Circular dichroism study of interactions of Fungizone or AmBisome forms of amphotericin B with human low density lipoproteins

Amphotericin B (AmB), a potent antifungal agent used to treat invasive fungal infections, is still employed more than 40 years after its introduction in the pharmacopea. When injected into the blood stream, this antibiotic is carried by low density lipoproteins (LDLs) to which it induces the formation of oxidation products responsible in part for some of the severe adverse effects of the drug. However, the oxidative damages induced to LDLs are not yet understood. We present here the effects of the Fungizone and AmBisome forms of AmB on LDLs as compared to those of CuSO(4), a well-known powerful oxidant of LDLs. We use circular dichroism (CD) spectroscopy, which is particularly useful because it allows the investigation of the structural integrity of the proteic moiety of LDL upon interaction with AmB. The CD spectra also yield information on the drug itself because in its oligomer form it presents a strong dichroic signal in a spectral region different from that of the protein. Our results show that neither form of AmB changes the secondary structure of the protein while the helical content of the LDL is increased either in the presence of CuSO(4) alone or in the presence of CuSO(4) and AmBisome or Fungizone. On the other hand, the CD spectra of the antibiotic indicate that Fungizone AmB suffers important oxidative damage in the presence of LDLs and CuSO(4) while this damage is not present with AmBisome AmB. These observations lead us to propose that the structural modifications of the proteic part of LDLs induced by the Cu(2+) ions are involved in the important oxidative damage suffered by Fungizone AmB, which in this form is much more susceptible to interaction with its environment than AmBisome.     

Amphotericin B interactions with a DOPC monolayer. Electrochemical investigations

A model lipid membrane consisting of a monolayer of dioleoyl phosphatidylcholine (DOPC) adsorbed onto a Hg electrode has been used to study the interaction between the lipid and different formulations of Amphotericin B (AmB) [Fungizone (FZ), Heated Fungizone (HFZ), and Abelcet]. The lipid organizational order was measured by electrochemical methods [capacitance and metal ion (Tl(+)) reduction], characterizing the change in lipid order due to interaction with the drug. The mean size and number density of pores formed in the monolayer were estimated by fitting the reduction current transients to a random array of microelectrode model. This method was shown sensitive for investigation of the interaction of drugs with the DOPC monolayer. Abelcet was found to have a smaller disruptive effect on lipid order than FZ and HFZ. The formulations used to solubilize the AmB were also studied. Sodium deoxycholate used as a solubilizer in FZ displayed significant influence on lipid order similar to that observed for Abelcet. The lipid complex, used in Abelcet, did not significantly perturb the DOPC monolayer order. The lipid complex used in Abelcet may have an annealing or healing effect that buffers the disruption possible due to AmB.     

Optimizing efficacy of amphotericin B through nanomodification

Fungal infections and leishmaniasis are an important cause of morbidity and mortality in immunocompromised patients. The macrolide polyene antibiotic amphotericin B (AmB) has long been recognized as a powerful fungicidal and leishmanicidal drug. A conventional intravenous dosage form of AmB, AmB- deoxycholate (Fungizone or D-AmB), is the most effective clinically available for treating fungal and parasitic (leishmaniasis) infections. However, the clinical efficacy of AmB is limited by its adverse effects mainly nephrotoxicity. Efforts to lower the toxicity are based on synthesis of AmB analogues such as AmB esters or preparation of AmB-lipid associations in the forms of liposomal AmB (L-AmB or AmBisome), AmB lipid complex (Abelcet or ABLC), AmB colloidal dispersion (Amphocil or ABCD), and intralipid AmB. These newer formulations are substantially more expensive, but allow patients to receive higher doses for longer periods of time with decreased renal toxicity than conventional AmB. Modifications of liposomal surface in order to avoid RES uptake, thus increased targetability has been attempted. Emulsomes and other nanoparticles are special carrier systems for intracellular localization in macrophage rich organs like liver and spleen. Injectable nano-carriers have important potential applications as in site-specific drug delivery.     

