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Current and Future Concepts in Hepatitis C Therapy

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      Current and Future Concepts in Hepatitis C Therapy

      Jean-Michel Pawlotsky, M.D., Ph.D.

      Semin Liver Dis. 2005; 25 (1): 72-83. �2005 Thieme Medical Publishers

      Abstract and Introduction

      Abstract

      The goal of hepatitis C virus (HCV) therapy is permanent viral eradication. This requires the use of drug combinations with multiple modes of action. Steady-state HCV replication kinetics can be disrupted by drugs that inhibit virus production (antiviral molecules), inhibit de novo cell infection, and/or accelerate the clearance of infected cells. Pegylated interferon-#945; and ribavirin combine all of these mechanisms of action when used together, yet fail to clear HCV from a significant number of patients. New therapeutic approaches are needed. The next generation of anti-HCV therapeutic agents will fall into four main categories: new interferons and interferon inducers, alternatives to ribavirin, specific HCV inhibitors, and immune therapies. Ideally, these new treatments will increase the rate of sustained viral eradication and improve tolerability and acceptability. Drug combinations will be tailored to the individual patient, based on baseline parameters and viral kinetics
      during therapy.

      Introduction

      Hepatitis C virus (HCV) infection is curable. The main end point of treatment is a sustained virological response, defined as undetectable HCV RNA in peripheral blood 24 weeks after the end of treatment,[1] which generally corresponds to permanent cure.[2] Viral eradication results from reduced viral production and gradual clearance of infected cells.[3] Treatment of chronic hepatitis C is currently based on a combination of pegylated interferon (IFN)-#945; and ribavirin.[1] Even under the favorable conditions of clinical trials, failure of IFN-#945;-based treatment is relatively rare in patients with genotype 2 or 3 infection, but remains frequent in genotype 1 and 4 infection.[4-6] The higher failure rate in patients with HCV genotype 1 and 4 infection appears to be due to multiple factors, including virological factors.[7] The concepts underlying current and future treatments for HCV infection are discussed in this article.

      The Challenge of Hepatitis C Therapy

      Steady-State HCV Kinetics

      Fig. 1 is a schematic representation of steady-state HCV kinetics during chronic infection, as initially described by Neumann et al[3] based on mathematical modeling of viral decay during IFN-#945; therapy. The liver of the chronically infected patient is composed of infected and uninfected hepatocytes. Collectively, the infected hepatocytes continuously produce large amounts of HCV virions, the vast majority of which are released into the peripheral circulation. Meanwhile, a small proportion of newly produced virions infect new liver cells that join the pool of infected hepatocytes. Both infected and uninfected hepatocytes die, essentially by apoptosis. Circulating virions are continuously degraded in a virtual degradation compartment by unknown mechanisms. The recent demonstration that HCV can also replicate in extrahepatic cells, such as subsets of B lymphocytes and dendritic cells, suggests that smaller compartments with similar properties may exist outside the liver.[8-12] These
      compartments probably contribute only a small fraction of daily virus production.



      Figure 1. Schematic representation of steady-state hepatitis C virus (HCV) kinetics during chronic infection, based on mathematical modeling of viral decay during interferon (IFN)-#945; therapy.[3]

      The steady-state viral kinetics observed during the chronic phase of HCV infection is characterized by (1) equilibrium between infection of new cells and death of infected cells, ensuring that the size of the pool of infected hepatocytes remains stable; and (2) equilibrium between the release of newly produced HCV virions into the peripheral circulation and their degradation, ensuring stable viral load.[3] The half-life of free HCV virions in peripheral blood has been estimated to be approximately 2.7 hours, meaning that half of all circulating virions are cleared and replaced by new virions in less than 3 hours. An estimated 1012 virions are produced and cleared each day in an infected individual.[3] Given the estimated number of hepatocytes in the liver, this means that every infected hepatocyte probably has a modest daily production rate.

