Novel Hepatitis C Drugs in Current Trials

Clin Liver Dis 12 (2008) 529–555

Novel Hepatitis C Drugs in Current Trials Bernd Kronenberger, MD, Christoph Welsch, MD, Nicole Forestier, MD, Stefan Zeuzem, MD* Zentrum der Inneren Medizin, Medizinische Klinik 1, Klinikum der Johann Wolfgang Goethe-Universita¨t, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany

Almost half of the patients who have chronic hepatitis C cannot be cured with the current standard treatment consisting of pegylated interferon-alfa and ribavirin. For those patients who have chronic hepatitis C and did not respond to interferon-alfa–based antiviral therapy, there is currently no approved treatment option available. ‘‘Difficult-to-cure’’ patient populations include patients infected with hepatitis C virus (HCV) genotype 1, HIV/HCV-coinfected patients, patients who have advanced liver cirrhosis, and patients who have recurrent hepatitis C after liver transplantation. Recent progress in structure determination of HCV proteins and development of a subgenomic replicon system [1] and, more recently, of a cell culture infectious HCV clone [2,3] enables the development of a specifically targeted antiviral therapy for hepatitis C (STAT-C). Many HCV-specific compounds are under investigation in preclinical and clinical trials. It is anticipated that HCV-specific inhibitors can improve treatment opportunities for patients who have chronic hepatitis C, especially in patients who are ‘‘difficult to cure.’’ Therapeutic approaches for specifically targeted antiviral therapy for hepatitis C virus The HCV genome is a positive-sense 9.6-kb RNA molecule, comprising 50 and 30 untranslated regions (UTRs) flanking a single open reading frame encoding for a polyprotein of approximately 3100 amino acids. Translation of the HCV polyprotein is initiated by an internal ribosome entry site (IRES)

* Corresponding author. E-mail address: [email protected] (S. Zeuzem). 1089-3261/08/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cld.2008.03.001 liver.theclinics.com

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located in the 50 UTR of the HCV genome (Fig. 1). The HCV polyprotein is co- and posttranslationally processed by host- and virally encoded proteases into structural (core, envelope E1, E2, and p7) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins (Fig. 2). The functions of the HCV proteins and therapeutic approaches for specifically targeted antiviral therapy are listed in Table 1. The core protein binds and packages the viral RNA genome and forms the viral nucleocapsid [4,5]. In addition, the core protein interacts with the envelope protein E1. In infected cells, the core protein is found on membranes of the endoplasmic reticulum, in membranous webs, and on the surface of lipid droplets. The core protein interacts with various cellular proteins. The association with lipid droplets may have a role during viral replication or virion morphogenesis. The deletion of the C-terminal hydrophobic region of the core protein causes translocation to the nucleus. Because of the various interactions with cellular proteins and the consecutive effects on signal transduction pathways and transcription, the core protein has been implicated in hepatocarcinogenesis and the alteration of apoptosis. Blocking of the core protein could alter viral morphogenesis, alter viral replication, and reverse liver steatosis. To date, small-molecule inhibitors for the core protein are not available. The envelope proteins E1 and E2 are essential for host cell entry and are potential targets of antiviral therapy. Blocking of HCV entry could be achieved by blocking the envelope proteins or by blocking cellular receptors of HCV, including the tetraspanin CD81, the scavenger receptor class B type

(+)RNA

1. binding and internalization 7. assembly and release

2. release and uncoating

3. IRES mediated translation 4. polyprotein processing

6. replication (+)RNA

(-)RNA

5. membraneous web formation

Endoplasmic reticulum

Fig. 1. Life cycle of HCV. IRES-mediated translation, polyprotein processing, membraneous web formation, and replication are illustrated as separate steps; however, they might occur in a tightly coupled fashion. Note that IRES-mediated translation and polyprotein processing occur at the endoplasmic reticulum.

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C

E1

E2

p7 NS2

NS3

NS4A NS4B

NS5A

NS5B

HCV polyprotein co- and posttranslational polyprotein processing NS2/3 Cysteine Protease

C Core

E1

E2

p7

Envelope Glycoproteins

NS2

NS3

NS4A NS4B

Cystein Serine Helicase Protease Protease

Ion Channel

structural proteins

NS3/4A Serine Protease

NS5A

Mebr. web

Serine Protease co-factor

NS5B RNA-dependent RNA-Polymerase

IFN-resistance?

non-structural proteins

Fig. 2. Polyprotein processing occurs co- and posttranslationally. The structural proteins and the p7 protein are cleaved by the endoplasmic reticulum signal peptidase. Mebr, membranous.

I (SR-BI), heparan sulfate (HS), dentritic cell and liver and lymph node-specific ICAM-3 grabbing non-integrin (DC-SIGN/L-SIGN), the low-density lipoprotein (LDL) receptor, and claudin-1 [6–8]. Testing of entry inhibitors is still at the preclinical stage. P7 is a small hydrophobic structural protein [9,10] that homo-oligomerizes into a circular hexamer and is believed to form an ion channel in its center [11]. The role of p7 in HCV replication is still unclear. In chimpanzees, p7 has been shown to be important for infectivity. An antiviral compound that has been shown in one study to block the p7 ion channel in vitro is amantadine [12]; however, the results on antiviral activity of amantadine in patients who have chronic hepatitis C are conflicting. A recent study of the antiviral effect of amantadine on cell culture infectious HCV particles showed that across a spectrum of HCV isolates and genotypes, amantadine affected neither RNA replication nor the release or infectivity of HCV particles [13]. Furthermore, the same study showed that the p7 ion channel activity was not affected by amantadine. Overall, the recent results demonstrate that amantadine is not an HCV-selective antiviral. The NS proteins include enzymes necessary for protein maturation (NS2/ 3 cysteine protease and NS3/4A serine protease) and viral replication (NS3 helicase/nucleoside triphosphatase and NS5B RNA-dependent RNA-polymerase). The NS2/3 protease mediates a single cleavage at the NS2/NS3 junction and releases the mature NS3 protease. NS4A enhances the proteolytic activity of the NS3 serine protease domain (Fig. 3). The NS3/4A protease cleaves at four downstream sites in the polyprotein to generate the N-termini of the NS4A, NS4B, NS5A, and NS5B proteins. The structure

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Table 1 Hepatitis C virus structural and nonstructural proteins and potential targets for specifically targeted antiviral therapy for hepatitis C virus HCV protein

Function

Therapeutic approach

50 -UTR

Contains the IRES necessary for initiation of translation of the HCV polyprotein

Core

Nucleocapsid, viral morphogenesis, RNA-binding and replication, association with lipid droplets Envelope, attachment, entry, interferon-alfa resistance (?)

Inhibition of IRES blocks initiation of translation; inhibitors are antisense oligonucleotides, ribozymes, and small molecules (eg, VGX-410C, siRNA) in vitro Inhibition might influence viral replication and viral morphogenesis and inhibit the development of liver steatosis Blocking of envelope proteins or corresponding receptors may inhibit HCV entry and reduce de novo infection of susceptible cells Blocking may reduce infectivity

E1/E2

p7 NS2

NS3

NS4A

NS4B

NS5A

NS5B

30 -UTR

Contains an ion channel potentially involved in viral infection Forms with NS3, a dimeric cysteine protease, for cleavage of the NS2/3 junction  NS2/3 cysteine protease  NS3/4A serine protease  Part of the viral RNA complex  RNA helicase  Nucleotide triphosphatase  Inhibition of the innate antiviral response  Cofactor of the NS3/4A serine protease  Enhances proteolytic activity of the NS3 protease  Membrane protein localized at the endoplasmatic reticulum  Forms a membranous web potentially harboring the HCV replication complex  Potential effect on interferon resistance  Involved in RNA replication  Modulation of cellular signaling pathways (apoptosis, cell growth) RNA-dependent RNA-polymerase

 RNA replication  Stimulation of IRES-dependent translation  Potential 30 -50 end interaction necessary for a switch between translation and RNA replication

Abbreviation: siRNA, small interfering RNA.

