All-oral therapy with nucleotide inhibitors sofosbuvir and GS-0938 for 14 days in treatment-naive genotype 1 hepatitis C (NUCLEAR)

Journal of Viral Hepatitis

Volume 20, Issue 10, pages 699–707, October 2013

Original Article

E. J. Lawitz^1,*, M. Rodriguez-Torres², J. Denning³, A. Mathias³, H. Mo³, B. Gao³, M. T. Cornpropst⁴, M. M. Berrey⁵, W. T. Symonds³

Article first published online: 31 MAR 2013

DOI: 10.1111/jvh.12091

© 2013 Blackwell Publishing Ltd

Abstract

Keywords: antiviral agents; direct-acting antiviral agents; nucleotide analogue; viral drug resistance

Summary

Sofosbuvir and GS-0938 are distinct nucleotide analogues with activity against hepatitis C virus (HCV) in vitro. We evaluated the antiviral activity and safety of sofosbuvir and GS-0938 alone and in combination in HCV genotype 1 patients. In this double-blind study, 40 treatment-naïve patients were randomly assigned to 4 treatment cohorts: (i) GS-0938 for 14 days, (ii) GS-0938 for 7 days followed by GS-0938 plus sofosbuvir for 7 days, (iii) sofosbuvir for 7 days followed by GS-0938 plus sofosbuvir for 7 days and (iv) GS-0938 plus sofosbuvir for 14 days. In each arm, 8 patients received active drug and 2 placebo. After 7 days of dosing, patients in all 4 dose groups experienced substantial reductions in HCV RNA, with median declines (Q1, Q3) of −4.50 (−4.66, −4.24) in Cohort 1, −4.55 (−4.97, −4.13) in Cohort 2, −4.65 (−4.78, −4.17) in Cohort 3 and −4.43 (−4.81, −4.13) in Cohort 4; patients receiving placebo had essentially no change in HCV RNA (+0.07 log₁₀ IU/mL). Seven days after the end of treatment, the proportions of patients with HCV RNA <15 IU/mL were 4 (50%), 8 (100%), 7 (88%) and 5 (63%) for Cohorts 1–4, respectively, vs 0 for placebo. No viral breakthrough or resistance mutations were observed. No serious adverse events or Grade 3 or 4 adverse events were reported. Sofosbuvir and GS-0938—alone and in combination—were well tolerated and led to substantial reductions in viral load. Sofosbuvir is undergoing further investigation as a possible backbone of an all-oral regimen for chronic HCV.

Source

Analysis of Genotype 2 and 3 Hepatitis C Virus Variants in Patients Treated With Telaprevir Demonstrates a Consistent Resistance Profile Across Genotypes

Journal of Viral Hepatitis

S. De Meyer, A. Ghys, G. R. Foster, M. Beumont, B. Van Baelen, T.-I. Lin, I. Dierynck, H. Ceulemans, G. Picchio

J Viral Hepat. 2013;20(6):395-403.

Abstract and Introduction

Abstract

Study C209 evaluated the activity of telaprevir in treatment-naïve patients with genotypes 2 or 3 (G2, G3) hepatitis C virus (HCV) infection. Telaprevir monotherapy showed potent activity against HCV G2, but limited activity against G3. This analysis was performed to characterize HCV viral variants emerging during telaprevir-based treatment of G2/G3 HCV-infected patients. Patients were randomized to receive 2 weeks of treatment with telaprevir (telaprevir monotherapy), telaprevir plus peginterferon alfa-2a and ribavirin (triple therapy), or placebo plus peginterferon alfa-2a and ribavirin (control), followed by 22–24 weeks of peginterferon/ribavirin alone. Viral breakthrough was defined as an increase >1 log₁₀ in HCV RNA from nadir, or HCV RNA >100 IU/mL in patients previously reaching <25 IU/mL. Twenty-three patients (47%) had G2 and 26 (53%) had G3 HCV. Viral breakthrough occurred during the initial 2-week treatment phase in six G2 patients (66.7%; subtypes 2, 2a and 2b) and three G3 patients (37.5%; all subtype 3a), all in the telaprevir monotherapy arm. Four breakthrough patients (three G2, one G3) subsequently achieved sustained virologic response (SVR). In all patients with breakthrough and available sequence data, mutations associated with reduced susceptibility to telaprevir in genotype 1 (G1) HCV were observed. No novel G2/G3-specific mutations were associated with telaprevir resistance. The telaprevir resistance profile appeared consistent across HCV genotypes 1, 2 and 3. Although viral breakthrough with resistance occurred in patients receiving telaprevir monotherapy, half of these patients achieved an SVR upon addition of peginterferon/ribavirin highlighting the importance of combination therapy.

Introduction

Chronic hepatitis C virus (HCV) infection is a major global healthcare burden.^[1] Six HCV genotypes and over 100 subtypes are documented,^[2] each with a distinct geographical distribution. Genotype 1 (G1) HCV predominates in Europe, the United States and Japan, G2 predominates in Mediterranean countries and the Far East, and G3 is common in the Indian subcontinent.^[3,4] An increase in the proportion of patients with G3 HCV infection was recently observed in Europe.^[5]

Two direct-acting antiviral (DAA) agents that target the HCV NS3-4A serine protease, telaprevir^[6] and boceprevir,^[7] showed substantial efficacy against chronic HCV G1 infection^[8–12] and were recently approved in the United States and Europe. Preliminary in vitro data suggested that telaprevir also has activity against G2/G3 HCV.^[13] Therefore, the Phase IIa C209 study evaluated the activity of telaprevir, administered for 15 days with or without peginterferon/ribavirin, on G2/G3 HCV infection. When administered as monotherapy, telaprevir had significant and rapid antiviral activity against G2 HCV, but little or no activity against G3.^[14]

G1 HCV variants associated with decreased susceptibility to telaprevir were previously identified in patients not achieving an SVR. The single amino acid changes V36A/M, T54A/S, R155K/T and A156S confer lower-level in vitro resistance to telaprevir (3- to 25-fold increase in replicon 50% inhibitory concentration [IC₅₀]), whereas single change A156T/V and double change V36M+R155K confer higher-level resistance (>25-fold increase in replicon IC₅₀).^[15] Whether similar variants are associated with lack of response to telaprevir in G2/G3 HCV infection is unknown. Therefore, this subanalysis of the C209 study characterized HCV viral variants emerging with telaprevir-based therapy in G2/G3 HCV-infected patients.

Materials and Methods

The methodology for this Phase IIa, multicentre, partially blinded, randomized, multiple-dose trial in treatment-naïve patients with chronic G2 or G3 HCV infection, was described elsewhere (ClinicalTrials.gov NCT00561015).^[14]

Study Design

After a 6-week screening period, patients were randomized 1:1:1 to receive 2 weeks of telaprevir 750 mg every 8 h (q8 h; telaprevir monotherapy), telaprevir 750 mg q8 h plus peginterferon alfa-2a 180 μg once-weekly and ribavirin 400 mg twice daily (bid; triple therapy), or placebo q8 h plus peginterferon alfa-2a 180 μg once-weekly and ribavirin 400 mg bid (control). This 2-week investigational treatment period was followed by a standard-treatment phase where patients received peginterferon alfa-2a plus ribavirin for an additional 24 weeks (telaprevir monotherapy arm) or 22 weeks (other arms). After treatment completion, patients were followed up for 24–48 weeks.

Primary and secondary endpoints are reported elsewhere.^[14] Viral breakthrough (vBT) was defined as an increase >1 log₁₀ in HCV RNA from nadir or HCV RNA >100 IU/mL in patients who previously reached HCV RNA <25 IU/mL, confirmed by two consecutive samples. Patients who had an unconfirmed vBT during telaprevir monotherapy were still considered to have vBT. SVR was defined as undetectable HCV RNA at end of treatment (EOT) and 24 weeks afterwards.

The study was approved by each centre's institutional review board and conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. Written informed consent was obtained from all patients.

Virologic Assessments

Hepatitis C virus RNA levels were evaluated using the Roche COBAS TaqMan HCV test version 2.0 (Roche Molecular Systems Inc., Branchburg, NJ, USA), with a limit of quantification (LOQ) of 25 IU/mL. Samples containing HCV RNA below the LOQ were reported as '<25 IU/mL detected'; those with no HCV RNA as '<25 IU/mL, target not detected' (also described as undetectable HCV RNA).

