutants were detected to the combination of ALV and NS5Ai after extensive in vitro drug selection (>6 months), whereas NS5Ai-resistant and ALV-resistant mutants emerged after two weeks and two months, respectively. In conclusion, these results suggest that the combination of the HTA Alisp orivir with NS5A inhibitors represents an attractive strategy, and is potentially an effective IFN-free combination regimen for treatment of HCV patients.

Disclosures:

Kelly A. Wong - Employment: Novartis Weidong Zhong - Employment: Novartis

Clifford Brass - Employment: Merck, Novartis; Patent Held/Filed: Merck, Novartis; Stock Shareholder: Merck, Novartis

Nikolai V. Naoumov - Employment: Novartis Pharma AG, Novartis Pharma AG The following people have nothing to disclose: Udayan Chatterji, Philippe Gallay

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Interferon- and Ribavirin-free Regimen of ABT-450/r + ABT-267 in HCV Genotype 1b-infected Treatment-naϊve Patients and Prior Null Responders

Eric Lawitz1, Christophe Hezode2, Peter Varunok3, Paul J. Thuluvoth4, Tolga Baykal5, Mudra Kapoor5, Sandra S. Lovell5, Tianli Wang5, Tomi Pilof-Mofios5, Regis A. Vilchez5, Barry Bernstein5
1The Texas Liver Institute, University of Texas Health Science Center, Son Antonio, TX; 2Assistance Publique Hopitaux de Paris' Paris, France; 3Premier Medical Group of the Hudson Valley' PC' Poughkeepsie, NY; 4The Institute for Digestive Health ond Liver Disease at Mercy, Baltimore, MD; 5AbbVie Inc., North Chicago, IL

Purpose: ABT-450 is an HCV protease inhibitor(dosed with ritonavir 100mg, ABT-450/r) identified by AbbVie and Enanta; ABT267 is an NS5A inhibitor. Both compounds have shown potent antiviral activity in vitro against HCV genotypes(GT)1-4 and 6.In the phase 2b study AVIATOR, all 27 treatment-naϊve patients and 19 prior null responders with HCV GT1b infection who received ABT-450/r+ABT267 with ribavirin(RBV) achieved SVR24.The PEARL-I study is evaluating the safety and efficacy of an IFN- and RBV-free regimen of ABT-450/r+ABT267 in patients with HCV GT1 b infection. Methods: Non-cirrhotic, treatment-naϊve patients and prior pegIFN/RBV null responders received ABT-450/r(150/100mg QD) and ABT267(25mg QD) for 12 wks. Observed rates of HCV RNA<25 Iし/mL at treatment wks 4 and 12, and observed SVR4 rates (percent of patients with HCV RNA<25 IU/mL at post-treatment wk 4) are reported. SVR12 rates will be presented. Results: 42 treatment-naϊve patients and 40 prior null responders with chronic HCV GT1b infection were enrolled. Baseline characteristics and efficacy are in the table. Observed SVR4 rates were 100% among treatment-naϊve patients and 87.9% among prior null responders. There were no discontinuations due to adverse events(AE) or laboratory abnormalities. 2 subjects interrupted study drug due to AEs. 1 interruption was considered probably related to study drug(increased ALT, AST, and bilirubin); these values improved after study drug interruption. AEs with >10% incidence in either arm were headache, nausea, dry skin, fatigue, pruritus, and diarrhea. 1 patient had a Grade 3 ALT elevation; 2 had Grade 3 AST elevations. Conclusions: The IFN- and RBV-free regimen of ABT-450/r+ABT267 for 12 wks was generally well-tolerated by patients in this study. Greater than 87% of HCV GT1 b-infected treatment-naϊve patients and prior null responders receiving ABT-450/r+ABT267 achieved SVR4.Larger studies are needed to further characterize response to this regimen.