      Objectives of Therapy

      The goal of hepatitis C therapy is to disrupt this steady-state HCV kinetics, by significantly reducing virion production and allowing all infected cells to be gradually cleared (or eventually cured). The steady state of HCV kinetics offers several potential targets, which ideally should be targeted simultaneously to achieve viral eradication within a reasonable time. The three targets of HCV therapy are described in the following list (Fig. 1). All three targets must be hit if the infection is to be cured.

      Inhibition of virion production. Antiviral intervention may target any step of the HCV life cycle, such as RNA replication, polyprotein translation, viral protein maturation or viral particle assembly. A significant reduction in viral production lowers the viral content of infected cells and diminishes the number of virions released into the circulation, while degradation continues at the same rate. In turn, infection of new cells is reduced, while the clearance of infected cells continues at the same rate. This gradually depletes the pool of infected cells and creates a virtual spiral of diminishing virus production.


      Inhibition of de novo HCV infection. Inhibition of de novo cell infection reduces the pool of infected cells (when infected cell clearance remains constant), resulting in lesser release of infectious virions into the pericellular space and peripheral blood. This, in turn, may reduce de novo cell infection and thus further deplete the pool of infected hepatocytes. Overall, the pool of uninfected hepatocytes expands.


      Acceleration of infected cell clearance. Infected cell clearance relies principally on apoptosis related to local cytotoxic responses and cytokine production. Strategies targeting this phenomenon are unlikely to have a durable impact on HCV infection unless combined with efficient inhibition of virus production. In that case, infected cell clearance depletes the pool of infected hepatocytes because de novo cell infection is reduced. It is also possible that some infected cells are cured from HCV infection, although this has never been demonstrated.



      Achievements and Failures of Current Treatments

      The combination of IFN-#945; and ribavirin appears to combine potent inhibition of viral replication, inhibition of de novo cell infection, and enhancement of cellular immune responses. This combination cures a significant proportion of patients.

      Antiviral Mechanisms of IFN-#945;

      Interferons are natural cellular proteins with a variety of actions, including induction of an antiviral state in their target cells, cytokine secretion, recruitment of immune cells, and induction of cell differentiation. After subcutaneous administration, IFN-#945; binds specifically to high-affinity receptors that are present on the surface of most cells, including hepatocytes. Binding of IFN to its receptor triggers a cascade of intracellular reactions that activate numerous IFN-inducible genes. The products of these genes mediate the cellular actions of IFN-#945;.[13] They are responsible for the antiviral effects of IFN-#945;, through two distinct but complementary mechanisms: (1) induction of a non-virus-specific antiviral state in infected cells, resulting in direct inhibition of viral replication (and potentially also in noninfected cells, reducing the chances that they will become infected); and (2) immunomodulatory effects that enhance the host's specific antiviral immune
      responses and may accelerate the death of infected cells.[14]

      Direct inhibition of HCV replication by IFN-#945; has been demonstrated in vitro in the subgenomic replicon, a synthetic in vitro replication system using HCV nonstructural proteins in HuH7 cell cultures.[15-17] We have also shown that this direct inhibition occurs in primary cultures of normal human hepatocytes, the model closest to the naturally infected human liver.[18] The IFN-induced proteins and enzymatic pathways involved in establishing the antiviral state are not entirely defined. The numerous candidates, which probably act together, include the 2�-5�-oligoadenylate synthetase system, Mx proteins, and double-stranded RNA-dependent protein kinase, as well as other, less well characterized or unknown, IFN-induced intracellular pathways.[14]

      IFN-#945; binding to its receptors at the surface of immune cells also triggers complex and intricate effects, such as the induction of class I major histocompatibility complex antigen expression, activation of effector cells (macrophages, natural killer cells, and cytotoxic T lymphocytes), and complex interactions with the cytokine cascade.[19,20] It also stimulates the production of type 1 T-helper (Th1) cells and reduces the production of Th2 (suppressor) cells.[19,20] Together, these properties mean that IFN-#945; could theoretically accelerate infected cell clearance in the presence of an adapted immune response, although this has not yet been experimentally demonstrated in HCV infection.

