Keywords

8.1 Introduction

Direct-acting antiviral (DAA) drugs strongly inhibit replication of hepatitis C virus (HCV) by directly targeting essential viral proteins. Triple combination therapy with pegylated interferon (PEG-IFN) plus ribavirin (RBV) and a protease inhibitor, e.g., telaprevir [1], simeprevir [2], or vaniprevir [3], is currently used in Japan for treatment of genotype 1 chronic HCV infection. However, several interferon-free all-oral DAA combination therapies have recently been approved, including daclatasvir plus asunaprevir therapy [4] and sofosbuvir plus ledipasvir [5] for treatment of genotype 1 infections and sofosbuvir plus RBV for treatment of genotype 2 infections [6]. DAAs have potent antiviral effects, and each of these therapies has been shown to have high safety and efficacy. However, drug resistance has nonetheless occurred in some patients, leading to treatment failure and raising questions about the best approach to re-treating these patients. Fortunately, a variety of new drugs are currently under development. Given the large number of DAAs in various stages of clinical development, the number of treatment options is only expected to increase, and effective treatment for all patients is a major goal. This chapter outlines current drug resistance challenges and discusses trends in ongoing and future drug development.

8.1.1 NS3/NS4A Protease Inhibitors

The first DAAs to be approved were the protease inhibitors telaprevir and boceprevir. The 9.6 kb HCV RNA genome contains a single open reading frame coding for a single 3000-amino-acid polypeptide, which must then be cleaved into three structural and six nonstructural proteins. Cellular proteases cleave the three structural proteins, which include the core protein and two envelope proteins, and the nonstructural proteins are cleaved at four sites by the virally encoded proteases NS2 and NS3 along with the NS4A cofactor. Telaprevir mimics the carboxy-terminal region of the HCV NS3/NS4 serine protease [7] and interferes with viral replication by preventing cleavage of the polyprotein. While telaprevir triple therapy improves SVR rates to around 70 %, aside from its inconvenient thrice-daily dosing regimen, the therapy is associated with a high frequency of adverse events, including pruritus, rash, and nausea [8], and has been reported to lead to treatment discontinuation in 18 % of patients [9]. Clinical trials in Japan reported comparable SVR rates but a higher incidence of adverse events, due in part to the relatively high fixed dose relative to body weight in Japanese patients [10].

The defining characteristic of DAAs is their high target specificity for viral proteins, but a consequence of this specificity is the relatively low barrier to resistance it presents to this highly adaptable RNA virus. Inter-genotypic variation in the NS3 domain restricts the use of first-generation protease inhibitors to HCV genotype 1 [11], but even within genotype 1, resistance occurs more frequently in genotype 1a than 1b due to a synonymous codon at R155 that reduces the number of nucleotide changes needed to achieve a favorable amino acid substitution [12]. Compensatory mutations such as V36M that restore viral fitness may also occur, allowing the virus to compete with wild-type virus in the absence of the drug [13], and cross-resistance prevents the use of related protease inhibitors.

8.1.2 Second-Wave Protease Inhibitors

Second-wave PIs, such as simeprevir, asunaprevir, faldaprevir, and paritaprevir, attempt to overcome these problems by increasing the barrier to resistance and broadening the antiviral activity to other genotypes. Although the historically difficult-to-treat genotype 1 is the most prevalent genotype worldwide with 46.2 % of cases, it has also received the most focus in drug development efforts, whereas genotype 4, which is overrepresented in low-income countries, remains difficult to treat with current therapies [14]. Another goal of second-wave PIs is to improve patient compliance by reducing the dosing schedule and reducing side effects [15]. In light of these improvements, telaprevir and boceprevir should be avoided as a first-line treatment.

