Emerging therapeutic options for the management of hepatitis C infection

World J Gastroenterol. 2014 June 21; 20(23): 7079-7088.

Published online 2014 June 21. doi: 10.3748/wjg.v20.i23.7079.

Copyright ©2014 Baishideng Publishing Group Inc. All rights reserved.

J Richard Thompson.

J Richard Thompson, Department of Pharmacy Practice, One University Park Drive, Lipscomb University College of Pharmacy, Nashville, TN 37204, United States

Author contributions: Thompson JR reviewed the literature and wrote the entire content for this manuscript.

Correspondence to: J Richard Thompson, PharmD, MBA, BCPS, Department of Pharmacy Practice, One University Park Drive, Lipscomb University College of Pharmacy, Nashville, TN 37204, United States. richard.thompson@lipscomb.edu

Telephone: +1-615-9667172 Fax: +1-615-9667163

Received October 10, 2013; Revised January 22, 2014; Accepted May 1, 2014;

Abstract

Until recently the traditional treatment for hepatitis C infection included pegylated interferon and ribavirin combination therapy. The sustained virological response (SVR) seen with this combination is poor and requires lengthy treatment to achieve. Additionally, significant side effects and numerous contraindications prevented many patients from being successfully treated with this therapy. In 2011, two new protease inhibitors, telaprevir and boceprevir, were approved for use with pegylated interferon and ribavirin in the United States by the United States Food and Drug Administration. These agents have significantly improved SVR rates; however significant problems with toxicity remain including severe skin rash and neutropenia. There are a wide range of compounds in late stage development for the future treatment of hepatitis C that exploit many different mechanisms of viral inhibition. Some of these compounds include additional protease inhibitors, like telaprevir and boceprevir, as well as inhibitors of other nonstructural proteins in the viral genome such as NS5A and NS5B, and compounds that target host proteins within the virus. Some of these agents are being developed for oral administration once daily and various combinations are being assessed for use without the need for pegylated interferon and ribavirin. This paper reviews agents in late phase development that may be commercially available within 1-2 years.

Keywords: Hepatitis C, Pharmacotherapy, Interferon, Ribavirin, Protease inhibitors, Polymerase inhibitors, Cyclophilin inhibitors

Core tip: A plethora of new agents for the management of hepatitis C promising higher response rates and better tolerated side effect profiles is upon us. Many of these new drugs in development utilize novel pharmacologic mechanisms and may replace older more toxic therapies such as interferon and ribavirin. In addition, once daily dosing and shorter treatment durations should help improve adherence and optimize therapeutic outcomes for hepatitis C infection.

INTRODUCTION

The hepatitis C virus (HCV) is a major cause of gastrointestinal morbidity and mortality worldwide. According to the World Health Organization (WHO), approximately 170 million people have chronic HCV infection with 3-4 million new infections occurring each year. In the United States, up to four million people have chronic HCV and 18-20000 new infections are diagnosed annually per the Centers for Disease Control (CDC).

Until recently treatment for HCV infection relied upon pegylated-interferon (PEG-IFN) and ribavirin combination therapy. The sustained virological response (SVR) seen with this combination is poor and requires lengthy treatment to achieve[¹]. Additionally, significant side effects and numerous contraindications, prevent most patients from being successfully treated with these agents.

Recently two new protease inhibitors, telaprevir and boceprevir, have been approved by the Food and Drug Administration in the United States and the use of direct-acting antiviral (DAA) triple therapy has substantially improved SVRs[^2,3]. In addition, a plethora of new chemical entities directed against various components of the nonstructural proteins in the hepatitis C virion are in late phase development and offer the promise of greater efficacy and fewer side effects, with once or twice daily oral administration and perhaps no longer requiring interferon and ribavirin. This paper will review telaprevir and boceprevir as well as agents that are in late phase development and may come available within the next 1-2 years.

TRADITIONAL TREATMENT APPROACH TO HEPATITIS C

The traditional treatment approach to hepatitis C relies heavily on combination therapy with pegylated interferon and ribavirin[^4,5]. For genotype I disease, patients less than 75 kg receive peginterferon alpha-2a 180 μg by injection per week plus ribavirin 1000 mg daily in divided doses for 48 wk or peginterferon alpha-2b 1.5 μg/kg per week by injection plus ribavirin 800-1000 mg (based on weight) daily in divided doses for 48 wk. Patients greater than 75 kg receive peginterferon alpha-2a 180 μg by injection per week plus ribavirin 1200 mg daily in divided doses for 48 wk or peginterferon alpha-2b 1.5 μg/kg per week by injection plus ribavirin 1000-1400 mg (based on weight) daily in divided doses for 48 wk. Patients with genotypes 2 or 3 disease may be treated with peginterferon alpha-2a 180 μg by injection per week plus ribavirin 800 mg daily in divided doses for 24 wk or peginterferon alpha-2b 1.5 μg/kg per week by injection plus ribavirin 800-1400 mg (based on weight) daily in divided doses for 24 wk. Dose reductions may be necessary for patients with significant renal disease or those experience serious adverse reactions. For patients with contraindications or intolerance to ribavirin, monotherapy with peginterferon alpha-2a 180 μg by injection per week for 48 wk or peginterferon alpha-2b 1 μg/kg per week by injection for one year may be used.

Clinical success is greater with combination therapy, but SVR rates remain less than optimal at 54%-56%[⁵]. Numerous contraindications to ribavirin preclude some patients from receiving combination therapy. These contraindications include autoimmune hepatitis, decompensated liver disease, pregnancy, hemoglobinopathy, renal insufficiency, hemodialysis, thyroid disease, diabetes, rheumatoid arthritis, asthma or chronic obstructive pulmonary disease, and ischemic cardiovascular or cerebrovascular disease. Also, side effects associated with both pegylated interferon and ribavirin are substantial and include fatigue, fever, headache, nausea, arthralgia, musculoskeletal pain, insomnia, depression, neutropenia, thrombocytopenia, and anemia. For these reasons, only about 10% of patients with hepatitis C are successfully treated with traditional therapy.

TRIPLE THERAPY WITH PROTEASE INHIBITORS FOR HEPATITIS C

The hepatitis C virus contains six nonstructural HCV proteins that are processed by both viral and host proteases[⁶]. These nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) are primarily enzymes that are essential in the HCV life cycle (Figure 1). A number of compounds have been developed which target these proteases involved in HCV polyprotein procession. They are divided into two chemical classes, macrocyclic inhibitors and linear tetra-peptide α-ketoamid derivatives. The NS3/4A protease inhibitors are potent anti-viral agents as monotherapy against HCV replication, but may cause selection of resistance species. However, this resistance appears to be attenuated when the drugs are used in combination with standard peginterferon/ribavirin therapy. The first two drugs to reach the commercial marketplace in this class are telaprevir and boceprevir.

Figure 1 Hepatitis C genomic targets for drug development. HCV: Hepatitis C virus; IFN: Interferon.

Telaprevir

Telaprevir is an orally bioavailable NS3 protease inhibitor of the α-ketoamid class that binds the enzyme in a covalent but reversible manner with an enzyme-inhibitor complex half-life of 58 min. A phase I, placebo-controlled dose ranging study compared telaprevir 450 mg or 750 mg every 8 h with 1250 mg every 12 h in treatment-naïve genotype I patients and found the 750 mg dose to be most effective with a median reduction in HCV RNA of 4.4 log₁₀ after 14 d[⁷]. A subsequent similar phase I study compared this dose following a 1250 mg loading dose either alone or in combination with peginterferon alpha-2a with peginterferon alpha-2a monotherapy for 14 d. The reduction in HCV RNA was 1.09 log₁₀ in the peginterferon alpha-2a/placebo group, 3.99 log₁₀ in the telaprevir/placebo group, and 5.49 log₁₀ in the peginterferon alpha-2a/telaprevir group at the end of therapy. The development of resistant mutants was significantly lower in the peginterferon combination treatment group and no breakthrough of virus was seen throughout the study period[⁸]. An additional 28 d trial in treatment-naïve genotype I patients showed undetectable HCV RNA serum levels following telaprevir 750 mg every 8 h combined with peginterferon alpha-2a and weight-based dosing of ribavirin[⁹].

