In the last few years, numerous directly acting antiviral agents (DAAs) have been implemented successfully in treatment algorithms of chronic hepatitis C virus (HCV) infection. Initially, as combination therapy with pegylated interferon (PEG-IFN) α / ribavirin, and – more recently and most importantly – as IFN-free combination therapies, DAA-based regimens result in HCV eradication in the vast majority of patients with chronic hepatitis C (Lange 2014).
In principle, each of the four HCV structural and six non-structural proteins, HCV-specific RNA structures such as the IRES, as well as host factors on which HCV depends, are suitable targets for DAA agents (Figure 1).
In this chapter, the historical development of DAA compounds as well as new regimens which are currently in clinical development are presented, whereas already approved DAA-based regimens are discussed in chapter 12.
|Drug name||Company||Target / Active site||Phase|
|NS3/4A protease inhibitors|
|Simeprevir (TMC435)||Janssen / Medivir||Active site / macrocyclic||IV|
|Vaniprevir (MK-7009)||Merck||Active site / macrocyclic||IV|
|Grazoprevir (MK-5172)||Merck||Active site / macrocyclic||IV|
|Asunaprevir (BMS-650032)||Bristol-Myers Squibb||Active site||IV|
|Voxilaprevir GS-9857||Gilead||Active site||IV|
|Paritaprevir (ABT-450)||Abbott||Active site||IV|
|Glecaprevir (ABT-493)||Abbott||Active site||IV|
|Nucleoside analogue NS5B polymerase inhibi-tors (NI)|
|Sofosbuvir (GS-7977)||Gilead||Active site||IV|
|Uprifosbuvir (MK-3682 formerly IDX20963)||Merck||Active site||III - stopped|
|AL335||Janssen||Active site||II - stopped|
|Non-nucleoside NS5B polymerase inhibitors (NNI)|
|Dasabuvir (ABT-333)||Abbott||NNI site 3 / palm 1||IV|
|Daclatasvir (BMS-790052)||Bristol-Myers Squibb||NS5A domain 1 inhibitor||IV|
|Pibrentasvir (ABT-530)||Abbvie||NS5A protein||IV|
|Ledipasvir (GS-5885)||Gilead||NS5A protein||IV|
|Velpatasvir (GS-5816)||Gilead||NS5A protein||IV|
|Ombitasvir (ABT-267)||Abbott||NS5A protein||IV|
|Odalasvir (ACH-2928)||Achillion||NS5A protein||II - stopped|
|Elbasvir (MK-8742)||Merck||NS5A protein||IV|
|Ruzasvir (MK-8408)||Merck||NS5A protein||III - stopped|
HCV is a positive-sense single-stranded RNA virus of approximately 9600 nucleotides. The HCV genome contains a single large open reading frame encoding for a polyprotein of about 3100 amino acids. From this initially translated polyprotein, the structural HCV protein core (C) and envelope glycoproteins 1 and 2 (E1, E2), p7, and the six non-structural HCV proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B, are processed by both viral and host proteases. The core protein forms the viral nucleocapsid carrying E1 and E2, the receptors for viral attachment and host cell entry. The non-structural proteins are multifunctional proteins essential for the HCV life cycle (Barten-schlager 2004, Moradpour 2007). P7 is a small hydrophobic protein that oligomerises into a circular hexamer, most likely serving as an ion channel through the viral lipid membrane. The large translated section of the HCV genome is flanked by the strongly conserved HCV 3’ and 5’ untranslated regions (UTR). The 5’UTR is comprised of four highly structured domains forming the internal ribosome entry site (IRES), which plays an important role in HCV replication (Figure 2).
After receptor-mediated endocytosis, the fusion of HCV with cellular membranes and uncoating of the viral nucleocapsid, the single-stranded positive-sense RNA genome of the virus is released into the cytoplasm to serve as messenger RNA for the HCV polyprotein precursor. HCV mRNA translation is under the control of the internal ribosome entry site (IRES) (Bartenschlager 2004, Moradpour 2007). IRES mediates the HCV polyprotein translation by forming a stable complex with the 40S ribosomal subunit, eukaryotic initiation factors and viral proteins.
