**9. Current standard of care and future therapies for HCV infection**

In the past few years, HCV therapy has quickly changed the natural history of this disease. Before 2011, the gold standard of therapy was based on the combination of peg-IFN and RBV that, however, acts by unspecific and not completely known mechanisms and exhibited low efficacy in some subgroup of population. The improvement of the knowledge on HCV life cycle allowed to identify innovative therapeutic targets and to develop new drugs known as direct acting antivirals (DAAs). These drugs target three of the main proteins involved in viral replication: the NS3/4A protease, the NS5B polymerase, and the NS5A. The addition of DAAs to peg-IFN and RBV and the development of new interferon-free regimen have dramatically increased clinical outcome leading SVR rates from 90 to 100% (Figure 2).


**Figure 2.** HCV protein products, mechanism of action, and activity of anti-HCV drugs. NIs: nucleoside inhibitors; NNIs: nonnucleoside inhibitors; n.a.: not available.

#### **9.1. Endpoints of treatment**

The goal of HCV therapy is to eradicate infection, thus limiting or preventing the development of disease complications. The most important endpoint, accepted by regulatory agencies for assessing the efficacy of the therapy, is the sustained virological response (SVR) (Table 3). SVR is defined as undetectable HCV RNA 12 weeks (SVR12) or 24 weeks (SVR24) after treatment completion. Achieving this result is associated with a reduced risk of disease progression in patients without cirrhosis, while those with cirrhosis remain at risk of life-threatening complications [30,31].


**Table 3.** Definition of responses to therapy according to the European Association for the Study of the Liver (EASL) (extracted from Conteduca et al. [75]).

#### **9.2. Dual therapy: Pegylated-interferon and ribavirin**

**Figure 2.** HCV protein products, mechanism of action, and activity of anti-HCV drugs. NIs: nucleoside inhibitors;

The goal of HCV therapy is to eradicate infection, thus limiting or preventing the development of disease complications. The most important endpoint, accepted by regulatory agencies for assessing the efficacy of the therapy, is the sustained virological response (SVR) (Table 3). SVR is defined as undetectable HCV RNA 12 weeks (SVR12) or 24 weeks (SVR24) after treatment completion. Achieving this result is associated with a reduced risk of disease progression in patients without cirrhosis, while those with cirrhosis remain at risk of life-threatening

NNIs: nonnucleoside inhibitors; n.a.: not available.

**9.1. Endpoints of treatment**

32 Recent Advances in Liver Diseases and Surgery

complications [30,31].

Until recently, the combination of peg-IFN and RBV was the "historical" standard of care for patients with HCV, and many regimens still contain one or both of these agents [8]. The IFNs are a family of proteins, naturally produced by cells of the immune system with antiviral, antiproliferative, and immunomodulatory activities. After administration, IFNs bind specifi‐ cally to high-affinity receptors that are present on the surface of most cells, triggering a cascade of intracellular signaling responsible for the antiviral functions and immunomodulatory effects that enhance the host-specific antiviral immune responses [63]. However, in clinical practice, the efficacy of IFN is limited by short half-life and frequent administration (at least three times weekly, even better daily). These limitations have been resolved by developing a modified IFN conjugated with the polymer polyethylene glycol (peg). The introduction of pegylated forms of IFN-α has substantially improved SVR rates and pharmacokinetic profile, allowing once-weekly dosing without changing the safety profile [64]. RBV is an oral guano‐ sine analog with broad antiviral activity against several RNA and DNA viruses. The exact mechanism of action has not yet been totally elucidated, although several hypotheses suggest that its biological action occurs through modest inhibition of viral replication, depletion of cellular guanosine triphosphate, immunomodulatory effects, and possible induction of viral mutagenesis [65]. The duration of combined therapy depends on genotype, viral load, and stage disease, with variable regimens from 24 to 48 weeks. Results from clinical practice showed that 45% of patients with GT-1 and GT-4 infection, 70–80% of those infected with GT-2 or GT-3, and 45–70% of patients with other genotypes achieved the SVR [66–72]. However, there were several limitations in treating patients with peg-IFN and RBV due to drug toxicities, low tolerability, or low efficacy (many patients do not respond or became intolerant) [69,70]. The safety profile is one of the limitations leading to dose reduction or treatment discontinu‐ ation. Adverse events caused by peg-IFN are fatigue, flu-like symptoms, depression, anemia, neutropenia, and thrombocytopenia, while those caused by RBV are blood and lymphatic disorder, nausea and vomiting, headache, anorexia, rash, and skin irritation [24,69].

#### **9.3. NS3/4A inhibitors class**

Protease inhibitors (PIs) act through reversible and covalent inhibition of the serine protease NS3/4A responsible for processing of HCV polyprotein and production of new infectious virions (Figure 2). These drugs can be structurally divided into two groups: linear tetrapeptide α-ketoamide derivatives and macrocyclic inhibitors. Generally, PIs have a remarkable antiviral activity and a low barrier to resistance and are selective against GT-1 infection. Furthermore, the most NS3/4A inhibitors interact with the cytochrome CYP3A4, one of the main enzymes responsible for drug metabolism, and this results in increased drug–drug interactions that can limit treatment regimen [8,73]. These limitations have been partially overcome by the advent of a new generation of PIs, which are also effective against genotypes other than the GT-1, and possess a higher barrier to viral resistance as well as lower propensity for toxicity and drug– drug interactions [8,74].

