Evaluation of the potential of phenothiazinium dyes for Toxoplasma gondii control

Abstract

Toxoplasma gondii is an obligatory intracellular parasite and the causative agent of severe disturbances in human and animals, mostly in pregnant women and immunosuppressed patients. The treatment and prevention for toxoplasmosis are complex and non-completely solved processes, once there are no forms for the effective control of all parasite transmission routes and proliferation. Among the main forms of parasite transmission, the ingestion of cysts in tissues of infected animals (swine have an important role in Brazil) or water and food contaminated with oocysts from felid feces are prevalent. Despite their antiparasitic potential against Plasmodium or Neospora caninum, the phenothiazinium dyes, such as Methylene Blue and its derivatives have not been tested against Toxoplasma gondii. Four commercial phenothiazinium dyes (Methylene Blue, New Methylene Blue, Toluidine Blue O and 1,9 - Dimethyl Methylene Blue ) and four non-commercial tetra and pentacyclic derivatives (DO15, DO16, DO37 and DO43), synthesized by Prof. Mark Wainwright from Liverpool Jonh Moores University (UK), will be studied. These molecules are able to inactivate the Apicomplexan Neospora caninum in nanomolar concentrations and are compatible with drugs applied against T. gondii, such as Pyrimethamine. In this project, we propose to evaluate the antiparasitic potential of Methylene Blue and the non-commercial phenothiazinium dyes against T. gondii, using in vitro and in vivo models. Firstly, the dyes will be tested in vitro in tachyzoites forms in order to determine the reference inhibitory concentrations (IC50) for each compound. The mechanisms of action of the treatments with different compounds will be investigated by the evaluation of reactive oxygen species production, parasite viability, mitochondria integrity, lipid peroxidation and measurement of reduced glutathione levels. The phenothiazinium dyes with better in vitro performance (IC50 below 0.5 mM) will be evaluated in an in vivo model of toxoplasmosis, combined or not with Pyrimethamine, which is the reference drug for the disease treatment. In the case of confirmation of the antiparasitic potential of phenothiazinium dyes in this project, these compounds would represent a low-cost therapeutic strategy to be tested in anti-Toxoplasma formulations.