New drugs for hepatitis C virus (HCV)

BaillieÁre's Clinical Gastroenterology Vol. 14, No. 2, pp. 293±305, 2000

doi:10.1053/bega.1999.0077, available online at http://www.idealibrary.com on

8 New drugs for hepatitis C virus (HCV) Berwyn E. Clarke

BSc, PhD

Disease Programme Leader Virology Research Unit, GlaxoWellcome Medicine Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, UK

Lack of ecacy and signi®cant side e€ects have severely limited the use of interferon-a (IFN-a) as the standard therapy for non-A non-B hepatitis (NANBH) caused by hepatitis C virus (HCV) and alternative, improved therapies are urgently sought. Attempts have been made to improve the potency and tolerability of IFN-a by adjusting dosing regimens, methods of delivery and length of treatment. Furthermore, a number of di€erent agents have been used in combination with IFN-a and, from these studies, therapeutic options have been galvanized by the synergistic e€ects of IFN-a and ribavirin. Nevertheless, the majority of patients with HCV still do not sustain lasting therapeutic bene®t from this combination and continuing research is required to identify new therapeutic candidates that will have more potent antiviral activity and less severe side e€ects. This review focuses on the progress that has been made in this area and the prospects for new e€ective therapies in the near future. Key words: hepatitis C virus; a-interferon; immunomodulation; anti-viral.

INTRODUCTION Infection with the hepatitis C virus (HCV) results in the majority of cases of non-A non-B hepatitis (NANBH) and is the most common cause of viral hepatitis in the Western world. Moreover, prevalence data indicates that in global terms approximately 3% of the world's population are infected with HCV.1 The insidious nature of this disease is such that the majority of these patients have sub-clinical disease and yet 20±30% of these individuals will eventually progress to cirrhosis and a large proportion to hepatocellular carcinoma.2,3 The consequence of this is that HCV-induced end-stage liver disease is the most common cause for orthotropic liver transplantation. Nevertheless, the slow progressive nature of the disease is such that only now is the magnitude of the healthcare problem becoming apparent.1,4 Recent improvements in therapeutic approaches to the treatment of chronic NANBH have shown sustained viral elimination in only 30±40% of treated patients.5±7 Nevertheless, it is true to say that all of the therapeutic regimens currently available are associated with considerable toxicity and the majority of patients (60±70%) show no sustained therapeutic bene®t. The purpose of this chapter is to discuss the limitations of current therapies and to review the new approaches that show promise for improvements in current drugs and the development of more potent and less toxic medicines. 1521±6918/00/020293+13 $35.00/00

c 2000 Harcourt Publishers Ltd *

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INTERFERON-a a Monotherapy Until recently, interferon-a (IFN-a) was the only therapy with proven bene®t for the treatment of HCV infection and standard recommended protocols used 3 million units (3 MU) three times weekly (t.i.w.) for 6 months (See Chapter 5). Using this regimen, typically 33±50% of patients showed a response to treatment, but these were not sustainable and long-term responses after discontinuation of therapy were only observed in 10±15% of patients.8±11 Several approaches have been taken to improve the numbers of patients who achieve viral eradication including increasing dose, dosing frequency and duration of therapy. Data on more radical new approaches to IFN-a monotherapy are now beginning to appear and these will now be reviewed. Duration and dose of therapy Until 1993 the standard regimen for IFN-a therapy was 9 MU (3 MU three times) per week for 6 months. However, the low levels of sustained response in these patients led to studies in which higher doses were administered for longer periods of time. These studies showed that sustained response could be improved by increasing the duration of therapy from 6 to 12 months.5,6,12 As well as increasing the length of IFN-a therapy, there are now many reports of HCV therapeutic trials using IFN-a doses in excess of the standard 9 MU per week. These have included studies of daily dosing (e.g. see13), high-dose induction therapy (e.g. see14) and escalation therapy (e.g. see 15,16). Although several studies have suggested an improvement in the end of treatment response, this response was usually not sustained (for a review see17) and the side e€ects associated with higher doses of IFN-a were predictably worse. It seems unlikely that high-dose IFN-a monotherapy will have a major role to play in future therapy of HCV. Prospects for the future use of IFN-a monotherapy Although an extended course of therapy and the use of high-dose IFN-a do show some bene®t in some patient groups, recent developments in combination therapy mean that the use of IFN-a as monotherapy will have little future utility. Nevertheless, there is still scope for improvement in terms of the IFN-a component of the combination. One possibility is the use of consensus interferon (CIFN), which is a synthetic IFN representing the consensus sequence of naturally occurring IFN-a molecules. This IFN has been used in clinical trials for HCV to assess any improvements in ecacy or side e€ects. Disappointingly, the results obtained indicated no signi®cant di€erence over standard recombinant IFN-a and the side-e€ect pro®le remained unaltered.18 Along similar lines, it is clear that the IFN-a gene family is a collection of closely related but distinct molecules that exhibit multiple and diverse biological activities. Moreover, some of the IFN-a subtypes exhibit signi®cantly di€erent anti-viral properties19, which can be speci®cally mapped to particular domains within the protein.20 Therefore, it may be envisaged that future IFN-a molecules could be speci®cally designed to enhance the anti-viral properties but to eliminate the associated toxicities. If this were achieved then this synthetic IFN-a could potentially be the cornerstone of all combination regimens. Finally, a more recent report suggests that addition of a polyethylene glycol (PEG) moiety to the IFN-a may result in slower clearance of the molecule from the bloodstream thus prolonging the exposure to the molecule and potentially improving the clinical outcome while decreasing the dosing frequency.21

