Activity Of Amphotericin Research Articles

A newly identified derivative of amphotericin B: isolation, structure determination and primary evaluation of the activity and toxicity

A new derivative of amphotericin B named amphotericin B(5) has been obtained under the following condition: amphotericin B powder was first dissolved in N,N-dimethylformamide at room temperature to obtain a solution of concentration of 2.0 mg ml(-1). Then, the solution was kept at 60 °C in an electric-heated thermostatic water bath for 48-60 h for the purpose of accelerating the degradation. Amphotericin B(5) was isolated by preparative HPLC. Its structure has been identified by means of UV, NMR and LCMS-IT-TOF as (1R, 3S, 5R, 6R, 9R, 11R, 15S, 16R, 17R, 18S, 19E, 21E, 23E, 25E, 27E, 29E, 31E, 33R, 35S, 36R, 37S)-33-[3-form-amide-3,6-dideoxy-β--mannopy-ran-osyl)oxy]-1,3,5,6,9,11,17,37-octahydroxy-15,16,18-trimethyl-13-oxo-14,39-di-oxabicyclo [33.3.1] nonatriaconta-19,21,23,25,27,29,31-heptaene-36-carboxylic acid named according to the International Union of Pure and Applied Chemistry. The activity of amphotericin B(5) was tested against Saccharomyces cerevisiae based on microbiological potency using cylinder-plate method, and the toxicities of amphotericin B and amphotericin B(5) were determined simultaneously in zebrafish embryo. The results showed that the toxicity of amphotericin B(5), as revealed by its LD(50) value, was lower than amphotericin B.

Antifungal Activity of Essential Oils and Their Constituents against <i>Candida</i> Spp. and Their Effects on Activity of Amphotericin B

Candidiasis is a common opportunistic fungal infection that responds well to amphotericin B (AMPH) treatment. However, AMPH often causes adverse effects such as kidney injury and hypokalemia. Because some essential oils have been reported to have antifungal effects, we investigated the antifungal activity of various essential oils and their major constituents against Candida spp. Most essential oils examined in this study showed antifungal activity, and several enhanced the antifungal effect of AMPH. Clove oil in particular, and its major constituent eugenol, had potent effects. These findings suggest that combining certain essential oils or their constituents with AMPH may be useful for suppressing the adverse effects of AMPH treatment.

In vitro combined activity of amphotericin B, caspofungin and voriconazole against clinical isolates of Trichosporon asahii

Disseminated infections caused by Trichosporon asahii are difficult to resolve. Combination regimens with synergistic drugs could provide additional options for treating trichosporonosis. The aim of this study was to evaluate the antifungal activities of voriconazole (VCZ), caspofungin (CAS) and amphotericin B (AMB) alone or in combination in vitro against clinical isolates of T. asahii. The combined antifungal activities of VCZ, CAS and AMB against 18 clinical isolates were assessed by a chequerboard microdilution method. CAS combined with AMB showed the highest percentage of synergistic effects (89%), much higher than those of the other two combinations (AMB/VCZ and CAS/VCZ both 17%). No antagonistic effect was observed in any case. This study demonstrates that the activity of two combined antifungals, especially the combination of CAS and AMB, against T. asahii is more effective than that of a drug alone against this fungus, suggesting that combined antifungal therapy may be a potential strategy for treating disseminated trichosporonosis.

A sensitive method for thin-layer chromatographic detection of amphotericin B

Thin-layer chromatography with microbiological detection (direct bioautography) of amphotericin B has never been reported. The combination of these methods can be used advantageously, especially when not only chemical identification of samples is required, but also when antifungal activity is of interest. In this paper a fast and easy-to-perform method is introduced in which major ( R F 0.46) and minor ( R F 0.31) components can be separated from amphotericin B, which itself is not a homogenous substance but mixture of polyenes. Thin-layer chromatography is performed on silica gel layers with chloroform-methanol-borate buffer 4:5:1 ( v/v ) as optimized mobile phase, and the microbiological activity of amphotericin B can be measured sensitively by direct bioautography. Candida albicans (ATCC 90028) and Saccharomyces cerevisiae (ATCC 9763) fungus strains were tested. Among the detection methods investigated, direct bioautography with Candida albicans proved to be the most sensitive, with a detection limit o...

