May 2012
Hepatitis C – BI-201335The standard treatment for hepatitis C infection is with an interferon plus the antiviral drug ribavirin; not only does this unpleasant treatment last for many months, only around half of all those who are treated respond, leaving few options if it fails.

April 2012
EASL 2012 -BI201335, a (HCV) protease inhibitor and BI207127, a non-nucleoside RNA polymerase inhibitor

Interferon-Free Combination Regimen Shows Promise for the Treatment of Chronic HCV
By Chris Berrie
Source: DGNews

BARCELONA, Spain -- April 25, 2012 -- An interferon-free combination regimen
consisting of BI201335, a hepatitis C virus (HCV) protease inhibitor and
BI207127, a non-nucleoside RNA polymerase inhibitor, with or without ribavirin,
shows potent antiviral activity in patients with chronic HCV.

Sustained virological response (SVR) rates were achieved by 60% of patients
receiving interferon-free combination therapy with BI201335, BI207127, and
ribavirin at 12 weeks, researchers said here on April 21 at the 47th Annual
Meeting of the European Association for the Study of the Liver (EASL).

In the study, 362 treatment-naïve patients were randomised to 1 of 5
treatment arms: BI201335 120 mg QD plus BI207127 600 mg TID and ribavirin for 16
weeks (arm A), 28 weeks (arm B), or 40 weeks (arm C); BI201335 120 mg QD plus
BI207127 600 mg BID plus ribavirin for 28 weeks (arm D); or BI201335 120 mg QD
plus BI207127 600 mg BID without ribavirin for 28 weeks (arm E).

Patient baseline characteristics were well balanced between groups. However,
there were more patients in arm D with liver cirrhosis (17%).

SVR Rates for arms A, B, C, and D were 59%, 61%, 56%, and 68%,
respectively.

Only 39% of patients in arm E -- the group not receiving ribavirin --
achieved SVR, and thus randomisation in this arm was closed early.

Arm D received the most benefit from treatment, according to Stefan Zeuzem,
MD, Department of Medicine, Johann Wolfgang Goethe University Hospital,
Frankfurt, Germany, and colleagues.

Lower efficacy was seen among patients with genotype 1a and 1b without
cirrhosis.

The safety and tolerability profile of BI201335, BI207127, and RBV was
comparable to other direct acting antiviral regimens.

“This data justifies phase 3 studies investigating the BID regimen in HCV
patients,” said Dr. Zeuzem.

Funding for this study was provided by Boehringer Ingelheim.

[Presentation title: SVR4 and SVR12 With the Interferon-Free Regimen of BI
201335 and BI 207127 ± Ribavirin, in Treatment-Naïve Patients With Chronic
Genotype-1 HCV Infection: Interim Results of SOUND-C2. Abstract 101]


SVR4 and SVR12 with an interferon-free regimen of BI 201335 AND BI 207127,  +/- ribavirin, in
treatment-naïve patients with chronic genotype-1 HCV infection:  Interim results
of SOUND-C2
  
 
Reported by Jules Levin
 Source:Coverage of the EASL @ NATAP
Slides Provided By NATAP

EASL 2012
Stefan Zeuzem,1 Vicente Soriano,2 Tarik Asselah,3 Jean-Pierre Bronowicki,4  Ansgar W. Lohse,5 Beat Mullhaupt,6 Marcus Schuchmann,7 Marc Bourliere,8  Maria Buti,9 Stuart Roberts,10 Ed Gane,11 Jerry O. Stern,12
George Kukolj,12  Luyan Dai,12 Wulf O. Böcher,13 Federico J. Mensa13 1Klinikum der J. W. Goethe-Universitat, Frankfurt am Main, Germany; 2Department of Infectious Diseases, Hospital Carlos III, Madrid, Spain;
3Hopital Beaujon, Clichy, France; 4Hopital de Brabois, Vandoeuvre, France; 5University Hospital Hamburg-Eppendorf, Hamburg; 6University Hospital of Zurich, Zurich, Switzerland; 7University Hospital Mainz, Mainz, Germany; 8Hopital Saint
Joseph, Marseille Cedex, France; 9Hospital Vall d'Hebron, Barcelona, Spain; 10Alfred Hospital, Department of Gastroenterology, Melbourne, Australia; 11Auckland Clinical Studies, Auckland, New Zealand; 12Boehringer Ingelheim
Pharmaceuticals, Ridgefield, CT, USA; 13Boehringer Ingelheim Pharma GmbH & Co KG, Biberach, Germany  

