Hepatitis C. Development of new drugs and clinical trials: Promises and pitfalls. Summary of an AASLD hepatitis single topic conference, Chicago, IL, February 27-March 1, 2003
Authors: Jean-Michel Pawlotsky, John G. McHutchison
Journal: Hepatology (2004)

Chronic hepatitis C virus (HCV) infection affects a large number of patients worldwide. While our current therapies (Table 1) are effective in approximately 50% of patients, therapy is costly, prolonged, and associated with significant side effects, and it is not suitable for certain groups of patients. For these reasons, improved treatment regimens are necessary for patients with this disease. In February 2003, the annual American Association for the Study of Liver Diseases (AASLD) Single Topic Conference devoted to viral hepatitis and to fostering development of research in this field focused on recent advances in the process of antiviral drug design and new anti-HCV drugs currently in development. The drug design process, how these agents should be evaluated in future clinical trials, the potential problems and pitfalls related to pharmacodynamics and pharmacokinetics, viral resistance, clinical trial end points and design, and drug interactions, as well as HCV targets for novel drug development and the HCV drug pipeline were discussed. The preface and aims of the conference were to provide investigators with new knowledge and some of the necessary tools to aid in planning future rational clinical trial design in this rapidly changing field. HCV, hepatitis C virus ; AASLD, American Association for the Study of Liver Diseases; PCR, polymerase chain reaction; IFN, interferon; NS3, nonstructural 3; IRES, internal ribosome entry segment; NC, noncoding; RdRp, HCV RNA-dependent RNA polymerase; DC-SIGN, dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin; L-SIGN, liver/lymph node-specific intercellular adhesion molecule 3-grabbing nonintegrin; LDL, low-density lipoprotein; TGF beta, transforming growth factor beta; PDGF, platelet derived growth factor; Th, T-helper; IL, interleukin; IMPDH, inosine monophosphate dehydrogenase; NK, natural killer; ALT, alanine aminotransferase; RNAi, RNA interference; siRNA, silencing RNA; BVDV, bovine viral diarrhea virus; HIV, human immunodeficiency virus; NNI, Nonnucleoside inhibitors; HCIg, hyperimmune anti-HCV immunoglobulin; ISCOMs, immunostimulating complexes; HGF, hepatocyte growth factor; PPAR, peroxisome proliferator activated nuclear receptors; FGF, fibroblast growth factor; TIMPs, tissue inhibitors of metalloproteinases. The first step is the screening of already existing libraries of potential compounds or the design and synthesis of new molecules based on potential target site structure knowledge. It is then necessary to rapidly test the potency and toxicity of potential agents—i.e. the selectivity of the targets and actions. The stability of the chemical entity and protein binding must be evaluated under various conditions. The mechanisms of action of the compound must be explored with appropriate in vitro tools. Toxicology studies must be run in at least two animal species. If the toxicity profile is acceptable, then the compound joins the "hot list" of compounds to proceed. The metabolism of the compound must be understood, and pharmacokinetics studies must be performed in animals. Efficacy studies must be performed in animals. The ultimate preclinical steps include various studies testing drug combinations in vitro and in vivo, selection of resistant viruses, viral fitness, pyrophosphorolysis, and others. At the present time, the relative potency of anti-HCV agents currently can be determined rapidly and reproducibly by means of enzymatic and cellular systems coupled with quantitative real-time polymerase chain reaction (PCR). Efficacy models include tissue culture and animal models in which HCV replicates efficiently and antiviral molecules can be tested for their capacity to inhibit HCV replication. Two models based on tissue culture appear to be of great interest for antiviral drug development. First, primary cultures of human hepatocytes isolated from uninfected patients, and infected in vitro with serum samples from HCV-infected patients represent the model closest to the physiologically infected cell.1 They have been shown to be sensitive to infection and permissive to HCV replication.1, 2 This system has been used to show that interferon (IFN) alfa inhibits HCV replication in a dose-dependent manner.2 It provides a very good model by which to study the antiviral effects of antiviral molecules in infected hepatocytes of human origin (communication by P. Maurel, Montpellier, France). Second, RNA replicons are subgenomic or genomic HCV RNA molecules that are capable of initiating and maintaining replication in the human Huh7 hepatoma line at relatively high copy numbers.3-11 The subgenomic or genomic replicon systems provide excellent cell-based assays to evaluate inhibitors of HCV enzymes or nucleic-acid targeting strategies and are