Amphotericin B interactions with a DOPC monolayer. Electrochemical investigations

A model lipid membrane consisting of a monolayer of dioleoyl phosphatidylcholine (DOPC) adsorbed onto a Hg electrode has been used to study the interaction between the lipid and different formulations of Amphotericin B (AmB) [Fungizone® (FZ), Heated Fungizone (HFZ), and Abelcet®]. The lipid organizational order was measured by electrochemical methods [capacitance and metal ion (Tl⁺) reduction], characterizing the change in lipid order due to interaction with the drug. The mean size and number density of pores formed in the monolayer were estimated by fitting the reduction current transients to a random array of microelectrode model. This method was shown sensitive for investigation of the interaction of drugs with the DOPC monolayer. Abelcet was found to have a smaller disruptive effect on lipid order than FZ and HFZ. The formulations used to solubilize the AmB were also studied. Sodium deoxycholate used as a solubilizer in FZ displayed significant influence on lipid order similar to that observed for Abelcet. The lipid complex, used in Abelcet, did not significantly perturb the DOPC monolayer order. The lipid complex used in Abelcet may have an annealing or healing effect that buffers the disruption possible due to AmB.     

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.     

Mechanism of Action of Amphotericin B at the Cellular Level. Its Modulation by Delivery Systems

Abstract

Amphotericin B (AmB) is the drug of choice for the treatment of systemic fungal infections, but its use is hampered by its severe side-effects. A better understanding of its mechanisms of action is needed to develop new AmB formulations with an optimal selectivity between fungal and mammalian cells. Interactions between AmB and cells depend on the concentration of the drug. Stimulatory effects, modulation of the activity of immunocompetent cells and inhibition of yeast adherence are early events that precede the actual cellular toxicity. If membrane permeability alterations are considered to be the first toxic step, cell death results not only from osmotic imbalances, but also from additional mechanisms, such as lipid peroxidation, inhibition of membrane enzymes and blockade of endocytosis. The selectivity between fungal and mammalian cells takes its origin from the difference in the nature of the membrane sterol: ergosterol in fungi, cholesterol in mammalian cells. Transmembrane pores result from different mechanisms according to the sterol: ergosterol-AmB complexes are formed from monomelic AmB in solution, which is the only form present in aqueous medium at low AmB concentrations, whereas pores in the cholesterol containing membrane result from the adsorption onto the membrane surface of aqueous self-associated AmB, that appears in medium when AmB concentration increases. The liposomes seem to sequester AmB in a manner which makes it unavailable for mammalian cells, but maintains its access to fungal cells. The transfer of AmB by progressive diffusion of free AmB through the aqueous phase could explain the enhancement of the therapeutic index of the drug by liposomes, since the induction of pore formation needs a higher threshold of drug for host cell than for fungal cell membranes. The closed structure of the vehicle is not required to enhance the selectivity of the drug: esters of sucrose or high concentration of sodium deoxycholate afford a protective effect as well. Macrophages, after phagocytosis of liposomal AmB, may be considered as a reservoir of AmB, from which the drug is progressively released. Finally, the strong binding of AmB to the delivery system reduces the amount of drug bound to serum components and thus the endocytosis of AmB through the LDL receptor, resulting in lower toxicity.     

Interactions of amphotericin B derivative of low toxicity with biological membrane components--the Langmuir monolayer approach

Amphotericin B (AmB)--a polyene macrolide antibiotic--exhibits strong antifungal activity, however, is known to be very toxic to mammalian cells. In order to decrease AmB toxicity, a number of its derivatives have been synthesized. Basing on in vitro and in vivo research, it was evidenced that one of AmB derivatives, namely N-methyl-N-D-fructopyranosylamphotericin B methyl ester (in short MF-AME) retained most of the antifungal activity of the parent antibiotic, however, exhibited dramatically lower animal toxicity. Therefore, MF-AME seems to be a very promising modification product of AmB. However, further development of this derivative as potential new antifungal drug requires the elucidation of its molecular mechanism of reduced toxicity, which was the aim of the present investigations. Our studies were based on examining the binding energies by determining the strength of interaction between MF-AME and membrane sterols (ergosterol-fungi sterol, and cholesterol-mammalian sterol) and DPPC (model membrane phospholipid) using the Langmuir monolayer technique, which serves as a model of cellular membrane. Our results revealed that at low concentration the affinity of MF-AME to ergosterol is considerably stronger as compared to cholesterol, which correlates with the improved selective toxicity of this drug. It is of importance that the presence of phospholipids is essential since--due to very strong interactions between MF-AME and DPPC--the antibiotic used in higher concentration is "immobilized" by DPPC molecules, which reduces the concentration of free antibiotic, thus enabling it to selectively interact with both sterols.     

