Simeprevir microbiology

# Microbiology

## Antiviral Activity
The median simeprevir EC50 and EC90 values against a HCV genotype 1b replicon were 9.4 nM (7.05 ng/mL) and 19 nM (14.25 ng/mL), respectively. Activity of simeprevir against a selection of genotype 1a (N=78) and genotype 1b (N=59) chimeric replicons carrying NS3 sequences derived from HCV NS3/4A protease-inhibitor-naive subjects resulted in median fold change (FC) in EC50 values of 1.4 (interquartile range, IQR: 0.8 to 11) and 0.4 (IQR: 0.3 to 0.7) compared to reference genotype 1b replicon, respectively. Genotype 1a (N=33) and 1b (N=2) isolates with a baseline Q80K polymorphism resulted in median FC in simeprevir EC50 value of 11 (IQR: 7.4 to 13) and 8.4, respectively. The presence of 50% human serum reduced simeprevir replicon activity by 2.4-fold. Combination of simeprevir with interferon, ribavirin, NS5A inhibitors, NS5B nucleoside analog polymerase inhibitors or NS5B non-nucleoside analog polymerase inhibitors, including NS5B thumb 1-, thumb 2-, and palm-domain targeting drugs, was not antagonistic.

## Resistance in Cell Culture
Resistance to simeprevir was characterized in HCV genotype 1a and 1b replicon-containing cells. Ninety-six percent (96%) of simeprevir-selected genotype 1 replicons carried one or multiple amino acid substitutions at NS3 protease positions F43, Q80, R155, A156, and/or D168, with substitutions at NS3 position D168 being most frequently observed (78%). Additionally, resistance to simeprevir was evaluated in HCV genotype 1a and 1b replicon assays using site-directed mutants and chimeric replicons carrying NS3 sequences derived from clinical isolates. Amino acid substitutions at NS3 positions F43, Q80, S122, R155, A156, and D168 reduced susceptibility to simeprevir. Replicons with D168V or A, and R155K substitutions displayed large reductions in susceptibility to simeprevir (FC in EC50 value greater than 50), whereas other substitutions such as Q80K or R, S122R, and D168E displayed lower reductions in susceptibility (FC in EC50 value between 2 and 50). Other substitutions such as Q80G or L, S122G, N or T did not reduce susceptibility to simeprevir in the replicon assay (FC in EC50 value lower than 2). Amino acid substitutions at NS3 positions Q80, S122, R155, and/or D168 that were associated with lower reductions in susceptibility to simeprevir when occurring alone, reduced susceptibility to simeprevir by more than 50-fold when present in combination.

## Resistance in Clinical Studies
In a pooled analysis of subjects treated with 150 mg OLYSIO in combination with peginterferon alfa and ribavirin who did not achieve SVR in the controlled Phase 2b and Phase 3 clinical trials, emerging amino acid substitutions at NS3 positions Q80, S122, R155 and/or D168 were observed in 180 out of 197 (91%) subjects. Substitutions D168V and R155K alone or in combination with other substitutions at these positions emerged most frequently (Table 8). Most of these emerging substitutions have been shown to reduce susceptibility to simeprevir in cell culture replicon assays.
HCV genotype 1 subtype-specific patterns of simeprevir treatment-emergent amino acid substitutions were observed in subjects not achieving SVR. Subjects with HCV genotype 1a predominately had emerging R155K alone or in combination with amino acid substitutions at NS3 positions Q80, S122 and/or D168, while subjects with HCV genotype 1b had most often an emerging D168V substitution (Table 8). In subjects with HCV genotype 1a with a baseline Q80K amino acid substitution an emerging R155K substitution was observed most frequently at failure.

## Persistence of ResistanceAssociated Substitutions
The persistence of simeprevir-resistant NS3 amino acid substitutions was assessed following treatment failure in the pooled analysis of subjects receiving 150 mg OLYSIO in combination with peginterferon alfa and ribavirin in the Phase 2b and Phase 3 trials. The proportion of subjects with detectable levels of treatment-emergent, resistance-associated variants was followed post treatment for a median time of 28 weeks (range 0 to 70 weeks). Resistant variants remained at detectable levels in 32 out of 66 subjects (48%) with single emerging R155K and in 16 out of 48 subjects (33%) with single emerging D168V.
The lack of detection of virus containing a resistance-associated substitution does not necessarily indicate that the resistant virus is no longer present at clinically significant levels. The long-term clinical impact of the emergence or persistence of virus containing OLYSIO-resistance-associated substitutions is unknown.

## Effect of Baseline HCV Polymorphisms on Treatment Response
Analyses were conducted to explore the association between naturally-occurring baseline NS3/4A amino acid substitutions (polymorphisms) and treatment outcome. In the pooled analysis of the Phase 3 trials QUEST 1 and QUEST 2, and in the PROMISE trial, the efficacy of OLYSIO in combination with peginterferon alfa and ribavirin was substantially reduced in subjects infected with HCV genotype 1a virus with the NS3 Q80K polymorphism at baseline .
The observed prevalence of NS3 Q80K polymorphic variants at baseline in the overall population of the Phase 2b and Phase 3 trials was 14%; while the observed prevalence of the Q80K polymorphism was 30% in subjects infected with HCV genotype 1a and 0.5% in subjects infected with HCV genotype 1b. The observed prevalence of Q80K polymorphic variants at baseline in the U.S. population of the Phase 2b and Phase 3 trials was 35% overall, 48% in subjects infected with HCV genotype 1a and 0% in subjects infected with HCV genotype 1b. With the exception of the NS3 Q80K substitution, baseline polymorphic variants at NS3 positions F43, Q80, S122, R155, A156, and/or D168, which were associated with reduced simeprevir activity in replicon assays, were generally uncommon (1.3%) in subjects with HCV genotype 1 infection in the Phase 2b and Phase 3 trials (n=2007).

## Cross-Resistance
Cross-resistance is expected among NS3/4A protease inhibitors. Some of the treatment-emergent NS3 amino acid substitutions detected in OLYSIO-treated subjects who did not achieve SVR in clinical trials, including R155K, which emerged frequently, and I170T, which emerged infrequently, have been shown to reduce the anti-HCV activity of the NS3/4A protease inhibitors, boceprevir and/or telaprevir.
The most frequently occurring boceprevir or telaprevir treatment-emergent NS3 amino acid substitutions that are expected to impact subsequent treatment with OLYSIO include R155K and A156T or V. The NS3 amino substitutions V36A or G and I170A or T, which displayed slight shifts in susceptibility to simeprevir in replicon cultures, may emerge in patients who do not achieve SVR with boceprevir or telaprevir, and may therefore also impact subsequent treatment with OLYSIO.