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New therapeutic strategies for hepatitis C
SPECIAL ARTICLE New Therapeutic Strategies for Hepatitis C Adrian M. Di Bisceglie,1 John McHutchison,2 and Charles M. Rice3
V
iral hepatitis is the single most important cause of liver disease in the United States and worldwide. With this in mind, the AASLD has launched an annual single topic conference devoted exclusively to viral hepatitis with the intention of bringing together researchers in viral hepatitis to foster progress in this important area. The first of these conferences, held June 15-16, 2001 in Chicago, IL, focused on new, evolving, and future approaches to therapy of hepatitis C. Dr. Eugene Schiff (University of Miami) reviewed the history and impact of hepatitis C. It appears that the hepatitis C virus (HCV) emerged in the U.S. population beginning in the 1960s, related to blood transfusion and injection drug use, although the extent of the problem was only apparent after 1990 when reliable blood tests first became available for hepatitis C.1 Studies of the natural history have been somewhat contradictory but indicate that over the first 20 years of chronic HCV infection, 20% of chronically infected patients will develop cirrhosis, and many of those will progress to hepatocellular carcinoma.2 HCV-associated end-stage liver disease is now recognized as a leading indication for liver transplantation in the United States and the developed western world.3
Virology Dr. Charles Rice (The Rockefeller University) described the virology of HCV; it has an RNA genome about 10,000 nucleotides in length.4 A hallmark of this RNA is the presence of a long open reading frame (ORF) that encodes a polyprotein that is processed into at least 10 discrete polypeptides (Fig. 1). Translation initiation is mediated by the internal ribosome entry site (IRES). Progress made in understanding HCV IRES structure and function was reviewed by Dr. Stanley Lemon (University of Texas at Galveston). The HCV IRES resides within the 5⬘ nontranslated region between nucleotides 44 and 342. Key structural features include two large stem-loops and a pseudoknot that involves sequences near the initiator codon. These structures have been deduced from thermo-
Abbreviations: HCV, hepatitis C virus; ORF, open reading frame; IRES, internal ribosome entry site; NS, nonstructural; HCIG, hyperimmune C immunoglobulin; HBIG, hyperimmune B immunoglobulin; PEG, polyethylene glycol; RdRp, RNA– dependent RNA polymerase; IMPDH, inosine monophosphate dehydrogenase; IL-10, interleukin 10. From 1Saint Louis University, St. Louis MO; 2Scripps Clinic, La Jolla, CA; and 3The Rockefeller University, New York, NY. Received August 24, 2001; accepted October 29, 2001. This is a report of the first AASLD Hepatitis Single Topic Conference, held in Chicago, June 15 and 16, 2001. Address reprint requests to: Adrian M. Di Bisceglie, M.D., Division of Gastroenterology and Hepatology, Department of Internal Medicine, Saint Louis University School of Medicine, 3635 Vista Avenue, St. Louis, MO 63110. E-mail: [email protected]; fax: 314-577-8125. Copyright © 2002 by the American Association for the Study of Liver Diseases. 0270-9139/02/3501-0030$35.00/0 doi:10.1053/jhep.2002.30531 224
dynamic modelling, phylogenetic analysis, chemical and enzymatic structure probing, X-ray crystallography, and nuclear magnetic resonance. The IRES forms a binary complex with the 40S ribosomal subunit in the absence of other translation initiation factors (unlike other viruses).5 This high-affinity complex results in a conformational change in the 40S subunit and binding of eukaryotic initiation factor 3. Via this process, the AUG initiator codon of the viral RNA is positioned near the ribosome decoding site. A specific sequence is not then required to initiate translation, but a stem loop must “melt” to allow this process to proceed. This complex viral RNA element could thus be a rich target for compounds that inhibit translation. Processing of the polyprotein is mediated by the cellular enzyme, host signal peptidase, and two viral proteases, the NS2-3 autoprotease and the NS3-4A serine protease. This results in the production of the core or capsid protein (C), two envelope glycoproteins (E1 and E2), a small hydrophobic protein (p7), and 6 nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS6A, and NS5B).4 Although it was thought that this was the only ORF in the HCV genome, two laboratories have recently reported the existence of an alternative ORF that overlaps the C protein gene.6,7 This protein, termed ARFP (for alternative reading frame protein) or F (for frameshift protein) appears to be expressed during HCV infection because some patients are seropositive for antibodies to this antigen. The function of ARFP/F is unknown but it is not required for RNA replication in cell culture. Numerous studies have examined HCV diversity and its possible importance in clinical progression. HCV RNA replication is error prone because of the lack of proofreading by the viral RNA polymerase. Four hierarchical strata of HCV diversity have been identified: genotypes, subtypes, isolates, and quasispecies. Patients infected with HCV genotype 1 clearly have a poorer response to current antiviral therapy than those with genotype 2 or 3.8 By contrast, the clinical significance of HCV genotypes in the natural history of hepatitis C is controversial. Dr. Patrizia Farci (University of Cagliari, Italy) presented data supporting a role for HCV quasispecies variability in determining (or correlating with) the outcome of HCV infection. Increased viral diversity during the acute phase of hepatitis was found to be associated with progression to chronicity whereas resolving acute hepatitis was associated with relative stasis of the quasispecies.9 Patients who respond to interferon therapy also have a decrease in viral genetic diversity and in the number of viral quasispecies. HCV replicates in hepatocytes and perhaps other cell types. Studies of viral dynamics after antiviral treatment or in the acute phase of infection indicate high turnover and a production rate in the range of 1012 virus particles per day.10 The mechanisms of HCV attachment and entry are still not known. As discussed by Dr. Sergio Abrignani (Chiron Corporation, Emeryville, CA), the HCV E2 protein binds avidly to CD81, a tetraspanin molecule
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Fig. 1. The horizontal strip indicates the structural and nonstructural proteins encoded by the HCV genome (c, capsid; E, envelope; NS, nonstructural). The X-ray crystallographic structures of specific HCV enzymes have also been identified as shown (not to scale). The HCV protease encoded within the genome requires NS2 to NS4 for full activity. HCV helicase is also a component of the NS3 protein. NS5b encodes an RNA-dependent RNA polymerase. The unique molecular structures of these specific viral enzymes should allow the identification and development of specific and potent inhibitors. As in other viral diseases, once developed and if given alone, rapid viral resistance may develop to each of these future compounds. Combination therapeutic approaches will probably be required to prevent the emergence of rapid viral resistance.
found on the surface of many cell types including hepatocytes.11 Recent data indicate that human CD81 by itself may not be sufficient to mediate virus attachment and entry. Instead it may serve only to attach HCV to the surface of cells allowing subsequent interaction with a more specific entry receptor.
Animal Models and Cell Culture Systems The study of HCV replication and the development of new therapies have been greatly advanced by the recent development of cell culture models for HCV. At present, the most effective of these models is the replicon system.12 The prototype replicon was a subgenomic HCV RNA in which the HCV structural region was replaced by the neomycin phosphotransferase gene, and translation of the HCV proteins NS3 to NS5B was directed by the IRES from encephalomyocarditis virus. The Huh-7 hepatoma cell line has been found to support replication of this replicon. Easily measurable levels of HCV RNA are produced and replication can be inhibited by antivirals such as interferon alfa.13 However, there are still several unexplained paradoxes associated with this system. Replication is thus far restricted to Huh7 cells and does not occur in other human hepatoma cell lines. Efforts to establish functional replicons for genotypes 1a and 2a have not met with success. In the case of the subtype 1b replicons, limited changes including even single amino acid substitutions can increase the efficiency of replication initiation by more than 100,000-fold. Such adaptive mutations tend to cluster in the NS5A protein but can also be found in NS3, NS4B, and NS5B. These advances provide a system for molecular genetic studies on HCV replication and facilitate rapid modification of replicons, via genetic engineering, to tailor assays for high throughput cell-based drug screening. The holy grail of HCV cell culture remains the establishment of a robust replication
system that includes virus assembly, release, and infection of naive cells. Dr. Keril Blight (The Rockefeller University) presented new data showing that full-length HCV genotype 1b RNAs harboring adaptive mutations could also replicate in Huh7 cells, albeit at lower levels than the replicon. Whether particle assembly, release, and spread occur in this system remains to be established. Dr. Jens Bukh (National Institute of Allergy and Infectious Diseases) reviewed progress using infectious cDNA clones to explore HCV biology in the chimpanzee model. Until recently,14 this was the only robust animal model supporting HCV replication. Functional cDNA clones exist for genotypes 1a, 1b, and 2a, and chimp-titered challenge stocks have been developed for most of the common HCV genotypes. Genetic studies using infectious clones have shown the essential nature of the HCV enzymes and the conserved elements of the 3⬘ nontranslated region. Remarkably, the E2 hypervariable region (HVR1) could be deleted without abrogating infectivity, although the mutant virus replicated poorly and compensating changes in E1 and E2 were selected upon passaging.15 On the vaccine front, challenge of animals with resolved HCV infection using homologous or heterologous virus has provided evidence for protective immunity. This is an important proof of principle for vaccine development.16,17 Dr. Mark Kay (Stanford University) described gene therapy approaches and methods for efficient RNA transfection of mouse hepatocytes in vivo. Using the latter method, no evidence was obtained for replication of chimpanzee infectious HCV RNAs in liver resident mouse hepatocytes. This suggests a requirement for human/chimpanzee-specific intracellular factors in one or more steps of the HCV replication cycle. As HCV’s closest relative, GB virus-B is being explored as a surrogate model. Molecular clones infectious in tamarins have
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been developed as have cultures of primary tamarin hepatocytes that support a complete cycle of virus replication.18
Immunology The immune response to HCV appears to be critical both in determining viral clearance or persistence and in contributing to liver injury. Dr. Barbara Rehermann (National Institute of Diabetes and Digestive and Kidney Diseases) pointed out that the frequency of HCV specific cytotoxic T cells is low, representing only 0.01% to 0.5% of circulating CD8 positive T cells.19 The proportion of HCV-specific T cells is substantially higher (about 30-fold) within the liver. Recovery from HCV infection is associated with an increased T-cell proliferative response whereas chronic infection is marked by impaired production of gamma interferon by lymphocytes. These functionally impaired CD8-positive T cells are CD27 positive, but CD28 negative and appear to suffer from lack of CD4 T helper activity. Dr. Christopher Walker (Children’s Hospital Research Foundation, Columbus, OH) noted an important recent observation common to both human and chimpanzee studies—that mutations within recognized HCV T-cell epitopes may allow HCV variants to escape immune recognition and contribute to persistence. The humoral response to HCV has not been well studied but investigation of the therapeutic use of immune serum globulin and hyperimmune C immunoglobulin (HCIG) suggests that it may also play an important role in HCV clearance. Historical studies in humans have shown that both immune serum globulin and hyperimmune B immunoglobulin (HBIG) prepared before elimination of anti-HCV positive blood reduce both the incidence and severity of post-transfusion non A, non B hepatitis.20,21 Dr. Krystof Krawczynki (Centers for Disease Control and Prevention) described chimpanzee studies utilizing HCIG conducted at the Centers for Disease Control and Prevention. Early studies showed that a single dose of HCIG delayed the onset of HCV infection and was associated with a reduction in the level of HCV viremia in the chimpanzee animal model. More recent studies have used HCIG prepared from as many as 500 HCV seropositive donors, all with normal serum aminotransferases. The plasma was purified by detergent treatment, ethanol fractionation, and ultrafiltration. Chronic administration of this preparation over a 15-week period was associated with a significant reduction in HCV titers in 2 of 3 chimpanzees tested. The optimal use for this type of product would be for postexposure prophylaxis and perhaps to prevent recurrence of hepatitis C after liver transplantation, as is done with HBIG after transplantation for hepatitis B.
