Antiviral efficacy of NS3-serine protease inhibitor BILN-2061 in patients with chronic genotype 2 and 3 hepatitis C


  • Conflict of interest: Nothing to report.

  • Presented in part in abstract form at the 54th annual meeting of the American Association for the Study of Liver Diseases, Boston, MA, 2003.


BILN-2061, a specific and potent peptidomimetic inhibitor of the HCV NS3 protease, has recently been shown to markedly lower serum hepatitis C virus (HCV)-RNA levels in patients chronically infected with HCV genotype 1 in three 2-day proof of principle studies. The aim of the current study was to assess the antiviral efficacy of BILN-2061 in patients with genotypes 2 and 3 HCV infection. The antiviral efficacy, pharmacokinetics, and tolerability of 500 mg twice-daily BILN-2061 given as monotherapy for 2 days in 10 patients chronically infected with non–genotype 1 HCV (genotype 2: n = 3; genotype 3: n =7) and minimal liver fibrosis (Ishak score 0-2) were assessed in a placebo-controlled (placebo n = 2), double-blind pilot study. HCV-RNA levels decreased by ≥1 log10 copies/mL in 4 of 8 patients treated with BILN-2061. One patient showed a weak response of <1 log10 copies/mL. Three of 8 treated patients showed no response. There was no correlation between baseline viral concentration or genotype and response. BILN-2061 exhibited good systemic exposure after oral administration and was well tolerated. In conclusion, the antiviral efficacy of the HCV serine protease inhibitor BILN-2061 is less pronounced and more variable in patients with HCV genotype 2 or 3 infection compared with previous results in patients with HCV genotype 1. A lower affinity of BILN-2061 for the NS3 protease of genotypes 2 and 3 HCV is most likely a major contributor to these findings. (HEPATOLOGY 2005.)

Despite advances in the therapy of chronic hepatitis C virus (HCV) infection, the side effects of currently available, interferon-based treatment regimens are substantial, and the number of patients who have not responded to these therapies is increasing.1–3 New effective antiviral therapeutics and strategies for the treatment of chronic HCV infection are therefore needed.4 The HCV-encoded serine protease (NS3 serine protease) is essential for viral replication and, hence, is an attractive target for HCV-specific antiviral therapy.5 BILN-2061 is a potent peptidomimetic inhibitor of the NS3 protease. It is a small, orally bioavailable molecule identified during a substrate-based development program conducted by Boehringer Ingelheim.6 Based on its potent inhibitory effects in Huh-7 hepatoma cells containing the subgenomic HCV type 1b NS2-NS5b replicon, BILN-2061 was first investigated in patients infected with HCV genotype 1 in 3 consecutive 2-day proof-of-concept studies. BILN-2061 administered twice daily at a dose of 25 to 500 mg was well tolerated and highly effective, inducing a rapid decline in virus concentration in all treated patients, as previously reported.7

In the current study, we investigated the antiviral effect of BILN-2061 in 10 patients infected with HCV genotype 2 or 3.


HCV, hepatitis C virus; AUC, area under the curve.

Materials and Methods

This prospective, multicenter, randomized, double-blind, placebo-controlled study was similar in design to the previously published study in patients infected with HCV genotype 1.7 In brief, patients were eligible if they were 18 years of age or older, with chronic non–genotype 1 HCV infection (Line Probe assay), a serum HCV-RNA concentration of at least 50,000 copies/mL (Amplicor assay) at screening, a liver biopsy with no or only mild fibrosis (Metavir score ≤ 1) within the previous 12 months. The study protocol, informed consent, subject information form, and amendments were approved by the institutional ethics committees at each center, and all the patients provided written informed consent. BILN-2061 500 mg or placebo was administered twice daily to 10 patients for 2 days; the randomization was 4:1. The patients were hospitalized for the duration of drug administration (a total of 3 nights). Blood was drawn for HCV-RNA determination and pharmacokinetic analysis on treatment days (0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 6, 8, 14 hours) and on days 3 and 4, as well as between days 5 and 7 and days 10 and 14, as described previously. HCV viral concentration was measured with bDNA version 3.0 assay (Bayer, limit of detection, 3,200 RNA copies or 615 IU/mL). Plasma samples for pharmacokinetics, including peak plasma concentration, peak time, and area under the curve (AUC0-8h), were analyzed by liquid/mass spectrometry (Darnow J et al., Boehringer Ingelheim, unpublished observations, 2002).


