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HCV Clinical Trials Abstracts

LB-1. Dual, Triple, and Quadruple Combination Treatment with a Protease Inhibitor (GS-9256) and a Polymerase Inhibitor (tegobuvir-GS-9190) alone and in Combination with Ribavirin (RBV) or PegIFN/RBV for up to 28 days in Treatment Naïve, Genotype 1 HCV Subjects. .  S. Zeuzem; P. Buggisch; K. Agarwal; M. P. Manns; P. Marcellin; G. R. Foster; D. Sereni; H. Klinker; C. Moreno; J. H. Zarski; M. J. Shelton; S. West; J. Zong; J. Harris; J. G. McHutchison; W. Lee; W. E. Delaney; D. Oldach

Background and Aims: Available data on combinations of direct-acting HCV agents are limited to 14 days of dual treatment.  The aim of the study was to test the efficacy, safety and tolerability the following:

·        GS-9256 plus tegobuvir

·        GS-9256 combined with tegobuvir and ribavirin

·        GS-9256 combined with tegobuvir, ribavirin, and pegylated interferon

Note:  GS-9256 75 mg twice a day (BID):  tegobuvir 40 mg BID;  pegylated interferon alpha 2 a 180 ug (once a week;  ribavirin 1001200 mg/day.

The viral resistance during and following treatment was also evaluated. 

Methods:

There were three arms in the study:

·        Group 1: GS-9256 plus tegobuvir for 4 weeks followed by pegylated interferon plus ribavirin (standard of care (SOC)) for a total treatment duration of 48 weeks.  (16 patients—2 patients with  IL28B cc genotype)

·        Group 2:  GS-9256 plus tegobuvir and ribavirin for 4 weeks followed by SOP for a total treatment duration of 48 weeks (15 patients—6 patients with IL28B cc genotype)

·        Group 3: GS-9256 plus tegobuvir, pegylated interferon and ribavirin for 4 weeks followed by SOC for a total treatment duration of 48 weeks (15 patients—4 pts with IL28B cc genotype )

The patient characteristics were well match.  All patients were HCV genotype 1 and subtype 1a and 1b,  but some rare genotype sub-types were also identified. 

Results:

·        1 of 14 pts (7%) of group 1; 5 of 13 pts (39%) of group 2 and 14 of 14 patients (100%)  of group 3 were HCV RNA undetectable at Day 28 (≤ 25 IU/mL)> 

Safety:

·        There were no grade 4 adverse events or lab values

·        One patient discontinued treatment due to adverse events due to fatigue after starting pegylated interferon/ribavirin therapy

·        2 serious adverse events reports:

o   1 patient fainted due to gastroenteritis from group 3

o   1 patient had bursitis of the knee after discontinuing the study drug, but while receiving pegylated interferon plus ribavirin

Conclusion:

·        GS-9256 plus tegobuvir has robust antiviral activity

·        The addition of ribavirin to GS-9256 plus tegobuvir demonstrated greater virologic suppression, decreased resistance emergence, and was well tolerated

·        GS-9256 plus tegobuvir plus pegylated interferon/ribavirin was well-tolerated, with typical pegylated interferon/ribavirin safety profile

·        Phase 2b evaluation of a 4 month regimen of GS-9256 plus tegobuvir, pegylated interferon/ribavirin is ongoing.

 


LB-11. Clinical Virology Results from Telaprevir Phase 3 Study ADVANCE.  T. L. Kieffer; D. J. Bartels; J. Sullivan; B. S. Adiwijaya; E. Z. Zhang; A. Tigges; J. Dorrian; J. Spanks; S. De Meyer; G. Picchio; N. Adda; A. D. Kwong

Background: The ADVANCE Phase 3 study assessed telaprevir (TVR)-based regimens in treatment-naïve patients with genotype 1 HCV infection. Patients were randomized to 48 wks of PR (control arm), or TVR for 8 wks (T8/PR) or 12 wks (T12/PR) in combination with PR for 24 or 48 wks based on eRVR (undetectable HCV RNA at wks 4 and 12). AEs in ADVANCE were similar in frequency and severity to those presented for Phase 2 studies.

Methods: HCV RNA measurements (LOQ 25 IU/mL) and population sequencing of NS3-4A (LOD >1000 IU/mL) were performed at baseline, during treatment, and follow-up in patients who did not achieve SVR. Amino acid substitutions potentially associated with TVR resistance were identified. On-treatment virologic failure was defined as HCV RNA >1000 IU/mL at wks 4 or 12, and detectable HCV RNA at wks 24-48. Viral dynamic modeling analyses were developed from Phase 2 data (PROVE1, PROVE2).

Results: The SVR rate for T8/PR (n=364) and T12/PR (n=363) arms was 69% and 75%, respectively, which was significantly higher (p<0.0001) than the PR control arm (44%, n=361). Virologic failure at wks 4 and 12 during TVR/placebo treatment was similar between arms (T8; 2.7% and T12; 3.3%). In contrast, the rate of virologic failure after wk 12 during PR treatment was somewhat higher in T8/PR (10.2%) compared to T12/PR (5.0%), and associated with wild-type (WT) and lower-level TVR-resistant variants, which suggest that 4 additional wks of TVR may further reduce virologic failure. Consistent with these results, modeling analyses suggested 12 wks of TVR may be sufficient to clear all WT and low-level resistant variants, but that a small population of these variants may still exist after 8 weeks of treatment.

Of 91 patients with sequencing data available and having TVR-resistant variants after not achieving SVR, 55 (60%) no longer had resistant variants detected at the end of study (median follow-up = 45 wks). The median time to loss of detectability of resistant variants for T54, A156, V36, and R155 variants was 13, 24, 36 and 44 wks, respectively.

Conclusions: The SVR rates in the T8/PR (69%) and T12/PR (75%)regimens were significantly higher than PR alone (44%). There was a ~5% higher rate of on-treatment virologic failure during the PR treatment phase in the T8/PR arm compared to the T12/PR arm, predominantly with lower-level TVR-resistant variants. These results suggest that TVR continued to exert antiviral pressure on WT and lower-level variants between wks 8 and 12, thus reducing subsequent virologic failure during the PR treatment phase in the T12 arm. Additionally, the majority of patients no longer had detectable TVR-resistant variants at the end of study.

 


LB-13. Non response to peginterferon alfa and ribavirin in IL28B CC & CT patients can be overcome by high dose continuous interferon alfa-2b administration in combination with ribavirin for chronic hepatitis C.  R. Roomer; R. J. de Knegt; J. F. Bergmann; R. M. Scherer; B. Van Antwerp; J. Lande; E. Grovender; H. L. Janssen; A. Boonstra

Background: The pegylation of interferon (IFN) improved the pharmacokinetic profile with higher sustained virological response (SVR) rates in naïve chronic HCV patients compared to standard IFN. However, the SVR rates remain low in patients who experienced a previous failure to therapy (in the 8-12% range). We hypothesized that continuous delivery via an external pump of high doses of IFN may improve the SVR rates in previous non-responders. Recently, single nucleotide polymorphisms (SNP) in the IL28B region have been shown to correlate with the response to IFN/ribavirin therapy in HCV patients naïve to IFN. We now present the first analysis of the IL28B genotype in a cohort of patients who were classified as previous nonresponders to treatment.

Method: In this study, we delivered IFN-alfa-2b continuously via subcutaneous infusion using a modified insulin pump (Medtronic Minimed). Thirty subjects were randomly assigned to receive a daily dose of 6, 9 or 12 MU IFN combined with weight-based ribavirin. Viral kinetics was measured at baseline, weekly until week 4, and then at week 8, 12, 24 and 48 weeks. The “Duke” SNP rs12979860 was determined using competitive allele-specific PCR genotyping.

Results: In this cohort 20 patients had IL28B genotype CT, 3 patients had genotype CC and 6 patients had genotype TT. In 1 patient genotype determination failed. In our study there were differences among the CC, CT and TT groups with respect to viral decay at week 4, which was independent of the IFN-dose. CC subjects had an average viral decay of 2.9 log at 4 weeks, while CT subjects showed a 1.65 log reduction and TT subject showed a 1.25 log reduction (p=0.119). Of the 6 TT subjects however, only 2 subjects showed more than 1 log reduction at 4 weeks. More importantly, in the high dose group receiving 12 MU IFN/day, all 10 patients had more than 2 logs viral decay at week 4 of treatment regardless of their IL28B genotype. Of all 30 patients 6 achieved SVR, 2 in the CC group (one in each of the 6 and 9 MU group) and 4 subjects in the CT group, who all received 12 MU IFN/day. In the multivariate linear regression analysis both IL28B (p=0.036) and IFN dose (p=0.002) were significantly associated with viral decay at week 4.

Conclusion: These results show that the IL28B genotype can be a strong predictor of both viral decay rates and subsequent SVR. Moreover, high doses of IFN can potentially overcome the innate lack of IFN sensitivity that the IL28B naïve data predicts. Finally, these results also support the concept that continuous delivery of IFN in previous therapy failures can be a successful treatment strategy, especially those with CT and CC genotypes.

 


LB-14. Adverse Event Reporting During HCV Treatment Via Weekly Clinic Visits Substantially Underestimates Flu Frequency & Severity Compared To Daily ePRO: Results From 133 Patients In EMPOWERW. A. Long; E. Lawitz; Y. Lurie; Z. Krastev; D. Takov; K. G. Tchernev; A. Rigney; N. Assy; Z. M. Younossi

Background and Aim: Flu symptoms (FluSxs) are well-known side effects of IFNa during HCV treatment (Rx). FluSxs are a disincentive to initiating Rx, but do not commonly cause early discontinuation of Rx. Most patients tolerate FluSxs if given proper support & management. The goal of this trial was to compare FluSxs frequency and severity recorded during wkly clinic visits (clv) to FluSxs frequency and severity collected by an electronic daily diary (ePRO).

Methods: EMPOWER, a 12-wk, randomized, open-label Phase 2b study derived prospectively from pts. enrolled in two contributing trials (SELECT-2 & 480 STUDY), was designed to compare FluSxs (headache, fever, muscle pain, joint pain, chills) in pts. treated with 480ug CR2b [Locteron®, Biolex Therapeutics, Pittsboro, NC, USA] to FluSxs in pts. treated with PEG2b [PegIntron®, Merck, Whitehouse Station, NJ, USA], both while receiving weight-based ribavirin. A key secondary goal was to compare FluSxs recorded at wkly clv to FluSxs recorded by ePRO.

Results: In the 133 pts. in EMPOWER, FluSxs frequency and severity were less by clv than by ePRO (Table 1, p<0.001). Both methods showed fewer FluSxs on CR2b than PEG2b (p<0.001 for lower total FluSxs event counts during Weeks 1-12 on CR2b by clv; p=0.041 for lower total FluSxs severity scores during Weeks 1-12 on CR2b by ePRO; p=0.058 for lower FluSxs severity scores during Weeks 1-6 on CR2b by ePRO; data not shown).

Conclusions: FLuSxs event counts and severity are substantially higher by ePRO than by clv. Explanations might include differences in daily vs. wkly pt. recall, suboptimal physician-pt. communications during clv, or other factors. Both methods suggest 480ug CR2b carries a smaller FluSxs burden than PEG2b, but confirmation in Phase 3 is required.

Flu Event Counts

Clinic Visit Reports (n=133)

ePRO Reports (n=133)

 

Fever

185

359

 

Headache

109

363

 

Joint Pains

58

465

 

Muscle Pains

101

534

 

All Events

507

2366

 

Flu Severity Counts

Clinic Visit Reports (n=133)

ePRO Reports (n=133)

 

Mild

442

477

 

Moderate

61

1196

 

Severe

4

693

 


LB-15. Response-Guided Therapy (RGT) with Boceprevir (BOC) + Peginterferon alfa-2b/Ribavirin (P/R) for Treatment-Naïve Patients with Hepatitis C Virus (HCV) Genotype (G) 1 Was Similar to a 48-Wk Fixed-Duration Regimen with BOC + P/R in SPRINT-2.  J. Bronowicki; J. McCone; B. R. Bacon; S. Bruno; M. P. Manns; M. S. Sulkowski; I. M. Jacobson; K. Reddy; N. Boparai; V. Sniukiene; C. A. Brass; J. K. Albrecht; F. Poordad

Background: In SPRINT-2, BOC added to P/R given for 44 wks or as RGT after a 4-wk lead-in (LI) treatment period with P/R significantly improved the sustained virologic response (SVR) in treatment-naïve G1 patients compared to standard of care. We examined whether RGT based on the HCV RNA response during Wks 8-24 for a total duration of 28 or 48 wks (but including only 24 wks of BOC from Wks 4 to Wk 28 regardless of the Wk 8-24 HCV RNA response) produced similar results to the 4-wk LI & then BOC/P/R for 44 wks.

Methods: SPRINT-2 compared a 4-week lead in period with pegylated interferon plus ribavirin, followed by:

·        Group 1) pegylated interferon plus ribavirin and placebo for 44 wks (48P/R);

·        (2) response guided therapy:  boceprevir, pegylated interferon, ribavirin for 24 wks, with an additional 20 wks of P/R alone only if HCV RNA was detected during Wks 8-24 (LI+24BOC/P/R+/-20P/R); or

·        (3) boceprevir/pegylated interferon/ribavirin for 44 wks (LI+44BOC/P/R).

 

Study therapy was stopped for futility in patients with detectable HCV RNA at Wk 24. The primary efficacy endpoint was SVR in all randomized patients treated with ≥1 dose of any study medication. Non-black (Cohort 1) & black (Cohort 2) patients were enrolled & analyzed separately per protocol.

Results:

·        SVR  results are as follows:

o   Non-black patients:

§  Group 1 –40%  SVR (control arm)

§  Group 2 – 68% SVR (response guided-triple therapy)

§  Group 3 – 67% SVR (48 weeks triple therapy)

o   Black patients

§  Group 1–23%  SVR (control arm)

§  Group 2–42% SVR (response guided – triple therapy)

§  Group 3–53% SVR (48 weeks triple therapy)

Conclusions:

·        Response guided therapy yielded high response rates equal to those in people who were treated for 48 weeks with boceprevir/pegylated interferon/ribavirin

·        Response guided therapy minimized boceprevir exposure in all patients

o   Week 8 is a good time point to assess whether a patient should receive 28-week or 48-week boceprevir response guided therapy since it is a good predictor of SVR

o   Approximately 60% of patients would be eligible for the shortened treatment duration of 28 weeks of the triple therapy of boceprevir, pegylated interferon and ribavirin.

 


LB-2. Telaprevir in Combination with Peginterferon Alfa2a and Ribavirin for 24 or 48 weeks in Treatment-Naïve Genotype 1 HCV Patients who Achieved an Extended Rapid Viral Response: Final Results of Phase 3 ILLUMINATE Study .  K. E. Sherman; S. L. Flamm; N. H. Afdhal; D. R. Nelson; M. S. Sulkowski; G. T. Everson; M. W. Fried; K. Kleber; M. Martin; A. J. Sankoh; R. S. Kauffman; S. George; C. I. Wright; F. Poordad

Background: The Phase 3 open-label study, ILLUMINATE, evaluated patients randomized to two durations of therapy (24 OR 48 week treatment duration among those who achieved extended rapid viral response (eRVR).

Methods: HCV genotype 1 treatment-naïve patients (pts) were treated with:

·        Telaprevir (12 weeks – 750 mg three times a day) with peginterferon alfa2a ( 180 μg/week) and ribavirin (1000-1200 mg/day) followed by peginterferon alfa2a and ribavirin.   Patients who achieved eRVR (undetectable HCV RNA at Weeks 4 and 12) were randomized at week 20 to continue receiving pegylated interferon and ribavirin for 24 or 48 weeks of total treatment. Patients not achieving eRVR were assigned 48 wks of treatment.

·        Patients who failed to achieve a 2log10 drop at 12 weeks or had detectable HCV RNA by 24 weeks were discontinued as virologic failures.

·        The primary endpoint was the proportion of randomized patients achieving SVR 24 weeks after planned treatment completion.

·        The study was powered to rule out non-inferiority (NI) of 24-wk to 48-wk treatment among randomized pts with an NI of -10.5%.

·        Analyses were based on the intent-to-treat (ITT) randomized population.

Results:

·        540 patients were treated at 74 sites: 60.2% male, 79.1% Caucasian, 13.5% Black, median HCV RNA log10 6.5 IU/ml, 11.3% cirrhosis.

·        Efficacy: 72% (n=389) of pts achieved RVR; 65.2% (n=352) of pts achieved eRVR.

·        322 (59.6%) pts were randomized (1:1) to either a 24-week or 48-week arm

o   SVR was 92% among patients randomized to 24 weeks (n=162).

§  Black patients achieved 88% SVR

§  Hispanic patients achieved 94% SVR

o   SVR was 87.5% (Δ4.5%, 2-sided 95% C.I. = -2.1% to +11.1%) among pts randomized to 48 wks (n=160).

§  Black patients achieved 88% SVR

§  Hispanic patients achieved 82% SVR

·        Overall, SVR was 71.9% (ITT analysis).

·         36 pts (6.7%) discontinued treatment due to virologic failure.

Safety:

·        94 pts (17.4%) had permanent discontinuation of all study drugs (D/C) for adverse events. 

o   Fatigue (n=22) and anemia (n=12) were the most common adverse events leading to discontinuation of treatment. Adverse events led to treatment discontinuation after Week 20 in 1 (0.6%) and 20 (12.5%) of eRVR+ pts randomized to 24 wks and 48 wks of treatment, respectively.

o   Treatment discontinuation due to anemia and rash were 3 (0.6%) and 6 (1.1%) pts, respectively, during the telaprevir treatment phase.