These early results were further validated in two phase II trials, PROVE 1 (American) and PROVE 2 (European) in treatment naïve, genotype I patients. In the PROVE 1 trial, combination therapy with telaprevir 1250 mg loading dose followed by 750 mg every 8 h or placebo, peginterferon alfa-2a 180 μg weekly, and ribavirin 1000-1200 mg/d based on weight for 12 wk was followed by interferon alfa-2a and ribavirin at the same dosages for an additional 0, 12, or 36 wk. Treatment was stopped after 12 or 24 wk only when a rapid virologic response (RVR) was achieved. This produced SVR rates of 35%, 61% and 67% at 12, 24, and 48 wk, respectively, vs 41% SVR with 48 wk standard therapy[¹⁰]. The PROVE 2 trial compared telaprevir + peginterferon alone for 12 wk, telaprevir + peginterferon and ribavirin for 12 wk, and telaprevir + peginterferon and ribavirin for 12 wk followed by an additional 12 wk of peginterferon and ribavirin alone, against standard peginterferon and ribavirin therapy. SVRs of 36%, 60%, and 69% respectively, vs 46% with standard therapy were documented[¹¹]. thus, ribavirin appears important to achieve high SVRs and 12 wk of therapy appears insufficient to prevent relapse. In these trials skin rash, anemia, and gastrointestinal disorders were the most common side effects, causing up to 18% of patients to discontinue therapy.

A third trial, PROVE 3, evaluated telaprevir-based combination therapy in patients who had prior non-response or relapse with standard peginterferon and ribavirin therapy[¹²]. SVRs in patients retreated with telaprevir, interferon and ribavirin for 12 or 24 wk followed by peginterferon and ribavirin alone for up to 24 wk were 51% and 53%, respectively, compared with standard therapy at 14%. However, retreatment with telaprevir and interferon alone for 24 wk followed by peginterferon and ribavirin alone for an additional 24 wk gave only a 24% SVR rate, again demonstrating the need for ribavirin in initial therapy with telaprevir.

Telaprevir has since been evaluated in three large, randomized, controlled trials in both treatment naïve and standard treatment failure patients. The ADVANCE trial enrolled a total of 1095 patients with treatment naïve genotype 1 chronic hepatitis C and compared three treatment arms: telaprevir 750 mg three times daily for 8 wk followed by peginterferon and ribavirin or placebo for an additional 4 wk and then peginterferon and ribavirin in both groups for 12 subsequent weeks (or 36 subsequent weeks in patients who did not have a RVR at 24 wk); telaprevir 750 mg three times daily for 12 wk followed by peginterferon and ribavirin for 12 subsequent weeks (or 36 subsequent weeks in patients who did not have a RVR at 24 wk); and peginterferon and ribavirin or placebo for 12 wk followed by peginterferon and ribavirin for the subsequent 36 wk. SVRs were seen in 72% of the telaprevir 8-wk arm, 79% in the telaprevir 12-wk arm, but only 46% in the peginterferon and ribavirin or placebo 48-wk group (P < 0.001)[¹³]. A 24-wk treatment period appeared sufficient for patients who achieved an early RVR.

The REALIZE trial evaluated telaprevir in 662 patients of which 354 were prior relapsers and 308 were prior non-responders to standard treatment with peginterferon and ribavirin. This study compared telaprevir 750 mg three times daily with peginterferon and ribavirin for 12 wk followed by peginterferon and ribavirin for an additional 36 wk; peginterferon and ribavirin for 4 wk followed by telaprevir 750 mg three times daily with peginterferon and ribavirin for an additional 12 wk, and then peginterferon and ribavirin alone for a subsequent 32 wk; or standard therapy with peginterferon and ribavirin for a full 48 wk course. In patients who had previously relapsed, SVR rates were 84%-88% in the telaprevir groups compared with only 22% in the placebo or peginterferon and ribavirin standard therapy groups[¹⁴]. In patients who had previously partially responded to standard therapy, SVR rates were 56%-61% in telaprevir treated patients compared with 15% in placebo or peginterferon and ribavirin treated patients. Previous nonresponders to standard therapy achieved SVRs of 31%-33% with telaprevir compared with only 5% with placebo or peginterferon and ribavirin standard therapy. The 4 wk lead-in exposure to interferon and ribavirin did not produce substantially different results than starting simultaneously with telaprevir. Patients with on-treatment virologic failure were fewer in telaprevir groups and relapse rates were lower than the control subjects for prior relapsers and prior non-responders.

A third phase III clinical trial, ILLUMINATE, evaluated the efficacy of telaprevir therapy for 12 wk with either 24 or 48 wk of peginterferon and ribavirin based upon an achievement of extended RVR (eRVR) at 24 wk. Treatment naïve patients with genotype I chronic hepatitis C were given telaprevir 750 mg three times daily for 12 wk with peginterferon and ribavirin for at least 24 wk. Patients who achieved an extended rapid virologic response as evidenced by undetectable HCV RNA levels at weeks 4 and 12 were randomized to either stop treatment at week 24 or continue peginterferon and ribavirin therapy for a full 48 wk. Patients who did not achieve an eRVR continued peginterferon and ribavirin therapy for the full 48 wk as well. SVRs were at least 90% in both groups and total treatment for 24 wk was non-inferior to 48 wk for those achieving an extended rapid virologic response. The SVR rate was > 70% for all groups, compared with historical standards of 46%-52%, and this study population included patients with historically lower SVRs with standard therapy. In addition, the relapse rate was low for both eRVR⁺ and eRVR^- patients at an overall rate of 9.2%[¹⁵].

The safety profile of telaprevir was evaluated in a pooled analysis of adverse events reported in all five phase II and III placebo-controlled trials[¹⁶]. During these trials, 2012 patients received at least one dose of telaprevir and 1346 patients were randomized to receive telaprevir 750 mg three times daily for 12 wk with peginterferon and ribavirin for 24-48 wk and 764 patients were randomized to receive placebo with peginterferon and ribavirin. A total of 73% of telaprevir-treated patients and 49.1% of placebo-treated patients completed the full duration of therapy. The most frequently occurring adverse events in the telaprevir group (> 20%) included pruritus, nausea, rash, anemia, and diarrhea. Hemorrhoids, anorectal discomfort, anal pruritus, dysgeusia, and generalized pruritus occurred less frequently. Anemia caused discontinuation of participation in 2.7% of telaprevir treated patients and 0.5% of placebo treated patients. Hemoglobin concentrations decreased rapidly over the first four weeks of treatment in both groups, but continued to decrease to a greater extent thereafter in telaprevir treated patients. The initial onset of rash occurred at any time following treatment with telaprevir, but most commonly occurred within the first four weeks. Progression of severity was reported for < 10% of cases, and many cases of rash resolved over the first 24 wk of therapy. A Dermatology Expert Panel reviewed the rashes and determined that the visual appearance of rash in telaprevir-treated patients was virtually indistinguishable from that seen in peginterferon and ribavirin-treated patients. One case of rash was suggestive of Stevens-Johnson syndrome, but it was not thought to be drug related as it occurred 11 wk after the last dose of telaprevir.

Boceprevir

Boceprevir is another orally bioavailable NS3 protease inhibitor of the α-ketoamid class that binds the enzyme in a covalent but reversible manner. In an early dose ranging trial of 100-400 mg daily for 14 d as monotherapy in genotype I patients with prior treatment failure on standard therapy, the maximum dose achieved a 2.06 log₁₀ reduction in HCV RNA load and was well tolerated. Viral breakthrough occurred in some patients, however[¹⁷]. A later phase I trial compared boceprevir 200 or 400 mg every 8 h for 7 d alone or in combination with peginterferon alpha 2b for 14 d with peginterferon alpha 2b monotherapy for 14 d in genotype I patients who were nonresponders to standard therapy. This approach achieved maximal reductions in HCV RNA of 2.45 and 2.88 log₁₀ for boceprevir 200 and 400 mg, respectively, with peginterferon alpha 2b, 1.08 and 1.61 log₁₀ for boceprevir 200 and 400 mg monotherapy, and 1.08 and 1.26 log₁₀ for peginterferon alpha 2b alone in the respective boceprevir dose groups[¹⁸]. Boceprevir was well tolerated in this trial as well, both alone and in combination with peginterferon alpha-2b, but viral breakthrough was observed again, primarily in patients receiving monotherapy.