Samples for viral sequencing were obtained at baseline; days 2, 3, 4, 8, 12 and 15; weeks 4, 6 and 14; at EOT; 4, 8, 12 and 24 weeks after EOT; and 24 weeks after relapse (if any). Population-based NS3 sequence analysis was conducted in all baseline samples and in postbaseline samples from telaprevir-treated patients with vBT and/or not achieving an SVR. Population-based sequencing of the HCV NS3 region used subtype-specific amplification and sequencing protocols (Virco BVBA, Beerse, Belgium). Clonal sequencing of the NS3 region was conducted in a subset of samples. For both population-based and clonal sequencing, total RNA was extracted from plasma. The RNA was reverse transcribed, and cDNA was amplified in a one-step polymerase chain reaction (PCR) using the SuperScriptTM III One-Step Reverse Transcriptase (RT)-PCR System (Life Technologies, Carlsbad, CA, USA). An amplicon encompassing the NS3 region was further amplified during a nested PCR (Expand high Fidelity PCR system, Roche Applied Science, Penzberg, Germany). For population-based sequencing, PCR products were purified and directly sequenced in both directions using Big Dye terminator sequencing chemistry (Life Technologies). In case of clonal sequencing, the PCR product was first cloned using a TOPO TA cloning kit (Life Technologies). DNA from 20 to 30 clones per sample was purified and sanger sequenced as described above. The genotype and subtype of each sample were determined based on the NS5B region. The lower limit of detection for the sequencing assay was ~1000 IU/mL HCV RNA.

For phenotypic analyses, cDNA encoding NS3 amino acids 1–181 was cloned and expressed using Escherichia coli Rosetta2 (DE3) (Novagen, Madison, WI, USA). Telaprevir susceptibility was tested using a fluorescence resonance energy transfer cleavage assay with the retS1 peptide substrate (Anaspec, San Jose, CA, USA). Briefly, NS3-4A was pre-incubated with telaprevir for 10 min. The retS1 substrate was added, and fluorescence continuously measured for 20 min (excitation 355 nm, emission 510 nm). The IC₅₀ value was calculated from the inhibition values of a series of telaprevir concentrations. Fold change in IC₅₀ values (FC), compared with wild-type (Con1b) HCV, was calculated.

Statistical Analyses

To identify mutations potentially associated with reduced susceptibility to telaprevir, the frequency of every amino acid at every position was compared between telaprevir-exposed and -unexposed samples. Statistical significance was defined as P < 0.05 in Fisher's exact test; Bonferroni correction was used when multiple amino acid values or positions were explored simultaneously. For G3 analyses, unexposed samples comprised 102 G3a HCV sequences from combined public Los Alamos and internal HCV sequence databases. No formal statistical analyses were performed for G2, due to the low number of available sequences from unexposed samples.

Results

Patient disposition and baseline characteristics were reported in detail elsewhere^[14] and were generally well-balanced across treatment groups and genotypes. Twenty-three patients (47%) had G2 HCV and 26 (53%) had G3 HCV. The HCV G2 subtypes were as follows: 2 (n = 7), 2c (n = 6), 2b (n = 4), 2a (n = 3), 2i (n = 1) and 2k (n = 1); G3 subtypes were 3a (n = 22) and 3b (n = 3). No sequence information was available for one G2 and one G3 patient. Forty patients (82%) completed the study, including follow-up.

G2 HCV-infected Patients

Antiviral Activity. During the investigational phase, vBT was observed in 6/9 G2 patients (66.7%) in the telaprevir monotherapy arm, compared with no vBT in the triple therapy and control arms (). One patient experienced vBT during the standard-treatment phase (control arm). One patient in the telaprevir monotherapy arm relapsed. HCV RNA profiles during telaprevir monotherapy are shown in Fig. 1a.

Table 1.  Telaprevir antiviral activity in patients infected with either G2 or G3 HCV. Data are shown as n (%)

	Patients infected with G2 HCV			Patients infected with G3 HCV
	T (n = 9)	T/PR (n = 5)	PR (n = 9)	T (n = 8)	T/PR (n = 9)	PR (n = 9)
Virologic response*
By end of telaprevir/placebo treatment	0	2 (40.0)	2 (22.2)	0	2 (22.2)	1 (11.1)
By end of treatment	8 (88.9)	5 (100)	8 (88.9)	6 (75.0)	9 (100)	9 (100)
Cumulative vBT
By end of telaprevir/placebo treatment	6 (66.7)	0	0	3 (37.5)	0	0
By end of treatment	6 (66.7)	0	1 (11.1)	3 (37.5)	0	0
Relapse	1 (12.5)†	0	0	2 (33.3)§	3 (33.3)¶	2 (22.2)¶
Missing follow-up data	2 (22.2)‡	0	0	0	0	3 (33.3)**
SVR	5 (55.6)	5 (100)	8 (88.9)	4 (50.0)	6 (66.7)	4 (44.4)

G, genotype; HCV, hepatitis C virus; T, telaprevir monotherapy, telaprevir 750 mg every 8 h (q8 h); T/PR, triple combination therapy, telaprevir 750 mg q8 h plus peginterferon alfa-2a 180 μg once-weekly and ribavirin 400 mg bid; PR, peginterferon/ribavirin, placebo q8 h plus peginterferon alfa-2a 180 μg once-weekly and ribavirin 400 mg bid; SVR, sustained virologic response; vBT, viral breakthrough. *Undetectable HCV RNA; ^†Eight patients had undetectable HCV RNA at end of treatment; ^‡One patient discontinued due to noncompliance and one patient lost to follow-up; ^§Six patients with undetectable HCV RNA at end of treatment; ^¶Nine patients with undetectable HCV RNA at end of treatment; **Three patients lost to follow-up.

Figure 1.

HCV RNA levels in individual patients over time. HCV RNA profiles during telaprevir monotherapy in patients infected with (a) G2 HCV and (b) G3 HCV are shown. LOQ was 25 IU/mL; HCV RNA values below LOQ were imputed with an arbitrary value of 17.5 for <25 IU/mL detectable and five for undetectable HCV RNA. LOQ, limit of quantification; HCV, hepatitis C virus

Baseline Virologic Data. Baseline population-based sequences were available for 22/23 G2 HCV-infected patients; no patient had mutations associated with decreased telaprevir susceptibility in G1 HCV. Clonal sequence analysis was conducted in the six patients with vBT: 1/27 clones in one patient had the T54A or R155K mutation at baseline; 1/22 clones in another patient had the A156V mutation at baseline (Fig. 2a).

Figure 2.

Variants detected by clonal sequencing in patients with vBT. Clonal sequence data are shown for (a) G2 and (b) G3 HCV-infected patients with vBT in the telaprevir monotherapy arm (20–40 clones/timepoint). Mutations at positions 36, 54, 155 and 156 were considered for this analysis. G, genotype

The mean G2 HCV telaprevir FC at baseline (vs wild type) was 2.4 (range, 0.2–6.6). For the seven telaprevir monotherapy patients with available phenotypic data, there was no clear relationship between baseline telaprevir FC and the change in HCV RNA from baseline to day 3 (Fig. 3a). However, the two patients with the lowest telaprevir FC at baseline had the greatest change in HCV RNA at day 3.

Figure 3.

HCV RNA reduction vs in vitro telaprevir susceptibility. Change in HCV RNA from baseline to day 3 is shown against baseline telaprevir FC in telaprevir monotherapy patients infected with (a) G2 HCV and (b) G3 HCV. G, genotype; HCV, hepatitis C virus; FC, fold change in IC₅₀

Genotypic Characterization of Viral Variants. Figure 4 shows HCV RNA, genotypic and phenotypic profiles over time for the six G2 HCV-infected patients (two G2b, one G2a and three G2 unknown subtype) in the telaprevir monotherapy arm with vBT.

Figure 4.

HCV RNA, genotypic and phenotypic profiles for individual G2 HCV-infected patients. Profiles are shown for (a) G2a and G2b and (b) G2 unknown subtype HCV-infected patients in the telaprevir monotherapy arm with vBT, during both the investigational (panel i) and standard (panel ii) treatment phases. HCV; hepatitis C virus; TVR disc, discontinuation of telaprevir; PR disc, discontinuation of peginterferon/ribavirin; LOQ, limit of quantification; FC, fold change in IC₅₀

In all five patients with vBT for whom sequences were available, emerging mutations associated with reduced telaprevir susceptibility in G1 HCV were observed using population sequencing. No sequence could be obtained for the day 15 sample of patient 5 due to low HCV RNA levels (101 IU/mL). At the time of vBT, T54A alone (n = 2), R155K alone (n = 1), A156S alone (n = 1) and the T54A+R155K combination (n = 1) were found. T54A was detected in one patient (patient 3) before vBT and A156S was detected in another patient (patient 1) after vBT. Clonal sequence analysis confirmed that in patients with vBT, mutations associated with reduced telaprevir susceptibility in G1 HCV emerged and became the dominant quasispecies (Fig. 2a).

