Hepatitis C virus (HCV) is a major cause of chronic liver disease, with an estimated 170 million people infected worldwide . HCV, identified in 1989, is an enveloped virus with a 9.6 kb single-stranded RNA genome , a member of the Flaviviridae family, genus Hepacivirus. The development of new molecules such as HCV enzyme inhibitors (called direct acting antivirals (DAA)) is ongoing . The aim of this review is to summarize results obtained with DAA.
Chronic hepatitis C is one of the leading causes of chronic liver disease with approximately 170 million people infected worldwide. Sustained virological response (SVR) is equivalent to viral eradication and associated with a reduction in the risk of cirrhosis. Nowadays the treatment for hepatitis C virus (HCV) genotype 1 chronic infection is the addition of direct acting antivirals (DAA) with a protease inhibitor (telaprevir or boceprevir) to the pegylated interferon (PEG-IFN) plus ribavirin (RBV) regimen. The future management of patients with these new molecules will require good clinical practice, knowledge of indications, management of side effects and monitoring for antiviral resistance. Certain major medical needs are still unmet and require studies in special populations (HIV-HCV coinfected patients, transplanted patients, etc.…) and also in HCV non-1 genotype patients and in non-responders. Second generation DAA are in development. Combinations of antivirals with additive potency that lack cross resistance and with a good safety profile may provide new regimens in the future to make HCV the first chronic viral infection eradicated worldwide with a finite duration of combination DAA therapy without IFN. The aim of this review is to summarize mechanisms of action and results obtained with DAAs.
direct acting antivirals
end of treatment Response
early virological response
rapid virological response
sustained virological response
The HCV lifecycle begins with virion attachment to its specific receptor The HCV RNA genome serves as a template for viral replication and as a viral messenger RNA for viral production. It is translated into a polyprotein that is cleaved by proteases. Then, viral assembly occurs. Potentially, each step of the viral cycle is a target for drug development (Fig. 1). The knowledge of the structures of HCV protease and HCV polymerase has allowed structure-based drug design to develop inhibitors to these enzymes [4, 5]. Several findings suggest that HCV modulation of IFN induction and signalling attenuates the expression of IFN-stimulated genes, allowing HCV to escape the antiviral actions of the host response [6, 7].
Viral targets for drug development
All the HCV enzymes – NS2-3 and NS3-4A proteases, NS3 helicase and NS5B RdRp – are essential for HCV replication, and are potential drug discovery targets (Fig. 2). Therefore DAA with different viral targets, including NS3 protease inhibitors, nucleoside/nucleotide analogue and non-nucleoside inhibitors of the RNA-dependent RNA polymerase, and NS5A inhibitors are under development. General characteristics of different classes of DAA are indicated in Table 1.
|Viral efficacy||Genotype specificity||Genetic barrier|
|Non Nucleoside |
The NS3 serine protease is located in the N-terminal region of NS3. The NS3 serine protease domain associates with the NS4A cofactor to cleave four specific sites. This enzyme has been extensively characterized at the biochemical level and its structure is known [4, 5]. The serine protease activity of NS3 is an attractive target for new drugs that could effectively block viral replication.
The NS3/4A protease inhibitors can be divided into two chemical classes: macrocyclic inhibitors and linear tetra-peptide α-ketoamid derivatives. In 2003, a protease inhibitor (BILN 2061) that blocks HCV replication in the replicon model was shown to be effective in humans [8-10].
Although proteases inhibitors have a high anti-viral efficacy, they have several potential limitations. Protease inhibitors are highly specific and since the amino acid sequence of the NS3 protease domain differs significantly between HCV genotypes, their antiviral efficacy differs in different genotypes. Indeed, telaprevir is less effective in treatment-naïve patients infected with genotypes other than genotype 1.
Furthermore, since HCV has a high mutation replication rate, with a lack of proof reading, resistance is an issue.
The genetic barrier to resistance is defined as the number of amino acid substitutions required to confer full resistance to a drug. Usually, DAA with a low genetic barrier to resistance require only one or two amino acid substitutions for high resistance. DAA with a high barrier of resistance usually require three or more amino acid substitutions in the same region.
The genetic barrier to protease inhibitors is usually low. Resistance differs significantly between HCV subtypes. Viral resistance to telaprevir occurred much more frequently in genotype 1a compared to genotype 1b. This result of nucleotide differences at position 155 in HCV subtype 1a (AGA, encodes R) vs. 1b (CGA, also encodes R). The mutation most frequently associated with resistance to telaprevir was R155K; changing R to K at position 155 requires one nucleotide change in HCV subtype 1a and two nucleotide changes in subtype 1b isolates .
As illustrated with the R155K mutation, which reduces replication capacity in the replicon model , resistance mutations frequently impair viral fitness. However, under antiviral pression, during therapy second site mutations are selected that restore fitness, explaining why the R155K primary mutation is frequently found in association with V36M in genotype 1a viruses. Therefore, it is recommended to stop rapid treatment in patients with viral breakthrough and good adherence to therapy.
Polymerase inhibitors interfere with viral replication by binding to the NS5B RNA-dependent RNA polymerase. NS5B RNA polymerase inhibitors can be divided into two distinct categories–nucleosides inhibitors and non-nucleotides inhibitors.
Nucleoside analogue inhibitors mimic the natural substrates of the polymerase and are incorporated into the RNA chain causing direct chain termination [13, 14]. Nucleoside analogue polymerase inhibitors are compounds that require conversion to an active triphosphate form. Since the active site of NS5B is highly conserved, nucleoside analogue inhibitors are potentially effective against all the different genotypes. Moreover, single amino acid substitutions in every position of the active site may result in loss of function. Resistance to nucleoside analogue inhibitors.
Non-nucleoside inhibitors is usually low bind to several discrete sites on the HCV polymerase, which results in conformational protein change before the elongation complex is formed [13, 14]. NS5B is structurally organized in a characteristic ‘right-hand motif’ containing finger, palm and thumb domains, and offers at least four NNI-binding sites, a benzimidazole (thumb 1)-binding, thiophene (thumb 2)-binding, benzothiadiazine (palm 1)-binding and benzofuran-(palm 2)-binding site. Resistance is more frequent with non-nucleoside inhibitors. Furthermore, mutations at non-nucleoside inhibitor-binding sites do not necessarily lead to impaired function of the enzyme.