      Antiviral Mechanisms of Ribavirin

      Ribavirin is a synthetic guanosine analogue. Like other antiviral nucleoside analogues, ribavirin undergoes intracellular phosphorylation. Ribavirin triphosphate is the main intracellular metabolite and is responsible for ribavirin's effects. Ribavirin has only a modest antiviral action on HCV in vivo. Indeed, ribavirin monotherapy has a significant but moderate (< 0.5 log HCV RNA IU/mL reduction on average) and transient (days 2 and 3 of administration) inhibitory effect on HCV replication in approximately 50% of patients.[21] This could be related to the weak inhibitory properties of ribavirin on the RNA-dependent RNA polymerase of Flaviviridae in vitro, and/or to inhibition of inosine monophosphate dehydrogenase (IMPDH), which transiently depletes intracellular guanosine triphosphate (GTP) pools.[22] However, a mathematical model of HCV kinetics constructed during IFN-#945; plus ribavirin therapy in 17 patients suggests that the principal mode of ribavirin action during
      combination therapy is to make HCV virions less infectious:[23] de novo hepatocyte infection would therefore be diminished in the context of efficient IFN-#945; inhibition of virus production. This hypothesis remains to be confirmed in a larger number of patients, and also experimentally in vivo or in vitro. The mechanisms by which ribavirin might render HCV less infectious are unclear. Recent studies of various viral models (including HCV) suggest that ribavirin is an RNA mutagen, driving viral quasispecies to error catastrophe (i.e., loss of fitness by lethal accumulation of nucleotide mutations during replication).[24-26] However, studies of human HCV infection have shown no acceleration of mutagenesis during ribavirin therapy.[27,28] Another hypothesis is that ribavirin accelerates the clearance of infected cells, given that it prevents relapse when added to IFN-#945;. Ribavirin also has putative immunomodulatory properties, such as the ability to tilt the Th1/Th2 balance toward
      Th1.[29,30]

      Achievements of IFN-#945; plus Ribavirin Therapy

      The combination of pegylated IFN-#945; and ribavirin yields sustained HCV eradication in a substantial number of patients. Fig. 2 summarizes the rates of sustained virological responses, defined by undetectable HCV RNA (i.e., < 50 HCV RNA IU/mL) 24 weeks after treatment completion.[4-6] HCV clearance is also associated with an improvement in liver lesions (potentially including cirrhosis) and prevention of the classical complications of chronic hepatitis C (cirrhosis and possibly hepatocellular carcinoma).[1] Successful therapy also significantly improves patients' quality of life.



      Figure 2. Rates of sustained virological response to pegylated interferon (IFN)-#945; plus ribavirin combination according to the hepatitis C virus (HCV) genotype in three pivotal trials.

      Failures of IFN-#945; plus Ribavirin Therapy

      IFN-#945; and ribavirin frequently fail to eradicate HCV infection, especially in patients infected by genotypes other than 2 and 3.[4-6] The only option for these patients is to enroll in clinical trials of more frequent injections and/or higher doses of pegylated IFN-#945;. It is important to understand the mechanisms underlying IFN-#945; plus ribavirin failure, to avoid the same pitfalls when new therapies become available.[7] The following factors are known to play a role in treatment outcome.

      The treatment regimen. The sustained high concentrations obtained after a single weekly injection of pegylated IFN-#945;, together with body weight adjustment of the ribavirin dose and, in some instances, of the pegylated IFN-#945; dose, have considerably improved treatment efficacy compared with three times weekly standard IFN-#945; monotherapy.[4-6,31,32] Thus, the drugs themselves have changed little during the last 20 years, but a significantly higher cure rate has been obtained by optimizing their use and increasing the steady-state levels of IFN-#945; and ribavirin.


      Host factors. Patient characteristics such as older age, male gender, and race (black) are associated with higher treatment failure rates.[7] Overweight can also negatively influence the chances of successful IFN-#945; plus ribavirin treatment, as can active alcohol or intravenous drug use,[4-6,31,32] as discussed by Dore and Thomas[33] (this issue). Adherence to therapy remains a major determinant of outcome.


      Disease-related factors. Advanced fibrosis and cirrhosis are associated with lower response rates.[31,32] Patients dually infected by HCV and human immunodeficiency virus also have lower response rates,[34-36] as discussed by Brau[37] (this issue).


