Hepatitis Experts Accelerating the Development of Targeted Therapies

Hepatitis Experts Create Roadmap for Accelerating the Development of Targeted Therapies for Hepatitis C Virus

WASHINGTON, Dec. 14, 2010 /PRNewswire-USNewswire/ --

To improve the care for individuals infected with the hepatitis C virus, a major health problem and a leading cause of chronic liver disease around the world, nearly 200 international hepatitis experts have taken an important step in escalating the introduction of a new class of targeted therapies for HCV – direct-acting antivirals (DAAs).


December 6 at a major scientific meeting – Advancing HCV Drug Development: A Collaborative Approach – convened by the Forum for Collaborative HIV Research, researchers, hepatitis advocates, members of industry and representatives from the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) created the roadmap for accelerating the development of DAAs, agreeing that this new class of drugs targeting specific hepatitis C virus proteins has the same potential to improve treatment outcomes for people with HCV as antiretroviral drugs changed the standard of care in HIV. Currently, two DAA compounds have advanced into phase 3 development in the United State and EU, and many more are in phase 2 trials and likely to advance to phase 3 research in the near future.
"If there was ever a time when we can change the course of HCV, it is now," said Veronica Miller, Ph.D., Director of the Forum. "We are now where we were with HIV more than a decade ago and can apply many of the lessons learned from HIV drug development to significantly accelerate the progress in bringing new and better HCV therapies to market."


DAAs directly attack the ability of the hepatitis C virus to replicate and can increase the cure rate in certain HCV patients to between 60 and 70 percent– a major advance over the 40 percent success rate associated with the currently recommended treatment for chronic HCV infection, the combination of pegylated interferon and ribavirin. Although the first DAAs still require concomitant use with current HCV medications, these new compounds will shorten the length of time on pegylated interferon and ribavirin therapy, which hepatitis specialists noted is often difficult to tolerate and has significant adverse event profiles that limit treatment in many patients. According to the latest data, between 15 and 30 percent of HCV patients started on current HCV therapy are unable to complete the year of treatment now required because they cannot tolerate the side effects.


Charting the future of HCV drug development, the meeting participants applied FDA's new guidance on conducting clinical trials on DAAs, which was issued in September 2010 in draft form and expected to be finalized in 2011. According to Jeffrey S. Murray, M.D., M.P.H., Deputy Director of the Division of Antiviral Drug Products in FDA's Center for Drug Evaluation and Research, the draft guidance follows the same approach FDA uses in developing HIV and oncology drugs. For early clinical testing, FDA recognizes that most if not all DAAs for HCV will be used in combination with other approved drug and therefore, recommends studies examining the relationship between the new DAA agent and both pegylated interferon and ribavirin as well as testing the combination antiviral activity. FDA's draft guidance also calls for using the results from proof-in-concept trials (meaning a study in HCV infected patients that demonstrates initial activity as measured by reductions in the HCV viral load) to guide dose selection for subsequent Phase 2 trials in which DAAs are studied for longer durations as part of a combination regimen. FDA is further encouraging drug sponsors to design development plans for combinations of two or more DAAs.


"The good news for the HCV community is that more drugs are coming," said Jur Strobos, MD, JD, FACEP, Deputy Director of the Forum. "The bad news is we don't know how to combine them and that is what we need to study."
With FDA's guidance as the framework, the hepatitis experts also identified the major factors researchers must take into account when designing clinical trials for DAAs and other new HCV therapies. Among the major issues cited are the emergence of resistant virus and its potential management, and including in future DAA clinical trials those special populations with significant unmet needs in HCV therapy. These patients include individuals co-infected with HIV, liver transplant recipients, patients with decompensated cirrhosis, opioid users and those on opiate substitution therapy, and children. According to the Centers for Disease Control and Prevention (CDC), between 5 and 6 percent of infants born to HCV infected women contract the infection from their mothers and the majority of those infants will develop a chronic infection.
Focusing on the special needs of pediatric patients, leaders from both FDA and EMA agreed that the time to start investigating DAAs in children is when sufficient safety data exist in adults. As explained by specialists in pediatric liver disease, children with HCV often tolerate drug therapy better than adults, which is why the ideal age to start children in pediatric trials for DAAs is when they are 3 years old. According to hepatitis experts, the beneficial impact of a 'cure' for children, preferably before they start school, cannot be overestimated.
Reducing Disparities in HCV Clinical Trials


Because identifying potential differences among groups treated with a therapeutic regimen is an important goal of human studies, the HCV community singled out the under-representation of women, older people and different ethnic subgroups in clinical trials as the problem requiring immediate attention and change at a systemic level. Although there is a higher prevalence of HCV in men than women, women metabolize HCV drugs differently and are more affected by autoimmune diseases, which share similar symptoms with HCV. Women also are twice as likely as men to suffer from depression, which is a common side effect of treatment with HCV medications.


Even more challenging for the HCV community is increasing the representation of older HCV-infected adults in HCV clinical trials, even though Baby Boomers constitute the majority of hepatitis C infections in the United States and are often less responsive than younger generations to antiviral treatment. Compounding the problem, older HCV patients are more difficult to treat, due to the increased prevalence of co-morbid conditions, such as diabetes, dyslipidemia, and other metabolic conditions that are correlated to chronic liver disease. Aging is also strongly associated with liver fibrosis progression, which means older HCV patients are likely to have advanced liver disease and a high risk for impending liver complications. But despite this reality, few studies have examined the age-specific factors of chronic HCV infection and the clinical management of the infection in this patient population.
More than an issue of fairness, HCV experts associate better designed clinical research studies with the increased ability of scientists to catalog and understand the influence of genetic and non-genetic factors on individual and group responses to new treatments. Findings from the large amount of genetic data generated to date show that more than 90 percent of the observed genetic variations occur within, rather than between groups. This underscores the fact that gender and ethnicity have biomedical consequences when evaluating patients with more resistant virus and with more severe disease.


Designing the Research Roadmap to Address a Growing Public Health Threat
Accelerating the development of DAAs to improve HCV treatment outcomes is especially warranted now that the hepatitis C virus has become the most common chronic blood borne infection in the U.S. According to new government estimates, approximately 4.1 million Americans are infected with HCV, of whom 60 to 70 percent will develop chronic liver disease. Currently, almost half of all liver transplants in the U.S. are performed for end-stage hepatitis C. Moreover, because liver disease is one of the leading causes of death in the U.S., the CDC predicts that deaths from chronic liver disease attributed to hepatitis C will double or triple over the next 15 to 20 years.