#### *9.3.1. Telaprevir and Boceprevir*

Telaprevir and boceprevir are the first generation of PIs approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA). Telaprevir and boceprevir have been licensed in combination with peg-IFN and RBV, for the treatment of GT-1 infection in naive and experienced patients with compensated liver disease. Telaprevir and boceprevir improved SVR from 49% to 75% in naive patients as compared to the dual therapy [7,75]. Although these therapies have increased clinical outcome, their use is limited by increased rate of adverse effects, including hemolymphopoietic disorders and other reactions related to gastrointestinal system (nausea, diarrhea, vomiting, hemorrhoids, proctalgia, and pruritus). Furthermore, the drugs have a low genetic barrier to resistance [68] and extensive drug–drug interactions that limit their use in transplanted or coinfected patients [76–78].

#### *9.3.2. Simeprevir*

Simeprevir is a once-daily, second-wave protease inhibitor, licensed recently by the FDA and the EMA. This agent is indicated in association with peg-IFN and RBV for the treatment of GT-1 and GT-4 infection. This drug can be associated with sofosbuvir regardless of prior patient treatment history [79]. Simeprevir has a broad spectrum of activity against multiple HCV genotypes except for GT-3 [80]. Data from different trials show that it is highly effective and well tolerated. The most common adverse events are nausea, rash, pruritus, dyspnea, increment in bilirubin blood levels, and photosensitivity [8,74,79]. The NS3 Q80K polymor‐ phism is commonly found in GT-1a viruses and is associated with resistance *in vitro* and impaired response to simeprevir. It is therefore recommended that patients infected with GT-1a must be screened for the presence of Q80K to evaluate the use of another agent in case of positive result [81]. The activity of simeprevir has been validated in several phase II/III studies: QUEST I, QUEST II, PROMISE, ASPIRE, and RESTORE.

In the QUEST I and QUEST II studies, 785 naive patients with GT-1 infection were randomized to placebo or simeprevir plus peg-IFN and RBV for 12 weeks. Eighty percent of patients treated with simeprevir achieved SVR12 compared with 50% in the placebo arm [81,82]. The PROMISE study randomized 393 relapsers with GT-1 infection to simeprevir or placebo for 12 weeks with peg-IFN plus RBV or RBV alone for additional 12–36 weeks, on a response-guided therapy basis. In this trial, 79% of simeprevir treated patients achieved an SVR at 12 weeks compared with 37% of patients in the placebo arm [83]. The efficacy of simeprevir in patients with GT-1 infection was evaluated also in the ASPIRE study that confirmed these results [84]. Finally, in the RESTORE trial, the efficacy of simeprevir in GT-4 infection was established [85].

#### *9.3.3. Paritaprevir*

or GT-3, and 45–70% of patients with other genotypes achieved the SVR [66–72]. However, there were several limitations in treating patients with peg-IFN and RBV due to drug toxicities, low tolerability, or low efficacy (many patients do not respond or became intolerant) [69,70]. The safety profile is one of the limitations leading to dose reduction or treatment discontinu‐ ation. Adverse events caused by peg-IFN are fatigue, flu-like symptoms, depression, anemia, neutropenia, and thrombocytopenia, while those caused by RBV are blood and lymphatic

Protease inhibitors (PIs) act through reversible and covalent inhibition of the serine protease NS3/4A responsible for processing of HCV polyprotein and production of new infectious virions (Figure 2). These drugs can be structurally divided into two groups: linear tetrapeptide α-ketoamide derivatives and macrocyclic inhibitors. Generally, PIs have a remarkable antiviral activity and a low barrier to resistance and are selective against GT-1 infection. Furthermore, the most NS3/4A inhibitors interact with the cytochrome CYP3A4, one of the main enzymes responsible for drug metabolism, and this results in increased drug–drug interactions that can limit treatment regimen [8,73]. These limitations have been partially overcome by the advent of a new generation of PIs, which are also effective against genotypes other than the GT-1, and possess a higher barrier to viral resistance as well as lower propensity for toxicity and drug–

Telaprevir and boceprevir are the first generation of PIs approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA). Telaprevir and boceprevir have been licensed in combination with peg-IFN and RBV, for the treatment of GT-1 infection in naive and experienced patients with compensated liver disease. Telaprevir and boceprevir improved SVR from 49% to 75% in naive patients as compared to the dual therapy [7,75]. Although these therapies have increased clinical outcome, their use is limited by increased rate of adverse effects, including hemolymphopoietic disorders and other reactions related to gastrointestinal system (nausea, diarrhea, vomiting, hemorrhoids, proctalgia, and pruritus). Furthermore, the drugs have a low genetic barrier to resistance [68] and extensive drug–drug

