The antimalarial compound qinghaosu (artemisinin) was tested in vitro for the ability to inhibit plaque formation by Toxoplasma gondii in fibroblasts. Qinghaosu at 0.4 ,ug/ml for 5 days eliminated all plaques and microscopic foci of T. gondii. At 1.3 ,ug/ml for 14 days, qinghaosu completely eliminated T. gondii. Pretreatment of host cells or T. gondii with qinghaosu had no effect on T. gondii growth. There was no apparent toxicity to human fibroblasts in long-term studies. Of the six qinghaosu derivatives tested, dihydroqinghaosu, 1-propyl-ether-qinghaosu, and 1-butyl-ether-qinghaosu were comparable to qinghaosu. Ethyl-ether-qinghaosu (arteether) and sec-butyl-ether-qinghaosu were more effective. Methyl-ether-qinghaosu (artemether) was the most effective, with a potency approximately 10-fold greater than that of qinghaosu.
MATERIALS AND METHODSGrowth of tissue cultures and organisms. The RH strain of T. gondii, obtained from J. D. Schwartzman (originally from E. R. Pfefferkorn), was maintained in locally obtained human embryonic lung fibroblasts of less than 30 passages. The fibroblasts were grown in VA-13 medium (minimal essential medium (MEM) with Earle salts, 2x MEM essential amino acids, lx MEM nonessential amino acids, 2x MEM vitamins, 3 x glucose, 1 mM sodium pyruvate, 100 U of penicillin G per ml, 100 ,ug of streptomycin sulfate per ml), pH 7.2. VA-13 medium was supplemented with 10% adult bovine serum for nonparasitized fibroblasts. For growth of T. gondii in stationary-phase fibroblasts, VA-13 was used with 0.3% bovine serum albumin (Cohn fraction V). When both the fibroblasts and T. gondii were grown concurrently, the VA-13 was supplemented with fetal bovine serum (10%). All cultures were maintained in 5% C02-95% air at 37°C.Quantitation of QHS. QHS (artemisinin), dihydro-QHS (dihydroartemisinin), methyl-ether-QHS (artemether), ethylether-QHS (arteether), 1-propyl-ether-QHS, 1-butyl-ether-QHS, and sec-butyl-ether-QHS were obtained from Hauser Chemical Research, Inc. (Boulder, Colo.). Stock solutions were prepared in DMSO (dimethyl sulfoxide). The concentration of QHS was verified by using the method of Zhao and Zeng (28). Briefly, 1 part of QHS solution was mixed with 4 parts of 50 mM NaOH and heated for 30 min at 45°C. The mixture was rapidly cooled to room temperature, and 5 parts of 100 mM acetic acid in 20% ethanol was added. The final reaction mixture was diluted 1/20 with 20 mM potassium phosphate buffer at pH 6.0, and the optical density was measured at 259 nm with a Beckman DU-7 spectrophotometer. The blank used for the spectrophotometer was the DMSO solvent processed in parallel. The extinction coefficient used was 1.3 x 104 M-1 cm-1 (28).The QHS derivatives were formulated on a weight-tovolume basis.Five-day plaque assay. The 5-day plaque assay is based on the method of Chaparas and Schlesinger (2)
