Inclusion complex of O-allyl-lawsone with 2-hydroxypropyl-ß-cyclodextrin: Preparation, physical characterization, antiparasitic and antifungal activity.

The subclass naphthoquinone represents a substance group containing several compounds with important activities against various pathogenic microorganisms. Accordingly, we evaluated O-allyl-lawsone (OAL) antiparasitic and antifungal activity free and encapsulated in 2-hydroxypropyl-ß-cyclodextrin (OAL MKN) against Trypanosoma cruzi and Sporothrix spp. OAL and OAL MKN were synthesized and characterized by physicochemical methods. The IC50 values of OAL against T. cruzi were 2.4 µM and 96.8 µM, considering epimastigotes and trypomastigotes, respectively. At the same time, OAL MKN exhibited a lower IC50 value (0.5 µM) for both trypanosome forms and low toxicity for mammalian cells. Additionally, the encapsulation showed a selectivity index approximately 240 times higher than that of benznidazole. Regarding antifungal activity, OAL and OAL MKN inhibited Sporothrix brasiliensis growth at 16 µM, while Sporothrix schenckii was inhibited at 32 µM. OAL MKN also exhibited higher selectivity toward fungus than mammalian cells. In conclusion, we described the encapsulation of O-allyl-lawsone in 2-hydroxypropyl-ß-cyclodextrin, increasing the antiparasitic activity compared with the free form and reducing the cytotoxicity and increasing the selectivity towardSporothrix yeasts and the T. cruzi trypomastigote form. This study highlights the potential development of this inclusion complex as an antiparasitic and antifungal agent to treat neglected diseases.

Synthesis and Biological Activity of Novel Zinc-Itraconazole Complexes in Protozoan Parasites and Sporothrix spp.

The new complexes Zn(ITZ)2Cl2 (1) and Zn(ITZ)2(OH)2 (2) were synthetized by a reaction of itraconazole with their respective zinc salts under reflux. These Zn-ITZ complexes were characterized by elemental analyses, molar conductivity, mass spectrometry, 1H and 13C{1H} nuclear magnetic resonance, and UV-vis and infrared spectroscopies. The antiparasitic and antifungal activity of Zn-ITZ complexes was evaluated against three protozoans of medical importance, namely, Leishmania amazonensis, Trypanosoma cruzi, and Toxoplasma gondii, and two fungi, namely, Sporothrix brasiliensis and Sporothrix schenckii The Zn-ITZ complexes exhibited a broad spectrum of action, with antiparasitic and antifungal activity in low concentrations. The strategy of combining zinc with ITZ was efficient to enhance ITZ activity since Zn-ITZ-complexes were more active than the azole alone. This study opens perspectives for future applications of these Zn-ITZ complexes in the treatment of parasitic diseases and sporotrichosis.

The Antifungal Activity of Naphthoquinones: An Integrative Review.

Naphthoquinones are the most commonly occurring type of quinones in nature. They are a diverse family of secondary metabolites that occur naturally in plants, lichens and various microorganisms. This subgroup is constantly being expanded through the discovery of new natural products and by the synthesis of new compounds via innovative techniques. Interest in quinones and the search for new biological activities within the members of this class have intensified in recent years, as evidenced by the evaluation of the potential antimicrobial activities of quinones. Among fungi of medical interest, yeasts of the genus Candida are of extreme importance due to their high frequency of colonization and infection in humans. The objective of this review is to describe the development of naphthoquinones as antifungals for the treatment of Candida species and to note the most promising compounds. By using certain criteria for selection of publications, 68 reports involving both synthetic and natural naphthoquinones are discussed. The activities of a large number of substances were evaluated against Candida albicans as well as against 7 other species of the genus Candida. The results discussed in this review allowed the identification of 30 naphthoquinones with higher antifungal activities than those of the currently used drugs.

Current Progress on Epidemiology, Diagnosis, and Treatment of Sporotrichosis and Their Future Trends.

