Isobolographic analysis of pharmacodynamic interactions between antifungal agents and ciprofloxacin against Candida albicans and Aspergillus fumigatus

Abstract

Patients suffering from invasive mycoses often receive concomitant antifungal therapy and antibacterial agents. Assessment of pharmacodynamic interactions between antifungal and antibacterial agents is complicated by the absence of a common antifungal end point for both agents. Ciprofloxacin has no intrinsic antifungal activity but may interact with antifungal agents, since it inhibits DNA gyrase (topoisomerase II), which is abundant in fungi. We therefore employed isobolographic analysis adapted to incorporate a nonactive agent in order to analyze the potential in vitro interaction between the fluoroquinolone ciprofloxacin and several representative antifungal agents against Candida albicans and Aspergillus fumigatus strains by using a microdilution checkerboard technique. In agreement with earlier in vitro studies, conventional fractional inhibitory concentration index analysis was unable to detect interactions between ciprofloxacin and antifungal agents. However, isobolographic analysis revealed significant pharmacodynamic interactions between antifungal agents and ciprofloxacin against C. albicans and A. fumigatus strains. Amphotericin B demonstrated concentration-dependent interactions for both species, with synergy (interaction indices, 0.14 to 0.81) observed at ciprofloxacin concentrations of <10.64 microg/ml. Synergy (interaction indices, 0.10 to 0.86) was also found for voriconazole and caspofungin against A. fumigatus. Isobolographic analysis may help to elucidate the pharmacodynamic interactions between antifungal and non-antifungal agents and to develop better management strategies against invasive candidiasis and aspergillosis.

FIG. 1.

Isobolographic analysis of pharmacodynamic interactions between amphotericin B and ciprofloxacin against Aspergillus fumigatus 2025. The concentration-effect relationships of amphotericin B alone (AMB regression curve) and in combination with ciprofloxacin in mixtures with an amphotericin B proportion of 0.24 (0.24 P_AMB regression curve) or 0.02 (0.02 P_AMB regression curve) are presented for one of three replicates. In order to assess the interactions at 15%, 50%, and 85% growth (dashed horizontal lines), the concentration-effect curves of the mixtures were compared with theoretical indifferent concentration-effect curves of the mixtures with 0.24 P_AMB (0.24 P_AMB additive curve) and 0.02 P_AMB (0.02 P_AMB additive curve). The concentration-effect curve of the mixture with 0.24 P_AMB is on the left of the corresponding indifferent curve (i.e., a smaller drug concentration is required in combination in order to produce the same effect as the drug alone), indicating synergistic (S) interactions for all growth levels. The concentration-effect curve of the mixture with 0.02 P_AMB is on the left of the corresponding indifferent curve at 85% of growth and on the right at 15% of growth (i.e., a higher drug concentration is required in combination in order to produce the same effect as the drug alone), indicating synergistic and antagonistic (A) interactions, respectively.

FIG. 2.

Two-dimensional isobolograms showing the interaction at a 15% growth level between amphotericin B and ciprofloxacin against A. fumigatus 2025 for one replicate. All points (shown as small circles) on the 15% isobologram represent drug concentrations, alone and in combination, that resulted in 15% growth. The isobol of indifference is the solid line, which starts from the EC₁₅ of amphotericin B on the x axis (solid circle) and runs parallel to the y axis, because ciprofloxacin has no antifungal activity. The dashed lines around the indifferent isobol line represent the 95% confidence limits. Open circles represent the experimentally derived EC_15,MIXs obtained from the regression analysis, and the error bars represent the 95% confidence limits. The experimental EC_15,MIX of the mixtures with amphotericin B proportions (P_AMB) of 0.01 and 0.02 were statistically significantly higher than the theoretical isobol of indifference, indicating antagonistic interactions, with interaction indices of 1.25 and 2.73, respectively. On the other hand, for mixtures with a P_AMB of >0.14, statistically significant synergy was found, with interaction indices of 0.6 to 0.45. The shaded dashed lines starting from the origin of the axes represent mixtures with different P_AMB. These interactions were considered significant if the interaction indices obtained from all three replicates were statistically significantly different from 1. For the exact magnitude of synergy and antagonism for mixtures with different P_AMB, see Table 3.