currently widely used for this purpose. Replicon-containing Huh7 cells have also been used to produce cell-free replication complex preparations that allow testing of inhibitors uncovered in screens using purified enzymatic components, and they can also be used to determine whether "hits" in cell-based replicon screens are capable of directly inhibiting RNA replication.9-11 (communication by C.M. Rice, New York, NY). Animal models provide a crucial link between in vitro experimentation and the establishment of human clinical trials. The chimpanzee model has contributed to many advances in the HCV field,12 but major impediments to the utilization of chimpanzees in medical research include the cost, availability, and ethical issues. In the tupaia, a tree shrew indigenous to Southeast Asia, HCV viremia is rare and, when it occurs, low and either transient or intermittent.13 In the "Trimera" mouse model,14 the practical window for drug administration is limited to a few days within the second week after transplantation, making this system difficult to utilize in drug screening experiments. Mercer's group recently "humanized" a murine liver through hepatocyte transplantation.15 These mice with "humanized" livers are capable of de novo infection with HCV, generate viral RNA levels equivalent to those of humans (105 to 106 viral RNA copies/mL), may support stable viral production for weeks or months, and can transmit the virus to other noninfected murine recipients.15 The efficacy of IFN alfa-2b, immunotherapies, antiviral gene therapy approaches, and pharmaceuticals that have shown promise in in vitro systems has been or is currently being tested in this model (unpublished data; communication by D.F. Mercer, Omaha, NE). The emphasis of pharmacological studies now has shifted to the early demonstration of pharmacological activity ("proof of concept") or efficacy. This shift has been aided by the development of sophisticated methods of assessment, focusing on the use of biomarkers in decision-making.16 At the preclinical stage, such models may assist initial human studies by identifying relevant exposure levels (communication by P. Glue, New York, NY). There are two main types of safety risk: (1) mechanism-based toxicity, in which adverse effects are due to modulation of the same target or pathway that produces the desired therapeutic effect, and (2) compound-based toxicity, in which adverse effects are due to secondary actions of the drug candidate and are not related to the therapeutic mechanism. Both mechanisms must be addressed prior to further development by means of in vitro and in vivo preclinical toxicity screens. In addition, many drug-drug interactions can be correlated with the inhibition and–or induction of cytochrome P450 enzyme activity. Quantitative in vitro prediction of clinical interactions, however, remains difficult (communication by K.R. Romines, Research Triangle Park, NC).17-19 After the long and arduous preclinical process, a few "survivor" compounds may reach the list of "lead candidate drugs" for clinical evaluation. Clinical evaluation then generally includes four types of trials, from phase I to phase IV. Additional issues, including viral kinetics and resistance, must be addressed at these stages. Using viral load monitoring and mathematical models to analyze longitudinal data yields estimates of the antiviral efficacy of therapy. Several studies have described the kinetics of HCV during IFN alfa therapy in vivo.20-25 Overall, these principles allow one to use viral kinetics to deduce features of the patient's response to therapy. They will prove to be particularly useful in evaluating the early efficacy of new HCV drugs, and understanding their mechanisms of action and eventual failure. They may also be important to predict the outcome of future therapy and derive appropriate clinical decision guidelines (communication by A.S. Perelson, Los Alamos, NM).26 Our current knowledge of HCV resistance to therapy includes partial understanding of the mechanisms underlying IFN alfa-based therapy failure to eradicate infection,27 and a clearer view of the mechanisms underlying the resistance of other viruses (such as hepatitis B virus, human immunodeficiency virus, or herpes viruses) to specific inhibitor molecules. Viral resistance to specific antiviral drugs is characterized by the selection of minor viral populations bearing mutations conferring resistance to the specific drug. Treatment withdrawal is usually followed by restoration of the "sensitive" genotype and phenotype. Combined therapy with multiple drugs with different targets and mechanisms of action delays the emergence of resistant viruses, but multiresistant viruses may emerge after several years of treatment.28 The natural variability of the HCV targets for new drugs, such as the nonstructural viral protein 3 (NS3) serine proteinase and internal ribosome entry segment (IRES),29 suggests that resistant variants could be selected by future HCV inhibitors. Such events