Amphotericin B Molecules Research Articles

One Platform Comparison of Polymeric and Lipidic Nanoparticles for the Delivery of Amphotericin B.

Amphotericin B (AmB) is a membrane-acting antibiotic used for the treatment of fungal and protozoal infections. AmB exists in various molecular forms, i.e., monomeric, super-aggregated, and oligomeric forms, where oligomeric forms are highly toxic because of their relative affinity toward cholesterol present over human cell membrane. Hence, the objective of our research work was to study the aggregation state of AmB in two different nanoformulations, i.e., solid lipid nanoparticles (SLNs) and zein-based nanoparticles (PNPs), with the aim of enhancing the fraction of less toxic form of AmB, and a comparative study was performed. The zein and glyceryl monostearate can intercalate the polyenic domain of AmB and thereby hinder the hydrophobic attractions between the AmB molecules, which allows their existence in monomeric forms. The particle size of AmB-SLNs and AmB-PNPs were 378.90 ± 9.50 nm and 184.90 ± 6.00 nm, while zeta potential was -34.97 ± 0.51 mV and +28.93 ± 2.29 mV, respectively. In vitro release studies showed more controlled release of AmB from PNPs (52.48 ± 1.07%) as compared to SLNs (86.33 ± 0.93%). The predominant aggregation state of AmB in both formulations was determined by UV-visible and circular dichroism spectrophotometry, where a higher degree of monomerization of AmB was reported in AmB-SLNs as compared to AmB-PNPs. Toxicity of the nanoformulations was evaluated through hemolysis test, where the results suggested that AmB-SLNs and AmB-PNPs were less hemolytic as compared to pure AmB. The nanoformulations demonstrated the predominant monomeric form of AmB, which may offer higher selectivity index toward microbial membrane.

Enhanced Antifungal Activity of Amphotericin B Bound to Albumin: A "Trojan Horse" Effect of the Protein.

Amphotericin B (AmB) is a life-saving and widely used antifungal antibiotic, but its therapeutic applicability is limited due to severe side effects. Here, we report that the formulation of the drug based on a complex with albumin (BSA) is highly effective against Candida albicans at relatively low concentrations, which implies lower toxicity to patients. This was also concluded based on the comparison with antifungal activities of other popular commercial formulations of the drug, such as Fungizone and AmBisome. Several molecular spectroscopy and imaging techniques, e.g., fluorescence lifetime imaging microscopy (FLIM), were applied to understand the phenomenon of enhanced antifungal activity of the AmB-BSA complex. The results show that the drug molecules bound to the protein remain mostly monomeric and are most likely bound in the pocket responsible for the capture of small molecules by this transport protein. The results of molecular imaging of single complex particles indicate that in most cases, the antibiotic-protein stoichiometry is 1:1. All of the analyses of the AmB-BSA system exclude the presence of the antibiotic aggregates potentially toxic to patients. Cell imaging shows that BSA-bound AmB molecules can readily bind to fungal cell membranes, unlike drug molecules present in the aqueous phase, which are effectively retained by the cell wall barrier. The advantages and prospects of pharmacological use of AmB complexed with proteins are discussed.

Cu(II)-Triggered Ion Channel Properties of a 2,2'-Bipyridine-Modified Amphotericin B.

