Recent advances and future directions in the management of hepatitis C infections.

JPT-06714; No of Pages 11 Pharmacology & Therapeutics xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Pharmacology & Therapeutics journal homepage: www.elsevier.com/locate/pharmthera

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Associate editor: F. Tarazi

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Victoria Belousova a, Ahmed A. Abd-Rabou a,b,c, Shaker A. Mousa a,⁎ a

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Keywords: Hepatitis C virus Hepatitis C prevention Hepatitis C treatment HCV genotypes Interferon free regimens Vaccine

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The Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, One Discovery Drive, Rensselaer, NY 12144, USA Hormones Department, Medical Research Division, National Research Center, Cairo, Egypt Center for Aging and Associated Diseases, Zewail City of Science and Technology, 6th of October, Egypt

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Current estimates indicate that the hepatitis C virus is the leading cause of death in the United States with infection rates steadily increasing. Successful treatment is made difficult by the presence of various host, virus, and treatment-related factors, warranting the development of new approaches to combat the silent epidemic. The addition of telaprevir and boceprevir to the pharmacotherapeutic arsenal drastically improved success rates in genotype 1 infected patients, but rapid development of resistance mechanisms increases in adverse effects, and a low spectrum activity proved to be barriers to efficacious treatment. In late 2013, two new agents were approved – sofosbuvir and simeprevir – that have higher barriers to resistance, favorable safety profiles, and profoundly improved success rates; however higher costs associated with the new medications could limit their wider utilization. Further strategies to combat the virus are under development, ranging from interferon-free regimens as well as prophylactic and therapeutic vaccines to applications of nanotechnology, helping us get closer to improved treatment of patients infected with hepatitis C. © 2014 Published by Elsevier Inc.

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Recent advances and future directions in the management of hepatitis C infections

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1. Introduction . . . . . . . . . . . . . . 2. Hepatitis C virus structure . . . . . . . . 3. Current hepatitis C virus treatment options 4. Reasons for treatment failure . . . . . . . 5. Drug targets . . . . . . . . . . . . . . 6. Sofosbuvir . . . . . . . . . . . . . . . 7. Simeprevir . . . . . . . . . . . . . . . 8. Future directions . . . . . . . . . . . . 9. Conclusion . . . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . References . . . . . . . . . . . . . . . . . .

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1. Introduction

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The hepatitis C virus (HCV), one of the main causes of morbidity and mortality globally, was first identified 25 years ago (Choo et al., 1989). Since then, the disease has spread, affecting 2–3% of the world's

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Abbreviations: GT, genotype; HA, hyaluronic acid; HCV, hepatitis C virus; RVR, rapid virologic response; SVR, sustained virologic response. ⁎ Corresponding author. Tel.: 518 694 7397; fax: 518 694 7567. E-mail address: [email protected] (S.A. Mousa).

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population (~130–170 million people) (Centers for Disease Control and Prevention, 2014). HCV infection is endemic globally, with a steady increase in prevalence rates between 1990 and 2005 from 2.3% to 2.8% (Mohd Hanafiah et al., 2013). The distribution of the infection varies broadly among various geographic areas. Industrialized areas like North America, Northern and Western Europe, and Australia have lower infection prevalence, while Africa and Asia have the highest reported rates (Zidan et al., 2012). In the United States, ~2.7–3.9 million people are infected, with prevalence and incidence rates steadily rising. Epidemiological data indicates that in 2011, there were a total of 1229

http://dx.doi.org/10.1016/j.pharmthera.2014.09.002 0163-7258/© 2014 Published by Elsevier Inc.

Please cite this article as: Belousova, V., et al., Recent advances and future directions in the management of hepatitis C infections, Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.09.002

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3. Current hepatitis C virus treatment options

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The main goal of HCV therapy is to clear or lower the virus below the level of detection, defined as the SVR. The SVR can be measured through a simple blood draw and is checked at 12 and 24 weeks post-treatment, with SVR 24 h post-treatment (SVR24) rates being indicative of treatment success. The goal of the primary efficacy endpoints in most of the treatment trials is to achieve undetectable HCV-RNA at weeks 4 and 12 (extended rapid virologic response, eRVR) and at 24 weeks posttreatment among infected patients. Several long-term follow-up studies have shown that the majority (nearly 100%) of patients who achieve SVR24 continue to remain “HCV negative” several years post-treatment (Maylin et al., 2009; Swain et al., 2010; Kim, 2011; Pearlman & Traub, 2011; Trapero-Marugán et al., 2011). Along with SVR, other markers of virologic response are often utilized to guide treatment (Table 1). Antiviral therapy is recommended for all patients who test HCV RNA positive, with treatment depending on genotype and co-morbid conditions; agents available for the treatment of HCV include peginterferon alfa-2a (PEGASYS®) or alfa-2b (PEGINTRON®), ribavirin (Copegus®, Rebetol®, Ribasphere®, Vilona®, and Virazole®), telaprevir (Incivek®) , boceprevir (Victrelis®), sofosbuvir (Sovaldi®), and simeprevir (Olysio®). It should be noted that in August 2014, Vertex Pharmaceuticals, the manufacturer of telaprevir, announced it would discontinue the sale of its hepatitis C medication (Incivek) in the United States in October. This comes as a result of a rise of alternative agents with higher success rates. Despite this, patients who are currently on telaprevir-based regimens will be able to continue taking the medication until they finish their treatments (Humer, 2014; Silverman, 2014). Recommended treatment regimens (American Society for the Study of Liver Diseases and Infectious Disease Society of America, 2013) are detailed in Table 2.

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4. Reasons for treatment failure

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The addition of the two protease inhibitors – telaprevir and boceprevir – has increased SVR rates among GT 1 patients from roughly 40% in treatment-naïve patients with dual therapy to 60.8–74.7% with telaprevir-based therapy and 54.2%–74.8% with boceprevir-based therapy. Despite these increases, overall SVR rates remain low due to a variety of host-, viral-, and treatment-related factors, contributing to the pursuit of new regimens for the treatment of HCV (Asselah et al., 2010; Manns & von Hahn, 2013).

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The viral particle that causes hepatitis C infection is an enveloped, single-stranded RNA genome containing roughly 9600 nucleotides (Bartenschlager & Lohmann, 2000). There are 11 genetically distinct genotypes (GTs) of the virus, and more than 50 subtypes have been identified (World Health Organization, 2002). The most prevalent GTs are 1–6, with a genetic variance of around 30% between them. Recent estimations in a selected United States population indicate that the GT percentages are: GT 1 (70%), GT 2 (16%), GT 3 (12%), GT 4 (1%), GT 5 (b1%), and GT 6 (1%) (Manos et al., 2012). Each genotype can be subdivided into a series of subtypes (1a, 1b, etc.), with a genetic

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variance of 20–25% between them, and in an individual person several quasispecies may exist. Despite these genetic differences, each genotype has a similar disease progression and similar mechanism of infection and replication (Simmonds, 2004). The primary mode of virus transmission appears to be percutaneous, with exposures resulting from injection drug use, blood transfusions and transmission via unsanitary conditions, and risky sexual behavior (Division of Viral Hepatitis, 2011). Once in the bloodstream, the virus' envelope proteins (E1 and E2) interact with several receptors on hepatic cells, including CD81, and a variety of low-density lipoproteins (Bartenschlager & Lohmann, 2000). After internalization, the viral genome is released and translated via host mechanisms to produce a 3000 amino acid polyprotein consisting of both structural and non-structural components (Fig. 1). The N-terminal (5′ end) of the polyprotein contains structural proteins C (the core), E1 and E2 (envelope glycoproteins), and p7 (a membrane protein that serves as an ion channel). The non-structural proteins are towards the 3′ end: NS2, NS3–NS4A, NS4B, NS5A, and NS5B. The NS2/3 cysteine protease starts a cascade of enzymatic reactions leading to the release of all subsequent proteins: NS3 serine protease and RNA helicase, NS3-4A serine protease, NS4B and NS5A RNA-binding proteins, and NS5B RNA-dependent RNA polymerase (Bartenschlager & Lohmann, 2000).

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newly reported cases of hepatitis C, representing a 44% increase over acute cases reported in 2010 (Division of Viral Hepatitis, 2011). For recent HCV epidemiological data, diagnosis, and prevention see Ansaldi et al. (2014). It is currently estimated that HCV is among the leading causes of death in the United States (Ly et al., 2012). A recent analysis of mortality data estimated that between 1999 and 2007 HCV-related deaths increased by 0.18 deaths per 100,000 persons per year. Comparatively, HIV-associated deaths for the same timeframe decreased by 0.21 deaths per 100,000 persons per year (Ly et al., 2012). Those between ages 20 to 29 had the highest increase in incidence rates (ranging from 0.75 to 1.18 cases per 100,000 population) (Division of Viral Hepatitis, 2011), indicating a growing number of patients needing treatment and increasing the burden of disease. The current medical costs associated with hepatitis C are estimated to be $6.5 billion and are expected to peek at $9.1 billion in 2024 (Razavi et al., 2013). Of those who become infected, 75–85% will go on to develop chronic infections (Division of Viral Hepatitis, 2011); however, the time between acute and chronic disease manifestation can take 20 to 30 years (Klevens et al., 2012), which makes identification of those infected difficult. During this time, the majority of patients remain either asymptomatic or experience non-specific symptoms, including nausea, anorexia, fatigue, abdominal pain, depression, and decreased overall physical and social functioning. Dark urine and jaundice are also common, though they are representatives of possible liver diseases (Klevens et al., 2012; Centers for Disease Control and Prevention, 2014). Not only is infection associated with lower health-related quality of life, but it is also one of the major causes of hepatocellular cancer and cirrhosis, and is the leading cause for liver transplantation (Bezemer et al., 2012; Centers for Disease Control and Prevention, 2014). HCV has also been associated with other disease states including diabetes, certain types of lymphomas, and renal disease (Klevens et al., 2012). Hepatitis C represents a growing health concern both in the United States and around the world. Treatment of the disease has been difficult due to a variety of host- and virus-related factors, and rates of clearing or lowering the virus below detection levels (termed sustained virologic responses, SVRs) remain low, and many patients do not tolerate treatment due to undesirable adverse events. However, in 2013 two new agents entered the market: the nucleoside NS5B polymerase inhibitor sofosbuvir (Sovaldi) and the NS3/4A protease inhibitor simeprevir (Olysio), which have demonstrated promising results for all patient populations, even those who have failed previous treatment. Also in 2013 the American Association for the Study of Liver Diseases released new guidelines for the management of HCV (American Society for the Study of Liver Diseases and Infectious Disease Society of America, 2013), providing an update to the previous recommendations. Additionally, novel treatment approaches are being investigated, including the development of a vaccine and encapsulation of therapies within nanoparticles that are targeted directly to the infected hepatocyte to reduce adverse events. This review focuses on the potential utilization of sofosbuvir and simeprevir, with an emphasis on the future prospects of improved managing and eventually eradication of this “silent epidemic”.