To change these statistics, hepatitis specialists focused on ways to advance HCV drug development so DAAs and other new classes of drugs for HCV can reach the market quickly. Here, the experts reached agreement on a number of issues:
Exposure to new single agents – because HCV remains sensitive to ribavirin and pegylated interferon, longer initial studies may be recommended to evaluate single drugs and novel combinations of drugs


Composition of patients in early studies (phase 1 and 2a) – early studies should be large enough so results with one type of virus or one group of patients can be easily discerned. Focusing on specific genetic sub-populations will also ensure that early studies do not produce confusing results


Drug resistance in HCV patients – unlike HIV, drug resistance in HCV may not be as large a concern because HCV does not integrate into host DNA as HIV does. Thus, resistant strains are not archived and there is the potential that resistant patients can be retreated with different combinations regimens, as and when they become available
Baseline parameters – there is the need to develop predictive algorithms based on baseline characteristics such as gender, body weight, HCV genotypes and subtypes
Exclusion of former and current drug users in clinical trials – exclusion is unnecessary and does not serve the field well. Over 60 percent of patients with HCV are infected through drug use, indicating the need to have quality data to guide treatment decisions in this patient population
As a next step, the Forum for Collaborative HIV Research will publish the consensus of this scientific meeting to advance the research agenda. Once published, the report will be distributed widely to the Forum's many constituencies – government, industry, patient advocates, healthcare providers, foundations, health insurers and academia – with the goal of advancing research on HCV and driving public policy.


About the Forum for Collaborative HIV Research
Now part of the University of California (UC), Berkeley School of Public Health and based in Washington, DC, the Forum was founded in 1997 as the outgrowth of a White House initiative which called for an ongoing collaboration among stakeholders to address emerging issues in HIV/AIDS and set the research strategy. Representing government, industry, patient advocates, healthcare providers, foundations and academia, the Forum is a public/private partnership that is guided by an Executive Committee that sets the research agenda. The Forum organizes roundtables and issues reports on a range of global HIV/AIDS issues, including treatment-related toxicities, immune-based therapies, health services research, co-infections, prevention, and the transference of research results into care. Forum recommendations have changed how clinical trials are conducted, accelerated the delivery of new classes of drugs, heightened awareness of TB/HIV co-infection, and helped to spur national momentum toward universal testing for HIV. http://www.hivforum.org

Hepatitis C :challenges in the management of STAT-C therapy

Forthcoming challenges in the management of STAT-C therapy
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Raffaele Bruno, Serena Cima, Laura Maiocchi, Paolo Sacchi
Received 22 July 2010; accepted 9 September 2010.

published online

27 October 2010.

Corrected Proof
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Abstract
Agents that specifically target the replication cycle of the virus [specifically targeted antiviral therapies for hepatitis C (STAT-Cs)] by directly inhibiting the NS3/4A serine protease, the NS5B polymerase and NS5A are currently in clinical development. The need to achieve serum drug concentrations able to suppress viral replication is a key factor for a successful antiviral therapy and the prevention of resistance. Thus pharmacokinetics parameters became important issues for drugs used in the therapy of hepatitis C. The ratio of Cmin/IC50 (inhibitory quotient or IQ) can provide a surrogate measure of a drug's ability to suppress HCV replication, by taking into account the relationship between plasma drug levels and viral susceptibility to the drug. Ritonavir boosting may be a useful strategy to improve pharmacokinetic parameters. Characterising resistance to STAT-Cs in clinical trials is essential for the management of a drug regimen to reduce the development of resistance and thereby maximise SVR rate. The lesson of HIV therapy, provide a compelling case for the exploration of combinations of direct-acting antiviral agents.
Keywords: STAT-C, Pharmacokinetics, Ritonavir boosting, Resistance

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1. Introduction

Hepatitis C virus (HCV) chronically infects 180 million people worldwide with an estimated incidence of new cases of 3–4 million each year [1], [2]. In the developed world, HCV accounts for 50–76% of all primary liver cancer cases and 30% of all liver transplants, 1 and has been estimated to result in a reduction in overall life expectancy in infected individuals of between 8 and 12 years [3]. The current recommended treatment, consisting of a peginterferon plus ribavirin (Peg-IFN/RBV) combination achieves response rates ranging from 76% to 80% in patients infected with HCV genotypes 2 and 3 and from 40% to 50% in those with genotype 1 [3]. Although this kind of therapy will remain the Standard of Care (SOC) for the next years, more effective therapeutic options with shorter treatment durations are needed to increase the response rate in difficult to treat patients (mainly genotype 1) and reduce the impact of HCV infection and its associated complications.

So far, agents that specifically target the replication cycle of the virus [specifically targeted antiviral therapies for hepatitis C (STAT-Cs)] by directly inhibiting the NS3/4A serine protease (which processes the HCV polyprotein to generate mature viral proteins), the NS5B polymerase (which replicates the viral RNA genome) and NS5A (which functions as a part of the replicase complex) are currently in clinical development [4].

The aim of this paper is to discuss the implications of viral kinetics, pharmacokinetics and resistance in the management of STAT-C therapy.

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2. Viral kinetics

2.1. Mechanisms of HCV replication

HCV is the only member of the hepacivirus genus of Flaviviridae. As with other Flaviviridae, the 9.4kb (+) sense RNA genome is translated into a single long polyprotein that is cleaved by both host 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 replicative intermediate RNA. As there is no DNA intermediate (i.e. no reverse transcriptase activity), HCV is not known to be capable of a latent phase [5].

The successful development of HCV replicons, autonomous RNAs, the replication of which is directed by the viral replication machinery (nonstructural proteins), has been a major advance not only for the elucidation of HCV RNA replication but also for the screening of candidate antiviral compounds that inhibit replication [6]

(see Fig. 1).

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Fig. 1. HCV replication cycle and site of action of STAT-C.