The development of stimuli-responsive synthetic channels that open and close in response to physical and chemical changes in the surrounding environment has attracted attention because of their potential bioapplications such as sensing, drug release, antibiotics, and molecular manipulation tools to control membrane transport in cells. Metal coordination is ideal as a stimulus for stimuli-responsive channels because it allows for reversible gating behavior through the addition and removal of metal ions and fine-tuning of channel structure through coordination geometry defined by the type of the metal ion and ligand. We have previously reported on transition metal-ion dependent ion permeability control of Amphotericin B (AmB) modified with a metal coordination site, 2,2'-bipyridine ligand (bpy-AmB). AmB is one of the polyene macrolide antibiotics, and it is known that the interaction between AmB and ergosterol molecules is required for AmB channel formation. In contrast, the Cu2+ coordination to the bpy moiety of bpy-AmB induces formation of Ca2+ ion-permeable channels in the ergosterol-free POPC membrane. However, the details of bpy-AmB properties such as channel stability, ion selectivity, pore size, and the effect of ergosterol on channel formation remain unclear. Here, we investigate bpy-AmB channels triggered by transition metal coordination in POPC or ergosterol-containing POPC liposomes using an HPTS assay, electrophysiological measurements, and time-resolved UV-vis spectral measurements. These analyses reveal that bpy-AmB channels triggered by Cu2+ ions are more stable and have larger pore sizes than the original AmB channels and enable efficient permeation of various cations. We believe that our channel design will lead to the construction of metal coordination-triggered synthetic ion channels.

Solubility, aggregation and stability of Amphotericin B drug in pure organic solvents: Thermodynamic analysis and solid form characterization

The solubility of amphotericin B (AmB) has been studied between temperatures of 298–343 K in a range of pure organic solvents including methanol, ethanol, isopropanol, butanol, acetone, tetrahydrofuran, and 1,4-dioxane. The initial solid form used in solubility determinations has been characterized by a series of techniques including powder X-ray diffraction (PXRD), scanning electron microscopy (SEM), Fourier transform infrared (FTIR) and Raman spectroscopy, thermogravimetric analysis and differential scanning calorimetry. Pure AmB melts at 444.5 K with an associated enthalpy of fusion of 177.2 kJ/mol. The equilibrated solids in suspension have been characterized by SEM and PXRD and additionally, the equilibrated saturated solutions have been characterized by in-situ UV–vis to probe the saturation and the aggregation state of AmB. In the explored solvents, in terms of mass ratio, the solubility has been found to be remarkably low, decreasing in the order: ethanol > tetrahydrofuran > butanol ≃ 1,4-dioxane ≃ isopropanol > acetone. The relative order of solubility obtained in the alcohols correlates well with their polarity, revealing important interactions of the solvents’ hydroxyl group with the polyol chain and the micosamine sugar carboxylic moiety of amphotericin B. Except in dioxane and isopropanol, the obtained Van’t Hoff enthalpies of solution reveal an inverse proportionality to the experimental molar fraction solubility values obtained experimentally, indicating a larger energy requirement to solvate AmB molecules. Aggregation of AmB in the equilibrated solutions was not detected in any of the solvents studied yet HPLC analyses revealed that the API degraded in methanol during the equilibration time for the solubility determinations.

In-silico approach as a tool for selection of excipients for safer amphotericin B nanoformulations.

Safer and efficacious Amphotericin B (AmB) nanoformulations can be designed by augmenting AmB in the monomeric or super-aggregated state, and restricting the aggregated state, by choosing the appropriate excipient, which can be facilitated by employing in-silico prediction as a tool. Excipients selected for the study included linear fatty acids from caprylic (C8) to stearic(C18) and the stearate based amphiphilic surfactants polyoxyl-15-hydroxystearate (PS15) and polyoxyl-40-stearate (PS40). Blend module was employed to determine the two miscibility parameters mixing energy (Emix) and interaction parameter (χ). AmB-excipient interactions were modelled using molecular docking software. The fatty acids revealed a decrease in Emix and χ values with increase in carbon chain length, suggesting enhanced affinity with increase in fatty acid hydrophobicity. Significantly higher affinity was observed with amphiphilic surfactants, in particular PS40 which exhibited negative values of Emix and χ proposing very high degree of miscibility. Molecular docking study confirmed extensive interaction of all the excipients with the AmB polyene chain. PS15 and PS40 displayed in addition hydrophilic interactions with the mycosamine and polyol moieties with PS40 exhibiting complete wrapping of the AmB molecule. PS15 demonstrated only partial wrapping, attributed to the shorter ethylene oxide chain. AmB nanosuspensions (NS) were prepared by in situ nanoprecipitation using the excipients and the AmB state identified by UV scanning between 300-500nm. AmB NS with fatty acids and PS15-AmB NS revealed a high intensity peak between 330-350nm of aggregated AmB and low intensity monomeric peaks between 405-415nm reflecting predominance of the aggregated state. PS40-AmB NS on the other hand revealed complete absence of aggregated state and a high intensity peak between 321-325nm which corresponded to the super-aggregated state. Also, the super-aggregated state slowly released the safe monomeric form without aggregate formation. Furthermore, very low hemolysis seen with PS40-AmB NS confirmed low toxicity attributed to the safer super-aggregated state and while higher hemolysis as anticipated was seen with PS15-AmB NS (aggregated state). The basis for selection of the appropriate excipient for design of safer AmB nanoformulations would be those excipients that exhibit negative values of miscibility parameters Emix and χ, exhibit interaction with the hydrophobic and hydrophilic regions of AmB and demonstrate complete wrapping of AmB in the molecular docking study. Our study thus demonstrates feasibility of in-silico prediction as a practical tool for excipient selection for safer AmB nanoformulations.