Structural characterization of the interaction of the polyene antibiotic Amphotericin B with DODAB bicelles and vesicles

Amphotericin B (AmB) is widely used in the treatment of systemic fungal infections, despite its toxic effects. Nephrotoxicity, ascribed as the most serious toxic effect, has been related to the state of aggregation of the antibiotic. In search of the increase in AmB antifungal activity associated with low toxicity, several AmB-amphiphile formulations have been proposed. This work focuses on the structural characterization of a specific AmB formulation: AmB associated with sonicated dioctadecyl dimethylammonium bromide (DODAB) aggregates. Here, it was confirmed that sonicated DODAB dispersion is constituted by DODAB bicelles, and that monomeric AmB is much more soluble in bicelles than in DODAB vesicles. A new optical parameter is proposed for the estimation of the relative amount of amphiphile-bound monomeric AmB. With theoretical simulations of the spectra of spin labels incorporated in DODAB bicelles it was possible to prove that monomeric AmB binds preferentially to lipids located at the edges of DODAB bicelles, rigidifying them, and decreasing the polarity of the region. That special binding of monomeric AmB along the borders of bicelles, where the lipids are highly disorganized, could be used in the formulation of other carriers for the antibiotic, including mixtures of natural lipids which are known to form bicelles.     

Molecular organization of antibiotic amphotericin B in dipalmitoylphosphatidylcholine monolayers induced by K(+) and Na(+) ions: the Langmuir technique study

The effect of potassium (K(+)) and sodium (Na(+)) ions on the self-association of antibiotic amphotericin B (AmB) in the lipid membrane was reported. Mixed Langmuir monolayers of AmB and dipalmitoylphosphatidylcholine (DPPC) were investigated by recording surface pressure-area isotherms spread on aqueous buffers containing physiological concentration of K(+) and Na(+) ions. The analyses of the π-A isotherms and compressional modulus curves indicate the interactions in the AmB-DPPC system. The strength of the AmB-DPPC interactions and the stability of the mixed monolayers were examined on the basis of the excess free energy of mixing values. The obtained results proved a high affinity of AmB towards lipids induced by the presence of K(+) than Na(+) ions. The most stable monolayers in the presence of K(+) and Na(+) ions were formed by AmB and DPPC with the 1:1 and 2:1 stoichiometry. The understanding of the AmB aggregation processes at the molecular level should contribute to elucidate the mechanisms of action and toxicity of this widely used drug. The presented results are potentially valuable in respect to develop more efficient and less toxic AmB formulations.     

Effect of aggregation of amphotericin B on lysophosphatidylcholine micelles as related to its complex formation with cholesterol or ergosterol

The effect of aggregation of amphotericin B (AmB), as well as the complex formation of AmB with cholesterol or ergosterol, was investigated in micelles and vesicles. AmB in lysophosphatidylcholine (LPC) micelles adopted a more favorable monomeric form than that in other drug formulations. At an LPC/AmB ratio of 200, AmB existed only in monomeric form. Such monomeric behavior is likely dependent upon the fluidity and size of the micelles. In LPC micelles composed of 90% monomeric AmB, AmB-ergosterol complex formation occurred with an increase in the sterol concentration, but the complex formation of AmB-cholesterol was slight. On the other hand, in LPC micelles composed of 40% monomeric AmB, the complex formation of AmB-cholesterol as well as AmB-ergosterol was extensive. These results suggest that the complex formation of AmB with both sterols is highly dependent upon the aggregated state of AmB. In addition, using monolayers, mixtures of AmB/LPC/ergosterol were became more stable with rising temperature, while the stability of mixtures of AmB/LPC/cholesterol remained unchanged, implying that complex formation of AmB with cholesterol is different from that of AmB with ergosterol.     

