Review
`
`Antiviral Chemistry & Chemotherapy 16:69–90
`
`Host cell targets in HCV therapy: novel strategy or
`proven practice?
`Bert M Klebl1*, Alexander Kurtenbach1, Kostas Salassidis1, Henrik Daub1 and Thomas Herget2
`
`1Axxima Pharmaceuticals AG, Munich, Germany
`2New Business Chemicals Bio, Merck KGaA Germany, Darmstadt, Germany
`
`*Corresponding author: Tel: +49 89 55065 460; Fax: +49 89 55065 461; E-mail: bert.klebl@axxima.com
`
`The development of novel antiviral drugs against
`hepatitis C is a challenging and competitive area of
`research. Progress of this research has been
`hampered due to the quasispecies nature of the
`hepatitis C virus, the absence of cellular infection
`models and the lack of easily accessible and highly
`representative animal models. The current combi-
`nation therapy consisting of interferon-α and
`ribavirin mainly acts by supporting host cell
`defence. These therapeutics are the prototypic
`representatives of indirect antiviral agents as they
`act on cellular targets. However, the therapy is not
`a cure, when considered from the long-term
`perspective, for almost half of the chronically
`infected patients. This draws attention to the
`urgent need for more efficient treatments. Novel
`anti-hepatitis C treatments under study are directed
`against a number of so-called direct antiviral
`targets such as polymerases and proteases, which
`are encoded by the virus. Although such direct
`antiviral approaches have proven to be successful
`in several viral indications, there is a risk of
`
`resistant viruses developing. In order to avoid resis-
`tance, the development of
`indirect antiviral
`compounds has to be intensified. These act on host
`cell targets either by boosting the immune
`response or by blocking the virus host cell interac-
`tion. A particularly interesting approach is the
`development of inhibitors that interfere with signal
`transduction, such as protein kinase inhibitors. The
`purpose of this review is to stress the importance of
`developing indirect antiviral agents that act on host
`cell targets. In doing so, a large source of potential
`targets and mechanisms can be exploited, thus
`increasing the likelihood of success. Ultimately,
`combination therapies consisting of drugs against
`direct and indirect viral targets will most probably
`provide the solution to fighting and eradicating
`hepatitis C virus in patients.
`
`Keywords: HCV, interferon, ribavirin, indirect
`(cellular) and direct (viral) antiviral targets,
`(in)direct antiviral agent/compound, protein kinase,
`signal transduction, glutathione peroxidase
`
`Introduction
`
`HCV: the disease and its consequences
`
`Hepatitis C is a serious threat to a significant percentage
`(1–2%) of the global population. This review summarizes
`the course of the disease and existing treatments, describes
`products under investigation and explains their mode of
`action on direct and indirect antiviral targets. Direct viral
`targets are those that are encoded by the viral genome.
`Indirect viral targets are those encoded by the host cell
`genome and are functions that are either usurped by the
`virus to allow its propagation or play important roles in the
`immune response and immunomodulation. We discuss
`here some exciting approaches to discovering and
`exploiting novel, cellular or indirect antiviral targets as
`potential therapies. These targets might be exploited to
`serve as a basis for the generation of new and powerful
`medications against hepatitis C.
`
`Chronic viral hepatitis is a common disease. More than 500
`million people suffer from chronic viral hepatitis world-
`wide, due to chronic infection with hepatitis B virus,
`hepatitis D virus or hepatitis C virus (HCV). Chronic viral
`hepatitis is the main cause of cirrhosis and hepatocellular
`carcinoma (HCC), which are responsible for major
`morbidity and mortality worldwide. There are 350 million
`cases of chronic hepatitis B infection and 170 million cases
`of chronic hepatitis C infection (Marcellin & Boyer, 2003).
`With no prophylactic or therapeutic vaccines available to
`date, this will create a major health crisis during the next
`decade. HCV escapes the immune system and establishes a
`persistent infection in approximately 75% of cases. These
`chronic carriers are at risk of developing life-threatening
`liver disease, such as cirrhosis and HCC. Five million
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`©2005 International Medical Press 0956-3202
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`BM Klebl et al.