In addition to the mutations associated with reduced susceptibility to telaprevir in G1 HCV, a number of other amino acid changes from baseline were identified in telaprevir monotherapy patients with vBT: Q32H, S146P, V163I, S166A and T179A (Fig. 4). However, no mutation appeared consistently across patients.

Of the six patients with vBT in the telaprevir monotherapy arm, five had undetectable HCV RNA at the end of peginterferon/ribavirin treatment, showing that the emerging variants remained sensitive to peginterferon/ribavirin. Three of these patients, including two with emerging mutations, achieved an SVR. For the patient who relapsed, no mutations associated with reduced susceptibility to telaprevir or other HCV protease inhibitors were detected at follow-up (week 24).^[15]

Phenotypic Characterization of Viral Variants. Paired genotypic and phenotypic data were available for 3/6 G2 patients with vBT. At the time of vBT, two patients (patients 3 and 4, both G2b) had an emerging R155K mutation associated with a >9-fold increase from baseline in telaprevir FC. The same two patients had an emerging T54A mutation. In patient 3, T54A was not associated with an increase in telaprevir FC; while in patient 4, it was not present in the expression construct used for phenotyping. At the time of vBT, patient 6 (G2) had an emerging A156S mutation associated with a >48-fold increase from baseline in FC.

Introducing the R155K mutation into the baseline NS3 sequence of patients 3 and 4 by site-directed mutagenesis increased the telaprevir FC from 6.4 and 5.4 (without R155K) to >76.9 and >100, respectively. Introducing T54A into the baseline NS3 sequence of patient 4 increased the FC from 5.4 to 39.5. Introducing the A156S mutation into the baseline NS3 sequence of patients 1 (G2a) and 6 increased the FC from 1.2–1.6 to >58.8–>71.4.

G3 HCV-infected Patients

Antiviral Activity. During the investigational phase, vBT was observed in 3/8 G3 patients (37.5%) in the telaprevir monotherapy arm, compared with no vBT in the triple therapy and control arms (). As telaprevir monotherapy only produced slight decreases in HCV RNA levels (Fig. 1b), most patients did not meet the definition for vBT. Relapse was observed in two patients in the telaprevir monotherapy arm, three patients in the triple therapy arm and two control patients.

Table 1.  Telaprevir antiviral activity in patients infected with either G2 or G3 HCV. Data are shown as n (%)

	Patients infected with G2 HCV			Patients infected with G3 HCV
	T (n = 9)	T/PR (n = 5)	PR (n = 9)	T (n = 8)	T/PR (n = 9)	PR (n = 9)
Virologic response*
By end of telaprevir/placebo treatment	0	2 (40.0)	2 (22.2)	0	2 (22.2)	1 (11.1)
By end of treatment	8 (88.9)	5 (100)	8 (88.9)	6 (75.0)	9 (100)	9 (100)
Cumulative vBT
By end of telaprevir/placebo treatment	6 (66.7)	0	0	3 (37.5)	0	0
By end of treatment	6 (66.7)	0	1 (11.1)	3 (37.5)	0	0
Relapse	1 (12.5)†	0	0	2 (33.3)§	3 (33.3)¶	2 (22.2)¶
Missing follow-up data	2 (22.2)‡	0	0	0	0	3 (33.3)**
SVR	5 (55.6)	5 (100)	8 (88.9)	4 (50.0)	6 (66.7)	4 (44.4)

G, genotype; HCV, hepatitis C virus; T, telaprevir monotherapy, telaprevir 750 mg every 8 h (q8 h); T/PR, triple combination therapy, telaprevir 750 mg q8 h plus peginterferon alfa-2a 180 μg once-weekly and ribavirin 400 mg bid; PR, peginterferon/ribavirin, placebo q8 h plus peginterferon alfa-2a 180 μg once-weekly and ribavirin 400 mg bid; SVR, sustained virologic response; vBT, viral breakthrough. *Undetectable HCV RNA; ^†Eight patients had undetectable HCV RNA at end of treatment; ^‡One patient discontinued due to noncompliance and one patient lost to follow-up; ^§Six patients with undetectable HCV RNA at end of treatment; ^¶Nine patients with undetectable HCV RNA at end of treatment; **Three patients lost to follow-up.

Baseline Virologic Data. Baseline population-based sequences were available for 24/26 G3 HCV-infected patients; no patient had mutations associated with decreased telaprevir susceptibility in G1 studies. No patient with vBT had known baseline mutations by clonal sequence analysis (Fig. 2b).

The mean telaprevir FC at baseline for G3 HCV (vs wild type) was 7.0 (range, 2.5–14.5). Unexpectedly, patients with the highest baseline telaprevir FC tended to show a greater change in HCV RNA between baseline and day 3 (Fig. 3b). However, patient numbers were small.

Genotypic Characterization of Viral Variants. All three telaprevir monotherapy patients with vBT had HCV subtype 3a (Fig. 5); R155K was present at vBT in two patients. In the third patient, no mutations associated with reduced telaprevir susceptibility in G1 studies were detected at vBT, but R155K was detected later. Clonal sequence analysis confirmed the results obtained by population sequencing (Fig. 2b). Two of the three patients with vBT had undetectable HCV RNA at the end of peginterferon/ribavirin treatment; one subsequently achieved an SVR, the other relapsed.

Figure 5.

HCV RNA, genotypic and phenotypic profiles for individual G3 HCV-infected patients. Profiles are shown for G3 HCV-infected patients in the telaprevir monotherapy arm with vBT, for both the investigational (panel i) and standard (panel ii) treatment phases. HCV; hepatitis C virus; TVR disc, discontinuation of telaprevir; PR disc, discontinuation of peginterferon/ribavirin, LOQ, limit of quantification; FC, fold change in IC₅₀

For patients without vBT, sequences of samples collected during the investigational phase were available for 4/5 patients in the telaprevir monotherapy arm and 1/7 patients in the triple therapy arm. No mutations associated with decreased telaprevir susceptibility in G1 HCV were detected by population sequencing. For patients who relapsed, sequences of samples were available for 4/5 patients in the telaprevir monotherapy and triple therapy arms. In patient 9, the R155K mutation was detected at time of relapse.

In addition to mutations associated with reduced susceptibility to telaprevir in G1, several other amino acid changes from baseline (T47A/T, A67A/V, K92K/N, A98A/T, S101A/T and P146S) were identified in G3 telaprevir monotherapy patients during or at the end of the investigational phase or at time of relapse. None of the NS3 region amino acid changes associated with telaprevir exposure reached statistical significance by Fisher's exact test.

Phenotypic Characterization of Viral Variants. Paired genotypic and phenotypic data were available for the three G3 patients with vBT. The emergence of R155K was associated with a >5-fold increase from baseline in FC.

Introduction of the R155K mutation into the NS3 baseline sequence of patients 7 and 9 (both G3a) by site-directed mutagenesis led to an FC increase from 9.4-10 (without R155K) to >58.8–>90.9 (with R155K).

Discussion

In treatment-naïve patients, telaprevir monotherapy had significant antiviral activity against G2 HCV but little or no activity against G3 HCV.^[14] All vBTs occurred during telaprevir monotherapy, and most NS3 mutations emerging during breakthrough occurred at amino acid positions previously reported to be associated with reduced telaprevir susceptibility in G1 HCV. The finding that telaprevir monotherapy is associated with a high rate of vBT in G2/G3 HCV is similar to previous observations in G1 HCV,^[15,16] and highlights the importance of combining telaprevir with peginterferon/ribavirin to control the emergence of resistant variants that remain susceptible to these agents.

In the telaprevir monotherapy arms, vBT was reported in six of nine patients with G2 HCV and three of eight patients with G3 HCV. Seven of these nine patients with vBT had undetectable HCV RNA at the end of subsequent treatment with peginterferon/ribavirin, and four achieved an SVR. The fact that G2 and G3 HCV variants with reduced telaprevir susceptibility remained sensitive to, and can be eradicated by, peginterferon/ribavirin extends similar observations with G1 HCV.^[15]

Three mutations associated with reduced telaprevir susceptibility in G1 HCV (R155K, T54A, A156S) were seen in G2 HCV patients with vBT; two (R155K and T54A) were seen in G3 HCV patients with vBT. As in G1a HCV,^[16] all emergent mutations required only a single nucleotide change from baseline. Several additional mutations were identified by the present analyses, but these did not occur consistently across patients and were not previously reported for other HCV protease inhibitors.^[2,15,17] Moreover, none of these additional mutations occurred with significantly higher frequency than in HCV samples from patients not exposed to telaprevir. However, small sample sizes and a restricted diversity of subtypes limited the robustness of our findings and, for G2, statistical analyses could not be performed. Furthermore, in samples with these additional mutations, resistance could be explained by known telaprevir-resistant mutations using site-directed mutagenesis, although we had not formally introduced the additional mutations into baseline samples to determine their impact on telaprevir susceptibility. In spite of this, our data suggest a lack of G2- or G3-specific mutations for telaprevir resistance. Therefore, the resistance profile appears consistent across G1, G2 and G3.

