Genotypic and Phenotypic Analysis of Samples From HCV-Infected Subjects

AASLD
60th Annual Meeting of the American Association for the Study of Liver Diseases
Boston, MA, Hynes Convention Center
October 30-November 3, 2009 Back grey_arrow_rt.gif





Genotypic and Phenotypic Analysis of Samples From HCV-Infected Subjects Treated with BMS-650032 in a Single Ascending Dose Study


Reported by Jules Levin
AASLD Oct 31-Nov 2 2009, Boston, MA

Fiona McPhee1, Dennis Hernandez1, Amy Sheaffer1, Min Lee1, Paul Falk1, Fei Yu1, Guangzhi Zhai1, Susan Chaniewski1, Claudio Pasquinelli1 1Bristol-Myers Squibb Company, Research and Development, Wallingford, CT, USA


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ABSTRACT
Background: The HCV NS3 protease inhibitor (PI) BMS-650032, is a potent and selective inhibitor that has demonstrated antiviral activity in both single and multiple ascending dose studies in subjects chronically infected with HCV genotype 1. Actively treated subjects experienced a mean decline in HCV RNA of ∼2.5 log10 at 24 hours after a single 600 mg dose of BMS-650032. Since pre-existence or emergence of resistance to a PI in monotherapy has been reported, we examined baseline sequences and how they changed over time after single doses of BMS-650032. Further, the susceptibility of any emerging viral variants to inhibition by BMS-650032 was evaluated in exploratory phenotypic assays.

Methods: Serum samples from treatment-naïve or experienced HCV-infected subjects administered 10, 50, 200 or 600 mg were collected at baseline (pre-dose), 24, 48, and 144 hours post-dose. Total HCV RNA was isolated from 10 and 50 mg baseline samples and from all time points for the 200 or 600 mg samples. The NS3 protease domain was amplified and analyzed by population sequencing. Viral sequences derived from the HCV-infected subjects were introduced into chimeric shuttle vectors representing either genotype 1a or 1b. One set of shuttle vectors was used to express the full-length NS3/4A protease enzyme and assess susceptibility to BMS-650032 inhibition while another set enabled the measurement of HCV replication.

Results: Enrichment of known PI-resistant variants (substitutions at positions V36, R155, A156, and D168) was not detected at the doses tested. Changes in sequence polymorphism were observed in select samples over time. Phenotypic analysis of these samples in NS3 protease enzyme-based and/or chimeric replicon cell-based assays revealed that the detected polymorphisms did not appear to confer resistance relative to sub-type references. A known resistance variant to BMS-650032 was detected at baseline in a subject who received placebo (1b-NS3-E168). Phenotypic analysis of the NS3 protease population sequence derived from this subject revealed that it indeed conferred resistance to BMS-650032 (EC50 = 70 nM) relative to a sub-type reference (EC50 = 1.1 nM).

Conclusions: In HCV-infected subjects administered single doses of BMS-650032, enrichment of variants known to confer resistance to BMS-650032 was not detected by population sequencing. Pre-existence of a known HCV NS3 protease resistance variant to BMS-650032 was detected. This finding highlights the need for combination therapies with pegylated interferon/ribavirin, an NS5A inhibitor or other direct acting antivirals to suppress resistant viral variants.

INTRODUCTION

The treatment of genotype 1 chronic hepatitis C (HCV) is evolving with the development of new treatments specifically targeting the HCV molecule

The HCV NS3/4A protease is an essential enzyme for viral replication and has been validated as a target for anti-HCV therapy in clinical trials

BMS-650032 is a potent and selective oral NS3/4A protease inhibitor that has demonstrated antiviral activity in both single and multiple ascending studies in HCV subjects infected with genotype 1 (GT 1)
· Achieved ∼2.5 log10 mean decline in HCV RNA following a single 600 mg dose
· Single and multiple ascending dose results to be presented (Abstract 225, Tuesday)
· Major signature resistance substitutions selected at R155 (GT 1a) and D168 (GT 1b) in vitro

Since pre-existence or emergence of resistance to a PI in monotherapy has been previously reported1-4, we examined baseline sequences and how they changed over time after subjects were administered single doses of BMS-650032


REFERENCES

1. Sarrazin et al, Gastroenterology. 2007; 132:1767-77
2. Kuntzen et al, Hepatology. 2008; 1769-78
3. Kukolj et al, EASL April 23-26 Copenhagen 2009; poster 954
4. Qiu et al, Nucleic Acids Res. 2009; 37(10):e74
5. Lallos et al, EASL April 23-26 Copenhagen 2009; poster 344
6. Sheaffer et al, 16th International Symposium on HCV and Related Viruses, October 3-7 Nice 2009; poster 235