Azoles de antes y ahora: una revisión / Azoles of then and now: a review

Resumen Los azoles son fármacos que inhiben la enzima 14α-esteroldemetilasa, impidiendo la unión de ergosterol; esto altera la estructura y función de la pared celular fúngica. Especialmente el grupo de los triazoles: fluconazol, itraconazol, voriconazol, posaconazol e isavuconazol, son una alternativa farmacológica para el tratamiento de la enfermedad fúngica invasora causada por Aspergillus spp, Candida spp, Cryptococcus spp, patógenos emergentes como los Mucorales, y de micosis endémicas como las ocasionadas por Histoplasma spp y Coccidioides spp. Los efectos adversos de los triazoles son menos frecuentes comparados con los ocasionados por anfotericina B, un antifúngico de uso común para estas micosis. Los principales efectos adversos de los triazoles son hepáticos, gastrointestinales y cardiovasculares como la prolongación del intervalo QT. Las interacciones farmacológicas son usuales y se presentan con moléculas que usan sustratos del citocromo CYP3A4, lo que incluye anti-retrovirales, anti-tuberculosos e inmunomoduladores. En este trabajo se revisan la historia, características farmacológicas y los ensayos clínicos que evidencian su eficacia clínica en los diferentes escenarios clínicos.

Abstract The azoles are drugs that inhibit the 14α-sterol-demethylase enzyme preventing the binding of ergosterol, altering the functionality and structure of the fungal cell wall. Especially the group of triazoles: fluconazole, itraconazole, voriconazole, posaconazole and isavuconazole, are a pharmacological alternative for the treatment of the invasive fungal disease, caused by Aspergillus spp, Candida spp, Cryptococcus spp, by emerging pathogens for example, the Mucoral and finally of endemic mycosis as those caused by Histoplasma spp. and Coccidioides spp. The adverse effects of the triazoles are less frequent compared to those caused by amphotericin B, the main ones being hepatics, gastrointestinals and cardiovasculars, such as the prolongation of the QT interval. The pharmacological interactions are common and occur with molecules that use the substrates of the CYP3A4 cytochrome, for example: antiretroviral, anti-tuberculous and immunomodulators. The history, pharmacological characteristics and clinical trials are reviewed.

A new coumarin derivative, 4-acetatecoumarin, with antifungal activity and association study against Aspergillus spp

Abstract Fungal infections have become a concern for health professionals, and the emergence of resistant strains has been reported for all known classes of antifungal drugs. Among the fungi causing disease, we highlight those that belong to the genus Aspergillus. For these reasons, the search for new antifungals is important. This study examines the effects of a coumarin derivative, 4-acetatecoumarin (Cou-UMB16) both alone and together with antifungal drugs, and its mode of action against Aspergillus spp. Cou-UMB16 was tested to evaluate its effects on mycelia growth, and germination of Aspergillus spp. fungal conidia. We investigated its possible action on cell walls, on the cell membrane, and also the capacity of this coumarin derivative to enhance the activity of antifungal drugs. Our results suggest that Cou-UMB16 inhibits Aspergillus spp. virulence factors (mycelia growth and germination of conidia) and affects the structure of the fungal cell wall. When applying Cou-UMB16 in combination with azoles, both synergistic and additive effects were observed. This study concludes that Cou-UMB16 inhibits mycelial growth and spore germination, and that the activity is due to its action on the fungal cell wall, and that Cou-UMB16 could act as an antifungal modifier.

In vitro anti-tuberculosis activity of azole drugs against Mycobacterium tuberculosis clinical isolates / Actividad in vitro de azoles contra aislamientos clínicos de Mycobacterium tuberculosis

Background: Latent tuberculosis has been associated with the persistence of dormant Mycobacterium tuberculosis in the organism of infected individuals, who are reservoirs of the bacilli and the source for spreading the disease in the community. New active anti-TB drugs exerting their metabolic action at different stages and on latent/dormant bacilli are urgently required to avoid endogenous reactivations and to be part of treatments of multi- and extensively-drug resistant tuberculosis (M/XDR-TB). It was previously reported that azole drugs are active against M. tuberculosis. For that reason, the aims of this study were to determine the in vitro activity of azole drugs, imidazole (clotrimazole, CLO and econazole, ECO) and nitroimidazole (metronidazole, MZ and ipronidazole, IPZ), against a collection of MDR M. tuberculosis clinical isolates; and to analyze their potential use in both the LTB and the active forms of M/XDR-TB treatments. Methods: A total of 55 MDR M. tuberculosis isolates and H37Rv were included. MZ and IPZ activity against M. tuberculosis isolates were tested using anaerobic culture conditions. The activity of ECO and CLO was measured by the minimal inhibitory concentration (MIC) using a microdilution colorimetric method. Results: MZ and IPZ showed bacteriostatic activity against M. tuberculosis strains. MIC5o and MIC90 to ECO was 4.0 µg/ml, while MIC50 to CLO was 4.0 µg/ml and MIC90 was 8.0 µg/ml respectively. Conclusion: All azole compounds tested in the study showed inhibitory activity against MDR M. tuberculosis clinical isolates.