Patient Demographics.

Ten patients were enrolled in the study; all were men. Their demographics and treatment histories are summarized in Table 1. All 10 patients completed the study. Adherence to the study medication was 100%, as BILN-2061 was administered as directly observed therapy.

Table 1. Patient Disposition and Treatment History
  • *

    Discontinuation of therapy because of nonresponse.

Total treated (N)82
Age (years)  
Previously treated for hepatitis C  

Virological Efficacy.

The HCV concentration decreased by ≥1 log10 copies/mL, as measured by the bDNA assay, in 4 of 8 patients treated with BILN-2061 (two genotype 2 and two genotype 3 HCV). Three of eight patients had a viral concentration reduction of ≥2 log10 copies/mL. A HCV-RNA reduction of 3 or more log10 copies/mL was not observed. One patient showed a weak response of <1 log10 copies/mL (genotype 3). No changes in viral concentrations were observed in the remaining 3 BILN-2061–treated patients (three genotype 3 HCV) nor in the 2 control patients (one genotype 2, one genotype 3) (Fig. 1). Viral responses were similar in patients who had high or low viral concentrations at baseline. There was no correlation between genotype and response. The HCV viral concentration returned to baseline within 1 to 7 days of completing 2 days of BILN-2061 treatment. Substantial changes in the secondary efficacy parameters, alanine aminotransferase and aspartate aminotransferase levels, were not observed.

Figure 1.

Viral concentration changes (bDNA assay). Dotted lines represent control patients; solid lines represent treated patients; upper intermittent line and lower solid line represent the upper and lower limits of quantification of the bDNA assay; black triangles represent administration of BILN-2061.


The pharmacokinetics after the first and third doses of the compound are presented in Table 2 and Fig. 2. An increase was observed in plasma concentrations of BILN-2061 as well as the Cmax, AUC0-8h, and trough concentrations between the first and third dose. Peak plasma concentration and AUC increased between 3- and 4-fold on day 2 compared with day 1 values. On average, the pharmacokinetic data resembled the findings observed in patients with HCV genotype 1 infection; interpatient variability was higher, however: the coefficient of variance (CV) of AUC0-8h was 121% on day 1 and112% on day 2. The best antiviral responses were observed in 2 patients with the highest AUC0-8h on days 1 and 2, whereas the 3 patients with the lowest AUC0-8h consistently showed no response.

Table 2. Pharmacokinetic Parameters—Descriptive Statistics
 tmax, 1 (hr)tmax, 2 (hr)AUC0–8h, 1 (hr *ng/mL)AUC0–8h, 3 (hr *ng/mL)Cmax, 1 (ng/mL)Cmax, 3 (ng/mL)C8h, 1 (ng/mL)C8h, 3 (ng/mL)
  1. Abbreviations: tmax, 1, time to maximum concentration after first dose; tmax, 3, time to maximum concentration after third dose; AUC0–8h, 1, area under the curve between 0 and 8 hours after first dose; AUC0–8h, 3, area under the curve between 0 and 8 hours after third dose; Cmax, 1, maximum concentration after first dose; Cmax, 3, maximum concentration after third dose; C8h, 1, concentration at 8 hours after first dose; C8h, 3, concentration at 8 hours after third dose; SD, standard deviation; G.Mean, geometric mean.

Figure 2.

