Potency, safety, and pharmacokinetics of the NS3/4A protease inhibitor BI201335 in patients with chronic HCV genotype-1 infection.

Abstract

BI201335 is a highly specific and potent HCV protease inhibitor. This multiple rising dose trial evaluated antiviral activity and safety in chronic HCV genotype-1 patients.
Thirty-four treatment-naïve patients were randomized to monotherapy with placebo or BI201335 at 20-240 mg once-daily for 14 days, followed by combination with pegylated interferon alfa/ribavirin (PegIFN/RBV) through Day 28. Nineteen treatment-experienced patients received 48-240 mg BI201335 once-daily with PegIFN/RBV for 28 days. HCV-RNA was measured with Roche COBAS TaqMan.
In treatment-naïve patients, median maximal viral load (VL) reductions during 14-day monotherapy were -3.0, -3.6, -3.7, and -4.2 log(10) for the 20, 48, 120, and 240 mg groups. VL breakthroughs (≥1 log(10) from nadir) were seen in most patients on monotherapy and were caused by NS3/4A variants (R155K, D168V) conferring in vitro resistance to BI201335. Adding PegIFN/RBV at Days 15-28 led to continuous viral load reductions in most patients. In treatment-experienced patients, treatment with BI201335 and PegIFN/RBV achieved VL<25 IU/ml at Day 28 in 3/6, 4/7, and 5/6 patients in the 48, 120, and 240 mg dose groups. VL breakthroughs were observed during triple combination in only 3/19 patients. BI201335 was generally well tolerated. Mild rash or photosensitivity was detected in four patients. Mild unconjugated hyperbilirubinemia was the only dose-dependent laboratory abnormality of BI201335. BI201335 elimination half-life supports once-daily dosing.
BI201335 combined with PegIFN/RBV was well tolerated and induced strong antiviral responses. These results support further development of BI201335 in HCV genotype-1 patients.

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Potency, safety, and pharmacokinetics of the NS3/4A protease
inhibitor BI201335 in patients with chronic HCV genotype-1 infection
Michael P. Manns1,⇑, Marc Bourlière2, Yves Benhamou3, Stanislas Pol4, Maurizio Bonacini5,
Christian Trepo6, David Wright7, Thomas Berg8, José L. Calleja9, Peter W. White10, Jerry O. Stern11,
Gerhard Steinmann12, Chan-Loi Yong11, George Kukolj10, Joe Scherer11, Wulf O. Boecher12
1Hannover Medical School, Department of Gastroenterology, Hepatology, and Endocrinology, Center for Internal Medicine, Hannover, Germany;
2Hopital Saint Joseph, Marseille, France;3Hopital Pitie Salpetriere, Paris, France;4Hopital Cochin, Paris, France;5California Pacific Medical
Center Research Institute, San Francisco, CA, USA;6Hopital Hotel Dieu, Lyon, France;7Central Texas Clinical Research, Austin, TX, USA;8Charité
Berlin Campus Virchow – Klinikum, Berlin, Germany;9Hospital Universitano Puerta de Hierro, Madrid, Spain;10Boehringer Ingelheim(Canada)
Ltd., Laval, Canada;11Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT, USA;12Boehringer Ingelheim Pharma GmbH & Co. KG,
Biberach, Germany
See Editorial, Pages 1087–1089
Background & Aims:BI201335 is a highly specific and potent
HCV protease inhibitor. This multiple rising dose trial evaluated
antiviral activity and safety in chronic HCV genotype-1 patients.
Methods:Thirty-four treatment-naïve patients were randomized
to monotherapy with placebo or BI201335 at 20–240 mg
once-daily for 14 days, followed by combination with pegylated
interferon alfa/ribavirin (PegIFN/RBV) through Day 28. Nineteen
treatment-experienced patients received 48–240 mg BI201335
once-daily with PegIFN/RBV for 28 days. HCV-RNA was measured
with Roche COBAS TaqMan.
Results:In treatment-naïve patients, median maximal viral load
(VL) reductions during 14-day monotherapy were ?3.0, ?3.6,
?3.7, and ?4.2 log10for the 20, 48, 120, and 240 mg groups. VL
breakthroughs (P1 log10from nadir) were seen in most patients
on monotherapy and were caused by NS3/4A variants (R155K,
D168V) conferring in vitro resistance to BI201335. Adding Peg-
IFN/RBV at Days 15–28 led to continuous viral load reductions
in most patients. In treatment-experienced patients, treatment
with BI201335 and PegIFN/RBV achieved VL <25 IU/ml at Day
28 in 3/6, 4/7, and 5/6 patients in the 48, 120, and 240 mg dose
groups. VL breakthroughs were observed during triple combina-
tion in only 3/19 patients. BI201335 was generally well tolerated.
