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Digestive and Liver Disease 43 (2011) 337–344

Contents lists available at ScienceDirect

Digestive and Liver Disease

journa l homepage: www.e lsev ier .com/ locate /d ld

eview article

orthcoming challenges in the management of direct-acting antiviralgents (DAAs) for hepatitis C

affaele Bruno ∗, Serena Cima, Laura Maiocchi, Paolo Sacchiepartment of Infectious Diseases, Foundation IRCCS San Matteo Hospital - University of Pavia, Pavia, Italy

r t i c l e i n f o

rticle history:eceived 22 July 2010ccepted 9 September 2010vailable online 25 October 2010

a b s t r a c t

Agents that specifically target the replication cycle of the virus direct-acting antiviral agents (DAAs) bydirectly inhibiting the NS3/4A serine protease, the NS5B polymerase and NS5A are currently in clinicaldevelopment. The need to achieve serum drug concentrations able to suppress viral replication is a keyfactor for a successful antiviral therapy and the prevention of resistance. Thus pharmacokinetics parame-

eywords:AAharmacokineticsitonavir boostingesistance

ters became important issues for drugs used in the therapy of hepatitis C. The ratio of Cmin/IC50 (inhibitoryquotient or IQ) can provide a surrogate measure of a drug’s ability to suppress HCV replication, by takinginto account the relationship between plasma drug levels and viral susceptibility to the drug. Ritonavirboosting may be a useful strategy to improve pharmacokinetic parameters. Characterising resistance toDAAs in clinical trials is essential for the management of a drug regimen to reduce the development ofresistance and thereby maximise SVR rate. The lesson of HIV therapy, provide a compelling case for the

ons o Gast

exploration of combinati© 2010 Editrice

. Introduction

Hepatitis C virus (HCV) chronically infects 180 million peopleorldwide with an estimated incidence of new cases of 3–4 million

ach year [1,2]. In the developed world, HCV accounts for 50–76%f all primary liver cancer cases and 30% of all liver transplants,and has been estimated to result in a reduction in overall life

xpectancy in infected individuals of between 8 and 12 years [3].he current recommended treatment, consisting of a peginterferonlus ribavirin (Peg-IFN/RBV) combination achieves response ratesanging from 76% to 80% in patients infected with HCV genotypes 2nd 3 and from 40% to 50% in those with genotype 1 [3]. Althoughhis kind of therapy will remain the Standard of Care (SOC) for theext years, more effective therapeutic options with shorter treat-ent durations are needed to increase the response rate in difficult

o treat patients (mainly genotype 1) and reduce the impact of HCVnfection and its associated complications.

So far, agents that specifically target the replication cycle of theirus direct-acting antiviral agents (DAAs) by directly inhibiting the

S3/4A serine protease (which processes the HCV polyprotein toenerate mature viral proteins), the NS5B polymerase (which repli-ates the viral RNA genome) and NS5A (which functions as a partf the replicase complex) are currently in clinical development [4].

∗ Corresponding author at: Department of Infectious Diseases, Hepatology Out-atients Unit, University of Pavia, Fondazione IRCCS Policlinico San Matteo, Viaaramelli, 5, 27100 Pavia, Italy. Tel.: +39 0382 501080; fax: +39 0382 501080.

E-mail addresses: raffaele.bruno@unipv.it, r.bruno@smatteo.p (R. Bruno).

590-8658/$36.00 © 2010 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevieroi:10.1016/j.dld.2010.09.007

f direct-acting antiviral agents.roenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

The aim of this paper is to discuss the implications of viral kinet-ics, pharmacokinetics and resistance in the management of DAAtherapy.

2. Viral kinetics

2.1. Mechanisms of HCV replication

HCV is the only member of the hepacivirus genus of Flaviviri-dae. As with other Flaviviridae, the 9.4 kb (+) sense RNA genomeis translated into a single long polyprotein that is cleaved by bothhost signal peptidases and virally encoded proteases (NS2, NS3/4A)into 10 functional peptides (structural and nonstructural proteins).One of these proteins, the NS5B RNA-dependent RNA polymerase,catalyzes the direct copying of the viral genome into a replicativeintermediate RNA. As there is no DNA intermediate (i.e. no reversetranscriptase activity), HCV is not known to be capable of a latentphase [5].

