Boceprevir microbiology

# Microbiology

## Activity in Cell Culture
The EC50 and EC90 values for boceprevir against an HCV replicon constructed from a single genotype 1b isolate were approximately 200 nM and 400 nM, respectively, in a 72-hour cell culture assay. Boceprevir cell culture anti-HCV activity was approximately 2-fold lower for an HCV replicon derived from a single genotype 1a isolate, relative to the 1b isolate-derived replicon. In replicon assays, boceprevir had approximately 2-fold reduced activity against a genotype 2a isolate relative to genotype 1a and 1b replicon isolates. In a biochemical assay, boceprevir had approximately 3- and 2-fold reduced activity against NS3/4A proteases derived from single isolates representative of HCV genotypes 2 and 3a, respectively, relative to a genotype 1b-derived NS3/4A protease. The presence of 50% human serum reduced the cell culture anti-HCV activity of boceprevir by approximately 3-fold.
Evaluation of varying combinations of boceprevir and interferon alfa-2b that produced 90% suppression of replicon RNA in cell culture showed additivity of effect without evidence of antagonism.

### Resistance
In HCV Replicon Cell Culture and Biochemical Studies
The activity of boceprevir against the HCV genotype 1a replicon was reduced (2- to 6-fold) by the following amino acid substitutions in the NS3 protease domain: V36A/L/M, Q41R, T54A/S, V55A, R155K and V158I. A greater than 10-fold reduction in boceprevir susceptibility was conferred by the amino acid substitutions R155T and A156S. The V55I and D168N single substitutions did not reduce sensitivity to boceprevir. The following double amino acid substitutions conferred more than 10-fold reduced sensitivity to boceprevir: V55A+I170V, T54S+R155K, R155K+D168N, R155T+D168N and V36M+R155K.
The activity of boceprevir against the HCV genotype 1b replicon was reduced (2- to 8-fold) by the following amino acid substitutions in the NS3 protease domain: V36A/M, Q41R, F43S, T54A/G/S, V55A/I, R155K, V158I, V170M and M175L. A greater than 10-fold reduction in boceprevir susceptibility was conferred by the amino acid substitutions A156S/T/V, V170A and V36M+R155K. The D168V single substitution did not reduce sensitivity to boceprevir.
Additional NS3 protease domain substitutions that have not been evaluated in the HCV replicon but have been shown to reduce boceprevir activity against the HCV NS3/4A protease in a biochemical assay include F43C and R155G/I/M/Q.
Resistance-associated amino acid substitutions for HCV genotype 1a and 1b observed in clinical trials are presented in Table 8.
In Clinical Studies
An as-treated, pooled genotypic resistance analysis was conducted for subjects who received four weeks of PegIntron/REBETOL followed by VICTRELIS 800 mg three times daily in combination with PegIntron/REBETOL in two Phase 3 studies, SPRINT-2 and RESPOND-2. Among subjects treated with VICTRELIS who did not achieve a sustained virologic response, and for whom samples were analyzed, 53% had one or more specific post-baseline, treatment-emergent NS3 protease domain amino acid substitutions detected by a population-based sequencing assay (Table 8). Similar patterns of treatment-emergent substitutions were observed in P06086, a Phase 3 clinical trial in previously untreated CHC subjects with genotype 1 infection comparing the use of ESA to ribavirin dose reduction for initial management of anemia during therapy with VICTRELIS in combination with PegIntron/REBETOL. Nearly all of these substitutions have been shown to reduce boceprevir anti-HCV activity in cell culture or biochemical assays. Among subjects treated with VICTRELIS in SPRINT-2 and RESPOND-2 who did not achieve SVR and for whom post-baseline samples were analyzed, 31% of PegIntron/REBETOL-responsive subjects, as defined by greater than or equal to 1-log10 decline in viral load at Treatment Week 4 (end of 4-week PegIntron/REBETOL lead-in period), had detectable treatment-emergent substitutions, compared to 68% of subjects with less than 1-log10 decline in viral load at Treatment Week 4. Clear patterns of boceprevir treatment-emergent substitutions in the NS3 helicase domain or NS4A coding regions of the HCV genome were not observed.

### Persistence of Resistance-Associated Substitutions
Data from an ongoing, long-term follow-up study of subjects who did not achieve SVR in Phase 2 trials with VICTRELIS, with a median duration of follow-up of approximately 2 years, indicate that HCV populations harboring certain post-baseline, treatment-emergent substitutions may decline in relative abundance over time. However, among those subjects with available data, one or more treatment-emergent substitutions remained detectable with a population-based sequencing assay in 25% of subjects after 2.5 years of follow-up. The most common NS3 substitutions detected after 2.5 years of follow-up were T54S and R155K. The lack of detection of a substitution based on a population-based assay does not necessarily indicate that viral populations carrying that substitution have declined to a background level that may have existed prior to treatment. The long-term clinical impact of the emergence or persistence of boceprevir-resistance-associated substitutions is unknown. No data are available regarding the efficacy of VICTRELIS among subjects who were previously exposed to VICTRELIS, or who previously failed treatment with a regimen containing VICTRELIS.

### Effect of Baseline HCV Polymorphisms on Treatment Response
A pooled analysis was conducted to explore the association between the detection of baseline NS3/4A amino acid polymorphisms and treatment outcome in the two Phase 3 studies, SPRINT-2 and RESPOND-2.
Baseline resistance associated polymorphisms were detected in 7% of subjects by a population-based sequencing method. Overall, the presence of these polymorphisms alone did not impact SVR rates in subjects treated with VICTRELIS. However, among subjects with a relatively poor response to PegIntron/REBETOL during the 4-week lead-in period, the efficacy of VICTRELIS appeared to be reduced for those who had V36M, T54A, T54S, V55A or R155K detected at baseline. Subjects with these baseline polymorphisms and reduced response to PegIntron/REBETOL represented approximately 1% of the total number of subjects treated with VICTRELIS.

### Cross-Resistance
Many of the treatment-emergent NS3 amino acid substitutions detected in subjects treated with VICTRELIS who did not achieve SVR in the Phase 3 clinical trials have been demonstrated to reduce the anti-HCV activity of other HCV NS3/4A protease inhibitors. The impact of prior exposure to VICTRELIS or treatment failure on the efficacy of other HCV NS3/4A protease inhibitors has not been studied. The efficacy of VICTRELIS has not been established for patients with a history of exposure to other NS3/4A protease inhibitors. Cross-resistance is not expected between VICTRELIS and interferons, or VICTRELIS and ribavirin.