Summary:

·        24-week telaprevir-based regimen non-inferior to a 48-week regimen in patients with eRVR

o   92% vs. 88% SVR

·        65% of patients eligible for a shorter duration of treatment

·        72% overall SVR in ITT analysis

o   63% in patients with bridging fibrosis and cirrhosis

o   60% in Black patients

o   67% in Hispanic patients

Conclusions:

·        Among patients who achieved eRVR, a 24-week telaprevir-based regimen was non-inferior to 48-week telaprevir-based regimen (92% SVR compared to 87.5%).

·        Response-guided treatment led to 71.9% SVR overall and nearly two-thirds of the patients were eligible for shorter duration of treatment.

·        Permanent discontinuation of all study drugs due to adverse events occurred in 17.4% of patients.

·        Among eRVR randomized patients, there were more adverse events and adverse event-related treatment discontinuations in the 48-week versus 24-week arm. These results support response-guided therapy for telaprevir-based regimens in treatment-naïve patients.

 


LB-4. Boceprevir (BOC) Combined with Peginterferon alfa-2b/Ribavirin (P/R) for Treatment-Naïve Patients with Hepatitis C Virus (HCV) Genotype (G) 1: SPRINT-2 Final Results.  F. Poordad; J. McCone; B. R. Bacon; S. Bruno; M. P. Manns; M. S. Sulkowski; I. M. Jacobson; K. Reddy; N. Boparai; V. Sniukiene; C. A. Brass; J. K. Albrecht; J. Bronowicki

Background: Sustained virologic responses (SVR) have been <50% in HCV G1 patients, especially in those of African descent. BOC is a HCV-NS3 protease inhibitor. SPRINT-2 assessed the safety & efficacy of P/R ± BOC in G1 patients.

Methods: This Phase 3 international double-blind randomized study compared a 4-wk lead-in (LI) treatment period with P/R, followed by (1) P/R plus placebo for 44 wks (48P/R); (2) response-guided therapy (RGT): BOC plus P/R for 24 wks, with an additional 20 wks of P/R only if detectable HCV RNA during Wk 8-24 (LI+24 BOC/P/R±20 P/R); or (3) BOC plus P/R for 44 wks (LI+44 BOC/P/R). P was dosed 1.5 μg/kg SC weekly, R dose was weight-based (600-1400 mg/day) PO divided BID, & the BOC dose was 800 mg PO TID. Patients with detectable HCV RNA at Wk 24 were discontinued for futility. The primary efficacy endpoint was SVR 24 wks post-therapy in all randomized patients treated with ≥1 dose of any study medication. Non-black (Cohort 1) & black (Cohort 2) patients were enrolled & analyzed separately per protocol.

Results: 938 non-black & 159 black patients were enrolled: 92% >400,000 IU/mL HCV RNA & 9% biopsy-proven F3/4. SVR in non-black patients was 40% for 48P/R & significantly higher (p<.0001) in both BOC arms: RGT (67%) & LI+44 BOC/P/R (68%); corresponding SVR in black patients were 23%, 42% (p=.044) & 53% (p=.004) (Table). For non-black patients receiving ≥1 dose of BOC or placebo, respective SVR were 42%, 70% & 71%. At the end of LI period, a high percent (25%) of patients had <1 log decline in baseline HCV RNA; regardless of Wk 4 decline, SVR were consistently higher in the BOC arms than controls. Discontinuation for adverse events occurred in 16%, 12% & 16% in the 3 arms, respectively. Anemia was reported in 29% of controls vs. 49% in the BOC arms, leading to dose reduction in 13% & 21% & discontinuation in 1% & 2%, respectively.

Conclusions: BOC/P/R significantly increased SVR in both the RGT & 48-wk treatment arms over standard of care by ~70%. BOC was well tolerated; although reported more often in BOC recipients, anemia rarely led to treatment discontinuation. Compared to 44 wks of triple therapy after the LI period, RGT with LI+24BOC/P/R±20P/R produced comparable SVR.

 

Cohort 1

Cohort 2

 

48P/R

RGT

LI+44BOC/P/R

48P/R

RGT

LI+44BOC/P/R

N

311

316

311

52

52

55

SVR, n(%)

125 (40)

211 (67)

213 (68)

12 (23)

22 (42)

29 (53)

End of therapy response, n (%)

176 (57)

235 (74)

241 (77)

15 (29)

26 (50)

36 (65)

Relapse, n/m (%)

37/162 (23)

21/232 (9)

18/230 (8)

2/14 (14)

3/25 (12)

6/35 (17)

SVR by decline in HCV RNA after 4-wk LI, n/m (%)

<1 log

3/62 (5)

21/73 (29)

31/79 (39)

0/21 (0)

6/24 (25)

5/16 (31)

1 log

121/234 (52)

187/228 (82)

178/218 (82)

12/26 (46)

16/24 (67)

22/36 (61)

 


LB-5. Efficacy and safety of TMC435 in combination with peginterferon α-2a and ribavirin in treatment-naïve genotype-1 HCV patients: 24-week interim results from the PILLAR study.  M. W. Fried; M. Buti; G. J. Dore; P. Ferenci; I. Jacobson; P. Marcellin; S. Zeuzem; O. Lenz; M. Peeters; V. J. Sekar; G. . De Smedt

Background and aim: TMC435, an oral investigational hepatitis C virus (HCV) NS3/4A protease inhibitor, has shown potent antiviral activity against genotype-1 (G1) HCV in Phase IIa. PILLAR (TMC435-C205; NCT00882908) is an ongoing international Phase IIb randomized, double-blind study to assess efficacy and safety of TMC435 in combination with peginterferon α-2a (PegIFN) and ribavirin (RBV) in treatment-naïve HCV G1 patients.

Methods: Patients (n=386) were randomly assigned to 1 of 5 study arms: TMC435 (75 or 150mg qd)+PegIFN (180μg/wk)/RBV (1000-1200mg/day) for 12 weeks, followed by PegIFN/RBV for 12 weeks; TMC435 (75 or 150mg qd)+PegIFN/RBV for 24 weeks; or placebo+PegIFN/RBV for 24 weeks followed by PegIFN/RBV for 24 weeks. Patients in TMC435 arms who achieved HCV RNA <25IU/mL detectable or undetectable at Week 4 and <25IU/mL undetectable at Weeks 12, 16 and 20 (Roche Taqman v2) were able to end treatment at Week 24. All other patients continued PegIFN/RBV for up to 48 weeks. To evaluate the primary endpoint (SVR), HCV RNA was measured regularly after end of treatment up to Week 72. Results of a Week 24 interim analysis are reported. Secondary endpoints included assessment of antiviral activity, viral breakthrough, safety, and tolerability. IL-28B genotype was assessed in all consenting patients.

Results: Between 79% and 86% of patients in TMC435 arms were able to end therapy at Week 24 as per protocol defined response criteria. Viral breakthrough was observed in 2.5%-7.8% of patients across different treatment arms. Addition of TMC435 to PegIFN/RBV increased response rates in all IL-28B genotypes. Adverse events (AEs) did not differ substantially between TMC435 and placebo arms, and no differences were reported in frequency of rash, gastrointestinal events, or anemia. AEs leading to treatment discontinuation were reported in 7.1% of TMC435 arms and 7.8% of placebo arm. Mild and reversible increases in bilirubin (direct and indirect) were noted in TMC435 150mg dose arms.

Conclusions: This interim analysis demonstrates that TMC435 in addition to PegIFN/RBV achieves RVR and cEVR for the majority of patients. Available data indicate that high SVR12 rates are observed in patients who completed therapy. In this analysis TMC435 was well tolerated in all treatment arms.

HCV RNA
(<25IU/mL undetectable)

TMC12-PR24
75mg
(n=78)

TMC24-PR24
75mg
(n=75)

TMC12-PR24
150mg
(n=77)

TMC24-PR24
150mg
(n=79)

Pbo24-
PR48
(n=77)

Week 4 (RVR)

77%
59/77

68%
51/75

76%
58/76

79%
59/75

5%
4/75

Week 12 (cEVR)

91%
71/78

96%
70/73

94%
72/77

97%
75/77

58%
43/74

Week 24

94%
72/77

97%
70/72

94%
65/69

95%
69/73

82%
60/73

Follow-up after planned end of treatment

 

 

 

 

SVR4

91%
59/65

93%
56/60

93%
57/61

91%
62/68

n/a

SVR12

97%
32/33

93%
27/29

89%
32/36

88%
28/32

n/a

 


LB-6. GI-5005 Therapeutic Vaccine Plus Peg-IFN/Ribavirin Improves Sustained Virologic Response Versus Peg-IFN/Ribavirin In Prior Non-Responders With Genotype 1 Chronic HCV Infection.  P. Pockros; I. Jacobson; T. D. Boyer; E. R. Schiff; G. T. Everson; W. M. Lee; J. M. Vierling; E. Lawitz; M. Kugelmas; N. Tsai; M. L. Shiffman; R. S. Brown; B. R. Armstrong; A. Mattson; T. C. Rodell; D. Apelian

Background and aims: The GI-5005 therapeutic vaccine has been shown to improve sustained virologic response in naïve subjects with the greatest effect observed in IL28B T/T subjects. We now report the sustained virologic response (SVR) data from prior IFN/ribavirin non-responders (NR).

Methods: HCV genotype 1 patients were randomized 1:1, and stratified by prior treatment status; Arm 1- GI-5005 monotherapy run-in of five weekly followed by 2 monthly subcutaneous (SC) doses of 40YU (1 YU = 10 yeast) GI-5005 over 12 weeks, followed by triple therapy of monthly 40YU GI-5005 doses plus 48 weeks pegIFN α-2a/ribavirin (SOC), Arm 2- SOC alone. NRs received 72 weeks of triple therapy versus SOC. Prior NRs were defined as poor responders (> 1 log10 and < 2 log10 reduction) or partial responders (> 2 log10 reduction without clearance at any time during therapy). Prior null response, relapse, and breakthrough were exclusionary.

Results: Triple therapy was well tolerated with an equivalent number of discontinuations due to adverse events in each group; Triple 8/68(11.8%) and SOC 8/65(12.3%). Improvement in end of treatment response (ETR) (Triple 6/18 [33%] vs SOC 2/19 [11%]) and SVR (Triple 3/18[17%] vs SOC 1/19[5%]) was observed in NR patients. Due to the small number of patients in each treatment arm, these differences were not statistically significant (see table). SVR in NRs occurred only in IL28B C/T subjects (Triple 3/13[23%] vs SOC 1/13[8%]). In summary, GI-5005 triple therapy delivered improved ETR and SVR (Δ ranging from 10-22%) in all patient subgroups (see table).

Conclusions: GI-5005 plus SOC is well tolerated and improved SVR rates compared to SOC in genotype 1 NR patients. ETR and SVR rates were improved by GI-5005 triple therapy for all subgroups (all, naïve, and NR). These data support further investigation of GI-5005 triple therapy in naïve and NR patients as well as novel combination strategies for GI-5005 with other HCV inhibitory agents in larger numbers of patients.

http://aasld2010.abstractcentral.com/user_images/aasld2010/1909/937579/table8-4-10.jpg

 


LB-7. Strong antiviral activity and safety of IFN-sparing treatment with the protease inhibitor BI 201335, the HCV polymerase inhibitor BI 207127 and ribavirin in patients with chronic hepatitis C.  S. Zeuzem; T. Asselah; P. W. Angus; J. H. Zarski; D. Larrey; B. Mullhaupt; E. J. Gane; M. Schuchmann; A. W. Lohse; S. Pol; Y. Benhamou; J. Bronowicki; S. K. Roberts; K. Arastéh; F. Zoulim; M. H. Heim; J. O. Stern; G. Kukolj; G. Nehmiz; C. Haefner; W. O. Boecher

Background: BI 201335 and BI 207127 are potent and specific inhibitors of the HCV NS3/4A protease and the NS5B RNA-dependent RNA polymerase, respectively. An IFN-free combination of both antivirals with ribavirin (RBV) to eradicate HCV infection would create a major paradigm shift in HCV treatment.

Methods: In this randomized open-label trial, 32 treatment-naïve HCV genotype-1 patients were treated over 4 weeks with 400 or 600 mg three times a day (TID) BI 207127, BI 201335 120 mg daily (QD) and RBV (1000/1200 mg daily in two doses). Plasma HCV RNA virus load (VL) was measured by Roche COBAS TaqMan assay with a lower limit of quantification of 25 IU/ml.

Results: At baseline, mean age was 51±11 years, mean BMI 23.8±3.4 kg/m, mean VL 6.48 LOG10. All patients had a rapid and sharp VL decline during the first two days, followed by a slower second phase decline in all except 2 patients. One patient experienced VL breakthrough (increase by >1 LOG10 from nadir during treatment) and one other experienced a 0.7 LOG10 VL increase. Both were in the lower dose group and were GT1a patients with high baseline VL. On day 29, all patients were switched per protocol to treatment with BI 201335 and PegIFN/RBV. Virological response rates (VL <25 IU/ml) after 1, 2, 3 and 4 weeks of oral treatment are shown in the table. At the higher dose level, there was no difference between GT1a and 1b, while GT1a patients at 400 mg TID had a lower response rate than those with GT1b. The PegIFN sparing treatment was well tolerated. The most common adverse events (AEs) were mostly mild gastro-intestinal effects (diarrhea, nausea, vomiting), rashes or photosensitivity. There were no severe AEs, SAEs or treatment discontinuations within the 4-week study period. Laboratory parameters did not indicate any relevant changes from baseline, except for a continuous drop in ALT in all patients, a decrease of haemoglobin (median -1.9 and -2.8 g/dL) and increase of unconjugated bilirubin (median +9.8 and +11.5 umol/L).

Conclusions:

·        PegIFN sparing treatment with the the NS3/4A inhibitor BI 201335, NS5B inhibitor BI 207127, and RBV, demonstrated strong early antiviral activity against HCV genotype 1 with good safety and tolerability.

·        No virologic breakthrough occurred at the 600 mg dose level and only one virologic breakthrough occurred at the lower dose level, demonstrating that the rapid and uniform slecti0n of resistance mutations associated with protease inhibitor monotherapy is effectively reduced or delayed in the PegINF-free regimen.

·        The rapid virological response rate in patients of the 600 mg dose group was comparable to that of PegIFN/RBV-based triple combinations with new DAAs (eg BI 201335.

·        The safety profile was good with predominance of the expected AEs of mild rashes and GI symptoms, which did not impact treatment continuation

·        A phase IIb trial testing different dose regimens of this combination, with longer durations, is planned to evaluate sustained virologic response rates.

Table: Proportion of patients with viral load <25 IU/ml

 

Day 8

Day 15

Day 22

Day 29

400 mg TID BI 207127 + BI 201335 + RBV

4/15

7/15

10/15

11/15

600 mg TID BI 207127 + BI 201335 + RBV

3/17

14/17

17/17

17/17

 


LB-8. Combination therapy with BMS-790052 and BMS-650032 alone or with pegIFN/RBV results in undetectable HCV RNA through 12 weeks of therapy in HCV genotype 1 null responders.  A. S. Lok; D. F. Gardiner; E. Lawitz; C. Martorell; G. T. Everson; R. H. Ghalib; R. Reindollar; V. K. Rustgi; P. Wendelburg; K. Zhu; V. Shah; D. Sherman; F. McPhee; M. Wind-Rotolo; M. Bifano; T. Eley; T. Guo; A. Persson; R. Hindes; D. M. Grasela; C. Pasquinelli

BMS-790052 is a potent NS5A inhibitor with broad genotypic coverage while BMS-650032 is a potent HCV NS3 protease inhibitor with coverage of HCV genotypes (GT) 1a and 1b. Clinical studies combining these compounds alone and with pegIFN/RBV are underway in HCV infected null responders to determine their safety and efficacy.

Methods: AI447011 is a randomized, open label, Phase 2a study comparing the antiviral activity and safety of BMS-790052 (60 mg QD) and BMS-650032 (600 mg BID) alone (Group A) or with pegIFN/RBV (Group B) for 24 weeks in HCV GT 1 null responders.

Aim: The primary aim was to determine the proportion of subjects achieving undetectable HCV RNA (<10 IU/mL) at Weeks 2 and 4 of therapy and 24 weeks post-treatment. A Week 12 interim analysis was performed.

Results: Twenty-one patients (11 Group A, 10 Group B) were randomized in a sentinel cohort. Median age was 55 years, 13 were male, and 16 were white. Virologic responses are presented in Table 1. 6 (54.5%) Group A subjects experienced viral breakthrough while all subjects in Group B maintained viral suppression. Viral breakthrough occurred exclusively in individuals infected with GT 1a occurring as early as Week 3 and as late as Week 12. The two GT 1b subjects in Group A remained HCV RNA undetectable. The 6 subjects with breakthrough had pegIFN/RBV added to their regimen. HCV RNA fell to UD in 2 subjects and to < 25 IU/mL in another 2 subjects while the other 2 had ≥ 1.5 log decreases in HCV RNA. No deaths, SAEs, or discontinuations due to AEs were recorded during the analysis period. Diarrhea was the most common AE and was mainly mild to moderate in severity.

Conclusions:

·        BMS-790052 plus BMS-650032 is generally well-tolerated when coadministered for 12 weeks in HCV-infected patients who were null responders to pegIFN/RBV

·        BMS-790052 plus BMS-650032 provided potent early antiviral activity; however, 6/11 cases of viral breakthrough were observed with the 2 drugs when given alone

·        BMS-790052 plus BMS-650032 in combination with pegIFN/RBV resulted in undetectable HCV RNA in 9/10 patients by week 12

·        Should the antiviral activity demonstrated by 4-drug therapy predict SVR, the results would have significant implications for future therapy

·        Study expansion with additional arms is planned based on future data

Table 1: Virologic responses by treatment

 

Group A
BMS-650032 and BMS-7HGroup90052
n=11

Group B
BMS-650032, BMS-790052, pegIFN/RBV
n=10

Genotype 1a
n

9

9

Median baseline HCV RNA IU/mL

6.9 log10

6.7 log10

Median HCV RNA decline Week 2 IU/mL

-5.1 log10

-5.3 log10

RVR (a)
n (%)

7 (63.6%)

6 (60%)

eRVR(a)
n (%)

4 (36.4%)

6 (60%)

cEVR(a,b)
n (%)

5 (45.5%)

9 (90%)(c)

Viral break through (d)

6/11 (55)

0

 

(a) Intent-to-treat analysis, breakthrough = failure.

