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Efficacy of Re-treatment With TMC435 as Combination Therapy in Hepatitis C Virus–Infected Patients Following TMC435 Monotherapy

2012; Elsevier BV; Volume: 143; Issue: 5 Linguagem: Inglês

10.1053/j.gastro.2012.07.117

1528-0012

Oliver Lenz, Joep de Bruijne, Leen Vijgen, Thierry Verbinnen, Christine J. Weegink, Herwig Van Marck, Ina Vandenbroucke, Monika Peeters, Kenneth Simmen, G. Fanning, René Verloes, Gastón Picchio, Hendrik W. Reesink,

Liver Disease Diagnosis and Treatment

In the TMC435-C101 study, 6 patients infected with hepatitis C virus genotype 1 were treated with the protease inhibitor TMC435 (200 mg once daily) as monotherapy for 5 days. Approximately 1.5 years later, 5 of these patients were re-treated with TMC435 (200 mg once daily) plus pegylated interferon alfa-2a and ribavirin (PegIFNα-2a and RBV) for 4 weeks, followed by PegIFNα-2a and RBV until week 48 (in the Optimal Protease inhibitor Enhancement of Response to therApy [OPERA-1] study). TMC435-resistant variants, which emerged in all 5 patients during the TMC435-C101 study, were no longer detected at the beginning of the OPERA-1 study based on virus population sequencing. During the OPERA-1 study, 3 patients had a sustained virologic response; deep sequencing indicated low-level persistence of resistant variants in the remaining 2 patients, which might have affected their response to re-treatment. Clinical trials.gov Number, NCT00561353. In the TMC435-C101 study, 6 patients infected with hepatitis C virus genotype 1 were treated with the protease inhibitor TMC435 (200 mg once daily) as monotherapy for 5 days. Approximately 1.5 years later, 5 of these patients were re-treated with TMC435 (200 mg once daily) plus pegylated interferon alfa-2a and ribavirin (PegIFNα-2a and RBV) for 4 weeks, followed by PegIFNα-2a and RBV until week 48 (in the Optimal Protease inhibitor Enhancement of Response to therApy [OPERA-1] study). TMC435-resistant variants, which emerged in all 5 patients during the TMC435-C101 study, were no longer detected at the beginning of the OPERA-1 study based on virus population sequencing. During the OPERA-1 study, 3 patients had a sustained virologic response; deep sequencing indicated low-level persistence of resistant variants in the remaining 2 patients, which might have affected their response to re-treatment. Clinical trials.gov Number, NCT00561353. New treatments for patients infected with hepatitis C virus (HCV) genotype 1 are now available, including HCV NS3/4A protease inhibitors (PIs).1Jacobson I.M. et al.N Engl J Med. 2011; 364: 2405-2416Crossref PubMed Scopus (2196) Google Scholar, 2Poordad F. et al.N Engl J Med. 2011; 364: 1195-1206Crossref PubMed Scopus (2285) Google Scholar Drug-resistant viral variants can emerge in patients treated with direct-acting antivirals who do not achieve a sustained virologic response.3Pawlotsky J.M. Hepatology. 2011; 53: 1742-1751Crossref PubMed Scopus (296) Google Scholar, 4Sarrazin C. et al.Gastroenterology. 2010; 138: 447-462Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar Although it has been described that variants become undetectable after treatment failure in most patients, it is unclear whether low-level variants persist and affect re-treatment options.5Zeuzem S. et al.Hepatology. 2010; 52 (Abstract 227): 436ACrossref PubMed Scopus (96) Google Scholar, 6Barnard R.J. et al.Hepatology. 2011; 54 (Abstract 164)Google Scholar TMC435 is an investigational HCV PI administered orally once daily.7Fried M.W. et al.American Association for the Study of Liver Diseases. 2011; Google Scholar, 8Lenz O. et al.Antimicrob Agents Chemother. 2010; 54: 1878-1887Crossref PubMed Scopus (199) Google Scholar, 9Manns M. et al.Antivir Ther. 2011; 16: 1021-1033Crossref PubMed Scopus (57) Google Scholar In study TMC435-C101, 6 patients infected with HCV genotype 1 received TMC435 (200 mg once daily) as monotherapy for 5 days.10Reesink H.W. et al.Gastroenterology. 