From the initially translated HCV polyprotein, the three structural and seven non-structural HCV proteins are processed by both host and viral proteases (Bartenschlager 2004, Moradpour 2007). NS2 is a metalloproteinase that cleaves itself from the NS2/NS3 protein, leading to its own loss of function and to the release of the NS3 protein (Lorenz 2006), which activates the serine protease, located in a small groove, and the helicase/NTPase (Kim 1998, Kim 1996). NS3 forms a tight, non-covalent complex with its cofactor and enhancer NS4A, which is essential for proper protein folding (Figure 3). The NS3/4A protease cleaves the junctions between NS3/NS4A, NS4A/NS4B, NS4B/NS5A and NS5A/NS5B. Besides its essential role in protein processing, NS3 is integrated into the HCV RNA replication complex, supporting the unwinding of viral RNA by its helicase activity. Moreover, NS3 may play an important role in HCV persistence via blocking TRIF-mediated toll-like receptor signaling and Cardif-mediated RIG-I signaling, subsequently resulting in impaired induction of type I interferons (Meylan 2005). Thus, pharmacologic NS3 inhibition might support viral clearance by restoring the innate immune response.
The location of the active site of the NS3/4A protease, the shallow groove mentioned previously, has made the design of compound inhibitors relatively difficult. Nevertheless, many NS3/4A protease inhibitors have been developed and they can be divided into two classes, the macrocyclic inhibitors and the linear tetrapeptide-based α-ketoamide derivatives. In general, NS3/4A protease inhibitors have been shown to strongly inhibit HCV replication during monotherapy but usually cause the selection of resistant mutants followed by viral breakthrough. The additional administration of pegylated interferon plus ribavirin or of other DAAs, however, was shown to reduce the frequency of development of resistance.
Telaprevir and Boceprevir are first generation protease inhibitors for the use in combination with pegylateded interferon alfa and ribavirin which are no longer available. In a second wave other first generation protease inhibitors like, simeprevir, paritaprevir, vaniprevir, asunaprevir and grazoprevir have been approved for the treatment of chronic HCV with limited antiviral activity mainly against HCV genotypes 1 and 4 infection (Lange 2014), as described in detail in chapter 12. In addition and most recently the two second generation protease inhibitors glecaprevir (ABT-493) and voxilaprevir (GS-9857) with pan-genotypic antiviral activity have recently been approved, which substantially expand the DAA repertoire.
Because of the high replication rate of HCV and the poor fidelity of its RNA-dependent RNA polymerase, numerous variants (quasispecies) are continuously produced during HCV replication. Among them, variants carrying mutations altering the conformation of the binding sites of DAA compounds can develop. During treatment with specific antivirals, these pre-existing drug-resistant variants have a fitness advantage and can be selected to become the dominant viral quasispecies. Many of these resistant mutants exhibit an attenuated replication with the consequence that, after termination of exposure to specific antivirals, the wild type may displace the resistant variants (Sarrazin 2007, Sarrazin 2010).
HCV quasispecies resistant to NS3/4A protease inhibitors or non-nucleoside polymerase inhibitors can be detected at low levels in some patients (approximately 1%) who have never been treated with these specific antivirals before (Gaudieri 2009). The clinical relevance of these pre-existing mutants is not completely understood, although there is evidence that they may reduce the chance of achieving an SVR with older DAA-based triple therapies (Boceprevir, Telaprevir) if the patient’s individual sensitivity to pegylated-interferon α plus ribavirin is low but do not affect the outcome of potent IFN-free combination regimens with newer protease inhibitors. A notable exception is the Q80R/K variant, which has been described as conferring low-level resistance to simeprevir (TMC435), a macrocyclic protease inhibitor. Of note, the Q80K variant can be detected in up to 50% of HCV genotype 1a-infected patients (approximately 20% in Europe and 50% in the US) at baseline (a much higher percentage than for other common baseline RAVs), while in <1% in 1b isolates, and a slower viral decline and lower SVR rates on simeprevir-based triple therapy have been observed (Jacobson 2013, Lenz 2011, Zeuzem 2013). The importance of the Q80K variant for other DAA regimen containing NS3 PI whith very low level resistance to Q80K (<2-fold in HCV replicon systems in vitro ) is not fully understood. Interestingly, in HCV genotype 1a infected patients who were treated with the 3D regimen without ribavirin virologic treatment failure was associated with the presence of Q80K in addition with pre-existing NS5A RAVs (Sarrazin et al., Paris HCV EASL/AASLD meeting September 2016)
Table 2 summarises the resistance profile of selected NS3/4A inhibitors. In general, different resistance profiles between linear tetrapeptide and macrocyclic inhibitors binding to the active site of the NS3 protease have been shown. R155 is the main resistance codon and different mutations at this amino acid site within the NS3 protease confer cross-resistance to most NS3/4A inhibitors (Sarrazin 2010), though some novel NS3/4A inhibitors display different resistance profiles and higher genetic barriers to the development of resistance.