(b)Complete early virologic response (cEVR): undetectable HCV RNA by week 12.

(c) One subject in group B (1/10) did not meet cEVR (week 12 HCV RNA <25 IU/mL); however, on retesting his HCV RNA was undetectable

(<10 IU/mL).

(d)Viral breakthrough: a) any increase in HCV RNA 1 log10 from nadir, or b) any detectable HCV RNA >25 IU/mL on or after week 4, or c) any

detectable HCV RNA <25 IU/mL on or after week 4 confirmed by retesting


31. Safety and Antiviral Activity of ANA598 in Combination With Pegylated Interferon α2A Plus Ribavirin in Treatment-Naïve Genotype-1 Chronic HCV Patients.  E. Lawitz; M. Rodriguez-Torres; V. K. Rustgi; T. Hassanein; M. H. Rahimy; C. A. Crowley; J. L. Freddo; A. J. Muir; J. G. McHutchison

Background: ANA598 is a potent non-nucleoside inhibitor of HCV polymerase. ANA598 was well tolerated in HCV patients at 200 mg, 400 mg and 800 mg bid for 3 days, and resulted in rapid reduction in HCV RNA (median EOT range 2.3 to 2.9 log10). This study evaluates safety and antiviral activity of ANA598 with PEG-IFN and ribavirin (standard of care—SOC).  SVR12 weeks reported. 

Methods: ANA598-504 is an ongoing double-blind, placebo-controlled Phase 2 study to assess safety, antiviral activity and PK of ANA598 in genotype-1 patients. Patients receive ANA598 or placebo with SOC for 12 weeks at 200 mg bid or 400 mg bid, both given with a loading dose of 800 mg q12h on day 1. Patients who achieve undetectable virus at weeks 4 and 12 are randomized to SOC alone for 12 or 36 additional weeks. Patients who did not meet these criteria are to continue SOC for a total of 48 weeks.

Results: 29 patients received the 200 mg bid (twice a day) arm, 34 in the 400 mg bid arm and 32 in the placebo control arm. One patient discontinued ANA598 (200 mg bid) due to rash and 6 patients discontinued placebo due to failure to reach a 1 log10 decline in HCV RNA by Week 4, but all continued to receive SOC. Both doses of ANA598 demonstrated a favorable safety and tolerability profile through 12 weeks. Safety laboratory values were comparable between the ANA598 and control arms. The most common AEs were those associated with SOC. Occurrence of rash was similar between ANA598 200 mg bid (12/29, 41%) and placebo (10/32, 31%) groups. In the 400 mg bid arm, 59% of patients (20/34) developed rash (80% were grade 1). SVR12 results in eight out of 11 patients (73%) who stopped treatment at week 24.  

Conclusions:

·        The combination of ANA598 with pegylated interferon plus ribavirin increased the rate of SVR in patients who completed 24 weeks of treatment. 

·        The triple combination of ANA598/PEG/RBV was generally well tolerated with one treatment discontinuation due to rash.

·        Complete results will be available in the near future


32. Phase II randomised, partially-blind, parallel-group study of oral danoprevir (RG7227) with PegIFNα-2a (PEGASYS) plus ribavirin in treatment-naive genotype 1 patients with CHC: Results of planned Week 12 interim analysis of the ATLAS study.  N. Terrault; C. Cooper; L. A. Balart; D. G. Larrey; T. D. Box; E. M. Yoshida; E. Lawitz; P. Buggisch; P. Ferenci; M. Weltman; E. Labriola-Tompkins; Y. Zhang; M. T. Navarro; C. Lim; E. S. Yetzer; P. Marcellin

Background:  Danoprevir (RG7227: DNV) is a potent, selective, oral HCV protease inhibitor. The efficacy and safety of DNV at 3 doses in combination with 180µg/week PegIFNα-2a plus 1000/1200mg/day ribavirin (SOC) was evaluated.

Methods:  Treatment-naive, non-cirrhotic adults with HCV G1 infection and serum HCV RNA ≥50 000 IU/mL were randomized to 12 wks of treatment with:

a.     DNV 300 mg q8h,

b.     600 mg q12h

c.      900 mg q12h or,

d.      to  placebo (Pla),

All groups received SOC.  SOC was continued after wk 12 to complete 24 wks (pts with wk 4 RVR) or 48 wks of treatment (non-RVR but undetectable HCV RNA at wk 24). Efficacy at weeks 4 and 12 was defined as undetectable HCV RNA (<15 IU/mL by Roche COBAS TaqMan HCV Test). Pts with missing HCV RNA results were considered non-responders.

Results:  In this planned interim analysis 225 pts were evaluable for safety and 212 for efficacy. Overall 60% were male, 10% were Black, 82% had a baseline HCV RNA ≥800 000 IU/mL and 66% were infected with HCV G1a.

Sixty-two (93%), 61 (94%) and 8 (16%) pts in arms A, B and C, respectively, completed 12 wks of treatment with DNV. Considering only administration of SOC, 63 (94%), 62 (95%), 50 (100%) and 24 (80%) pts in arms A, B, C and D completed ≥12 wks of treatment.

Rates of RVR were substantially higher in the DNV arms (73–86%) compared to the SOC arm (7%). Similarly, rates of undetectable HCV RNA at wk 12 were higher in the DNV arms (88–92%) compared to the SOC arm (43%).

 SAEs up to the clinical cut-off date were similar across all arms (4–6%) and there were no deaths. Five pts (1 each in A, B, and C and 2 in D) withdrew from the study for safety events. Reversible Grade 4 (>10x ULN) ALT elevations were noted in 0%, 1%, 6% and 0% of pts in arms A, B, C, and D, respectively and as a consequence treatment with DNV in arm C was terminated early. Neutropenia (<750/mm3) was reported in 24%, 25%, 31% and 16% and anemia (Hb <10 g/dL) in 14%, 19%, 8% and 23% in arms A–D respectively. The incidence of rash was not increased by addition of DNV to the SOC.

Conclusion:

·        The combination of DNV plus SOC produces rapid and profound reductions in HCV RNA.

·        With the exception of dose-dependent G4 ALT elevations, DNV was well tolerated.

·        Current and future studies with DNV will explore ritonavir boosting to maintain high efficacy with reduced overall drug exposure.

http://aasld2010.abstractcentral.com/user_images/aasld2010/1909/889959/Table_for_submission.JPG

 


33. IMO-2125, a TLR9 Agonist, Induces Immune Responses which Correlate with Reductions in Viral Load in Null Responder HCV Patients.  M. Rodriguez-Torres; R. H. Ghalib; S. C. Gordon; E. Lawitz; K. Patel; R. Pruitt; A. M. Sheikh; R. D. Arbeit; A. S. Bexon; E. R. Kandimalla; M. Precopio; T. Sullivan; A. J. Muir; J. G. McHutchison

Purpose: Evaluate the clinical pharmacodynamics of IMO-2125, an agonist of Toll-like Receptor 9 (TLR9) designed to treat chronic hepatitis C virus (HCV) infection by inducing high levels of endogenous interferons and other Th1-type immune responses.

Methods: In this phase 1 trial, IMO-2125 was administered weekly by subcutaneous injection for four weeks to null responder HCV patients, defined as patients who had failed to achieve a 2 log10 reduction in viral load under pegylated-IFN plus ribavirin treatment of at least 12 weeks duration. Forty of 41 patients enrolled had genotype 1 HCV. Each escalating dosage cohort was to include 8 patients on IMO-2125 and 2 patients on placebo (saline). A comprehensive blood sampling schedule evaluated immunological responses to IMO-2125, including serum IFN-α, interferon-γ-inducible protein-10 (IP-10), 2’,5’-oligoadenylate synthetase (2,5-OAS), and surface marker expression on peripheral blood mononuclear cells.

Results: IMO-2125 treatment of null responder HCV patients achieved dose-dependent increases in serum IFN-α, IP-10, 2,5-OAS, expression of CD69 activation marker on CD4+ and CD8+ T cells, and in the frequency of NK cells. Serum IFN-α concentrations induced by IMO-2125 treatment inversely correlated with dose-dependent decreases in HCV viral load. IMO-2125 treatment was well tolerated with no serious adverse events through the 0.32-mg/kg/week dosage.

Conclusions: IMO-2125 induced strong Th1-type immune responses in null responder HCV patients. The induction of endogenous IFN-α correlated with viral load reductions. Results are consistent with the intended mechanism of action of IMO-2125.

IMO-2125 Dosage,
mg/kg/w
(N=8 or 9)

Median Serum Concentrations

Mean Fold Change % CD69+ T Cells

Mean Fold Change % NK Cells

Decrease in HCV RNA (log10)

 

IFN-α pg/mL 

IP-10 pg/mL 

2,5-OAS pmol/dL 

CD4+

CD8+

Pts with ≥ 1 log10 decrease (range)

Median decrease (25-75% quartiles), all pts

 

Placebo

ND

387

66

1.69 ± 0.39

1.44 ± 0.39

1.06 ± 0.13

0/6

-0.29 (-0.16 to -0.67)

 

0.04

32 (N=2)

1734

82

1.70 ± 0.56

2.12 ± 0.54

1.00 ± 0.11

0/8

-0.47 (-0.25 to -0.62)

 

0.08

47 (N=3)

2285

172

3.18 ± 0.68

4.69 ± 1.44

1.00 ± 0.12

4/9
(-1.1 to -2.3)

-0.96 (-0.70 to -1.23)

 

0.16

38 (N=5)

2465

133

4.68 ± 1.93

5.41 ± 1.32

1.49 ± 0.18

5/8
(-1.2 to -2.0)

-1.33 (-0.62 to -1.60)

 

0.32

110 (N=7)

4534

131

5.90 ± 1.05

8.46 ± 1.75

1.69 ± 0.10

6/8
(-1.0 to -3.5)

-1.60 (-0.89 to -2.37)

 Dose 1 medians above limit of detection (25 pg/mL); number detectable in parentheses  Dose 1 maximums from 8-48 h post-dose  Pre-dose 4  24 h post-dose 1 compared to pre-dose  Maximum decrease during treatment period

 


34. A Phase IIa Study of IDX184 in Combination with Pegylated Interferon (pegIFN) and Ribavirin (RBV) in Treatment-Naïve HCV Genotype 1-Infected Subjects.  J. P. Lalezari; W. O'Riordan; F. Poordad; T. T. Nguyen; G. Dubuc Patrick; J. Chen; X. Zhou; J. Sullivan-Bólyai; D. L. Mayers

Background: IDX184 is a liver-targeted, oral nucleotide prodrug of 2’-methylguanosine (2’-MeG) monophosphate. After single and multiple doses for three days, IDX184 was safe and well tolerated up to 100 mg/day in both healthy volunteers and treatment-naïve HCV genotype 1-infected subjects, respectively.

Methods: This is a randomized, double-blind, placebo-controlled ascending dose study to assess safety, tolerability, antiviral activity and pharmacokinetics (PK) of IDX184 in combination with pegIFN/RBV in treatment-naïve subjects with genotype 1 chronic HCV infection. Sequential cohorts of 20 subjects, randomized 16:4 (IDX184:placebo), receive 50, 100, 150 or 200 mg of IDX184 (or placebo) daily for the first 14 days in combination with pegIFN/RBV which is then continued for an additional 14 days. HCV RNA levels are quantified by the Roche COBAS Taqman assay. Plasma concentrations of IDX184, 2’-MeG and RBV are determined using a validated LC-MS/MS method

Results: An analysis was conducted after completion of the IDX184 treatment period for the 150 mg cohort. Fatigue, headache, myalgia, nausea, neutropenia and anemia were the most common adverse events (AEs) and laboratory abnormalities and were comparable across treatment groups, including placebo. One subject in the 150 mg IDX184 group discontinued the study during the 14-day IDX184 treatment period due to acute cholecystitis, an SAE which was considered not to be related to IDX184. A second subject in the 150 mg IDX184 group discontinued due to non-compliance and was replaced. Mean ALT and AST levels improved in IDX184 recipients. A significant PK/PD relationship was found between the Day 14 viral load response and steady-state trough concentration of 2’-MeG (p<0.0001).

Conclusions: At daily doses of 50, 100 and 150 mg IDX184 in combination with pegIFN/RBV for 14 days, the side effect profile of the three drug combination was consistent with pegIFN/RBV alone. IDX184 demonstrated potent antiviral activity when combined with pegIFN/RBV. At daily IDX184 doses of 100 and 150 mg in combination with pegIFN/RBV, mean HCV RNA reductions were ≥ 4 log10 IU/mL with 40%-50% of subjects achieving undetectable viral load at Day 14. Full data from all cohorts will be presented.

Day 14 HCV RNA Results

Cohort

Mean Change ± SD in HCV RNA (log10 IU/mL) at Day 14

Subjects with Undetectable
Viral Load at Day 14 (<15 IU/mL)

Placebo + P/R (N=12)

-1.1 ± 0.9

0 (0%)

50 mg IDX184 QD + P/R (N=16)

-2.7 ± 1.3

2 (13%)

50 mg IDX184 BID + P/R (N=8)

-4.0 ± 1.7

4 (50%)

100 mg IDX184 QD + P/R (N=8)

-4.2 ± 1.9

4 (50%)

150 mg IDX184 QD + P/R (N=15)*

-4.1 ± 1.2

6 (40%)

*2 subjects who discontinued the study were excluded from the efficacy analysis;one was replaced. P/R=pegIFN/RBV

 


35. Cell-to-cell transmission of small silencing RNA in the liver extends the therapeutic reach of RNAi.  Q. Pan; V. Ramakrishnaiah; P. E. de Ruiter; H. W. Tilanus; H. L. Janssen; L. J. van der Laan

Background: In plants and invertebrate, RNA interference (RNAi) provides an important mechanism of cellular defense against pathogens and involves the spread of small interfering RNA (siRNA) to neighboring cells. In this study, we investigated whether endogenous expressed liver-specific microRNA (miRNA) and vector-delivered siRNA can transfer between hepatic cells in vitro and in mice and whether this exchange could extend the therapeutic effect of RNAi against hepatitis C virus (HCV) infection.

Methods and Results: We observed that human hepatoma cells, Huh7, highly expressing miRNA miR-122 also release this hepatocyte-abundant miRNA into culture medium (Huh7-CM). Electron micrographic imaging showed the presence of exosomes in Huh7-CM and RT-PCR analysis showed high levels of miR-122 in purified exosome preparations. Incubation of Huh7 cells with fluorescently labeled (Rhodamine) exosomes, showed that binding was rapid and endocytosis resulted in accumulation of exosomes in the cells within 30 min. Upon incubation of different cell lines with Huh7-CM, the levels of miR-122 in these cells dramatically increased, confirming uptake of miRNAs. Cell-to-cell transfer was not only seen with endogenious miRNA but also with siRNA derived from lentiviral vectors designed for therapeutic RNAi. In coculture of transduced and nontransduced Huh7 cells at a 1:1 ratio up to 52% gene silencing of HCV and CD81 was observed in nontransduced cells (P<0.01). To further explore the evidence of RNAi transmission in vivo, NOD/SCID mice were engrafted with Huh7 cells, stably expressing shCD81 and GFP, and which form solid tumors in the liver. Flowcytometric analysis of dissociated liver cells from these mice showed a significant reduction of CD81 expression on mouse hepatocytes and non-parenchymal cells (mean reduction of 57.9% ± 24.0, P<0.01), compared with mice engrafted with Huh7 cells expressing control shRNA. Moreover, treatment with CM from shCD81 expressing Huh7 cells also significantly reduced CD81 expression in mouse liver cell (mean reduction of 31.6% ± 15.6) as compared to the shCon-CM controls (P<0.05).

Conclusion: We demonstrated that transmission of small RNA in human and mouse liver cells which can both mediate the exchange of microRNA and vector-derived siRNA, thereby extending the therapeutic effects of RNAi on HCV.

36. The Non-Immunosuppressive Cyclophilin Inhibitor SCY-635 Inhibits the Association of NS5A and Cyclophilin A.  S. Hopkins; Z. Huang; S. Mosier; U. Chatterji; P. Gallay

Purpose: SCY-635 is a novel, non-immunosuppressive cyclophilin inhibitor that suppresses HCV-specific RNA replication in vitro and when administered as monotherapy to adults with Chronic Hepatitis C infection. Serial passage studies performed in con 1b-derived replicon cells indicate that mutations that confer decreased sensitivity to SCY-635 map to several positions within NS5A and NS5B. These results suggest that SCY-635 exerts anti-viral effects through multiple, cyclophilin A-dependent mechanisms. The following studies were performed in order to assess the sensitivity of the cyclophilin A (CypA)/NS5A interaction to SCY-635. Highly purified NS5A proteins were isolated from wild type viruses representing genotypes 1, 2, 3, and 4 as well as several engineered mutant species of NS5A that were identified during in vitro serial passage studies.

Methods: Recombinant full-length NS5A-His and GST-CypA proteins were induced, expressed, and isolated from bacterial lysates using nickel and glutathione beads. Pull down assays were conducted and followed by Western blotting analyses using anti-His and anti-CypA antibodies. The CypA/NS5A binding interaction was further examined by ELISA.

