ABSTRACT
OBJECTIVE: Amphotericin B (AMB), a potent antifungal agent, has been employed as topical and systemic therapy for sinonasal fungal infections. A novel formulation of nanodisc (ND) containing super aggregated AMB (ND-AMB) for the treatment of fungal infections has been recently developed to provide greater protection from AMB toxicity than current, clinically approved lipid-based formulations. The objective of the current study was to evaluate the safety and potency of ND-AMB for sinonasal delivery using an in vitro model. METHODS: Human sinonasal tissue was harvested during endoscopic sinus surgery and grown at air-liquid interface until well-differentiated. Cultures were exposed to ND-AMB vs AMB and changes in K+ permeability and resistance were measured and recorded via Ussing chamber assay. Ciliary beat frequency (CBF) was analyzed in parallel as well as cytotoxic assay. Potency was assessed using real-time PCR measurement of the Aspergillus fumigatus 18S rRNA. RESULTS: Ussing chamber studies revealed K+ currents that increased rapidly within 30 s of adding AMB (10 µg/mL) to the apical side, indicating apical membranes had become permeable to K+ ions. In contrast, negligible induction of K+ current was obtained following addition of ND-AMB [AMB = (107.7 ± 15.9) µA/cm2 AMB vs ND-AMB = (2.3 ± 0.7) µA/cm2 ND-AMB; P = 0.005]. ND-AMB also protected nasal epithelial cells from cytotoxicity of AMB (P < 0.05). There was no difference in ciliary beat frequency between the two groups (P = 0.96). The expression of A. fumigatus 18S rRNA with exposure of lower dose of ND-AMB was significantly lower compared to that with AMB (P < 0.05). CONCLUSIONS: Data from the present study suggests ND-AMB protects human nasal epithelia membranes from AMB toxicity by protecting against apical cell K+ permeability while maintaining uncompromised antifungal property compared to AMB. ND-AMB could provide a novel topical therapy for sinonasal fungal diseases.
ABSTRACT
The anticancer ruthenium complex trans-[tetrachlorobis(1H-indazole)ruthenate(III)], otherwise known as KP1019, has previously been shown to inhibit proliferation of ovarian tumor cells, induce DNA damage and apoptosis in colon carcinoma cells, and reduce tumor size in animal models. Notably, no dose-limiting toxicity was observed in a Phase I clinical trial. Despite these successes, KP1019's precise mechanism of action remains poorly understood. To determine whether Saccharomyces cerevisiae might serve as an effective model for characterizing the cellular response to KP1019, we first confirmed that this drug is internalized by yeast and induces mutations, cell cycle delay, and cell death. We next examined KP1019 sensitivity of strains defective in DNA repair, ultimately showing that rad1Δ, rev3Δ, and rad52Δ yeast are hypersensitive to KP1019, suggesting that nucleotide excision repair (NER), translesion synthesis (TLS), and recombination each play a role in drug tolerance. These data are consistent with published work showing that KP1019 causes interstrand cross-links and bulky DNA adducts in mammalian cell lines. Published research also showed that mammalian cell lines resistant to other chemotherapeutic agents exhibit only modest resistance, and sometimes hypersensitivity, to KP1019. Here we report similar findings for S. cerevisiae. Whereas gain-of-function mutations in the transcription activator-encoding gene PDR1 are known to increase expression of drug pumps, causing resistance to structurally diverse toxins, we now demonstrate that KP1019 retains its potency against yeast carrying the hypermorphic alleles PDR1-11 or PDR1-3. Combined, these data suggest that S. cerevisiae could serve as an effective model system for identifying evolutionarily conserved modulators of KP1019 sensitivity.