Importantly, many resistance mutations can be detected in vivo only. For example, mutations at four positions conferring telaprevir resistance have been characterised so far (V36A/M/L, T54A, R155K/M/S/T and A156S/T), but only the A156 was identified initially in vitro in the replicon system (Lin 2005). These mutations, alone or as double mutations, conferred low (V36A/M, T54A, R155K/T, A156S) to high (A156T/V, V36M + R155K, V36M + 156T) levels of resistance to telaprevir (Sarrazin 2007). A comparable resistance profile to telaprevir has been described for boceprevir (Susser 2009). It is thought that the resulting amino acid changes of these mutations alter the configuration of the catalytic pocket of the protease, which impedes binding of the protease inhibitor (Welsch 2008).
Newer NS3/4A inhibitors have other, though overlapping, resistance profiles to telaprevir and boceprevir. Besides the important variant Q80K, the emergence of variants at positions S122, R155, and D168 has been observed in patients treated with simeprevir-based therapies (Lenz 2014). These variants confer moderate to high levels of simeprevir-resistance in vitro (Lenz 2014).
Mutations at position R155, A156, and D168 significantly reduce the antiviral activity of paritaprevir in vitro (Pilot-Matias 2014). The R155 and D168 variants have been observed in virtually all patients with viral relapse after paritaprevir-based all-oral therapy (Pilot-Matias 2014).
The most important variants conferring resistance to asunaprevir are mutations at position Q80, R155 and D168, which have been observed in patients with viral relapse both after asunaprevir-based triple therapy as well as after asunaprevir + daclatasvir all-oral therapy (Manns 2014, McPhee 2013).
Grazoprevir has an outstanding resistance profile. In contrast to most other NS3/4A inhibitors, mutations at position R155 only slightly reduce the in vitro efficacy of grazoprevir (Summa 2012). In patients with virologic failure after grazoprevir-based therapies, resistance mutants at position D168 were most frequently observed (Howe 2014). For several NS3/4A inhibitors, resistance differs significantly between HCV subtypes. For example, in all clinical studies of telaprevir alone or in combination with PEG-IFN α plus RBV, viral resistance and breakthrough occurs much more frequently in patients infected with HCV genotype 1a compared to genotype 1b. This difference was shown to result from nucleotide differences at position 155 in HCV subtype 1a (aga, encodes R) versus 1b (cga, also encodes R). The mutation most frequently associated with resistance to telaprevir is R155K. Changing R to K at position 155 requires 1 nucleotide change in HCV subtype 1a, and 2 nucleotide changes in subtype 1b isolates (McCown 2009). In addition, HCV genotype 1a isolates generally display a higher fitness compared to HCV genotype 1b isolates, which explains a higher risk of resistance development at other positions within NS3/4A and other genomic regions of HCV genotype 1a (Romano 2012).
However, it is important to note that the second-generation PIs glecaprevir and voxilaprevir have high barriers to resistance against common NS3 RAVs. However, in the very few patients with failure to second generation PIs like Glecaprevir and Voxilaprevir in principle the variants at the same positions as for first generation PIs have been selected (Y56, R155, A156, D168). For example selection of RAVs in NS3 has been observed at low frequencies in few patients after short-term therapy (6 weeks) with sofosbuvir, velpatasvir and voxilaprevir (Gane 2016). Yet, RAVs appear to play only a minor role for the treatment with these novel, highly potent DAA regimens if appropriate treatment-durations (8–12 weeks) are chosen.