Mild rash or photosensitivity was detected in four patients. Mild
unconjugated hyperbilirubinemia was the only dose-dependent
laboratory abnormality of BI201335. BI201335 elimination half-
life supports once-daily dosing.
Conclusions:BI201335 combined with PegIFN/RBV was well
tolerated and induced strong antiviral responses. These results
support further development of BI201335 in HCV genotype-1
patients.
? 2010 European Association for the Study of the Liver. Published
by Elsevier B.V. All rights reserved.
Introduction
Hepatitis C virus (HCV) infection is a leading cause of chronic hep-
atitis, liver cirrhosis, and liver cancer worldwide. Genotype-1
(GT-1) represents the most prevalent and also most difficult-to-
treat HCV subtype. Treatment with the current standard-of-care
(SOC) i.e. 48 weeks of pegylated interferon alfa (PegIFN) and
ribavirin (RBV), leads to a sustained virologic response (SVR) in
40–50% of patients, indicative of the resolution of the infection
[1–5]. However, more than 50% of those treated do not respond
or relapse. In addition, many patients cannot tolerate interferon
and/or RBV-based therapies. Furthermore, SVR rates are much
lower forso-calleddifficult-to-treatpopulationslike patientswith
decompensated liver cirrhosis, human immunodeficiency virus
(HIV)-HCV co-infected patients, post-transplant HCV infection,
hemodialysis patients, and others [3].
The HCV protease inhibitor BILN 2061 was the first agent in
man to specifically target HCV replication and thus provided
the proof-of-concept for directly inhibiting HCV NS3/NS4A prote-
ase as a means of suppressing viral replication [6]. This agent
showed 3–4 log10reductions in HCV RNA within 48 h of treat-
Journal of Hepatology 2011 vol. 54j1114–1122
Keywords: Hepatitis C virus; Antiviral; Pegylated interferon; Ribavirin; Resistant
variants.
Received 26 March 2010; received in revised form 18 August 2010; accepted 19
August 2010; available online 11 November 2010
DOI of original articles: 10.1016/j.jhep.2010.12.016,10.1016/j.jhep.2010.11.001.
⇑Corresponding author. Address: Hannover Medical School, Department of
Gastroenterology, Hepatology, and Endocrinology, Center for Internal Medicine,
Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany. Tel.: +49 511 532 3305;
fax: +49 511 532 4896.
E-mail address: Manns.Michael@mh-hannover.de (M.P. Manns).
Abbreviations: HCV, hepatitis C virus; GT-1, genotype-1; SOC, standard-of-care;
PegIFN, pegylated interferon alfa; RBV, ribavirin; SVR, sustained virologic respo-
nse; HIV, human immunodeficiency virus; qd, once-daily; TN, treatment-naïve;
TE, treatment-experienced; HBV, hepatitis B virus; LLOQ, lower limit of quanti-
fication; LLOD, lower limit of detection; VL, viral load; AEs, adverse events; ALT,
alanine aminotransferase; AST, aspartate transaminase; CV%, coefficient of
variation.
Research Article

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ment in HCV GT-1 patients [7], as well as a smaller but significant
activity against GT-2/3 infection [8]. However, the development
of BILN 2061 was halted due to ultrastructural cardiotoxic
changes observed in animals treated with supratherapeutic
doses. The encouraging viral response to this first protease
inhibitor, however, has led to the development of several fol-
low-up agents, many of which are now in different stages of drug
development. Two developmental protease inhibitors, telaprevir
and boceprevir, are now being tested in phase III trials, after suc-
cessful completion of phase II studies [9–11]. In these trials, tela-
previr is given for 8 or 12 weeks on a background of 24 or
48 weeks of PegIFN/RBV, while boceprevir is given for 24 or
44 weeks in combination with 28 to 48 weeks of PegIFN/RBV.
However, both drugs are being given every 8 h or three times
per day and have significant side effects including skin rash, ane-
mia, and gastrointestinal symptoms. Therefore, HCV protease
inhibitors with improved pharmacokinetics, safety, and tolerabil-
ity are needed.