Simeprevir is a once-daily, second-wave protease inhibitor, licensed recently by the FDA and the EMA. This agent is indicated in association with peg-IFN and RBV for the treatment of GT-1 and GT-4 infection. This drug can be associated with sofosbuvir regardless of prior patient treatment history [79]. Simeprevir has a broad spectrum of activity against multiple HCV genotypes except for GT-3 [80]. Data from different trials show that it is highly effective and well tolerated. The most common adverse events are nausea, rash, pruritus, dyspnea, increment in bilirubin blood levels, and photosensitivity [8,74,79]. The NS3 Q80K polymor‐ phism is commonly found in GT-1a viruses and is associated with resistance *in vitro* and

interactions that limit their use in transplanted or coinfected patients [76–78].

disorder, nausea and vomiting, headache, anorexia, rash, and skin irritation [24,69].

**9.3. NS3/4A inhibitors class**

34 Recent Advances in Liver Diseases and Surgery

drug interactions [8,74].

*9.3.2. Simeprevir*

*9.3.1. Telaprevir and Boceprevir*

Paritaprevir is an NS3/NS4A protease inhibitor that has been licensed by the FDA and the EMA in combination with ritonavir, ombitasvir, and dasabuvir with or without RBV. Pari‐ taprevir is metabolized primarily by cytochrome CYP3A4 and is used in combination with ritonavir, a potent CYP3A4 inhibitor, in order to improve the exposures at acceptable dosing frequency [86,87].

#### **9.4. NS5A inhibitors class**

The nonstructural NS5A protein is critical for the virus functions, having a role in viral replication and assembly, and performing complex interactions with cellular functions. Because of this crucial role, NS5A has been identified as a suitable target for viral inhibition (Figure 2). NS5A inhibitors have a high antiviral potency, a pan-genotypic activity, and a genetic barrier to resistance from medium to high. They also possess a good pharmacokinetic and a safety profile that allow once-daily dosing [8,75,88]. Although several NS5A inhibitors are in clinical development or already approved, the exact mechanism is not yet completely known [89]. Recent evidence reported that some of these drugs inhibit formation of the *membranous web* (Figure 2) that is thought to be the site of viral RNA replication [88,90]; other hypotheses suggest that NS5A inhibitors induce rearrangement of NS5A from endoplasmic reticulum-derived foci and limit hyperphosphorylation of this nonstructural protein [91–93].

#### *9.4.1. Daclatasvir*

Daclatasvir is the first NS5A inhibitor that is active at picomolar concentrations with broad coverage of HCV genotypes [89]. Daclatasvir has been recently approved in combination with sofosbuvir with or without RBV for the treatment of GT-1, GT-3, and GT-4 chronic hepatitis C in naive and experienced patients. Daclatasvir has a pharmacokinetic profile that allows oncedaily dosing, and a low potential of causing drug–drug interactions with other medications [94]. Daclatasvir was studied in various combinations with NS3 and NS5B inhibitors and with peg-IFN and RBV.

In a phase II study, 395 naive patients with GT-1 and GT-4 infection were randomized to receive two doses of daclatasvir (20 or 60 mg) in combination with peg-IFN and RBV compared with peg-IFN and RBV plus placebo. The SVR24 was achieved by 59.2% of patients receiving 20 mg, 59.6% in those who received 60 mg, and 37.5% in the placebo group. In patients with GT-4 infection, the SVR24 was achieved by 66.7% and 100% of those who received 20 mg or 60 mg daclatasvir, respectively, vs 50.0% in the placebo group [95].

In the COMMAND trial, 151 treatment-naive patients with GT-2 and GT-3 infection were randomly assigned to receive daclatasvir or placebo plus peg-IFN and RBV for 24 weeks. SVR24 was achieved by 83% in GT-2 infection and by 69% in GT-3 infection, vs 63% in control arm [96]. The treatment is well tolerated, and the main adverse events reported are diarrhea, fatigue, headache, and nausea. The most significant resistant associated variants are 31V and Y93H for GT-1b, and 31V, Y93H M28, and Q30 for GT-1a [97].

#### *9.4.2. Ledipasvir*

Ledipasvir is a potent NS5A inhibitor against GT-1, GT-4, and GT-5 infection but has lower activity against GT-2 and GT-3 infection [89]. Ledipasvir was recently approved in combina‐ tion with sofosbuvir with or without RBV for the treatment of GT-1-, GT-3-, and GT-4-infected patients, naive or experienced, and for the advanced liver disease [98]. This combination is one of the most emerging interferon-free therapies that present a better safety profile than standard therapy and an elevated efficacy with SVR rates from 90% to 100%.