|Vaniprevir MK-7009* (macrocyclic)|
|Simeprevir (TMC435*) (macrocyclic)|
|Asunaprevir (BMS-650032*) (macrocyclic)|
|Paritaprevir (ABT-450*) (macrocyclic)|
|Voxilaprevir (GS-9857) (macrocyclic)|
|Glecaprevir (ABT-493) (linear)|
|Grazoprevir (MK-5172***) (macrocyclic)|
HCV replication is initiated by the formation of the replication complex, a highly structured association of viral proteins and RNA, of cellular proteins and cofactors, and of rearranged intracellular lipid membranes derived from the endoplasmic reticulum (Moradpour 2007). The key enzyme in HCV RNA replication is NS5B, an RNA-dependent RNA polymerase that catalyses the synthesis of a complementary negative-strand RNA by using the positive-strand RNA genome as a template (Lesburg 1999) (Figure 5). From this newly synthesised negative-strand RNA, numerous RNA strands of positive polarity are produced by NS5B activity that serve as templates for further replication and polyprotein translation. Because of poor fidelity leading to a high rate of errors in its RNA sequencing, numerous different isolates are generated during HCV replication in any given patient, termed HCV quasispecies. Due to the lack of proofreading of the NS5B polymerase together with the high replication rate of HCV, every possible mutation is generated every day.
NS5B RNA polymerase inhibitors can be divided into two distinct categories. Nucleoside analogue inhibitors (NIs) like sofosbuvir, mericitabine or ALS-220 mimic the natural substrates of the polymerase and are incorporated into the growing RNA chain, thus causing direct chain termination by blocking the active site of NS5B (Koch 2006). Because the active centre of NS5B is a highly conserved region of the HCV genome, NIs are potentially effective against different genotypes. Single amino acid substitutions in every position of the active centre may result in loss of function or in extremely impaired replicative fitness. Thus, there is a relatively high barrier to the development of resistance to NIs.
In contrast to NIs, the heterogeneous class of non-nucleoside inhibitors (NNIs) achieves NS5B inhibition by binding to different allosteric enzyme sites, which results in conformational protein change before the elongation complex is formed (Beaulieu 2007). For allosteric NS5B inhibition high chemical affinity is required. NS5B is structurally organised in a characteristic “right hand motif”, containing finger, palm and thumb domains, and offers at least four NNI binding sites, a benzimidazole-(thumb 1)-, thiophene-(thumb 2)-, benzothiadiazine-(palm 1)- and benzofuran-(palm 2)-binding site (Lesburg 1999) (Figure 6). Because of their distinct binding sites, different NNI (polymerase) inhibitors can theoretically be used in combination or in sequence to manage the development of resistance.
But because NNIs bind relatively distantly from the active centre of NS5B (Figure 7), their application may rapidly lead to the development of resistant mutants in vitro and in vivo. Moreover, mutations at the NNI binding sites do not necessarily lead to impaired function of the enzyme. Figure 6 shows the structure of selected nucleoside and non-nucleoside inhibitors as well as the active centre.
Sofosbuvir is a nucleoside analogue NS5B inhibitor strongly effective against all HCV genotypes. It has a very high genetic and fitness barrier to development of resistance. Thus far, S282T is the only known variant in NS5B which is associated with a reduced susceptibility to sofosbuvir. However, S282T has been identified in less than 1% of patients who have failed sofosbuvir-based antiviral therapy, and the frequency of S282T decreased rapidly after treatment termination in these patients (Gane 2015). Due to these features, sofosbuvir is a key compound of current antiviral treatment regimens, as described in chapter 12.
Uprifosbuvir (MK-3682 or previously IDX21437) is another uridine nucleotide analogue NS5B inhibitor with potent antiviral activity against all HCV genotypes in vitro. In a phase 1/2a study, MK-3682 was safe and well tolerated and led to a mean maximum decrease in HCV viral load (HCV gentoypes 1, 2 and 3) of approximately 4.3 log10 IU/mL after 7 days of monotherapy (Gane 2014). Another nucleoside analogue NS5B inhibitor is AL-335, which is currently in phase II clinical development. However, further development of both polymerase inhibitors has been stopped by Merck and Janssen, respectively, due to limited antiviral efficacy in combination regimens (see below).