2010; 138: 913-921Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar Approximately 1.5 years later, 5 of the 6 patients participated in the Optimal Protease inhibitor Enhancement of Response to TherApy (OPERA-1; TMC435-C201) study and were treated with TMC435 (200 mg once daily) plus peginterferon alfa-2a/ribavirin (PegIFNα-2a/RBV) for 4 weeks, followed by PegIFNα-2a/RBV up to week 48 (Supplementary Materials and Methods and Supplementary Figure 1). Patients were white men previously treated with interferon-based therapy who did not respond to treatment or experienced a relapse (Table 1).Table 1Baseline Patient Characteristics and Virologic Response in the OPERA-1 Study (Cohort 5)Patient 140Patient 141aSubhemophilia.,bPatient 141 stopped treatment at day 14 due to elevated bilirubin levels.Patient 142cPatient 142 experienced viral breakthrough at week 28 during PegIFN/RBV treatment.Patient 143Patient 144Patient/disease characteristics Body mass index (kg/m2)24.626.323.820.527.5 METAVIR scoreF2F4F1F1F1 IL28B genotypeCCTTTTCTCT HCV genotype subtype1a1b1a1b1a Previous IFN-based therapydDifferent doses of PegIFN and RBV were used in previous therapy.IFN α-2a/RBVPegIFN/RBVPegIFN/RBVPegIFN/RBVPegIFN/RBV/amantadine Response to previous IFN-based therapyeCategorized by investigator.RelapserNonresponderNonresponderRelapserNonresponderHCV RNA plasma concentration Baseline6.64 log10IU/mL7.10 log10IU/mL6.85 log10IU/mL7.34 log10IU/mL6.87 log10IU/mL Day 14<25 IU/mL (detectable)4.59 log10IU/mL244 IU/mL<25 IU/mL (detectable)<25 IU/mL (detectable) Day 28<25 IU/mL (undetectable)NA<25 IU/mL (detectable)<25 IU/mL (undetectable)<25 IU/mL (undetectable) Week 12<25 IU/mL (undetectable)NA<25 IU/mL (detectable)<25 IU/mL (undetectable)<25 IU/mL (undetectable) Week 72 (sustained virologic response)<25 IU/mL (undetectable)NANA<25 IU/mL (undetectable)<25 IU/mL (undetectable)IL28B, polymorphism rs12979860; IFN, interferon; NA, not applicable.a Subhemophilia.b Patient 141 stopped treatment at day 14 due to elevated bilirubin levels.c Patient 142 experienced viral breakthrough at week 28 during PegIFN/RBV treatment.d Different doses of PegIFN and RBV were used in previous therapy.e Categorized by investigator. Open table in a new tab IL28B, polymorphism rs12979860; IFN, interferon; NA, not applicable. In TMC435-C101, all patients had a rapid and pronounced decline in HCV RNA during TMC435 monotherapy.10Reesink H.W. et al.Gastroenterology. 2010; 138: 913-921Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar Although no viral breakthrough was observed, mutations at NS3 amino acid positions 80, 155, 156, and/or 168 emerged in all patients. Deep sequencing at baseline of TMC435-C101 showed that there had been no additional preexisting NS3 mutations at positions known to affect TMC435 activity in vitro (80, 155, 156, and 168).11Verbinnen T. et al.J Virol. 2010; 84: 11124-11133Crossref PubMed Scopus (63) Google Scholar At baseline of OPERA-1, these variants that emerged during C101 were no longer detectable using population sequencing (Supplementary Table 1). With deep sequencing, Q80L and R155G were observed at baseline of OPERA-1 at low frequency (1%–2%) in 2 of 3 patients (patients 140 and 144, respectively) who achieved undetectable HCV RNA after 4 weeks of triple therapy and ultimately achieved a sustained virologic response (Table 1 and Supplementary Figures 2A and 3). Q80L had been previously detected in patient 140 as an emerging minority variant in TMC435-C101 (frequency <5%), while in patient 144, R155G had not been previously observed. In the remaining 2 patients who did not achieve a sustained virologic response and who were previous nonresponders to PegIFN/RBV and of IL28B TT genotype (polymorphism rs12979860), decreases in HCV RNA levels from baseline were slower in OPERA-1, in which TMC435 was given in combination with PegIFN/RBV, compared with TMC435-C101, in which TMC435 was given alone. In patient 141, decreases at day 7 were −2.8 IU/mL (OPERA-1) vs −3.5 log10 IU/mL (TMC435-C101) (Supplementary Figure 1B). In patient 142, decreases at day 3 were −2.9 IU/mL (OPERA-1) vs −3.7 log10 IU/mL (TMC435-C101) (Supplementary Figure 2B). Patient 142 achieved an HCV RNA level <25 IU/mL at the end of triple therapy (OPERA-1) and experienced viral breakthrough at week 28 during PegIFN/RBV treatment. Population sequencing identified emerging D168A/V and R155K mutations during TMC435-C101 and R155K at day 7, day 14, and time of viral breakthrough in OPERA-1. Based on deep sequencing (Figure 1), R155K became detectable (frequency of 9%) at day 3 of TMC435-C101 before becoming the major variant and was detected 4 weeks after the end of TMC435 dosing (frequency of 64%). R155K was still detectable at baseline of OPERA-1 (frequency of 1%) and became the major variant from day 7 to time of viral breakthrough, 24 weeks after the end of TMC435 dosing. The ability of R155K to persist is consistent with reports showing the relatively high fitness of variants harboring this mutation. In addition to R155K, multiple mutations emerged at position 80 and 168 (Q80L, K, R and D168V, A, E, H, N, G) as well as in TMC435-C101 and/or OPERA-1 as minor variants. Patient 141 had a TMC435 dose reduction at day 10 from 200 to 100 mg once daily and stopped all treatment at day 14 due to elevated bilirubin levels. HCV RNA levels did not decline further from day 7 until day 14. Q80R + D168E and Q80K + D168E mutations were detected at day 7 and 4 weeks after the end of the 14-day treatment period in OPERA-1, respectively. In TMC435-C101, population-based sequencing showed the emergence of D168V (Supplementary Table 1). Based on deep sequencing (Figure 1), D168V became detectable (frequency of 2%) at day 4 of TMC435-C101 before becoming the major variant, and D168E emerged (frequency of 6%) at the last follow-up visit, 4 weeks after the end of dosing with TMC435. At baseline of OPERA-1, only the wild-type residue aspartate was observed at position 168, while during OPERA-1 D168E became the major variant (frequency of 83% at day 7) and D168V was only transiently detectable (frequency of 12% at day 7). In parallel to the emergence of D168E, Q80R and subsequently Q80K became detectable. This suggests that previous exposure to TMC435 facilitated the emergence of a Q80R/K + D168E double mutant variant, which confers a similar level of resistance to TMC435 as the single D168V mutation but displays higher fitness. In summary, although only 5 patients were analyzed and their first exposure to TMC435 was short, all patients had TMC435-resistant variants at the end of their first treatment that were no longer detectable (by population sequencing) at baseline of a second study 1.5 years later. A similar pattern may occur in patients who fail to respond to a PI-based regimen and are later evaluated for re-treatment. The data presented here suggest that in some patients the frequency of emerging resistant viral variants may decrease over time, in the absence of selective pressure imposed by a direct-acting antiviral, to levels that do not negatively affect outcome on subsequent re-treatment with a regimen containing the same direct-acting antiviral. However, in other patients, resistant variants might persist at low levels, potentially below the detection limit of deep sequencing assays, which may affect the efficacy of a second course of treatment. The impact of preexisting resistant variants on treatment outcome is intimately linked to the intrinsic PegIFN/RBV responsiveness of the patient, as shown in this and other studies, because their sole presence does not automatically translate into treatment failure.12Lenz O. et al.American Association for the Study of Liver Diseases. 2011; Google Scholar, 13Marcellin P. et al.Gastroenterology. 