Therapeutic Advances in Gastroenterology
`
`Review
`
`Hepatitis C virus replication and potential
`targets for direct-acting agents
`
`Jacqueline G. O’Leary and Gary L. Davis
`
`Ther Adv Gastroenterol
`(2010) 3(1) 43—53
`
`DOI: 10.1177/
`1756283X09353353
`! The Author(s), 2010.
`Reprints and permissions:
`http://www.sagepub.co.uk/
`journalsPermissions.nav
`
`Abstract: We finally stand at the brink of novel, oral, direct-acting antivirals for the treatment
`of hepatitis C virus (HCV) infection. Basic science research has lead to a greater understanding
`of the viral life cycle and identified numerous potential targets for therapy. Early compounds
`were plagued by inconsistent in vivo activity and side effects that led to discontinuation of
`investigational efforts. However, several agents have now progressed to phase 2 human
`studies and two protease inhibitors have completed enrolment for their phase 3 clinical trials
`and look promising. Thus, while it appears that protease inhibitors will likely be the next
`available drugs for the treatment of HCV infection, the quest for additional therapeutic agents
`will continue. The future of HCV therapy lies in multidrug cocktails of several agents targeted
`against a variety of targets. In the near future these agents will be added to the current standard
`therapy consisting of pegylated interferon and ribavirin; however, the ultimate and probably
`realistic goal will be to develop multidrug oral regiments to replace the need for interferon.
`
`Keywords: HCV replication, new treatment, polymerase inhibitors, protease inhibitors, therapy
`
`Introduction
`Hepatitis C infects 170 million people worldwide
`and 1.6% of
`the United States population
`[Armstrong et al. 2006; Davis et al. 2003; Alter
`et al. 1999; WHO, 1999]. After acute infection,
`55% to 85% of patients develop chronic disease.
`The natural history of chronic hepatitis C varies
`significantly because of host, viral and environ-
`mental factors. Chronic infection leads to cirrho-
`sis in approximately 20% of patients after 20 years
`of infection [Freeman et al. 2001]. Thereafter,
`other complications including hepatic decompen-
`sation (ascites, encephalopathy, variceal hemor-
`rhage, hepatorenal syndrome, or hepatic synthetic
`dysfunction) and hepatocellular carcinoma ensue
`at a rate of about 3% per year [Sangiovanni et al.
`2006; El-Serag, 2004; Serfaty et al. 1998;
`Fattovich et al. 1997]. Without liver transplanta-
`tion, decompensated cirrhosis leads to death in
`50—72% of patients after 5 years [Fattovich et al.
`2002]. As a result of the high prevalence of hepa-
`titis C virus (HCV) infection and resultant com-
`plications, HCV is the leading indication for liver
`transplantation in the United States and the world
`as a whole [Wasley and Alter, 2000].
`
`Chronic hepatitis C is the only chronic viral
`infection that can be cured with antiviral therapy.
`
`Unlike human immunodeficiency virus and
`hepatitis B, a sustained virologic response
`(SVR), defined as HCV-RNA undetectable by a
`sensitive amplification test 6 months after the
`completion of therapy, is equivalent to a cure in
`>99% of cases [Fried et al. 2002; Manns et al.
`2001]. Patients with compensated cirrhosis who
`achieve an SVR essentially eliminate their subse-
`quent risk of decompensation, may achieve his-
`tologic regression, and decrease their risk of
`hepatocellular carcinoma by two thirds [Bruno
`et al. 2007; Di Bisceglie et al. 2007; Camma
`et al. 2004].
`
`Correspondence to:
`Jacqueline G. O’Leary
`4th Floor Roberts,
`Hepatology-Transplantation,
`Baylor University Medical
`Center,
`3500 Gaston Ave, Dallas,
`TX 75246, USA
`Jacquelo@
`BaylorHealth.edu
`
`Gary L. Davis
`Department of Medicine,
`Baylor University Medical
`Center, Dallas,
`TX, USA
`
`The current standard of care for the treatment of
`HCV infection remains the combination of pegy-
`lated interferon and ribavirin [Fried et al. 2002;
`Manns et al. 2001]. This therapy eradicates HCV
`in 40—50% of genotype 1 non-cirrhotic patients
`and 70—80% of genotype 2 and 3 non-cirrhotic
`patients. The response to treatment is lower in
`obese,
`insulin-resistant, or African-American
`patients and in those with advanced hepatic
`fibrosis or high viral loads. Of greatest concern
`are patients with decompensated cirrhosis and
`immunosuppressed patients, such as liver trans-
`plant recipients, who are rarely able to tolerate
`Gilead 2004
`full doses of therapy.