Introducción: La tuberculosis (TB) latente ha sido asociada a la persistencia de Mycobacterium tuberculosis durmientes en el organismo de las personas infectadas, las cuales constituyen un reservorio del bacilo y una fuente de diseminación de la enfermedad en la comunidad. Urge la necesidad de contar con nuevos fármacos antituberculosos con acción sobre el bacilo en estado latente/durmiente, a fin de evitar reactivaciones endógenas y para ser incluidas en el tratamiento de la TB multirresistente y extensivamente resistente (M/XDR-TB). Se ha reportado que los azoles son activos contra M. tuberculosis. Por esta razón, los objetivos del presente estudio fueron determinar la actividad in vitro sobre aislamientos clínicos de M/XDR-TB de distintos azoles, incluyendo los imidazoles econazol (ECO) y clotrimazol (CLO) y los 5-nitro-imidazoles ipronidazol (IPZ) y metronidazol (MZ), así como analizar su potencial uso contra las formas latente y activa de esta enfermedad. Métodos: Fueron incluidos 55 aislamientos clínicos de M. tuberculosis MDR y la cepa de referencia H37Rv. Se evaluó la actividad del MZ y el IPZ sobre los aislamientos en condiciones de cultivo anaeróbico, mientras que la actividad del ECO y el CLO fue estimada determinando la concentración inhibitoria mínima (CIM) mediante el método colorimétrico de microdilución en placa. Resultados: El MZ y el IPZ presentaron actividad bacteriostática frente a las cepas de M. tuberculosis. La CIM50 y CIM90 del ECO fue de 4 µg/ml, mientras que el CLO presentó una CIM50 de 4 µg/ml y una CIM90 de 8 µg/ml. Conclusión: Todos los compuestos azólicos evaluados presentaron actividad inhibitoria frente a aislamientos clínicos de M. tuberculosis.

Extracellular phospholipase production of oral Candida albicans isolates from smokers, diabetics, asthmatics, denture wearers and healthy individuals following brief exposure to polyene, echinocandin and azole antimycotics

Abstract Objective Candida albicans is the primary causative agent of oral candidosis, and one of its key virulent attributes is considered to be its ability to produce extracellular phospholipases that facilitate cellular invasion. Oral candidosis can be treated with polyenes, and azoles, and the more recently introduced echinocandins. However, once administered, the intraoral concentration of these drugs tend to be sub-therapeutic and rather transient due to factors such as the diluent effect of saliva and cleansing effect of the oral musculature. Hence, intra-orally, the pathogenic yeasts may undergo a brief exposure to antifungal drugs. We, therefore, evaluated the phospholipase production of oral C. albicans isolates following brief exposure to sub-therapeutic concentrations of the foregoing antifungals. Materials and methods Fifty C. albicans oral isolates obtained from smokers, diabetics, asthmatics using steroid inhalers, partial denture wearers and healthy individuals were exposed to sub-therapeutic concentrations of nystatin, amphotericin B, caspofungin, ketoconazole and fluconazole for one hour. Thereafter the drugs were removed and the phospholipase production was determined by a plate assay using an egg yolk-agar medium. Results The phospholipase production of these isolates was significantly suppressed with a percentage reduction of 10.65, 12.14, 11.45 and 6.40% following exposure to nystatin, amphotericin B, caspofungin and ketoconazole, respectively. This suppression was not significant following exposure to fluconazole. Conclusions Despite the sub-therapeutic, intra oral, bioavailability of polyenes, echinocandins and ketoconazole, they are likely to produce a persistent antifungal effect by suppressing phospholipase production, which is a key virulent attribute of this common pathogenic yeast.

Novel point mutations in the ERG11 gene in clinical isolates of azole resistant Candida species

The azoles are the class of medications most commonly used to fight infections caused by Candida sp. Typically, resistance can be attributed to mutations in ERG11 gene (CYP51) which encodes the cytochrome P450 14α-demethylase, the primary target for the activity of azoles. The objective of this study was to identify mutations in the coding region of theERG11 gene in clinical isolates of Candidaspecies known to be resistant to azoles. We identified three new synonymous mutations in the ERG11 gene in the isolates of Candida glabrata (C108G, C423T and A1581G) and two new nonsynonymous mutations in the isolates of Candida krusei - A497C (Y166S) and G1570A (G524R). The functional consequence of these nonsynonymous mutations was predicted using evolutionary conservation scores. The G524R mutation did not have effect on 14α-demethylase functionality, while the Y166S mutation was found to affect the enzyme. This observation suggests a possible link between the mutation and dose-dependent sensitivity to voriconazole in the clinical isolate of C. krusei. Although the presence of the Y166S in phenotype of reduced azole sensitivity observed in isolate C. kruseidemands investigation, it might contribute to the search of new therapeutic agents against resistant Candida isolates.