### *9.4.3. Ombitasvir*

Ombitasvir is a novel potent NS5A inhibitor with a promising efficacy particularly in difficultto-treat patients, in association with other DAAs [99]. This drug has been licensed by the FDA and the EMA in combination with paritaprevir/ritonavir and Dasabuvir with or without RBV. The efficacy of this drug was proved in several clinical trials both in association with peg-IFN/ RBV and in interferon-free regimens. In a study of treatment-naive GT-1-infected patients, ombitasvir in combination with peg-IFN and RBV showed an early virological response in 25 out of 28 patients receiving the NS5A inhibitor compared with 6 out of 9 patients in the placebo group [89,99,100].

#### **9.5. NS5B inhibitors class**

NS5B protein is responsible for replication of HCV RNA and represents one of DAAs thera‐ peutic target (Figure 2). NS5B RNA polymerase inhibitors can be divided into two distinct categories: the nucleoside inhibitors (NIs) and the nonnucleoside inhibitors (NNIs). NIs act by binding to the active site of the enzyme and are integrated into the growing RNA chain, causing chain interruption. Nonnucleoside inhibitors (NNIs) bind outside the active site, leading to the allosteric inhibition of RNA polymerase activity [8,75]. NIs have pan-genotypic activity and a medium–high barrier to resistance; NNIs have a low–medium activity against different HCV genotypes as well as a low barrier to resistance. These differences are explicated on the basis of different mechanisms of action because NIs act in a highly conserved region of the HCV genome while NNIs bind only one of the four binding sites, and this results in a lower efficacy against the different HCV genotypes [7,75].

#### *9.5.1. Sofosbuvir*

[94]. Daclatasvir was studied in various combinations with NS3 and NS5B inhibitors and with

In a phase II study, 395 naive patients with GT-1 and GT-4 infection were randomized to receive two doses of daclatasvir (20 or 60 mg) in combination with peg-IFN and RBV compared with peg-IFN and RBV plus placebo. The SVR24 was achieved by 59.2% of patients receiving 20 mg, 59.6% in those who received 60 mg, and 37.5% in the placebo group. In patients with GT-4 infection, the SVR24 was achieved by 66.7% and 100% of those who received 20 mg or 60 mg

In the COMMAND trial, 151 treatment-naive patients with GT-2 and GT-3 infection were randomly assigned to receive daclatasvir or placebo plus peg-IFN and RBV for 24 weeks. SVR24 was achieved by 83% in GT-2 infection and by 69% in GT-3 infection, vs 63% in control arm [96]. The treatment is well tolerated, and the main adverse events reported are diarrhea, fatigue, headache, and nausea. The most significant resistant associated variants are 31V and

Ledipasvir is a potent NS5A inhibitor against GT-1, GT-4, and GT-5 infection but has lower activity against GT-2 and GT-3 infection [89]. Ledipasvir was recently approved in combina‐ tion with sofosbuvir with or without RBV for the treatment of GT-1-, GT-3-, and GT-4-infected patients, naive or experienced, and for the advanced liver disease [98]. This combination is one of the most emerging interferon-free therapies that present a better safety profile than standard

Ombitasvir is a novel potent NS5A inhibitor with a promising efficacy particularly in difficultto-treat patients, in association with other DAAs [99]. This drug has been licensed by the FDA and the EMA in combination with paritaprevir/ritonavir and Dasabuvir with or without RBV. The efficacy of this drug was proved in several clinical trials both in association with peg-IFN/ RBV and in interferon-free regimens. In a study of treatment-naive GT-1-infected patients, ombitasvir in combination with peg-IFN and RBV showed an early virological response in 25 out of 28 patients receiving the NS5A inhibitor compared with 6 out of 9 patients in the placebo

NS5B protein is responsible for replication of HCV RNA and represents one of DAAs thera‐ peutic target (Figure 2). NS5B RNA polymerase inhibitors can be divided into two distinct categories: the nucleoside inhibitors (NIs) and the nonnucleoside inhibitors (NNIs). NIs act by binding to the active site of the enzyme and are integrated into the growing RNA chain, causing chain interruption. Nonnucleoside inhibitors (NNIs) bind outside the active site, leading to the allosteric inhibition of RNA polymerase activity [8,75]. NIs have pan-genotypic activity and a medium–high barrier to resistance; NNIs have a low–medium activity against different

daclatasvir, respectively, vs 50.0% in the placebo group [95].

Y93H for GT-1b, and 31V, Y93H M28, and Q30 for GT-1a [97].

therapy and an elevated efficacy with SVR rates from 90% to 100%.

peg-IFN and RBV.

36 Recent Advances in Liver Diseases and Surgery

*9.4.2. Ledipasvir*

*9.4.3. Ombitasvir*

group [89,99,100].

**9.5. NS5B inhibitors class**

Sofosbuvir is the first NI approved by the FDA and the EMA in combination with other antiviral drugs for the treatment of all HCV genotypes in adults [101]. Recently, sofosbuvir was approved as a fixed-dose combination in a single tablet with ledipasvir [98]. Sofosbuvir has a potent activity against all HCV genotypes, a high barrier to resistance, an excellent tolerability, and a very favorable pharmacokinetic profile. The addition of sofosbuvir to peg-IFN and RBV did not increase the frequency or severity of side effects [101,102]. The main adverse events reported in clinical trials are fatigue, headache, and nausea. *In vitro* resistance is linked to the development of an S282T mutation in the NS5B gene, although this should be confirmed in higher numbers of patients [7,8]. The efficacy of sofosbuvir was evaluated in patients infected with GT-1 to GT-6 chronic hepatitis C and was licensed on the basis of the following three studies: NEUTRINO, PROTON, and ATOMIC.