2011; 140: 459-468Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar The significance of preexisting variants on sustained virologic response may be different when considering interferon-free regimens. O.L. and J.D. contributed equally to this work. Editorial support was provided by Phil Matthews, PhD, of Complete Medical Communications, funded by Janssen Research and Development. The authors thank the patients and the study staff, as well as all the colleagues from Janssen Infectious Diseases who contributed to this work. The authors thank C. J. Schinkel and R. Molenkanp (Department of Clinical Virology, Academic Medical Center, Amsterdam, The Netherlands) for performing IL28B testing. Results for cohorts 1–4 of OPERA-1 (TMC435-C201, NCT00561353) have been reported previously.1Manns M. et al.Antivir Ther. 2011; 16: 1021-1033Crossref PubMed Scopus (57) Google Scholar In cohort 5 of OPERA-1, subjects who were nonresponders to and experienced a relapse after previous interferon-based therapy who had subsequently received 5 days of treatment with TMC435 (200 mg once daily) in study C1012Reesink H.W. et al.Gastroenterology. 2010; 138: 913-921Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar were enrolled and received TMC435 (200 mg once daily) in combination with PegIFN/RBV for 4 weeks, followed by up to 44 weeks of PegIFN/RBV only. The cohort 5 study was conducted in The Netherlands (Department of Gastroenterology and Hepatology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands). PegIFNα-2a was given subcutaneously at a dosage of 180 μg once weekly, and RBV was orally administered at a dosage of 1000–1200 mg twice daily (body weight dependent). The objective of cohort 5 was to assess the antiviral activity, safety, and tolerability of a TMC435-containing treatment regimen in patients who had previously received TMC435 monotherapy. Plasma samples were obtained throughout the study and HCV RNA levels were assessed using TaqMan HCV/HPS assay v2.0 (Roche Molecular Diagnostics, Basel, Switzerland), with a lower limit of quantification of 25 IU/mL and a lower limit of detection of approximately 10–15 IU/mL. Samples in which no target HCV RNA was detected below the lower limit of quantification were reported as 1 log10 IU/mL from nadir (lowest HCV RNA level obtained before breakthrough) or >100 IU/mL in patients whose HCV RNA had previously been <25 IU/mL undetectable. HCV RNA was extracted from plasma samples, and the HCV NS3/4A region was sequenced at the population level using reverse-transcription polymerase chain reaction (PCR) and standard Sanger sequencing. HCV genotypes/subtypes were determined by sequencing and basic local alignment search tool (BLAST) analysis of a 329–base pair region within the HCV NS5B gene. In addition, selected samples were analyzed using clonal single genome sequencing or were subjected to massively parallel pyrosequencing (deep sequencing) using the 454 GS-FLX platform (454 Life Sciences, Roche Applied Science) essentially as previously described.3Verbinnen T. et al.J Virol. 2010; 84: 11124-11133Crossref PubMed Scopus (64) Google Scholar For clonal sequencing, NS3/4A amplicons were prepared and bacterially cloned, and 20–80 clones were sequenced. For 454 sequencing, complementary DNA was generated using random primers, followed by PCR generating 2 amplicons covering NS3 amino acid positions 1–99 and 85–192, respectively. Sequence adaptors and additional multiplex identifiers (ie, short barcode sequences) were attached to the primers and PCR using Phusion Hot Start High-Fidelity DNA polymerase (Finnzymes, Vantaa, Finnland) was performed. To maximize the amount of input templates and to minimize variation due to PCR drift, 7 replicate reverse-transcription PCRs were performed for each sample and subsequently pooled for sequence analysis using the 454 GS-FLX amplicon approach followed by analyses with Amplicon Variant Analysis software (Roche). The median (Q1–Q3) number of sequence reads per position (coverage) for NS3 positions of interest (36, 43, 54, 80, 138, 155, 156, 168, 169, and 170) for the subjects and visits included in this report was 5849 (3611–9176). The sensitivity limit was set as a frequency of 0.5% (data not shown). Consent for IL28B testing4Ge D. et al.Nature. 