T cytotoxicity was measured with either compound alone or with the above combinations (unshown data). These results suggest that even relatively low concentrations of clemizole have a dramatic effect on viral replication when added to SCH503034. To formally characterize the SCH503034-clemizole combination’s interaction, a data subset where the drugs were mixed at a fixed molar ratio matching their equipotent concentrations (SCH503034 to clemizole ratio of 1:10), was analyzed using CalcuSynTM. As shown in the resulting isobolgram (Fig. 1B), the calculated EC50, EC75, and EC90 values for the SCH503034-clemizole combinations plotted far to the left of the corresponding lines of additivity, suggesting that the tested combinations are indeed synergistic [12,13]. At an equipotent ratio of 1:10, the CIs at the EC50, EC70, and EC90 were 0.61, 0.479 and 0.397, respectively (Fig. 1C) [15]. Being below 0.9, these indices confirm that the interaction is synergistic. These CIs are similar in magnitude to the most potent synergistic interaction measured by others between HCV PIs and polymerase inhibitors [20]. While the interaction was found to be synergistic at any tested ratio, lowest CIs were measured at SCH503034 to clemizole ratio of 1:4 (Fig. 1C). To confirm the nature of this interaction, to adjust for the various concentration ratios, and to better quantify the degree of the observed synergy, we further analyzed the data by a mathematical model, MacSynergy [16,17]. 4 by 3 matrix data sets in four replicates were assessed at the 95 confidence level for each of the three experiments performed. The clemizole-SCH503034 combination had antiviral effects that were significantly more potent than the theoretical additive effects, supporting that this combination was indeed synergistic (Fig 1D). No evidence of antiviral antagonism was seen with any of the tested doses. The calculated synergy and log volume were 210M2 and 19, respectively. According to the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/20587871 criteria suggested by Prichard et al. [17] (see methods), the clemizole-SCH503034 combination is considered strong and will likely be important in vivo. Importantly, since there was no cellular toxicity with either drug alone at the studied concentrations and no increase in cytotoxicity when used in combinations, the measured synergy is indeed specific and does not reflect synergistic toxicity. The synergy of clemizole-SCH503034 combination is not genotype-specific To determine whether the observed synergy of the clemizole-SCH503034 combination is genotype-specific, the experiments outlined above were repeated using a CAL-120 custom synthesis subgenomic genotype 1b Bart79I HCV replicon harboring a luciferase reporter gene [8]. In contrast to its effect in genotype 2a, very mild concentration-dependent inhibition of HCV replication was measured following 72hr treatment with clemizole alone, with an average EC50 of 24?M (p=0.01, R2=0.85) and an average CC50 of 40?M (p<0.05) (Table 1). It is possible that the lower sensitivity of this assay compared with the 2a luciferase reporter gene assay (resulting from lower level of genotype 1b replication compared with the genotype 2a clone) accounts for the difference in clemizole's EC50s between genotypes. Alternatively, the difference might result from differential antiviral activity of clemizole against the two genotypes. Selection of clemizole-resistant mutants in 1b genotype replicon cells [6], suggests that clemizole does have an antiviral effect against genotype 1b. We thus fav.