New drugs for hepatitis C virus 295

This is an exciting prospect and the data to indicate whether there is a genuinely improved clinical outcome should be available in the near future. Interferon combination therapy The low ecacy of current regimens of IFN-a monotherapy combined with the low numbers of patients showing sustained therapeutic bene®t have severely restricted e€ective treatment of hepatitis C. This has led to the investigation of alternative therapies that could be used to supplement IFN-a and a number of these have now been clinically evaluated. In particular, the combination that has recently transformed HCV therapy has been the addition of the synthetic nucleoside analogue, ribavirin (see Chapter 5). Ribavirin Ribavirin is well-known to have broad-spectrum anti-viral activity against several RNA and DNA viruses and it was an obvious candidate for therapeutic evaluation against HCV. Unfortunately, pilot studies using ribavirin as monotherapy revealed that there was no anti-viral e€ect, although there was some evidence of disease modi®cation as shown by reduction in liver enzyme levels.22 Nevertheless, a number of small, randomized, controlled trials were carried out in patients with chronic HCV infection, comparing virological, biochemical and histological responses after 6 months of therapy with IFN-a alone or in combination with ribavirin. As show in Table 1, there was evidence of signi®cant enhancement of sustained virological response in the combination arms of all studies. Furthermore, in patients with a sustained virological response there was also marked improvement in both biochemical and histological parameters. Table 1. Comparison of IFN-a versus IFN-a plus ribavirin in patients with chronic HCV. EOT response (%)

Study

IFN

No. of patients

[23] [24] [25]

a-2b Nat. a-2b

50 : 50 15 : 15 21 : 19

‡ RVN

Sustained response (%)

ÿ RVN

‡ RVN

ÿ RVN

ALT

RNA

ALT

RNA

ALT

RNA

ALT

RNA

66 67 76

52 60 90

56 60 37

52 53 42

44 47 43

36 47 43

24 13 11

18 7 6

Abbreviations: EOT, end of treatment; RVN, ribavirin; ALT, alanine aminotransferase; NAT, natural.

Based on the encouraging data that emerged from these studies two large multicentre studies have now been completed in Europe and the USA. Both studies con®rmed radically improved sustained response rates in combination therapy compared with IFN-a monotherapy.26,27 Similar trials have also been conducted in patients who have relapsed after previously successful IFN monotherapy and have shown that almost 50% of these patients achieved sustained clearance of viral RNA.28 Overall, therefore, combination of IFN-a with ribavirin a€ords around 40% of chronic HCV patients a change of long-term sustained virological response and its consequent biochemical and histological improvements. However, caution must be exercised when using these

296 B. E. Clarke

agents in combination because ribavirin itself is associated with a signi®cant side-e€ect pro®le that is distinct from that of IFN-a. Current studies indicate that these side e€ects are not synergistic and criteria are now in place regarding dose modi®cation should the drugs be poorly tolerated.29 Clearly, therefore, the use of this combination has signi®cantly improved the available therapy for chronic HCV and it now remains to be established whether the clinical data can be further improved by incorporating modi®ed IFN-a components (e.g. high dose/PEG-IFN) into the combination. Other IFN-a combinations In parallel with the IFN and ribavirin studies a number of other potential clinical combinations have also been explored. One of the more intriguing is the use of amantadine and rimantadine as potential partners for IFN. These drugs were developed as therapeutic agents against in¯uenza virus and there is no rational mechanism by which they would be expected to have anti-viral ecacy against HCV. Indeed, recent data examining the clinical e€ects of amantadine monotherapy against HCV show little evidence of ecacy alone.30 In combination therapy with IFN small scale studies in IFN non-responders suggested that IFN plus amantadine was ine€ective31, but one recent small study of triple combination therapy with ribavirin, amantadine and IFN-a showed signi®cant end of treatment virological responses and clearly warrants further investigation.32 Although none of the combinations has, so far, yielded results as promising as ribavirin, the combination of IFN with other anti-viral, anti-in¯ammatory or immunomodulatory agents may be advantageous. Indeed, several immunomodulatory agents, e.g. ursodeoxycholic acid (UDCA), thymosin-a 1 and non-steroidal antiin¯ammatory agents (NSAIDs), have already been explored in pilot studies (see33) but no improvement has been observed over the use of IFN-a as monotherapy. Nevertheless, considerable research is in progress to understand the mechanisms of immunopathogenesis in HCV-related disease and the mechanisms by which the virus is able to establish a chronic infection.34 This information, in the longer term, may allow radical new approaches to the development of new therapies. In the shorter term, the focus for new drug discovery must be the identi®cation of direct inhibitors of virus replication that may be ecacious alone or as components of combination therapies with existing agents.