`
`people in Europe and 4 million people in the USA are
`chronically infected, with an estimated 20–50% of these
`patients likely to develop cirrhosis within the next 20–30
`years (Vogel, 2003). The entire spectrum of outcome ranges
`from a very slow progression over 50 years to an accelerated
`progression to cirrhosis within 5 years (Zoulim et al., 2003).
`In retrospective studies of post-transfusion hepatitis C,
`some 20–50% developed cirrhosis and 5–25% developed
`HCC after 10–30 years (Durantel et al., 2003) (Figure 1).
`Cases of decompensated liver cirrhosis ultimately undergo
`liver transplantation. Even after liver transplantation, the
`rate of relapses is high, which is most probably due to
`extrahepatic infection, subsequently resulting in re-
`infection of the transplanted liver. Several reports indicate
`that HCV can also infect organs and cell types other than
`the liver, particularly lymphoid cells (Dammacco et al.,
`2000). Furthermore, HCV RNA can persist at very low
`levels in the serum and peripheral lymphoid cells and can
`persist in peripheral blood monocytes for many years after
`spontaneous or antiviral therapy-induced resolution of
`chronic hepatitis C (Pham et al., 2004). Negative-strand
`HCV RNA has been reported in the brain, providing
`evidence that the central nervous system is an additional
`site of HCV replication (Forton et al., 2004). This may
`explain the cerebral dysfunction found in chronically
`
`HCV-infected patients. These extrahepatic infections
`might contribute to the immune-mediated pathogenesis of
`chronic liver disease and/or the development of auto-
`immune diseases, including mixed cryoglobulinaemia. The
`presence of abnormal serum levels of cryoglobulins can
`damage the kidneys and cause glomerulonephritis, a kidney
`disease affecting the capillaries of the glomeruli (the
`compact cluster of capillaries in the kidney that filter
`blood). The protein is characterized by oedema, raised
`blood pressure and excess protein in the urine.
`A prognosis for chronically infected HCV patients can
`be made, relying on biochemical and histological parame-
`ters (Marcellin & Boyer, 2003). Important parameters for
`the long-term prognosis of chronically infected patients are
`the initial level of viral load in the serum and the early viro-
`logical response towards interferon (IFN) treatment
`(Durantel et al., 2003). In general, ~25% of patients have
`normal serum alanine aminotransferase (ALT) levels
`despite detectable HCV RNA in serum; liver histological
`lesions are generally mild, cirrhosis is rare and their prog-
`nosis is good. The majority, ~50%, of chronically infected
`patients progress very slowly and the long-term risk of
`developing cirrhosis is low. Approximately 25% of the
`chronically infected patients have moderate to severe
`chronic hepatitis. Liver biopsies are used to diagnose
`
`Figure 1. Progression of HCV infection
`
`Responders
`(45–46%)
`
`Non-responders
`(35–54%)
`
`Treatment:
`PEG-IFNα/ribavirin
`
`Acute hepatitis C
`(45–46%)
`
`Chronic
`(75%)
`
`Cirrhosis
`(20–50%)
`
`Liver failure,
`HCC, transplant
`(25%)
`
`Resolved
`(25%)
`
`Stable disease
`(50–80%)
`
`Slowly progressive
`(75%)
`
`Flow chart for the progression and prognosis of acute hepatitis C infection. Patients suffering from acute hepatitis C have a 25% chance of
`resolving the disease and a 75% chance of developing chronic hepatitis C. Without treatment, chronic hepatitis C patients develop either
`stable disease or cirrhosis. Of cirrhotic patients, 75% are slowly progressive and 25% develop hepatocellular carcinoma (HCC), ultimately
`undergoing liver failure requiring transplantation. Of chronic patients, 46–65% who are treated with PEG-IFN-α/ribavirin combination are
`long-term responders. The remaining 35–54% are non-responders and subject to the same conditions as non-treated chronic patients.
`
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`moderate or severe chronic hepatitis. These patients are at
`elevated risk of developing HCV-related cirrhosis, which
`can lead to mortality due to portal hypertension, hepatic
`failure or HCC. These numbers are representative of non-
`treated HCV patients. Approximately 250000 chronically
`infected HCV patients receive therapy per year. Only
`46–65% of these patients respond to the current drug
`regimen. The remaining 35–54%, the so-called non-
`responders, face disease comparable with non-treated
`chronically infected patients,
`leading to a significant
`number of cirrhotic patients with an elevated risk of devel-
`oping HCC and liver failure (Figure 1).