Simeprevir has been approved in the USA (150 mg dose) and Japan (100 mg dose) for use in triple therapy with PEG-IFN and ribavirin. Simeprevir is a once-daily macrocyclic PI active against genotypes 1, 2, 4, 5, and 6 [16]. Simeprevir triple therapy achieves SVR rates of up to approximately 80 % [17–19], with similar incidence and severity of adverse events to PEG-IFN and ribavirin alone. With response-guided therapy, up to 96 % of prior relapsers were reported to achieve SVR, but viral breakthrough or relapse was common [20, 21]. Despite these promising results, the simeprevir-resistant Q80K mutation occurs frequently (9–48 %) in genotype 1a patients [22], potentially requiring screening of genotype 1a patients for the Q80 mutation and an alternative therapy if necessary.

8.1.3 Second-Generation Protease Inhibitors

Second-generation PIs attempt to go a step further and provide pan-genotypic activity against all HCV genotypes as well as resistance mutations affecting first-generation PIs. Grazoprevir (MK-5172) is a much anticipated once-daily second-generation PI currently undergoing advanced clinical testing. Grazoprevir is not sensitive to most variants affecting first-generation inhibitors and has a higher barrier to resistance, with SVR rates ranging from 89 % to 100 % [23]. In the randomized, open-label phase II C-WORTHY clinical trial, treatment-naive HCV genotype 1 patients were treated with grazoprevir plus the NS5A inhibitor elbasvir (MK-8742) with or without ribavirin for 12 weeks [24]. The SVR12 rate was 93 % for patients without ribavirin and 93 % with ribavirin. While no patients discontinued due to adverse events, virological failure occurred in 4 % of patients due to emergence of resistance-associated variants against grazoprevir or elbasvir. In another C-WORTHY study examining treatment duration in treatment-naive cirrhotic patients and prior null responders, SVR12 rates up to 100 % were achieved among prior null responders treated for 18 weeks with grazoprevir, elbasvir, and ribavirin [25]. The regimen is now undergoing phase III clinical testing in the C-Edge series of studies examining safety and efficacy of the therapy in a variety of difficult-to-treat patient populations, including those with HIV coinfection, chronic kidney disease, severe liver damage, or blood disorders. Results of a phase III C-Edge HIV coinfection study involving 12 weeks of grazoprevir plus elbasvir therapy indicate that 96 % of patients achieved SVR12, including all 35 patients with cirrhosis [26], while several patients relapsed, in two cases due to reinfection. Adverse events were mild and included fatigue, headache, and nausea. Improvements in drug development will continue to extend the reach of DAA therapy to patients with previously unmet treatment needs, although achieving 100 % SVR may prove challenging.

8.2 Second-Generation NS5A Inhibitors

NS5A inhibitors are among the most effective antivirals available, with picomolar efficacy and pan-genotypic activity, and might serve as the backbone of future HCV therapies. However, first-generation NS5A inhibitors are highly vulnerable to resistance variants, such as L31M and Y93H. Compared to the first-generation NS5A inhibitors daclatasvir and ledipasvir, second-generation inhibitors under development, such as ACH-3120, GS-5816, and MK-8742, have a higher genetic barrier and are expected to be highly effective against resistance mutations that affect first-generation drugs. Tables 8.1 and 8.2 show sensitivity of first- and second-generation NS5A inhibitors to wild-type and first-generation resistance mutations for genotype 1a and 1b using an HCV replicon [27]. The second-generation NS5A inhibitor ACH-3120 has been shown to have overall higher efficacy against NS5A mutations compared to ledipasvir and daclatasvir. For example, ledipasvir and daclatasvir show EC50 fold changes of 119 and 18 against NS5A-L31V variants relative to wild type, whereas ACH-3102 shows no change in sensitivity. Similarly, ledipasvir and daclatasvir show EC50 changes of 2727 and 40 against the NS5A-Y93H wild type, while ACH-3102 shows only a slight reduction in sensitivity with a fold change of 2. Even against the double variant NS5A-L31M-Y93H, ACH-3120 remains relatively sensitive, with an EC50 fold change of 12, whereas ledipasvir and daclatasvir become almost completely ineffective, with fold changes of 272,727 and 54,333, respectively. This result suggests that treatment with a second-generation NS5A inhibitor may be an effective re-treatment option for patients who experienced treatment failure with daclatasvir and asunaprevir therapy due to NS5A-L31 and NS5A-Y93 mutations. Clinical trials are currently underway to examine the combination of ACH-3120 and sofosbuvir. In a phase 2 clinical trial, 36 treatment-naive genotype 1 HCV patients were treated with 50 mg ACH-3120 and 40 mg sofosbuvir once daily for 6 or 8 weeks [28]. All patients achieved SVR regardless of treatment period. In the future, ACH-312 and sofosbuvir combination therapy should be examined as a potential re-treatment option for patients who failed to clear the virus during daclatasvir and asunaprevir therapy.

