New copper(II) complexes with isoconazole: Synthesis, structures and biological properties

There is an increasing demand for novel metal-based complexes with biologically relevant molecules in technology and medicine. Three new Cu(II) coordination compounds with antifungal agent isoconazole (L), namely mononuclear complexes CuCl2(L)(2) (1), and Cu(O2CMe)(2)(L)(2)center dot 2H(2)O (2) and coordination polymer Cu(pht)(L)(2)(n) (3) (where H(2)pht - o-phthalic acid) were synthesized and characterized by IR spectroscopy, thermogravimetric analysis and X-ray crystallography. X-ray analysis showed that in all complexes, the isoconazole is coordinated to Cu(II) centres by a N atom of the imidazole fragment. In complex I, the square-planar environment of Cu(II) atoms is completed by two N atoms of isoconazole and two chloride ligands, whereas the Cu(II) atoms are coordinated by two N atoms from two isoconazole ligands and two O atoms from the different carboxylate residues: acetate in 2 and phthalate in 3. The formation of an infinite chain through the bridging phthalate ligand is observed in 3. The biosynthetic ability of micromycetes Aspergillus niger CNMN FD 10 in the presence of the prepared complexes 1-3 as well as the antifungal drug isoconazole were studied. Complexes 2 and 3 accelerate the biosynthesis of enzymes (beta-glucosidase, xylanase and endoglucanase) by this fungus. Moreover, a simplified and improved method for the preparation of isoconazole nitrate was developed.

Obtenção de gel mucoadesivo de nistatina para o tratamento da candidíase oral. Desenvolvimento e caracterização de dispersões sólidas de nistatina

A nistatina (NYS) é o fármaco de primeira escolha no tratamento da candidíase oral, que frequentemente acomete mais os indivíduos imunocomprometidos e pacientes com outras desordens (diabetes não tratada, neoplasias, imunodeficiências). No mercado brasileiro, a NYS é encontrada na forma de suspensão oral aquosa, onde o procedimento para sua administração consiste em bochechar o medicamento. Apesar de haver a indicação de que se mantenha o contato direto entre fármaco e a mucosa oral, na qual se encontra a Candida spp., o que aumentaria expressivamente o sucesso terapêutico, a suspensão não apresenta tal propriedade. Assim, a NYS que é fármaco com ação efetiva contra a candidíase oral, é considerada pertencente à Classe IV do Sistema de Classificação Biofarmacêutica, ou seja, apresenta baixa solubilidade e baixa permeabilidade. A baixa solubilidade pode comprometer sua disponibilidade na cavidade oral, e consequentemente, sua ação farmacológica. Diante desse quadro, o objetivo do presente trabalho foi o desenvolvimento de dispersões sólidas de NYS para o tratamento da candidíase oral, e sua posterior incorporação em gel mucoadesivo oral, favorecendo a formulação no local de ação. As dispersões sólidas são sistemas farmacêuticos, onde um fármaco pouco solúvel em água encontra-se dispersado em um carreador, no estado sólido. Os carreadores normalmente são hidrofílicos, o que permite que esses sistemas sejam empregados para aumentar a solubilidade aquosa do fármaco. Assim, foram desenvolvidas as dispersões sólidas de NYS, pelo método de eliminação do solvente, empregando como carreadores, lactose, HPMC, poloxamer 407 e poloxamer 188. Essas foram submetidas à caracterização por análise térmica, usando os ensaios de calorimetria exploratória diferencial (DSC) e termogravimetria/termogravimetria derivada (TG/DTG). Dentre essas dispersões sólidas, aquelas que se mostraram com comportamento térmico sugerindo a formação de um novo \"sistema\", foram analisadas por meio de ensaio de solubilidade. Dessa forma, a formulação NYS DS G2 (49) se destacou, pois apresentou maior solubilidade em água (4,484 mg/mL); em pH 5,5 (4,249 mg/mL) e em pH 7,0 (4,293 mg/mL), ou seja, houve um aumento de 1,426 vezes em água; 4,227 vezes em pH 5,5; e 2,743 vezes em pH 7,0. Essa formulação foi, por fim avaliada por difração de raio-X e espectroscopia de infravermelho com transformada de Fourier, técnicas que corroboraram com a análise térmica quanto à indicação de formação da dispersão sólida. Por sua vez, essa dispersão sólida foi incorporada em 4 bases de géis mucoadesivos de carbopol ® 934 PNF, alterando apenas a concentração do polímero (0,5; 1,0; 1,5; 2,0 %p/p). Foi observado que a liberação de NYS DS G2 (49) foi superior, quando comparada à liberação de NYS MP a partir do gel, e através do ensaio de mucoadesão, percebeu-se que os géis desenvolvidos apresentaram propriedades mucoadesivas compatíveis com relatos na literatura, independentemente da quantidade de carbopol ® empregada. As características reológicas foram distintas, e foi observado que as formulações Gel A e Gel B, que possuem menor quantidade de polímero, tiverem um indicativo de comportamento de fluido newtoniano, diferente dos demais, o que pode não ser desejado para esse tipo de forma farmacêutica tópica e semi-sólida. Ao final desse trabalho, pode-se concluir que foi possível desenvolver um sistema farmacêutico na forma de dispersão sólida com maior solubilidade que a NYS pura, e sua incorporação em uma forma farmacêutica mucoadesiva, e que a liberação da NYS na forma DS foi muito superior que o fármaco na forma \"convencional\", o que permite que a NYS esteja mais disponível na cavidade oral, e também junto à mucosa bucal, o que levaria a efeito farmacológico mais efetivo do antifúngico.