The addition of ribavirin to boceprevir and peginterferon alpha-2b was evaluated in a phase II trial (SPRINT-1) in treatment naïve, genotype I patients. In this trial patients received 28 or 48 wk of boceprevir 800 mg three times daily, peginterferon alpha-2b, and ribavirin or a 4 wk lead-in treatment of peginterferon alpha-2b with ribavirin followed by 24 or 44 wk of boceprevir 800 mg three times daily, peginterferon alpha-2b, and ribavirin compared with standard therapy with peginterferon alpha-2b and ribavirin for a full 48 wk. The four week lead-in treatment with peginterferon alpha-2b and ribavirin boosted SVR rates from 54% to 56% at 28 wk and from 67% to 75% at 48 wk, compared with 38% with standard peginterferon alpha-2b and ribavirin therapy for 48 wk[¹⁹]. However, RVR rates with boceprevir triple therapy were only 38% compared with 70% seen in telaprevir triple therapy trials. The most common side effects seen with boceprevir in this trial were anemia, nausea, vomiting, and dysgeusia. A subsequent phase II trial evaluated boceprevir triple therapy in HCV genotype I nonresponders, but SVR rates were poor, ranging from 2% for control to 14% with boceprevir[²⁰].

Boceprevir has subsequently been evaluated in two large, well-controlled, multicenter clinical trials in both untreated and previously treated patients with genotype I chronic HCV infection. The SPRINT-2 trial enrolled a total of 1099 patients with untreated genotype I HCV infection who were randomized to receive triple therapy with boceprevir 800 mg three times daily with peginterferon alpha-2b and ribavirin for 24 or 44 wk following a lead-in treatment of 4 wk with standard peginterferon alpha-2b and ribavirin therapy and compared with a full 48 wk course of standard peginterferon alpha-2b and ribavirin therapy. One arm of the trial was allowed to discontinue therapy at 24 wk if HCV RNA levels were undetectable, while those with detectable levels received additional interferon alpha-2b and ribavirin with placebo from weeks 28 to 48. Black patients and nonblack patients were enrolled and analyzed separately. In the larger, nonblack cohort, an SVR rate of 40% was achieved with standard pegylated interferon alpha-2b and ribavirin for 48 wk, compared with 67% in patients treated with boceprevir for 24 wk and 68% in patients treated with boceprevir for 44 wk (P < 0.001)[²¹]. In the black cohort, an SVR rate of 23% was achieved with standard pegylated interferon alpha-2b and ribavirin for 48 wk, compared with 42% in patients treated with boceprevir for 24 wk (P < 0.04) and 53% in patients treated with boceprevir for 44 wk (P < 0.004). In the variable duration arm, 44% of patients had nondetectable HCV RNA levels and were able to discontinue therapy at 24 wk. Adverse events occurred in 98% of patients and were similar in number across groups. Fatigue, headache, and nausea were the most common clinical adverse events. Dysgeusia was twice as frequent in boceprevir treated patients and anemia occurred in 49% of boceprevir treated patients compared with 29% of those receiving peginterferon alpha-2b only. This led to dose reduction in 13% of the control patients and 21% of boceprevir treated patients and discontinuation of therapy in 1% and 2%, respectively.

The RESPOND-2 trial provided a similar evaluation of boceprevir in patients with chronic HCV genotype I infection, who had been previously treated with standard therapy and experienced either a nonresponse or relapse. This trial enrolled 403 patients and randomized them in a 1:2:2 fashion to standard therapy with peginterferon alpha-2b and ribavirin for 48 wk, boceprevir 800 mg three times daily with peginterferon alpha-2b and ribavirin for 32 wk, or boceprevir 800 mg three times daily with peginterferon alpha-2b and ribavirin for 44 wk. Both boceprevir triple therapy arms were preceded by 4 wk of treatment with standard peginterferon alpha-2b and ribavirin therapy. One arm of the trial was allowed to discontinue therapy at 32 wk if HCV RNA levels were undetectable, while those with detectable levels received additional interferon alpha-2b and ribavirin with placebo from weeks 36 to 48. The overall rate of SVR for boceprevir treated patients was significantly higher in this trial (59% at 32 wk and 66% at 44 wk) compared with standard therapy (21%, P < 0.001) for the full 48 wk[²²]. Patients who had undetectable HCV RNA levels at week 8 had SVR rates of 86% after 32 wk of boceprevir triple therapy and 88% after 44 wk. Patients whose HCV RNA level decreased by less than 1 log₁₀ at 4 wk had SVR rates of 33% and 34% after boceprevir triple therapy compared with 0% after standard therapy. Side effects seen in this trial were similar to those seen in SPRINT-2, with anemia being more common in boceprevir treated patients (43%-46%) compared with standard therapy (20%). Erythropoeitin was required to manage the anemia in 41%-46% of boceprevir treated patients compared with 21% with standard therapy.

DEVELOPMENTAL PROTEASE INHIBITORS

Protease inhibitors are classified as 1^st generation or 2^nd generation based upon resistance profiles. The generations are further subdivided into waves based on improved potency and dosing[²³]. Telaprevir and boceprevir represent the first wave of the first generation of protease inhibitors. A number of agents are currently being investigated, which constitute the second wave. These agents have resistance profiles similar to telaprevir and boceprevir. Second generation protease inhibitors have pan-genotypic activity and a higher barrier for resistance than first generation PIs. Table 1 lists protease inhibitors currently under development.

Table 1 Protease inhibitors currently under development

• First generation (wave 2)
• Simeprevir
• Faldaprevir
• Danoprevir
• Vaniprevir
• ABT-450/ABT-450r
• Asunaprevir
• Second generation
• MK-5172
• ACH-2684

Simeprevir

Simeprevir is a macrocyclic NS3/4A protease inhibitor that is active against all genotypes of hepatitis C except genotype 3. It undergoes hepatic metabolism through cytochrome p4503A with an elimination half-life of 40 h, which makes it suitable for once daily dosing[²⁴]. In a large phase III clinical trial, simeprevir 150 mg once daily with pegylated interferon and ribavirin for 12 wk followed by pegylated interferon and ribavirin alone for an additional 12-36 wk produced SVRs at 12 wk post-treatment (SVR₁₂) of 81% in genotype 1, treatment naïve patients[²⁵]. Response rates were even higher in patients with the IL28B polymorphism and lower stage liver fibrosis. Frequently reported adverse effects included rash and indirect hyperbilirubinemia.

Faldaprevir

Faldaprevir is another potent NS3 protease inhibitor that can be dosed on a once daily basis. However, its activity is limited to genotype 1 disease. In a large phase III trial of genotype 1, treatment-naïve patients, faldaprevir 120 or 240 mg once daily with pegylated interferon and ribavirin for 12 wk achieved 80% SVR₁₂ with even higher responses seen in patients with the IL28B polymorphism[²⁶]. Indirect hyperbilirubinemia was reported in this trial as well.

Danoprevir

Danoprevir is also a potent macrocyclic protease inhibitor with activity against HCV genotypes 1, 4, and 6. Unlike simeprevir and faldaprevir, however, danoprevir requires twice daily dosing. In a recent phase IIb study, danoprevir 300 mg every 8 h, 600 mg every 12 h, 900 mg every 12 h or placebo was given with pegylated interferon and ribavirin for 12 wk in genotype 1 patients and followed with pegylated interferon/ribavirin alone for an additional 48 wk[²⁷]. Treatment was stopped at 24 wk if an extended rapid virologic response (eRVR) with HCV RNA below 15 IU/mL during weeks 4-20 was achieved. This occurred in 65% of the 300 mg group and 70% of the 600 mg group. Unfortunately, the 900 mg group was discontinued early due to reversible, grade 4 increases in alanine aminotransferase in three patients. SVR at 24 wk post-treatment was 68%, 85%, and 76% in the 300 mg, 600 mg, and 900 mg groups, respectively, vs 42% in the placebo group. Serious adverse effects occurred in 19% of patients in the placebo group vs 7%-8% of patients in the active treatment groups.

Vaniprevir

Vaniprevir is a potent NS3/4A protease inhibitor in genotypes 1 and 2 that has shown efficacy in phase II trials when given twice daily to non-cirrhotic patients who failed previous pegylated interferon/ribavirin therapy. Vaniprevir 300 mg and 600 mg twice daily for 24-48 wk with pegylated interferon/ribavirin therapy produced SVR rates at 24 wk post-treatment ranging from 66.7%-78% vs only 19% with placebo and pegylated interferon/ribavirin[²⁸]. Higher rates of gastrointestinal adverse events were seen with vaniprevir, but no difference in rash or anemia occurred between the two groups.

ABT-450/ABT-450r

ABT-450 is an NS3/4A protease inhibitor that is metabolized by cytochrome p4503A and coadministered with ritonavir 100 mg (ABT-450r) to allow once daily dosing. It has been studied in combination with ABT-333, an NS5B nonnucleoside polymerase inhibitor, as part of an interferon-free regimen[²⁹]. Clinical trial results of this combination will be discussed later.