Table 8.1 NS5A inhibitor susceptibility to amino acid substitutions in genotype 1a (modified from reference 7 [27])
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Table 8.2 NS5A inhibitor susceptibility to amino acid substitutions in genotype 1b (modified from reference 7 [27])
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8.3 Non-nucleoside Polymerase Inhibitors

Although both targeting the HCV RNA-dependent RNA polymerase, the polymerase inhibitor DAAs are divided into two drug classes, nucleoside and non-nucleoside inhibitors, that target different steps in RNA synthesis and have different mechanisms and resistance profiles. Following the success of the nucleoside inhibitor sofosbuvir, clinical testing of non-nucleoside inhibitors such as beclabuvir has begun. Beclabuvir, which inhibits polymerase activity by targeting the thumb 1 domain of the NS5B polymerase, has exhibited pan-genotypic antiviral activity against genotypes 1, 3, 4, 5, and 6 in vitro [29]. In a phase II clinical trial, 66 patients were treated for 12 or 24 weeks with beclabuvir in combination with daclatasvir (NS5A inhibitor) and asunaprevir (protease inhibitor), resulting in an SVR rate of 92 % [30]. However, beclabuvir is susceptible to mutations at NS5B-A421 and NS5B-P495 [31, 32]. A breakthrough occurred in genotype 1a patients with treatment-emergent NS3-R155K + NS5A-Q30R-L31M + NS5B-P495L mutations, and NS3-V36M-R155K+ NS5A-M28A-Q30R-H58P + NS5B-P495L mutations emerged at the time of relapse in a patient with preexisting NS3-V36M + NS5A-H58P mutations. In a phase III clinical trial, an SVR rate of 98 % (81/83) was observed for treatment-naive genotype 1b patients, and an SVR rate of 100 % (28/28) was observed for previously treated patients after 12 weeks of treatment [33]. Even among genotype 1a patients, SVR rates of 90 % (206/229) and 85 % (64/75) were observed in treatment-naive patients and previously treated patients, respectively. Furthermore, even genotype 1b patients with preexisting NS5A-L31I/M or NS5A-Y93H mutations were able to achieve SVR (Table 8.3). In an HCV replicon study involving genotype 1b NS5A-L31M-Y93H double mutants, no inhibitory effect was observed for daclatasvir and asunaprevir alone, but the three-way combination of daclatasvir, asunaprevir, and beclabuvir effectively suppressed HCV replication [34]. This result suggests that addition of beclabuvir might be effective in re-treating patients who fail to respond to daclatasvir and asunaprevir therapy. On the other hand, the frequency of resistance mutations affecting non-nucleoside polymerase inhibitors and their effects on combination therapy are not well understood and must be further examined to determine the safest course of treatment for these patients.