Lichtheimia Infection in a Lymphoma Patient: Case Report and a Brief Review of the Available Diagnostic Tools

We describe the case of a patient with a T-lymphoblastic lymphoma whose disseminated mucormycosis was diagnosed with delay, and we address the diagnostic and therapeutic decision-making process and review the diagnostic workup of patients with potential IFD. The diagnosis was delayed despite a suggestive radiological presentation of the patient's pulmonary lesion. The uncommon risk profile (T-lymphoblastic lymphoma, short neutropenic phases) wrongly led to a low level of suspicion. The diagnosis was also hampered by the lack of indirect markers for infections caused by Mucorales, the low sensitivity of both fungal culture and panfungal PCR, and the limited availability of species-specific PCR. A high level of suspicion of IFD is needed, and aggressive diagnostic procedures should be promptly initiated even in apparently low-risk patients with uncommon presentations. The extent of the analytical workup should be decided on a case-by-case base. Diagnostic tests such as the galactomannan and β-D-glucan test and/or PCR on biological material followed by sequencing should be chosen according to their availability and after evaluation of their specificity and sensitivity. In high-risk patients, preemptive therapy with a broad-spectrum mould-active antifungal agent should be started before definitive diagnostic findings become available.

Refinement of a cell line based artificial diet for rearing the parasitoid wasp, Trichogramma pretiosum

An artificial diet incorporating insect cells originally developed for Trichogramma australicum Girault (Hymenoptera: Tricho-grammatidae) was successfully used to rear Trichogramm pretiosum Riley (Hymenoptera: Trichogrammatidae). To refine the diet, individual components were removed. Chicken egg yolk and the insect cells were identified as the most important components for T. pretiosum development. Their removal resulted in few pupae and no adults. Removal of Grace's insect medium, a common component of artificial diets, was found to markedly improve the development of T pretiosum, producing 60% larva to pupa transition and 19% pupa to adult transition. There was no significant difference in T pretiosum development on diets in which milk powder, malt powder or infant formula were interchanged, despite differences in nutrient composition. The use of yeast extract resulted in significantly higher survival to the adult stage when compared with yeast hydrolysate enzymatic and a combination of yeast extract and yeast hydrolysate enzymatic. Comparison of four antimicrobial agents showed the antibacterial agent Gentamycin and the antifungal agent Nystatin had the least detrimental effect on T pretiosum development. The use of insect cell line diets has the potential to simplify artificial diet production and significantly reduce T pretiosum production costs in Australia compared to diets using insect hemolymph or the use of natural or factitious hosts. (c) 2005 Elsevier Inc. All rights reserved.

Calbistrin E and two other new metabolites from an Australian isolate of Penicillium striatisporum

An Australian isolate of Penicillium striatisporum collected near Shalvey, New South Wales, exhibited selective antifungal activity against Candida albicans versus Saccharomyces cerevisiae. Bioassay-directed fractionation yielded members of the rare class of fungal metabolites known as the calbistrins. These included a new example of this structure class, calbistrin E (1), as well as the known polyenes calbistrin C (2) and deformylcalbistrin A (3). Also recovered from P. striatisporum were new triene and butenolide acids, striatisporin A (4) and striatisporolide A (5), together with the known fungal metabolites versiol (6) and (+)-hexylitaconic acid (7). Structures for all metabolites were determined by detailed spectroscopic analysis.

