In Vitro-In Vivo Correlation of Posaconazole-Amphotericin B Combination against Candida albicans: In Vitro Interacting Concentrations Are Associated with In Vivo Free Drug Levels.

The in vitro/in vivo correlation of antifungal combination testing is necessary in order to assess the efficacy of combination regimens. We, therefore, attempted to correlate in vitro chequerboard testing of posaconazole (POS) and amphotericin B (AMB) with the in vivo outcome of combination therapy against experimental candidiasis in a neutropenic murine model. The AMB + POS combination was tested against a Candida albicans isolate. In vitro, a broth microdilution 8 × 12 chequerboard method with serial two-fold drug dilutions was used. In vivo, CD1 female neutropenic mice with experimental disseminated candidiasis were treated with i.p. AMB and p.o. POS alone and in combination at three effective doses (ED20, ED50 and ED80 corresponding to 20%, 50% and 80% of maximal effect, respectively). CFU/kidneys after 2 days were determined. The pharmacodynamic interactions were assessed based on Bliss independence interaction analysis. In vitro, a Bliss antagonism of -23% (-23% to -22%) was observed at 0.03-0.125 mg/L of AMB with 0.004-0.015 mg/L of POS, while a Bliss synergy of 27% (14%-58%) was observed at 0.008-0.03 mg/L of AMB with 0.000015-0.001 mg/L of POS. In vivo, Bliss synergy (13 ± 4%) was found when an AMB ED20 of 1 mg/kg was combined with all POS ED 0.2-0.9 mg/kg, while Bliss antagonism (35-83%) was found for the combinations of AMB ED50 2 mg/kg and ED80 3.2 mg/kg with POS ED80 of 0.9 mg/kg. Free drug serum levels of POS and AMB in in vivo synergistic and antagonistic combinations were correlated with the in vitro synergistic and antagonistic concentrations, respectively. Both synergistic and antagonistic interactions were found for the AMB + POS combination. POS compromised the efficacy of high effective AMB doses and enhanced low ineffective AMB doses. In vitro concentration-dependent interactions were correlated with in vivo dose-dependent interactions of the AMB + POS combination. In vivo interactions occurred at free drug serum levels close to in vitro interacting concentrations.

Amphotericin B- and voriconazole-echinocandin combinations against Aspergillus spp.: Effect of serum on inhibitory and fungicidal interactions.

Antifungal combination therapy with voriconazole or amphotericin B and an echinocandin is often employed as primary or salvage therapy for management particularly of refractory aspergillosis. The pharmacodynamic interactions of amphotericin B- and voriconazole-based combinations with the three echinocandins caspofungin, micafungin, and anidulafungin in the presence of serum were tested against 15 Aspergillus fumigatus complex, A. flavus complex, and A. terreus complex isolates to assess both their growth-inhibitory and fungicidal activities. The in vitro activity of each drug alone and in combination at a 1:1 fixed concentration ratio was tested with a broth microdilution colorimetric method, and interactions were assessed by isobolographic analysis. Synergy was found for all amphotericin B- and voriconazole-based combinations, with amphotericin B-based combinations showing strong inhibitory synergistic interactions (interaction indices of 0.20 to 0.52) and with voriconazole-based combinations demonstrating strong fungicidal synergistic interactions (interaction indices of 0.10 to 0.29) (P < 0.001). Drug- and species-specific differences were found, with caspofungin and the A. fumigatus complex exhibiting the weakest synergistic interactions. In the presence of serum, the synergistic interactions were reduced in the order (from largest to smallest decrease) micafungin > anidulafungin > caspofungin, and A. flavus complex > A. fumigatus complex > A. terreus complex, resulting in additive interactions, particularly for inhibitory activities of amphotericin B-echinocandin combinations and fungicidal activities of voriconazole-echinocandin combinations. Drug- and species-specific differences were found in the presence of serum for inhibitory activities of antifungal drugs, with the lowest interaction indices being observed for amphotericin B-caspofungin (median, 0.77) and for the A. terreus complex (median, 0.56). The present in vitro data showed that serum had a major impact on synergistic interactions of amphotericin B-echinocandin and voriconazole-echinocandin combinations, resulting in additive interactions and explaining the indifferent outcomes usually observed in vivo.

Inhibitory and fungicidal effects of antifungal drugs against Aspergillus species in the presence of serum.

Given the high protein binding rates of antifungal drugs and the effect of serum proteins on Aspergillus growth, we investigated the in vitro pharmacodynamics of amphotericin B, voriconazole, and three echinocandins in the presence of human serum, assessing both inhibitory and fungicidal effects. In vitro inhibitory (IC) and fungicidal (FC) concentrations against 5 isolates of Aspergillus fumigatus, Aspergillus flavus, and Aspergillus terreus were determined with a CLSI M38-A2-based microdilution method using the XTT methodology after 48 h of incubation at 35 °C with a medium supplemented with 50% human serum. In the presence of serum, the IC and FC of amphotericin B and the IC of echinocandins were increased (1.21- to 13.44-fold), whereas voriconazole IC and FC were decreased (0.22- to 0.90-fold). The amphotericin B and voriconazole FC/IC ratios did not change significantly (0.59- to 2.33-fold) in the presence of serum, indicating that the FC increase was due to the IC increase. At echinocandin concentrations above the minimum effective concentration (MEC), fungal growth was reduced by 10 to 50% in the presence of human serum, resulting in complete inhibition of growth for some isolates. Thus, the in vitro activities of amphotericin B and echinocandins were reduced, whereas that of voriconazole was enhanced, in the presence of serum. These changes could not be predicted by the percentage of protein binding, indicating that other factors and/or secondary mechanisms may account for the observed in vitro activities of antifungal drugs against Aspergillus species in the presence of serum.