Asunaprevir

Asunaprevir is also a potent NS3 inhibitor that is dosed twice daily, but is limited in efficacy to genotype 1 disease. It has been studied in combination with daclatasvir, a potent NS5A replication complex inhibitor, as a part of an interferon-free regimen[³⁰]. Clinical trial results of this combination will be discussed later.

MK-5172

MK-5172 is a second generation protease inhibitor with pan-genotypic activity. In a phase II trial, MK-5172 100, 200, 400 and 800 mg once daily combined with pegylated interferon and ribavirin for 12 wk gave SVRs of 86%, 92%, 91%, and 87%, respectively, compared with 54% in the control group receiving triple therapy with pegylated interferon/ribavirin and boceprevir[³¹]. Elevations in bilirubin and serum transaminases were seen mostly in the higher dose groups. Rates of serious adverse events were similar between all groups but half as many patients discontinued therapy due to adverse events in the MK-5172 treatment arms (7% vs 14%).

ACH-2684

ACH-2684 is also a pan-genotypic, highly potent second generation protease inhibitor. This compound can be dosed orally once daily and does not inhibit cytochrome p450 microsomal enzymes or activate transcription[³²]. ACH-2684 has completed phase I trials and is currently being evaluated in phase II.

NS5A HCV REPLICATION INHIBITORS

Daclatasvir

Unlike protease inhibitors that generally interfere with protein processing within the HCV genome, a number of compounds in development target nonstructural proteins involved in viral replication. (Table 2) Daclatasvir works by inhibiting the function of a viral replication complex by binding to the NS5A protein. It is highly potent orally, pan-genotypic in coverage and can be dosed once daily, but offers a lower barrier to resistance so will likely be used as combination therapy. Initial studies in combination with pegylated interferon/ribavirin showed 10 and 60 mg doses once daily produced SVRs at 24 wk post-treatment of 83% compared to 25% with standard therapy alone[³³]. More recently, daclatasvir combined with sofosbuvir, an NS5B polymerase inhibitor discussed below, was shown to produce a 100% SVR at 24 wk in genotype 1 patients who had failed triple therapy with pegylated interferon/ribavirin and telaprevir or boceprevir without ribavirin[³⁴]. The combination was generally well tolerated without serious adverse events or discontinuations related to adverse events. Daclatasvir has also been studied in combination with asunaprevir and another NS5B polymerase inhibitor (BMS-791325) in treatment-naïve, genotype 1 non-cirrhotic patients for 12 and 24 wk with SVRs of 88% and 94%, respectively[³⁰].

Table 2 Additional drugs in development for hepatitis C

• NS5A hepatitis C virus replication inhibitors
• Daclatasvir
• Ledipasvir
• ACH-3102
• ABT-267
• NS5B RNA dependent RNA polymerase inhibitors
• Sofosbuvir
• Mericitabine
• ABT-333
• Host targeted agents
• Alisporivir
• Miravirsen
• Interferon-λ

Ledipasvir

Ledipasvir is also an NS5A replication inhibitor that has been studied in combination with sofosbuvir, an NS5B polymerase inhibitor, in phase II trials. Ledipasvir 90 mg orally once daily in combination with both sofosbuvir and ribavirin in genotype 1 treatment-naïve and prior nonresponse patients increased SVRs to 100% at 12 wk post-treatment vs 84% in naïve patients given sofosbuvir and ribavirin alone and 10% in sofosbuvir/ribavirin nonresponders[³⁵]. These agents are currently in phase III trials in a fixed-dose, once daily oral combination formulation.

ACH-3102

ACH-3102 is a structurally distinct, pan-genotypic, second generation NS5A replication inhibitor with a high barrier to resistance. An ongoing phase II clinical trial is evaluating an oral, interferon-free combination regimen of ACH-3102 and sovaprevir with and without ribavirin for 12 and 8 wk durations of treatment in genotype 1 patients with HCV[³⁶]. ACH-3102 has been granted fast-track status from the United States Food and Drug Administration.

ABT-267

ABT-267 is an additional NS5A replication inhibitor that has been studied at a dose of 25 mg once daily in combination with ABT-450/r and ABT-333, an NS5B polymerase inhibitor, and ribavirin in non-cirrhotic, treatment-naïve patients with genotype 1 disease and prior pegylated interferon/ribavirin nonresponders. Phase II trial data showed a greater than 90% SVR in both groups at both 12 and 24 wk post-treatment[³⁷]. This combination regimen is currently being evaluated in a large phase III trial.

NS5B RNA DEPENDENT RNA POLYMERASE INHIBITORS

Nucleoside/tide inhibitors (NIs) block HCV RNA transcription and elongation by acting as chain terminators. Because of the highly conserved nature of the polymerase catalytic site, NIs as a class are pan-genotypic in coverage and they have the highest barrier to resistance. Non-NIs (NNIs) bind allosteric polymerase sites away from the catalytic site and, while potent, have a much lower barrier to resistance.

Sofosbuvir

Sofosbuvir is an NS5B nucleoside inhibitor that is pan-genotypic, highly potent, and suitable for once daily oral dosing. This compound has now completed study in four large, phase III trials and has been awarded priority review status by the United States Food and Drug Administration. Additionally, phase II studies of sofosbuvir combination therapies as interferon-free regimens are currently underway.

Sofosbuvir 400 mg once daily was initially studied in combination with pegylated interferon/ribavirin therapy in 327 HCV patients with genotypes 1, 4, 5, or 6 for 12 wk (98% of patients were genotype 1 or 4)[³⁸]. A SVR of 90% was achieved in these patients. A subsequent follow up study compared 499 patients with HCV genotypes 2 or 3 receiving sofosbuvir 400 mg once daily plus ribavirin or pegylated interferon plus ribavirin for 12 wk. The sofosbuvir/ribavirin regimen was shown to be non-inferior to the pegylated interferon/ribavirin regimen achieving as SVR of 67%[³⁸]. Adverse events were less common with sofosbuvir than pegylated interferon and consisted primarily of headache, fatigue, nausea, and neutropenia.

Sofosbuvir has since been evaluated in HCV patients with genotype 2 or 3 who were not eligible to receive pegylated interferon or who had previous failed therapy with this agent. Sofosbuvir 400 mg once daily in combination with ribavirin or matching placebos was studied in 278 patients who had previously discontinued pegylated interferon therapy secondary to side effects, who had a current medical condition precluding treatment with pegylated interferon, or who decided against treatment with pegylated interferon for other reasons. Sofosbuvir produced a SVR at 12 wk of 78% vs 0% with placebo in these patients[³⁹]. In a study of 201 similar patients who had failed previous therapy with pegylated interferon, sofosbuvir 400 mg once daily plus ribavirin was compared in 12 and 16 wk treatment groups. Sofosbuvir achieved SVR rates of 50% and 73% in these treatment arms respectively, compared with historical controls of 25%[³⁹]. The drug was well tolerated in both of these trials with primary side effects of fatigue and insomnia occurring in 3%-5% of patients and few patients discontinuing use because of adverse effects.

Mericitabine

Mericitabine is also an NS5B nucleoside inhibitor with pan-genotypic activity, but requiring twice daily dosing. Mericitabine 1000 mg twice daily or placebo was given with pegylated interferon/ribavirin to 166 patients with HCV genotypes 1 or 4 for 24 wk. Patients who achieved an HCV RNA level < 15 IU/mL (eRVR) from weeks 4 to 22 stopped all treatment at that time, but all other patients continued to receive pegylated interferon/ribavirin for a full 48 wk of therapy. Mericitabine-treated patients achieved a SVR at 24 wk post-treatment of 56.8%, compared with 36.5% of placebo-treated patients[⁴⁰]. Relapse rates were 27.7% and 32% in patients treated with mericitabine and placebo, respectively. The safety profile was similar in both groups, but fewer patients in the mericitabine group discontinued therapy for reasons of safety.

ABT-333

ABT-333 is a NS5B non-nucleoside polymerase inhibitor that has been evaluated in combination with the protease inhibitor, ABT450/r, and ribavirin in HCV genotype 1 treatment-naïve patients, as well as null or partial prior responders to pegylated interferon and ribavirin. ABT-333 at a dose of 400 mg twice daily plus ABT-450/r 150 or 250 mg and ribavirin produced SVRs of 93%-95% at 12 wk post-treatment in previously untreated patients[⁴¹]. Only 47% of prior null or partial responders to pegylated interferon and ribavirin achieved an SVR₁₂, however. As mentioned previously, this drug is currently being studied in a phase III trial in combination with ABT-450/r and ABT-267, with or without ribavirin in genotype 1b patients with HCV.

