Artemisone and artemiside control acute and reactivated toxoplasmosis in a murine model

Abstract

Immunocompromised patients are at risk of developing toxoplasmosis, and although chemotherapy is available, standard treatments are often complicated by severe side effects. Artemisinin is a new highly potent antimalarial drug that has activity against Toxoplasma gondii in vitro. However, artemisinin derivatives have previously been ineffective in vivo using a rat model of toxoplasmosis. In the present study, the efficacy of several new artemisinin derivates was investigated for treatment of mice infected with the parasite Toxoplasma gondii. Artemiside and artemisone displayed better inhibition than either artemisinin or artesunate against the parasite in vitro. Artemiside and artemisone treatment controlled parasite replication in vivo, and mice survived the acute infection. In a murine model of reactivated toxoplasmosis, both drugs increased survival, although artemiside was more effective. These results indicate that these newer derivatives of artemisinin may have potential for treatment of toxoplasmosis.

FIG. 1.

Structure diagrams of artemisinin and related compounds. Artemisinin, 1; artesunate, 2; DHA, 3; artemether, 4; artemisone, 5; artemiside, 6; sulfamide derivative, 7; benzylpiperazine derivative, 8; pyrimidylpiperazine derivative, 9; and 4′-fluorophenyl derivative, 10.

FIG. 2.

Inhibition of in vitro growth of T. gondii by artemisinin derivatives. Parasite growth was monitored by measurement of β-galactosidase activity (absorbance at 570 nm) following 72 h of incubation with the different compounds (see Materials and Methods). Artemiside and artemisone showed slightly better activity in vitro compared to the other compounds. 7, sulfamide derivative; 8, benzylpiperazine derivative; 9, pyrimidylpiperazine derivative; and 10, 4′-fluorophenyl derivative.

FIG. 3.

Inhibition of in vivo growth of T. gondii by artemisinin derivatives. (A) Survival of mice following oral challenge with T. gondii and administration of artemisinin derivates. CD1 mice were infected i.p. with 10⁶ tachyzoites of type II (PRU-Luc-GFP) T. gondii, and compounds were injected (s.c. with 10 mg/kg) every day for 8 days. Survival of mice was monitored for 25 days. The majority of the artemiside- and artemisone-treated mice survived the infection, while artemisinin-treated and control mice died. Combined results from the three experiments, n = 3 or 4 animals per group, are shown. (B) Parasite burdens in mice during acute infection as measured by luciferase imaging. CD1 mice were infected with 10⁶ tachyzoites of type II (PRU-Luc-GFP) parasites, treated with compounds (10 mg/kg s.c.) every day for 8 days, and imaged every second day. Artemiside- and artemisone-treated mice were able to control the infection. Experiments were repeated four times; n = 3 or 4 animals per group. Graph shows one representative experiment. † indicates 4 of 5 animals succumbed to infection; thus, the average luciferase (Luc) value dropped after this point. †† indicates all animals succumbed to infection. Luc values represent relative light output. (C) Chronic infection in mice as monitored by cyst formation in the brains of CD1 mice. Artemiside- and artemisone-treated mice developed lower cyst numbers in the brain compared to artemisinin-treated mice. Data shown here are combined results from three experiments; n = 2 animals per group. Numbers represent an estimate of the total number of cysts per brain. Numbers are not available for control mice, which succumbed to infection (†).

FIG. 4.

Inhibition of reactivation of chronic toxoplasmosis by artemisinin derivatives. (A) Murine model of reactivated toxoplasmosis. At day 0, IFN-γ^−/− mice were infected orally with 20 cysts of PRU-Luc-GFP, and 2 days later sulfadiazine (400 mg/kg) was administered for 3 weeks in the drinking water. Two days after discontinuation of the drug, compounds were administered for 8 days (s.c., 10 mg/kg), and survival was monitored. (B) Treatment with artemiside or artemisone resulted in increased survival following reactivation. Data shown are combined results from three individual experiments; n = 3 to 5 animals per group.