| |
Deep sequencing analysis of baseline and on-treatment samples from HCV genotype-1 patients treated for 5 days with TMC435 monotherapy and subsequently re-treated with TMC435 in combination with PegIFNα-2a/ribavirin - poster presentation
|
| |
| |
Reported by Jules Levin
O Lenz,1 L Vijgen,1 T Verbinnen,1 H van Marck,1 I Vandenbroucke,1 M Peeters,1 G De Smedt,1 K Simmen,1 G Fanning,1 H Reesink,2 G Picchio3
1Tibotec BVBA, Mechelen, Belgium; 2Academic Medical Center, Amsterdam, The Netherlands; 3Tibotec Inc., Yardley, PA, USA
Author Conclusions
Treatment with TMC435 200 mg QD resulted in potent antiviral activity in study TMC435-C101, during five days of monotherapy, and subsequently in the same patients when combined with PegIFN/RBV in the OPERA-1 study.
Emerging viral variants were associated with a high fold change in EC50 values to TMC435 in in vitro replicon assays.
Viral variants that emerged during first exposure to TMC435 in study TMC435-C101 were no longer detectable in most cases over time, while in some they persisted at low frequencies based on ultra-dee sequencing analysis.
Persistence of viral variants in two patients at low frequency was associated with slightly reduced antiviral activity of TMC435 during the first seven days of the OPERA-1 study.
The newly identified F169L mutation reduced activity of TMC435 in vitro when combined with the Q80R and/or D168E mutation in a genotype-1b replicon.
Introduction
· TMC435 is a potent and selective macrocyclic inhibitor of the hepatitis C virus (HCV) 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 TMC435 is currently in Phase IIb development.
· 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 TMC435.2
· In a Phase I study (TMC435-C101), six HCV genotype-1-infected patients who failed previous interferon (IFN)-based therapy received TMC435 200 mg once
daily (QD) for 5 days.3 Approximately 1.5 years later, 5/6 patients 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 withTMC435 (200 mg QD) in combination with pegylated
(Peg)IFNα-2a/ribavirin (RBV) for 28 days followed by PegIFNα-2a/RBV up to Week 48.4
· Median/mean trough TMC435 concentrations observed following doses of 75 mg and 200 mg QD ranged from approximately 25-50 and 350-950 fold, respectively, of the EC50 values obtained in the genotype-1b replicon cell line (6 ng/mL).3,5,6
· Here we present a detailed genotypic and phenotypic analysis of HCV isolates obtained from patients participating in the TMC435-C101 study who were re-treated in OPERA-1.
METHODS
· Plasma samples were collected during the TMC435-C101 and OPERA-1 studies.
· HCV ribonucleic acid (RNA) was extracted from plasma samples and the NS3/4A region was sequenced using standard population sequencing. In addition, selected samples were analysed using clonal single genome sequencing (20-80 clones per sample) or were subjected to massive
parallel pyrosequencing (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 and the antiviral activity of TMC435 was tested and compared with the parental replicon in a standard transient replicon assay.
Figure 1. Schematic representation of genotypic and phenotypic assessments.
RESULTS
Antiviral activity and genotypic/phenotypic changes in the HCV NS3 region
· A consistent and rapid drop in plasma HCV RNA was observed in all six patients, with a maximal median reduction from baseline of 3.9 log10 IU/mL at Day 6 in study TMC435-C101. All four subjects who completed the 28-day treatment in the OPERA-1 study reached HCV RNA levels of <25 IU/mL.
· Although overall changes in HCV RNA were similar between studies, in two patients the RNA decrease from baseline was slower in OPERA-1 compared with study TMC435-C101: in patient 142 at Day 3 (-2.9 versus -3.7 log10 IU/mL) and patient 141 at Day 7 (-2.8 versus -3.5 log10 IU/mL).
· At baseline of study TMC435-C101, four patients had no detectable variations at positions identified in vitro to confer reduced susceptibility to TMC435, while in two patients (177 and 140), a Q80K and D168D/E variation was observed, respectively. Initial HCV RNA reductions in these two patients were comparable to those observed in patients without baseline variations. Change from baseline to Day 3 was 3.1 and 3.6 log10 IU/mL for these two patients in study TMC435-C101, respectively.
· Mutations at one or more of the amino acid positions 80, 155, 156 and 168 were newly detected in all subjects as early as Day 3 of study TMC435-C101.
· Population sequencing and phenotypic analysis of the HCV isolates obtained from 5/6 patients at baseline of OPERA-1 suggested that the viral variants returned to the characteristics observed prior to study TMC435-C101 in all five patients.
