Efficacy of a Binuclear Cyclopalladated Compound Therapy for Cutaneous Leishmaniasis in the Murine Model of Infection with Leishmania amazonensis and Its Inhibitory Effect on Topoisomerase 1B

Abstract

Leishmaniasis is a disease found throughout the (sub)tropical parts of the world caused by protozoan parasites of the Leishmania genus. Despite the numerous problems associated with existing treatments, pharmaceutical companies continue to neglect the development of better ones. The high toxicity of current drugs combined with emerging resistance makes the discovery of new therapeutic alternatives urgent. We report here the evaluation of a binuclear cyclopalladated complex containing Pd(II) and N,N'-dimethylbenzylamine (Hdmba) against Leishmania amazonensis The compound [Pd(dmba)(μ-N₃)]₂ (CP2) inhibits promastigote growth (50% inhibitory concentration [IC₅₀] = 13.2 ± 0.7 μM) and decreases the proliferation of intracellular amastigotes in in vitro incubated macrophages (IC₅₀ = 10.2 ± 2.2 μM) without a cytotoxic effect when tested against peritoneal macrophages (50% cytotoxic concentration = 506.0 ± 10.7 μM). In addition, CP2 was also active against T. cruzi intracellular amastigotes (IC₅₀ = 2.3 ± 0.5 μM, selective index = 225), an indication of its potential for use in Chagas disease therapy. In vivo assays using L. amazonensis-infected BALB/c showed an 80% reduction in parasite load compared to infected and nontreated animals. Also, compared to amphotericin B treatment, CP2 did not show any side effects, which was corroborated by the analysis of plasma levels of different hepatic and renal biomarkers. Furthermore, CP2 was able to inhibit Leishmania donovani topoisomerase 1B (Ldtopo1B), a potentially important target in this parasite. (This study has been registered at ClinicalTrials.gov under identifier NCT02169141.).

Keywords: Chagas disease; Leishmania amazonensis; Leishmania donovani; Trypanosoma cruzi; cyclopalladated complex; leishmaniasis; topoisomerase 1B.

FIG 1

Structure of binuclear cyclopalladated complex CP2, [Pd(dmba)(μ-N₃)]₂ (61). A better model for [Pd(dmba)(N₃)]₂ is a blend of Lewis structures I and II with N1-N2 and N4-N5 intermediate in properties between a single and a double bond, with N2-N3 and N5-N6 between a double and a triple bond. This blending of structures is called resonance. The blended structure is a resonance hybrid of the contributing Lewis structures. A molecule does not flicker between different structures: a resonance hybrid is a blend of structures.

FIG 2

In vitro effect of CP2, pentamidine, and amphotericin B on L. amazonensis intracellular amastigotes (A) and in vitro effect of CP2 and benznidazole on T. cruzi intracellular amastigotes (B). The infection index was calculated after 72 h of treatment with the IC₅₀s of each compounds. The negative control is L. amazonensis or T. cruzi intracellular amastigotes not treated with CP2. Data are expressed as averages plus the standard deviations (SD) for three independent experiments (P < 0.05).

FIG 3

In vivo efficacies of CP2 and amphotericin B treatment in BALB/c mice infected with L. amazonensis. Development of foot lesions in L. amazonensis-infected BALB/c mice treated with CP2. The treatment was started 60 days after infection and continued for 35 days. Data points represent the average measurements for seven groups of eight mice each. The development of foot lesions was monitored three times a week. Values indicate the mean volume (volume = D × d × e, where D is the larger diameter, d is the minor diameter, and e is the thickness) of lesions in each group, and bars represent the SD. α, statistically significant difference from infected, nontreated animals (P < 0.05); β, statistically significant difference from infected animals treated with PBS (P < 0.05).

FIG 4

Quantitation of tissue parasite load of L. amazonensis in skin lesions of infected BALB/c mice using the LDU index (i.e., the number of Leishmania amastigotes in 1,000 nucleated cells per organ weight). The data are expressed as averages plus the SD. *, α, statistically significant difference relative to the control group (P ≤ 0.001, P ≤ 0.01); β, statistically significant difference relative to the group treated with PBS (P < 0.05). The negative controls were infected animals not treated with CP2.

FIG 5

Plasma levels of biomarkers of liver and renal function in BALB/c mice noninfected and infected with L. amazonensis and treated with 0.20 or 0.35 mg/kg/day of CP2. (A) Total bilirubin and ALP levels; (B) ALT and AST levels; (C) urea and creatinine levels. The data are expressed as averages plus the SD. *, statistically significant difference with the noninfected animals (healthy animals) (P < 0.05); α, statistically significant difference with infected, untreated animals (P < 0.05); β, statistically significant difference with infected animals treated with PBS (P < 0.05).

FIG 6

(A) Relaxation of negative supercoiled plasmid DNA by L. donovani topoisomerase 1B (Ldtopo1B) in the presence of increasing concentrations of CP2 (lanes 4 to 13). Lane 1, only substrate; lane 2, substrate plus 300 μM CP2; lane 3, substrate plus Ldtopo1B enzyme. (B) Plasmid relaxation as a function of time in DMSO (at a concentration identical to that used to dissolve the CP2 compound) (lanes 2 to 5), 12.5 μM CP2 (lanes 6 to 9), 12.5 μM CP2 preincubated for 5 min with enzyme before DNA addition (lanes 10 to 13), 12.5 μM CP2 preincubated for 5 min with DNA before enzyme addition (lanes 14 to 17). NC, nicked circular plasmid DNA; SC, supercoiled plasmid DNA.

FIG 7

(A) CL1/CL2 suicide substrate used to measure the cleavage kinetics of the enzyme. *, preferential cleavage site (CL1); °, second cleavage site (CL2). (B) Gel analysis of Ldtopo1B cleavage kinetics in the absence (lanes 2 to 10) or presence (lanes 11 to 19) of 150 μM CP2 at various time points (0, 0.25, 0.5, 0.75, 1, 2, 4, 8, 16, and 30 min). CL1, DNA fragment cleaved at the preferred site; CL2, second cleavage site.