      Viral factors. HCV genotypes 1 and 4 are intrinsically more resistant than genotypes 2 and 3 to the antiviral action of IFN-#945;, but within each genotype, different HCV strains have very different sensitivity to IFN-#945;.[38,39] The underlying mechanisms of IFN-#945; action remain largely unknown, but it is noteworthy that treatment does not select intrinsically resistant viruses.[40,41] For unknown reasons, clearance of infected cells in patients who respond to IFN-#945; is often delayed and is slower in patients infected by HCV genotype 1 or 4 than in those infected by genotype 2 or 3.[40,41] Possible explanations are direct interactions with cellular mechanisms and/or genotype-specific immune modulation. Likewise, ribavirin administration does not appear to be associated with selection of specifically resistant strains. A recently identified amino acid substitution putatively conferring resistance to the antiviral effect of ribavirin[42] in fact appears to be a relatively
      common polymorphism of the RNA polymerase (Dev et al, unpublished data).



      Pitfalls on the Way to New Therapies

      Very few of the multitudes of new molecules synthesized every day in public and private laboratories reach the clinical development phase, much less attain clinical use. The many pitfalls on the way to creating clinically useful new therapies are discussed in the following sections.

      Drug Design and Selection

      The first step in drug development is to screen libraries of potential compounds or to design and synthesize new molecules matching potential target sites.[43] Specific HCV inhibitors are selected by systematic screening of a very large number of small molecules, based on their predicted physical ability to bind to a functional site of an HCV target structure (RNA or protein) and to inhibit its vital function in the virus life cycle. Such screening or design may be based on a precise knowledge of the three-dimensional structure of the target (available for several HCV enzymes and some parts of the internal ribosome entry site [IRES]), generally derived from published prototype or consensus sequences. Unfortunately, the natural genetic and structural variability of these structures is not generally known,[44] and this may explain why drugs that are efficacious in vitro or in animal models often have little if any activity in humans. It may also explain the sometimes rapid selection of
      resistant strains.

      Efficacy Testing in Experimental Models

      There is no consensus on the sequence of experiments required before launching clinical development. One of the necessary steps that needs to be taken at some point is to select a model system. Several HCV model systems are available.

      In vitro models include culture models of surrogate viruses, such as bovine viral diarrhea virus (a virus belonging to the same Flaviviridae family as HCV) in cell culture;[45] in vitro cell-free models of specific enzymatic protein or RNA functions; the replicon system, a cell culture model of HCV replication in the presence of HCV IRES and nonstructural proteins;[17] primary cultures of human hepatocytes isolated from uninfected patients that replicate HCV after in vitro infection with serum samples from HCV-infected patients;[18] and a recently described cell culture system that appears to produce relatively large amounts of HCV viral particles.[46,47]


      Animal models include experimentally infected chimpanzees, the use of which is limited by cost, availability, and ethical issues;[48] various mouse models, such as Trimera mice (short-lived viremia makes this system difficult to use for drug screening)[49] and severe combined immunodeficient SCID/uPA mice, the liver of which is humanized by hepatocyte transplantation and is therefore susceptible to de novo HCV infection, with serum viral RNA levels equivalent to those seen in humans.[50]



      Experimental models are used to establish the inhibitory concentration 50 or 90 (i.e., the drug concentration that inhibits 50% or 90% of the target activity, respectively). It should be stressed, however, that the results can vary from one model to another and may not be predictive of efficacy in the clinical setting. Candidate drugs lacking efficacy in preclinical models are usually dropped, yet even those with significant efficacy in tissue culture and/or animal models are in no way guaranteed to have clinical efficacy.

      Genotype Specificity

      Most currently developed HCV inhibitors display maximum efficacy for genotype 1, because genotype 1 is generally used for screening.[51,52] This may not be a major issue, given that the main problem currently is the high rate of treatment failure in HCV genotype 1 infection, which is also by far the most frequent genotype worldwide. Genotype 1-specific drugs could thus easily find a place on the anti-HCV drug market. One potential drawback is that reduced efficacy against genotype 1 variants differing slightly from the prototype or consensus sequences used for drug design would greatly favor viral resistance.