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Fig. 1. Hepatitis C polyprotein structure and selected targets. The HCV viral particle contains roughly 9600 nucleotides, which are translated into a 3000 amino acid polypeptide using host machinery. The polypeptide consists of both structural and non-structural components. The N-terminal (5′ end) of the polyprotein contains structural proteins C (the core), E1 and E2 (envelope glycoproteins), and p7 (a membrane protein that serves as an ion channel). The non-structural proteins are towards the 3′ end: NS2, NS3–NS4A, NS4B, NS5A, and NS5B. The NS2/3 cysteine protease starts a cascade of enzymatic reactions leading to the release of all subsequent proteins: NS3 serine protease and RNA helicase, NS3–4A serine protease, NS4B and NS5A RNA-binding proteins, and NS5B RNA-dependent RNA polymerase. The majority of these viral components have been investigated as targets for anti-HCV therapy, primarily NS3/4A and NS5B inhibitors and more recently NS5A and p7. Additionally, the envelope and core proteins are being utilized as potential targets for both prophylactic and therapeutic vaccines.

4.1. Host-related factors

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Several patient characteristics have been linked with worsening response rates, including age ≥40, male gender, cirrhosis, steatosis, insulin resistance, diabetes, African American ethnicity, obesity, alcohol consumption, co-infection with HIV and/or hepatitis B virus and the IL28B gene, which has been associated with predicting response to peginterferon and ribavirin (Asselah et al., 2010; Zhu & Chen, 2013).

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4.2. Viral-related factors

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HCV genotype, high baseline viral load (N800,000 IU/mL), and virologic response during treatment have all been implicated in reducing

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Table 1 Definitions of virologic response for hepatitis C virus (HCV).

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treatment response. The fast rate of replication of the virus combined with highly error-prone NS5B polymerase contributes not only to the high range of virus diversity, but also to the necessity of genotypespecific treatment regimens. The addition of the protease inhibitors has also added a new dimension for treatment failure and worsening response rates in the form of resistance mechanisms. Both treatment-naïve (Kuntzen et al., 2008; Palanisamy et al., 2013) and treatment-experienced patients have shown resistance strains; resistance can develop quickly and persist for up to 4 years post-treatment with both telaprevir and boceprevir treatments (Susser et al., 2011). Moreover, telaprevir and boceprevir are effective against GT 1 infections with limited efficacy in other genotypes because of their specific effectiveness against HCV NS3 protease, which is dominant in HCV genotype 1 infections.

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Definition

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Rapid virologic response Early virologic response Partial early virologic response Extended rapid virologic response

RVR eRVR – eRVR

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End-of-treatment response Sustained virologic response

ETR SVR12

Undetectable viral load at week 4 of treatment ≥2 log10 IU/mL reduction in HCV RNA at week 12 compared with pretreatment HCV RNA level Detectable HCV viral load despite ≥2 log IU/mL reduction in HCV RNA at week 12 of treatment Undetectable viral load between weeks 4 and 12 or 8 and 24 when treated with telaprevir or boceprevir, respectively Undetectable viral load at the end of treatment Negative viral load 12 weeks post-treatment This is the currently accepted end point in clinical trials Negative viral load at 24 weeks post-treatment Generally considered as patient achieving cure Reappearance of detectable viral load while on therapy or following end of therapy Failure to achieve negative viral load by week 24 of treatment Failure to achieve N2 log10 IU/mL reduction in HCV RNA by week 24 of treatment ≥2 log10 IU/mL reduction in HCV RNA level by week 24 but still HCV RNA positive

SVR24 t1:10 t1:11 t1:12 t1:13

Breakthrough or relapse Non-responder Null responder Partial responder

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Table 2 Recommended treatment regimens for treatment-naïve patients based on genotype [American Society for the Study of Liver Diseases and Infectious Disease Society of America (2013). Recommendations for Testing, Managing, and Treating Hepatitis C. Accessible via. http://hcvguidelines.org/full-report-view)]. Dosages common among all genotypes are: sofosbuvir, 400 mg daily; PegIFN alfa-2a, 180 μg SC once weekly; PegIFN alfa-2b 1.5 μg/kg SC once weekly; ribavirin, b75 kg body weight, 1000 mg daily (400 mg in the morning, 600 mg in the evening); ribavirin, ≥75 kg body weight, 1200 mg daily (600 mg in the morning, 600 mg in the evening). IFN = interferon.

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IFN eligible Sofosbuvir Ribavirin PegIFN alfa-2a PegIFN alfa-2b IFN ineligible Sofosbuvir +/− ribavirin Simeprevir (150 mg daily) Sofosbuvir + ribavirin Sofosbuvir + ribavirin IFN eligible Sofosbuvir Ribavirin PegIFN alfa-2a PegIFN alfa-2b IFN ineligible Sofosbuvir + ribavirin Sofosbuvir Ribavirin PegIFN alfa-2a PegIFN alfa-2b

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The replacement of interferon with peginterferon in regimens has reduced a considerable amount of side effects (Table 3), however, discontinuation due to adverse events such as neutropenia, anemia, headache, and fatigue remains high in therapies containing peginterferon (Manns et al., 2001; Torriani et al., 2004). Many patients are not able to receive therapy due to contraindications and drug interactions, leaving them without treatment options. The addition of telaprevir and boceprevir increased rates of side effects typically associated with peginterferon and ribavirin. Moreover, the administration requirements (multiple-daily dosing, take with food) can lead to unfavorable weight gain in patients.

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5. Drug targets

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Uncovering the viral genome paved the way for investigation of direct-acting anti-retroviral agents. Studies in chimpanzees demonstrated that the enzymatic activity of NS2/3, NS3/4A, and NS5B is

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Table 3 Common adverse events associated with peginterferon, ribavirin, telaprevir, and boceprevir therapies.

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The actions of the NS5B RNA-dependent polymerase may be halted via nucleoside or non-nucleoside derived inhibitors. Both prevent the initiation of RNA transcription and elongation of nascent RNA via differing mechanisms (Ranjith-Kumar & Cheng Kao, 2006). Nucleoside-derived analogs are considered pro-drugs because they must first be converted into an active triphosphate form (Asselah et al., 2009). Then they will bind directly to the active site, preventing further function of the enzyme. Resistance to nucleoside-based polymerase inhibitors is expected to be low due to lack of tolerance to amino acid substitutions in this area (Manns & von Hahn, 2013). Additionally, because of this lack of diversity within the active site, these polymerase inhibitors are expected to have similar efficacy throughout the hepatitis C virus spectrum (Zhu & Chen, 2013). In contrast, non-nucleoside derived analogs bind to one of five allosteric sites on the NS5B polymerase, causing conformational changes that inhibit its activity (Zhu & Chen, 2013). Mutations may be more common in non-nucleoside NS5B polymerase inhibitors because they might not necessarily lead to impairment of enzymatic function; furthermore these mutations may confer different efficacy depending on genotype (Kwo & Vinayek, 2011). Because of the different mechanisms of actions, there is potential for utilizing a combination of nucleoside and non-nucleoside polymerase inhibitors in the treatment of hepatitis C, however the use of this combination hasn't been evaluated yet. Humans lack RNA-dependent RNA polymerases, allowing treatment to be very specific and reduce unwanted side effects that otherwise might be attributed to treatment. Sofosbuvir is the only NS5B polymerase inhibitor currently FDA-approved for use; several other nucleoside and non-nucleoside agents are currently being developed.

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5.2. Polymerase inhibitors

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The hepatitis C genome encodes for two proteases, NS2/3 autoprotease and NS3/4A serine protease. The NS2/3 cysteine protease is less studied, and there are conflicting reports on its importance for viral replication (Lohmann et al., 1999; Kolykhalov et al., 2000). A recent study evaluated the use of a cysteine protease inhibitor, cystatin C, in children with hepatitis C. Results indicated increases in cystatin C levels correlated with reductions in viral load, suggesting that inhibition of NS2/3 may be effective in inhibiting HCV replication (Behairy et al., 2012). Further research should be conducted with NS2/3 inhibitors to determine the magnitude of effect and potential usefulness for treatment of HCV. The NS3/4A protease consists of a catalytic site (NS3) and a co-factor (NS4A), which are both required for optimal function of the NS3 protease. Not only is this serine protease responsible for cleaving subsequent enzymes, but it has also been shown to act as an antagonist to host immunity by suppressing interferon production and subsequently preventing degradation of viral RNA (Foy et al., 2003). Telaprevir and boceprevir are so-called ‘first-generation NS3/4A inhibitors’. Though they have led to substantial increases in SVR rates among GT 1 patients, the main concern is the emergence of resistant strains, many of which have been identified in treatment-naïve patients. Because of the specific amino acid sequence of NS3/4A, inhibitors have varying effects in different genotypes (Schinazi et al., 2014). To combat this disparity, several second-generation/second-wave protease inhibitors are being developed. These new agents will be higher potency, effective against first-generation effective strains, and have more favorable pharmacokinetic profiles (Clark et al., 2013).

