15th International HIV Drug Resistance Workshop 13-17 June 2006, Sitges, Spain

SYNERGY OF SMALL MOLECULAR INHIBITORS OF HEPATITIS C VIRUS REPLICATION DIRECTED AT DIFFERENT VIRAL TARGETS

Antivir Ther. 2006, 11:S4 (abstract no. 2)

DL Wyles, K Kaihara and RT Schooley
University of California, San Diego, La Jolla, CA, USA

BACKGROUND: Limitations in interferon based therapy for hepatitis C virus (HCV) have spurred an intense drug discovery effort. Early reports of HCV protease and RNA polymerase inhibitors have yielded encouraging results; however, resistance to these compounds has been described in vitro and in vivo. We have developed a HCV-replicon based luciferase reporter system as an in vitro model for the study of combination therapy for HCV infection.

METHODS: Huh 7.5.1 cells (10,000/well) stably expressing the luciferase replicon were incubated with compounds for 48 hours in 96 well plates. Luciferase activity was determined using a micro-plate luminometer. Compounds tested included three peptidomimetic HCV protease inhibitors (BILN 2061 and a Vertex PI kindly provided by Ann Kwong; GSK PI kindly provided by Karen Romines), two nucleoside analogue HCV polymerase inhibitors (kindly provided by Karen Romines and Norbert Bischofberger of GSK and Gilead respectively) and interferon-α. The doses of each compound were determined based on the IC₅₀ of the compound alone. An isobologram analysis was used to determine the additivity, synergism, or antagonism of inhibitor combinations. Combination indices were determined using Calcusyn (Biosoft).

RESULTS: Alone HCV protease inhibitors and polymerase inhibitors exhibited IC₅₀'s ranging from 5–160 and 200–1200 nM, respectively. Combinations of two protease inhibitors were additive (CI: 0.94). Combination of the two RNA polymerase inhibitors also displayed additivity (CI: 0.99). In contrast, strong synergy was consistently found between protease inhibitors and RNA polymerase inhibitors (CI: 0.25–0.29). All compounds tested were synergistic with interferon-α CI: 0.32–0.74). None of the compound combinations showed cytotoxicity at the concentrations tested.

CONCLUSIONS: Small molecular inhibitors of the HCV protease and polymerase show antiviral activity in our genotype 1 replicon system. Combinations of inhibitors targeting different viral proteins act synergistically; while combinations targeting the same viral protein are additive. Synergistic combinations of HCV inhibitors are likely to produce greater viral load decreases in vivo and substantially increase the resistance barrier. These effects should result in the increased durability and efficacy of small molecule-based HCV antiviral therapy. This system provides a useful approach for the in vitro testing of antiviral combinations in anticipation of rationally designed clinical studies of combination chemotherapy directed at HCV.

This research was funded in part by a 2005 developmental grant from the UC San Diego Center for AIDS Research, an NIH funded program #5P30 AI-36214.

2006-06-13
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