The way forward in HCV treatment - finding the right path: New HCV Drugs, resist

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The way forward in HCV treatment - finding the right path: New HCV

Drugs, resistance (full report attached)

“Resistance to new HCV antivirals…. …… Innovative agents in

clinical development--- Protease inhibitors.….. inhibitors of

cyclophilin B (for example, NIM-811 and DEBIO-025), a host factor

involved in viral replication…. Polymerase inhibitors…. Clinical

trials of NS3 (protease) and NS5B (polymerase) inhibitors…. Lessons

from HIV combination therapy?.... HCV vaccine development

Nature Reviews Drug Discovery 6, 991-1000 (December 2007) |

doi:10.1038/nrd2411

P. Manns1, Graham R. 2, Jürgen K. Rockstroh3, Stefan

Zeuzem4, Fabien Zoulim5 & Houghton6 About the authors

Infection with the hepatitis C virus (HCV) represents an important

health-care problem worldwide. The prevalence of HCV-related disease

is increasing, and no vaccine is yet available. Since the

identification of HCV as the causative agent of non-A, non-B

hepatitis, treatment has progressed rapidly, but morbidity and

mortality rates are still predicted to rise. Novel, more efficacious

and tolerable therapies are urgently needed, and a greater

understanding of the viral life cycle has led to an increase in the

number of possible targets for antiviral intervention. Here we review

the specific challenges posed by HCV, and recent developments in the

design of vaccines and novel antiviral agents.

“…..the success of future HCV antiviral agents will be influenced

by their resistance profiles; that is, their ability to inhibit viral

variants and prevent the emergence of resistance mutants….

combination therapy using multiple small molecules designed to

inhibit different virus-specific targets and producing diverse

resistance patterns may improve response rates; for example, protease

inhibitors with polymerase inhibitors or nucleoside inhibitors with

non-nucleoside inhibitors96. Development of new combination

strategies and the use of short-term therapy will potentially allow

improved treatment success while minimizing the potential for

developing resistance to any single agent….

Response to therapy is dependent on several factors including

treatment-related factors, host characteristics (including the

ability of host cells to respond to IFN, induce antiviral defences

and clear infected cells), viral-related factors and disease-related

factors76, 78, 79. In addition, the genetic heterogeneity or

quasispecies nature of HCV has important therapeutic implications, as

the generation and selection of resistant variants can allow the

virus to escape the antiviral pressure exerted by treatment….. As

previously discussed, many emerging HCV treatments are targeted

against specific HCV enzymes; among the most promising are the NS3

serine protease inhibitors and the NS5B RNA-dependent RNA polymerase

inhibitors. As the active site for protease inhibitors is a long

shallow groove, a single-point mutation in this enzyme might be

sufficient to hinder the binding of these antivirals, with different

mutations conferring low-level or high-level resistance (Fig. 4). For

example, sequencing studies using samples from patients treated with

telaprevir have identified several mutations that confer low-level

and high-level resistance80. Resistant isolates are selected rapidly

and therefore combination therapy with pegIFN-a2a or other antiviral

agents will be required to limit the development of resistance to

telaprevir. As far as we know, telaprevir-resistant mutants are

sensitive to IFN-a…… The active site of the NS5B RNA-dependent

RNA polymerase is a highly conserved region in all HCV genotypes and

any amino-acid mutations in this region may inhibit the ability of

the virus to replicate (Fig. 5). This suggests that resistance to

nucleoside polymerase inhibitors by mutation in the enzyme may not

readily develop….. In vitro data suggest a low probability of cross-

resistance between some of the different nucleoside polymerase

inhibitors or between nucleoside and non-nucleoside inhibitors…â€

Hepatitis C virus (HCV) infection represents an important global

health-care burden, which is likely to increase over the coming

years. There are approximately 3-4 million new cases of HCV infection

each year, and current estimates suggest that a minimum of 3% of the

world's population (approximately 170 million people) are chronically

infected, and are at risk of developing liver cirrhosis and/or

hepatocellular carcinoma1. Today, in developed countries, most cases

are acquired through the sharing of infected needles whilst injecting

drugs or, to a much lesser extent, via sexual and perinatal

transmission2. However, in a significant number of patients the route

of infection remains unknown. Before the routine screening of blood

for HCV, many patients were infected by blood transfusions or

treatment with infected blood products. At present, most new cases of

HCV infection occur in the developing world3, and it is believed that

immigration will impact on HCV prevalence and subsequent disease

burden in the developed world. In the developed world, infection with

HCV is responsible for 50-76% of all cases of liver cancer and

accounts for two-thirds of all liver transplants4.

