Triple antifungal combinations are used against refractory invasive aspergillosis without an adequate understanding of their pharmacodynamic interactions. We initially studied the in vitro triple combination of voriconazole, amphotericin B, and caspofungin against Aspergillus fumigatus, A. flavus, and A. terreus by a spectrophotometric microdilution broth method after 48 h of incubation. We then analyzed these results with a recently described nonlinear mixture response surface E max -based model modified to assess pharmacodynamic interactions at various growth levels. The new model allows flexibility in all four parameters of the E max model and is able to describe complex pharmacodynamic interactions. Concentration-dependent pharmacodynamic interactions were found within the triple antifungal combination. At the 50% growth level, synergy Invasive aspergillosis is an important cause of morbidity and mortality in immunocompromised patients (20). New therapeutic approaches are needed to improve outcome (30). The introduction of newer antifungal agents with different mechanisms of action has made combination therapy a possibility and an area of compelling investigational interest (38). Because of their different mechanisms of action, triazoles, echinocandins, and polyenes are likely candidates for combination therapy. Echinocandins inhibit the synthesis of 1,3--D-glucan, a key component of the cell walls of most fungi; triazoles inhibit the synthesis of ergosterol by inhibiting the enzyme lanosterol C-14 demethylase; and polyenes act directly at the fungal cell membrane to alter its integrity (10).Triple combination therapy using all three classes of antifungal agents may be used in refractory aspergillosis (8,35,39). However, this practice has not been well studied, and the in vitro pharmacodynamic interactions are unknown. Preclinical studies are therefore required to understand the pharmacodynamic interactions and appropriately adjust in vivo dosing regimens in order to maximize synergistic effects and minimize the antagonistic ones. In vitro pharmacodynamic interactions within a triple combination can be complex (24); powerful analytical tools are required to accurately describe the effects of the triple combination in a wide range of drug concentrations and to determine the nature and intensity of antifungal pharmacodynamic interactions. Response surface methodologies provide the necessary tools to capture the information present in the full concentration-effect data set for two or more agents and to quantify synergy and antagonism (9).A new nonlinear mixture-amount response surface model for analyzing three-drug combinations was recently described (40). By contrast with other fully parametric response surface models, the new model is flexible enough to describe complicated response surfaces and to determine complex patterns of synergy and antagonism. With this model, response surfaces are modeled using the sigmoid E max concentration-effect relationship, and pharmacodynamic interactions are assessed based o...