Synergistic effects of tacrolimus and azole antifungal compounds in fluconazole-susceptible and fluconazole-resistant Candida glabrata isolates

In vitro interaction between tacrolimus (FK506) and four azoles (fluconazole, ketoconazole, itraconazole and voriconazole) against thirty clinical isolates of both fluconazole susceptible and -resistant Candida glabrata were evaluated by the checkerboard microdilution method. Synergistic, indifferent or antagonism interactions were found for combinations of the antifungal agents and FK506. A larger synergistic effect was observed for the combinations of FK506 with itraconazole and voriconazole (43%), followed by that of the combination with ketoconazole (37%), against fluconazole-susceptible isolates. For fluconazole-resistant C. glabrata, a higher synergistic effect was obtained from FK506 combined with ketoconazole (77%), itraconazole (73%), voriconazole (63%) and fluconazole (60%). The synergisms that we observed in vitro, notably against fluconazole-resistant C. glabrata isolates, are promising and warrant further analysis of their applications in experimental in vivo studies.

Chloroquine sensitizes biofilms of Candida albicans to antifungal azoles

Biofilms formed by Candida albicans, a human pathogen, are known to be resistant to different antifungal agents. Novel strategies to combat the biofilm associated Candida infections like multiple drug therapy are being explored. In this study, potential of chloroquine to be a partner drug in combination with four antifungal agents, namely fluconazole, voriconazole, amphotericin B, and caspofungin, was explored against biofilms of C. albicans. Activity of various concentrations of chloroquine in combination with a particular antifungal drug was analyzed in a checkerboard format. Growth of biofilm in presence of drugs was analyzed by XTT-assay, in terms of relative metabolic activity compared to that of drug free control. Results obtained by XTT-metabolic assay were confirmed by scanning electron microscopy. The interactions between chloroquine and four antifungal drugs were determined by calculating fractional inhibitory concentration indices. Azole resistance in biofilms was reverted significantly (p < 0.05) in presence of 250 µg/mL of chloroquine, which resulted in inhibition of biofilms at very low concentrations of antifungal drugs. No significant alteration in the sensitivity of biofilms to caspofungin and amphotericin B was evident in combination with chloroquine. This study for the first time indicates that chloroquine potentiates anti-biofilm activity of fluconazole and voriconazole.

Proteomic analysis of cytosolic proteins associated with petite mutations in Candida glabrata

The incidence of superficial or deep-seated infections due to Candida glabrata has increased markedly, probably because of the low intrinsic susceptibility of this microorganism to azole antifungals and its relatively high propensity to acquire azole resistance. To determine changes in the C. glabrata proteome associated with petite mutations, cytosolic extracts from an azole-resistant petite mutant of C. glabrata induced by exposure to ethidium bromide, and from its azole-susceptible parent isolate were compared by two-dimensional polyacrylamide gel electrophoresis. Proteins of interest were identified by peptide mass fingerprinting or sequence tagging using a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer. Tryptic peptides from a total of 160 Coomassie-positive spots were analyzed for each strain. Sixty-five different proteins were identified in the cytosolic extracts of the parent strain and 58 in the petite mutant. Among the proteins identified, 10 were higher in the mutant strain, whereas 23 were lower compared to the parent strain. The results revealed a significant decrease in the enzymes associated with the metabolic rate of mutant cells such as aconitase, transaldolase, and pyruvate kinase, and changes in the levels of specific heat shock proteins. Moreover, transketolase, aconitase and catalase activity measurements decreased significantly in the ethidium bromide-induced petite mutant. These data may be useful for designing experiments to obtain a better understanding of the nuclear response to impairment of mitochondrial function associated with this mutation in C. glabrata.

Species-Specific Differences in Rhodamine 6G Accumulation of Candida Isolates Detected by Flow Cytometric Analysis / 대한진단검사의학회지