REPLICATION INHIBITORS Apart from the interferons, the majority of anti-viral drugs that are currently clinically available are all molecules that directly inhibit viral replication, e.g. Aciclovir for Herpes simplex, Lamivudine for hepatitis B virus (HBV), reverse transcriptase and protease inhibitors for HIV and Relenza for in¯uenza. It is therefore not surprising that the majority of research e€orts for developing novel therapies against HCV have focused on the development of similar agents. However, the search for replication inhibitors of the HCV has been complicated by the fact that no reliable and reproducible tissue culture system is available, unlike the other viral systems where replicative inhibitors have been successfully developed. Alternative strategies have therefore been developed to overcome these limitations and the crucial factor has been the detailed understanding of the molecular virology of the virus.

New drugs for hepatitis C virus 297

MOLECULAR TARGETS HCV is a single-stranded positive sense RNA virus with a genome of approximately 9000 bases that is translated into a single polyprotein 3010 amino acid residues in length.35 A detailed molecular map of the viral genome is shown in Figure 1. and extensive research has revealed detailed biochemical characteristics of several of the viral proteins (see Chapter 4), which are all putative targets for anti-viral attack, as well as the untranslated regions.36 The replication cycle of the HCV virus is now understood, at least in outline, and Figure 2 illustrates the viral life cycle and identi®es possible targets for therapeutic intervention. Nucleocapsid Envelope 1 Envelope 2 5’UTR

C

E1

E2

Metalloprotease Serine protease RNA helicase Co-factor

NS2

NS3

4A

4B

RNA polymerase 5A

5B

P7

3’UTR

Figure 1. Genomic organization of hepatitis C virus RNA. UTR, untranslated region..

The viral structural proteins are the core (C) and the two envelope proteins, E1 and E2, which are present at the amino-terminus of the polyprotein and are nascently released from the polyprotein by the action of host cell proteases. Functions for the small P7 protein and the NS2 protein are currently unclear but the remainder of the polyprotein is clearly involved in viral RNA replication and these replicative proteins are released from the polyprotein by the action of two viral proteases. An unusual feature of the viral genome is the presence of rather long and highly ordered untranslated regions at both the 50 and 30 ends, which recent data has suggested may play key roles in viral replication. All of these viral functions are candidates for antiviral attack and considerable progress has been made in several areas that will now be reviewed. Untranslated regions (UTR) 50 UTR The 50 UTR of HCV is unusually long for a positive strand RNA virus, being 341 nucleotides in length. Computer modelling and biochemical mapping have shown that it folds into a complex secondary and tertiary structure (see36). Sequence comparison of many HCV isolates has revealed that this region possesses the highest degree of sequence conservation across the whole genome, implying an important replicative function that is not susceptible to mutation. These features of sequence conservation and highly ordered structure have been observed in other viral families (e.g. Picornaviruses) but are very rarely found in mammalian cell mRNAs. The major function of this region is as an internal ribosomal entry site (IRES) and this has been well characterized by several groups. Classical translational mechanisms allow ribosomes to bind to the 50 end of the RNA, usually via a specialized cap structure, followed by migration of the ribosome to the most proximal AUG codon where translation is initiated. There is no evidence that HCV-RNA possesses such a structure and it is thermodynamically impossible for a ribosome to bind to the 50 end of the RNA and disrupt the extensive

298 B. E. Clarke

1 (+) RNA

5’

3’

2 Translation 3 Processing

6

4

5 C C

Golgi

E1 E2

p7

E1 E2

2

(-) RNA

3’

3

5A

4B

5’

5B

4A

Endoplasmic reticulum

Nucleus

Figure 2. Replication of HCV and possible targets for novel therapeutics. 1. Viral particles enter the cell, possibly by an interaction with CD81. Inhibition of viral entry may be used to reduce viral spread but, at present, there is insucient data regarding the entry process to allow formal development of entry inhibitors. 2. After uncoating the viral RNA is translated to produce viral proteins. The translation process involves novel interactions between the viral 50 UTR and the host ribosomes and is an attractive target for therapeutic intervention that is currently under investigation. 3. The newly translated viral polyprotein is cleaved (processed) by the viral proteases to produce viral proteins that bind to the endoplasmic reticulum to form the replication complex. This cleavage process is a potential target for novel anti-virals. 4. Negative strand viral RNA is used as a template for viral replication and the production of new plus-strand viral RNA. This occurs at a replication complex and involves a number of viral proteins, all of which may be seen as therapeutic targets. 5. Newly synthesized viral RNA is encapsidated. This unique process is poorly understood and, although theoretically a good target for intervention, is not currently the subject of investigation. 6. Viral particles exit from the cell via the Golgi. This process may be interupted by drugs that interfere with Golgi transport and is being investigated as a potential therapeutic target.37.

structure to migrate to the translational start site. Instead, there is an interaction between the IRES and the ribosome that speci®cally orientates the ribosome onto the RNA so that it is directed exactly towards the correct AUG residue and translation can proceed.38 In many respects this mechanism resembles the process by which translation occurs in prokaryotes39 and there are already several examples of anti-bacterial drugs that inhibit bacterial replication by interference with this process. Although drugs that target RNAs in mammalian systems are not well precedented, the unique nature of this viral function means that it is an extremely attractive target in terms of selectivity compared to mammalian functions. Apart from the IRES function itself, the high degree of conservation in this region of the genome has also been exploited in other, di€erent, therapeutic strategies. Several laboratories have designed anti-sense RNA molecules that bind to speci®c parts of the 50 UTR and are able to disrupt its function.40 As an extension to this approach other groups have used ribozyme molecules (RNA molecules that catalyse cleavage of RNA) ¯anked by anti-sense sequences that target the ribozyme to the viral genome, whereon the viral genome is speci®cally cleaved by the endonuclease activity. This system has the advantage that the ribozyme is subsequently released from the inactivated genome and is then able to repeat the process on another genome.41 Although both the antisense and ribozyme approaches have technical

