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 μM), and amphotericin B showed slightly higher potency against intracellular amastigotes (IC ₅₀ , 0.1 to 0.4 μM). A similar trend was observed for miltefosine, with comparable efficacies against the extracellular (IC ₅₀ , 0.4 to 3.8 μM) and intracellular (IC ₅₀ , 0.9 to 4.3 μM) stages. Sodium stibogluconate, used either as Pentostam or as a crystalline substance, was inactive against all axenic stages (IC ₅₀ , >64 μg Sb ^V /ml) but showed good efficacy against intracellular amastigotes (IC ₅₀ , 22 to 28 μg Sb ^V /ml); the crystalline substance was about two to three times more potent (IC ₅₀ , 9 to 11 μg 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 μg/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.

Diagnostic material from patients with leishmaniasis is generally available as promastigotes, and proper testing for susceptibility to first-line drugs by the intracellular amastigote assay is frequently hampered by the poor infectivity of the promastigotes for the macrophage host cell. Several conditions for optimization of the in vitro metacyclogenesis and cell infectivity of Leishmania donovani , L. guyanensis , and L. braziliensis field strains obtained from patients receiving standard antimony medication were investigated. Triggering log-phase promastigotes to become amastigote-like by increasing the temperature or acidifying the culture medium was not successful. Adequate metacyclogenesis and the highest levels of macrophage infection were obtained after 5-day-old late-log-phase promastigote cultures were preconditioned at 25°C to pH 5.4 for 24 h in Schneider's medium prior to infection. The susceptibility assay with primary peritoneal mouse macrophages included pentavalent antimony (Sb ^V ; sodium stibogluconate), trivalent antimony (Sb ^III ; potassium antimonyl tartrate), miltefosine, and the experimental drug PX-6518. All strains were sensitive to miltefosine (50% inhibitory concentration [IC ₅₀ ] < 10 μM) and PX-6518 (IC ₅₀ < 2 μg/ml) but showed distinct susceptibility to Sb ^V and/or Sb ^III , depending on whether they were derived from cured, relapse, or nonresponder patients. Within the available set of Leishmania species and strains, simultaneous Sb ^V -Sb ^III resistance was clearly associated with treatment failure; however, a larger set of isolates is still needed to judge the predictive value of Sb ^V -Sb ^III susceptibility profiling on treatment outcome. In conclusion, the proposed conditioning protocol further contributes toward a more standardized laboratory model for evaluation of the drug sensitivities of field isolates.

The in vitro and in vivo activities of a mixture of six oleane triterpene saponins, recovered from the methanolic extract of the leaves of the Vietnamese plant Maesa balansae (PX-6518), were evaluated against drug-sensitive visceral Leishmania strains. The in vitro 50% inhibitory concentration (IC ₅₀ ) against intracellular Leishmania infantum amastigotes was 0.04 μg/ml. The cytotoxic concentrations causing 50% cell death (CC ₅₀ s) were about 1 μg/ml in murine macrophage host cells and >32 μg/ml in human fibroblasts (MRC-5 cell line). Evaluation in the Leishmania donovani BALB/c mouse model indicated that a single subcutaneous administration of 0.4 mg/kg at 1 day after infection reduced liver amastigote burdens by about 95% in all treated animals. If treatment was delayed until 14 days after infection, a dose of 1.6 mg/kg of body weight was required to maintain the same level of activity. Single 250-mg/kg doses of sodium stibogluconate (Pentostam) 1 and 14 days after infection produced comparable efficacies. A single dose of PX-6518 at 2.5 mg/kg administered 5 days before infection was still 100% effective in preventing liver infection, suggesting a particularly long residual action. Spleen and bone marrow could not be cleared by PX-6518 nor sodium stibogluconate. PX-6518 did not show activity after oral dosing at up to 200 mg/kg for 5 days. This study concludes that triterpenoid saponins from M. balansae show promising in vitro and in vivo antileishmanial potential and can be considered as new lead structures in the search for novel antileishmanial drugs.