Current Optimal Therapy The most effective and available initial therapy for these patients is the combination of interferon and ribavirin (Fig. 2). Dr. John McHutchison (Scripps Clinic) noted that the sustained virologic response rate with this regimen is approximately 35% to 40% after a 24- or 48-week course of this therapy, respectively.8 The 2-drug regimen increases the number of patients who clear HCV RNA during treatment, and decreases the number of patients who relapse. In general, patients with genotype 1 infection should receive 48 weeks of therapy and those with genotype non-1 infection, only 24 weeks. Patients with genotype 2 or 3, with low viral
Fig. 2. Sustained virologic response rates reported from key studies that evaluated the effect of interferon and ribavirin in therapy of chronic hepatitis C. Study A: Comparison of interferon alfa-2b with ribavirin versus interferon alfa-2b alone, both for 48 weeks (McHutchison et al.8). Study B: pegylated interferon alfa-2b versus interferon alfa-2b (Lindsay et al.26). Study C: pegylated interferon alfa-2a versus interferon alfa-2a (Zeuzem et al.24). Study D: pegylated interferon alfa-2b with ribavirin versus interferon alfa-2b with ribavirin (Manns et al.27). Study E: pegylated interferon alfa-2a with ribavirin versus interferon alfa-2b with ribavirin (Fried et al.28). In each case, the study group mentioned first is represented by the black bar.
load, who are less than 40 years of age, who are women, and who do not have advanced fibrosis are most likely to respond. Lack of response can be reliably predicted at week 24 of therapy, such that genotype 1–infected patients who have not responded and cleared virus at this time can cease treatment, as less than 2% of these patients will achieve a sustained response. Studies of the predictive value of week 12 responses are underway. Side effects more frequently observed when ribavirin is combined with interferon include cough, dyspnea, rash, itch, insomnia, and nausea. Ribavirin also accumulates in red blood cells and causes a dose-dependant hemolytic anemia of moderate severity that requires dose reduction in 7% to 10% of patients.22 Despite these side effects and the greater proportion of patients who require discontinuation or modification of therapy, combination therapy with interferon has also been shown to be the most cost-effective approach to therapy. Although the optimal dose of ribavirin is currently unknown, available data suggest that higher doses increase efficacy (albeit with a greater degree of anemia). The dose of 800 mg/d may be the most appropriate lower dose for those patients who require dosage modification for anemia or other side effects. As in human immunodeficiency virus therapy, compliance or adherence during combination therapy also enhances response rates. Patients who achieve a sustained virologic response have been shown to have improvements in their quality of life, continued improvement in histology, and durable responses lasting 3 or more years in over 95% of these patients. Efficacy of drugs is often limited by the relatively short time that they are present in circulation or the target tissue at inhibitory concentrations. Dr. Marlene Modi (Hoffmann La Roche, Inc.) described the pharmacology of pegylated interferon. Addition of a polyethylene glycol (PEG) molecule to a protein results in a product with better physical and thermal stability and that is protected against rapid enzyme degradation.23 This results in a biologically
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active drug with a more convenient longer half-life and in general, more favorable pharmacokinetics. Early attempts to develop pegylated interferons were hampered by compounds with relatively short half-lives with little if any enhancement in efficacy. The subsequently developed pegylated interferons alfa-2a and alfa-2b were initially studied in randomized clinical trials as monotherapy. Although these two different pegylated interferons differ in their structural properties (chain size and type, linkage, amino acid sequence, attachment site, half-lives, and dose) both have been shown to be more effective than their unmodified interferon counterparts. Dr. Mitchell Shiffman (Medical College of Virginia) described clinical trials conducted with pegylated interferons alone. Initial phase II dose-finding studies with both forms of pegylated interferon identified subsequent doses for phase III registration trials. pegylated interferon alfa-2a has been administered to patients with chronic hepatitis C with elevated liver tests and shown to enhance response rates from 19% to 39%.24 In another study of patients with cirrhosis, sustained response was observed in 30% of patients (180 g injected subcutaneously once a week for a 48-week treatment period).25 Like standard interferons, histologic improvement and biochemical improvement were observed in both of these studies, and it was noted that the side effect profile was similar to standard interferons apart from a greater incidence of neutropenia, which more frequently required dose reductions. A large phase III randomized controlled trial of pegylated interferon alfa-2b in similar patients with chronic hepatitis C also indicated a higher sustained response rate in patients receiving 1.0 g/kg/wk or 1.5 g/kg/wk compared with standard interferon administered for 48 weeks.26 This has subsequently led to FDA approval, licensing, and availability of pegylated interferon alfa 2b monotherapy in the United States. As in prior interferon studies, genotype non-1 infected patients had higher sustained response rates than those infected with genotype 1, and histologic improvement was also noted in patients treated with pegylated interferons. Dose reductions and dose discontinuations appear to be more common with pegylated interferons, frequently due to neutropenia. In general, although there are slightly higher incidences of side effects and rates of discontinuation, pegylated interferon monotherapies double the sustained response rates compared with their standard interferon counterparts, but they do not decrease relapse and are ineffective in the majority of genotype 1–infected patients. As such, they may be useful alone in patients with contraindications to ribavirin. Although they are more effective than their regular interferon counterparts, they are, in general, not as effective as the combination of interferon and ribavirin and are unlikely to replace this combination. A direct comparison of the efficacy of pegylated interferon alfa-2a to pegylated interferon alfa-2b has not been performed. Dr. Christian Trepo (INSERM, Lyon, France) discussed a recently completed trial of pegylated interferon alfa-2b plus ribavirin involving 1,530 patients.27 The group that received pegylated interferon alfa-2b at a dose of 1.5 g/kg/wk plus ribavirin for 48 weeks had a significantly higher sustained response rate of 54% compared with 47% with either a standard interferon alfa-2b plus ribavirin or a lower dose of pegylated interferon alfa-2b (0.5 g/
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kg/wk) plus ribavirin. The response rate in genotype 1–infected patients treated with the higher dosage of pegylated interferon alfa-2b plus ribavirin was 42% (9% better than with standard combination therapy). Data also indicated that both pegylated interferon alfa-2b and ribavirin should be dosed according to body weight to maximize response rates. The side effect profiles of pegylated interferon alfa-2b plus ribavirin and interferon alfa-2b plus ribavirin were also similar; with no new side effects. There was an increased incidence of flu-like symptoms in the pegylated interferon alfa-2b 1.5 g/kg group. Injection site reactions were more frequently noted with pegylated interferon alfa-2b, but were generally mild and did not limit treatment. Asthenia, cough, and alopecia were also noted more frequently, but there was no greater degree of anemia. Dose reductions for neutropenia with a pegylated interferon alfa-2b 1.5-g/kg regimen was 21% versus 8% for interferon alfa-2b plus ribavirin. Discontinuation rates among the treatment groups were similar (13%14%). As in prior trials, adherence to or compliance with therapy was also associated with enhanced sustained response rates. Overall, 63% of patients who received more than 80% of both pegylated interferon alfa-2b 1.5 g/kg plus ribavirin achieved a sustained response (compared with 54%). A second recent phase III controlled trial evaluated the efficacy of 180 g weekly of pegylated interferon alfa-2a plus ribavirin (1,000-1,200 mg) compared with pegylated interferon alfa-2a alone or interferon alfa-2b plus ribavirin in 1,121 patients.28 The best efficacy was obtained with pegylated interferon alfa-2a plus ribavirin with an overall sustained response rate of 56%. The sustained response rate for genotype 1–infected patients treated with this regimen was 46% (again 9% better than with standard interferon alfa-2b plus ribavirin in this study). Similar to pegylated interferon alfa-2b plus ribavirin, treatment was generally well tolerated with no new adverse events. Rates of discontinuation of therapy with pegylated interferon alfa-2a plus ribavirin were approximately 10%, and similar to standard combination therapy. Thus, the combination of pegylated interferon and ribavirin offers the next available best option and chance of response in patients with chronic hepatitis C infection. While there are differences in the regimens studied to date, they seem comparable and have an acceptable safety profile. These regimens will provide an incremental benefit for suitable patients with chronic hepatitis C.