2.2. Hepatitis C virus mutations, replicative fitness

Each HCV-infected patient carries a heterogeneous population of HCV, including preexisting variants with decreased sensitivity to direct-acting antiviral drugs. The emergence of clinically relevant resistant variants depends on several factors such as the selective pressure applied by the drug, the genetic barrier to resistance, and the replication fitness of the resistant variants.
As for human immunodeficiency virus (HIV) and hepatitis B virus (HBV), HCV- resistant variants are usually not as fit as wild type viruses, particularly if drugs to which they are resistant bind directly to the active site of a viral enzyme. However, a resistant variant can improve fitness by the accumulation of additional compensatory mutations.

The fitness of these HCV variants is typically estimated in vitro by measuring replication capacity (by transient replication in the replicon system) and enzymatic fitness (by measuring catalytic efficiency) [7], [8]. The resistant variants have varying degrees of decreased replication capacity. The NS3 A156T mutation, which confers resistance to many Protease Inhibitors (PIs), has significantly reduced NS3/4A catalytic efficiency and replication capacity [9], [10]. The NS5B nucleoside inhibitor-resistant mutation, S282T, also has decreased replication capacity [8]. The non-nucleoside inhibitor-resistant mutation P495A/L has decreased replication capacity, but fitness can be restored by compensatory mutations elsewhere in NS5B.
Recently, the in vivo fitness of viral variants with decreased sensitivity to the HCV PI telaprevir (TVR) has been estimated using a novel method that assessed growth rate in the absence of TVR selective pressure. The replicative fitness of different viral variants was inversely correlated with their degree of resistance to TVR [11]. Fitness of resistant variants is not only important in determining the probability of emerging resistance but also to predict if they will revert to wild type in the absence of drug selective pressure. Resistant variants with significantly impaired fitness will be replaced by wild-type viruses more rapidly in the absence of drug selective pressure [11].

2.3. STAT-C in clinical development

Inhibitors of the HCV NS3/4a serine protease and the NS5b RNA-dependent RNA polymerase (RdRp) and have progressed to the more advanced stages of clinical development [12]. A common theme in the development of these agents is that combination therapy with pegIFNa and RBV will continue to be important to increase anti-viral efficacy and limit the selection of drug resistant mutants [13].

2.4. NS3/4a protease inhibitors

The HCV NS3 protein is a multifunctional protein that consists of an amino-terminal serine protease and a carboxy-terminal helicase/nucleoside triphosphatase domain [14] and is necessary for post-translational processing of the NS3–NS5 region of the HCV polyprotein to generate components of the viral RNA replication complex [14]. NS4a acts as a cofactor to facilitate the serine protease function. The helicase is thought to have a role in viral replication by unwinding the viral RNA [14]. The NS3/4a protease has been shown to be a key regulator of intracellular type I IFN pathways. Inhibitors of the NS3/4a protease therefore act to inhibit directly viral replication. We briefly reviewed the clinical characteristics of STAT-C agents, currently in phase of clinical development.

2.5. NS5B polymerase inhibitors

The HCV NS5B RNA-dependent RNA polymerase is a key enzyme involved in HCV replication, catalyzing the synthesis of the complementary minus-strand RNA and subsequent genomic plus-strand RNA from the minus-strand template. Both nucleos(t)ide and non-nucleos(t)ide polymerase inhibitors (NI/NNI) are currently in development. In addition, the replicative activity of the RdRp has recently been reported to be augmented by direct binding to cyclophilin B, a host cell isomerase [15]. A cyclophilin B inhibitor has also progressed to a phase 2 clinical development programme.

To describe the characteristics and the results obtained in clinical trial of each molecules is beyond the goal of this article which is focused on specific issues of the management.

A list of the drugs so far tested in clinical trials and the side effects of them are summarized in Table 1, Table 2.

Table 1,

Site 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 I
................................................................................VX-222.......Phase II
...............................................................................ABT-072.....Phase I
.............................................................................MK-3281......Phase I
...............................................................................PSI-7851.....Phase I
................................................................................ABT-333....Phase I
..................................................................................IDX184.....Phase II
................................................................................ANA598......Phase II
.................................................................................GS9190......Phase II
...................................................................................VX-759.....Phase II
...............................................................................PSI-7977......Phase IIa
...............................................NS5A inhibitor.........PPI-461.......Phase I
....................................................................................A-832......Phase II
.......................................................................BMS-790052......Phase II
................................................HCV polymerase..........VX-916.....Phase I
....................................HCV polymerase inhibitor......Filibuvir....Phase I
...............................................Cyclophilin inhibitor.....SCY-635....Phase I
.................................................................................Debio 025.....Phase II
........................................................NS4B inhibitor....Clemizole.....Phase I
......Post-translation processing...Protease inhibitor..RG7227....Phase II
.................................................................VX950 (telaprevir).......Phase III
.................................................................................IDX320.......Phase I
..............................................................................ACH-1625.......Phase I
...................................................................................VX-813.......Phase I
................................................................................PHX1766.......Phase I
..................................................................................GS-9256......Phase II
..............................................................................BI 201335.......Phase II
.........................................................SCH900518 (narlaprevir).......Phase II
.............................................................................TMC435......Phase IIa
................................................SCH 503034 (boceprevir).....Phase III
........................HCV protease inhibitor..............VX-500......Phase I
...................................................MK-7009 (vaniprevir)........Phase II


Table 2
Main side effects of STAT-C tested in clinical trials.

Class.................Drug..............Side effects
........................NS3/4a protease inhibitors......Boceprevir
Headache, rigor, myalgia and fever fatigue, anemia, nausea; adverse events were also the most commonly for PEG-IFNα2b monotherapy [46]
..............................................................................Telaprevir...Influenza-like illness, fatigue, headache, nausea, anemia, depression, and pruritus constipation, abdominal pain, gastroesophageal reflux disease, nausea, vomiting, headache, acute otitis media, and seasonal allergy. Decreases in hemoglobin, total white blood cell count, neutrophils, and platelets occurred during the study drug dosing period [13]
................................NS5B NIs......R1626....Vomiting and diarrhoea, moderate leokopenia; cytopenia, haematological toxicity, neutropenia, anemia, rash [13]
.......................................................R7128 4...............Haematological, toxicity, Headache, fatigue and chills [13]
.......................................................R7128 4.............Mild-moderate diarrhoea [13]

3. Pharmacokinetics
The need to achieve serum drug concentrations able to suppress viral replication is a key factor for a successful antiviral therapy and the prevention of resistance. Thus pharmacokinetics parameters became important issues for drugs used in the therapy of hepatitis C.
Although the HCV protease inhibitors may have a major clinical impact as a drug class, they have a relatively narrow therapeutic index because they require highly suppressive drug concentrations to prevent the emergence of resistance. The concept of protease inhibitor (PI) boosting was developed to overcome these issues [16].
When considering the pharmacokinetics of antivirals several parameters must be taken into account.

