Defining fractional inhibitory concentration index cutoffs for additive interactions based on self-drug additive combinations, Monte Carlo simulation analysis, and in vitro-in vivo correlation data for antifungal drug combinations against Aspergillus fumigatus

Abstract

The fractional inhibitory concentration (FIC) index range of 0.5 to 4 that is commonly used to define additivity results in no interactions in most combination studies of antifungal agents. These results may differ from those of in vivo studies, where positive and negative interactions may be observed. We reassessed this in vitro FIC index range based on (i) the experimental variation of the checkerboard technique using multiple replicates, (ii) the ability to correctly determine purely additive self-drug and two-drug antagonistic combinations of amphotericin B (AMB) and voriconazole (VRC), (iii) Monte Carlo simulation analysis, and (iv) in vitro-in vivo correlation using experimental models of invasive pulmonary aspergillosis against the same Aspergillus fumigatus isolate based on visual, spectrophotometric, and colorimetric determinations of FICs after 24 and 48 h of incubation. FICs obtained after 24 h of incubation ranged from 0.5 to 1.25 for the self-drug additive combinations of AMB plus AMB and VRC plus VRC and from 2.25 to 4.25 for the antagonistic combination of AMB plus VRC. Monte Carlo simulation analysis showed that self-drug combinations were correctly classified as additive and that the combination of AMB plus VRC was correctly classified as antagonistic for >85% of the simulated FICs when deviation of the 95% confidence interval (CI) of replicate FICs from the additivity range of 1 to 1.25 was used to assess interactions after 24 h. In vitro-in vivo correlation analysis showed that the 95% CIs of the FICs of the in vivo synergistic combination anidulafungin plus VRC determined after 24 h were lower than 1 and the 95% CIs of the FICs of the in vivo antagonistic combination AMB plus ravuconazole were higher than 1.25. Adequate insight into weak pharmacodynamic interactions with in vivo relevance may be obtained by demonstrating that triplicate FICs at 24 h are outside an inclusive additivity range of 1 to 1.25.

FIG. 1.

Self-drug combination of AMB by the XTT COL method (a classical example of an additive interaction) with an FIC_min of 1 and an FIC_max of 1.25. Of note, the commonly used additivity range of 0.5 to 4 encompasses a great area of the checkerboard and it contains two series of clear wells and one series of wells with growth, i.e., a stricter criterion for antagonistic interactions than for synergistic interactions. Because of experimental variation, the suggested additivity range encompasses ∑FICs of 1 to 1.25 inclusive. Values inside the wells are the ∑FICs if the wells were clear.

FIG. 2.

Schematic representation of the in vitro and in vivo results obtained with the synergistic combination of anidulafungin plus VRC and the antagonistic combination of AMB plus ravuconazole. In vitro checkerboards based on data obtained by the SPE method after 24 and 48 h of incubation and by the COL assay using XTT after 48 h of incubation with A. fumigatus strain 4215 are shown. The numbers in the checkerboards represent growth percentages calculated by dividing the OD of each well with the average OD of the drug-free wells of the last column after subtracting the corresponding background OD. The percentages were normalized to span a range of 0 to 100%. For details about the FIC indices, see Table 3. The lung weights obtained in the experimental rabbit model of invasive pulmonary aspergillosis with A. fumigatus strain 4215 from references and are also shown as a measure of the in vivo fungal burdens for the synergistic combination of anidulafungin plus VRC and the antagonistic combination of AMB plus ravuconazole.