Table 8.3 SVR rates for combination therapy with daclatasvir, asunaprevir, and beclabuvir in patients with NS5A amino acid mutations (Modified from [33])
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8.4 Paritaprevir/Ritonavir, Ombitasvir, and Dasabuvir

Although many sofosbuvir-based therapies are being evaluated, several clinical trials have examined AbbVie’s alternative DAA combination therapy consisting of paritaprevir with ritonavir (ABT-450/r, a protease inhibitor), ombitasvir (ABT-267, an NS5A inhibitor), and dasabuvir (ABT-333, an NNI polymerase inhibitor), with or without ribavirin. In a phase III clinical trial, treatment-naive genotype 1 patients who were treated with paritaprevir/r, ombitasvir, dasabuvir, and ribavirin for 12 weeks achieved an SVR12 rate of 96 % (genotype 1a, 95 %; genotype 1b, 98 %) [35]. In another phase III trial, 99.5 % of genotype 1b patients and 97 % of genotype 1a patients achieved SVR12 (36).

The combination therapy also showed promise for patients with cirrhosis or nonresponse to prior interferon therapy. In a phase III trial, genotype 1 patients who failed to achieve SVR during prior interferon therapy were treated with paritaprevir/r, ombitasvir, dasabuvir, and ribavirin for 12 weeks and achieved an SVR rate of 96 % [37]. In a phase III clinical study of patients with Child-Pugh class A compensated cirrhosis who were treated with paritaprevir/r, ombitasvir, dasabuvir, and ribavirin for 12 or 24 weeks, 91 % of patients in the 12-week arm achieved SVR, and 96 % of patients in the 24-week arm achieved SVR [38].

In December 2014, the FDA approved the Viekira Pak (co-formulated ombitasvir, paritaprevir, and ritonavir co-packaged with dasabuvir tablets) for treatment of genotype 1 infection, including patients with compensated cirrhosis. In September 2015, Japan approved AbbVie’s VIEKIRAX (paritaprevir/ritonavir co-formulated with ombitasvir) for treatment of genotype 1 HCV infection, partly on the basis of results of the GIFT-1 clinical phase III clinical trial in which 94 % of non-cirrhotic patients and 91 % of cirrhotic patients were able to achieve SVR12 [39]. These approvals, along with the FDA’s approval of Gilead’s Harvoni (ledipasvir co-formulated with sofosbuvir) for genotype 1 in October 2014 and its expanded approval for genotypes 4, 5, and 6 in November 2015, signal an important trend toward fixed, once-daily dosing. While this change should simplify therapy implementation and improve patient compliance, it also limits the discretion of physicians to adjust the dosage or substitute an alternative DAA in response to patient needs, especially among cirrhotic patients. Shortly after approving the Viekira Pak, the FDA warned of serious liver injury or death in some patients with cirrhosis. While therapy was contraindicated or not recommended for many of these patients, treating cirrhotic patients remains a priority, and early discontinuation of DAA therapy could promote antiviral resistance. Interferon-free therapy for patients with advanced liver disease is a recent and unprecedented development, and the long-term outcomes and risks have yet to be determined.

8.5 Host Factor-Targeting Antivirals

8.5.1 Entry Inhibitors

While DAAs have improved treatment prospects for most patients, their safety and effectiveness is less clear in immunocompromised or coinfected patients, as well as patients with advanced liver disease. Prevention of graft reinfection in patients who receive liver transplantation is another concern that may require alternative or complementary approaches. These patients may benefit from HCV entry inhibitors, which interfere with early interactions between the HCV envelope proteins and host factors, by disrupting attachment to hepatocyte receptors or by interfering with post-binding events or viral fusion [40]. Saikosaponins, particularly SSb2, derived from Bupleurum kaoi root, have been shown to prevent HCV entry by targeting HCV E2 and inhibiting viral attachment and fusion events [41]. Synergistic interactions between DAAs and host-targeting agents such as antibodies against CD81, SR-B1, or CLDN1 might also help to improve efficacy in difficult-to-treat patients while reducing toxicity [42].

8.5.2 Cyclophilin A Inhibitors

While entry inhibitors prevent entry of HCV into uninfected cells, cyclophilin inhibitors act against already infected cells by disrupting the interaction between HCV NS5A and the HCV replication complex [43, 44]. Addition of the cyclophilin inhibitor SCY-635 was shown to restore interferon-stimulated gene activity in HCV-infected cells by reducing phosphorylation of two negative regulators of ISG activity, PKR and eIF2α [45]. Although the mechanism of action of cyclophilin inhibitors is incompletely understood, Chatterji et al. showed that cyclophilin A and NS5A are essential for the creation of double-membrane vesicles required for HCV RNA replication [46].