After multiple doses of a drug are administered over several days of treatment, the maximum and minimum concentrations of drug achieved after each dose reach constant levels.

This is referred to as the “steady state.” The key measures of pharmacokinetics that impact in clinical practice are the following:

Cmax=the peak or highest plasma concentration achieved during a dosing interval.
Tmax=the time taken to reach the highest observed plasma concentration.
Cmin=the lowest observed plasma concentration achieved during a dosing interval. Cmin is also called the ‘trough concentration’, and generally occurs at the end of the dosing interval

(see Fig. 2).

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

The activity of PIs is dependent on the maintenance of circulating concentrations that suppress viral maturation. Indeed, if treatment regimens produce drug trough concentrations allowing persistent low-level viral replication, at the same time they permit the accumulation of mutations required for significant resistance. Thus, for PIs the Cmin is likely to correlate most closely with antiviral 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 concentration of drug required to inhibit viral replication by 50%/90%.
IC50 and IC90 vary depending upon a number of factors, including the viral strain, cell type and assay used, and on adjustments made for plasma protein binding. Drug-resistant strains tend to have higher IC50 values compared to wild type.

The ratio of Cmin/IC50 (inhibitory quotient or IQ) can provide a surrogate measure of a drug's ability to suppress HCV replication, by taking into account the relationship between plasma drug levels and viral susceptibility to the drug. Higher values would provide a degree of pharmacologic “forgiveness”, a characteristic which minimizes the impact of less than perfect adherence, an heterogeneous viral population or variable drug absorption [17]

(see Fig. 3).

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Fig. 3.

For antiviral therapy to be successful, drug levels need to be always well above the IC50, to avoid “blips” of viral replication and the selection of resistance.

4. Protease inhibitor boosting
The goal of PI boosting is to increase the exposure to these agents, ensuring sustained and effective concentrations throughout the dosing interval. PI boosting is best achieved by administering ritonavir (usually low dose 100mg once or twice daily) along with the PI.
Ritonavir may increase exposure of a concomitantly administered PI by exerting the following effects:

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

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

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

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Thus, inhibition of P-glycoprotein and CYP3A4 by ritonavir affects the following PK parameters of coadministered PIs, giving at the same time some clinical advantages, as shown in Table 3.

Table 3.

Key pharmacokinetics (PK) parameters and clinical significance of antiviral drugs.
PK parameters..............................................Clinical advantages
Decreased systemic clearance(by ritonavir PK boosting)......Lower likelihood of resistance
Increased trough (Cmin)..................................Improved antiviral activity
Decreased peak (Cmax)...................................Reduced drug toxicity
Reduced pharmacokinetic variability............Amelioration of food restrictions
Increased AUC..................................................Improves adherence

4.1. PK of clinically tested STAT-C and ritonavir boosting

Telaprevir has a half-life of 0.8–3.2h [20], [21]. Its area under the curve (AUC) ratio for liver/plasma concentration is 2.3–35 [20], [21]. 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 24–48h of the start of dosing. The concentration of telaprevir was higher than the concentration that exerts antiviral activity on the replicon system for as long as 12h [21].

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

The concentration of telaprevir 8h after dosing was increased by>50 fold. Based on these results, twice-daily dosing of telaprevir/ritonavir at 250/100mg is predicted to provide a mean plasma concentration equivalent to that achieved with telaprevir 750mg every 8h [22].
Pharmacokinetics parameters for boceprevir were evaluated on day 1 of monotherapy and on days 1 and 8 of combination therapy with PEG-IFN α-2b. The AUC on day 1 of monotherapy and combination therapy of both dose groups are similar, which suggests that there was no interaction between boceprevir and PEG-IFN.

The combination therapy with boceprevir at either dose level and PEG-IFN α-2b resulted in greater decreases in HCV RNA than PEG-IFN α-2b alone. Furthermore, the degree of virological response was related to the dose of boceprevir in monotherapy and combination therapy regimens (Sarrazin et al.). A maximum mean change in HCV RNA of was observed for PEG-IFN α-2b plus boceprevir 400mg 3 times daily (overall mean maximum decline, −2.88/−0.22) [23].

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5. Development of resistance to protease inhibitors
HCV replication is characterized by a short virion half-life and a high production and clearance rate of free virions [24]. The estimated average half-life of the hepatitis C virus is 2.7h, with pretreatment production and clearance of 1012 virions per day. The HCV/NS5B RNA-dependent RNA polymerase has a low fidelity and no proofreading function.
From treatment of HIV and HBV with specific inhibitors, it is known that rapid selection of preexisting drug-resistant variants occurs during treatment. During selection pressure, the relative replication fitness of a selected drug-resistant variant determines whether the variant will grow out. Without selection pressure, a drug resistant variant may be generated but will simply vanish if its relative replication fitness is lower than that of non-drug-resistant variants.
The speed of selecting drug resistance depends mainly on the turnover of the viral nucleic acid; the HCV RNA strands present in infected hepatocytes serve as templates for producing new HCV virions that are released soon after [24]. As a result, the viral genetic half-life is shorter for HCV than for HIV and HBV and the time required for selecting drug-resistant mutants and for their expansion to become the major part of the viral population is shorter for HCV than for HIV and HBV. Thus, resistance to HCV antivirals is more likely than resistance to antiretrovirals [24], [25].

The rapid selection of viral variants displaying drug-resistant phenotypes has been observed in patients experiencing viral rebound during treatment as well as in replicon experiments.

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Variants resistant to STAT-C reported to date are summarized in Table 4

**Please Click To See Table 4.

Two recent reports suggested that resistant variants may already be present at a 1% frequency in the quasispecies population in treatment naıve patients [26], [27], consistent with their dominant emergence only days after treatment initiation [28], [11]. However, drug treatment in the setting of resistance mutations may still be beneficial, because a decreased in vitro replicative capacity has been demonstrated for many viral strains resistant to protease or RdRp inhibitors [29], [30], [7], [31], [10], [32], [33].

