In Vitro Susceptibilities of Leishmania donovani Promastigote and Amastigote Stages to Antileishmanial Reference Drugs: Practical Relevance of Stage-Specific Differences

Abstract

The in vitro susceptibilities of the reference strain Leishmania donovani MHOM/ET/67/L82 to sodium stibogluconate, amphotericin B, miltefosine, and the experimental compound PX-6518 were determined for extracellular log-phase promastigotes, established axenic amastigotes, fresh spleen-derived amastigotes, and intracellular amastigotes in primary mouse peritoneal macrophages. Susceptibility to amphotericin B did not differ across the various axenic models (50% inhibitory concentrations [IC 50 ], 0.6 to 0.7 M), and amphotericin B showed slightly higher potency against intracellular amastigotes (IC 50 , 0.1 to 0.4 M). A similar trend was observed for miltefosine, with comparable efficacies against the extracellular (IC 50 , 0.4 to 3.8 M) and intracellular (IC 50 , 0.9 to 4.3 M) stages. Sodium stibogluconate, used either as Pentostam or as a crystalline substance, was inactive against all axenic stages (IC 50 , >64 g Sb V /ml) but showed good efficacy against intracellular amastigotes (IC 50 , 22 to 28 g Sb V /ml); the crystalline substance was about two to three times more potent (IC 50 , 9 to 11 g Sb V /ml). The activity profile of PX-6518 was comparable to that of sodium stibogluconate, but at a much higher potency (IC 50 , 0.1 g/ml). In conclusion, the differential susceptibility determines which in vitro models are appropriate for either drug screening or resistance monitoring of clinical field isolates. Despite the more complex and labor-intensive protocol, the current results support the intracellular amastigote model as the gold standard for in vitro Leishmania drug discovery research and for evaluation of the resistance of field strains, since it also includes host cell-mediated effects. Axenic systems can be recommended only for compounds for which no cellular mechanisms are involved, for example, amphotericin B and miltefosine.Current first-line chemotherapy of leishmaniasis relies on a rather limited arsenal of drugs including sodium stibogluconate, meglumine antimoniate, amphotericin B, and miltefosine, but these entail either problems of emerging resistance, severe side effects, or high costs (5). Since vaccines are not yet on the horizon (23), maintenance and improvement of existing treatment regimens, combined with new drug discovery initiatives, appear to be the only ways to guarantee continued control of this important tropical disease (3,11,27).Both for drug screening and for determination of the susceptibility of field strains, different laboratory methods are being used that focus on the promastigote, the axenic amastigote, or the intracellular amastigote stage. However, it remains unclear how these models cross-validate each other. The differences in environmental conditions between promastigotes and amastigotes in vivo are reflected in their needs for in vitro cultivation. While promastigotes are easily cultured in suspension (8), amastigotes are more difficult to maintain in vitro, since they require macrophages as host cells to meet the highly acidic intracellular environment (15). Cultivat...

Antileishmanial activity and evaluation of the mechanism of action of strychnobiflavone flavonoid isolated from Strychnos pseudoquina against Leishmania infantum

“…(De Muylder et al, 2011). Compounds active against axenic parasites might be unable to reach the intracellular amastigotes, because of their inability to cross the host cell membranes, or maintain stability under low pH, whereas other compounds may need to be metabolized by macrophages to gain activity (Vermeersch et al, 2009). This is in accordance with previous studies, which found that only 4% of their hits identified in a promastigote primary screening were actually active in an intracellular context (Siqueira-Neto et al, 2010).…”

Effect of 3‐Alkylpyridine Marine Alkaloid Analogues in Leishmania Species Related to American Cutaneous Leishmaniasis

“…Despite the more complex and labor-intensive protocol, the intracellular amastigote model has been cited as the golden standard for in vitro Leishmania drug discovery research (9,28). The results showed in this work reinforce the potential leishmanicidal activity of the oxygenated analogues of marine 3-alkylpyridine alkaloids.…”

“…Vermeersch et al (28) recently recommended that the intracellular amastigote model could be employed as the 'gold standard' for antileishmanial in vitro evaluation. Amastigote forms are the stage of parasite found in the mammalian host and responsible for the human disease.…”

Toxicity of betulin derivatives and in vitro effect on promastigotes and amastigotes of Leishmania infantum and L. donovani

“…On the other hand, the lack of correlation between the results against promastigotes and amastigotes points to the need for using at least the intracellular stage in macrophages as the screening method to identify lead compounds against Leishmania. This need has been recognized, 25,31,32 and is possibly critical for reducing the attrition rates of drug discovery.…”