Effect of Aggregation of Amphotericin B on Lysophosphatidylcholine Micelles as Related to Its Complex Formation with Cholesterol or Ergosterol

Kawabata, Manami; Onda, Maki; Mita, Tomoyoshi

doi:10.1093/oxfordjournals.jbchem.a002912

Cited by 15 publications

(13 citation statements)

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“…Since AMB-exposed biofilms maintain metabolic activity (see above), this change in biofilm color was unlikely to reflect a loss of viability of the biofilm cells following exposure to AMB. AMB is yellow with absorption maxima around 390, 415 (monomeric), 365, and 336 nm (dimeric or aggregate) in organic or aqueous solutions (33,66), suggesting that the yellow color of the biofilm might reflect binding of AMB to the biofilm. Since AMB is soluble in DMSO, AMB-exposed and control biofilms were subjected to two rounds of 100% DMSO extraction, and the resulting extracts were scanned for UV-visible spectral properties (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Interaction of Candida albicans Biofilms with Antifungals: Transcriptional Response and Binding of Antifungals to Beta-Glucans

Vediyappan

Rossignol

d’Enfert

2010

Antimicrob Agents Chemother

171

117

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Candida albicans can form biofilms that exhibit elevated intrinsic resistance to various antifungal agents, in particular azoles and polyenes. The molecular mechanisms involved in the antifungal resistance of biofilms remain poorly understood. We have used transcript profiling to explore the early transcriptional responses of mature C. albicans biofilms exposed to various antifungal agents. Mature C. albicans biofilms grown under continuous flow were exposed for as long as 2 h to concentrations of fluconazole (FLU), amphotericin B (AMB), and caspofungin (CAS) that, while lethal for planktonic cells, were not lethal for biofilms. Interestingly, FLU-exposed biofilms showed no significant changes in gene expression over the course of the experiment. In AMB-exposed biofilms, 2.7% of the genes showed altered expression, while in CAS-exposed biofilms, 13.0% of the genes had their expression modified. In particular, exposure to CAS resulted in the upregulation of hypha-specific genes known to play a role in biofilm formation, such as ALS3 and HWP1. There was little overlap between AMB-or CAS-responsive genes in biofilms and those that have been identified as AMB, FLU, or CAS responsive in C. albicans planktonic cultures. These results suggested that the resistance of C. albicans biofilms to azoles or polyenes was due not to the activation of specific mechanisms in response to exposure to these antifungals but rather to the intrinsic properties of the mature biofilms. In this regard, our study led us to observe that AMB physically bound C. albicans biofilms and beta-glucans, which have been proposed to be major constituents of the biofilm extracellular matrix and to prevent azoles from reaching biofilm cells. Thus, enhanced extracellular matrix or beta-glucan synthesis during biofilm growth might prevent antifungals, such as azoles and polyenes, from reaching biofilm cells, thus limiting their toxicity to these cells and the associated transcriptional responses.

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Section: Resultsmentioning

confidence: 99%

“…AMB was removed by centrifugation (4,000 rpm, 45 s), and the pellets were washed twice each with 1 volume of water. Polymer-bound AMB was extracted once with 1 volume (0.5 ml) of 100% DMSO, and the extracts were scanned for absorption spectra using a spectrophotometer (33,66).…”

Section: Methodsmentioning

confidence: 99%

Interaction of Candida albicans Biofilms with Antifungals: Transcriptional Response and Binding of Antifungals to Beta-Glucans

Vediyappan

Rossignol

d’Enfert

2010

Antimicrob Agents Chemother

171

117

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“…Amphotericins A and B normally sediment with the mycelial fraction of producing cultures. Aggregation and low water-solubility increase the toxicity of amphotericin B (Kawabata et al, 2001).…”

Section: Analysis Of Products Made By M57 Mutantmentioning

confidence: 99%

Analysis and manipulation of amphotericin biosynthetic genes by means of modified phage KC515 transduction techniques