Protease inhibitors given as monotherapy also show rapid
selection of drug-resistant variants. These variants result from
the poor replication fidelity of the virus and are selected due to
the pressure placed on the virus. Consequently, combination of
a HCV protease inhibitor with PegIFN/RBV is currently required
in order to minimize the risk of virologic breakthrough due to
resistance [9–11].
BI201335 is a peptidomimetic HCV-specific protease inhibitor
with high in vitro activity against GT-1a and -1b subtypes, with
EC50 values of 6.5 and 3.1 nM, respectively. Preclinical and
preliminary human pharmacokinetic studies suggested that
BI201335 maintains sufficient plasma concentrations at steady-
state to allow once-daily (qd) dosing (Boehringer Ingelheim,
unpublished data).Amultiplerising dosestudyin volunteersindi-
cated that BI201335 was safe and well tolerated at doses of 20–
240 mg qd for 21–28 days (Boehringer Ingelheim, unpublished
data). These observations allowed progression to the phase Ib trial
(1220.2) reported here.
Patients and methods
Study design
This was a randomized, multi-center, multiple rising-dose trial, performed
between December 2007 and June 2008 at 16 sites in France, Germany, Spain,
and USA. The trial was double-blind and placebo-controlled for groups with treat-
ment-naïve (TN) patients. The trial was conducted in full compliance with the
Guidelines of Good Clinical Practice and the Declaration of Helsinki and approved
by all competent institutional review boards, ethical committees, and national
authorities. All patients gave written informed consent. An overview of the trial
design is provided in Fig. 1.
Study treatments
TN patients were randomized to receive, in successive cohorts, either BI201335
monotherapy (20, 48, 120, and 240 mg qd) for 14 days, or matching placebo in a
3:1 ratio within each cohort. The data from each cohort was reviewed by a Data
Monitoring Committee prior to enrolling the next dose cohort. In patients with
a HCV RNA decrease P1 log10from baseline (on Day 10), BI201335 treatment
was combined with PegIFN alfa 2a (180 lg/week) and RBV (1000 or 1200 mg/
day for body weight 6 or >75 kg) from Days 14 to 28. Treatment-experienced
(TE) patients received triple combination treatment for 28 days: BI201335 at
48, 120, or 240 mg qd, combined with PegIFN alfa 2a and RBV as above. The
data from each cohort were reviewed by a Data Monitoring Committee prior
to enrolling the next dose cohort. All patients were offered to extend SOC to
Week 48, with an additional 24 weeks of follow-up, at the discretion of the
investigator.
Inclusion and exclusion criteria
Patients with chronic HCV infection of GT-1 were recruited to the study, if they
were TN (no prior therapy with interferon, PegIFN, or RBV) or TE (virologic fail-
ure during or after treatment with an approved dose of PegIFN combined with
RBV), had HCV RNA P100,000 IU/ml and were aged 18 years or older. Patients
with liver cirrhosis, hyperbilirubinemia (>1.5? upper limit of normal; patients
with Gilbert’s disease were accepted), HIV, or hepatitis B virus (HBV) co-infec-
tion were excluded. Furthermore, patients who had previously received any
treatment with a protease inhibitor and females of child-bearing potential not
agreeing or able to use medically accepted contraception throughout the study
were excluded.
Pre-treatment response as reported by the investigator was classified in accor-
dance with recent FDA definitions [12]; relapse: re-appearance of HCV RNA in
serum after treatment discontinuation; break-through: re-appearance of HCV
RNA during treatment; null response: maximum reduction in HCV RNA <2 log10
at Week 12 of treatment; partial response: maximal reduction in HCV RNA at Week
12 >2 log10 but HCV RNA never undetectable. All patients provided written
informed consent before study participation.
Assessments
Analysis of HCV RNA and genotype
Plasma samples (10 ml) for HCV RNA analysis were obtained from all patients at
for Days 1, 2, 3, 4, 6, 10, and 14. Additionally, for TN patients, for Days 15, 16, 17,
18, and 28, samples were taken from those having achieved an HCV RNA reduc-
tion by P1 log10at Day 10. Similarly, for TE patients with a P1 log10HCV RNA
decline at Day 10, additional samples were taken on Days 21 and 28. Plasma sam-
ples were processed in a central laboratory using the Roche COBAS TaqMan HCV/
HPS assay (Roche Diagnostics, Indianapolis, IN). The lower limit of quantification
(LLOQ) was 25 IU/ml; the lower limit of detection (LLOD) approximates 10 IU/ml,
although not validated by the manufacturer. HCV genotype was determined using
the Trugene HCV assay (Bayer, Leverkusen, Germany). Viral load (VL) break-
through was defined as HCV RNA rebound P1 log10from the HCV RNA nadir or
to P1000 IU/ml if HCV RNA was previously undetectable.