      Toxicity

      Thorough preclinical toxicity studies are vital. They include both in vitro and in vivo (animal) preclinical toxicity screening tests that must address the two principal risks: toxicity due to modulation of the same target or pathway that produces the desired therapeutic effect, and toxicity due to secondary actions unrelated to the therapeutic mechanism of the candidate drug.[43] A substantial number of potentially highly active candidate drugs have already disappeared from the HCV drug pipeline because of worrisome preclinical toxicity profiles.[53,54] This includes toxic or even lethal effects, but also adverse effects that would be unacceptable given the natural prognosis of untreated hepatitis C infection. In addition, quantitative in vitro prediction of potential drug-drug interactions is useful but difficult.

      Resistance

      Experience in the treatment of human immunodeficiency virus or hepatitis B virus infection with specific inhibitors of viral enzymes shows that, apart from nonadherence to therapy, the principal cause of treatment failure is viral resistance.[55,56] Viral resistance is characterized by the selection of minor viral populations bearing mutations that confer resistance to the specific drug. Treatment withdrawal is usually followed by recovery of the sensitive wild-type genotype and phenotype. Viral resistance is a major clinical issue. Combination therapy with multiple drugs with different targets and mechanisms of action offers a solution, but multidrug-resistant viruses may emerge after several years of treatment.[55-58] In the case of HCV, there are already strong arguments that specific HCV inhibitors will select resistant variants when these compounds are used in the clinic. Indeed, HCV is a highly variable virus with a quasispecies distribution.[59,60] Some minor variants have
      been shown to bear mutations within or close to functionally important drug binding sites.[61] Several drugs currently in preclinical development were recently reported to select variants bearing mutations that confer resistance in the replicon system in Huh7 cell lines.[62-65] Rapid development of viral resistance to specific HCV inhibitors is therefore foreseeable if these drugs are used for single-agent therapy; the question is how well combination therapies involving several mechanisms will control this problem.

      Cure of Infection Versus Reduction of Liver Damage

      The goal of future therapies is to increase the rate of cure (i.e., permanent viral eradication). It is conceivable, however, that some or even most of those patients who do not eradicate the infection during current standard therapy are not immunologically equipped to clear infected cells. If so, there is little hope that new therapies will drastically improve the overall cure rate. If it is shown to slow liver disease progression and prevent the complications of chronic hepatitis C, long-term suppressive, noncurative treatment might remain the only option for certain subgroups of patients in whom all potentially curative treatments have failed.

      The Pipeline of Drugs Targeting Steady-State HCV Kinetics

      Four major categories of drugs are currently in the development stage: new interferons and interferon inducers, alternatives to ribavirin, specific anti-HCV inhibitors, and immune therapies.[43] They can be classified according to their anticipated impact on steady-state HCV kinetics, as schematically outlined in the following sections (note that some approaches combine several antiviral mechanisms, whereas others act through unknown mechanisms).

      Reducing Virus Production

      New IFNs. Various approaches can be envisaged to improve the antiviral performance of IFN molecules. Modifications of the primary amino acid sequence can improve antiviral activity. For instance, consensus IFN-#945; is designed to contain the most frequently occurring amino acids among the nonallelic IFN-#945; subtypes. Consensus IFN-#945; is a more potently antiviral molecule than other type 1 IFNs in vitro and could also be more effective in HCV-infected patients.[66-68] Another example is gene-shuffled IFN-#945;, which was produced by gene shuffling the family of 20 human IFN-#945; species.[69] This molecule has been shown to be far more potently antiviral than conventional IFN-#945; in vitro but has not yet been given to patients.