Results: CypA interacts directly with NS5A proteins derived from viruses representing multiple genotypes as well as all mutant species of NS5A. SCY-635 inhibited the association of CypA and NS5A in a dose-dependent manner. SCY-635 also promoted the dissociation of the CypA/NS5A complex. SCY-635 is approximately 3-fold more potent than cyclosporin A (CsA) with respect to inhibiting the formation of the CypA/NS5A complex. These observations are consistent with the differences in anti-viral potency determined for CsA and SCY-635 in con 1b-derived bi-cistronic replicon cells and for the replication of JFH1 genomic RNA in Huh-7 cells. Interestingly, engineered mutant NS5A proteins are indistinguishable from wild type NS5A with respect to their affinity for CypA and to the sensitivity of the mutant-NS5A/CypA complex to dissociation caused by SCY-635.

Conclusions: The CypA/NS5A interaction is highly conserved across viruses representing diverse genotypes and among engineered mutant species of NS5A. Differences in the sensitivity of the wild type CypA/NS5A complex to CsA and SCY-635 indicate that this interaction may account for the anti-viral activity of SCY-635. The sensitivity of the formation and dissociation of the mutant-NS5A/CypA complex to SCY-635 suggests that resistance to SCY-635 involves the participation of other proteins beyond the minimal NS5A/CypA complex.


1852. IL28B Polymorphism and Kinetics of Antiviral Activity for ANA598 in Combination With Pegylated Interferon α2A Plus Ribavirin in Treatment-Naïve Genotype-1 Chronic HCV Patients.  A. J. Muir; E. Lawitz; M. Rodriguez-Torres; V. K. Rustgi; T. Hassanein; J. R. Appleman; C. A. Crowley; J. L. Freddo; J. G. McHutchison

Background: ANA598 is a potent non-nucleoside inhibitor of HCV polymerase. ANA598 was well tolerated in HCV patients at 200 mg, 400 mg and 800 mg bid for 3 days, and resulted in rapid reduction in HCV RNA (median EOT range 2.3 to 2.9 log10). An ongoing phase 2 study is evaluating safety and antiviral activity of ANA598 with PEG-IFN and ribavirin (SOC). 24-week results for the first cohort (200 mg bid) and 12-week results for the second cohort (400 mg bid) have been reported. The CC IL28B-type has been associated with improved early viral kinetics and greater likelihood of RVR and cEVR in HCV-1 patients treated with SOC alone. 66 of 93 patients in this trial have been genotyped (CC, CT or TT) at the polymorphic site rs8099917 to determine the effect on response when a non-nucleoside inhibitor is added to SOC.

Methods: ANA598-504 is an ongoing double-blind, placebo-controlled Phase 2 study to assess safety, antiviral activity and PK of ANA598 in genotype-1 patients. Patients receive ANA598 or placebo with SOC for 12 weeks at 200 mg bid or 400 mg bid, both given with a loading dose of 800 mg q12h on day 1. Patients who achieve undetectable virus at weeks 4 and 12 are randomized to SOC alone for 12 or 36 additional weeks. Patients who did not meet these criteria are to continue SOC for 36 additional weeks. Comparable antiviral response data (cEVR 73-75%) has been reported for ANA598 at 200 mg bid and 400 mg bid. Viral kinetics and rates of RVR and cEVR were compared by IL28B-type between ANA598 and placebo control groups.

Results: Table shows a summary of the results.

Conclusions: The addition of ANA598 to SOC improves the early viral kinetics and percent of patients with genotype-1 HCV who respond and reach undetectable levels of virus for CC, CT and TT patients. ANA598 dramatically accelerates the rate of achieving undetectable levels of HCV RNA in the CT and TT patients where SOC alone is considerably less efficacious.

IL28B

Group

N

Week 1

Week 2

Week 3

Week 4

Week 6

Week 8

Week 10

Week 12

CC

Placebo

11

0

18

27

27

36

64

73

82

CC

ANA598

10

40

70

80

80

80

100

90

90

(C/T)T

Placebo

10

0

0

0

10

10

20

20

50

(C/T)T

ANA598

35

3

11

23

31

51

60

69

69

 

1853. Genotypic and Phenotypic analysis of HCV NS5A Inhibitor Resistance Variants: Correlations Between In vitro and In vivo Observations.  M. Gao; C. Wang; J. Sun; D. R. O'Boyle; R. Hindes; P. Yin; S. M. Schnittman; P. T. Nower; L. Valera; J. Lemm; S. A. Voss; F. McPhee; D. Hernandez; J. Kadow; M. Belema; N. A. Meanwell; M. Cockett; R. A. Fridell; R. Nettles; M. Bifano; H. Sevinsky; X. Huang; B. Kienzle; P. Patel

Background: NS5A plays a central role in HCV viral replication. BMS-790052 is a first-in-class and potent NS5A Inhibitor with broad genotypic coverage. The in vitro picomolar potency seen with BMS-790052 has translated in vivo effects in clinical studies.

Methods: We assessed the genotypic and phenotypic HCV resistance observed in Phase I (14-day monotherapy) and Phase IIa (BMS-790052 + pegylated interferon alpha [Peg] and ribavirin [Rib]) studies using HCV replicon system.

Results: In Phase I, HCV infected subjects generally experienced rapid and marked viral load declines, and in several subjects HCV RNA dropped below the level of quantitation (below LOQ, 25 IU/mL). Viral breakthrough (VBT) was observed on-treatment, however, in the majority of genotype 1a-infected subjects, but less often in subjects infected with genotype 1b. In the first 12 weeks of Phase IIa, VBT was observed in 2 of 12 subjects who received the lowest dose of BMS-790052 (3 mg) in combination with PegIFN/Rib and was associated with resistance substitutions previously identified in vitro. Twenty-three of 24 subjects who received BMS-790052 (10 mg or 60 mg) in combination with PegIFN/Rib achieved HCV RNA levels below detection (less than 10 IU/mL) through week 12, and VBT was not observed, demonstrating the efficacy of BMS-790052. Baseline variants with resistance substitutions were observed in 3 of the 23 subjects, but were suppressed below LOQ at week 4 when BMS-790052 was combined with PegIFN/Rib, demonstrating the impact of combination therapy.

Conclusion: Our results indicate that (i) the majority of resistance substitutions observed in vivo have been previously identified in vitro, demonstrating the relevance of the in vitro replicon system; (ii) resistant variants may pre-exist at baseline, but the rapid and marked suppression of HCV RNA (viral kinetic analysis) suggests these variants were inhibited by BMS-790052; and (iii) since BMS-790052 belongs to a novel class of HCV inhibitor with a potent antiviral effect in the clinic, it is an excellent candidate for combination therapy which should reduce the selection of resistant variants.

 


1854. In Vitro Activity of the Combination of Pegylated Interferon-Lambda (PEG-IFN-λ) with Direct-Acting Antivirals in the HCV Replicon Model.  F. McPhee; A. K. Sheaffer; J. A. Freeman; S. Chaniewski; S. Levine; C. Chen; S. A. Voss; J. Lemm; M. Gao; A. Majumdar; D. M. Miller; J. Friborg

Background and Aims: IFN-λ is a novel interferon that exerts an intracellular antiviral response through a unique receptor. A pegylated form of IFN-λ (PEG-IFN-λ) has demonstrated antiviral activity in subjects infected with HCV. In these proof-of-concept studies, PEG-IFN-λ appeared to exhibit an improved safety profile compared to PEG-IFN-α which may be a consequence of the limited distribution of the IFN-λ receptor. Combination of PEG-IFN-α with novel direct-acting antivirals (DAA) has significantly enhanced antiviral responses in HCV-infected patients. HCV replicon models were employed to assess the potential synergy of PEG-IFN-λ plus potent NS5A and NS3 inhibitors (BMS-790052 and BMS-650032, respectively).

Methods: The antiviral activity and cytotoxicity profiles of PEG-IFN-λ were tested in HCV replicon models, representing genotypes (G) 1a, 1b, and 2a, as well as in HCV G2a and yellow fever virus (YFV) models. HCV replicon activity was evaluated by measuring a change in fluorescence or luciferase signal. Activity in the virus models was evaluated by measuring either a change in luciferase signal or cell viability. Cytotoxicity in was assessed using a Cell Titer Blue assay. Combination data were assessed using either the combination index method of Chou or the method of Prichard and Shipman (MacSynergy II). Drug-resistant HCV replicons were selected in the presence of neomycin, 10% fetal bovine serum, and IFN-λ and/or IFN-α and/or DAA each at 1 to 30 times their respective EC50 values; colony formation was examined after approximately 25 days incubation.

Results: In HCV replicon models representing G1a, G1b, or G2a, PEG-IFN-λ suppressed HCV replication with EC50 values of 4.5 to 11.7 ng/mL. Against HCV JFH-1 (G2a) virus and YFV, the potencies were also comparable (EC50 values ranging from 2.2 to 4.5 ng/mL). In 2- and 3-drug combination studies, PEG-IFN-λ acted in a synergistic manner with ribavirin or in a mixed additive/synergistic manner with PEG-IFN-α or DAA clinical candidates BMS-650032 and/or BMS-790052. The emergence of DAA-resistant HCV replicons was suppressed when HCV replicons were exposed to combinations of IFN-λ with 2 DAA. The combination of IFN-α with 2 DAA also suppressed the emergence of DAA-resistant replicons.

Conclusions: These results demonstrate that PEG-IFN-λ has robust antiviral activity. Furthermore, these data suggest that the combination of PEG-IFN-λ with DAA may act in a mixed additive/synergistic manner and suppress the emergence of DAA-resistant virions. The combination of PEG-IFN-λ with ribavirin or novel DAA may represent effective treatment regimens for patients infected with HCV.

 


1855. Initial antiviral activity of the HCV NS3 protease inhibitor ABT-450 when given with low dose ritonavir as 3-day monotherapy: Preliminary results of study M11-602 in genotype-1 (GT1) HCV-infected treatment-naïve subjects.  E. Lawitz; I. Gaultier; F. Poordad; D. E. Cohen; R. Menon; L. M. Larsen; T. J. Podsadecki; B. Bernstein

Background: ABT-450, a potent HCV protease inhibitor, has been identified as a lead compound by Abbott and Enanta and is being developed for the treatment of HCV GT1 infection in combination with other anti-HCV agents. ABT-450 plasma concentrations are dramatically increased when coadministered with ritonavir (ABT-450/r), supporting once-daily dosing. This abstract presents the first HCV RNA response data on ABT-450/r in GT1 HCV-infected subjects.

Methods:  M11-602 study is an on-going randomized, double blinded (site and subject), placebo-controlled, dose ranging study exploring safety, pharmacokinetics and antiviral activity of ABT-450/r as monotherapy for 3 days, followed by 81 days of ABT-450/r + pegylated interferon alfa-2a (pegIFNα2a) + ribavirin (RBV). All subjects then receive pegIFNα2a + RBV for 36 weeks and are followed to sustained virologic response (SVR24). Safety is monitored by adverse events (AEs) and laboratory abnormalities. Up to 30 GT1 HCV-infected treatment-naïve, non-cirrhotic subjects with HCV viral load > 100,000 IU/mL will be randomized to one of 3 ABT-450/r doses: 50/100, 100/100 and 200/100 mg once-daily, or matching placebo.

Results: The current preliminary analysis presents the safety and 3-day monotherapy HCV RNA response for the first 24 Genotype 1a/b treatment-naïve subjects randomized to ABT-450/r or placebo: 8 subjects were female, 3 subjects were black, and 20 were white including  10 of Latino ethnicity. At baseline, mean age was 50 years, mean BMI was 26.6 kg/m2 and mean HCV RNA was 6.74  log10 IU/mL (range: 5.21 to 7.49).

ABT-450/r was well tolerated at all doses, with no serious or severe AEs to date. AEs were not different from those reported with placebo, and all but one (headache) were mild. No clinically significant changes in vital signs or ECGs were observed.

Efficacy:

         The primary endpoint of the study was the mean maximum change in HCV RNA during the 3-day monotherapy with ABT-450/r or placebo

         Profound decreases in HCV RNA were observed at all ABT-450/r doses during the 3-day monotherapy

         From day 1 through day 4, the mean maximum HCV RNA decrease from baseline was 4.02 log10 IU/mL (SD = 0.43) for subjects receiving ABT-450/r versus 0.36 log10 IU/mL (SD = 0.13) for subjects receiving placebo (P<0.001)

         Similar HCV RNA changes from baseline were observed in the 3 ABT-450/r dose groups

Conclusions:

         ABT-450 is a potent NS3 protease inhibitor, producing profound, rapid and dose-dependent HCV RNA reduction in all subjects on ABT-450/r to date

         ABT-450/r resulted in a profound decrease in HCV RNA during 3 days of monotherapy at all doses studied

         Through 3 days of monotherapy response was similar in the 3 ABT-450/r dose groups. The mean maximum HCV RNA decrease from baseline was 4.02 log10 IU/mL (SD=0.43) for subjects receiving ABT-450/r versus 0.36 log10 IU/mL (SD = 0.13) for subjects receiving placebo (P<0.001).

         ABT-450/r was safe and well tolerated when taken as monotherapy for 3 days, and no subjects discontinued during the monotherapy period

 

 


1857. Insulin resistance and obesity is improved by aerobic exercise that can be safely performed even by patients with hepatitis C virus.  I. Konishi; Y. Hiasa; Y. Tokumoto; T. Mashiba; M. Abe; S. Furukawa; B. Matsuura; M. Onji

Background and aims:  Hepatitis C virus infection often complicates glucose intolerance. Insulin resistance is one mechanism that causes glucose intolerance in patients with chronic hepatitis C (CH-C). We recently reported that glucose intolerance is a risk factor for hepatocarcinogenesis, and thus improving insulin resistance would be important to prevent this pathology. Aerobic exercise is recommended for patients with type 2 diabetes to improve insulin sensitivity and obesity. We conducted a pilot study to clarify whether aerobic exercise combined with diet therapy improves insulin resistance and obesity in 17 patients with CH-C.

Methods: The patients received nutrition education at entry and every two months thereafter. Target dietary energy intake was defined as standard body weight (kg) × 30 kcal/day. Starting from two months after enrollment, the patients were monitored for 6 months using a pedometer while walking at least 8,000 steps/day on a flat field. The following parameters were evaluated before and after exercise: octapolar bioimpedance obtained using an InBody 720 multifrequency analyzer, liver functions, homeostasis model assessment of insulin resistance (HOMA-IR), visceral (VFA) and subcutaneous (SFA) fat areas measured using computed tomography, adipocytokines such as leptin and adiponectin, and the Short Form-36 (SF-36) as a QOL score.

Results: Fifteen patients completed the study protocol. One dropped out due to knee pain and another because of depression. By the end of the study, 47% of the patients achieved an average of 8,000 steps/day and significantly decreased body weight, body mass index, VFA and SFA (P < 0.01, < 0.01, < 0.05 and < 0.01, respectively). However, muscle weight did not decrease. Alanine amino transferase significantly decreased, whereas serum albumin and prothrombin time did not change. The HOMA-IR was significantly decreased by the end of the study (P < 0.01). Serum levels of adiponectin did not change during the study period, whereas those of leptin significantly decreased (P < 0.01). The vitality score on the SF-36 scale tended to improve after exercise (P = 0.087), whereas no other parameters changed.

Conclusions: Simply walking as aerobic exercise can safely improve obesity and insulin resistance in patients with CH-C. Our results indicate that aerobic exercise can be recommended for such patients. Further investigation should identify whether aerobic exercise could prevent the development of hepatocellular carcinoma by improving obesity and insulin resistance and by decreasing serum levels of leptin.

 


1858. BMS-824393 is a Potent Hepatitis C Virus NS5A Inhibitor with Substantial Antiviral Activity when given as Monotherapy in Subjects with Chronic G1 HCV Infection.  R. Nettles; X. Wang; S. Quadri; Y. Wu; M. Gao; M. Belema; E. Lawitz; R. Goldwater; M. p. DeMicco; T. C. Marbury; A. Vutikullird; E. Fuentes; A. Persson; D. M. Grasela

Background: NS5A plays a central role in HCV viral replication. BMS-824393 is a potent NS5A Inhibitor with broad genotypic coverage, including picomolar in vitro potency against genotypes 1a and 1b. In a combined single and multiple ascending dose study with healthy subjects, BMS-824393 was shown to be well-tolerated and had a pharmacokinetic profile supportive of once-daily dosing.

Methods: The objectives of this open label, multiple ascending/descending dose, monotherapy study were to evaluate the antiviral activity, safety, tolerability and pharmacokinetics of BMS-824393 in treatment-naive subjects with genotype 1 chronic hepatitis C. Men or women, 18 to 60 years of age with HCV RNA ≥ 105 IU/ml with non-cirrhotic compensated liver disease received either 1, 10, 50 or 100 mg of BMS-824393 for 3 days [10 subjects (7 G1a and 3 G1b) per dose group].

Results –

Safety:

·        All doses were generally well-tolerated

·        No BMS-824393related deaths, serious adverse events, or discontinuations due to adverse events

·        No clinically relevant effect on ECGs, laboratory tests, vital signs, or physical examinations

·        There were 17 adverse events in 11 of 37 subjects (30%)

o   The majority of adverse events were mild

o    Only headache (2 of 37, 5%), atypical chest pain (2 of 37, 5%) and back pain (2 of 37, 5%) occurred in more than 1 subject

§  One of the back pain and both of the atypical chest pain events occurred more than 60 days after the last dose of BMS-824393 during the preplanned, prolonged follow-up period

 

Pharmacokinetics:

·        Following oral administration, BMS-824393 was readily absorbed

·        The mean terminal half-life ranged from 15 to 25 hours

·        BMS-824393 produced exposures comparable to those observed in a previous study in healthy volunteers (AI451001)

 


1859. ACH-2684: HCV NS3 Protease Inhibitor with Potent Activity against Multiple Genotypes and Known Resistant VariantsM. Huang; S. Podos; D. Patel; G. Yang; J. L. Fabrycki; Y. Zhao; C. Marlor; P. Kapoor; X. Wang; A. Hashimoto; V. Gadhachanda; G. Pais; D. Chen; A. Agarwal; M. Deshpande; K. L. Stauber; A. Phadke

Background: HCV NS3 protease is a clinically validated target. Most NS3 protease inhibitors in clinical development are ineffective against some non-genotype 1 HCV and HCV variants carrying NS3 protease inhibitor-resistant mutations. These NS3 protease inhibitor-resistant variants preexist in HCV-infected patients, a factor that could contribute to virologic breakthrough. ACH-2684 is derived from our efforts to design NS3 protease inhibitors with potent activity against different genotypes and against known resistant variants.