Interaction between polyene antifungal drug and saturated phospholipid monolayer regulated by calcium ions at the air-water interface

Amphotericin B (AmB) is a polyene antifungal drug, which could directly form pores on the sterol-free phospholipid monolayers. The interaction between AmB and phospholipid can be affected by calcium ions, but the mechanism is still unclear. DPPC is a saturated phospholipid with -PC group, which is often used to simulate the outer cell membrane leaflet. And DPPC is also the main constituent of pulmonary surfactants. In this work, the DPPC monolayer was used as a model membrane to study the effect of calcium ions on the interaction between AmB and phospholipid. The influence of different concentration of calcium ions on the elastic modulus, mean molecular area increment, excess Gibbs free energy and stability of the AmB/DPPC mixed monolayer has been researched at the surface pressure of 7.5 mN/m, 12.5 mN/m and 22.5 mN/m. The AmB/DPPC monolayers at the air-water interface have been observed in real-time by Brewster angle microscope and the microstructure of the Langmuir-Blodgett monolayer films transferred onto the mica have been researched by scanning electron microscope and atomic force microscope. The results showed that calcium ions had a significant influence on the elastic modulus, mean molecular area increment, excess Gibbs free energy, stability and microstructure of the AmB/DPPC monolayer. It has been indicated that the influence of calcium ions on the interaction between AmB and DPPC molecules mainly depended on the effect of calcium ions on the orientation of AmB molecules. The calcium ions could regulate the effect of AmB to the stability of the DPPC monolayer. This regulatory role changed with the different concentrations of calcium ions and the different phase states of the monolayer. This work provides useful information to further understand the influence mechanism of calcium ions on the interaction between AmB and saturated phospholipid with -PC group, which is helpful to find out the effect mechanism of calcium ion on the interaction between AmB and the outer layer of cell membrane or pulmonary surfactants in different phase states and to understand the toxicity mechanism of AmB on the cell membrane or lungs.

Interaction of amphotericin B and saturated or unsaturated phospholipid monolayers containing cholesterol or ergosterol at the air-water interface