Treatment-naϊve Patients (N=42)Prior Null Responders (N=40)
  1. *Observed data. Excludes patients with data missing for reasons besides virlogic failure

Baseline characteristics
Male, n (%) 25 (59.5)15 (37.5)
White race, n (%)27 (65.9)39 (97.5)
Age <50 yr, n (%) 7(16.7)13(32.5)
IL28B CC, n (%)13(31.7)2 (5.0)
Efficacy
HCV RNA <25 IU/mL at treatment week 4,n/N (%)i 42/42 (100)39/40 (97.5)
HCV RNA <25 IU/mL at treatment week 12' n/N (%)*40/40(100)39/40 (97.5)
SVR4, n/N(%)*39/39 (100)29/33 (87.9)
On-treatment failure, n01
Relapse, n03

Disclosures:

Eric Lawitz - Advisory Committees or Review Panels: AbbVie, Achillion Pharmaceuticals, BioCryst, Biotica, Enanta, Idenix Pharmaceuticals, Janssen, Merck & Co, Novartis, Santaris Pharmaceuticals, Theravance, Vertex Pharmaceuticals; Grant/Research Support: AbbVie, Achillion Pharmaceuticals, Boehringer Ingelheim, Bristol-Myers Squibb, Gilead Sciences, GlaxoSmithKline, Idenix Pharmaceuticals, Intercept Pharmaceuticals, Janssen, Merck & Co, Novartis, Presidio, Roche, Santaris Pharmaceuticals, Vertex Pharmaceuticals; Speaking and Teaching: Gilead, Kadmon, Merck, Vertex

Christophe Hezode - Speaking and Teaching: Roche, BMS, MSD, Janssen, abbvie, Gilead

Peter Varunok - Speaking and Teaching: Vertex Pharmaceuticals, Merck, Forest Pharmaceuticals

Paul J. Thuluvath - Advisory Committees or Review Panels: Bayer, Gilead, Vertex; Grant/Research Support: Gilead, Abbott, BMS, Boehringer, Salix; Speaking and Teaching: Bayer/Onyx, Vertex, Gilead

Tolga Baykal - Employment: AbbVie

Mudra Kapoor - Employment: AbbVie Inc.

Sandra S. Lovell - Employment: AbbVie

Tianli Wang - Employment: AbbVie Inc.

Tami Pilot-Matias - Employment: AbbVie; Stock Shareholder: AbbVie Regis A. Vilchez - Employment: AbbVie Inc.

Barry Bernstein - Employment: AbbVie; Stock Shareholder: AbbVie

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High Efficacy and Safety of the All-Oral Combination Regimen, MK-5172/MK-8742 +/- RBV for 12 Weeks in HCV Genotype 1 Infected Patients: The C-WORTHY Study

Eric Lawitz1, John M. Vierling2, Abel Murillo3, Marcelo Kugelmas4, Jan Gerstoft5, Peter Winkle6, Luis A. Balart7, Peer B. Christensen8, Reem H. Ghalib9, Ronald Nahass10, Melissa Shaughnessy11, Xiao Sun11, Peggy Hwang11, Janice Wahl11, Michael Robertson11, Barbara Haber11
1University of Texas Health Science Center, Texas Liver Institute, San Antonio, TX; 2Baylor College of Medicine, Houston, TX; 3Advanced Intervention & Pain Management Research Clinic, Miami, FL; 4South Denver Gastroenterology, PC, Englewood, CO; 5Epidemiklinikken, Rigshospitalet, Copenhagen, Denmark; 6Anaheim Clinical Trials, Anaheim, CA; 7Tulane University Medical Center, New Orleans, LA; 8Infektionsmed. Afd Q 2 sal, Odense Universitets Hospital, Odense, Denmark; 9Texas Clinical Research Institute, Arlington, TX; 10D Care, Hillsborough, NJ; 11Merck Research Laboratories, Upper Gwynedd, PA