Sporotrichosis, a human and animal disease caused by Sporothrix species, is the most important implantation mycosis worldwide. Sporothrix taxonomy has improved in recent years, allowing important advances in diagnosis, epidemiology, and treatment. Molecular epidemiology reveals that S. brasiliensis remains highly prevalent during the cat-transmitted sporotrichosis outbreaks in South America and that the spread of S. brasiliensis occurs through founder effects. Sporothrix globosa and S. schenckii are cosmopolitan on the move, causing major sapronoses in Asia and the Americas, respectively. In this emerging scenario, one-health approaches are required to develop a creative, effective, and sustainable response to tackle the spread of sporotrichosis. In the 21st century, it has become vital to speciate Sporothrix, and PCR is the main pillar of molecular diagnosis, aiming at the detection of the pathogen DNA from clinical samples through multiplex assays, whose sensitivity reaches remarkably three copies of the target. The treatment of sporotrichosis can be challenging, especially after the emergence of resistance to azoles and polyenes. Alternative drugs arising from discoveries or repositioning have entered the radar of basic research over the last decade and point to several molecules with antifungal potential, especially the hydrazone derivatives with great in vitro and in vivo activities. There are many promising developments for the near future, and in this review, we discuss how these trends can be applied to the Sporothrix-sporotrichosis system to mitigate the advance of an emerging and re-emerging disease.

Characterization of Aspergillus nidulans Biofilm Formation and Structure and Their Inhibition by Pea Defensin Psd2.

Approximately four million people contract fungal infections every year in Brazil, primarily caused by Aspergillus spp. The ability of these fungi to form biofilms in tissues and medical devices complicates treatment and contributes to high rates of morbidity and mortality in immunocompromised patients. Psd2 is a pea defensin of 5.4 kDa that possesses good antifungal activity against planktonic cells of representative pathogenic fungi. Its function depends on interactions with membrane and cell wall lipid components such as glucosylceramide and ergosterol. In the present study, we characterized Aspergillus nidulans biofilm formation and determined the effect of Psd2 on A. nidulans biofilms. After 4 hours, A. nidulans conidia adhered to polystyrene surfaces and formed a robust extracellular matrix-producing biofilm at 24 h, increasing thickness until 48 h Psd2 inhibited A. nidulans biofilm formation in a dose-dependent manner. Most notably, at 10 µM Psd2 inhibited 50% of biofilm viability and biomass and 40% of extracellular matrix production. Psd2 significantly decreased the colonized surface area by the biofilm and changed its level of organization, causing a shortening of length and diameter of hyphae and inhibition of conidiophore formation. This activity against A. nidulans biofilm suggests a potential use of Psd2 as a prototype to design new antifungal agents to prevent biofilm formation by A. nidulans and related species.

A novel naphthoquinone derivative shows selective antifungal activity against Sporothrix yeasts and biofilms.

Sporotrichosis is a subcutaneous mycosis that affects humans and animals, with few therapeutic options available in the pharmaceutical market. We screened the in vitro antifungal activity of fourteen 1,4-naphthoquinones derivative compounds against Sporothrix brasiliensis and Sporothrix schenckii, the main etiological agents of sporotrichosis in Latin America. The most active compound was selected for further studies exploring its antibiofilm activity, effects on yeast morphophysiology, interaction with itraconazole, and selectivity to fungal cells. Among the fourteen 1,4-naphthoquinones tested, naphthoquinone 5, a silver salt of lawsone, was the most active compound. Naphthoquinone 5 was able to inhibit Sporothrix biofilms and induced ROS accumulation, mitochondrial disturbances, and severe plasmatic membrane damage in fungal cells. Furthermore, naphthoquinone 5 was ten times more selective towards fungal cells than fibroblast, and the combination of itraconazole with naphthoquinone 5 improved the inhibitory activity of the azole. Combined, the data presented here indicate that the silver salt naphthoquinone 5 exerts promising in vitro activity against the two main agents of sporotrichosis with important antibiofilm activity and a good toxicity profile, suggesting it is a promising molecule for the development of a new family of antifungals.

