Comparison of commercial itraconazole to aerosolized nanoparticle itraconazole in a murine model for the prevention of invasive pulmonary aspergillosis (IPA)

PURPOSE: The primary purpose of this study was to compare commercial itraconazole solution (Sporanox) to placebo and three itraconazole nanoparticle formulations based on lung tissue burden, survival, and steady-state pharmacokinetics in a murine model infected with Aspergillus flavus. The secondary purpose was to utilize the tissue burden, survival, and pharmacokinetic (PK) data to determine among the nanoparticle formulations which formulation is the most efficacious as a prophylaxis regimen in mice infected with A. flavus. METHODS: In this 2-phase trial (Phase I - tissue burden and pharmacokinetic data and Phase II - tissue burden, survival and pharmacokinetic study), five week-old ICR/Swiss male mice, weighing approximately 13 gm, were randomly assigned in groups of 10, to receive 30mg/kg/dose of one of the following itraconazole formulation regimens: commercial Sporanox via gavage three-times-daily (TID), Spray-Freezing into Liquid (SFL) via gavage TID, Evaporative Precipitation into Aqueous Solution (EPAS) via aerosolization twice-daily (BID), SFL via aerosolization BID, or control (distilled, sterile water) via aerosolization BID. After 1 day of treatment, the mice were infected via inhalation with approximately 10⁶ condida of itraconazole susceptible A. flavus (MIC = 0.125mcg/ml). Prophylaxis with each regimen continued daily for a total of 10 days for the tissue burden study and 12 days for the survival study. Survival data and tissue burden data were collected on all infected mice. Additionally, after 6 days of itraconazole dosing, serum and lung tissue were collected from 2 mice per group at the following timepoints: 0.5, 1, 2, 6, 10, and 24 hours for determination of steady state pharmacokinetics parameters. Itraconazole serum and lung concentrations were determined via a validated high performance liquid chromatography (HPLC). Pharmacokinetic parameters were determined using non-compartmental analysis. Statistical comparisons were conducted via one-way analysis of variance (ANOVA) and Tukey-Kramer. Statistical significance was set at p < 0.05. RESULTS: The overall starting inoculum (log₁₀ mean ± SEM) of Aspergillus flavus was 5.69 ± 0.14 per lung and 6.20 ± 0.17 per gram in Phase I and 4.73 ± 0.21 per lung and 5.21 ± 0.25 per gram in Phase II. No statistical difference was identified among the overall starting inoculum when each phase was examined independently. However, Phase I starting inoculum was significantly higher than Phase II by approximately one log (p < 0.0001). In the tissue burden study, no statistical differences were identified between the groups in either phase. For Phase I, the colony counts (in CFUs per gram) ranged from 4.97 ± 0.20 in the SFL aerosolized group to 5.18 ± 0.08 in the control group and in Phase II, ranged from 4.34 ± 0.19 in those mice treated with SFL oral to 5.06 ± 0.06 for the control group. In the survival study, the SFL aerosolized group had a statistically significant longer survival than the control group (14.7 ± 2.30 vs. 6.30 ± 1.08 days survival post-infection, p = 0.0046), SFL oral (5.80 ± 1.69, p < 0.05), and commercial itraconazole (5.60 ± 1.28, p < 0.05). However, no significant differences existed between the SFL aerosolized group and the EPAS aerosolized group (14.7 ± 2.30 vs. 11.7 ± 2.44 days survival post-infection). Serum and lung tissue concentrations of itraconazole were below the limit of quantification for all groups except for the lung tissue concentrations in the EPAS and SFL aerosolization groups. For the EPAS aerosolization formulation, the maximum concentration (Cmax), half-life (T₁/₂) and area under the curve (AUC₀₋₂₄hrs) were 25.89 [microgram]/gm, 7.17 hours, and 70.89 [microgram]h/ml, respectively. The SFL aerosolized formulation demonstrated a Cmax of 5.28 [microgram/gm, a T₁/₂ of 2.95 hours and AUC₀₋₂₄hrs of 28.02 [microgram]h/ml. CONCLUSIONS: Although no significant difference in tissue burden was observed among the formulations of itraconazole administered for the prevention of A. flavus in this murine model, survival was highest with aerosolization of itraconazole nanoparticles compared to oral administration or control groups. In addition to the increased duration of survival, the aerosolized itraconazole formulations produced by EPAS and SFL nanoparticle technology demonstrated superior pharmacokinetic characteristics. Notwithstanding the promising pharmacokinetic results, the lack of correlation between increased survival and tissue burden among these itraconazole formulations requires further investigation