Properties of amphotericin B channels in a lipid bilayer

Properties of individual ionic channels formed by polyene antibiotic Amphotericin B were studied on brain phospholipid membranes containing cholesterol. The ionic channels have a closed state and an open one (with conductance of about 6.5 pS in 2 M KCl). The conductance value of an open channel is independent of cholesterol concentration in the membrane and of pH in the range from 3.5 to 8.0. The voltage-current characteristics of a single channel are superlinear. Zero current potential value in the case of different KCl concentrations in the two solutions indicates preferential but not ideal anionic selectivity of a single channel. Channel conductivity grows as the electrolyte concentration is increased and tends to a limiting value at high concentrations. A simple model having only one site for an ion was shown to represent satisfactorily an open channel behaviour under different conditions. An individual ionic channel performs a large number of transitions between the open and closed states during its life-time of several minutes. Rate constants of these transitions depend on the kind and concentration of salt in aqueous solutions. The switching system functioning is not influenced by an ion situated inside the pore.     

In vitro and in vivo antifungal activities of liposomal amphotericin B,and amphotericin B lipid complex

The in vitro and in vivo antifungal activities of liposomal amphotericin B (L-AMPH) and amphotericin B lipid complex (ABLC), which is composed of amphotericin B and the phospholipids dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol, were compared with those of conventional amphotericin B (Fungizone®, AMPH). The acute intravenous toxicity was markedly lower in BALB/c mice; 50% lethal doses (LD_50s) were 2.75 mg/kg in AMPH, 32.9 mg/kg in L-AMPH and >75 mg/kg in ABLC. In vitro antifungal activities againstCandida albicans, C. parapsilosis, C. tropicalis, C. glabrata, andC. krusei were evaluated by the agar plate dilution method. The activities were unchanged againstC. albicans, but MICs increased more than four fold in 18 of the 20 strains other thanC. albicans in L-AMPH and in 9 of the 20 in ABLC. L-AMPH and ABLC were as efficacious as AMPH in the treatment of mice infected withC. albicans, and at a dose of 0.5 and 1.0 mg/kg of body weight, ABLC was more efficacious on survival. A ten-times larger dose (10 mg/kg) of L-AMPH and ABLC was administered to mice with 100% survival, suggesting improved tolerability as compared to amphotericin B.     

Effect of nystatin,amphotericin B and amphotericin B methyl ester on Saccharomyces cerevisiae with different lipid composition

Saccharomyces cerevisiae was cultured under anaerobiosis in semi-complete medium to which either palmitoleic or oleic acid was added. Cells were grown at 20 °C or 30 °C. The levels of total lipids, total sterols, and phospholipids were higher in cells grown at 20 °C than at 30 °C. The effects of nystatin (NYS), amphotericin B (AMB), and amphotericin B methyl ester (AME) were evaluated by determining cell viability and liberation of intracellular compounds. The loss of cell viability is higher in the first 30 minutes of incubation with the drugs and is the same regardless of the type of cells obtained. Low molecular weight compounds and ions such as K⁺ are liberated a few minutes after incubation with the drugs whereas proteins and substances absorbing at 260 nm are liberated later. Phosphate liberation comes after K⁺ and before compounds of higher molecular weights.     

Structure of amphotericin B aggregates based on calculations of optical spectra

The Degenerate ground state approximation was used to calculate the optical absorption and CD spectra for helical polymer models of amphotericin B aggregates in aqueous solution. Comparisons with experimental spectra indicate that a two-molecule/unit cell helical polymer model is a possible structure for aggregates of amphotericin B.     