Mechanism of Actions of Current Therapies Presently, 50% to 70% of patients with hepatitis C do not respond to type 1 interferons, which have pleiotropic antiviral and immunomodulatory actions. Dr. Michael Katze (University of Washington) presented data related to the mechanism of action of interferon in this disease. Available data indicate that the nonstructural NS5A protein likely plays a critical role in viral pathogenesis and may allow HCV to evade the host interferon system.29 Whether this is in part by interacting with and inhibiting interferon-induced protein kinase, GRB-2, or other as yet unknown interactions is yet to be determined. To more carefully elucidate the actual actions of interferons and to develop new generations of antiviral therapies, a global analysis of the total host response to hepatitis C infection and more particularly to interferon therapy will be important. Current techniques using DNA microarray
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techniques, proteomics, and computational biology and merging these technologies with virology and the clinical aspects of hepatitis C should improve our understanding of the mechanisms and pathogenesis of interferon resistance. Ribavirin, a purine nucleoside analog, was discovered in 1972 and first approved for use in severe respiratory syncitial virus infection in 1985 and subsequently for use in combination with ribavirin for patients with hepatitis C in 1998. A trial to establish its efficacy against West Nile virus infection is also in progress. Ribavirin has a broad spectrum of antiviral activities against various RNA and DNA viruses.30 While many hypotheses have been proposed to explain the diverse actions of this drug, the exact mechanism of action in patients with hepatitis C in combination with interferon is unknown. Currently two theories exist: (1) that it has direct antiviral activities targeting the viral polymerase and (2) that the beneficial effects occur through indirect mechanisms including immunomodulation and potentially others. Possible direct antiviral effects of ribavirin are supported by studies that indicate that ribavirin is readily taken up by cells and phosphorylated by cellular kinases to mono-, di-, and triphosphate derivatives. As reviewed by Dr. Zhi Hong (ICN Pharmaceuticals), ribavirin 5⬘ triphosphate, a major metabolite of ribavirin, mimics natural purine nucleotides and also weakly inhibits the HCV RNA– dependent RNA polymerase (RdRp). As tissue and intracellular levels may be quite high, this may lead to direct inhibition of the viral polymerase. Another recent study suggests that ribavirin can act as a mutagen. Ribavirin triphosphate is a substrate for viral RNA polymerases and the monophosphate derivative can be incorporated into viral genomes. Studies measuring mutation rates of poliovirus in the presence of ribavirin show that this rate is increased in a dose-dependent manner. This higher mutation rate leads to “error catastrophe” and diminished viral fitness and infectivity. For poliovirus, treatment of infected cells with high concentrations of ribavirin leads to a 5-log reduction in infectivity. Such observations, while still experimental, provide a conceptual framework for using this therapeutic strategy to reduce viral fitness.31 Both ribavirin and levovirin (a second generation L-isomer of ribavirin) have been shown in tissue culture systems to enhance the type-1 cytokine response and to broaden the HCV-specific cytotoxic T lymphocyte proliferative response.32 Human studies have also indicated that ribavirin induces a type 1 cytokine bias (elevated gamma interferon levels and reduced interleukin 10 levels), which correlates with a favorable response. Taken together, these observations probably indicate that ribavirin is acting at least partially via an immune modulatory effect in chronically infected individuals. A second potential indirect antiviral effect of ribavirin is through its inhibition of inosine monophosphate dehydrogenase (IMPDH), an enzyme that catalyzes a rate-limiting step in guanosine triphosphate biosynthesis. Inhibition of this enzyme leads to depletion of intracellular GTP levels and thus nonspecifically reduces viral RNA synthesis.
New Approaches to Therapy Inhibitors of Virus Replication. One potential therapeutic approach to HCV infection, as described by Dr. Jack Wands (Brown University), involves antisense oligonucleotides, which are short synthetic sequences of 15 to 40 nucleotides stabilized by the
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addition of phosphorothioates to protect these molecules against cellular nuclease degradation or other modifications to enhance binding to target RNAs.33 By hybridizing to RNA (via WatsonCrick base pairing), such compounds can inhibit protein expression by a variety of mechanisms. In vitro data suggest that antisense molecules, designed to target the 5⬘ noncoding region of HCV, exert inhibitory effects on subsequent protein expression, as predicted. A 20 nucleotide phosphorothioate oligonucleotide with a sequence complementary to the HCV translation initiation region is currently undergoing clinical trials in patients with HCV infection who have failed to respond to available antiviral therapies. Preliminary results suggest that at higher doses, some of these refractory patients have viral load reductions of ⱖ1 log HCV RNA after 28 days of therapy. Dr. Lawrence Blatt (Ribozyme Pharmaceuticals) described an alternative approach to therapy that is currently being investigated and involves synthetic nuclease-resistant ribozymes designed to cleave the HCV IRES. These molecules also contain modified nucleotides and phosphorothioate linkages. Cell culture studies using an HCV/poliovirus chimera that contains the HCV IRES have identified ribozymes that target sequences within the IRES and inhibit replication.34 This inhibition is potentiated by the addition of interferon to the system. Cleavage occurs efficiently at the expected site in HCV RNA, and the ribozymes are taken up rapidly by the liver in murine studies. Clinical studies are currently being planned with these ribozymes in patients with chronic HCV infection. While these new molecular approaches provide hope and excitement for treatment of HCV-infected patients, further studies are required to determine the efficacy and safety of such approaches, to determine the precise mechanism of antiviral effects, and to evaluate whether such agents may need to be administered in combination with our available antiviral therapies. The NS3 protein of HCV has long been considered an attractive target for antiviral therapy of hepatitis C. NS3 encodes a serine protease domain in its N-terminal one third and an NTPase/helicase domain in the C-terminal portion. The N-terminal third of NS3 is also an essential component of the NS2-3 autoprotease. The NS3 enzymatic activities are required for HCV replication in vivo. The NS3 serine protease is responsible for cleavage of the HCV polyprotein, specifically of NS4A from NS3 (in cis) and NS4B from NS4A, NS5A from NS4B, and NS5B from NS5A in trans.4 NS4A acts as a cofactor for the serine protease, is found as a stable heterodimer in vivo, and appears to function in part by stabilizing the protease molecule. The protease has a prominent zinc-binding site that stabilizes the molecule. Of the possible mechanisms of inhibition of the NS3 protease, the one that holds the most promise is inhibition of substrate binding. Unfortunately, the substrate binding site of the NS3 protease is long, shallow, and flat in configuration making it difficult to design molecules that would bind to, and inhibit, the protease. Dr. Raffaele de Francesco (I.R.B.M., P. Angeletti, Italy) described his efforts, and those of colleagues at Merck, to identify inhibitors of the NS3 protease. They found that the protease is susceptible to feedback inhibition by the products released from the polyprotein substrate after enzymatic cleavage. He described the structure of molecules based on products of enzymatic cleavage, based on the
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DEMEEC amino acid motif. Stylized molecules based on this amino acid structure were manufactured and shown to inhibit the NS3 protease effectively in vitro. However, as currently configured, these molecules are likely to have poor bioavailability because of their large size. Although the HCV helicase is covalently tethered to the protease, the two enzymes have spatially separate active sites.34 The amino acid motif DEAH or DEXH/D is specific for helicases. HCV NS3 has seven conserved signature motifs characteristic of DEAH or DEXH/D helicases that line the active site of the enzyme in its 3-dimensional structure. It is important to emphasize that the HCV helicase is a mobile molecule and that this movement is critical for its function to unwind double-stranded RNA. Dr. Patricia Weber (Schering-Plough) summarized recent work on the helicase and indicated that helicase inhibitors are under development.35 The 3-dimensional structure of the NS5B RdRp of HCV has been recently solved by several groups and its function studied.36,37 This enzyme target appears to offer great potential for the development of new antiviral agents. Nonetheless, as discussed by Dr. C. Cheng Kao (Indiana University), the RdRp has very complex functions. Evidence from other viral systems suggests that it functions as part of a replicase (a multiunit complex, usually membrane-associated, which may contain host factors as well as the helicase). This complex binds substrate RNA, often resulting in a conformational change in the RdRp. RNA strand elongation begins with a nucleotide triphosphate rather than a primer. Initiation is followed by elongating RNA synthesis, termination of synthesis, and release of nascent RNA. To date, the search for candidate inhibitors of the RdRp has focused on the elongation reaction using biochemical screens. The efficacy of inhibitors is now being evaluated in cells using the HCV replicon model system, and promising compounds will undergo testing in animals for bioavailability and toxicity before being tested in humans. Strategies to Minimize Hepatic Injury and Fibrosis. The ultimate goal of antiviral therapy for hepatitis C has long been the elimination of HCV from serum and liver during and after therapy. However, it must be recognized that it is not the viral infection per se that must be treated but the liver disease that results from chronic infection. This recognition has led to consideration of therapies that are aimed at minimizing hepatic injury and fibrosis. For example, interferon alfa therapy results in improvement in liver histopathology in patients with chronic hepatitis C, even if they do not experience a sustained virologic response. Thus, in most patients who do not clear the virus from serum, a decrease can be seen in the degree of necroinflammatory change in the liver. It has also been suggested that interferon alfa therapy may decrease or slow progression of hepatic fibrosis. This approach would probably require long-term therapy, and there are few data available of the benefits of long-term therapy with interferon alfa. A decrease in hepatic necrosis, inflammation, and even fibrosis has been noted in patients with chronic hepatitis C treated for 3 years with interferon.38 Dr. Adrian Di Bisceglie (Saint Louis University) described the NIH-funded HALT-C trial, which aims to study the effects of long-term therapy (for 4 years) with pegylated interferon alfa in patients with advanced hepatic fibrosis who are at greatest risk for development of cirrhosis and its complications. In this trial ap-