The NS5A is a membrane-associated phosphoprotein present in basally phosphorylated (p56) and hyperphosphorylated (p58) forms . It was previously reported that only p58-defective mutants could be complemented in trans, and NS5A is involved in HCV virion production, suggesting that different forms of NS5A exert multiple functions at various stages of the viral life cycle. The N terminus of NS5A (domain I) has been crystallized in alternative dimeric forms and contains both zinc- and RNA-binding domains, properties that have been demonstrated in vitro. NS5A has been shown to interact with a number of host proteins and plays a role in interferon resistance in vivo. This compound is active at picomolar concentrations in vitro towards replicons expressing a broad range of HCV genotypes and acts in an additive to synergistic fashion with interferon and other small molecule antiviral compounds [15, 16]. The resistance profile of BMS-790052 reveals inhibitor sensitivity maps to the N terminus of domain 1 of NS5A . In addition, as well as being an active-site inhibitor that specifically binds NS3 protease, it has been demonstrated that NS5A inhibitors could block hyperphosphorylation of NS5A, which is believed to play an essential role in the viral life cycle.
First generation protease inhibitors
Proteases inhibitors that have been approved or are currently under development in phase II or III trials are indicated in Table 2.
|Telaprevir||Vertex / Tibotec||Approved|
|BI 201335||Boehringer Ingelheim Pharma||Phase III|
|Vaniprevir (MK-7009)||Merck||Phase II|
|RG7227 (Danoprevir)||InterMune / Genentech||Phase II|
|ABT-450||Abbott / Enanta||Phase II|
|BMS 650032||Bristol-Myers Squibb||Phase II|
Triple therapy with telaprevir, PEG-IFN plus RBV
In 2011, two NS3/4A protease inhibitors, telaprevir and boceprevir, were approved in Europe and the United States for use in combination with PEG-IFN/RBV [([17-21], commented in [16, 22]] for the treatment of genotype 1 chronic hepatitis C in both treatment-naïve and experienced patients. The Advance study was a 3-arm double-blind, randomized, placebo-controlled Phase 3 study assessing the efficacy and safety of two telaprevir-based response-guided regimens compared with PEG-IFN alfa-2a/RBV in treatment-naïve patients with chronic genotype 1 HCV infection . A significantly greater proportion of patients achieved SVR with the 12-week and 8-week telaprevir-based combination regimens (75% and 69% respectively) compared to the PEG-IFN alfa-2a/RBV 48 week control arm (44%, P < 0.001) (Fig. 3).
The most common adverse events in the telaprevir arms were fatigue, pruritus, nausea, headache, anaemia, rash, influenza-like illness, insomnia, pyrexia and diarrhoea. Discontinuation of treatment because of adverse events occurred in 7% and 8% of patients in the telaprevir regimens and 4% in PEG-IFN alfa-2a/RBV.
Finally, SVR rates improved with telaprevir-based therapy in genotype 1 treatment-naïve patients. The benefit-risk profile with a 12-week telaprevir-based regimen was better than with an 8-week regimen. With response guided treatment (RGT) nearly two-thirds of treatment-naïve patients were eligible for a 24-week treatment regimen, and achieved high SVR rates. Discontinuation of treatment because of rash was minimized by stopping medication sequentially.
The Illuminate study was a phase 3 open label study which randomized patients who achieved an eRVR into two durations of therapy . RVR was achieved in 72% of patients; 65.2% of patients achieved an eRVR. A total of 322 (59.6%) patients were randomized (1:1) to either a 24 or 48-week arm. The SVR rate was 92% in patients randomized to 24 weeks and 87.5% in patients randomized to 48 weeks (Fig. 4). Thirty-six patients (6.7%) discontinued treatment owing to virological failure. Ninety-four patients (17.4%) permanently discontinued all study drugs because of adverse events. Fatigue and anaemia were the most common adverse events leading to discontinuation.
Finally, the 24-week telaprevir-based regimen was non-inferior to the 48-week telaprevir-based regimen in patients who achieved an eRVR, (92% SVR compared to 87.5%). Thus, RGT resulted in an overall SVR of 71.9% SVR and nearly two-thirds of the patients were eligible for shorter treatment. These results support the use of RGT for telaprevir-based regimens in treatment-naïve patients.
What have we learned from the telaprevir studies in treatment-naïve genotype 1 patients?
Firstly, the duration of therapy can be shortened in treatment-naive genotype 1 patients who achieve an eRVR. One major result of these studies was that approximately two-thirds of patients achieved an RVR and remained HCV RNA negative through 24 weeks, thus benefiting from 24 weeks of treatment. Treatment-naïve patients with cirrhosis who have undetectable HCV RNA at weeks 4 and 12 may benefit from an additional 36 weeks of PEG-IFN/RBV (48 weeks total).
Secondly, knowledge of the stopping rules is important. Discontinuation of therapy is recommended in all patients with HCV RNA levels of 1000 IU/ml or more at treatment week 4 or 12; or those with confirmed detectable HCV RNA levels at treatment week 24, because of the high risk of resistance.
Can either interferon (2a or 2b) be used with either protease inhibitor?
Yes, certainly. In a prospective, multicentre, randomized, open label, phase 2 clinical trial study including 161 HCV genotype 1 patients, a high proportion (> 80%) of patients achieved a SVR regardless of the frequency of telaprevir dosing (q8 or q12 h) or type of PEG-IFN alfa used (alfa-2a or alfa-2b) . Since each PEG-IFN results in approximately the same SVR , either IFN can be utilized with either protease inhibitor.