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essential for HCV replication (Kolykhalov et al., 2000). The non- 227 enzymatic NS5A protein has also shown importance in viral replication 228 (Bukh et al., 2002). 229

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t2:1 t2:2 t2:3 t2:4 t2:5 t2:6 t2:7 t2:8 t2:9


Central nervous system • Fatigue • Headache • Fever • Insomnia • Depression • Dizziness Dermatologic • Alopecia • Pruritus • Dry skin • Injection site reactions (peginterferon)

Gastrointestinal • Nausea • Anorexia • Diarrhea • Weight loss Hematologic • Hemolytic anemia • Neutropenia General • Myalgia • Arthralgia • Back pain • Cough • Flu-like syndrome


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Sofosbuvir (Sovaldi®, Gilead Sciences, Inc.) is a novel nucleoside analog NS5B RNA-dependent polymerase inhibitor approved in late 2013 for the treatment of chronic hepatitis C GT 1, 2, 3, and 4 infections in mono-infected and HIV/HCV co-infected persons in combination with either peginterferon interferon and/or ribavirin (Gilead Sciences, Inc., 2013a). Phase 2 and 3 trials have evaluated its safety and efficacy in a variety of patient populations and have successful results. Its high SVR rates (upwards of 90%), high barrier to resistance, and low rates of unfavorable adverse effects make it a worthwhile contender for being the new backbone of therapy. Other agents with similar mechanisms include mericitabine and ABT-333 that are in development.

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6.1. Phase 2 trials

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Four phase 2 trials have demonstrated the effectiveness of sofosbuvir in the treatment of various genotypes in a variety of patient populations when either combined with ribavirin, or peginterferon and ribavirin. The safety, tolerability, and efficacy of sofosbuvir in 147 noncirrhotic, treatment-naïve patients with GT 1, 2, and 3 infections were examined in a randomized, double-blind phase 2 trial by Lawitz et al. (2013a, 2013b, 2013c, 2013d, 2013e). Though there was no cohort evaluating current standard of care for GT 1 (peginterferon + ribavirin + telaprevir or boceprevir), sofosbuvir achieved SVR rates of 85–89% compared to placebo (Lawitz et al., 2013a, 2013b, 2013c, 2013d, 2013e). The ELECTRON trial sought to determine the safety and efficacy of sofosbuvir and ribavirin in various interferon-sparing and interferon-free regimens in 95 patients with HCV GTs 1, 2, and 3 (Gane et al., 2013). All patients who received sofosbuvir succeeded in achieving a rapid virologic response. Additionally, all patients receiving sofosbuvir dual therapy for 12 weeks or sofosbuvir triple therapy for 8 weeks continued to achieve undetectable viral loads at 24 weeks post-treatment. Despite the small sample size, several conclusions could be drawn. For treatment-naïve GT 2 and 3 patients, dual therapy with sofosbuvir and ribavirin for 12 weeks may be sufficient to achieve successful SVR rate. For GT 1 patients, especially those who are treatment-experienced, the addition of a third agent may be warranted (Gane et al., 2013). The ATOMIC trial built off of ELECTRON trial and aimed to determine whether a 12-week regimen of sofosbuvir and ribavirin would benefit from the addition of peginterferon and if a 24-week regimen of this triple therapy would provide any additional benefit. The majority of patients in all three cohorts (96%, 98%, and 97%, respectively) achieved

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The major sofosbuvir phase 3 trials have been NEUTRINO, FISSION, POSITRON, FUSION, and VALENCE. NEUTRINO examined a 12-week regimen of sofosbuvir with peginterferon and ribavirin in 327 treatment-naïve, chronic hepatitis C patients with GTs 1, 4, 5, and 6, with the majority of patients being GT 1. SVR rates of 90% were recorded and patients with historically low SVR rates managed to achieve higher than expected rates, suggesting the added benefit of sofosbuvir in this patient population (Lawitz et al., 2013a, 2013b, 2013c, 2013d, 2013e). The FISSION trial aimed to determine the non-inferiority of a 12-week regimen containing sofosbuvir and ribavirin and a 24-week regimen of peginterferon and ribavirin for GTs 2 and 3. Results successfully demonstrated the non-inferiority of the 12-week regimen, because both treatment groups achieved 67% SVR rates. While a 12-week course of sofosbuvir and ribavirin might be sufficient for GT 2 patients who can't tolerate peginterferon therapy, patients with GT 3 infections might need an additional agent or longer treatment duration because their SVR rates remained lower (Lawitz et al., 2013a, 2013b, 2013c, 2013d, 2013e). POSITRON and FUSION aimed to determine the efficacy of sofosbuvir and ribavirin in GT 2 and 3 patients who either have contraindications to peginterferon therapy, or who have failed to achieve response with such therapies, and are often left without other treatment options. In those patients for whom interferon therapy is not an option due to safety profile or contraindications, the utilization of a 12-week regimen of sofosbuvir and ribavirin proved to be effective, leading to SVR24 rates of 78% in all patients, with minimal safety concerns. In those patients who had previously failed to achieve response on an interferon-based treatment regimen, achievement of SVR depended upon genotype and treatment duration but was higher than historical values; 50% GT 2 and 73% GT 3, vs. 25%. Both trials demonstrated that the presence of GT 3 requires either an additional agent or longer treatment duration (Jacobson et al., 2013a, 2013b). The VALENCE trial aimed to determine if longer duration of therapy is effective in treating GT 3 patients because previous trials demonstrated that either an additional agent or longer treatment duration would be needed in this patient population. When compared to results from previous trials that used 12 weeks of therapy, increasing treatment duration to 24 weeks in GT 3 patients substantially increased SVR rates, jumping from 56% (as demonstrated in FISSION) to 85%. Additionally, results indicated that for GT 2 patients, dual therapy for 12 weeks is sufficient because 93% of patients achieved SVR (Zeuzem et al., 2013).

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6.3. Sofosbuvir evaluation

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6.3.1. Rapid virologic response (RVR) rates Achieving an undetectable viral load at week 4 of treatment has been positively correlated to achieving sustained virologic responses and is a currently accepted method of guiding treatment response in genotype 1 patients (Mira et al., 2007; Fried et al., 2011). Phase 2 and 3 data indicate

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The exact role of the NS5A protein is still under investigation. Several functions have been proposed, including promoting viral replication, mediating interferon-response, and preventing apoptosis signals (Macdonald & Harris, 2004; Fridell et al., 2011). Loss of function and inhibition leads to inefficient replication (Bukh et al., 2002; Gao et al., 2010; Wang et al., 2012, 2013), making it a potential target for therapies. Studies performed on the NS5A inhibitor daclatasvir have shown promising results, with SVR rates upwards of 98% when used in combination with the polymerase inhibitor sofosbuvir in GT 1 patients. Importantly, though mutant strains were identified, patients were still able to attain a sustained virologic response (Sulkowski et al., 2014). While more information is needed about the exact function of this nonenzymatic protein, its demonstrated necessity in the HCV life cycle makes it a viable target for therapy. Examples of NS5A HCV replication inhibitors in development include daclatasvir, ledipasvir, ACH-3102, ABT-267, and others.

P

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SVR regardless of duration of treatment, indicating that extending duration to 24 weeks provided no added benefit (Kowdley et al., 2013). In order to establish a firm conclusion as to the effectiveness of sofosbuvir in all patient populations, the NIH SPARE study was conducted in GT 1 patients with unfavorable characteristics. Increased rates of SVR were achieved with weight-based ribavirin compared to low-dose (68% vs. 48%), which is comparable to SVR rates achieved by interferon-containing therapies. Because of this, it may be warranted to examine the effects of sofosbuvir and interferon-containing therapies for potential increases in outcomes. Additional benefits were seen in 27 out of the 29 patients with paired liver biopsies in areas of fibrosis scores and normalizations of liver enzymes ALT (alanine transaminase) and AST (aspartate aminotransferase) by day 14 of treatment, suggesting that even with low SVR rates, treatment may still be beneficial (Osinusi et al., 2013).

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364 365 366 367 368 369 370 Q5 371 372 373 374 375 376 377 378 379 Q6 380 381 382 383 384 385 386 387 388 389 390 391 392 393 Q7 394 395 396 397 398 399 400 401 402

406 407 408

434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471

486 487

7. Simeprevir

525

Simeprevir (OLYSIO™, Medevir and Janssen Pharmaceuticals) is a second-wave NS3/4A protease inhibitor approved in late 2013 for the treatment of chronic hepatitis C GT 1 in combination with peginterferon and ribavirin; treatment duration is based on patient characteristics and response to therapy. Although most studies have compared simeprevir's efficacy to placebo, SVR rates appear higher and comparable to telaprevir- and boceprevir-containing regimens. In treatment-naïve patients, simeprevir

526

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6.3.5. Special patient populations Patients with HCV have a variety of co-morbid conditions that influence treatment options, including co-infections with HIV and/or HBV, compensated cirrhosis, history of liver transplantations, and renal disease. The only FDA-approved treatments for HIV/HCV co-infection are peginterferon–ribavirin for 48 weeks, with SVR rates varying from 35% for GT 1 to 72% for GT 2 (Núñez et al., 2007). HIV treatment regimens incur high pill burdens and undesirable side effects; the addition of peginterferon–ribavirin to an already difficult regimen introduces a new burden for these patients. Additionally, several anti-retroviral therapies (such as didanosine and zidovudine) are contra-indicated with peginterferon/ribavirin, making treatment difficult. These factors necessitate investigation of a new treatment for these patients. With the success of the combination of sofosbuvir and ribavirin, the phase 3 PHOTON-1 trial aimed to evaluate the safety and efficacy of this therapy for HCV/HIV co-infections for patients with GTs 1 (24 weeks treatment), 2 (12 weeks treatment), and 3 (12 weeks treatment). At 4 weeks of treatment, between 96% and 100% of all patients had no detectable virus; at 12 weeks post-treatment, 76% of patients with GT 1, 88% of patients with GT 2, and 67% of patients with GT 3 achieved SVR. Additionally, patients were on a variety of HIV medications, with no HIV breakthroughs due to HCV treatment. Though there were decreases in absolute CD4-T cell counts, these were consistent with what is known to occur with ribavirin therapy. In patients who did not achieve SVR12, no S282T mutations were detected. PHOTON-1 demonstrated the potential for an all-oral regimen for patients with HCV/HIV co-infection who might not be able to tolerate the standard of care with peginterferon and ribavirin, and it is now an approved indication (Lohmann et al., 1999). The LONESTAR-2 trial aimed to determine the efficacy of sofosbuvir, peginterferon, and ribavirin in GT 2 and 3 treatment-experienced patients with and without cirrhosis. The overall SVR rate was 89%, which is consistent with what has been seen with previous sofosbuvircontaining regimens. SVR rates were comparable in patients with and without cirrhosis: GT 2, 93% (with cirrhosis) vs. 100% (without cirrhosis) and GT 3, 83% for both with and without cirrhosis. Discontinuations due to side effects were low (~1%), and most common adverse events were consistent with those seen in previous studies (Lawitz et al., 2013a, 2013b, 2013c, 2013d, 2013e).