Interactions of amphotericin B derivatives with lipid membranes--a molecular dynamics study

Amphotericin B (AmB) is a well-known polyene macrolide antibiotic used to treat systemic fungal infections. AmB targets more efficiently fungal than animal membranes. However, there are only minor differences in the mode of action of AmB against both types of membranes, which is a source of AmB toxicity. In this work, we analyzed interactions of two low toxic derivatives of AmB (SAmE and PAmE), synthesized in our laboratory, with lipid membranes. Molecular dynamics simulations of the lipid bilayers containing ergosterol (fungal cells) or cholesterol (animal cells) and the studied antibiotic molecules were performed to compare the structural and dynamic properties of AmB derivatives and the parent drug inside the membrane. A number of differences was found for AmB and its derivatives' behavior in cholesterol- and ergosterol-containing membranes. We found that PAmE and SAmE can penetrate deeper into the hydrophobic region of the membrane compared to AmB. Modification of the amino and carboxyl group of AmB also resulted in the conformational transition within the antibiotic's polar head. Wobbling dynamics differentiation, depending on the sterol present, was discovered for the AmB derivatives. These differences may be interpreted as molecular factors responsible for the improved selectivity observed macroscopically for the studied AmB derivatives.     

New lipid formulation of amphotericin B: spectral and microscopic analysis

UV-visible and dichroic spectrum analysis and electron microscopy have been used to characterize a new amphotericin B (AmB) lipid formulation prepared by a solvent displacement process. The composition was dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) in molar ratio DMPC/DMPG/AmB 7:3:5, a similar composition to that of Abelcet®. Although the latter has a “ribbon-like” structure, our process gave a thin disc-like structure. Analysis of circular dichroism (CD) and UV-visible spectra of formulations containing different percentages of AmB revealed that a minimum of AmB self-association was observed with 7:3:5 molar ratio. Varying the lipid ratio (DMPC/DMPG) while maintaining the fixed ratio of AmB yielded similar results when DMPC was in excess (DMPC/DMPG from 10:0 to 6:4). However, when the ratio was between 5:5 to 3:7, AmB self-aggregation increased. For compositions rich in DMPG (2:8 and 0:10), inversion of the CD spectrum was observed. The influence of the lipid composition on the morphology of the complex was also evident in electron microscopy. DMPC/DMPG/AmB (10:0:5) gave large unfracturable lamellae. The presence of DMPG shortened the lamellae, which often appeared as disc-like structures. AmB content, the presence of DMPG and the preparation process all contribute to generating these original structures with particular CD spectra.     

Mechanism of blockage of amphotericin B channels in a lipid bilayer.

A number of organic compounds (non-electrolytes, tetraalkylammonia, etc.) with a molecular size of 6--8 angstrom decrease the conductance of ionic channels formed in the lipid bilayer by a polyene antibiotic amphotericin B. It is suggested that these compounds, upon entering the channel, block the passage of inorganic ions. The extent of conductance blockage by organic ions depends on the membrane potential and electrolyte concentration. In the presence of ionic blockers, for instance tetraethylammonium, amphotericin B-containing membranes assume some properties characteristic of excitable membranes, i.e. the current-voltage characteristic acquires the negative resistance region, and in response to a potential step activation followed by inactivation of conductance is observed. It is shown that the potential dependence of the blockage is due to interaction inside the channel of the blocker ion with penetrating ions, by a mechanism similar to that described by Armstrong ((1979) Q. Rev. Biophys. 7, 179--210) for blockage of squid axon potassium channels by ammonium derivatives.     

Transient permeability induced by alkyl derivatives of amphotericin B in lipid membranes

Individual ionic channels were shown to be formed in the brain cholesterol containing phospholipid membranes by two-sided addition of the amphotericin B alkyl derivatives. At concentrations between 10(-8) and 10(-7) M, the resulting conductance appeared to be transient. Existence of different antibiotic assemblies was justified by the kinetic analysis of the membrane conductance decline following the antibiotic washing out. In order to account for the transient characteristics of the induced conductance, it was proposed that the antibiotic oligomers incorporate into the membrane from the aqueous phase, form channels aggregating with cholesterol, and then dissociate in the bilayer into non-active degraded oligomeric or monomeric forms.     