`
`Incidences and transmission
`De novo infections are still considered to occur at the rate of
`20–25 cases per 100000 persons per year (Vogel, 2003).
`HCV is mainly transmitted through contact with blood
`and blood products, with blood transfusions and sharing of
`non-sterilized needles and syringes being the main routes
`(Poynard et al., 2003). With the advent of routine blood
`screening for HCV antibodies in 1991, transfusion-related
`hepatitis C has almost disappeared and the incidence has
`been in decline (Marcellin & Boyer, 2003).
`
`Treatments and perspectives
`Because of the small number of symptomatic patients,
`randomized trials to identify new regimens in acute
`hepatitis C have been rare. So far, the standard treatment
`for chronic HCV infection, IFN-α, has been found to be
`effective for acute hepatitis C (Poynard et al., 2003). The
`aim of the antiviral therapy is to cure viral infection and
`thereby prevent the progression of liver disease towards
`cirrhosis and HCC. Ten years ago, IFN-α monotherapy
`was approved by the Food and Drug Administration
`(FDA) as the basis of therapies for chronic viral hepatitis
`(Tan et al., 2002). Subsequently, a combination treatment
`(Davis et al., 1998; McHutchinson et al., 1998), consisting
`of IFN-α and ribavirin (Sidwell et al., 1972) was intro-
`duced 6 years ago. The IFN-α/ribavirin combination has
`been considerably improved by the introduction of pegy-
`lated interferons (PEG-IFNs) (Vogel, 2003). Using either
`PEG-IFN-α2b
`(PEG-Intron®;
`Schering-Plough,
`Kenilworth, NJ, USA) or PEG-IFN-α2a (Pegasys®;
`Hoffmann-LaRoche, Basel, Switzerland) in combination
`with ribavirin (Rebetol®; Schering-Plough or Copesus®;
`Hoffmann-LaRoche), sustained viral responses (SVRs) as
`high as 46–65% have been achieved for chronically
`infected patients (Figure 1). These clinically manifested
`results have turned the combination treatment into the
`most efficacious therapy for HCV currently available
`(Vogel, 2003).
`Only a minor fraction of chronically infected patients
`receive medication, indeed, less than 1%. The costs of
`
`Host cell targets in HCV therapy
`
`complications, including decompensated cirrhosis and liver
`transplantation, may far exceed the medication costs for
`PEG-IFN-α/ribavirin treatment. In the absence of a more
`affordable combination treatment, we expect a pharma-
`coeconomic health crisis for the industrialized world, let
`alone in the major areas of virus dissemination in Asia, with
`a total of 9 million people infected with HCV in the USA
`and Europe (Vogel, 2003). Since 35–54% of patients are
`still non-responders to the currently existing and expensive
`therapies, it has become obvious that there is a huge need
`for novel treatment alternatives and medications. Without
`effective treatment strategies, HCV-related morbidity and
`mortality are expected to increase nearly threefold by the
`year 2015. The age-adjusted death rate in 1999 was 1.8 per
`100000 persons in the USA (Kim, 2002). The develop-
`ment of small molecule drugs will become particularly
`important as they are typically associated with lower
`production and development costs. Novel treatment
`options should not only address the patient population of
`IFN non-responders, but also try to shorten the overall
`treatment period, which currently takes up to 24 months.
`Equally important, although difficult to achieve due to the
`quasispecies nature of HCV (a quasispecies is a family of
`closely related, but slightly different, viral genomes; viral
`genetic variants, derived from the original infecting virus,
`which are present during an infection) and the large
`number of HCV genotypes, is the development of preven-
`tive and therapeutic vaccines, which are currently being
`tested
`in Phase II clinical
`trials
`(Pawlotsky &
`McHutchison, 2004).