Since the discovery of the virus in 1989 (Ref. 5), the development of

HCV therapy has progressed significantly (Fig. 1). With the

introduction of IFN monotherapy, and the current recommendation of

pegIFN-a and ribavirin, the proportion of patients achieving

sustained antiviral response (SVR) has increased significantly6, 7,

8, 9, 10, 11, 12, 13, 14, 15. The mechanism of action of IFN-a and

ribavirin is still incompletely understood. IFN has a direct

antiviral effect and acts on the immune system of the host, and

ribavirin alone does not inhibit HCV replication significantly but

augments the antiviral action of IFN. Importantly, ribavirin prevents

relapse after the end of antiviral treatment. Despite this, the

morbidity and mortality rates associated with HCV are predicted to

rise in the coming years, and more efficacious and tolerable

therapies are urgently required, particularly for the increasing

proportion of patients who are refractory to treatment with IFN- and

ribavirin. Numerous studies have estimated the extent to which the

burden of the disease will increase, but these projections may prove

to be an underestimate. Consequently, HCV-related annual mortality is

set to increase in most Western countries over the next two decades.

In France, for example, the likely future mortality of HCV has been

examined using the back-calculation method; this predicted a rise

from 3,000 in 1998 to 4,500 in 2022 (Ref. 16). This is unlikely to

change unless at least 50% of the HCV-infected population is treated

effectively. For this, HCV carriers have to be readily identified.

Projections in the United States suggest that if half of all patients

infected with HCV are identified, even with the most aggressive

treatment at optimal doses and durations, the best possible outcome

is a 24% reduction in the incidence of decompensated cirrhosis after

20 years17. By 2020, the proportion of all US HCV cases with liver

cirrhosis is estimated to increase from 16% to 32%, and

decompensation will increase by 106% over current levels17 resulting

in an increased need for liver transplantation.

This Review is based on a satellite meeting held at the 12th

International Symposium on Viral Hepatitis and Liver Disease in

Paris, 2006, and is updated to include the current knowledge and

recent developments in the field of HCV therapy at the time of

publication. Given the shortcomings of current HCV treatment, we

examine emerging new therapies for HCV, the impact of viral

resistance, and key lessons from HIV management, in particular the

potential of combination treatment strategies.

Obstacles in current HCV management

Recent studies have highlighted the barriers and challenges that

exist in ensuring patients newly diagnosed with HCV receive

appropriate treatment18, 19. In a US study of patients infected with

HCV in primary care, obstacles to receiving appropriate treatment

included negative views of patients regarding treatment, inadequate

patient follow-up, a tendency for providers not to consider treatment

of past drug abusers, and delays in obtaining specialist input18. An

observational study in the UK found that among all patients diagnosed

with HCV over a 2-year period, only about half of all patients were

appropriately referred for further management and only 10% began

treatment19. Conversely, in France, the Ministry of Health has

implemented a nationwide viral hepatitis prevention and control

programme aimed at increasing both detection of seropositive

individuals and provision of antiviral treatment20. By 2002, it was

estimated that 60% of new HCV patients had been diagnosed through

improved HCV screening programmes, and the number of patient

referrals to hepatology reference centres had more than doubled from

2,063 in 2000 to 4,259 in 2002. Despite this success, the programme

recommended that additional efforts and new strategies were needed to

improve treatment compliance and for treating non-responders20.

Nationwide screening for HCV began in 2002 in Japan, and as a

consequence, a reduction in hepatocellular carcinoma and in the

number of candidates requiring liver transplantation is anticipated21.

