Interaction Of Amphotericin B Research Articles

Effect of high sodium ion level on the interaction of AmB with a cholesterol-rich phospholipid monolayer.

Invasive fungal infections are a primary reason for high mortality in immunocompromised people, especially in critically ill patients, such as intensive care unit (ICU) patients, advanced cancer patients, or severe burn patients. Hypernatremia also can increase mortality in severely ill patients. Amphotericin B (AmB) is the gold standard for treating infections, but in severely ill patients, AmB can cause hematotoxicity when administered intravenously due to its interaction with cholesterol on red blood cell membranes. This results in limited doses of AmB and affects the treatment of infections. The proportion of cholesterol molecules in membrane lipids in red blood cells is as high as 50mol%, and the sodium ions can influence the interaction between AmB and lipids on the membrane. Therefore, in the complex clinical situation of a severely ill patient with a fungal infection and hypernatremia, the interaction between amphotericin B and the red blood cell membranes is worth studying in depth. In this work, the interaction between AmB and the dipalmitoyl phosphatidylcholine (DPPC)/cholesterol mixed monolayer in the presence of high sodium ion levels was studied when the proportion of cholesterol was 50%. The results show that the effect of AmB on reducing the monolayer's area at a high level of sodium ions is slightly stronger at 30mN/m. The effect of AmB on reducing the elastic modulus of the DPPC/Chol monolayer is significantly weakened by a high sodium ion level, compared with the level of sodium ions at normal physiological concentration. The higher the sodium ion concentration, the weaker the intermolecular force of the DPPC/Chol/AmB mixed monolayers. The scanning electron microscope (SEM) and atomic force microscopy (AFM) observations suggest that at a high sodium ion level, the presence of AmB significantly reduces the surface roughness of the DPPC/Chol monolayer. AmB may bind to cholesterol molecules, and it isolates cholesterol from the monolayer, resulting in a reduced height of the cholesterol-rich monolayer and an increasingly dispersed monolayer region. The results are beneficial to understanding the mechanism of impact of a high sodium ion level on the relationship between AmB and red blood cell membranes rich in cholesterol and are valuable for understanding the hemolytic toxicity of AmB to red blood cells at a high sodium ion level.

Atomic Resolution Studies of Sterol Interactions by Solid-State NMR Spectroscopy

Sterols, vital and abundant components of plasma membranes, are involved in numerous cellular biochemical processes such as regulation of membrane properties and signaling events. However, these sterol interactions are not completely elucidated at the atomic level; this impedes further investigations into disorders related to these biological activities. Combining solid-state NMR (SSNMR), high yield biosynthesis of isotopically labeled sterols, and molecular dynamics, we aim to understand the key interactions that determine the sterol-specificity to amphotericin B (AmB), a powerful but toxic antifungal drug used to treat life-threatening fungal infections. AmB forms large aggregates that are neither crystalline nor soluble, making structural information difficult or impossible to obtain via x-ray crystallography or solution-state NMR. We have recently determined that AmB kills yeast cells primarily by binding to ergosterol1 (Erg) and forming a large extramembranous sterol sponge.2 These findings were enabled by the biosynthesis of fractionally 13C labeled Erg and state-of-the-art SSNMR techniques. The sterol sponge model hypothesizes that interactions of AmB with Erg determine its ability to kill yeast, whereas binding to cholesterol is responsible for determining its toxicity in human cells. SSNMR spectroscopy is uniquely able to detect and quantify the binding of sterols to AmB in the sterol sponge in atomistic detail. These studies provide a roadmap towards an improved therapeutic index3 for this drug while simultaneously elucidating additional details about the molecular mechanism of AmB-sterol interactions.(1)“Amphotericin primarily kills yeast by simply binding ergosterol”, Gray et al. Proc. Natl. Acad. Sci. 2011, 109, 2234-2239.(2)“Amphotericin forms an extramembranous sterol sponge”, Anderson et al. Nat. Chem. Bio. 2014, 10, 400-406.(3)“C2’-OH of amphotericin B plays an important role in binding the primary sterol of human cells but not yeast cells”, Wilcock et al. J. Am. Chem. Soc. 2013, 135, 8488-8491.

Recent progress in the study of the interactions of amphotericin B with cholesterol and ergosterol in lipid environments.

