Figure 1.
Chemical structures of edelfosine, miltefosine, perifosine and erucylphosphocholine.
Table 1.
Inhibition of proliferation of different Leishmania spp. (IC50 values) by ALPs.
Figure 2.
Differential ability of ALPs to induce apoptosis-like cell death in Leishmania spp.
(A) Promastigotes from different Leishmania spp. were treated with 10 µM edelfosine, miltefosine, perifosine or erucylphosphocholine (ErPC) at 26°C for 24 h. Apoptosis-like cell death was then quantitated as percentage of parasites in the sub-G0/G1 region by flow cytometry. (B) L. infantum promastigotes were incubated with different concentrations of edelfosine, miltefosine, perifosine and erucylphosphocholine (ErPC) at 26°C for 24 h, and then apoptosis-like cell death was determined by flow cytometry. (C) Representative histograms of cell cycle analysis of L. panamensis promastigotes treated with 5 and 10 µM edelfosine and miltefosine at different incubation times. The position of the sub-G0/G1 peak, integrated by parasites undergoing apoptosis-like cell death, is indicated by arrows. Percentages of apoptotic parasites are shown in each histogram. (D) L. panamensis promastigotes were treated with 5 and 10 µM edelfosine or miltefosine at different incubation times, and then apoptosis-like cell death was determined by flow cytometry. Untreated Leishmania promastigotes were run in parallel, and apoptosis-like cell death was less than 1.5% in untreated parasites in all cases shown in panels A–D. Data are means ± SD or representative of four independent experiments. Asterisks indicate that the differences between edelfosine- and miltefosine-treated cells are statistically significant. (*) P<0.05. (**) P<0.01.
Figure 3.
Antileishmanial activity of edelfosine against intracellular Leishmania amastigotes within macrophage-like J774 cells.
(A) J774 cells, incubated with the blue-emitting fluorescent analog PTE-ET (left panel), or with L. panamensis (Lp) and then with PTE-ET (middle panel), or with PTE-ET and then with L. panamensis (Lp) (right panel), were analyzed by fluorescence microscopy to examine drug localization. White arrows point to the intracellular amastigotes. (B) Parasite burden in L. panamensis-infected J774 cells untreated (Control) and treated with 5 or 10 µM edelfosine for 24 h. Data are means ± SD or representative of four independent experiments. Asterisks indicate that the differences between control and edelfosine-treated groups are statistically significant. (*) P<0.05. (**) P<0.01.
Figure 4.
In vivo antileishmanial action of edelfosine in L. major-infected mice.
BALB/c mice were infected with 2×106 L. major promastigotes in the left hind footpad, and after swelling was perceptible, mice were randomized into drug-treated (15 mg edelfosine/kg of body weight, daily oral administration for 28 days) and control (water vehicle) groups of 7 mice each. After completion of the 4-week treatment, lesions were evaluated by measuring the footpad swelling (A) and determining the parasite load (B), using caliper measurements and limiting dilution assays respectively. Data are means ± SD (n = 7). Asterisks indicate that the differences between control and edelfosine-treated groups are statistically significant. (**) P<0.01.
Figure 5.
In vivo antileishmanial action of edelfosine in L. panamensis- and L. braziliensis-infected hamsters.
Golden hamsters were infected with 1×106 L. panamensis or L. braziliensis promastigotes in the nose, and after swelling was perceptible, hamsters were randomized into drug-treated (26 mg edelfosine/kg of body weight, daily oral administration for 28 days) and control (water vehicle) groups of 7 hamsters each. (A, D) Lesion development was monitored by measuring nose thickness at regular intervals, and comparison to values obtained before treatment (evolution index). (B, E) Parasite load was determined by limiting dilution assays after completion of the 4-week in vivo assays. Data are means ± SD (n = 7). Asterisks indicate that the differences between control and edelfosine-treated groups are statistically significant. (*) P<0.05. (**) P<0.01. (C, F) Edelfosine treatment led to a dramatic decrease and amelioration in parasite-induced nose thickness and damage, as shown by representative photographs from drug-free control and edelfosine-treated hamsters.
Figure 6.
Sensitivity of SbV-resistant L. panamensis parasites to edelfosine.
(A) Two groups of eight golden hamsters each were infected in the nose with wild type and SbV-resistant L. panamensis (Lp) promastigotes, and after the sixth week post-infection, they were treated with a daily intramuscular injection of Glucantime (SbV) for 4 weeks. Disease evolution rate was measured along the whole process through determining nose thickness as compared to figures obtained before infection. (B) Golden hamsters inoculated with SbV-resistant (SbV-R) L. panamensis (Lp) did not respond to treatment with Glucantime (inflamed nose) (upper left panel), and nose smears showed amastigotes within macrophages (arrow) following Giemsa staining (lower left panel). However, hamsters infected with wild type (wt) L. panamensis (Lp) fully responded to Glucantime treatment, showing uninflamed nose and negative staining for amastigotes in nose smears (upper and lower right panels). (C) IC50 values of edelfosine, miltefosine, perifosine, and erucylphosphocholine (ErPC) for in vitro growth inhibition of wild type (wt) and SbV-resistant (SbV-R) L. panamensis (Lp) promastigotes were determined by XTT assays. (D–F) Golden hamsters were infected with 1×106 SbV-resistant L. panamensis promastigotes in the nose, and after nose inflammation was evident, hamsters were randomized into drug-treated (26 mg edelfosine/kg of body weight, daily oral administration for 28 days) and control (water vehicle) groups of 7 hamsters each. (D) Lesion development was monitored by measuring nose thickness at regular intervals and comparison to values obtained before treatment (evolution index). (E) Parasite load was determined by limiting dilution assays after completion of the 4-week in vivo assay. (F) Edelfosine treatment led to a dramatic decrease and amelioration in parasite-induced nose thickness and damage, as shown by photographs from drug-free control and edelfosine-treated hamsters. Data are means ± SD or representative experiments (n = 7). Asterisks indicate that differences between control and edelfosine-treated groups, or between wild type and SbV-resistant parasites treated with SbV, are statistically significant. (*) P<0.05. (**) P<0.01.
Table 2.
Differential incubation times required for drug resistance generation.