The antimicrobial activity of amphotericin B (AmB) depends on its interaction with ergosterol-containing cell membranes of fungus. Cholesterol is a sterol in mammalian cell membrane, and its structure is very similar to ergosterol, which caused to the toxic of amphotericin B to mammalian or human cell membranes. Even so, it is still the gold standard for the treatment of fungal infections. The mechanism of its toxicity to mammalian cell membrane has become a hot topic. The toxicity mechanism of amphotericin B on the cell membrane is also related to the phospholipids on the membrane. The effects of saturated and unsaturated fat chains on the interaction of amphotericin B with phospholipid monolayers containing cholesterol or ergosterol were studied at the molecular level using an air-water interface monolayer model. Both atomic force microscope and Brewster angle microscope were used to observe the surface morphology of the monolayer. The analysis of limiting molecular area suggested that the interaction between AmB and the two kinds of sterol is significantly different on the unsaturated lipid monolayer. According to the elastic modulus, the AmB molecules can increase the compressibility or viscoelasticity of the phospholipid/sterol monolayer. However, this impact of AmB on the DOPC/sterol monolayer containing ergosterol was stronger than that containing cholesterol at 25 ~ 50 mN/m. While this impact of AmB on the DPPC/sterol monolayer containing cholesterol was stronger than that containing ergosterol at 32 ~ 56 mN/m. The excess Gibbs free energy of the monolayer showed that, in the presence of saturated fat chain, amphotericin B could make the molecules of the DPPC/cholesterol monolayer and the DPPC/ergosterol monolayer arrange more closely and make intermolecular interaction stronger. There was no significant difference between DPPC/cholesterol monolayer and DPPC/ergosterol monolayer. However, in the presence of unsaturated chain, the effects of amphotericin B on the DOPC/cholesterol monolayer and the DOPC/ergosterol monolayer were significantly different. Amphotericin B made the molecular arrangement of DOPC/ergosterol monolayer more loosed, and the intermolecular force weakened at 5-35 mN/m. AFM images reflect that AmB can perforate the phospholipid-ergosterol monolayer, which was no significant correlation with saturation of the lipid monolayer. But the areas of dark areas shaped holes on the DPPC/ergosterol monolayer were larger than that on the DOPC/ergosterol monolayer. The adsorption of amphotericin B on lipid/sterol monolayer suggests that the orientation of amphotericin B may be different when it is inserted into the monolayer of phospholipid-sterol in the presence of saturated or unsaturated chains. The results are helpful to understand the complex mechanism of toxicity of amphotericin B to cell membrane.

Influence of metal cations and cholesterol on lipid-amphotericin membrane

Amphotericin B(AmB) has been widely used in antifungal therapy. AmB molecules combine with cholesterol to form pores that can be toxic to human cells, thus greatly limiting its clinical application. The interaction between AmB and the cell membrane may be influenced by potassium, sodium and calcium ions. In this study, the bilayer in large unilamellar lipid-drug liposomes with or without cholesterol was employed as a model membrane. N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine(NBD-PE) and 1-palmitoyl-2-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminodoclecanoyl]-sn-glysero-3-phosphocholine(6-NBD-PC) are two kinds of fluorescent lipid probes, and the NBD group is attached to the polar lipid headgroup in the former, but to the sn-2 fatty acyl chain in the latter. The effect of these metal cations on the lipid-drug membrane was monitored by red edge excitation shift(REES), fluorescence polarization, and the fluorescence lifetime of lipid probes in hydrophilic and hydrophobic areas of the membrane. These ions have different effects on the lipid-AmB membrane. Cholesterol can strengthen the packing ability of the membrane, which is influenced differently by potassium, sodium and calcium ions. Moreover, the influence of these ions on the membrane may be relative to the method of ion transportation through the membrane. This study is significant to understand the reduction of AmB’s cellular toxicity.

De Novo Pyrimidine Biosynthesis Connects Cell Integrity to Amphotericin B Susceptibility in Cryptococcus neoformans.