Transient permeability induced by alkyl derivatives of amphotericin B in lipid membranes

Individual ionic channels were shown to be formed in the brain cholesterol containing phospholipid membranes by two-sided addition of the amphotericin B alkyl derivatives. At concentrations between 10⁻⁸ and 10⁻⁷ M, the resulting conductance appeared to be transient. Existence of different antibiotic assemblies was justified by the kinetic analysis of the membrane conductance decline following the antibiotic washing out. In order to account for the transient characteristics of the induced conductance, it was proposed that the antibiotic oligomers incorporate into the membrane from the aqueous phase, form channels aggregating with cholesterol, and then dissociate in the bilayer into non-active degraded oligomeric or monomeric forms.     

Ion channels, induced by amphotericin B introduced unilaterally into lipid bilayers

Single ionic channels of approximately 10 pS in magnitude and approximately 100 ms duration (in 2 M KCl solution) were recorded when amphotericin B (AB) was added to one side of a lipid bilayer. Using blocking ions it has been shown that these channels are asymmetrical half-pores (similar to those postulated by Marty and Finkelstein) which are capable of forming long-living symmetrical pores if AB is added to both sides of the membrane.     

Deuterium NMR investigation of an amphotericin B derivative in mechanically aligned lipid bilayers

The methyl-d(3) amide derivative of the polyene antibiotic amphotericin B was synthesized, assayed for biological activity, incorporated into mechanically aligned bilayers of dipalmitoylphosphatidylcholine (DPPC), and examined by deuterium and phosphorus NMR. The amide derivative has a lesser, but qualitatively similar, biological activity relative to amphotericin B. Incorporation of the amide derivative and ergosterol into aligned DPPC bilayers resulted in a single, stable bilayer phase, as shown by phosphorus NMR of the DPPC headgroups. Deuterium NMR spectra revealed one major (2)H quadrupolar splitting and one major (2)H-(1)H dipolar splitting in the liquid-crystalline phase, consistent with a high degree of alignment and a single, averaged physical state for amphotericin B methyl-d(3) amide in the bilayer. Variations of the quadrupolar and dipolar splittings as a function of macroscopic sample orientation and temperature indicated that the amide derivative undergoes fast rotation about a motional axis that is parallel to the bilayer normal.     

Interactions of amphotericin B derivatives with lipid membranes—A molecular dynamics study

Amphotericin B (AmB) is a well-known polyene macrolide antibiotic used to treat systemic fungal infections. AmB targets more efficiently fungal than animal membranes. However, there are only minor differences in the mode of action of AmB against both types of membranes, which is a source of AmB toxicity. In this work, we analyzed interactions of two low toxic derivatives of AmB (SAmE and PAmE), synthesized in our laboratory, with lipid membranes. Molecular dynamics simulations of the lipid bilayers containing ergosterol (fungal cells) or cholesterol (animal cells) and the studied antibiotic molecules were performed to compare the structural and dynamic properties of AmB derivatives and the parent drug inside the membrane. A number of differences was found for AmB and its derivatives' behavior in cholesterol- and ergosterol-containing membranes. We found that PAmE and SAmE can penetrate deeper into the hydrophobic region of the membrane compared to AmB. Modification of the amino and carboxyl group of AmB also resulted in the conformational transition within the antibiotic's polar head. Wobbling dynamics differentiation, depending on the sterol present, was discovered for the AmB derivatives. These differences may be interpreted as molecular factors responsible for the improved selectivity observed macroscopically for the studied AmB derivatives.     