`
`HCV biology
`
`HCV has been classified as the sole member of a distinct
`genus, Hepacivirus, in the family Flaviviridae, which also
`includes flaviviruses and pestiviruses. Originally cloned in
`1989 (Choo et al., 1989), the viral genome is now well
`characterized. HCV is an enveloped particle harbouring a
`plus-strand RNA molecule that is ~9600 nucleotides in
`length (Bartenschlager & Lohmann, 2000; Bartenschlager,
`2002). The initiation of translation is mediated by the
`interplay of host and viral factors. An internal ribosome
`entry side (IRES), a complex RNA structure located at the
`5′ non-coding region, serves to bind directly to ribosomes
`to initiate protein synthesis (Durantel et al., 2003;
`Bartenschlager, 2002). The open reading frame (ORF)
`encodes a polyprotein of ~3000 amino acids in length,
`which is processed by both cellular and viral proteases into
`at least 10 discrete polypeptides (Figure 2A). The structural
`proteins (core or capsid, gpE1 and gpE2) are used for the
`assembly of new-progeny virus particles, whereas most of
`the non-structural (NS) proteins (p7, NS2, NS3, NS4A,
`NS4B, NS5A and NS5B) participate in the replication of
`
`Antiviral Chemistry & Chemotherapy 16:2
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`BM Klebl et al.
`
`Figure 2. Schematic representation of (A) the HCV genome and (B) the sub-genomic replicon
`
`A
`
`5‘
`
`NTR
`IRES
`
`Structural proteins
`
`Non-structural proteins
`
`C
`
`E1
`
`E2
`
`p7
`
`2
`
`3
`
`4A 4B
`
`5A
`
`5B
`
`NTR
`
`3‘
`
`Translation and processing
`
`Signal peptidase
`
`NS2–NS3
`proteinase
`
`NS3 proteinase
`
`C
`
`E1
`
`E2
`
`p7
`
`2
`
`3
`
`4A
`
`4B
`
`5A
`
`5B
`
`Core
`
`Envelope
`
`Proteinase
`Helicase
`
`Phospho-
`protein
`
`RNA-dependent
`RNA polymerase
`
`B
`
`5‘
`
`NTR
`
`neo
`
`EMCV
`
`IRES
`
`Non-structural proteins
`
`3
`
`4A 4B
`
`5A
`
`5B
`
`NTR
`
`3‘
`
`(A) The major structural proteins include the core (C) and the envelope proteins (E1 and E2). The non-structural proteins p7, NS2, NS3,
`NS4A/NS4B and NS5A/NS5B are indicated. At the 5′ non-translated region (NTR) resides the internal ribosomal entry site (IRES), which is high-
`ly conserved and represents a site for development of translation inhibitors such as antisense oligonucleotides, ribozymes and small intefer-
`ing RNAs (siRNAs). NS3 encodes a protein with a specific protease and helicase activity. The NS5A region encodes for a phosphoprotein and
`the NS5B for an RNA-dependent RNA polymerase enzyme, both important for viral replication. They also represent sites for the development
`of specific viral enzyme inhibitors. Other potential enzyme targets include the HCV-specific proteases (NS2/3 and NS3). These enzymes are
`involved in processing the viral polyprotein at specific sites as indicated. (B) The subgenomic replicon has been generated by replacing the
`region that encodes the core protein up to the NS2-encoding region by the neomycin phosphotransferase gene (neo) and the IRES of the
`encephalomyocarditis virus (EMCV). This IRES drives the translation of the HCV polypeptide from NS3 to NS5B, whereas the selectable marker
`neo is expressed under the control of the original HCV IRES of the 5′ non-translated region.
`
`the viral genome. During viral replication, the viral genome
`acts as a template for the synthesis of negative-strand
`RNA, which, in turn, is a template for the production of
`excess amounts of positive-strand RNA progeny. Details
`concerning the initiation of the synthesis of the positive-
`strand RNA are not clearly known and only sparse infor-
`mation is available in terms of initial and late phases, for
`example, entry and morphogenesis of viral particles, since
`the field lacks a reproducible and efficient cell culture
`system for viral replication (Bartenschlager & Lohmann,
`2000; Bartenschlager, 2002; Dymock et al., 2000).
`
`HCV consists of six different genotypes (genotypes 1–6).