The use of amphotericin B (AmB) in conjunction with 5-fluorocytosine (5-FC) is known to be the optimal therapy for treating cryptococcosis, but the mechanism by which 5-FC synergizes with AmB is unknown. In this study, we generated a Cryptococcusneoformans ura1Δ mutant lacking dihydroorotate dehydrogenase (DHODH), which demonstrated temperature-sensitive growth due to a defect in cell integrity and sensitivity to cell wall-damaging agents. In addition, sensitivity to AmB was greatly increased. Inclusion of uracil or uridine in the medium did not suppress the cell wall or AmB phenotype, whereas complementation with the wild-type URA1 gene complemented the mutant phenotype. As a measure of membrane accessibility, we assayed the rate of association of the lipid-binding dye 3,3'-dihexyloxacarbocyanine iodide (DiOC6) and saw more rapid association in the ura1Δ mutant. We likewise saw an increased rate of DiOC6 association in other AmB-sensitive mutants, including a ura- spontaneous URA5 mutant made by 5-fluoroorotic acid (5-FOA) selection and a bck1Δ mutant defective in cell integrity signaling. Similar results were also obtained by using a specific plasma membrane-binding CellMask live stain, with cell integrity mutants that exhibited increased and faster association of the dye with the membrane. Chitin synthase mutants (chs5Δ and chs6Δ) that lack any reported cell wall defects, in turn, demonstrate neither any increased susceptibility to AmB nor a greater accessibility to either of the dyes. Finally, perturbation of the cell wall of the wild type by treatment with the β-1,6-glucan synthase inhibitor caspofungin was synergistic with AmB in vitro. IMPORTANCE Synergy between AmB and nucleotide biosynthetic pathways has been documented, but the mechanism of this interaction has not been delineated. Results from this study suggest a correlation between uridine nucleotide biosynthesis and cell integrity likely mediated through the pool of nucleotide-sugar conjugates, which are precursor molecules for both capsule and cell wall of C.neoformans. Thus, we propose a mechanism by which structural defects in the cell wall resulting from perturbation of pyrimidine biosynthesis allow faster and increased penetration of AmB molecules into the cell membrane. Overall, our work demonstrates that impairment of pyrimidine biosynthesis in C.neoformans could be a potential target for antifungal therapy, either alone or in combination with AmB.

Membrane Sterols Modulate the Binding Mode of Amphotericin B without Affecting Its Affinity for a Lipid Bilayer.

Membrane-active antibiotics are known to selectively target certain pathogens based on cell membrane properties, such as fluidity, lipid ordering, and phase behavior. These are in turn modulated by the composition of a lipid bilayer and in particular by the presence and type of membrane sterols. Amphotericin B (AmB), the golden standard of antifungal treatment, exhibits higher activity toward ergosterol-rich fungal membranes, which permits its use against systemic mycoses; however, the selectivity for fungal membranes is far from satisfactory leading to severe side effects. Despite decades of research, no consensus has emerged on the origin of AmB specificity for fungal cells and its actual mode of action at the molecular level. Previously, it has been proposed that the specific action of AmB is related to differences in its affinity for membranes of different composition. In this work, we investigate this relationship by employing molecular dynamics simulations to compare the free energy of insertion of AmB into three types of membranes: a pure DMPC bilayer and DMPC bilayers containing 30% of cholesterol or ergosterol. We analyze the orientation of AmB molecules within the bilayer in order to unambiguously establish their membrane binding mode and relate the orientational freedom to the sterol-dependent tightness of lipid packing. Our results strongly indicate that the membrane insertion of AmB proceeds virtually to completion independent of membrane type, and hence the higher toxicity against fungal membranes may rather result from differences in subsequent oligomerization in the membrane and assembly of monomers into functional transmembrane pores. In particular, the latter could be facilitated by sterol-induced ordering of AmB molecules along the membrane normal, revealed by our free energy profiles. Moreover--in contrast to certain claims--we find no stable binding mode corresponding to the horizontal adsorption of AmB on the membrane surface.

Catalytic effect of free iron ions and heme-iron on chromophore oxidation of a polyene antibiotic amphotericin B