Inhibition blocks HCV polyprotein processing Inhibition blocks polyprotein procession and viral replication; furthermore, blocking may restore the innate antiviral response; inhibitors (telaprevir, boceprevir) are in phase 2 clinical trials

Inhibitor in phase 1 trial; inhibition may overcome interferon resistance and reduce HCV replication

Inhibition blocks HCV replication; inhibitors (nucleoside/nonnucleoside) in phase 2 clinical trials

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Fig. 3. Structure of the HCV NS3/4A serine protease. The protease subdomains are marked in light and dark gray with residues of the catalytic triad in the crevice between the subdomains. An inhibitor (ball-and-stick model) is covalently bound to the active site catalytic triad. The NS4A cofactor (ball-model) is bound to NS3.

of both proteases has been determined. Therefore, NS2/3 and NS3/4A are promising targets for the development of specific inhibitors. The NS3/4A serine protease has also been shown to cleave and inactivate the host proteins (Toll-like receptor domain-containing adapter inducing IFNb [Trif] and Cardif). Both proteins have important roles in the interferon response mediated by Toll-like receptor 3 (TLR3) and retinoic-acid inducible gene I (RIG-I), respectively [14,15]. Inactivation of both proteins blocks the double-stranded (ds) RNA–dependent innate immune response and indicates that the NS3/4A serine protease may have an important role in evasion of the innate immune response. Furthermore, it has been shown that NS3 is not only a protease but an integral part of the viral RNA replication complex and functions as an RNA helicase and a nucleotide triphosphatase (NTPase). Because of the multiple functions, it can be assumed that inhibitors against NS3 are highly efficient in blocking HCV replication. Several antiviral drugs directed against the NS3/4A protease have been developed and are listed in Table 2. NS4B is a membrane protein with four transmembrane regions localized at the endoplasmatic reticulum membrane. The function of NS4B is not well understood. NS4B is assumed to form a membranous web that is necessary for the HCV replication complex [16]. The functions of NS4B need to be understood better before it can be considered a promising target of antiviral therapy. NS5A is a pleiotropic protein with key roles in viral RNA replication and modulation of the physiology of the host cell. NS5A is mostly known because of its potential effect on interferon-alfa signaling. Furthermore, NS5A has been shown to affect cell growth of target cells and apoptosis. The crystal structure of domain I of NS5A has recently been solved [17], and specific inhibitors are currently being developed.

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Table 2 Emerging therapies against hepatitis C virus Name

Producer

Study phase

Long-acting interferons Albinterferon Pegamax

Human Genome Sciences/Novartis Maxygen/Roche

Phase 3 Preclinical/phase 1 (stopped) Phase 2 Phase 2

Locteron (BLX-883) Biolex Interferon-omega Intarcia Therapeutics Oral interferon Belerofon Nautilus Biotech STAT-C NS3/4 serine protease inhibitors Ciluprevir (BILN 2061) Boehringer Ingelheim Telaprevir (VX-950) Vertex Boceprevir (SCH 503034) Schering-Plough ITMN-191 InterMune GS9132/ACH-806 Gilead Sciences/Achillion NS5B RNA-dependent RNA-polymerase inhibitors Nucleoside analogues Valopicitabine (NM283) Idenix/Novartis R1626 (prodrug of R1479) Roche R1656/R7128 Pharmasset/Roche XTL-2125 XTL-Biopharmaceuticals MK-608 Merck Nonnucleoside polymerase inhibitors HCV-796 ViroPharma/Wyeth BILB 1941 Boehringer Ingelheim A-837093 Abbott GS-9190 Gilead NS5A inhibitors A-831 Arrow Therapeutics/AstraZeneca A-689 Arrow Therapeutics/AstraZeneca Cyclophilin inhibitors DEBIO-25 Debiopharm NIM811

Novartis

Stopped Phase 2 Phase 2 Phase 1 Stopped

Stopped Phase 2 Phase 1 Stopped Preclinical Stopped Stopped Preclinical Phase 1 Phase 1 Preclinical Phase 1 in HCV/HIVcoinfected patients Preclinical

The NS5B RNA-dependent RNA-polymerase is another promising target for the development of HCV-specific compounds. NS5B reveals the typical polymerase structure, a classic ‘‘right-hand’’ shape of thumb, palm, and finger domains encircling the active site (Fig. 4) [18]. To date, many inhibitors against the NS5B RNA-dependent RNA-polymerase have been developed, including nucleoside analog polymerase inhibitors and nonnucleoside polymerase inhibitors (see Table 2). The IRES located in the 50 -UTR is required for the initiation of HCV polyprotein translation. The IRES has been targeted by the antisense oligonucleotide inhibitor ISIS 14803 and the small-molecule organic drug mifepristone (VGX 410C). The development of ISIS 14803 was stopped

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Fig. 4. Structure of the HCV NS5B polymerase. Ligands are given as ball-and-stick models. The active site is located in the palm domain. Regions important for inhibitor binding are indicated for nucleoside and nonnucleoside compounds.

because of weak and highly variable antiviral activity and substantial increases of aminotransferases in a phase 1 trial in patients who had chronic hepatitis C [19]. Mifepristone is currently being investigated in a phase 2 trial in patients who have chronic hepatitis C, and data are pending [20]. The 30 -UTR is necessary for RNA replication and amplification of IRESdependent protein translation and also seems to be a promising antiviral target. To date, most new antiviral drugs for HCV focus on the NS3/4A serine protease and on the NS5B RNA-dependent RNA-polymerase. The results of clinical trials are presented in the following sections. Current therapies for hepatitis C virus Pegylated interferon-alfa and ribavirin are the current standard of care for treatment of patients who have chronic hepatitis C. Great efforts have been made for the individualization of antiviral therapy to reduce adverse events and to improve sustained virologic response rates. HCV genotype and early virologic response during treatment are important factors for individualization of antiviral therapy. An extended treatment duration of 72 weeks has been shown to reduce relapse rates significantly in patients who have chronic HCV genotype 1 infection and a slow virologic response compared with the standard duration of 48 weeks [21,22]. Conversely, HCV genotype 1–infected patients with a low baseline HCV RNA concentration who become HCV RNA-negative at week 4 may be treated for 24 weeks without compromising sustained virologic response rates [23].

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In patients who have HCV genotype 2 or 3 infection, who have a better response to interferon-alfa than patients infected with HCV genotype 1, the standard treatment duration is 24 weeks. Several trials investigated whether a shorter treatment duration is possible in patients who have genotype 2 or 3 infection without compromising the sustained virologic response. Smaller trials showed that a shorter treatment duration of 12 to 16 weeks is equally effective as the standard treatment duration in those patients infected with HCV genotype 2 or 3 who achieve a rapid virologic response after 4 weeks of therapy [24,25]. The large ACCELERATE trial comparing 16 versus 24 weeks of treatment in patients who had HCV genotype 2 or 3 infection showed that a shorter treatment duration of 16 weeks results in reduced sustained virologic response rates compared with the standard treatment duration, however [26]. In the ACCELERATE trial, a shorter course of therapy over 16 weeks has been shown to be as effective as a 24-week course in those patients who have genotype 2 or 3 infection, have a baseline viral load of 400,000 IU/mL or less, and achieve an early virologic response at week 4 [26]. In patients who have genotype (2 and) 3 infection without a rapid virologic response (!50 IU/mL) at week 4, a longer treatment duration may be necessary to optimize sustained virologic response rates [27]. The ribavirin dose may also influence the rate of sustained virologic response. The Weight-based dosing of pegINterferon alfa-2b and Ribavirin (WIN-R) trial investigated pegylated interferon alfa-2b plus weight-based ribavirin (800–1400 mg) or flat-dose ribavirin (800 mg) in patients who had chronic hepatitis C [28]. Sustained virologic response but not end-of-treatment rates were significantly higher in patients treated with weight-based ribavirin than in patients treated with flat-dose ribavirin (44.2% versus 40.5%). Sustained virologic response rates by intention-to-treat analysis were 34.0% and 28.9%, respectively, in genotype 1–infected patients. In genotype 2– or 3–infected patients, sustained virologic response rates were not significantly different (61.8% and 59.5%, respectively), regardless of treatment duration [28]. From the current point of view, further individualization of standard therapy seems unlikely to improve the convenience and outcome of antiviral therapy markedly. New interferons have been developed, however, and are currently being investigated in clinical trials.

Further development of interferon therapy Albinterferon Albinterferon is a novel long-acting form of interferon-alfa that results from the genetic fusion of interferon-alfa with human albumin. Albinterferon has a longer half-life than pegylated interferon-alfa. A recent phase 2b clinical trial investigated antiviral efficacy and tolerability of albinterferon in combination with ribavirin in patients who had chronic hepatitis C genotype 1 infection and were naive to

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interferon-alfa–based treatment regimens [29]. In this trial, the virologic response rates 12 weeks after treatment were 58% and 55% in patients treated with albinterferon, 900 mg and 1200 mg every 2 weeks, plus ribavirin versus 58% in patients treated with pegylated interferon-alfa-2a, 180 mg once weekly, plus ribavirin. The health-related quality-of-life scores were more favorable in the albinterferon group than in the pegylated interferon-alfa2a group. Overall, these interim results indicate that albinterferon plus ribavirin may offer efficacy comparable to pegylated interferon-alfa-2a plus ribavirin with half of the injections and the potential for less impairment of quality of life. Albinterferon is currently being investigated in two randomized, openlabel, active controlled, multicenter, noninferiority phase 3 trials to evaluate the efficacy, safety, and impact on health-related quality of life of albinterferon in combination with ribavirin versus pegylated interferonalfa-2a in combination with ribavirin. The ACHIEVE 1 trial is conducted in patients infected with HCV genotype 1 randomized into three treatment groups, including two groups with subcutaneously administered albinterferon once every 2 weeks (900 or 1200 mg), and a control group with pegylated interferon-alfa-2a administered once every week at a dose of 180 mg. All patients receive oral ribavirin (1000–1200 mg) concomitantly and are treated for 48 weeks. The ACHIEVE 2/3 trial is being conducted in patients infected with HCV genotype 3. The ACHIEVE 2/3 trial has the same design as the ACHIEVE 1 trial with a shorter treatment duration of 24 weeks and a lower ribavirin dose of 800 mg. The primary efficacy end point in both trials is a sustained virologic response, defined as an undetectable HCV RNA level (!10 IU/mL) at weeks 72 and 48, respectively. Higher doses of albinterferon administered every 4 weeks, in combination with ribavirin, are to be explored in separate trials, which are expected to begin in 2007. Interferon-omega Interferon-omega is a type 1 interferon that shares 70% homology of the amino-acid sequence to interferon-alfa and is derived from Chinese hamster ovary cells. Interferon-omega, which is fully glycosylated, has been broadly studied in phase 1 and 2 clinical studies. These studies have demonstrated that interferon-omega is safe and has potent antiHCV activity. Preliminary results from a phase 2 study comparing interferon-omega (25 mg/d) with the combination of interferon-omega and ribavirin (1000–1200 mg/d) have shown virologic response rates 12 weeks after the end of treatment of 6% and 36%, respectively [30]. It is planned to administer interferon-omega by an implantable osmotic minipump, requiring changes every 3 months, which delivers consistent drug levels through the device outlet.