R O

430 431

474 475

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428 429

6.3.4. Cost Sofosbuvir is expected to cost ~$1000 per pill, because the wholesaler acquisition cost of a 4-week supply is $28,000 (Gilead Sciences, Inc., 2013b). Depending on genotype, sofosbuvir treatment costs can be between $84,000 (12 weeks) and $168,000 (24 weeks), without taking into consideration costs of ribavirin and peginterferon. Not only does this add an additional burden to the health care system, but could also prevent patients from getting access. Gilead's Support Path program offers prospective patients who might not be able to afford therapy with services to help pay for the medication, including case managers and coupons. However this program is only offered to citizens of the United States, globally hindering those who can't pay.

D

426 427

correlation can't be established due to simple coincidence as other fac- 472 tors might have been in play. 473

T

424 425

C

422 423

E

420 421

R

418 419

R

416 417

6.3.2. Resistance mechanisms In vitro studies in GT 1–6 replicon cells identified that the S282T mutation confers low levels of resistance to sofosbuvir, with 2.4- to 18.1-fold reductions in susceptibility (Simin et al., 2013). Further analysis demonstrated that the mutation has reduced replicative ability, suggesting that the chances of attaining this mutation are low. In strains resistant to common agents used in the treatment of HCV (NS3 protease inhibitors, NS5A and NS5B non-nucleoside polymerase inhibitors), sofosbuvir demonstrated full activity. Additionally, there was no decreased susceptibility when sofosbuvir was exposed to ribavirin- or nucleoside inhibitor-mutations (Simin et al., 2013). These results are consistent with the mechanism of action of sofosbuvir because nucleoside polymerase inhibitors are less likely to have resistance mechanisms due to the importance of the active site of the polymerase. Unlike the presence of resistance mechanisms to NS3 protease inhibitors in treatment-naïve patients, the presence of the S282T mutation was not identified in this patient population (Castilho et al., 2011; Franco et al., 2013), suggesting a possible superiority of sofosbuvir to protease inhibitors. Patients who did not achieve a sustained virologic response at 12 weeks post-treatment in the phase 3 clinical trials (225 out of 226) – NEUTRINO, FISSION, POSITRON, and FUSION – were evaluated for resistance mechanisms (Svarovskaia et al., 2013). Deep sequencing indicated that there were no S282T substitutions either at baseline or post-treatment, and while population sequencing did indicate several substitutions, these were not associated with decreased susceptibility to sofosbuvir or ribavirin. The only S282T mutation was found through population sequencing in a GT 2 patient who relapsed in the ELECTRON trial, however it is unknown if this mutation was present prior to the start of therapy. The fact that only one mutation has been identified in a handful of patients who have received sofosbuvir therapy indicates the low “replicative fitness” of the mutation. In contrast with protease inhibitors, whose mutations have high replicative fitness, sofosbuvir has an advantage. However, until there are head-to-head studies evaluating the therapies, no direct conclusions can be drawn. Since patients still have relapses without any detectable mutations suggests a yet-unknown mechanism or factor that prevents treatment success. Despite results indicating that there were no significant reductions in sofosbuvir sensitivity with other nucleoside polymeraseinduced resistance mechanisms, a recent report seems to conflict with this. Examination of mericitabine, the pro-drug of the nucleoside polymerase inhibitor PSI-6130, showed the presence of a double mutant L159/L320F, which was found to confer cross-resistance to sofosbuvir (Tong et al., 2014). Further examination of the data will help determine the significance of cross-resistance and potentially examine the presence of this mutation in patients who failed treatment with sofosbuvir.

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that sofosbuvir-based treatment regimens allow patients to achieve rapid declines in HCV RNA and achieve rapid virologic responses, which correlated to high SVR rates, strengthening the relationship between RVR and SVR. Results of POSITRON indicated a 100% concordance between RVR and SVR at 12 and 24 weeks.

U

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6

6.3.3. Safety profile The most common adverse events were those that have been found in previous patients undergoing peginterferon and ribavirin therapies: headache, fatigue, insomnia, nausea, rash, and anemia. Discontinuation rates due to adverse events were low, and the most common events were associated with peginterferon and ribavirin therapies. In NEUTRINO, the rate of discontinuation due to adverse effects was roughly 2% when patients received sofosbuvir and peginterferon/ribavirin, whereas typical discontinuation rates due to peginterferon-ribavirin therapies range from 12 to 14% (Manns et al., 2001; Torriani et al., 2004; Arase et al., 2007). This could indicate that the addition of sofosbuvir may increase tolerability to or alleviate adverse events; however this


476 Q8 477 478 479 480 481 482 483 484 485

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558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597

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E

554 555

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R

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N C O

548 549

U

7.2. Phase 3 trials

630

The major phase 3 trials are QUEST-1, QUEST-2, and PROMISE. QUEST-1 and QUEST-2, conducted in treatment-naïve GT 1 patients, evaluated the efficacy and safety of simeprevir (150 mg) in combination with peginterferon and ribavirin for 12 weeks, followed by peginterferon and ribavirin therapies. Patients enrolled in QUEST-1 received peginterferon alfa-2a, while patients in QUEST-2 received peginterferon alfa-2b. In a pooled analysis, roughly 78% of all patients receiving simeprevir achieved RVR, compared with 12% of placebo. Overall, 80% of patients receiving simeprevir achieved SVR12, of whom 90% achieved RVR (Jacobson et al., 2013a, 2013b; Janssen Therapeutics, 2013; Poordad et al., 2013). Though PROMISE had the same treatment regimen and duration, it was conducted in patients who had failed on peginterferon and ribavirin therapies. RVR was achieved in 77% of simeprevir-treated patients and in only 3% of placebo-treated patients. Subsequently SVR12 was achieved in 80% of simeprevir-treated patients and 37% of placebo-treated patients (Lawitz et al., 2013a, 2013b, 2013c, 2013d, 2013e).

631

7.3. Simeprevir evaluation

648

7.3.1. Rapid virologic response (RVR) rates Between 53% and 78% of patients in all trials achieved RVR, and QUEST-1 and QUEST-2 trials demonstrated a 90% concordance between RVR and SVR. Telaprevir and boceprevir have response-guided therapy algorithms, allowing treatment duration to be reduced due to viral load at certain time points; because simeprevir is a protease inhibitor, it is also eligible for a therapy algorithm. The majority of patients in all simeprevir-based regimens were able to shorten treatment duration to 24 weeks, which decreases exposure to therapy; this can decrease discontinuation rates due to adverse events and subsequently increase tolerability of therapy.

649 650

F

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O

Phase 2 studies have evaluated simeprevir-based treatment regimens in both treatment-naïve and treatment-experienced patients. The proof-of-concept TMC435-C202 trial evaluated the safety and efficacy of 7 days of monotherapy with simeprevir (200 mg) in 37 treatment-naïve GT 2–6 patients. After monotherapy, patients received peginterferon and ribavirin for up to 35 days. A rapid decline in viral load occurred at day 3 in all patients except GT 3. Compared to baseline, change in viral loads at day 7 was greatest for GT 6 (−5.4 ± 0.29), followed by GT 4 (−3.52 ± 0.43), GT 2 (−2.73 ± 0.71), and GT 5 (−2.19 ± 0.39); there was no visible response for GT 3 patients during simeprevir monotherapy, which is consistent with an in vitro assay that showed GT 3 is less responsive to simeprevir. After peginterferon and ribavirin therapies were initiated, viral loads continued to decrease for all patients with the exception of GT 4. At the end of the study 83% of GT 2, 38% of GT 3, 68% of GT 4, 14% of GT 5, and 75% of GT 6 had undetectable HCV RNA levels (Moreno et al., 2012). The results of the TMC435-C202 study suggest that simeprevir has activity against GTs 2, 4, 5, and 6 because monotherapy for 7 days resulted in decreases in viral loads. This could be a promising development, because telaprevir and boceprevir have little-to-no efficacy in GTs 2–6, and there are limited treatment options for GTs 4–6 (Manns & von Hahn, 2013). The OPERA-1 trial investigated the efficacy of simeprevir, peginterferon, and ribavirin in GT 1 patients in three main cohorts. Treatment-naïve patients received 7 days of simeprevir (25, 75, or 200 mg) or placebo followed by triple therapy for either 3 or 4 weeks; treatment-experienced patients received 4 weeks of simeprevir (25, 150, or 200 mg) or placebo with peginterferon and ribavirin, followed by peginterferon and ribavirin for up to 48 weeks. Patients who had never received treatment experienced rapid declines in HCV RNA within the first 7 days and between 70 and 88% of all patients receiving simeprevir achieved an RVR, compared to 22% with placebo. All treatment-experienced patients who had relapsed achieved an RVR at both 150 and 200 mg dosages, suggesting no added benefit of the increased dose; however, treatment-experienced patients who were categorized as non-responders had RVR rates ranging from 28 to 67%, with higher rates corresponding with higher dosages. Importantly, treatment response was comparable between those with and without cirrhosis and treatment-naïve and treatment-experienced (Manns et al., 2011). Five patients who had previously received simeprevir were also included in the study in a separate cohort, and three of these patients achieved SVR at week 72 (Lenz et al., 2012). Because previous studies have shown that treatment-experienced patients have difficulties achieving SVR and OPERA-1 showed the effectiveness of simeprevir in prior relapsers, the ASPIRE trial aimed to further investigate the utilization of simeprevir in treatment-experienced patients. A total of 423 patients with GT 1 who were classified as null responders, partial responders, and previous relapsers were enrolled. RVR was achieved in 53.0%–67.6% of all patients and SVR24 was achieved in 60.6%–80.0% of all patients; prior relapse patients had the highest rates of RVR and SVR24 (Zeuzem et al., 2014). When compared with traditional response rates of treatment-experienced patients on telaprevirand boceprevir-containing regimens (Park et al., 2014), these rates can be considered an improvement and suggest simeprevir's efficacy in this patient population. Additionally, 24–73% of patients with