`Knowledge of the genotype or serotype is helpful for predic-
`tion of SVR and the choice of treatment duration. Genotypes
`do not change during the course of an infection. Response
`rates to treatment with the combination of PEG-IFN and
`ribavirin are 88% for genotypes 2 and 3, and 48% for geno-
`types 1, 4, 5 and 6 (Poynard et al., 2003). Unfortunately,
`genotype 1 is the most frequent genotype in Europe and the
`USA and is present in 60–80% of cases (Marcellin & Boyer,
`2003). These insufficient response rates underscore the need
`for the development of novel and efficient genotype-
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`

`independent treatments and also argue for the further
`improvement of existing cellular and animal models.
`
`Tissue culture model: the replicon system
`
`Many aspects of HCV disease can only be addressed in
`vivo, for example, the influence of the immune system. For
`many investigatiors, however, it is sufficient and desirable
`to work with a less complex, well-controlled system in
`vitro. The recently generated HCV-replicon system, in
`which expression of the HCV non-structural proteins
`drives the replication of a subgenomic HCV RNA, fulfills
`these criteria (for reviews, see Bartenschlager & Lohmann,
`2001; Bartenschlager, 2002).
`The human hepatoma cell line (Huh-7) was trans-
`fected with subgenomic HCV RNAs called replicons
`(Lohmann et al., 1999). These were derived from a cloned
`full-length HCV genome of genotype 1b (Figure 2A) by
`replacing the reading frame for the N-terminal proteins
`(including p7 or NS2) with the neomycin phosphotrans-
`ferase gene (neo) downstream of the HCV IRES.
`Translation of the HCV NS2-5B or NS3-5B region was
`directed by the IRES of the encephalomyocarditis virus
`(EMCV) inserted downstream of the neo gene (Figure
`2B). Upon transfection of Huh-7 cells, only those in
`which HCV RNA replication occurs develop continuous
`resistance against the drug geniticin due to neo expression.
`Cell lines obtained from such resistant colonies contain
`high
`levels of replicon RNAs and viral proteins
`(Lohmann et al., 1999; Pietschmann et al., 2001). This
`allows the study of HCV RNA replication as well as of
`translation and processing of those HCV proteins present
`in the system (Figure 2).
`The development of subgenomic replicons containing
`HCV non-structural proteins that replicate in human
`hepatoma cells, now provides a system to test or screen
`candidate drugs that target the non-structural gene prod-
`ucts. Thus, the antiviral efficacy of BILN 2061, a NS3
`protease inhibitor, the development of which was put on
`hold after entering Phase II clinical trials (see Table 1), was
`determined in the HCV replicon cell culture system. BILN
`2061 has an IC50 of 4 nM (genotype 1a) and 3 nM (geno-
`type 1b), while cytotoxicity analysis in parental Huh-7 cells
`produced a cytotoxic concentration (CC50) of 16–35 μM
`(Pause et al., 2003; Lamarre et al., 2003). As these sub-
`genomic replicons do not produce infectious virions, the
`option of studying encapsidation or cell-to-cell viral trans-
`mission is not provided. Furthermore, the replicon cell lines
`are of clonal origin and have, apart from adaptive muta-
`tions,
`identical HCV sequences (Krieger et al., 2001).
`However, the genetic heterogeneity of HCV suggests that
`future drugs should display activity against a broad range of
`HCV genotypes, subtypes and quasispecies.
`
`Host cell targets in HCV therapy
`
`In summary, the HCV-replicon RNA replicates to fairly
`high levels in Huh-7 cells and provides, for the first time, a
`genetic system to study HCV RNA replication and a cell-
`based assay screen for HCV inhibitors. Such a replicon-
`based screen for anti-HCV substances has been described in
`a recent study on the nucleoside antimetabolite-mediated
`reduction of HCV RNA (Stuyver et al., 2003). In this study,
`a specific anti-HCV replicon effect was defined as minimal
`interference with the exponential cell growth, minimal
`reduction in cellular host RNA levels and reduction of the
`HCV RNA copy number per cell compared with that of the
`untreated control (Stuyver et al., 2003).