HOST TARGETED AGENTS FOR HEPATITIS C

Alisporivir

Alisporivir is a nonimmunosuppressive form of cyclosporine that blocks HCV replication by neutralizing the peptidyl-prolyl isomerase activity of the host protein, cyclophilin, which is required by NS5B for maximum RNA binding[⁴²]. Alisporivir 600 mg twice daily for one week followed by 600 mg daily thereafter or placebo combined with pegylated interferon and ribavirin was evaluated in 288 patients with treatment-naïve genotype 1 HCV for 24 and 48 wk of therapy[⁴³]. SVR rates at 24 wk post-treatment were 76% in patients receiving triple therapy with alisporivir and pegylated interferon/ribavirin for 48 wk vs 55% in the control arm. The drug was well tolerated overall with serious adverse events occurring in 7% of patients treated with alisporivir for 24 wk and 10% of patients treated with alisporivir for 48 wk. Subsequent to this trial, 3 patients developed pancreatitis, including one who died, and the FDA asked the manufacturer to place further trials on clinical hold until it can be determined if alisporivir potentiates the risk of hepatitis that is known with interferon therapy.

Miravirsen

Miravirsen is an antisense oligonucleotide that works by inhibiting miR-122, a micro-RNA found in the liver essential to the stability and propagation of HCV RNA. Miravirsen can be dose subcutaneously once daily, is pan-genotypic in activity, and has a high barrier to resistance. In a phase IIa dose-ranging trial, miravirsen resulted in dose-dependent reduction in HCV RNA levels that were still not present 14 wk post-treatment[⁴⁴]. No dose-limiting adverse events or escape mutations were noted in this small trial.

Interferon-λ

Pegylated interferon lambda is a type 3 interferon that signals through a different receptor than type 1 interferon, but can inhibit HCV viral replication in vitro. In early trials it was well tolerated without the usual flu-like syndrome and hematopoietic effects typically seen with interferon alpha. A phase II trial comparing pegylated interferon lambda with pegylated interferon alpha each in combination with ribavirin found comparable SVR rates but less interferon side effects with pegylated interferon lambda[⁴⁵]. This could prove to be a useful option for patients who do not respond to interferon-free regimens currently in development.

PLACE IN THERAPY OF NEW AGENTS FOR HEPATITIS C

Clearly the availability of both telaprevir and boceprevir, as well as other anti-HCV drugs in development, will vastly improve our antiviral armamentarium for patients with hepatitis C. Telaprevir and boceprevir must be used in conjunction with standard pegylated interferon and ribavirin therapy. However, the duration of treatment may be reduced substantially for many patients, based upon the clinical trial results from patients receiving a rapid virologic response. Substantial improvement in the SVR should be seen with these new agents for both treatment-naïve as well as prior relapsed or non-responsive patients. The further development of non-protease inhibitor based therapy offers the potential for interferon and ribavirin free therapy with once or twice daily oral dosing and a better tolerated side-effect profile. Additional studies are needed to determine which agent or agents should be used initially and the optimal combination regimen for patients needing additional therapy.

At this point in time, it appears that second generation protease inhibitors will supplant current protease inhibitor therapy on the basis of broader genotypic coverage and a higher barrier to resistance. Both NS5A replication inhibitors and NS5B RNA dependent RNA polymerase inhibitors offer greater potency than currently available drugs, but a lower barrier to resistance with the NS5As will likely require combination therapy. The availability of these new agents should preclude the continued need for interferon therapy and possibly for ribavirin therapy as well.

Footnotes

P- Reviewer: Scuteri A S- Editor: Ma YJ L- Editor: A E- Editor: Zhang DN

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Source

Naturally occurring resistance mutations to inhibitors of HCV NS5A region and NS5B polymerase in DAA treatment-naive patients

Virology Journal 2013, 10:355 doi:10.1186/1743-422X-10-355

Stefania Paolucci (s.paolucci@smatteo.pv.it)
Loretta Fiorina (loretta75@libero.it)
Bianca Mariani (b.mariani@smatteo.pv.it)
Roberto Gulminetti (r.gulminetti@smatteo.pv.it)
Stefano Novati (s.novati@smatteo.pv.it)
Giorgio Barbarini (g.barbarini@smatteo.pv.it)
Raffaele Bruno (r.bruno@smatteo.pv.it)
Fausto Baldanti (f.baldanti@smatteo.pv.it)

ISSN 1743-422X

Article type Research
Submission date 23 September 2013
Acceptance date 3 December 2013
Publication date 17 December 2013

Article URL http://www.virologyj.com/content/10/1/355
This peer-reviewed article can be downloaded, printed and distributed freely for any purposes (see
copyright notice below).

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Abstract

Background

Direct-acting antiviral (DAA) agents target HCV proteins; some of these have already been approved for the treatment of HCV infection, while others are in development. However, selection of DAA-resistant viral variants may hamper treatment. The aim of this study was to illustrate potential natural DAA-resistance mutations in the HCV NS5A and NS5B regions of HCV genotypes 1a and 1b from DAA-naïve patients.

Methods

Direct sequencing of HCV NS5A and NS5B regions was performed in 32 patients infected with HCV genotype 1a and 30 patients infected with HCV genotype 1b; all subjects were naïve to DAAs.

Results

In genotype 1a strains, resistance mutations in NS5A (M28V, L31M and H58P) were observed in 4/32 (12.5%) patients, and resistance mutations in NS5B (V321I, M426L, Y448H, Y452H) were observed in 4/32 (12.5%) patients. In genotype 1b, resistance mutations in NS5A (L28V, L31M, Q54H, Y93H and I280V) were observed in 16/30 (53.3%) patients, while resistance mutations in NS5B (L159F, V321I, C316N, M426L, Y452H, R465G and V499A) were observed in 27/30 (90%) patients.

Conclusions

Mutations conferring DAA resistance were detected in NS5A and NS5B of HCV genotypes 1a and 1b from DAA-naïve patients. Although some mutations confer only a low level of resistance, the presence at baseline of mutated HCV variants should be taken into consideration in the context of DAA therapy.

Keywords Hepatitis C virus, HCV baseline resistance, NS5A and NS5B genes, DAA inhibitors

Background

Hepatitis C virus (HCV) is classified into six genotypes (1–6) and more than 100 subtypes. The most common genotypes in Western countries are 1a and 1b [1]. Peginterferon/ribavirin (PegIFN/RBV) for the treatment of HCV infection is burdened by adverse reactions in at least 10% of patients [2]. Moreover, a sustained virological response is achieved in only 50% of patients infected with HCV genotype 1 [3]. PegIFN/RBV treatment failure is mainly attributed to its low efficacy against genotypes 1 and 4, but also, to some extent to its side effects [3,4]. Recently developed direct-acting antiviral agents (DAAs) are predicted to have a major impact both in combination with PegIFN/RBV, as well as in IFN-free regimens and telaprevir and boceprevir have now been approved as standard of care treatment [5]. Targets for DAA include HCV NS3 protease, NS5B polymerase and NS5A protein which are essential for virus replication. [6-12].

Nevertheless, the combination of a high HCV replication rate, the low fidelity of HCV polymerase and selective pressures by the immune system and drug treatment lead to the in vivo development of viral quasispecies with high sequence diversity among various genotypes and subtypes [13,14] with the potential accumulation of virus variants showing mutations with varying degrees of resistance to DAAs [11-13,15-22], even in the absence of pre-existing drug-exposure [17,23-26]. In particular, natural changes in HCV NS5A and NS5B amino acids (aa) associated with reduced drug susceptibility have been observed in treatment naïve patients [17,27,28].

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Hepatitis C Virus NS3 Inhibitors: Current and Future Perspectives

BioMed Research International
Volume 2013 (2013), Article ID 467869, 9 pages
http://dx.doi.org/10.1155/2013/467869

Review Article

Kazi Abdus Salam and Nobuyoshi Akimitsu

Radioisotope Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan

Received 29 April 2013; Accepted 8 September 2013

Academic Editor: Fumio Imazeki

Copyright © 2013 Kazi Abdus Salam and Nobuyoshi Akimitsu. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Currently, hepatitis C virus (HCV) infection is considered a serious health-care problem all over the world. A good number of direct-acting antivirals (DAAs) against HCV infection are in clinical progress including NS3-4A protease inhibitors, RNA-dependent RNA polymerase inhibitors, and NS5A inhibitors as well as host targeted inhibitors. Two NS3-4A protease inhibitors (telaprevir and boceprevir) have been recently approved for the treatment of hepatitis C in combination with standard of care (pegylated interferon plus ribavirin). The new therapy has significantly improved sustained virologic response (SVR); however, the adverse effects associated with this therapy are still the main concern. In addition to the emergence of viral resistance, other targets must be continually developed. One such underdeveloped target is the helicase portion of the HCV NS3 protein. This review article summarizes our current understanding of HCV treatment, particularly with those of NS3 inhibitors.