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Locteron (BLX-883) Locteron is a recombinant interferon-alfa that is released by a special biodegradable polymeric drug delivery system. The polymeric drug delivery system consists of polyester or polyether copolymers that are degraded by hydrolysis and oxidation and enables linear release of compounds. Locteron is designed to be administered every 2 weeks. A phase 1 clinical study investigating the safety, pharmacokinetics, and pharmacodynamics of locteron in healthy volunteers was completed [31]. Results of this phase 1 clinical trial showed that a single dose of locteron was safe and well tolerated. In particular, groups receiving locteron reported fewer, less severe, and shorter lasting flu-like symptoms than those subjects receiving pegylated interferon-alfa-2b. Maxy-alpha (R7025/RO5014583, pegamax) Maxy-alpha (R7025) is an interferon-alfa variant that has been created through a directed molecular evolution gene shuffling technology to have stronger antiviral activity against the hepatitis C virus and to be more effective in stimulating immune responses than standard interferon-alfa. A pegylated form of maxy-alpha has been developed using the same pegylation technology as for pegylated interferon-alfa-2a. Preclinical data comparing maxy-alpha with pegylated interferon-alfa-2a show that maxy-alpha has increased antiviral activity in the HeLa encephalomyocarditis virus assay and stronger immune stimulatory activity on T helper 1 (Th1) cytokine induction and dendritic cell maturation than pegylated interferon-alfa-2a [32]. A double-blind, dose-escalation, controlled phase 1 study of a single subcutaneous administration of maxy-alpha has been initiated in healthy volunteers [32], and data are pending. Oral interferon (belerofon) Belerofon is a variant of human interferon-alfa with a single amino-acid replacement that has been designed to lower the susceptibility of interferonalfa to proteolytic degradation and to make it longer lasting in serum [33]. In animal models, appropriate oral doses have shown that belerofon can be absorbed from the intestine into the bloodstream and reaches blood levels comparable to those obtained by subcutaneously injected interferon-alfa [33]. Oral belerofon is formulated as enteric-coated tablets containing the lyophilized belerofon protein. Phase 1 results on the safety, tolerability, and pharmacokinetics of oral belerofon are not yet available. Further development of ribavirin Taribavirin (previously known as viramidine) is a prodrug of ribavirin with a distinct pharmacologic profile. Taribavirin is preferentially taken

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up by the liver and is converted to ribavirin by hepatic adenosine deaminase. In contrast to ribavirin, taribavirin is poorly taken up by red blood cells. The efficacy and safety of taribavirin versus ribavirin combined with pegylated interferon in patients who have chronic hepatitis C was investigated in a phase 2 open-label study [34]. Patients were stratified according to HCV genotype and randomized to receive pegylated interferon-alfa-2a at a dosage of 180 mg/wk plus taribavirin at a dosage of 800, 1200 or 1600 mg/d or ribavirin at a dosage of 1000 or 1200 mg/d. Patients infected with HCV genotype 1, 4, 5, or 6; mixed genotypes; or an indeterminate genotype were treated for 48 weeks, and those infected with HCV genotype 2 or 3 were treated for 24 weeks. All patients were followed for 24 weeks after the end of treatment to determine the rate of sustained virologic response. The rates of sustained virologic response were 23%, 37%, and 29% for the different taribavirin dose groups, respectively, and 44% for pegylated interferon alfa-2a plus ribavirin. Fewer patients on any dose of taribavirin had severe anemia (hemoglobin !10 g/dL) than on ribavirin (4% versus 27%). Two large-scale randomized phase 3 clinical trials comparing the safety and efficacy of taribavirin plus pegylated interferon-alfa-2b (VISER-1) and pegylated interferon-alfa-2a (VISER-2) versus pegylated interferonalfa-2b/2a plus ribavirin, respectively, were recently completed. In both trials, patients were stratified according to genotype, baseline viral load, and weight. The VISER-1 trial showed a significantly lower rate of anemia among patients treated with taribavirin compared with ribavirin; however, the overall rate of sustained virologic response in the VISER-1 trial was lower in taribavirin-treated patients versus ribavirin-treated patients (38% versus 52%) [35]. Similar results were obtained in the VISER-2 study (40% versus 55% for taribavirin-treated versus ribavirin-treated patients) [36]. Future studies may be warranted to examine higher weight-based doses of taribavirin in combination with pegylated interferon [34].

Specifically targeted antiviral therapy for hepatitis C virus Protease inhibitors Ciluprevir (BILN 2061) Ciluprevir is the first potent and specific inhibitor of the NS3/4A serine protease [37] that was tested within a randomized, multiple-dose, doubleblind, placebo-controlled pilot study in patients who had chronic hepatitis C [38]. In this study, the oral administration of ciluprevir (25–500 mg twice per day) to patients who had chronic hepatitis C genotype 1 infection for 2 days resulted in viral RNA reductions of 2- to 3-log10 copies/mL in most of the patients. The study provided proof of concept that HCV NS3/4A protease inhibitors are a therapeutic option for patients who have chronic hepatitis C.

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The HCV genotype is currently the most important baseline predictive factor for virologic response to interferon-alfa–based treatment. The antiviral efficacy of ciluprevir was also investigated in patients who had chronic HCV genotype 2 or 3 infection [39]. The antiviral efficacy of ciluprevir was less pronounced and more variable in HCV genotype 2– or genotype 3–infected patients compared with HCV genotype 1–infected patients, showing that treatment response to protease inhibitors may also depend on HCV genotype [38,39]. Safety and tolerability are important issues in the development of new antiviral drugs. In rhesus monkeys treated with high doses of ciluprevir for 4 weeks, histologic signs of cardiotoxicity were observed. Because of this cardiotoxicity, the clinical development of ciluprevir was stopped. The development of resistance against ciluprevir was studied in HCV genotype 1b replicon cells [40]. Several mutations in the NS3 protease were identified that were associated with resistance against ciluprevir (Table 3). Modeling studies indicate that all mutations are located in close proximity to the inhibitor binding site [40]. Telaprevir (VX-950) Telaprevir is a peptidomimetic inhibitor of the NS3/4A serine protease that has been developed for the specific treatment of hepatitis C. Compared with ciluprevir, telaprevir exhibits a longer half-life of the bound enzyme inhibitor complex [41]. The first placebo-controlled double-blind phase 1 study with telaprevir was started in June 2004. In this study, 34 patients who had chronic genotype 1 infection were randomized to receive placebo or telaprevir at a dosage of 450 mg or 750 mg every 8 hours or 1250 mg every 12 hours for 14 days [42]. Most of the included patients in this study (27 of 34 patients) had failed prior interferon-based treatment. During treatment with telaprevir, all Table 3 Mutations in the protease conferring resistance to various protease inhibitors BILN 2061

Telaprevir

Boceprevir

ITMN-191

In vitro

R155Q A156V/T D168V/A/Y

A156S/V/T

T54A A156S/T V170A

In vivo

No data

V36M/A T54A R155K/T 36/155 A156S/V/T 36/156

T54A

V23A Q41R S138T D168A/V/E D168V/A156S/V S489L No data

Data from Refs. [43,45,76].