Limitations of current treatment options

Long-term studies have shown that SVR indicates clearance of virus

and cure of the disease22, 23. However, the response to therapy is

dependent on several factors, including viral genotype (Fig. 1) and

patient characteristics. There are six different genotypes of HCV,

with numerous subtypes. Genotype 1 is the most prevalent and most

difficult to treat viral strain in Europe and North America, and

represents the greatest unmet treatment need24. Genotypes 2 and 3

appear to be more prevalent in the Far East. Of the other genotypes,

genotype 4 is common in Africa and the Middle East, whereas genotypes

5 and 6 are predominant in South Africa and South-East Asia,

respectively3.

Certain patient populations are difficult to treat; these include non-

responders to prior treatment with IFN-based therapies, patients with

severe liver fibrosis or cirrhosis, those of African-American

ethnicity, individuals co-infected with HIV, and patients with

comorbidities, such as alcohol consumption, fatty liver or insulin

resistance25, 26, 27, 28, 29, 30, 31, 32. For example, response rates

in African-American patients with genotype 1 HCV have been shown to

be as low as 6-26%, and 50% in those with genotypes 2 or 3 (Refs

29,33). This is compared with overall cure rates of 40-50% for

genotype 1 and more than 75% in patient groups with genotypes 2 and 3

(Refs 8,11-13).

There are no approved treatment options available for patients who

have failed to respond to previous treatments. Studies suggest that

in non-responders to IFN monotherapy, re-treatment with pegIFN and

ribavirin can achieve SVR rates of 25-40%; and in non-responders to

IFN and ribavirin, re-treatment can achieve SVR rates of up to 10%28,

34. It has also been shown that extending the treatment duration in

slow responders infected with HCV genotype 1 might increase the rate

of SVR to the current standard of care for this patient

population105, 106. Trials investigating re-treatment of non-

responders with current standard of care are ongoing, but the results

available so far are not promising.

Studies in patients co-infected with HIV have shown SVR rates of 17-

62% (17-29% for genotypes 1 or 4 and 44-62% for genotypes 2, 3 and 5)

31, 32. These responses may, in part, be explained by viral kinetics -

the response to therapy generally being delayed in patients with co-

infection31, 32. For example, Torriani et al. state that although

patients who are mono-infected with HCV genotype 2 or 3 require 24

weeks of pegIFN-a plus ribavirin therapy, those co-infected with HCV

and HIV probably need 48 weeks of treatment31. This could be due to

the higher viral load in co-infected patients, as well as host immune

deficiency. It should be noted that in initial trials for HIV-HCV co-

infected patients, lower ribavirin dosages were used than dosages

commonly recommended for treatment of HCV infection. Subsequent

studies were able to demonstrate significantly higher SVR rates in

HIV-HCV co-infected patients with genotype 1 infection if standard

weight-adapted ribavirin dosing was used35.

In addition to inadequate response rates, current therapies are

associated with numerous side effects, including flu-like illness,

fever, fatigue, haematological disease, anaemia, leucopaenia,

thrombocytopaenia, alopecia and depression. Treatment-associated side

effects are an important consideration in the management of patients

with HCV. A review of current treatments indicates that side effects

may reduce adherence to therapy, resulting in 10-20% of premature

treatment discontinuations36. Consequently, improvements in

tolerability and the addition of supportive strategies, such as

patient-focused treatment education, may drive overall success rates.

As the ultimate goal of HCV therapy is the complete elimination of

the virus in all patients, new strategies for treatment are needed.

Prophylactic and therapeutic vaccines are in development, and new

approaches include the development of innovative new agents targeting

different stages of the viral life cycle, as well as improvements to

current strategies. Furthermore, a combination of complementary

approaches and individualization based on genotype, viral load and

early virological response will improve outcomes.

HCV vaccine development

As yet, no prophylactic vaccine is available for HCV, but extensive

studies of a recombinant vaccine in chimpanzees showed encouraging

results. Based on the viral envelope proteins E1/E2 (see Fig. 2a), it

protected more than 80% of the animals from developing chronic

infection following the experimental challenge with either homologous

or heterologous HCV-1a viral strains107. A T-cell vaccine eliciting

broad cellular responses to HCV-1b non-structural proteins 3, 4 and

5, was also shown to exhibit prophylactic activity in chimpanzees

after heterologous HCV-1a challenge108.

