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The phosphodiesterase inhibitor tetrahydrophthalazinone NPD-008 was explored by phenotypic in vitroscreening, target validation, and ultrastructural approaches against Trypanosoma cruzi. NPD-008 displayed activity against different forms and strains of T. cruzi(50% effective concentration [EC50], 6.6 to 39.5 μM). Increased cAMP levels of T. cruziand its combination with benznidazole gave synergistic interaction.

ABSTRACTAlthough the exact role of quorum sensing (QS) in various stages of biofilm formation, maturation, and dispersal and in biofilm resistance is not entirely clear, the use of QS inhibitors (QSI) has been proposed as a potential antibiofilm strategy. We have investigated whether QSI enhance the susceptibility of bacterial biofilms to treatment with conventional antimicrobial agents. The QSI used in our study target the acyl-homoserine lactone-based QS system present in Pseudomonas aeruginosaand Burkholderia cepaciacomplex organisms (baicalin hydrate, cinnamaldehyde) or the peptide-based system present in Staphylococcus aureus(hamamelitannin). The effect of tobramycin (P. aeruginosa, B. cepaciacomplex) and clindamycin or vancomycin (S. aureus), alone or in combination with QSI, was evaluated in various in vitroand in vivobiofilm model systems, including two invertebrate models and one mouse pulmonary infection model. In vitrothe combined use of an antibiotic and a QSI generally resulted in increased killing compared to killing by an antibiotic alone, although reductions were strain and model dependent. A significantly higher fraction of infected Galleria mellonellalarvae and Caenorhabditis eleganssurvived infection following combined treatment, compared to treatment with an antibiotic alone. Finally, the combined use of tobramycin and baicalin hydrate reduced the microbial load in the lungs of BALB/c mice infected with Burkholderia cenocepaciamore than tobramycin treatment alone. Our data suggest that QSI may increase the success of antibiotic treatment by increasing the susceptibility of bacterial biofilms and/or by increasing host survival following infection.

ABSTRACTDiagnostic 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. braziliensisfield 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 (SbV; sodium stibogluconate), trivalent antimony (SbIII; potassium antimonyl tartrate), miltefosine, and the experimental drug PX-6518. All strains were sensitive to miltefosine (50% inhibitory concentration [IC50] < 10 μM) and PX-6518 (IC50< 2 μg/ml) but showed distinct susceptibility to SbVand/or SbIII, depending on whether they were derived from cured, relapse, or nonresponder patients. Within the available set of Leishmaniaspecies and strains, simultaneous SbV-SbIIIresistance was clearly associated with treatment failure; however, a larger set of isolates is still needed to judge the predictive value of SbV-SbIIIsusceptibility 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.

ABSTRACTThe in vitro susceptibilities of the reference strain Leishmania donovaniMHOM/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 [IC50], 0.6 to 0.7 μM), and amphotericin B showed slightly higher potency against intracellular amastigotes (IC50, 0.1 to 0.4 μM). A similar trend was observed for miltefosine, with comparable efficacies against the extracellular (IC50, 0.4 to 3.8 μM) and intracellular (IC50, 0.9 to 4.3 μM) stages. Sodium stibogluconate, used either as Pentostam or as a crystalline substance, was inactive against all axenic stages (IC50, >64 μg SbV/ml) but showed good efficacy against intracellular amastigotes (IC50, 22 to 28 μg SbV/ml); the crystalline substance was about two to three times more potent (IC50, 9 to 11 μg SbV/ml). The activity profile of PX-6518 was comparable to that of sodium stibogluconate, but at a much higher potency (IC50, 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 Leishmaniadrug 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.

ABSTRACTMaesabalide III (MB-III), an oleane triterpene saponin isolated from the Vietnamese plant Maesa balansae, is a new antileishmanial lead compound whose activity against Leishmania donovani(MHOM/ET/67/L82) in groups of five golden hamsters was evaluated after administration of a single subcutaneous dose on either day 1 (prophylactic treatment) or day 28 (curative treatment) after infection. Liposomal amphotericin B (AmBisome), administered intravenously at 5 mg/kg of body weight, was used as the reference drug. Amastigote burdens in liver, spleen, and bone marrow were determined either 7 days (early effects) or 56 days (late effects) after treatment. Prophylactic administration of MB-III at 0.2 mg/kg reduced liver amastigote burdens by 99.8 and 83% within 7 and 56 days after treatment, respectively. In the latter group, however, all animals became ill and some died. Both MB-III at 0.8 mg/kg and liposomal amphotericin B were 100% effective against liver stages, but clearance from the spleen and bone marrow was not achieved. Curative administration of MB-III at 0.2 and 0.4 mg/kg was not protective, as no survivors were left at the termination of the experiment on day 84. Despite the high level of reduction of the liver amastigote burden after treatment with MB-III at 0.8 mg/kg (94.2%) or liposomal amphotericin B (99.4%), clinical protection could not be obtained in either group, with two deaths occurring and the residual liver burdens persisting. It is concluded that administration of a single dose of MB-III at 0.8 mg/kg has efficacy potential comparable to that of a single dose of liposomal amphotericin B at 5 mg/kg and is therefore considered a promising new antileishmanial lead compound. However, multiple-dose pharmacological, toxicological, and pharmacokinetic studies are still needed before it can become a valid drug candidate for development.

ABSTRACTThe 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 Leishmaniastrains. The in vitro 50% inhibitory concentration (IC50) against intracellular Leishmania infantumamastigotes was 0.04 μg/ml. The cytotoxic concentrations causing 50% cell death (CC50s) 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 donovaniBALB/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. balansaeshow promising in vitro and in vivo antileishmanial potential and can be considered as new lead structures in the search for novel antileishmanial drugs.

ABSTRACTThe triazole antifungal pramiconazole (Stiefel, a GSK company) was compared with itraconazole, miconazole, and terbinafine in vitroand in vivo. Potent in vitroactivities against Candidaspp. (50% inhibitory concentration [IC50], 0.04 to 1.83 μM) and Microsporumand Trichophytonspp. (IC50, 0.15 to 1.34 μM) were obtained but not, however, against other filamentous molds and zygomycetes. In the M. canisguinea pig model and C. albicansvulvovaginitis rat model, pramiconazole was superior to the reference compounds after oral and topical administration.