544 545

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7.1. Phase 2 trials

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cirrhosis achieved a SVR, representing a substantial increase over traditional therapies (Bourlière et al., 2013). The PILLAR study evaluated two different dosages of simeprevir (150 mg and 75 mg) in combination with peginterferon and ribavirin in treatment-naïve GT 1 patients. A total of 368 patients received either 12 or 24 weeks of triple therapy, followed by 24 weeks of peginterferon and ribavirin. Between 68% and 75.3% of patients achieved RVR; of these patients, 87.9%–94.9% went on to achieve SVR24. Overall, 74.7%–86.1% of those patients receiving either dose of simeprevir achieved SVR24, with a greater proportion belonging in the 150 mg group (Fried et al., 2013). TMC435-C202, OPERA-1, ASPIRE, and PILLAR showed substantial benefits of simeprevir therapy in both treatment-naïve and treatmentexperienced patients when combined with peginterferon and ribavirin. With the success of sofosbuvir-based therapies, the COSMOS trial was initiated to examine the effect of 12 and 24 weeks of simeprevir and sofosbuvir with and without ribavirin in treatment-naïve and treatment-experienced-patients. While all patients achieved RVR regardless of treatment regimen or duration, SVR rates were dependent on treatment duration and regimen. For null responders without cirrhosis, 24 weeks of simeprevir and sofosbuvir therapy was more beneficial than triple therapy (93.3% vs. 79.2%) for achieving SVR12. In contrast, 12 weeks of triple therapy was more beneficial than dual therapy (96.3% vs. 92.9%) for achieving SVR12. For treatment-naïve patients, triple and dual therapy resulted in 100% SVR rates, suggesting no added benefit of ribavirin in this patient population. For null responders with cirrhosis, SVR24 rates were higher with dual therapy than triple therapy (100% vs. 93.3%). This result questions the necessity of ribavirin in null responders because 24 weeks of dual therapy resulted in similar SVR rates with 12 weeks of triple therapy (93.3%), regardless of the presence of cirrhosis (Large et al., 1999; Lawitz et al., 2013a, 2013b, 2013c, 2013d, 2013e).

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achieved SVR of 80% vs. telaprevir (60.8–74.8%) vs. boceprevir (54.2– 74.8%), and in those who failed prior treatment simeprevir achieved SVR of 77–79% vs. telaprevir (51.3–66.3%) vs. boceprevir (58.6–66.5%) (Janssen Therapeutics, 2013; Park et al., 2014). Though it has a low barrier to resistance, evaluation of SVR rates and the adverse event profile suggests that simeprevir could replace telaprevir and boceprevir for treatment of GT 1 patients. However, until a head-to-head study is conducted, the superiority of simeprevir cannot be concluded with confidence.

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7


600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 Q10 629

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651 652 653 654 655 656 657 658 659

677 678 679 680

681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696

697

7.3.3. Safety profile Across phase 2 and 3 trials, simeprevir was generally well tolerated, with the most common adverse events associated with simeprevir monotherapy being influenza-like illness, headache, and diarrhea (Moreno et al., 2012). When combined with peginterferon and ribavirin therapies, the most common adverse events were those that are historically associated with this combination therapy, including fatigue, neutropenia, rash, and pruritus. In contrast to telaprevir- and boceprevir-based regimens, the presence of simeprevir did not significantly increase the rate of adverse events including anemia and neutropenia, indicating simeprevir's benefit over these current protease inhibitors. Despite the promising safety profile, there were reports of higher frequencies of rash and pruritus (Zeuzem et al., 2014) compared to peginterferon and ribavirin therapies alone as well as reports of transient increases in bilirubin level. These necessitate further investigation.

729

Vaccines developed using the recombinant E1 and E2 envelope proteins primarily target humoral responses, leading to antibody responses. Recombinant E1E2 proteins adjuvant with MF59 have been shown to be safe and demonstrated a dose-dependent humoral and cell-mediated immune response in healthy volunteers (Frey et al., 2010); in vitro it has also demonstrated an ability to neutralize the HCV pseudo-particles (Stamataki et al., 2011). The ability of these recombinant proteins to neutralize the virus suggests potential for a prophylactic vaccine. However, larger studies must be conducted to gain a better understanding of the effectiveness and safety of these recombinant particles. A number of recombinant viruses have also been used as delivery mechanisms, and animal models (Makimura et al., 1996; Large et al., 1999; Ip et al., 2014) demonstrated their ability to elicit both humoral and cellular immune responses, but their safety and efficacy in humans have yet to be determined.

730 731

8.4. DNA technology

745

Compared to traditional vaccines, which are aimed to target humoral responses, DNA vaccines are able to elicit humoral and cell-mediated responses through production of viral particles within the host (Forns et al., 2002). This dual mechanism leads to activation of T-cell responses, which is more favorable for viral diseases. Specifically, activating cytotoxic T-cells provides a direct mechanism for clearance of the virus (Ullah et al., 2012). Promising results have been seen with DNA vaccines in lab animals, targeting both structural and non-structural proteins (Lin et al., 2008; Youn et al., 2008; Masalova et al., 2010). There are three DNA vaccines that have entered clinical trials: CIGB230, ChronVac-C, and INO-8000/VGX-6150. CIGB-23 and ChronVac-C have been used as therapeutic vaccines in trials (Alvarez-Lajonchere et al., 2009; Fournillier et al., 2013; Amador-Cañizares et al., 2014) and have demonstrated promising results that suggest the potential for their use as adjuvants to current treatment options. A phase 1 trial has recently been initiated with INO-8000/VGX-6150 to determine its safety, tolerability, immunogenicity, and virologic response as a second-line therapeutic agent in patients with hepatitis C.

746

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8.3. Recombinant proteins and recombinant viruses

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673 674

719 720

R O

671 672

Due to the growing population of those infected with HCV, primary preventive measures are being sought in the forms of vaccines. Development is difficult due to the high virus mutation rate, genetic diversity between genotypes, and the ability of the virus to cause re-infection. Several approaches are currently being investigated to battle these barriers, including using recombinant proteins, recombinant viruses, and DNA technology. Vaccines can be further subdivided into those that aim to target T-cell responses (targeting non-structural proteins) vs. humoral responses (targeting envelope glycoproteins) and prophylactic vs. therapeutic (Forns et al., 2002; Ullah et al., 2012).

P

669 670

718

D

667 668

8.2. Vaccine

T

666

C

664 665

E

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7.3.2. Resistance In simeprevir-treated replicon GT 1 cells, mutations at NS3 positions F43, Q80, R155, A156, and D168 were the most common. Though all mutations demonstrated reduction in susceptibility, D168 was associated with the greatest reduction, ranging from b10-fold to ~2000-fold reductions (Lenz et al., 2010). Patients who did not achieve SVR in phase 2 and 3 trials were investigated for substitutions in the NS3 amino acid sequence and 91% demonstrated substitutions at the aforementioned sites. Further analysis demonstrated that, at baseline, Q80K was present in 14% of all patients, while variants associated with greatest impact were present at a lower rate (~1.3%). Interestingly, Q80K was associated with reduced response in patients enrolled in QUEST-1, QUEST-2, and PROMISE, even though it has been shown to have the least impact on response in vitro (Lenz et al., 2010; Janssen Therapeutics, 2013). This warrants further investigation and suggests that there might be other factors contributing to reducing response. Even though simeprevir is a ‘second-wave’ protease inhibitor, it still demonstrates resistance-associated amino acid substitutions in treatment-naïve patients, just like telaprevir and boceprevir. However, it retained its activity in the presence of telaprevir- and boceprevirassociated resistance mutations (Lenz et al., 2010).

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8

721 722 723 724 725 726 727 728

732 733 734 735 736 737 738 739 740 741 742 743 744

747 748 749 750 751 752 753 754

706

8. Future directions

8.5. Nanotechnology and hepatitis C virus therapy

764

707

8.1. Interferon-free regimens

765

708 709

Interferon has long been the backbone of hepatitis C therapy due to its ability to stimulate an immune response. Nonetheless, contraindications, drug interactions, and an unfavorable safety profile have limited its use in certain patient populations. The era of interferon-free regimens is fast approaching because a variety of interferon-free regimens that are currently being evaluated (Schinazi et al., 2014) may provide treatment options for those who currently have none. The availability of several effective new agents against the different or all HCV genotypes and the higher barrier to resistance should eliminate the continued need for interferon and ribavirin.

There has been a growing interest in nanotechnology with potential applications in drug delivery for treatment of many diseases (Shoo & Parveen, 2007; Park et al., 2008; Sumer & Gao, 2008), especially for hepatitis C therapy by either targeting specific nanoparticles to the viral infected hepatocytes in early-stages of HCV infections (Lee et al., 2012; Abo-zeid et al., 2013), or transplanting liver tissues using a nanogel platform in end-stage liver disease (ESLD) (Hubbell, 2004). Ribavirin, a nucleoside inhibitor used for hepatitis viral genome treatment, is accompanied by accumulation inside red blood cells (RBCs) causing hemolytic anemia (Schekman & Singer, 1976; Rothen-Rutishauser et al., 2006). Therefore, scientists designed poly(glycerol-adipate) nanoparticles labeled with rhodamine B

710 711 712 713 714 715 716 717

O

C

702 703

N

700 701

U

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7.3.4. Cost Janssen Therapeutics has set a comparable price for a 12-week course of simeprevir to 12 weeks of telaprevir therapy and 24– 44 weeks of boceprevir therapy. To assist patients in attaining simeprevir therapy, Janssen created the “OLYSIO Savings Program,” which will allow patients to pay a maximum of $25 per prescription, with a maximum of $25,000 per year. The program would only be open for patients with private insurance; however, several foundations are available for those patients with Medicare and Medicaid.