Spectrophotometric analysis of organisation of dipalmitoylphosphatidylcholine bilayers containing the polyene antibiotic amphotericin B

Amphotericin B (AmB) is a polyene antibiotic widely used in the treatment of deep-seated fungal infections. The mode of action of AmB is directly related to the effect of the drug on the lipid phase of biomembranes. In the present work the effect of AmB on the properties of lipid bilayers formed with dipalmitoylphosphatidylcholine (DPPC) and the effect of the lipid phase on the molecular organisation of AmB were studied with application of spectrophotometry in the UV-Vis region. The absorption spectra of AmB in lipid membranes display a complex structure with hypsochromically and bathochromically shifted bands indicative of formation of molecular aggregates of the drug. Formation of molecular aggregates was analysed at different concentrations of the drug in the lipid phase in the range 0.05--5 mol% and at different temperatures in the range 5--55 degrees C. The aggregation level of AmB in the ordered phase of DPPC displayed a minimum corresponding to a concentration of 1 mol% with respect to the lipid. An increase in the aggregation level was observed in the temperature region corresponding to the main phase transition. The structure of molecular aggregates of AmB is analysed on the basis of spectroscopic effects in terms of the exciton splitting model. Analysis of the position of the absorption maximum of AmB in the lipid phase of DPPC in terms of the theory of solvatochromc effects makes it possible to ascribe the refractive indices n=1.40 and n=1.49 to the hydrophobic core of the membrane in the L(alpha) and the P(beta)' phase respectively. Analysis of the aggregation of AmB in the lipid phase in relation to the physical state of the membrane reveals that the temperature range of the main phase transition of a lipid cluster in the immediate vicinity of AmB depends on its concentration. The termination of the phase transition temperature, as read from the AmB aggregation, varies between 42 degrees C at 1 mol% AmB in DPPC and 49 degrees C at 5 mol% AmB in DPPC. The exciton splitting theory applied to the analysis of the spectroscopic data makes it possible to calculate the diameter of the AmB pore as 2.8 A in the gel phase and 3.6 A in the fluid phase of the DPPC membrane, on the assumption that the pore is formed by nine AmB molecules.     

Organization of antibiotic amphotericin B in model lipid membranes. A mini review

Amphotericin B (AmB) is a polyene antibiotic frequently applied in the treatment of fungal infections. According to the general understanding, the mode of action of AmB is directly related to the molecular organization of the drug in the lipid environment, in particular to the formation of pore-like molecular aggregates. Electronic absorption and fluorescence techniques were applied to investigate formation of molecular aggregates of AmB in the lipid environment of liposomes and monomolecular layers formed at the argon-water interface. It appears that AmB dimers, stabilized by van der Waals interactions, are present in the membrane environment along with the aggregates formed by a greater number of molecules. Linear dichroism measurements reveal that AmB is distributed between two fractions of molecules, differently oriented with respect to the bilayer. Molecules in one fraction remain parallel to the plane of the membrane and molecules in the other one are perpendicular. Scanning Force Microscopy imaging of the surface topography of the monolayers formed with AmB in the presence of lipids reveals formation of pore-like structures characterized by the external diameter close to 17 A and the internal diameter close to 6 A. All the findings are discussed in terms of importance of the molecular organization of AmB in the pharmacological action, as well as of the toxic side effects of the drug.     

The effects of amphotericin B on lipid metabolism in cultured human skin fibroblasts

Summary Amphotericin B inhibits hydroxymethylglutarylcoenzyme A (HMGCoA) reductase activity and incorporation of [³H]acetate into sterols and fatty acids of human skin fibroblasts. Delivery of cholesterol to cells via the low density lipoprotein receptor pathway is not altered. The effects of amphotericin B on cell lipid metabolism are partially reversed by both potassium and insulin. Portions of this work were presented at the Southern Regional Meeting of the American Federation for Clinical Research, January 1983.     

Fonsecaea pedrosoi: lipid composition and determination of susceptibility to amphotericin B.

Conidia and mycelial cells of Fonsecaea pedrosoi ATCC 46428 were obtained for analyses of lipid composition. Total lipids, phospholipids, sterols, and qualitative sterols and fatty acid composition were determined. A higher lipid content was detected in conidia than in mycelial cells of Fonsecaea pedrosoi, which could not be attributed to total sterols and phospholipids. In both forms of this fungus, ergosterol was the only sterol detected. The minimal inhibitory concentration of amphotericin B was lower for conidia than for mycelium.     