The successful development of HCV replicons, autonomousRNAs, the replication of which is directed by the viral replicationmachinery (nonstructural proteins), has been a major advance notonly for the elucidation of HCV RNA replication but also for thescreening of candidate antiviral compounds that inhibit replication[6] (see Fig. 1).

2.2. Hepatitis C virus mutations, replicative fitness

Each HCV-infected patient carries a heterogeneous populationof HCV, including preexisting variants with decreased sensitiv-

Ltd. All rights reserved.

338 R. Bruno et al. / Digestive and Liver Disease 43 (2011) 337–344

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ty to direct-acting antiviral drugs. The emergence of clinicallyelevant resistant variants depends on several factors such ashe selective pressure applied by the drug, the genetic bar-ier to resistance, and the replication fitness of the resistantariants.

As for human immunodeficiency virus (HIV) and hepatitis Birus (HBV), HCV- resistant variants are usually not as fit as wildype viruses, particularly if drugs to which they are resistant bindirectly to the active site of a viral enzyme. However, a resistantariant can improve fitness by the accumulation of additional com-ensatory mutations.

The fitness of these HCV variants is typically estimated in vitroy measuring replication capacity (by transient replication in theeplicon system) and enzymatic fitness (by measuring catalyticfficiency) [7,8]. The resistant variants have varying degrees ofecreased replication capacity. The NS3 A156T mutation, whichonfers resistance to many Protease Inhibitors (PIs), has signifi-antly reduced NS3/4A catalytic efficiency and replication capacity9,10]. The NS5B nucleoside inhibitor-resistant mutation, S282T,lso has decreased replication capacity [8]. The non-nucleosidenhibitor-resistant mutation P495A/L has decreased replicationapacity, but fitness can be restored by compensatory mutationslsewhere in NS5B.

Recently, the in vivo fitness of viral variants with decreased sen-itivity to the HCV PI telaprevir (TVR) has been estimated using aovel method that assessed growth rate in the absence of TVR selec-ive pressure. The replicative fitness of different viral variants wasnversely correlated with their degree of resistance to TVR [11]. Fit-ess of resistant variants is not only important in determining therobability of emerging resistance but also to predict if they willevert to wild type in the absence of drug selective pressure. Resis-ant variants with significantly impaired fitness will be replacedy wild-type viruses more rapidly in the absence of drug selectiveressure [11].

.3. DAA in clinical development

Inhibitors of the HCV NS3/4a serine protease and the NS5b RNA-ependent RNA polymerase (RdRp) and have progressed to the

ore advanced stages of clinical development [12]. A common

heme in the development of these agents is that combination ther-py with pegIFNa and RBV will continue to be important to increasenti-viral efficacy and limit the selection of drug resistant mutants13].

iral agents. Adapted from Lindenbach BD et al., Nature 2005.

2.4. NS3/4a protease inhibitors

The HCV NS3 protein is a multifunctional protein that con-sists of an amino-terminal serine protease and a carboxy-terminalhelicase/nucleoside triphosphatase domain [14] and is necessaryfor post-translational processing of the NS3–NS5 region of theHCV polyprotein to generate components of the viral RNA repli-cation complex [14]. NS4a acts as a cofactor to facilitate the serineprotease function. The helicase is thought to have a role in viralreplication by unwinding the viral RNA [14]. The NS3/4a pro-tease has been shown to be a key regulator of intracellular typeI IFN pathways. Inhibitors of the NS3/4a protease therefore actto inhibit directly viral replication. We briefly reviewed the clin-ical characteristics of DAAs, currently in different phases of clinicaldevelopment.