2009; 461: 399-401Crossref PubMed Scopus (3148) Google Scholar was obtained retrospectively. IL28B polymorphism (rs12979860 and rs17809917) was determined from patient serum by high-resolution melting curve analysis using the LightCycler 480 (Roche Applied Science, Penzberg, Germany) and High Resolution Melting Master (Roche Applied Science). Adverse events were monitored during all study visits. Vital signs, electrocardiogram recordings, and laboratory parameters were assessed at screening and on days 1, 7, 14, 21, and 28. Final analysis was performed when all patients had completed week 72 of treatment or discontinued earlier. Descriptive analyses were performed. During TMC435 dosing, there were no serious adverse events. Influenza-like illness was the most frequently reported adverse event, reported in 4 of 5 patients. All but one of the observed adverse events was mild to moderate in severity and not, or doubtfully, related to TMC435. One patient discontinued treatment after 14 days (following a reduction of the dose of TMC435 at day 10 to 100 mg once daily) due to an increase in blood bilirubin level (grade 4). This patient had an elevated bilirubin level (grade 2) at baseline, although the value at screening was within normal limits. Bilirubin level normalized after discontinuation of treatment. There were no other grade 3 or 4 adverse events during treatment with TMC435. No clinically relevant changes were observed in any other laboratory parameters, electrocardiogram parameters, or vital signs. During the PegIFNα-2a/RBV treatment from day 28 onward, aspartate aminotransferase and alanine aminotransferase levels decreased in all 5 patients during therapy.Supplementary Table 1Amino Acid Substitution in NS3 Protease Domain (Defined as Changes From Reference Sequence: H77 for Genotype 1a and con1 for Genotype 1b) Detected in Studies C101 and OPERA-1 Using Population-Based SequencingGenotype 1aI018T054V055P067Q080S122S133R155D168I170Patient 140 (R)C101 Day 1VSI/VSN/SF/SD/EC101 Day 3VSISQ/RN/SEI/TC101 Day 6VSIP/SQ/RK/REC101 4 weeks after last doseVSIP/SQ/REOPERA-1 Day 1VSI/VSN/SD/EGenotype 1bS007R026V048Y056Q80P089A150D168F169Patient 141 (NR)C101 Day 1AKIFSVC101 Day 3AKIFSVC101 Day 6AKIFSVVC101 4 weeks after last doseAKIFSVVOPERA-1 Day 1AKIFSVOPERA-1 Day 7AKIFRSVEOPERA-1 Day 14AKIFRSVELOPERA-1 Day 21 (1-week follow-up)AKIFR/K/QSVEF/LOPERA-1 Week 6 (4-week follow-up)AKIFKSEGenotype 1aG15V029S122I132R155D168Patient 142 (NR)C101 Day 1AC101 Day 3AD/VC101 Day 5AK/RA/DC101 Day 6AV/AC101 4 weeks after last doseAK/ROPERA-1 Day 1AOPERA-1 Day 7AKOPERA-1 Day 14G/WALKOPERA-1 Week 32 (4-week follow-up)ARKGenotype 1bR026D168Patient 143 (R)C101 Day 1KC101 Day 3K/RD/VC101 Day 6KVC101 4 weeks after last doseKOPERA-1 Day 1KGenotype 1aP067K068S122R155D168Patient 144 (NR)C101 Day 1SK/NN/SC101 Day 3SK/NN/SC101 Day 6SNND/VC101 1 week after last doseSN/SK/RA/VC101 4 weeks after last doseSN/SKD/EOPERA-1 Day 1SNOTE. Mutations T40A, S91T/A, and L153I were found in all HCV genotype 1b patients at all time points analyzed, and the mutation R26K was found in all HCV genotype 1b patients at all time points analyzed. Dark grey indicates NS3 position at which mutations have been shown to reduce TMC435 activity in vitro.R, relapse; NR, nonresponse. Open table in a new tab Supplementary Figure 2(A) Individual HCV RNA levels in cohort 5 of OPERA-1 over the entire study period and (B) individual changes in HCV RNA levels from baseline to day 8 in study C101 compared with OPERA-1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure 2(A) Individual HCV RNA levels in cohort 5 of OPERA-1 over the entire study period and (B) individual changes in HCV RNA levels from baseline to day 8 in study C101 compared with OPERA-1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure 3Presence of viral variants at baseline in studies C101 and OPERA-1 based on 454 deep sequencing.View Large Image Figure ViewerDownload Hi-res image Download (PPT) NOTE. Mutations T40A, S91T/A, and L153I were found in all HCV genotype 1b patients at all time points analyzed, and the mutation R26K was found in all HCV genotype 1b patients at all time points analyzed. Dark grey indicates NS3 position at which mutations have been shown to reduce TMC435 activity in vitro. R, relapse; NR, nonresponse.