1. Introduction

In the mid-1970s, it was noticed that supply of blood was contaminated with an unidentified agent causing posttransfusion non-A, non-B hepatitis [1]. This unknown infectious agent struck intravenous drug users and blood transfusion recipients. The offender agent identified in 1989 was hepatitis C virus (HCV) and the first sequences of HCV were reported [2]. HCV is one of the leading agents that cause liver failure, and hepatocellular carcinoma and is the most relevant reason for liver transplantation. HCV infects about 3% of the world population; 130–200 million people are estimated to be chronically infected globally. Alarming news is that 350,000 people worldwide die from HCV-related disease every year [3]. For more than 20 years, HCV has been taking the attention of the health professionals, and now, well recognized that HCV is actually a major global health problem. Recently, health professionals determined the worldwide prevalence of HCV in comparison with HIV. The global prevalence of HCV estimates is 400,000 chronically infected subjects in Australia and Oceania, 14 million in the United States of America, 16 million in the Middle East, 17.5 million in Europe, 28 million in Africa, and 83 million in Asia [4]. Therefore, novel and effective inventions with fewer adverse effects are required for the prevention and control of HCV. The main goal of this review article is to be updated with the current treatments of HCV, putting an emphasis on the HCV NS3 protease and NS3 helicase inhibitors.

2. HCV Translation and Polyprotein Processing

HCV belongs to the founding member Hepacivirus genus of the family Flaviviridae [2, 5]; it is a positive sense single-stranded RNA virus with seven genotypes and more than 90 different subtypes [6]. The viral genome is 9600 nucleotides (nt) in length, which contains a 5′-nontranslated region (NTR) with an internal ribosome entry site (IRES), 3′-NTR and encode a single polyprotein containing 3000 amino acids, and is positioned between 5′-NTR and 3′-NTR. The translation of the polyprotein is initiated by an internal ribosome entry site (IRES) present at the 5′-NTR [7]. Unlike eukaryotic mRNA, HCV genome which lacks a 5′ cap translation depends on IRES that directly binds with 40S ribosomal subunits, inducing conformational changes in the 40S subunits [8]. The IRES-40S complex then recruits eukaryotic initiation factor (eIF) 3 and the ternary complex of Met-tRNA-eIF2-GTP to form a noncanonical 48S intermediate before a kinetic slow transition to the translationally active 80S complex [9, 10]. Once the formation of initiation complex takes place, the genome of HCV is translated to produce a large polyprotein that undergoes proteolytic cleavages with specific viral and cellular proteases to form 10 individual viral proteins, each of which has specific functions in viral life cycle (Figure 1). The N-terminal one-third of the polyprotein encodes the virion structural proteins; the core protein (C) forms the viral nucleocapsid and envelopes glycoproteins E1 and E2, involved in receptor binding required for viral entry into the hepatocyte [11]. A small integral membrane protein, p7, functions as an ion channel [12, 13]. The remaining portion of the genome encodes 6 important nonstructural (NS) proteins: NS2, NS3, NS4A, NS4B, NS5A, and NS5B, which coordinate the intracellular processes of the viral life cycle. Host endoplasmic reticulum (ER) derived signal peptidase cleavages the mature structural proteins among the junctions C/E1, E1/E2, and E2/p7. Signal peptide peptidase releases core from E1 signal peptide. The p7/NS2 junction is also cleaved by signal peptidase within the NS region. Two viral enzymes, the NS2 autoprotease and the NS3-4A serine protease, are involved further in the proteolytic processing of NS proteins. The NS2 autoprotease cleaves at the NS2/3 site, whereas the NS3-4A serine protease, which requires the NS4A protein as cofactor for functioning properly, cleaves at all downstream junctions. Another small protein that encodes HCV genome is called F (frame shift) or ARFP (alternative reading frame protein), but its precise roles in viral life cycle are unknown [14].

Figure 1: HCV genome and polyprotein processing. (a) Open arrow, closed arrows, closed circle, and open circles indicated signal peptide peptidase, signal peptidase, NS2 autoprotease, and NS3-4A serine protease cleavage site(s), respectively. (b) This figure was drawn by UCSF Chimera (http://www.cgl.ucsf.edu/chimera/), a software program for visualizing molecules, with the structural data from Protein Data Bank (PDB) ID 3O8R. Each domain of NS3 was color-coded. Both blue and purple represent helicase core domain, and green and yellow indicate C-terminal region and protease domain, respectively. ADP and RNA were drawn in red as ligands.

3. The Functions of HCV NS3 Proteins

NS3 is a multifunctional protein (amino acids 1–631) with serine protease activity at the N-terminal (aa 1–180) and a nucleoside-triphosphatase- (NTPase-) dependent RNA helicase activity (NS3 NTPase/helicase) at the C-terminal (aa 181–631). Both enzyme activities have been well defined and high-resolution structures have been solved [15]. The C-terminus of NS3 encodes a DExH/D-box RNA helicase. NS3 helicase hydrolyzed NTP as an energy source to unwind double-stranded RNA in a 3′ to 5′ direction during replication of viral genomic RNA [16]. Structural analysis of NS3 revealed the unidirectional translocation and proposed a new function of NS3 as translocase, considering feasible strategies for developing specific inhibitors to block the action of NS3 helicase [17]. The activity of NS3 helicase can be regulated by interactions between the serine protease and helicase domains of NS3 [18,19], indicating that these two enzyme activities may be somehow coordinated during replication. The function of the HCV helicase is unknown; it has been shown that without functional helicase domains, HCV cannot replicate in cells. It may be involved in the initiation of RNA synthesis on the HCV genome RNA, which contains stable 3′-terminal secondary structure in dissociation of nascent RNA strands from their template during RNA synthesis or in displacement of proteins or other trans-acting factors from the RNA genome. It has been now well recognized that both activities of NS3 protein are required for the replication of virus; they are considered as attractive target sites for the development of direct-acting antivirals (DAAs) therapies. NS5B is the viral RNA-dependent RNA polymerase [20], another promising anti-HCV target site. NS5A is a phosphoprotein specifically [21] capable of interacting with the 3′-NTR of the HCV genome [22], other nonstructural proteins [23], and numerous cellular proteins [24, 25]. NS5A also functions in virus assembly [26, 27]. NS4B is an integral membrane protein that is required for the assembly of the “membranous web,” the organelle used for RNA replication [28, 29]. NS4A is a cofactor for NS3 that directs the localization of NS3 and modulates its enzymatic activities [30].

4. Current Treatment for HCV Infection

A combination of pegylated interferon and ribavirin is still the only choice for the treatment of hepatitis C. Depending on the genotypes, this standard of care (SOC) increased the sustained virologic response (SVR) and defined the HCV RNA levels undetectable in the blood 24 weeks posttreatment, from ~5% to ~40–80%. In HCV genotype 1 infected patients, those with high viral loads, mostly null responders or relapsers, the SOC treatment with pegylated interferon plus ribavirin for 48 weeks achieves 50% SVR [31–35]. On the other hand, the SOC treatment with pegylated interferon plus ribavirin for 24 weeks up to 80% achieves SVR in the HCV genotype 2 infected patients. However, current SOC is associated with severe side effects including rash, nausea, anemia, and depression.

The preventive measures against HCV include the development of HCV vaccine which may be one good idea. This is a challenging job because HCV has a great ability to change its amino acid and evade the immune response, which is-10 fold higher than HIV [3]. The development of HCV vaccine is now in progress [36]. In 2011, the US Food and Drug Administration approved two new antivirals, boceprevir and telaprevir, which was a milestone in HCV research. They inhibit an important viral protein, the NS3-4A protease. The drugs are designed in such a way that specifically attack HCV genotype 1, which is considered one of the most prevalent genotypes, accounting for about 60% of global infections, and the least responsive to current treatment. This new standard of care, a combination of boceprevir or telaprevir with peg-IFN plus ribavirin, has been approved for elimination of HCV infection in the USA, Europe, and Japan [37–40].

Anti-HCV DAAs can be classified into several categories: (1) HCV NS3-4A serine protease inhibitors, (2) HCV NS3 NTPase/helicase inhibitors, (3) HCV NS5B polymerase inhibitors, (4) HCV NS5A inhibitors, and others.