New drugs for hepatitis C virus 299

limitations in terms of pharmacokinetics and uptake into mammalian cells, the experimental data are suciently encouraging that clinical trials in chronic HCV patients are currently being planned. These studies are likely to be the ®rst of the next generation of HCV inhibitors and the results of these studies are eagerly awaited. 30 UTR Molecular studies on the 30 UTR are far less well advanced than the 50 UTR. Nevertheless, there are similarities in that the sequence in this region is again highly conserved between viral isolates and is predicted to have a highly ordered structure.42 Recent evidence has shown that the presence of the whole of this region is absolutely essential for viral replication but it is still unclear exactly how this functions.43 As for the 50 UTR the high degree of conservation would make this an attractive target for an antisense approach but so far there have been no reports of activity in this area. NS3 The NS3 protein is the most extensively studied enzyme in the viral polyprotein. Sequence alignments between the HCV NS3 sequence and other related viral sequences from the Flavivirus family indicated that this 70 kDa protein could potentially encode multiple enzymatic functions.44 Firstly, the amino terminal third of the molecule possesses sequence characteristics and an absolutely conserved catalytic triad, characteristic of a serine protease. Second, similar analysis predicted that the remainder of the molecule would possess RNA helicase activity essential for unwinding ordered structures within the genome and separating the positive and negative strands of the virus during RNA replication. Further supporting evidence came from the fact that similar enzymatic functions had been well characterized in related viral systems, particularly yellow fever virus, Dengue virus and bovine viral diarrhoea virus. For ease of experimentation, the majority of enzyme characterization has been undertaken on the two domains as separate entities and, signi®cantly, it has recently been reported that the two domains may be physically separated during viral replication.45 It is therefore appropriate to consider the properties of each as distinct potential targets for anti-viral drug development. NS3 protease Serine proteases are probably the most well characterized enzyme class currently available and there are several existing biochemical tools that have been well validated as inhibitors of serine protease activity. For these reasons the NS3 protease represents an attractive enzyme target for developing new anti-virals for HCV. This protease was initially shown, using in vitro systems, to be responsible for the proteolytic cleavage of the majority of the cleavages of the polyprotein releasing the non-structural replicative proteins (see Figure 1). Interestingly, it was also demonstrated that the eciency of these cleavages could be signi®cantly stimulated by the presence of a small peptide derived from the NS4A protein.46 Following these observations the protein has been produced in a variety of expression systems and the puri®ed protein subjected to exhaustive biochemical characterization. In particular, assay systems have been established using peptide substrates that allow rapid identi®cation of potential inhibitors in high throughput screening campaigns (e.g. see47). Disappointingly, initial observations indicated that, although the enzyme appeared to be a classical serine protease, it was

300 B. E. Clarke

particularly refractory to inhibition by conventional serine protease inhibitors so completely new inhibitor templates were required. These approaches have mainly focused on the use of peptide fragments corresponding to the regions of the polyprotein around the target cleavage sites. Although such peptide inhibitors require signi®cant chemical development to produce genuine pharmaceutical molecules, considerable progress has been made towards optimizing these template inhibitors (e.g. see48). In particular, these studies have been galvanized by the resolution of the Xray structure of the enzyme by several groups. These studies have allowed a remarkable insight into the molecule and revealed several features that had been previously unexpected (e.g. see49). The basic structure of the molecule showed a typical chymotrypsin-like fold but, unlike any other serine protease, the NS3 backbone folded intimately around the NS4A co-factor in such a way that the presence of the cofactor was essential for optimal alignment of the enzyme active site. Not only did this explain the biochemical observations on the requirement for NS4A, but also it e€ectively presented the interaction of NS3 and NS4A as a potential secondary antiviral target, albeit a protein±protein interaction that would be dicult to disrupt. A further interesting feature of the structure was the presence of a tetrahedral zinc ion that also played an important role in the structural integrity of the enzyme. However, the most revealing feature in terms of anti-viral drug development was the conformation of the protein around the active site. Unlike the other serine protease structures that were available, the active site of the HCV enzyme was very shallow, with very little surface topography. This feature partly explained the lack of activity of conventional serine protease inhibitors but it also posed signi®cant hurdles for the rational design of novel inhibitors that could bind to the active site of the enzyme with high anity. Nevertheless, further crystallographic analysis of the interaction of the enzyme with its substrate has revealed detailed information on how the substrate docks into the active site.50 This information is now being used to rationally design new inhibitors that will be further re®ned by an iterative process of chemical modi®cation and re-crystallization. Given the current rate of progress in this area, the prospect of a potential clinical candidate emerging from this approach in the near future seems promising. NS3 helicase Sequence analysis of the remainder of the NS3 protein revealed the presence of several motifs characteristic of RNA helicases that had been well documented in other viruses. The function of this portion of the protein, presumably, is to unwind the positive and negative strands of the genome during viral replication and, possibly, to disrupt areas of local secondary structure (e.g. the IRES, 30 UTR) allowing the viral polymerase to replicate its template. As for the protease, this part of NS3 has now been expressed in several di€erent forms and detailed biochemical analysis performed.51 The helicase is not speci®c for RNA helices but has been shown to function similarly on DNA substrates. Given this information it has been relatively straightforward to develop high throughput screening systems in which displacement of radiolabelled DNA strands from small duplex DNA substrates can easily be monitored without separation of the free strands. However, these random screening approaches have, again, been signi®cantly enhanced by the detailed elucidation of the crystal structure of the helicase.52 The structure indicates that the helicase is composed of three domains one of which encompasses the ATPase activity of the enzyme and one of which possesses the nucleic acid binding region. Hydrolysis of ATP by the ATPase provides the energy