Genotypic and phenotypic resistance monitoring
Viral RNA was isolated from the plasma of HCV-infected subjects, at baseline and
indicated time-points, using the QiaAmp Viral RNA extraction kit (Qiagen, Hilden,
Germany). A DNA fragment containing the complete NS3/NS4A region was syn-
thesized using Superscript III one step reverse transcription polymerase chain
reaction system and two gene-specific primers. Two second-round, semi-nested
polymerase chain reaction products spanning either the entire NS3/NS4A region
or the NS3 protease domain were generated using Novagen KOD Hot Start DNA
polymerase (Merck, Darmstadt, Germany). The LLOD of the reverse transcription
polymerase chain reaction amplification method restricted the analysis to patient
samples with a VL P1000 IU/ml. The NS3/NS4A nucleotide sequence was
obtained by direct DNA sequencing of the amplified product using Big Dye Termi-
nator V3.1 and the ABI 3100 Genetic Analyzer (Applied Biosystems, CA, USA)
detection system in combination with 3100 Genetic Analyzer Data collection soft-
ware V1.1.
0244872
Time (weeks)
SOC BI201335
SVR
BI201335 +
PegIFN/RBV
No treatment
SVR End treatment
End treatment
<1 log
discontinue
TE patients
TN patients
BI201335 +
PegIFN/RBV
No treatment SOC
Fig. 1. Trial design.
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For phenotypic resistance testing, polymerase chain reaction products from
HCV-infected patient plasma samples were used to amplify DNA fragments con-
taining the NS3 protease domain. NS3 amplicons were ligated into a HCV replicon
shuttle vector (pIT2) containing a luciferase reporter gene, and reconstituted plas-
mid DNA was used to generate HCV subgenomic replicon RNA transcripts (T7
Ribomax kit; Promega, WI, USA). The in vitro transcribed RNA was electroporated
into Huh-7.5 cells which were then seeded in 96-well plates for 24 h and treated
with a range of BI201335 (or interferon alfa) concentrations for a period of 72 h.
At the end of the incubation, luciferase activity was measured with Bright-Glo
substrate, as a marker for HCV RNA replication, and luminescence quantified.
The concentration of BI201335 giving 50% inhibition of HCV RNA replication
(EC50) was determined.
Safety assessments
Adverse events (AEs) were recorded descriptively for all patients receiving study
drugs and coded using the Medical Dictionary for Drug Regulatory Affairs. Vital
signs (blood pressure, pulse rate), electrocardiogram and safety laboratory
parameters were also evaluated.
Assessment of drug plasma concentrations
Plasma samples were collected and stored at ?20 ?C at various times during the
course of the study. Plasma concentrations of BI201335 were determined by a
validated high performance liquid chromatography and tandem mass spectrom-
etry assay method with a LLOQ of 0.2 ng/ml. Pharmacokinetic parameters cal-
culated from the plasma concentration–time data on Day 28 following the
last BI201335 dose administration were obtained by non-compartmental
analysis.
Statistical assessments
Activity and safety
Due to the exploratory multiple rising dose design of this trial, sample sizes per
cohort sufficient and typical for early phase Ib trials were chosen. Frequency
tables for categorical endpoints and summary statistics including mean, med-
ian, quartiles, and minimum and maximum for continuous endpoints are
presented.
Results
Patient disposition and baseline characteristics
In 2007 and 2008, a total of 171 patients were screened: 53
patients were randomized to treatment in this study; 75 patients
did not meet the inclusion criteria, 43 patients were entered into
later cohorts that will be reported in a separate manuscript. The
rate of study completion was high; 52 out of 53 patients random-
ized to treatment completed the study. The reason for the discon-
tinuation of one subject was the diagnosis of an unexpected
pregnancy of his partner representing an exclusion criterion for
treatment with RBV. Baseline demographics were similar across
all cohorts (Table 1).