Enhancement of the Fungicidal Activity of Amphotericin B by Allicin: Effects on Intracellular Ergosterol Trafficking

In Saccharomyces cerevisiae, ergosterol was visible in the plasma membrane of untreated cells and was enriched in the vacuole membrane in response to amphotericin B (AmB) treatment at a non-lethal concentration. The simultaneous addition of allicin was inhibitory to AmB-induced ergosterol enrichment in the vacuole membrane, resulting in increased sensitivity of the organelles to the disruptive action of AmB. Allicin was also inhibitory to ergosterol enrichment in the vacuole membrane that was achieved by its external addition to cells. The combined fungicidal activity of AmB and allicin was suppressed together with suppression of vacuole membrane damage in cells where ergosterol had been fully enriched in the vacuole membrane. AmB caused direct disruptive damage of the isolated vacuoles, but the antibiotic was apparently less effective in disrupting the organelles that were isolated after ergosterol enrichment. These findings suggest that allicin enhances AmB-induced vacuole membrane damage by inhibiting ergosterol trafficking from the plasma membrane to the vacuole membrane.

In Vitro Activity of Amphotericin B Against Aspergillus terreus Isolates from Different Countries and Regions

In Vitro Activity of Amphotericin B Against Aspergillus terreus Isolates from Different Countries and Regions

Enhancement of the in vitro activity of amphotericin B against the biofilms of non-albicans Candida spp. by rifampicin and doxycycline

The in vitro activity of amphotericin B (AMB) alone and in combination with rifampicin (RIF) and doxycycline (DOX) was tested against the biofilms of 30 clinical isolates of non-albicans Candida (NAC) species namely, Candida parapsilosis, Candida krusei and Candida glabrata. The killing activity of AMB at 10 x MIC was significantly increased in combination with either antibiotic. With RIF, the killing activity increased by 20.6, 23.5 and 14 % against the biofilms of C. parapsilosis, C. krusei and C. glabrata, respectively; with DOX, the killing activity increased by 30.64, 35.28 and 31.13 %, respectively. Pre-exposure of the isolates to the same combinations significantly reduced the number of colonized cells in the biofilms by 20, 25.14 and 13.07 % with RIF for C. parapsilosis, C. krusei and C. glabrata, respectively, and by 18.94, 24.52 and 29.15 % with DOX, respectively. The data showed that combination of RIF or DOX with AMB enhanced the killing activity of the antifungal agent against biofilms of NAC species. Whether such an effect operates against biofilm-associated infections needs to be clarified by further in vivo studies.

P1956 In vitro activity of amphotericin B and anidulafungin against Candida albicans biofilms

P1956 In vitro activity of amphotericin B and anidulafungin against Candida albicans biofilms

Antifungal resistance in Candida spp. isolated in Italy between 2002 and 2005 from children and adults

The activity of amphotericin B, fluconazole, flucytosine, itraconazole and voriconazole was tested in vitro against 618 clinical Candida spp. isolates, using the broth microdilution or the disk diffusion method (voriconazole). Amphotericin B and voriconazole were the most potent antifungal agents assayed (100% of susceptible strains). Resistance to fluconazole and itraconazole was detected in three (0.7%) and 11 (2.7%) isolates of Candida albicans and in four (3.7%) isolates of Candida glabrata. Flucytosine intermediate, resistant strains, or both, were observed in C. albicans (0.3% and 0.7%), C. glabrata (2.8% intermediate) and C. tropicalis (15.2% and 15.2%). C. krusei was the least susceptible species to azoles. No statistically significant differences in the rates of resistant isolates depending on site of infection and age of the patient were observed, with the exception of C. albicans and itraconazole (higher percentage of resistance in children). At present, acquired antifungal resistance represents an uncommon finding in most Candida spp. circulating in Northern Italy.