Strategies for local drug delivery targeting the oesophagus

Localised, targeted drug delivery to the oesophagus offers the potential for more effective delivery and reduced drug dosages, coupled with increased patient compliance. This thesis considers bioadhesive liquids, orally retained tablets and films as well as chewable dosage forms as drug delivery systems to target the oesophagus. Miconazole nitrate was used as a model antifungal agent. Chitosan and xanthan gum hydrogels were evaluated as viscous polymer viables with the in vitro retention, drug release and minimum inhibitory concentration values of the formulations measured. Xanthan showed prolonged retention on the oesophageal surface in vitro yet chitosan reduced the MIC value; both polymers offer potential for local targeting to the oesophagus. Cellulose derivatives were investigated within orally retained dosage forms. Both drug and polymer dissolution rates were measured to investigate the drug release mechanism and to develop a formulation with concomitant drug and polymer release to target the oesophagus with solubilised drug within a viscous media. Several in vitro dissolution methods were evaluated to measure drug release from chewable dosage forms with both drug and polymer dissolution quantified to investigate the effects of dissolution apparatus on drug release. The results from this thesis show that a range of drug delivery strategies that can be used to target drug to the oesophagus. The composition of these formulations as well as the methodology used within the development are crucial to best understand the formulation and predict its performance in vivo.

Capacidade de adesão e formação de biofilme de Candida ssp. isoladas da cavidade oral de pacientes transplantados renais na presença do extrato de Eugenia uniflora

Candidiasis is a major oral manifestation in kidney transplant patients. Candida spp. possess essential virulence factors which contribute for the infectious process, including the ability to adhere to epithelial cells and biofilm formation. The extract obtained from the leaves of Eugenia uniflora [acetone: water (7:3, v/v)] has demonstrated antifungal activity against Candida spp. This study evaluated the influence of the extract of E. uniflora in adhesion to human buccal epithelial cells (HBEC) and biofilm formation of 42 strains of Candida spp. isolated from the oral cavity of kidney transplant patients. Candida spp. strains belonging to a culture collection were reactivated and phenotypically re-identified by classical and molecular methods (genotyping ABC and RAPD), when necessary, to complete the identification to the species level. For the virulence tests evaluated in vitro, yeasts were grown in the presence and absence of 1000 g/mL of the extract. A ratio of 10: 1 (Candida spp. cells x HBECs) was incubated for 1 hour at 37 ° C, 200 rpm, fixed with 10% formalin and the number of Candida cells adhered to 150 HBEC determined by optical microscope. Biofilms were formed on polystyrene microplates in the presence or absence of the extract. The quantification was performed with crystal violet staining at 570 nm. All isolates were viable and exhibited phenotypic characteristics suggestive of each species identified. Two strains presumptively identified as Candida dubliniensis belonged to this species as determined with genotyping ABC, while strains identified as belonging to the Candida parapsilosis species complex were differentiated by RAPD genotyping. Candida albicans was found to be the most adherent species to the buccal epithelia, while C. tropicalis showed remarkable biofilm formation.We could detect that the extract of E. uniflora was able to reduce adhesion to HBEC for both Candida albicans and non-Candida albicans Candida species. On the other hand, only 16 Candida spp. strains (36 %) showed reduced biofilm formation. However, two highly biofilm producer strains of C. tropicalis had an expressive reduction in biofilm formation. This study reinforces the idea that besides growth inhibition, E. uniflora may interfere with the expression of some virulence factors of Candida spp., and may be possibly applied in the future as a novel antifungal agent.

Similarity between the in vitro activity and toxicity of two different fungizone™ / lipofundin™ admixtures