The NS3 region of HCV shows some differences between genotypes. For example, position 36 of the NS3 region, identified as a site for telaprevir-resistant G1 mutations, contains leucine (L) in patients with G2 and G3 HCV, but valine (V) in G1 HCV. The impact of this amino acid change, if any, on telaprevir susceptibility is not fully understood and may be genotype and subtype dependent. For example, some G2 NS3 proteins carrying an 'L' at position 36 exhibited telaprevir FC values similar to those observed with G1 proteins carrying a 'V' at position 36 (Janssen Infectious Diseases BVBA, data on file). G3 NS3 proteins carrying an 'L' at position 36 exhibited on average higher FC values, although changes at other amino acid positions could have caused this decrease in susceptibility. This type of finding may underlie the differences observed in this study between G2 and G3 in baseline telaprevir FC, as well as the differences in viral activity of telaprevir against the two genotypes.

G2-infected patients with vBT during telaprevir therapy had subtype 2b, 2a or an unknown subtype, but none had 2c. Although patient numbers were small, this suggests that vBT may be less common with subtype 2c. All G3-infected patients with vBT had subtype 3a. The lack of vBT among subtype 3b patients could relate to the lack of telaprevir activity. The R155K mutation only emerged in G2-infected patients with subtype 2b; in G2a viruses, the emergence of a 'K' at position 155 would require a double nucleotide change. The R155K mutation was dominant in G3a HCV patients with vBT.

For patients with G2 HCV in the telaprevir monotherapy arm, the greatest HCV RNA decline from baseline to day 3 was observed in those with the highest telaprevir susceptibility at baseline. Conversely, in G3 patients, the greatest HCV RNA decline from baseline to day 3 was in those with the lowest baseline telaprevir susceptibility. Patient numbers were small, and the phenotype data were obtained with a biochemical assay, which is less reliable than a cell-based assay. However, this inverse correlation might relate to the low efficacy of telaprevir monotherapy in G3 patients. As previously observed in G1 HCV, emergence of the R155K mutation at vBT was associated with a high FC increase, and site-directed mutagenesis confirmed that R155K is associated with reduced susceptibility to telaprevir in G2/G3 patients. In addition, the A156S mutation was associated with reduced telaprevir susceptibility in G2 HCV.

Clonal sequence analysis confirmed that in most instances of vBT in patients with G2/G3 HCV, mutations associated with reduced telaprevir susceptibility in G1 HCV emerged and became the dominant quasispecies. In two patients, a mutation associated with reduced telaprevir susceptibility was detected at baseline in one clone. However, a mutation found in one clone of 22–27 clones can be the result of a PCR amplification error and not a true mutation present in the clonal population.

In addition to the limitations already mentioned for this study, the sample size was small, and a proportion of patients were unfortunately lost to follow-up. Small samples, however, are a common characteristic of proof-of-principle studies exploring the intrinsic activity of an antiviral agent.

In summary, telaprevir showed activity against G2 HCV in treatment-naïve patients, but limited or no activity against G3 HCV. The resistance profile of telaprevir in G2 and G3 HCV appears to involve similar amino acid substitutions as previously observed in G1 HCV. All cases of vBT occurred in the telaprevir monotherapy arms, highlighting the importance of combining telaprevir with peginterferon/ribavirin to avoid the emergence of resistant variants. A high proportion of patients who experienced vBT with telaprevir monotherapy still achieved an SVR after peginterferon/ribavirin treatment, indicating that the emergence of variants with reduced susceptibility to telaprevir can be controlled with these agents.

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7. Berman K, Kwo PY. Boceprevir, an NS3 protease inhibitor of HCV. ClinLiver Dis 2009; 13(3): 429–439.

8. Bacon BR, Gordon SC, Lawitz E, et al. Boceprevir for previously treated chronic HCV genotype 1 infection. N Engl J Med 2011; 364(13): 1207–1217.

9. Poordad F, McCone J Jr, Bacon BR, et al. Boceprevir for untreated chronic HCV genotype 1 infection. N Engl JMed 2011; 364(13): 1195–1206.

10. Jacobson IM, McHutchison JG, Dusheiko G, et al. Telaprevir for previously untreated chronic hepatitis C virus infection. N Engl J Med 2011; 364(25): 2405–2416.

11. Sherman KE, Flamm SL, Afdhal NH, et al. Response-guided telaprevir combination treatment for hepatitis C virus infection. N Engl J Med 2011; 365(11): 1014–1024.

12. Zeuzem S, Andreone P, Pol S, et al. Telaprevir for retreatment of HCV infection. N Engl J Med 2011; 364 (25): 2417–2428.

13. Lin C, Hanzelka BL, Müh U, et al. Telaprevir (VX-950) is a potent inhibitor of HCV NS3 proteases derived from genotype non-1 HCVinfected patients. J Hepatol 2007; 46 (Suppl 1): S8.

14. Foster GR, H_ezode C, Bronowicki J-P, et al. Telaprevir alone or with peginterferon and ribavirin reduces HCV RNA in patients with chronic genotype 2 but not genotype 3 infections. Gastroenterology 2011; 141(3): 881–889.

15. Kieffer TL, Kwong AD, Picchio GR. Viral resistance to specifically targeted antiviral therapies for hepatitis C (STAT-Cs). J AntimicrobChemother 2010; 65(2): 202–212.

16. Sarrazin C, Kieffer TL, Bartels D, et al. Dynamic hepatitis C virus genotypic and phenotypic changes in patients treated with the protease inhibitor telaprevir. Gastroenterology 2007; 132(5): 1767–1777.

17. Qiu P, Sanfiorenzo V, Curry S, et al. Identification of HCV protease inhibitor resistance mutations by selection pressure-based method. Nucleic Acids Res 2009; 37(10): e74.22.

Abbreviations
HCV, hepatitis C virus; G, genotype; SVR, sustained virologic response; DAA, direct-acting antiviral; IC50, 50% inhibitory concentration; q8h, every 8 h; bid, twice daily; vBT, viral breakthrough; EOT, end of treatment; LOQ, limit of quantification; FC, fold change in IC50; L, leucine; V, valine; T, telaprevir monotherapy; T/PR, triple combination therapy; PR, peginterferon/ribavirin; TVR disc, discontinuation of telaprevir; PR disc, discontinuation of PR; PCR, polymerase chain reaction.

Acknowledgements
We express our gratitude to the patients who participated in the study, as well as the study centre staff and Janssen Infectious Diseases personnel. We acknowledge Tom Westgate and Joanne Williams (Medical Writers, Gardiner-Caldwell Communications, Macclesfield, UK) for assistance in drafting the manuscript and collating author contributions. The clinical trial was sponsored by Janssen Pharmaceuticals and Vertex Pharmaceuticals.

J Viral Hepat. 2013;20(6):395-403. © 2013 Blackwell Publishing

Source

Importance of Pharmacokinetics and Adherence to Avoid Emergence of Resistant HCV Variants

Jean-Michel Pawlotsky, MD, PhD
Professor, Department of Virology
Henri Mondor Hospital
Université Paris Est
Créteil, France

Introduction

Hepatitis C virus (HCV) resistance to a direct-acting antiviral (DAA) agent corresponds to the selection during treatment of viral variants that bear amino acid substitutions that alter the drug target; therefore, they are less susceptible to the inhibitory activity of the drug. These drug-resistant variants preexist as minor populations within the patient’s HCV quasispecies. Drug exposure profoundly inhibits replication of the dominant “wild-type” drug-sensitive viral population, and the resistant variants gradually occupy the vacant replication space. Moreover, viruses with low-level or partial resistance that can continue to replicate in the presence of drug, often favored by suboptimal drug exposure, may accumulate further mutations, leading to stepwise decreases in drug susceptibility, albeit often at a cost of reduced replicative capacity. If insufficient antiviral activity is provided because of suboptimal dosing or adherence, inadequate virologic suppression and the selection of resistance is inevitable. Therefore, to reduce the development of resistance, it is essential to achieve optimal drug concentrations through proper dosing and maximal adherence.