Pharmocokinetics of BILN-2061. Geometric mean plasma BILN-2061 concentration-time profiles for the 500 mg BID dose groups in patients with genotpye 1 (GT1 as previously shown)6 and non–genotype 1 (Non-GT1) hepatitis C.


As observed in previous trials, BILN-2061 was well tolerated. No relevant drug-induced changes in vital signs, routine laboratory findings including troponin-T (3rd generation), or electrocardiogram were observed during the study. Possible drug-related adverse events included mild constitutional symptoms, such as a drunken feeling (n = 1), fatigue (n = 1), and somnolence (n = 1); and mild gastrointestinal symptoms, such as diarrhea (n = 2). All adverse events had resolved by the end of the follow-up period.


This study demonstrates a variable antiviral efficacy of the NS3 serine protease inhibitor BILN-2061 in 8 patients with non–genotype 1 HCV infection. Compared with our previous study, which showed a consistent viral decline in all treated patients with HCV genotype 1 infection, the antiviral effects were less pronounced and exhibited a marked variability between patients. Several issues must be taken into account to explain these results: First, the catalytic activities of the NS3 proteases of HCV genotypes 2 and 3 compared with genoype 1; second, variations in the replication kinetics for different genotypes and for different patients; third, the affinity of BILN-2061 for the NS3 proteases of different genotypes; and fourth, the pharmacokinetic characteristics in different patients.

Cloning and expression of the NS3-NS4 protease complexes of HCV genotypes 1, 2, and 3 displayed no major differences in the protease activity (all within 3-fold) as recently shown by Thibeault et al.,8 making high catalytic activities in HCV genotype 2 and 3 an unlikely cause of a blunted response to BILN-2061. Because viral concentrations of patients with non–genotype 1 HCV infection were not different from the previous trial of patients with HCV genotype 1 and no clear correlation between baseline viral concentration and response to BILN-2061 was observed, differences in replication kinetics between genotypes or patients also seem an unlikely explanation for the observed responses in non–genotype 1 infections. The average plasma concentrations of BILN-2061 in patients with HCV genotype 2 or 3 infection were not different from those in the previously described patients with genotype 1 infection (Fig. 2). The 2 patients with the highest AUC0-8h on days 1 and 2 had the greatest response to treatment, and the 3 patients with the lowest AUC0-8h consistently showed no response. Therefore, pharmacokinetics may contribute to the lesser decrease of serum HCV-RNA in patients with genotype 2 and 3 infection. The number of observations, however, is too low to support any generalization.

The affinity of BILN-2061 for the NS3 protease of genotypes 2 and 3 has been shown to be 50 to 60 times lower than for genotype 1, therefore giving the best explanation for the overall less significant antiviral effects observed in the current study. The substantial patient-to-patient variability of antiviral responses observed in genotype 2 and 3 infections, even within the same genotype, however, can not easily be explained and are most likely the result of a complex interplay of multiple host and viral factors.

In summary, this 2-day proof-of-principle study demonstrates a variable and less pronounced effect of BILN-2061 on HCV genotype 2 and 3 compared with genotype 1 viremia. Because BILN-2061 was specifically designed to inhibit the NS3 protease of HCV genotype 1, variations in the effect on other genotypes were not unexpected and may be the “price” for the high efficacy observed in genotype 1 HCV infection. Individual differences across genotypes represent a problem for the development of the ideal anti-HCV drug, which would be active across different genotypes and prevent resistance. Until recently no genotype 2 or 3 HCV replicon systems have been available for drug candidate testing and optimization, which underscores the significance of the data presented.

A cardiac histological toxicity was identified in rhesus monkeys receiving high doses of BILN-2061 for 4 weeks' duration. Boehringer Ingelheim is continuing to evaluate various aspects of these toxicological findings and the options regarding further development of this compound.


The authors thank Dr. Jens Crönlein, whose guidance and comments were fundamental to this study.