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Conflict of interest statement

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804 805 806 807 808 809 810 811 812

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The authors declare that there are no conflicts of interest.

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We have come a long way since the first introduction of an antihepatitis C regimen. As we learn more about the viral life cycle and develop new chemical moieties for treatment, we are one step closer to fully eradicating the virus. The next decade looks promising, as we enter a world of new drug classes, interferon-free regimens, nanotechnology, and vaccines. The knowledge that is gained through these advances could pave the way for development of more effective treatments and even vaccines against other chronic disease states caused by viruses, such as HIV. The combined efforts of the entire global community will allow us to get control of the virus and reduce its burden not only on our health care system, but also on those who are living with the disease.

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References

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Abo-zeid, Y., Irving, W., Thomson, B., & Garnett, M. (2013). Nanoparticle delivery systems for HCV treatment: do nanoparticles avoid uptake by erythrocytes? Viral Hepatitis Congress 2013 September 26–28, 2013; Frankfurt, Germany. Alter, M. J., Kruszon-Moran, D., Nainan, O. V., McQuillan, G. M., Gao, F., Moyer, L. A., et al. (1999). The prevalence of hepatitis C virus infection in the United States, 1988 through 1994. N Engl J Med 341, 556–562. Alvarez-Lajonchere, L., Shoukry, N. H., Grá, B., Amador-Cañizares, Y., Helle, F., Bédard, N., et al. (2009). Immunogenicity of CIGB-230, a therapeutic DNA vaccine preparation, in HCV-chronically infected individuals in a Phase I clinical trial. J Viral Hepat 16, 156–167. Amador-Cañizares, Y., Martínez-Donato, G., Álvarez-Lajonchere, L., Vasallo, C., Dausá, M., Aguilar-Noriega, D., et al. (2014). HCV-specific immune responses induced by CIGB230 in combination with IFN-α plus ribavirin. World J Gastroenterol 20, 148–162. American Society for the Study of Liver Diseases and Infectious Disease Society of America (2013). Recommendations for testing, managing, and treating hepatitis C. Accessible via. http://hcvguidelines.org/full-report-view Ansaldi, F., Orsi, A., Sticchi, L., Bruzzone, B., & Icardi, G. (2014). Hepatitis C virus in the new era: perspectives in epidemiology, prevention, diagnostics and predictors of response to therapy. World J Gastroenterol 20, 9633–9652. Arase, Y., Suzuki, F., Suzuki, Y., Akuta, N., Kawamura, Y., Kobayashi, M., et al. (2007). Side effects of combination therapy of peginterferon and ribavirin for chronic hepatitis-C. Intern Med 46, 1827–1832. Asselah, T., Benhamou, Y., & Marcellin, P. (2009). Protease and polymerase inhibitors for the treatment of hepatitis C. Liver Int 29, 57–67.

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Asselah, T., Estrabaud, E., Bieche, I., Lapalus, M., De Muynck, S., Vidaud, M., et al. (2010). Hepatitis C: viral and host factors associated with non-response to pegylated interferon plus ribavirin. Liver Int 30, 1259–1269. Bartenschlager, R., & Lohmann, V. (2000). Replication of hepatitis C virus. J Gen Virol 81, 1631–1648. Behairy, B. E., Saber, M. A., Elhenawy, I. A., Abou-Zeinah, S. S., El-Sharawy, A. A., & Sira, M. M. (2012). Serum cystatin C correlates negatively with viral load in treatment-naïve children with chronic hepatitis C. J Pediatr Gastroenterol Nutr 54, 364–368. Bezemer, G., Van Gool, A. R., Verheij-Hart, E., Hansen, B. E., Lurie, Y., Esteban, J. I., et al. (2012). Long-term effects of treatment and response in patients with chronic hepatitis C on quality of life. An international, multicenter, randomized, controlled study. BMC Gastroenterol 12, 11. Bourlière, M., Wendt, A., Fontaine, H., Hézode, C., Pol, S., & Bronowicki, J. P. (2013). How to optimize HCV therapy in genotype 1 patients with cirrhosis. Liver Int 33, 46–55. Bukh, J., Pietschmann, T., Lohmann, V., Krieger, N., Faulk, K., Engle, R., et al. (2002). Mutations that permit efficient replication of hepatitis C virus RNA in Huh-7 cells prevent productive replication in chimpanzees. Proc Natl Acad Sci U S A 99, 14416–14421. Castilho, M. C. B., Martins, A. N., Horbach, I. S., Perez, R. d. M., Figueiredo, F. A. F., Pinto,, P. d. T. A., et al. (2011). Association of hepatitis C virus NS5B variants with resistance to new antiviral drugs among untreated patients. Mem Inst Oswaldo Cruz 106, 968–975. Centers for Disease Control and Prevention (2014). CDC Health Information for International Travel 2014. Accessible via. http://wwwnc.cdc.gov/travel/yellowbook/2014/ chapter-3-infectious-diseases-related-to-travel/hepatitis-c Choo, Q. L., Kuo, G., Weiner, A. J., Overby, L. R., Bradley, D. W., & Houghton, M. (1989). Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 244, 359–362. Clark, V. C., Peter, J. A., & Nelson, D. R. (2013). New therapeutic strategies in HCV: secondgeneration protease inhibitors. Liver Int 33, 80–84. Division of Viral Hepatitis, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention (2011). Surveillance for viral hepatitis — United States, 2011. Accessible via. http://www.cdc.gov/hepatitis/Statistics/2011Surveillance/index.htm Forns, X., Bukh, J., & Purcell, R. H. (2002). The challenge of developing a vaccine against hepatitis C virus. J Hepatol 37, 684–695. Fournillier, A., Frelin, L., Jacquier, E., Ahlén, G., Brass, A., Gerossier, E., et al. (2013). A heterologous prime/boost vaccination strategy enhances the immunogenicity of therapeutic vaccines for hepatitis C virus. J Infect Dis 208, 1008–1019. Foy, E., Li, K., Wang, C., Sumpter, R., Jr., Ikeda, M., Lemon, S. M., et al. (2003). Regulation of interferon regulatory factor-3 by the hepatitis C virus serine protease. Science 300, 1145–1148. Franco, S., Casadellà, M., Noguera-Julian, M., Clotet, B., Tural, C., Paredes, R., et al. (2013). No detection of the NS5B S282T mutation in treatment-naïve genotype 1 HCV/HIV-1 coinfected patients using deep sequencing. J Clin Virol 58, 726–729. Frey, S. E., Houghton, M., Coates, S., Abrignani, S., Chien, D., Rosa, D., et al. (2010). Safety and immunogenicity of HCV E1E2 vaccine adjuvanted with MF59 administered to healthy adults. Vaccine 28, 6367–6373. Fridell, R. A., Qiu, D., Valera, L., Wang, C., Rose, R. E., & Gao, M. (2011). Distinct functions of NS5A in hepatitis C virus RNA replication uncovered by studies with the NS5A inhibitor BMS-790052. J Virol 85, 7312–7320. Fried, M. W., Buti, M., Dore, G. J., Flisiak, R., Ferenci, P., Jacobson, I., et al. (2013). Once-daily simeprevir (TMC435) with pegylated interferon and ribavirin in treatment-naïve genotype 1 hepatitis C: the randomized PILLAR study. Hepatology 58, 1918–1929. Fried, M. W., Hadziyannis, S. J., Shiffman, M. L., Messinger, D., & Zeuzem, S. (2011). Rapid virological response is the most important predictor of sustained virological response across genotypes in patients with chronic hepatitis C virus infection. J Hepatol 55, 69–75. Gane, E. J., Stedman, C. A., Hyland, R. H., Ding, X., Svarovskaia, E., Symonds, W. T., et al. (2013). Nucleotide polymerase inhibitor sofosbuvir plus ribavirin for hepatitis C. N Engl J Med 368, 34–44. Gao, M., Nettles, R. E., Belema, M., Snyder, L. B., Nguyen, V. N., Fridell, R. A., et al. (2010). Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect. Nature 65, 96–100. Gilead Sciences, Inc. (2013a). SOVALDI full prescribing information. Accessible via. http:// www.gilead.com/~/media/Files/pdfs/medicines/liver-disease/sovaldi/sovaldi_pi.pdf Gilead Sciences, Inc. (2013b). U.S. Food and Drug Administration approves Gilead's Sovaldi™ (sofosbuvir) for the treatment of chronic hepatitis C. Accessible via. http:// www.gilead.com/news/press-releases/2013/12/us-food-and-drug-administrationapproves-gileads-sovaldi-sofosbuvir-for-the-treatment-of-chronic-hepatitis-c#sthash. uKOXItHl.dpuf Hubbell, J. A. (2004). Tissue and cell engineering. Curr Opin Biotechnol 15, 381–382. Humer, C. (2014). Vertex to end sales of hepatitis C drug Incivek. Accessible via. http:// www.reuters.com/article/2014/08/13/us-vertex-hepatitiscidUSKBN0GD1S520140813 Ip, P. P., Boerma, A., Regts, J., Meijerhof, T., Wilschut, J., Nijman, H. W., et al. (2014). Alphavirus-based vaccines encoding nonstructural proteins of hepatitis C virus induce robust and protective T-cell responses. Mol Ther 22, 881–890. Jacobson, I. M., Dore, G. J., Foster, G., Fried, M. W., Radu, M. N., Rafalskiy, V. V., et al. (2013). Sa2072 simeprevir (TMC435) with peginterferon/ribavirin for chronic HCV genotype-1 infection in treatment-naïve patients: results from QUEST-1, a phase III trial. Gastroenterology 144, S-374. Jacobson, I. M., Gordon, S. C., Kowdley, K. V., Yoshida, E. M., Rodriguez-Torres, M., Sulkowski, M. S., et al. (2013). Sofosbuvir for hepatitis C genotype 2 or 3 in patients without treatment options. N Engl J Med 368, 1867–1877. Janssen Therapeutics (2013). OLYSIO prescribing information. Accessible via. http:// www.olysio.com/shared/product/olysio/prescribing-information.pdf Kim, K. -A. (2011). Durability of a sustained virologic response in patients with chronic hepatitis C treated with peginterferon and ribavirin. Korean J Hepatol 17, 84–86.