Factors That Influence Viral Resistance in vivo

In vivo, viral resistance is influenced by 3 major related factors: the genetic barrier to resistance, the in vivo fitness of the resistant viral population, and drug exposure.^[1]

The genetic barrier to resistance is defined as the number of amino acid substitutions needed for a viral variant to acquire full resistance to the drug in question. If a single substitution is sufficient to confer high-level resistance to a specific drug, the drug is considered to have a low genetic barrier to resistance, whereas 3 or more substitutions are required to confer resistance to a drug with a high genetic barrier. There is a low likelihood that variants bearing a large number of resistance substitutions will preexist in a given patient and be fit enough to replicate at high levels when an antiviral drug is administered. Therefore, drugs with a high genetic barrier to resistance are less likely to be associated with clinically meaningful resistance.

The in vivo fitness of the viral variant is defined as its ability to survive and grow in the replicative environment. A selected resistant variant must have the capacity to propagate to fill in the replication space left vacant by the elimination of a susceptible wild-type virus during drug exposure. Thus, a highly resistant but poorly “fit” virus will be less clinically significant than a less resistant but “fitter” virus that can replicate efficiently in the presence of the drug. The acquisition of compensatory mutations may restore the fitness of a resistant variant and allow it to replicate efficiently in the presence of the drug, possibly allowing it to persist after drug withdrawal.

Finally, drug exposure affects the development of drug resistance. The degree of drug resistance of a variant can be measured in vitro as the fold increase in the 50% and 90% inhibitory concentrations (IC₅₀ and IC₉₀ in cell-free assays) or the 50% and 90% effective concentrations (EC₅₀ and EC₉₀ in cell-culture systems), that is, the drug concentrations that inhibit the tested enzyme function or viral replication by 50% and 90%, respectively. Drug exposure is defined as the drug concentration achieved in vivo relative to the IC₅₀, IC₉₀, EC₅₀, or EC₉₀ of resistant variants. This measurement is a key determinant of the development of resistance in vivo. Indeed, if drug levels achieved in vivo are far above these IC/EC values, then resistant variants will be effectively inhibited even if they are far less susceptible than the wild-type virus in vitro. Therefore, the pharmacokinetics of the antiviral drugs and adherence to therapy are key in preventing treatment failure due to viral resistance.

Importance of Pharmacodynamics/Adherence to Interferon-Containing DAA Regimens

The importance of achieving high blood DAA concentrations to prevent the emergence of resistance was demonstrated in the first phase Ib trial with telaprevir, an NS3/4A protease inhibitor (PI) with a low genetic barrier to resistance, administered as monotherapy for 14 days.^[2] In this trial, patients were more likely to achieve either an HCV RNA plateau or virologic breakthrough during the dosing period due to selection of telaprevir-resistant variants if they received lower telaprevir doses (450 mg every 8 hours or 1250 mg every 12 hours) than if they received the higher dose (750 mg every 8 hours).^[2] However, outgrowth of resistant viral populations was only delayed in the latter group. In vivo fitness assessments showed that less resistant, but “fitter” variants were more likely to become the dominant species than more resistant, less “fit” HCV variants.^[2] These findings led the American and European regulatory agencies to limit monotherapy studies involving DAAs with low genetic barriers to resistance to only a few days. Therefore, no other studies provided sufficiently long administration to accurately assess the effect of drug exposure on the emergence of resistance.

Resistance to antiviral drugs is classically prevented by combining several drugs with potent antiviral activity and no cross-resistance. Indeed, HCV resistance to DAAs is observed significantly less frequently when one of these drugs is administered in combination with peginterferon and ribavirin.^[3,4] Therefore, the triple combination of peginterferon, ribavirin, and a PI—telaprevir or boceprevir—has become the new standard-of-care therapy for both treatment-naive and treatment-experienced patients with genotype 1 HCV infection.^[5-8] For the reasons defined above, it is crucial that optimal exposure to all 3 drugs in the regimen be achieved for these patients. Telaprevir must be taken at a dose of 750 mg every 8 hours with fatty food, whereas boceprevir must be taken at the dose of 800 mg every 8 hours with food. Patients must fully adhere to the regimen for the entire treatment period because any prolonged interruption in PI administration would inevitably result in a resurgence of wild-type viruses and the opportunity for resistant variants to acquire fitness, especially if the antiviral pressure exerted by peginterferon and ribavirin is only modest. However, if the patient does miss a dose of telaprevir or boceprevir, the package inserts provide guidance on how to manage these short interruptions. For telaprevir, the prescribing information recommends that a missed dose should be skipped if more than 4 hours have passed since the time it is usually taken; however, if it is within 4 hours of the time that it is usually taken, the missed dose should be taken immediately with high-fat food.^[5,6] The recommendation for boceprevir is similar, but the timing is different. A missed boceprevir dose should be skipped if it is fewer than 2 hours before the next dose is scheduled; if it is more than 2 hours before the next scheduled dose, the missed dose should be taken immediately with food.^[7,8]

The importance of adherence to peginterferon and ribavirin has been demonstrated in the absence of DAAs; these data showed that optimal response rates were observed in patients who achieved more than 80% of their prescribed peginterferon and ribavirin doses for more than 80% of the time.^[9] The impact of poor adherence to, or dose reductions of, peginterferon, ribavirin, or both has not been extensively studied in clinical trials with the triple combination of peginterferon, ribavirin, and telaprevir or boceprevir. Nevertheless, maintaining the dose of peginterferon is likely to be key in interferon-responsive patients treated with triple therapy because treatment failure primarily results from an inadequate response to peginterferon, leading to the uncontrolled outgrowth of resistant variants selected by the PI.^[1,10-12] By contrast, a retrospective analysis of patients completing 48 weeks of peginterferon/ribavirin therapy suggested that reducing the dose of ribavirin has a negative impact on the outcome of therapy only before HCV RNA becomes undetectable, whereas the impact is modest after HCV replication is controlled.^[13] More recent studies in patients receiving PI-based therapy have shown that modest ribavirin dose reductions do not impair the likelihood of treatment success.^[14,15] Finally, a recent study showed that the cost effectiveness of triple therapy is dependent on optimal adherence.^[16]

There are several strategies that can be used to optimize adherence rates in patients receiving DAA-based therapy, some of which are under investigation. Current strategies include patient education on the importance of adherence, interventions to reduce the adverse effects of therapy, addressing comorbidities that may affect adherence to therapy, and using a multidisciplinary team to help physicians implement all of the aforementioned strategies. Regarding patient education, a prospective, multicenter study was conducted to determine the influence of patient education on adherence to peginterferon plus ribavirin therapy in patients infected with HCV.^[17] Investigators showed that patient education can significantly influence adherence to treatment as evidenced by adherence rates to ribavirin of 56% at 6 months in 175 patients not receiving education vs 70% in 208 patients receiving education (P = .006).^[17] Therapeutic education included intervention by healthcare professionals other than the prescribing physician as well as the distribution of support documents and educational materials. Another study found that physician’s treatment experience and patient motivation were associated with improved adherence to HCV therapy, indicating that empowering patients to take charge of their own treatment can impact adherence.^[18] It should be noted, however, that no data are yet available on how these strategies may affect adherence to triple combinations with the HCV PIs.

Importance of Pharmacodynamics/Adherence to Interferon-Free DAA Regimens

Maintaining optimal adherence has been shown to be an extremely effective way of minimizing the development of resistance in the HIV field.^[19] Strategies under investigation that may help optimize adherence rates to interferon-free DAA therapy include less frequent dosing and ritonavir boosting. Many DAA agents in development have half-lives that may allow for twice- or even once-daily dosing, which is encouraging.

In HIV therapy, the use of low-dose ritonavir to improve the pharmacokinetics of HIV PIs (so-called ritonavir boosting) has become standard practice. NS3/4A PIs are cytochrome P450 3A substrates; therefore, their plasma concentrations can also be substantially improved when coadministered with low-dose ritonavir, which may allow for prolonged dosing intervals and subsequent increased adherence rates. Boosting has been studied with 3 PIs—danoprevir, narlaprevir, and ABT-450—with encouraging results that suggest such strategies may support once-daily dosing, reduce adverse events (by lowering the required dose of the HCV PI), and reduce the risk of resistance.^[20-23] Results with ABT-450 showed that higher plasma trough levels were associated with a lower likelihood of selecting resistant HCV variants over a short course of administration of 3 days.^[23] However, a number of first-generation PIs can achieve high and well-tolerated plasma concentrations with once- or twice-daily dosing without ritonavir boosting.