Purpose: To assess the efficacy and safety of a 12-week regimen of MK-5172 (a potent hepatitis C virus [HCV] NS3/4A protease inhibitor) and MK-8742 (a potent HCV NS5A replication complex inhibitor) ± ribavirin (RBV) in patients with genotype (GT)1 HCV infection. In in vitro studies, both agents have demonstrated a high barrier to resistance and activity against GT1 variants that are resistant to other first-generation agents in the same classes. This is the first report of the combination of MK-5172 and MK-8742 in an all-oral regimen with or without RBV. Methods: Treatment-naive, non-cirrhotic (F0-F2) patients with HCV GT1 infection were randomized to one of three 12-week treatment arms: 1.MK-5172 (100 mg QD), MK-8742 (20 mg QD) plus RBV (600-1400 mg/d); 2.MK-5172 (100 mg QD), MK-8742 (50 mg QD) plus RBV; 3.MK-5172 (100 mg QD) and MK-8742 (50 mg QD). Arms 1 and 2 were stratified by GT1a vs. GT1b. Arm 3 (RBV-free) included only GT1binfected patients. Virologic response was assessed each week during treatment and at 2, 4, 8, 12, and 24 weeks after end of treatment. HCV RNA samples were assessed using COBAS TaqMan v2.0 (lower limit of quantitation [LLOQ] <25 IU/mL). Results: A total of 65 patients were enrolled: 45% were male, 11% were African American, and 58% had GT1a infection. Mean baseline HCV RNA was 6.1 log10 IU/mL. The most frequently reported adverse events (>10%) were headache (13/65, 20%), fatigue (15/65, 23%), and nausea (8/65, 12%). ALT was elevated at baseline in 50 patients and normalized on treatment in all but 1 patient (ALT = 42 IU/mL). Twenty patients had transient, mild (grade1-2) total bilirubin elevation on treatment. 〇 n-treatment response (HCV RNA <LLOQ) at weeks 1, 2, 4, and 8 are presented in the table. The trial is ongoing: end-of-treatment response, SVR4, and SVR12 will be presented in November. Conclusion: Treatment with MK5172 / MK-8742 ± RBV was associated with robust virologic suppression. No viral breakthrough was detected and in all patients HCV RNA was <LL〇Q by week 4.Although some differences were noted at treatment week 1, there were no differences in on-treatment response at treatment week 4 in GT1a compared to GT1b patients, or in GT1b patients according to the presence or absence of RBV. These data warrant further evaluation of MK-5172 + MK-8742 in IFN-free and IFN/RBVfree regimens.

GenomeRegimenTW1TW2TW4TW8
  1. *Excludes one patient who received RBV due to a dosing error. TW, treatment week.

laMK-5172/MK-8742 (both doses) + RBV (N = 38)19/3733/3736/3632/32
(51%)(89%)(100%)(100%)
lbMK-5172/MK-8742 (both doses) + RBV(N=14)10/1314/1414/148/8
(77%)(100%)(100%)(100%)
MK-5172/MK-8742 50 mg (N = 12*)5/1210/1111/115/5
(42%)(91%)(100%)(100%)

Disclosures:

Eric Lawitz - Advisory Committees or Review Panels: AbbVie, Achillion Pharmaceuticals, BioCryst, Biotica, Enanta, Idenix Pharmaceuticals, Janssen, Merck & Co, Novartis, Santaris Pharmaceuticals, Theravance, Vertex Pharmaceuticals; Grant/Research Support: AbbVie, Achillion Pharmaceuticals, Boehringer Ingelheim, Bristol-Myers Squibb, Gilead Sciences, GlaxoSmithKline, Idenix Pharmaceuticals, Intercept Pharmaceuticals, Janssen, Merck & Co, Novartis, Presidio, Roche, Santaris Pharmaceuticals, Vertex Pharmaceuticals; Speaking and Teaching: Gilead, Kadmon, Merck, Vertex

John M. Vierling - Advisory Committees or Review Panels: National Institutes of Health, US Food and Drug Administration CDER, Centers for Disease Control and Prevention, Abbott, Bristol-Myers Squibb, Excalenz, Gilead, Globeimmune, HepQuant, Idenix, Immuron, Janssen, Novartis, Roche, Salix, Schering (now Merck), Sundise, Vertex, HepaLife Technologies, Herbalife, Ocera, SciGen; Grant/Research Support: Abbott, Bristol-Myers Squibb, Conatus, Excalenz, Genfit, Gilead, Globeimmune, Hyperion, Idenix-Novartis, Ikaria, Intercept, Merck, Mochida, Novartis, Ocera, Pfizer, Pharmasset, Roche

Marcelo Kugelmas - Advisory Committees or Review Panels: Merck, Vertex, Genentech; Consulting: Vertex, Merck, Gilead; Grant/Research Support: Vertex, Bristol Myers Squibb, Boehringer-Ingelheim, Gilead, Janssen, Roche, Anadys, Merck; Speaking and Teaching: Vertex, Gilead, Merck, Genentech