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proximately 900 patients who are nonresponders to retreatment with a 6-month course of pegylated interferon and ribavirin will be randomized to receive interferon alone for an additional 3.5 years or to serve as an untreated control. The end points of this study are the development of cirrhosis on liver biopsy or the development of hepatic decompensation or hepatocellular carcinoma. A similar clinical trial, dubbed the COPILOT study, is also underway and is aimed at examining the role of pegylated interferon for prolonged therapy compared with a colchicine-treated control group. The Liver Diseases Section of the National Institute of Diabetes and Digestive and Kidney Diseases was among the first to study ribavirin for chronic hepatitis C. Using ribavirin by itself, they found that about one third of patients experience a return to normal of serum aminotransferases, associated with improvement in the degree of necroinflammatory activity on liver biopsy.39 Dr. Marc Ghany (National Institute of Diabetes and Digestive and Kidney Diseases) provided an update on the status of studies with ribavirin as monotherapy in chronic hepatitis C. He described a study in which nonresponders to interferon and ribavirin were randomly allocated to either ribavirin or placebo for 48 weeks. After this period of time, the histologic activity index had decreased significantly in those treated with ribavirin but not those on placebo. Furthermore, the fibrosis score was unchanged in those on ribavirin but had increased in placebo recipients, suggesting that ribavirin may prevent progression of fibrosis. Other therapies aimed at preventing or treating fibrosis were also reviewed. Although there are many agents with the potential to minimize fibrosis through antioxidant activity, few had been thoroughly tested in controlled clinical trials. A pilot study suggested that interleukin-10 (IL-10) may decrease hepatic fibrosis in patients with hepatitis C.40 Dr. Gary Davis (University of Florida) reported on a follow-up to that pilot study involving individuals with chronic hepatitis C and bridging hepatic fibrosis or cirrhosis who were treated with IL-10 for 12 months. Over this time, serum aminotransferase levels decreased in association with a decrease in hepatic inflammation. Only 4 of 27 treated patients had a decrease in the degree of hepatic fibrosis. Furthermore, levels of HCV RNA in serum tended to rise, by as much as 3-fold, when the period of IL-10 therapy was extended beyond 3 or 4 months. IMPDH Inhibitors. The development of specific inhibitors of the enzyme IMPDH may provide an alternative treatment for patients with chronic HCV infection when combined with interferon. This enzyme, which is also inhibited by ribavirin, is essential for the modulation of host cellular pathways. Inhibition of this enzyme, which is responsible for catalyzing an essential step in the de novo biosynthesis of guanosine nucleotides, leads to depletion of intracellular GTP levels. Other inhibitors of IMPDH include mycophenolic acid and mizoribine monophosphate, a drug approved in Japan for certain autoimmune diseases. Dr. Anne Kwong (Vertex Pharmaceuticals) summarized data on VX-497, a small orally bioavailable drug that specifically inhibits IMPDH and has broad spectrum antiviral activity including activity against HCV. In terms of IMPDH inhibition, the non-nucleoside VX-497 is approximately 20-fold more potent than ribavirin. A number of early phase I and II studies in patients with chronic hepatitis C have suggested ALT reductions in patients receiving VX-497, similar to earlier studies with ribavirin monotherapy.
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Trials combining VX-497 with interferon suggest a similar clinical effect, although data regarding long-term sustained response rates are unavailable at this time. Further development of more potent and specific IMPDH inhibitors including VX-497 will require randomized controlled clinical trials to determine their efficacy and future place in the management of patients with chronic hepatitis C infection.
References 1. Wasley A, Alter MJ. Epidemiology of hepatitis C: Geographic differences and temporal trends. Semin Liver Dis 2000;20:1-13. 2. Di Bisceglie AM. Natural history of hepatitis C: its impact on clinical management. HEPATOLOGY 2000;31:1014-1018. 3. Keeffe EB. Liver transplantation: current status and novel approaches to liver replacement. Gastroenterology 2001;120:749-762. 4. Liang TJ, Thomson M. Molecular biology of hepatitis C virus. In, Liang TJ, Hoofnagle JH, eds. Hepatitis C. San Diego: Academic Press, 2000;1-24. 5. Rijnbrand R, Bredenbeek PJ, Haasnoot PC, Kieft JS, Spaan WJ, Lemon SM. The influence of downstream protein-coding sequence on internal ribosome entry on hepatitis C virus and other flavivirus RNAs. RNA 2001;7:585-597. 6. Walewski JL, Keller TR, Stump DD, Branch AD. Evidence for a new hepatitis C virus antigen encoded in an overlapping reading frame. RNA 2001;7: 710-721. 7. Xu Z, Choi J, Yen TS, Lu W, Strohecker A, Govindarajan S, Chien D, Selby MJ, Ou, J. Synthesis of a novel hepatitis C virus protein by ribosomal frameshift. EMBO J 2001;20:3840-3848. 8. McHutchison JG, Gordon SC, Schiff ER, Shiffman ML, Lee WM, Rustgi VK, Goodman ZD, et al. Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. N Engl J Med 1998;339:1485-1492. 9. Farci P, Shimoda A, Coiana A, Diaz G, Peddis G, Melpolder JC, Strazzera A, et al. The outcome of acute hepatitis C predicted by the evolution of the viral quasispecies. Science 2000;288:339-344. 10. Neumann AU, Lam NP, Dahari H, Gretch DR, Wiley TE, Layden TJ, Perelson AS. Hepatitis C viral dynamics in vivo and the antiviral efficacy of interferon-alpha therapy. Science 1998;282:103-107. 11. Hadlock KG, Lanford RE, Perkins S, Rowe J, Yang Q, Levy S, Pileri P, Abrignani S, Foung SK. Human monoclonal antibodies that inhibit binding of hepatitis C virus E2 protein to CD81 and recognize conserved conformational epitopes. J Virol 2000;74:10407-10416. 12. Lohmann V, Korner F, Koch J, Herian U, Theilmann L, Bartenschlager R. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 1999;285:110-113. 13. Blight KJ, Kolykhalov AA, Rice CM. Efficient initiation of HCV RNA replication in cell culture. Science 2000;290:1972-1974. 14. Mercer DF, Schiller DE, Elliott JF, Douglas DN, Hao C, Rinfret A, Addison WR, et al. Hepatitis C virus replication in mice with chimeric human livers. Nat Med 2001;7:927-933. 15. Forns X, Thimme R, Govindarajan S, Emerson SU, Purcell RH, Chisari FV, Bukh J. Hepatitis C virus lacking the hypervariable region 1 of the second envelope protein is infectious and causes acute resolving or persistent infection in chimpanzees. Proc Natl Acad Sci U S A 2000;97:13318-13323. 16. Bassett SE, Guerra B, Brasky K, Miskovsky E, Houghton M, Klimpel GR, Lanford RE. Protective immune response to hepatitis C virus in chimpanzees rechallenged following clearance of primary infection. HEPATOLOGY 2001;33:1479-1487. 17. Weiner AJ, Paliard X, Selby MJ, Medina-Selby A, Coit D, Nguyen S, Kansopon J, et al. Intrahepatic genetic inoculation of hepatitis C virus
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RNA confers cross- protective immunity. J Virol 2001;75:71427148. Bukh J, Apgar CL, Yanagi M. Toward a surrogate model for hepatitis C virus: an infectious molecular clone of the GB virus-B hepatitis agent. Virology 1999;262:470-478. He XS, Rehermann B, Boisvert J, Mumm J, Maecker HT, Roederer M, Wright TL, et al. Direct functional analysis of epitope-specific CD8⫹ T cells in peripheral blood. Viral Immunology 2001;14:5969. Seeff LB, Zimmerman HJ, Wright EC, Finkelstein JD, Garcia-Pont P, Greenlee HB, Dietz AA, et al. A randomized, double blind controlled trial of the efficacy of immune serum globulin for the prevention of post-transfusion hepatitis. A Veterans Administration cooperative study. Gastroenterology 1977;72:111-121. Sanchez-Quijano A, Pineda JA, Lissen E, Leal M, Diaz-Torres MA, Garcia De Pesquera F, Rivera F, et al. Prevention of post-transfusion non-A, non-B hepatitis by non-specific immunoglobulin in heart surgery patients. Lancet 1988;1:1245-1249. Maddrey WC. Safety of combination interferon alfa-2b/ribavirin therapy in chronic hepatitis C-relapsed and treatment-naive patients. Semin Liver Dis 1999;19(Suppl 1):67-75. Glue P, Fang JWS, Rouzier-Panis R, Raffanel C, Sabo R, Gupta SK, Salfi M, Jacobs S. Pegylated interferon-␣2b: pharmacokinetics, pharmacodynamics, safety, and preliminary efficacy data. Clin Pharmacol Ther 2000;68:556-567. Zeuzem S, Feinman SV, Rasenack J, Heathcote EJ, Lai M-Y, Gane E, O’Grady J, et al. Peginterferon alfa-2a in patients with chronic hepatitis C. N Engl J Med 2000;343:1666-1672. Heathcote EJ, Shiffman