An axenic amastigote system for drug screening

Abstract

Currently available primary screens for selection of candidate antileishmanial compounds are not ideal. The choices include screens that are designed to closely reflect the situation in vivo but are labor-intensive and expensive (intracellular amastigotes and animal models) and screens that are designed to facilitate rapid testing of a large number of drugs but do not use the clinically relevant parasite stage (promastigote model). The advent of successful in vitro culture of axenic amastigotes permits the development of a primary screen which is quick and easy like the promastigote screen but still representative of the situation in vivo, since it uses the relevant parasite stage. We have established an axenic amastigote drug screening system using a Leishmania mexicana strain (strain M379). A comparison of the 50% inhibitory concentration (IC50) drug sensitivity profiles of M379 promastigotes, intracellular amastigotes, and axenic amastigotes for six clinically relevant antileishmanial drugs (sodium stibogluconate, meglumine antimoniate, pentamidine, paromomycin, amphotericin B, WR6026) showed that M379 axenic amastigotes are a good model for a primary drug screen. Promastigote and intracellular amastigote IC50s differed for four of the six drugs tested by threefold or more; axenic amastigote and intracellular amastigote IC50s differed by twofold for only one drug. This shows that the axenic amastigote susceptibility to clinically used reference drugs is comparable to the susceptibility of amastigotes in macrophages. These data also suggest that for the compounds tested, susceptibility is intrinsic to the parasite stage. This contradicts previous hypotheses that suggested that the activities of antimonial agents against intracellular amastigotes were solely a function of the macrophage.

Parasite Susceptibility to Amphotericin B in Failures of Treatment for Visceral Leishmaniasis in Patients Coinfected with HIV Type 1 andLeishmania infantum

“…Remarkably, the IC 50 to AmB of all primary isolates varied very little between strains, with karyotypes having IC 50 values ranging from 0.26 to 0.6 mg/mL. Similar IC 50 values were observed between promastigotes and amastigotes, further supporting the idea that the stage of the parasite does not seem to influenc AmB susceptibility [40,41]. One of the most important finding of this study is that parasites that have been in contact with AmB for many episodes are not less susceptible to the drug than are primary isolates (table 1).…”

Reduction of Sb(V) in a Human Macrophage Cell Line Measured by HPLC-ICP-MS

“…Several studies [11][12][13][14] have found that Sb(V) is inefficient against amastigotic Leishmania parasites cultured axenically, whereas it has effect on parasites when intracellular; this may indicate that the macrophages are the primary site for activation of the Sb drug. Shaked-Mishan [17] found reduction of Sb(V) within the parasite which could explain that other authors have reported on axenically amastigotes where Sb(V) did show activity [15,16]. Different parasite strains is likely an explanation of these discrepancies, but even for 'self-reducing' strains, a reduction in the macrophages will most likely contribute significantly to the Sb(V) drugs' effect in the clinical situation, as the parasite will take up Sb(III) if present (except for certain Sb-resistant strains) [9,17].…”

Evaluation of the leishmanicidal activity of plants used by Peruvian Chayahuita ethnic group

“…The differences in sensibility of promastigotes compared to amastigotes is well documented (Callahan et al, 1997;Berman et al, 1982;Croft et al, 1981). Callahan et al (1997) and us herein showed that Amphotericin B did have an activity on promastigotes of Leishmania mexicana equivalent to the one on axenic amastigotes (IC 50 around 0.2 g/ml). On the contrary, Glucantime was shown to be almost inactive on promastigotes (IC 50 : 1100 g/ml) but active on amastigotes (IC 50 : 30 g/ml).…”