      Fixing IFN to large molecules can considerably improve its pharmacokinetic and pharmacodynamic properties and, as a result, its long-term efficacy. For example, addition of a polyethylene glycol molecule to the IFN-#945; molecule (pegylation) has significantly improved the results of HCV therapy compared with standard IFN-#945;.[4-6] Pegylated consensus IFN-#945; and pegylated IFN-#946; are currently being tested in clinical trials.[70] Another way to improve the pharmacokinetic and pharmacodynamic properties of IFN-#945; is to fuse it with a large molecule such as human serum albumin. This approach, implemented in Albuferon, improved the pharmacokinetic properties of IFN-#945;,[69,71] and a recent study showed significant viral load reductions when this drug was given every 2 to 4 weeks.[72] A large-scale phase III clinical trial of Albuferon will start soon.

      New IFN delivery systems are also being developed, such as disposable infusion pumps, controlled release from an injectable polymer matrix, polyamino acid-based oral delivery, and liposome encapsulation. An inhaled formulation of consensus IFN-#945; is being tested, and adaptation to the pegylated form is envisaged.

      Finally, the utility of type non-1 IFNs is being explored. Preliminary in vitro and in vivo studies suggest that IFN-#947; could potentiate the effect of coadministered IFN-#945;, and this possibility is being tested in prospective clinical trials.[73] IFN-omega is also being studied in a phase III trial.

      Specific HCV Inhibitors. A very large number of specific HCV inhibitors are currently at the preclinical or early clinical development stage.[43] Although all functional HCV structures theoretically represent potential targets for such molecules, the three most promising targets appear to be the IRES, which drives polyprotein translation; NS3 serine proteinase, which plays a major role in posttranslational poly-protein processing; and NS5B RNA-dependent RNA polymerase, the enzyme that catalyzes RNA replication.

      Approaches using nucleotide base complementarity to inhibit IRES function have produced promising results in vitro. Ribozyme and antisense oligonucleotides have been tested in phase I and II trials but gave disappointing results.[74-77] The reasons are unclear but could relate principally to the difficulty of achieving sufficient active drug concentrations at the target site, and to the poor accessibility of IRES nucleotide sequences because of secondary and tertiary structures and association with several canonical and noncanonical translation factors in infected cells. Similarly, silencing RNAs are potent HCV inhibitors in vitro,[78-80] but the concept that RNA silencing could have antiviral efficacy in humans remains to be proven. The future of IRES inhibition appears to lie in targeting the three-dimensional functional structure of the HCV IRES jointly with ribosome units and host cell factors involved in the translational process. A better understanding of the IRES
      three-dimensional structure during the HCV life cycle is needed to implement these approaches.

      Several approaches are being followed to inhibit the NS3 serine proteinase, including peptide-based and peptidomimetic molecules. The design of low molecular weight NS3 serine proteinase inhibitors is challenging, however, because the enzyme's active site is long, shallow, and exposed, offering little grasp for small inhibitor molecules.[81-83] A peptidomimetic molecule, BILN 2061, potently inhibited NS3 proteinase and HCV replication in various in vitro models and also reduced HCV replication by at least 2 to 3 log IU/mL in all HCV genotype 1-infected patients at doses of 200 mg or more after only 2 days of administration, with excellent tolerability.[52,53] Antiviral efficacy in non-genotype 1 HCV infection was less good.[51,84] Unfortunately, animal studies suggested cardiac toxicity, and the clinical development of BILN 2061 was not pursued.[53] It is unclear whether other drugs from the same family will be developed in the near future. Another NS3 proteinase inhibitor, VX-950,
      has been shown to exert potent antiviral activity in various preclinical models and will reach the clinical developmental phase soon.[63] Several other NS3 proteinase inhibitors are undergoing preclinical evaluation. NS3 helicase has been considered for years to be a potential target for anti-HCV drugs, but most attempts to develop inhibitors have failed.

      The HCV RNA-dependent RNA polymerase is another excellent target for specific HCV inhibitors. Like other viral polymerases, it can be inhibited by nucleoside/nucleotide analogues that directly target the catalytic site.[62] A nucleoside analogue, NM 283, has shown significant antiviral efficacy in the HCV replicon system and in infected chimpanzees. The first clinical study of this drug showed a significant viral load reduction of less than 1 log on average.[85] Combination with IFN-#945; with or without ribavirin will be tested soon and more potent molecules belonging to this class of drugs are expected. Nonnucleoside inhibitors are also being developed. Several drugs, such as benzimidazoles, thiophenes, and benzothiadiazines are currently in preclinical development.[65,86-91] These molecules bind to allosteric sites on the enzyme surface, some distance from the active site, and distort its fine geometry and function.