The Realize study was a phase 3, randomized, double-blind, placebo-controlled study in 662 genotype 1 experienced chronic hepatitis C patients . SVR rates for the telaprevir simultaneous start arm and the delayed start arm were 64% and 66% respectively, overall, based on an intent-to-treat analysis. Primary analysis showed that the SVR rates for the telaprevir simultaneous start arm, delayed start arm and control arm respectively, were 83%, 88% and 24% in relapsers; 59%, 54% and 15% in partial responders; and 29%, 33% and 5% in non-responders (Fig. 5). Prior relapsers could be treated for 24 weeks if they achieved an eRVR. About 76% of prior relapsers achieved an eRVR and 95% of these achieved a SVR. Partial responders and non-responders should be treated for 48 weeks. Non-responders could wait for future combination treatments. Although the mechanisms of non-response to IFN are not well understood the addition of a protease inhibitor could partially restore IFN responsiveness .
Approximately 90% of all rashes were mild or moderate (grades 1 and 2), although severe rash (grade 3) developed in 6% of patients, leading to discontinuation of telaprevir . Grade 3–4 skin lesions require immediate treatment discontinuation and consultation with an experienced dermatologist. Frequent physical examinations and laboratory testing for anaemia must be performed to adapt dosing in these regimens. HCV RNA must be measured frequently with real-time PCR to monitor drug resistance. A viral breakthrough in a compliant patient is very likely to be drug resistance and is a stopping rule.
Triple therapy with boceprevir, PEG-IFN plus ribavirin
Boceprevir is a linear peptidomimetic ketoamide serine protease inhibitor that binds reversibly to the HCV non-structural 3 (NS3) active site.
The Sprint-2 study was a phase 3 international double-blind randomized study including genotype 1 treatment-naïve patients that compared triple therapy with boceprevir (with or without a 4-week lead-in treatment with PEG-IFN alfa-2b/RBV) to PEG-IFN alfa-2b/RBV plus placebo . The SVR in non-Black patients was 40% for 48 weeks of PEG-IFN alfa-2b/RBV and was significantly higher in both boceprevir arms: 67% for RGT and 68% for the lead-in phase followed by 44 weeks of boceprevir plus PEG-IFN alfa-2b/RBV. The corresponding SVR in Black patients was 23%, 42% and 53% respectively. SVR rates in non-Black patients receiving ≥ 1 dose of boceprevir or placebo were 42%, 70% and 71% (Fig. 6). Anaemia was reported in 29% of controls vs. 49% in the boceprevir arms, leading to a dose reduction in 13% and 21% and discontinuation in 1% and 2% respectively.
In conclusion, boceprevir plus PEG-IFN alfa-2b/RBV significantly increased the SVR (approximately 70%) in both arms compared to PEG-IFN alfa-2b/RBV. Although anaemia occurred more often with boceprevir it rarely led to treatment discontinuation. Compared to 44 weeks of triple therapy after the lead-in period, RGT with lead-in plus 24 weeks of boceprevir plus PEG-IFN alfa-2b/RBV ± 20 PEG-IFN alfa-2b/RBV resulted in comparable SVR rates.
What have we learned from this study of boceprevir in genotype 1 treatment-naïve patients?
Firstly, there was a very high response rate. Boceprevir plus PEG-IFN alfa-2b/RBV significantly increased SVR (to approximately 70%) in both treatment arms compared to SOC. The treatment duration in treatment-naïve genotype 1 patients who achieve an RVR can be shortened. Approximately two-thirds of patients achieved an RVR and remained HCV RNA negative through 24 weeks of treatment, thus benefiting from 24 to 28 weeks of treatment.
Baseline predictors of non-response were age over 40 years, Black ethnicity, baseline HCV RNA levels > 400 000 IU/ml, the presence of cirrhosis and not receiving boceprevir. A decrease in HCV RNA levels by 1 log10 IU/ml or more at the end of the 4-week lead-in period was strongly predictive of a SVR. In an expanded model that included response data during the treatment period, the highest odds ratio was found when HCV RNA levels were undetectable at week 4 or had decreased by 1 log10 IU/ml or more from baseline. SVR rates in patients with advanced fibrosis were lower than in those with mild fibrosis, although the numbers of patients with a Metavir fibrosis score of 3 or 4 (indicating bridging fibrosis or cirrhosis) were small, particularly in the cohort of Black patients.
The RVR was a strong predictor of SVR and failure to achieve an EVR was a strong predictor of non-SVR, independent of the patients’ pretreatment status . When RVR was added to baseline characteristics, prediction of SVR was more accurate. The combination of RVR and EVR provided complementary information, and thus provides a key opportunity to individualize treatment and improve the benefit/risk ratio of therapy.
Secondly, lessons obtained from the strategy of the lead-in phase. In one arm, boceprevir was added after lead-in treatment with PEG-IFN/RBV alone. Theoretically, a lead-in phase could lower HCV RNA levels before exposure to the protease inhibitor, thus reducing the risk of viral breakthrough or resistance to DAA, as noted in a phase 2 study which compared lead-in plus boceprevir to boceprevir without a lead-in. Patients with a poor response to IFN, defined as a reduction in HCV RNA levels of less than 1log10 IU/ml after 4 weeks could wait for multiple DAA combinations and avoid protease monotherapy, unless they have cirrhosis. The SVR rates in patients with an RVR are high and they might not require a protease inhibitor, but could benefit from PEG-IFN/RBV alone.
Thirdly, the safety profile. Dysgeusia, and anaemia were more frequent in the boceprevir groups than in the control group. Boceprevir regimens were associated with increased rates of anaemia, and nearly twice as many boceprevir recipients as controls had a haemoglobin level of less than 9.5 g/dl or received erythropoietin (43% vs. 24%). In patients receiving erythropoietin, the average duration of use was shortest in group 2. Neither the incidence of serious adverse events nor the frequency of discontinuation due to adverse events differed significantly between patients receiving boceprevir and those receiving SOC.
The SVR rates in patients with a poor response to IFN, defined as a reduction in HCV RNA of less than 1log10 IU/ml after 4 weeks of PEG-IFN/RBV therapy, were sufficiently high to dispel concern that the addition of a protease inhibitor to the treatment regimen would be equivalent to functional monotherapy. However, these patients were less likely than patients with a strong response to IFN to have a SVR after boceprevir. Thus, patients with a poor response to IFN should be monitored closely to determine who can benefit from better therapies, once they are available.