2.5. NS5B polymerase inhibitors

The HCV NS5B RNA-dependent RNA polymerase is a key enzymeinvolved in HCV replication, catalyzing the synthesis of the comple-mentary minus-strand RNA and subsequent genomic plus-strandRNA from the minus-strand template. Both nucleos(t)ide andnon-nucleos(t)ide polymerase inhibitors (NI/NNI) are currentlyin development. In addition, the replicative activity of the RdRphas recently been reported to be augmented by direct binding tocyclophilin B, a host cell isomerase [15]. A cyclophilin B inhibitorhas also progressed to a phase 2 clinical development programme.

To describe the characteristics and the results obtained in clin-ical trial of each molecules is beyond the goal of this article whichis focused on specific issues of the management. A list of the drugsso far tested in clinical trials and the side effects of them are sum-marized in Tables 1 and 2.

3. Pharmacokinetics

The need to achieve serum drug concentrations able to suppressviral replication is a key factor for a successful antiviral therapy andthe prevention of resistance. Thus pharmacokinetics parametersbecame important issues for drugs used in the therapy of hepatitis

C.

Although the HCV protease inhibitors may have a major clini-cal impact as a drug class, they have a relatively narrow therapeuticindex because they require highly suppressive drug concentrationsto prevent the emergence of resistance. The concept of protease

R. Bruno et al. / Digestive and Liver Disease 43 (2011) 337–344 339

Table 1Site of action and stage of development of molecules tested so far in clinical trials.

Life cycle step Category Drug name Phase of development

HCV replication Polymerase inhibitor RG7128 Phase IVX-222 Phase IIABT-072 Phase IMK-3281 Phase IPSI-7851 Phase IABT-333 Phase IIDX184 Phase IIANA598 Phase IIGS9190 Phase IIVX-759 Phase IIPSI-7977 Phase IIa

NS5A inhibitor PPI-461 Phase IA-832 Phase IIBMS-790052 Phase II

HCV polymerase VX-916 Phase IHCV polymerase inhibitor Filibuvir Phase ICyclophilin inhibitor SCY-635 Phase I

Debio 025 Phase IINS4B inhibitor Clemizole Phase I

Post-translation processing Protease inhibitor RG7227 Phase IIVX950 (telaprevir) Phase IIIIDX320 Phase IACH-1625 Phase IVX-813 Phase IPHX1766 Phase IGS-9256 Phase IIBI 201335 Phase IISCH900518 (narlaprevir) Phase II

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nhibitor (PI) boosting was developed to overcome these issues16].

When considering the pharmacokinetics of antivirals severalarameters must be taken into account.

After multiple doses of a drug are administered over several daysf treatment, the maximum and minimum concentrations of drugchieved after each dose reach constant levels. This is referred to

s the “steady state.” The key measures of pharmacokinetics thatmpact in clinical practice are the following:

Cmax = the peak or highest plasma concentration achieved duringa dosing interval.

able 2ain side effects of direct-acting antivral agents tested in clinical trials.

Class Drug Side effects

NS3/4a proteaseinhibitors

Boceprevir Headache, rigor, myalgia and feverfatigue, anemia, nausea; adverseevents were also the most commonlyfor PEG-IFN�2b monotherapy [46]

Telaprevir Influenza-like illness, fatigue,headache, nausea, anemia, depression,and pruritus constipation, abdominalpain, gastroesophageal reflux disease,nausea, vomiting, headache, acuteotitis media, and seasonal allergy.Decreases in hemoglobin, total whiteblood cell count, neutrophils, andplatelets occurred during the studydrug dosing period [13]

NS5B NIs R1626 Vomiting and diarrhoea, moderateleokopenia; cytopenia, haematologicaltoxicity, neutropenia, anemia, rash [13]

R7128 4 Haematological, toxicity, Headache,fatigue and chills [13]

R7128 4 Mild-moderate diarrhoea [13]

TMC435 Phase IIaSCH 503034 (boceprevir) Phase IIIVX-500 Phase IMK-7009 (vaniprevir) Phase II

Tmax = the time taken to reach the highest observed plasma con-centration.Cmin = the lowest observed plasma concentration achieved duringa dosing interval. Cmin is also called the ‘trough concentration’, andgenerally occurs at the end of the dosing interval (see Fig. 2).