In the past decade substantial progress has been made in understanding the organization and biological activity of amphotericin B (AmB) in the presence of sterols in lipid environments. This review concentrates mainly on interactions of AmB with lipids and sterols, AmB channel formation in membranes, AmB aggregation, AmB modifications important for understanding its biological activity, and AmB models explaining its mechanism of action. Most of the reviewed studies concern monolayers at the water–gas interface, monolayers deposited on a solid substrate by use of the Langmuir–Blodgett technique, micelles, vesicles, and multi-bilayers. Liposomal AmB formulations and drug delivery are intentionally omitted, because several reviews dedicated to this subject are already available.

Molecular modelling of membrane activity of amphotericin B, a polyene macrolide antifungal antibiotic.

Amphotericin B (AmB) is a well known polyene macrolide antibiotic used to treat systemic fungal infections. Despite its toxicity AmB is still regarded as a life-saving drug. The lack of adequate knowledge of the AmB mechanism of action is a serious obstacle to efficient development of new less toxic derivatives. Complementary to various experimental approaches, computational chemistry methods were used to study AmB mechanism of action. A programme lasting for a decade, that was run by our group covered studies of: i) molecular properties of AmB and its membrane targets, ii) structure and properties of AmB membrane channels, and iii) interaction of AmB with the membrane.

Interaction of amphotericin B and its low toxic derivative, N-methyl- N-D-fructosyl amphotericin B methyl ester, with fungal, mammalian and bacterial cells measured by the energy transfer method

Amphotericin B (AMB) derivative, N-methyl- N- D-fructosyl amphotericin B methyl ester (MFAME) retains the broad antifungal spectrum and potency of the parent antibiotic, whereas its toxicity towards mammalian cells is reduced by about two orders of magnitude. The purpose of this work was to find out whether the differences observed in the toxicity of MFAME and native AMB are due to the differential drugs affinity to fungal and mammalian cell membranes. Comparative studies on AMB and MFAME biological activity and their affinity to fungal, mammalian and bacterial cells were performed. The interaction of AMB and MFAME with cells have been studied by fluorescence method based on the energy transfer between membrane fluorescent probe (donor) and the polyenic chromophore of the antibiotic (acceptor) simultaneously present in the cell membrane. The amount of the antibiotic bound to cells was indicated by the extent of fluorescence quenching of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) or 1,6-diphenyl-1,3,5-hexatriene (DPH) by polyenic chromophore of the antibiotic. The results obtained indicate that binding extent and characteristics for both antibiotics are comparable in the three types of cells studied. Dramatically lower toxicity of MFAME as compared to AMB towards mammalian cells is not related to the antibiotic-cell affinity, but rather to different consequences of these interactions for cells, reflected in membrane permeabilization. MFAME is definitely less effective than parent AMB in the permeabilizing species formation in mammalian cell membrane.

Comparative study of the effects of amphotericin B on the glucose metabolism in Saccharomyces cerevisiae in K +- and Na +-rich media

In order to elucidate the effects of amphotericin B (AMB) on the glycolytic pathway, the metabolism of [1- 13C]glucose in glucose-grown repressed Saccharomyces cerevisiae was studied. The cells were aerobically suspended in pyrophosphate solutions of high potassium concentration with or without 10 −6 M amphotericin B and measurements were made using 1H-, 13C-NMR spectroscopy and biochemical methods. The results were compared with those obtained under the same experimental conditions but in a medium rich in sodium salts containing the same antibiotic concentration. In general the presence of 10 −6 M AMB reduces the glucose consumption and the ethanol production while favouring the glycerol and trehalose formation. These effects are greatly reduced when a high K + concentration was used. The AMB effects on the glucose consumption and the production of ethanol, glycerol and trehalose, observed in a suspension rich in Na +, can be fairly well explained by the leakage of K + through AMB membrane channels. This outflux induces a substantial decrease in the activity of some K +-dependent enzymes, such as aldolase, phosphofructokinase and pyruvate kinase. The intensities of the glutamate C2 and C4 signals are higher with a suspension rich in Na + than with a suspension rich in K +, suggesting that the Krebs cycle operates more effectively in a solution rich in Na +. In the absence of AMB, the passive diffusion of glycerol through the cell membrane is relatively slow and apparently depends on the ionic external medium: it is more efficient in solutions with a high K + than with a high Na + concentration. In the presence of 10 −6 M AMB, the glycerol C1,3 resonance drastically decreases at 20 min and then disappears in the noise. This rapid disappearance suggests that glycerol can easily pass through the pores arising from the interaction of AMB with the membrane sterols. However, the rate of pore formation is slow, independent of the external medium (Na + or K +) and this process is not completed within 20 min.