Several approaches are also being taken to develop therapeutic

vaccines. For example, a clinical-grade HCV E1 protein produced and

purified from mammalian cells (InnoVac-C) has been evaluated in

clinical trials37, 38. In a Phase IIa study involving 35 patients

with chronic HCV infection, cellular immune responses were boosted

with a recombinant E1 vaccine, including a significant T-cell

response. However, these cellular immune responses were not

accompanied by any significant reductions in serum HCV RNA37. Another

peptide-based therapeutic HCV vaccine, IC-41, also induced

significant T-cell responses, but HCV decay was not more than 1 log10

in individual patients39. The only parameter that was shown to

correlate with RNA response to IC-41 was an increase in HCV-specific

IFN-y secreting CD8+ cytotoxic T cells above a critical threshold. A

clinical trial was initiated with the aim to increase T cell

responses, and an optimized schedule increased responder rates,

caused a fivefold stronger CD8+ response (sustained for at least 20

weeks), and a broader induction of cytotoxic T-cell responses40. The

optimized regimen is currently being tested in a clinical trial of

treatment-naive HCV patients. Such immune boosting in HCV carriers is

likely to be most effective when used as an adjunct therapy with

standard-of-care antiviral drugs. Other approaches to therapeutic HCV

vaccines include the use of the recombinant core protein adjuvanted

with Iscomatrix. This combination elicited an unusually strong T-

helper and cytotoxic T-cell response to HCV in rhesus macaques109,

and a clinical trial in HCV patients who previously failed IFN

therapy is underway.

Innovative agents in clinical development

For the development of new, specific anti-HCV drugs, an understanding

of the HCV life cycle (Fig. 2b), in particular the genomic

organization and polyprotein processing, is essential. It has

resulted in the development of several agents that target specific

stages of the life cycle, the so-called specifically targeted

antiviral therapy for HCV (STAT-C) drugs. Potential processes for

viral inhibition include virus entry into the host cell, proteolytic

processing, RNA replication, and the assembly and release of the new

virions. Among the most promising new agents in development are the

protease and polymerase inhibitors, as discussed below. RNA-targeted

therapies, such as antisense oligonucleotides41, ribozymes42 and

small interfering RNA (siRNA)-targeting structures43, have shown

substantial success at inhibiting the HCV life cycle in vitro, but

not in vivo. The structural viral envelope proteins E1 and E2, as

well as their assembly, represent other potential antiviral

targets44, 110. Analogous to a recently developed HIV cell fusion

inhibitor, detailed understanding of HCV cell fusion and cell entry

could permit the development of specific HCV entry inhibitors.

Protease inhibitors. The non-structural protein NS3 possesses a

protease domain that is responsible for polyprotein processing and is

a potential target for antiviral intervention. Despite the catalytic

site being a shallow and largely hydrophobic groove, making it

difficult to target, several compound inhibitors of the NS3 protease

have been successfully designed and are currently in preclinical and

clinical development (for example, telaprevir (VX-950), boceprevir

(SCH503034) BI12202, MK-7009, TMC435350 and ITMN-191). The proof-of-

principle for this class of compounds was provided by BILN 2061, an

NS3 protease inhibitor that provides at least a 2-3 log10 decrease in

HCV load within 48 hours45. However, the clinical development of BILN

2061 was stopped owing to significant side effects.

Protease inhibitors have been associated with substantial reductions

in serum HCV RNA in clinical studies when given alone or in

combination with pegIFN-46, 47, 48, 49 (see also clinical trials

section below). NS3 possesses a helicase domain that has multiple

functions, including RNA-stimulated nucleoside 5'-triphosphate

hydrolase (NTPase) activity, RNA binding and unwinding of RNA regions

with extensive secondary structure. Other potential targets include

the NTP binding site and the binding site for single-stranded RNA50.