Dec 2011

BI 201335-Boehringer announced final patient entry for Phase III Trial
Boehringer Ingelheim today announced that the final patient has been randomised to treatment in the large-scale Phase III clinical trial programme for BI 201335, its investigational, oral protease inhibitor for the treatment of chronic hepatitis C virus (HCV).
The extensive study programme is underway at more than 350 sites in 15 countries and together encompasses nearly 2,000 treatment-experienced as well as treatment-naïve patients. Key regions in the programme include the European Union, Japan, U.S., Canada, Taiwan, Korea and Russia......


Novel Hep C Treatment Excludes Peginterferon Alfa
By: DENISE NAPOLI, Internal Medicine News Digital Network
Therapy with a novel nonnucleoside polymerase inhibitor, in combination with a protease inhibitor and ribavirin, achieved a 100% virologic response rate at 29 days among patients with hepatitis C genotype 1, Dr. Stefan Zeuzem and colleagues reported in the December issue of Gastroenterology.
Moreover, this novel alternative to the standard peginterferon alfa regimen was safe, with no serious adverse events, and was highly tolerable, the investigators said.
"This indicates that HCV [hepatitis C virus] can be eradicated in chronically infected patients with a PegIFN-free DAA [direct-acting antiviral agent] combination regimen," the authors wrote.
Dr. Zeuzem, of the Johann Wolfgang Goethe University Hospital in Frankfurt, Germany, and colleagues studied 34 adult patients (mean age 51 years) who were naive to interferon, PegIFN, ribavirin, or any DAA for acute or chronic hepatitis C infection.
All patients had plasma HCV RNA levels of at least 100,000 IU/mL at screening and did not have cirrhosis.
Patients with hepatitis B virus, human immunodeficiency virus, previous or ongoing rash or photosensitivity, decompensated liver disease, or hyperbilirubinemia greater than 1.5 times the upper limit of normal were excluded.
Participants were randomized in a 1:1 fashion to receive a thrice-daily 400-mg or 600-mg dose of BI 207127, "an orally bioavailable, reversible, thumb pocket 1 nonnucleoside inhibitor (NNI) of the HCV NS5B polymerase with potent and specific antiviral activity in vitro," according to the authors (Gastroenterology 2011 [doi:10.1053/j.gastro.2011.08.051]).
"This indicates that HCV can be eradicated in chronically infected patients with a PegIFN-free DAA combination regimen."
All participants also received BI 201335, "a second-generation HCV NS3/4A protease inhibitor with highly potent in vitro activity against GT-1a/1b subtypes," wrote the authors, at a dose of 120 mg per day, as well as daily weight-dosed ribavirin, all taken with food, for 4 weeks.
"Drug-resistance studies in cell culture demonstrate that BI 201335 and BI 207127 have different resistance profiles, and previous observations using NS3/4A protease inhibitors and NS5B thumb pocket 1 NNI compounds demonstrate that 2-drug combinations profoundly reduce the selection of drug-resistant variants," explained Dr. Zeuzem.
The researchers found that in the cohort taking the larger, 600-mg doses of the polymerase inhibitor BI 207127, virologic response rates (defined as plasma HCV RNA levels of less than 25 IU/mL) were 18%, 82%, 100%, and 100% at days 8, 15, 22, and 29, respectively.
Slightly less impressive results were seen in the 400-mg group, with rates of 27%, 47%, 67%, and 73% at days 8, 15, 22, and 29 respectively.
Also in the 400-mg cohort, "higher response rates were observed in genotype 1b infected patients, compared with genotype 1a infected patients," wrote the authors, whereas results in the 600 mg group were not contingent on sub-genotypes.
"Most patients in the 600 mg TID [three times a day] dose group even had undetectable HCV RNA at days 22 and 29 (53% and 71%, respectively), while these rates did not exceed 20% in the 400 mg TID dose group," added the investigators.
Looking at safety and tolerability, Dr. Zeuzem and colleagues noted that most patients in both dosing cohorts complained of mild diarrhea, nausea, and vomiting.
Additionally, at the 600-mg dose, 42% developed mild rashes or photosensitivities, they said, and four patients developed "transient and very mild paresthesias of very different localizations."
"However, there were no severe AEs [adverse events], serious AEs or AE-related premature treatment discontinuations within the 4-week study period."
The "crucial next step," according to the investigators, will be achievement of longer-term SVR on the novel peginterferon-free regimen.
"Moreover, eliminating not only PegIFN but also RBV [ribavirin] from future HCV treatment would undoubtedly improve tolerability and would potentially allow for the treatment of patients with RBV contraindications," they added.
Indeed, at the time of this study’s publication, a phase 2b study was ongoing.
Dr. Zeuzem, along with several of his coinvestigators, disclosed financial and consulting relationships with multiple pharmaceutical companies, including the makers of the novel drugs used in this study, Boehringer Ingelheim. Boehringer Ingelheim also funded editorial assistance provided for this article.