`I-MAK v. Gilead
`IPR2018-00211
`
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`
`43
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`

`

`Therapeutic Advances in Gastroenterology 3 (1)
`
`Figure 1. A graphic depiction of the viral life cycle with the potential antiviral targets listed. NS, nonstructural
`proteins; ER, endoplasmic reticulum; IRES, internal ribosome entry side.
`
`Fortunately, we are entering a new era of HCV
`therapy. A greater understanding of the HCV
`replication machinery has led to a large list of
`potential targets for therapy (Figure 1). One
`such target is the nonstructural 3 (NS3) viral
`protease,
`an
`enzyme
`that
`is
`critical
`to
`post-translational processing of the viral polypro-
`tein. Drugs that effectively inhibit this enzyme are
`in the final stages of clinical trials, and it appears
`that the combination of a protease inhibitor with
`pegylated interferon and ribavirin will signifi-
`cantly improve the SVR rate, perhaps even with
`a shorter duration of treatment. In addition,
`these medications will allow us to retreat previous
`relapsers and nonresponders to pegylated inter-
`feron and ribavirin with some success. However,
`while such drugs will be a tremendous addition to
`our therapeutic armamentarium, it is important
`to recognize that they will not eradicate infection
`in all patients and barriers to using interferon or
`ribavirin in many patients will still be a problem.
`
`This article will review the most promising
`potential targets for new direct-acting antiviral
`agents (DAA). Drugs for some of these targets
`are well along in clinical development while
`others are only hypothetical and supported by
`in vitro studies (Table 1). It is important for the
`reader to understand that the high replication
`and nucleic acid substitution rate of HCV will
`
`likely lead to rapid emergence of viral drug resis-
`tance if a single one of these replicative steps is
`targeted. Thus, the future of HCV therapy lies in
`the combination of multiple agents against differ-
`ent targets such as receptor binding, cell entry,
`viral transcription, translation, polyprotein pro-
`cessing, particle assembly and export of virus
`progeny. These DAAs might also be combined
`with non-specific antiviral agents such as those
`that enhance the endogenous immune response
`to the virus, e.g.
`interferons or
`therapeutic
`vaccines, or neutralize extracellular virus, e.g.
`hyperimmune globulins. The goal, of course,
`is to seek combinations that will both increase
`efficacy and improve tolerability.
`
`Early inhibitors: receptor binding and
`cell entry
`Entry of HCV into the hepatocyte involves a
`series of sequential interactions with soluble and
`cell surface host factors, and remains incomple-
`tely understood. Plasma-derived HCV is com-
`plexed with low-density and very-low-density
`lipoproteins (LDL and VLDL), which probably
`facilitates the initial attraction and concentration
`of virus on the cell surface via interaction with
`the low-density lipoprotein receptor (LDL-R)
`[Andre et al. 2005]. The highly glycosylated
`viral
`envelope
`proteins
`E1
`and
`E2
`
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`

`

`Table 1. Direct-acting agents to treat hepatitis C in phase 2 and 3 clinical trials.
`
`Class
`
`Drug name
`
`Drug Company
`
`JG O’Leary and GL Davis
`
`Entry and RNA-Binding Inhibitors
`Neutralizing Antibodies
`Glycosylation Inhibitors
`
`IRES Inhibitors
`
`Protease Inhibitors
`NS2 Inhibitors
`NS3 Inhibitors
`
`NS4A Inhibitors
`RNA Transcription
`NS4B Inhibitors
`Helicase Inhibitors
`Cyclophilin A Inhibitors
`
`NS5B Polymerase Inhibitors
`
`NS5A Inhibitors
`Other
`TLR-9 Agonists
`NKT Cell Agonist
`Other
`
`Civacir
`Celgosivir
`*UT-231B
`*Heptazyme
`*ISIS-14803
`VGX-410C
`
`none
`Telaprevir
`Boceprevir
`ITMN-191
`TCM435
`MK-7009
`BMS-650032
`BMS-791325
`*ACH-806
`
`none
`none
`Debio-025
`NIM-811
`R7128
`*R1626
`*NM283
`*HCV-796
`VCH-759
`PF-868554
`IDX184
`GS 9190
`BMS790052
`
`*CPG 10101
`KRN7000
`Nitazoxanide
`
`NABI Pharmaceuticals
`Migenix, Inc.