· Emerging mutations R155K, and Q80R/K, D168E plus F169L, were detected in two subjects in OPERA-1, respectively.
· 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 in OPERA-1.
Figure 2. A) Individual changes in plasma HCV RNA from baseline in genotype-1-infected IFN-experienced patients during five days of dosing with 200 mg QD of TMC435 alone in study TMC435-C101 and in combination with PegIFNα-2a/RBV in OPERA-1. B) Mutations detected in the NS3 protease domain defined as changes from reference sequence (Con1 for genotype-1b and H77 for genotype-1a). C) Fold changes in EC50 values compared with wild-type assessed in a transient replicon assay using chimeric replicons harbouring the NS3 protease domain derived from patient samples. Day 1 = Baseline; FUP1 = follow-up 1 (2 weeks after end of dosing); FUP2 = follow-up 2 (4 weeks after end of dosing).
*Patient discontinued TMC435 due to adverse event at Day 14 (dose reduction to 100 mg at Day 10)
C201=OPERA-1
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.
C201=OPERA-1
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; PegIFN, pegylated interferon; QD, once daily; RBV, ribavirin; R, relapser; RNA, ribonucleic acid; wt, wild type
Presence of viral variants at baseline in studies TMC435-C101 and OPERA-1 based on ultra-deep sequencing
· No additional pre-existing NS3 mutations were detected at baseline of TMC435-C101 using ultra-deep sequencing (Q80K and D168D/E were also detected by population sequencing).
· At baseline of OPERA-1, additional mutations were observed at low frequency (<2%) in three patients: Q80L, R155G and R155K, respectively.
· Alternative codon usage was noted for wild-type and mutated amino acid, respectively.
Figure 3. The sequence of the HCV NS3 protease domain was determined for baseline samples of study TMC435-C101 and OPERA-1 using ultra-deep sequencing. Percentage of codon frequency at NS3 positions 80, 155, 156 and 168 are shown. Grey dots represent codons encoding wild-type amino acids. Coloured dots represent codons encoding mutations (defined as changes from reference sequence: Con1 for genotype-1b and H77 for genotype-1a).
C201=OPERA-1
HCV, hepatitis C virus; OPERA, Optimal Protease inhibitor Enhancement of Response to TherApy
Single genome (clonal) sequencing of NS3 protease domain and effect of isolated mutations on in vitro activity of TMC435
· In addition to single mutations, variants harbouring double and triple mutations were observed.
· In general, when introduced into the replicon, most of the single mutations detected resulted in lower fold change in EC50 values than double or triple mutations.
· Mutation F169L, which has no effect on TMC435 activity alone, further decreased susceptibility to TMC435 when combined with Q80R and/or D168E.
Figure 4. A) NS3 protease domain was amplified, cloned into standard cloning vectors and sequenced to determine linkage of mutations. Mutations at position 80, 155, 156 and 168 (169) were considered for this analysis. Frequency of clones is represented. B) Single and multiple mutations were introduced in a genotype-1b replicon backbone and EC50 values were determined using a standard transient replicon assay. Fold change in EC50 values versus the parental wild-type replicon are indicated.
Evolution of amino acid changes over time
· In patient 141, ultra-deep sequencing detected a Q80R and D168E mutation four weeks after end of dosing for study TMC435-C101, which were no longer detectable at baseline of the OPERA-1 study
- These two mutations emerged as major variants upon re-exposure to TMC435 in the OPERA-1 study.
· Different mutational patterns were observed in response to exposure to TMC435.
· Variants with different mutations at a given position were detected.
Figure 5. The sequence of the HCV NS3 domain was analysed from multiple samples using ultra-deep sequencing. Changes of codon frequency over time for NS3 at positions 80, 155, 156 and 168 are shown for patient 142 (A) and 141 (B).
C201=OPERA-1
FUP, follow-up; HCV, hepatitis C virus; AA, amino acid
References
1. Lin TI et al. Antimicrob Agents Chemother 2009;53:1377-1385.
2. Lenz O et al. Antimicrob Agents Chemother 2010;54:1878-1887.
3. Reesink HW et al. Gastroenterology 2010;138:913-921.
4. Reesink HW et al. Presented at the 60th American Association for the Study of Liver
Diseases (AASLD) meeting, Boston, MA, USA, 30 October-3 November, 2009.
5. Sekar V et al. Presented at the 45th Annual Meeting of the European Association for
the Study of the Liver (EASL), Vienna, Austria, 14-18 April, 2010.
6. Data on file. Tibotec Inc. 2010.
|
|
| |
| |
|
|
|
