Douglas J. Bartels

Quorum Sensing in Staphylococcus aureus Biofilms

Several serious diseases are caused by biofilm-associated Staphylococcus aureus, infections in which the accessory gene regulator (agr) quorum-sensing system is thought to play an important role. We studied the contribution of agr to biofilm development, and we examined agr-dependent transcription in biofilms. Under some conditions, disruption of agr expression had no discernible influence on biofilm formation, while under others it either inhibited or enhanced biofilm formation. Under those conditions where agr expression enhanced biofilm formation, biofilms of an agr signaling mutant were particularly sensitive to rifampin but not to oxacillin. Time lapse confocal scanning laser microscopy showed that, similar to the expression of an agr-independent fluorescent reporter, biofilm expression of an agr-dependent reporter was in patches within cell clusters and oscillated with time. In some cases, loss of fluorescence appeared to coincide with detachment of cells from the biofilm. Our studies indicate that the role of agr expression in biofilm development and behavior depends on environmental conditions. We also suggest that detachment of cells expressing agr from biofilms may have important clinical implications.

Characterization of telaprevir treatment outcomes and resistance in patients with prior treatment failure: Results from the REALIZE trial

In the Phase 3 REALIZE study, 662 genotype 1 hepatitis C virus (HCV)‐infected patients with prior peginterferon/ribavirin treatment failure (including relapsers, partial, and null responders) were randomized to 12 weeks of telaprevir given immediately (T12/PR48) or following 4 weeks of peginterferon/ribavirin (lead‐in T12/PR48), or 12 weeks of placebo (PR48), combined with a total of 48 weeks of peginterferon alfa‐2a/ribavirin. Sustained virologic response (SVR) rates were 64% (T12/PR48), 66% (lead‐in T12/PR48), and 17% (PR48). This analysis aimed to characterize treatment outcomes and viral variants emerging in telaprevir‐treated patients not achieving SVR. HCV NS3·4A population sequencing was performed at baseline, during treatment, and follow‐up. Telaprevir‐resistant variants were classified into lower‐level (3‐ to 25‐fold 50% inhibitory concentration [IC50] increase: V36A/M, T54A/S, R155I/K/M/T, and A156S) and higher‐level (>25‐fold IC50 increase: V36M+R155K and A156T/V) resistance. Resistant variants were uncommon at baseline. Overall, 18% (52%, 19%, and 1% of prior null and partial responders and relapsers, respectively) of telaprevir‐treated patients had on‐treatment virologic failure, with no significant difference with or without a lead‐in. Virologic failure during the telaprevir‐treatment phase was predominantly associated with higher‐level resistance; virologic failure during the peginterferon/ribavirin‐treatment phase was associated with higher‐ or lower‐level, or wildtype variants, depending on genotype. Relapse occurred in 9% of patients completing assigned treatment and was generally associated with lower‐level resistant variants or wildtype. Resistant variants were no longer detectable by study end (median follow‐up of 11 months) in 58% of non‐SVR patients. Conclusion: In REALIZE, variants emerging in non‐SVR, telaprevir‐treated patients were similar irrespective of the use of a lead‐in and were consistent with those previously reported. In most patients, resistant variants became undetectable over time. (HEPATOLOGY 2012;56:2106–2115)

Patterns of viral load decline with telaprevir-based therapy in patients with genotype 1 chronic HCV infection

BACKGROUND Telaprevir-based therapy is associated with rapid decline in HCV RNA, enabling the application of early futility rules. OBJECTIVES To familiarize physicians with this paradigm, a comprehensive analysis of the most frequent HCV viral load profiles observed during treatment with telaprevir/Peg-IFN/RBV in Phase III trials is provided. DESIGN HCV RNA profiles were analyzed from 320 HCV genotype 1 treatment-naïve patients enrolled in the ADVANCE study, and 225 prior Peg-IFN/RBV treatment-experienced patients enrolled in the REALIZE study. Patients received 12 weeks of telaprevir with either 24 or 48 weeks of Peg-IFN alfa-2a/RBV. Patients with missing SVR assessments during follow-up, detectable HCV RNA at end of treatment but who did not have viral breakthrough (vBT), or with early vBT who discontinued telaprevir before time of failure were excluded. RESULTS All analyzed patients experienced a rapid decline in HCV RNA (>2.0 log(10)) by Day 14, irrespective of baseline characteristics and/or prior response to Peg-IFN/RBV (relapse, partial response and null response). Subsequently, HCV RNA continued to decline to undetectable levels in most patients. These patients went on to have one of the following outcomes: sustained virologic response, late vBT (after Week 12, i.e. during the Peg-IFN/RBV phase), or relapse. In the small subset of patients with early vBT or meeting a futility rule before Week 12, HCV RNA usually never became undetectable and/or increased rapidly after reaching the nadir. CONCLUSIONS HCV RNA profiles with telaprevir/Peg-IFN/RBV are different from those with Peg-IFN/RBV alone. It is important that clinicians understand these HCV RNA profiles and monitor patient viral load in order to apply futility rules correctly.