The NEUTRINO was a phase III, single-arm study that investigated the efficacy and safety of sofosbuvir with peg-IFN and RBV in 327 naive patients with GT-1, GT-4, GT-5, or GT-6 infection. SVR rates at 12 weeks were 90% for GT-1 infection, 97% for GT-4/GT-5/GT-6 infections, and 80% in patients with cirrhosis [103]. In the PROTON study, 147 GT-1-infected patients were treated with sofosbuvir or placebo in combination with peg-IFN and RBV for 12 weeks. SVR12 rates were achieved by 91% in sofosbuvir arm and 58% in the placebo group [8,104]. Finally, results from ATOMIC study confirmed the high efficacy of sofosbuvir in these populations [105]. The introduction of this drug in clinical practice has changed the clinical outcome achieving SVR over 90% especially in difficult to treat population as the GT-1-infected one.

#### *9.5.2. Dasabuvir*

Dasabuvir is a nonnucleoside inhibitor and will be used as a part of the all-oral interferon-free HCV therapy in combination with ombitasvir and paritaprevir/ritonavir. This combined therapy has been recently approved by the FDA and the EMA. This combination has shown high efficacy in several clinical trials and is one of the most promising interferon-free regimen. Dasabuvir was developed to treat GT-1-infected patients while is inactive toward GT-2, GT-3, and GT-4 infection. Dasabuvir is well tolerated, and the main adverse events recorded, when in combination with other DAAs, were mild such as headache and fatigue [106,107].

### **9.6. Future therapies for HCV infection: interferon-free regimen**

During the last year, the advent of interferon-free regimen has dramatically changed the standard of care of anti-HCV therapy. These therapies include molecules with different mechanisms of action, pan-genotypic activity that improve their safety, and efficacy profile, simplifying treatment duration. Several interferon-free combinations have been recently approved, and other trials are in ongoing with different DAAs. Results from recent clinical studies established that a permanent cure from infection could be achieved with interferonfree combinations [20].

#### *9.6.1. Sofosbuvir plus ribavirin*

Sofosbuvir was the first drug licensed by the FDA and the EMA as part of interferon-free regimen. Currently, sofosbuvir is indicated in combination with RBV for the treatment of patients with GT-2 and GT-3 infection, even at advanced stages of the disease, while for all the other genotypes, it is recommended only in patients ineligible or intolerant to peg-IFN. Sofosbuvir-based treatment has been evaluated in several clinical trials [101]: FISSION, POSITRON, VALENCE, and FUSION.

FISSION was a randomized study that evaluated 12 weeks of treatment with sofosbuvir and RBV compared with 24 weeks of treatment with peg-IFN and RBV in 499 treatment-naive patients with GT-2 or GT-3 infection. The SVR rates were 95% and 56% in GT-2- and GT-3 infected patients, respectively, for the treatment with sofosbuvir/RBV, vs 78% and 63% in the peg-IFN/RBV arm [103]. POSITRON study confirmed the clinical results obtained in FISSION study [108].

In the FUSION trial, the combination of sofosbuvir/RBV was evaluated in GT-2- or GT-3 infected patients, nonresponders to prior interferon-based treatment. SVR rates were 86–94% in patients with GT-2 infection and 30–62% in GT-3 infection, for 12 or 24 weeks of treatment, respectively [108]. The results obtained in the FISSION study for patients with GT-2 or GT-3 infection have been confirmed by the VALENCE trial [109].

Based on these studies, the combination of sofosbuvir and RBV showed high efficacy with SVR >90% in patients with GT-2 infection, while lower SVR rates were recorded in patients with GT-3 infection. This last population remains the most challenging group of patients to treat with interferon-free regimen.

#### *9.6.2. Sofosbuvir/ledipasvir ± ribavirin*

Recently, the FDA and the EMA approved the fixed combination of sofosbuvir/ledipasvir with or without RBV for 12 or 24 weeks for the treatment of GT-1, GT-3, and GT-4 chronic hepatitis C in naive and experienced patients and in patients who had liver peritransplant [98]. The efficacy of sofosbuvir/ledipasvir was evaluated in three phase III studies: ION-3, ION-2, and ION-1.