Activity of Metal-Azole Complexes Against Biofilms of Candida albicans and Candida glabrata.

BACKGROUND: Onychomycosis is a chronic nail infection caused by fungi frequently resistant to antifungal treatments. Recalcitrance in nail infections is a result of reduced antifungal penetration due to biofilm formation, combined with poor patient compliance with the treatment, which can be as long as 18 months. OBJECTIVE: Metal-drug complexation is a widely used strategy to increase drug efficacy. Therefore, the aim of this work was to evaluate the antifungal and anti-biofilm activity of several metal-azole complexes against Candida albicans and Candida glabrata. METHODS: Susceptibility assays and scanning electron microscopy were performed to determine the anti-biofilm activity of eight metal-azole complexes in vitro and ex-vivo, using human nail fragments. RESULTS: In vitro susceptibility assays showed that complexation of both Au(I) and Zn(II) to clotrimazole and ketoconazole improved the anti-biofilm activity compared to the azole alone. Using an ex-vivo model of biofilm formation on fragments of human nails, we also demonstrate the improved efficacy of metal-azole complexes against biofilms of C. albicans and C. glabrata that resembles the onychomycosis structure. Noteworthy, biofilms of C. glabrata were more susceptible to the optimized complexes than those of C. albicans. CONCLUSION: In conclusion, metal-azole complexes used in this work show promising anti-biofilm activity and further clinical studies should confirm its potential for the treatment of Candida-associated onychomycosis.

Formulation and Evaluation of a Novel Itraconazole-Clotrimazole Topical Emulgel for the Treatment of Sporotrichosis.

In recent years, the development of new pharmaceutical formulations for the treatment of sporotrichosis has become a relevant research field. In this work, we aimed to develop an emulgel containing itraconazole and clotrimazole to ensure therapeutic effectiveness against Sporothrix brasiliensis. The topical use of a formulation that combines both drugs represents an interesting option for the complementary treatment of sporotrichosis. The emulgel formulation was prepared and evaluated for its zeta potential, viscosity, in vitro antifungal activity and stability at different storage conditions. The results showed that the newly developed emulgel displayed promising physicochemical characteristics, as well as a good in vitro inhibitory activity against S. brasiliensis yeasts. The results obtained in this work suggest that the emulgel containing itraconazole and clotrimazole might highly be efficient and a complementary therapy to oral administration in the treatment of sporotrichosis.

Investigation of a Microemulsion Containing Clotrimazole and Itraconazole for Transdermal Delivery for the Treatment of Sporotrichosis.

The aim of this study was to develop a microemulsion (ME) formulation containing an association of itraconazole (ITC) and clotrimazole (CLT) as a transdermal delivery system for the treatment of sporotrichosis. Pseudoternary phase diagrams were constructed to optimize the ME formulation. The ME formulation selected contained 1% (w/w) ITC and 1% (w/w) CLT and was composed of 23.07% Tween® 60 (surfactant), 23.07% propylene glycol (cosurfactant/cosolvent), 30.77% benzyl alcohol (oil), and 21.09% water. The ITC/CLT-loaded ME (ITC/CLT-ME) had a droplet size value of 217 ± 0.9 nm, with a polydispersity index of 0.5 ± 0.1. Permeation experiments on pig ear skin were conducted for ITC/CLT-ME, and the results indicated that the drug permeation performance was influenced by CLT, indicating that CLT acts as a promoter enhancer. In the in vitro antifungal activity assay using Sporothrix brasiliensis yeast, the inhibition halo produced by ITC/CLT-ME exhibited a mean diameter of 43.67 ± 2.31 mm. The ITC/CLT-ME formulation did not cause skin irritation in mice. The results suggest that ITC/CLT-ME is a promising tool for the transdermal treatment of sporotrichosis.