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patients showed a decline in viral load of 2 log10 or greater. Twenty-six of 28 patients had a decline in viral load of 3 log10 or greater. In the 750-mg dose group, the median reduction of HCV RNA was 4.4 log10 after 14 days. In the 450-mg dose group and the 1250-mg dose group, the maximum effect was seen between days 3 and 7 of dosing, followed by an increase of HCV RNA [42]. The increase of HCV RNA between days 7 and 14 indicates the selection of variants with reduced sensitivity to telaprevir [42,43]. Several mutations conferring resistance to telaprevir were detected in the replicon system and during monotherapy with telaprevir [43]. The development of resistant variants was investigated in patients during telaprevir monotherapy [43]. The known in vitro and in vivo mutations conferring resistance to telaprevir are listed in Table 3. Mutations that confer low-level resistance (V36A/M, T54A, R155K/T, and A156S) and high-level resistance (A156V/T, 36þ155, 36þ156) to telaprevir were detected and correlated with telaprevir exposure and virologic response. Viral fitness generally refers to the relative replication competence of a virus under defined circumstances. Sarrazin and colleagues [43] generated an algorithm to calculate viral fitness using the HCV RNA data and sequence data at the end of treatment and at follow-up. Changes in the frequency of mutations after the end of dosing showed an inverse relation between in vivo viral fitness and resistance. In the absence of telaprevir selective pressure, most resistant variants were replaced by wild-type virus within 3 to 7 months. The rapid development of resistance during telaprevir monotherapy indicates that combination therapy with pegylated interferon-alfa or other direct antiviral drugs seems to be necessary to avoid the development of resistance [43]. Subsequently, telaprevir was investigated in combination with pegylated interferon-alfa-2a [44]. Treatment-naive patients who had chronic hepatitis C genotype 1 infection were randomized for treatment with pegylated interferon-alfa-2a plus placebo (n ¼ 4), telaprevir monotherapy (n ¼ 8), or a combination of telaprevir and pegylated interferon-alfa-2a (n ¼ 8) for 2 weeks. The median changes in HCV RNA level from baseline to day 15 were 1.09 log10 IU/mL, 3.99 log10 IU/mL, and 5.49 log10 IU/mL in the pegylated interferon alfa-2a plus placebo, the telaprevir monotherapy, and the telaprevir plus pegylated interferon alfa-2a combination groups, respectively. The results of this study demonstrated at least additive antiviral effects of telaprevir in combination with pegylated interferon-alfa-2a. The treatment was well tolerated, all patients completed dosing, and no serious adverse events were reported. A detailed kinetic analysis of variants in patients treated with telaprevir alone or with telaprevir plus pegylated interferon-alfa-2a for 14 days was performed [45]. This analysis indicates that the initial antiviral response to telaprevir is attributable to a sharp reduction in wild-type virus, which uncovers preexisting telaprevir-resistant variants. The combination of telaprevir and pegylated interferon-alfa-2a inhibited wild-type and resistant

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variants, indicating that telaprevir-resistant variants are sensitive to pegylated interferon-alfa-2a. Combination therapy with pegylated interferon-alfa-2a plus ribavirin was offered to the patients after the initial 14-day dosing period [44,45]. Nineteen of the 20 patients began standard therapy after completing the dosing period (1 patient of the previous telaprevir monotherapy group declined treatment). Twelve weeks after starting standard therapy, HCV RNA was undetectable in 5 patients of the previous telaprevir group and in all 8 patients of the previous telaprevir plus pegylated interferon-alfa-2a group. At 24 weeks, HCV RNA was undetectable in all patients who started standard therapy in the previous telaprevir monotherapy group (n ¼ 7) and the previous telaprevir plus pegylated interferon-alfa-2a group (n ¼ 8) [44,45]. In consecutive studies, telaprevir is being combined with pegylated interferon-alfa-2a and ribavirin [46,47] and is currently being further investigated in this combination in two phase 2 studies (PROVE-1 and -2). In the PROVE-1 trial (conducted in the United States), treatment-naive patients infected with HCV genotype 1 (n ¼ 260) have been randomized into four groups: 1. Twelve weeks of therapy with telaprevir (750 mg every 8 hours) in combination with pegylated interferon-alfa-2a and ribavirin (total duration of 12 weeks, n ¼ 20) 2. Twelve weeks of therapy with telaprevir (750 mg every 8 hours) in combination with pegylated interferon-alfa-2a and ribavirin, followed by pegylated interferon-alfa-2a and ribavirin alone for 12 weeks (total duration of 24 weeks, n ¼ 80) 3. Telaprevir (750 mg every 8 hours) in combination with pegylated interferon-alfa-2a and ribavirin for 12 weeks, followed by pegylated interferon-alfa-2a and ribavirin alone for 36 weeks (total duration of 48 weeks, n ¼ 80) 4. Control arm with pegylated interferon-alfa-2a and ribavirin for 48 weeks (total duration of 48 weeks, n ¼ 80) Only patients in the 12- and 24-week treatment arms who achieve a rapid viral response (HCV RNA level !10 IU/mL) by the end of week 4 and who maintain this status through to week 10 or 20, respectively, are planned to stop all treatment at the 12- or 24-week time point (according to the study arms) and are to be followed after treatment to evaluate whether they achieve a sustained virologic response. Patients in these treatment arms without a rapid virologic response are required to continue on pegylated interferon-alfa-2a and ribavirin for a total of 48 weeks, however. The PROVE-2 study is conducted in Europe (n ¼ 320, n ¼ 80 for each treatment arm) and has a similar study design as the PROVE-1 study. In the PROVE-2 trial, the 12-week treatment arm with telaprevir plus pegylated interferon-alfa-2a plus ribavirin followed by 36 weeks of treatment with pegylated interferon-alfa-2a plus ribavirin is replaced by 12 weeks of

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treatment with telaprevir plus pegylated interferon-alfa-2a only. Another important difference is that patients within the PROVE-2 trial can stop treatment according to study arm assignment independent of a rapid virologic response criterion. The interim analysis of the PROVE-1 trial showed that a rapid virologic response after 4 weeks of treatment (HCV RNA level !10 IU/mL) was achieved in 79% of patients in the triple-therapy arm, including telaprevir, and in 11% of patients in the standard combination arm. The virologic response rate after 12 weeks of treatment (HCV RNA level !10 IU/mL) was 70% in the triple-therapy arm and 39% in the standard combination arm. Nine patients who were treated with pegylated interferon-alfa-2a, ribavirin, plus telaprevir in study arm A and achieved a rapid virologic response at week 4 (!10 IU/mL) discontinued triple therapy after 12 weeks. Twenty weeks after discontinuation of triple therapy, six of nine patients had undetectable HCV RNA in serum. This is the first result indicating that specifically targeted antiviral therapy against HCV may shorten the required treatment duration and improve the sustained virologic response rate. In the PROVE-1 trial, the total incidence of adverse events in patients treated with telaprevir, interferon-alfa-2a, and ribavirin was similar to that of the control group. Discontinuation attributable to adverse events was more frequent in the telaprevir arm compared with the control arm (9% versus 3%), however. Gastrointestinal events, rashes (severe in several cases), and anemia were more common in the triple-therapy arms, which included telaprevir, than in the standard double-combination treatment arm. The efficacy of telaprevir in patients infected with HCV genotype 1 who have not achieved a sustained virologic response with a previous interferonbased treatment is to be investigated in the PROVE-3 trial (n ¼ 400 with 60% nonresponders and 40% relapsers to previous interferon-based treatment). The PROVE-3 trial is a double-blind randomized phase 2 study investigating telaprevir or placebo with peginterferon-alfa-2a, with or without ribavirin. In the PROVE-3 trial, patients are to be randomized into four groups [48]: 1. Twelve weeks of therapy with telaprevir, pegylated interferon-alfa-2a and ribavirin, followed by 12 weeks of treatment with placebo, pegylated interferon-alfa-2a, and ribavirin (total duration of 24 weeks) 2. Twenty-four weeks of therapy with telaprevir and pegylated interferonalfa-2a (total duration 24 weeks) 3. Twenty-four weeks of therapy with telaprevir plus pegylated interferonalfa-2a and ribavirin, followed by 24 weeks of treatment with pegylated interferon-alfa-2a and ribavirin (total duration of 48 weeks) 4. Control arm with placebo plus pegylated interferon-alfa-2a and ribavirin for 24 weeks, followed by 24 weeks of treatment with pegylated interferon-alfa-2a and ribavirin only (total duration of 48 weeks)

