New therapeutic approaches to hepatitis C virus
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Year 201X will see a huge battle against hepatitis C virus (HCV) infection. HCV, a leading cause of end stage diseases and hepatocellular malignancies, is a negative legacy of the past in many regions worldwide, and has long been refractory to conventional treatments. The most effective peginterferons and ribavirin-based antiviral therapies can eliminate the virus in only half of patients treated, and the treatments are often poorly tolerated. Recently, the development of an HCV cell culture system has become a turning point of basic research. At present, novel therapeutic agents with different mechanisms of action are under development or on clinical trials. Some of these drugs have been proven to be effective when used with the conventional treatments, and may constitute antiviral therapies without being used in combination with interferons. This article reviews the current status of preclinical drug development, ongoing clinical trials, and near future perspectives in the field of HCV therapeutics.
KeywordsProtease inhibitor Polymerase inhibitor Cyclosporine HCV replicon system
Hepatitis C virus (HCV), infecting 170 million people worldwide, is characterized by chronic liver inflammation and fibrogenesis leading to end-stage liver failure and hepatocellular malignancy . Since the first report in 1986, interferon-alpha and -beta formulations have continued to be a mainstay against HCV infection . However, even combination treatments of ribavirin plus peginterferon can achieve sustained viral response (SVR) rates of only 40–50% [3, 4, 5, 6]. Furthermore, these therapies carry a substantial risk of serious side effects, and in quite a considerable proportion of patients require premature therapy discontinuation . Given these situations, the development of safe and effective therapies against HCV has been our first priority.
Basics of HCV infection and replication
Drug development and clinical trials of anti-HCV drugs
Excerpt: ongoing clinical trials of anti-HCV agents (exclude interferon-alpha, peginterferons, and ribavirin)
NS5A inhibitor, A-831
cIFN + RBV
Interferon lambda (IL-29)
Thymosin alpha 1
Fluvastatin + IFN/RBV
The HCV proteins are post- or co-translationally processed by host and viral proteases. The nonstructural NS3 protein is a bifunctional protein that has serine protease and helicase (Fig. 2). NS3 serine protease, which the protease inhibitors target, cleaves junctions between NS3 and NS4A in cis and subsequently cleaves junctions between the other nonstructural proteins in trans. In addition to the effects on the viral proteins, NS3 protease targets host proteins, TRIF and Cardif (a.k.a. VISA, IPS-1 or MAVS) that mediate innate antiviral signaling to induce production of endogenous interferon in response to cellular virus replication . Based on the findings, targeting of NS3 protease not only inhibits virus expression but also may help impaired cells recover their ability to produce antiviral proteins.
Protease inhibitors were designed from crystal structures of the NS3 protease domain . The first in vivo proof-of-concept report was that of BILN2061 , which showed rapid and potent antiviral effects by 4-day twice daily oral administration. Further development of the drug, however, has been halted because of cardiotoxicity in pre-clinical animal tests. At present, two protease inhibitors are in late phase II and III, telaprevir (VX-950) and boceprevir (SCH405054).
A 14-day short-term administration showed that the plasma HCV titer decreased 1.1-log by peginterferon-alpha2a alone, 4.0-log by telaprevir, and 5.5-log by a combination of peginterferon and telaprevir [20, 21, 22]. Two phase II trials, PROVE1 and PROE2, have been independently conducted [23, 24]. In PROVE 1, 61% achieved SVR by the triple combination therapy for 24 weeks and 67% for 48 weeks, while only 41% achieved SVR standard of care treatment . In PROVE 2, 68 and 62% achieved SVR by 12- or 24-week triple combination therapy, while 48% achieved standard of care . The above results from the two trials showed that treatment period of 24 weeks is sufficient to improve the outcome of the triple combination by over 20% compared to the conventional 48-week peginterferon and ribavirin treatment and that the combination with ribavirin is necessary to achieve optimal results.
Emergence of resistant mutations has been reported in vitro [26, 27] and in vivo [20, 28]. After 14 days of telaprevir monotherapy, 50–75% of serum quasispecies acquired resistant mutations . The viral breakthrough occurred in 5 of 163 patients during 12 weeks of the triple combination therapy and in 24 out of 78 patients who were treated with telaprevir and peginterferon. The telaprevir-resistant mutations were present in most breakthrough cases . A phase III trial is currently being conducted in Japan.