June 10
BI201335 Shows Potent Activity against HCV
The advent of direct-acting antiviral agents active against different steps of the hepatitis C virus (HCV) lifecycle will dramatically change therapy, especially for difficult-to-treat patients. Current drugs, however, still must be used in combination with pegylated interferon alfa (Pegasys or PegIntron) plus ribavirin. The first 2 HCV protease inhibitors -- boceprevir (Victrelis) and telaprevir (Incivek) -- were approved in May; next-generation agents are currently moving through the development pipeline.

As described in the June 2011 Journal of Hepatology, Michael Manns from Hannover Medical School and colleagues evaluated the safety and efficacy of Boehringer-Ingelheim's candidate, BI201335, in a dose-escalation trial of treatment-naive and experienced participants.

A total of 34 previously untreated genotype 1 chronic hepatitis C patients were randomly assigned to receive BI201335 at doses of 20 mg to 240 mg once-daily, or else placebo, for 14 days, followed by pegylated interferon/ribavirin through day 28. In addition, 19 treatment-experienced people received 48 mg to 240 mg BI201335 once-daily in combination with pegylated interferon/ribavirin for 28 days.
Read More http://www.hivandhepatitis.com/hep_c/news/2011/0610_2011_a.html

April 2011

BI 201335 for Hepatitis C Moves into Phase 3 Trials

Hepatitis C;Phase 3 trials recruiting to evaluate BI 201335 Plus SOC

SVR and pharmacokinetics of the HCV protease inhibitor BI 201335 with PegIFN/RBV in HCV genotype-1 patients with compensated liver cirrhosis and non-response to previous PegIFN/RBV -  

BI 201335 pharmacokinetics and early effect on viral load in HCV genotype-1 patients



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

Michael P. Manns1, , Marc Bourliere2, Yves Benhamou3, Stanislas Pol4, Maurizio Bonacini5, Christian Trepo6, David Wright7, Thomas Berg8, Jose 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; 8Charite 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 

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 20-240mg once-daily for 14days, followed by combination with pegylated interferon alfa/ribavirin (PegIFN/RBV) through Day 28. Nineteen treatment-experienced patients received 48-240mg BI201335 once-daily with PegIFN/RBV for 28days. 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 -4.2log10 for the 20, 48, 120, and 240mg groups. VL breakthroughs (>1log10 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<25IU/ml at Day 28 in 3/6, 4/7, and 5/6 patients in the 48, 120, and 240mg 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. 

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. 