Patients may therefore benefit from elimination of the dominant, drug susceptible viral quasispecies with more than 1000-fold reductions in their viral load, as long as only drug-resistant but replication-deficient quasispecies constitute the residual viral population [28], [11]. Although in some cases replication levels may later be restored by compensatory mutations, it seems possible that this effect could suffice to achieve treatment success if several drugs were combined to suppress viral replication before compensatory mutations or additional resistance mutations could evolve.

The presence of pre-existing mutations in treatment naïve patients may be a factor affecting the response to therapy. Indeed, the PI-resistant mutation R155K was recently detected as the dominant quasispecies in a treatment-naıve patient [34].

In a study by Kuntzen et al. dominant mutations were mostly observed as sporadic, unrelated cases at frequencies between 0.3% and 2.8% in the population. Taken together, however, 8.6% of patients infected with genotype 1a and 1.4% of patients infected with genotype 1b exhibited at least one drug resistance mutation, including two cases with possible multidrug resistance. Viral loads were high in the majority of patients carrying these mutations, suggesting that resistant viruses might achieve replicative capacities comparable to nonresistant strains in vivo [35].

Treatment success as measured by decreasing viral loads represents a balance between the achievable drug concentration in the plasma, the frequency and degree of drug resistance among the viral quasispecies [36], and the replication efficiency of drug-resistant viral strains. Numerous studies have investigated the impact of amino acid substitutions on viral replication and drug susceptibility to investigational NS5B polymerase or NS3/4A protease inhibitors in the replicon model. A pronounced reduction in the replicative capacity was described for the highly resistant A156 variants in NS3 and M423 variants in NS5B, and also to a lesser extent for the low-level PI-resistant R155, T54, and V36 mutations [11], [29], [33], [35]. It has been speculated that these lower replication levels could facilitate eradication through combination treatment [28], because STAT-C resistant viral strains appear to remain sensitive to interferon and ribavirin [33]. In a recent clinical trial, the in vivo relevance of these findings was supported by the observation that weakly telaprevir-resistant variants rose predominantly in patients who only achieved lower plasma drug levels, whereas increasing drug concentrations selected for mutants that were also highly resistant in vitro [11].

Preliminary results indicate that dominant resistance mutations can potentially reduce the early treatment response to STAT-C drugs [36], and it must be assumed that low-level resistant strains will sustain viral replication in patients who do not achieve optimal drug levels with standard dosing, in cases with poor adherence, or when dose reductions are inevitable due to adverse events. Importantly, continued viral replication in the presence of the selecting drug would put the patient at risk of developing additional resistance mutations. Here, observations from HIV infection suggest that baseline resistance even against only one drug in a multidrug antiviral regimen may affect treatment success [11], and further indicate that apart from single mutations conferring high level resistance, stepwise accumulation of subtle but synergistically acting resistance mutations may also eventually lead to treatment failure [37]. Data from a recent study using telaprevir has indicated a similar pathway in HCV infections, where combination of the low-level resistance mutations V36M and R155K resulted in a highly drug resistant phenotype, the appearance of which coincided with viral breakthrough [11], [28].

In HIV infection, resistance testing was calculated to be cost-effective when the prevalence of drug resistance becomes 1% at baseline and is currently recommended in areas with more than 5% prevalence of resistant strains, and in all cases of treatment failure [38]. For novel STAT-C drugs, important factors such as their cost and treatment response rates are not yet available to derive similar calculations, but further studies are needed to address these issues.

6. Tools for monitoring viral resistance
Characterizing resistance to STAT-C in clinical trials is essential for the management of a drug regimen to reduce the development of resistance and thereby maximize SVR rate. So far, methods to characterize viral resistance works in a complementary way and are distinct in genotypic and phenotypic assays [2].

6.1. Genotypic assays
Genotypic assays examine the genetic sequence of a target region involved directly or indirectly in the interaction of a drug with its target [11], [28], [39]. Initial characterization of the resistance profile for a drug requires comparing viral sequences before, during 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 they cannot determine linkage between different mutations in a single variant, or detect variants with mutations that are present in less than 25% of the population.
More sensitive methods such as clonal sequencing or the TaqMan™ mismatch amplification mutation assay (TaqMAMA) [40], may be more costly and time consuming.

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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 in vitro using a transient replicon system [7], or by comparing colony formation efficiency of the mutant replicon RNA with that of WT variants in co-culture growth competition assays.
Fitness has also been determined in vivo [11] by using HCV RNA levels and clonal sequencing to calculate the frequency of a given variant over time after the end of dosing to assess the growth rate compared with WT in the absence of drug-selective pressure.

6.3. Phenotypic assays
In clinical research, viral variants identified by genotypic testing should be tested with a phenotypic assay, both to confirm that the mutation confers resistance to the drug and to assess the degree of resistance [41], [42], [43]. Phenotypic assays measure the IC50 in an enzyme or replicon assay. By testing the HCV variants for drug susceptibility in vitro, the fold change in sensitivity can be calculated as 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 protease inhibitors is the finding that genetic barrier and resistance profiles substantially differ between the different genotype 1 subtypes. The reason is that only one nucleotide substitution is needed to generate a subtype 1a sequence variant, whereas two substitutions are needed to the 1b sequence. In vivo, different resistance profiles in patients infected by HCV subtypes 1a and 1b have been demonstrated. In the former, the V36 and R155 substitutions represent the backbone of resistance, whereas in the latter resistance is less frequent as it is preferentially associated with substitutions at position A156 that are associated with a decreased fitness of the variants [10], [33], [41].
A correct identification of HCV subtypes 1a and 1b is hence crucial in clinical trials designated for new HCV drugs to avoid misinterpretation of efficacy and resistance data. It may also become important in future clinical practice, when therapy schedule needs to be tailored in genotype 1 patients according to genotype subtype. To identify the HCV genotype and subtype both in clinical trials and practice, commercial assays have been developed, most of them targeting the 59 noncoding region (59NCR) of the HCV genome [43].

.

