Proinflammatory and cytotoxic effects of hexadecylphosphocholine (miltefosine) against drug-resistant strains of Trypanosoma cruzi

Abstract

The increased resistance of the protozoan parasite Trypanosoma cruzi to nitro derivatives is one of the major problems for the successful treatment of Chagas' disease. In the present study, we have tested the effects of 1-O-hexadecylphosphocholine (miltefosine) against strains of T. cruzi that are partially resistant (strain Y) and highly resistant (strain Colombiana) to the drugs in clinical use. As expected, epimastigotes of strain Colombiana showed higher levels of resistance to benznidazole than those of strain Y. However, the level of resistance to miltefosine was the same for both strains. This alkylphospholipid was also extremely toxic against intracellular amastigotes of both strains. This ether-lipid analogue induced in a dose-dependent manner the production of tumor necrosis factor alpha and nitric oxide (NO) radicals by infected and noninfected macrophages, suggesting that miltefosine may activate macrophages in vitro. Nevertheless, the cytotoxic effect of miltefosine against intracellular amastigotes was independent of the amount of NO produced by the infected macrophages since the same dose-response curves for miltefosine were observed when the NO production was blocked by the NO synthase inhibitor N(G)-monomethyl-L-arginine monoacetate. Preliminary in vivo studies with BALB/c mice infected with strain Y indicated that oral miltefosine promoted survival and reduced the parasitemia to levels comparable to those observed when benznidazole was used. Four months after treatment, no parasites were detected in the blood or spleen tissue sections maintained in culture. Together, these results support the hypothesis that miltefosine may be used for the treatment of Chagas' disease, including cases caused by resistant strains of T. cruzi.

FIG. 1.

Dose-dependent inhibition of epimastigote viability by miltefosine and benznidazole in axenic culture. Parasites from strains Y and Colombiana were cultivated in 96-well plates in BHI-FCS medium with different concentrations of miltefosine or benznidazole. The viability of each culture was determined after 72 h at 28°C by direct counting of the number of parasites per milliliter, and each experimental point corresponds to the mean ± standard deviation for duplicates from three independent experiments. ▪, controls; •, strain Y cultivated with miltefosine; ▴, strain Colombiana cultivated with miltefosine; ○, strain Y cultivated with benznidazole; ▵, strain Colombiana cultivated with benznidazole.

FIG. 2.

Miltefosine inhibits the percentage of infected Mφ and the number of amastigotes per Mφ. Murine peritoneal Mφ plated onto coverslips were infected with strain Y (A) or Colombiana (B) TCT forms (ratio of five parasites per Mφ) for 2 h, washed, and incubated with medium alone or medium containing increasing amounts of miltefosine, as indicated. After 3 days, the coverslips were fixed and stained with Giemsa, and the percentage of infected Mφ as well as the number of amastigotes per Mφ were determined by direct counting, as described in Materials and Methods. The graphs represent the means of duplicates from four independent experiments.

FIG. 3.

Miltefosine stimulates the synthesis of TNF-α and NO by infected and noninfected Mφ. Plated murine peritoneal Mφ were infected (+) or not infected (−) with Y-strain MCT forms for 24 h, washed, and then incubated with fresh medium containing (+) or not containing (−) LPS plus INF-γ or increasing amounts of miltefosine, as indicated at the bottom. After 2 days of culture, aliquots of the supernatants were taken for measurement of the levels of production of NO (B) and TNF-α (C); and after 5, 7, and 10 days of culture, the number of trypomastigotes found in the supernatants was determined (A). The graphs represent the mean of duplicates from two independent experiments.

FIG. 4.

l-NMMA inhibits the synthesis of NO by T. cruzi-infected macrophages but does not change the cytotoxicity of miltefosine against the parasites. Plated Mφ were infected with T. cruzi as described in the legend to Fig. 3 and incubated in the absence (−) or the presence (+) of LPS plus INF-γ, l-NMMA, or miltefosine, as indicated at the bottom. After 2 days of infection the levels of NO were similar to those observed in Fig. 3B, except that in the presence of l-NMMA there was 85 to 90% inhibition in the level of NO production (data not shown). The graph shows the number of trypomastigotes released to the culture supernatants after 5, 7, and 10 days after infection and represents the mean of duplicates from two independent experiments.

FIG. 5.

Miltefosine promotes a dramatic decrease in the level of parasitemia and parasitological cure rate in BALB/c mice infected with T. cruzi strain Y. Results are from a single experiment in which a total of 15 BALB/c mice were infected intraperitoneally with 10⁵ Y-strain MCTs and groups of 5 mice each were left untreated (○) or were immediately treated orally for 20 days with 100 mg of benznidazole kg⁻¹ (▵) or 25 mg of miltefosine kg⁻¹ (▪). As indicated, the number of parasites in the blood was determined at days 6, 7, 8, 13, 14, and 15 after infection (A); and the rate of survival was monitored for 30 days (B).