will need to be monitored with appropriate virological tools early in the clinical development of these drugs. If viral resistance becomes a clinical issue, multiple therapy combining drugs with different targets and modes of actions will probably become the standard of care (communication by J.M. Pawlotsky, Créteil, France). RNA structures and viral proteins playing a critical role in the HCV lifecycle represent ideal targets for specific HCV inhibitors. The short 5′ and 3′ noncoding (NC) segments of the HCV genome contain structured RNA elements that are critically important for the initiation of protein translation, as well as for specific recognition of the termini of positive- and negative-strand RNAs by the HCV RNA-dependent RNA polymerase (RdRp) and subsequent initiation of RNA transcription.30, 31 Two general approaches to the inhibition of these key steps can be envisioned. First, molecules that cleave the 5′ NC segment or otherwise destabilize its higher order structure will abrogate translation of the viral polyprotein. Such drug candidates may also disrupt initiation of plus-strand RNA synthesis, since the involved RNA signals are overlapping within the genome. Similar approaches could be adopted for the 3′ NC segment, potentially eliminating the first step in viral RNA synthesis. Alternatively, the growing understanding of the structures of these RNA segments at atomic level resolution31-33 should make it possible to rationally design small molecules that bind specifically to structures within the 5′ NC and 3′ NC segments and that could serve as inhibitors of translation, translation initiation, and/or RdRp recognition. In both cases, the target would be a relatively large RNA structure that most likely has multiple contacts with macromolecular protein assemblies (i.e., the 40S ribosome subunit or the replication complex31, 32). It remains to be seen whether small molecules could effectively disrupt the resulting high affinity RNA-protein interaction (communication by S.M. Lemon, Galveston, TX). Although its function is partly unknown, it has been suggested that the p7 protein could form ion channels in the endoplasmic reticulum membrane, and that this may be necessary to HCV replication. It therefore provides a potential target for antiviral intervention. The NS3 serine protease belongs to the chymotrypsin class of proteases and is involved in HCV polyprotein maturation in which it cleaves an initially synthesized viral polyprotein into functional proteins. Three-dimensional (3-D) structures of the NS3 protease with and without the requisite viral cofactor NS4A were determined by x-ray crystallography.34, 35 Extended contacts are made between the polyprotein and NS3 protease during recognition of polyprotein processing sites. These contact regions potentially provide distinct types of inhibitor binding sites on NS3 (Fig. 1A).36, 37 Some NS3 protease inhibitors bind at subsites utilized by the polyprotein region on the N-terminal side of the scissile bond. Many of these inhibitors achieve increased affinity for the NS3 protease through additional interactions with the active site catalytic machinery including the active site serine and oxyanion hole. Inhibitors that span the active site and thus more closely mimic the polyprotein substrate have also been synthesized (communication by P.C. Weber, Yardley, PA). (A) Inhibitor sites on the NS3/4A protease/helicase. The backbone trace of the bifunctional NS3/4A is drawn from the crystallographic coordinates (1CU1). NS3 protease subdomains are shown in yellow and red, the central portion of the NS4A cofactor is shown in green, and the essential Zn is indicated by a sphere. The three helicase subdomains are shown in shades of blue. The NTPase subdomain is colored cyan and one of the NTP phosphate binding locations is indicated by a pink sphere. The arginine-rich subdomain that is structurally-homologous to the NTPase domain is colored dark blue, and the third helicase subdomain is colored light purple. Sites for competitive inhibitors include the protease active site (1), and here the serine and histidine side chains of the catalytic triad are shown. Arrows (2) and (3) indicate the polyprotein substrate P and P' recognition sites, respectively, and (4) and (5) indicate the NTPase- and ssRNA-binding sites of the helicase. Locations of additional RNA-recognition sites are unknown. Molecules that prevent productive binding of essential cofactors, such as the Zn atom (6) or NS4A cofactor peptide (7), also act as inhibitors. Inhibitors of requisite interdomain motions of the helicase subdomains bind near the end of the arrow (8). The general region of membrane attachment of the bifunctional protease/helicase is denoted by an arrow (9), and interference with attachment could also prevent formation of a competent replication complex. Finally, inhibitors preventing substrate recognition by the NS2/3 protease might bind near the NS3 N-terminus (10) prior to cleavage of the polyprotein (figure