8. The need for combination therapy
As monotherapy, Direct Antiviral Agents (DAA) of several classes have been shown to induce significant viral suppression, with the addition of PEG-IFN/RBV or even PEG-IFN alone conferring additional viral suppression and/or markedly inhibiting the development of resistance to the direct-acting antiviral agent [28]. In the PROVE1 and PROVE2 studies of genotype 1 treatment-naive patients treated with the PI telaprevir, viral breakthrough was an infrequent event, especially in patients who experienced RVR [44], [45]. Similarly, in the SPRINT-1 boceprevir trial in treatment-naive patients, only a small number of patients discontinued for viral breakthrough [46]. These observations, together with in vitro experiments demonstrating the capacity of two or three drug combinations to induce additive or synergistic viral suppression [47], as well as the history of HIV therapy, provide a compelling case for the exploration of combinations of direct-acting antiviral agents. The ultimate goal of this approach is to build regimens able to overcome the development of resistance. As suggested in a interesting review by Pereira & Jacobson, the height of the “genetic barrier”, such as the relative likelihood to acquire conferring resistance mutations, varies among the different STAT-C drugs. Nucleoside analogues seem to have the highest barrier when compared to protease and non-nucleosides inhibitors. The combination of drug with different resistance profiles may become an interesting strategy to avoid the development of overlapping resistances [48]. So far, combinations have been studied with IFN as a cornerstone, but ultimately the development of IFN-free regimens is a major goal. Very recently, the INFORM-1 study, an intriguing dose-ranging, exploratory study of R7227, a PI, combined with R7128, a nucleoside polymerase inhibitor, for up to 2 weeks, demonstrated marked viral suppression [49]. No viral breakthroughs were reported. Data from the RBV-free arm of PROVE2 [47], the RBV-free arm of PROVE3, a study on telaprevir in earlier nonresponders [44] and the low dose RBV arm of SPRINT-1 [46] have provided a compelling case for the role of RBV in preventing the emergence of resistant variants. Such data provide a foundation for the early exploration of IFN-free regimens consisting of two DAA agents plus RBV. Given the universal interest in studying combinations of DAA agents, the timeline for the development of such combinations is a critical issue.

9. Summary and conclusion
The success of STAT-C agents will depend on their ability to inhibit the replication of a broad range of viral quasispecies and prevent emergence of drug-resistant mutants. Present and forthcoming drugs should have a pharmacokinetic profile which could warrant plasma levels able to inhibit the viral replication along the inter-dose period. Moreover, treatment regimens based on the combination of drugs with different resistance profile may be the best strategy for improving the response rate in difficult to treat patients in the years to come.

Conflict of interest statement


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

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Department of Infectious Diseases, Foundation IRCCS San Matteo Hospital - University of Pavia, Pavia, Italy
Corresponding author at: Department of Infectious Diseases, Hepatology Outpatients Unit, University of Pavia, Fondazione IRCCS Policlinico San Matteo, Via Taramelli, 5, 27100 Pavia, Italy. Tel.: +39 0382 501080; fax: +39 0382 501080.
PII: S1590-8658(10)00314-2
doi:10.1016/j.dld.2010.09.007
© 2010 Published by Elsevier Inc.

Hepatitis C Treatment: current and future perspectives

Hepatitis C Treatment: current and future perspectives

Saira Munir¤, Sana Saleem¤, Muhammad Idrees*, Aaliyah Tariq, Sadia Butt, Bisma Rauff, Abrar Hussain, Sadaf Badar, Mahrukh Naudhani, Zareen Fatima, Muhmmad Ali, Liaqat Ali, Madiha Akram, Mahwish Aftab, Bushra Khubaib and Zunaira Awan


Received: 22 September 2010
Accepted: 1 November 2010
Published: 1 November 2010


Abstract
Hepatitis C virus (HCV) is a member of Flaviviridae family and one of the major causes of liver disease. There are about 175 million HCV infected patients worldwide that constitute 3% of world's population. The main route of HCV transmission is parental however 90% intravenous drug users are at highest risk. Standard interferon and ribavirin remained a gold standard of chronic HCV treatment having 38-43% sustained virological response rates.

Currently the standard therapy for HCV is pegylated interferon (PEG-INF) with ribavirin. This therapy achieves 50% sustained virological response (SVR) for genotype 1 and 80% for genotype 2 & 3. As pegylated interferon is expensive, standard interferon is still the main therapy for HCV treatment in under developed countries. On the other hand, studies showed that pegylated IFN and RBV therapy has severe side effects like hematological complications. Herbal medicines (laccase, proanthocyandin, Rhodiola kirilowii) are also being in use as a natural and alternative way for treatment of HCV but there is not a single significant report documented yet. Best SVR indicators are genotype 3 and 2, < 0.2 million IU/mL pretreatment viral load, rapid virological response (RVR) rate and age <40 years. New therapeutic approaches are under study like interferon related systems, modified forms of ribavirin, internal ribosome entry site (HCV IRES) inhibitors, NS3 and NS5a inhibitors, novel immunomodulators and specifically targeted anti-viral therapy for hepatitis C compounds. More remedial therapies include caspase inhibitors, anti-fibrotic agents, antibody treatment and vaccines. Background Hepatitis C virus (HCV) is a meticulous factor of liver disease and one of the most important health issues worldwide [1,2] . Hepatitis C has approximately 175 million Global Disease Burden which represent almost 3% of the whole population in the world, each year 3 to 4 million new patients with HCV are diagnosed. HCV remains endemic in many countries of the world [3-5] .

Statistics based on general healthy population revealed that HCV has 5.3% seroprevalence in Pakistan, 2.2% in Turkey and 7.7% in Zimbabwe [6-8] . Hepatitis C virus infection is not a main factor of mortality in the first decade of infection [9] . Even though, the biological aspects of HCV are revealed to a great extent in recent years, an absolute therapy of hepatitis C remains problematic in a large majority of patients [10] and about 50% HCV patients does not attain sustained virological Responses [11-13] .

A few years back, it was not easy to study HCV in invitro because there was no proficient system present but fortunately Heller et al got success in establishing in vitro model of HCV virions. This system proves good for high level production and secretion of HCV virions hence this system expands the scope of tools present for HCV study [14,15] .

Many patients remain asymptomatic for years and are only detected on health screening or at the time of blood transfer [16] . Peg. INF and ribavirin therapy is still the therapy of choice for
HCV patients besides having many side affects [17,12] .