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isothiocyanate (RBITC-PGA) for targeting the ribavirin specifically to the liver and subsequently decreasing the RBC's uptake rate. Hence, these ribavirin-nanoparticles could ameliorate the HCV therapy by enhancing the drug delivery targetability to hepatocytes and improving endocytic mechanisms (Abo-zeid et al., 2013). It has also been found that hyaluronic acid (HA) in combination with gold nanoparticles (AuNPs) has a high biological activity and chemical stability in serum compared to poly(ethylene glycol) (PEG) nanoparticles (Lee et al., 2012). This HA– AuNP nano-complex was therefore investigated as a delivery vehicle of interferon-α for HCV infection therapy. Seven days after injection of interferon-α encapsulated within HA–AuNP, it was observed that this complex was successfully and specifically delivered to the murine hepatic tissue, while PEG-interferon-α nanoparticles were not observed in the hepatocytes. Finally, it was concluded that the target-specific HA–AuNP/interferon-α nano-complex was successfully applied to the systemic treatment of HCV infection (Lee et al., 2012). On the other hand, hepatic transplantation, the currently available therapy for ESLD mainly caused by HCV, has critical obstacles with respect to the transplantable liver tissue shortage and the graft rejection (Alter et al., 1999). In one study PEG di-acrylate (PEG-DA)-based nanogel, a non-toxic material, was synthesized to create a platform of three dimensional artificial human liver tissues by entrapment of human progenitor cells in this platform that maintained the hepatic features (Hubbell, 2004). This novel nanogel platform has exciting implications for tissue engineering and HCV therapeutic studies.

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Mohd Hanafiah, K., Groeger, J., Flaxman, A. D., & Wiersma, S. T. (2013). Global epidemiology of hepatitis C virus infection: new estimates of age-specific antibody to HCV seroprevalence. Hepatology 57, 1333–1342. Moreno, C., Berg, T., Tanwandee, T., Thongsawat, S., Van Vlierberghe, H., Zeuzem, S., et al. (2012). Antiviral activity of TMC435 monotherapy in patients infected with HCV genotypes 2–6: TMC435-C202, a phase IIa, open-label study. J Hepatol 56, 1247–1253. Núñez, M., Miralles, C., Berdún, M. A., Losada, E., Aguirrebengoa, K., Ocampo, A., et al. (2007). Role of weight-based ribavirin dosing and extended duration of therapy in chronic hepatitis C in HIV-infected patients: the PRESCO trial. AIDS Res Hum Retroviruses 23, 972–982. Osinusi, A., Meissner, E. G., Lee, Y. J., Bon, D., Heytens, L., Nelson, A., et al. (2013). Sofosbuvir and ribavirin for hepatitis c genotype 1 in patients with unfavorable treatment characteristics: a randomized clinical trial. JAMA 310, 804–811. Palanisamy, N., Danielsson, A., Kokkula, C., Yin, H., Bondeson, K., Wesslén, L., et al. (2013). Implications of baseline polymorphisms for potential resistance to NS3 protease inhibitors in hepatitis C virus genotypes 1a, 2b and 3a. Antiviral Res 99, 12–17. Park, C., Jiang, S., & Lawson, K. A. (2014). Efficacy and safety of telaprevir and boceprevir in patients with hepatitis C genotype 1: a meta-analysis. J Clin Pharm Ther 39, 14–24. Park, J. H., Lee, S., Kim, J. -H., Park, K., Kim, K., & Kwon, I. C. (2008). Polymeric nanomedicine for cancer therapy. Prog Polym Sci 33, 113–137. Pearlman, B. L., & Traub, N. (2011). Sustained virologic response to antiviral therapy for chronic hepatitis C virus infection: a cure and so much more. Clin Infect Dis 52, 889–900. Poordad, F., Manns, M. P., Marcellin, P., de Araujo, E. S. A., Buti, M., Horsmans, Y., et al. (2013). Simeprevir (TMC435) with peginterferon/ribavirin for treatment of chronic HCV genotype-1 infection in treatment-naive patients: results from QUEST-2, a phase III trial. Gastroenterology 144, S-151. Ranjith-Kumar, C. T., & Cheng Kao, C. (2006). Biochemical activities of the HCV NS5B RNAdependent RNA polymerase. In T. SL (Ed.), Hepatitis C Viruses: Genomes and Molecular Biology. Norfolk (UK): Horizon Bioscience. Razavi, H., ElKhoury, A. C., Elbasha, E., Estes, C., Pasini, K., Poynard, T., et al. (2013). Chronic hepatitis C virus (HCV) disease burden and cost in the United States. Hepatology 57, 2164–2170. Rothen-Rutishauser, B. M., Schürch, S., Haenni, B., Kapp, N., & Gehr, P. (2006). Interaction of fine particles and nanoparticles with red blood cells visualized with advanced microscopic techniques. Environ Sci Tech 40, 4353–4359. Schekman, R., & Singer, S. J. (1976). Clustering and endocytosis of membrane receptors can be induced in mature erythrocytes of neonatal but not adult humans. Proc Natl Acad Sci U S A 73, 4075–4079. Schinazi, R., Halfon, P., Marcellin, P., & Asselah, T. (2014). HCV direct-acting antiviral agents: the best interferon-free combinations. Liver Int 34, 69–78. Shoo, S. K., & Parveen, S. (2007). The present and future of nanotechnology in human health care. Nanotechnology 3, 20–31. Silverman, E. (2014). From riches to rags: vertex discontinues Incivek as sales evaporate. Accessible via. http://blogs.wsj.com/pharmalot/2014/08/12/from-riches-to-ragsvertex-discontinues-incivek-as-sales-evaporate/ Simin, X., Rajyaguru, S., Chiu, S., Hebner, C., Svarovskaia, E. S., Gontcharova, V., et al. (2013). Sofosbuvir selects the NS5B S282T mutation in vitro in genotype 1–6 replicons and is not cross-resistant to resistance-associated variants selected by other classes of antiviral inhibitors. 64th Annual Meeting of the American Association for the Study of Liver Diseases (Washington, DC). Simmonds, P. (2004). Genetic diversity and evolution of hepatitis C virus — 15 years on. J Gen Virol 85, 3173–3188. Stamataki, Z., Coates, S., Abrignani, S., Houghton, M., & McKeating, J. A. (2011). Immunization of human volunteers with hepatitis C virus envelope glycoproteins elicits antibodies that cross-neutralize heterologous virus strains. J Infect Dis 204, 811–813. Sulkowski, M. S., Gardiner, D. F., Rodriguez-Torres, M., Reddy, K. R., Hassanein, T., Jacobson, I., et al. (2014). Daclatasvir plus sofosbuvir for previously treated or untreated chronic HCV infection. N Engl J Med 370, 211–221. Sumer, B., & Gao, J. (2008). Therapeutic nanomedicine for cancer. J Nanomed 3, 137–140. Susser, S., Vermehren, J., Forestier, N., Welker, M. W., Grigorian, N., Füller, C., et al. (2011). Analysis of long-term persistence of resistance mutations within the hepatitis C virus NS3 protease after treatment with telaprevir or boceprevir. J Clin Virol 52, 321–327. Svarovskaia, E. S., Dvory-Sobol, H. S., Hebner, C., Doehle, B., Gontcharova, V., Martin, R., et al. (2013). No resistance detected in four phase 3 clinical studies in HCV genotypes 1–6 of sofosbuvir + ribavirin with or without peginterferon. 64th Annual Meeting of the American Association for the Study of Liver Diseases, Nov 1–5, 2013; Washington, DC. Swain, M. G., Lai, M. Y., Shiffman, M. L., Cooksley, W. G. E., Zeuzem, S., Dieterich, D. T., et al. (2010). A sustained virologic response is durable in patients with chronic hepatitis C treated with peginterferon alfa-2a and ribavirin. Gastroenterology 139, 1593–1601. Tong, X., Le Pogam, S., Li, L., Haines, K., Piso, K., Baronas, V., et al. (2014). In vivo emergence of a novel mutant L159F/L320F in the NS5B polymerase confers low-level resistance to the HCV polymerase inhibitors mericitabine and sofosbuvir. J Infect Dis 209, 668–675. Torriani, F. J., Rodriguez-Torres, M., Rockstroh, J. K., Lissen, E., Gonzalez-García, J., Lazzarin, A., et al. (2004). Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection in HIV-infected patients. N Engl J Med 351, 438–450. Trapero-Marugán, M., Mendoza, J., Chaparro, M., González-Moreno, L., MorenoMonteagudo, J. A., Borque, M. J., et al. (2011). Long-term outcome of chronic hepatitis C patients with sustained virological response to peginterferon plus ribavirin. World J Gastroenterol 17, 493–498. Ullah, S., Shah, M. A. A., & Riaz, N. (2012). Recent advances in development of DNA vaccines against hepatitis C virus. Indian J Virol 23, 253–260. Wang, C., Jia, L., Huang, H., Qiu, D., Valera, L., Huang, X., et al. (2012). In vitro activity of BMS-790052 on hepatitis C virus genotype 4 NS5A. Antimicrob Agents Chemother 56, 1588–1590.