Jan Gerstoft - Advisory Committees or Review Panels: msd, medivir, gilead; Speaking and Teaching: msd, abbivie, bms, viiv, gilead

Luis A. Balart - Advisory Committees or Review Panels: Genentech, Genentech; Grant/Research Support: Merck, Genentech, Bayer, conatus, Ocera, Hyperion, Gilead Sciences, Bristol Myers Squibb, Mochida, Eisai, Vertex, Merck, Genentech, Bayer, Conatus, Ocera, Hyperion, Gilead Sciences, Bristol Myers Squibb, Mochida, Eisai, Vertex, takeda, GI Dynamics; Speaking and Teaching: Merck, Merck, Merck, Merck

Reem H. Ghalib - Grant/Research Support: Bristol Myers Squibb Pharmaceuticals, Vertex Pharmaceuticals, Merck, Genentech, Zymogenetics, Pharmasset, Anadys, Duke Clinical Research Institute, Achillion, Boehringer Ingelheim, Gilead Pharmaceuticals, Virochem Pharmaceuticals, Abbott, Medtronic Inc, Novartitis, Roche, Schering Plough, tibotec, Inhibitex; Speaking and Teaching: Merck, Genentech, Vertex Pharmaceuticals

Ronald Nahass - Advisory Committees or Review Panels: Gilead; Grant/Research Support: Gilead; Speaking and Teaching: Gilead

Xiao Sun - Employment: Merck

Peggy Hwang - Employment: Merck, Merck

Janice Wahl - Employment: Merck & Co,

Michael Robertson - Employment: Merck; Stock Shareholder: Merck Barbara Haber - Employment: Merck

The following people have nothing to disclose: Abel Murillo, Peter Winkle, Peer B. Christensen, Melissa Shaughnessy

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Preclinical Evaluation of TT-034 - Safe and Durable Hepatic Expression of Anti-HCV shRNA in a NonHuman Primate Model

Shih Chu Kao1, Tin Mao1, John F. Wright2,3, Katherine High4,2, Per Lindell1, Peter French5, David Suhy1,5
1Tacere Therapeutics, San Francisco, CA; 2The Children's Hospital of Philadelphia, Philadelphia, PA; 3University of Pennsylvania, Philadelphia, PA; 4Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, Philadelphia, PA; 5Benitec Biopharma, Sydney, NSW, Australia

The Hepatitis C virus (HCV) chronically infects over 170 million individuals worldwide and despite the recent addition of DAAs, the current standard of care treatment is limited by long duration and significant side effects, as well as viral escape and incomplete genotype coverage. Because the viral genome is comprised of a single strand of RNA and its replication occurs strictly within the cytoplasm, HCV is an ideal candidate for therapeutics based upon RNA interference (RNAi). We have developed an RNAi-based therapeutic against HCV, termed TT-034, which uses three independent transcriptional cassettes to express three shRNAs that simultaneously target multiple wellconserved regions of the HCV genome. Intended as a “one-shot cure”, TT-034 uses an Adeno-Associated Virus (AAV) capsid to deliver the recombinant genome preferentially into hepatocytes; following intravenous administration, greater than 90% of vector was measured liver tissues in biodistribution studies. Once inside the cell, the TT-034 expression cassette uses the cell's own transcriptional machinery to produce a constantly replenishing pool of shRNA. We have characterized the therapeutic potential of TT-034 in several in vitro studies using replicon systems as well as an infectious tissue culture model of HCV and demonstrate that as little as 20-200 copies of shRNA per cell, as measured by quantitative PCR, was required for complete inhibition of replicon activity. Systemic administration via a single intravenous injection in a non-human primate model resulted in complete transduction of hepatocytes, as assessed by in situ hybridization analysis of shRNA, and resulted in the persistent expression of shRNA out to 180 days, the duration of the study. Acute toxicity studies performed in non-human primates with clinically relevant doses of TT-034 demonstrate that upwards of 8300 copies of shRNA per cell, far in excess of the predicted effective dose, could be produced in the absence of hepatocellular toxicity. No other adverse events were noted in the animals. Follow-on IND-enabling, GLP toxicology studies on 520 mice and 80 cynomolgus monkeys, followed over 180 days, showed an excellent safety profile, supporting the safety of a planned Phase I/II clinical study in subjects chronically infected with HCV. An open label phase I/II first-in-man dose escalation safety study in chronic HCV patients that have failed the current Standard of Care is planned to start in 2H 2013.