4.1. HCV NS3 Protease Inhibitors

HCV NS3-4A protease inhibitors (NS3-4A PIs) are classified into two groups. (1) The first generations PIs (boceprevir and telaprevir) are the linear α-ketoamide derivatives. These two inhibitors formed a covalent bond with the active site of the enzyme in a reversible way. Boceprevir and telaprevir are considered the first two DAAs that come to the HCV drug market, and are approved by FDA for the treatment of HCV genotype 1 infected patients as triple therapy with conventional approaches. (2) The second generations of PIs are mostly linear and macrocyclic noncovalent inhibitors of the NS3-4A enzyme. To date, both generations of PIs are highly potent inhibitors of the NS3-4A enzyme. It is known that the advantages of the second generations of PIs over the first generations are their convenience and improved side effects profile. As the resistance mutations are crucial issues in HCV therapy, unfortunately, they share the same basic resistance mutations that are generated by the first generations of PIs. Only two exceptional drugs, MK-5272 and ACH-2684, do not share the same resistance mutations, are now in clinical investigations (Table 1). ABT-450/r with potent clinical effects achieved SVR through 36 weeks of posttreatment observation, raising the possibility to treat hepatitis C with interferon-free regimens in HCV genotype 1 infected patients [41]. Simeprevir (TMC435) is being under investigated macrocyclic noncovalent NS3-4A protease inhibitor that is currently in Phase III clinical development. Clinical data showed that the addition of TMC435 to the SOC significantly increased the SVR [42]. Faldaprevir (BI 201335) is an inhibitor of HCV NS3-4A protease and is undergoing Phase III clinical trials [43, 44]. The major pharmacologic properties of clinically developed NS3-4A protease inhibitors are summarized in Table 2.

Table 1: Current HCV NS3-4A protease inhibitors/drugs in pipeline (/r means boosted by ritonavir).

Mechanism	Inhibitor name	Genotypic coverage	Daily dosing	Company	Status
Reversible covalent inhibitor	Incivek (telaprevir, VX-950)	1	Three times	Vertex	Approved
	Victrelis (boceprevir, SCH503034)	1	Three times	Merck	Approved
Noncovalent inhibitor	ABT-450/r	1	Once	Abbott	Phase III
	Simeprevir (TMC435)	1, 2, 5, and 6	Once	Janssen	Phase III
	Faldaprevir (BI201335)	1	Once	Boehringer Ingelheim	Phase III
	Danoprevir (RG7227)	1	Twice	Genentech	Phase II
	Vaniprevir (MK-7009)	1	Twice	Merck	Phase II
	MK-5172	1, 2	Once	Merck	Phase II
	Asunaprevir (BMS-650032)	1, 4	Once	Bristol-Myers Squibb	Phase II
	ACH-1625	1	Once	Achillion	Phase II
	GS-9256	1	Twice	Gilead	Phase II
	ACH-2684	1, 3	Once	Achillion	Phase II
	GS-9451	1a, 1b	Once	Gilead	Phase II
	Narlaprevir/r	1	Once	Merck	Phase II
	IDX320	1, 1b, 3a, and 4a	Once	Idenix	Phase II

Table 2: Pharmacologic properties of direct-acting anti-HCV agents in clinical development, modified by Liang and Ghany [71].

Property	NS3-4A protease inhibitors
Efficacy	High
Genotypic coverage	Narrow (second generation drugs have broader coverage)
Probability of drug resistance	High
Side effects	Substantial
Drug-drug interactions	Substantial

Currently, many NS3 protease inhibitors with various combinations of NS5A and polymerase inhibitors, with or without ribavirin, are being clinically investigated. For example, a study of the protease inhibitor asunaprevir in combination with the NS5A inhibitor daclatasvir, administered for genotype 1a or 1b infected patients, showed the eradication of the virus in 4 out of 11 patients (36%) [45]. Another report, which used the same regimen but only in patients with genotype 1b infection, achieved SVR 90% [46]. These two studies clearly demonstrated the effects of HCV subtype on the response to a regimen that consists entirely of direct-acting antiviral agents. Therefore, it may be feasible to treat HCV without interferon or ribavirin.

4.2. HCV NS3 NTPase/Helicase Inhibitors

The structure of the NS3 helicase is also available and well characterized. However, the developments of NS3 helicase inhibitors have been slow. This target is traditionally difficult as evidenced by the fact that no helicase inhibitors have been approved for clinical use. The main issue might be toxicity because the motor domains of HCV helicase are conserved to that of cellular proteins. As a result, more attention should be given to find inhibitors that bind sites rather than the conserved regions of cellular enzymes without affecting cellular ATPases or GTPases. Recently, a good number of high-throughput screening systems (HTS) have been developed to screen potential inhibitors that specifically inhibit essential activities of NS3. Many world renowned laboratories are engaged to study the helicase portion of NS3 as a possible HCV drug target over the last 17 years. Several studies have revealed that NS3 is essential for viral replication, both in whole animal and replicon model [47, 48]. Mutations in HCV RNA are unable to replicate in subgenomic replicons, which further validates the necessities of NS3 helicase in viral life cycle. NS3 helicase has unique property that plays a more complex role in viral replication. NS3 helicase unwinds both double-stranded DNA and duplex RNA, but typically most helicases do not unwind both. It is known that there is no DNA stage in HCV replication and replication occurs outside the nucleus; the biological importance of the NS3 helicase’s ability to unwind DNA remains elucidate.

The ATP and RNA binding sites are the most promising targets on HCV. To the best of our knowledge, very limited numbers of small molecules have been reported in the literature over the past years and fewer structure-activity relationships data are available. Because NS3 helicase seems to key cellular motor proteins, monitoring ATP hydrolysis is the early screening assays to screen potential inhibitors that yielded few specific hits. However, recent screens of small chemical libraries through HTS have identified some valuable compounds that inhibit HCV catalyzed DNA unwinding, NTPase-dependent RNA helicase, and RNA binding ability, some of which also prevent HCV replicon in cells. Major NS3 helicase inhibitors with their helicase inhibitory activity employing both DNA or RNA substrate and ATPase activities are discussed in Table 3.

Table 3: Inhibitory effects of some NS3 helicase inhibitors.

NS3 helicase inhibitor	IC50 (μM)			References
	Helicase
	DNA	RNA	ATPase
DRBT	1.5	500	No inhibition	[49]
TBBT	20	60	No inhibition	[49]
Soluble blue HT	40	Inhibition	23.8	[51]
Ring-expanded (fat) nucleoside analogues	7–11	5.5–12	Activation	[52]
AICAR analogue (compound 4)	37	No inhibition	ND	[53]
QU663	, 0.75	ND	No inhibition	[54]
p14	0.2	ND	No inhibition	[55]
DBMTr	17.6	No inhibition	No inhibition	[56, 57]
Acridone derivatives	1.5–20	ND	No inhibition	[58, 59]
Thiazolpiperazinyl derivative (compound 23)	110	ND	1000	[60]
(BIP)2B	5.4	0.7	Inhibition (in the presence of RNA)	[61]
Tropolone derivatives	3.4–17.8	ND	ND	[62]
Tetrahydroacridine derivative, 3a	, 0.02	ND	ND	[63]
Manoalide	ND	15	70	[64]
Thioflavin S	10	12	ND	[65]
SG1-23-1	ND	11.7 μg/mL	No inhibition	[66]
LOPAC compounds	0.6–3.7	0.8–8.9	ND	[67]
C-29EA	ND	18.9 μg/mL	No inhibition	[68]
Psammaplin A	ND	17	32	[69]
Cholesterol sulfate	ND	1.7	No inhibition	[70]

ND: not determined.

Halogenated benzimidazoles and benzotriazoles such as dichloro(ribofuranosyl) benzotriazole (DRBT) and tetrabromobenzotriazole (TBBT) both inhibit HCV helicase catalyzed DNA unwinding with IC₅₀of 1.5 and 20 μM, respectively. When employing RNA substrate, only TBBT inhibits RNA unwinding with IC₅₀ of 60 μM [49]. In another report, the efficacies of TBBT and DRBT were tested in four different HCV genotype 1b replicon systems. Depending on the cell line, TBBT inhibits HCV replicons with IC₅₀ ranging from 40 to 65 μM and DRBT inhibits HCV replicons with IC₅₀ ranging from 10 to 53 μM [50].

Soluble blue HT inhibits NS3 catalyzed DNA unwinding with an IC₅₀ of 40 μM [51]. After several rounds of structural refinement, discovered one of the soluble blue HT derivatives, compound 12, which is a good anti-HCV agent with an IC₅₀ of 10.1 μM and EC₅₀ value of 2.72 μM against HCV NS3 catalyzed DNA unwinding and replicon Ava.5/Huh-7 cells, respectively [51].