The phase III ION-3 study evaluated 8 weeks of treatment with ledipasvir/sofosbuvir with or without RBV and 12 weeks of treatment with ledipasvir/sofosbuvir, in 647 treatment-naive noncirrhotic patients with GT-1 infection. The SVR12 was 94% in ledipasvir/sofosbuvir, 93% ledipasvir/sofosbuvir plus RBV in patients who received 8 weeks, and 95% in patients who received 12 weeks of ledipasvir/sofosbuvir. These results showed no benefits with the addition of RBV in the regimen or with extension of the treatment duration to 12 weeks [110]. A similar rate of SVR was achieved in experienced patients with GT-1 infection in ION-2 and ION-1 studies, with clinical outcome ranging from 94% to 99% for subjects treated with ledipasvir/ sofosbuvir ± ribavirin [111,112]. The treatment is well tolerated, and the most common side effects are fatigue and headache [98].

### *9.6.3. Sofosbuvir plus daclatasvir ± ribavirin*

mechanisms of action, pan-genotypic activity that improve their safety, and efficacy profile, simplifying treatment duration. Several interferon-free combinations have been recently approved, and other trials are in ongoing with different DAAs. Results from recent clinical studies established that a permanent cure from infection could be achieved with interferon-

Sofosbuvir was the first drug licensed by the FDA and the EMA as part of interferon-free regimen. Currently, sofosbuvir is indicated in combination with RBV for the treatment of patients with GT-2 and GT-3 infection, even at advanced stages of the disease, while for all the other genotypes, it is recommended only in patients ineligible or intolerant to peg-IFN. Sofosbuvir-based treatment has been evaluated in several clinical trials [101]: FISSION,

FISSION was a randomized study that evaluated 12 weeks of treatment with sofosbuvir and RBV compared with 24 weeks of treatment with peg-IFN and RBV in 499 treatment-naive patients with GT-2 or GT-3 infection. The SVR rates were 95% and 56% in GT-2- and GT-3 infected patients, respectively, for the treatment with sofosbuvir/RBV, vs 78% and 63% in the peg-IFN/RBV arm [103]. POSITRON study confirmed the clinical results obtained in FISSION

In the FUSION trial, the combination of sofosbuvir/RBV was evaluated in GT-2- or GT-3 infected patients, nonresponders to prior interferon-based treatment. SVR rates were 86–94% in patients with GT-2 infection and 30–62% in GT-3 infection, for 12 or 24 weeks of treatment, respectively [108]. The results obtained in the FISSION study for patients with GT-2 or GT-3

Based on these studies, the combination of sofosbuvir and RBV showed high efficacy with SVR >90% in patients with GT-2 infection, while lower SVR rates were recorded in patients with GT-3 infection. This last population remains the most challenging group of patients to treat

Recently, the FDA and the EMA approved the fixed combination of sofosbuvir/ledipasvir with or without RBV for 12 or 24 weeks for the treatment of GT-1, GT-3, and GT-4 chronic hepatitis C in naive and experienced patients and in patients who had liver peritransplant [98]. The efficacy of sofosbuvir/ledipasvir was evaluated in three phase III studies: ION-3, ION-2, and

The phase III ION-3 study evaluated 8 weeks of treatment with ledipasvir/sofosbuvir with or without RBV and 12 weeks of treatment with ledipasvir/sofosbuvir, in 647 treatment-naive noncirrhotic patients with GT-1 infection. The SVR12 was 94% in ledipasvir/sofosbuvir, 93% ledipasvir/sofosbuvir plus RBV in patients who received 8 weeks, and 95% in patients who received 12 weeks of ledipasvir/sofosbuvir. These results showed no benefits with the addition

free combinations [20].

study [108].

ION-1.

*9.6.1. Sofosbuvir plus ribavirin*

38 Recent Advances in Liver Diseases and Surgery

with interferon-free regimen.

*9.6.2. Sofosbuvir/ledipasvir ± ribavirin*

POSITRON, VALENCE, and FUSION.

infection have been confirmed by the VALENCE trial [109].

The combination of sofosbuvir plus daclatasvir with or without RBV for 12 or 24 weeks was evaluated in the AI444040 study in 211 patients infected with GT-1, GT-2, or GT-3, including treatment-naive individuals and who had failed prior therapy with boceprevir or telaprevir. SVR12 was achieved in 98% naive and experienced patients with GT-1 infection, 96% of those with GT-2 infection and 89% of those with GT-3 infection. The treatment was well tolerated, and the most common adverse events reported are fatigue, nausea, and headache. This results indicated that sofosbuvir plus daclatasvir is efficacious in GT-1-, GT-2-, or GT-3-infected patients and in nonresponders with GT-1 infection [94,113]. This therapeutic approach is now being tested in a phase III study, in subjects with GT-3 infection [114].

#### *9.6.4. Sofosbuvir plus simeprevir ± ribavirin*

The safety and efficacy of combined oral sofosbuvir plus simeprevir was evaluated in the COSMOS study. In this trial, 168 patients (treatment-naive patients and previous nonrespond‐ ers) were randomized in two cohorts on the base of METAVIR scores (F0–F2 in cohort 1, F3– F4 in cohort 2) to receive 12 or 24 weeks of simeprevir and sofosbuvir with or without RBV. SVR was achieved by 92% in cohort 1 and 94% in cohort 2. This study suggested that the addition of RBV and treatment duration for 24 weeks did not clearly improve SVR rates. This combination therapy was well tolerated, and the most common adverse events were fatigue, headache, and nausea [115].