Owing to the presence of a chromophore in the amphotericin B (AmB) structure, the molecule can undergo the oxidation process. In this research, AmB chromophore oxidation was catalysed by iron ions (iron(III) chloride (FeCl3), pH 2.5) and by heme-iron (methemoglobin (HbFe(III)), and hemin (heme-Fe(III)) at pH 7.0). Additionally, we compared oxidation processes induced by the aforementioned oxidizing agents with autoxidation by dioxygen (O2) naturally occurring in a sample. The effects of the interaction of the oxidizing agents with AmB were analysed using molecular spectroscopies (electronic absorption (UV–Vis), fluorescence) and LC-MS. The use of a 1,10-phenanthroline (phen) chelator facilitated unambiguous determination of the oxidative effect of free iron(III) ions (FeIII) in an acidic solution on the AmB molecules. Also, the changes in the spectra of fluorescence emission centred at ∼470 nm indicate iron-catalysed processes of AmB chromophore oxidation. Unexpectedly, we found a similar spectroscopic effect for AmB induced by methemoglobin and hemin at pH 7.0. Methemoglobin and hemin at a concentration of 8 × 10−7 M (physiological) significantly increases the rate of the processes of AmB chromophore oxidation relative to the process of autoxidation.

Self-association of amphotericin B: spontaneous formation of molecular structures responsible for the toxic side effects of the antibiotic.

Amphotericin B (AmB) is a lifesaving antibiotic used to treat deep-seated mycotic infections. Both the pharmaceutical activity and highly toxic side effects of the drug rely on its interaction with biomembranes, which is governed by the molecular organization of AmB. In the present work, we present a detailed analysis of self-assembly of AmB molecules in different environments, interesting from the physiological standpoint, based on molecular spectroscopy techniques: electronic absorption, circular dichroism, steady state and time-resolved fluorescence and molecular dynamic calculations. The results show that, in the water medium, AmB self-associates to dimeric structures, referred to as "parallel" and "antiparallel". AmB dimers can further assemble into tetramers which can play a role of transmembrane ion channels, affecting electrophysiological homeostasis of a living cell. Understanding structural determinants of self-assembly of AmB opens a way to engineering preparations of the drug which retain pharmaceutical effectiveness under reduced toxicity.

Two-step impact of Amphotericin B (AmB) on lipid membranes: ESR experiment and computer simulations

In this study, the electron spin resonance (ESR) method was used to examine the effect of Amphotericin B (AmB) molecules on the fluidity of model membranes made of dipalmitoylphosphatidylcholine (DPPC). The changes occurring under increased AmB concentrations in the spectroscopic parameters of spin probes placed in liposomes were determined. Three probes were used, penetrating the membrane at different depths which allowed the changes in its fluidity to be found in the transverse section. A computer model of the surface layer of membrane, with AmB admixture, was developed and subjected to computer simulation. The effect of changing concentration of the admixture on the binding energy in the system of dipoles representing the surface of the membrane was examined. The ESR studies showed that the process of accumulation of AmB molecules in the membrane has two stages, marked by local maxima in the ESR spectra. The first appears for concentrations of ca. 0.25–0.5% and the second appears for ca. 2.5–3% AmB of its molar ratio to DPPC. The computer simulations permitted reconstructing the two-stage mechanism of interaction between the molecules and the membrane. They demonstrated that, at low concentrations, the AmB molecules position themselves flat on the membrane surface. After the threshold concentration is exceeded, they re-orientate to a vertical position. This process leads to the perforation of the membrane.

The Effect of Sterols on Amphotericin B Self-Aggregation in a Lipid Bilayer as Revealed by Free Energy Simulations

Amphotericin B (AmB) is an effective but toxic antifungal drug, known to increase the permeability of the cell membrane, presumably by assembling into transmembrane pores in a sterol-dependent manner. The aggregation of AmB molecules in a phospholipid bilayer is, thus, crucial for the drug’s activity. To provide an insight into the molecular nature of this process, here, we report an atomistic molecular dynamics simulation study of AmB head-to-head dimerization in a phospholipid bilayer, a possible early stage of aggregation. To compare the effect of sterols on the thermodynamics of aggregation and the architecture of the resulting AmB-AmB complexes, free energy profiles for the dimerization in ergosterol- or cholesterol-containing and sterol-free membranes are derived from the simulations. These profiles demonstrate that although AmB dimers are formed in all the systems studied, they are significantly less favorable in the bilayer with ergosterol than in the cholesterol-containing or sterol-free ones. We investigate the structural and energetic determinants of this difference and discuss its consequences for the AmB mechanism of action.