Ring-expanded “fat” nucleosides (RENs) inhibit HCV and related Flavivirus helicase, including the West Nile virus (WNV) and Japanese encephalitis virus (JEV). They catalyzed HCV DNA unwinding with IC₅₀ in the 7–11 μM range and HCV helicase catalyzed RNA unwinding with IC₅₀ of 5.5–12 μM. In this paper, RENs demonstrated different selectivity profiles between the viral enzymes [52].

Another nucleoside, the compound 4 (4-carbamoyl-5-[4,6-diamino-2,5-dihydro-1,3,5-triazin-2-yl]imidazole-1-β-D-ribofuranoside), inhibits helicase catalyzed DNA unwinding against WNV and HCV with IC₅₀ of 23 and 37 μM, respectively, but it had no effect on helicase catalyzed RNA unwinding. It was a surprise that no activity was observed against the NTPase/helicase of either DENV or JEV irrespective of whether RNA or a DNA substrate was employed [53].

QU663 inhibits HCV helicase catalyzed DNA unwinding with a <?XML:NAMESPACE PREFIX = "[default] http://www.w3.org/2000/svg" NS = "http://www.w3.org/2000/svg" /> of 750 nM, competing with the nucleic acid substrate without affecting ATPase function, even at high concentrations. Docking studies showed that by interacting with the putative binding site QU663 induced a similar conformational shift [54].

Small peptide inhibitor, 14 amino acid-long peptide (p14), revealed a basic amino acid stretch corresponding to motif VI of HCV, WNV, and JEV of NTPase/helicase. This peptide inhibited the HCV unwinding activity of the enzyme with an IC₅₀ of 0.2 μM employing DNA substrate. The order of inhibitory effects was HCV > WNV > JEV. The binding of the peptides does not interfere with the NTPase activity of the enzymes [55].

Tropolone derivatives have been screened as inhibitors of HCV helicase catalyzed DNA unwinding. The derivative of tropolone, called 3,7-dibromo-5 morpholinomethyltropolone (DBMTr), acts with an IC₅₀of 17.6 μM. It has no effect on HCV helicase catalyzed ATP hydrolysis [56] nor HCV helicase catalyzed RNA unwinding [57]. The authors also mentioned that DBMTr might be developed as potent inhibitor of the HCV helicase due to its low toxicity to yeast cells [56].

Acridone derivatives have also been screened as inhibitors of HCV helicase catalyzed DNA unwinding with IC₅₀ between 1.5 and 20 μM. These compounds also inhibit replication of HCV (EC₅₀ 1–10 μM) and are not particularly toxic to cells [58, 59].

The thiazolpiperazinyl derivative compound 23 inhibits the helicase activity with an IC₅₀ of 110 μM, using DNA substrate. None of the compounds were able to inhibit the NS3 NTPase activity. Testing in the subgenomic HCV replication, it exhibited EC₅₀ of 3 μg/μL and CC₅₀ > 50 μg/μL [60].

1-N,4-N-bis[4-(1H-Benzimidazol-2-yl)phenyl]benzene-1,4-dicarboxamide, designed as (BIP)₂B, is a potent and selective inhibitor of HCV NS3 helicase, which inhibits unwinding reaction regardless of DNA or RNA substrate, but not ATP hydrolysis without RNA or at saturated level of RNA. (BIP)₂B inhibited NS3 helicase from HCV genotypes 1a, 1b, 2a, and 3a. Evidence presented here shows that it directly and specifically binds to NS3 protein [61].

Other new tropolone derivatives, compounds 2, 6, and 7, inhibit HCV catalyzed DNA unwinding (IC₅₀= 3.4–17.8 μM). They are also effective in RNA replication (EC₅₀ = 32.0–46.9 μM) and exhibit the lowest cytotoxicity. The derivatives 2 and 7 have been shown to be resistant mutants. The effects of the compound 2 plus IFN-γ and compound 2 plus ribavirin combinations were evaluated in cell culture, indicating that both combinations result in an additive effect with a very slight tendency to synergy [62]. The tetrahydroacridinyl derivative 3a is the most potent inhibitor reported to date ( nM). It did not show inhibition towards the ATPase activity of NS3 up to 100 μM [63].

Manoalide was originally identified as an inhibitor of phospholipase A₂, but later it was reported that it inhibits HCV NS3 helicase activity with RNA substrate (IC₅₀ = 15 μM). In addition, it inhibits the NS3 ATPase and RNA binding to NS3. A direct interaction between manoalide and NS3 was presented to explain the inhibition of NS3 activities through the structural change upon its binding [64].

The commercially available dye thioflavine S is identified as the most potent inhibitor of NS3 catalyzed DNA and RNA unwinding. After separating into their active components, P4 inhibits unwinding, subgenomic replication with IC₅₀ of 2 and 10 μM, respectively, and was not toxic [65].

SG1-23-1, isolated from ethyl acetate extract from marine feather star, Alloeocomatella polycladia, exhibits the strongest inhibition of NS3 helicase activity using RNA substrate (IC₅₀ = 11.7 μg/mL). Interestingly, the extract inhibits interaction between NS3 and RNA but not ATPase of NS3. Moreover, it also inhibits the RNA replication with EC₅₀ of 23 to 44 μg/mL [66].

Four LOPACs Sigma’s library of pharmacologically active compounds (ATA, AG 538, NF 023, and Suramin) were identified. All but AG 538 have the ability to unwind DNA (IC₅₀ = 0.6–3.7 μM) and RNA (IC₅₀ = 0.8–8.9 μM). All but NF 023 inhibited replication of subgenomic HCV replicons (EC₅₀ = 18–98 μM). Unfortunately, none of these inhibitors were specific to NS3 helicase [67].

Recently, it has been reported that an ethyl acetate extract from marine sponge Amphimedon sp., called C-29EA, inhibits both protease (IC₅₀ = 10.9 μg/mL) and helicase (IC₅₀ = 18.9 μg/mL) activities of HCV, but not ATPase activity. Importantly, it has been shown that the highest inhibition on viral replication is derived from genotypes 1b and 2a with EC₅₀ values of 1.5 and 24.9 μg/mL, respectively [68].

Psammaplin A (PsA) has antibacterial and antitumor activity and also inhibits a wide range of enzymes reported to date. PsA has the ability to inhibit HCV helicase catalyzed RNA unwinding (IC₅₀ = 17 μM) in addition to ATPase and RNA binding activity. PsA inhibited the subgenomic viral replication derived from genotype 1b and genotype 2a, with EC₅₀ 6.1 and 6.3 μM, respectively [69].

Cholesterol sulfate might be a potential inhibitor of HCV NS3 helicase, with IC₅₀ of 1.7 μM using RNA substrate. However, it exerted no ATPase and serine protease activity. A structure-activity study revealed that anion binding and hydrophobic region in NS3 may be targets of cholesterol sulfate [70].

Despite the great efforts, no potent and selective NS3 helicase inhibitors have been entered for clinical use. However, some good candidates, for example, soluble blue HT derivative, compound 12 [51], QU663 [54], and acridone derivatives [58, 59] have been identified to be suitable for further development as NS3 helicase inhibitors. It is not a surprise to imagine that NS3 helicase inhibitors will dominate HCV research in the near future.

5. Conclusions and Future Remarks

The direct-acting antiviral agents (DAAs), particularly NS3 protease inhibitors, telaprevir and boceprevir, which were approved in combination with current SOC (peg-IFN and ribavirin) for the treatment of HCV infection that significantly increased SVR, have opened a new window in HCV therapy. However, the side effects associated with this new therapy are a questionable maker. Anemia is the most frequent adverse effects with either telaprevir or boceprevir. They also exhibit strong inhibitory effect against an important drug metabolism enzyme, cytochrome P4503A4 (CYP3A4) resulting in the development of drug-drug interactions. In addition to drug resistance, the efficacies of these inhibitors differ significantly between HCV genotypes. It is well known that IFN itself has significant side effects. Another important issue arises with their short half-life and frequent dosing. With the advent of different small classes of DAAs, the future aim is to introduce an IFN-free regimen, oral cocktails of DAAs. The proof-of-concept studies presented some promising data confirming that the achievements of SVR without introducing IFN may be feasible. Thus, the combination of host and viral targeted inhibitors could be an attractive strategy in maximizing antiviral efficacy.

Conflict of Interests

The authors declare that they have no conflict of interests.

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