HCV Drug Resistance

HCVADVOCATE.COM Newsletter — Liz Highleyman

A new approach to hepatitis C treatment – specifically targeted antiviral therapy for hepatitis C, or “STAT-C” – promises to improve the likelihood of achieving a cure for hard-to-treat patients. But these new therapies also come with a drawback: HCV can develop drug resistance, making them less effective.

HCV Replication and Mutation
There are two basic approaches to fighting HCV: strengthening the body’s immune response and attacking the virus directly. Interferon-based therapy uses a manufactured version of a natural cytokine to boost immune function. HCV does not develop resistance to interferon over time.

STAT-C drugs block specific steps of the viral lifecycle. For example, HCV protease inhibitors (such as telaprevir and boceprevir) interfere with an enzyme encoded by the NS3/4A gene that processes proteins before they can be assembled into new virions (virus particles). HCV polymerase inhibitors disrupt the action of another enzyme, encoded by the NS5B gene, that copies viral genetic material. (The HCV lifecycle and how drugs work is explained more fully in the December 2009 HCV Advocate.)

Drug resistance can develop when a virus mutates, or changes its genetic code. HCV replicates very rapidly – at an estimated rate of 1012 virions per day – and is prone to errors as it copies its genetic material. The genetic code of mutated HCV is essentially an altered blueprint. Viral proteins consist of a chain of building blocks called amino acids. When the altered blueprint is used to make new proteins and enzymes, the directions call for insertion of a wrong amino acid.

For example, the NS3 protease enzyme produced by mutated HCV might have the usual amino acid valine (V) replaced by alanine (A) at position 107 of the chain – designated V107A. (See chart below for list of amino acids letter codes.)

Amino Acids

In many cases, proteins containing the wrong amino acids do not work properly. This can make mutant HCV less “fit” than wild-type (non-mutated) virus, so it stays at a very low level or dies out. But in other cases, a “wrong” amino acid turns out to be right for the virus, giving it an evolutionary advantage. In fact, this is how viruses manage to survive constant attack by the immune system.

Mechanisms of Resistance
One such advantage is drug resistance – mutations that allow the virus to keep replicating despite the presence of a drug. Amino acid substitutions in the HCV protease or polymerase enzyme can change the protein’s shape and interfere with a drug’s action. For example, a small structural change to the shallow binding pocket of the NS3 protease makes it difficult for a protease inhibitor to fit in and perform its intended function.

HCV polymerase inhibitors are of two types, nucleoside analogs – which act as defective building blocks when the virus tries to copy its genetic material – and non-nucleoside inhibitors, which work by other mechanisms. The HCV NS5B polymerase has at least five sites that are potential drug targets.

Ribavirin, which is used with pegylated interferon to help prevent HCV relapse, is also a nucleoside analog, but it has additional mechanisms of action, and resistance due to viral mutation has not been a concern in hepatitis C treatment.

The emergence of drug resistant mutations sometimes occurs randomly as HCV mutates to evade immune defenses, so an individual who has never been treated may harbor some strains, or quasispecies, that are naturally resistant (known as primary resistance). Most genetic screening studies have detected protease and polymerase resistance mutations at rates of <1% to 5% in previously untreated individuals. These variants are usually only a small minority of the total virus population, and so far they have not been linked to treatment failure – though some research does suggest reduced potency.

More often, resistance is due to continued viral replication and mutation in the presence of a drug. This can happen in two ways. A pre-existing naturally resistant minority variant can become the dominant strain if a drug knocks out the majority wild-type virus. Or, if a drug reduces but does not completely halt viral replication, wild-type HCV can evolve new mutations to evade the drug’s effects. Sometimes a mutation that allows the virus to escape the action of one drug will also make similar agents less effective, a phenomenon known as cross-resistance.

Resistance Studies
Experience treating hepatitis B and HIV led researchers to suspect that using single directly targeted anti-HCV agents – known as monotherapy – would likely promote resistance. As such, resistance testing is part of hepatitis C drug development from the earliest laboratory studies through the final clinical trials.

One or more amino acid substitutions that reduce antiviral potency have been identified for all the HCV protease and polymerase inhibitors currently in development. At the outset, STAT-C agents used as monotherapy may rapidly and dramatically decrease HCV RNA. But before long – days to months, depending on the specific agent – viral load may start to rise again (known as viral breakthrough), indicating that drug-resistant variants are gaining the upper hand.

But drug resistance does not necessarily lead to treatment failure. Even when a mutant virus is less susceptible, a drug still may be potent enough to keep replication under control. Sometimes multiple mutations must coexist to cause a notable decrease in effectiveness. And because HCV treatment is relatively short – typically 24-48 weeks – it does not present the risk of long-term resistance after taking a drug for years (as with hepatitis B) or even for life (as with HIV).

In the November 10, 2009 advance online edition of the Journal of Viral Hepatitis, A.J.V. Thompson and J.G. McHutchison presented an overview of HCV drug resistance, including data from laboratory studies and clinical trials.