`
`Animal models
`
`Developing robust animal model systems for HCV is highly
`desirable and some progress has recently been achieved
`(reviewed by Pietschmann & Bartenschlager, 2003). Besides
`the fact that ethical considerations do not permit experi-
`ments with humans, an animal system may allow for moni-
`toring of the entire cycle of viral replication, from infection
`of naive tissues to full-blown viraemia. Since acute HCV
`infection often happens without any obvious symptoms,
`these patients do not consult a physician. Thus, valuable
`information about early events of HCV infection and
`remission is lacking. A model system will allow the study of
`each phase of the disease under controlled conditions.
`Establishing a model system is hampered by the fact that
`HCV infects only humans and chimpanzees, primarily
`targeting hepatocytes. The determinants of the restricted
`host and tissue specificity are not understood. Despite the
`extremely robust replication rate of HCV in humans, efforts
`to propagate this virus in cell culture have been frustratingly
`unsuccessful. Therefore, the use of surrogate virus models
`closely related to HCV, such as the bovine viral diarrhoea
`virus (BVDV) and the tamarin GB virus-B (GBV-B), both
`which belong
`to Flaviviridae, provides alternative
`approaches. GBV-B is most closely related to HCV and is,
`therefore, a good surrogate model. For instance, one poten-
`tial mechanism of action of the pleiotropic antiviral agent,
`ribavirin, was verified using the GBV-B model. The
`antiviral effect of ribavirin does not seem to be solely based
`on the inhibition of inosine 5′-monophosphate dehydroge-
`nase (IMPDH) and reduction of intracellular pools of GTP
`and dGTP, but also by the incorporation of ribavirin
`triphosphate into viral RNA and induction of error-prone
`replication (Lanford et al., 2001). However, the biological
`activity of a tested compound in these systems does not
`necessarily translate into efficacy for human HCV infection.
`
`Transplant mouse models
`Mice are the preferred models for scientific studies for a
`number of reasons (for example, short breeding cycles,
`
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`BM Klebl et al.
`
`Table 1. Current HCV development pipeline
`
`Phase I
`
`Phase II
`
`Phase III
`
`Phase IV
`
`New interferons
`Albuferon* (Human Genome Sciences)
`
`PEG alphacon* (InterMune)
`Transfersome containing IFN-α* (IDEA)
`
`Other biologicals (antisense, vaccines, etc)
`HCV/MF59† (Chiron)
`
`Immunomodulators
`
`Oral IFN-α*
`(Amarillo Biosciences)
`IFN-γ1b* (InterMune)
`Omega Interferon* (BioMedicine)
`Multiferon* (Viragen)
`
`ISIS 14803† (ISIS Pharmaceuticals/Elan)
`E-1† (Innogenetics)
`Civacir† (NABI)
`HepeX™-C† (XTL)
`Rituximab† (Rituxam)
`(Genentech/IDEC)
`HCV vaccine† (Intercell)
`
`Merimebodib-VX-497‡ (Vertex)
`Ceplene‡ (Maxim)
`ANA245‡ (Anadys)
`Viramidine‡,§ (Valeant Pharmaceuticals)
`CPG 10101‡ (Coley
`Pharmaceuticals)
`
`REBIF*
`(Ares-Serono)
`
`Infergen*
`(InterMune)
`
`Zadaxin‡ (SciClone)
`
`Direct antiviral small molecules
`HCV-086 (ViroPharma/Wyeth)
`VX-950¶ (Vertex)
`SCH-7¶ (Schering)
`R803§,£ (Rigel Pharmaceuticals)
`JTK-109§ (AKROS Pharma/Japan Tobacco)
`R1479§ (Roche)
`
`Antifibrotics
`
`NM283§ (Idenix Pharmaceuticals)
`BILN 2061¶,r (Boehringer Ingelheim)
`JTK-003§ (AKROS Pharma/Japan Tobacco)
`KPE02003002§ (Kemin Pharma)
`UT-231-B$ (United Therapeutics)
`
`Amantadine$
`(Endo Labs Solvay)
`
`IDN-6556♠(Idun Pharmaceuticals)
`
`IP-501u (Indevus)
`
`*Interferon family, covering long-lasting interferons such as albuferon, which is a fusion of human interferon and albumin, and multiferon.
`REBIF is IFN-β1a and omega interferon is a new formulation intended to target the liver specifically. IFN-γ1b is aimed at treating liver fibrosis.
