Ann D. Kwong

Telaprevir and pegylated interferon-alpha-2a inhibit wild-type and resistant genotype 1 hepatitis C virus replication in patients

Telaprevir (VX‐950) is an orally active, specifically targeted antiviral therapy for hepatitis C virus (HCV) that has been shown to profoundly reduce plasma HCV RNA in genotype 1 patients. Using a highly sensitive sequencing assay that detects minor populations of viral variants (≥5%), mutations were identified that conferred low‐level (V36M/A, T54A, or R155K/T) or high‐level (A156V/T and 36/155) resistance to telaprevir in vitro. We report a detailed kinetic analysis of these variants in 16 patients given telaprevir or telaprevir + pegylated interferon–alpha‐2a (PEG‐IFN–alpha‐2a) for 14 days. In 4 patients who had a viral rebound on telaprevir alone, the R155K/T and A156V/T variants were detected during the initial steep decline in HCV RNA. During the rebound phase, the R155K/T and A156V/T variants were replaced by V36(M/A)/R155(K/T) double mutant variants. In the remaining 12 patients given telaprevir alone or with telaprevir/PEG‐IFN–alpha‐2a, the A156V/T variant was detected in some patients, but viral levels continued to decline in all patients. Conclusion: These studies suggest that the initial antiviral response to telaprevir is due to a sharp reduction in wild‐type virus, which uncovers pre‐existing telaprevir‐resistant variants. In patients given telaprevir alone, viral rebound can result from the selection of variants with greater fitness. However, the combination of telaprevir and PEG‐IFN–alpha‐2a inhibited both wild‐type and resistant variants. In the present study, every patient who began PEG‐IFN–alpha‐2a and ribavirin after the 14‐day dosing period had undetectable HCV RNA levels at 24 weeks, indicating that telaprevir‐resistant variants are sensitive to PEG‐IFN–alpha‐2a and ribavirin. (HEPATOLOGY 2007.)

Preclinical Profile of VX-950, a Potent, Selective, and Orally Bioavailable Inhibitor of Hepatitis C Virus NS3-4A Serine Protease

ABSTRACT VX-950 is a potent, selective, peptidomimetic inhibitor of the hepatitis C virus (HCV) NS3-4A serine protease, and it demonstrated excellent antiviral activity both in genotype 1b HCV replicon cells (50% inhibitory concentration [IC50] = 354 nM) and in human fetal hepatocytes infected with genotype 1a HCV-positive patient sera (IC50 = 280 nM). VX-950 forms a covalent but reversible complex with the genotype 1a HCV NS3-4A protease in a slow-on, slow-off process with a steady-state inhibition constant (Ki*) of 7 nM. Dissociation of the covalent enzyme-inhibitor complex of VX-950 and genotype 1a HCV protease has a half-life of almost an hour. A >4-log10 reduction in the HCV RNA levels was observed after a 2-week incubation of replicon cells with VX-950, with no rebound of viral RNA observed after withdrawal of the inhibitor. In several animal species, VX-950 exhibits a favorable pharmacokinetic profile with high exposure in the liver. In a recently developed HCV protease mouse model, VX-950 showed excellent inhibition of HCV NS3-4A protease activity in the liver. Therefore, the overall preclinical profile of VX-950 supports its candidacy as a novel oral therapy against hepatitis C.

