icon-folder.gif   Conference Reports for NATAP  
  EASL 46th Annual Meeting
March 30th - April 3rd 2011
Berlin, Germany
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Treatment outcome and resistance analysis in HCV genotype 1 patients previously exposed to TMC435 monotherapy and re-treated with TMC435 in combination with PegIFNa-2a/ribavirin
  Reported by Jules Levin EASL 2011 March 30-Apr 2 Berlin Germany
O Lenz,1 J de Bruijne,2 L Vijgen,1 T Verbinnen,1 C Weegink,2 H van Marck,1 I Vandenbroucke,1 M Peeters,1 G De Smedt,1 K Simmen,1 G Fanning,1 G Picchio,3 H Reesink2 1Tibotec, Beerse, Belgium; 2Academic Medical Center, Amsterdam, The Netherlands; 3Tibotec Inc., Yardley, PA, USA


· TMC435 is a once daily (QD) oral NS3/4A protease inhibitor currently in Phase III clinical development for the treatment of hepatitis C virus (HCV) infection.
· TMC435 is an investigational potent and selective inhibitor of the NS3/4A protease, with an in vitro 50% effective concentration (EC50) of 8 nM (6 ng/mL) in a genotype 1b replicon cell line.1
· Findings from Phase I and II studies have shown that TMC435 is well tolerated, with promising efficacy reported in combination with pegylated interferon a-2a/ribavirin (PegIFNa-2a/RBV).2-6
· In vitro resistance studies identified changes at amino acid positions 43, 80, 155, 156 and 168 within the NS3 protease domain that confer variable degrees of reduced susceptibility to TMC435.7
· Median/mean trough TMC435 concentrations observed following doses of 75 mg and 200 mg QD were approximately 25-50 and 350-950 fold higher, respectively, than the EC50 values obtained in the genotype 1b-replicon cell line (6 ng/mL).2,8,9
· In a Phase I study (TMC435-C101; NCT00938899), six HCV genotype 1-infected patients who failed previous IFN-based therapy received TMC435 200 mg QD for 5 days.2 Approximately 1.5 years later 5/6 patients (1 patient died 1 year after study TMC435-C101; unrelated to HCV therapy) were enrolled into Cohort 5 of the Phase IIa Optimal Protease inhibitor Enhancement of Response to TherApy (OPERA)-1 study (TMC435-C201; NCT00561353) and were re-treated with TMC435 (200 mg QD) in combination with PegIFNa-2a/RBV for 28 days followed by PegIFNa-2a/RBV up to Week 48.3
· Here we report the final clinical outcomes for the 5 patients included in the TMC435-C101 study who were re-treated in TMC435-C201, and present a detailed genotypic and phenotypic analysis of HCV genotype 1 isolates obtained from these patients.
· Plasma samples were collected during the TMC435-C101 and TMC435-C201 studies and HCV ribonucleic acid (RNA) levels were assessed with a Roche COBAS® TaqMan® HCV/HPS v2.0 assay. This assay has a lower limit of quantification (LLOQ) of <25 IU/mL. HCV RNA results below the LLOQ are reported as either <25 IU/mL detectable or <25 IU/mL undetectable.
· HCV RNA was extracted from plasma samples and the NS3/4A protease region was sequenced using standard population sequencing, if HCV RNA was >100 IU/mL. In addition, selected samples were analysed using clonal single genome sequencing (20-80 clones per sample) or were subjected to massive parallel pyrosequencing (ultra-deep sequencing) using the 454 life science platform (GS-FLX, Roche Applied Science).
· For phenotypic analysis, selected mutations or patient-derived NS3 protease regions were introduced into a genotype 1b replicon backbone. The antiviral activity of TMC435 was tested and compared with the parental replicon in a standard transient replicon assay.
Antiviral Activity and Efficacy

· At Day 6 in study TMC435-C101, a consistent and rapid decline in plasma HCV RNA was observed in all six patients, with a maximal median reduction from baseline of 3.9 log10 IU/mL.2
· Of the five patients who were re-treated in study TMC435-C201,3 one (patient 141, previous non-responder) stopped all treatment at Day 14 due to an adverse event (AE) (increase in bilirubin level). Three of the remaining four patients (one non-responder and two patients with viral relapse) who completed the 28-day TMC435 in combination with PegIFNa-2a/RBV treatment had undetectable HCV RNA levels at Day 28 and ultimately achieved a sustained virologic response (SVR). One patient (patient 142) achieved HCV RNA <25 IU/mL detectable at Day 28, followed by a viral breakthrough during PegIFNa-2a/RBV therapy at Week 28 (Figure 1a).
· Overall changes in HCV RNA during the first week of therapy were similar between study TMC435-C101 and TMC435-C201. In two patients, the HCV RNA reduction from baseline was slower in TMC435-C201 versus study TMC435-C101: in patient 141 at Day 3 (-2.9 versus -3.7 log10 IU/mL) and patient 142 at Day 7 (-2.8 versus -3.5 log10 IU/mL) (Figure 1b). This difference could be attributable to persistent virus and/or suboptimal response to PegIFNa-2a/RBV in study C201.