Introduction  
Hepatitis C virus (HCV) infection is a leading cause of chronic hepatitis, 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. 48weeks 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], [2], [3], [4], [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 for so-called difficult-to-treat populations like patients with 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 protease as a means of suppressing viral replication [6]. This agent showed 3-4log10 reductions in HCV RNA within 48h of treatment 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 follow-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 successful completion of phase II studies [9], [10], [11]. In these trials, telaprevir is given for 8 or 12weeks on a background of 24 or 48weeks of PegIFN/RBV, while boceprevir is given for 24 or 44weeks in combination with 28 to 48weeks of PegIFN/RBV. However, both drugs are being given every 8h or three times per day and have significant side effects including skin rash, anemia, and gastrointestinal symptoms. Therefore, HCV protease inhibitors with improved pharmacokinetics, safety, and tolerability 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], [10], [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 3.1nM, 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). A multiple rising dose study in volunteers indicated that BI201335 was safe and well tolerated at doses of 20-240mg qd for 21-28days (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 treatment-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 240mg qd) for 14days, 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 >1log10 from baseline (on Day 10), BI201335 treatment was combined with PegIFN alfa 2a (180μg/week) and RBV (1000 or 1200mg/day for body weight ≤ or >75kg) from Days 14 to 28. Treatment-experienced (TE) patients received triple combination treatment for 28days: BI201335 at 48, 120, or 240mg 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 24weeks 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 failure during or after treatment with an approved dose of PegIFN combined with RBV), had HCV RNA >100,000IU/ml and were aged 18years or older. Patients with liver cirrhosis, hyperbilirubinemia (>1.5x upper limit of normal; patients with Gilbert's disease were accepted), HIV, or hepatitis B virus (HBV) co-infection 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 accordance 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<2log10 at Week 12 of treatment; partial response: maximal reduction in HCV RNA at Week 12 >2log10 but HCV RNA never undetectable. All patients provided written informed consent before study participation. 

Assessments  Analysis of HCV RNA and genotype  

Plasma samples (10ml) 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 reduction by >1log10 at Day 10. Similarly, for TE patients with a >1log10 HCV RNA decline at Day 10, additional samples were taken on Days 21 and 28. Plasma samples 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 25IU/ml; the lower limit of detection (LLOD) approximates 10IU/ml, although not validated by the manufacturer. HCV genotype was determined using the Trugene HCV assay (Bayer, Leverkusen, Germany). Viral load (VL) breakthrough was defined as HCV RNA rebound 1log10 from the HCV RNA nadir or to 1000IU/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 synthesized 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 >1000IU/ml. The NS3/NS4A nucleotide sequence was obtained by direct DNA sequencing of the amplified product using Big Dye Terminator V3.1 and the ABI 3100 Genetic Analyzer (Applied Biosystems, CA, USA) detection system in combination with 3100 Genetic Analyzer Data collection software V1.1. For phenotypic resistance testing, polymerase chain reaction products from HCV-infected patient plasma samples were used to amplify DNA fragments containing the NS3 protease domain. NS3 amplicons were ligated into a HCV replicon shuttle vector (pIT2) containing a luciferase reporter gene, and reconstituted plasmid 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 24h and treated with a range of BI201335 (or interferon alfa) concentrations for a period of 72h. 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 spectrometry assay method with a LLOQ of 0.2ng/ml. Pharmacokinetic parameters calculated 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, median, 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 randomized to treatment completed the study. The reason for the discontinuation 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 20mg treatment group, all patients receiving BI201335 (n=25, 96.2%) achieved the primary efficacy endpoint of >2log10 plasma 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 2-3days after starting monotherapy (Fig. 2B). However, in the majority of patients in all dose groups, virologic breakthrough during treatment was seen in the first 14days 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 240mg qd of BI201335, respectively. Following 2weeks of monotherapy, SOC (PegIFN/RBV) treatment was added in patients with a >1log10 reduction 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 treatment, 4/6, 4/7, 6/7, and 6/6 patients treated with 20, 48, 120, or 240mg BI201335, respectively, showed VL reductions by >1log from Days 15 to 28. 