What about resistance?
During monotherapy, viral variants with substitutions at the six main positions (36, 54, 55, 155, 156 and 170) within the NS3 protease have been selected. Phenotypic analyses of resistance based on in vitro replicons showed different levels of resistance conferred by substitutions at these different positions. Changes at positions 36, 54, 55, 155 and 156 conferred moderate resistance . Cross resistance studies have shown that most of the known resistance mutations confer resistance to most of the protease inhibitors under development.
HCV resistance to boceprevir is significantly less frequent when administered in combination with PEG-IFN/RBV. The Sprint-1 phase II study showed that administering a standard dose PEG-IFN/RBV and boceprevir are crucial to improve SVR in genotype 1 treatment-naïve patients, and that RBV is needed to prevent resistance . In the past with the PEG-IFN plus RBV treatment, premature discontinuation of RBV was associated with a significant increase in relapse , and even though the mechanism of action of RBV is poorly understood, this drug appears to be important in future and near future combinations.
Respond 2 study
The final results of this trial showed that combination therapy with boceprevir resulted in higher SVR rates in patients with HCV genotype 1 who did not respond to or relapsed after treatment with PEG-IFN and RBV . In this trial, three arms were randomly defined from 403 HCV genotype 1 patients who previously failed treatment–partial/non-responders or relapsers. The SVR was significantly higher in the two boceprevir groups (group 2, 59%; group 3, 66%) than in the control group (21%). The SVR in patients with undetectable HCV RNA levels at week 8 was 86% after 32 weeks of triple therapy and 88% after 44 weeks of triple therapy. The SVR in the 102 patients whose HCV RNA levels decreased by less than 1 log10 IU/ml at treatment week 4 were 0%, 33%, and 34% in groups 1, 2, and 3 respectively. Anaemia was significantly more common in the boceprevir groups than in the control group, and erythropoietin was administered to 41–46% of boceprevir-treated patients and 21% of controls.
The SVR in patients with prior relapse was 29% in group 1, vs. 69% and 75% in groups 2 and 3 respectively. The corresponding rates in patients with a prior non-response were 7% vs. 40% and 52% respectively. A total of 102 patients (15%, 28%, and 27% in groups 1, 2 and 3 respectively) had a poor response to IFN, defined as a decrease in HCV RNA levels of less than 1log10 IU/ml after the 4-week lead-in (Fig. 7). None of the patients in group 1 of this subgroup achieved a SVR, although 33% and 34% achieved an SVR in groups 2 and 3 respectively. The SVR in patients with a response to IFN (a decrease in HCV RNA level of at least 1 log10 IU/ml at week 4), was 25%, 73% and 79% in groups 1, 2 and 3 respectively.
An assessment for amino acid variants associated with reduced susceptibility to boceprevir was performed in 114 patients in groups 2 or 3 who did not achieve an SVR. Post-baseline data were available for 98 of the 114 patients (86%) and variants were detected in 43 (44%). The rate of amino acid variants associated with reduced susceptibility to boceprevir was higher in patients with a poor response to IFN than in those with a good response.
The EVR (i.e., undetectable HCV RNA at week 8) was associated with a high SVR rate in all three groups. The proportion of patients with undetectable HCV RNA levels at week 8 in the boceprevir groups was approximately six times that in group 1. The factors associated with SVR were: receiving boceprevir, previous relapse, low viral load at baseline and the absence of cirrhosis. When the decline in viral load at week 4 was added to the model, the week 4 response was a stronger predictor of SVR than the historical response.
There was a higher incidence of anaemia in the boceprevir groups (43–46%) than in the control group (20%). However, discontinuation owing to anaemia was infrequent. Dysgeusia, rash, and dry skin were more frequent in the boceprevir than in the control groups.
The SVR rate in patients with undetectable HCV RNA levels at treatment week 8 was similar whether boceprevir was taken for 32 or 44 weeks. Thus, an early response identified patients who could benefit from shorter treatment.
The authors identified 102 patients with a poor response to IFN, defined as a decrease in HCV RNA of less than 1log10 IU/ml at week 4. This is important to identify changes over time in patients who have previously been treated and are awaiting retreatment. The possible explanations for these changes include an increase in body weight, development of glucose intolerance, an increase in hepatic steatosis and the progression of fibrosis, all of which could decrease the responsiveness to PEG-IFN/RBV. In particular SVR was achieved after boceprevir was added to SOC in 33–34% of patients with a poor response to IFN, compared to 0% of patients who were retreated with PEG-IFN/RBV alone.
Furthermore, certain IFN stimulated genes were shown to be highly expressed in non-responders, thus pre-activation of the IFN system in patients appears to limit the effect of IFN antiviral therapy. Failure to respond to exogenous PEG-IFN in non-responders could indicate a blunted response to IFN .
Genetic prediction of treatment response
Independent groups have identified SNPs, near the IL-28B (IFN-3k) region, which are associated with a treatment response, thus opening a window for personalized medicine . A strong association was reported between rs12979860 and SVR in genotype 1 treatment-naive patients treated with PEG-IFNalfa-2a/RBV. Although all the identified variants lie in or near the IL-28B gene, none of them has an obvious effect on gene function . The power of IL-28B testing to predict SVR to triple therapy has not been sufficiently evaluated and cannot be recommended at this time.
Finally, promising results have been reported in genotype 1 when the protease inhibitors telaprevir or boceprevir are added to PEG-IFN plus RBV. However, despite the high expectations, caution must be taken. It will be important to focus on compliance. In addition to PEG-IFN/RBV boceprevir or telaprevir is administered orally three times daily, making compliance an issue, since poor compliance is associated with failure and resistance. Finally management of side effects will be important.