Genotypic/Phenotypic Changes in the HCV NS3 Protease
Region During Study TMC435-C101 and TMC435-C201

· Mutations at one or more of the amino acid positions 80, 155, 156 and 168, which were absent at baseline, were detected in all subjects as early as on Day 3 of study TMC435-C101 using population sequencing (Figure 2a).
· Population sequencing and phenotypic analysis of the HCV isolates obtained at baseline of TMC435-C201 suggested that the dominant viral variants returned to the characteristics observed prior to study TMC435-C101 in all 5 patients.
· Mutations S122R plus R155K (patient 142), and Q80R plus D168E plus F169L (patient 141), were detected in TMC435-C201 during treatment.
· The in vitro activity of TMC435 against the treatment emerging NS3 variants identified revealed fold changes in EC50 values (FC) >100 in all patients in study TMC435-C101 and in patient 141 and 142 in TMC435-C201 (Figure 2b).
FIGURE 2: a) Mutations detected in the NS3 protease domain defined as changes from reference sequence (Con1 for genotype 1b and H77 for genotype 1a). b) Fold changes in EC50 values compared with wild-type assessed in a transient replicon assay using chimeric replicons harbouring the NS3 protease region derived from patient samples.


*Patient discontinued due to hyperbilirubinemia. Mutations T40A, S91T/A and L153I were found in all HCV genotype 1a patients at all time points analysed; the mutation R26K was found in all HCV genotype 1b patients at all time points analysed; A156A/V was present in patient 143 at Day 4.
NR, non-responder to previous IFN-based therapy; R, relapser to previous IFN-based therapy. Day 1 = Baseline; C101 follow-up 1 = 2 weeks after end of dosing; C101 follow-up 2 = 4 weeks after end of dosing. Missing values were below the lower limit of the assay


*Fold change determined using site directed Mutants S122R and R155K. Patient discontinued at Week 32 C201=OPERA-1 D, day; EC50, 50% effective concentration; FC, fold change; FUP, follow-up; HCV, hepatitis C virus; NR, non-responder; OPERA, Optimal Protease inhibitor Enhancement of Response to TherApy; R, relapser; WT, wild type
Presence of Viral Variants at Baseline in Studies TMC435-C101 and TMC435-C201 based on Deep Sequencing
· No additional NS3 mutations at key binding pocket residues (80, 155, 156 and 168) were detected at baseline of the TMC435-C101 study using ultra-deep sequencing (D168D/E was also detected by population sequencing).
· At baseline of the TMC435-C201 study, mutations at key binding positions were observed at low frequency (<2%) in 3 patients: Q80L, R155G and R155K, in patients 140, 144 and 142, respectively (Figure 3).
- Patient 142 showed a slower initial viral decline in TMC435-C201 compared with TMC435-C101, while patients 140 and 144 showed a comparable viral decline in both studies.


Single Genome (Clonal) Sequencing of NS3 Protease Domain
· In addition to single mutations, variants encoding 2 or 3 amino acid changes on the same genome were observed (Figure 4).
· In general, when introduced into the replicon, most of the single mutations detected resulted in a lower fold change in EC50 value compared with replicons with 2 or 3 mutations (data not shown).
Evolution of Amino Acid Changes over Time
Based on Deep Sequencing

· In patient 141, ultra-deep sequencing detected a Q80R and a D168E mutation four weeks after the end of dosing for study TMC435-C101, which were no longer detectable at baseline of the TMC435-C201 study. These two mutations emerged as major variants upon re-exposure to TMC435 in the TMC435-C201 study (Figure 5a). This patient also showed a slower initial viral decline in TMC435-C201 compared with in TMC435-C101.
· In patient 142, ultra-deep sequencing detected variant R155K persisting at low frequency (<2%) at baseline of the TMC435-C201 study. This mutation became detectable by population sequencing at Day 7 and 14 of the study while HCV RNA was still declining. An S122R and an R155K mutation became detectable by population and 454 sequencing at the time of viral breakthrough (Week 28) (Figure 5b).
FIGURE 4: NS3 protease domain was amplified, cloned into standard cloning vectors and sequenced to determine linkage of mutations. Mutations at position 80, 155, 156, 168 and 169 were considered for this analysis. Frequency of clones is represented.


FIGURE 5: The sequence of the HCV NS3 protease domain was analysed from multiple samples using ultra-deep sequencing. Changes of codon frequency over time for NS3 at positions 80, 122, 155 and 168 are shown for two patients a) 141 b) 142.


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