Polymerase inhibitors target viral NS5B RNA-dependent RNA polymerase. There are both nucleoside and non-nucleoside type agents. In vitro studies showed that the nucleoside type polymerase inhibitors have a higher barrier to viral drug resistance than the non-nucleoside type . Treatment of patients with polymerase inhibitors developed no viral resistance after 14-day monotherapy with R1626 or NM283.
R1626 is a prodrug of NS5B polymerase inhibitor R1479. A 4-week administration of R1626 in combination with peginterferon alfa-2a plus ribavirin in HCV genotype 1 patients showed that HCV RNA was undetectable in 69 and 74% of patients at the end of treatment with R1626 plus peginterferon and triple combination, respectively. In contrast, standard of care therapy eliminated HCV-RNA in only 5% within 4 weeks. There was no evidence of development of viral resistance. Grade 4 neutropenia developed in 78, 39, and 10% of the dual, triple and standard of care, respectively, and was the main reason for dose reductions. There are other polymerase inhibitors in early clinical and preclinical development, PF-00868554 , BILB 1941 , AG-021541 . Two polymerase inhibitors, NM283  and HCV796 , have been cancelled or halted in their clinical trials for gastrointestinal adverse effects and for hepatotoxicity, respectively .
New interferon formulations
New formulations of type I interferons and their relatives have been developed. These include Albuferon , IL-29, Locteron and interferon-omega . Because these type-I interferons share a common cell surface receptor, IFNAR, most new formulations are designed in combination with controlled release drug delivery devices.
IL-29 (interferon lambda)
An interferon-related cytokine IL-29, also known as interferon-lambda 1, inhibits virus replication by inducing a cellular antiviral response similar to that activated by interferon-alpha/beta. Because IL-29 binds to its unique receptor, this cytokine might also be useful therapeutically in combination with interferons to treat HCV infection. In vitro studies have shown that IL-29 has potent and specific anti-viral activities against HCV and other flaviviruses . A combination of IL-29 with interferon-alpha or interferon-gamma induced antiviral gene expression was more effective than when used alone and was more effective at blocking vesicular stomatitis virus and HCV replication in vitro . A phase I study of a pegylated IL-29 formulation showed that the drug was save and well tolerated.
Locteron is a controlled-release formulation of unpegylated recombinant interferon-alpha2b in microspheres, which enables injections at a rate of every two weeks. Locteron has been evaluated in 27 volunteers . After injection of Locteron, serum interferon-alpha2b remained elevated through 14 days. The serum half-life of Locteron was more than 2-fold that of peginterferon-alpha2b. A phase IIa study for treatment naïve HCV patients has shown that the early virological response (EVR, less than two-log drop of virus titer in 12 weeks) was 88 to 100% with doses of 320 through 640 mg per 2 weeks (EASL 2008).
Toll-like receptor (TLR) agonists
TLRs are transmembrane receptors that recognize pathogen-associated molecular patterns of pathogens and mediate protective responses against microorganisms. Single-stranded viral RNA and bacterial DNA containing unmethylated CpG motifs are the ligands for TLR7 and TLR8 and 9 respectively [41, 42]. Binding of PAMP onto TLRs recruits an appropriate adaptor protein to the TIR domain and leads to the activation of NF-kB and IRF3 resulting in the induction of high and sustained levels of interferons and related genes. Isatoribine is a selective agonist of TLR7. A 7-day treatment with intravenous isatoribine caused significant reduction of plasma HCV RNA in patients [43, 44]. Viral load reduction occurred in patients infected with HCV genotype 1 as well as non-1 genotypes. IMO-2125, a TLR9 agonist, has started phase II for study in prior null-responder HCV patients .
Thymosin alpha 1
A phase III study of thymosin alpha 1 used in combination with interferon and ribavirin showed only a modest improvement in treatment outcome and liver histology. Final results from the complete study have found that thymosin alpha 1 in combination with pegylated interferon failed to produce significant results over pegylated interferon alone (46).