New drugs for hepatitis C virus 301

for the domains to conformationally shift and it is this movement that causes the disruption of the local secondary structure. During this process the enzyme moves along the substrate and, thus, repeats the activity on the next part of the helix. This structural information allows rational design of new inhibitor templates but the interaction of new inhibitors identi®ed by random screening campaigns can also be assessed, and optimized, by co-crystallization as enzyme±inhibitor complexes. One advantage of the helicase over the protease as a target is that the helicase functions of viruses such as yellow fever are, intrinsically, very similar. This allows inhibitors produced against the HCV enzyme to be further validated against these surrogate viruses and this ability will signi®cantly assist the development of drugs against this target. A further factor that will enhance the development of drugs against the NS3 domains will be an understanding of the ways in which the protease and helicase domains interact with each other. It is already clear that the protease and helicase activities of the whole NS3 protein are subtly di€erent biochemically than the isolated domains.53 Elucidation of the crystal structure of the whole NS3 protein is likely to reveal more information that should allow further optimization of inhibitor molecules targeted to either the protease or helicase domains of NS3. NS5B RNA-dependent RNA polymerase In some respects the most attractive viral target is the RNA-dependent RNA polymerase activity present in the NS5B protein. Firstly, this polymerase is clearly an essential component of the viral replication machinery so interference in its function will have a dramatic e€ect on viral replication. Second, the majority of anti-viral drugs that are currently marketed all target the polymerase (replicase) functions from their respective viruses e.g. Aciclovir (Herpes), AZT (HIV) and Lamivudine (HBV). Not only is there signi®cant experience in developing such inhibitors, but many reagents are also available that may provide templates for developing inhibitors against the HCV polymerase. The enzyme itself was ®rst identi®ed as the viral polymerase by alignment with other viral RNA polymerases and by the presence of a conserved characteristic GDD motif in the proposed active site. As for the other enzymes, production of recombinant enzyme and characterization of its biochemical properties subsequently con®rmed this activity (e.g. see54) and recently the resolution of the crystal structure of the enzyme was reported.55 As would be expected, the enzyme is capable of incorporating radioactive ribonucleotide triphosphates into nascent RNA molecules using speci®c primer/template systems. This incorporation can be easily monitored and allows the establishment of high throughput robotic systems for rapid identi®cation of inhibitory molecules. The limitation of this system lies in the fact that nucleoside analogues require phosphorylation to their respective triphosphates by host or viral kinases in order to be active. This can only occur in cellular systems and, in some instances, in the presence of replicating virus. The ®rst generation high throughput systems that have been developed for the HCV polymerase are all in vitro systems that do not possess these characteristics and so the development of a cellular replication system for HCV remained a high priority. Furthermore, it is important to recognize that HCV, like other positive strand RNA viruses, is likely to replicate its genome via enzyme-bound multimolecular replication complexes comprising all the virus replicative enzymes, as well as any required host co-factors. Although studies of individual viral enzymes in isolation has given enormous insight into their function, these systems are all arti®cial and it is essential to study the ways these enzymes function in the context of the whole