Second generation protease inhibitors
BI 201335 (Boehringer Ingelheim)
The BI 201335 is an HCV NS3 protease inhibitor being developed by Boehringer Ingelheim Pharma (Ridgefield, CT, USA). BI 201335 has completed clinical trials through Phase 2b (SILEN-C studies) [32, 33]. Phase 3 trials with BI 201335 plus PEG-IFN/RBV are ongoing in treatment-naïve and experienced genotype 1 infected patients. In treatment-naive patients (Study 1220.47), BI 201335 will be dosed once daily at either 120 or 240 mg for 12 or 24 weeks in combination with 24 or 48 weeks of PEG-IFN/RBV, the current HCV SOC. In treatment-experienced patients (Study 1220.7) BI 201335 will be dosed once daily at 240 mg for 12 or 24 weeks in combination with SOC for 48 weeks for prior partial and non-responder patients. Patients with prior relapse will be dosed with BI 201335 once daily at 240 mg for 12 or 24 weeks in combination with SOC for a total duration of 24 or 48 weeks. The primary endpoint of each trial is a SVR, which is considered a viral cure.
In a phase IIB study, TMC435 75 or 150 mg QD in combination with P/R led to significantly higher SVR rates compared to P/R alone, with a total of 24 weeks of treatment in most patients. TMC435 150 mg QD will be administered in Phase III trials . Tibotec Pharmaceuticals has announced that two global, registrational phase 3 trials are recruiting patients to examine TMC435 in treatment-naive adults with chronic genotype 1 hepatitis C virus (HCV) . A third global phase 3 trial is being performed in genotype 1 HCV relapse patients who have received prior IFN-based treatment. The first global, phase 3, double-blind, randomized study will evaluate a single TMC435 once daily oral tablet (150 mg) vs. placebo in treatment-naïve HCV patients. Both groups will also receive PEG-IFNalfa-2a/RBV as part of their treatment. The second global, phase 3, double-blind, randomized study will also evaluate a single TMC435 once daily oral tablet (150 mg) vs. placebo in treatment-naïve HCV patients. However, patients in this trial will either receive PEG-IFNalfa-2a or 2b/RBV as part of their treatment. A third global, phase 3, double-blind randomized study will evaluate a single TMC435 once daily oral tablet (150 mg) vs. placebo in HCV relapse patients who have received previous IFN-based therapy. Both groups will receive PEG-IFNalfa-2a/RBV. Treatment will last 24 or 48 weeks in all three trials depending upon patient response.
MK7009 (Vaniprevir; Merck)
The MK-7009 is a rapidly reversible non-covalent competitive inhibitor of the non-structural 3/4A protease of the HCV, developed by Merck & Co., Inc. (Whitehouse Station, NJ, USA) that exhibits good inhibitory potency against genotypes 1 and 2. Results from an ongoing Phase IIa trial in which all patients received MK-7009 or placebo and PEG-IFN/RBV) showed that combination therapy with MK-7009 significantly improved RVR within 28 days, compared to placebo in previously untreated (treatment-naïve) patients with chronic HCV genotype 1. A Phase 2b study of MK-7009 (vaniprevir) in patients with genotype 1 HCV infection who failed previous PEG-IFN/RBV treatment is ongoing .
Danoprevir (RG7227; ITMN-191) is a potent inhibitor of the HCV NS3/4A serine protease. A double-blind, placebo-controlled, multiple-ascending dose phase Ib study was performed to evaluate safety, tolerability, antiviral activity, resistance and pharmacokinetics of once- and twice daily danoprevir in the presence of low dose ritonavir (danoprevir/r) and in combination with PEG-IFNalfa-2a/RBV in treatment-naïve HCV genotype 1 patients . Thirty eligible patients were enrolled into three cohorts and treated with danoprevir/r or placebo/r all in combination with PEG-IFNalfa-2a/RBV for 15 days. Cohort 1 received danoprevir/r at 100/100 mg twice daily; Cohort 2 200/100 mg once-daily; and Cohort 3 200/100 mg twice daily. The median reductions in HCV RNA from baseline after 14 days of treatment (day 15) were –5.1, –4.8 and –4.6 log10 IU/ml in Cohorts 1, 2 and 3 respectively, and –2.7 log10 in placebo/r and PEG-IFNalfa-2a/RBV recipients. Viral breakthrough was not observed in any patients. On day 15, HCV RNA was undetectable (< 15 IU/ml) in 6/9 (67%), 4/8 (50%), and 8/8 (100%) patients in Cohorts 1, 2, and 3 respectively. When co-administered with low dose ritonavir, danoprevir concentrations reached a steady state between 6 and 10 days of dosing. Danoprevir exposures increased more than dose proportionally between 100/100 and 200/100 mg. Danoprevir/r plus PEG-IFNalfa-2a (40KD)/RBV was well tolerated with no safety-related discontinuations. The authors concluded that danoprevir/r plus PEG-IFNalfa-2a (40KD)/RBV provides profound and robust reductions in serum HCV RNA, at substantially lower systemic exposures than with higher doses of danoprevir alone. These results support further studies with danoprevir/r. A study evaluating RGT with danoprevir (DNV; RG7227) plus PEG-IFNalfa-2a (40KD) and RBV (P/R) in treatment-naïve HCV genotype 1 (G1) patients is ongoing .
Nucleoside/nucleotide analogue inhibitors of RdRp
Polymerase inhibitors currently under development in phase II are indicated in Table 3.
|Nucleoside/nucleotide analogue inhibitors|
|RG7128 (Mericitabine)||Roche / Genentech||Phase II|
|BMS 791325||Bristol-Myers Squibb||Phase II|
|ANA598 (Setrobuvir)||Anadys Pharmaceuticals||Phase II|
|BI 207127||Boehringer Ingelheim Pharma||Phase II|
In September 2011, Pharmasset announced the SVR results of its phase 2b PROTON study with PSI-7977 400 mg once-daily in combination with PEG-IFNalfa- 2a and RBV in treatment-naïve subjects with HCV genotype 1. Pharmasset reported a SVR 12 weeks after treatment (SVR12) of 91%. Ninety-five treatment-naïve patients with HCV genotype 1 were enrolled into two open label arms of the trial, to receive either PSI-7977 200 mg QD (n = 48) or 400 mg QD (n = 47) for 12 weeks. Both arms received PEG-IFN/RBV for 24 weeks and were followed post-treatment to assess SVR12. Forty-three out of 44 (98%) subjects achieved an SVR12, defined as HCV RNA below the limit of detection (< 15 IU/ml) 12 weeks after completing treatment. All subjects will be followed-up to determine SVR24, the primary efficacy endpoint of this study. Interim results from the PROTON trial show that 43/47 subjects receiving the 400 mg dose of PSI-7977 achieved an eRVR, defined as HCV RNA below the limit of detection (< 15 IU/ml) at weeks 4–12. Three of the patients who did not achieve an eRVR discontinued therapy early as a result of unrelated adverse events and one was lost to follow-up. In particular one of these individuals achieved a SVR12 in spite of the shorter course of therapy. The combination of PSI-7977, PEG-IFN/RBV was generally safe and well tolerated. A study with PSI-7977 once a day, plus PEG-IFN/RBV in treatment-naïve patients with HCV genotype1 is ongoing . Interestingly this compound has pan-genotype antiviral activity.