Cochleates: New Lipid-Based Drug Delivery System

Abstract

Cochleates are a lipid-based tailored drug delivery system formed by the precipitation of a negatively charged lipid and a cation, for example phosphatidylserine and calcium. Hydrophobic, amphiphilic, negatively or positively charged moieties are suitable candidates to be delivered via cochleates. Various procedures have been developed allowing the control of cochleate particle size, including the trapping and hydrogel methods, which use either a direct addition or a slow diffusion of calcium into the negatively charged liposome/drug suspension. The efficacy of cochleates to encapsulate and deliver drugs was evaluated using amphotericin B as a model. Amphotericin B cochleates (CAMB) were compared to Fungizone® and AmBisome®, two commercially available AmB products. Parenterally, CAMB was given IP to ICR mice infected with Candida albicans. 100% survival was observed with low doses of CAMB (0.5 mg/kg/day, 10 days) compared to 60% for Fungizone, at the same dose. Tissue burden studies were conducted in parallel. Mice were treated daily from day 1 to day 7 post challenge and tissue burden assessed at day 8. In the kidneys, all three formulations were comparable in reducing colony counts. In the spleen, CAMB at 10 mg/kg/day was comparable to AmBisome given IV at the same dose. At 1 mg/kg/day, CAMB was more potent than Fungizone and AmBisome. Oral administration of CAMB in C57BL/6 mice, at 10 mg/kg results in high levels of AmB in target tissues. Multiple daily doses (10) showed accumulation of AmB in key tissues (liver, lungs, spleen, and kidneys) and AmB tissue concentrations are raised to therapeutic levels. Orally administered CAMB are highly effective against fungal infections in mice at very low doses. Balb/C mice were infected with Candida albicans and were given oral CAMB as a daily dose for 15 days. Comparison was done to AmBisome given orally at 10 mg/kg and Fungizone IP. 100% survival was obtained with CAMB at doses as low as 0.5 mg/kg/day (15 days). CAMB eradicate Candida from lungs when given at 2.5 mg/kg/day and was comparable to Fungizone given IP at almost the same dose (2 mg/kg/day). The comparison between CAMB and AmBisome shows that oral CAMB is 10 times more effective than oral AmBisome in reducing colony counts in both kidneys and lungs. Orally administered CAMB were non-toxic even at the highest dose of 50 mg/kg/day (14 days). This was demontrated by 100% survival of the animals and normal histopathology analysis. No lesions in the kidneys, GI tract, lungs, liver and spleen was observed despite the substantial amount of AmB in these organs. AmB cochleate promise to be a safe, broad spectrum, effective and orally available, antifungal formulation.     

Comparative molecular dynamics simulations of amphotericin B-cholesterol/ergosterol membrane channels

Amphotericin B (AmB) is a very effective anti-fungal polyene macrolide antibiotic whose usage is limited by its toxicity. Lack of a complete understanding of AmB's molecular mechanism has impeded attempts to design less toxic AmB derivatives. The antibiotic is known to interact with sterols present in the cell membrane to form ion channels that disrupt membrane function. The slightly higher affinity of AmB toward ergosterol (dominant sterol in fungal cells) than cholesterol (mammalian sterol) is regarded as the most essential factor on which antifungal chemotherapy is based. To study these differences at the molecular level, two realistic model membrane channels containing molecules of AmB, sterol (cholesterol or ergosterol), phospholipid, and water were studied by molecular dynamics (MD) simulations. Comparative analysis of the simulation data revealed that the sterol type has noticeable effect on the properties of AmB membrane channels. In addition to having a larger size, the AmB channel in the ergosterol-containing membrane has a more pronounced pattern of intermolecular hydrogen bonds. The interaction between the antibiotic and ergosterol is more specific than between the antibiotic and cholesterol. These observed differences suggest that the channel in the ergosterol-containing membrane is more stable and, due to its larger size, would have a higher ion conductance. These observations are in agreement with experiments.     

Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy

Amphotericin B (AMB) is a highly hydrophobic antifungal, whose use is limited by its toxicity and poor solubility. To improve its solubility, AMB was reacted with a functionalized polyethylene glycol (PEG), yielding soluble complex AmB-PEG formulations that theoretically comprise of chemically conjugated AMB-PEG and free AMB that is physically associated with the conjugate. Reverse-phase chromatography and size exclusion chromatography methods using HPLC were developed to separate conjugated AMB-PEG and free AmB, enabling the further characterization of these formulations. Using HPLC and dynamic light scattering analyses, it was observed that the AMB-PEG 2 formulation, having a higher molar ratio of 2 AMB: 1 PEG, possesses more free AMB and has relatively larger particle diameters compared to the AMB-PEG 1 formulation, that consists of 1 AMB: 1 PEG. The identity of the conjugate was also verified using mass spectrometry. AMB-PEG 2 demonstrates improved antifungal efficacy relative to AMB-PEG 1, without a concurrent increase in in vitro toxicity to mammalian cells, implying that the additional loading of free AMB in the AMB-PEG formulation can potentially increase its therapeutic index. Compared to unconjugated AMB, AMB-PEG formulations are less toxic to mammalian cells in vitro, even though their MIC₅₀ values are comparatively higher in a variety of fungal strains tested. Our in vitro results suggest that AMB-PEG 2 formulations are two times less toxic than unconjugated AMB with antifungal efficacy on Candida albicans and Cryptococcus neoformans.     