Antiviral response
TN patients
With the exception of one patient in the 20 mg treatment group,
all patients receiving BI201335 (n = 25, 96.2%) achieved the pri-
mary efficacy endpoint of P2 log10plasma VL reduction at any
time up to Day 14. No significant change in VL was observed in
any patients in the placebo-control groups. Maximum changes
from baseline to Day 14 (for naïve patients) were dose-dependent
and are summarized in Table 2A and median VL changes from
baseline are shown in Fig. 2A.
In the active treatment groups, HCV RNA decline was rapid,
with a nadir that typically reached 2–3 days after starting
monotherapy (Fig. 2B). However, in the majority of patients in
all dose groups, virologic breakthrough during treatment was
seen in the first 14 days of monotherapy (Fig. 2). The proportion
of patients with viral breakthrough by Day 14 was 83.3%, 71.4%,
71.4%, and 83.3% with 20, 48, 120, and 240 mg qd of BI201335,
respectively.
Following 2 weeks of monotherapy, SOC (PegIFN/RBV) treat-
ment was added in patients with a P1 log10reduction in VL at
Days 6 or 10. This criterion was met in none of the placebo
patients, but in all patients on BI201335. After start of SOC treat-
ment, 4/6, 4/7, 6/7, and 6/6 patients treated with 20, 48, 120, or
240 mgBI201335, respectively,
P1 log from Days 15 to 28.
showed VL reductionsby
TE patients
In all TE triple combination groups, rapid, strong VL reductions
were observed from Days 1 to 28 during triple-combination ther-
apy (Fig. 3A). All TE patients achieved the primary efficacy end-
point of P2 log10 reduction in VL from baseline during
treatment. As shown in Fig. 3A, median VL reduction appeared
to be dose-dependent. The numbers of patients in the 120 and
240 mg dose groups attaining rapid viral response at Day 28
showed a trend to decreased breakthroughs and increased rapid
viral response rates compared to the 48 mg group, as shown in
Table 2B.
In contrast to monotherapy, VL breakthrough was rare
under triple therapy with BI201335 and PegIFN/RBV, despite
enrollment of pre-treatment failures including null-responders,
as only three patients with VL breakthrough were observed:
two in the 48 mg and one in the 120 mg dose groups (two
previous null-responders, one partial responder). There were
no breakthroughs at the highest dose level of 240 mg qd
(Fig. 3b).
Across all dose groups, 3 of 6 null-responders, 4 of 7 partial
responders, 0 of 1 breakthrough and 5 of 5 relapsers to
previous PegIFN/RBV treatment experienced a RVR (VL <25 IU/
ml at Day 28) during re-treatment with BI201335 plus Peg-
IFN/RBV.
Safety
Monotherapy (TN patients)
During the 14-day monotherapy period, the frequency of AEs was
similar with all doses of BI201335, compared with placebo. Most
AEs were mild-to-moderate in intensity and not dose-dependent
in frequency or intensity. There were no reports of rash or ery-
thema during monotherapy with BI201335.
Safety laboratory analyses indicated that there was no nega-
tive impact of monotherapy on blood counts, liver enzymes, or
other safety parameters in TN patients (Table 3A). Median alanine
aminotransferase (ALT) and aspartate transaminase (AST) con-
centrations decreased in all dose groups from baseline to Day
14, accompanying the decline in HCV RNA. There was a slight
and dose-dependent increase of the median unconjugated biliru-
bin (?0.1, +0.1, +0.3, +0.8 mg/dl in the 20, 48, 120, and 240 mg
dose groups, respectively).
Research Article
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Combination therapy (TN and TE patients)
Under combination therapy of BI201335 with PegIFN/RBV, the
most common AEs in both, TN or TE patients, were fatigue, nau-
sea, headache, gastrointestinal tract disorders, and anemia. Three
cases of mild rash or erythema occurred in TN patients during
treatment with 20, 48, or 240 mg BI201335 and SOC, that all
Table 1. Summary of baseline characteristics for (A) treatment-naïve and (B) treatment-experienced patient cohorts.