The activity of PIs is dependent on the maintenance of cir-culating concentrations that suppress viral maturation. Indeed, iftreatment regimens produce drug trough concentrations allowingpersistent low-level viral replication, at the same time they permitthe accumulation of mutations required for significant resistance.Thus, for PIs the Cmin is likely to correlate most closely with antivi-ral efficacy. The higher the Cmin above the inhibitory concentration,the higher the potential for viral suppression [16,17].

3.1. Understanding parameters of antiviral efficacy

Inhibitory Concentration (IC)50/IC90 are the in vitro concentra-tion of drug required to inhibit viral replication by 50%/90%.

IC50 and IC90 vary depending upon a number of factors, includ-ing the viral strain, cell type and assay used, and on adjustmentsmade for plasma protein binding. Drug-resistant strains tend tohave higher IC50 values compared to wild type.

TheCmin

IC50ratio

The ratio of Cmin/IC50 (inhibitory quotient or IQ) can provide asurrogate measure of a drug’s ability to suppress HCV replication,

by taking into account the relationship between plasma drug levelsand viral susceptibility to the drug. Higher values would provide adegree of pharmacologic “forgiveness”, a characteristic which min-imizes the impact of less than perfect adherence, an heterogeneousviral population or variable drug absorption [17] (see Fig. 3).

340 R. Bruno et al. / Digestive and Liver Disease 43 (2011) 337–344

Fig. 2. Theoretical plasma concentration/time curve showing fundamental pharmacokinetics parameters.

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vir/ritonavir at 250/100 mg is predicted to provide a mean plasmaconcentration equivalent to that achieved with telaprevir 750 mgevery 8 h [22].

Pharmacokinetics parameters for boceprevir were evaluated onday 1 of monotherapy and on days 1 and 8 of combination therapy

Table 3Key pharmacokinetics (PK) parameters and clinical significance of antiviral drugs.

PK parameters Clinical advantages

Decreased systemic clearance(by ritonavir PK boosting)

Lower likelihood of resistance

Fig. 3. For antiviral therapy to be successful, drug levels need to be always we

. Protease inhibitor boosting

The goal of PI boosting is to increase the exposure to thesegents, ensuring sustained and effective concentrations throughouthe dosing interval. PI boosting is best achieved by administeringitonavir (usually low dose 100 mg once or twice daily) along withhe PI.

Ritonavir may increase exposure of a concomitantly adminis-ered PI by exerting the following effects:

1) Ritonavir inhibits P-glycoprotein transport in the intestine,which increases absorption of the second PI.

2) Ritonavir is one of the most powerful inhibitors of the metabolicenzyme CYP3A4 in the intestinal wall and liver, which reducesthe extent of first-pass metabolism of the second PI.

3) Ritonavir inhibits metabolism by CYP3A4 in the liver, whichreduces the rate of systemic clearance of the second PI [18,19].

hus, inhibition of P-glycoprotein and CYP3A4 by ritonavir affectshe following PK parameters of coadministered PIs, giving at theame time some clinical advantages, as shown in Table 3.

.1. PK of clinically tested DAAs and ritonavir boosting

Telaprevir has a half-life of 0.8–3.2 h [20,21]. Its area under theurve (AUC) ratio for liver/plasma concentration is 2.3–35 [20,21].

ve the IC50, to avoid “blips” of viral replication and the selection of resistance.

Hence, telaprevir is taken up by the liver on first pass metabolism,resulting in higher concentrations in the liver than in plasma.Steady state was reached within 24–48 h of the start of dosing. Theconcentration of telaprevir was higher than the concentration thatexerts antiviral activity on the replicon system for as long as 12 h[21].

In one study on rats and human liver microsomes, telaprevirwas greatly boosted by ritonavir co-dosing.