Amphotericin B (AmB), an antifungal agent that presents a broad spectrum of activity, remains the gold standard in the antifungal therapy. However, sometimes the high level of toxicity forbids its clinical use. The aim of this work was to evaluate and compare the efficacy and toxicity in vitro of Fungizon™ (AmB-D) and two new different AmB formulations. Methods: three products were studied: Fungizon™, and two Fungizon™ /Lipofundin™ admixtures, which were diluted through two methods: in the first one, Fungizon™ was previously diluted with water for injection and then, in Lipofundin™ (AmB-DAL); the second method consisted of a primary dilution of AmB-D as a powder in the referred emulsion (AmB-DL). For the in vitro assay, two cell models were used: Red Blood Cells (RBC) from human donors and Candida tropicallis (Ct). The in vitro evaluation (K+ leakage, hemoglobin leakage and cell survival rate-CSR) was performed at four AmB concentrations (from 50 to 0.05mg.L-1). Results: The results showed that the action of AmB was not only concentration dependent, but also cellular type and vehicle kind dependent. At AmB concentrations of 50 mg.L-1, although the hemoglobin leakage for AmB-D was almost complete (99.51), for AmB-DAL and AmB-DL this value tended to zero. The p = 0.000 showed that AmB-D was significantly more hemolytic. Conclusion: The Fungizon™- Lipofundin™ admixtures seem to be the more valuable AmB carrier systems due to their best therapeutic index presented

Similarity between the in vitro activity and toxicity of two different fungizone™ / lipofundin™ admixtures

Amphotericin B (AmB), an antifungal agent that presents a broad spectrum of activity, remains the gold standard in the antifungal therapy. However, sometimes the high level of toxicity forbids its clinical use. The aim of this work was to evaluate and compare the efficacy and toxicity in vitro of Fungizon™ (AmB-D) and two new different AmB formulations. Methods: three products were studied: Fungizon™, and two Fungizon™ /Lipofundin™ admixtures, which were diluted through two methods: in the first one, Fungizon™ was previously diluted with water for injection and then, in Lipofundin™ (AmB-DAL); the second method consisted of a primary dilution of AmB-D as a powder in the referred emulsion (AmB-DL). For the in vitro assay, two cell models were used: Red Blood Cells (RBC) from human donors and Candida tropicallis (Ct). The in vitro evaluation (K+ leakage, hemoglobin leakage and cell survival rate-CSR) was performed at four AmB concentrations (from 50 to 0.05mg.L-1). Results: The results showed that the action of AmB was not only concentration dependent, but also cellular type and vehicle kind dependent. At AmB concentrations of 50 mg.L-1, although the hemoglobin leakage for AmB-D was almost complete (99.51), for AmB-DAL and AmB-DL this value tended to zero. The p = 0.000 showed that AmB-D was significantly more hemolytic. Conclusion: The Fungizon™- Lipofundin™ admixtures seem to be the more valuable AmB carrier systems due to their best therapeutic index presented

Use of green fluorescent protein-based reporters to monitor balanced production of antifungal compounds in the biocontrol agent Pseudomonas fluorescens CHA0.

AIMS: To develop reporter constructs based on stable and unstable variants of the green fluorescent protein (GFP) for monitoring balanced production of antifungal compounds that are crucial for the capacity of the root-colonizing Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soil-borne pathogenic fungi. METHODS AND RESULTS: Pseudomonas fluorescens CHA0 produces the three antifungal metabolites 2,4-diacetylphloroglucinol (DAPG), pyoluteorin (PLT) and pyrrolnitrin (PRN). The gfp[mut3] and gfp[AAV] reporter genes were fused to the promoter regions of the DAPG, PLT and PRN biosynthetic genes. The reporter fusions were then used to follow the kinetics of expression of the three antifungal metabolites in a microplate assay. DAPG and PLT were found to display an inverse relationship in which each metabolite activates its own biosynthesis while repressing the synthesis of the other metabolite. PRN appears not to be involved in this balance. However, the microbial and plant phenolic metabolite salicylate was found to interfere with the expression of both DAPG and PLT. CONCLUSIONS: The results obtained provide evidence that P. fluorescens CHA0 may keep the antifungal compounds DAPG and PLT at a fine-tuned balance that can be affected by certain microbial and plant phenolics. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, the present study is the first to use stable and unstable GFP variants to study antibiotic gene expression in a biocontrol pseudomonad. The developed reporter fusions will be a highly valuable tool to study in situ expression of this bacterial biocontrol trait on plant roots, i.e. at the site of pathogen suppression.

Characterization of PhlG, a hydrolase that specifically degrades the antifungal compound 2,4-diacetylphloroglucinol in the biocontrol agent Pseudomonas fluorescens CHA0.