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All patients who do not achieve an early virologic response at week 4 discontinue treatment. Patients in the control group 4 have the option to receive telaprevir through a rollover study after week 24. Final data are awaited in 2008. Boceprevir (SCH 503034) Boceprevir binds reversibly to the NS3 protease active site and has potent activity in the replicon system alone [49] and in combination with interferonalfa-2b [49]. In a phase 1 open-label combination study, boceprevir was evaluated in combination with pegylated interferon-alfa-2b versus either agent alone in a crossover design in adult patients who have HCV genotype 1 and were previous nonresponders to pegylated interferon-alfa-2b–based therapy [50]. Patients were randomized to receive in random sequence (1) boceprevir (200 mg or 400 mg every 8 hours) as a monotherapy for 7 days, (2) pegylated interferon-alfa-2b (1.5 mg/kg/wk) as monotherapy for 14 days, and (3) boceprevir plus pegylated interferon-alfa-2b combination therapy for 14 days in a three-period crossover design with a 3-week washout between treatments. Mean maximum log10 changes in HCV RNA were 2.45  0.22 and 2.88  0.22 for pegylated interferon-alfa-2b plus boceprevir at a rate of 200 mg or 400 mg, respectively, compared with 1.08  0.22 and 1.61  0.21 for boceprevir at a rate of 200 mg and 400 mg, respectively, and 1.08  0.22 and 1.26  0.20 for pegylated interferon-alfa-2b alone in the groups with boceprevir administered at 200 mg and 400 mg, respectively [50]. Several mutations conferring resistance to boceprevir were observed in the replicon system, but only a single mutation has to date been described in one patient by direct sequencing (see Table 3). Based on the results of the phase 1 clinical program and extensive preclinical safety and pharmacology studies, a large randomized phase 2 dose-finding study has been initiated [51]. This study was designed to evaluate the safety and efficacy of boceprevir in combination with pegylated interferon-alfa-2b, with and without added ribavirin, for 24 or 48 weeks in patients infected with chronic HCV genotype 1 who were nonresponders to previous pegylated interferon and ribavirin combination therapy. The primary objective of this study was to determine the safe and effective dose range of boceprevir in combination with pegylated interferon-alfa-2b in this patient population. A secondary objective was to explore whether ribavirin provides an additional benefit when combined with boceprevir plus pegylated interferon-alfa-2b. The study was completed in 2007, and the results are pending [51]. The safety and efficacy of boceprevir in treatment-naive patients who have chronic HCV genotype 1 infection is currently being investigated in a phase 2, randomized, 5-arm, comparative, open-label safety and efficacy study. The study compares treatment with pegylated interferon-alfa-2b plus ribavirin with treatment with boceprevir plus pegylated interferonalfa-2b and ribavirin.

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Optimizing pharmacokinetics: ritonavir boosting Ritonavir is a protease inhibitor approved for treatment of HIV with potent inhibitory effects against cytochrome P450 3A (CYP3A). Codosing with ritonavir leads to pharmacokinetic enhancement (boosting) of peptidomimetic HIV protease inhibitors, which are metabolized by CYP3A. The interaction of the investigational HCV protease inhibitors telaprevir and boceprevir with ritonavir was studied in vitro and in vivo [52]. In rat and human liver microsomes, the metabolism of telaprevir and boceprevir was strongly inhibited by ritonavir. On codosing of telaprevir or boceprevir with ritonavir in rats, plasma exposure of the HCV protease inhibitors was increased by greater than 15-fold and plasma concentrations 8 hours after dosing were increased by greater than 50-fold. ITMN-191 ITMN-191 is another inhibitor of the NS3/4A protease with potent antiviral activity in vitro [53]. Several mutations associated with resistance to ITMN-191 were found in the replicon system (see Table 3). A phase 1a trial in healthy volunteers was completed in May 2007. The phase 1b study in patients who have chronic hepatitis C is designed to assess the effect on viral kinetics, viral resistance, pharmacokinetics, safety, and tolerability of multiple ascending doses of ITMN-191 given as monotherapy for 14 days. Twice per day and three times per day dosage regimens are to be studied. [54]. NS4A inhibitors ACH-806, ACH-1095. ACH-806 is an antagonist of the NS4A protein, which is a cofactor of the NS3 protease. Thus, ACH-806 is a protease inhibitor with a distinct mechanism of action compared with the previously described NS3/4A protease inhibitors. ACH-806 prevents the formation of the replicase complex after viral protein processing, a necessary step in viral replication that occurs before copying the viral RNA genome. This unique mechanism may contribute to a lack of cross-resistance between ACH-806 and other HCV NS3/4A protease inhibitors in vitro. Data from a phase 1b trial indicated that ACH-806 has antiviral activity against HCV, validating the novel anti-HCV mechanism. Based on elevations of serum creatinine, which were reversible after completion of dosing, however, further development of ACH-806 was stopped [55]. ACH-1095 is one of a series of next-generation compounds with potent antiviral potency in the replicon system [56]. Results from clinical trials on this compound are not yet available. Polymerase inhibitors Two classes of polymerase inhibitors, nucleoside and nonnucleoside polymerase inhibitors, have been developed. Nucleoside analog polymerase inhibitors are converted to triphosphates by cellular kinases and

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incorporated into the elongating RNA strand as chain terminators. Generally, they show similar efficacy against all HCV genotypes. Nucleoside analog polymerase inhibitors Valopicitabine (NM283) Valopicitabine is an orally bioavailable prodrug of a nucleoside analogue inhibiting the HCV NS5B RNA-dependent RNA-polymerase [57]. Valopicitabine inhibits the viral polymerase directly and is incorporated into growing strands of viral RNA, with subsequent termination of RNA chain extension. Human polymerases do not seem to be affected by valopicitabine. Valopicitabine was investigated in patients who had chronic hepatitis C alone and in combination with pegylated interferon. Patients infected with HCV genotype 1 and prior nonresponse to interferon-based antiviral treatment showed a mean reduction of 0.15 to 1.21 log10 IU/mL after 14 days of treatment with different doses of valopicitabine ranging from 50 to 800 mg/d [58]. Combination therapy was generally well tolerated; however, higher doses of valopicitabine were associated with gastrointestinal side effects that were severe in some patients [59]. Therefore, the maximum dose of valopicitabine was reduced from 800 to 400 mg/d by protocol amendment during the progress of the phase 2 trials. An interim analysis showed that the combination of pegylated interferon-alfa-2a plus valopicitabine in treatment-naive patients who had chronic hepatitis C genotype 1 infection was associated with a mean decline of HCV RNA of 3.90 to 4.56 log10 IU/mL and 3.75 to 4.41 log10 IU/mL after 24 and 36 weeks of treatment, respectively [59]. Valopicitabine in combination with pegylated interferon-alfa-2a was also investigated in patients with prior nonresponse to interferon-alfa–based treatment [60]. The interim analysis of this study after 24 weeks of treatment showed a significantly stronger decline of HCV RNA in the combination of valopicitabine (800 mg) and pegylated interferon-alfa-2a versus pegylated interferon-alfa-2a and ribavirin treatment (3.32 versus 2.31 log10 IU/mL) [60]. After 40 weeks of treatment, the maximum dose of valopicitabine also had to be reduced to 400 mg/d by protocol amendment [61]. After 48 weeks of treatment, the decline of HCV RNA in the previous high-dose valopicitabine plus pegylated interferon-alfa-2a arms was still 0.8 log10 IU greater than in the pegylated interferon-alfa-2a plus ribavirin combination arm; however, the difference was not significant [61]. Based on the overall risk-benefit profile observed in clinical testing, the development program of valopicitabine for the treatment of hepatitis C has been placed on clinical hold. Resistance to valopicitabine was investigated using the replicon system. Replicon variants bearing an S282T mutation in the viral polymerase showed resistance to the active metabolite of valopicitabine [62,63]. Replicon variants bearing this mutation showed reduced fitness compared with the wild-type replicon.

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Nonsynergistic interactions of antiviral drug combinations could be a problem for future therapies. It was found that ribavirin antagonizes the in vitro anti-HCV activity of 20 -C-methylcytidine, the active metabolite of valopicitabine. These findings may have implications in future clinical studies with specific nucleoside analog polymerase inhibitors [64]. R1479 and R1626 The nucleoside analogue R1479 (40 -azidocytidine) is a potent inhibitor of NS5B-dependent RNA synthesis and hepatitis C virus replication in cell culture [65]. R1626 is a prodrug of R1479 [66]. A multiple dose ascending phase 1 study was designed to evaluate the safety, tolerability, pharmacokinetics, and antiviral activity of R1626 in previously untreated patients infected with chronic hepatitis C genotype 1 [66]. In this study, patients were randomized for treatment with different doses of R1626 ranging from 500 to 4500 mg of R1626 or placebo twice daily. Patients were treated for 14 days and followed up for another 14 days. Mean viral load reductions of 1.2, 2.6, and 3.7 log10 IU/mL were observed with R1626 at doses of 1500, 3000, and 4500 mg, respectively. A phase 2 trial evaluates safety and efficacy of R1626 in combination with peginterferon-alfa-2a and ribavirin. Development of resistance to R1479 was investigated in the replicon system. Resistance was associated with the presence of amino-acid substitutions S96T and S96T/N142T in the NS5B polymerase [67]. PSI-6130 and R7128 R7128 is another nucleoside type polymerase inhibitor that has been developed for the treatment of chronic hepatitis C [68]. R7128 is a prodrug of PSI-6130, an oral cytidine nucleoside analogue. In preclinical studies, no toxicity was observed in various human cells, including liver cells, bone marrow cells, and white blood cells. When compared in laboratory studies with several other compounds in development for the treatment of HCV, PSI6130 was found to be more active at low concentrations or less toxic. In combination with interferon, PSI-6130 was active and additive to the activity of interferon alone in these preclinical assays. Results from phase 1 studies on R7128 are pending. Nonnucleoside polymerase inhibitors The mechanisms of action of nonnucleoside polymerase inhibitors are different from those of nucleoside polymerase inhibitors. Therefore, crossresistance between these two classes is unlikely to occur. Several structurally distinct nonnucleoside inhibitors of the HCV RNA–dependent RNApolymerase NS5B have been reported to date, including benzimidazole, benzothiadiazine, and disubstituted phenylalanine or thiophene or dihydropyranone derivatives. They target different sites within the thumb domain of