The concentration of telaprevir 8 h after dosing was increasedby > 50 fold. Based on these results, twice-daily dosing of telapre-

Increased trough (Cmin) Improved antiviral activityDecreased peak (Cmax) Reduced drug toxicityReduced pharmacokinetic

variabilityAmelioration of food restrictions

Increased AUC Improves adherence

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Disease 43 (2011) 337–344 341

with PEG-IFN �-2b. The AUC on day 1 of monotherapy and com-bination therapy of both dose groups are similar, which suggeststhat there was no interaction between boceprevir and PEG-IFN. Thecombination therapy with boceprevir at either dose level and PEG-IFN �-2b resulted in greater decreases in HCV RNA than PEG-IFN�-2b alone. Furthermore, the degree of virological response wasrelated to the dose of boceprevir in monotherapy and combinationtherapy regimens (Sarrazin et al.). A maximum mean change in HCVRNA of was observed for PEG-IFN �-2b plus boceprevir 400 mg 3times daily (overall mean maximum decline, −2.88/−0.22) [23].

5. Development of resistance to protease inhibitors

HCV replication is characterized by a short virion half-life anda high production and clearance rate of free virions [24]. Theestimated average half-life of the hepatitis C virus is 2.7 h, withpretreatment production and clearance of 1012 virions per day.The HCV/NS5B RNA-dependent RNA polymerase has a low fidelityand no proofreading function.

From treatment of HIV and HBV with specific inhibitors, it isknown that rapid selection of preexisting drug-resistant variantsoccurs during treatment. During selection pressure, the relativereplication fitness of a selected drug-resistant variant determineswhether the variant will grow out. Without selection pressure, adrug resistant variant may be generated but will simply vanish if itsrelative replication fitness is lower than that of non-drug-resistantvariants.

The speed of selecting drug resistance depends mainly on theturnover of the viral nucleic acid; the HCV RNA strands presentin infected hepatocytes serve as templates for producing new HCVvirions that are released soon after [24]. As a result, the viral genetichalf-life is shorter for HCV than for HIV and HBV and the timerequired for selecting drug-resistant mutants and for their expan-sion to become the major part of the viral population is shorter forHCV than for HIV and HBV. Thus, resistance to HCV antivirals ismore likely than resistance to antiretrovirals [24,25].

The rapid selection of viral variants displaying drug-resistantphenotypes has been observed in patients experiencing viralrebound during treatment as well as in replicon experiments. Vari-ants resistant to DAAs reported to date are summarized in Table 4.

Two recent reports suggested that resistant variants mayalready be present at a 1% frequency in the quasispecies popu-lation in treatment naıve patients [26,27], consistent with theirdominant emergence only days after treatment initiation [28,11].However, drug treatment in the setting of resistance mutations maystill be beneficial, because a decreased in vitro replicative capacityhas been demonstrated for many viral strains resistant to proteaseor RdRp inhibitors [29,30,7,31,10,32,33]. Patients may thereforebenefit from elimination of the dominant, drug susceptible viralquasispecies with more than 1000-fold reductions in their viralload, as long as only drug-resistant but replication-deficient qua-sispecies constitute the residual viral population [28,11]. Althoughin some cases replication levels may later be restored by compen-satory mutations, it seems possible that this effect could suffice toachieve treatment success if several drugs were combined to sup-press viral replication before compensatory mutations or additionalresistance mutations could evolve.

The presence of pre-existing mutations in treatment naïvepatients may be a factor affecting the response to therapy. Indeed,the PI-resistant mutation R155K was recently detected as the dom-

inant quasispecies in a treatment-naıve patient [34].

In a study by Kuntzen et al. dominant mutations were mostlyobserved as sporadic, unrelated cases at frequencies between 0.3%and 2.8% in the population. Taken together, however, 8.6% ofpatients infected with genotype 1a and 1.4% of patients infected

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ith genotype 1b exhibited at least one drug resistance mutation,ncluding two cases with possible multidrug resistance. Viral loads

ere high in the majority of patients carrying these mutations, sug-esting that resistant viruses might achieve replicative capacitiesomparable to nonresistant strains in vivo [35].