Polymerase inhibitors. The protein NS5B is cleaved from the HCV

polyprotein by the NS3 serine protease, and functions as a RNA-

dependent RNA polymerase. It is the key enzyme for synthesis of a

complementary minus-strand RNA, using the genome as a template, and

the subsequent synthesis of genomic plus-strand RNA from this minus-

strand RNA template. Several compound inhibitors of the NS5B

polymerase are, or have been, in clinical development. Two separate

classes of compounds have shown inhibitory effects on the NS5B

through two distinct mechanisms: first, nucleoside polymerase

inhibitors, which directly inhibit the active site causing chain

termination (for example, valopicitabine (NM-283), MK-0608, R1626,

PSI-6130 and its prodrug R7128), and second, non-nucleoside

polymerase inhibitors, which cause allosteric inhibition resulting in

a conformational change of the protein (for example, BILB 1941 and

HCV-796)50. Preclinical studies have shown that agents targeting the

HCV RNA polymerase are associated with significant reductions in

serum HCV RNA51 and clinical studies have demonstrated the promising

antiviral effects of NS5B inhibitors when used either as monotherapy

or in combination with pegIFN- (Refs 52-54). However, due to safety

concerns and unfavourable risk-benefit profiles, the development of

several polymerase inhibitors, including HCV-796, BILB 1941 and

valopicitabine, is on hold.

Immune modulators. Other mechanisms that are under investigation

include immune modulators targeting the cellular immune response,

which plays a major role in HCV infection. Examples include agents

that generate and/or promote an effective immune response by inducing

or modulating cytokine responses, such as the toll-like receptor

(TLR) agonists (for example, CPG 10101 and ANA 975), which have shown

antiviral efficacy in initial clinical studies55. CPG 10101 (Coley

Pharmaceuticals) is a synthetic oligodeoxynucleotide TLR9 agonist

that also induces T-helper type 1 cytokine responses, resulting in

high levels of type 1 IFN, natural killer (NK) cell stimulation and

other viral-specific immunomodulatory responses. In a Phase 1b

clinical trial, patients with HCV genotype 1 who received at least 1

mg CPG 10101 twice a week for 4 weeks experienced increases in IFN-

and other markers of immune response along with a mean 1 log10

decline in HCV RNA levels55, 111. However, improved SVR results have

not been reported so far. The clinical development of the TLR7 and

TLR9 agonists is currently on hold - Coley Pharmaceuticals has

stopped further development of CPG 10101 for viral hepatitis and are

concentrating their efforts towards the more promising use of CPG

10101 as an anticancer drug. The development of the TLR7 agonist ANA

975 (Anadys Pharmaceuticals) was stopped owing to preclinical safety

issues, as it was found to induce a general inflammatory response in

animals.

Further novel investigational agents. The effectiveness of inhibitors

of cyclophilin B (for example, NIM-811 and DEBIO-025), a host factor

involved in viral replication, is being evaluated in patients with

HCV. NIM-811, a cyclosporin A analogue, suppresses HCV genome

replication in a cell culture system and may provide a novel strategy

for anti-HCV treatment56, 57. DEBIO-025 has demonstrated strong

antiviral activity in vitro against HCV genotype 1 and HIV-1. In a

Phase Ib study of HCV-HIV co-infected patients, those receiving

treatment with DEBIO-025 achieved a mean HCV viral load reduction of

3.6 log10 after 15 days compared with 0.7 log10 for patients

receiving placebo58.

Recently, it has also been reported that NS4A, a cofactor for the NS3

protease, is a valid therapeutic target for chronic HCV infection.

ACH-806 (GS-9132) binds to HCV NS4A, inhibiting the correct

proteolytic processing of the HCV polyprotein and thereby the

formation of a functional replication complex, consequently

decreasing viral RNA synthesis. Results of a randomized, double-

blind, placebo-controlled, dose-escalation study demonstrated

clinical proof-of-concept, although reversible nephrotoxicity

precludes further development of ACH-806 (Ref. 59). Furthermore,

glucosidase inhibitors have been in development for many years albeit

with slow progress.