R7128 is a prodrug of PSI-6130, a cytidine analogue inhibitor of the HCV polymerase inhibitor. In a phase I trial, the combination of R7128 1500 mg b.i.d. plus PEG-IFNalfa-2a/RBV reduced serum HCV RNA levels by a mean 5.12 log10 IU/ml in 20 treatment-naïve patients with HCV genotype 1 infection (vs. 2.95 log10 IU/ml in 10 patients treated with placebo plus SOC). A total of 17/20 (85%) patients in the triple therapy group had undetectable HCV RNA (o15 IU/ml) at week 4 compared to 1/10 (10%) in the control group. Headache, chills, fatigue, nausea and fever were the most common adverse events. Grade 4 neutropenia was reported in 1/20 patients in each dosing cohort and in 1/10 placebo recipients. Furthermore, a study was designed to evaluate R7128 in combination with SOC in genotype 2 patients and genotype 3 patients . Twenty-five (20 active/5 placebo) patients with genotypes 2 (n = 10) and genotype 3 (n = 15) who had previously not achieved a SVR with IFN-based therapy were enrolled. Preliminary data available for 25 patients through day 14 suggest that R7128 (1500 mg b.i.d.), combined with PEG-IFN/RBV in prior genotype 2/3 non-responders to IFN-based therapy, provides a high RVR (Z86%), with an acceptable side-effect profile.
The IDX184, a novel liver-targeted nucleotide prodrug of 2′-methyl guanosine monophosphate has been developed by Idenix Pharmaceuticals (Cambridge, MA, USA) . A phase IIa clinical randomized, double-blind, placebo-controlled, sequential dose-escalation study was begun at the end of 2009 to evaluate the safety, tolerability, pharmacokinetics and antiviral activity of IDX184 in combination with PEGIFN/RBV in treatment-naive HCV genotype 1-infected patients. Patients receive a daily dose of IDX184 or placebo, plus PEG-IFN/RBV for 14 days and then continue PEG-IFN/RBV for an additional 14 days. This study is evaluating four dosing regimens of IDX184 from 50 to 200 mg/day. In the 100 mg and 200 mg cohorts, once daily (QD) and twice daily (BID) regimens are compared. Each cohort includes 20 patients randomized 4:1, IDX184: placebo. In the data presented, IDX184 demonstrated potent dose-dependent antiviral activity when combined with PEG-IFN/RBV. At Day 14, mean viral load reductions were 1.2, 2.7, 4.0 and 4.2 log10 IU/ml in the placebo (n = 8), 50 mg IDX184 QD (n = 16), 50 mg IDX184 BID (n = 8) and 100 mg IDX184 QD (n = 8) cohorts respectively. Half of the subjects receiving a total daily dose of 100 mg IDX184 achieved undetectable viral loads (< 15 IU/ml) by Day 14. Antiviral activity and safety parameters measured after a total daily dose of 100 mg IDX184 did not differ between a QD or BID dosing regimen. The side effect profile of the three-drug combination was consistent with the known side effect profile of PEG-IFN/RBV alone. The most common adverse events reported were fatigue, myalgia, headache and nausea. No virological breakthrough was observed during treatment with IDX184 in combination with PEG-IFN/RBV.
Non-nucleoside inhibitors of RdRp
Filibuvir [FBV; formerly PF 00868554 developed by Pfizer Global Research and Development (New London, CT, USA)] is a non-nucleoside inhibitor of the HCV polymerase . FBV monotherapy (100–450 mg BID or 300 mg TID × 8 days) demonstrated dose-dependent inhibition of viral replication, with mean maximum reductions in HCV RNA ranging from –0.97 to –2.13 (log10 IU/ml). The safety and efficacy of FBV combined with PEG-IFNalfa-2a/RBV(RBV) was evaluated in this study. Results of PF-00868554, were recently presented . Thirty-one healthy male subjects completed this study with no serious adverse events, drug discontinuation or dose reductions when the drug was given twice a day for 14 days. Another study in treatment-naïve patients infected with HCV, is ongoing.
Phase II clinical studies combining BMS-790052 with the NS3 protease inhibitor BMS-650032 are ongoing and interim results have shown that this combination therapy alone or with PEG-IFN/RBV results in undetectable HCV RNA after 12 weeks of therapy in HCV genotype 1 non-responders . Clinical proof-of-concept has recently been obtained with NS5A inhibitors, indicating that small molecules targeting a non-traditional viral protein without any known enzymatic activity can also have profound antiviral effects in HCV infected subjects. Achieving high potency and selectivity against anon-mammalian target, the traditional goal of antiviral medicinal chemistry, has in the past translated into a wider therapeutic index in the clinic. Although preliminary, these data indicate that inhibitors of HCV NS5A offer considerable promise for the treatment of HCV.
The PEG-IFN lambda (IL-29) is a novel interferon under development for hepatitis C. PEG-IFN lambda is a member of the Type III lambda IFN family, which includes IL-28A, IL-28B and IL-29 (also known as IFN lambda 2, 3, and 1 respectively). Type III IFNs signal through a different receptor than type I IFNs, such as IFN α. The native human IFN lambda proteins are generated by the immune system in response to viral infection. In a Phase 1b clinical trial in relapse patients PEG-IFN lambda was administered for 4 weeks in combination with RBV .
