Axenically grown amastigotes of Leishmania infantum used as an in vitro model to investigate the pentavalent antimony mode of action

The mechanism(s) of activity of pentavalent antimony [Sb(V)] is poorly unde rstood, In a recent study, we have shown that potassium antimonyl tartrate, a trivalent antimonial [Sb(III)], was substantially more potent than Sb(V) against both promastigotes and axenically grown amastigotes of three Leish mania species, supporting the idea of an in vivo metabolic conversion of Sb (V) into Sb(III), We report that amastigotes of Leishmania infantum culture d under axenic conditions were poorly susceptible to meglumine [Glucantime; an Sb(V)], unlike those growing inside THP-1 cells (50% inhibitory concent rations [IC(50)s], about 1.8 mg/ml and 22 mu g/ml, respectively). In order to define more precisely the mode of action of Sb(V) agents in vivo, we fir st induced in vitro Sb(III) resistance by direct drug pressure on axenicall y grown amastigotes of L. infantum. Then we determined the susceptibilities of both extracellular and intracellular chemoresistant amastigotes to the Sb(V)-containing drugs meglumine and sodiun stibogluconate plus m-chlorocre sol (Pentostam), The chemoresistant amastigotes LdiR2, LdiR10, and LdiR20 w ere 14, 26, and 32 times more resistant to Sb(III), respectively, than the wild-type one (LdiWT), In accordance with the hypothesis described above, w e found that intracellular chemoresistant amastigotes were resistant to meg lumine [Sb(V)] in proportion to the initial level of Sb(III)-induced resist ance. By contrast, Sb(III)-resistant cells were very susceptible to sodium stibogluconate, This lack of cross-resistance is probably due to the presen ce in this reagent of m-chlorocresol, which we found to be more toxic than Sb(III) to L. infantum amastigotes (IC(50)s, of 0.54 and 1.32 mu g/ml, resp ectively). Collectively, these results were consistent with the hypothesis of an intramacrophagic metabolic conversion of Sb(V) into trivalent compoun ds, which in turn became readily toxic to the Leishmania amastigote stage.