FTIR spectroscopic study of molecular organization of the antibiotic amphotericin B in aqueous solution and in DPPC lipid monolayers containing the sterols cholesterol and ergosterol

Langmuir monolayers of amphotericin B (AmB) were investigated by recording π-A isotherms under different pH conditions. To gain a better insight into antibiotic-membrane interactions they were monitored by use of the ATR-FTIR spectroscopy. It was observed for AmB monolayers that the limiting molecular area was larger at high than at neutral pH. Analysis of FTIR spectra at different pH revealed substantial differences, depending on ionic state, for different orientations of AmB molecules. These results enable better understanding of the participation of functional groups in the interactions between AmB and sterol-containing DPPC membranes. AmB molecules incorporated into two-component lipid monolayers bind strongly to the ergosterol-rich membrane (maximum penetration surface pressures ca 35 mN/m). The FTIR spectra revealed that the ionic state of AmB and the presence of sterols led to changes in membrane fluidity and molecular packing of the AmB molecules in the lipid membranes. These investigations should be further investigated to discover the molecular mechanism responsible for the mode of action AmB in biological systems.

Amphotericin B Channel-Forming Activity Depends on Membrane Dipole Potential

Amphotericin B (AmB) is the most effective polyene antibiotic used in treatment of systemic fungal infections. The activity of AmB results from its interaction with sterol-containing membranes and consequent pore formation. As some sterols influence membrane dipole potential (Vd) one can assume that Vd may regulate the ability of AmB to form ion channels. The aim of the present work was to study the channel-forming activity of AmB as well as the properties of its single pores in planar lipid bilayers of different dipole potentials. AmB (0.1-0.2 µM) was added on both sides of a membrane prepared from diphytanoylphosphocholine (67 mol %) and cholesterol (33 mol %) in 2 M KCl (5 mM Hepes, pH 7.0) solutions. A reduction of Vd by addition of 20 µM phloretin to the bilayer bathing solutions is accompanied by ∼3-fold decrease of a single channel conductance and ∼20-fold increase of a steady-state AmB-induced membrane conductance. Increasing the membrane dipole potential by adding 5 µM RH421 produces ∼2-fold increase of the conductance of AmB channels and practically does not affect the steady-state bilayer conductance. The observed changes in the channel-forming activity of AmB in the presence of dipole modifying agents correspond to ∼60-fold increase and ∼2 fold decreases in steady-state open channel number in the presence of phloretin and RH421, respectively. The obtained results allow us to speculate that AmB molecule possess a certain dipole moment. Therefore, the decrease of membrane dipole potential produces a reduction of the energy barrier for interfacial accumulation of AmB dipoles. These results may be instructive in understanding of the membrane activity of amphotericin B. The study was supported in part by RFBR (# 09-04-00883), SS-3796.2010.4, the Program “Molecular and Cell Biology”, RAS, and the State contract # P1372 (MES, FTP“SSEPIR”).

Spectroscopic studies of molecular organization of antibiotic amphotericin B in monolayers and dipalmitoylphosphatidylcholine lipid multibilayers

Amphotericin B (AmB) is considered the gold-standard in the treatment of serious systemic mycoses despite its numerous adverse effects. Both the mechanism of antifungal action and the toxicity of this drug are dependent on its molecular organization. The effect of AmB on the organization of lipid membranes formed with dipalmitoylphosphatidylcholine (DPPC) was studied with application of the Langmuir–Blodgett technique and ATR-FTIR spectroscopy. The aim of this research was to analyze the physical interactions leading to the formation of aggregated forms of AmB molecules in one-component monolayers and lipid multibilayers. Analysis of FTIR spectra of two-component multibilayers suggests the possibility the mutual reorientation of the amino-sugar moiety (mycosamine) and macrolide ring. This effect may be significant in the explanation of the aggregation processes of AmB in biological systems.