      Even if small-molecule inhibitors of HCV functions are easy to design and synthesize, most will never reach clinical use. The antiviral efficacy of some is disappointing, but the biggest problem is their toxicity. In addition, the place of molecules that reduce viral replication without modulating the immune response is questionable because of the risk of HCV resistance, already shown to occur in vitro with several of these molecules.

      Reducing de novo HCV Infection

      Three drug categories are discussed here: ribavirin and analogues, which appear to make HCV virions less infectious; inhibitors of HCV entry into target cells; and hyperimmune anti-HCV immunoglobulins, intended to neutralize infectious HCV virions.

      Ribavirin and Analogues. As discussed previously, the mechanisms of antiviral action of ribavirin are largely unknown. It has been suggested that ribavirin could act by IMPDH inhibition, thus depleting intracellular GTP pools. However, selective and specific IMPDH inhibitors such as mycophenolate mofetil (Cellcept) and merimepodib (VX-497) have been disappointing, suggesting that this is not the main mechanism of ribavirin action. Mathematical modeling of viral decay during IFN-#945; plus ribavirin combination therapy recently suggested that ribavirin's principal mode of action is to make produced virions less infectious (i.e., less prone to infect new host cells).[23] This would help explain how ribavirin prevents relapses by accelerating the depletion of the infected hepatocyte pool while HCV replication is inhibited by IFN-#945;, and would also explain why ribavirin monotherapy is ineffective in the long term.

      Hemolytic anemia is a frequent dose-limiting side effect of ribavirin,[4-6] and this has stimulated a search for alternatives with similar efficacy but less toxicity. Levovirin, the L-sugar analogue of ribavirin, does not undergo metabolism to phosphate metabolites and does not therefore inhibit host IMPDH or accumulate in erythrocytes (the mechanism responsible for hemolytic anemia). However, a recent trial comparing the combination of pegylated IFN-#945; with levovirin versus ribavirin was interrupted (together with levovirin's development) when the combination showed lesser antiviral efficacy at 12 weeks of treatment compared with ribavirin.[92]

      Viramidine is a ribavirin prodrug. It can be converted to ribavirin by adenosine deaminase. Given that the liver is rich in deaminases, viramidine is preferentially converted to ribavirin and its phosphorylated metabolites in the liver, whereas erythrocytes display lower ribavirin concentrations. A phase II study in combination with pegylated IFN-#945; is ongoing. Interim analysis showed similar end-of-treatment virological response rates in patients receiving ribavirin and those receiving 600 mg of viramidine twice a day, with a significantly reduced incidence of anemia (especially severe anemia) in the latter.[93] The final results of this trial (i.e., the sustained virological response rates) will be known soon and a large phase III trial is ongoing.

      Cell Entry Inhibitors. Major efforts are being made to determine the three-dimensional structure of HCV envelope glycoproteins, to identify receptor complex structures, and to understand the molecular mechanisms of cell entry and the early steps of the HCV life cycle, to derive inhibitor molecules. Current approaches are restricted to the development of anti-HCV monoclonal antibodies,[94] but small inhibitor molecules are also being tested in vitro.

      Hyperimmune Anti-HCV Immunoglobulins. With the recent development of a neutralization assay based on infectious retroviral pseudoparticles expressing folded HCV envelope glycoproteins, it is clear that the serum of HCV-infected patients expresses neutralizing responses that block HCV cell entry.[95,96] Chimpanzee studies showed that infusion of hyperimmune anti-HCV immunoglobulins prepared from pooled virus-inactivated HCV RNA-negative plasma could prevent or abrogate acute hepatitis.[97,98] For example, anti-HCV immunoglobulins could be valuable in preventing recurrent hepatitis C in HCV-infected liver transplant recipients. A clinical trial is currently underway to investigate the safety, pharmacokinetics, and efficacy of hyperimmune anti-HCV immunoglobulins in such patients.