`Unither Pharmaceuticals
`Ribozyme Pharmaceuticals, Inc.
`ISIS Pharmaceuticals
`VGX Pharmaceuticals
`
`Vertex Pharmaceuticals
`Schering-Plough Corporation
`Intermune, Inc.
`Tibotec Pharmaceuticals Limited
`Merck
`Bristol-Myers Squibb
`Bristol-Myers Squibb
`Achillion Pharmaceuticals Inc.
`
`DebioPharm Group
`Novartis
`Pharmasset, Inc. and Roche
`Roche
`Idenix Pharmaceuticals, Inc.
`ViroPharm, Inc.
`Vertex Pharmaceuticals
`Pfizer
`Idenix Pharmaceuticals, Inc.
`Gilead
`Bristol-Myers Squibb
`
`Pfizer
`Kyowa Hakko Kirin Company, Limited
`Romark Laboratories L.C.
`
`Data available at: www.clinicaltrials.gov and www.hcvdrugs.com. NS, nonstructural; IRES, internal ribosome entry site;
`TLR-9, toll-like receptor-9.
`*Future investigation of these compounds has been aborted.
`
`conformationally attach to glycosamioglycans at
`the hepatocyte cell surface and to the c-type lec-
`tins DC-SIGN (dendritic cell-specific intercellu-
`lar adhesion molecule-3-grabbing nonintegrin;
`CD209) and L-SIGN (DC-SIGNr;
`liver and
`lymph node specific; CD209L) on neighboring
`dendritic
`cells
`and
`liver
`sinusoidal
`cells
`[Cormier et al. 2004a]. E2 then sequentially
`attaches to the tetraspanin CD81 and scavenger
`receptor class B type 1 (SR-B1) to form a recep-
`tor complex that utilizes the tight junction clau-
`din
`proteins,
`particularly CLDN1,
`for
`internalization [Zeisel et al. 2007; Cormier et al.
`2004b; Pileri et al. 1998]. Occludin (OCLN), a
`tight junction protein, is also essential for cell
`entry but its exact role remains to be defined
`[Lanford et al. 2009; Ploss et al. 2009; Evans
`et al. 2007]. Interestingly, EWI-2wint, a small
`
`protein that is associated with CD81 in several
`cell
`lines and efficiently blocks HCV entry, is
`absent in hepatocytes and this probably explains
`the selective susceptibility of hepatocytes to HCV
`infection
`[Schuster
`and Baumert,
`2009;
`Rocha-Perugini et al. 2008].
`
`The role of the humoral immune response in con-
`trolling HCV infection is not clear. Monoclonal
`anti-CD81 antibodies have been utilized to effec-
`tively block HCV infection of mice with huma-
`nized livers [Lanford et al. 2009]. However, they
`do not affect HCV infection after it has been
`established. Monoclonal and polyclonal antibo-
`dies with HCV envelope neutralizing capacity
`have been tested in humans at the time of liver
`transplant but have been ineffective in preventing
`reinfection of
`the donor liver; however,
`this
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`

`Therapeutic Advances in Gastroenterology 3 (1)
`
`remains a potential perioperative strategy during
`transplantation [Davis et al. 2005]. The currently
`available antibodies’
`lack of efficacy may be
`attributed to the heterogeneity of
`the virus,
`its association with apolipoproteins, or other
`factors.
`
`Another approach to blocking cellular infection is
`to inhibit binding and processing of HCV via the
`cell surface receptor proteins involved in cell
`entry. Glycosylation inhibitors may alter
`the
`structure of cell
`surface glycosaminoglycans
`thereby decreasing or eliminating viral concentra-
`tion at the cell surface [Pawlotsky et al. 2007].
`MX-3256 (Celgosivir; Migenix Inc.) is an oral
`alpha-glucosidase I inhibitor that acts through
`host-directed glycosylation to prevent proper
`folding of the HCV envelope. In preclinical stu-
`dies, celgosivir demonstrated strong synergy with
`pegylated interferon plus ribavirin, but a phase
`IIa monotherapy study did not show any reduc-
`tion in HCV-RNA [Kaita et al. 2007; Yoshida
`et al. 2006]. Unfortunately, a 12-week study of
`another
`glycosylation
`inhibitor, UT-231B
`(Unither Pharmaceuticals),
`in HCV-infected
`patients who previously failed interferon-based
`therapy also failed to reduce virus
`levels.