A
TN
Placebo
(n = 8)
Gender, n (%)
Male
Female
Genotype, n (%)
1
1a
1b
Race, n (%)
Caucasian
6 (75)
2 (25)
1 (12.5)
2 (25.0)
5 (62.5)
8 (100)
Asian 0 (0)
49.9 (8.4)
27.2 (6.0)
58 (24)
6.8
(6.0–7.3)
Age, years (SD)
Body mass index, kg/m2
ALT, U/L (SD)
HCV RNA, log10IU/ml
Median (range)
BI201335
20 mg qd
(n = 6)
6 (100)
0 (0)
0 (0)
3 (50.0)
3 (50.0)
5 (83.3)
1 (16.7)
51.0 (8.0)
23.4 (2.2)
67 (40)
7.1
(6.4–7.7)
BI201335
48 mg qd
(n = 7)
5 (71.4)
2 (28.6)
0 (0)
2 (28.6)
5 (71.4)
7 (100)
0 (0)
47.1 (13.8)
25.4 (5.6)
101 (60)
6.7
(4.7–7.3)
BI201335
120 mg qd
(n = 7)
5 (71.4)
2 (28.6)
2 (28.6)
2 (28.6)
3 (42.9)
7 (100)
0 (0)
48.6 (16.3)
26.6 (3.0)
87 (47)
6.5
(5.8–7.0)
BI201335
240 mg qd
(n = 6)
5 (83.3)
1 (16.7)
1 (16.7)
3 (50.0)
2 (33.3)
6 (100)
0 (0)
47.8 (9.3)
26.8 (3.8)
53 (11)
6.8
(6.0–7.2)
TE
Gender, n (%)
Male
Female


Genotype, n (%)
1

1a
1b


Race, n (%)
Caucasian
Age, years (SD)
Body mass index, kg/m2
ALT, U/L (SD)
HCV RNA, log10 IU/ml
Median (range)
Pre-treatment response, n
Relapse
Break-through
Null-response
Partial response





dqgm84533102 IB
+ PegIFN/RBV
(n = 6)
4 (66.7)
2 (33.3)
) 0 . 0 (0
3 (50.0)
3 (50.0)
6 (100.0)
46.8 (11.8)
26.4 (4.4)
74 (37)
7.1
(5.9–7.3)
2
1
2
1
BI201335 120 mg qd
+ PegIFN/RBV
(n = 7)
4 (57.1)
3 (42.9)
1 (14.3)
4 (57.1)
2 (28.6)
7 (100.0)
46.7 (4.8)
25.4 (6.1)
86 (34)
7.0
(6.2–7.4)
1
0
4
2
BI201335 240 mg qd
+ PegIFN/RBV
(n = 6)
3 (50.0)
3 (50.0)
1 (16.7)
4 (66.7)
1 (16.7)
6 (100.0)
49.3 (10.1)
28.0 (4.0)
89 (64)
6.8
(6.3–7.1)
2
0
0
4
B
Abbreviations: TN, treatment-naïve; TE, treatment-experienced; ALT, alanine aminotransferase; HCV, hepatitis C virus; SD, standard deviation; qd, once-daily; relapse
[re-appearance of HCV RNA in serum after treatment discontinued], break-through [re-appearance of HCV RNA in serum during treatment], null response: <2 log10
maximum reduction in HCV RNA at week 12 of treatment; partial response: maximal reduction in HCV RNA at week 12 P2 log10, but never achieved HCV RNA below level
of detection.
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recovered spontaneously. In addition, one case of mild photosen-
sitivity reaction emerged in a TE patient treated with 240 mg
BI201335 and SOC. Following the initiation of therapy with
PegIFN/RBV, two patients experienced serious AEs (asthenia,
cataract); neither were considered to be related to the study drug.
There were no AEs leading to pre-term discontinuation of
treatment. The only other changes were decreases of blood
counts from baseline to Day 28 and a slight dose-dependent
increase of unconjugated bilirubin across the three dose groups
(Table 3B). The highest total bilirubin values were 4 mg/dl in
one patient treated with 240 mg qd and 3.2 mg/dl in two individ-
uals treated with 120 and 240 mg QD. Unconjugated hyperbiliru-
binemiawasisolated,asymptomatic
completion of BI201335 in all affected patients. However, there
were no reports of jaundice during treatment with BI201335
and no signs of liver toxicity or hemolysis in individuals with ele-
vated unconjugated bilirubin.
andreversible after
Viral resistance
Population sequencing of viral isolates obtained at baseline
revealed in one patient a variant encoding an NS3V/I170T substi-
tution that conferred a sevenfold reduction in BI201335 sensitiv-
ity (increased EC50) relative to the subtype reference; the patient
harboring this variant was in the lowest dose group (20 mg TN)
who had failed to achieve P2 log10VL reduction within the first
14 days. HCV subgenomic replicon based phenotyping of all other
baseline-derived NS3 amplicons resulted in mean EC50values for
GT-1a (10 ± 8 nM) or -1b (9 ± 4 nM) that were within threefold of
the reference values.
