**6. Experimental approaches for drug testing in Leishmania**

Several *in vitro* assays to test *Leishmania* susceptibility to new potential inhibitors have been developed for the two stages of *Leishmania*, namely promastigotes and amastigotes. These two stages are morphologically and biochemically different and these differences are likely responsible for their differing susceptibility to proven anti-leishmanial compounds. Assays developed with intracellular amastigotes have the advantage of being more "disease

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**9. References** 

appropriate" since this is the stage responsible for mammalian disease. In addition, an intracellular *in vitro* model resembles the natural event when the parasite is in the mammalian host. Axenic amastigotes are also employed, although a more efficient alternative is the use of intracellular fluorescent or bioluminescent amastigotes.

The effectiveness of compounds to kill the parasites has been evaluated using different methodological approaches. Several years ago, direct parasite counting was the most used method (Gaspar., et al. 1992; Chan-Bacab., et al. 2003; Khan., et al. 2003). However, this method lacked accuracy and precision, likely due to human errors. This made necessary to develop new automated methods based on colorimetric, radioactive, fluorescent and luminometer detection (Fumarola., et al. 2004). Colorimetric methods like MTT [3-(4,5 dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide] have been used frequently. Recently, these methods are being replaced by transgenic parasites with reporter genes that do not interfere with cellular mitochondria. Parasites genetically engineered to express green fluorescent protein (GFP) or luciferase, have been developed and are currently used in automatized protocols involving flow cytometer or luminometer.

On the other hand, it is important to measure cytotoxicity to evaluate the possibility that a compound might produce side effects in humans. Mammalian or mouse cell lines are usually employed in these assays. The most used cells are U-937 human histiocytes, TPH-1 human peripheral blood monocytes, and hamster peritoneal macrophages (Robledo., et al. 1999; Weniger., et al. 2001; Varela., et al. 2009; Taylor., et al. 2010). To increase the selectivity of promising drugs, liposomal formulations of the compounds may be evaluated in order to reduce the toxicity as was observed for amphotericin B (Mehta., et al. 1985; Lopez-Berestein 1987).

The leishmanicidal activity of compounds that show high anti-leishmanial activity and low toxicity for mammalian cells *in vitro*, is next evaluated *in vivo*. This is normally performed in mouse or the golden hamster (*Mesocricetus auratus*) models depending on the *Leishmania* subgenus (Travi., et al. 2002). Monkey models can also used but these studies are limited to only a few laboratories worldwide (Grimaldi., et al. 2010).