The potent antimicrobial compound 2,4-diacetylphloroglucinol (DAPG) is a major determinant of biocontrol activity of plant-beneficial Pseudomonas fluorescens CHA0 against root diseases caused by fungal pathogens. The DAPG biosynthetic locus harbors the phlG gene, the function of which has not been elucidated thus far. The phlG gene is located upstream of the phlACBD biosynthetic operon, between the phlF and phlH genes which encode pathway-specific regulators. In this study, we assigned a function to PhlG as a hydrolase specifically degrades DAPG to equimolar amounts of mildly toxic monoacetylphloroglucinol (MAPG) and acetate. DAPG added to cultures of a DAPG-negative DeltaphlA mutant of strain CHA0 was completely degraded, and MAPG was temporarily accumulated. In contrast, DAPG was not degraded in cultures of a DeltaphlA DeltaphlG double mutant. To confirm the enzymatic nature of PhlG in vitro, the protein was histidine tagged, overexpressed in Escherichia coli, and purified by affinity chromatography. Purified PhlG had a molecular mass of about 40 kDa and catalyzed the degradation of DAPG to MAPG. The enzyme had a kcat of 33 s(-1) and a Km of 140 microM at 30 degrees C and pH 7. The PhlG enzyme did not degrade other compounds with structures similar to DAPG, such as MAPG and triacetylphloroglucinol, suggesting strict substrate specificity. Interestingly, PhlG activity was strongly reduced by pyoluteorin, a further antifungal compound produced by the bacterium. Expression of phlG was not influenced by the substrate DAPG or the degradation product MAPG but was subject to positive control by the GacS/GacA two-component system and to negative control by the pathway-specific regulators PhlF and PhlH.

Silver nanoparticles: influence of stabilizing agent and diameter on antifungal activity against Candida albicans and Candida glabrata biofilms

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Alterations in growth and branching of Neurospora crassa caused by sub-inhibitory concentrations of antifungal agents

Six antifungal agents at subinhibitory concentrations were used for investigating their ability to affect the growth and branching in Neurospora crassa. Among the antifungals herein used, the azole agent ketoconazole at 0.5 mu g/ml inhibited radial growth more than fluconazole at 5.0 mu g/ml while amphotericin B at 0.05 mu g/ml was more effective than nystatin at 0.05 mu g/ml. Morphological alterations in hyphae were observed in the presence of griseofulvin, ketoconazole and terbinafine at the established concentrations. The antifungal agents were more effective on vegetative growth than on conidial germination. Terbinafine markedly reduced growth unit length (GU) by 54.89%, and caused mycelia to become hyperbranched. In all cases, there was a high correlation between hyphal length and number of tips (r > 0.9). All our results showed highly significant differences by ANOVA, (p < 0.001, alpha = 0.05). Considering that the hyphal tip is the main interface between the fungus and its environment/through which enzymes and toxins are secreted and nutrients absorbed, it would not be desirable to obtain a hyperbranched mycelia with inefficient doses of antifungal drugs.

The "in vitro" antifungal activity evaluation of propolis G12 ethanol extract on Cryptococcus neoformans

Cryptococcosis is a worldwide disease caused by the etiological agent Cryptococcus neoformans. It affects mainly immunocompromised humans. It is relatively rare in animals only affecting those that have received prolonged antibiotic therapy. The propolis is a resin that can present several biological properties, including antibacterial, antifungal and antiviral activities. The standard strain C. neoformans ATTC 90112 was used to the antifungal evaluation. The tests were realized with propolis ethanol extract (PEE) G12 in concentrations from 0.1 to 1.6 mg mL-1. The evaluation of MIC and MFC were done according to DUARTE (2002)5. The inhibitory effect of PEE G12 on the fungal growing was seen at the concentration of 0.2 mg mL-1 and 1.6 mg mL-1 was considered a fungicidal one.

ANTIFUNGAL ACTIVITY OF SILVER NANOPARTICLES OBTAINED BY GREEN SYNTHESIS

Silver nanoparticles (AgNPs) are metal structures at the nanoscale. AgNPs have exhibited antimicrobial activities against fungi and bacteria; however synthesis of AgNPs can generate toxic waste during the reaction process. Accordingly, new routes using non-toxic compounds have been researched. The proposal of the present study was to synthesize AgNPs using ribose as a reducing agent and sodium dodecyl sulfate (SDS) as a stabilizer. The antifungal activity of these particles against C. albicans and C. tropicalis was also evaluated. Stable nanoparticles 12.5 ± 4.9 nm (mean ± SD) in size were obtained, which showed high activity against Candida spp. and could represent an alternative for fungal infection treatment.