Five xanthone derivatives and one flavanol were isolated from the dichloromethane extract of Garcinia mangostana. Dichloromethane, ethyl acetate extract and the major xanthone (α-mangostin) were evaluated in vitro against erythrocytic schizonts of Plasmodium falciparum, intracellular amastigotes of Leishmania infantum and Trypanosoma cruzi and free trypomastigotes of T. brucei. The major constituent α-mangostin was also checked for antimicrobial potential against Candida albicans, Escherichia coli, Pseudomonas aeruginosa, Bacillius subtilis, Staphylococcus aureus, Mycobacterium smegmatis, M. cheleneoi, M. xenopi and M. intracellulare. Activity against P. falciparum (IC₅₀ 2.7 μg/mL) and T. brucei (IC₅₀ 0.5 μg/mL) were observed for the dichloromethane extract, however, with only moderate selectivity was seen based on a parallel cytotoxicity evaluation on MRC-5 cells (IC₅₀ 9.4 μg/mL). The ethyl acetate extract was inactive (IC₅₀ > 30 µg/mL). The major constituent α-mangostin showed rather high cytotoxicity (IC₅₀ 7.5 µM) and a broad but non-selective antiprotozoal and antimicrobial activity profile. This in vitro study endorses that the antiprotozoal and antimicrobial potential of prenylated xanthones is non-conclusive in view of the low level of selectivity.

The present study investigated the in vitro antiprotozoal activity of sixteen selected medicinal plants. Plant materials were extracted with methanol and screened in vitro against erythrocytic schizonts of Plasmodium falciparum, intracellular amastigotes of Leishmania infantum and Trypanosoma cruzi and free trypomastigotes of T. brucei. Cytotoxic activity was determined against MRC-5 cells to assess selectivity. The criterion for activity was an IC₅₀ < 10 µg/mL (4. Antiplasmodial activity was found in the extracts of Prosopis juliflora and Punica granatum. Antileishmanial activity against L. infantum was demonstrated in Caralluma sinaica and Periploca aphylla. Amastigotes of T. cruzi were affected by the methanol extract of Albizia lebbeck pericarp, Caralluma sinaica, Periploca aphylla and Prosopius juliflora. Activity against T. brucei was obtained in Prosopis juliflora. Cytotoxicity (MRC-5 IC₅₀ < 10 µg/mL) and hence non-specific activities were observed for Conocarpus lancifolius.

Background: Worldwide particularly in developing countries, a large proportion of the population is at risk for tropical parasitic diseases. Several medicinal plants are still used traditionally against protozoal infections in Yemen and Saudi Arabia. Thus the present study investigated the in vitro antiprotozoal activity of twenty-five plants collected from the Arabian Peninsula. Methods: Plant materials were extracted with methanol and screened in vitro against erythrocytic schizonts of Plasmodium falciparum, intracellular amastigotes of Leishmania infantum and Trypanosoma cruzi and free trypomastigotes of T. brucei. Cytotoxic activity was determined against MRC-5 cells to assess selectivity. The criterion for activity was an IC₅₀ < 10 μg/ml (4. Results: Antiplasmodial activity was found in the extracts of Chrozophora oblongifolia, Ficus ingens, Lavandula dentata and Plectranthus barbatus. Amastigotes of T. cruzi were affected by Grewia erythraea, L. dentata, Tagetes minuta and Vernonia leopoldii. Activity against T. brucei was obtained in G. erythraea, L. dentata, P. barbatus and T. minuta. No relevant activity was found against L. infantum. High levels of cytotoxicity (MRC-5 IC₅₀ < 10 μg/ml) and hence non-specific activities were noted in Cupressus sempervirens, Kanahia laniflora and Kniphofia sumarae. Conclusion: The results endorse that medicinal plants can be promising sources of natural products with antiprotozoal activity potential. The results support to some extent the traditional uses of some plants for the treatment of parasitic protozoal diseases.