In vitro combinatory effect of HCV NS3/4A protease inhibitor ABT-450, NS5A inhibitor ABT-267, and non-nucleoside NS5B polymerase inhibitor ABT-333
Speaker: Tami Pilot-Matias
Author: T. Pilot-Matias*, G. Koev, P. Krishnan, J. Beyer, T. Reisch, R. Mondal, L. Lu, T. Middleton, C. Maring, W. Kati, C. Collins, A. Molla
Affiliation: Antiviral Research, Abbott Laboratories, Abbott Park, IL, USA. *tami.pilot-matias@abbott.com
Background and aims: Abbott currently has several direct-acting antiviral agents (DAAs) targeting HCV in clinical development, including NS3/4A protease inhibitor ABT-450 (in collaboration with Enanta Pharmaceuticals), NS5A inhibitor ABT-267, and non-nucleoside polymerase inhibitor ABT-333. In vitro combination studies were conducted with these agents in order to better understand their potential to be combined in HCV-infected patients.
Methods: Each pair of inhibitors was combined based on the checkerboard method and added to a stable HCV replicon cell line for 3 days. The extent of HCV replicon replication was assessed for synergistic, additive, or antagonist effects using the Prichard direct method. Colony formation assays were performed using replicon cell lines incubated in the presence of G418 for approximately three weeks with each inhibitor alone, each pair, and all three together. Replicon RNA reduction assays were performed by incubating replicon cell lines in the presence of each inhibitor alone or in combination in the absence of G418 for up to 6 passages.
Results: Each pair-wise combination of ABT-450, ABT-267, and ABT-333 displayed additive to synergistic interactions in a checkerboard combination assay. In colony formation assays, a greater than 100-fold reduction in colony number was observed in the presence of each pair-wise combination of inhibitors as compared to each inhibitor alone. There were no surviving colonies (< 0.0001%) when all three inhibitors were included, even at only 10-fold above their respective EC50, indicating that inclusion of each additional DAA leads to an increase in the genetic barrier to resistance. In replicon RNA reduction assays, the combination of two inhibitors at 10-fold over their respective EC50 resulted in a more profound reduction in replicon RNA than when either inhibitor was present alone at 100-fold over EC50.
Conclusions: These results indicate that the combination of any two of these DAAs will likely lead to an improved in vivo resistance barrier, while the combination of all three should lead to an even more significant improvement. Furthermore, when these DAAs are used in combination in vivo, it may be possible to overcome viral resistance at doses that are significantly lower than might be required with single drug alone.