Antitrypanosomal activity of fexinidazole metabolites, potential new drug candidates for Chagas disease

Abstract

This study was designed to verify the in vivo efficacy of sulfoxide and sulfone fexinidazole metabolites following oral administration in a murine model of Chagas disease. Female Swiss mice infected with the Y strain of Trypanosoma cruzi were treated orally once per day with each metabolite at doses of 10 to 100 mg/kg of body weight for a period of 20 days. Parasitemia was monitored throughout, and cures were detected by parasitological and PCR assays. The results were compared with those achieved with benznidazole treatment at the same doses. Fexinidazole metabolites were effective in reducing the numbers of circulating parasites and protecting mice against death, compared with untreated mice, but without providing cures at daily doses of 10 and 25 mg/kg. Both metabolites were effective in curing mice at 50 mg/kg/day (30% to 40%) and 100 mg/kg/day (100%). In the benznidazole-treated group, parasitological cure was detected only in animals treated with the higher dose of 100 mg/kg/day (80%). Single-dose pharmacokinetic parameters for each metabolite were obtained from a parallel group of uninfected mice and were used to estimate the profiles following repeated doses. Pharmacokinetic data suggested that biological efficacy most likely resides with the sulfone metabolite (or subsequent reactive metabolites formed following reduction of the nitro group) following administration of either the sulfoxide or the sulfone and that prolonged plasma exposure over the 24-h dosing window is required to achieve high cure rates. Fexinidazole metabolites were effective in treating T. cruzi in a mouse model of acute infection, with cure rates superior to those achieved with either fexinidazole itself or benznidazole.

FIG 1

In vitro assay with Trypanosoma cruzi. The comparative concentration-dependent antiparasitic effects of fexinidazole metabolites and benznidazole were evaluated in murine macrophages infected with T. cruzi Y strain, in a 72-h assay. Data represent the mean values of percent inhibition, which were obtained by dividing the number of infected cells in treated cultures by the mean value for infected control cultures and multiplying the result by 100. The results represent the means of results from two independent experiments.

FIG 2

Activity of fexinidazole metabolites against intracellular amastigotes of Trypanosoma cruzi. Peritoneal murine macrophages were infected with T. cruzi Y strain and incubated with different concentrations of fexinidazole metabolites or benznidazole for 72 h. Methanol-fixed cells were stained with Giemsa. Arrows, intracellular parasites. (A, C, and E) Drug activity at the 90% inhibitory concentration (IC₉₀) (note the absence of parasites); (B, D, and F) drug activity at the IC₅₀; (G) uninfected cells; (H) infected and untreated cells. No cytotoxic effects were observed in host cells.

FIG 3

Weight loss or gain for animals infected with Trypanosoma cruzi Y strain and treated with fexinidazole sulfoxide or fexinidazole sulfone. Infected mice were treated once daily with 10, 25, 50, or 100 mg/kg of fexinidazole sulfoxide, fexinidazole sulfone, or benznidazole for 20 consecutive days, and results were compared with those for infected and untreated control (IC) and noninfected control (NIC) groups. The weight loss or gain was calculated as the difference between the weights of animals before and after treatment. The weights of animals in the uninfected control group were assessed over the same period. The weights of the animals in the infected and untreated control group were assessed 14 days after infection (all animals died before the end of the treatment). a, significant difference relative to infected control (P < 0.005); b, significant difference relative to uninfected control (P < 0.005).

FIG 4

Parasitemia levels during and after oral administration of fexinidazole sulfoxide or fexinidazole sulfone. Bars represent the means of the maximum parasitemia values obtained in peripheral blood samples from mice (n = 10) infected with 5,000 trypomastigotes of Trypanosoma cruzi Y strain during and up to 30 days after 20 days of treatment with doses of 10, 25, 50, or 100 mg/kg/day of fexinidazole sulfoxide, fexinidazole sulfone, or benznidazole. IC, infected and untreated control. a, b, and c, bars with similar letters are not significantly different, and bars with different letters are significantly different (P < 0.05).

FIG 5

Anti-Trypanosoma cruzi activity of fexinidazole metabolites in mice infected with Y strain. Mice were inoculated intraperitoneally with 5,000 trypomastigotes of T. cruzi Y strain and treated with doses of 10, 25, 50, or 100 mg/kg fexinidazole sulfoxide or fexinidazole sulfone or 100 mg/kg benznidazole once daily for 20 consecutive days. (A) IgG antibodies in treated mice as detected by ELISA. The reactivity index was obtained by dividing the absorption value of each serum sample by the mean value of the uninfected control sample. (B) Myocardial inflammatory cell counts in mice infected with T. cruzi Y strain at 6 months posttreatment. (C) Hematoxylin/eosin-stained slides. Magnification, ×40. NIC, noninfected control; −50 and +50, mice treated with 50 mg/kg/day of fexinidazole sulfoxide or fexinidazole sulfone with negative (−50) or positive (+50) results for parasites in fresh blood examinations and qPCR assays. a and b, bars with similar letters are not significantly different, and bars with different letters are significantly different (P < 0.05).

FIG 6

Plasma concentration-time profiles following a single oral administration of fexinidazole sulfoxide or fexinidazole sulfone to mice. Mice were dosed orally with 10 (●), 25 (△), 50 (◼), or 100 (◇) mg/kg of fexinidazole sulfoxide or fexinidazole sulfone. (A) Fexinidazole sulfoxide concentrations after administration of fexinidazole sulfoxide; (B) fexinidazole concentrations after administration of fexinidazole sulfoxide; (C) fexinidazole sulfone concentrations after administration of fexinidazole sulfoxide; (D) fexinidazole sulfone concentrations after administration of fexinidazole sulfone.

FIG 7

Simulated plasma concentration-time profiles following repeated once-daily administration of 100 mg/kg benznidazole (A), fexinidazole sulfoxide (B), or fexinidazole sulfone (C) to mice. For ease of interpretation, only the first 3 days of the 20-day dosing regimen are shown. Symbols represent the experimental data, and lines represent the repeated-dose simulated profiles. Dashed lines indicate the in vitro IC₅₀ for each compound.