Combination of a hepatitis C virus NS3-NS4A protease inhibitor and alpha interferon synergistically inhibits viral RNA replication and facilitates viral RNA clearance in replicon cells

Abstract

The present standard of care for hepatitis C virus (HCV) infection is pegylated alpha interferon (IFN-alpha) in combination with ribavirin. However, specific antivirals such as HCV NS3-NS4A protease inhibitors are now in clinical development, and these agents can potentially be used in combination with the present treatments. Therefore, it is important to investigate the potential benefits or adverse effects of these new combinations by using available in vitro HCV culture systems first. In the present study we demonstrate that the combination of a specific HCV NS3-NS4A protease inhibitor and IFN-alpha synergistically inhibits HCV RNA replication in replicon cells, with little or no increase in cytotoxicity. Furthermore, the benefit of the combination was sustained over time, such that a greater than 3-log reduction in HCV RNA levels was achieved following 9 days of treatment. The viral RNA appeared to be cleared from the replicon cells after 14 days of treatment, and no viral RNA rebound was observed upon withdrawal of the inhibitors. In each case, the antiviral effects obtained with higher concentrations of either the protease inhibitor alone or IFN-alpha alone can be achieved by a combination of both agents at lower concentrations, which may potentially reduce the risk of possible adverse effects associated with high doses of either agent.

FIG. 1.

Chemical structure of PI-1, a specific HCV NS3-NS4A protease inhibitor.

FIG. 2.

Dose-dependent inhibition of HCV replicon by PI-1 or IFN-α alone. HCV replicon cells were treated with PI-1 (A) or IFN-α (B) for 48 h. At the end of the 48-h treatment, total cellular RNA was extracted with the RNeasy-96 kit. The levels of HCV replicon RNA remaining were then determined by quantitative RT-PCR, as described in Materials and Methods, and are shown as a percentage of the levels of replicon RNA in cells treated with 0.5% DMSO (control). Each bar represents the average of five cell culture replicates with the standard deviation.

FIG. 3.

Combination of PI-1 with IFN-α in the 2-day HCV replicon cell assay. HCV replicon cells were treated with various concentrations of PI-1 (indicated on the x axis) in combination with various concentration of IFN-α (indicated at the top) for 48 h. (A) The level of HCV RNA remaining in replicon cells treated with PI-1 and IFN-α in combination was determined by quantitative RT-PCR and is shown as a percentage of the level in cells treated with 0.5% DMSO (control). Each bar represents the average of six cell culture replicates with the standard deviation. (B) The viability of HCV replicon cells following compound treatment for 48 h was determined by an MTS-based cell viability assay and is presented as a percentage of the viability of cells treated with 0.5% DMSO (control). Each bar represents the average of four cell culture replicates with the standard deviation.

FIG. 4.

Analysis of the combination of PI-1 and IFN-α by use of the Bliss independence model. A three-dimensional graph was used to illustrate the difference between the experimental effect and the theoretical additive effect for each combination. The predicted theoretical additive effect was calculated by using the MacSynergy program, as described in Materials and Methods, and was subtracted from the experimental effect (shown in Fig. 3A), and then the difference between the two effects was plotted in the graph shown here. The zero plane (the gray plane) across the z axis represents the theoretical additive effect, a positive value displayed as a peak above the plane (the height is correlated with increasing grayness) indicates synergy, and a negative value shown as a valley below the plane indicates antagonism. Each experimental datum point represents the average of six cell culture replicates, and 95% confidence intervals were used to evaluate the data.

FIG. 5.

Analysis of the combination of PI-1 and IFN-α by use of the Loewe additivity model. (A) The data for the combination shown in Fig. 3A were also analyzed by using CalcuSyn program and are presented as a traditional isobologram, in which the lines represent the EDs of the two drugs required to achieve 50, 75, or 90% inhibition if the effects of the two compounds were simply additive. The dots are the actual experimental doses used to achieve those effects. (B) The CI can be calculated for each combination, as described in the Materials and Methods, and is plotted as the solid line versus the percent inhibition (i.e., the fractional effect). The 95% confidence intervals (1.96 standard deviations) of the CI are shown as two curved dotted lines. The theoretic additive effect (CI = 1) is represented by a straight dotted line.

FIG. 6.

Nine-day treatment of HCV replicon cells with PI-1, IFN-α, or ribavirin. The cells were treated with compound for 3, 6, or 9 days. At the end of each treatment period, cell numbers were determined by an MTS-based cell viability assay with an established standard curve, and the level of HCV RNA in the cells was determined by the quantitative RT-PCR assay. The copy number of HCV RNA per cell in each sample is plotted as the percentage (on a log scale) of the copy number in replicon cells incubated with 0.2% DMSO (control) for the same period of time. Each datum point represents the average of five cell culture replicates with the standard deviation.

FIG. 7.

Rebound of HCV replicon after a 14-day treatment with PI-1, IFN-α, or ribavirin. HCV replicon cells were treated with PI-1, IFN-α, or ribavirin alone or the combination of IFN-α and either PI-1 or ribavirin in the absence of G418. After 14 days of treatment, the compounds were withdrawn and 250 μg of G418 per ml was added to enrich the remaining HCV replicon-positive cells that are capable of growing in the presence of G418 (rebound). The cultures were monitored for another 21 days in the presence of G418, and cell samples were collected whenever the cell monolayer reached confluence. The cell number was determined by the Guava ViaCount assay, as described in the Materials and Methods, and the level of HCV RNA in the cells was determined by the quantitative RT-PCR assay. The absolute numbers of HCV replicon RNA copies per viable cell are shown.