At least 4 different allosteric binding sites have been identified for inhibition of the NS5B polymerase by non-nucleoside inhibitors. Numerous non-nucleoside inhibitors have been developed and assessed in clinical studies (e.g., thumb 1 inhibitors BI-207127, BMS-791325; thumb 2 inhibitors filibuvir and VX-222; palm I inhibitor ANA598 and ABT-333; palm 2 inhibitors tegobuvir and IDX-375) (Ali 2008, Cooper 2007, Erhardt 2009, Kneteman 2009, Larrey 2012). In general, these non-nucleoside analogues display variable antiviral activity and a low genetic barrier to resistance, evidenced by frequent viral breakthrough during monotherapy studies and selection of resistance mutations at variable sites of the enzyme. In line with these experiences in phase 1 studies, a phase 2 triple therapy study with filibuvir in combination with pegylated interferon plus RBV showed high relapse and relative low SVR rates (Jacobson 2010). In contrast to nucleoside-analogues, non-nucleoside analogues in general do not display antiviral activity against different HCV genotypes (Sarrazin 2010). Due to their low antiviral efficacy and low genetic barrier to resistance, non-nucleoside analogues are only suitable as components of multidrug all-oral regimens (see below). Dasabuvir has been approved for the treatment of HCV genotype 1 infection as component of combination therapies including NS3/4A and NS5A inhibitors (details in chapter 12).
The HCV NS5A protein seems to play a manifold role in HCV replication, assembly and release (Moradpour 2007). It was shown that NS5A is involved in the early formation of the replication complex by interacting with intracellular lipid membranes, and it initiates viral assembly at the surface of lipid droplets together with the HCV core (Shi 2002). NS5A may also serve as a channel that helps to protect and direct viral RNA within the membranes of the replication complex (Tellinghuisen 2005). Moreover, it was demonstrated that NS5A is able to interact with NS5B, which results in an enhanced activity of the HCV RNA polymerase. Besides its regulatory impact on HCV replication, NS5A has been shown to modulate host cell signalling pathways, which has been associated with interferon resistance (Wohnsland 2007). Furthermore, mutations within the NS5A protein have been clinically associated with resistance / sensitivity to IFN-based antiviral therapy (Wohnsland 2007).
The NS5A inhibitors daclatasvir, ledipasvir, velpatasvir, elbasvir, ombitasvir and pibrentasvir were approved for the treatment of hepatitis C. Even low doses of NS5A inhibitors display high antiviral efficacy against all HCV genotypes in vitro , with the notable exception of HCV genotype 3, were higher EC50 concentrations were observed for some agents (ledipasvir, elbasvir, ombitasvir); details below and in chapter 12 (Lange 2014).
For example, monotherapy with daclatasvir led to a sharp initial decline of HCV RNA concentrations, though its genetic barrier to resistance is relatively low (Gao 2010). During monotherapy, rapid selection of variants resistant to daclatasvir occurred (Nettles 2011). The most common resistance mutations in HCV genotype 1a patients were observed at residues M28, Q30, L31, and Y93 of NS5A. In HCV genotype 1b patients, resistance mutations were observed less frequently, predominantly at positions L31 and Y93. These resistance mutations increased the EC50 to daclatasvir moderately to strongly (Fridell 2011). Interestingly and different to NS3 protease inhibitor resistance mutations, variants conferring resistance to NS5A inhibitors are not associated with impaired replication fitness and thus during follow-up after the end of treatment do not disappear. Indeed, in a first follow-up study for approximately 1 year, persistence of the majority of NS5A resistance mutations was seen (McPhee 2013). Therefore, NS5A inhibitors can only be used in combination with other DAAs or (theoretically) with PEG-IFN α and ribavirin.
Other NS5A inhibitors (e.gRuzasvir (MK-8408) and odalasvir were in clinical development. Like daclatasvir, these NS5A inhibitors are characterised by broad genotypic coverage, high antiviral activity, and partially overlapping resistance profiles (cross-resistance). However, further development was terminated due to insufficient broad antiviral activity in combination regimens against several HCV geno-/subtypes.
The tetraspanin protein CD81, claudin-1, occludine, scavenger receptor class B type 1 (SR-B1), the low-density lipo-protein (LDL) receptor, glycosaminoglycans and the dendritic cell- /lymph node-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN/L-SIGN) have been identified as putative ligands for E1 and E2 during viral attachment and entry (Moradpour 2007).
HCV entry inhibition might enrich future hepatitis C treatment opportunities, in particular in the prevention of HCV liver graft reinfection. HCV entry inhibition can be theoretically achieved by the use of specific antibodies or small molecule compounds either blocking E1 or E2 or their cellular receptors. So far, only results from clinical trials using polyclonal (e.g., civacir) (Davis 2005) or monoclonal (e.g., HCV-AB 68) (Schiano 2006) HCV-specific antibodies are available. The clinical benefit of these antibodies has been poor, however.
