FIG 1 .

Anti-cryptococcal activity of triphenylethylenes. (A) MICs of the indicated molecules against C. neoformans var. grubii strain H99 as determined by CLSI standard methods. CLM, clomiphene; TAM, tamoxifen; TOR, toremifene; 4-HO-TAM, 4-hydroxy-tamoxifen; ENDO, endoxifen; IDX, idoxifene; M-IDX, methylene-idoxifene. The chemical structures for each molecule are shown in Fig. S1A. The y axis of the graph is a modified log₂ scale. (B) The combination of subinhibitory concentrations of toremifene (TOR; 2 µg/ml) and fluconazole (FLU; 2 µg/ml) reduces the initial inoculum of C. neoformans (4 log₁₀ CFU/ml) more than 2 log₁₀ CFU/ml at 24 h and is therefore a fungicidal combination. (C) J774 mouse macrophages were infected with opsonized C. neoformans, washed to remove nonadherent/nonphagocytosed cells, and incubated with fresh medium containing either DMSO (1%) or drug for 24 h. After 24 h, macrophages were lysed and the number of C. neoformans CFU per well determined as described in Materials and Methods. Data are means of triplicates, and error bars represent standard deviations. Differences between groups were statistically significant (Student’s t test; P < 0.05). (D) Male A/JCr mice (7 per group) were infected with serotype A C. neoformans H99 by tail vein injection. The control group (CTRL) received saline by intraperitoneal injection and peanut oil by oral gavage. The fluconazole group (FLU) received 5 mg/kg FLU by intraperitoneal injection and peanut oil by oral gavage. The tamoxifen group (TAM) received saline by intraperitoneal injection and 200 mg/kg tamoxifen suspended in peanut oil by oral gavage. The combination treatment group (TAM+FLU) received 5 mg/kg fluconazole by intraperitoneal injection and 200 mg/kg tamoxifen suspended in peanut oil by oral gavage. The bars represent the median for each group, and each dot represents an individual animal. The number of CFU/gram of brain tissue was determined and transformed into log₁₀ units, and differences between groups were analyzed by ANOVA; statistical significance was set at a P value of <0.05 (SigmaPlot software).