#### *9.6.5. 3D regimen: paritaprevir/ritonavir, ombitasvir, dasabuvir ± ribavirin*

The multitarget therapy, which includes all-oral combination of paritaprevir/ritonavir, ombitasvir, and dasabuvir, is one of the most promising interferon-free therapies. Paritaprevir/ ritonavir and ombitasvir are coformulated as fixed combination in a single tablet. The thera‐ peutic regimen "all in one" is completely oral, without interferon, and is the unique that provides three antiviral agents with direct action, each characterized by a different mechanism of action. The 3D regimen ± RBV is indicated for 12 or 24 weeks for the treatment of patients with GT-1 infection, while only paritaprevir/ritonavir and ombitasvir are indicated in GT-4 infection. The 3D regimen is also indicated in combination with RBV for 24 weeks in liver transplant recipients with GT-1 infection, in patients coinfected with HIV-1, and in patients receiving replacement therapy with opioids [116,117]. The safety and the efficacy of this regimen were based on the results of six clinical trials: SAPPHIRE I, SAPPHIRE II, PEARL II, PEARL III, PEARL IV, and TORQUOISE II.

In the phase III SAPPHIRE I study, 631 treatment-naive adults with GT-1 infection were treated for 12 weeks with 3D regimen in combination with RBV. The overall SVR12 was 96% [118].

The SAPPHIRE-II trial was conducted in 394 experienced patients with GT-1 infection without cirrhosis. The SVR rates were 95.3% among patients with a prior relapse, 100% among patients with a prior partial response, and 95.2% among patients with a prior null response [119].

The PEARL-III and PEARL-IV studies assessed the needing to include RBV in the 3D regimen in treatment-naive adults with GT-1 infection. Clinical results showed that SVRs are similar in GT-1a infection (99.5% *vs* 99% with or without RBV, respectively), while patients with GT-1b infection achieved higher SVR12 in RBV group (97.0% vs 90.2%, with or without RBV, respectively) [120]. Similar results were obtained in the PEARL-II, in experienced patients with GT-1b infection [121]. The efficacy of paritaprevir/ritonavir and ombitasvir in treatment-naive or experienced patients with GT-4 infection was proved in the PEARL-I. In this trial, 90.9% of naive patients treated with 3D regimen without RBV and 100% of naive and experienced patients treated with 3D regimen plus RBV achieved SVR12 [122].

In the TURQUOISE-II study, the efficacy and the safety of 12 or 24 weeks with 3D regimen with RBV were assessed in patients with advanced disease and GT-1 infection. Ninety-two percent of patients achieved SVR rates at 12 weeks, vs 96% at 24 weeks. Experienced patients with GT-1a infection had a better response from 24 weeks of treatment [123]. The resistance profile observed in these clinical trials seems to have little impact on the likelihood of achieving SVR, given the low virological failure rates recorded. The 3D regimen has shown high efficacy in patients with GT-1 infection (90–100%) and is well tolerated. The main adverse events reported are moderate, mainly pruritus, fatigue, and headache [117,118]. This interferon-free regimen is now being tested in different clinical trials in association with other DAAs.

New interferon-free combinations are under investigation in phase II/III clinical trials. New compounds seem to have a more potent activity vs different genotypes than the DAAs of second generation. The aim of these new therapies is to treat HCV infection through shorter regimen. Grazoprevir and elbasvir ± sofosbuvir and sofosbuvir and ledipasvir plus GS-9451 are the most promising combination in clinical development. A six-week interferon-free oral treatment regimen for HCV GT-1 infection will be likely available in the near future [124–127].

### **9.7. Special population**

### *9.7.1. Liver transplanted patients*

HCV infection is one of the risk factors of liver transplantation and an important cause of morbidity and mortality in these patients [128]. HCV infection recurrence occurs in 50% of subjects with detectable HCV RNA at the time of liver transplantation [31,129]. Dual therapy based on peg-IFN/RBV was the standard of care and is associated with low SVR rates at 24 weeks (20–25%). Telaprevir and boceprevir improved SVR until 67%, but drug–drug interac‐ tion with immunosuppressive agents and serious adverse events can limit their use [8,130,131]. The introduction of DAAs has improved the efficacy of HCV therapy in patients before and after liver transplantation.

The first interferon-free regimen evaluated in pretransplant setting was 48 weeks of sofosbuvir and RBV for all HCV genotypes. The posttransplant follow-up showed that sofosbuvir and RBV prevented recurrence of HCV infection in 70% of patients [132]. Similar SVRs were obtained in patients that had received liver transplant and then relapsed. The safety profile is better than standard therapy on the base of adverse events reported [133,134].