`Despite these early setbacks, this remains an
`interesting target for future therapies.
`
`In addition, the lectin cyanovirin-N interacts with
`HCV envelope glycoproteins and blocks the asso-
`ciation between E2 and CD81 [Helle et al. 2006].
`Therefore, targeting the cell surface and/or viral
`glycans could be a promising approach to anti-
`viral therapy.
`
`Virus uncoating, HCV-RNA release and
`attachment to the endoplasmic reticulum
`The HCV and receptor complex fuses with the
`hepatocyte
`cell membrane
`and undergoes
`clatharin-mediated endocytosis. Acidification of
`the vesicle leads to fusion of the viral and vesicle
`membranes resulting in uncoating and release of
`the viral RNA into the cytoplasm. The presence
`of viral RNA in the cytosol has pathogenic and
`potential therapeutic implications.
`
`The cell recognizes the presence of single-strand
`RNA (ssRNA) in the cytosol as abnormal and
`initiates a pathogen-associated molecular pattern
`(PAMP)-associated response via toll-like recep-
`tors [Sen, 2001]. This process involves activation
`of
`retinoic
`acid inducible gene-1 (RIG-1)
`and Toll-IL-1 receptor domain containing
`
`that
`adaptor-inducing interferon-beta (TRIF)
`under normal circumstances leads to cellular
`production of
`type 1 interferons and tumor
`necrosis
`factor-related
`apoptosis-inducing
`ligand (TRAIL) mediated apoptosis [Saito and
`Gale, 2008]. However, HCV is capable of down-
`regulating this
`step of
`the innate immune
`response. The HCV NS3/4 protease cleaves and
`inactivates the RIG-1 adaptor protein IFN-beta
`promoter stimulator-1 (IPS-1) and TRIF itself
`thereby blocking downstream activation of the
`interferon regulatory genes [Zhu et al. 2007;
`Foy et al. 2005; Li et al. 2005]. Therefore,
`NS3/4 inhibition (see later), in addition to its
`direct effect on viral polyprotein processing, has
`the potential to restore the RIG-1 and TRIF
`pathways of innate immunity.
`
`The cytosolic viral ssRNA is also a vulnerable
`potential target for therapeutic oligonucleotides
`such as antisense nucleotides (small non-coding
`strands of RNA that hybridize and inactivate
`mRNA) or ribozymes (RNA molecules that cat-
`alyze cleavage of a target RNA). However, these
`agents require an absolutely conserved target in
`an otherwise very heterogeneous virus in order to
`have their effect. The internal ribosomal entry
`site (IRES) at the 5’ end of the viral RNA is
`that highly conserved target. IRES is the landing
`pad that directs the positive strand HCV-RNA to
`the endoplasmic reticulum (ER) for protein
`translation. Thus,
`inhibition of attachment of
`IRES to both cellular and viral proteins by oligo-
`nucleotides could effectively inhibit HCV repli-
`cation. While several class-specific problems with
`oligonucleotides such as drug delivery, instability,
`proinflammatory effects, and other unintended
`‘off-target’ side effects have been partially over-
`come by modifications of the compounds, all
`candidate drugs to date have been plagued by
`safety concerns. Development of Heptazyme
`(Ribozyme Pharmaceuticals, Inc.), a ribozyme
`that cleaved IRES, was stopped secondary to
`toxicity.
`ISIS-14803 (ISIS Pharmaceuticals),
`a 20 base pair antisense oligodeoxynucleotide,
`led to a 1.2 to 1.7 log decline in HCV RNA
`when given as monotherapy three times a week
`for 4 weeks in three out of 28 patients; however,
`asymptomatic liver function test abnormalities
`also
`occurred
`in
`five
`treated
`patients
`[McHutchison et al. 2006]. VGX-410C (VGX
`Pharmaceuticals) was a small molecule that also
`targeted HCV IRES binding. It appeared to be
`safe in phase 2 clinical trials, but was not effec-
`tive. Despite these early setbacks, this approach
`
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`

`

`JG O’Leary and GL Davis
`
`intriguing
`remains
`exploration.