Methods: Structure-guided rational drug design employed both molecular modeling and protein-inhibitor complex X-ray structures. Biochemical and replicon assays were used to determine activity against NS3 variants. ADME characteristics, pharmacokinetic properties, and off-target activities were examined by standard procedures.

Summary/Conclusions

·        ACH-2684 is a potent NS3 protease inhibitor against genotypes 1 to 6.

·        ACH-2684 retains its activity against the most common NS3 protease mutants that are highly resistant to multiple NS3 protease inhibitors.

·        ACH-2684 forms a long-lived complex with NS3 enzyme that may prolong suppression of HCV replication. 

·        ACH-2684 potently inhibits HCV replication in genotype 1a and 1b replicon systems, with broad coverage of natural variants and resistant mutants.

·        ACH-2684 shows low potential for off-target activity and drug-drug interaction.

·        ACH-2684 is metabolically stable and shows favorable pharmacokinetic properties including preferential distribution into the liver.

·        ACH-2684 has been selected for clinical development for hepatitis C treatment.

 


1861. Clinical synergy of an Anti-HCV Nucleoside Analog with SOC: Viral Kinetics of PSI-7977 with SOC E. Lawitz; J. P. Lalezari; M. Rodriguez-Torres; K. V. Kowdley; D. Nelson; E. DeJesus; J. G. McHutchison; A. De La Rosa; W. Symonds; M. Berrey

Background: PSI-7977 is a novel uridine nucleotide analog in development for the treatment of HCV which demonstrated a 0.5-2.0 log10 IU/mL decline in a 3-day monotherapy trial. Nucleoside/tide analogs: 1) require intracellular phosphorylation which may result in a slower initial antiviral decline in monotherapy assessments; 2) in vitro select for unfit resistant variants which do not seem to be readily present in the treatment-naïve viral population; and, 3) retain activity against HCV variants with resistance against other classes of antivirals. These characteristics are consistent and predictable, and clinically may be the explanation for the demonstrated potent antiviral activity in combination with alfa-interferon and/or DAA.

Methods: Sixty-three treatment-naïve non-cirrhotic patients infected with HCV genotype 1 (GT-1) were enrolled in a randomized study of PSI-7977 100mg, 200mg, 400mg QD or matching placebo, administered with PEG-IFN/RBV for 28 days. Based upon the 1.0 log10 antiviral decline of PSI-7977 200mg QD in the 3-day monotherapy study, we hypothesized that an additive effect in combination with Peg-IFN/RBV could be predicted by combining the monotherapy response with the observed HCV RNA decline in peg-IFN/RBV alone in the 28-day study.

Results:

·        Administration of PSI-7977 with SOC resulted in a greater than predicted decline in HCV RNA as early as Day 1 based on the PSI-7977 monotherapy and placebo plus SOC data

·        The HCV RNA reduction with 100mg, 200mg, and 400 mg PSI-7977 plus SOC is -1.27, -1.43 and -0.78 log10 greater than predicted on Day 3, respectively, assuming additive activity of SOC and PSI-7977

·        The increased activity of PSI-7977 plus SOC does not appear to result from increased exposure to PSI-7977 metabolites when co-administered with SOC

·        Near maximal HCV RNA suppression was achieved with 400 mg PSI-7977 plus SOC resulting in significantly greater HCV undetectability (88-94%) at Day 28 relative to placebo (21%)

·        RVR rates with PSI-7977 plus SOC are as good or better than those reported for other DAAs plus SOC

Conclusions:

·        PSI-7977 demonstrated synergistic antiviral activity when combined with Peg-IFN/RBV.

·        These data highlight the potential for a nucleotide-containing combination regimen to produce significant antiviral activity, the extent of which may be underestimated by monotherapy antiviral responses

·        Addition combinations fo PSI-7977 with other DAAs are needed to explore the potential for clinical synergy in an interferon-sparing regimen

 


1862. Genotypic and phenotypic analysis of the NS5B polymerase region from viral isolates of HCV chronically infected patients treated with BI 207127 for 5-days monotherapyL. Lagace; M. Cartier; G. Laflamme; C. Lawetz; M. Marquis; I. Triki; M. Bernard; R. Bethell; D. G. Larrey; S. Lueth; C. Trepo; J. O. Stern; W. O. Boecher; J. Steffgen; G. Kukolj

Background: BI 207127, a specific and potent non-nucleoside inhibitor of the HCV RNA-dependent RNA polymerase in vitro, has been studied in patients with chronic genotype (GT) 1 HCV infection for 5 days as monotherapy. The antiviral activity of BI 207127 reached a maximal effect (median 3.8 log10 VL decline) for the group receiving 800 mg q8h with no viral breakthrough observed during treatment. NS5B genotypic and phenotypic characterization of viral isolates at baseline and after BI 207127 dosing has been performed.

Methods: HCV RNA was collected and extracted at baseline and at time points following the last dose from 60 patients treated with either placebo, 100, 200, 400, 800 or 1200 mg BI 207127 q8h. Population sequencing was performed on the NS5B region. Clonal sequencing permitted the quantification and identification of major and minor variants (LLOQ: 5%) in the NS5B region. Chimeric HCV sub-genomic replicons were constructed to determine BI 207127 EC50 values against patient-derived NS5B polymerase sequences.

Summary/Conclusions:

·        In this study with 5 days monotherapy, slight differences in mean BI 207127 GT-1a and GT-1b susceptibility were observed among diverse GT-1a and Gt-1b NS5B from the isolates in this study, consistent with the preclinical profile of BI 207127 and with the observed clinical response

·        HCV NS5B resistant variants that confer resistance to BI 207127 were selected in 6/46 patients treated with BI 207127 monotherapy.  The predominant mutation in five of these GT-1b viruses encoded changes at P495

·        The lack of persistence of P495 variant and rapid outgrowth of WT P495 during follow-up suggest the fitness of this variant in the absence of drug pressure is substantially lower than that of WT-potential for a higher barrier to resistance

·        The NS5B variants do not alter the sensitivity to IFSN-a or NS3 protease inhibitor and support the further investigation of BI 207127 in combination with pegylated interferon and ribavirin and/or other direct antivirals for the treatment of HCV infection

 


1867. Enhanced in vitro Antiviral Activity by Combining GS-9256, a Novel Protease Inhibitor, with GS-9190, a Non-nucleoside NS5B Inhibitor .  H. Mo; K. S. Ku; H. Yang; M. Robinson; A. Bae; M. D. Miller; W. E. Delaney

Background: GS-9256, an HCV protease inhibitor (PI), and GS-9190, a non-nucleoside NS5B inhibitor (NNI), have demonstrated potent antiviral activity in genotype 1 HCV infected subjects during monotherapy studies. Previous in vitro resistance selection studies identified A156T and D168A/G/E/N/V in NS3 protease as primary GS-9256 resistance mutations and C316Y, C445F, Y448H and Y452H in NS5B in GS-9190 treated replicons.

Objectives: To characterize cross-resistance profiles of GS-9256 and GS-9190 and to determine the antiviral activity of GS-9256 and GS-9190 in short- and long-term in vitro combination assays.

Methods: GS-9256 and GS-9190 associated mutations were introduced into 1b replicons and antiviral susceptibility was tested in transient replication assays. The antiviral activity of GS-9256 in combination with GS-9190 was monitored by reduction of luciferase signal after 3 days of treatment or by the suppression of HCV replicon RNA following long-term passage in the presence of drugs.

Results: GS-9256 is a potent HCV inhibitor with an EC50 value of ~20 nM against the HCV 1b replicon. Reduced GS-9256 susceptibility was observed with the replicons carrying the NS3 A156T and D168A/G/E/N/V mutations. In contrast, no change in susceptibility to GS 9190 was observed in replicons carrying GS 9256 or other HCV PI resistance mutations. Likewise, GS 9256 retained wild-type potency against mutations that confer resistance to GS 9190 and other classes of HCV NNIs. Furthermore, combination of GS 9256 with GS-9190 in 3-day replicon assays produced an additive antiviral interaction without detectable cytotoxicity. Finally, long-term treatment of HCV replicons with combinations of GS 9256 and GS-9190 resulted in substantial reductions in HCV RNA that were significantly greater than those observed with treatment with either compound alone.

Conclusions: These findings support the clinical strategy of combining GS-9256 with GS-9190 to more effectively suppress the HCV replication and reduce resistance development. Accordingly, the combination of GS-9256 and GS-9190 is in active clinical development.

 


1868. Interferon lambda plays a critical role on antiviral response by hepatotropic inducer of innate immunity in human hepatocyte.  Y. Hirata; S. Nakagawa; Y. Tokunaga; Y. Tanaka; M. Mizokami; K. Inoue; M. Kohara

Type I interferon (IFN-α/β) is currently used for clinical treatment for viral infections. Polyinosinic-polycytidylic acid (pIC) known as an inducer of innate immunity is evaluated as adjuvants and immunomodulatory devices. Here, we developed pIC complexed with a hepatotropic cationic liposome (LIC) to induce innate immunity in the liver specifically. Then, we examined whether antiviral response induced by pIC/LIC complex eliminate the hepatotropic pathogens such as hepatitis C virus (HCV) or hepatitis B virus (HBV) causing chronic infection in human hepatocyte. In chimeric mice with humanized liver infected by HCV, treatment with pIC/LIC daily at 0.1 mg/kg led to dose-dependent and stronger reductions in HCV than PegIFN-α treatment (30 μg/kg twice weekly: a dose that is 20 times greater than the clinical dose).

We further explored the effects of pIC/LIC against HBV. Chimeric mice in the pIC/LIC group were intravenously treated with pIC/LIC daily, and chimeras in the entecavir (ETV) group were treated orally with 17 μg/kg of ETV daily (the same as the clinical dose). The pIC/LIC treatment more effectively reduced HBV-DNA levels than the ETV treatment by Day 14.

So far, pIC has been used as an effective activator of innate immunity in human culture cells or mouse bodies. However, we found that IFN-lambdas were more strongly induced than IFN-α/β in human hepatocyte from comprehensive gene expression analysis. On repeated administration of pIC/LIC, we observed that the kinetics of IFN-lambdas induction were consistent with the duration of antiviral effects of pIC/LIC, while kinetics of type I interferon were inconsistent. Additionally, these antiviral effects of pIC/LIC were attenuated by neutralizing antibodies against human IFN-lambdas. We demonstrated that pIC/LIC elicited antiviral effect on HCV and HBV via inducing IFN-lambdas rather than IFN-α/β, indicating that IFN-lambda plays a critical role on antiviral response in human hepatocyte.

http://aasld2010.abstractcentral.com/user_images/aasld2010/1909/893982/Figure_1_anti-HCV_effect.jpg


1870. Differential in vitro Effects of Intravenous Versus Oral Formulations of Silibinin on the HCV Life Cycle and Inflammation.  J. Wagoner; C. Morishima; T. N. Graf; N. Oberlies; E. Pécheur; J. Tavis; S. J. Polyak

Silymarin is extracted from the seeds of the milk thistle plant, prevents liver fibrosis progression in many experimental rodent models, and is the most popular botanical medicine consumed by patients with hepatitis C. It is therefore imperative to determine if and how silymarin protects the liver.

Silibinin is a major component of silymarin, consisting of the flavonolignans silybin A and silybin B, which are insoluble in aqueous solution. However, the chemically modified, soluble version of silibinin, SIL, has been shown to potently reduce HCV RNA levels in vivo when administered intravenously.

By contrast, silymarin and silibinin do not appear to reduce HCV RNA levels in patients when ingested orally, although they inhibit HCV infection in cell culture.

Silymarin, silibinin, and pure flavonolignans inhibit HCV entry and fusion, NS5B polymerase activity, HCV RNA and protein expression and virus transmission. Since silibinin and SIL are chemically different, we hypothesized that their biological effects would vary. We compared the hepatoprotective profiles of the natural product silibinin and SIL in assays that measure antiviral, anti-inflammatory, and immunomodulatory functions. Both inhibited fusion of HCV pseudoparticles with fluorescent liposomes in a dose-dependent fashion.

SIL inhibited 5 clinical genotype 1b isolates of NS5B RNA dependent RNA polymerase (RdRp) activity better than silibinin, with IC50 values of 40-85 μM. However, inhibition of the RdRps by both compounds plateaued at 43-73%, suggesting that these products are poor inhibitors of RdRp.

Like silymarin, silibinin did not inhibit HCV RNA and protein expression from subgenomic genotype 1b or 2a replicons, but it did inhibit JFH-1 infection. In contrast, SIL inhibited 1b but not 2a subgenomic replicons and also inhibited JFH-1 infection at much higher doses than required for RdRp inhibition. Silibinin but not SIL inhibited NF-κB-dependent transcription and inflammatory T cell functions.

The data indicate that SIL and silibinin have very different effects on liver, immune cells, and HCV replication. The data further suggest that silymarin and its constituents elicit hepatoprotection primarily by targeting host cell components.


1871. Impact of ribavirin on HCV replicon RNA decline during treatment with Interferon alfa and the protease inhibitors boceprevir or telaprevir.  W. P. Hofmann; T. Chung; C. Osbahr; S. Susser; U. Karey; C. Welsch; J. Lötsch; C. Sarrazin; S. Zeuzem; E. Herrmann

Background: Ribavirin increases sustained virologic response rates in patients chronically infected with the hepatitis C virus (HCV) who receive pegylated interferon alfa and novel HCV protease inhibitors.

Methods: To better characterize antiviral efficacies of these upcoming therapies, Huh7 cells harboring a subgenomic HCV replicon system were cultivated with various doses and combinations of ribavirin, interferon alfa, and the protease inhibitors boceprevir and telaprevir. Antiviral efficacy parameters were estimated from HCV RNA decay and synergistic effects of combination therapies were analysed with the Bliss independency model.

Results: Single drug antiviral activities showed dose dependent HCV RNA reductions in replicon cells (IC50 of 386.16 µM, 81.67 U/ml, 0.44 µM and 0.81 µM after 48 h for ribavirin, interferon alfa, boceprevir, and telaprevir, respectively). For the dual combination of ribavirin with either boceprevir or telaprevir, no deviation from additivity was observed whereas the reduction of HCV RNA was synergistic for ribavirin with Interferon alfa (p<0.001). Triple combinations with ribavirin, interferon alfa and protease inhibitors showed the most profound HCV RNA decay.

Conclusions: The beneficial in vitro antiviral effect of ribavirin with interferon alfa and novel HCV protease inhibitors demonstrates that ribavirin may be required as antiviral backbone in the near future.

 


1873. Pharmacokinetic-pharmacodynamic analyses of TMC435 in patients infected with hepatitis C virus (HCV) genotypes 2 to 6.  V. Sekar; M. Peeters; O. Lenz; M. Simonts; G. . De Smedt

Aim: TMC435, a macrocyclic NS3/4A protease inhibitor, has demonstrated potent antiviral activity in treatment-naïve and -experienced patients infected with hepatitis C virus (HCV) genotype 1 (GT 1). This Phase IIa, open-label, proof-of-concept study (TMC435-C202) evaluated the antiviral activity, safety, tolerability, and pharmacokinetics (PK) of TMC435 in patients infected with HCV GT 2 to 6; here we describe the PK of TMC435 and the relationship to antiviral activity and safety parameters.

Methods: Treatment-naïve patients infected with HCV GT 2-6 were included in five cohorts by genotype and received seven consecutive days of monotherapy with oral TMC435 200 mg once daily (QD). From Day 8, patients received standard of care. PK profiles of TMC435 were determined up to 96 hours after TMC435 intake on Day 7. Relationships between TMC435 PK and change from baseline in plasma HCV ribonucleic acid (RNA; log10 IU/mL) were explored at Day 7. Safety assessments included change from baseline in alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and serum bilirubin.

Results: Following 7 days of monotherapy with TMC435 200 mg QD, steady-state trough, minimum, and maximum concentrations, and TMC435 exposure (area under the curve from 0-24 hours), were comparable for the GT 4 (n=8), GT 5 (n=7), and GT 6 (n=8) cohorts. Lower values were observed for the GT 2 (n=6) and GT 3 (n=8) cohorts, with the lowest values seen in the latter cohort. Subgroup analyses showed that TMC435 exposure appeared to be approximately two-fold higher in patients without cirrhosis (n=26) versus with cirrhosis (n=11), and was comparable between the different races (Asian [n=9], Black [n=3], Caucasian/White [n=25]). Age, weight, and gender did not appear to influence TMC435 exposure. TMC435 demonstrated potent antiviral activity in GT 2 and GT 4-6. At Day 8, mean change from baseline in HCV RNA was highest for GT 6 (-4.35) and GT 4 (-3.52), followed by GT 2 (-2.73) and GT 5 (-2.19); no antiviral activity was seen against GT 3 (-0.04). No relevant relationship was observed between TMC435 exposure and antiviral activity on Day 7 for the different genotypes. Higher exposure to TMC435 was associated with increases in direct, indirect, and total bilirubin; but no relevant relationship was observed between TMC435 exposure and changes in ALP, AST, or ALT.