The Phase 2b study will include approximately 600 patients with genotypes 1–4 chronic HCV infection (ZymoGenetics and BMS). This study will assess the safety and antiviral efficacy of the three specified doses of PEG-IFN lambda compared to PEG-IFNalfa-2a. Each cohort of approximately 150 patients will include at least 100 genotype 1 patients. Weekly subcutaneous doses of PEG-IFN lambda or PEG-IFNalfa-2a will be administered for 48 weeks in HCV genotype 1 or 4 patients and for 24 weeks in genotype 2 or 3 patients. All patients will also receive daily RBV.
Future perspectives: Interferon free combination trials
At present, several studies of DAA combinations are ongoing in treatment-naïve HCV patients (Table 4). All studies include different HCV targets: NS3/4a protease inhibitor, combined with an agent targeting the HCV polymerase complex – either a non-nucleoside NS5B, nucleoside NS5B, or NS5A inhibitor.
|Drug (class)||Drug (class)||Company|
|RG7128 (NI)||InterMune / Roche|
|BI 201335 (PI)||BI 207127 (NNI)||Boehringer |
|GS-9256 (PI)||GS-9190 |
|PSI-7977 (NI)||PSI-938 (NI)||Pharmasset|
|ABT-450 (PI)||ABT-333 (NNI)||Abbott / Enanta|
|ABT-450 (PI)||ABT-072 (NNI)||Abbott / Enanta|
|BMS 790052 |
|PSI-7977 (NI)||BMS / Pharmasset|
|PSI-7977 (NI)||TMC435 (PI)||Pharmasset / Tibotec|
The proof-of-concept INFORM-1 study evaluated combination DAA . In this randomized, placebo-controlled double-blind trial, 87 patients with HCV genotype 1 infection were randomized to receive up to 13 days of either oral combination therapy with RG7227/danoprevir, an NS3/4A protease inhibitor, and RG7128, a nucleoside polymerase inhibitor, or with matched placebos. Both agents had already been administered to patients for 12 weeks in combination with PEG-IFN/RBV. The DAA combination resulted in profound antiviral suppression that was greater than the additive effects of either treatment alone (Fig. 8). No evidence of resistance to either compound was observed during this study. No serious adverse events were reported. The antiviral efficacy was similar in treatment-naïve and experienced patients, including non-responders. Since the total duration of therapy was only 13 days, all patients rolled over into PEG-IFN/RBV treatment. The rates of RVR, EVR and ETR were markedly increased by the 2 weeks of pretreatment. In the final cohort of patients who received the highest dose of RG7227 and RG7128, 100% achieved ETR after 24 weeks PEG-IFN and RBV treatment.
BI 201335 and BI 207127 (Boehringer Ingelheim)
Recently, the antiviral effect and safety of BI 201335 (an inhibitor of the NS3/4A protease) and BI 207127 (an inhibitor of the NS5B non-nucleoside polymerase) with RBV were evaluated [46-48]. In a recent publication, 32 treatment-naïve patients with chronic HCV genotype 1 infection were randomly assigned to groups that were given 400 or 600 mg BI 207127 three times daily plus 120 mg BI 201335 once daily and 1000–1200 mg/day RBV for 4 weeks . In the group given BI 207127 400 mg three times daily, the SVR rates were 47%, 67% and 73% at days 15, 22 and 29 (Fig. 9); a higher rate of response was observed in patients with genotype 1b than in genotype 1a infections. In the group that received BI 207127 600 mg three times daily, the SVR were 82%, 100% and 100% respectively, and did not differ among genotypes. One patient in the group who received 400 mg three times daily had a virological breakthrough (≥ 1 log rebound in HCV RNA) at day 22. The most frequent adverse events were mild gastrointestinal disorders, rash and photosensitivity. There were no severe or serious adverse events; none of the patients discontinued therapy prematurely. In conclusion, the combination of the protease inhibitor BI 201335, the polymerase inhibitor BI 207127, and RBV has rapid and strong activity against HCV genotype 1 and does not cause serious adverse events. In SOUND-C2, 362 treatment-naïve patients with chronic genotype-1 HCV infections were randomized into five IFN-free treatment arms, each with 120 mg BI 201335 once daily (QD) but with different dosings of BI 207127 and RBV.
GS-9256 and tegobuvir (Gilead)
Tegobuvir (GS-9190), a non-nucleoside NS5B polymerase inhibitor, and GS-9256, an NS3 serine protease inhibitor, were developed by Gilead. The antiviral activity of tegobuvir and GS-9256 as oral combination therapy, or together with RBV or PEG-IFN and RBV, was assessed in a phase 2, randomized, open label trial . Treatment-naïve patients with genotype 1 HCV were assigned 28 days of tegobuvir 40 mg twice daily and GS-9256 75 mg twice daily (n = 16), tegobuvir and GS-9256 plus RBV 1000–1200 mg daily (n = 15), or tegobuvir and GS-9256 plus PEG-IFNalfa-2a/RBV (n = 15). The primary efficacy endpoint was RVR at Day 28. After 28 days, all patients received PEG-IFN/RBV. Median maximal reductions in HCV RNA were –4.1 log10 IU/ml for tegobuvir/GS-9256, –5.1 log10 IU/ml for tegobuvir/GS-9256/RBV, and –5.7 log10 IU/ml for tegobuvir/9256/PEG-IFN/RBV. RVR was observed in 7% (1/15) of patients receiving tegobuvir/GS-9256, 38% (5/13) receiving tegobuvir/GS-9256/RBV, and 100% (14/14) receiving tegobuvir/9256/PEG-IFN/RBV. The addition of PEG-IFN/RBV at Day 28 or earlier resulted in HCV RNA < 25 IU/ml at Week 24 in 67% (10/15), 100% (13/13) and 94% (13/14) of patients in the three treatment groups. Transient elevations in serum bilirubin occurred in all treatment groups. In conclusion, in genotype 1 HCV, adding RBV or RBV with PEG-IFN provides additive antiviral activity to combination therapy with tegobuvir and GS-9256.