Determination of the Number of Molecules Involved in Different Types of Amphotericin B Channels

Electrophysiological properties of Amphotericin B (AmB) pores in lipid bilayers have been studied extensively with the purpose of understanding membrane transport phenomena1. Also because AmB has a clinical relevance, it is the drug of choice for treating systemic fungal infection in spite of having strong collateral toxicity. One of the interests has been to determine the number of molecules involved in the formation of the ion channels reported. Reports in the literature present values that go from 4 to 12. This has been explained because channels with different conductivities are formed2 and therefore the determination with macroscopic currents corresponds to an average of the population of different channels. In this work, we determined the number of molecules of AmB involved in the formation of each one of these pores by single channel methods3. Our results show that different types of channels are formed by the same number of AmB molecules, which is very interesting, considering that these channels have certainly different radius. This suggests a model in which AmB pores are not the only component of the pores, having either lipids in a toroidal structure or sterols in a barrel structure.

On the Possibility of the Amphotericin B-Sterol Complex Formation in Cholesterol- and Ergosterol-Containing Lipid Bilayers: A Molecular Dynamics Study

Amphotericin B (AmB) is a well-known membrane-active antibiotic that has been used to treat systemic fungal infections for more than 45 years. Therapeutic application of AmB is based on the fact that it is more active against ergosterol-containing membranes of fungal cells than against mammalian membranes with cholesterol. In this paper, we examine the hypothesis according to which the selectivity of the AmB's membrane action originates from its different ability to form the binary complexes with the relevant sterols. To this end, molecular dynamics simulations were performed for systems containing the preformed models of AmB/sterol complexes embedded in lipid bilayers containing either cholesterol or ergosterol. The initial structures of the studied binary associates were selected on the basis of a systematic scan of all possible mutual positions and orientations of the two molecules. The results obtained demonstrate that in general the complexes with ergosterol are more stable on the 100 ns time scale. Furthermore, on the basis of motional correlation analysis, taking into account the effects of lipid environment, we propose that, within the sterol-enriched liquid-ordered membrane phases, AmB molecules exhibit a greater tendency to bind ergosterol than cholesterol. The analysis of the interactions suggests that this affinity difference is of enthalpic origin and may arise from the considerable difference in the energy of the van der Waals interactions between AmB and the two types of sterols. Thus, our current results: (i) support the hypothesis that binary AmB/sterol complexes form within a lipid membrane and (ii) suggest that the higher toxicity may at least partly be attributed to the higher affinity of AmB for ergosterol than for cholesterol within a lipid membrane environment.

Amphotericin B: side effects and toxicity

Amphotericin B (AmB) is a crucial agent in the management of serious systemic fungal infections. In spite of its proven track record, its well-known side effects and toxicity will sometimes require discontinuation of therapy despite a life-threatening systemic fungal infection. The mechanism of action of AmB is based on the binding of the AmB molecule to the fungal cell membrane ergosterol, producing an aggregate that creates a transmembrane channel, allowing the cytoplasmic contents to leak out, leading to cell death. Most of the efforts at improving AmB have been focused on the preparation of AmB with a lipid conjugate. AmB administration is limited by infusion-related toxicity, an effect postulated to result from proinflammatory cytokine production. The principal acute toxicity of AmB deoxycholate includes nausea, vomiting, rigors, fever, hypertension or hypotension, and hypoxia. Its principal chronic adverse effect is nephrotoxicity. AmB probably produces renal injury by a variety of mechanisms. Risk factors for AmB nephrotoxicity include male gender, higher average daily dose of AmB (≥35 mg/day), diuretic use, body weight ≥90 kg, concomitant use of nephrotoxic drugs, and abnormal baseline renal function. Clinical manifestations of AmB nephrotoxicity include renal insufficiency, hypokalemia, hypomagnesemia, metabolic academia, and polyuria due to nephrogenic diabetes insipidus. Human studies show convincingly that sodium loading in excess of the usual dietary intake notably reduces the incidence and severity of AmB-induced nephrotoxicity.