HCV depends on various host factors throughout its life cycle. Cyclophilin B is expressed in many human tissues and provides a cis-trans isomerase activity, which supports the folding and function of many proteins. Cyclophilin B enhances HCV replication by incompletely understood mechanisms, like the modulation of NS5B activity. Alisporivir (Debio-025) is an orally bioavailable cyclophilin B inhibitor exerting an antiviral impact on both HCV and HIV replication. In clinical trials in HCV/HIV-coinfected patients, treatment with 1200 mg alisporivir twice daily for two weeks led to a mean maximal log10 reduction of HCV RNA of 3.6 and of HIV DNA of 1.0 (Flisiak 2008). Alisporivir was well-tolerated and no viral breakthrough occurred during the 14 days of treatment.
Combination therapy of alisporivir 200 mg, 600 mg or 1000 mg and PEG-IFN α-2a was evaluated in a double-blind placebo-controlled phase 2 trial in treatment-naïve patients monoinfected with HCV genotypes 1, 2, 3 or 4. Treatment was administered for 29 days. Mean log10 reductions in HCV RNA at day 29 were 4.75 (1000 mg), 4.61 (600 mg) and 1.8 (200 mg) in the combination therapy groups compared to 2.49 (PEG-IFN α-2a alone) and 2.2 (1000 mg alisporivir alone) in the monotherapy groups. No differences in antiviral activity were observed between individuals infected with the different genotypes. Alisporivir was safe and well tolerated but led to a reversible bilirubin increase in some of the patients treated with 1000 mg alisporivir daily (Flisiak 2009). A high genetic barrier to resistance of alisporivir and a broad HCV genotypic activity highlight the potential of drugs targeting host proteins.
In a phase 2 clinical trial in treatment-naïve HCV genotype 1 patients, combination therapy with alisporivir, PEG-IFN α-2a plus ribavirin for 24–48 weeks resulted in SVR rates of 69–76% compared to 55% in the control group (Flisiak 2011). Furthermore, interesting first studies with interferon-free treatment regimens including alisporivir and ribavirin have been conducted. Despite these promising data, the development of alisporivir was put on hold due to rare cases of severe pancreatitis during combination therapy with alisporivir and PEG-IFN α-.
Nitazoxanide with its active metabolite tizoxanide is a thiazolide antiprotozoal approved for the treatment of Giardia lamblia and Cryptosporidium parvum infections. In vitro studies have revealed an essential inhibitory impact on HCV and HBV replication by still unknown mechanisms.
Results of two phase 2 studies evaluating 500 mg nitazoxanide twice daily for 12 weeks followed by nitazoxanide, PEG-IFN α-2a ± RBV for 36 weeks yielded conflicting results with SVR rates of 79% in treatment-naïve genotype 4 patients, but of only 44% in HCV genotype 1 patients (Rossignol 2009). However, additional studies revealed a less impressive gain in SVR rates with the addition of nitazoxanide to PEG-IFN α-2a plus RBV.
Silymarin, an extract of milk thistle (Silybum marianum) with antioxidant activity, has been empirically used to treat chronic hepatitis C and other liver diseases. Silibinin is one of the six major flavonolignans in silymarin. Surprisingly, recent reports demonstrated that silibinin inhibits HCV at various steps of its life cycle (Ahmed-Belkacem 2010, Wagoner 2010). In addition, intravenous silibinin in non-responders to prior IFN-based antiviral therapy led to a decline in HCV RNA between 0.55 to 3.02 log10 IU/mL after 7 days and a further decrease after an additional 7 days in combination with PEG-IFN α-2a+RBV in the range of 1.63 and 4.85 log10 IU/mL (Ferenci 2008). On a case report basis, it was shown that treatment with silibinin can prevent recurrent hepatitis C after liver transplantation in selected cases (Neumann 2010).
MicroRNA-122 (miRNA-122) is a liver-specific microRNA that has been shown to be a critical host factor for HCV (Landford 2010). MiRNA-122 binds to the 5’ NTR region of the HCV genome, which appears to be vital in the HCV replication process. Miravirsen is a modified antisense oligonucleotide that targets miRNA-122 and thereby prevents binding of miRNA-122 to the HCV genome. In a phase 2a proof-of-principle study, weekly subcutaneous injections of miravirsen led to a reduction of HCV RNA serum concentration of up to 2.7 log10 IU/mL, indicating that an antisense oligonucleotide-based approach of miRNA-122 inhibition could be a promising modality for antiviral therapy (Janssen 2013). No relevant side effects were seen in this study.