ML, Cooksley WGE, Dusheiko GM, Lee SS, Balart L, Reindollar R, et al. Peginterferon alfa-2a in patients with chronic hepatitis C and cirrhosis. N Engl J Med 2000;343:16731680. Lindsay KL, Trepo C, Heintges T, Shiffman ML, Gordon SC, Hoefs JC, Schiff ER, et al. A randomized, double blind trial comparing PEGylated interferon alfa-2b to interferon alfa-2b as initial treatment for chronic hepatitis C. HEPATOLOGY 2001;34:395-403. Manns MP, McHutchison JG, Gordon SC, Rustgi VK, Shiffman M, Reindollar R, Goodman ZD, et al. Peginterferon alfa-2b plus ribavirin compared with interferonalfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomized trial. Lancet 2001;358: 958-965. Fried MW, Shiffman ML, Reddy K, Smith C, Marino G, Goncales F, Haeussinger D, et al. Pegylated (40 kDa) interferon alfa-2a (PEGASYS) in combination with ribavirin: efficacy and safety results from a phase II, randomized, actively-controlled, multicenter study [Abstract]. Gastroenterology 2001;120:A-55. Tan SL, Katze MG. How hepatitis C virus counteracts the interferon response: the jury is still out on NS5A. Virology 2001;284:1-12. Patterson JL, Fernandez-Larson R. Molecular mechanisms of action of ribavirin. Rev Infect Dis 1990;12:1139-1146. Crotty S, Cameron CE, Andino R. RNA virus error catastrophe: direct molecular test by using ribavirin. Proc Natl Acad Sci U S A 2001;98:6895-6900. Tam RC, Pai B, Bard J, Lim C, Averett DR, Phan UT, Milovanovic T. Ribavirin polarizes human T cell responses towards a type 1 cytokine profile. J Hepatol 1999;30:376-382. Wakita T, Moradpour D, Tokushihge K, Wands JR. Antiviral effects of antisense RNA on hepatitis C virus RNA translation and expression. J Med Virol 1999;57:217-222. Usman N. Blatt LM. Nuclease-resistant synthetic ribozymes: developing a new class of therapeutics. J Clin Invest 2000;106:1197-1202. Yao N, Reichert P, Taremi SS, Prosise WW, Weber PC. Molecular views of viral polyprotein processing revealed by the crystal structure
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of the hepatitis C virus bifunctional protease-helicase. Structure 1999;7:1353-1363. 36. Lesburg CA, Cable MB, Ferrari E, Hong Z, Mannarino AF, Weber PC. Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site. Nat Struc Biol 1999;6:937-943. 37. Oh J-W, Ito T, Lai MMC. A recombinant hepatitis C virus RNAdependent RNA polymerase capable of copying the full-length viral RNA. J Virol 1999;73:7694-7702. 38. Shiffman ML, Hofmann CM, Contos MJ, Luketic VA, Sanyal AJ, Sterling RK, Ferreira-Gonzalez A, et al. A randomized, controlled
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trial of maintenance interferon therapy for patients with chronic hepatitis C virus and persistent viremia. Gastroenterology 1999;117: 1164-1172. 39. Di Bisceglie AM, Conjeevaram HS, Fried MW, Sallie R, Park Y, Yurdaydin C, Swain M, et al. Ribavirin as therapy for chronic hepatitis C: a randomized, double-blind, placebo-controlled trial. Ann Intern Med 1996;123:897-903. 40. Nelson DR, Lauwers GY, Lau JY, Davis GL. Interleukin 10 treatment reduces fibrosis in patients with chronic hepatitis C: a pilot trial of interferon nonresponders. Gastroenterology 2000;118: 655-660.
V
iral hepatitis is the single most important cause of liver disease in the United States and worldwide. With this in mind, the AASLD has launched an annual single topic conference devoted exclusively to viral hepatitis with the intention of bringing together researchers in viral hepatitis to foster progress in this important area. The first of these conferences, held June 15-16, 2001 in Chicago, IL, focused on new, evolving, and future approaches to therapy of hepatitis C. Dr. Eugene Schiff (University of Miami) reviewed the history and impact of hepatitis C. It appears that the hepatitis C virus (HCV) emerged in the U.S. population beginning in the 1960s, related to blood transfusion and injection drug use, although the extent of the problem was only apparent after 1990 when reliable blood tests first became available for hepatitis C.1 Studies of the natural history have been somewhat contradictory but indicate that over the first 20 years of chronic HCV infection, 20% of chronically infected patients will develop cirrhosis, and many of those will progress to hepatocellular carcinoma.2 HCV-associated end-stage liver disease is now recognized as a leading indication for liver transplantation in the United States and the developed western world.3
Virology Dr. Charles Rice (The Rockefeller University) described the virology of HCV; it has an RNA genome about 10,000 nucleotides in length.4 A hallmark of this RNA is the presence of a long open reading frame (ORF) that encodes a polyprotein that is processed into at least 10 discrete polypeptides (Fig. 1). Translation initiation is mediated by the internal ribosome entry site (IRES). Progress made in understanding HCV IRES structure and function was reviewed by Dr. Stanley Lemon (University of Texas at Galveston). The HCV IRES resides within the 5⬘ nontranslated region between nucleotides 44 and 342. Key structural features include two large stem-loops and a pseudoknot that involves sequences near the initiator codon. These structures have been deduced from thermo-
Abbreviations: HCV, hepatitis C virus; ORF, open reading frame; IRES, internal ribosome entry site; NS, nonstructural; HCIG, hyperimmune C immunoglobulin; HBIG, hyperimmune B immunoglobulin; PEG, polyethylene glycol; RdRp, RNA– dependent RNA polymerase; IMPDH, inosine monophosphate dehydrogenase; IL-10, interleukin 10. From 1Saint Louis University, St. Louis MO; 2Scripps Clinic, La Jolla, CA; and 3The Rockefeller University, New York, NY. Received August 24, 2001; accepted October 29, 2001. This is a report of the first AASLD Hepatitis Single Topic Conference, held in Chicago, June 15 and 16, 2001. Address reprint requests to: Adrian M. Di Bisceglie, M.D., Division of Gastroenterology and Hepatology, Department of Internal Medicine, Saint Louis University School of Medicine, 3635 Vista Avenue, St. Louis, MO 63110. E-mail: [email protected]; fax: 314-577-8125. Copyright © 2002 by the American Association for the Study of Liver Diseases. 0270-9139/02/3501-0030$35.00/0 doi:10.1053/jhep.2002.30531 224
dynamic modelling, phylogenetic analysis, chemical and enzymatic structure probing, X-ray crystallography, and nuclear magnetic resonance. The IRES forms a binary complex with the 40S ribosomal subunit in the absence of other translation initiation factors (unlike other viruses).5 This high-affinity complex results in a conformational change in the 40S subunit and binding of eukaryotic initiation factor 3. Via this process, the AUG initiator codon of the viral RNA is positioned near the ribosome decoding site. A specific sequence is not then required to initiate translation, but a stem loop must “melt” to allow this process to proceed. This complex viral RNA element could thus be a rich target for compounds that inhibit translation. Processing of the polyprotein is mediated by the cellular enzyme, host signal peptidase, and two viral proteases, the NS2-3 autoprotease and the NS3-4A serine protease. This results in the production of the core or capsid protein (C), two envelope glycoproteins (E1 and E2), a small hydrophobic protein (p7), and 6 nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS6A, and NS5B).4 Although it was thought that this was the only ORF in the HCV genome, two laboratories have recently reported the existence of an alternative ORF that overlaps the C protein gene.6,7 This protein, termed ARFP (for alternative reading frame protein) or F (for frameshift protein) appears to be expressed during HCV infection because some patients are seropositive for antibodies to this antigen. The function of ARFP/F is unknown but it is not required for RNA replication in cell culture. Numerous studies have examined HCV diversity and its possible importance in clinical progression. HCV RNA replication is error prone because of the lack of proofreading by the viral RNA polymerase. Four hierarchical strata of HCV diversity have been identified: genotypes, subtypes, isolates, and quasispecies. Patients infected with HCV genotype 1 clearly have a poorer response to current antiviral therapy than those with genotype 2 or 3.8 By contrast, the clinical significance of HCV genotypes in the natural history of hepatitis C is controversial. Dr. Patrizia Farci (University of Cagliari, Italy) presented data supporting a role for HCV quasispecies variability in determining (or correlating with) the outcome of HCV infection. Increased viral diversity during the acute phase of hepatitis was found to be associated with progression to chronicity whereas resolving acute hepatitis was associated with relative stasis of the quasispecies.9 Patients who respond to interferon therapy also have a decrease in viral genetic diversity and in the number of viral quasispecies. HCV replicates in hepatocytes and perhaps other cell types. Studies of viral dynamics after antiviral treatment or in the acute phase of infection indicate high turnover and a production rate in the range of 1012 virus particles per day.10 The mechanisms of HCV attachment and entry are still not known. As discussed by Dr. Sergio Abrignani (Chiron Corporation, Emeryville, CA), the HCV E2 protein binds avidly to CD81, a tetraspanin molecule
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Fig. 1. The horizontal strip indicates the structural and nonstructural proteins encoded by the HCV genome (c, capsid; E, envelope; NS, nonstructural). The X-ray crystallographic structures of specific HCV enzymes have also been identified as shown (not to scale). The HCV protease encoded within the genome requires NS2 to NS4 for full activity. HCV helicase is also a component of the NS3 protein. NS5b encodes an RNA-dependent RNA polymerase. The unique molecular structures of these specific viral enzymes should allow the identification and development of specific and potent inhibitors. As in other viral diseases, once developed and if given alone, rapid viral resistance may develop to each of these future compounds. Combination therapeutic approaches will probably be required to prevent the emergence of rapid viral resistance.