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the polymerase. Nonnucleoside inhibitors based on the structure of a benzimidazole or an indole core bind to an exposed guanosine triphosphate (GTP) site on the protein surface, whereas inhibitors with a phenylalanine, thiophene, or dihydropyranone scaffold bind 1.0–1.5 nm from that external GTP site in the region of a hydrophobic cleft at the base of the thumb domain (see Fig. 4). Different resistance profiles attributable to distinct target sites can be expected for the class of nonnucleoside inhibitors. As with protease inhibitors, however, a single mutation may already confer resistance to nonnucleoside polymerase inhibitors. In contrast to nucleoside polymerase inhibitors, a restricted spectrum of activity of nonnucleoside polymerase inhibitors against different HCV genotypes and subtypes has been described. In addition to classic nonnucleoside analog inhibitors, several pyrophosphate mimics have been described that interact with catalytic metal ions in the active site of the enzyme. HCV-796 HCV-796 is a nonnucleoside polymerase inhibitor of the NS5B RNAdependent RNA-polymerase that has demonstrated potent antiviral activity in vitro and in patients who have chronic hepatitis C. Monotherapy showed a maximum antiviral effect after 4 days of treatment with a mean reduction of HCV RNA of 1.4 log10 IU/mL. Viral load started to increase thereafter, however, indicating that resistance might be an issue. The emergence of a C316Y amino-acid substitution in NS5B in isolates of patients treated with HCV-796 was associated with the development of resistance to HCV-796. The combination of HCV-796 and pegylated interferon-alfa-2b was investigated in treatment-naive patients who had chronic hepatitis C. The combination of HCV-796 and pegylated interferon-alfa-2b produced a mean viral reduction of 3.3 to 3.5 log10 IU/mL after 14 days of treatment compared with 1.6 log10 IU/mL with pegylated interferon-alfa-2b alone [69]. The antiviral activity of HCV-796 differed by HCV genotype. Mean reductions at day 14 for patients infected with HCV genotype 1 ranged from 2.6 to 3.2 log10 IU/mL in the combination groups versus l.2 log10 IU/mL for pegylated interferon-alfa-2b alone. For HCV genotype non–1-infected patients, the respective reductions of HCV RNA were 3.5 to 4.8 log10 IU/mL versus 2.6 log10 IU/mL. Common adverse events in all groups were those typically associated with interferons, including headache, chills, rash, and myalgia [69]. In a consecutive phase 2 study evaluating HCV-796 in combination with pegylated interferon and ribavirin, clinically significant elevations of liver enzymes were observed in approximately 8% of patients receiving HCV796, including two patients who experienced serious adverse events leading to withdrawal from active therapy with HCV-796, pegylated interferon, and ribavirin [70]. Because of this potential safety issue, HCV-796 treatment was discontinued in the phase 2 program.

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GS-9190 GS-9190 is another nonnucleoside polymerase inhibitor with potent antiviral activity in the replicon system [71]. The antiviral activity of GS-9190 is higher in HCV genotype 1 replicons compared with HCV genotype 2 replicons. GS-9190 is currently being investigated in phase 1 clinical trials. BILB 1941 BILB 1941 is an orally bioavailable reversible nonnucleoside inhibitor of the RNA-dependent RNA-polymerase of the hepatitis C virus. The compound exhibits potent and specific inhibition of the HCV RNA–dependent RNA-polymerase in enzymatic- and cell-based assays. In a phase 1 trial, BILB 1941 was given as monotherapy in a liquid formulation for 5 days and demonstrated significant antiviral activity in patients infected with HCV genotype 1 [72]. Increased virologic response was limited by gastrointestinal intolerance that precluded testing at higher doses. The contribution to the gastrointestinal side effects by BILB 1941 versus the constituents of the liquid formulation remains uncertain. NS5A antagonists A-831 and A-689 A-831 targets the NS5A protein and has shown potent activity in the replicon assay. The drug has an excellent therapeutic index and good pharmacokinetic properties. A-831 is currently being investigated in a phase 1 trial, and results are pending. A-689 is another NS5A inhibitor currently in preclinical development. A-689 has a different chemical structure from A-831 and binds to the NS5A target at a different site [73]. Cyclophilin inhibitors DEBIO-25, NIM811 Cyclophilins are ubiquitous proteins in human cells that are involved in protein folding. Moreover, cyclophilins participate in HCV replication. It was shown that cyclophilin B binds to the HCV NS5B polymerase and stimulates its RNA-binding activity. Cyclophilin inhibitors show strong antiviral activity in vitro and in vivo. Cyclophilin inhibitors may not only be effective against HCV but against HIV. The cyclophilin inhibitor DEBIO-025 showed a strong dual antiviral activity against HCV and HIV in a phase 1 trial with HCV/HIV-coinfected patients [74]. In this study, the mean maximal decrease in HIV-1 viral load was 1.0 log10 IU/mL. A pronounced effect on HCV RNA was found, with a mean maximal decrease of 3.6 log10 IU/ mL. All patients except one showed an HCV RNA reduction of more than 2 log10 IU/mL, with differences depending on genotype. DEBIO-025 was well tolerated. Laboratory abnormalities observed were hyperbilirubinemia and low platelet count. Clinical data for another cyclophilin inhibitor, NIM811, are currently not available.

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Alpha-glucosidase I inhibitor Celgosivir (MX-3253) Celgosivir (MX-3253) is a new class of antiviral in clinical development for treatment of patients who have chronic hepatitis C. The active metabolite of celgosivir is castanospermine, which is a potent inhibitor of the alphaglucosidase I that is a host enzyme required for viral assembly and release. Celgosivir is potentially synergistic with other mechanistically diverse antiHCV drugs. A phase 2, multicenter, double-blind, controlled study on the efficacy of celgosivir in patients who had chronic hepatitis genotype 1 infection with prior nonresponse to interferon-based antiviral treatment was performed [75]. In this study, virologic nonresponders to previous interferon-alfa–based antiviral treatment showed a 1.2-log10 decline of HCV RNA after treatment with celgosivir, pegylated interferon-alfa-2b, and ribavirin compared with a 0.4-log10 decline after treatment with placebo plus pegylated interferon-alfa-2b and ribavirin (one-sided P!.05).

Summary Research in the past years has focused on individualization of interferonalfa–based treatment regimens for patients who have chronic hepatitis C. New long-acting interferons may improve convenience of application and potentially improve the adverse event profile. Further modification of interferon therapy is unlikely to improve sustained virologic response rates markedly. Specific targeted antiviral therapy for HCV is a new perspective in the treatment of chronic hepatitis C especially for patient populations that are difficult to treat. The results from recent clinical trials indicate that several compounds have potent antiviral activity; however, not all were considered as safe and well tolerated. A central question remains whether the new antiviral compounds not only increase the virologic response during treatment but increase the rate of sustained virologic response after treatment. A first interim analysis on sustained virologic response after treatment with telaprevir in combination with pegylated interferon-alfa and ribavirin has recently been presented, and the results are promising. The results suggest that combination therapy with telaprevir with pegylated interferon-alfa plus ribavirin may not only increase the rate of sustained virologic response compared with standard therapy but may allow a reduction of treatment duration. The viral RNA-polymerase of HCV has a high error rate. As a result, different HCV variants are continuously produced during replication. HCV does not integrate into host DNA. Therefore, every infected cell has the potential of producing multiple drug-resistant mutants over time. The emergence of resistance might possibly limit the success of HCV-specific antiviral compounds, and is therefore a highly clinically relevant issue.

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Selection of drug-resistant HCV strains may occur when viral replication continues while drugs are taken. Not all viral strains have the same ability to replicate. The inherent ability of a virus to replicate is termed viral fitness. The fitness of variants present in a patient may lead to differences in viral response. The fitness of viral variants was investigated in patients who had chronic hepatitis C after stopping monotherapy with telaprevir [43]. In this study, high-level resistant strains were replaced more rapidly than low-level resistant strains by wild-type virus. The results indicate that telaprevir resistance is inversely correlated with viral fitness to replicate [43]. Innate and adaptive immune responses play an important role in the control of viral diseases. In most patients infected with the hepatitis C virus, the innate and adaptive immune responses are too weak for complete elimination of HCV-infected cells, which is the prerequisite for a cure from HCV. The consequence of an impaired immune response is the persistence of infected cells and the development of chronic hepatitis C. Several mechanisms have been identified by which the hepatitis C virus attenuates innate and adaptive immune responses. It can be anticipated that inhibition of HCV replication by new HCV-specific antiviral compounds leads to a reconstitution of the innate and adaptive immune response against HCV during antiviral therapy. A potential reconstitution of the immune response against HCV could enhance the infected cell loss during treatment, and thereby improve the antiviral efficiency of HCV-specific compounds [50]. Primary or secondary resistance to HCV specific inhibitors is potentially a serious problem. Combination of (non–cross-resistant) antiviral compounds inhibiting different viral and cellular mechanisms may reduce the problem of resistance. Therefore, future research should not only focus on the development of new compounds but on optimal drug combinations. A recent study on cross-resistance of telaprevir and interferon-alfa indicates that telaprevir-resistant strains are sensitive to interferon-alfa [45]. This study supports the concept that combination therapy may reduce the development and selection of resistant strains. The development of agents in different classes may allow construction of antiviral combinations that enhance the effectiveness of antiviral treatment, reduce the duration of treatment, and, eventually, may even avoid the use of interferon-alfa. References [1] Lohmann V, Korner F, Koch J, et al. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 1999;285(5424):110–3. [2] Wakita T, Pietschmann T, Kato T, et al. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 2005;11(7):791–6. [3] Lindenbach BD, Evans MJ, Syder AJ, et al. Complete replication of hepatitis C virus in cell culture. Science 2005;309(5734):623–6. [4] Irshad M, Dhar I. Hepatitis C virus core protein: an update on its molecular biology, cellular functions and clinical implications. Med Princ Pract 2006;15(6):405–16.