Conclusions: No relevant relationship between TMC435 PK and antiviral activity was observed for the different genotypes. Consistent with previous reports, increases in serum bilirubin were exposure-related, but there was no relevant relationship between TMC435 PK and other hepatic laboratory parameters.


1874. Metabolite Characterization of INX-189, a Potent HCV Inhibitor, in Fresh Human Primary Hepatocytes and Human Liver and Kidney Cell Lines.  A. Hall; N. Raja; S. D. Chamberlain; E. Gorovits; G. W. Henson; J. T. Hutchins; J. Muhammad; J. Patti; J. Vernachio; J. Wang; K. Madela; M. Aljarah; S. l. Jones; C. McGuigan

INX-189 is a potent phosphoramidate analog of the nucleoside 2’-C-methylguanosine currently in clinical development for the treatment of HCV. The compound is designed to deliver the monophosphate form of the nucleotide intracellularly, by-passing the first rate-limiting phosphorylation step and facilitating rapid conversion to the active triphosphate.

The aim of this study was to identify and quantitate the metabolites generated in vitro after INX-189 incubation with primary human hepatocytes and cell lines. Fresh plated hepatocytes from human donors and cell lines such as human liver cells (HepG2) and human embryonic kidney cells (293) were incubated at 37°C in the presence of INX-189. The intracellular and extracellular metabolites were detected and quantified using LC-MS/MS methodology.

The proposed metabolic pathway of INX-189 includes enzymatic cleavage of the carboxylester group followed by release of 1-Naphthol. 1-Naphthol is rapidly metabolized in the liver to the glucuronidated and sulphated derivatives. The cyclic phosphoramidate created after the processing of the ester group undergoes spontaneous hydrolytic ring opening to create the aminoacyl phosphoramidate which is converted to 2’-C methyl guanosine monophosphate via cleavage of the amino acid group and loss of the O6-methyl group. The monophosphate is metabolized to the di- and triphosphates by cellular kinases. The active triphosphate is catabolized by cellular 5’-nucleotidases to the diphosphate, the monophosphate, and finally to the nucleoside 2’- C-methyl guanosine.

To confirm this proposed pathway, LC-MS/MS analytical methods and synthesized analytical standards were used to quantitate key intermediates. In primary human hepatocytes and cell lines, the parent protide, 2’-methyl guanosine and O6-Methoxy 2’-C-Methyl guanosine nucleosides, aminoacyl metabolites of INX-189 as well as mono-and triphosphate forms of 2’-methyl guanosine and O6-Methoxy 2’-C-Methyl guanosine were quantitated. The pharmacologically active triphosphate was identified as a predominant intracellular metabolite in vitro. Triphosphate was detected early in the time course (within 15 minutes of incubation with cells) and peaked within 8 hours.

We have established analytical methods to identify and quantitate the key metabolites of the proposed metabolic pathway of INX-189. These methods have been applied to the analyses of samples from in vitro studies to confirm the proposed metabolic scheme.

 


1876. Characterization of HCV Resistance from Single and Multiple Dose Clinical Trials of GS-9256, a novel NS3 Protease InhibitorK. A. Wong; A. Bae; X. Qi; K. S. Ku; A. Worth; J. Waters; S. C. Sun; J. Harris; M. D. Miller; H. Mo

Background: GS-9256 is a novel NS3 active site protease inhibitor with an EC50 of ~20nM in HCV genotype (GT) 1b replicon assays. GS-9256 also displayed potent activity in HCV GT1-infected subjects during single ascending dose (SAD, 150mg, 300mg, and 450 mg) and multiple ascending dose (MAD, 25mg, 75mg, and 200mg BID, 300mg QD for 3 days) trials. This study aims to characterize the virologic resistance from phase I SAD and MAD studies of GS-9256.

Methods: The full-length NS3/4A gene was amplified and population sequenced at baseline, the final treatment day, and follow-up timepoints. NS3 protease domains from patient samples with emerging mutations were cloned into an NS3 shuttle vector and their susceptibilities to GS-9256 and other HCV inhibitors were determined using a transient replication assay.

Results: In the SAD study, single conserved site resistance mutations in NS3 (R155K, D168E or D168V) were observed as mixtures with wild-type HCV in 4/8 subjects at 450mg GS-9256, but not in patients receiving lower GS-9256 doses or placebo. Subjects with resistance mutations had a greater viral load reduction than those without resistance mutations (mean -3.4 vs. -2.1 log10). One subject had ~14% D168E mutant at baseline by clonal analysis, which correlated with a suboptimal antiviral response. In the MAD study, R155K, D168G/E/V, or A156A/V mutations were identified. NS3 resistance mutations were more common in subjects receiving higher doses of GS-9256; these subjects also had greater declines in HCV RNA. There was no evidence of HCV rebound during 3 days of monotherapy. The R155K and D168G/E/V mutations conferred significant reductions in GS-9256 susceptibility in vitro. These NS3 resistance mutations were cross-resistant to GS-9451 and ITMN-191. The D168G/E/V mutants remained fully susceptible to VX-950, while the R155K mutant conferred 7-fold resistance to VX-950. All GS-9256 mutations showed wild-type sensitivity to IFN-α and ribavirin, as well as to the Gilead NS5B inhibitor GS-9190.

Summary: In both single and multi-dose studies, NS3 resistance mutations to GS-9256 were rapidly selected at higher doses, indicating the presence of pre-existing resistance mutations and the potent inhibition of GS-9256 against wild-type HCV. The lack of cross-resistance between GS-9256 resistance mutants and GS-9190, IFN-α, and ribavirin makes GS-9256 a candidate for use in combination anti-HCV therapy.

 


1877. HCV resistance To Nitazoxanide is not due to Changes in the Viral Sequence.  B. Korba

Background and Aims: Nitazoxanide (NTZ, Alinia®, Romark Laboratories, LC) is a licensed thiazolide anti-infective, and is currently in advanced stage clinical trials for the treatment of HCV infection. We and others have demonstrated that NTZ, and its active metabolite, tizoxanide (TIZ), exhibit potent antiviral activity against multiple genotypes of HCV in cell culture, and that while resistant HCV replicon-containing cell lines to NTZ/TIZ can be selected, attempts to transfer NTZ-resistance by transfection of HCV genomes in whole cell RNA preparations from NTZ-resistant cells to naive cell cultures were unsuccessful (Korba, et al. 2008, Antivir. Res. 77:56; AAC 52: 4069). The aim of this current study was to further explore the nature of the resistance of HCV to NTZ.

Methods: Full length HCV replicon sequences (at least 10 per cell line) were obtained from two NTZ-resistant (NTZ-11, TIZ-9), and the parental (RP7) cell lines, followed by transfection with indivudal HCV replicon constructs.

Results: A consensus sequence for the wild-type (CON1) HCV from RP7 was developed. Numerous nucleotide changes were observed in individual HCV genomes from both NTZ-11 and TIZ-9 relative to the RP7 HCV consensus sequence, but no conserved mutations in the HCV non-structural genes or the 3’UTR from both cell lines were detected. A cluster of single nucleotide mutations was observed in all HCV replicon sequences from both resistant cell lines in the stem of the 5’-proximal stem-loop structure of the 5’-UTR. Three individual mutations (5’UTR G17A, G18A, C20T) were individually inserted into wild-type CON1 HCV replicons. Colony formation efficiency for the mutants was reduced (6 to 20-fold) relative to wild-type HCV, but none of these mutants conferred resistance to NTZ. In addition, TIZ was found to be inactive against the activity of HCV polymerase, protease, and helicase in enzymatic assays. To determine if NTZ-resistance was conferred primarily by cellular factors, RP7, NTZ-11, and TIZ-9 were cured of HCV genomes by serial passage under high dose interferon alpha (1000IU/ml). The antiviral sensitivity patterns of HCV in whole RNA from RP7, NTZ-11, TIZ-9, and the three engineered HCV mutants following transfection into the cured cell lines are currently being determined.

Conclusions: These data confirm previous conclusions that HCV resistance to NTZ is not due to mutations in the virus. Together with the observation that TIZ does not directly affect HCV enzymatic activities, these data are consistent with the earlier hypothesis that antiviral activity and HCV resistance to TIZ are due to its interactions with a cellular target.


1878. Augmentation of Interferon signaling pathway by Nitazoxanide: A novel therapeutic strategy for Relapsers to Peg-interferon and Ribavirin therapy.  C. Wang; X. Zhang; A. Osinusi; H. Masur; D. Fishbein; S. Kottilil

Background: Pegylated-interferon alpha (peg-IFN) and ribavirin (RBV) remain the current standard for treatment of HCV among HIV-infected patients, however, there remains an increased rate of relapse in those infected with HIV. The mechanism of HCV relapse to IFN-based therapy is not completely understood even though their host response to IFN appears similar to those with sustained virologic response (SVR). In this study, we investigated a novel approach in an attempt to maximize HCV antiviral effect, in vitro and ex vivo by using an agent, nitazoxanide (NTZ) to enhance the host response to the IFN signaling pathway.

Methods: In vitro, the Huh7.5/JFH-1 HCV continuous cell culture system was used to study the effects of serial concentrations of NTZ on HCV replication in the presence and absence of IFN. Cell viability was determined by Calcein assay. HCV replication levels were determined by RT-PCR on RNA from cell culture supernatants. Phosphorylation profile of STAT (1 to 6) post-NTZ treatment was determined by western blot using cell lysates of JFH-1 infected and uninfected Huh7.5 cells. Ex vivo, PBMCs were treated with NTZ, in the presence or absence of IFN, from HIV/HCV coinfected subjects who were null responders (NR), relapsers and SVRs (N=10 each) to prior peg-IFN and RBV treatment. IFN-inducible gene (IFIG) expression was measured using multiplex PCR. ANOVA was used for statistical analysis.

Results: We found a dose-dependent NTZ direct suppression of HCV replication in vitro when compared to untreated cells (p<0.002) and a synergistic antiviral effect with IFN, compared to IFN alone (p<0.01) and NTZ alone (p<0.003). Pre-treatment of NTZ for 3 days prior to IFN treatment resulted in the maximal suppression of HCV replication in vitro (p<0.001). NTZ treatment of PBMCs, ex vivo, resulted in a direct induction (fold change) of IFIGs in PBMCs of relapsers (2.6 ± 0.9, p<0.01) and SVRs (3.1 ± 1.1, p<0.003), but not in null responders (0.6 ± 0.3; p>0.05). NTZ treatment also augmented IFN–mediated IFIG induction seen in PBMCs from relapsers (4.6 ± 1.2, p<0.002) and SVRs (5.1 ± 1.3, p<0.001), but not in null responders (1.5 ± 0.9, p>0.3) when compared to IFN alone, although relapsers and SVR had no differences in IFIG induction when treated with IFN alone.

Conclusions: Combination therapy of peg-IFN/RBV, with the addition of an agent that stimulates the IFN signaling cascade, such as NTZ may be most effective for IFN relapsers, but not for NR, who may have a blunted IFN signaling pathway. NTZ has potent direct and synergistic (with IFN) anti-HCV activity and may have a role in combination therapy using directly acting antiviral drugs.

 


1879. IL28B rs12979860 SNP is associated with lipids metabolism, viral genotype and spontaneous viral clearance in Hepatitis C.  M. Maraver Zamora; J. A. Del Campo; R. Ramírez-Lorca; B. Pardo-Yules; M. E. Sáez; L. M. Real; M. Diago; F. J. Morón; I. Carmona; R. J. Andrade; M. F. González-Escribano; M. A. Montes-Cano; M. Cuaresma; A. Rojas; J. Aguilar-Reina; M. Serrano-Ríos; A. Ruiz; M. Romero-Gomez

Aims: We sought to determine if the IL28B rs12979860 gene variant was associated with viral genotypes, serum lipid levels and spontaneous viral clearance (SVC) in hepatitis C patients in comparison with non-infected healthy people from Spain.

Methods: We have analyzed the IL28B rs12979860 polymorphism in 1100 Spanish individuals: 240 chronic hepatitis C, 69 SVC and 791 healthy controls from the general population using real time-PCR coupled to Fluorescence Resonance Energy Transfer (FRET). We divided them in two genotype groups (CC versus CT+TT) and then we analyzed the genotype effect on treatment outcome, lipid and glucose profiles and laboratory measurements using conventional statistical analyses.

Results: CC genotype was associated with higher serum LDL (CC=116 mg/dl; CT+TT=95 mg/dl, p=0.012) and total cholesterol (CC=188.6 mg/dl, CT+TT=171.4 mg/dl, p=0.007, adjusted by age, sex, viral load, weight and height) but not with HDL (CC=51 mg/dl, CT+TT=57 mg/dl, p=0.11) and VLDL (CC=18.9 mg/dl, CT+TT=17.5 mg/dl). This association was not replicated in the general population: total cholesterol (CC=219 mg/dl, CT+TT=221 mg/dl, p=0.43), LDL (CC=143 mg/dl, CT+TT=144 mg/dl, p=0.67). IL28B polymorphism was associated with SVC and viral genotype: prevalence of CC genotype in SVC (n=69, 72.5%) and in patients infected by genotypes 2 and 3 (n=89, 57.3%), genotype 1 (n=151, 32.5%) and general population (n=791, 50.3%); p<0.01.

Conclusion: Our results suggest that observed associations are directly related to hepatitis C virus-host interactions instead of a direct effect of this locus on lipid metabolism. This effect seems to be on a genotype-dependent manner. Host factor (IL28b SNP) could select virus infection and promote chronic infection or spontaneous clearance according to viral genotype and lipid metabolism.

 


1880. In Vitro Combination Studies of ACH-1625 (HCV NS3 Protease Inhibitor) and ACH-2928 (HCV NS5A inhibitor) in Presence and Absence of Ribavirin.  Y. Zhao; G. Yang; J. L. Fabrycki; D. Patel; J. Wiles; X. Wang; A. Phadke; M. Deshpande; M. Huang

Background: Due to high replication rate and poor fidelity of RNA polymerase, preexisting variants resistant to any single direct-acting anti-HCV agent are deemed inevitable. Therefore, therapy with direct antiviral agents will require combination treatment to achieve sustained virologic response. ACH-1625 is an NS3 protease inhibitor that exhibits potent antiviral activity (a 3.81 log10 mean maximum drop of plasma HCV RNA) after dosing hepatitis C patients at 600 mg QD for 5 days. ACH-2928 is an NS5A inhibitor that displays low picomolar potency against HCV replicons. In this in vitro study, the combinatory effects of two drugs for antiviral activity and for prevention of emergence of resistant variants were investigated in the presence and absence of ribavirin.

Methods: Huh-7 cells harboring HCV replicons were utilized for evaluation of combination treatment. These replicons were derived from a laboratory strain or a lab strain within which the inhibitor-targeted region was replaced with a corresponding region of HCV RNA obtained from clinical isolates. For evaluation of antiviral activity, inhibitors were added to cells in serial dilutions of concentrations in a checker board manner. Three days after addition of compounds, the level of HCV RNA replication was quantified with luciferase reporter assay. For evaluation of prevention of emergence of resistant variants, cells were treated with inhibitors either alone or together at several concentrations in the presence of G418 till the cells formed visible colonies.

Results: Combination of ACH-1625 and ACH-2928 in the presence or absence of ribavirin yielded an additive to synergistic antiviral effect. Combination of these two agents blocked significantly the emergence of resistant variants and this effect is more pronounced in the presence of ribavirin. More importantly, such an effect in preventing the emergence of resistant variants was also observed with cells harboring a population of chimeric replicons from HCV-infected patients, a system representing the quasispecies nature of HCV virions in patients.

Conclusion:

·        Combination of ACH-1655 and ACH-2928 results in a synergistic antiviral effect

·        Combination of ACH-1625 and ACH-2928 blocks the emergence of the resistant colonies

·        In presence of ribavirin, the synergistic antiviral activity as well as the effect on preventing the emergence of the resistant colonies is enhanced

·        These data support clinical evaluation of combination of NS3 protease inhibitor ACH-1625, NS5A inhibitor ACH-2928 and ribavirin.

 


1881. BMS-790052, a First-in Class Potent Hepatitis C Virus NS5A Inhibitor, Demonstrates Multiple-Dose Proof-of-Concept in Subjects with Chronic GT1 HCV infection.  R. Nettles; H. Sevinsky; E. Chung; D. Burt; H. Xiao; T. C. Marbury; R. Goldwater; M. p. DeMicco; M. Rodriguez-Torres; E. Fuentes; A. Vutikullird; E. Lawitz; A. Persson; M. Bifano; D. M. Grasela

Background: NS5A plays a central role in HCV viral replication. BMS-790052 is a first-in-class and potent NS5A inhibitor with broad genotypic coverage. In a multiple-ascending dose (MAD) study with healthy subjects, BMS-790052 was shown to be well-tolerated, and had a pharmacokinetic (PK) profile supportive of once-daily dosing.

Methods: The objectives of this randomized, double-blind, placebo-controlled, MAD monotherapy study were to evaluate the antiviral activity, safety, tolerability, and PK of BMS-790052 in treatment-naive subjects infected with genotype 1 HCV. Men and women, 18 to 60 years of age with HCV RNA ≥105 IU/mL with non-cirrhotic compensated liver disease were eligible to participate in the study. Patients were randomized to receive 14 days of 1, 10, 30, 60, or 100 mg once daily (QD) or 30 mg twice-daily (BID) of BMS-790052 or placebo (5 patients per dose; active:placebo=4:1).

