Delivering small molecule chemical series that give full cure in the Chagas disease mouse model of infection is proving challenging, despite compounds having high potency against the parasite and good oral pharmacokinetics. It is therefore proving important to try and identify where remaining parasites are residing directly following ending of drug pressure in order to identify those tissues/organs compounds need further optimisation for to deliver therapeutic levels.
The potential reasons for a new drug treatment to not deliver ‘cure’ in the disease model are:
- Inability to kill all forms of the parasite i.e. ‘wrong’ mode of action. There is growing evidence that there are persister/dormant parasites, less susceptible to drug treatment. Our new wash-out assay described above should help identify these compounds but success in vitro is not always followed by success in vivo.
- Poor drug distribution properties to those sites where parasites reside. It is clear from the bioluminescent mouse model of disease that the parasites are highly motile between tissues and extensively distributed throughout the body, potentially located in tissues and organs that create significant drug delivery challenges, such as immunoprivileged sites (testes, ovaries and brain) and large, poorly perfused tissues (visceral fat).
To better understand where parasites reside we are developing sensitive assays that allow detection of single parasites within organs. A method that makes organs ‘see-through’. CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktail) uses detergents that allow preservation of fluorescent proteins expressed by transgenic parasites within a tissue or organ, so they will be easily visualised without the need to dissect and thus disrupt the sample. Once the tissue or organ has been cleared components such as the cell nucleus or other cellular structures can be labelled to help identify where the parasites are located. Using specialist microscopy, such as confocal or light-sheet microscopy, a 3D image of the tissue or organ can be built.
Once we know which organs (and areas of these organs) the parasites are located we then need to check we are getting the correct amount of compound to these locations. Non-uniform drug distribution could still account for sub-therapeutic tissue levels where the parasites reside even though whole tissue drug free concentration would advise otherwise. As example, in Figure 2, there is non-uniform distribution of this lead compound in brain yet total brain homogenate analysis suggested sufficient brain-free exposure to deliver cure.
Matrix-assisted laser desorption / ionization mass spectrometry imaging (MALDI MSI) has become a very powerful tool for studying the localization of endogenous and exogenous compounds, in particular drug distribution in biological tissues. Knowledge of the deposition and distribution of drug compounds and their metabolites is essential in drug discovery and development as it provides important information to the understanding of the efficacy, toxicology and pharmacokinetics of the drug. We intend to use mass spectrometry imaging, predominantly MALDI MS imaging, and associated histopathological techniques to understand:
- Compound distributional properties in those organs and tissues where remaining parasites are identified following drug treatment and,
- To attempt to identify and visualize parasite-specific biological markers, e.g. lipid or small peptide markers, and thus directly provide molecular information on the parasite distributions within the host(s), complementary to high-spatial resolution staining and pathology approaches. The analysis of an inactivated, in-vitro culture of the parasite could be a primary source of sufficient material to identify unique, specific lipid and peptides. The specific biological markers will then be used for the detection of parasites in infected animals
This project is supported by an MRC iCASE PhD in partnership with GlaxoSmithKline with WCAIR support for the animal disease model.