`IFN-α is administered in low doses, which get absorbed through mucosal membranes (oral IFN-α). Infergen® is a consensus interferon, which
`was generated through comparing the amino acid sequences of the then-known subtypes of INF-α and assigning the most commonly occurring
`amino acid sequence to a new protein, thereby creating a ‘consensus’ interferon.
`†Biologicals, such as vaccines (HCV/MF59, E-1), antibodies (Rituximab, Civacir) and antisense (ISIS 14803).
`‡Immunomodulator, boosting the immune system, like the IMPDH inhibitor VX-497 (merimebodib) and viramidine, the toll-like receptor 7
`agonist ANA245 (isatoribine), the toll-like receptor 9 agonist CPG 10101, histamine (Ceplene) and thymosin α-1 (Zadaxin).
`§Small molecule nucleosidic and non-nucleosidic polymerase inhibitors (JTK-003, JTK-109, R803, R1479, NM283, viramidine). Viramidine pre-
`sents a special case, since it is a prodrug of ribavirin, which is specifically targeted towards liver cells. The molecular target for the antiviral
`compound KPE02003002 has not been revealed to our knowledge.
`¶Small molecule inhibitors of the viral protease (BILN2061, SCH-7, VX-950).
`$Small molecule inhibitors of the viral p7 ion channel. UT-231-B belongs to the iminosugar derivatives, for which activity on p7 has been
`demonstrated. Amantadine is a pleiotropic agent that has been launched for the treatment of influenza. It blocks the M2 ion channel of the
`influenza A virus and prevents the passage of H+ ions (DeClercq, 2004). Therefore, it seems likely that amantadine might act via inhibiting the
`p7 ion channel.
`♠IDN-6556 is a caspase inhibitor, which is believed to preserve cell structure and protect liver from damage caused by HCV.
`uPurified phospholipid.
`rPhase II trials have been halted and further development of this drug is reportedly suspended owing to cardiac toxicity in non-human pri-
`mates at a supra-efficacious dose.
`£Rigel have just announced insignificant clinical effects for R803 in a Phase I/II HCV trial due to poor bioavailability
`(http://www.rigel.com/rigel/corporate).
`Underlined text represents indirect antiviral targets or host cell targets, which are modulated through the treatment. Italic text represents
`direct antiviral targets. There are two exceptions: viramidine since it is a modified version of ribavirin. Ribavirin is a pleiotropic agent, exert-
`ing direct and indirect antiviral effects. The same is true for amantadine, since amantadine does not only target the virally encoded M2 pro-
`ton channel (Lear, 2003). Amantadine is an antagonist of the N-methyl D-aspartate receptor as well (Magnet et al., 2004).
`
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`simple handling and genetic characterization). Mice cannot
`be infected directly with HCV due to the restricted host
`range of the virus. Therefore, transgenic mice expressing
`one or several viral proteins were created to analyse their
`effects on liver pathology, but their meaningfulness for
`drug development remains somewhat unclear.
`Significantly more progress has been reported on the
`generation of transplant mouse models. These transplant
`mouse models might lead to the development of more suit-
`able small animal models in the future. Propagation of
`HCV in these chimeric mouse models has recently been
`achieved (Mercer et al., 2001; Ilan et al., 2002).
`The principle of the first model is that immunocompro-
`mised mice, unable to develop murine hepatocytes due to a
`transgene, were engrafted with human hepatocytes isolated
`from fresh livers and shown to be susceptible to HCV infec-
`tion and replication. To achieve this system, Mercer and
`colleagues transplanted human hepatocytes into immunod-
`eficient transgenic mice (SCID, homozygous for the SCID
`trait) carrying the urokinase-type plasminogen-activator
`gene controlled by the albumin promoter (Alb-uPA mice).
`The Alb-uPA transgenic mouse, developed in 1990 to study
`neonatal bleeding disorders, carries a tandem array of four
`murine urokinase genes controlled by an albumin promoter.
