Ravuconazole

Sensitivity of the fungal strains to different antifungal agents was examined in a 96-well microtiter plate assay. The susceptibility test was performed according to the CLSI recommendation (59 ) in three biological replicates. Ketoconazole (Alfa Aesar), itraconazole (Across Organics), fluconazole (Alfa Aesar), ravuconazole (Sigma-Aldrich), posaconazole (Sigma-Aldrich), isavuconazole (Sigma-Aldrich), and amphotericin B (Sigma-Aldrich) were dissolved in dimethyl sulfoxide to prepare the stock. These stocks were then diluted with liquid RPMI 1640 medium. Final concentrations of azoles and AmB in the wells ranged from 0.125 to 16 μg/mL. Inocula were prepared and diluted in liquid RPMI 1640. Plates were incubated for 48 h at 25°C. For the antifungal susceptibility test, Candida krusei ATCC 6258 was used as a reference strain.

Sensitivity of the fungal strains to different antifungal agents, and H₂O₂ was examined in a 96-well microtiter plate assay. The susceptibility test was performed according to the CLSI recommendation (Rex and Clinical, 2008 ) in three biological replicates. Ketoconazole (Alfa Aesar), itraconazole (Across Organics), fluconazole (Alfa Aesar), ravuconazole (Sigma Aldrich), posaconazole (Sigma Aldrich), isavuconazole (Sigma Aldrich), amphotericin B (Sigma Aldrich), hygromycin B (Sigma Aldrich), terbinafine (Sigma Aldrich), micafungin (Sigma Aldrich) and cyclohexymide (PanReac AppliChem) were dissolved in DMSO to prepare the stock. These stocks were then diluted with liquid RPMI-1640 medium. Final concentrations of ketoconazole, itraconazole, fluconazole, terbinafine and micafungin in the wells ranged from 0.125 to 64 μg/ml while those of ravuconazole and isavuconazole ranged from 0.125 to 16 μg/ml. In the case of posaconazole and amphotericin B, the range was 0.125 to 8 μg/ml. Cyclohexymide were diluted in RPMI-1640 to obtain a range of 250-0,78 μg/ml. Hydrogen peroxide was diluted in liquid RPMI-1640 to prepare a stock solution of 100 mM. Final concentrations of the hydrogen peroxide in the wells ranged from 0 to10 mM. Inocula were prepared and diluted in liquid RPMI-1640. Plates were incubated for 48 h at 25°C.

In vitro antifungal susceptibility testing was performed according to the recommendations proposed in the Clinical and Laboratory Standards Institute (CLSI) M38-A2 protocol [25 ] with modifications. AMB, FLC, ketoconazole (KTC), POS, ITC, VRC, ravuconazole (RVC), 5FC, TRB, caspofungin (CAS) (all from Sigma-Aldrich Chemical Corporation, St. Louis, MO, USA) and micafungin (MFG) (Astellas Pharma Tech Corporation, Takaoka city, Toyama, Japan) were tested. The inoculum was prepared from a seven-day old PDA culture; the cells were harvested in RPMI medium and diluted to approximately 0.4–5 × 10⁴ cells/mL. The plates were incubated at 35°C for five days [14 (link)]. The minimal inhibitory concentration (MIC) for AMB, FLC, KTC, POS, ITC, VRC, RVC, 5FC, and TRB; and the minimal effective concentration (MEC) for CAS and MFG were determined according to the CLSI M38-A2 protocol [25 ]. The reference strains Aspergillus flavus (ATCC 204304), Aspergillus fumigatus (ATCC 204305), Candida krusei (ATCC 6258) and Candida parapsilosis (ATCC 22019) were used for quality control.

Bifonazole, butoconazole, clotrimazole, econazole, itraconazole, ketoconazole, miconazole, posaconazole, ravuconazole, terconazole, tioconazole, voriconazole and benzyl-4-hydroxybenzoate (Internal Standard, IS) (>98% purity grade), sodium phosphate monobasic and sodium phosphate dibasic (>99% purity grade), phosphoric acid (90% purity grade) and trichloroacetic acid (>99% purity grade) were purchased from Sigma-Aldrich (Milan, Italy).
The commercial drugs were obtained from a local Pharmacy after medical prescription. Both HPLC-grade Acetonitrile (AcN) and methanol (MeOH) were supplied by Carlo Erba (Milan, Italy) and were used without further purification. The water for HPLC analysis was generated by using a Millipore Milli-Q Plus water treatment system (Millipore Bedford Corp., Bedford, MA). Oasis HLB (1 mL, 30 mg) and Sep-Pak (1 mL, 50 mg) were obtained from Waters (Milford, MA), while Strata-X (1 mL, 30 mg) was purchased from Phenomenex (Torrance, CA). MEPS device (syringe) and replacement needle with C₁₈ stationary phase were purchased from SGE Analytical Science (Melbourne, Australia).