TE patients  

In all TE triple combination groups, rapid, strong VL reductions were observed from Days 1 to 28 during triple-combination therapy (Fig. 3A). All TE patients achieved the primary efficacy endpoint of >2log10 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 240mg dose groups attaining rapid viral response at Day 28 showed a trend to decreased breakthroughs and increased rapid viral response rates compared to the 48mg 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 48mg and one in the 120mg dose groups (two previous null-responders, one partial responder). There were no breakthroughs at the highest dose level of 240mg 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<25IU/ml at Day 28) during re-treatment with BI201335 plus PegIFN/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 erythema during monotherapy with BI201335. Safety laboratory analyses indicated that there was no negative 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) concentrations 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 bilirubin (-0.1, +0.1, +0.3, +0.8mg/dl in the 20, 48, 120, and 240mg dose groups, respectively).

 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, nausea, headache, gastrointestinal tract disorders, and anemia. Three cases of mild rash or erythema occurred in TN patients during treatment with 20, 48, or 240mg BI201335 and SOC, that all recovered spontaneously. In addition, one case of mild photosensitivity reaction emerged in a TE patient treated with 240mg 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 4mg/dl in one patient treated with 240mg qd and 3.2mg/dl in two individuals treated with 120 and 240mg QD. Unconjugated hyperbilirubinemia was isolated, asymptomatic and reversible after 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 elevated unconjugated bilirubin. 

Viral resistance  

Population sequencing of viral isolates obtained at baseline revealed in one patient a variant encoding an NS3V/I170T substitution that conferred a sevenfold reduction in BI201335 sensitivity (increased EC50) relative to the subtype reference; the patient harboring this variant was in the lowest dose group (20mg TN) who had failed to achieve >2log10 VL reduction within the first 14days. HCV subgenomic replicon based phenotyping of all other baseline-derived NS3 amplicons resulted in mean EC50 values for GT-1a (10±8nM) 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 conferred reductions in sensitivity to BI201335 with EC50 values of 1.8-6.5μM, whereas the EC50 values for D168V variants were 3.6-15μM (Table 4). However, all BI201335 resistant mutant replicons remained susceptible to inhibition by interferon alfa. The details of these phenotyping and genotyping studies, including 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 2-6h (tmax) and increased supra-proportionally with dose as seen for mean Cmax, Cmin and AUC0-τ. Mean elimination half-life of BI201335 was approximately 20-30h, suggesting suitability for qd dosing. No apparent difference was found between TN and TE HCV patients in plasma concentrations and exposure. Inter-individual variability was moderately high with a coefficient of variation (CV%) for AUC0-τ of up to 65%. 

Discussion  

This trial investigated the antiviral activity, safety and pharmacokinetics of BI201335 in 53 patients with HCV GT-1 infection treated with BI201335±PegIFN and RBV for 28days. When given qd as monotherapy in TN patients for 14days, 48-240mg BI201335 qd induced a rapid, dose-dependent decrease in plasma HCV RNA by >2log10 from baseline in all patients. Maximal VL declines from baseline occurred within 2-3days of first administration. At the 240mg qd dose, the median maximal decline from baseline was -4.4log10IU/ml. The magnitude of VL declines was similar to those recorded for other highly potent protease inhibitors in development (typical range -3.5 to -4.5log10IU/ml). Notably, compared with telaprevir (maximal median HCV RNA decline -4.4log10 at 750mg every 8h for 14days [13]) and boceprevir (maximal mean HCV RNA decline -2.06log10IU/ml at 400mg three times per day for 14days [14]), the virologic responses for BI201335 were achieved from single daily doses as opposed to multiple daily dosing.

During BI201335 monotherapy, the initial rapid VL drop was followed by VL breakthrough in most patients. When BI201335 was given in combination with PegIFN/RBV, a rapid and more profound dose-related reduction in HCV RNA was found even in patients failing previous PegIFN/RBV treatment. At the dose of 240mg BI201335 qd, the decline in HCV RNA achieved by triple combination treatment was -5.3log10IU/ml. The rapid virological response rates (HCV RNA<25IU/ml at Day 28) in TE patients increased with dose up to 5/6 (83.3%) in the 240mg dose group. Notably, virologic breakthroughs were infrequent and confined to previous non-responders treated in the lower 48 and 120mg dose groups (total n=3). No virologic breakthrough was observed over 28days of triple therapy in the 240mg dose group in TE patients. All relapsers to previous PegIFN/RBV treatment achieved RVR, whereas previous null or partial responders across all dose groups had similar response and breakthrough rates. Thus, in this study relapsers had on-treatment response rates similar to TN patients, while there were no differences in RVR rates between partial and null responders. However, the small numbers for these comparisons limit strong conclusions. 