Interferon-free regimens will reduce toxicity and adverse effects associated with peginterferon and ribavirin therapy, possibly improving adherence rates. The results of a short-term study combining the NS3/4A PI GS-9256 and the nonnucleoside RNA-dependent RNA polymerase inhibitor tegobuvir has been disappointing because this combination of agents has a low genetic barrier to resistance and frequent early virologic breakthroughs were observed.^[24] Additional studies which have combined agents with low genetic barriers to resistance include the SOUND-C1 and SOUND-C2 studies. These trials combined the NS3/4A protease inhibitor BI 201335 and nonnucleoside polymerase inhibitor BI 207127, with or without ribavirin.^[25,26] The ZENITH study combined the NS5B polymerase inhibitor VX-222 plus telaprevir.^[27] In the SOUND-C2 study, treatment response rates, and likely the selection of resistant HCV variants, appeared to be influenced by the genetic background of the host (IL28B genotype).^[26] Regimens comprising agents with higher barriers to resistance, such as the cyclophilin inhibitor alisporivir,^[28] the combination of the nucleoside analogue inhibitor mericitabine and the PI danoprevir,^[29] and the combination of the nucleotide inhibitor PSI-7977 with ribavirin have shown more promising results.^[30] Indeed, the latter combination resulted in undetectable HCV RNA at 12 weeks post-therapy (SVR12) for 10 out of 10 genotype 2/3 HCV–infected treatment-naive patients receiving this regimen for 12 weeks.

Another interferon-free regimen comprising agents with low barriers to resistance—the first-generation NS3/4A PI asunaprevir and the NS5A inhibitor daclatasvir—has provided valuable information on the importance of drug exposure and how this differs depending on HCV subtype.^[31,32] Protease inhibitors have a low genetic barrier to resistance; they can select fit variants that are poorly controlled at the drug concentrations achieved by doses used in clinical trials and practice. This is also true for NS5A inhibitors in genotype 1a HCV, as suggested by in vitro by studies showing a major shift in the IC₅₀s induced by single amino acid substitutions in the NS5A sequence.^[33] As a result, virologic breakthrough due to the selection of HCV variants resistant to both drugs was observed within a few days to weeks in 6 of the 9 patients infected with genotype 1a HCV.^[31] By contrast, in genotype 1b HCV models in vitro, daclatasvir retains subnanomolar potency against all variants with single amino acid substitutions; the fold-change in IC₅₀s conferred by these substitutions in the presence of the inhibitor was substantially lower than in genotype 1a.^[33] Consequently, the drug concentrations achieved in vivo in genotype 1b HCV–infected patients were able to control NS5A variants carrying these substitutions, both in the absence or presence of associated substitutions conferring resistance to the PI. As a result, the combination of asunaprevir and daclatasvir was shown to lead to high sustained viral eradication rates (~ 90%) in patients infected with genotype 1b HCV.^[31,32] To date, the available data on interferon-free regimens are more informative regarding pharmacodynamics and exposure than adherence. Additional data are awaited.

Conclusions

Although data are scarce regarding drug exposure and adherence in the context of HCV treatment with new DAA-based therapies, preliminary data and lessons learned from the HIV field suggest that optimal drug exposure and maximal adherence will be crucial to success with DAA-based therapies. In addition, the data from interferon-free regimens emphasize the importance of drug exposure, through optimal dosing and strict adherence to the prescribed regimen, when using combinations of drugs with a low to moderate barrier to resistance. Drugs with a higher barrier to resistance, such as nucleos(t)ide analogues, cyclophilin inhibitors, or second-generation PIs may theoretically be more tolerant to weaker adherence, but data are lacking thus far. When these therapies are available in clinical practice, virologic failures may be observed more often than in strictly controlled clinical trials. Vigilance and thorough patient education will be required to ensure high cure rates.

References

1. Pawlotsky JM. Treatment failure and resistance with direct-acting antiviral drugs against hepatitis C virus. Hepatology. 2011;53:1742-1751.

2. Sarrazin C, Kieffer TL, Bartels D, et al. Dynamic hepatitis C virus genotypic and phenotypic changes in patients treated with the protease inhibitor telaprevir. Gastroenterology. 2007;132:1767-1777.

3. Hézode C, Forestier N, Dusheiko G, et al. Telaprevir and peginterferon with or without ribavirin for chronic HCV infection. N Engl J Med. 2009;360:1839-1850.

4. McHutchison JG, Everson GT, Gordon SC, et al. Telaprevir with peginterferon and ribavirin for chronic HCV genotype 1 infection. N Engl J Med. 2009;360:1827-1838.

5. Incivek [package insert]. Cambridge, MA: Vertex Pharmaceuticals; 2011.

6. Incivo [telaprevir]. European Medicines Agency. Available at: http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Product_Information/human/002313/WC500115529.pdf. Accessed February 16, 2012.

7. Victrelis [package insert]. Whitehouse Station, NJ: Merck & Company; 2011.

8. Victrelis [boceprevir]. European Medicines Agency. Available at: http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Product_Information/human/002332/WC500109786.pdf. Accessed February 16, 2012.

9. McHutchison JG, Manns M, Patel K, et al. Adherence to combination therapy enhances sustained response in genotype-1-infected patients with chronic hepatitis C. Gastroenterology. 2002;123:1061-1069.

10. Poordad F, McCone J Jr, Bacon BR, et al. Boceprevir for untreated chronic HCV genotype 1 infection. N Engl J Med. 2011;364:1195-1206.

11. Bacon BR, Gordon SC, Lawitz E, et al. Boceprevir for previously treated chronic HCV genotype 1 infection. N Engl J Med. 2011;364:1207-1217.

12. Zeuzem S, Andreone P, Pol S, et al. Telaprevir for retreatment of HCV infection. N Engl J Med. 2011;364:2417-2428.

13. Reddy KR, Shiffman ML, Morgan TR, et al. Impact of ribavirin dose reductions in hepatitis C virus genotype 1 patients completing peginterferon alfa-2a/ribavirin treatment. Clin Gastroenterol Hepatol. 2007;5:124-129.

14. Sulkowski MS, Poordad F, Manns MP, et al. Peginterferon alfa-2b/ribavirin with or without boceprevir is associated with higher SVR rates: analysis of previously untreated and previous treatment-failure patients. Program and abstracts of the 46th Annual Meeting of the European Association for the Study of the Liver; March 30 - April 3, 2011; Berlin, Germany. Poster 476.

15. Poordad F, Sulkowski MS, Reddy R, et al. Anemia had no effect on efficacy outcomes in treatment-naive patients who received telaprevir-based regimens in the ADVANCE and ILLUMINATE phase 3 studies. Program and abstracts of the Digestive Disease Week; May 7-10, 2011; Chicago, Illinois. Abstract 626.

16. Shan Liu SM, Cipriano LE, Holodniy M, et al. New protease inhibitors for the treatment of chronic hepatitis C. Ann Intern Med. 2012;156:279-290.

17. Cacoub P, Ouzan D, Melin P, et al. Patient education improves adherence to peg-interferon and ribavirin in chronic genotype 2 or 3 hepatitis C virus infection: a prospective, real-life, observational study. World J Gastroenterol. 2008;14:6195-6203.

18. Tanioka D, Iwasaki Y, Araki Y, et al. Factors associated with adherence to combination therapy of interferon and ribavirin for patients with chronic hepatitis C: importance of patient's motivation and physician's treatment experience. Liver Int. 2009;29:721-729.

19. Mocroft A, Phillips AN, Soriano V, et al. Reasons for stopping antiretrovirals used in an initial highly active antiretroviral regimen: increased incidence of stopping due to toxicity or patient/physician choice in patients with hepatitis C coinfection. AIDS Res Hum Retroviruses. 2005;21:743-752.

20. Gane E, Rouzier R, Stedman C, et al. Ritonavir boosting of low dose RG7227/ITMN-191, HCV NS3/4A protease inhibitor, results in robust reduction in HCV RNA at lower exposures than provided by unboosted regimens. Program and abstracts of the 45th Annual Meeting of the European Association for the Study of the Liver; April 14-18, 2010; Vienna, Austria. Abstract 38.

21. Rouzier R, Larrey D, Gane EJ, et al. Activity of danoprevir plus low-dose ritonavir (DNV/R) in combination with peginterferon alfa-2a (40KD) plus ribavirin (PEGIFNα-2A/RBV) in previous null responders. Program and abstracts of the 46th Annual Meeting of the European Association for the Study of the Liver; March 30 - April 3, 2011; Berlin, Germany. Abstract 62.

22. de Bruijne J, Bergmann JF, Reesink HW, et al. Antiviral activity of narlaprevir combined with ritonavir and pegylated interferon in chronic hepatitis C patients. Hepatology. 2010;52:1590-1599.