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[5] Moradpour D, Penin F, Rice CM. Replication of hepatitis C virus. Nat Rev Microbiol 2007; 5(6):453–63. [6] Evans MJ, von Hahn T, Tscherne DM, et al. Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry. Nature 2007;446(7137):801–5. [7] Barth H, Liang TJ, Baumert TF. Hepatitis C virus entry: molecular biology and clinical implications. Hepatology 2006;44(3):527–35. [8] Diedrich G. How does hepatitis C virus enter cells? FEBS J 2006;273(17):3871–85. [9] Lin C, Lindenbach BD, Pragai BM, et al. Processing in the hepatitis C virus E2-NS2 region: identification of p7 and two distinct E2-specific products with different C termini. J Virol 1994;68(8):5063–73. [10] Carrere-Kremer S, Montpellier-Pala C, Cocquerel L, et al. Subcellular localization and topology of the p7 polypeptide of hepatitis C virus. J Virol 2002;76(8):3720–30. [11] Clarke D, Griffin S, Beales L, et al. Evidence for the formation of a heptameric ion channel complex by the hepatitis C virus p7 protein in vitro. J Biol Chem 2006;281(48): 37057–68. [12] Griffin SD, Beales LP, Clarke DS, et al. The p7 protein of hepatitis C virus forms an ion channel that is blocked by the antiviral drug, amantadine. FEBS Lett 2003;535(1–3):34–8. [13] Steinmann E, Whitfield T, Kallis S, et al. Antiviral effects of amantadine and iminosugar derivatives against hepatitis C virus. Hepatology 2007;46(2):330–8. [14] Meylan E, Curran J, Hofmann K, et al. Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus. Nature 2005;437(7062):1167–72. [15] Li K, Foy E, Ferreon JC, et al. Immune evasion by hepatitis C virus NS3/4A proteasemediated cleavage of the Toll-like receptor 3 adaptor protein TRIF. Proc Natl Acad Sci U S A 2005;102(8):2992–7. [16] Elazar M, Liu P, Rice CM, et al. An N-terminal amphipathic helix in hepatitis C virus (HCV) NS4B mediates membrane association, correct localization of replication complex proteins, and HCV RNA replication. J Virol 2004;78(20):11393–400. [17] Tellinghuisen TL, Marcotrigiano J, Rice CM. Structure of the zinc-binding domain of an essential component of the hepatitis C virus replicase. Nature 2005;435(7040):374–9. [18] Lesburg CA, Cable MB, Ferrari E, et al. Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site. Nat Struct Biol 1999;6(10):937–43. [19] Soler M, McHutchison JG, Kwoh TJ, et al. Virological effects of ISIS 14803, an antisense oligonucleotide inhibitor of hepatitis C virus (HCV) internal ribosome entry site (IRES), on HCV IRES in chronic hepatitis C patients and examination of the potential role of primary and secondary HCV resistance in the outcome of treatment. Antivir Ther 2004; 9(6):953–68. [20] VGX Pharmaceuticals. VGX-410C. Available at: http://www.viralgenomix.com/VGX410. html. Accessed December 13, 2007. [21] Berg T, von Wagner M, Nasser S, et al. Extended treatment duration for hepatitis C virus type 1: comparing 48 versus 72 weeks of peginterferon-alfa-2a plus ribavirin. Gastroenterology 2006;130(4):1086–97. [22] Sanchez-Tapias JM, Diago M, Escartin P, et al. Peginterferon-alfa2a plus ribavirin for 48 versus 72 weeks in patients with detectable hepatitis C virus RNA at week 4 of treatment. Gastroenterology 2006;131(2):451–60. [23] Zeuzem S, Buti M, Ferenci P, et al. Efficacy of 24 weeks treatment with peginterferon alfa-2b plus ribavirin in patients with chronic hepatitis C infected with genotype 1 and low pretreatment viremia. J Hepatol 2006;44(1):97–103. [24] von Wagner M, Huber M, Berg T, et al. Peginterferon-alpha-2a (40 KD) and ribavirin for 16 or 24 weeks in patients with genotype 2 or 3 chronic hepatitis C. Gastroenterology 2005;129(2):522–7. [25] Mangia A, Santoro R, Minerva N, et al. Peginterferon alfa-2b and ribavirin for 12 vs. 24 weeks in HCV genotype 2 or 3. N Engl J Med 2005;352(25):2609–17.

NOVEL HEPATITIS C DRUGS IN CURRENT TRIALS

553

[26] Shiffman ML, Suter F, Bacon BR, et al. Peginterferon alfa-2a and ribavirin for 16 or 24 weeks in HCV genotype 2 or 3. N Engl J Med 2007;357(2):124–34. [27] Zeuzem S, Hultcrantz R, Bourliere M, et al. Peginterferon alfa-2b plus ribavirin for treatment of chronic hepatitis C in previously untreated patients infected with HCV genotypes 2 or 3. J Hepatol 2004;40(6):993–9. [28] Jacobson IM, Brown RS Jr, Freilich B, et al. Peginterferon alfa-2b and weight-based or flatdose ribavirin in chronic hepatitis C patients: a randomized trial. Hepatology 2007;46(4): 971–81. [29] Zeuzem S, Benhamou Y, Bain V, et al. Antiviral response at week 12 following completion of treatment with albinterferon alfa-2b plus ribavirin in genotype 1, IFN-naive, chronic hepatitis C patients. J Hepatol 2007;46:293A. [30] Novozhenov V, Zakharova N, Vinogradova E, et al. Phase 2 study of omega interferon alone or in combination with ribavirin in subjects with chronic hepatitis C genotype 1 infection. J Hepatol 2007;46:8A. [31] Octoplus. Locteron. Available at: http://www.octoplus.nl/index.cfm/site/OctoPlus/pageid/ AC35865A-91AC-590B-FFB3F45418E98C27/index.cfm. Accessed July 26, 2007. [32] Maxygen. 2006 EASL presentations. Available at: http://www.maxygen.com/pdf/EASL_ Roche_2006.pdf. Accessed November 7, 2007. [33] Nautilus Biotech. Press release. Available at: http://www.nautilusbiotech.com/news_ 100507.html. Accessed April 2, 2007. [34] Gish RG, Arora S, Rajender RK, et al. Virological response and safety outcomes in therapy-naive patients treated for chronic hepatitis C with taribavirin or ribavirin in combination with pegylated interferon alfa-2a: a randomized, phase 2 study. J Hepatol 2007; 47(1):51–9. [35] Benhamou Y, Pockros P, Rodriguez-Torres M, et al. The safety and efficacy of viramidine plus PegIFN alfa-2b versus ribavirin plus PegIFN alfa-2b in therapy-naive patients infected with HCV: phase 3 results (VISER1). J Hepatol 2006;44:273A. [36] Marcellin P, Lurie Y, Rodriguez-Torres M, et al. The safety and efficacy of taribavirin plus pegylated interferon alfa-2a versus ribavirin plus pegylated interferon alfa-2a in therapynaive patients infected with HCV: phase 3 results. J Hepatol 2007;46:7A. [37] Lamarre D, Anderson PC, Bailey M, et al. An NS3 protease inhibitor with antiviral effects in humans infected with hepatitis C virus. Nature 2003;426(6963):186–9. [38] Hinrichsen H, Benhamou Y, Wedemeyer H, et al. Short-term antiviral efficacy of BILN 2061, a hepatitis C virus serine protease inhibitor, in hepatitis C genotype 1 patients. Gastroenterology 2004;127(5):1347–55. [39] Reiser M, Hinrichsen H, Benhamou Y, et al. Antiviral efficacy of NS3-serine protease inhibitor BILN-2061 in patients with chronic genotype 2 and 3 hepatitis C. Hepatology 2005; 41(4):832–5. [40] Lu L, Pilot-Matias TJ, Stewart KD, et al. Mutations conferring resistance to a potent hepatitis C virus serine protease inhibitor in vitro. Antimicrob Agents Chemother 2004;48(6): 2260–6. [41] Perni RB, Almquist SJ, Byrn RA, et al. Preclinical profile of VX-950, a potent, selective, and orally bioavailable inhibitor of hepatitis C virus NS3-4A serine protease. Antimicrob Agents Chemother 2006;50(3):899–909. [42] Reesink HW, Zeuzem S, Weegink CJ, et al. Rapid decline of viral RNA in hepatitis C patients treated with VX-950: a phase Ib, placebo-controlled, randomized study. Gastroenterology 2006;131(4):997–1002. [43] Sarrazin C, Kieffer TL, Bartels D, et al. Dynamic hepatitis C virus genotypic and phenotypic changes in patients treated with the protease inhibitor telaprevir. Gastroenterology 2007; 132(5):1767–77. [44] Forestier N, Reesink HW, Weegink CJ, et al. Antiviral activity of telaprevir (VX950) and peginterferon alfa-2a in patients with hepatitis C. Hepatology 2007;46(3): 640–8.