Tacrolimus Increases the Effectiveness of Itraconazole and Fluconazole against Sporothrix spp.

Calcineurin inhibitors - such as the clinically used drug tacrolimus - are active against important fungal pathogens, particularly when combined with azoles. However, tacrolimus has not been tested against sporotrichosis, an endemic subcutaneous mycosis with worldwide distribution. Here, we evaluated the activity of tacrolimus and cyclosporine A in vitro - as monotherapy and in combination with itraconazole or fluconazole - against yeasts of Sporothrix brasiliensis and S. schenckii, the main sporotrichosis agents in Brazil. We also analyzed the effect of tacrolimus treatment on intracellular neutral lipid levels, which typically increase after azole treatment. Tacrolimus inhibited the growth of yeasts from S. brasiliensis and S. schenckii reference isolates, with minimum inhibitory concentration (MIC) values (required for ≥50% growth inhibition) of 1 and 2 mg/L, respectively. Importantly, the combination of tacrolimus and azoles exhibited high synergy toward reference Sporothrix isolates. Tacrolimus combined with itraconazole significantly increased neutral lipid accumulation in S. brasiliensis, but not in S. schenckii. Clinical isolates of S. brasiliensis and S. schenckii were more sensitive to tacrolimus as monotherapy than feline-borne isolates, however, synergy between tacrolimus and azoles was only observed for feline-borne isolates. Cyclosporine A was effective against S. brasiliensis and S. schenckii as monotherapy (MIC = 1 mg/L), but exhibited no synergy with itraconazole and fluconazole. We conclude that tacrolimus has promising antifungal activity against sporotrichosis agents, and also increases the activity of the current anti-sporotrichosis therapy (itraconazole and fluconazole) in combination assays against S. brasiliensis feline-borne isolates.

Δ(24)-Sterol Methyltransferase Plays an Important Role in the Growth and Development of Sporothrix schenckii and Sporothrix brasiliensis.

Inhibition of Δ(24)-sterol methyltransferase (24-SMT) in Sporothrix schenckii sensu stricto and Sporothrix brasiliensis was investigated in vitro. The effects on fungal growth and sterol composition of the 24-SMT inhibitor 22-hydrazone-imidazolin-2-yl-chol-5-ene-3ß-ol (H3) were compared to those of itraconazole. MIC and MFC analysis showed that H3 was more effective than itraconazole against both species in both their filamentous and yeast forms. H3 showed fungistatic activity in a time-kill assay, with inhibitory activity stronger than that of itraconazole. GC analysis of cell sterol composition showed that sterols present in control cells (ergosterol and precursors) were completely replaced by 14α-methylated sterols after H3 exposure. Itraconazole only partially inhibited ergosterol synthesis but completely arrested synthesis of other sterols found in control cells, promoting accumulation of nine 14α-methyl sterols. Based on these results, we propose a schematic model of sterol biosynthesis pathways in S. schenckii and S. brasiliensis. Effects on cell morphology due to 24-SMT inhibition by H3 as analyzed by SEM and TEM included irregular cell shape, reduced cytoplasmic electron-density, and reduced thickness of the microfibrillar cell wall layer. Moreover, 24-SMT inhibition by H3 promoted mitochondrial disturbance, as demonstrated by alterations in MitoTracker(®) Red CMXRos fluorescence intensity evaluated by flow cytometry. When used in conjunction with itraconazole, H3 enhanced the effectiveness of itraconazole against all tested strains, reducing at least half (or more) the MIC values of itraconazole. In addition, cytotoxicity assays revealed that H3 was more selective toward these fungi than was itraconazole. Thus, 24-SMT inhibition by H3 was an effective antifungal strategy against S. schenckii and S. brasiliensis. Inhibition of the methylation reaction catalyzed by 24-SMT has a strong antiproliferative effect via disruption of ergosterol homeostasis, suggesting that this enzyme is a promising target for novel antifungal therapies against sporotrichosis, either as sole treatments or in combination with itraconazole.