T-24 Retreatment with telaprevir/Peg-IFN/RBV after a short exposure to telaprevir in Phase I studies: interim results from a Phase IIIb rollover trial (C219)

35 Retreatment with telaprevir/Peg-IFN/RBV after a short exposure to telaprevir in Phase I studies: interim results from a Phase IIIb rollover trial (C219) C. Sarrazin; H.W. Reesink; S. Zeuzem; C.J. Weegink; D. Luo; J. Witek; T.L. Kieffer; D.J. Bartels; I. Dierynck; S. De Meyer; G. Picchio C219: Phase IIIb, Open-label, Roll-over Study *All 9 patients had completed Week 8 of treatment at the time of the analysis; Peg-IFN: P: Peg-IFN alfa-2a = 180μg/week; RBV: R = 1000–1200mg/day; TVR = telaprevir 750mg every 8 hours; HCV RNA determined using Roche COBAS TaqMan® assay version 2.0 (lower limit of quantification 25 IU/mL, lower limit of detection approximately 10 IU/mL) 48 4 16 0 12 8 Weeks 72 Peg-IFN + RBV TVR + Peg-IFN + RBV Follow-up SVR assessment Interim analysis at Week 8 for 9 patients from Phase I studies (101/103)* REALIZE Phase III (Relapser, Non-responder) N=81 (PR control; TVR naïve) 101/103 Phase I (Naïve, Relapser, Non-responder) N=9 (TVR exposed) Serrazin C, et al. 62nd AASLD; San Francisco, CA; November 04-08, 2011. Abst. 35. Changes in HCV RNA Over Time During Previous Phase I Studies (101/103) *Patient started Peg-IFN/RBV after Day 14 C ha ng e in H C V R N A fr om ba se lin e (lo g 1 0 IU /m L) 1 0 –1 –2 –3 –4 –5 –6 –7 Baseline 3 7 14 17 21 28 TVR discontinuation Time (days) Genotype 1a Genotype 1b Study 101 (TVR only) Study 103 (TVR + Peg-IFN) * * Serrazin C, et al. 62nd AASLD; San Francisco, CA; November 04-08, 2011. Abst. 35. H C V R N A (IU /m L) 108 107 106 105 104 103 102 101 100 Baseline 1 2 4 6 8 Time (weeks) LOQ = 25 IU/mL Patient had a confirmed viral breakthrough at Week 8 (V36M/V + R155K/R) Genotype 1a Genotype 1b C219: HCV RNA Values Over Time (Week 8 Interim Analysis, TVR-exposed) LOQ: limit of quantification HCV RNA values below LOQ are imputed with an arbitrary value: 17.5 for <25 IU/mL detectable and 5 for <25 IU/mL undetectable Serrazin C, et al. 62nd AASLD; San Francisco, CA; November 04-08, 2011. Abst. 35. 1 RAV Detected 2 RAVs Detected 3 RAVs Detected > 3 RAVs Detected RAV Patients, n RAV Patients, n RAV Patients, n RAV Patients, n V36M 18 V36M, R155K 37 V36M, T54S, R155K 4 V36M, T54S, R155K, R155T 1 R155K 11 T54S, R155K 8 T54A, T54S, A156S 2 V36M, T54S, R155K, A156S 1 T54A 10 R155K, V158I 4 V36L, R155K, V158I 2 V36L, R155K, V158I, I170F 1 R155T 5 T54S, A156S 4 T54A, V170A, M175L 1 T54A, T54C, T54S, V55A, A156S 1 n=63 n=72 n=15 n=5 0 10 20 30 40 50 60 70 80 1 RAV 2 RAVs 3 RAVs > 3 RAVs Pa tie nt s, n SPRINT-2 and RESPOND-2 Data From Population Sequencing (linkage of RAVs not known) Analysis of Resistance-Associated Amino Acid Variants (RAVs) in Non-SVR Patients Enrolled in a Retrospective Long-term Follow-up Analysis of Boceprevir Phase 3 Clinical Studies Bernard R, et al. 62nd AASLD; San Francisco, CA; November 04-08, 2011. Abst. 164. (SPRINT-2 and RESPOND-2) 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% End Treatment 2 4 6 8 10 12 14 Time Since the End of Treatment (Months) Pa tie nt s w ith T 54 A D et ec te d 16 % Patients with T54S (by pop sequencing) Kaplan-Meier Plot of Patients with Viruses Harboring RAV T54A Detected During Follow-up Bernard R, et al. 62nd AASLD; San Francisco, CA; November 04-08, 2011. Abst. 164. Comparative Activity of Pls Against HCV NS3 Proteases from Genotypes 1-6 (SEAP Assay)