As HCV is mainly a chronic disease and progress very slowly therefore persistent infection is a typical characteristic of disease which can be found in approximately 75% patient at primarily stage. Prospective studies conducted on natural history suggest that HCV take almost 20 years to develop cirrhosis and only 20% of cirrhotic patient can develop Hepatocellular Carcinoma (HCC) after 40 years of preliminary infection [18,10] .

HCV genotypes and treatment response Patients with different HCV genotypes react in a different way to alpha interferon because genotype is one of the strongest prognostic aspects of sustained virological response [19,20] . This clinical importance of HCV genotype was revealed by clinical studies based on interferon treatment response account [5] . Patients show more sustained virological response when suffered from HCV genotype 2 and 3 as compared to HCV infected persons of genotype1 [6] .

Patients infected with HCV genotype 2 and 3 show 65% SVR and patients with HCV genotype 1 show 30% Sustained Virological Response (SVR) [7,8] . Thus genotype of patients must not be over looked when giving standard interferon therapy. Different ethnic groups respond differently to standard therapy of HCV and hence there is variation in Early Treatment Response (ETR) and SVR rates [21] .

Mechanism of Pathogenesis and interferon resistance Now a number of mechanisms associated with escape of the pathogen from the host's immune response, hepatocyte damage and molecular oncogenesis of hepatocellular carcinoma have been elucidated. Inefficient clearance of virus from patient's body is basically due to the hyper-variability of virus envelope protein that enables HCV to neutralize antibody [22,23] .

Once the virus enters the hepatocytes through receptor mediated endocytosis and starts replication, it initiate damaging of hepatocyte, the major component of which is through the host's own immune response [24,23] . Interferon is the most potent natural weapon of the host against intra-cellular viral infection. HCV, however, owing to intricate actions of its genomic proteins is equipped with ability to evade the natural interferon-mediated clearance. HCV core protein has been reported to decrease the robustness of the host's immune response by decreasing transcription of interferon induced antiviral genes [25,23] . HCV NS3/4A protease also has been concerned in inhibiting the interferon amplification loop which otherwise results in suppression of HCV replication. Inhibition of HCV protease can reverse the effects of HCV infection that make protease inhibitors one of the most noteworthy potential therapeutic agents for HCV [26,25] .

Route of transmission and treatment response At first, it was believed that most frequent route of transmission of HCV was blood transfusion and intravenous drug abuse. But recent epidemiological studies suggest further routes of transmission [27] . The main route of HCV transmission is parental. However 90% intravenous drug users are at highest risk of getting HCV infection such as those who require multiple blood transfusions and blood products (hemophiliacs) or those who go through major surgery [28,29] . Unlike HBV, HCV infection transfer less frequently by sexual or intimate contact (0.4 to 3%).

Domestic contacts are also at low risk [30] . Almost 5% HCV infections are caused by needle stick injury [29,30] . 3% to 5% infants acquire HCV from infected mother by perinatal transmission [31] . HCV is present in saliva and milk but transfer of HCV infection through breast milk has not been reported [32,33] . Community barbershops also play a key role in HCV transmission in under development countries [27] .

Some other reported risk factors of disease transmission are dental and surgical treatments, circumcision, ear piercing, tattooing and dialysis [34-36] . In a study conducted on 3351 patients of HCV in Pakistan it has been documented that more than 70% hepatitis C infections are spread in hospitals by the use of same needle several times and major or minor operations that are extremely frequent in Pakistan. Globally reuse of needles is also common source of transmission [37] .

Studies show that RVR and SVR are independent of transmission routes of HCV. Base line diagnosis Detection of anti HCV by ELISA is the initial step in diagnosis of HCV infection and it is more than 99% sensitive and specific [38] . PCR is the second main step in the analysis of chronic HCV infection and exposure of virus is usually detectable within 7 to 21 days [39,40] . Liver biopsy is also an important parameter in diagnosis of chronic HCV infection but as persons infected with genotype 2/3 respond well to standard therapy, treatment can be started without liver biopsy [40] .

Therapy for HCV infection Chronic HCV is treated with a glycoprotein commonly known as interferon (INF) alpha and it is considered the backbone of therapy because it efficiently increases the immune response against virus [41] . Afterward interferon plus ribavirin become a gold standard (3 MIU thrice weekly along with ribavirin 800 to1200 mg per day). This treatment enhances SVR rate up to 38-43%. As SVR greatly depend on HCV genotype so genotype 1 needs treatment for 48 weeks to achieve SVR of 29% and genotype 2 and 3 needs treatment up to 24 weeks to attain SVR rate of 66% [42] . Currently the regular treatment of HCV is pegelated interferon (PEG-INF) in combination with ribavirin.

This therapy achieves SVR of about 50% for genotype 1 and 80% for genotype 2 & 3 [43] . There are two types of pegylated interferon; PEG-IFN-alpha-2a and PEG-IFN-alpha-2b. These are dissimilar only by size and configuration of the polyethylene glycol molecules that has binding sites for interferon. The functioning of these two formulated interferon not compared still but both are equally good for HCV treatment [44] .

Current HCV therapy for genotypes 2a to 2b, 3a to 3d, 5a, 6a and mixed genotypes infected patients is 3 subcutaneous injections of 3 MU of recombinant interferon alpha and ribavirin (10 mg per day per kg body weight) in one week for 6 months. Individuals infected from HCV genotype 1a to 1c, 4 and mixture of 1 and 4 HCV genotypes should receive three 3 MU subcutaneous injections of recombinant IFN alpha and ribavirin that are given orally (for individuals with ≤ 75 kg body weight) require 1,000 mg per day, for patients with > 75 kg body mass require 1,200 mg per day) in a week for total 48 weeks [45] .

Conventional interferon (C-INF) therapy is used for HCV treatment in poor countries because of financial reasons and Pakistan Society of Gastroenterology and GI Endoscopy also recommend the use of C-INF therapy for HCV genotype 3 in Pakistan [46,40] . In under developed and developing countries including Pakistan, pegylated interferon therapy is beyond the reach of common poor patients [47,40] . In 2001, FDA permitted two kinds of PEG-INF (i) PEG-INF Alpha 2a (40 KD) and (ii) PEG-INF Alpha 2b (12 KD). These are administered only once a week because they have long half life of plasma (almost 10 times) in comparison with conventional INF. Liver primarily metabolizes PEG-INF Alpha 2a and kidney excretes out PEG-INF Alpha 2b. Recent studies and clinical trials confirmed that SVR rates could be increased by the using mono therapy with PEG-INF 2a or PEG-INF 2b in comparison with conventional interferon [48,40] .