The SOLAR-1 Phase II study analyzed the combination of sofosbuvir and ledipasvir for 12 or 24 weeks, in naive and experienced patients with a relapse of GT-1/GT-4 infection after liver transplantation. The results showed that 96–98% of patients with F0-F3 fibrosis, 96% with Child–Pugh–Turcotte A cirrhosis, 85–83% with Child–Pugh–Turcotte B cirrhosis, and 60–67% with Child–Pugh–Turcotte C achieved the SVR12. The treatment was generally safe and well tolerated [135]. Finally, in the CORAL-I study, the safety and the efficacy of 24 weeks of 3D regimen with RBV were studied in 34 GT-1-infected liver transplant recipients with none or mild fibrosis. The SVR was achieved in 97.1% of patients [136].

#### *9.7.2. HIV-coinfected patients*

In the phase III SAPPHIRE I study, 631 treatment-naive adults with GT-1 infection were treated for 12 weeks with 3D regimen in combination with RBV. The overall SVR12 was 96% [118].

The SAPPHIRE-II trial was conducted in 394 experienced patients with GT-1 infection without cirrhosis. The SVR rates were 95.3% among patients with a prior relapse, 100% among patients with a prior partial response, and 95.2% among patients with a prior null response [119].

The PEARL-III and PEARL-IV studies assessed the needing to include RBV in the 3D regimen in treatment-naive adults with GT-1 infection. Clinical results showed that SVRs are similar in GT-1a infection (99.5% *vs* 99% with or without RBV, respectively), while patients with GT-1b infection achieved higher SVR12 in RBV group (97.0% vs 90.2%, with or without RBV, respectively) [120]. Similar results were obtained in the PEARL-II, in experienced patients with GT-1b infection [121]. The efficacy of paritaprevir/ritonavir and ombitasvir in treatment-naive or experienced patients with GT-4 infection was proved in the PEARL-I. In this trial, 90.9% of naive patients treated with 3D regimen without RBV and 100% of naive and experienced

In the TURQUOISE-II study, the efficacy and the safety of 12 or 24 weeks with 3D regimen with RBV were assessed in patients with advanced disease and GT-1 infection. Ninety-two percent of patients achieved SVR rates at 12 weeks, vs 96% at 24 weeks. Experienced patients with GT-1a infection had a better response from 24 weeks of treatment [123]. The resistance profile observed in these clinical trials seems to have little impact on the likelihood of achieving SVR, given the low virological failure rates recorded. The 3D regimen has shown high efficacy in patients with GT-1 infection (90–100%) and is well tolerated. The main adverse events reported are moderate, mainly pruritus, fatigue, and headache [117,118]. This interferon-free regimen is now being tested in different clinical trials in association with other DAAs.

New interferon-free combinations are under investigation in phase II/III clinical trials. New compounds seem to have a more potent activity vs different genotypes than the DAAs of second generation. The aim of these new therapies is to treat HCV infection through shorter regimen. Grazoprevir and elbasvir ± sofosbuvir and sofosbuvir and ledipasvir plus GS-9451 are the most promising combination in clinical development. A six-week interferon-free oral treatment regimen for HCV GT-1 infection will be likely available in the near future [124–127].

HCV infection is one of the risk factors of liver transplantation and an important cause of morbidity and mortality in these patients [128]. HCV infection recurrence occurs in 50% of subjects with detectable HCV RNA at the time of liver transplantation [31,129]. Dual therapy based on peg-IFN/RBV was the standard of care and is associated with low SVR rates at 24 weeks (20–25%). Telaprevir and boceprevir improved SVR until 67%, but drug–drug interac‐ tion with immunosuppressive agents and serious adverse events can limit their use [8,130,131]. The introduction of DAAs has improved the efficacy of HCV therapy in patients before and

patients treated with 3D regimen plus RBV achieved SVR12 [122].

**9.7. Special population**

*9.7.1. Liver transplanted patients*

40 Recent Advances in Liver Diseases and Surgery

after liver transplantation.

Due to shared modalities of transmission, the infection with HCV is often widespread among HIV-infected people. In the last decade, the rate of HCV coinfection was increased, and it has been estimated that about 15–30% of HIV-infected patients are also infected with HCV. HIV/ HCV-infected patients are more difficult to treat since the coinfection decrease HCV clearance. The standard of care of these patients was the combination of peg-IFN and RBV, but the coadministration of several agents leads to increased drug–drug interaction and adverse events and requires dose adjustment [137,138]. Similarly to that reported for HCV monoin‐ fected patients, the development of DAAs and interferon-free regimens has substantially increased the treatment outcome. The combination of sofosbuvir and RBV was explored in two studies. PHOTON-1 showed that 76%, 88%, and 67% of treatment-naive patients with GT-1, GT-2, or GT-3 infection, respectively, achieved the SVR12. Sofosbuvir has minimal or none interactions with a wide range of antiretroviral drugs, and treatment was well tolerated [139,140]. Similar results have been obtained from PHOTON-2 [141]. The combination of sofosbuvir and ledipasvir was evaluated in the ERADICATE study. In this trial, 100% of untreated and antiretroviral-treated patients achieved the SVR12 [142]. Finally, the results from TURQUOISE-I study showed that 93.5% of patients achieved SVR12 with 3D regimen plus RBV [143].