prepared by P.C. Weber). (B) Inhibitor sites on the NS5B polymerase. The backbone trace of the NS5B enzyme is drawn from the crystallographic coordinates of its binary complex with UTP (1GX6). The polymerase is shown in red, gold, and blue for the fingers, palm, and thumb subdomain, respectively. Sites for competitive inhibitors include the polymerase active site (1), and here the side chains of the catalytic aspartic acid residues are shown. Arrows (2) and (3) indicate the nucleotide binding sites for polymerization and for de novo initiation of RNA synthesis, respectively. Bound nucleotides are indicated in black. An arrow (4) indicates the location of the allosteric surface GTP binding site, a region at the connection between fingers and thumb where several nonnucleotide inhibitors have been shown to bind. In this region, the side chains of residues that contact the allosteric GTP are indicated in black (Pro 495 and 499, Arg 502). Finally, an arrow (5) shows the template RNA binding groove (communication by F.A. Rey). The NS3 helicase primary function is to unwind the viral genomic RNA during replication. Studies probing the mechanism of polynucleotide unwinding have led to observations that may suggest new strategies for drug discovery.38 In addition, an increasing number of 3-D helicase structure determinations, including a structure of the full-length NS3 protein having both protease and helicase domains39 (Fig. 1A), has helped delineate the functionally important regions of the NS3 helicase. Structural comparisons revealed helicase domain motions that accompany the progressive unwinding of nucleic acid duplexes and that may serve as targets for specific inhibitors (Fig. 1A).40 The NS3 helicase is characterized by the location of an NTP binding site near the interface between two structurally homologous nucleotide binding domains, the existence of a third domain composed primarily of alpha-helices, and the occurrence of a pronounced substrate binding groove, which binds an extended polynucleotide (communication by P.C. Weber). The HCV RdRp (NS5B protein) is a 68 kDa protein that catalyzes RNA synthesis during replication. Several 3-D structures of active, water-soluble domains missing the hydrophobic C-terminal tail that localizes the RdRp to internal cellular membranes have been determined.41-43 The HCV RdRp displays the classical "Finger/Palm/Thumb" motif observed in nucleic acid polymerases and a number of molecular interaction sites that can be targeted by specific inhibitors (Fig. 1B). It is, however, unique by its fully encircled active site into which nucleotides can bind in the absence of the template. The active site of the enzyme is a target for nucleoside/nucleotide analogue inhibitors. In addition, a specific GTP binding site has been identified at the surface of the enzyme, more than 30 Å away from the active site.44 Nonnucleoside inhibitors of HCV RdRp were recently shown to bind very close to the surface GTP site, suggesting that a possible mode of action of these drugs is by inhibiting the conformational change that would be needed for RNA elongation.45 Finally, indirect evidence suggests that some conformational changes must occur upon simultaneous binding of template and nucleotide to create the replication initiation platform. This mechanism could also serve as a target for antiviral drugs (communication by F.A. Rey, Gif-sur-Yvette, France). Currently the most promising targets for new drug development appear to be the NS3 serine protease and RdRp (Fig. 1). Other mechanisms of infection and disease may be suitable for future drug targets. The objectives may be to inhibit virus entry into cells by targeting the cellular receptor structures or their interactions with surface viral structures, to neutralize the virus or accelerate infected cell death by interacting with host immune responses, or to prevent liver disease progression by slowing or reversing fibrogenesis. The HCV genome encodes two envelope glycoproteins, E1 and E2, which are thought to be expressed as noncovalent heterodimers at the virus surface. The mechanism by which HCV enters target cells is currently unknown. The E2 glycoprotein is thought to be responsible for initiating virus attachment, and the E1 glycoprotein has been hypothesized to contain the fusion peptide responsible for the fusion of the virus and cell membranes.46 An early interaction of envelope glycoproteins with glycosaminoglycans has been suggested to play a role in cell recognition and tropism. Truncated soluble versions of E2 have been reported to bind specifically to human cells and were used to identify interactions with tetraspanin CD81,47 with scavenger receptor class B type I,48 and with dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and liver/lymph node-specific intercellular adhesion molecule 3-grabbing nonintegrin (L-SIGN).49 A role has also been suggested for the low-density lipoprotein (LDL) receptor.50 The relevance of these molecules as receptors for HCV