Limitations of Recent HCV Therapy
It has been reported that 40% to 50% patients with HCV genotypes 1 and or 4 early attain SVR in comparison with 80% patients infected with genotypes 2 and or 3 [4,49] . However PEG-IFN and ribavirin treatment has severe side effects. Major complications of standard interferon and ribavirin therapy are anemia, cytopenias, neutropenia and thrombocytopenia as elucidated in table 1.

Table 1. Contraindications situations for pegylated interferon and ribavirin therapy

Novel types of interferon alpha (albinterferon) are under study; these might be very suitable anti-viral therapy because these can be given just once or twice a month as compared to standard PEG-IFN therapy [4,49] . Taribavirin, a recently introduced drug, is tested in various randomized trials that show low efficacy but also has a few complains of anemia and the side effects are easily manageable [50,4] . There are also several side affects associated with conventional interferon and ribavirin therapy including Influenza like sign and symptoms. For example headache, myalgias or arthralgias, fever, anorexia, nausea or vomiting, fatigue, abdominal pains, insomnia, suicide attempt, pruritis, anaemia, redness at injection site, dry skin, leucopoenia, irritability, thrombocytopoenia, anxiety, psychosis and laryngitis [51] .

Herbal treatment
There is no effective vaccine developed or excellent drug available for the treatment of HCV. Standard INF therapy in combination with ribavirin show sustained virological response with efficacy of not more than 50%, therefore most of the patients try herbal medicine and conventional medicine all over the world particularly in poor countries. Laccase are largely used as herbal medicine that is extracted from oyster mushroom (Pleurotus ostreatus). Studies showed that laccase is proficient in inhibiting the HCV replication rate [52] however the mechanism of action of this medicine is not known.

Herbal treatment can open a natural and alternative way for treatment of HCV. As Hepatitis C virus infects liver and this infection requires two or more decades to extend into substantial disease, a nutritional supplement might facilitate to decrease or stop disease development. More recent studies regarding herbal treatment provoke a hope for HCV patient that is based on a chemical known as proanthocyandin, extracted from blueberry leaves. It has been reported that proanthocyandin can stop HCV replication in infected patients [53] . According to another study rhizomes of the Chinese medicinal herb Rhodiola kirilowii may also act as possible inhibitor of HCV [54] .

Factors affecting treatment response
Treatment response is better in patient of less than 40 years of age in comparison with elderly. Young females respond well to the treatment. High intensity of viremia is related with deprived response. Immunodeficiency, excessive use of alcohol and co-infection with HIV or HBV, all harmfully cause the result to HCV infection [55,16] .

HCV therapy is not suitable for people suffering from severe HCV related cirrhosis, undergone organ transplant, children of < 3 years and specific contraindication to the medication. Interferon causes severe side effect includes, anxiety, irritability personality changes, even suicide, depression or acute psychosis. Ribavirin side effect included anemia, renal dysfunction of coronary artery. Fetal abnormality and fatality are important side effects of ribavirin, a well-known teratogen.

Due to the distinctive character of the virus to develop vaccine against HCV leftovers, a disappointment has been seen due to its high mutation rate. It has already been reported that the rate of HCV reproduction is high and the error-prone polymerase causes mutation continuously. The high HCV replication rate provides sufficient chance of mutation that occurs in the viral population inside an infected person. Production of virus has been estimated at 1012 (one trillion) new HCV virions per day [56] . Studies on chronically infected HCV patients show that rate of mutation in HCV genome has been approximately 0.001 substitutions per genomic site in one year. Such high rate of mutation could result into 8-18 mutations within the RNA of 9.6 kb genomic size. It has also been reported that envelop protein E2 has highly mutated sites known as hypervariable region HVR1. High variation in E2 causes immune escape mutants of the virus as of the neutralizing antibodies and therefore describes the constant viremia. In addition to E2 gene, P7 region has also been shown with increased variability [16] .

Future perspectives
New therapeutic approaches are under study like interferon related systems, modified forms of ribavirin, siRNA, internal ribosome entry site (IRES) inhibitors, NS3 and NS5a inhibitors and novel immunomodulators. These are particularly for those patients who show low SVR rate by traditional therapies. More remedial therapies include antifibrotic agents, caspase inhibitors and antibody treatment and vaccines. Particularly targeted antiviral compounds like specifically targeted anti-viral therapy for hepatitis C' (STAT-C) compounds are now under study by scientists that are used along with standard interferon therapy. Reports confirm improved SVR rate at least in HCV genotype 1 patients. Further studies are required to confirm its significance in the clearance of HCV RNA if used as a single therapy without interferon and ribavirin [57,58] .

Conclusion
Currently chronic HCV treatment consists of pegelated interferon alpha and a nucleoside analogue ribavirin for 3 to 18 months. However several side effects are associated with this treatment. New therapeutic approaches are under study and recent clinical trials are being focused on inhibitors of HCV NS3 and NS5a RNA polymerase. Parameters that increase SVR rate for HCV are genotype 2 and 3, age < 40 years and low viral load before treatment.

Abbreviations
HCV: hepatitis C virus; PEG-INF: pegylated interferon; RVR: rapid virological response; SVR: sustained virological response; RBV: ribavirin; ETR: end of treatment response; ELISA: enzyme linked immunosorbant assay; PCR: polymerase chain reaction; MIU: million international units; SDINF: standard interferon; HVR: hiper variable region; IRES: internal ribosome entry site; STAT-C: specifically targeted anti-viral therapy for hepatitis C.

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
SM and SS reviewed the literature, and wrote the manuscript. MI edited the manuscript. AT, SB, BR, AH, SB, ZA, MN, ZF, MA, LA, MA, MA, BK, helped SM & SS in literature review. All the authors read and approved the final manuscript.

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¤ Equal contributors Author Affiliations Division of Molecular Virology & Molecular Diagnostics, National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan For all author emails, please log on. Virology Journal 2010, 7:296 doi:10.1186/1743-422X-7-296 The electronic version of this article is the complete one and can be found online at: http://www.virologyj.com/content/7/1/296

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