23. Pilot-Matias T, Tripathi R, Dekhtyar T, et al. Genotypic and phenotypic characterization of NS3 variants selected in HCV-infected patients treated with ABT-450. Program and abstracts of the 46th Annual Meeting of the European Association for the Study of the Liver; March 30 - April 3, 2011; Berlin, Germany. Abstract 1229.

24. Zeuzem S, Buggisch P, Agarwal K, et al. The protease inhibitor GS-9256 and non-nucleoside polymerase inhibitor tegobuvir alone, with RBV or peginterferon plus RBV in hepatitis C. Hepatology. 2011;[Epub ahead of print].

25. Zeuzem S, Asselah T, Angus PW, et al. High sustained virologic response following interferon-free treatment of chronic HCV GT1 infection for 4 weeks with HCV protease inhibitor BI201335, polymerase inhibitor BI207127 and ribavirin, followed by BI201335 and pegIFN/ribavirin—the SOUND-C1 study. Program and abstracts of the 62nd Annual Meeting of the American Association for the Study of Liver Diseases; November 5-8, 2011; San Francisco, California. Abstract 249.

26. Zeuzem S, Soriano V. Asselah T, et al. Virologic response to an interferon-free regimen of BI 201335 and BI 207127, with and without ribavirin, in treatment-naive patients with chronic genotype-1 HCV infection: Week 12 interim analysis of the SOUND-C2 study. Program and abstracts of the 62nd Annual Meeting of the American Association for the Study of Liver Diseases; November 5-8, 2011; San Francisco, California. Abstract LB15.

27. Nelson DR, Gane EJ, Jacobson IM, et al. VX-222/telaprevir in combination with peginterferon-alfa and ribavirin in treatment-naive genotype 1 HCV patients treated for 12 weeks: ZENITH study, SVR12 interim analysis. Program and abstracts of the 62nd Annual Meeting of the American Association for the Study of Liver Diseases; November 5-8, 2011; San Francisco, California. Abstract LB14.

28. Pawlotsky JM, Flisiak R, Rasenack J, et al. Once-daily alisporivir interferon (IFN)-free regimens achieve high rates of early HCV clearance in previously untreated patients with HCV genotype (G) 2 or 3. Program and abstracts of the 62nd Annual Meeting of the American Association for the Study of Liver Diseases; November 5-8, 2011; San Francisco, California. Abstract LB11.

29. Gane EJ, Roberts SK, Stedman CA, et al. Oral combination therapy with a nucleoside polymerase inhibitor (RG7128) and danoprevir for chronic hepatitis C genotype 1 infection (INFORM-1): a randomised, double-blind, placebo-controlled, dose-escalation trial. Lancet. 2010;376:1467-1475.

30. Gane EJ, Stedman CA, Hyland RH, et al. PSI-7977: ELECTRON. Interferon is not required for sustained virologic response in treatment-naive patients with HCV GT2 or GT3. Program and abstracts of the 62nd Annual Meeting of the American Association for the Study of Liver Diseases; November 5-8, 2011; San Francisco, California. Abstract 34.

31. Lok AS, Gardiner DF, Lawitz E, et al. Preliminary study of two antiviral agents for hepatitis C genotype 1. N Engl J Med. 2012;366:216-224.

32. Chayama K, Takahashi S, Kawakami Y, et al. Dual oral combination therapy with the NS5A inhibitor daclatasvir (DCV; BMS-790052) and the NS3 protease inhibitor asunaprevir (ASV; BMS-650032) achieved 90% sustained virologic response (SVR12) in Japanese HCV genotype 1b–infected null responders. Program and abstracts of the 62nd Annual Meeting of the American Association for the Study of Liver Diseases; November 5-8, 2011; San Francisco, California. Abstract LB4.

33. Fridell RA, Qiu D, Wang C, Valera L, Gao M. Resistance analysis of the hepatitis C virus NS5A inhibitor BMS-790052 in an in vitro replicon system. Antimicrob Agents Chemother. 2010;54:3641-3650.

Source

Importance of Pharmacokinetics and Adherence to Avoid Emergence of Resistant HCV Variants

Jean-Michel Pawlotsky, MD, PhD
Professor, Department of Virology
Henri Mondor Hospital
Université Paris Est
Créteil, France

Introduction

Hepatitis C virus (HCV) resistance to a direct-acting antiviral (DAA) agent corresponds to the selection during treatment of viral variants that bear amino acid substitutions that alter the drug target; therefore, they are less susceptible to the inhibitory activity of the drug. These drug-resistant variants preexist as minor populations within the patient’s HCV quasispecies. Drug exposure profoundly inhibits replication of the dominant “wild-type” drug-sensitive viral population, and the resistant variants gradually occupy the vacant replication space. Moreover, viruses with low-level or partial resistance that can continue to replicate in the presence of drug, often favored by suboptimal drug exposure, may accumulate further mutations, leading to stepwise decreases in drug susceptibility, albeit often at a cost of reduced replicative capacity. If insufficient antiviral activity is provided because of suboptimal dosing or adherence, inadequate virologic suppression and the selection of resistance is inevitable. Therefore, to reduce the development of resistance, it is essential to achieve optimal drug concentrations through proper dosing and maximal adherence.

Factors That Influence Viral Resistance in vivo

In vivo, viral resistance is influenced by 3 major related factors: the genetic barrier to resistance, the in vivo fitness of the resistant viral population, and drug exposure.^[1]

The genetic barrier to resistance is defined as the number of amino acid substitutions needed for a viral variant to acquire full resistance to the drug in question. If a single substitution is sufficient to confer high-level resistance to a specific drug, the drug is considered to have a low genetic barrier to resistance, whereas 3 or more substitutions are required to confer resistance to a drug with a high genetic barrier. There is a low likelihood that variants bearing a large number of resistance substitutions will preexist in a given patient and be fit enough to replicate at high levels when an antiviral drug is administered. Therefore, drugs with a high genetic barrier to resistance are less likely to be associated with clinically meaningful resistance.

The in vivo fitness of the viral variant is defined as its ability to survive and grow in the replicative environment. A selected resistant variant must have the capacity to propagate to fill in the replication space left vacant by the elimination of a susceptible wild-type virus during drug exposure. Thus, a highly resistant but poorly “fit” virus will be less clinically significant than a less resistant but “fitter” virus that can replicate efficiently in the presence of the drug. The acquisition of compensatory mutations may restore the fitness of a resistant variant and allow it to replicate efficiently in the presence of the drug, possibly allowing it to persist after drug withdrawal.

Finally, drug exposure affects the development of drug resistance. The degree of drug resistance of a variant can be measured in vitro as the fold increase in the 50% and 90% inhibitory concentrations (IC₅₀ and IC₉₀ in cell-free assays) or the 50% and 90% effective concentrations (EC₅₀ and EC₉₀ in cell-culture systems), that is, the drug concentrations that inhibit the tested enzyme function or viral replication by 50% and 90%, respectively. Drug exposure is defined as the drug concentration achieved in vivo relative to the IC₅₀, IC₉₀, EC₅₀, or EC₉₀ of resistant variants. This measurement is a key determinant of the development of resistance in vivo. Indeed, if drug levels achieved in vivo are far above these IC/EC values, then resistant variants will be effectively inhibited even if they are far less susceptible than the wild-type virus in vitro. Therefore, the pharmacokinetics of the antiviral drugs and adherence to therapy are key in preventing treatment failure due to viral resistance.

Importance of Pharmacodynamics/Adherence to Interferon-Containing DAA Regimens

The importance of achieving high blood DAA concentrations to prevent the emergence of resistance was demonstrated in the first phase Ib trial with telaprevir, an NS3/4A protease inhibitor (PI) with a low genetic barrier to resistance, administered as monotherapy for 14 days.^[2] In this trial, patients were more likely to achieve either an HCV RNA plateau or virologic breakthrough during the dosing period due to selection of telaprevir-resistant variants if they received lower telaprevir doses (450 mg every 8 hours or 1250 mg every 12 hours) than if they received the higher dose (750 mg every 8 hours).^[2] However, outgrowth of resistant viral populations was only delayed in the latter group. In vivo fitness assessments showed that less resistant, but “fitter” variants were more likely to become the dominant species than more resistant, less “fit” HCV variants.^[2] These findings led the American and European regulatory agencies to limit monotherapy studies involving DAAs with low genetic barriers to resistance to only a few days. Therefore, no other studies provided sufficiently long administration to accurately assess the effect of drug exposure on the emergence of resistance.