      Accelerating Infected Cell Clearance

      New IFNs. The new IFN molecules described will likely be selected for their increased antiviral potency. However, these molecules may also have enhanced immunomodulatory properties that will accelerate infected cell clearance. Such properties are difficult to test in preclinical models, and only clinical experimentation (especially viral kinetics studies) can show whether these molecules indeed accelerate infected cell clearance.

      Therapeutic Vaccines. Recent indirect data suggest that a therapeutic vaccine capable of stimulating functional CD4+ and CD8+ T-cell responses in patients with chronic hepatitis C may be of benefit.[99-104] Various HCV recombinant polypeptide and plasmid DNA vaccines have been shown to be capable of priming broad, functional CD4+ and CD8+ T-cell responses in nonhuman primates. In addition, a recombinant vaccine using the HCV E1 envelope glycoprotein successfully stimulated both humoral and cellular immune responses in healthy volunteers. Intriguingly, when this vaccine was administered to infected patients, a significant decline in alanine aminotransferase levels and liver fibrosis was reported, together with a decline in liver HCV E2 antigen levels, with no effect on serum HCV RNA levels.[105] Other studies with recombinant E2 envelope glycoprotein and nonstructural polyproteins are ongoing. It should be noted, however, that proof of the concept that therapeutic vaccination could be
      of any benefit in chronic viral infections remains to be obtained.

      Other Drugs

      Oral IFN inducers have been developed to induce an effective immune response by modulation of cytokine responses. Isatoribine is a guanosine analogue that activates innate immunity through toll-like receptor 7 stimulation. Isatoribine was recently reported to be well tolerated and to reduce the HCV RNA load by 1 log on average at the highest dose tested in infected patients.[106] IFN effectors appeared to be induced, but the precise mechanism by which isatoribine reduced viral load is unknown (possible mechanisms include an antiviral effect, modulation of innate immunity, or enhancement of cellular responses).

      Several other drugs are being tested in HCV-infected patients for which no clear potential mechanism of action has been identified. They include amantadine, thymosin-#945;1, and histamine dihydrochloride, which have not yet been shown to improve the rate of sustained virological response compared with the current standard of care. Other immunomodulatory molecules are currently in phase 1 trials, such as EHC-18 and HE-2000. Iminosugars have been suggested to act through inhibition of p7, a viral protein that might act as an ion channel in infected cells. CpG-10101 is a toll-like receptor 9 agonist currently in phase 1 trials. Finally, IDN-6556 is a pan caspase inhibitor that interacts with apoptosis mechanisms.

      Conclusion

      The goal of HCV therapy is permanent viral eradication. This cannot be achieved by treatments targeting a single mechanism, and combination strategies with drugs that have multiple modes of action will therefore be needed, ideally to improve the sustained viral eradication rate, and at least to improve the tolerability and acceptability of therapy for patients in whom the peginterferon plus ribavirin combination is currently effective. The drug cocktail will need to be tailored to the individual patient, based on baseline parameters and viral kinetics during therapy. Several questions remain. Could a combination of potent inhibitors of HCV replication achieve sustained viral clearance without the help of an immunomodulatory drug? Should specific HCV inhibitors be combined with peginterferon-#945; and ribavirin, or with immune therapies? Could HCV inhibitors replace ribavirin or peginterferon in the standard of care? These crucial issues will be addressed through a careful,
      step-by-step approach in future clinical trials.

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      Abbreviation Notes

      IFN = interferon; HCV = hepatitis C virus; Th1 = type 1 T-helper cell; IMPDH = inosine monophosphate dehydrogenase; GTP = guanosine triphosphate; IRES = internal ribosome entry site

      Reprint Address

      Professor Jean-Michel Pawlotsky, Service de Virologie, H�pital Henri Mondor, 51 avenue du Mar�chal de Lattre de Tassigny, 94010 Cr�teil, France. E-mail: jean-michel.pawlotsky@...



      Jean-Michel Pawlotsky, M.D., Ph.D. , Professor, Department of Virology, INSERM U635, H�pital Henri Mondor, Universit� Paris XII, Cr�teil, France









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