Results: Following oral administration, BMS-790052 was readily absorbed with largely dose-proportional exposures over the studied dose range. The mean terminal half-life of BMS-790052 was approximately 13 to 15 hours. Mean maximum declines in HCV RNA in G1a and 1b infected subjects after multiple 1, 10, 30, 60, 100 mg QD and 30 mg BID doses of BMS-790052 are below. The antiviral effect was not sustained for the entire 14 days of dosing and some subjects experienced viral rebound on or before Day 7 of dosing. All BMS-790052 multiple doses were well-tolerated and had a safety profile similar to that of placebo. There were no serious adverse events or discontinuations due to adverse events. The only adverse event that occurred in > 10% of subjects was headache; reported in 5 (20.8%) BMS-790052 treated subjects and 2 (33.3%) placebo recipients.

Conclusions: BMS-790052 is a potent NS5A inhibitor that produces a robust decline in HCV RNA following multiple doses in subjects chronically infected with either HCV genotype 1a or genotype 1b. A subsequent clinical study of BMS-790052 in treatment-naïve G1 HCV subjects demonstrated potent early antiviral activity against both genotypes 1a and 1b when combined with pegIFN/RBV. BMS-790052 was well-tolerated in multiple doses of up to 100 mg and has a PK profile that supports once-daily dosing. These results support the importance of inhibiting NS5A-mediated HCV replication in the treatment of HCV. Further studies are underway to confirm the role of NS5A inhibition in HCV therapy.

Mean Maximum Decline in HCV RNA (log10)

Genotype

QD Dose Range (mg)

BID Dose (mg)

1

10

30

60

100

30

1a (n)

2.4 (2)

3.0 (2)

3.3 (4)

3.8 (4)

3.6 (3)

2.6 (2)

1b (n)

3.3 (2)

4.3 (2)

n/a (0)

n/a (0)

4.5 (1)

5.7 (2)

 


1883. Nonclinical Profile and Phase I Results in Healthy Volunteers for the Novel and Potent HCV NS5A Inhibitor GS-5885J. O. Link; R. Bannister; L. D. Beilke; G. Cheng; M. Cornpropst; A. Corsa; E. Dowdy; H. Guo; D. Kato; T. Kirschberg; H. Liu; M. Mitchell; M. Matles; E. Mogalian; E. Mondou; C. Ohmstede; B. Peng; R. W. Scott; J. W. Findlay; G. E. Chittick; F. Wang; J. Alianti; J. Sun; J. Taylor; Y. Tian; L. Xu; C. Yang; G. J. Yuen; K. Wang; E. J. Eisenberg

Background: HCV NS5A has emerged as an important target for small molecule antiviral drugs. GS-5885 is a highly potent HCV NS5A replication inhibitor and is under development for the treatment of HCV infection. Here we describe the nonclinical profile, and the first-in-human safety and pharmacokinetic (PK) data from an ongoing phase I study of GS-5885 in healthy subjects.

Methods: Potency and selectivity were studied using cell based HCV replicon assays. Cross-resistance was determined in a GT 1b transient replicon assay. PK was assessed in rats, dogs and cynomolgus monkeys following intravenous and oral administration. Metabolic stability was determined in cryopreserved human hepatocytes. Safety pharmacology and repeat dose toxicology studies were conducted in rats and dogs. Safety, tolerability and PK are being studied in a phase I escalating single oral dose trial in healthy volunteers randomized to GS-5885 or placebo (8:2) that is underway, with dosing completed at 3, 10 and 30 mg under fasted conditions. Further dose escalation is ongoing.

Results: GS-5885 inhibits HCV RNA replication in replicon cell lines with an EC50 of 41 picomolar (pM) for GT 1a, and 5 pM for GT 1b. The selectivity index in GT 1a replicon cells is >800,000. GS-5885 exhibits potency shifts for signature resistance mutations in NS5A (L31V and Y93H) and is fully active against signature HCV NS3 protease (e.g. R155K and A156T) or NS5B polymerase (e.g. M423T and S282T) mutants. GS-5885 is highly stable in cryopreserved human hepatocytes (predicted clearance is <0.07 L/hr/kg). GS-5885 has good PK properties in rat, dog and cynomolgus monkey with half lives (t1/2) of 4.7, 7.4 and 10.3 hours respectively and oral bioavailability ranging from 32-53%. Nonclinical safety studies showed that GS-5885 was well tolerated in repeat dose studies in rats and dogs. Clinical results for the first three dose levels (3, 10 and 30 mg) in healthy subjects have shown GS-5885 to be safe and well-tolerated with no serious AEs, no treatment-emergent Grade 3/4 chemistry or hematology abnormalities, and few AEs to date (all AEs mild in the 30-mg cohort). The PK in the first cohort of subjects dosed at 3 mg resulted in a long median t1/2 of 32 hours and 24 hour plasma levels above the plasma protein-binding adjusted GT 1a EC50 for all subjects.

Conclusion: GS-5885 is a potent inhibitor of HCV replication with a favorable nonclinical profile. PK and safety data in healthy subjects indicate that once daily dosing of GS-5885 has potential utility in novel treatment regimens for HCV infection.


1884. High exposure to danoprevir (RG7227) increases the probability of ALT elevations in patients treated with danoprevir plus PegIFN α-2a (40KD) (PEGASYS) plus ribavirinM. Levi; N. Frey; J. C. Hsu; K. Jorga; J. Tran; E. Labriola-Tompkins; E. S. Yetzer

Background: Danoprevir (RG7227; DNV) is a potent, selective, orally administered hepatitis C virus (HCV) protease inhibitor. Reversible grade 4 alanine aminotransferase (ALT) elevations have been reported at high DNV doses (600 and 900 mg q12h) in a Phase 2b study. The objective of this analysis was to assess the relationship between DNV exposure and ALT elevations.

Methods: DNV pharmacokinetic (PK) samples were obtained from treatment-naive, non-cirrhotic adults with HCV genotype 1 infection randomized to 12 weeks of (A) DNV 300 mg q8h, (B) 600 mg q12h or (C) 900 mg q12h in combination with PegIFN α-2a 180 µg/week plus ribavirin 1000/1200 mg/day. Sparse PK samples including predose and postdose samples were collected at baseline and at weeks 1, 2, 4, 6, 8 and 12 for most patients. Serial PK samples up to 8 hours (for q8h regimen) or 12 hours (for q12h regimens) postdose were obtained at weeks 4 and 12 in approximately 20% of patients. Population PK methodology was used to describe the concentration time profiles in all patients with evaluable PK data. The area under the curve (AUC) was estimated for all patients (sparse and intense PK) using the Maximal a Posteriori Bayesian individual model PK parameter estimates. The relationship between DNV exposure and the probability of ALT elevations was estimated by multiple logistic regressions. For the logistic regression the inter-dose interval exposure (AUC0-24) is used as an independent variable and the occurrence of ALT elevation (defined using ACTG grading) during DNV therapy as a categorical dependant variable.

Results: A total of 1748 PK observations from 198 patients were available for analysis. Reliable AUC estimates were obtained from 129 patients. High variability was observed in the AUC0-24 of the three dosage groups. This high variability in DNV PK was observed in previous studies. DNV PK was described reasonably well by a two-compartment disposition with a complex zero- and first-order absorption model. Non-compartmental estimates of AUC0-24 in the densely sampled patients were in agreement with corresponding model predictions. The logistic regression indicates that higher DNV exposure (AUC0-24) increased the probability of ALT elevations. No ALT grade 4 elevations were observed at AUC0-24 of 782 ng*hr/ml and lower.

Conclusion: The probability of ALT elevations increases with increasing DNV AUC0-24 in patients with chronic hepatitis C. RTV boosting of low DNV doses is being explored to maintain potent antiviral activity while reducing DNV exposure (AUC0-24) which would consequently reduce the probability of ALT elevations at effective concentrations.


1885. Safety, Tolerability, and Pharmacokinetics after Single and Multiple Doses of MK-5172, a Novel HCV NS3/4a Protease Inhibitor with Potent Activity Against Known Resistance Mutants, in Healthy Subjects.  D. M. Brainard; A. Petry; M. S. Anderson; A. Mitselos; T. Laethem; I. Heirman; L. Caro; J. A. Stone; P. Sun; P. Panorchan; L. M. Van Bortel; M. Iwamoto; J. A. Wagner

Introduction: MK-5172 is a novel, competitive inhibitor of the HCV NS3/4a protease with selective, potent in vitro activity against a broad range of HCV genotypes (GTs) and known viral variants that are resistant to other protease inhibitors in development. Pharmacokinetic and safety data were collected after single-dose (SD) and multiple-dose (MD) administration in healthy subjects.

Methods: SD: Alternating panel, multiple period, dose escalation study in 24 healthy males who received 2 to 1600 mg single doses of MK-5172 or placebo in the fed or fasted state; MD: Serial panel study in 40 healthy males who received 100 to 1000 mg MK-5172 or placebo once daily for 10 days. Safety evaluations were performed throughout the studies. Plasma samples for MK-5172 concentration determination and pharmacokinetics were collected.

Results: There were no serious adverse experiences (AEs) reported. One subject was discontinued from the study for flu-like illness classified by the investigator as definitely not related to study drug. SD: Following oral administration, MK-5172 increased in plasma with median Tmax values of 2.0 – 5.0 hours. Thereafter, concentrations declined in a biphasic manner with mean terminal t½ ~15.0 – 34.4 hours. Administration of 50 mg with a high-fat meal had no clinically meaningful effect on the pharmacokinetic parameters. Mean AUC0-∞, Cmax and C24hr values appeared to increase in a dose proportional fashion through 200 mg and in a greater than dose proportional manner at doses greater than 200 mg. MD: Steady state was achieved after approximately 6 days. At steady state, approximate 3-fold accumulations of MK-5172 for AUC0-24hr and Cmax were observed for doses of 100-400 mg. At higher doses, the extent of accumulation was less (~1.5-fold) for AUC0-24hr and Cmax due to a greater contribution of AUC0-24hr to the overall exposure. The C24hr geometric mean accumulation ratio (Day 10/ Day 1) was approximately 1.5 for most dose levels. Mean AUC0-24hr and Cmax appeared to increase in a greater than dose proportional manner at steady-state. The median Tmax (2.5 - 4.0 hours) and apparent t½ (~20 hours) of MK-5172 on Day 10 after once daily dosing were consistent with values from the SD study.

Conclusions: MK-5172 is generally well tolerated and exhibits a pharmacokinetic profile supportive of once daily dosing.

 


1886. Altered hepatic energy metabolism and a lack of obvious adverse findings after miR-122 inhibition in miceC. Esau; S. Davis; C. Li; A. Chang; J. Murray; R. A. Capaldi; E. Marcusson; B. Bhat; C. Newgard; P. Linsley

Background: microRNA-122 (miR-122), a conserved microRNA abundantly and specifically expressed in the adult liver, is a key regulator of cholesterol metabolism and serves as an essential host factor for hepatitis C viral replication. Because miR-122 inhibition is being investigated as a novel therapeutic strategy for chronic hepatitis C infection, we examined the effects of miR-122 inhibition on hepatic gene expression patterns and metabolic parameters in mice. We also examined the safety of long-term inhibition of miR-122 in mice.

Methods: In the short-term study, mice were dosed with an oligonucleotide that inhibits miR-122 (anti-miR-122) at 30 mg/kg/week for 2 weeks. Hepatic gene expression was measured by Affymetrix array profiling. Mitochondrial respiration rate was measured in isolated mitochondria by MitoXpress. ATP/ADP ratios in the liver were measured by ATP-dependent luciferase assay. Metabolite profiling of amino acids, organic acids, and acylcarnitines in the liver and plasma was performed by GC/MS and MS/MS. The long-term safety of inhibiting miR-122 was assessed by treating CD-1 mice (n=8/group) weekly with 25 mg/kg/week or 75 mg/kg/week anti-miR-122 for up to 50 weeks.

Results: After anti-miR-122 treatment for 2 weeks, we identified a gene expression signature pointing to changes in energy utilization in the mitochondria. ATP/ADP ratio in the liver was reduced after anti-miR-122 treatment in the fasted but not fed state, but mitochondrial coupling was not affected. Broad metabolite profiling in the liver identified localized perturbations in the citric acid cycle and branched chain amino acid catabolism, which could be correlated with modulation of direct target genes in those pathways by anti-miR-122. All of these target genes are prominently expressed in highly oxidative tissues such as heart and muscle. In the 50-wk dosing study, anti-miR-122 treatment resulted in a significant and sustained decrease in total plasma cholesterol, consistent with the pharmacodynamic effects of miR-122 inhibition. However, there were no obvious deleterious effects based on clinical chemistry and histopathology evaluations.

Overall, the data show that miR-122 has a role in shaping hepatic energy metabolism in the adult mouse that may involve suppressing metabolic pathways important in oxidative tissues such as heart and muscle. Such changes, however, are not associated with obvious adverse findings, suggesting that miR-122 inhibition will be well tolerated as a therapy for hepatitis C infection.

 


1890. Pharmacokinetics, Safety, and Tolerability of PSI-352938, a Novel Nucleotide Polymerase Inhibitor for HCV, Following Single Ascending Oral Doses in Healthy Subjects.  W. Symonds; J. M. Denning; E. Albanis; R. Wright; A. Lai; M. Berrey

Background: PSI-352938 is a novel nucleotide prodrug of a purine nucleotide analog polymerase inhibitor in clinical development for the treatment of chronic HCV infection. PSI-352938 demonstrates potent antiviral activity in vitro, maintains activity in the presence of the S282T mutation, achieves high liver:plasma ratios in preclinical studies and has the potential to be dosed once daily. As with other nucleoside/tides, PSI-352938 has the potential for a high genetic barrier to resistance, pan-genotype potency, and ease of combination with compounds from other DAA classes with minimal risk of drug interaction. PSI-352938 was selected as a candidate for its complementary attributes for combination with the pyrimidine nucleotide analog, PSI-7977. This study assessed the safety, tolerability and pharmacokinetics of PSI-352938 in healthy volunteers, compared capsule and tablet formulations and evaluated the potential effect of food.

Methods: Single oral doses of PSI-352938 or placebo administered to 3 alternating panels of healthy subjects to receive PSI-352938 in capsules at doses of 25, 50, 100, 200, 400 or 800mg and a tablet formulation administered with or without food at the 200mg dose level and 1600 mg (without food). Forty-five subjects (14 females); White (36 pts), Black (8 patients) and 1 Asian patients.   Mean age was 31-5 yrs (19-52 yo); mean weight was 76.9 kg (45.9-105.2), and mean BMI was 25.12 (19.2-29.7). 

Summary/Conclusions

·        The various doses were safe and well-tolerated with no sever or serious adverse events reported.  All reported adverse events were mild to moderate.

·        Singles ascending doses of PSI-352938 up to 1600 mg were generally safe and well-tolerated

·        The PK results demonstrate a systemic exposure profile consistent with a prodrug exhibiting extended plasma stability, allowing for continued uptake by the liver over time

·        Based upon the long terminal half-life, daily dosing is likely

·        Based upon these results, a monotherapy multiple ascending dose study of PSI-352938 has been initiated in genotype 1 HCV-infected subjects to assess safety, tolerability, and antiviral activity

·        Exploratory combination studies with complementary nucleosides/tides and/or other DAAs are planned for the near term. 

 


1891. Phase I Study in Healthy Volunteers and Patients with IDX375, a Novel Non-Nucleoside HCV Polymerase Inhibitor.  J. de Bruijne; J. van de Wetering de Rooij; A. A. van Vliet; J. Leempoels; X. Zhou; C. J. Weegink; R. Molenkamp; J. Schinkel; M. Temam; J. Molles; J. Chen; K. Pietropaolo; J. Sullivan-Bólyai; D. L. Mayers; H. W. Reesink

Background: IDX375 is a potent and selective non-nucleoside inhibitor of HCV genotype (GT) 1 polymerase with low nanomolar EC50 in the HCV 1a and 1b replicon system.

Methods: A Phase I study of the choline salt (CS) form of IDX375 was conducted using doses ranging from 25 mg single dose to 200 mg BID for one day in healthy volunteers (HV, N=40). Three HCV GT 1-infected patients received 200 mg IDX375 BID for one day.

A free acid (FA) form was developed with improved stability properties of the drug product. The Phase I study is being extended to higher doses of IDX375 using the FA form. Single doses of 200, 400 (with food effect), 800 and 1200 mg IDX375 (FA) are being sequentially administered to cohorts of HV. Additional cohorts will receive 800 mg BID for one day and 200 and 400 mg BID for three days. Plasma and urine IDX375 levels are measured using validated LC/MS-MS methods.

Summary/Conclusions

·        At the doses evaluated, IDX375 was generally well-tolerated and achieved pharmacologically relevant drug concentrations

·        PK/PD analyses suggest that target trough levels of IDX375 between 2-10 ug/mL may be optimal for treatment of HCV-infected patients

·        High drug levels were achieved with 800 mg IDX375 FA BID after one day and resulted in asymptomatic grade ½ increases in indirect bilirubin in HV.  None of the increases in indirect bilirubin were associated with increases in ALT, AST, or alkaline phosphatase.  All values returned to baseline levels upon completion of dosing.  These observations are consistent with in vitro UGT1A1 inhibition of IDX375

·        A 3-day proof-of-concept study with IDX375 FA in treatment-naïve, genotype 1 HCV-infected patients is ongoing

 


 

36. The Non-Immunosuppressive Cyclophilin Inhibitor SCY-635 Inhibits the Association of NS5A and Cyclophilin A. S. Hopkins; Z. Huang; S. Mosier; U. Chatterji; P. Gallay

Purpose: SCY-635 is a novel, non-immunosuppressive cyclophilin inhibitor that suppresses HCV-specific RNA replication in vitro and when administered as monotherapy to adults with Chronic Hepatitis C infection. Serial passage studies performed in con 1b-derived replicon cells indicate that mutations that confer decreased sensitivity to SCY-635 map to several positions within NS5A and NS5B. These results suggest that SCY-635 exerts anti-viral effects through multiple, cyclophilin A-dependent mechanisms.