In patients experiencing viral breakthroughs, the predominant
GT-1a resistance mutations in on-treatment samples encoded an
R155K substitution, whereas GT-1b viruses mainly encoded
changes at D168, with valine (D168V) as a predominant and
glutamate (D168E) as a rare substitution. R155K variants con-
ferred reductions in sensitivity to BI201335 with EC50values of
1.8–6.5 lM, whereas the EC50values for D168V variants were
3.6–15 lM (Table 4). However, all BI201335 resistant mutant
replicons remained susceptible to inhibition by interferon alfa.
The details of these phenotyping and genotyping studies, includ-
ing longitudinal clonal sequence analyses, will be reported in a
follow-up paper.
Pharmacokinetics
Steady-state pharmacokinetic parameters of BI201335 following
the last dose on Day 28 are summarized in Table 5. Plasma
BI201335 concentrations peaked at 2–6 h (tmax) and increased
supra-proportionally with dose as seen for mean Cmax, Cminand
AUC0–s.Mean elimination
approximately 20–30 h, suggesting suitability for qd dosing. No
apparent difference was found between TN and TE HCV patients
in plasma concentrations and exposure. Inter-individual variabil-
ity was moderately high with a coefficient of variation (CV%) for
AUC0–sof up to 65%.
half-lifeofBI201335was
Discussion
This trial investigated the antiviral activity, safety and pharmaco-
kinetics of BI201335 in 53 patients with HCV GT-1 infection trea-
ted with BI201335 ± PegIFN and RBV for 28 days.
When given qd as monotherapy in TN patients for 14 days,
48–240 mg BI201335 qd induced a rapid, dose-dependent
decrease in plasma HCV RNA by P2 log10from baseline in all
patients. Maximal VL declines from baseline occurred within 2–
3 days of first administration. At the 240 mg qd dose, the median
maximal decline from baseline was ?4.4 log10IU/ml. The magni-
tude of VL declines was similar to those recorded for other highly
Table 2. HCV RNA maximum change from (A) baseline to day 14 of BI201335 monotherapy in treatment-naïve patients and (B) baseline to day 28 of combination
therapy with BI201335 and PegIFN/RBV in treatment-experienced patients; and proportion of patients achieving HCV RNA <LLOD (RNA <10 IU/ml) and <LLOQ (RNA
<25 IU/ml) at Week 4 (intention-to-treat cohort).
A
Placebo
(n = 8)
BI201335
20 mg qd
(n = 6)
BI201335
48 mg qd
(n = 7)
BI201335
120 mg qd
(n = 7)
BI201335
240 mg qd
(n = 6)
Max. change from baseline
Median (min, max), log10 IU/ml
RNA LLOD, n (%)
RNA LLOQ, n (%)
B
Max. change from baseline
Median (min, max), log10 IU/ml
RNA LLOD, n (%)
RNA LLOQ, n (%)
BI201335 48 mg qd
+ PegIFN/RBV
(n = 6)
2 (33.3)
3 (50.0)
BI201335 120 mg qd +
PegIFN/RBV
(n = 7)
5.2
( 6.0, 3.9)
-
1 (14.3)
4 (57.1)
BI201335 240 mg qd +
PegIFN/RBV
(n = 6)
5.3
( 6.1, 4.7)
-
) 7. 61 (1
5 (83.3)
-
-
4.4
( 4.9, 3.8)
-
1 (16.7)
3 (50.0)
-
-
3.7
( 4.1, 3.3) -
1 (14.3)
2 (28.6)
-
-
3.6
( 3.8, 3.1)
-
2 (28.6)
) 6 . 8 2 (2
-
-
3.0
( 3.9, 1.5)
-
) 0 (
1 (
0
1 ) 7 . 6
-
-
-
-
( 5.9, 3.4)
--
5.0
0.06
, 7 . 0(-
0 (0)
0 (0)
-
-
) 1 . 0
Abbreviations: HCV, hepatitis C virus; LLOD, lower limit of detection (RNA <10 IU/ml); LLOQ, lower limit of quantification (RNA <25 IU/ml); PegIFN/RBV, pegylated interferon
alfa/ribavirin; qd, once-daily.
Research Article
1118
Journal of Hepatology 2011 vol. 54j1114–1122