Cyclosporins and cyclophilin inhibitors
Cyclosporin A (CsA) is a neutral cyclic undecapeptide that was isolated from the fungus Hypocladium inflatum gams. CsA has been used widely for post-transplantation immune modulation and various autoimmune and inflammatory diseases. It has been reported that CsA suppressed HIV replication in vitro by blocking the function of cellular cyclophilin A to bind to viral gag protein [47, 48]. We and another group have reported that CsA substantially and specifically inhibits intracellular HCV replication in vitro [49, 50] using HCV replicon . The 50% replicon inhibitory concentration of CsA is ~0.5 μg/ml, which is within clinically achievable concentrations. The antiviral effect of CsA is mediated by intracellular ligand proteins, cyclephillins [51). Non-immunosuppressive cyclosporin analogues, cyclosporin D  and NIM811  induced a similar suppression of HCV as well as of HIV replication .
DEBIO-025, a non-immunosuppressive cyclosporine analogue, inhibited as efficiently as CsA in vitro . Clinical trial of DEBIO-025 monotherapy in HCV/HIV coinfection patients showed serum viral decline by 3.6-log10 14-day oral treatment with 1200 mg twice daily . In vitro synergic effects of Debio 025 have been reported when used in combination with STAT-C agents, polymerase or protease inhibitors . Results from a double-blind, placebo-controlled study of Debio 025 in combination with peginterfeorn-alpha2a in HCV patients showed that their combination achieved a 4.6–4.8 log10 early HCV-RNA decline, compared to a 2.5 log10 decline in peginterferon monotherapy.
Nitazoxanide was originally developed and widely used as an antiparasite agent for cryptosporidiosis and helminthic infections. This drug has been recently shown to be effective against a wide range of organisms, including bacteria and viruses . Nitazoxanide and its active metabolite, tizoxanide (TIZ), suppressed HBV and HCV replication in vitro . Nitazoxanide was equally effective on lamivudine- or adefovir-resistant HBV mutants and on telaprevir-resistant HCV mutants . Monotherapy of nitazoxanide in HCV genotype 4, low virus titer patients showed that 7 of 23 patients (30.4%) became serum HCV-RNA negative during therapy and 17.4% achieved SVR. Adverse events were similar to the placebo group . Combination therapy of nitazoxanide, peginterferon plus ribavirin showed that the SVR rate was 79% in triple combination therapy, which was significantly higher than that of the standard of care patients of 50% . Combination treatment with nitazoxanide plus peginterferon had a still higher SVR rate of 61%. The therapies were well tolerated except for higher rates of anemia in the groups receiving ribavirin. An in vitro study demonstrated a resistance to nitazoxanide by prolonged treatment, which was directed by adaptation of host cells but not by virus mutation . Currently, a phase II trial is ongoing.
Other host-targeted drugs
NA255 prevents the de novo synthesis of sphingolipids, major lipid raft components, by inhibiting serine palmitoyltransferase activity. NA255 inhibited HCV replication by disrupting anchoring of HCV nonstructural proteins on the lipid rafts. .
Bavituximab, a chimeric antibody against exposed anionic phospholipids, showed antiviral action against a wide variety of viruses. Bavituximab selectively binds to virus-infected cells in which anionic phospholipids are exposed on the cell surface presumably by virus replication-induced ER-stress response . A phase I clinical study is being prepared .
Celgosivir is an alpha-glucosidase I inhibitor that has been shown to inhibit the replication of various enveloped viruses. In vitro studies using flaviviruses, bovine viral diarrhea virus (BVDV) as well as hepatitis C virus, showed that celgosivir had potent and specific activities [66, 67].
Given the current situation of the absence of singly effective, proven antiviral agents against HCV other than interferon formulations, combinations of interferon with agents that possess potential antiviral effects will continue to dominate the therapy. Various anti-HCV agents have been developed and have shown antiviral activities and safety of use. These situations suggest that the next field of anti-HCV therapy will be interferon treatment potentiated by combinations of certain drugs. Continuing the search for more potent, singly effective, and less toxic antiviral drugs is mandatory to improve clinical anti-HCV chemotherapeutics.
A part of this study was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan, the Japan Society for the Promotion of Science, Ministry of Health, Labour and Welfare, Japan, Japan Health Sciences Foundation, and the National Institute of Biomedical Innovation.