N

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O

R

R

E

C

T

Klevens, R. M., Hu, D. J., Jiles, R., & Holmberg, S. D. (2012). Evolving epidemiology of hepatitis C virus in the United States. Clin Infect Dis 55, S3–S9. Kolykhalov, A. A., Mihalik, K., Feinstone, S. M., & Rice, C. M. (2000). Hepatitis C virusencoded enzymatic activities and conserved RNA elements in the 3′ nontranslated region are essential for virus replication in vivo. J Virol 74, 2046–2051. Kowdley, K. V., Lawitz, E., Crespo, I., Hassanein, T., Davis, M. N., DeMicco, M., et al. (2013). Sofosbuvir with pegylated interferon alfa-2a and ribavirin for treatment-naive patients with hepatitis C genotype-1 infection (ATOMIC): an open-label, randomised, multicentre phase 2 trial. Lancet 381, 2100–2107. Kuntzen, T., Timm, J., Berical, A., Lennon, N., Berlin, A. M., Young, S. K., et al. (2008). Naturally occurring dominant resistance mutations to hepatitis C virus protease and polymerase inhibitors in treatment-naïve patients. Hepatology 48, 1769–1778. Kwo, P. Y., & Vinayek, R. (2011). The therapeutic approaches for hepatitis C virus: protease inhibitors and polymerase inhibitors. Gut Liver 5, 406–417. Large, M. K., Kittlesen, D. J., & Hahn, Y. S. (1999). Suppression of host immune response by the core protein of hepatitis C virus: possible implications for hepatitis C virus persistence. J Immunol 162, 931–938. Lawitz, E., Forns, X., Zeuzem, S., Gane, E., Bronowicki, J. -P., Andreone, P., et al. (2013). 869b Simeprevir (TMC435) with peginterferon/ribavirin for treatment of chronic HCV genotype 1 infection in patients who relapsed after previous interferon-based therapy: results from PROMISE, a phase III trial. Gastroenterology 144, S-151. Lawitz, E., Ghalib, R., Rodriguez-Torres, M., Younossi, Z. M., Corregidor, A., Jacobson, I. M., et al. (2013). Sa2073 SVR4 results of a once daily regimen of simeprevir (TMC435) plus sofosbuvir (GS-7977) with or without ribavirin (RBV) in HCV GT 1 null responders. Gastroenterology 144, S-374–S-375. Lawitz, E., Lalezari, J. P., Hassanein, T., Kowdley, K. V., Poordad, F. F., Sheikh, A. M., et al. (2013). Sofosbuvir in combination with peginterferon alfa-2a and ribavirin for noncirrhotic, treatment-naive patients with genotypes 1, 2, and 3 hepatitis C infection: a randomised, double-blind, phase 2 trial. Lancet Infect Dis 13, 401–408. Lawitz, E., Mangia, A., Wyles, D., Rodriguez-Torres, M., Hassanein, T., Gordon, S. C., et al. (2013). Sofosbuvir for previously untreated chronic hepatitis C infection. N Engl J Med 368, 1878–1887. Lawitz, E., Poordad, F., Brainard, D. M., Hyland, R. H., An, D., Symonds, W. T., et al. (2013). Sofosbuvir in combination with pegIFN and ribavirin for 12 weeks provides high SVR rates in HCV-infected genotype 2 or 3 treatment-experienced patients with and without compensated cirrhosis: results from the LONESTAR-2 study. 64th Annual Meeting of the American Association for the Study of Liver Diseases, Nov 1–5, 2013; Washington, DC. Lee, M. -Y., Yang, J. -A., Jung, H. S., Beack, S., Choi, J. E., Hur, W., et al. (2012). Hyaluronic acid–gold nanoparticle/interferon α complex for targeted treatment of hepatitis C virus infection. ACS Nano 6, 9522–9531. Lenz, O., de Bruijne, J., Vijgen, L., Verbinnen, T., Weegink, C., Van Marck, H., et al. (2012). Efficacy of re-treatment with TMC435 as combination therapy in hepatitis C virus-infected patients following TMC435 monotherapy. Gastroenterology 143, 1176–1178 (.e1176). Lenz, O., Verbinnen, T., Lin, T. -I., Vijgen, L., Cummings, M. D., Lindberg, J., et al. (2010). In vitro resistance profile of the hepatitis C virus NS3/4A protease inhibitor TMC435. Antimicrob Agents Chemother 54, 1878–1887. Lin, Y., Kwon, T., Polo, J., Zhu, Y. -F., Coates, S., Crawford, K., et al. (2008). Induction of broad CD4+ and CD8+ T-cell responses and cross-neutralizing antibodies against hepatitis C virus by vaccination with Th1-adjuvanted polypeptides followed by defective alphaviral particles expressing envelope glycoproteins gpE1 and gpE2 and nonstructural proteins 3, 4, and 5. J Virol 82, 7492–7503. Lohmann, V., Körner, F., Koch, J., Herian, U., Theilmann, L., & Bartenschlager, R. (1999). Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285, 110–113. Ly, K. N., Xing, J., Klevens, R. M., Jiles, R. B., Ward, J. W., & Holmberg, S. D. (2012). The increasing burden of mortality from viral hepatitis in the United States between 1999 and 2007. Ann Intern Med 156, 271–278. Macdonald, A., & Harris, M. (2004). Hepatitis C virus NS5A: tales of a promiscuous protein. J Gen Virol 85, 2485–2502. Makimura, M., Miyake, S., Akino, N., Takamori, K., Matsuura, Y., Miyamura, T., et al. (1996). Induction of antibodies against structural proteins of hepatitis C virus in mice using recombinant adenovirus. Vaccine 14, 28–34. Manns, M. P., McHutchison, J. G., Gordon, S. C., Rustgi, V. K., Shiffman, M., Reindollar, R., et al. (2001). Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial. Lancet 358, 958–965. Manns, M. P., Reesink, H., Berg, T., Dusheiko, G., Flisiak, R., Marcellin, P., et al. (2011). Rapid viral response of once-daily TMC435 plus pegylated interferon/ribavirin in hepatitis C genotype-1 patients: a randomized trial. Antivir Ther 16, 1021–1033. Manns, M. P., & von Hahn, T. (2013). Novel therapies for hepatitis C — one pill fits all? Nat Rev Drug Discov 12, 595–610. Manos, M. M., Shvachko, V. A., Murphy, R. C., Arduino, J. M., & Shire, N. J. (2012). Distribution of hepatitis C virus genotypes in a diverse US integrated health care population. J Med Virol 84, 1744–1750. Masalova, O. V., Lesnova, E. I., Grabovetskiĭ, V. V., Smirnova, O. A., Ulanova, T. I., Burkov, A. N., et al. (2010). DNA immunization with a plasmid containing gene of hepatitis C virus protein 5A (NS5A) induces the effective cellular immune response. Mol Biol 44, 275–283. Maylin, S., Martinot-Peignoux, M., Ripault, M. P., Moucari, R., Cardoso, A. C., Boyer, N., et al. (2009). Sustained virological response is associated with clearance of hepatitis C virus RNA and a decrease in hepatitis C virus antibody. Liver Int 29, 511–517. Mira, J. A., Valera-Bestard, B., Arizcorreta-Yarza, A., González-Serrano, M., Torre-Cisneros, J., Santos, I., et al. (2007). Rapid virological response at week 4 predicts response to pegylated interferon plus ribavirin among HIV/HCV-coinfected patients. Antivir Ther 12, 523–529.

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Wang, C., Valera, L., Jia, L., Kirk, M. J., Gao, M., & Fridell, R. A. (2013). In vitro activity of daclatasvir on hepatitis C virus genotype 3 NS5A. Antimicrob Agents Chemother 57, 611–613. World Health Organization (2002). Global Alert and Response (GAR), hepatitis C. Accessible via. http://www.who.int/csr/disease/hepatitis/whocdscsrlyo2003/en/index2. html Youn, J. -W., Hu, Y. -W., Tricoche, N., Pfahler, W., Shata, M. T., Dreux, M., et al. (2008). Evidence for protection against chronic hepatitis C virus infection in chimpanzees by immunization with replicating recombinant vaccinia virus. J Virol 82, 10896–10905. Zeuzem, S., Berg, T., Gane, E., Ferenci, P., Foster, G. R., Fried, M. W., et al. (2014). Simeprevir increases rate of sustained virologic response among treatment-experienced patients with HCV genotype-1 infection: a phase IIb trial. Gastroenterology 146, 430–441 (.e436).

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Zeuzem, S., Dusheiko, G. M., Salupere, R., Mangia, A., Flisiak, R., Hyland, R. H., et al. (2013). Sofosbuvir + ribavirin for 12 or 24 weeks for patients with HCV genotype 2 or 3: the VALENCE trial. 64th Annual Meeting of the American Association for the Study of Liver Diseases, Nov 1–5, 2013; Washington, DC. Zhu, Y., & Chen, S. (2013). Antiviral treatment of hepatitis C virus infection and factors affecting efficacy. World J Gastroenterol 19, 8963–8973. Zidan, A., Scheuerlein, H., Schüle, S., Settmacher, U., & Rauchfuss, F. (2012). Epidemiological pattern of hepatitis B and hepatitis C as etiological agents for hepatocellular carcinoma in Iran and worldwide. Hepat Mon 12, e6894.

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Youth, Technology, and HIV: Recent Advances and Future Directions.

On sleep and development: recent advances and future directions.

HIV Vaccine: Recent Advances, Current Roadblocks, and Future Directions.

Psychotherapy and Psychosocial Treatment: Recent Advances and Future Directions.

Clinical Research Informatics: Recent Advances and Future Directions.

Recent advances in understanding hepatitis C.

Hepatitis C in the Russian Federation: challenges and future directions.

Advances in and the future of treatments for hepatitis C.

Recent advances and future directions in the management of knee osteoarthritis: Can biological joint reconstruction replace joint arthroplasty and when?

Fish locomotion: recent advances and new directions.

Recent and future directions in CT imaging.

Beyond HER2: recent advances and future directions in targeted therapies in esophagogastric cancers.

Solid-State ¹⁷O NMR studies of organic and biological molecules: Recent advances and future directions.

Consciousness in humans and non-human animals: recent advances and future directions.

Behavioral cardiology: current advances and future directions.

Gastric Cancer Genomics: Advances and Future Directions.

Genetic psychophysiology: advances, problems, and future directions.

Advances and Future Directions in the Treatment of Hepatocellular Carcinoma.

Recent advances on hepatitis C virus in dialysis population.

Recent Advances in Antiviral Therapy for Chronic Hepatitis C.

Recent advances and future directions of hypothermia therapy for traumatic brain injury.

High-dose chemotherapy for resistant germ cell tumors: recent advances and future directions.

Pulmonary arterial hypertension associated with congenital heart disease: recent advances and future directions.

Recent advances and future prospects in choroideremia.