In vitro studies of cross-resistance mutations against two hepatitis C virus serine protease inhibitors, VX-950 and BILN 2061

VX-950 is a potent, small molecule, peptidomimetic inhibitor of the hepatitis C virus (HCV) NS3·4A serine protease and has recently been shown to possess antiviral activity in a phase I trial in patients chronically infected with genotype 1 HCV. In a previous study, we described in vitro resistance mutations against either VX-950 or another HCV NS3·4A protease inhibitor, BILN 2061 (Lin, C., Lin, K., Luong, Y.-P., Rao, B. G., Wei, Y.-Y., Brennan, D. L., Fulghum, J. R., Hsiao, H.-M., Ma, S., Maxwell, J. P., Cottrell, K. M., Perni, R. B., Gates, C. A., and Kwong, A. D. (2004) J. Biol. Chem. 279, 17508-17514). Single amino acid substitutions that conferred drug resistance (distinct for either inhibitor) were identified in the HCV NS3 serine protease domain. The dominant VX-950-resistant mutant (A156S) remains sensitive to BILN 2061. The major BILN 2061-resistant mutants (D168V and D168A) are fully susceptible to VX-950. Modeling analysis suggested that there are different mechanisms of resistance for these mutations induced by VX-950 or BILN 2061. In this study, we identified mutants that are cross-resistant to both HCV protease inhibitors. The cross-resistance conferred by substitution of Ala156 with either Val or Thr was confirmed by characterization of the purified enzymes and reconstituted replicon cells containing the single amino acid substitution A156V or A156T. Both cross-resistance mutations (A156V and A156T) displayed significantly diminished fitness (or replication capacity) in a transient replicon cell system.

Natural Prevalence of Hepatitis C Virus Variants with Decreased Sensitivity to NS3·4A Protease Inhibitors in Treatment-Naive Subjects

BACKGROUND The prevalence and clinical implications of naturally occurring variants that are resistant to hepatitis C virus (HCV) protease inhibitors in treatment-naive patients has not been reported. We report here the prevalence of such variants and their effect on clinical response. METHODS Population sequence analysis of the NS3.4A protease was conducted in 570 treatment-naive subjects. RESULTS Most subjects (98%) had wild-type virus. The remaining subjects had the following variants present in significant proportions (100%): V36M, 0.9%; R155K, 0.7%; V170A, 0.2%; and R109K, 0.2%. The V36M, R109K, and V170A substitutions confer low-level resistance (<7-fold) to protease inhibitors in replicon cells. The R155K substitution confers low-level resistance to telaprevir (TVR) and boceprevir and confers high-level resistance (>70-fold) to BILN 2061 and ITMN-191. Five subjects with the V36M or R109K variant were treated with 8-24 weeks of TVR and peginterferon-alpha2a (P) with or without ribavirin (R). Four achieved a sustained viral response, and 1 was lost to follow-up. In subjects with the R155K variant, TVR/PR provided greater antiviral activity than PR alone; however, the antiviral response was lower than that observed in subjects with wild-type virus. CONCLUSION High levels of naturally occurring protease inhibitor-resistant variants were uncommon (<1% each) in HCV treatment-naive patients. TVR/PR efficiently inhibited V36M and R109K variants and contributed partial antiviral activity against the R155K variant. As new HCV agents are evaluated in clinical trials, it will be important to monitor the effect of baseline variants on sensitivity.

Hepatitis C virus variants

HBV replicates through reverse transcription of an RNA intermediate; the inherent lack of proofreading causes a high mutation frequency. Mutations in the precore and core promoter regions that abolish or reduce the production of hepatitis B e antigen occur most commonly. Patients with these HBV variants remain viremic and can develop progressive liver disease. Mutations in the core promoter region are associated with an increased risk of hepatocellular carcinoma. Exogenous selection pressure might favor certain mutations. Mutations in the HBV polymerase that confer resistance to nucleoside and nucleotide analog treatments are a major barrier to the success of therapy for hepatitis B. The development of antiviral drug resistance negates the initial treatment response and can lead to hepatitis flares and hepatic decompensation. Prompt addition of another drug to which the virus is not cross-resistant is required. Mutations in the HBV surface protein that facilitate escape from host immunity are responsible for the failure of immune prophylaxis in infants who received HBV vaccine and in liver transplant recipients who received hepatitis B immune globulin.

