Treatment with gomesin effectively reduced Candida albicans in the kidneys, spleen, liver and vagina of infected mice. The biodistribution of gomesin labelled with technetium-99 m showed that the peptide is captured in the kidneys, spleen and liver. Enhanced production of TNF-α, IFN-γ and IL-6 was detected in infected mice treated with gomesin, suggesting an immunomodulatory activity. Moreover, immunosuppressed and C. albicans-infected mice showed an increase in survival after treatment with gomesin and fluconazole. Systemic administration of gomesin was also not toxic to the mic

Two strains of Candida albicans were used: isolate 78 [29] and the isolate ATCC 90028. Periodically, isolate 78 was inoculated into mice in order to maintain its virulence.

The antifungal activity of antimicrobial compounds was evaluated by using the protocol M-27A2, according to the Clinical and Laboratory Standards Institute (CLSI) [30]. Briefly, 80 μl of RPMI 1640 with 1.6 M MOPS pH 7 containing 10⁴ yeast/mL of C. albicans in logarithmic growth phase, were added to the wells of a polypropylene 96-well plate containing 20 μl of serial two-fold dilution of gomesin (starting at 44 μM), fluconazole (starting at 1,488 μM) or the combination of gomesin (starting at 11 μM) and fluconazole (starting at 115 μM). After 48 h of incubation at 37°C fungal growth was evaluated by determining the absorbance at 595 nm. The lowest concentration that inhibited 100% growth was considered the minimum inhibitory concentration (MIC). The fractional inhibitory concentration index (FICI) was determined following the methodology described previously [31].

BALB/c mice (6- to 8-week-old males or females) were bred at the Animal Facility at the Institute of Biomedical Science of University of São Paulo, Department of Immunology under specific pathogen-free conditions. Food and water were given ad libitum. All animals were handled in accordance with good animal practice as defined by the relevant national animal welfare bodies and all in vivo testing was approved by the Institutional Animal Care and Use Committee of the University of São Paulo, reference number: 87/42. For immunosuppression of animals, doses of 100 mg/kg cyclophosphamide were administered intraperitoneally 4 days and 1 day before infection with C. albicans, the third day after infection and, from this point on, every 4 days until the end of treatment [32]. The animals were kept in cages lined with wood shavings and closed with autoclaved filter, and served autoclaved food and water in order to maintain a sterile environment. Cages were exchanged twice a week in laminar flow hoods. The animals were considered anergic when the number of leukocytes was found to be less than 100 cells/mm³ [33]. The vaginal candidiasis model was developed by inducing the pseudo oestrus phase by the subcutaneous administration of 0.5 mg of 17 beta-estradiol valerate (Sigma Chemicals, St Louis), dissolved in sesame oil (Sigma Chemicals, St Louis) 3 days before the vagina's infection [34]. Swiss mice were provided by the Animal Facility of IPEN-CNEN for the biodistribution studies.

Mice with disseminated candidiasis were treated with gomesin and fluconazole. The drugs were administered intraperitoneally in a final volume of 500 μl at the following concentrations: gomesin (2.5 mg/kg, 5 mg/kg and 15 mg/kg), fluconazole (10 mg/kg and 20 mg/kg) and a combination of both (2.5 mg/kg to 5 mg/kg gomesin and 10 mg/kg to 20 mg/kg fluconazole). For mice with vaginal candidiasis, gomesin (0.02%, 0.2% and 0.5%), miconazole (2%) and a combination of both (0.2% gomesin and 2% miconazole) were incorporated into a vaginal cream (10% Wax self-nonionic emulsifier, 2% mineral oil, 5% propylene glycol and 84% distilled water, pH 4.5) for topical application. For all treatments, drugs were administered 1, 3 and 6 days after infection with C. albicans. An equivalent volume of PBS or cream was administered to the control animals. To evaluate the fungal burden, the kidneys, spleen, liver and vagina of the mice were dissected aseptically on the seventh day after infection, weighed and homogenised in 1 mL of PBS. Aliquots of the homogenate (100 μl) were inoculated on brain and heart infusion (Oxoid, England) containing 2% agar. After incubation for 18 h at 37°C, the number of CFUs was determined. The effectiveness of treatment was determined by comparing the number of CFUs per gram of tissue of treated animals with the number of CFUs per gram of tissue of control animals (untreated). For the survival curves, the animals were monitored daily for 30 days.

The kidneys of animals infected with C. albicans and treated with gomesin or fluconazole were dissected, washed in PBS and homogenised with an electric tissue homogeniser in 1 mL of PBS containing a cocktail of protease inhibitors (Sigma Chemicals, St Louis). This cocktail of protease inhibitors was composed of 1 μg/ml of pepstatin A (aspartyl protease inhibitor), 4 mM benzamidine (seine protease inhibitor), 1 mM ethylenediaminetetraacetic acid acetic (metallo-protease inhibitor) and 1 mM N-Ethylmaleimide (cysteine protease inhibitor). Non-infected animals or animals infected and not treated were used as controls. The concentrations of IFN-γ, TNF-α and IL-6 were evaluated on a flow cytometer (BD FACSCaliburTM, San José) using the kit Cytometric Bead Array and Mouse Inflammation™ (BD, San José) and the methodology described by the manufacturer.

Blood was collected by puncturing the brachial plexus of anesthetised mice using EDTA (1%) as an anticoagulant after 7 days of gomesin administration (15 mg/kg). Reticulocytes cells and leukocytes were counted by standard methods. The haemoglobin concentration was determined using the modified Drabkin method. Blood samples were prepared on microscopic glasses, dried and stained with May-Grünwald reagents for morphological examination of the blood. The number of reticulocytes was determined in blood smears stained with Supra Vital New Methylene Blue. We also determined the levels of bilirubin, creatinine and gamma GT biochemically using the Sims-Horm, Enzyme and Alkaline picrate methods, respectively.

HYNIC-gomesin was manually synthesised by solid phase methodology as described previously, except that pyroglutamic acid was substituted for 6-hydrazino nicotinamide (HYNIC) [6]. The HYNIC-gomesin conjugate was labelled with the radioisotope technetium-99 m obtained from an alumina-based 99Mo/99mTc generator, supplied by the Radiopharmacy Centre of the Institute of Energetic and Nuclear Research (IPEN/CNEN). Briefly, 20 mg of tricine and 5 mg of ethylenediamine N,N'-diacetic acid (EDDA) were dissolved in 0.5 ml of 0.1 M PBS, previously nitrogenated. Ten micrograms of HYNIC-gomesin, 5 μl of 8.9 mM SnCl₂·2H₂O solution in 0.1 N HCl (nitrogen-purged) and 500 μl of Na^99mTcO₄ was added to the vial. The reaction was conducted by heating the solution at 100°C for 20 min in a water bath and then allowing it to cool to room temperature. The pH of the reaction mixture was 7 [35]. The product ^99mTc-HYNIC-gomesin (0.1 mL), with an approximate activity of 74 MBq (2 mCi), was administered to the tail vein of the mice. The animals were sacrificed in a CO₂ chamber at 5, 30, 60, 120, 240, 360, and 1,440 min after injection of the radiolabeled gomesin. Six animals were used for each time point. The kidneys, spleen and liver of each animal was dissected and transferred to tubes to measure radioactivity. The radioactivity count was performed in the Gamma Counter Shaft type NaI, using the same standard dose in injected animals minus the radioactivity retained in the site injection (tail). The uptake of radiolabeled gomesin by each organ was calculated using the following equation: DI% = (CPM organ/standard CPM) × 100), where%DI = percentage of the injected dose and CPM = count per min [35].