302 B. E. Clarke

replication complex. Recent reports indicate that cellular RNA replication systems have now been successfully achieved and these will now be described. HCV replicons Ideally, the easiest way to study virus replication is if the virus is able to ful®l its full replicative cycle by infection of stable cell-lines that are used routinely in the laboratory. Virus replication can then be monitored by the cytopathic e€ect of the virus on the cells or by direct detection of viral proteins or replicating genomes. Although many attempts have been made to develop such systems for HCV none of these has been suciently reproducible or able to support high-level virus replication. It is currently unclear why it is so dicult to propagate HCV in tissue culture so alternative approaches have therefore been explored. The most obvious approach to resolving this problem lay in the generation of infectious molecular clones of HCV which, upon transfection into cell-lines, would allow translation of the viral genome and initiate the replicative cycle. Several groups have now reported the successful construction of such clones56±58, the RNA of which causes typical hepatitis when injected directly into the livers of chimpanzees. However, disappointingly, there have been no reports that any of these clones were able to initiate viral replication when they were studied in tissue culture systems. In a re®nement of this approach Bartenschlager and colleagues59 (see Chapter 4) generated a full-length copy of the viral genome and replaced the structural proteins of the virus with a marker protein coding for neomycin resistance (neo). This genome was then transfected into cell-lines of hepatocyte origin in the presence of a selectable drug, G418. Any cell that does not produce the neo protein is selectively removed by the toxicity of G418 so any clones that are resistant to G418 should be undergoing an RNA replication cycle dependent on the HCV replicative machinery. Using this system it was clearly shown that genuine HCV-RNA replication could be achieved, the cells that were replicating the RNA could be maintained stably for considerable periods of time and the replication was totally dependent on the activity of the HCV NS5B RNA polymerase enzyme. Moreover, the ratios of negative strand RNA produced compared to positive strand (1:10) suggested that the replication was typically asymmetrical indicating that natural RNA replication was occurring. Although this system does not represent a full natural virus replication cycle it does provide a huge breakthrough in the area and will signi®cantly assist the development of HCV replication inhibitors in the near future. IMMUNOMODULATORS The most successful anti-viral drugs developed to date have targetted viral replication. However, there is considerable interest in the possibility of developing immunomodulatory drugs that might enhance the host immune response and lead to viral clearance. For HCV, progress in this area has been slow since the absence of a small animal model makes it dicult to evaluate the key components of the immune response that may lead to viral clearance. Studies in human liver transplant recipients who received passive immunization with immunoglobulin to prevent hepatitis B infection have suggested that, in those co-infected with HCV, passive immunization with immunoglobulin prepared before screening for HCV was introduced ameliorated the resulting HCV related hepatitis but this bene®t was no longer seen when immunoglobulin donors were screened for HCV.60 This suggests that antibodies

New drugs for hepatitis C virus 303

against HCV, present in the unscreened sera, may ameliorate HCV and indicates that passive vaccination against HCV may be bene®cial. The costs and inconvenience of using sera from patients with resolved HCV clearly mitigate against this approach but the development of neutralizing monoclonal antibodies against appropriate viral epitopes may allow this form of therapy to be developed. For many viral infections viral eradication is dependent upon an appropriate cytotoxic T-cell response. In patients with chronic HCV it is clear that cytotoxic Tcells directed against HCV proteins are present but they are unable to eliminate all of the infected hepatocytes.61 The reasons for this immunological failure are not yet clear but it is to be hoped that a better understanding of the immune paralysis found in chronic HCV will lead to new immunomodulatory therapies.

SUMMARY Clinical data emerging from the use of IFN-a in combination with other drugs, particularly ribavirin, have signi®cantly improved the therapy of hepatitis C. In the near future, clinical studies will focus on improving the ways in which these existing combinations are used and optimizing the components of the combinations. However, given the signi®cant side e€ects associated with the existing drugs, there is considerable need for the development of new therapeutics for incorporating into the combinations. These new therapeutics may be direct inhibitors of viral replication or immunomodulators that are able to stimulate the host immune system and, possibly, induce therapeutic cure. Considerable progress is being made at a research level towards attaining these goals and the prospects of a new generation of HCV inhibitors in the near future looks increasingly optimistic.

REFERENCES 1. Alter MJ. Epidemiology of hepatitis C. Hepatology 1997; 26: 62±65. * 2. See€ LB, Buskell-Bales Z, Wright EC et al. Long-term mortality after transfusion-associated non-A non-B hepatitis. New England Journal of Medicine 1992; 72: 1906±1911. 3. Darby SC, Ewart DW, Giangrande PLF et al. Mortality from liver cancer and liver disease in haemophiliac men and boys given blood products contaminated with hepatitis C. Lancet 1997; 349: 1425±1431. 4. El-Serag HB & Mason AC. Rising incidence of hepatocellular carcinoma in the United States. New England Journal of Medicine 1999; 340: 745±750. 5. McHutchison JG, Gordon SC, Schi€ ER et al. Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. New England Journal of Medicine 1998; 339: 1485±1492. 6. Poynard T, Marcellin P, Lee S et al. Randomised trial of interferon alfa-2b and ribavirin for 48 weeks or for 24 weeks versus interferon alfa-2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis C virus. Lancet 1998; 352: 1426±1432. * 7. Davis GL, Esteban-Mur R, Rustgi V et al. Interferon alpha-2b alone or in combination with ribavirin for the treatment of relapse of chronic hepatitis C. New England Journal of Medicine 1998; 339: 1493±1499. 8. Davis GL, Balart LA, Schi€ ER et al. Treatment of chronic hepatitis C with recombinant interferon alfa. A multicentre randomized, controlled trial. New England Journal of Medicine 1989; 321: 1501±1506. 9. Di Bisceglie AM, Martin P, Kassianides C et al. Recombinant interferon alfa therapy for chronic hepatitis C. A randomized double blind placebo-controlled trial. New England Journal of Medicine 1989; 321: 1506±1510. 10. Marcellin P, Boyer N, Giostra E et al. Recombinant human alpha-interferon in patients with chronic nonA, non-B hepatitis: a multicentre randomized controlled trial from France. Hepatology 1991; 13: 393±397. 11. Causse X, Godinot H, Chevallier M et al. Comparison of 1 or 3 MU of interferon alfa-2b and placebo in patients with chronic non-A, non-B hepatitis. Gastroenterology 1991; 101: 497±502.