Treatment success as measured by decreasing viral loads rep-esents a balance between the achievable drug concentration inhe plasma, the frequency and degree of drug resistance amonghe viral quasispecies [36], and the replication efficiency of drug-esistant viral strains. Numerous studies have investigated thempact of amino acid substitutions on viral replication and drugusceptibility to investigational NS5B polymerase or NS3/4A pro-ease inhibitors in the replicon model. A pronounced reduction inhe replicative capacity was described for the highly resistant A156ariants in NS3 and M423 variants in NS5B, and also to a lesserxtent for the low-level PI-resistant R155, T54, and V36 mutations11,29,33,35]. It has been speculated that these lower replicationevels could facilitate eradication through combination treatment28], because DAA resistant viral strains appear to remain sensi-ive to interferon and ribavirin [33]. In a recent clinical trial, then vivo relevance of these findings was supported by the observa-ion that weakly telaprevir-resistant variants rose predominantlyn patients who only achieved lower plasma drug levels, whereasncreasing drug concentrations selected for mutants that werelso highly resistant in vitro [11]. Preliminary results indicate thatominant resistance mutations can potentially reduce the earlyreatment response to DAAs [36], and it must be assumed thatow-level resistant strains will sustain viral replication in patients

ho do not achieve optimal drug levels with standard dosing, inases with poor adherence, or when dose reductions are inevitableue to adverse events. Importantly, continued viral replication inhe presence of the selecting drug would put the patient at riskf developing additional resistance mutations. Here, observationsrom HIV infection suggest that baseline resistance even againstnly one drug in a multidrug antiviral regimen may affect treat-ent success [11], and further indicate that apart from singleutations conferring high level resistance, stepwise accumulation

f subtle but synergistically acting resistance mutations may alsoventually lead to treatment failure [37]. Data from a recent studysing telaprevir has indicated a similar pathway in HCV infec-ions, where combination of the low-level resistance mutations36 M and R155K resulted in a highly drug resistant phenotype, theppearance of which coincided with viral breakthrough [11,28].

In HIV infection, resistance testing was calculated to be cost-ffective when the prevalence of drug resistance becomes 1% ataseline and is currently recommended in areas with more than% prevalence of resistant strains, and in all cases of treatmentailure [38]. For novel DAAs, important factors such as their costnd treatment response rates are not yet available to derive sim-lar calculations, but further studies are needed to address thesessues.

. Tools for monitoring viral resistance

Characterizing resistance to DAAs in clinical trials is essentialor the management of a drug regimen to reduce the developmentf resistance and thereby maximize SVR rate. So far, methods toharacterize viral resistance works in a complementary way andre distinct in genotypic and phenotypic assays [2].

.1. Genotypic assays

Genotypic assays examine the genetic sequence of a targetegion involved directly or indirectly in the interaction of a drugith its target [11,28,39]. Initial characterization of the resistancerofile for a drug requires comparing viral sequences before, during

Disease 43 (2011) 337–344

and after treatment to detect changes that occur during treatment.Available genotypic assays have different levels of sensitivity.

Sequencing methods are relatively simple to conduct, but theycannot determine linkage between different mutations in a singlevariant, or detect variants with mutations that are present in lessthan 25% of the population.

More sensitive methods such as clonal sequencing or theTaqManTM mismatch amplification mutation assay (TaqMAMA)[40], may be more costly and time consuming.

6.2. Viral fitness assays

Although not a measure of resistance per se, the viral fitness(replicative capacity) of resistant variants is an important factor,with implications for clinical resistance.

The replication capacity of HCV variants is typically assessed invitro using a transient replicon system [7], or by comparing colonyformation efficiency of the mutant replicon RNA with that of WTvariants in co-culture growth competition assays.

Fitness has also been determined in vivo [11] by using HCV RNAlevels and clonal sequencing to calculate the frequency of a givenvariant over time after the end of dosing to assess the growth ratecompared with WT in the absence of drug-selective pressure.

6.3. Phenotypic assays

In clinical research, viral variants identified by genotypic testingshould be tested with a phenotypic assay, both to confirm that themutation confers resistance to the drug and to assess the degreeof resistance [41–43]. Phenotypic assays measure the IC50 in anenzyme or replicon assay. By testing the HCV variants for drug sus-ceptibility in vitro, the fold change in sensitivity can be calculatedas the IC50 value of the isolate/IC50 value of the reference strain (e.g.WT).

