Supplementary Material for: Protective Therapy with Novel Chromone Derivative against <i>Leishmania donovani</i> Infection Induces Th1 Response in vivo

<i>Background:</i> Visceral leishmaniasis is a chronic protozoan disease caused by <i>Leishmania donovani,</i> an obligatory intracellular parasite that resides and multiplies within macrophages of the reticuloendothelial system. The aim of this study was to evaluate the efficacy of nine novel synthetic chromone derivatives as antileishmanial molecules in experimental murine visceral leishmaniasis. <i>Methods:</i> In vitro activity of the molecules (2, 5 and 10 µg/ml) was assessed against promastigotes of both pentavalent antimonial-responsive strain AG83 and pentavalent antimonial-resistant strain GE1F8R at days 2 (48 h), 4 (96 h) and 6 (144 h). The efficacy of the most efficient chromone derivative [C-(6-Methyl-4-oxo-4<i>H</i>-1-benzopyran-3-yl)-N-(<i>p</i>-tolyl) nitrone], designated here as NP1, was also tested against intracellular amastigotes in vitro and in vivo. <i>Results:</i> NP1, 5 µg/ml, inhibited the growth of AG83 and GE1F8R promastigotes by 98.57% (day 4) and 75.75% (day 6), respectively, and also inhibited the growth of intracellular amastigotes by 85% (day 3), compared to DMSO control. Treatment of <i>L. donovani-</i>infected mice with NP1 resulted in a 70% significant decrease in parasite load in the spleen in the 7th week after infection (5 mice in each group), with associated induction of interferon-γ synthesis by dose 2 (37.5 mg/kg body weight) compared to DMSO control. Dose 2 was found efficient over dose 1 (25 mg/kg body weight). <i>Conclusions:</i> The novel synthetic chromone derivative is effective in the treatment of visceral leishmaniasis and induces the synthesis of interferon-γ in rodent models.