Resistance to antiviral drugs is classically prevented by combining several drugs with potent antiviral activity and no cross-resistance. Indeed, HCV resistance to DAAs is observed significantly less frequently when one of these drugs is administered in combination with peginterferon and ribavirin.^[3,4] Therefore, the triple combination of peginterferon, ribavirin, and a PI—telaprevir or boceprevir—has become the new standard-of-care therapy for both treatment-naive and treatment-experienced patients with genotype 1 HCV infection.^[5-8] For the reasons defined above, it is crucial that optimal exposure to all 3 drugs in the regimen be achieved for these patients. Telaprevir must be taken at a dose of 750 mg every 8 hours with fatty food, whereas boceprevir must be taken at the dose of 800 mg every 8 hours with food. Patients must fully adhere to the regimen for the entire treatment period because any prolonged interruption in PI administration would inevitably result in a resurgence of wild-type viruses and the opportunity for resistant variants to acquire fitness, especially if the antiviral pressure exerted by peginterferon and ribavirin is only modest. However, if the patient does miss a dose of telaprevir or boceprevir, the package inserts provide guidance on how to manage these short interruptions. For telaprevir, the prescribing information recommends that a missed dose should be skipped if more than 4 hours have passed since the time it is usually taken; however, if it is within 4 hours of the time that it is usually taken, the missed dose should be taken immediately with high-fat food.^[5,6] The recommendation for boceprevir is similar, but the timing is different. A missed boceprevir dose should be skipped if it is fewer than 2 hours before the next dose is scheduled; if it is more than 2 hours before the next scheduled dose, the missed dose should be taken immediately with food.^[7,8]

The importance of adherence to peginterferon and ribavirin has been demonstrated in the absence of DAAs; these data showed that optimal response rates were observed in patients who achieved more than 80% of their prescribed peginterferon and ribavirin doses for more than 80% of the time.^[9] The impact of poor adherence to, or dose reductions of, peginterferon, ribavirin, or both has not been extensively studied in clinical trials with the triple combination of peginterferon, ribavirin, and telaprevir or boceprevir. Nevertheless, maintaining the dose of peginterferon is likely to be key in interferon-responsive patients treated with triple therapy because treatment failure primarily results from an inadequate response to peginterferon, leading to the uncontrolled outgrowth of resistant variants selected by the PI.^[1,10-12] By contrast, a retrospective analysis of patients completing 48 weeks of peginterferon/ribavirin therapy suggested that reducing the dose of ribavirin has a negative impact on the outcome of therapy only before HCV RNA becomes undetectable, whereas the impact is modest after HCV replication is controlled.^[13] More recent studies in patients receiving PI-based therapy have shown that modest ribavirin dose reductions do not impair the likelihood of treatment success.^[14,15] Finally, a recent study showed that the cost effectiveness of triple therapy is dependent on optimal adherence.^[16]

There are several strategies that can be used to optimize adherence rates in patients receiving DAA-based therapy, some of which are under investigation. Current strategies include patient education on the importance of adherence, interventions to reduce the adverse effects of therapy, addressing comorbidities that may affect adherence to therapy, and using a multidisciplinary team to help physicians implement all of the aforementioned strategies. Regarding patient education, a prospective, multicenter study was conducted to determine the influence of patient education on adherence to peginterferon plus ribavirin therapy in patients infected with HCV.^[17] Investigators showed that patient education can significantly influence adherence to treatment as evidenced by adherence rates to ribavirin of 56% at 6 months in 175 patients not receiving education vs 70% in 208 patients receiving education (P = .006).^[17] Therapeutic education included intervention by healthcare professionals other than the prescribing physician as well as the distribution of support documents and educational materials. Another study found that physician’s treatment experience and patient motivation were associated with improved adherence to HCV therapy, indicating that empowering patients to take charge of their own treatment can impact adherence.^[18] It should be noted, however, that no data are yet available on how these strategies may affect adherence to triple combinations with the HCV PIs.

Importance of Pharmacodynamics/Adherence to Interferon-Free DAA Regimens

Maintaining optimal adherence has been shown to be an extremely effective way of minimizing the development of resistance in the HIV field.^[19] Strategies under investigation that may help optimize adherence rates to interferon-free DAA therapy include less frequent dosing and ritonavir boosting. Many DAA agents in development have half-lives that may allow for twice- or even once-daily dosing, which is encouraging.

In HIV therapy, the use of low-dose ritonavir to improve the pharmacokinetics of HIV PIs (so-called ritonavir boosting) has become standard practice. NS3/4A PIs are cytochrome P450 3A substrates; therefore, their plasma concentrations can also be substantially improved when coadministered with low-dose ritonavir, which may allow for prolonged dosing intervals and subsequent increased adherence rates. Boosting has been studied with 3 PIs—danoprevir, narlaprevir, and ABT-450—with encouraging results that suggest such strategies may support once-daily dosing, reduce adverse events (by lowering the required dose of the HCV PI), and reduce the risk of resistance.^[20-23] Results with ABT-450 showed that higher plasma trough levels were associated with a lower likelihood of selecting resistant HCV variants over a short course of administration of 3 days.^[23] However, a number of first-generation PIs can achieve high and well-tolerated plasma concentrations with once- or twice-daily dosing without ritonavir boosting.

Interferon-free regimens will reduce toxicity and adverse effects associated with peginterferon and ribavirin therapy, possibly improving adherence rates. The results of a short-term study combining the NS3/4A PI GS-9256 and the nonnucleoside RNA-dependent RNA polymerase inhibitor tegobuvir has been disappointing because this combination of agents has a low genetic barrier to resistance and frequent early virologic breakthroughs were observed.^[24] Additional studies which have combined agents with low genetic barriers to resistance include the SOUND-C1 and SOUND-C2 studies. These trials combined the NS3/4A protease inhibitor BI 201335 and nonnucleoside polymerase inhibitor BI 207127, with or without ribavirin.^[25,26] The ZENITH study combined the NS5B polymerase inhibitor VX-222 plus telaprevir.^[27] In the SOUND-C2 study, treatment response rates, and likely the selection of resistant HCV variants, appeared to be influenced by the genetic background of the host (IL28B genotype).^[26] Regimens comprising agents with higher barriers to resistance, such as the cyclophilin inhibitor alisporivir,^[28] the combination of the nucleoside analogue inhibitor mericitabine and the PI danoprevir,^[29] and the combination of the nucleotide inhibitor PSI-7977 with ribavirin have shown more promising results.^[30] Indeed, the latter combination resulted in undetectable HCV RNA at 12 weeks post-therapy (SVR12) for 10 out of 10 genotype 2/3 HCV–infected treatment-naive patients receiving this regimen for 12 weeks.

Another interferon-free regimen comprising agents with low barriers to resistance—the first-generation NS3/4A PI asunaprevir and the NS5A inhibitor daclatasvir—has provided valuable information on the importance of drug exposure and how this differs depending on HCV subtype.^[31,32] Protease inhibitors have a low genetic barrier to resistance; they can select fit variants that are poorly controlled at the drug concentrations achieved by doses used in clinical trials and practice. This is also true for NS5A inhibitors in genotype 1a HCV, as suggested by in vitro by studies showing a major shift in the IC₅₀s induced by single amino acid substitutions in the NS5A sequence.^[33] As a result, virologic breakthrough due to the selection of HCV variants resistant to both drugs was observed within a few days to weeks in 6 of the 9 patients infected with genotype 1a HCV.^[31] By contrast, in genotype 1b HCV models in vitro, daclatasvir retains subnanomolar potency against all variants with single amino acid substitutions; the fold-change in IC₅₀s conferred by these substitutions in the presence of the inhibitor was substantially lower than in genotype 1a.^[33] Consequently, the drug concentrations achieved in vivo in genotype 1b HCV–infected patients were able to control NS5A variants carrying these substitutions, both in the absence or presence of associated substitutions conferring resistance to the PI. As a result, the combination of asunaprevir and daclatasvir was shown to lead to high sustained viral eradication rates (~ 90%) in patients infected with genotype 1b HCV.^[31,32] To date, the available data on interferon-free regimens are more informative regarding pharmacodynamics and exposure than adherence. Additional data are awaited.

Conclusions
Although data are scarce regarding drug exposure and adherence in the context of HCV treatment with new DAA-based therapies, preliminary data and lessons learned from the HIV field suggest that optimal drug exposure and maximal adherence will be crucial to success with DAA-based therapies. In addition, the data from interferon-free regimens emphasize the importance of drug exposure, through optimal dosing and strict adherence to the prescribed regimen, when using combinations of drugs with a low to moderate barrier to resistance. Drugs with a higher barrier to resistance, such as nucleos(t)ide analogues, cyclophilin inhibitors, or second-generation PIs may theoretically be more tolerant to weaker adherence, but data are lacking thus far. When these therapies are available in clinical practice, virologic failures may be observed more often than in strictly controlled clinical trials. Vigilance and thorough patient education will be required to ensure high cure rates.

References

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