Because HCV is difficult to grow in vitro, most laboratory studies use models called replicons that may not respond to drugs exactly like whole virus in the body. Genotypic tests, which examine viral gene sequences for substitutions known to confer resistance, do not always reflect what happens when HCV variants are exposed to drugs in the laboratory (phenotypic testing), which in turn may not predict clinical outcomes in patients.

Nevertheless, some clear patterns have emerged. As noted, the structure of the NS3 protease allows HCV to easily evade protease inhibitors, especially with mutations at positions 155 and 156. For HCV genotype 1, the A156S/T/V and R155K/Q/T substitutions confer resistance to telaprevir, boceprevir, and RG7227 (ITMN-191), especially when additional mutations including V36M and T54A are also present. Other mutations confer resistance primarily to specific drugs, for example V170A for boceprevir and D168V/A for RG7227.

Current polymerase inhibitor candidates produce smaller viral load decreases than protease inhibitors, but they also promote less resistance. Nucleoside analogs such as RG7128 appear to have a particularly high barrier to resistance. Among the non-nucleosides, most agents target only one binding site, and there appears to be no cross-resistance – and perhaps even synergy – between drugs targeting different sites.

To date, no mutations have been found to confer resistance to both protease and polymerase inhibitors, though researchers have produced replicons that simultaneously carry separate protease and polymerase mutations. Fortunately, HCV with protease and/or polymerase resistant mutations appears to remain as responsive as wild-type virus to standard therapy with pegylated interferon plus ribavirin.

Preventing and Overcoming Resistance
The surest way to prevent drug resistance is by completely halting viral replication, or even better, eradicating HCV altogether. With current drugs, however, many people continue to have some level of ongoing viral replication.

Another way to overcome resistance is combination therapy. In order to evade the action of multiple drugs, HCV would have to develop simultaneous mutations, which typically reduces viral fitness in other ways.

Learning from the setbacks of hepatitis B and HIV treatment, researchers understand the benefits of using STAT-C agents in combination regimens from the outset, rather than adding additional drugs after resistance develops. Given how quickly resistance can emerge, the U.S. Food and Drug Administration now limits clinical trials of direct antiviral agents to three days of monotherapy.

Several studies have demonstrated that combining HCV protease inhibitors with pegylated interferon/ribavirin delays the emergence of resistance. In the PROVE trials, however, about 25% of participants who took telaprevir plus pegylated interferon without ribavirin developed resistance. Some trials start with a pegylated interferon/ribavirin “lead-in” period to drive down viral load before adding the direct antiviral agent.

Researchers are exploring combinations of STAT-C agents that work by different mechanisms in the hope of one day eliminating pegylated interferon/ribavirin. In the first such clinical trial (INFORM-1), presented last fall at the 2009 annual meeting of the American Association for the Study of Liver Diseases (AASLD), S. Le Pogam and colleagues showed that over 14 days, the protease inhibitor RG7227 plus the polymerase inhibitor RG7128 produced potent antiviral activity. Viral load declined by as much as 5 logs and no resistant mutations were detected, even in one patient in a low-dose arm who experienced viral breakthrough.

Using another novel approach, L. Delang and colleagues recently demonstrated that adding statin drugs (usually used to manage high cholesterol) to HCV protease and polymerase inhibitors enhanced antiviral activity and reduced emergence of resistant mutations.

In the coming years, hepatitis C therapy is likely to increasingly resemble HIV treatment, using complementary oral drugs that target different steps of the viral lifecycle. As such, it will draw on lessons from that field, such as the importance of good adherence, frequent viral load monitoring, and resistance testing to guide selection of the drugs most likely to be effective.

Selected References
L. Delang et al. Statins potentiate the in vitro anti-hepatitis C virus activity of selective hepatitis C virus inhibitors and delay or prevent resistance development. Hepatology 50(1): 6-16. July 2009.
S. Gaudieri et al. Hepatitis C virus drug resistance and immune-driven adaptations: relevance to new antiviral therapy. Hepatology 49(4): 1069-1082. April 2009.
T. Kuntzen et al. Naturally occurring dominant resistance mutations to hepatitis C virus protease and polymerase inhibitors in treatment-naive patients. Hepatology 48(6): 1769-1778. December 2008.
S. Le Pogam et al. Combination therapy with nucleoside polymerase R7128 and protease R7227/ITMN-191 inhibitors in genotype 1 HCV infected patients: interim resistance analysis of INFORM-1 cohorts A-D. 60th Annual Meeting of the American Association for the Study of Liver Diseases. Boston. October 30-November 1, 2009. Abstract 1585.
M.F. McCown et al. The hepatitis C virus replicon presents a higher barrier to resistance to nucleoside analogs than to nonnucleoside polymerase or protease inhibitors. Antimicrobial Agents & Chemotherapy 52(5): 1604-1612. May 2008.
A.J.V. Thompson and J.G. McHutchison. Antiviral resistance and specifically targeted therapy for HCV (STAT-C). Journal of Viral Hepatitis 16(6): 377-387. November 10, 2009 [Epub ahead of print].