8.5.3 Silymarin

The milk thistle extract silymarin is an antioxidant with hepatoprotective effects on the liver by promoting hepatocyte regeneration and reducing inflammation and fibrogenesis [47] and has long been used for treatment of Amanita phalloides-induced liver failure [48]. High-dose intravenous silymarin has been successfully used to treat HCV patients who fail to respond to PEG-IFN plus ribavirin therapy [49–51].

8.5.4 Miravirsen

Highly expressed in the liver, the microRNA miR-122 plays an essential role in HCV replication and presents a potential host antiviral drug target [52]. Miravirsen, a 15-nucleotide locked nucleic acid (LNA), is a modified phosphorothioate antisense oligonucleotide that binds to miR-122 and inhibits its function [53]. Miravirsen has shown pan-genotypic inhibitory effects on replication against genotypes 1 through 6. MiR-122 binds both to the 5′UTR (S1 and S2) [54] and to the 3′UTR (S3) [55] of the HCV RNA genome. Long-term HCV replicon studies of miravirsen activity have not revealed mutations in the miR-122-binding region [56]. Efficacy of miravirsen against chronic hepatitis C patients has been examined in a phase II clinical trial. In this study, 36 genotype 1 patients were assigned to receive placebo or 3, 5, or 7 mg/kg of miravirsen administered weekly by subcutaneous injection for 5 weeks, and HCV RNA levels were monitored for 18 weeks [57]. In the miravirsen-treated patients, blood HCV RNA titers were significantly reduced in a dose-dependent manner that persisted even after the end of therapy. The average HCV RNA reduction was 0.4 log IU/mL in the placebo group, 1.2 log IU/mL (P = 0.01) in the 3 mg/kg group, 2.9 log IU/mL (P = 0.003) in the 5 mg/kg group, and 3.0 log IU/mL (P = 0.002) in the 7 mg/kg group. In addition, after 14 weeks of follow-up, HCV RNA became undetectable in four patients in the 7 mg/kg group and one patient in the 5 mg/kg group. Following the end of treatment, HCV RNA increased again in some patients, but no mutations in the S1, S2, and S3 HCV miR-122-binding sites were observed. However, mutations at A4C and C3U in the full-length 5′UTR were recognized in these patients [56]. In an HCV replicon study, miravirsen sensitivity did not differ between A4C mutant and wild-type strains, but resistance in C3U mutants was sevenfold higher than wild type, which may be problematic during treatment with miravirsen. However, in combination with DAAs, IFN, or RBV, C3U mutations show comparable sensitivity to wild type (Table 8.4). The combination of miravirsen with these agents may help to effectively suppress emergence of resistance mutations.

Table 8.4 Susceptibility of various drugs to 5′UTR substitutions (Modified from [56])
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8.6 Conclusions

Development of new HCV drugs unaffected by resistance-associated variants that limit current DAA drugs is anticipated. In principle, an SVR rate for treatment of chronic hepatitis C approaching 100 % is possible. However, a number of issues remain with respect to the emergence of resistance mutations, re-treatment following treatment failure, and treatment of patients with renal function decline or decompensated cirrhosis. Although many new drugs are under development, recent clinical trials have focused on shortening the duration of therapy, identifying effective DAA combinations for genotype 1 as well as other genotypes, improving resistance and safety profiles, and evaluating the need for ribavirin. The recent trend toward co-formulated once-daily fixed-dose tablets simplifies the treatment landscape and should improve patient compliance but at the cost of reduced flexibility and potentially greater risk for cirrhotic patients. With the development of new drugs and more effective treatments, it is hoped that all patients with chronic hepatitis C can be successfully treated.