found on the surface of many cell types including hepatocytes.11 Recent data indicate that human CD81 by itself may not be sufficient to mediate virus attachment and entry. Instead it may serve only to attach HCV to the surface of cells allowing subsequent interaction with a more specific entry receptor.
Animal Models and Cell Culture Systems The study of HCV replication and the development of new therapies have been greatly advanced by the recent development of cell culture models for HCV. At present, the most effective of these models is the replicon system.12 The prototype replicon was a subgenomic HCV RNA in which the HCV structural region was replaced by the neomycin phosphotransferase gene, and translation of the HCV proteins NS3 to NS5B was directed by the IRES from encephalomyocarditis virus. The Huh-7 hepatoma cell line has been found to support replication of this replicon. Easily measurable levels of HCV RNA are produced and replication can be inhibited by antivirals such as interferon alfa.13 However, there are still several unexplained paradoxes associated with this system. Replication is thus far restricted to Huh7 cells and does not occur in other human hepatoma cell lines. Efforts to establish functional replicons for genotypes 1a and 2a have not met with success. In the case of the subtype 1b replicons, limited changes including even single amino acid substitutions can increase the efficiency of replication initiation by more than 100,000-fold. Such adaptive mutations tend to cluster in the NS5A protein but can also be found in NS3, NS4B, and NS5B. These advances provide a system for molecular genetic studies on HCV replication and facilitate rapid modification of replicons, via genetic engineering, to tailor assays for high throughput cell-based drug screening. The holy grail of HCV cell culture remains the establishment of a robust replication
system that includes virus assembly, release, and infection of naive cells. Dr. Keril Blight (The Rockefeller University) presented new data showing that full-length HCV genotype 1b RNAs harboring adaptive mutations could also replicate in Huh7 cells, albeit at lower levels than the replicon. Whether particle assembly, release, and spread occur in this system remains to be established. Dr. Jens Bukh (National Institute of Allergy and Infectious Diseases) reviewed progress using infectious cDNA clones to explore HCV biology in the chimpanzee model. Until recently,14 this was the only robust animal model supporting HCV replication. Functional cDNA clones exist for genotypes 1a, 1b, and 2a, and chimp-titered challenge stocks have been developed for most of the common HCV genotypes. Genetic studies using infectious clones have shown the essential nature of the HCV enzymes and the conserved elements of the 3⬘ nontranslated region. Remarkably, the E2 hypervariable region (HVR1) could be deleted without abrogating infectivity, although the mutant virus replicated poorly and compensating changes in E1 and E2 were selected upon passaging.15 On the vaccine front, challenge of animals with resolved HCV infection using homologous or heterologous virus has provided evidence for protective immunity. This is an important proof of principle for vaccine development.16,17 Dr. Mark Kay (Stanford University) described gene therapy approaches and methods for efficient RNA transfection of mouse hepatocytes in vivo. Using the latter method, no evidence was obtained for replication of chimpanzee infectious HCV RNAs in liver resident mouse hepatocytes. This suggests a requirement for human/chimpanzee-specific intracellular factors in one or more steps of the HCV replication cycle. As HCV’s closest relative, GB virus-B is being explored as a surrogate model. Molecular clones infectious in tamarins have
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been developed as have cultures of primary tamarin hepatocytes that support a complete cycle of virus replication.18
Immunology The immune response to HCV appears to be critical both in determining viral clearance or persistence and in contributing to liver injury. Dr. Barbara Rehermann (National Institute of Diabetes and Digestive and Kidney Diseases) pointed out that the frequency of HCV specific cytotoxic T cells is low, representing only 0.01% to 0.5% of circulating CD8 positive T cells.19 The proportion of HCV-specific T cells is substantially higher (about 30-fold) within the liver. Recovery from HCV infection is associated with an increased T-cell proliferative response whereas chronic infection is marked by impaired production of gamma interferon by lymphocytes. These functionally impaired CD8-positive T cells are CD27 positive, but CD28 negative and appear to suffer from lack of CD4 T helper activity. Dr. Christopher Walker (Children’s Hospital Research Foundation, Columbus, OH) noted an important recent observation common to both human and chimpanzee studies—that mutations within recognized HCV T-cell epitopes may allow HCV variants to escape immune recognition and contribute to persistence. The humoral response to HCV has not been well studied but investigation of the therapeutic use of immune serum globulin and hyperimmune C immunoglobulin (HCIG) suggests that it may also play an important role in HCV clearance. Historical studies in humans have shown that both immune serum globulin and hyperimmune B immunoglobulin (HBIG) prepared before elimination of anti-HCV positive blood reduce both the incidence and severity of post-transfusion non A, non B hepatitis.20,21 Dr. Krystof Krawczynki (Centers for Disease Control and Prevention) described chimpanzee studies utilizing HCIG conducted at the Centers for Disease Control and Prevention. Early studies showed that a single dose of HCIG delayed the onset of HCV infection and was associated with a reduction in the level of HCV viremia in the chimpanzee animal model. More recent studies have used HCIG prepared from as many as 500 HCV seropositive donors, all with normal serum aminotransferases. The plasma was purified by detergent treatment, ethanol fractionation, and ultrafiltration. Chronic administration of this preparation over a 15-week period was associated with a significant reduction in HCV titers in 2 of 3 chimpanzees tested. The optimal use for this type of product would be for postexposure prophylaxis and perhaps to prevent recurrence of hepatitis C after liver transplantation, as is done with HBIG after transplantation for hepatitis B.
Current Optimal Therapy The most effective and available initial therapy for these patients is the combination of interferon and ribavirin (Fig. 2). Dr. John McHutchison (Scripps Clinic) noted that the sustained virologic response rate with this regimen is approximately 35% to 40% after a 24- or 48-week course of this therapy, respectively.8 The 2-drug regimen increases the number of patients who clear HCV RNA during treatment, and decreases the number of patients who relapse. In general, patients with genotype 1 infection should receive 48 weeks of therapy and those with genotype non-1 infection, only 24 weeks. Patients with genotype 2 or 3, with low viral
Fig. 2. Sustained virologic response rates reported from key studies that evaluated the effect of interferon and ribavirin in therapy of chronic hepatitis C. Study A: Comparison of interferon alfa-2b with ribavirin versus interferon alfa-2b alone, both for 48 weeks (McHutchison et al.8). Study B: pegylated interferon alfa-2b versus interferon alfa-2b (Lindsay et al.26). Study C: pegylated interferon alfa-2a versus interferon alfa-2a (Zeuzem et al.24). Study D: pegylated interferon alfa-2b with ribavirin versus interferon alfa-2b with ribavirin (Manns et al.27). Study E: pegylated interferon alfa-2a with ribavirin versus interferon alfa-2b with ribavirin (Fried et al.28). In each case, the study group mentioned first is represented by the black bar.
