Hexadecylphosphocholine (Miltefosine) Has Broad-Spectrum Fungicidal Activity and Is Efficacious in a Mouse Model of Cryptococcosis

The alkyl phosphocholine drug miltefosine is structurally similar to natural substrates of the fungal virulence determinant phospholipase B1 (PLB1), which is a potential drug target. We determined the MICs of miltefosine against key fungal pathogens, correlated antifungal activity with inhibition of the PLB1 activities (PLB, lysophospholipase [LPL], and lysophospholipase-transacylase [LPTA]), and investigated its efficacy in a mouse model of disseminated cryptococcosis. Miltefosine inhibited secreted cryptococcal LPTA activity by 35% at the subhemolytic concentration of 25 μM (10.2 μg/ml) and was inactive against mammalian pancreatic phospholipase A2 (PLA(2)). At 250 μM, cytosolic PLB, LPL, and LPTA activities were inhibited by 25%, 51%, and 77%, respectively. The MICs at which 90% of isolates were inhibited (MIC(90)s) against Candida albicans, Candida glabrata, Candida krusei, Cryptococcus neoformans, Cryptococcus gattii, Aspergillus fumigatus, Fusarium solani, Scedosporium prolificans, and Scedosporium apiospermum were 2 to 4 μg/ml. The MICs of miltefosine against Candida tropicalis (n = 8) were 2 to 4 μg/ml, those against Aspergillus terreus and Candida parapsilosis were 8 μg/ml (MIC(90)), and those against Aspergillus flavus (n = 8) were 2 to 16 μg/ml. Miltefosine was fungicidal for C. neoformans, with rates of killing of 2 log units within 4 h at 7.0 μM (2.8 μg/ml). Miltefosine given orally to mice on days 1 to 5 after intravenous infection with C. neoformans delayed the development of illness and mortality and significantly reduced the brain cryptococcal burden. We conclude that miltefosine has broad-spectrum antifungal activity and is active in vivo in a mouse model of disseminated cryptococcosis. The relatively small inhibitory effect on PLB1 enzyme activities at concentrations exceeding the MIC by 2 to 20 times suggests that PLB1 inhibition is not the only mechanism of the antifungal effect

Interlaboratory Comparison of Results of Susceptibility Testing with Caspofungin against Candida and Aspergillus Species

Seventeen laboratories participated in a study of interlaboratory reproducibility with caspofungin microdilution susceptibility testing against panels comprising 30 isolates of Candida spp. and 20 isolates of Aspergillus spp. The laboratories used materials supplied from a single source to determine the influence of growth medium (RPMI 1640 with or without glucose additions and antibiotic medium 3 [AM3]), the same incubation times (24 h and 48 h), and the same end point definition (partial or complete inhibition of growth) for the MIC of caspofungin. All tests were run in duplicate, and end points were determined both spectrophotometrically and visually. The results from almost all of the laboratories for quality control and reference Candida and Aspergillus isolates tested with fluconazole and itraconazole matched the NCCLS published values. However, considerable interlaboratory variability was seen in the results of the caspofungin tests. For Candida spp. the most consistent MIC data were generated with visual “prominent growth reduction” (MIC(2)) end points measured at 24 h in RPMI 1640, where 73.3% of results for the 30 isolates tested fell within a mode ± one dilution range across all 17 laboratories. MIC(2) at 24 h in RPMI 1640 or AM3 also gave the best interlaboratory separation of Candida isolates of known high and low susceptibility to caspofungin. Reproducibility of MIC data was problematic for caspofungin tests with Aspergillus spp. under all conditions, but the minimal effective concentration end point, defined as the lowest caspofungin concentration yielding conspicuously aberrant hyphal growth, gave excellent reproducibility for data from 14 of the 17 participating laboratories