Improvements to current therapies. Longer-acting IFNs and IFN-

inducing molecules are in development. One example is albinterferon-

a2b (albIFN-a2b)[Albuferon], a fusion protein comprising albumin and

IFN-a2b, which has been shown to have antiviral activity in a

clinical trial setting, with a less frequent dosing regimen than

current pegIFNs60. A recent Phase IIb, active-controlled study

evaluated the efficacy and safety of three therapeutic dosage

regimens of albIFN-a2b (900 ug or 1200 ug every 2 weeks or 1200 ug

every 4 weeks) compared with pegIFN-a2a (180 ug once a week) in

treatment-naive patients with genotype 1 chronic HCV infection. All

treatments were in combination with ribavirin 1,000-1,200 mg per day

(based on body weight). SVR rates for the albIFN-2b arms were 58.5%

for 900 ug every 2 weeks, 55.5% for 1,200 ug every 2 weeks and 50.9%

for 1200 ug every 4 weeks, compared with 57.9% for the weekly pegIFN-

2a arm61. In addition, patients who received albIFN-a2b 900 ug every

2 weeks reported less impairment of quality of life (measured using

the SF-36 Health Survey62) than those who received weekly pegIFN-a2a.

These data suggest that albIFN-2b given every 2 weeks may offer

comparable efficacy to pegIFN-2a, with an improved dosing schedule

and the potential for less impairment of quality of life.

Other strategies to improve IFN efficacy include gene shuffle (this

compound was developed by Maxygen [development of Maxygen

discontinued by Roche] and was in development together with Roche),

IFN variants112 and the development of long-lasting IFNs, like albIFN-

a2b (Human Genome Sciences and Novartis Pharma), locteron (OctoPlus)

and omega IFN with a subcutaneous delivery device (Intarcia

Therapeutics) lasting 12 weeks. Furthermore, ribavirin derivatives

have been developed to improve efficacy and tolerability - these

include levovirin and viramidine (taribavirin). However, combination

of levovirin and pegIFN-a2a fails to generate virological responses

comparable with ribavirin-pegIFN-a2a combination therapy in patients

with chronic HCV63. Fixed-dose viramidine was shown to be less

efficacious than ribavirin in two Phase III clinical studies,

although anaemia rates were significantly lower in patients treated

with viramidine compared with those treated with ribavirin64, 65.

Weight-based dosing of viramidine is currently being evaluated in a

Phase IIb study of treatment-naive patients with HCV genotype 1.

Clinical trials of NS3 and NS5B inhibitors

The two novel innovative agents furthest in clinical development

(late Phase II) (Fig. 3) are the protease inhibitors telaprevir (VX-

950) and boceprevir (SCH503034). Valopicitabine (NM-283) was the

first polymerase inhibitor to reach Phase IIb clinical testing, but

was recently placed on clinical hold in the United States following a

review by the Food and Drug Administration (FDA)66. These three

agents have been shown to have significant antiviral activity in

patients with HCV genotype 1, including treatment-naive patients and

those not responding to other therapies54, 67, 68, 69, 70.

A Phase II clinical study in treatment-naive patients with genotype 1

evaluated valopicitabine 200-800 mg once a day with pegIFN-a2a for 12

weeks compared with pegIFN-a2a alone for the first 4 weeks, followed

by pegIFN-a2a and valopicitabine (400-800 mg) from week 5 onwards71.

At week 4, all combination therapy groups demonstrated greater

reductions in HCV RNA than the pegIFN-2a monotherapy group, and end-

of-treatment data indicated that valopicitabine maintained antiviral

activity for up to 48 weeks. In a Phase IIb study in non-responders

to pegIFN-2a and ribavirin, SVR data demonstrated comparable results

for valopicitabine plus pegIFN-a2a versus re-treatment with pegIFN-

and ribavirin; SVR was not achieved by any patient in the

valopicitabine plus pegIFN-2a arm and one patient (3%) in the pegIFN-

a2a and ribavirin arm72. The clinical hold imposed by the FDA was

based on the agency's overall assessment of the risk-benefit profile

observed to date. R1626, a prodrug of R1479, is a polymerase

inhibitor currently in Phase II development, which has shown a

maximum mean (median) HCV RNA reduction of 3.7 (4.1) log10 in

treatment-naive patients at a dose of 4,500 mg twice daily for 14

days73.

