Potency and Preclinical Evidence of Synergy of Oral Azole Drugs and Miltefosine in an Ex Vivo Model of Leishmania (Viannia) panamensis Infection

Abstract

Failure of treatment of cutaneous leishmaniasis with antimonial drugs and miltefosine is frequent. Use of oral combination therapy represents an attractive strategy to increase efficacy of treatment and reduce the risk of drug resistance. We evaluated the potency of posaconazole, itraconazole, voriconazole, and fluconazole and the potential synergy of those demonstrating the highest potency, in combination with miltefosine (HePC), against infection with Leishmania (Viannia) panamensis. Synergistic activity was determined by isobolograms and calculation of the fractional inhibitory concentration index (FICI), based on parasite quantification using an ex vivo model of human peripheral blood mononuclear cells (PBMCs) infected with a luciferase-transfected, antimony and miltefosine sensitive line of L. panamensis. The drug combination and concentrations that displayed synergy were then evaluated for antileishmanial effect in 10 clinical strains of L. panamensis by reverse transcription-quantitative (qRT-PCR) of Leishmania 7SLRNA. High potency was substantiated for posaconazole and itraconazole against sensitive as well as HePC- and antimony-resistant lines of L. panamensis, whereas fluconazole and voriconazole displayed low potency. HePC combined with posaconazole (Poz) demonstrated evidence of synergy at free drug concentrations achieved in plasma during treatment (2 μM HePC plus 4 μM Poz). FICI, based on 70% and 90% reduction of infection, was 0.5 for the sensitive line. The combination of 2 μM HePC plus 4 μM Poz effected a significantly greater reduction of infection by clinical strains of L. panamensis than individual drugs. Orally administrable miltefosine/posaconazole combinations demonstrated synergistic antileishmanial capacity ex vivo against L. panamensis, supporting their potential as a novel therapeutic strategy to improve efficacy and effectiveness of treatment.

Keywords: Leishmania; azoles; combined therapy; miltefosine; posaconazole.

FIG 1

Potency of posaconazole and itraconazole for intracellular amastigotes of L. (V.) panamensis. (A to D) Dose response of antileishmanial activity of Poz (A), Itra (B), HePC (C), and SbV (D) in PBMCs from healthy volunteers infected with drug-sensitive and HePC- or SbV-resistant lines. Data are based on 3 replicates for each condition per experiment and presented as the mean and standard error of the mean (SEM) of two independent experiments for resistant lines and three experiments for the susceptible line.

FIG 2

Potency of fluconazole and voriconazole against intracellular amastigotes of L. (V.) panamensis. (A and B) Dose response of antileishmanial activity of Fluc (A) and Vori (B) in the ex vivo PBMC model infected with drug-sensitive and HePC- or SbV-resistant laboratory-derived lines. Data are based on three replicates for each condition per experiment and presented as the mean and SEM of two independent experiments.

FIG 3

Isobolograms of miltefosine and posaconazole combinations. Representation of the EC₅₀, EC₇₀, and EC₉₀ of the drugs combined (F0.2, F0.35, F0.5, F0.65, and F0.8) or alone (F0, Poz [y axis]; F1, HePC [x axis]) for infection with the L. (V.) panamensis drug-sensitive line. The black triangle in each panel corresponds to the fraction F0.5, selected as the optimal drug concentration for combination therapy (A). Combination index score for each fractional concentration (B). Synergy is defined by a fractional inhibitory concentration index ≤0.5. Each data point is based on a dose response curve of 6 concentrations of each drug or combination. Data are presented as the mean of two independent experiments.

FIG 4

Antileishmanial activity of the selected miltefosine/posaconazole combination for intracellular amastigotes of clinical strains. Potency of the selected drug combination (2 μM HePC/4 μM Poz) and the same concentrations of each drug alone for clinical strains of L. (V.) panamensis using an ex vivo model of PBMCs. Ten clinical strains were evaluated, five sensitive (empty symbols) and five tolerant/resistant (filled symbols) to miltefosine based on previously conducted evaluations of susceptibility in U937 macrophages. Each data point corresponds with the mean parasite burden determined by quantitative PCR of Leishmania 7SLRNA in two replicate cultures of PBMCs from the same donor. Bars correspond with the geometric mean of the parasite burden of the 10 clinical strains of L. (V.) panamensis. Symbols represent individual clinical strains.