BACKGROUND: Fluorescent dye Rhodamine 6G (R6G) is a substrate of multidrug resistance pumps and its accumulation is reduced in some azole-resistant Candida isolates with the upregulation of multidrug efflux transporter genes. Despite reports on species-specific differences in azole susceptibility in various Candida species, only a few studies have been reported on the R6G accumulation among clinical isolates of Candida species. In this study, we compared R6G accumulation between six different Candida species. METHODS: The intracellular accumulation of R6G and minimal inhibitory concentrations (MICs) of three triazole agents were investigated in 48 strains of six Candida species (14 C. albicans, 9 C. tropicalis, 8 C. glabrata, 8 C. krusei, 7 C. parapsilosis, and 2 C. haemulonii). R6G accumulation was measured by using flow cytometry and the geometric mean of the fluorescence intensity (GMF) was used to compare the accumulation between the Candida isolates. RESULTS: The GMF values for the C. tropicalis, C. albicans, C. krusei, C. parapsilosis, and C. glabrata isolates were 167.3+/-18.5, 126.9+/-6.6, 88.5+/-18.5, 50.8+/-7.0, and 38.1+/-3.9, respectively. C. glabrata had a significantly lower mean GMF than all the other Candida species (P<0.05). While some Candida strains with trailing growth phenomenon and increased fluconazole MIC did not have a reduced GMF, three Candida strains with increased MICs to all three triazole agents had a reduced GMF. CONCLUSIONS: This study found species-specific differences in R6G accumulation in Candida. In addition, the intracellular R6G accumulation can be used to investigate the drug efflux mechanism in azole-resistant Candida strains.

Antimicóticos en niños / Antifungals in Children

Las micosis superficiales son padecimientos frecuentes en la infancia y para su manejo se pueden utilizar antimicóticos tanto tópicos como sistémicos. Sin embargo, en las últimas décadas, ha aumentado la población infantil susceptible a infecciones diseminadas o sistémicas por agentes oportunistas que ponen en riesgo la vida. Las principales son la candidosis y la aspergilosis. Se han desarrollado nuevos antimicóticos con espectros más amplios de acción y menor toxicidad, pero la mayoría de los reportes en la literatura se refieren a estudios hechos en adultos, cuyos resultados son extrapolados a la población pediátrica. La presente revisión tiene como objetivo condensar la información referente al uso de antimicóticos en niños, con énfasis en las diferencias farmacocinéticas con respecto a los adultos y las indicaciones principales para su uso (AU)

Superficial mycoses are common in childhood, either topical or systemic antifungals can be used for treatment. However, in the past decade, the pediatric population at risk of a disseminated or systemic infection by opportunistic fungi has increased. The most important are candidiasis and aspergillosis. New antifungals, with a wider spectrum of action and less toxicity have been developed, nevertheless, most of the studies and reports of the literature focus on adults and the findings are extrapolated to children. The objective of this paper is to review what has been published on the use of antifungals in the pediatric age, focusing in pharmacokinetic differences regarding adults and current indications for this group of drugs (AU)

Species Distribution and Susceptibility to Azole Antifungals of Candida Bloodstream Isolates from Eight University Hospitals in Korea

PURPOSE: The incidence of Candida bloodstream infections (BSI) has increased over the past two decades. The rank order of occurrence and the susceptibility to antifungals of the various Candida species causing BSI are important factors driving the establishment of empirical treatment protocols; however, very limited multi-institutional data are available on Candida bloodstream isolates in Korea. MATERIALS AND METHODS: We investigated the susceptibility to azole antifungals and species distribution of 143 Candida bloodstream isolates recovered from eight university hospitals over a six-month period. Minimal inhibitory concentrations (MICs) of fluconazole, itraconazole, and voriconazole for each isolate were determined by the broth microdilution method of the Clinical and Laboratory Standards Institute (CLSI). RESULTS: The Candida species recovered most frequently from the blood cultures was C. albicans (49%), followed by C. parapsilosis (22%), C. tropicalis (14%), and C. glabrata (11%). The MIC ranges for the Candida isolates were 0.125 to 64microgram/mL for fluconazole, 0.03 to 2microgram/mL for itraconazole, and 0.03 to 1microgram/mL for voriconazole. Overall, resistance to fluconazole was found in only 2% of the Candida isolates (3/143), while the dose-dependent susceptibility was found in 6% (8/143). The resistance and dose-dependent susceptibility of itraconazole were found in 4% (6/143) and 14% (20/143) of the isolates, respectively. All bloodstream isolates were susceptible to voriconazole (MIC, < or = 1microgram/mL). CONCLUSION: Our findings show that C. albicans is the most common cause of Candida-related BSI, followed by C. parapsilosis, and that the rates of resistance to azole antifungals are still low among bloodstream isolates in Korea.

Antimycotic effects of some antibiotics on the growth of some dermatophytes and other keratin degrading fungi.

Four antibiotics have been tested against the growth of some dermatophytes and keratin degrading fungi. A gradual decrease in growth was observed with increase in concentration of all antibiotics. All, but griseofulvin observed to inhibit > 50% mycelial weight even at a lower concentration of 50 ppm. Azole derivatives were most toxic to the growth of M. gypseum at all concentrations, whereas, to that of C. tropicum at above 100 ppm. Mycostatin was the most toxic antibiotic to the growth of M. gypsea at all concentrations.