Hepatitis C Virus Variants with Decreased Sensitivity to Direct-Acting Antivirals (DAAs) Were Rarely Observed in DAA-Naive Patients prior to Treatment

ABSTRACT The prevalence of naturally occurring hepatitis C virus (HCV) variants that are less sensitive to direct-acting antiviral (DAA) inhibitors has not been fully characterized. We used population sequence analysis to assess the frequency of such variants in plasma samples from 3,447 DAA-naive patients with genotype 1 HCV. In general, HCV variants with lower-level resistance (3- to 25-fold increased 50% inhibitor concentration [IC50]) to telaprevir were observed as the dominant species in 0 to 3% of patients, depending on the specific variant, whereas higher-level resistant variants (>25-fold-increased IC50) were not observed. Specific variants resistant to NS5A inhibitors were predominant in up to 6% of patients. Most variants resistant to nucleo(s/t)ide active-site NS5B polymerase inhibitors were not observed, whereas variants resistant to non-nucleoside allosteric inhibitors were observed in up to 18% of patients. The presence of DAA-resistant variants in NS5A, NS5B, or NS3 (including telaprevir-resistant variants), in baseline samples of treatment-naive patients receiving a telaprevir-based regimen in phase 3 studies did not affect the sustained viral response (SVR). Treatment-naive patients with viral populations containing the telaprevir-resistant variants NS3 V36M, T54S, or R155K at baseline achieved a 74% SVR rate, whereas patients with no resistant variants detected prior to treatment achieved a 76% SVR rate. The effect of specific resistant variant frequency on response to various DAA treatments in different patient populations, including interferon nonresponders, should be further studied.

Viral resistance to specifically targeted antiviral therapies for hepatitis C (STAT-Cs)

Promising results have been observed with an investigational drug class for hepatitis C (HCV), the specifically targeted antiviral therapies for hepatitis C (STAT-Cs), when combined with peginterferon plus ribavirin (Peg-IFN/RBV). This class has the potential to increase sustained virological response (SVR) rates and reduce therapy duration in genotype 1 chronic HCV patients compared with Peg-IFN/RBV alone. However, because of the remarkable sequence variation in HCV (resulting from the high viral replication rate and intrinsically error-prone nature of HCV polymerase), variants with reduced susceptibility to STAT-Cs can occur naturally before treatment, usually at low levels, and can be selected in patients not responding to potent STAT-C treatment. This review first describes how resistance to a STAT-C can develop and then provides an overview of mutations that confer varying levels of resistance to STAT-Cs, which have been identified and characterized using both genotypic and phenotypic tools. We will discuss why an understanding of the selection of variants with reduced susceptibility to a treatment regimen may be important in optimizing the use of this new class of HCV therapy. Strategies for optimizing treatment regimens to increase response rates, and thereby minimize resistance, will be discussed. Finally, although resistance can be a consequence of not achieving an SVR on an initial regimen, there may be alternative treatment options for patients to achieve an SVR in the future. Future potential therapeutic strategies to address patients who do develop resistance to STAT-Cs are discussed, including combination therapy with multiple STAT-Cs with non-overlapping resistance profiles.

Phenotypic Characterization of Resistant Val36 Variants of Hepatitis C Virus NS3-4A Serine Protease

ABSTRACT In patients chronically infected with hepatitis C virus (HCV) strains of genotype 1, rapid and dramatic antiviral activity has been observed with telaprevir (VX-950), a highly selective and potent inhibitor of the HCV NS3-4A serine protease. HCV variants with substitutions in the NS3 protease domain were observed in some patients during telaprevir dosing. In this study, purified protease domain proteins and reconstituted HCV subgenomic replicons were used for phenotypic characterization of many of these substitutions. V36A/M or T54A substitutions conferred less than eightfold resistance to telaprevir. Variants with double substitutions at Val36 plus Thr54 had ∼20-fold resistance to telaprevir, and variants with double substitutions at Val36 plus Arg155 or Ala156 had >40-fold resistance to telaprevir. An X-ray structure of the HCV strain H protease domain containing the V36M substitution in a cocomplex with an NS4A cofactor peptide was solved at a 2.4-Å resolution. Except for the side chain of Met36, the V36M variant structure is identical to that of the wild-type apoenzyme. The in vitro replication capacity of most variants was significantly lower than that of the wild-type replicon in cells, which is consistent with the impaired in vivo fitness estimated from telaprevir-dosed patients. Finally, the sensitivity of these replicon variants to alpha interferon or ribavirin remained unchanged compared to that of the wild-type.

