Efficacy of voriconazole-liposomal amphotericin B combination against azole-resistant Aspergillus fumigatus in an in vitro pharmacokinetic-pharmacodynamic model Maria Siopi1, Nikolaos Siafakas1, Sophia Vourli1, Johan Mouton2, Loukia Zerva1 and Joseph Meletiadis1,2 UNIVERSITY GENERAL HOSPITAL ATTIKON 1Clinical Microbiology Laboratory, Attikon University General Hospital, Athens, Greece, 2Dept of Med Microbiology Erasmus Medical Centre, Rotterdam, Netherlands

Correspondence: Joseph Meletiadis, 1 Rimini str, Chaidari 124 62, Athens Greece, Tel: +30-210-583-1909, Email: [email protected]

INTRODUCTION AND PURPOSE RESULTS Azole-resistant aspergillosis is associated with high We therefore investigated the pharmacodynamic  Pharmacokinetic analysis. VRC levels were close to 84% vs. 88%, 38%, 19%, respectively). mortality rate. Combination therapy may be used in effects of VRC plus L-AMB combination against VRC- the target values observed in human plasma with an  Pharmacodynamic interactions. When the optimal order to increase the efficacy of voriconazole (VRC) susceptible and VRC-resistant Aspergillus fumigatus average t1/2 6.2h with Cmax of 7.65±0.34, 1.67±0.04 and L-AMB dosing regimen (Cmax 0.32 mg/L) was combined which is the first-line antifungal agent against invasive isolates with a validated in vitro pharmacokinetic- 0.26±0.03 mg/L (Fig. 2). A biphasic time-concentration with VRC, independent interactions were found against aspergillosis. The benefit of combination therapy with pharmacodynamic (PK-PD) model simulating human profile of L-AMB was simulated in the in vitro model the VRC-susceptible strain, whereas antagonistic VRC and liposomal amphotericin B (L-AMB) is pharmacokinetics. attaining a Cmax of 0.44 and 0.06 mg/L with t1/2,α 2h and interactions (-14.5−-18.5%) were observed against the controversial due to potential antagonistic interactions. t1/2,β >24h (Fig. 2). VRC-resistant isolate. When the sub-optimal L-AMB

 Pharmacodynamic analysis. L-AMB was equally dosing regimen (Cmax 0.08 mg/L) was combined with METHODS effective against both isolates (100% and 47% GM VRC, independent interactions were found against the suppression at Cmax 0.32 and 0.08 mg/L, respectively), VRC-susceptible strain, whereas antagonism (-14.7−- In vitro PK-PD model. One VRC-susceptible wild-type alone and in combination (3x2) including drug-free while VRC suppressed GM production in a greater 34.8%) was found against the VRC-resistant strain at strain without mutation in the cyp51A gene (AZN8196) controls (Fig. 2). After inoculation with Aspergillus degree for the VRC-susceptible than for the VRC- low voriconazole exposures (≤1.5 mg/L) (Fig. 3). with VRC/AMB CLSI MIC 0.125/0.5 mg/L and one VRC- conidia (103 cfu/mL), drugs were injected alone and in resistant isolate at Cmax 7, 1.5 and 0.3 mg/L (94%, 93%, resistant strain harboring the TR35/L98H mutation combination in both compartments and the model was (V52-35) with VRC/AMB CLSI MIC 2/0.25 mg/L were incubated at 37oC on a magnetic stirrer for 72 hours. tested in a previously optimized two compartment PK- Drug levels were determined by microbiological agar PD dialysis/diffusion closed model (Siopi et al JAC 2014) diffusion assays. Fungal growth was assessed based on (Fig.1). The in vitro system has been adapted to galactomannan (GM) levels using a commercially Combination with high optimal accommodate two drugs with different t1/2 enabling available sandwich enzyme-linked immunoassay. L-AMB dose thus the study of drug combinations (Siopi et al AAC Analysis. GM-time curves were constructed and the 2015). nature of in vitro pharmacodynamic interactions were Pharmacokinetics. Different regimens of VRC with assessed using the Bliss independence model. All

Cmax 7, 1.5 and 0.3 mg/L and t1/2 6h dosed q12 were experiments were carried out in duplicate and were combined with L-AMB dosing regimens with Cmax 0.32 independently performed on two different days with and 0.08 mg/L with t1/2,α 2h and t1/2,β 24h dosed q24 individually prepared inocula.

Combination with low sub-optimal Peristaltic pump L-AMB dose

Figure 3. In vitro pharmacodynamics of VRC plus L-AMB combination against a wild-type VRC susceptible (left graphs) and a non-

wild-type VRC resistant harboring the cyp51A TR35/L98H (right graphs) A. fumigatus isolate.

CONCLUSIONS  Liposomal amphotericin B was equally effective found against the azole-resistant isolate particularly against azole-susceptible and azole-resistant at low sub-optimal voriconazole exposures. Aspergillus fumigatus isolates.  Therapeutic drug monitoring of antifungal  When liposomal amphotericin B was combined with combination therapy can be employed in order to voriconazole, no interaction was found against the target specific drug exposures that will minimize the Figure 1. In vitro PK-PD dialysis/diffusion closed model. Figure 2. In vitro pharmacokinetics of VRC and L-AMB. azole-susceptible isolate, whereas antagonism was likelihood of antagonistic interactions.