Phase Ib studies have evaluated telaprevir as monotherapy69 and in

combination with pegIFN-a2a and ribavirin in treatment-naive patients

with HCV genotype 1 (Ref. 46). Telaprevir was well tolerated as

monotherapy (750 mg every 8 hours) for 14 days and in combination

with pegIFN-a2a and ribavirin, and patients receiving telaprevir plus

pegIFN-a2a and ribavirin demonstrated the largest reduction in plasma

HCV RNA levels46. Telaprevir is currently being evaluated in three

Phase II studies. An interim analysis of one of these studies, PROVE

1, showed that 70% of patients who received telaprevir (750 mg every

8 hours) plus pegIFN-a2a and ribavirin had HCV RNA below 10 IU per ml

after 12 weeks of treatment compared with 39% of patients who were

treated with pegIFN-a2a and ribavirin alone (intention-to-treat

analysis)74. According to the study protocol, patients in one of the

study treatment arms (telaprevir plus pegIFN-2a plus ribavirin) were

eligible to stop all treatment at week 12 if they met certain on-

treatment criteria, including a rapid virological response (RVR,

defined as less than 10 IU per ml HCV RNA at week 4) and maintenance

of this response at week 10. Nine out of 17 patients achieved week-4

RVR and discontinued therapy at 12 weeks; six of these patients

continued to have undetectable HCV RNA 20 weeks post-treatment. Of

the remaining eight patients in this study arm, four discontinued

owing to adverse events before week 12 and four did not achieve RVR.

The first SVR data of the PROVE 1 study114, as well as first results

from PROVE 2, another Phase II trial of telaprevir with treatment

naive patients, have just been reported115.

A dose-ranging study of boceprevir (100-400 mg twice a day) in

patients with HCV genotype 1 that had previously failed pegIFN-a2a

therapy indicates that this protease inhibitor has dose-related

antiviral activity as monotherapy70. A Phase Ib 14-day study of

boceprevir (200 or 400 mg three times daily) administered in

combination with pegIFN-a2a (1.5 g per kg weekly) demonstrated a dose-

response relationship in non-responder patients with HCV genotype 1.

Mean maximum log10 reductions in HCV RNA were 2.45 and 2.88 for 200

and 400 mg boceprevir plus pegIFN-a2a, respectively49, and the

combination of agents provided greater antiviral activity than either

drug as monotherapy. Boceprevir 800 mg three times a day is currently

being evaluated in combination with pegIFN-2a and ribavirin in a

Phase II trial of non-responders. A further Phase II trial of

boceprevir 800 mg three times a day in combination with pegIFN-a2a

and ribavirin has also been initiated in treatment-naive patients.

Recent preliminary results from this so-called SPRINT (Serine

Protease Inhibitor Therapy) study are comparable to the two

telapravir Phase II studies in treatment naive patients113, 114, 115.

Many of the studies with novel agents conducted so far have focused

on the response in patients infected with genotype 1. Studies of the

agents in patients infected with other genotypes and in non-responder

populations with refractory disease are also required as clinical

programmes progress. For example, clinical studies with the now

discontinued protease inhibitor BILN-2061 highlighted that antiviral

activity may be less pronounced in patients infected with genotypes 2

or 3 compared with those infected with genotype 1 (Refs 48,75).

Resistance to new HCV antivirals

Response to therapy is dependent on several factors including

treatment-related factors, host characteristics (including the

ability of host cells to respond to IFN, induce antiviral defences

and clear infected cells), viral-related factors and disease-related

factors76, 78, 79. In addition, the genetic heterogeneity or

quasispecies nature of HCV has important therapeutic implications, as

the generation and selection of resistant variants can allow the

virus to escape the antiviral pressure exerted by treatment77.