load, who are less than 40 years of age, who are women, and who do not have advanced fibrosis are most likely to respond. Lack of response can be reliably predicted at week 24 of therapy, such that genotype 1–infected patients who have not responded and cleared virus at this time can cease treatment, as less than 2% of these patients will achieve a sustained response. Studies of the predictive value of week 12 responses are underway. Side effects more frequently observed when ribavirin is combined with interferon include cough, dyspnea, rash, itch, insomnia, and nausea. Ribavirin also accumulates in red blood cells and causes a dose-dependant hemolytic anemia of moderate severity that requires dose reduction in 7% to 10% of patients.22 Despite these side effects and the greater proportion of patients who require discontinuation or modification of therapy, combination therapy with interferon has also been shown to be the most cost-effective approach to therapy. Although the optimal dose of ribavirin is currently unknown, available data suggest that higher doses increase efficacy (albeit with a greater degree of anemia). The dose of 800 mg/d may be the most appropriate lower dose for those patients who require dosage modification for anemia or other side effects. As in human immunodeficiency virus therapy, compliance or adherence during combination therapy also enhances response rates. Patients who achieve a sustained virologic response have been shown to have improvements in their quality of life, continued improvement in histology, and durable responses lasting 3 or more years in over 95% of these patients. Efficacy of drugs is often limited by the relatively short time that they are present in circulation or the target tissue at inhibitory concentrations. Dr. Marlene Modi (Hoffmann La Roche, Inc.) described the pharmacology of pegylated interferon. Addition of a polyethylene glycol (PEG) molecule to a protein results in a product with better physical and thermal stability and that is protected against rapid enzyme degradation.23 This results in a biologically
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active drug with a more convenient longer half-life and in general, more favorable pharmacokinetics. Early attempts to develop pegylated interferons were hampered by compounds with relatively short half-lives with little if any enhancement in efficacy. The subsequently developed pegylated interferons alfa-2a and alfa-2b were initially studied in randomized clinical trials as monotherapy. Although these two different pegylated interferons differ in their structural properties (chain size and type, linkage, amino acid sequence, attachment site, half-lives, and dose) both have been shown to be more effective than their unmodified interferon counterparts. Dr. Mitchell Shiffman (Medical College of Virginia) described clinical trials conducted with pegylated interferons alone. Initial phase II dose-finding studies with both forms of pegylated interferon identified subsequent doses for phase III registration trials. pegylated interferon alfa-2a has been administered to patients with chronic hepatitis C with elevated liver tests and shown to enhance response rates from 19% to 39%.24 In another study of patients with cirrhosis, sustained response was observed in 30% of patients (180 g injected subcutaneously once a week for a 48-week treatment period).25 Like standard interferons, histologic improvement and biochemical improvement were observed in both of these studies, and it was noted that the side effect profile was similar to standard interferons apart from a greater incidence of neutropenia, which more frequently required dose reductions. A large phase III randomized controlled trial of pegylated interferon alfa-2b in similar patients with chronic hepatitis C also indicated a higher sustained response rate in patients receiving 1.0 g/kg/wk or 1.5 g/kg/wk compared with standard interferon administered for 48 weeks.26 This has subsequently led to FDA approval, licensing, and availability of pegylated interferon alfa 2b monotherapy in the United States. As in prior interferon studies, genotype non-1 infected patients had higher sustained response rates than those infected with genotype 1, and histologic improvement was also noted in patients treated with pegylated interferons. Dose reductions and dose discontinuations appear to be more common with pegylated interferons, frequently due to neutropenia. In general, although there are slightly higher incidences of side effects and rates of discontinuation, pegylated interferon monotherapies double the sustained response rates compared with their standard interferon counterparts, but they do not decrease relapse and are ineffective in the majority of genotype 1–infected patients. As such, they may be useful alone in patients with contraindications to ribavirin. Although they are more effective than their regular interferon counterparts, they are, in general, not as effective as the combination of interferon and ribavirin and are unlikely to replace this combination. A direct comparison of the efficacy of pegylated interferon alfa-2a to pegylated interferon alfa-2b has not been performed. Dr. Christian Trepo (INSERM, Lyon, France) discussed a recently completed trial of pegylated interferon alfa-2b plus ribavirin involving 1,530 patients.27 The group that received pegylated interferon alfa-2b at a dose of 1.5 g/kg/wk plus ribavirin for 48 weeks had a significantly higher sustained response rate of 54% compared with 47% with either a standard interferon alfa-2b plus ribavirin or a lower dose of pegylated interferon alfa-2b (0.5 g/
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kg/wk) plus ribavirin. The response rate in genotype 1–infected patients treated with the higher dosage of pegylated interferon alfa-2b plus ribavirin was 42% (9% better than with standard combination therapy). Data also indicated that both pegylated interferon alfa-2b and ribavirin should be dosed according to body weight to maximize response rates. The side effect profiles of pegylated interferon alfa-2b plus ribavirin and interferon alfa-2b plus ribavirin were also similar; with no new side effects. There was an increased incidence of flu-like symptoms in the pegylated interferon alfa-2b 1.5 g/kg group. Injection site reactions were more frequently noted with pegylated interferon alfa-2b, but were generally mild and did not limit treatment. Asthenia, cough, and alopecia were also noted more frequently, but there was no greater degree of anemia. Dose reductions for neutropenia with a pegylated interferon alfa-2b 1.5-g/kg regimen was 21% versus 8% for interferon alfa-2b plus ribavirin. Discontinuation rates among the treatment groups were similar (13%14%). As in prior trials, adherence to or compliance with therapy was also associated with enhanced sustained response rates. Overall, 63% of patients who received more than 80% of both pegylated interferon alfa-2b 1.5 g/kg plus ribavirin achieved a sustained response (compared with 54%). A second recent phase III controlled trial evaluated the efficacy of 180 g weekly of pegylated interferon alfa-2a plus ribavirin (1,000-1,200 mg) compared with pegylated interferon alfa-2a alone or interferon alfa-2b plus ribavirin in 1,121 patients.28 The best efficacy was obtained with pegylated interferon alfa-2a plus ribavirin with an overall sustained response rate of 56%. The sustained response rate for genotype 1–infected patients treated with this regimen was 46% (again 9% better than with standard interferon alfa-2b plus ribavirin in this study). Similar to pegylated interferon alfa-2b plus ribavirin, treatment was generally well tolerated with no new adverse events. Rates of discontinuation of therapy with pegylated interferon alfa-2a plus ribavirin were approximately 10%, and similar to standard combination therapy. Thus, the combination of pegylated interferon and ribavirin offers the next available best option and chance of response in patients with chronic hepatitis C infection. While there are differences in the regimens studied to date, they seem comparable and have an acceptable safety profile. These regimens will provide an incremental benefit for suitable patients with chronic hepatitis C.
Mechanism of Actions of Current Therapies Presently, 50% to 70% of patients with hepatitis C do not respond to type 1 interferons, which have pleiotropic antiviral and immunomodulatory actions. Dr. Michael Katze (University of Washington) presented data related to the mechanism of action of interferon in this disease. Available data indicate that the nonstructural NS5A protein likely plays a critical role in viral pathogenesis and may allow HCV to evade the host interferon system.29 Whether this is in part by interacting with and inhibiting interferon-induced protein kinase, GRB-2, or other as yet unknown interactions is yet to be determined. To more carefully elucidate the actual actions of interferons and to develop new generations of antiviral therapies, a global analysis of the total host response to hepatitis C infection and more particularly to interferon therapy will be important. Current techniques using DNA microarray
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techniques, proteomics, and computational biology and merging these technologies with virology and the clinical aspects of hepatitis C should improve our understanding of the mechanisms and pathogenesis of interferon resistance. Ribavirin, a purine nucleoside analog, was discovered in 1972 and first approved for use in severe respiratory syncitial virus infection in 1985 and subsequently for use in combination with ribavirin for patients with hepatitis C in 1998. A trial to establish its efficacy against West Nile virus infection is also in progress. Ribavirin has a broad spectrum of antiviral activities against various RNA and DNA viruses.30 While many hypotheses have been proposed to explain the diverse actions of this drug, the exact mechanism of action in patients with hepatitis C in combination with interferon is unknown. Currently two theories exist: (1) that it has direct antiviral activities targeting the viral polymerase and (2) that the beneficial effects occur through indirect mechanisms including immunomodulation and potentially others. Possible direct antiviral effects of ribavirin are supported by studies that indicate that ribavirin is readily taken up by cells and phosphorylated by cellular kinases to mono-, di-, and triphosphate derivatives. As reviewed by Dr. Zhi Hong (ICN Pharmaceuticals), ribavirin 5⬘ triphosphate, a major metabolite of ribavirin, mimics natural purine nucleotides and also weakly inhibits the HCV RNA– dependent RNA polymerase (RdRp). As tissue and intracellular levels may be quite high, this may lead to direct inhibition of the viral polymerase. Another recent study suggests that ribavirin can act as a mutagen. Ribavirin triphosphate is a substrate for viral RNA polymerases and the monophosphate derivative can be incorporated into viral genomes. Studies measuring mutation rates of poliovirus in the presence of ribavirin show that this rate is increased in a dose-dependent manner. This higher mutation rate leads to “error catastrophe” and diminished viral fitness and infectivity. For poliovirus, treatment of infected cells with high concentrations of ribavirin leads to a 5-log reduction in infectivity. Such observations, while still experimental, provide a conceptual framework for using this therapeutic strategy to reduce viral fitness.31 Both ribavirin and levovirin (a second generation L-isomer of ribavirin) have been shown in tissue culture systems to enhance the type-1 cytokine response and to broaden the HCV-specific cytotoxic T lymphocyte proliferative response.32 Human studies have also indicated that ribavirin induces a type 1 cytokine bias (elevated gamma interferon levels and reduced interleukin 10 levels), which correlates with a favorable response. Taken together, these observations probably indicate that ribavirin is acting at least partially via an immune modulatory effect in chronically infected individuals. A second potential indirect antiviral effect of ribavirin is through its inhibition of inosine monophosphate dehydrogenase (IMPDH), an enzyme that catalyzes a rate-limiting step in guanosine triphosphate biosynthesis. Inhibition of this enzyme leads to depletion of intracellular GTP levels and thus nonspecifically reduces viral RNA synthesis.
