In vivo efficacy analysis of the FK506 analogs. The C. neoformans cells cultured overnight were washed twice with PBS, and the number of cells was synchronized. (A) In vivo efficacy test using a Galleria mellonella insect model. Amphotericin B (AMB), FK506, and the FK506 analogs were used at 5 mg/kg, and insects were treated three times at 24, 48, and 72 h after inoculation. (B) In vivo efficacy test in a murine model. Cells (5 × 105) were used to infect each mouse via intravenous inoculation. The concentrations of fluconazole (FCZ), FK506, and 9-deoxo-31-O-demethyl-FK506 (9D31OD-FK506) were 3 mg/kg, and the combination group was treated with a mixture of 3 mg/kg of 9D31OD-FK506 with 3 mg/kg of FCZ. The log-rank (Mantel-Cox) test was used for statistical analysis.