Ocular amphotericin B delivery by chitosan-modified nanostructured lipid carriers for fungal keratitis-targeted therapy

Abstract

Context: Fungal keratitis, a corneal fungal infection of the eye caused mainly by Candida species, has become the leading cause of blindness resulting from corneal disease in China. Present limitations in the management of ophthalmic fungal infections include the inability to provide long-term extraocular drug delivery without compromising intraocular structures and/or systemic drug exposure.

Objective: The aim of this study was to construct amphotericin B (AmB) loaded, chitosan-modified, nanostructured lipid carriers (AmB-CH-NLC) for prolonged ocular application and for the improvement of the targeted delivery of AmB to the ocular mucosa.

Materials and methods: The AmB-CH-NLC was produced by the method of emulsion evaporation-solidification at low temperature. The particle size, zeta potential, and encapsulation efficiency, drug-release behavior, and corneal penetration ability were performed in vitro and in vivo.

Results and discussion: The prepared AmB-CH-NLC nanoparticles exhibited a measured size of 185.4?nm, a zeta potential of 27.1?mV, and an entrapment efficiency of 90.9%. Sustained drug release behavior was observed in vitro. The in vivo ocular pharmacokinetic study indicated improved bioavailability of AmB-CH-NLC. The corneal penetration study showed that the AmB-CH-NLC could successfully penetrate into the cornea with no obvious irritation to the rabbits’ eyes.

Conclusion: The results support that this novel nanomedicine could be a promising system for effective ocular delivery of amphotericin B for fungal keratitis-targeted therapy.     

Determination of amphotericin B, liposomal amphotericin B, and amphotericin B colloidal dispersion in plasma by high-performance liquid chromatography

Amphotericin B is a potent polyene antifungal drug for intravenous treatment of severe infections. It is used as amphotericin B-deoxycholate and in order to reduce amphotericin B toxicity as lipid-formulated complex (liposomal or colloidal dispersion). A sensitive and specific analytical method is presented for the separation of lipid-complexed and plasma protein-bound amphotericin B in human heparinized plasma. This separation, which is required for pharmacokinetic studies, is achieved by solid-phase extraction (SPE) via Bond Elut C₁₈. The protein-bound amphotericin B has a higher affinity to the SPE material and is therefore retained, whereas the lipid-complexed amphotericin B is eluted in the first step. The recovery of the SPE was >75% for high concentrations and >95% for low concentrations. Quantification was performed by reversed-phase HPLC using a LiChrosorb-RP-8 column, UV detection (λ=405 nm) and a mixture of acetonitrile–methanol–0.010 M NaH₂PO₄ buffer (41:10:49, v/v) as mobile phase. The retention time for amphotericin B under the given conditions was 6.7 min. The calibration curves were found to be linear (r≥0.999) in two different ranges (5.0–0.50 μg/ml and 0.50–0.005 μg/ml). Intra- and inter-day precision and accuracy fulfilled the international requirements. No interference from other drugs (typical broad medication for intensive-care patients) or common plasma components was detected in >400 samples analyzed.     

Binding of antibiotic amphotericin B to lipid membranes: a 1H NMR study

The (1)H NMR technique was applied to study binding of AmB, an antifungal drug, to lipid membranes formed with egg yolk phosphatidylcholine. The analysis of (1)H NMR spectra of liposomes, containing also cholesterol and ergosterol (at 40 mol%), shows that AmB binds preferentially to the polar headgroups. Such a binding restricts molecular motion of the choline fragment in the hydrophilic region at the surface of liposomes but increases the segmental motional freedom in the hydrophobic core. The same effects are also observed in the sterol-containing membranes, except that the effect on the hydrophobic core was exclusively observed in the membranes containing ergosterol.