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

Abstract

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.

Keywords: Candida albicans; amphotericin B; combination therapy; in vitro–in vivo correlation; posaconazole.

Figure 1

Posaconazole–amphotericin B in vitro interaction based on Bliss independence theory and posaconazole–amphotericin B free concentrations in mouse serum. The top panel depicts the percentage of fungal growth, while the bottom panel depicts statistically significant synergy (green color) or antagonism (red color) at each concentration of posaconazole and amphotericin B alone and in combination. The predicted free posaconazole and amphotericin B Cmax and Cmin concentrations in mouse serum of 3 × 3 dosing regimens used in combination therapy are also shown.

Figure 2

Interaction surface obtained from response surface analysis of Bliss-independence-based drug interaction model for the in vitro combination of amphotericin B and posaconazole against Candida albicans. The zero plane indicates Bliss-independent interactions, whereas values below the zero plane indicate statistically significant antagonistic interactions (negative ΔΕ ). The different tones in the 3-dimensional plot represent different percentile bands of antagonism.

Figure 3

In vivo dose–response curves for amphotericin B (AMB), posaconazole (POS) alone and in combination. Synergy was determined at lower amphotericin B doses combined with posaconazole, while antagonism occurred at higher amphotericin B doses in combination with posaconazole.

Figure 4

In vivo dose–response curves of amphotericin B and posaconazole monotherapy. Error bars correspond to SD.

Figure 5

Dose–serum concentrations relationship for amphotericin B and posaconazole in mice.