The effect of propyl gallate on the activity of various antifungal drugs against filamentous fungi in vitro

The influence of an antioxidant, propyl gallate (PG), on the activity of amphotericin B (AMB), terbinafine (TBF), butenafine (BTF) and ketoconazole (KCZ) against ocular pathogenic filamentous fungi in vitro was investigated to determine whether PG could increase the antifungal activity. Susceptibility tests were performed against 6 isolates of ocular pathogenic filamentous fungi (Fusarium solanae, Fusarium moniliforme, Fusarium poae, Fusarium oxysporum, Aspergillus fumigatus and Aspergillus flavus) and 2 quality control strains (Candida krusei ATCC 6258 and Cadida parapsilosis ATCC 22019) by the NCCLS M38-P broth microdilution method (MIC). PG was added to the incubation media at a final concentration of 400 microg/ml. Antifungal agents were serially two-fold diluted and final dilutions were made in 1640 and PG-1640 culture media to a concentration ranging from 0.0313 to 16 microg/ml for AMB, TBF, BTF and KCZ. One hundred microl of the corresponding diluted inoculum suspension was added to each well of the microdilution tray. The MIC end-point of AMB was determined as 100% growth reduction and the MIC end-point of TBF, BTF, KCZ and PG was determined as 75% growth reduction as compared with the turbidity produced by the control well. At a concentration of 400 microg/ml, PG did not show any antifungal activity under these experimental conditions. The combination of PG (400 microg/ml) with amphotericin B revealed a remarkably increased activity against all of the isolates of ocular pathogenic filamentous fungi and quality control strains. In the combination of PG with terbinafine, a remarkably increased activity was observed against Fusarium solanae, Fusarium poae, Fusarium oxysporum, Aspergillus fumigatu and Aspergillus flavus. The combination of PG with butenafine had remarkably synergistic effect against Fusarium solanae, but did not synergistic or even showed antagonistic effect for other isolates. The combination of PG with ketaconazole was synergistic against Fusarium solanae, but was antagonistic against all other isolates. Combination of PG and amphotericin has remarkably synergistic effect against all tested ocular pathogenic filamentous fungi isolates. Combination of PG and terbinafine has remarkably synergistic effect against some isolates. The PG-amphotericin combination and the PG-terbinafine combination may have a role in future studies of antifungal eye drops.

Evaluation of the Microdilution, Etest and Disk Diffusion Methods for Antifungal Susceptibility Testing of Clinical Strains of Trichosporon Spp.

Trichosporon spp are well recognized as pathogens capable of causing invasive disease. Despite the increasing frequency and severity of trichosporonosis, data on the antifungal susceptibility of Trichosporon spp. are limited and recommendations for in vitro testing of this fungus are not included in the guidelines of the National Committee for Clinical Laboratory Standards. The purpose of this study was to determine the in vitro susceptibility of clinical Trichosporon isolates to systemic antifungals. We evaluated the in vitro activity of amphotericin B, fluconazole, itraconazole and voriconazole against 27 clinical isolates of Trichosporon spp. (14 T. mucoides and 13 T. asahii) using NCCLS M27-A2 reference microdilution, Etest and disk diffusion methods. In the microdilution and Etest methods Trichosporon spp. demonstrated relatively high minimum inhibitory concentrations (MICs) for fluconazole (MIC90 4 and 6 μg/ml, respectively) and relatively low MICs for voriconazole (MIC90 0.125 and 0.125 μg/ml, respectively). MICs for amphotericin B determined on antibiotic medium 3 were lower (MIC90 0.06 μg/ml) than those on RPMI (MIC90 1 μg/ml). Observed agreements were 81-100% according to these drugs. Disk diffusion zone diameters correlate inversely with MICs from dilution tests except for amphotericin B. Validation of the clinical significance of these observations demands determination of MIC breakpoints for Trichosporon and in vitro- in vivo correlation studies.

Susceptibility of clinical isolates of Candida lusitaniae to five systemic antifungal agents.