In all cases, viral breakthroughs during BI201335 treatment were caused by the rapid emergence of drug-resistant viral variants, as observed with other protease inhibitors [15]. The predominant GT-1a NS3/NS4A resistance mutation in on-treatment viral breakthrough samples encoded an R155K substitution. GT-1b viruses mainly encoded changes at D168, with valine as a predominant substitution. R155K variants conferred reductions in sensitivity to BI201335 with EC50 values of 1.8-6.5μM, whereas the EC50 for D168V variants were 3.6-15μM. These variants among others have also been observed with other protease inhibitors such as telaprevir, boceprevir, ITMN-191, and TMC-435 [15], [16], [17], and are thus likely to confer cross-resistance and to eventually cause virologic breakthrough in case of combination or sequential treatment with different compounds of this class. However, the in vitro sensitivity of both variants to interferon was uncompromised (Boehringer Ingelheim, unpublished data).

This is consistent with the clinical findings of (i) substantial HCV RNA reductions found in patients with resistance mutations selected during monotherapy following add-on treatment with SOC; and (ii) by the rare frequency of HCV RNA breakthroughs under triple therapy with BI201335 and PegIFN/RBV even in patients failing previous PegIFN/RBV treatment. Thus, the combination of BI201335 with PegIFN/RBV successfully minimized the risk of virologic breakthrough during the treatment period. All three breakthroughs detected on triple treatment emerged at lower dose levels and in patients with previous null- or partial response to PegIFN/RBV. BI201335 was well tolerated in both TN and TE patient groups. Typical AEs were those commonly associated with PegIFN/RBV [5].

The majority of AEs were mild-to-moderate in severity, not dose-dependent and judged by the investigators to be unrelated to BI201335. In this study, the incidence of mild rash or photosensitivity (4/34 patients) during combination treatment of BI201335 with PegIFN and RBV was similar to the 22-28% incidence of such events reported for SOC treatment alone [1], [18]. However, interim analyses of ongoing phase II trials of BI201335 with PegIFN/RBV have indicated dose-dependent increases of mostly mild or moderate rash and phototoxicity at BI201335 doses of 240mg once or twice daily, while the incidence of such events in the 120mg dose group was similar to the SOC control [19], [20]. The lower incidence in this phase I trial is likely due to the shorter treatment duration and smaller sample size.

The only relevant safety laboratory effects were asymptomatic and isolated elevations in unconjugated bilirubin at higher doses of BI201335. However, this was reversible upon treatment completion and may be explained by the competitive inhibition of UGT1A1 by BI201335, which has been detected in vitro (Boehringer Ingelheim, unpublished data). This is supported by the finding of a strong correlation of frequency and extent of unconjugated hyperbilirubinemia with the presence of functional UGT1A1 polymorphisms, which has been observed in a healthy volunteer study (Boehringer Ingelheim, unpublished data). Importantly, no hemolysis or liver-related events of concern were reported (e.g. increase in direct bilirubin or liver enzyme markers). UGT1A1 inhibition has been identified previously as a mechanism for other antiviral drugs to cause indirect hyperbilirubinemia, such as atazanavir and indinavir [21], [22]. Isolated unconjugated hyperbilirubinemia associated with these drugs is generally regarded as harmless. 

Pharmacokinetic analysis shows that plasma BI201335 concentrations increase supra-proportionally with dose in TN and TE patients and are consistent with the virologic responses seen in these patient populations. Notably, BI201335 has a long elimination half-life with a steady-state achieved after 1week of dosing. This allows for a once-daily dosing schedule without risk of suboptimal plasma concentrations between doses. In conclusion, BI201335 is a potent and well tolerated protease inhibitor in both TN and TE HCV GT-1a and -1b patients. Its qd oral dosing schedule does not further complicate anti-HCV therapy. These data support the investigation of different treatment regimens testing doses from 120 to 480mg daily in phase IIb trials

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