RG-101 is another RNA targeting miRNA-122, which is modified by carbohydrate-conjugation leading to a very long intracellular half-life of this oligonucleotide. A single dose of RG-101 has been shown to suppress HCV replication for up to 28 weeks of follow-up in some patients (van der Ree 2015). Whether this long-term suppression is equivalent to cure of HCV infection remains to be awaited by final data of this highly innovative study. Also the safety profile of this treatment currently is unclear as in some patients features of cholesterol levels indicated long term persistence of siRNA.
TT-034 is another RNA interference therapeutic which is delivered by an adenovirus-associated capsid to hepatocytes. TT-034 encodes three short hairpin RNAs which specifically targets three conserved regions in the HCV RNA genome. A single dose of TT-034 has been assessed in 7 patients with chronic hepatitis C providing a proof of concept, that TT-034 delivery is successful and results in inhibition of HCV replication (Suhy 2015).
Though not further followed for the treatment of hepatitis C, the successes with RNA interference-based therapies have encouraged the development of such strategies for the treatment of chronic hepatitis B.
Due to a the approval of a number of highly potent DAA regimens with which almost all HCV infected patients can be cured, the development of novel DAA-based combination therapies is strongly declining.
As described in chapter 12 in detail, combination therapy of the NS3-4A inhibitor grazoprevir with the NS5A inhibitor elbasvir has been approved for the treatment of HCV genotype 1 and 4 infection. Ruzasvir (MK-8408) is a novel NS5A inhibitor effective against all HCV genotypes and higher potency against common NS5A RAVs than elbasvir. Uprifosbuvir (MK-3682) is a novel potent nucleoside-analogue NS5B inhibitor.
The combination of grazoprevir, ruzasvir and Uprifosbuvir for 8-16 weeks with or without ribavirin was investigated in DAA-naïve HCV genotype 1, 2 and 3 patients in the phase I and II CREST studies. Very high SVR rates were observed after 8 or 12 weeks of treatment in HCV genotype 1b patients, after 12 weeks of treatment in HCV genotype 1a patients, after 12-16 weeks in HCV genotype 2 patients, and after 8, 12 and 16 weeks in HCV genotype 3 patients. No benefit of additional ribavirin was observed in this study (Gane 2017).
Importantly, re-treatment of patients who had experienced a viral relapse after 8 weeks of treatment with grazoprevir and ruzasvir with the same regimen for 16 weeks plus additional ribavirin resulted in SVR of almost all patients (Wyles 2017). Re-treatment of patients with prior DAA failure with razoprevir, ruzasvir and uprifosbuvir for 16 with ribavirin or 24 weeks without ribavirin was also investigated in the phase 2 C-SURGE study. gSVR rates after 16 and 24 weeks of therapy were 98% and 100%, respectively, and no impact of baseline RAVs on treatment outcome was observed (Wyles 2017).
However, Merck has discontinued to further develop this regimen in September 2017.
Odalasvir is a 2nd generation NS5A inhibitor with pan-genotypic activity. AL-335 is a novel nucleoside uridine NS5B polymerase inhibitor. These drugs have been assessed in phase I clinical trials in combination with the NS3-4A inhibitor simeprevir. In treatment-naïve HCV genotype 1 patients without liver cirrhosis, SVR rates were 100% after 6 and 8 weeks of therapy, but only 90% after 8 weeks of therapy with odalasvir and AL-335 alone (Gane 2016). In the phase II Omega-1 study, the combination of simeprevir (75 mg), odalasvir (25 mg) and AL-335 (800 mg) once daily was assessed in 365 DAA-naïve HCV genotype 1, 2, 4-6 patients for 6 or 8 weeks (Zeuzem 2017). Overall, SVR rates were almost 100%, also one relapse in a HCV genotype 1 patient and 4 relapses in HCV genotype 2c (representing an SVR-rate of less than 85% in this subtype) patients occurred. Therefore, further development was stopped by Janssen.
A number of novel potent DAAhave been approved for the treatment of chronic hepatitis C, which with which the vast majority of HCV infected patients can be successfully treated. The further development of DAAs may potentially help to reduce treatment costs, reduce treatment durations or improve treatment opportunities of special patient populations like patients with decompensated liver cirrhosis and renal failure.
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