Disclosures:

John F. Wright - Advisory Committees or Review Panels: Avalanche Bio; Consulting: Tacere Therapeutics, Third Rock Ventures; Patent Held/Filed: Children's Hospital of Philadelphia

Katherine High - Advisory Committees or Review Panels: Genzyme, Bristol-MyersSquibb, Alnylam, Novo-Nordisk; Consulting: Tacere; Employment: Pfizer, Inc; Stock Shareholder: Bluebird bio

Per Lindell - Consulting: Benitec Ltd, Tacere Inc

Peter French - Management Position: Tacere Theraputics

David Suhy - Employment: Tacere Therapeutics

The following people have nothing to disclose: Shih Chu Kao, Tin Mao

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Kinetic Analyses of Antiviral Suppression by NS5A Inhibitors Reveal Early and Potent Inhibition of Viral Assembly and Release of Infectious Virus

David R. McGivern1, Takahira Masaki1, LiFang Ping1, Yan Yang1, Paul Ingravallo2, Sara Williford1, Anita Y. Howe2, Stanley M. Lemon1
1Dept. of Medicine, University of North Carolina af Chapel Hill, Chapel Hill, NC; 2Merck Research Laboratory, Kennilworth, NJ

Aim Inhibitors of NS5A are proving to have potent antiviral effects in clinical trials involving patients with chronic hepatitis C, but their mechanism of action remains obscure. To study the mechanism of action of NS5A inhibitors, we compared the kinetics of antiviral suppression with other classes of direct-acting antiviral (DAA) using the genotype 1a cell culture-infectious virus H77S. 3.Methods Detailed kinetic analyses of antiviral suppression were performed following treatment with representative protease inhibitors, polymerase inhibitors or NS5A inhibitors. Viral RNA replication was measured by monitoring Gaussia luciferase (GLuc) activity in culture medium from Huh7 cells infected with H77S. 3 expressing GLuc and also by quantitative RT-PCR. Infectious virus production from Huh7 cells infected with H77S. 3 (both intracellular and extracellular infectivity) was measured by focus forming unit assay. Results Kinetics of antiviral suppression by NS5A inhibitors, measured by GLuc or qRT-PCR, were strikingly different to other classes of antiviral. Despite their high potency, NS5A inhibitors were slow to inhibit viral RNA replication compared to protease or polymerase inhibitors. At 1d after addition of inhibitor, little reduction of RNA replication was observed in GLuc assays (typically less than 50%), even at micromolar concentrations. In contrast inhibition of virus production by NS5A inhibitors was potent and rapid. Following treatment of HCV-infected cell cultures with NS5A inhibitors, maximum inhibition of virus production was observed as early as 12 hours whereas maximum inhibition of RNA replication was not achieved until 3 days. Accumulation of intracellular infectious virus was also rapidly inhibited by NS5A inhibitor treatment, indicating that the early effect on virus production occurs at a stage in the virus life cycle prior to egress but after viral RNA replication – most likely on assembly. Conclusions Our analyses of the kinetics of antiviral suppression of the gt 1a strain H77S. 3 by NS5A inhibitors provide direct evidence for a two-pronged mechanism of action. First, NS5A inhibitors cause an extremely rapid drop in virus production by inhibiting virus assembly and release of infectious virus. This rapid effect is followed by a slower reduction in RNA replication.

Disclosures:

Anita Y. Howe - Employment: Merck Research Laboratory

Stanley M. Lemon - Advisory Committees or Review Panels: Merck, Santaris, Abbott, Gilead; Consulting: Achillion, Idenix; Grant/Research Support: Merck, Tibotec, Scynexis; Speaking and Teaching: Hoffman LaRoche

The following people have nothing to disclose: David R. McGivern, Takahiro Masaki, LiFang Ping, Yan Yang, Paul Ingravallo, Sara Williford