The following studies were performed in order to assess the sensitivity of the cyclophilin A (CypA)/NS5A interaction to SCY-635. Highly purified NS5A proteins were isolated from wild type viruses representing genotypes 1, 2, 3, and 4 as well as several engineered mutant species of NS5A that were identified during in vitro serial passage studies.

Methods: Recombinant full-length NS5A-His and GST-CypA proteins were induced, expressed, and isolated from bacterial lysates using nickel and glutathione beads. Pull down assays were conducted and followed by Western blotting analyses using anti-His and anti-CypA antibodies. The CypA/NS5A binding interaction was further examined by ELISA.

Results: CypA interacts directly with NS5A proteins derived from viruses representing multiple genotypes as well as all mutant species of NS5A. SCY-635 inhibited the association of CypA and NS5A in a dose-dependent manner. SCY-635 also promoted the dissociation of the CypA/NS5A complex. SCY-635 is approximately 3-fold more potent than cyclosporin A (CsA) with respect to inhibiting the formation of the CypA/NS5A complex. These observations are consistent with the differences in anti-viral potency determined for CsA and SCY-635 in con 1b-derived bi-cistronic replicon cells and for the replication of JFH1 genomic RNA in Huh-7 cells. Interestingly, engineered mutant NS5A proteins are indistinguishable from wild type NS5A with respect to their affinity for CypA and to the sensitivity of the mutant-NS5A/CypA complex to dissociation caused by SCY-635.

Conclusions: The CypA/NS5A interaction is highly conserved across viruses representing diverse genotypes and among engineered mutant species of NS5A. Differences in the sensitivity of the wild type CypA/NS5A complex to CsA and SCY-635 indicate that this interaction may account for the anti-viral activity of SCY-635. The sensitivity of the formation and dissociation of the mutant-NS5A/CypA complex to SCY-635 suggests that resistance to SCY-635 involves the participation of other proteins beyond the minimal NS5A/CypA complex.


 

82. Sustained Viral Response (SVR) Rates in Genotype 1 Treatment-naïve Patients with Chronic Hepatitis C (CHC) Infection Treated with Vaniprevir (MK-7009), a NS3/4a Protease Inhibitor, in Combination with Pegylated Interferon Alfa-2a and Ribavirin for 28 Days. M. P. Manns; E. J. Gane; M. Rodriguez-Torres; A. D. Stoehr; C. Yeh; P. Marcellin; R. T. Wiedmann; P. Hwang; R. J. Barnard; A. W. Lee

Background: Vaniprevir is a noncovalent competitive inhibitor of HCV NS3/4A protease which significantly improved rapid viral response (RVR) rates in CHC subjects when administered in combination with pegylated interferon alfa 2a (peg-IFN) and ribavirin (RBV) for 28 days. We now report on final efficacy and safety results for patients who continued peg-IFN/RBV treatment for a total of 48 weeks.

Methods: This was a randomized, placebo-controlled, double-blind study of vaniprevir in treatment-naïve CHC patients. Vaniprevir was administered for 28 days with peg-IFN/RBV in 1 of 5 regimens: placebo, 300 mg BID, 600 mg BID, 600 mg QD, or 800 mg QD; all patients continued peg-IFN/RBV for an additional 44 weeks. HCV RNA was determined by Roche Cobas Taqman PCR with a lower limit of detection (LLOD) ~10 IU/mL. RVR and SVR24 rates are defined as the percentage of treated patients below LLOD at 4 weeks of treatment, and 24 weeks after the end of treatment, respectively.

Results:

·        The RVR rates in the vaniprevir-containing arms ranged from 67% to 84%, vs. 5% for the control [p < 0.001 for each dose group, full analysis set (FAS) analysis].

·        High SVR24 rates in subjects treated with vaniprevir (plus peg/rbv)for 4 weeks followed by an additional 44 weeks of peg-IFN/RBV were achieved  the following SVR results:

o   600 mg BID (twice daily) = 80%

o   600 mg QD (once daily) = 78%

o   800 mg QD = 84%

o   Placebo (peg/rbv) = 63%

 

Resistant HCV variants at NS3/4a positions 155 and 168 were detected in viral breakthrough patients receiving vaniprevir. Vaniprevir in combination with peg-IFN/RBV was well tolerated, with no serious adverse events (SAEs) during the vaniprevir treatment and follow-up period (42 days). There were 9 SAEs reported during the peg-IFN/RBV continuation period, none of which were related to vaniprevir.

Conclusions:

·        In this first study of vaniprevir in combination with peg-IFN/RBV, vaniprevir was well-tolerated and achieved potent inhibition of HCV.

·         Subjects treated with vaniprevir in combination with standard therapy for the initial 28 days had significantly higher RVR compared to control. The majority of vaniprevir dose combinations had a numerically higher rate of SVR24 as compared to control. The results support further development of vaniprevir for HCV treatment.

 

MK7009
Dose group

RVR
(Full Analysis Set)

SVR24
(Full Analysis Set)

300 mg BID

12/18 (67%)

11/18 (61%)

600 mg BID

16/20 (80%)

16/20 (80%)

600 mg QD

12/17 (71%)

14/18 (78%)

800 mg QD

16/19 (84%)

16/19 (84%)

Placebo

1/19 (5%)

12/19 (63%)

 


 

LB-10.  4-week virologic response and safety of ABT-450 given with low-dose ritonavir (ABT-450/r) in combination with pegylated interferon alpha-2a and ribavirin (SOC) after 3-day monotherapy in genotype 1 (GT1) HCV-infected treatment-naïve subjects E. Lawitz; I. Gaultier; F. Poordad; E. DeJesus; K. V. Kowdley; G. Sepulveda; D. E. Cohen; R. Menon; L. M. Larsen; T. J. Podsadecki; B. Bernstein

Background: ABT-450 is a potent NS3 HCV protease inhibitor identified as lead compound by Abbott and Enanta. It is being developed for the treatment of HCV GT1 in combination with other anti-HCV agents. Co-administration with low-dose ritonavir results in increased ABT-450 plasma concentrations and longer half-life, supporting once-daily dosing.

Methods: This ongoing, randomized, blinded, placebo-controlled, dose-ranging study is assessing the safety, tolerability, pharmacokinetics, and antiviral activity of ABT-450/r monotherapy (at doses of 50/100, 100/100, and 200/100 mg once-daily, or matching placebo) for 3 days, followed by ABT-450/r + pegylated interferon alpha-2a 180 μg/week + weight-based ribavirin 1000-1200 mg/day for 12 weeks. At week 12, subjects receive SOC alone for 36 weeks.

Results: 24 subjects have been randomized to ABT-450/r or placebo.  In the group who received the study drug:  8 subjects were female, 20 patients were white (of whom 10 were Hispanic); 3 were black, 1was other race.   At baseline (BL), mean age was 50 years, mean BMI was 26.6 kg/m2, and mean HCV RNA was 6.74 log10 IU/mL.

19 subjects were infected with genotype 1a.  BL phenotypes, available for 24 patients. 

Efficacy through Week 4:

·        The primary endpoint of the study was the mean maximum change in HCV RNA during the 3-day monotherapy with ABT-450/ritonavir ®  or placebo.  Through 3 days of monotherapy, reponse was similar in the 3 ABT-450/r groups:  Mean maximum decreases from 3.89 to 4.11 log 10 IU/mL were observed in all 3 dose groups compared to 0.36 log10 IU/mL for the placebo group (p<0.001 for each comparison)

·        Beginning at 8 hours post the first dose on day 1 through week 4, all 3 ABT-450/r versus dose groups had mean HCV RNA deceases from baseline statistically significantly different from placebo at every time point

·        At week 4, the mean (± SD) HCV RNA decrease from baseline was 5.58 ± 0.65 log10 IU/mL for subjects on ABT-450/r versus 1.86 ± 1.90 log10 IU/mL for subjects on placebo (p<0.001)

·        The mean HCV RNA (range) at week  4 was 1.15 (1.00 – 2.87) log10 IU/mL for subjects on placebo

·        Through week 4, virologic response was similar in the 3 ABT-450/r dose groups

·        Although few subjects with genotype 1b enrolled, there were no apparent difference in virologic response to ABT-450./r or placebo between genotypes 1a and 1b

·        No subject receiving ABT-450/r experienced a virologic rebound (increase greater than or equal to 0.5 log10 IU/mL from nadir) through the 4 weeks of ABT/r plus standard of care treatment

Proportion of Subjects with HCV RNA <25 IU/ml at Week :

·        For each ABT-450/r dose group, mean HCV RNA was <25 IU/mL at week 4

·        21 of 23 (91.3%) subjects receiving ABT-450/r with data at week 4 had HCV

·        RNA <25 IU/mL versus 1 out of 8 (12.5%) subjects receiving placebo

·        In an Intent-to-Treat analysis, where discontinued subjects are counted as failures, 21/24 (87.5%) subjects randomized to ABT-450/r had HCV RNA <25IU/mL versus 1/11 (9.1%) subjects randomized to placebo

Conclusions:

·         At week 4, the mean (± SD) HCV RNA decrease from baseline was 5.58 ± 0.65 log10 IU/mL for subjects on ABT-450/r compared with 1.86 ± 1.90 log10 IU/mL for subjects on placebo (P<0.001); the 3 doses showed similar HCV RNA decreases from baseline

·         21 of 23 (91.3%) subjects receiving ABT-450/r had HCV RNA <25 IU/mL at week 4 compared with 1 of 8 (12.5%) subjects on placebo

·         ABT-450/r + SOC was safe and well tolerated during 4 weeks of treatment, with an adverse event profile comparable to SOC alone

 

LB-12. Safety and Pharmacokinetics of PPI-461, a Potent New Hepatitis C Virus (HCV) NS5A Inhibitor with Pan-Genotype Activity. N. A. Brown; P. Vig; E. Ruby; A. Muchnik; E. Pottorff; S. J. Knox; S. Febbraro; W. Wargin; T. Molvadgaard; A. Jones; R.

Purpose: HCV-related mortality is increasing. Most new direct-acting HCV antivirals are optimized for activity vs HCV genotype-1, comprising <50% of infections globally. PPI-461 is a novel HCV NS5A inhibitor with potent activity vs all 7 HCV genotypes, g1-7 (R Colonno, EASL 2010). The 50% inhibitory concentrations (EC50s) for PPI-461 are 0.21 nM and 0.01 nM for HCV g1a and g1b, and 0.1 to 9.3 nM for g2a-7a. Here we report the first human trial of PPI-461.

Methods: This Phase 1 trial was a randomized, double-blind study of safety and pharmacokinetics (PK) for 5 PPI-461 dosing regimens: single doses of 20, 50, 100 and 200 mg; and a multi-dose regimen, 200 mg QD x 5 days. The dose regimens were tested in sequential groups of 8 healthy subjects (total = 40). In each group subjects were randomized in a 6:2 ratio to blinded treatment with PPI-461 or matching placebo, with 7 days' follow-up. The PPI-461 bioanalytic assay had a plasma limit of detection of 10 ng/mL.

Results: All PPI-461 dosing regimens were well-tolerated. There were no adverse events (AEs) attributed by the investigator to PPI-461. Non-attributed AEs in the 30 PPI-461 recipients were mild headache (2), moderate backache (1), mild nausea (1), and 2 common colds. AEs in 10 placebo recipients were headache (2) and transient hearing impairment (1).

There were no patterns of change or significant abnormalities for any of the safety-related laboratory parameters (hematologic values, serum chemistries, urinalyses and ECGs).

Summary/Conclusions

·        In healthy volunteers, PPI-461 was well tolerated, clinical and laboratory safety monitoring did not indicate any pattern of clinical adverse events or abnormalities in the tested dosing range of 20-200 mg, with a treatment duration of up to 5 days

·        Substantial dose-related systemic exposures were consistently obtained in subjects doses with PPI-461

·        Cmax plasma levels achieved quickly in all subjects (1-4 hr), and consistently long PPI-461 half-life (ca 8-10 hr) resulted in C 24hr levels exceeding repliccon EC50 levels (all genotypes) in all subjects dosed with ≥50 mg

·        Steady-state concentration reached with 2 days in 5-day repeat-dose cohort (200 QD)

·        Dosing with high-fat meal resulted in 43% reduction in AUC 0-24 hr levels in subjects treated with 50 mg PPI-461, C24hr levels were minimally changed

·        Results support ongoing evaluation of PPI-461 in HCV patients with QD dosing. 

 


 

228. Clinical, Virological, Biochemical Outcomes After 20 Years of Sustained Virological Response (SVR) in Chronic Hepatitis C: The NIH Experience. C. Koh; T. Heller; V. Haynes-Williams; K. Hara; J. Feld; Y. Rotman; M. G. Ghany; T. Liang; J. H. Hoofnagle

Introduction: The short-term goal of therapy of hepatitis C is viral eradication; but the long-term goal is prevention of liver-related disability or death. Although the short-term benefits of an SVR after therapy are established, the long-term clinical benefits are less defined.

Aims: To assess changes in non-invasive markers of disease activity and fibrosis in a cohort of pts followed for up to 22 yrs after SVR.

Methods: The first 103 patients (pts) to achieve SVR after being treated at the National Institutes of Health, starting in 1984, using standard or peginterferon with or without ribavirin were evaluated. Serum markers of hepatic inflammation, synthetic function and fibrosis before treatment and at the last visit were compared. Pts evaluated since 2007 underwent transient elastography

Results: Three of the 103 pts relapsed; 0.7, 6.4 and 6.5 years after therapy (10-year relapse rate of 5.7% by Kaplan-Meier analysis). The 100 remaining pts included 56 men; 88 whites, 4 African Americans, 8 Asians; average age at last visit 56 years (range 17 - 84); HCV genotype 1 in 45%, 2 and 3 in 53%, other in 2%. Pretreatment liver histology (99 pts) showed mild fibrosis (Ishak score 0-2) in 64, moderate (3-4) in 25, and cirrhosis (5-6) in 10. After SVR, pts were followed for 0.5 to 22 (median = 7.6) yrs. There were no cases of hepatic decompensation or liver cancer. Serum markers improved in all long-term responders, including mean ALT (from 152 to 27 U/L), AST (86 to 24 U/L), alkaline phosphatase (78 to 69 U/L), globulin (3.2 to 2.8 gm/dL), IgG (1462 to 1113 mg/dL), alpha fetoprotein (4.6 to 2.9 ng/mL), GGT (47 to 28 U/L), rheumatoid factor (38% to 19% positive), platelet count (208,000 to 239,000/ μL) and AST-platelet ratio index (APRI: 0.99 to 0.25) (p < 0.001 for all). Transient elastography was successful in 75 pts and was normal (< 7.0 kPa) in 60%; moderately elevated (7.1-13.8) in 31%; and in the cirrhotic range (> 13.8) in 9%. Of 7 pts with cirrhosis before therapy, 6 had abnormal elastography at follow up. Elastography readings but not serum markers at the time of last follow up correlated with pre-treatment liver fibrosis (p=<0.001).

Conclusions: In long-term follow up, 97% of patients with an SVR maintained a virological response. No patients died of liver-related causes. Despite long-term SVR, patients with pre-existing cirrhosis still had evidence of hepatic fibrosis by transient elastography. Noninvasive markers of liver disease all improve over time. In chronic hepatitis C, SVR is associated with both short term and long-term benefits.

 


 

LB-3. Controlled Attenuation Parameter: a novel FibroScan®-based tool to detect and quantify steatosis in chronic hepatitis B

A. F. Cardoso; M. C. Sasso; V. Miette; C. Fournier; L. Sandrin; M. Beaugrand; C. Douvin; V. de Ledinghen; R. Poupon; M. Ziol; P. Bedossa; P. Marcellin

 

Background and Aims: Steatosis may contribute to the progression of liver fibrosis in patients with chronic hepatitis B (CHB) but its evaluation by non invasive means is still a challenge. Since fat affects ultrasound propagation, a novel Controlled Attenuation Parameter (CAP) evaluated on the signals acquired by the FibroScan® has been developed. The aim of this work was to validate the CAP performance for detection and quantification of steatosis in CHB patients.

 

Methods: 133 consecutive CHB patients were prospectively included (62% men, age = 39±13 years). All patients underwent both liver biopsy (LB) and FibroScan® in 5 liver units within 60 days. The CAP was retrospectively evaluated on the raw data acquired by the FibroScan® and corresponds to the ultrasonic attenuation value in dB/m at the centre frequency of the probe (3.5 MHz). LBs were analysed by the same pathologist. Activity and fibrosis were staged according to the METAVIR classification. Steatosis was graded according to the following scale: S0: steatosis ≤10% of hepatocytes, S1: 11~33%, S2: 34~66%, S3: ≥67%. Proportions in each steatosis grade were: 77%, 11%, 9% and 3%, respectively. Performance was evaluated in terms of Area Under the Receiver Operating Characteristic (AUROC).

 

Results: In univariate analysis, liver stiffness (LS) was significantly (p<0.05) correlated to fibrosis (Spearman ρ=0.60), gender, activity, steatosis, age and sinusoidal fibrosis. By multivariate analysis, liver stiffness was associated only with liver fibrosis (OR=19.9 [6.65-59.8]). In univariate analysis, CAP was mainly correlated with steatosis (ρ=0.50, p<107) but also with BMI, presence of NASH and liver fibrosis. In multivariate analysis CAP was associated only with steatosis (OR=7e13 [4e8-1e19]). Performances in terms of AUROC (i) for LS were: 0.80 (0.72-0.89) for F≥2, 0.91 (0.84-0.99) for F≥3 and 0.91 (0.78-1.00) for F=4; (ii) for CAP: 0.82 (0.74-0.89) for S≥10% and 0.81 (0.72-0.90) for S≥30%. Similar results were obtained for both LS and CAP using jack-knife cross validation.

 

Conclusion: The CAP is an accurate new non invasive tool to detect and quantify steatosis in CHB patients.