Ravuconazole self-emulsifying delivery system: in vitro activity against Trypanosoma cruzi amastigotes and in vivo toxicity

Pollyanna Álvaro Spósito,1 Ana Lia Mazzeti,1,2 Caroline de Oliveira Faria,1 Julio A Urbina,3 Gwenaelle Pound-Lana,1 Maria Terezinha Bahia,2 Vanessa Furtado Mosqueira1 1Laboratory of Pharmaceutics and Nanotechnology Research, Pharmacy Department, School of Pharmacy, Universidade Federal de Ouro Preto, Minas Gerais, Brazil; 2Parasite Diseases Research Laboratory, NUPEB, Medical School, Universidade Federal de Ouro Preto, MG, Brazil; 3Venezuelan Institute for Scientific Research, Apartado, Caracas, Venezuela Abstract: Self-emulsifying drug delivery systems (SEDDSs) are lipid-based anhydrous formulations composed of an isotropic mixture of oil, surfactant, and cosurfactants usually presented in gelatin capsules. Ravuconazole (Biopharmaceutics Classification System [BCS] Class II) is a poorly water-soluble drug, and a SEDDS type IIIA was designed to deliver it in a predissolved state, improving dissolution in gastrointestinal fluids. After emulsification, the droplets had mean hydrodynamic diameters <250 nm, zeta potential values in the range of −45 mV to −57 mV, and showed no signs of ravuconazole precipitation. Asymmetric flow field-flow fractionation with dynamic and multiangle laser light scattering was used to characterize these formulations in terms of size distribution and homogeneity. The fractograms obtained at 37°C showed a polydisperse profile for all blank and ravuconazole–SEDDS formulations but no large aggregates. SEDDS increased ravuconazole in vitro dissolution extent and rate (20%) compared to free drug (3%) in 6 h. The in vivo toxicity of blank SEDDS comprising Labrasol® surfactant in different concentrations and preliminary safety tests in repeated-dose oral administration (20 days) showed a dose-dependent Labrasol toxicity in healthy mice. Ravuconazole–SEDDS at low surfactant content (10%, v/v) in Trypanosoma cruzi-infected mice was safe during the 20-day treatment. The anti-T. cruzi activity of free ravuconazole, ravuconazole–SEDDS and each excipient were evaluated in vitro at equivalent ravuconazole concentrations needed to inhibit 50% or 90% (IC50 and IC90), respectively of the intracellular amastigote form of the parasite in a cardiomyocyte cell line. The results showed a clear improvement of the ravuconazole anti-T. cruzi activity when associated with SEDDS. Based on our results, the repurposing of ravuconazole in SEDDS dosage form is a strategy that deserves further in vivo investigation in preclinical studies for the treatment of human T. cruzi infections. Keywords: ravuconazole, self-emulsifying drug delivery, asymmetric flow field-flow fractionation, Trypanosoma cruzi, Chagas disease, in vitro activit

Comparison of statistical models in a meta-analysis of fungicide treatments for the control of citrus black spot caused by Phyllosticta citricarpa

Meta-analysis has been recognised as a powerful method to synthetize existing published data from different studies through a formal statistical analysis. Several statistical models have been proposed to evaluate the effectiveness of treatments against plant diseases using meta-analysis, but the sensitivity of the estimated treatment effects to the model chosen has not been investigated in detail in the context of plant pathology. In this paper, four different statistical models were defined to analyse fungicide control trials with binary outcomes. These models were used to conduct a meta-analysis on the effectiveness of fungicide treatments against citrus black spot, a fungal disease caused by the quarantine pathogen Phyllosticta citricarpa. The models differed in the assumption made on the variability of the treatment effect (constant or variable between experimental plots) and in the method used for parameter estimation (classical or Bayesian). Odds ratios were estimated for two groups of fungicides, copper compounds and dithiocarbamates, widely applied for CBS control using each model in turn. Classical and Bayesian statistical models led to similar results, but the estimated treatment effectiveness and their associated levels of uncertainty were sensitive to the assumption made about the variability of the treatment effect. Estimated odds ratios were different depending on whether the treatment effect was assumed to be constant or variable between experimental plots. The size of the confidence intervals was underestimated when the treatment effect was assumed constant while it was variable in reality. Because of the strong between-plot variability, the 90 % percentiles of the odds ratios were much higher than the point estimates, and this result revealed that, in some plots, treatment effectiveness could be much lower than expected. Based on our results, we conclude that it is not sufficient to calculate point estimates of odds ratio when the between-plot variability of the treatment effect is strong and that, in such case, it is recommended to compute the predictive distributions of the odds ratio