304 B. E. Clarke 12. Poynard T, Bedossa P, Chevallier M et al. A comparison of three interferon alfa-2B regimens for the longterm treatment of chronic non-A, non-B hepatitis. New England Journal of Medicine 1995; 332: 1457±1462. 13. Fried MW, Shi€mann ML, Sterling RK et al. A multicenter, randomized trial of daily high dose interferon alfa-2b for the treatment of chronic hepatitis C: prospective strati®cation by viral burden and genotype [Abstract L1080]. Gastroenterology 1998; 114: A1242.. 14. Ouzan D, Babany G, Valla D & Opolon P. Comparison of high initial and ®xed-dose regimens of interferon-alpha 2a in chronic hepatitis C: a randomized controlled trial. Journal of Viral Hepatitis 1998; 5: 53±59. 15. Bosch O, Tapia L, Quiroga JA & Carreno V. An escalating dose regime of recombinant interferon alpha2a in the treatment of chronic hepatitis C. Journal of Hepatology 1993; 17: 146±149. 16. Shi€mann ML, Hofmann CM, Luketic VA, Thompson EB & Sanyal AJ. Treatment of chronic hepatitis C with escalating doses of interferon alpha-2b increases both biochemical and virologic response [Abstract 184]. Hepatology 1995; 22: 152A. 17. Shi€man ML. Use of high-dose interferon in the treatment of chronic hepatitis C. Seminars in Liver Disease 1999; 19: 25±34. 18. Tong MJ, Reddy KR, Lee WM et al. Treatment of chronic hepatitis C with consensus interferon: a multicenter, randomized, controlled trial. Hepatology 1997; 26: 747±754. 19. Foster GR & Finter NB. Are all type 1 interferons equivalent? Journal of Viral Hepatitis 1998; 5: 143±152. 20. Meister A, Uze G, Mogensen KE et al. Biological activities and receptor binding of two human recombinant interferons and their hybrids. Journal of General Virology 1986; 67: 1633±1643. 21. Xu Z-X, Ho€mann J, Patel I et al. Single-dose safety/tolerability and pharmacokinetic/pharmacodynamics (PK/PD) following administration of ascending subcutaneous doses of pegylated-interferon (PEG-IFN) and interferon a-2a (IFN a-2a) to healthy subjects [Abstract 2157]. Hepatology 1998; 28: 702A. 22. Bodenheimer HC, Lindsay KL, Davis GL et al. Tolerance and ecacy of oral ribavirin treatment of chronic hepatitis C: a multicenter trial. Hepatology 1997; 26: 473±477. 23. Reichard O, Norkrans G, Fryden A et al. Randomised double-blind, placebo-controlled trial of interferon alpha-2b with and without ribavirin for chronic hepatitis C. Lancet 1998; 351: 83±86. 24. Chemello L, Cavalletto L, Bernardinello E et al. The e€ect of interferon alfa and ribavirin combination therapy in naõÈ ve patients with chronic hepatitis C. Journal of Hepatology 1995; 23: 8±12. 25. Lai MY, Kao JH, Yang PM et al. Long-term ecacy of ribavirin plus interferon alfa in the treatment of chronic hepatitis C. Gastroenterology 1996; 111: 1307±1312. *26. McHutchison JG, Gordon SC, Schi€ ER et al. Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. New England Journal of Medicine 1998; 339: 1485±1492. *27. Poynard T, Marcellin P, Lee S et al. Randomised trial of interferon alfa-2b and ribavirin for 48 weeks or for 24 weeks versus interferon alfa-2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis C virus. Lancet 1998; 352: 1426±1432. *28. Davis GL, Esteban-Mur R, Rustgi V et al. Interferon alpha-2b alone or in combination with ribavirin for the treatment of relapse of chronic hepatitis C. New England Journal of Medicine 1998; 339: 1493±1499. 29. Maddrey WC. Safety of combination interferon alfa-2b/ribavirin therapy in chronic hepatitis-C relapsed and treatment-naõÈ ve patients. Seminars in Liver Disease 1999; 19: 67±75. 30. Smith JP. Treatment of chronic hepatitis C with amantadine. Digestive Diseases and Sciences 1997; 42: 1681±1687. 31. Khalili M, Olmeda M, Yantsos V et al. Pilot study of intron-A and ribavirin vs intron-A and amantadine in interferon non-responders with chronic hepatitis C [Abstract L0303]. Gastroenterology 1998; 114: 1271. 32. Brillanti S, Foli M, Gramantieri L et al. Triple antiviral therapy for chronic hepatitis C in interferon alfa non-responders: a pilot randomized controlled study [Abstract 953]. Hepatology 1997; 26: 367A. 33. Younossi ZM & Perrillo RP. The roles of amantadine, rimantadine, ursodeoxycholic acid, and NSAIDs, alone or in combination with alpha interferons, in the treatment of chronic hepatitis C. Seminars in Liver Disease 1999; 19: 95±102. *34. Davis GL, Nelson DR & Reyes GR. Future options for the management of hepatitis C. Seminars in Liver Disease 1999; 19: 103±112. *35. Choo QL, Richman KH, Han JH et al. Genetic organisation and diversity of the hepatitis C virus. Proceedings of the National Academy of Sciences of the USA 1991; 88: 2451±2455. 36. Clarke BE. Molecular virology of hepatitis C virus. Journal of General Virology 1997; 78: 2397±2410. 37. Zitzmann N, Mehta AS, Carrouee S et al. Imino sugars inhibit the formation and secretion of bovine viral diarrhea virus, a pestivirus model of hepatitis C virus: implications for the development of broad spectrum anti-hepatitis virus agents. Proceedings of the National Academy of Sciences of the USA 1999; 96: 11878±11882 (In process citation). 38. Sizova DV, Kolupaeva VG, Pestova TV et al. Speci®c interaction of eukaryotic translation initiation factor 3 with the 50 nontranslated regions of hepatitis C virus and classical swine fever virus. Journal of Virology 1998; 72: 4775±4782.