New Approaches to Therapy Inhibitors of Virus Replication. One potential therapeutic approach to HCV infection, as described by Dr. Jack Wands (Brown University), involves antisense oligonucleotides, which are short synthetic sequences of 15 to 40 nucleotides stabilized by the
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addition of phosphorothioates to protect these molecules against cellular nuclease degradation or other modifications to enhance binding to target RNAs.33 By hybridizing to RNA (via WatsonCrick base pairing), such compounds can inhibit protein expression by a variety of mechanisms. In vitro data suggest that antisense molecules, designed to target the 5⬘ noncoding region of HCV, exert inhibitory effects on subsequent protein expression, as predicted. A 20 nucleotide phosphorothioate oligonucleotide with a sequence complementary to the HCV translation initiation region is currently undergoing clinical trials in patients with HCV infection who have failed to respond to available antiviral therapies. Preliminary results suggest that at higher doses, some of these refractory patients have viral load reductions of ⱖ1 log HCV RNA after 28 days of therapy. Dr. Lawrence Blatt (Ribozyme Pharmaceuticals) described an alternative approach to therapy that is currently being investigated and involves synthetic nuclease-resistant ribozymes designed to cleave the HCV IRES. These molecules also contain modified nucleotides and phosphorothioate linkages. Cell culture studies using an HCV/poliovirus chimera that contains the HCV IRES have identified ribozymes that target sequences within the IRES and inhibit replication.34 This inhibition is potentiated by the addition of interferon to the system. Cleavage occurs efficiently at the expected site in HCV RNA, and the ribozymes are taken up rapidly by the liver in murine studies. Clinical studies are currently being planned with these ribozymes in patients with chronic HCV infection. While these new molecular approaches provide hope and excitement for treatment of HCV-infected patients, further studies are required to determine the efficacy and safety of such approaches, to determine the precise mechanism of antiviral effects, and to evaluate whether such agents may need to be administered in combination with our available antiviral therapies. The NS3 protein of HCV has long been considered an attractive target for antiviral therapy of hepatitis C. NS3 encodes a serine protease domain in its N-terminal one third and an NTPase/helicase domain in the C-terminal portion. The N-terminal third of NS3 is also an essential component of the NS2-3 autoprotease. The NS3 enzymatic activities are required for HCV replication in vivo. The NS3 serine protease is responsible for cleavage of the HCV polyprotein, specifically of NS4A from NS3 (in cis) and NS4B from NS4A, NS5A from NS4B, and NS5B from NS5A in trans.4 NS4A acts as a cofactor for the serine protease, is found as a stable heterodimer in vivo, and appears to function in part by stabilizing the protease molecule. The protease has a prominent zinc-binding site that stabilizes the molecule. Of the possible mechanisms of inhibition of the NS3 protease, the one that holds the most promise is inhibition of substrate binding. Unfortunately, the substrate binding site of the NS3 protease is long, shallow, and flat in configuration making it difficult to design molecules that would bind to, and inhibit, the protease. Dr. Raffaele de Francesco (I.R.B.M., P. Angeletti, Italy) described his efforts, and those of colleagues at Merck, to identify inhibitors of the NS3 protease. They found that the protease is susceptible to feedback inhibition by the products released from the polyprotein substrate after enzymatic cleavage. He described the structure of molecules based on products of enzymatic cleavage, based on the
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DEMEEC amino acid motif. Stylized molecules based on this amino acid structure were manufactured and shown to inhibit the NS3 protease effectively in vitro. However, as currently configured, these molecules are likely to have poor bioavailability because of their large size. Although the HCV helicase is covalently tethered to the protease, the two enzymes have spatially separate active sites.34 The amino acid motif DEAH or DEXH/D is specific for helicases. HCV NS3 has seven conserved signature motifs characteristic of DEAH or DEXH/D helicases that line the active site of the enzyme in its 3-dimensional structure. It is important to emphasize that the HCV helicase is a mobile molecule and that this movement is critical for its function to unwind double-stranded RNA. Dr. Patricia Weber (Schering-Plough) summarized recent work on the helicase and indicated that helicase inhibitors are under development.35 The 3-dimensional structure of the NS5B RdRp of HCV has been recently solved by several groups and its function studied.36,37 This enzyme target appears to offer great potential for the development of new antiviral agents. Nonetheless, as discussed by Dr. C. Cheng Kao (Indiana University), the RdRp has very complex functions. Evidence from other viral systems suggests that it functions as part of a replicase (a multiunit complex, usually membrane-associated, which may contain host factors as well as the helicase). This complex binds substrate RNA, often resulting in a conformational change in the RdRp. RNA strand elongation begins with a nucleotide triphosphate rather than a primer. Initiation is followed by elongating RNA synthesis, termination of synthesis, and release of nascent RNA. To date, the search for candidate inhibitors of the RdRp has focused on the elongation reaction using biochemical screens. The efficacy of inhibitors is now being evaluated in cells using the HCV replicon model system, and promising compounds will undergo testing in animals for bioavailability and toxicity before being tested in humans. Strategies to Minimize Hepatic Injury and Fibrosis. The ultimate goal of antiviral therapy for hepatitis C has long been the elimination of HCV from serum and liver during and after therapy. However, it must be recognized that it is not the viral infection per se that must be treated but the liver disease that results from chronic infection. This recognition has led to consideration of therapies that are aimed at minimizing hepatic injury and fibrosis. For example, interferon alfa therapy results in improvement in liver histopathology in patients with chronic hepatitis C, even if they do not experience a sustained virologic response. Thus, in most patients who do not clear the virus from serum, a decrease can be seen in the degree of necroinflammatory change in the liver. It has also been suggested that interferon alfa therapy may decrease or slow progression of hepatic fibrosis. This approach would probably require long-term therapy, and there are few data available of the benefits of long-term therapy with interferon alfa. A decrease in hepatic necrosis, inflammation, and even fibrosis has been noted in patients with chronic hepatitis C treated for 3 years with interferon.38 Dr. Adrian Di Bisceglie (Saint Louis University) described the NIH-funded HALT-C trial, which aims to study the effects of long-term therapy (for 4 years) with pegylated interferon alfa in patients with advanced hepatic fibrosis who are at greatest risk for development of cirrhosis and its complications. In this trial ap-
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proximately 900 patients who are nonresponders to retreatment with a 6-month course of pegylated interferon and ribavirin will be randomized to receive interferon alone for an additional 3.5 years or to serve as an untreated control. The end points of this study are the development of cirrhosis on liver biopsy or the development of hepatic decompensation or hepatocellular carcinoma. A similar clinical trial, dubbed the COPILOT study, is also underway and is aimed at examining the role of pegylated interferon for prolonged therapy compared with a colchicine-treated control group. The Liver Diseases Section of the National Institute of Diabetes and Digestive and Kidney Diseases was among the first to study ribavirin for chronic hepatitis C. Using ribavirin by itself, they found that about one third of patients experience a return to normal of serum aminotransferases, associated with improvement in the degree of necroinflammatory activity on liver biopsy.39 Dr. Marc Ghany (National Institute of Diabetes and Digestive and Kidney Diseases) provided an update on the status of studies with ribavirin as monotherapy in chronic hepatitis C. He described a study in which nonresponders to interferon and ribavirin were randomly allocated to either ribavirin or placebo for 48 weeks. After this period of time, the histologic activity index had decreased significantly in those treated with ribavirin but not those on placebo. Furthermore, the fibrosis score was unchanged in those on ribavirin but had increased in placebo recipients, suggesting that ribavirin may prevent progression of fibrosis. Other therapies aimed at preventing or treating fibrosis were also reviewed. Although there are many agents with the potential to minimize fibrosis through antioxidant activity, few had been thoroughly tested in controlled clinical trials. A pilot study suggested that interleukin-10 (IL-10) may decrease hepatic fibrosis in patients with hepatitis C.40 Dr. Gary Davis (University of Florida) reported on a follow-up to that pilot study involving individuals with chronic hepatitis C and bridging hepatic fibrosis or cirrhosis who were treated with IL-10 for 12 months. Over this time, serum aminotransferase levels decreased in association with a decrease in hepatic inflammation. Only 4 of 27 treated patients had a decrease in the degree of hepatic fibrosis. Furthermore, levels of HCV RNA in serum tended to rise, by as much as 3-fold, when the period of IL-10 therapy was extended beyond 3 or 4 months. IMPDH Inhibitors. The development of specific inhibitors of the enzyme IMPDH may provide an alternative treatment for patients with chronic HCV infection when combined with interferon. This enzyme, which is also inhibited by ribavirin, is essential for the modulation of host cellular pathways. Inhibition of this enzyme, which is responsible for catalyzing an essential step in the de novo biosynthesis of guanosine nucleotides, leads to depletion of intracellular GTP levels. Other inhibitors of IMPDH include mycophenolic acid and mizoribine monophosphate, a drug approved in Japan for certain autoimmune diseases. Dr. Anne Kwong (Vertex Pharmaceuticals) summarized data on VX-497, a small orally bioavailable drug that specifically inhibits IMPDH and has broad spectrum antiviral activity including activity against HCV. In terms of IMPDH inhibition, the non-nucleoside VX-497 is approximately 20-fold more potent than ribavirin. A number of early phase I and II studies in patients with chronic hepatitis C have suggested ALT reductions in patients receiving VX-497, similar to earlier studies with ribavirin monotherapy.
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Trials combining VX-497 with interferon suggest a similar clinical effect, although data regarding long-term sustained response rates are unavailable at this time. Further development of more potent and specific IMPDH inhibitors including VX-497 will require randomized controlled clinical trials to determine their efficacy and future place in the management of patients with chronic hepatitis C infection.
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