infection in vivo and as targets for future therapies is currently unknown and awaits the development of systems to study HCV-cell entry. One approach to overcoming the lack of a conventional cell culture system for HCV is to generate retroviral pseudotypes bearing HCV glycoproteins, which are infectious for a restricted number of human hepatoma cell lines.51, 52 This system will allow further understanding of the early events of HCV infection in detail,51, 52 to identify cellular molecules used by HCV, and to screen for small molecules able to block viral entry (communication by J.A. McKeating, New York, NY). This system will also be useful to assess neutralizing responses in vitro and to test therapies based on virus neutralization, such as specific anti-HCV immunoglobulins. The cellular immune response plays a major role in HCV infection and could represent an attractive target for therapeutic intervention. Indeed, vigorous and multispecific CD4+ and CD8+ T-cell responses during acute hepatitis C are associated with viral clearance and recovery.53 Moreover, HCV-specific memory T cell responses remain detectable in the peripheral blood for decades54 and mediate rapid viral clearance upon reexposure to the virus.55, 56 In contrast, a lack of this response or a failure to maintain it for a sufficient time, particularly in the face of viral mutations, is associated with development of persistent infection and chronic hepatitis.57 Once persistent hepatitis is established, the frequency of HCV-specific T cells is typically low, and their proliferative, cytokine, and cytotoxic effector functions appear to be impaired.58 Whether successful antiviral therapy leads to transient and/or long-lasting reconstitution of cellular immune responses is controversial.59 With regard to the future role of immunotherapy, our current knowledge of HCV-specific cellular immune responses raises the following questions: Should antiviral therapy be combined with approaches to modulate and/or induce the cellular immune response in order to induce long-lasting immunity, and does immunomodulatory therapy accelerate infected cell clearance and improve sustained virological response rates in chronic hepatitis C? (communication by B. Rehermann, Bethesda, MD). If the virus cannot be eradicated, an alternative approach would be to slow the progression of liver disease. Hepatic fibrosis is the major histologic complication of chronic HCV infection, and it generally develops after many years, leading to cirrhosis within an estimated 10–50 years. A key pathogenic event in fibrosis progression is the transition of hepatic stellate cells from a quiescent to an "activated" state. This process is characterized by the production of increased amounts of extracellular matrix and de novo expression of smooth muscle alfa-actin, the latter characteristic consistent with cellular transformation to myofibroblasts.60-62 One of the most remarkable aspects of this response is enhanced extracellular matrix production, or fibrogenesis. Fibrogenesis after injury to the liver is characterized by significant increases in collagen (type I>III>IV) and other extracellular matrix constituents such as laminin, fibronectin, and proteoglycans (dermatan sulfate, chondroitin sulfate, heparan sulfate).61, 63, 64 This "wounding" process is complex and integrated, and it involves aspects of matrix synthesis and deposition, as well as degradation. Cross-talk between stellate cells and the extracellular matrix appears to play a critical role in fibrogenesis, as well as a number of cytokines and small peptides, including transforming growth factor (TGF) beta, platelet derived growth factor (PDGF), endothelin, and angiotensin II. New specific, antifibrotic therapies may target at reducing or removing the primary injury, inhibiting stellate cell activation, stimulating matrix degradation, stimulating stellate cell apoptosis, or blocking the downstream effects of stellate cell activation (communication by D.C. Rockey, Durham, NC). Various new directions are currently being explored in HCV drug development, and several specific and nonspecific anti-HCV drugs have already reached the clinical development phase (Table 2). Human IFNs have been classified according to the cell surface receptor with which they bind. Type 1 IFNs bind to the IFN alpha heterodimeric receptor IFNAR1/IFNAR2 and include the 21 nonallelic subtypes IFN alfa, IFN beta, IFN omega, and IFN tau.65 IFN gamma binds to a unique cell surface receptor, is classified as a type 2 IFN, and displays antiviral, antifibrotic, and immunomodulating activity that effects the T-helper (Th) 1 response.66 Recently, three novel cytokines have been identified that share sequence identity with type 1 IFNs and bind to a novel cell surface receptor. These putative type 3 IFN molecules have been called interleukin (IL) 28A, IL-28B and IL-29. They are induced by viral infections and exhibit marked antiviral activity in vitro.67 Challenges still exist with respect to IFN