C. White (University of Selleck GSK J4 Missouri-Kansas City, MO). MML610 is the azole-susceptible parental strain of the azole-resistant derivative MML611. The sensitive and resistant strains had FLC MICs (the minimum concentrations giving >80% growth inhibition compared to the no-drug control) of 0.5 and 64 μg mL−1, respectively (Holmes et al., 2008). Strain MML610 expressed basal levels of Cdr1p but MML611 expressed significant amounts of both Cdr1p and Cdr2p, and neither strain

expressed Mdr1p (Holmes et al., 2008). The strains did not contain ERG11 mutations previously shown to be associated with the acquisition of FLC resistance. The resistant strain did possess a mutation (T580C) that results in a Phe-to-Leu change at position 145 (F145L) in Erg11p. This mutation has been shown not to

be associated with azole resistance (Marr et al., 2001; Morio et al., 2010). Candida albicans cells were stored at −80 °C in Sabouraud dextrose broth (Becton Dickinson, MD) containing 0.5% yeast extract (Becton Dickinson) and 10% glycerol (v/v, final concentration). The strains were cultured on Sabouraud dextrose agar plates for 20 h at 37 °C before use in the mouse oral candidiasis model. Experimental procedures for the mouse oral candidiasis model have been described previously (Takakura et al., 2003), and all animal experiments were performed according to the guidelines for the care and use of animals approved by Teikyo University. Six-week-old female ICR mice (Charles River Japan, Inc., Kanagawa, Japan) were immunosuppressed by subcutaneous treatment with prednisolone (100 mg kg−1; Mitaka Pharmaceutical beta-catenin inhibitor Co., Palmatine Tokyo, Japan)

1 day prior to oral infection. Tetracycline hydrochloride (15 mg mL−1; Takeda Shering Purau Animal Health Co., Tokyo, Japan) was added to the mice’s drinking water, from 1 day before infection. Thirty minutes before Candida infection, and before the second round of oral administration (24 h later), the mice were anaesthetized for approximately 3 h by intramuscular injection of the foot with 100 μL of chlorpromazine chloride (14.4 mg kg−1; Wako Pure Chemical Industries Ltd, Osaka, Japan). The mice were infected orally by rolling a cotton swab (baby cotton buds; Johnson & Johnson Co., Tokyo, Japan) soaked in a suspension of C. albicans cells (2–3 × 108 viable cells mL−1 in RPMI 1640 medium containing 2.5% fetal calf serum) over all areas of the mouth. The number of Candida cells inoculated in the oral cavity was calculated to be about 1~1.5 × 106 cells per mouse based on the difference in viable cell number present on the cotton swabs before and after oral inoculation (Taguchi et al., 2011). The d-octapeptide derivative RC21 specifically inhibits Cdr1p (Holmes et al., 2008), and its active principal RC21v3 used in the present experiments was prepared by manual peptide synthesis, purified by HPLC and characterized by mass spectrometry at the Centre for Separation Science at Massey University (Palmerston North, New Zealand).