Novel Nystatin A1 derivatives exhibiting low host cell toxicity and antifungal activity in an in vitro model of oral candidosis

© 2014, Springer-Verlag Berlin Heidelberg. Opportunistic oral infections caused by Candida albicans are frequent problems in immunocompromised patients. Management of such infections is limited due to the low number of antifungal drugs available, their relatively high toxicity and the emergence of antifungal resistance. Given these issues, our investigations have focused on novel derivatives of the antifungal antibiotic Nystatin A1, generated by modifications at the amino group of this molecule. The aims of this study were to evaluate the antifungal effectiveness and host cell toxicity of these new compounds using an in vitro model of oral candidosis based on a reconstituted human oral epithelium (RHOE). Initial studies employing broth microdilution, revealed that against planktonic C. albicans, Nystatin A1had lower minimal inhibitory concentration than novel derivatives. However, Nystatin A1was also markedly more toxic against human keratinocyte cells. Interestingly, using live/dead staining to assess C.albicans and tissue cell viability after RHOE infection, Nystatin A1derivatives were more active against Candida with lower toxicity to epithelial cells than the parent drug. Lactate dehydrogenase activity released by the RHOE indicated a fourfold reduction in tissue damage when certain Nystatin derivatives were used compared with Nystatin A1. Furthermore, compared with Nystatin A1, colonisation of the oral epithelium by C. albicans was notably reduced by the new polyenes. In the absence of antifungal agents, confocal laser scanning microscopy showed that C. albicans extensively invaded the RHOE. However, the presence of the novel derivatives greatly reduced or totally prevented this fungal invasion

A novel in vitro assay for assessing efficacy and toxicity of antifungals using human leukemic cells infected with Candida albicans

Aims This study describes a novel in vitro assay that simultaneously determines antifungal efficiency and host cell toxicity using suspensions of human leukemic cells (HL-60) infected with Candida albicans. Methods and Results The effect of Candida infection on host cell viability was evaluated by microscopy of trypan blue stained cells and lactate dehydrogenase (LDH) activity. The in vitro ‘drug potency assay’ utilized the Cell Counting Kit-8 and measured post antifungal treatment viability of Candida-infected HL-60 cells and ability of the antifungal to prevent infection. LDH activity showed that 42% ± 4.0 and 85.3% ± 7.40 of HL-60 cells were killed following Candida infection at multiplicity of infection (MOI) of 1:1 and 1:5, respectively. Using the assay, the antifungal nystatin (0.78-25 μmol l−1) was found to inhibit C. albicans infection as seen by significantly increased viability of HL-60 cells. Using the assay, cytotoxicity of nystatin towards infected HL-60 cells was evident at higher concentrations and this was also confirmed by propidium iodide staining. Conclusions An assay using undisturbed cell suspension conditions was successfully developed for assessing selectivity of antifungal therapy in the host-Candida environment. Significance and Impact of the Study The assay employing Candida infection of host cell suspensions represents a promising method for testing interactions of antifungal compounds with both fungal and host cells