The aim of the present study was to expand the MIC database for Candida lusitaniae in order to further determine its antifungal susceptibility pattern. The activities of amphotericin B, fluconazole, itraconazole, voriconazole and flucytosine were determined in vitro against 80 clinical isolates of C. lusitaniae. A set of 59 clinical isolates of Candida albicans and of 51 isolates of Candida glabrata was included to compare the susceptibilities to amphotericin B. The MICs were determined by Etest with RPMI 1640 agar, and with both this medium and antibiotic medium 3 (AM3) agar for testing of amphotericin B. All isolates were highly susceptible to fluconazole. The susceptibility to itraconazole was good; only 4% of isolates had dose-dependent susceptibility (MICs 0.25-0.5 mg/L). Voriconazole was very active in vitro (100% of isolates were inhibited at < or =0.094 mg/L). Flucytosine MICs ranged widely (0.004->32 mg/L). The set included 19% of flucytosine-resistant isolates. For amphotericin B, 100% of isolates were inhibited at < or =0.75 mg/L (MIC(50) 0.047 mg/L; MIC(90) 0.19 mg/L) and at < or =4 mg/L (MIC(50) 0.25 mg/L; MIC(90) 0.75 mg/L) on RPMI and on AM3, respectively. A single isolate was categorized as resistant to amphotericin B (MIC 0.75 and 4 mg/L on RPMI and on AM3, respectively). Amphotericin B thus appeared very active in vitro against C. lusitaniae. Whatever the test medium, the level of susceptibility of C. lusitaniae to amphotericin B did not differ much from those of C. albicans and C. glabrata. C. lusitaniae appears to be susceptible to amphotericin B, azole antifungal agents, and, to a lesser extent, flucytosine.

Effect of CpG DNA Oligodeoxynucleotides and Echinacea on the Fungicidal Activity of Amphotericin B in a Disseminated Murine Candidal Infection Model

Effect of CpG DNA Oligodeoxynucleotides and Echinacea on the Fungicidal Activity of Amphotericin B in a Disseminated Murine Candidal Infection Model

The activity of amphotericin B against Candida albicans is not directly associated with extracellular calcium concentration

The ability of amphotericin B to increase intracellular calcium concentrations in human cells is associated with the toxicity of this antifungal agent. The present study was performed to determine whether amphotericin B affects the influx or efflux of calcium in Candida albicans, and whether the antifungal activity of amphotericin B is dependent upon extracellular calcium concentrations. Concentration-response studies demonstrated that the addition of up to 1 mM EGTA to standard growth medium, with a more than 4000-fold decrease in extracellular calcium concentration, had no effect on the activity of amphotericin B against C. albicans. Amphotericin B did affect the kinetics of calcium influx acutely (< or =10 min), but had no net effect on long-term (1-24 h) calcium accumulation. Calcium efflux was also not affected by amphotericin B. These results indicate that, unlike its effects on mammalian cells, the toxicity of amphotericin B against C. albicans is not dependent upon increased movement of calcium across the cell membrane or the presence of extracellular calcium.

Histone Deacetylase Inhibitors Enhance Candida albicans Sensitivity to Azoles and Related Antifungals: Correlation with Reduction in CDR and ERG Upregulation

Histone acetylation and deacetylation play important roles in eukaryotic gene regulation. Several histone deacetylase (HDA) inhibitors have been characterized, including trichostatin A (TSA), apicidin, and sodium butyrate. We tested their effects on Candida albicans in vitro growth, heat sensitivity, and germ tube formation; minimal effects were observed. However, there was a dramatic effect of TSA on C. albicans sensitivity to the azoles fluconazole, itraconazole, and miconazole. Similar effects were observed with other HDA inhibitors and with the antifungals terbinafine and fenpropimorph, which target, as do azoles, enzymes in the ergosterol biosynthetic pathway. In contrast, HDA inhibitors had minimal effect on the activities of amphotericin B, flucytosine, and echinocandin, which have unrelated targets. Specifically, addition of 3 micro g of TSA/ml lowered the itraconazole MIC for five susceptible C. albicans isolates an average of 2.7-fold at 24 h, but this increased to >200-fold at 48 h. Thus, the primary effect of TSA was a reduction in azole trailing. TSA also enhanced itraconazole activity against Candida parapsilosis and Candida tropicalis but had no effect with four less related yeast species. To examine the molecular basis for these effects, we studied expression of ERG genes (encoding azole and terbinafine targets) and CDR/MDR1 genes (encoding multidrug transporters) in C. albicans cells treated with fluconazole or terbinafine with or without TSA. Both antifungals induced to various levels the expression of ERG1, ERG11, CDR1, and CDR2; addition of TSA reduced this upregulation 50 to 100%. This most likely explains the inhibition of azole and terbinafine trailing by TSA and, more generally, provides evidence that trailing is mediated by upregulation of target enzymes and multidrug transporters.