`This transgene targets urokinase over-production to the
`liver resulting in a profoundly hypofibrinogaenemic state
`and accelerated hepatocyte death. Normal human hepato-
`cytes were transplanted into these SCID mice carrying the
`plasminogen activator transgene Alb-uPA, thus generating
`mice with chimeric human livers. After inoculation with
`infected human serum, viral infection persisted beyond 4–5
`weeks only in homozygous Alb-uPA mice. The shorter
`persistence of infection in Alb-uPA heterozygous mice is
`attributed to a much lower survival of transplanted human
`hepatocytes in these mice relative to homozygous mice.
`Approximately 75% of homozygous Alb-uPA mice inocu-
`lated with serum from hepatitis C patients developed HCV
`titres. The viral titres reached 3×104–3×106 copies/ml in the
`blood of infected mice and were equal to or higher than
`those present in patients with chronic hepatitis C. This
`novel mouse model supports prolonged HCV infection for
`15–17 weeks, and even for 35 weeks in one mouse. Infection
`could be serially passaged through three generations of
`mice, confirming both synthesis and release of infectious
`viral particles. Thus, this is the first murine model that may
`be suitable for studying human HCV in vivo (Mercer et al.,
`2001). However, this system is laborious and requires special
`expertise to isolate and transplant human hepatocytes and
`to maintain a colony of fragile immunodeficient mice with
`an approximately 35% mortality in newborns due to a defect
`in blood coagulation. This may be the reason why, 3 years
`after its introduction into the scientific community, this
`system has not reached a broad application. There is also a
`
`Host cell targets in HCV therapy
`
`scientific obstacle: in humans with chronic hepatitis C, the
`injury of hepatocytes is not directly caused by HCV infec-
`tion but rather is a consequence of the destruction of
`infected hepatocytes by cytotoxic lymphocytes. A crucial
`question is whether these mice will develop liver disease
`confined to the transplanted human hepatocytes or whether
`the immunosuppressed mice will be infected but free of
`disease.
`In another approach, immunodeficient BNX mice were
`irradiated with a lethal dose, rescued by transplantation of
`bone marrow cells originating from severe combined
`immunodeficient SCID mice and finally, had liver frag-
`ments from ex vivo HCV-infected humans transplanted
`under the kidney capsule (Galun et al., 1995). The resulting
`system, made up from three genetically disparate sources of
`tissue, was termed ‘Trimera’. Viraemia (positive-strand
`HCV-RNA levels) in HCV-Trimera mice peaked at
`approximately day 18 after liver transplantation, and viral
`replication in liver grafts was evidenced by the presence of
`specific negative-strand HCV RNA. Recently,
`it was
`claimed that up to 85% of the transplanted animals of this
`Trimera-mouse system were HCV-infected and that an
`antibody was effective in reducing the viraemia in this
`model (Ilan et al., 2002). This anti-HCV monoclonal anti-
`body (MAb) HCV ABXTL68 was developed from the
`peripheral blood lymphocytes of an HCV-positive patient.
`It was characterized as a fully human high-affinity IgG1
`subtype MAb against the HCV E2 envelope protein. The
`antibody was further developed and is currently being
`studied in clinical trials for chronic HCV patients
`(HepeX™-C in Table 1). So far, the antibodies have been
`shown to be safe, tolerable and could significantly reduce
`viral load (Ilan et al., 2002; Dagan & Eren, 2003).
`Another antiviral drug, I70,
`inhibiting HCV RNA
`translation, was also effective in reducing the viraemia in
`this model. The I70 molecule was selected via high-
`throughput screening in a cell-based IRES assay designed
`for screening of HCV IRES inhibitors (Ilan et al., 2002).
`In conclusion, both systems should help to expedite
`anti-HCV drug evaluation if available to the pharmaceu-
`tical industry without extreme costs and legal complica-
`tions, but they also have their limitations, especially in light
`of the role of host cell targets, which play an important role
`in the immune response and the HCV replication process.
`
`Chimpanzee model
`Until recently the only well-established animal model
`supporting HCV replication was the chimpanzee model.
`The successful infection of this animal was invaluable for
`the initial characterization of HCV, which finally led to the
`identification of infectious cDNA clones of the virus (Choo
`et al., 1989). The clinical course of virus infection observed
`in chimpanzee and humans shows some similarities. Apart
`
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`BM Klebl et al.
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`from humans,