Combination of a Hepatitis C Virus NS3-NS4A Protease Inhibitor and Alpha Interferon Synergistically Inhibits Viral RNA Replication and Facilitates Viral RNA Clearance in Replicon Cells

ABSTRACT The present standard of care for hepatitis C virus (HCV) infection is pegylated alpha interferon (IFN-α) in combination with ribavirin. However, specific antivirals such as HCV NS3-NS4A protease inhibitors are now in clinical development, and these agents can potentially be used in combination with the present treatments. Therefore, it is important to investigate the potential benefits or adverse effects of these new combinations by using available in vitro HCV culture systems first. In the present study we demonstrate that the combination of a specific HCV NS3-NS4A protease inhibitor and IFN-α synergistically inhibits HCV RNA replication in replicon cells, with little or no increase in cytotoxicity. Furthermore, the benefit of the combination was sustained over time, such that a greater than 3-log reduction in HCV RNA levels was achieved following 9 days of treatment. The viral RNA appeared to be cleared from the replicon cells after 14 days of treatment, and no viral RNA rebound was observed upon withdrawal of the inhibitors. In each case, the antiviral effects obtained with higher concentrations of either the protease inhibitor alone or IFN-α alone can be achieved by a combination of both agents at lower concentrations, which may potentially reduce the risk of possible adverse effects associated with high doses of either agent.

Hepatitis C Viral Evolution in Genotype 1 Treatment-Naïve and Treatment-Experienced Patients Receiving Telaprevir-Based Therapy in Clinical Trials

Background In patients with genotype 1 chronic hepatitis C infection, telaprevir (TVR) in combination with peginterferon and ribavirin (PR) significantly increased sustained virologic response (SVR) rates compared with PR alone. However, genotypic changes could be observed in TVR-treated patients who did not achieve an SVR. Methods Population sequence analysis of the NS3•4A region was performed in patients who did not achieve SVR with TVR-based treatment. Results Resistant variants were observed after treatment with a telaprevir-based regimen in 12% of treatment-naïve patients (ADVANCE; T12PR arm), 6% of prior relapsers, 24% of prior partial responders, and 51% of prior null responder patients (REALIZE, T12PR48 arms). NS3 protease variants V36M, R155K, and V36M+R155K emerged frequently in patients with genotype 1a and V36A, T54A, and A156S/T in patients with genotype 1b. Lower-level resistance to telaprevir was conferred by V36A/M, T54A/S, R155K/T, and A156S variants; and higher-level resistance to telaprevir was conferred by A156T and V36M+R155K variants. Virologic failure during telaprevir treatment was more common in patients with genotype 1a and in prior PR nonresponder patients and was associated with higher-level telaprevir-resistant variants. Relapse was usually associated with wild-type or lower-level resistant variants. After treatment, viral populations were wild-type with a median time of 10 months for genotype 1a and 3 weeks for genotype 1b patients. Conclusions A consistent, subtype-dependent resistance profile was observed in patients who did not achieve an SVR with telaprevir-based treatment. The primary role of TVR is to inhibit wild-type virus and variants with lower-levels of resistance to telaprevir. The complementary role of PR is to clear any remaining telaprevir-resistant variants, especially higher-level telaprevir-resistant variants. Resistant variants are detectable in most patients who fail to achieve SVR, but their levels decline over time after treatment.