Clinical microbiological case: chronic disseminated candidiasis unresponsive to treatment

Clinical microbiological case: chronic disseminated candidiasis unresponsive to treatment

In vitro studies on the activity of amphotericin B and lipid-based amphotericin B formulations against Aspergillus conidia and hyphae.

The minimum inhibitory concentrations (MICs) of amphotericin B and lipid-based amphotericin B formulations against isolates of Aspergillus spp. were tested using a broth microdilution method. Twelve isolates of Aspergillus fumigatus, eight of Aspergillus flavus, six of Aspergillus niger and seven of Aspergillus terreus were examined. In addition, an assay for hyphae of Aspergillus spp. was performed since the invasive form is manifested by the appearance of hyphal structures. MICs of hyphae against lipid-based amphotericin B formulations were within three dilutions higher than those against conidia for almost all isolates of Aspergillus spp. (P < 0.01). In contrast, the differences in the in vitro efficacies of amphotericin B were the lowest for Aspergillus spp. This study demonstrates the importance of the type of inoculum used to test antifungal susceptibilities of Aspergillus spp. The significance of these results for in vivo outcome needs to be determined.

Effect of Medium Composition on Static and Cidal Activity of Amphotericin B, Itraconazole, Voriconazole, Posaconazole and Terbinafine Against Aspergillus fumigatus: A Multicenter Study

The effect of the medium composition on the fungistatic (MIC) and fungici-dal (MLC) activity of amphotericin B, itraconazole, voriconazole, posaconazole and terbinafine against four Aspergillus fumigatus strains has been investigated by four European laboratories. MICs were determined by broth microdilu-tion, using RPMI 1640 and Antibiotic Medium 3 (AM3), three times in three independent determinations by the four laboratories. MLCs were determined for the three independent determinations by the four laboratories, subculturing 100 μl from each well showing no visible growth after 48 hours. Except for a 2-dilution difference observed in three cases, no differences were observed between MICs determined on the two media. In contrast, a 3- to 6-dilution discrepancy between the MLCs was observed for the azoles. Endpoints on RPMI were higher than those on AM3. A 1-2 dilution difference was noted between both the endpoints of amphotericin B and of terbinafine. The highest inter- and intra-laboratory agreements were reached on AM3. The azoles showed a medium-dependent fungicidal activity.

Block copolymer micelles for the encapsulation and delivery of amphotericin B.

To assess the effect of fatty acid substitution of a micelle-forming poly(ethylene oxide)-block-poly(N-hexyl stearate-L-aspartamide) (PEO-b-PHSA) on the encapsulation, hemolytic properties and antifungal activity of amphotericin B (AmB). PEO-b-PHSA with three levels of stearic acid substitution were synthesized and used to encapsulate AmB by a solvent evaporation method. Size exclusion chromatography and UV spectroscopy were used to confirm and measure levels of encapsulated AmB. The hemolytic activity of encapsulated AmB toward human red blood cells and its minimum inhibitory concentration against Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans were obtained and compared to AmB alone. An increase in the level of stearic acid substitution on PEO-b-PHSA improved the encapsulation of AmB while reducing its hemolytic activity. PEO-b-PHSA micelles having 50% and 70% stearic acid substitution (mol fatty acid: mol reacted and unreacted hydroxyls) were completely non-hemolytic at 22 microg/ml. At 11% stearic acid substitution, AmB caused 50% hemolysis at 1 microg/ml. AmB in PEO-b-PHSA micelles was as effective as AmB alone against pathogenic fungi. PEO-b-PHSA micelles with a high level of stearic acid side chain substitution can effectively solubilize AmB, reduce its hemolytic activity yet retain its potent antifungal effects.

Treatment of Absidia corymbifera infection in mice with amphotericin B and itraconazole

The activities of amphotericin B and itraconazole were studied in a temporarily neutropenic murine model of disseminated Absidia corymbifera infection, caused by two different strains. Amphotericin B MICs were 0.25 mg/L for both strains and itraconazole MICs were 1 and 2 mg/L. Amphotericin B was effective in vivo with both isolates. Itraconazole was less effective.