Atovaquone nanosuspensions show excellent therapeutic effect in a new murine model of reactivated toxoplasmosis

Abstract

Immunocompromised patients are at risk of developing toxoplasma encephalitis (TE). Standard therapy regimens (including sulfadiazine plus pyrimethamine) are hampered by severe side effects. While atovaquone has potent in vitro activity against Toxoplasma gondii, it is poorly absorbed after oral administration and shows poor therapeutic efficacy against TE. To overcome the low absorption of atovaquone, we prepared atovaquone nanosuspensions (ANSs) for intravenous (i.v.) administration. At concentrations higher than 1.0 microg/ml, ANS did not exert cytotoxicity and was as effective as free atovaquone (i.e., atovaquone suspended in medium) against T. gondii in freshly isolated peritoneal macrophages. In a new murine model of TE that closely mimics reactivated toxoplasmosis in immunocompromised hosts, using mice with a targeted mutation in the gene encoding the interferon consensus sequence binding protein, i.v.-administered ANS doses of 10.0 mg/kg of body weight protected the animals against development of TE and death. Atovaquone was detectable in the sera, brains, livers, and lungs of mice by high-performance liquid chromatography. Development of TE and mortality in mice treated with 1.0- or 0.1-mg/kg i.v. doses of ANS did not differ from that in mice treated orally with 100 mg of atovaquone/kg. In conclusion, i.v. ANSs may prove to be an effective treatment alternative for patients with TE.

FIG. 1

(A) Number of macrophages infected after incubation with T. gondii at a parasite-to-cell ratio of 2:1, rinsing off of free parasites 1 h later, and a 48-h treatment with ANS or free drug at different concentrations. (B) Viability of macrophages after 20 h of incubation with ANS or free drug, determined by MTT assay.

FIG. 2

Model of reactivated toxoplasmosis in ICSBP^−/− mice.

FIG. 3

Survival rates of ICSBP^−/− mice i.v. treated with surfactant solution (control) (n = 7) or ANS (0.1, 1, or 10 mg/kg of body weight) (n = 8) every other day from day 2 until day 16 after discontinuation of sulfadiazine. One group of mice was treated orally with 100 mg of atovaquone suspension/kg at the same time points (n = 7). Survival rates presented are representative of three independent experiments.

FIG. 4

Inflammatory foci (A) and corresponding parasite foci (parasitophorous vacuoles and parasitic antigen) (B) in brains of ICSBP^−/− mice at day 8 after discontinuation of sulfadiazine (magnification, ×400); intact brain tissue, without inflammation, under treatment with sulfadiazine (C) and under treatment with ANS (10 mg/kg of body weight) (D) at day 8 after discontinuation of sulfadiazine (magnification, ×100).

FIG. 5

Concentration of atovaquone in sera of ICSBP^−/− mice i.v. treated with surfactant solution (control) or ANS (0.1, 1, or 10 mg/kg) or treated orally with atovaquone suspension every other day. Serum samples were obtained 3 h after the last dosing. Values were derived from three mice per group that were sacrificed at day 8 after discontinuation of sulfadiazine (means ± SDs, left ordinate). Survival rates at day 8 refer to the right ordinate.

FIG. 6

Concentrations of atovaquone in brain, liver, and lung tissues of ICSBP^−/− mice i.v. treated with surfactant solution (control) or ANS (0.1, 1, or 10 mg/kg) every other day from day 2 until day 16 after discontinuation of sulfadiazine. One group of mice was treated orally with 100 mg of atovaquone suspension/kg at the same time points. Values were derived from three mice per group that were sacrificed at day 8 after discontinuation of sulfadiazine (means ± SDs) and are representative of three independently performed experiments.