PSI-7977 and PSI-938 (Pharmasset)
In September 2001, Pharmasset announced that screening had begun in a Phase 2b, international study of PSI-7977 and PSI-938, two nucleotide analogue polymerase inhibitors for the treatment of HCV. The QUANTUM trial will evaluate IFN-free regimens of PSI-7977 400 mg QD and PSI-938 300 mg QD with and without RBV for 12 or 24 weeks in treatment-naïve patients with HCV. The trial will also evaluate the use of PSI-938 monotherapy. HCV patients will be stratified by IL-28B status and baseline HCV RNA to ensure balance across cohorts. Patients with and without cirrhosis will be enrolled in this study.
ABT-333, ABT-450 (Abbott)
An open label phase II study that was launched in 2011 to study the efficacy of the combination of ABT-333, ABT-450/r and RBV (without IFN) to treat chronic hepatitis C genotype 1 treatment-naïve patient and prior non-responder patients. On Oct 21, 2011 Abbott announced preliminary results from this study in which all patients achieved an EVR –undetectable HCV RNA at week 12. After 24 weeks of therapy, nine had achieved a SVR or a cure.
BMS-790052 with PSI-7977 (BMS and Pharmasset)
This proof-of concept study will evaluate the potential to achieve SVR 24 weeks post-treatment (cure) with an oral, once daily treatment regimen in patients with HCV genotypes 1, 2 and 3. Specifically, this study will assess the safety, pharmacokinetics and pharmacodynamics of BMS-790052 in combination with PSI-7977, with and without RBV, in 84 treatment-naïve patients chronically infected with HCV genotypes 1, 2, and 3. The beginning of this study was announced by Pharmasset on 25 May 2011.
Quadruple therapy with BMS-790052, BMS-650032 and PEG-IFN/RBV (BMS)
The BMS-790052 is a potent HCV NS5A replication complex inhibitor although BMS-650032 is a potent HCV NS3 protease inhibitor. AI447011 is a randomized, open label, Phase 2a study exploring the antiviral activity and safety of BMS-790052 (60 mg QD) and BMS-650032 (600 mg BID) alone (Group A) or with PEG-IFN/RBV (Group B) for 24 weeks in a sentinel cohort of HCV GT 1 non-responders . The primary objective was to determine the proportion of subjects achieving undetectable HCV RNA (< 10 IU/ml) 12 weeks post-treatment (SVR12). Twenty-one subjects (11 Group A, 10 Group B), 19 with unfavourable IL-28B genotypes (rs12979860 CT/TT) were treated in the sentinel cohort. A non-response was defined as an HCV RNA decrease < 2 log10 after 12 weeks of PEG-IFN/RBV. An interim analysis was performed when all subjects not experiencing a viral breakthrough reached 12 weeks post-treatment. Seven (63.6%) Group A subjects had undetectable HCV RNA by week 4; five remained undetectable at the end of treatment; four (2/9 GT1a and 2/2 GT1b) achieved SVR12, whereas one experienced viral relapse 4 weeks post-therapy. Six subjects in Group A (all genotype1a) experienced viral breakthrough on therapy, all had PEG-IFN/RBV added to their regimen and four subsequently achieved undetectable HCV RNA. Analysis of post-breakthrough viral sequences revealed emergence of variants resistant to both antivirals in all cases. Six (60%) Group B subjects had undetectable HCV RNA by week 4 and all 10 achieved SVR12. Diarrhoea was the most common adverse event (71.4%) and was mainly mild to moderate; six subjects experienced ALT > 3x ULN all had total bilirubin < 2x ULN. Six subjects (all receiving PEG-IFN/RBV) experienced Grade 3/4 neutropenia. No severe adverse events or discontinuations owing to adverse events occurred.
In conclusion, two antivirals alone can lead to SVR in certain difficult-to-treat subjects. Inclusion of PEG-IFN/RBV with the 2 DAA suppresses the emergence of resistance variants resulting in a 100% rate of SVR12. Enrolment of additional cohorts of patients is ongoing to validate these results.
In March 2010 a phase II trial of telaprevir/VX-222 (two arms with and one arm without PEG-IFN/RBV) was begun. The three treatment arms included HCV genotype 1 treatment-naïve patients in each arm. The treatment duration (12 weeks, 24 weeks) will be guided by response at certain time points during the trial. On July, 2011 Vertex announced preliminary results –50% of patients (15 out of 30) who received 400 mg VX-22 (plus telaprevir, and PEG/RBV) were able to complete treatment at week 12 and 93% (14 out of 15 patients) achieved SVR12. The remaining patients received an additional 12 weeks of PEG-IFN/RBV – 100% (13 of 13 patients) were HCV RNA undetectable at the end of the 24 weeks . Interim safety results found that the side effects were mild.
Major breakthroughs have been achieved in the treatment of HCV chronic infection. Currently, the standard of care of treatment of HCV genotype 1 is the addition of a DAA protease inhibitor (telaprevir or boceprevir) to the PEG-IFN plus RBV regimen. The future management of patients with these new molecules will require good clinical practice, knowledge of indications, management of side effects and monitoring for antiviral resistance. Certain major medical needs are still unmet requiring studies in special populations (HIV-HCV coinfected patients, transplanted patients, etc.…) in HCV genotypes non-1 patients and in non-responders.
Second generation protease inhibitors and new classes of DAA are under development in clinical trials. Preliminary results of DAA combinations show increased antiviral efficacy, reduced resistance and a good safety profile. It is important to note that some of these drugs have pan-genotypic activity. This rapid progress strongly suggests that in the near future, IFN-free short duration DAA combinations will make HCV the first chronic viral infection to be eradicated worldwide.
Conflicts of interest
Tarik Asselah is a speaker and investigator for BMS, Boehringer-Ingelheim, Tibotec, Janssen, Gilead, Roche, and Merck. Patrick Marcellin is a speaker and investigator for BMS, Boehringer-Ingelheim, Tibotec, Janssen, Gilead, Roche, and Merck.