`
`and warrants
`
`further
`
`Polyprotein translation and protein processing
`Once the viral RNA attaches to the ER, a single
`large polyprotein is translated. This polyprotein
`is then co- and post-translationally processed by
`host and viral proteases into at least 11 viral pro-
`teins. Two host cellular pepsidases are required
`for cleaving HCV structural proteins. These are
`not targets for HCV therapy as they are essential
`for cellular function. NS2 complexes with NS3
`and zinc to form a cystine protease. This complex
`autocatalytically cleaves NS2 from NS3, and is
`then degraded by the proteasome. To date, no
`inhibitors of the NS2 protease have entered clin-
`ical development.
`
`In contrast, the NS3 protease has been the pri-
`mary focus of recent drug development. NS3
`complexes with NS4A and acts as a serine pro-
`tease to cleave the polyprotein at the NS3-NS4A,
`NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B
`sites. The NS3-NS4A complex’s catalytic triad
`lies adjacent to a shallow substrate binding area
`that has made design of potent inhibitors challen-
`ging. It is this active site that is the target for
`drugs
`that are
`currently in clinical
`trials.
`Telaprevir
`(VX-950; Vertex Pharmaceuticals)
`and boceprevir (SCH503034; Schering-Plough
`Corporation) are both in phase 3 clinical trials.
`While these agents are potent inhibitors of HCV
`replication, monotherapy leads to rapid selection
`of drug-resistant strains of the virus, typically
`within days [Kieffer et al. 2007]. Therefore, effec-
`tive treatment, where each drug is dosed three
`times per day, requires combination with other
`agents which means that all current studies
`include pegylated interferon and ribavirin. This
`significantly reduces the likelihood of drug resis-
`tance, but the requirement for frequent dosing
`with these first-generation agents may impede
`compliance and increase the chance of resistance
`outside the setting of clinical trials.
`
`Telaprevir administered during the first 12 weeks
`of a 24 week course of pegylated interferon
`and
`ribavirin
`in
`previously
`untreated
`genotype-1-infected patients resulted in an SVR
`in 61% compared with 41% in those treated with
`pegylated interferon and ribavirin alone for 48
`weeks (standard treatment) [McHutchison et al.
`2008]. A similar trial in Europe achieved an SVR
`in 68% with the 24-week triple-drug regimen
`compared to 48% with standard treatment
`
`[Dusheiko et al. 2008]. A lower dose or elimina-
`tion of ribavirin resulted in a lower chance of
`response. Thus, it appears that, at least for the
`time being, both pegylated interferon and riba-
`virin continue to be essential components of
`treatment. Telaprevir in combination with pegy-
`lated interferon and ribavirin is also effective in
`retreating genotype 1 patients who had relapsed
`or failed to respond to a prior course of pegylated
`interferon
`and
`ribavirin. The
`previously
`described 24 week regimen achieved an SVR in
`69% of prior relapsers and 39% of previous non-
`responders [Manns et al. 2009b]. Extension of
`the total length of treatment to 48 weeks did
`not appear to improve the response to retreat-
`ment. Efficacy and side effects are both increased
`with triple drug therapy. Rash is most common,
`but rarely leads to drug discontinuation. Pruritus,
`nausea, diarrhea and rectal discomfort are also
`more common.
`
`Twenty-eight weeks of therapy with Boceprevir,
`given orally three times a day, in combination
`with peginterferon and ribavirin, led to an SVR
`rate of 55% in previously untreated genotype
`1-infected patients [Kwo et al. 2008]. To address
`concerns about resistance, a second group of
`patients was treated with a 4-week lead-in phase
`of pegylated interferon and ribavirin to reduce
`HCV-RNA levels before the introduction of
`boceprevir; however,
`the SVR rate remained
`unchanged indicating that this is unnecessary.
`Unlike telaprevir, boceprevir
`treated patients
`may benefit from longer therapy. Patients treated
`with 4 weeks of pegylated interferon and ribavirin
`were then treated with either 44 weeks of pegy-
`lated interferon and ribavirin (38% SVR), 24
`weeks of boceprevir, pegylated interferon and
`ribavirin (56% SVR), or 44 weeks of triple ther-
`apy (75% SVR) [Kwo et al. 2009]. Side effects
`with boceprevir include headache, gastrointesti-
`nal complaints and anemia that may limit the
`ability to maintain the ribavirin dose.
`
`ITMN-191 (Intermune, Inc.), another protease
`inhibitor, has been used in combination with
`pegylated interferon and ribavirin for 14 days to
`achieve an undetectable HCV-RNA in 71% of
`treated patients [Kamal and Nasser, 2008].