Indeed, mutations in both the polymerase and protease enzymes have

already been identified (Table 1). In addition, the overall

prevalence of individual mutations changes over time, indicating that

the relative fitness (that is, the ability to replicate) of a

resistant variant will have a role in viral dynamics during treatment.

As previously discussed, many emerging HCV treatments are targeted

against specific HCV enzymes; among the most promising are the NS3

serine protease inhibitors and the NS5B RNA-dependent RNA polymerase

inhibitors. As the active site for protease inhibitors is a long

shallow groove, a single-point mutation in this enzyme might be

sufficient to hinder the binding of these antivirals, with different

mutations conferring low-level or high-level resistance (Fig. 4). For

example, sequencing studies using samples from patients treated with

telaprevir have identified several mutations that confer low-level

and high-level resistance80. Resistant isolates are selected rapidly

and therefore combination therapy with pegIFN-a2a or other antiviral

agents will be required to limit the development of resistance to

telaprevir. As far as we know, telaprevir-resistant mutants are

sensitive to IFN-a. The T54A mutation will confer resistance to both

telaprevir and boceprevir, whereas the A156S mutation leads to

resistance to telaprevir, but not boceprevir81, 82. There have been

several other reports of the selection of HCV-resistant mutants

against various protease inhibitors using the in vitro replicon

system70, 83, 84, 85, 86.

The active site of the NS5B RNA-dependent RNA polymerase is a highly

conserved region in all HCV genotypes and any amino-acid mutations in

this region may inhibit the ability of the virus to replicate (Fig.

5). This suggests that resistance to nucleoside polymerase inhibitors

by mutation in the enzyme may not readily develop. Although selection

of replicons resistant to 2'-C-methyl-nucleosides has shown that HCV

is rapidly able to discriminate between antiviral agents and natural

nucleosides81, in vitro studies have shown that replicons carrying

these mutations showed decreased replication fitness83, 87, 88, 89.

There are several binding sites for non-nucleoside analogues within

the NS5B polymerase (Fig. 5). Several mutations have been identified

as determinants for resistance to non-nucleosides. For example, it

has been demonstrated in vitro that replacement of P495 with alanine

or leucine strongly reduces affinity for non-nucleoside inhibitors83,

89. Such a mutation decreases the efficiency of viral replication,

but viral fitness can be restored by mutations elsewhere in the NS5B

coding region83.

In vitro data suggest a low probability of cross-resistance between

some of the different nucleoside polymerase inhibitors or between

nucleoside and non-nucleoside inhibitors (see also Table 1). For

example, production of mutant viral strains by an amino-acid

substitution at S96T alone or in combination with N142T confers

resistance to R1479 (for which R1626 is the pro-drug), but not

valopicitabine87, 90, and the S282T substitution confers resistance

to valopicitabine but not to R1479. Furthermore, molecular biology

suggests no cross-resistance between protease and polymerase

inhibitors83. There was also no cross-resistance observed between the

cyclophilin B inhibitor NIM-811 and NM-107, the active moiety of

valopicitabine91. These data suggest that NIM-811, an agent that

targets host-viral interactions, provides another option for

combination therapy with other antiviral agents, which would reduce

the emergence of resistance82, 87.

>From the results of in vitro studies we can anticipate drug

resistance in vivo and consider options to reduce it, such as the use

of agents with a low probability of cross-resistance in combination.

For example, telaprevir monotherapy in treatment-naive patients with

HCV genotype 1 produced subsets of patients that had a plateau in HCV

RNA decline or breakthrough response during 14 days of dosing92.

Sequencing assays of the viral RNA in these patients detected that

these responses correlated with the selection of viruses containing

one or two mutations in the NS3 protease region. In vitro analysis

demonstrated that specific mutations correlated with the level of

resistance; viruses with mutations at A156V/T conferred a high level

of resistance to telaprevir, whereas T54A conferred a low level of

resistance. In the absence of drug-selective pressure, high-level

resistant variants rapidly became undetectable and replaced with wild-

type variants92. Moreover, administration of telaprevir in

combination with pegIFN-a2a alone or with ribavirin appeared to

prevent the selection of inhibitor-resistant variants and, hence,

viral rebound46, 69.