New drugs for hepatitis C virus 305 39. Pestova TV, Shatsky IN, Fletcher SP et al. A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of hepatitis C and classical swine fever virus RNAs. Genes and Development 1998; 12: 67±83. 40. Brown-Driver V, Eto T, Lesnik E et al. Inhibition of translation of hepatitis C virus RNA by 20 -modi®ed antisense oligonucleotides. Antisense and Nucleic Acid Drug Development 1999; 9: 145±154. 41. Lieber A, He C-Y, Polyak S et al. Elimination of hepatitis C virus RNA in infected human hepatocytes by adenovirus-mediated expression of ribozymes. Journal of Virology 1996; 70: 8782±8791. 42. Tanaka T, Kato N, Cho MJ & Shimotohno K. A novel sequence found at the 30 terminus of the hepatitis C virus genome. Biochemical and Biophysical Research Communications 1995; 215: 744±749. *43. Kolykhalov AA, Agapov EV, Blight KJ et al. Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA. Science 1997; 277: 570±574. 44. Grakoui A, Wychowski C, Lin C et al. Expression and identi®cation of hepatitis C virus polyprotein cleavage products. Journal of Virology 1993; 67: 1385±1395. 45. Shoji I, Suzuki T, Sato M et al. Internal processing of hepatitis C virus NS3 protein. Virology 1999; 254: 315±323. 46. Bartenschlager R, Lohmann V, Wilkinson T et al. Complex formation between the NS3 serine-type proteinase of the hepatitis C virus and NS4A and its importance for polyprotein maturation. Journal of Virology 1995; 69: 7519±7528. 47. Kakiuchi N, Nishikawa S, Hattori M et al. A high throughput assay of the hepatitis C virus nonstructural protein 3 serine proteinase. Journal of Virological Methods 1999; 80: 77±84. 48. Cicero DO, Barbato G, Koch U et al. Structural characterization of the interactions of optimized product inhibitors with the N-terminal proteinase domain of the hepatitis C virus (HCV) NS3 protein by NMR and modelling. Journal of Molecular Biology 1999; 289: 385±396. 49. Kim JL, Morgenstern KA, Lin C et al. Crystal structure of the hepatitis C virus NS3 protease domain complexed with a synthetic NS4A cofactor peptide. Cell 1996; 87: 343±355. 50. LaPlante SR, Cameron DR, Aubry N et al. Solution structure of substrate-based ligands when bound to hepatitis C virus NS3 protease domain. Journal of Biological Chemistry 1999; 274: 18618±18624. 51. Preugschat F, Averett DR, Clarke BE & Porter DJT. A steady-state and pre-steady-state kinetic analysis of the NTPase activity associated with the hepatitis C virus NS3 helicase domain. Journal of Biological Chemistry 1996; 271: 24449±24457. 52. Yao N, Hesson T, Cable M et al. Structure of the hepatitis C virus RNA helicase domain. Nature Structural Biology 1997; 4: 463±467. 53. Morgenstern KA, Landro JA, Hsiao K et al. Polynucleotide modulation of the protease, nucleoside triphosphatase and helicase activities of a hepatitis C virus NS3±NS4A complex isolated from transfected COS cells. Journal of Virology 1997; 71: 3767±3775. 54. Oh J-W, Ito T & Lai MMC. A recombinant hepatitis C virus RNA-dependent RNA polymerase capable of copying the full-length viral RNA. Journal of Virology 1999; 73: 7694±7702. *55. Lesburg CA, Cable MB, Ferrari E et al. Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site. Nature Structural Biology 1999; 6: 937±943. 56. Major ME, Mihalik K, Fernandez J et al. Long-term follow-up of chimpanzees inoculated with the ®rst infectious clone for hepatitis C virus. Journal of Virology 1999; 73: 3317±3325. 57. Yanagi M, St Claire M, Shapiro M et al. Transcripts of a chimeric cDNA clone of hepatitis C virus genotype 1b are infectious in vivo. Virology 1998; 244: 161±172. 58. Beard MR, Abell G, Honda M et al. An infectious molecular clone of a Japanese genotype 1b hepatitis C virus. Hepatology 1999; 30: 316±324. *59. Lohmann V, Korner F, Koch J et al. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line [see comments]. Science 1999; 285: 110±113. 60. Feray C, Gigou M, Samuel D et al. Incidence of hepatitis C in patients receiving di€erent preparations of hepatitis B immunoglobulins after liver transplantation. Annals of Internal Medicine 1998; 128: 810±816. 61. Koziel M. The role of immune responses in the pathogenesis of hepatitis C virus infection. Journal of Viral Hepatitis 1997; 4S1: 31±41.