`ITMN-191 is active against HCV strains that
`are resistant to telaprevir and boceprevir. This
`drug is being studied in combination with pegy-
`lated interferon and ribavirin, as well as in com-
`bination with a polymerase inhibitor (R7128;
`Pharmasset,
`Inc. and Roche) without either
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`Therapeutic Advances in Gastroenterology 3 (1)
`
`interferon or ribavirin. The latter study is the first
`proof-of-concept study of an interferon-free reg-
`imen in humans. After 14 days of double therapy,
`virus levels had declined by 4.8 to 5.2 logs and
`63%—71% of patients had no detectable virus in
`serum [Gane et al. 2009].
`
`susceptible to resistance, particularly if there is
`not strict adherence to the dosing regimen.
`Therefore, the future of HCV therapy lies with
`improved pharmacodynamics and in combina-
`tions of agents targeting different sites and
`mechanisms of the viral life cycle.
`
`TCM435 (Tibotec Pharmaceuticals Limited and
`Medivir) is a once daily oral NS3 protease inhib-
`itor. A 4.3 to 5.5 log drop (depending on the
`dose) in viral load was achieved after 28 days
`when TCM435 was combined with pegylated
`interferon and ribavirin in genotype 1 HCV-
`infected patients who were previous nonrespon-
`ders or relapsers to interferon based therapy
`[Marcellin et al. 2009]. Three more clinical
`trials utilizing TCM435 with pegylated interferon
`and
`ribavirin
`have
`been
`registered
`at
`ClinicalTrials.gov, two in genotype 1 and one in
`other genotypes.
`
`testing,
`Although in the preclinical phase of
`MK-7009 (Merck) is another NS3 protease
`inhibitor with impressive potency. This drug
`caused a drop of over 5 log10 in HCV RNA in
`5 days in chimpanzees [Lanford et al. 2009].
`When combined with pegylated interferon and
`ribavirin 69 to 82% achieved a rapid virologic
`response compared with 6% who were treated
`with pegylated interferon and ribavirin [Manns
`et al. 2009a]. Like MK-7009, there are many
`other protease inhibitors in various stages of clin-
`ical trials.
`
`Another potential target at the translation step of
`replication is NS4A. NS4A complexes with NS3
`to stabilize the protease functions and anchor the
`protein complex to the endoplasmic reticulum.
`ACH-806 (Achillion Pharmaceuticals Inc.), a
`selective NS4A binder, in vitro caused synergistic
`inhibition of HCV viral replication when used in
`combination with NS3 protease inhibitors such
`as telaprevir [Wyles et al. 2008].
`
`The addition of a protease inhibitor to pegylated
`interferon and ribavirin remains the most prom-
`ising next step in the near future to improve SVR
`rates with HCV therapy. These drugs are potent
`HCV polyprotein processing inhibitors, may
`restore host innate immunity, may improve the
`sensitivity to interferon and are orally bioavail-
`able. These drugs are not without their limita-
`tions, however. They are genotype and probably
`subtype specific to various degrees, have their
`own unique side effects, and will likely remain
`
`HCV-RNA transcription
`Viral transcription occurs in a replicase complex
`built upon a membranous web within the hepa-
`tocyte that is comprised of host and viral ele-
`ments, including the viral polymerase. NS4B is
`essential to construction of the membranous web
`and has important RNA-binding activity facilitat-
`ing attachment of the positive strand viral RNA
`[Egger et al. 2002]. Cyclophilin A recruits NS5B,
`the RNA-dependent RNA polymerase, to the
`viral replication complex. After gathering these
`essential elements at the replication complex,
`the process of strand replication is directed by
`the viral polymerase and generates both the neg-
`ative strand template and positive strand progeny
`to incorporate into new virus particles. The
`newly transcribed positive strand is unwound
`from the template by the viral helicase and this
`single positive sense strand is now available for
`translation, transcription or packaging in new
`virions.
`
`The HCV RNA-dependent RNA polymerase can
`be inhibited by targeting the RNA-binding site or
`one of the other four nonnucleoside allosteric
`sites. Several agents are now in various stages of
`clinical development. R7128 is an active site
`NS5B inhibitor that, in combination with pegy-
`lated interferon and ribavirin, led to an 85% loss
`of detectable virus after just 4 weeks (rapid viro-
`logic response, RVR)
`in naı¨ve genotype 1
`patients. Only 10% of controls receiving pegy-
`lated interferon and ribavirin achieved a RVR
`[Lalezari et al. 2008]. R1626 (Roche), another
`active site NS5B inhibitor, given orally twice a
`day in combination with pegylated interferon
`and ribavirin led to a 74% RVR as compared to
`a 5% RVR in patients treated with pegylated
`interferon and ribavirin [Pockros et al. 2008].
