Female BALB/c mice (n = 24 mice, 6 to 8 weeks of age, weight of 16 to 18 g) were obtained from Charles River, Margate, United Kingdom. All mice were topically infected with C. Albicans 529L (4.5 × 106 CFU/mouse in 0.025 ml) by intravaginal injection under temporary inhaled anesthesia (2.5% isoflurane for 3 to 4 min). The mice were randomly divided into three groups (n = 8/group): the control group (0.05 ml of 65% [vol/vol] polyethylene glycol [PEG] 14000 administered vaginally once daily), the NP339 group (0.05 ml NP339 in 65% [vol/vol] PEG 14000 administered in sterile distilled water at 1 mg/kg vaginally once daily), and the Clotrimazole group (0.05 ml, as purchased at 1% [wt/vol], administered vaginally once daily). Clotrimazole (Canetsen) was from Bayer, Leverkusen, Germany. At 3 days postinfection, fungal burdens in vaginal lavage samples were determined as described above.
Clotrimazole
Manufactured by Bayer
Sourced in Germany, United Kingdom, New Zealand
Clotrimazole is a topical antifungal medication used in the treatment of fungal skin infections. It is a synthetic compound that belongs to the class of azole antifungals. Clotrimazole works by inhibiting the growth of fungal organisms by disrupting the fungal cell membrane.
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3 protocols using clotrimazole
1
Murine Vaginal Candidiasis Model
2
Antifungal Efficacy on Infected Porcine Skin
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Explanted porcine skin was sterilised and infected with conidia as above, with the porcine skin placed on mineral salts agar. This was incubated for 3 days at 30°C to induce fungal skin infection. The infected skin was then treated topically using clinically used antifungal creams, being 1% clotrimazole (Bayer, Reading, UK) or 1% terbinafine hydrochloride (GlaxoSmithKline, Brentford, UK). Both were applied as directed in the patient information leaflets. In brief, the creams were applied thinly on the skin (using a sterile swab) once per day (terbinafine) or twice per day (clotrimazole), for a period of 7 days. The explanted skin was kept at 30°C for the duration of the experiment and was rinsed once per day with sterile PBS to simulate washing. Control specimens were treated the same but were wiped with a sterile swab dipped in sterile water instead of with antifungal cream. After one week, all specimens were washed with sterile PBS and gently dried with sterile tissue paper. The specimens were then transferred to fresh mineral salts agar and incubated for a further 7 days at 30°C.
3
Substrate Specificity of Efflux Pumps
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The following compounds listed from smallest to largest (molecular weights in bracket) were used to determine the substrate specificity of the two possible efflux pumps: anisomycin (ANI; 265), acridine orange (AOR; 265), cycloheximide (CHX; 281), terbinafine (TRB; 291), trichodermin (TRD; 292), FLC (306; Diflucan; Pfizer Laboratories, Auckland, New Zealand), clotrimazole (CLT; 345; Bayer, Osaka, Japan), VRC (349; Cayman Chemical, MI, United States), difenoconazole (DFC; 406), rhodamine 6G (R6G; 479), ketoconazole (KTC; 531) and nigericin (NIG; 725). ANI, AOR, CHX, TRB, TRD, DFC, R6G, KTC, and NIG were purchased from Sigma-Aldrich, MO, United States. To test the drug susceptibilities of cells, 10 mL YPD overnight cultures of yeast cells were diluted 1:20 into 3 mL complete supplement mixture (CSM) pH 7.0 (0.69% YNB, 2% glucose, 0.079% CSM, 10 mM MOPS, 20 mM HEPES; pH 7.0) and grown to mid-logarithmic growth phase (OD600 ∼1; ∼107 cells/mL) at 30°C for ∼4 h. Broth microdilution assays of twofold serial dilutions of test compounds in CSM pH 7.0 were used to determine the minimum growth inhibitory concentrations (MIC) of test compounds. The MIC was defined as the lowest concentration of drug that inhibited growth by >90% (Niimi et al., 2004 (link)).
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