Stage-specific activity of pentavalent antimony against Leishmania donovani axenic amastigotes

Abstract

The standard treatment of human visceral leishmaniasis involves the use of pentavalent antimony (SbV) compounds. In recent years increasing numbers of clinical failures of treatment with SbV have been reported, probably due to the development of parasite resistance to this compound. The mode of action and mechanisms of resistance to SbV have not been fully elucidated. In the present study an axenic amastigote culture was used to study the in vitro responses of Leishmania donovani to SbV. Susceptibility to both sodium stibogluconate and meglumine antimoniate was found to be stage specific. Amastigotes were 73 to 271 times more susceptible to SbV than were promastigotes. As opposed to SbV, trivalent antimony (SbIII) was similarly toxic to both developmental stages. When promastigotes were transformed to amastigotes, susceptibility to meglumine antimoniate developed after 4 to 5 days, upon the completion of differentiation. In contrast, with transformation from amastigotes to promastigotes, resistance to meglumine antimoniate was acquired rapidly, within 24 h, before the completion of differentiation. The culture of promastigotes at an acidic pH (5.5) or at an elevated temperature (37 degrees C) alone did not lead to the appearance of SbV susceptibility, emphasizing the requirement of both these environmental factors for the development of SbV susceptibility. A previously isolated sodium stibogluconate (Pentostam)-resistant L. donovani mutant (Ld1S.20) is also resistant to meglumine antimoniate, indicating cross-resistance to SbV-containing compounds. In contrast, no cross-resistance was found with SbIII, suggesting a mechanism of SbV resistance different from that described in Leishmania tarentolae. These data show that L. donovani susceptibility to SbV is parasite intrinsic, stage specific, and macrophage independent.

FIG. 1

Stage-specific susceptibility of L. donovani to SbV. Promastigotes (○) and amastigotes (•) were incubated in the presence of increasing concentrations of sodium stibogluconate (A) and meglumine antimoniate (B) for 48 h and were assayed for ODC activity as described in Materials and Methods. The results are expressed as means ± standard deviations (n = 6).

FIG. 2

Dose-response curve for L. donovani to SbIII. Promastigotes (○) and amastigotes (•) were incubated in the presence of increasing concentrations of potassium antimonyl tartrate for 48 h and were assayed for ODC activity as described in Materials and Methods. The results are expressed as means ± standard deviations (n = 3).

FIG. 3

Dose-response curve for L. donovani promastigotes and meglumine antimoniate: effects of pH and temperature over time. Promastigotes were maintained at 26°C and pH 7.4 (■), at 37°C and pH 7.4 (•), or at 26°C and pH 5.5 (○). After ∼2 weeks, the parasites were incubated and were assayed for ODC activity.

FIG. 4

Development of L. donovani susceptibility to meglumine antimoniate during in vitro transformation of promastigotes to amastigotes. Promastigotes were transformed to amastigotes as follows: mid-logarithmic-phase promastigotes were transferred to 37°C for 24 h and then shifted to pH 5.5 as described in Materials and Methods. Meglumine antimoniate dose-response measurements were performed daily over 5 additional days: 1 day at 37°C and pH 7.4 (•) and 1 day (▴), 2 days (⧫), 3 days (■), 4 days (▾), and 5 days (✚) at 37°C and pH 5.5. The results are expressed as means ± standard deviations (n = 3).

FIG. 5

Loss of L. donovani susceptibility to meglumine antimoniate during in vitro transformation of amastigotes to promastigotes. Amastigotes (•) were transformed to promastigotes as follows: amastigotes were centrifuged, suspended in promastigote medium, and incubated at 26°C and pH 7.4 as described in Materials and Methods. Meglumine antimoniate dose-response measurements were performed daily for 5 additional days: day 1 (○), day 2 (▵), day 3 (□), and day 5 (▿). The results are expressed as means ± standard deviations (n = 3).