7. Identification of genotype/subtype

A major issue that limits the efficacy of NS3/4A proteaseinhibitors is the finding that genetic barrier and resistance profilessubstantially differ between the different genotype 1 subtypes. Thereason is that only one nucleotide substitution is needed to gener-ate a subtype 1a sequence variant, whereas two substitutions areneeded to the 1b sequence. In vivo, different resistance profiles inpatients infected by HCV subtypes 1a and 1b have been demon-strated. In the former, the V36 and R155 substitutions representthe backbone of resistance, whereas in the latter resistance is lessfrequent as it is preferentially associated with substitutions at posi-tion A156 that are associated with a decreased fitness of the variants[10,33,41].

A correct identification of HCV subtypes 1a and 1b is hencecrucial in clinical trials designated for new HCV drugs to avoid mis-interpretation of efficacy and resistance data. It may also becomeimportant in future clinical practice, when therapy schedule needsto be tailored in genotype 1 patients according to genotype subtype.To identify the HCV genotype and subtype both in clinical trials andpractice, commercial assays have been developed, most of themtargeting the 59 noncoding region (59NCR) of the HCV genome [43].

8. The need for combination therapy

As monotherapy, Direct Antiviral Agents (DAA) of several classeshave been shown to induce significant viral suppression, withthe addition of PEG-IFN/RBV or even PEG-IFN alone conferringadditional viral suppression and/or markedly inhibiting the devel-opment of resistance to the direct-acting antiviral agent [28]. In

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he PROVE1 and PROVE2 studies of genotype 1 treatment-naiveatients treated with the PI telaprevir, viral breakthrough wasn infrequent event, especially in patients who experienced RVR44,45]. Similarly, in the SPRINT-1 boceprevir trial in treatment-aive patients, only a small number of patients discontinued foriral breakthrough [46]. These observations, together with in vitroxperiments demonstrating the capacity of two or three drug com-inations to induce additive or synergistic viral suppression [47], asell as the history of HIV therapy, provide a compelling case for the

xploration of combinations of direct-acting antiviral agents. Theltimate goal of this approach is to build regimens able to over-ome the development of resistance. As suggested in a interestingeview by Pereira & Jacobson, the height of the “genetic barrier”,uch as the relative likelihood to acquire conferring resistanceutations, varies among the different DAAs. Nucleoside analogues

eem to have the highest barrier when compared to protease andon-nucleosides inhibitors. The combination of drug with differ-nt resistance profiles may become an interesting strategy to avoidhe development of overlapping resistances [48]. So far, combina-ions have been studied with IFN as a cornerstone, but ultimatelyhe development of IFN-free regimens is a major goal. Very recently,he INFORM-1 study, an intriguing dose-ranging, exploratory studyf R7227, a PI, combined with R7128, a nucleoside polymerasenhibitor, for up to 2 weeks, demonstrated marked viral suppres-ion [49]. No viral breakthroughs were reported. Data from theBV-free arm of PROVE2 [47], the RBV-free arm of PROVE3, atudy on telaprevir in earlier nonresponders [44] and the low doseBV arm of SPRINT-1 [46] have provided a compelling case forhe role of RBV in preventing the emergence of resistant variants.uch data provide a foundation for the early exploration of IFN-ree regimens consisting of two DAA agents plus RBV. Given theniversal interest in studying combinations of DAA agents, theimeline for the development of such combinations is a criticalssue.

. Summary and conclusion

The success of DAAs will depend on their ability to inhibit theeplication of a broad range of viral quasispecies and prevent emer-ence of drug-resistant mutants. Present and forthcoming drugshould have a pharmacokinetic profile which could warrant plasmaevels able to inhibit the viral replication along the inter-doseeriod. Moreover, treatment regimens based on the combinationf drugs with different resistance profile may be the best strategyor improving the response rate in difficult to treat patients in theears to come.

onflict of interest statementhe authors declare that they have no conflict of interest to disclose.

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