A sequential mechanism for the formation of aqueous channels by amphotericin B in liposomes. The effect of sterols and phospholipid composition

The kinetics of formation of amphotericin B (AmB) aqueous pores in orgosterol-containing DMPC or egg-PC liposomes was investigated using a stopped-flow method. The formation of aqueous pores by AmB occurred very rapidly (in milliseconds to seconds depending of the AmB concentration), and it was always preceded by the formation of transient, non-aqueous pre-pore structures. As anticipated, these non-aqueous pre-pore structures made the liposomes more permeable to urea without at the same time leading to a decrease of the reflection coefficient of urea or to an enhancement of glucose permeability. However, when liposomes were composed of egg-PC and cholesterol, the formation of non-aqeuous and aqueous channels by AmB occurred after a lag time of several minutes. Such a time lag for AmB action was not observed in cholesterol-containing DMPC liposomes, an indication that the phospholipid composition is an important parameter in the formation of non-aqueous channels by AmB. Both non-aqueous and aqueous channels were always formed at lower concentrations of AmB in liposomes containing ergosterol while higher concentrations were needed in cholesterol-containing liposomes. Measurements of the permeabilizing effect of AmB on liposomes prepared without sterols indicate that non-aqueous channels were formed in DMPC (but not in egg-PC) at polyene concentrations identical to that found for cholesterol-containing liposomes. No evidence of the formation of aqueous channels by AmB was found in pure DMPC liposomes. These data are consistent with the concept that AmB forms non-aqueous channels without the direct participation of sterol molecules. The initially formed non-aqueous channels subsequently interact with the sterols in the membrane to form aqueous channels, having an enlarged diameter. This sequential mechanism for the formation of AmB aqueous pores in liposomes provides a rationale for the understanding of the effect of both the phospholipid composition and type of sterol in the interaction of AmB with natural membranes and artificial bilayers.

Novel antifungal drug delivery: stable amphotericin B-cholesteryl sulfate discs

To improve the clinical utility of a potent fungicidal agent, amphotericin B (AB), we developed a novel drug dosage form based on the specific interaction of AB with sterols. AB and sodium cholesteryl sulfate at an equimolar ratio form a thermodynamically stable discoidal complex of uniform size. The therapeutic index of the drug is significantly improved (4–6-fold) as a direct result of the unique physical properties of this colloidal system. This drug/lipid complex does not hemolyze red blood cells, exhibits only low binding to plasma lipoproteins in vitro, alters plasma kinetics and tissue distribution compared to a deoxycholate/AB micellar formulation, and is effective in eradicating fungal infections in vivo.

The effect of surfactants on the aggregation state of amphotericin B

We have studied the effect of two surfactants, one non-ionic, lauryl sucrose (LS) and the other ionic, sodium deoxycholate (DOC), on the aggregation state of amphotericin B (AmB) and its selectivity towards ergosterol and cholesterol. It is shown that the addition of these surfactants has very similar effects on the AmB micelles. Below the critical micellar concentration of the surfactants, mixed micelles with AmB are first formed as a result of the penetration of the surfactant molecules into the AmB micelles. At higher concentrations of the surfactant molecules, the micellar structure is completely destroyed and AmB is found as monomers in solution. When the concentration of the surfactant is further increased, micelles of the surfactant molecules are built up, AmB remaining in monomeric form. However, the critical micellar concentration of LS is modified by the presence of AmB in solution, while that of DOC is not affected, thereby indicating that the interactions of AmB with LS are stronger than those of DOC with AmB. We also show that both surfactants enhance the selectivity of the AmB binding to sterols at exactly the concentrations of the surfactants which induce the monomerization of the antibiotic. It is observed that the maximal selectivity is found at a concentration of the surfactants corresponding to their particular CMC in presence of the antibiotic.