treatment optimization, including poor pharmacokinetic profiles, limited biological activity, suboptimal therapeutic indices, and limited knowledge on non-alfa, non-beta IFNs efficacy. Several groups have attempted to modify naturally occurring IFNs to improve their performance. These modifications include alteration of the primary amino acid sequences, the addition of polyethylene glycol, alterations of glycosylation patterns, and the production of fusion proteins. Consensus IFN alfa (IFN-alfacon1) is a second-generation cytokine that was engineered to contain the most frequently occurring amino acids among the nonallelic IFN alfa subtypes. IFN alfacon1 has been shown to be more effective than naturally occurring type 1 IFNs in cell culture models and equally effective in clinical trials.68, 69 Other novel alfa IFNs have been produced by gene-shuffling the family of 20 human IFN alfa DNA encoding sequences.70 Using this technique, a novel nonnaturally occurring type 1 IFN has been described with a 285,000-fold increase in antiviral activity when compared with IFN alfa-2b. The role of this highly active IFN in the treatment of chronic hepatitis C has yet to be elucidated. The addition of polyethylene glycol to therapeutic IFN alfa proteins has been shown to dramatically increase plasma exposure following dosing and has led to increased response rates.71, 72 Recently, an IFN beta molecule has been conjugated to a linear 20-kDa polyethylene glycol molecule targeted at a single site on the N-terminal amine.73 This molecule is now entering clinical trials for the treatment of chronic hepatitis C. A pegylated form of IFN-alfacon1 is also being clinically evaluated. Finally, a fusion protein of IFN alfa-2 and human serum albumin, has entered clinical trials for the treatment of chronic hepatitis C.74 This molecule displays similar in vitro antiviral and antiproliferative activity to unmodified IFN alfa-2, but has marked improvements in pharmacokinetic characteristics. Several new techniques for selection of novel second-generation IFNs have produced even more potent molecules, and clinical trials of these molecules are either underway or planned for the near future. Lastly, the role of additional naturally occurring IFN species, such as IFN gamma and IFN omega, is now being elucidated in clinical trials (communication by L.M. Blatt, Brisbane, CA). Oral IFN inducers represent a class of compounds that may allow the generation of an effective immune response by induction/modulation of cytokine responses at the site of infection, or that may supplement or replace parenteral administration of IFN. The central challenge in the use of such agents as oral therapy for chronic HCV infection is the delivery of effective doses to the liver. Agents known to induce IFN alfa and other cytokines include (among many) relatively high molecular-weight agents, such as double-stranded RNA (poly I:C), and CpG oligonucleotide derivatives. In contrast to the complexities associated with oral delivery of such large molecules, low-molecular-weight molecules may offer similarly useful immune-modulating properties and a reasonable probability of oral absorption. Two advanced chemical classes include the imidazoquinolones imiquimod and resiquimod,75 and the nucleoside analogs ANA245 and ANA971. Imiquimod is approved for use as a topical agent in dermatology. Toxicity probably resulting from cytokine induction was reported in human studies.76, 77 Resiquimod is currently in phase II studies for chronic viral hepatitis, and preliminary reports indicate a lack of specific detectable antiviral activity (Pockros et al., poster presented at the meeting). ANA245 is a low-molecular-weight nucleoside analog that induces multiple cytokines, including IFN alfa, and it showed immunologically mediated antiviral activity in a range of viral infection models (D.R. Averett, unpublished data, May 2003). However, its oral bioavailability is limited at high doses. Studies in humans are underway to characterize the safety and pharmacokinetics of ANA245 after intravenous administration, as a component of the development program for ANA971, a novel molecule that was shown to efficiently deliver ANA245 at levels associated with antiviral effects to the plasma of various animals (Averett et al., unpublished data) (communication by D.R. Averett, San Diego, CA). Ribavirin monotherapy is ineffective in inducing sustained viral clearance, but it significantly enhances the sustained viral clearance rate when combined with IFN. Until now, the mechanism by which ribavirin enhances IFN efficacy remains unknown. There are four proposed mechanisms of action: immune-mediated activity on the host Th1/Th2 balance; inhibition of host enzyme inosine monophosphate dehydrogenase (IMPDH) activity; weak inhibitory activity against the RdRp; and induction of RNA mutagenesis.78 Hemolytic anemia is a frequent side effect that limits ribavirin dosing, emphasizing the need for alternative molecules with similar mechanisms an