`Unfortunately, R1626 was
`associated with
`dose-limiting
`neutropenia. NM283
`(Idenix
`Pharmaceuticals, Inc.), a third active site inhibi-
`tor of NS5B, was aborted secondary to gastroin-
`testinal side effects.
`
`Several new drugs have also been developed
`against the HCV polymerase’s allosteric sites.
`HCV-796 (ViroPharma,
`Inc.) had entered
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`JG O’Leary and GL Davis
`
`phase 2 clinical trials in combination with pegy-
`lated interferon and ribavirin; however, further
`study of this drug was discontinued secondary
`to elevated liver
`function tests
`in treated
`patients [Evans et al. 2007]. VCH-759 (Vertex
`Pharmaceuticals), another HCV allosteric inhib-
`itor of the NS5B polymerase, was used in phase 1
`clinical trials as an oral agent dosed three times
`per day [Cooper et al. 2009]. Although drug
`resistance was seen, a 2.5 log10 drop in HCV
`viral
`load was documented after 10 days of
`monotherapy with the highest dose. Pfizer’s com-
`pound, PF-00868554,
`in monotherapy at the
`highest dose resulted in a 1.95 log drop in viral
`load in HCV-infected patients [Hammond et al.
`2008]. Therefore it has progressed to phase 2
`clinical trials.
`
`Polymerase inhibitors represent the second most
`attractive target for HCV therapy after protease
`inhibitors. In addition to the agents described
`above, numerous others are currently in early
`development. Perhaps more than with the pro-
`tease inhibitors, this class of drug has been pla-
`gued with unacceptable side effects that have led
`to discontinuation of investigation of several oth-
`erwise promising compounds. The predominant
`unacceptable side effects have been nausea,
`vomiting and diarrhea, but
`like the protease
`inhibitor side effects, these may be unique to
`each agent rather than class effects. Although
`the nucleoside inhibitors may be genotype spe-
`cific, they may have fewer problems with resis-
`tance than drugs against other
`targets. The
`non-nucleoside
`inhibitors have
`encountered
`rapid emergence of resistance, which may limit
`their usefulness unless they are part of a multi-
`drug cocktail.
`
`There are several potential targets at the step of
`viral transcription other than direct polymerase
`inhibitors. The NS4B, critical for both construc-
`tion of the replicase complex and RNA-binding,
`is a potential target for therapy that has yet to be
`exploited [Lanford et al. 2009]. Cyclophilin inhi-
`bitors, such as Debio-025 (Debiopharm Group)
`and NIM-811 (Novartis), inhibit RNA polymer-
`ase incorporation in the replicase complex.
`Debio-025 is a once-a-day oral agent that has
`already been utilized alone and in combination
`with pegylated interferon and ribavirin in HCV-
`infected patients [Flisiak et al. 2008]. When com-
`bined with pegylated interferon and ribavirin, it
`resulted in a 4.75-log drop in serum HCV RNA
`levels in 29 days, compared with a 2.49-log
`
`decline in HCV RNA levels with pegylated inter-
`feron alone and a 2.2-log decline in HCV RNA
`levels with DEBIO-025 alone. NS5A is a multi-
`function protein that is essential to genome rep-
`lication, particle assembly and the host response
`to the virus [Best et al. 2005]. Its precise role in
`replication is not entirely understood, but
`because it is a multifunctional protein it is an
`extremely attractive target for therapeutic inter-
`vention. Two novel NS5A inhibitors have been
`reported [Lanford et al. 2009]. BMS790052
`(Bristol-Myers Squibb) resulted in a 3.6 log
`decline in HCV RNA following a single dose.
`Like the protease inhibitors, resistance to NS5A
`inhibitors has already been reported. Finally, the
`viral helicase is a potential target. Helicase inhi-
`bitors must be specific for the viral helicase,
`avoiding inhibition of host cellular helicases
`[Dubuisson, 2007; Myong et al. 2007]. No
`HCV helicase inhibitors are currently in clinical
`trials, but preclinical studies of a herpes simplex
`virus helicase inhibitor have shown promise
`[Jankowsky and Fairman, 2007; Kwong et al.