554

KRONENBERGER

et al

[45] Kieffer TL, Sarrazin C, Miller JS, et al. Telaprevir and pegylated interferon-alpha-2a inhibit wild-type and resistant genotype 1 hepatitis C virus replication in patients. Hepatology 2007; 46:631–9. [46] Lawitz E, Rodriguez-Torres M, Muir A, et al. Tolerability and antiviral effects in a 28-day study of the hepatitis C protease inhibitor telaprevir (VX-950), in combination with peginterferon-alpha-2a and ribavirin. J Clin Virol 2006;36:239A. [47] Lawitz EJ, Rodriguez-Torres M, Muir A, et al. 28 Days of the hepatitis C protease inhibitor VX-950, in combination with PEG-interferon-alfa-2a and ribavirin, is well-tolerated and demonstrates robust antiviral effects. Gastroenterology 2006;131(3):950A. [48] Vertex Pharmaceuticals. Reports 2006 financial results. Available at: http://www.vpharm. com/Pressreleases2007/pr020107.html. Accessed October 26, 2007 [49] Malcolm BA, Liu R, Lahser F, et al. SCH 503034, a mechanism-based inhibitor of hepatitis C virus NS3 protease, suppresses polyprotein maturation and enhances the antiviral activity of alpha interferon in replicon cells. Antimicrob Agents Chemother 2006;50(3):1013–20. [50] Sarrazin C, Rouzier R, Wagner F, et al. SCH 503034, a novel hepatitis C virus protease inhibitor, plus pegylated interferon alpha-2b for genotype 1 nonresponders. Gastroenterology 2007;132(4):1270–8. [51] Clinical Trial gov. SCH 503034 plus peg-intron, with and without added ribavirin, in patients with chronic hepatitis C, genotype 1, who did not respond to previous treatment with peginterferon alfa plus ribavirin (Study P03659). Available at: http://clinicaltrials.gov/show/ NCT00160251. Accessed July 19, 2007. [52] Kempf DJ, Klein C, Chen HJ, et al. Pharmacokinetic enhancement of the hepatitis C virus protease inhibitors VX-950 and SCH 503034 by co-dosing with ritonavir. Antivir Chem Chemother 2007;18(3):163–7. [53] Seiwert SD, Andrews SW, Tan H, et al. 750 Preclinical characteristics of ITMN-B, an orally active inhibitor of the HCV NS3/4A protease nominated for preclinical development. J Hepatol 2006;44:278A. [54] InterMune. InterMune submits CTA amendment for ITMN-191 and provides update on clinical development program. Available at: http://www.corporate-ir.net/ireye/ir_site.zhtml? ticker¼itmn&script¼410&layout¼-6&item_id¼1030100. Accessed July 24, 2007. [55] Hepatitis C program. Available at: http://www.achillion.com/hepatitisc.php. Accessed August 1, 2007. [56] Achillion Pharmaceuticals. NS4A data overview. Available at: http://www.cheminus.com/ main.aspx?pn¼Ns4aData&fl¼l. Accessed August 1, 2007. [57] Pierra C, Amador A, Benzaria S, et al. Synthesis and pharmacokinetics of valopicitabine (NM283), an efficient prodrug of the potent anti-HCV agent 20 -C-methylcytidine. J Med Chem 2006;49(22):6614–20. [58] Zhou XJ, Afdhal N, Godofsky E, et al. Pharmacokinetics and pharmacodynamics of valopicitabine (NM283), a new nucleoside HCV polymerase inhibitor: results from a phase I/II dose-escalation trial in patients with HCV-1 infection. J Hepatol 2005;42:229A. [59] Lawitz E, Nguyen T, Younes Z, et al. Clearance of HCV RNA with valopicitabine (NM283) plus peg-interferon in treatment-naive patients with HCV-1 infection: results at 24 and 48 weeks. J Hepatol 2007;46:9A. [60] Pockros P, O’Brien C, Godofsky E, et al. Valopicitabine (NM283), alone or with peginterferon compared to peg-interferon/ribavirin retreatment in hepatitis C patients with prior non-response to pegIFN/RBV: week 24 results. Gastroenterology 2006;130:748A. [61] Afdhal N, O’Brien C, Godofsky E, et al. Valopicitabine (NM283), alone or with peginterferon compared to peg interferon/ribavirin retreatment in patients with HCV-1 infection and prior non-response to peg interferon/ribavirin: one-year results. J Hepatol 2007;46:9A. [62] Migliaccio G, Tomassini JE, Carroll SS, et al. Characterization of resistance to non-obligate chain-terminating ribonucleoside analogs that inhibit hepatitis C virus replication in vitro. J Biol Chem 2003;278(49):49164–70.

NOVEL HEPATITIS C DRUGS IN CURRENT TRIALS

555

[63] Ludmerer SW, Graham DJ, Boots E, et al. Replication fitness and NS5B drug sensitivity of diverse hepatitis C virus isolates characterized by using a transient replication assay. Antimicrob Agents Chemother 2005;49(5):2059–69. [64] Coelmont L, Paeshuyse J, Windisch MP, et al. Ribavirin antagonizes the in vitro antihepatitis C virus activity of 20 -C-methylcytidine, the active component of valopicitabine. Antimicrob Agents Chemother 2006;50(10):3444–6. [65] Klumpp K, Leveque V, Le Pogam S, et al. The novel nucleoside analog R1479 (40 -azidocytidine) is a potent inhibitor of NS5B-dependent RNA synthesis and hepatitis C virus replication in cell culture. J Biol Chem 2006;281(7):3793–9. [66] Roberts S, Cooksley G, Dore G, et al. Results from a phase 1B, multiple dose study of R1626, a novel nucleoside analog targeting HCV polymerase in chronic HCV genotype 1 patients. Hepatology 2006;44:692A. [67] Le Pogam S, Jiang WR, Leveque V, et al. In vitro selected Con1 subgenomic replicons resistant to 20 -C-methyl-cytidine or to R1479 show lack of cross resistance. Virology 2006;351(2): 349–59. [68] Pharmasset. R7128, a prodrug of PSI-6130. Available at: http://www.pharmasset.com/ pipeline/psi-6130.asp. Accessed August 15, 2007. [69] Villano S, Raible D, Harper D, et al. Antiviral activity of the non-nucleoside polymerase inhibitor, HCV-796, in combination with pegylated interferon alfa-2b in treatment naive patients with chronic HCV. J Hepatol 2007;46:24A. [70] ViroPharma. Potential safety issue identified in ongoing phase 2 clinical study of HCV-796. Available at: http://phx.corporate-ir.net/phoenix.zhtml?c¼92320&p¼irol-researchNewsArticle& ID¼1039323&highlight¼. Accessed August 10, 2007. [71] GILEAD. Corporate overview. Available at: http://www.gilead.com/corporate_ overview. Accessed November 15, 2007. [72] Erhardt A, Wedemeyer H, Benhamou Y, et al. Safety, pharmacokinetics and antiviral effect of BILB 1941, a novel HCV RNA polymerase inhibitor, after 4 days oral treatment in patients with chronic hepatitis C. J Hepatol 2007;44:222. [73] Arrow. Arrow product pipeline. Available at: http://www.arrowt.co.uk/product-hcv.asp. Accessed October 12, 2007. [74] Flisiak R, Horban A, Kierkus J, et al. The cyclophilin inhibitor DEBIO-025 has a potent dual anti-HIV and anti-HCV activity in treatment-naı¨ ve HIV/HCV co-infected subjects. Hepatology 2006;44:609A. [75] Kaita K, Yoshida E, Kunimoto D, et al. PhII proof of concept study of celgosivir in combination with peginterferon alpha-2b and ribavirin in chronic hepatitis C genotype-1 nonresponder patients. J Hepatol 2007;46:S56–7. [76] Seiwert SD, Hong J, Lim SR, et al. Sequence variation of NS3/4A in HCV replicons exposed to ITMN-191 concentrations encompassing those likely to be achieved following clinical dosing. J Hepatol 2007;46:244A–5A.