Carmody

Byrne

Murphy

et al. 2004

Gene

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Amphotericin B is a medically important antifungal antibiotic that is produced by Streptomyces nodosus. Genetic manipulation of this organism has led to production of the first amphotericin analogues by engineered biosynthesis. Here, these studies were extended by sequencing the chromosomal regions flanking the amphotericin polyketide synthase genes, and by refining the phage KC515 transduction method for disruption and replacement of S. nodosus genes. A hybrid vector was constructed from KC515 DNA and the Escherichia coli plasmid pACYC177. This vector replicated as a plasmid in E. coli and the purified DNA yielded phage plaques on Streptomyces lividans after polyethylene glycol (PEG)-mediated transfection of protoplasts. The left flank of the amphotericin gene cluster was found to include amphRI, RII, RIII and RIV genes that are similar to regulatory genes in other polyene biosynthetic gene clusters. One of these regulatory genes, amphRI, was found to have a homologue, amphRVI, located in the right flank at a distance of 127 kbp along the chromosome. However, disruption of amphRVI using the hybrid vector had no effect on the yield of amphotericin obtained from cultures grown on production medium. The hybrid vector was also used for precise deletion of the DNA coding for two modules of the AmphC polyketide synthase protein. Analysis by UV spectrophotometry revealed that the deletion mutant produced a novel pentaene, with reduced antifungal activity but apparently greater water-solubility than amphotericin B. This shows the potential for use of the new vector in engineering of this and other biosynthetic pathways in Streptomyces.

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“…Although the positions and intensities of the band may vary, the ratio of the intensities of absorption at the lowest (around 346): the highest (around 409) wavelength may be taken as a measure of the level of aggregation. This ratio is reported to be very low, (<0.25) for unaggregated or monomeric form and as high as 2.0 for highly aggregated form (17). The ratio of absorptivities at the lower and higher wavelengths for Amphotericin B in methanol and in the diluted AmB-ME were found to be 0.22 and 2.4 respectively, leading to the conclusion that the drug in microemulsion exists in aggregated form.…”

Section: Resultsmentioning

confidence: 98%

Formulation and Evaluation of Microemulsion Based Delivery System for Amphotericin B

2008

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Abstract. The present studies were designed to develop a formulation of amphotericin B in a lipid-based preparation as a microemulsion and to compare its toxicity with the commercial formulation Fungizone \ . The final product developed is a lyophilized amphotericin B, oil and surfactant blend for reconstitution in water to yield a microemulsion containing 5 mg/ml of the drug. Pseudoternary phase diagrams were constructed to identify areas of existence of microemulsion composed of Peceol \ (glyceryl monooleate) as oil phase and Mys 40 \ (polyethylene glycol 40 stearate) and Solutol HS 15 \ (polyethylene glycol 15 hydroxy stearate) as surfactants. Amphotericin B was co-evaporated with oil -surfactant mixture to produce a microemulsion pre-concentrate. The co-evaporate was diluted in water, filtered for sterilization and lyophilized to obtain the final product. The lyophilized as well as the reconstituted products were separately studied for stability and the latter was also characterized for various physicochemical aspects including droplet size of the dispersed phase, osmolarity and aggregation state of drug. The dispersion showed no evidence of precipitation of drug for 48 h, and resisted destabilization due to freeze-thaw cycles or centrifugation. The dispersed phase globules measured a mean size of 84 nm and uvspectrophotometric studies indicated the presence of self-aggregated amphotericin B. The present formulation showed a 92% decrease in haemolysis of human RBC in vitro when compared with the commercially available Fungizone \ . The LD 50 in mice was estimated to be 3.4 mg/kg. The results indicate that the formulation holds promise for development as a safer and efficacious alternative for amphotericin B therapy.

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Effect of Aggregation of Amphotericin B on Lysophosphatidylcholine Micelles as Related to Its Complex Formation with Cholesterol or Ergosterol

Cited by 15 publications

References 0 publications

Interaction of Candida albicans Biofilms with Antifungals: Transcriptional Response and Binding of Antifungals to Beta-Glucans

Interaction of Candida albicans Biofilms with Antifungals: Transcriptional Response and Binding of Antifungals to Beta-Glucans

Analysis and manipulation of amphotericin biosynthetic genes by means of modified phage KC515 transduction techniques

Formulation and Evaluation of Microemulsion Based Delivery System for Amphotericin B

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