`2005].
`
`Viral assembly and export
`Viral particle formation is initiated by the inter-
`action of the core protein with genomic RNA in
`the endoplasmic reticulum [Mizuno et al. 1995;
`Tanaka et al. 2000]. Viral particle assembly
`requires N-glycosylation of envelope proteins
`for proper envelope folding. This process of enve-
`lope folding and configuration is essential for
`assembly of new virions, virus export, antigeni-
`city
`and receptor binding
`for
`reinfection.
`Therefore, agents that alter envelope glycosyla-
`tion may interfere with numerous steps in the
`viral life cycle (see MX-3256 under ‘Early inhi-
`bitors: receptor binding and cell entry’).
`
`Other
`Interferons have been the cornerstone of hepatitis
`C treatment
`for more than two decades.
`Although interferons stimulate the host innate
`immune response and initiate numerous antiviral
`mechanisms within the cell, the precise effects
`responsible for its efficacy in HCV infection are
`not known. However, some specific host innate
`immune pathways have recently been identified
`and exploited as potential targets for therapeu-
`tics. For
`example,
`two toll-like
`receptor-9
`(TLR-9) agonists are in clinical trials. One such
`compound, CPG 10101 (Pfizer), is a synthetic
`oligodeoxynucleotide that activates TLR-9 lead-
`ing to stimulation of B cells and plasmacytoid
`
`http://tag.sagepub.com
`
`49
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`

`

`Therapeutic Advances in Gastroenterology 3 (1)
`
`dendritic cells that in turn secrete antiviral cyto-
`kines [McHutchison et al. 2007]. CPG 10101
`when given as monotherapy to HCV-infected
`patients decreased HCV viral
`loads in a dose
`dependent manner, with the highest dose achiev-
`ing a 1.7 log reduction after 4 weeks. One of its
`major effects is to increase cellular interferon.
`Whether this endogenous interferon will prove
`superior to administration of exogenous inter-
`feron is not clear.
`
`KRN7000 (Kyowa Hakko Kirin Company,
`Limited) is another immune activator; specifi-
`cally, a synthetic analog of a-galactosylceramide,
`which stimulates natural killer T cells (NKT)
`in mice and humans. Immune activation of
`NKT cells leads to cytokine production of inter-
`feron-g and tumor necrosis factor-a (TNF-a).
`Although a phase one clinical trial has been com-
`pleted no results have been published (http://
`clinicaltrials.gov/ct2/show/NCT00352235).
`It
`remains unclear if further development of drugs
`that target immune activation will lead to oral
`medications with minimal side effects, or if we
`might simply discover other ways of producing
`cytokines with unacceptable side effects.
`
`NS5A, described above in its role in viral repli-
`cation, also impairs host immune response to
`HCV infection
`in
`numerous ways. The
`N-terminal end of NS5A appears to prevent
`phosphorylation of the Janus Kinases Jak1 and
`Tyk2 and is essential for interferon signaling.
`NS5A also induces the pro-inflammatory cyto-
`kine interleukin-8 (IL-8), which as been asso-
`ciated with an impaired response to interferon
`treatment [Mihm et al. 2004; Polyak et al.
`2001]. In addition, NS5A inhibits apoptosis of
`infected hepatocytes. In other words, NS5A facil-
`itates viral evasion of the host innate immune
`response. In vitro replicon experiments have
`shown that knockout mutations of NS5A result
`in improved interferon sensitivity [Bonte et al.
`2004]. Thus, NS5A inhibitors could enhance
`the sensitivity of infected cells to interferon and
`restore the host’s innate antiviral response.
`
`(Romark Laboratories
`Finally, nitazoxanide
`L.C.) is an antiparasitic agent that was serendi-
`pitously discovered to have antiviral activity
`against hepatitis B and C infection. The pro-
`posed mechanism of action is the increase in
`phosphorylation of the protein kinase PKR that
`phosphorylates eukaryotic initiation factor 2a,
`which leads to the inhibition of HCV-RNA
`
`translation [Darling and Fried, 2009; Rossignol
`et al. 2009; Elazar et al. 2008]. The drug has been
`shown to improve SVR rates in patients infected
`with genotype 4 HCV, the predominant viral gen-
`otype in Egypt where these studies were con-
`ducted. Specifically, 79% of patients achieved
`an SVR when 12 weeks of nitazoxanide was fol-
`lowed by 36 weeks of