Factors Affecting Enhanced Permeation of Amphotericin B Across Cell Membranes and Safety of Formulation

The aim of this study was to determine amphotericin B (AmB) permeation across lipid bilayer membranes mounted on Transwell® and to observe the phagocytosis of the AmB and the AmB-lipid formulations by alveolar macrophage (AM) cell lines using a fluorescence microscope. The lipid bilayer membranes were prepared from phospholipid and ergosterol as well as phospholipid and cholesterol in a ratio (67:33 mol%). AmB-lipid formulations were prepared from AmB incorporated with four lipid derivatives during a lyophilization process. In vitro cytotoxicity studies were carried out on kidney cells by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The levels of nitric oxide production by AMs exposed to these AmB-lipid formulations were determined by the Griess reaction. Phagocytosis of the AmB-lipid formulations was carried out using AM cells. The lipid bilayer membranes and AmB-lipid formulations were successfully prepared. In vitro cytotoxicity results showed less toxicity to kidney cells than pure AmB, and a 1,000-fold less production of nitric oxide by NR8383 cell lines was obtained when compared to lipopolysaccharide. Permeation results were two- to fivefold higher than for pure AmB in the ergosterol containing lipid bilayer and two- to fourfold higher than AmB in the cholesterol containing compositions, both of which were enough to kill the fungi according to their MICs and MFCs. AM phagocytosed the AmB-lipid formulations. We suggest that these products especially the AmB-sodium deoxycholate sulfate are potential candidates for targeting AM cells for the treatment of invasive pulmonary aspergillosis.     

Amphotericin B encapsulated in micelles based on poly(ethylene oxide)-block-poly(L-amino acid) derivatives exerts reduced in vitro hemolysis but maintains potent in vivo antifungal activity

The core-forming blocks of amphiphilic diblock copolymers based on methoxypoly(ethylene oxide)-block-poly(L-aspartate), PEO-b-p(L-Asp), were derivatized to incorporate stearate side chains. The effects of stearate esterification were assessed in terms of micelle stability and amphotericin B (AmB) encapsulation/release. The level of stearate esterification modulates the relative self-aggregation state of encapsulated AmB as evidenced by absorption spectroscopy. When AmB is physically loaded into polymeric micelles, the onset of hemolytic activity toward bovine erythrocytes is delayed relative to that of the free drug. Furthermore, the extent of esterification (0, 46, or 91%) appears to have profound influence on the time-dependent hemolytic profile of AmB toward bovine erythrocytes. Particularly in the case of highly substituted stearate ester micelles, incomplete and gradual build-up of hemolysis was observed over a period of 24 h. On the basis of the corresponding absorption spectra, we speculate that encapsulated AmB may interact strongly with stearate side chains, resulting in sustained release. In a neutropenic murine model of disseminated candidiasis, kidney colony-forming unit determination revealed dose-dependent efficacy for the polymeric micelle/AmB formulation, which was not significantly different from that of Fungizone at doses of 0.2, 0.3, and 0.6 mg/kg (p = 0.7). Thus, AmB administered via a polymeric micelle formulation retained potent in vivo activity.     

The effect of amphotericin b derivatives on Leishmania and immune functions

The effects of a water-soluble amphotericin B (AmB)-arabinogalactan (AG) conjugate on several immune functions were investigated. The experiments measured the effects of AmB-AG on (1) release of tumor necrosis factor-alpha (TNF-alpha), nitric oxide (NO), and interferon-gamma (IFN-gamma) from phagocytic cells and (2) cell-mediated immune responses. AmB-AG increased TNF-alpha release from mouse peritoneal macrophages and human monocytes but had no effect on IFN-gamma and NO release. A commercial preparation of nonconjugated AmB (Fungizone) also increased TNF-alpha production, but to a lesser extent than AmB-AG. AG alone had no effect on TNF-alpha production, proving that AmB caused the increased TNF-alpha production. AmB-AG and Fungizone were also tested for their effect on B- and T-cell proliferation. Neither compound altered T-lymphocyte responses to concanavalin A, but both inhibited the stimulation of B lymphocytes by lipopolysaccharides. However, Fungizone showed a stronger inhibitory effect on B cells. Allocytotoxicity was also inhibited by AmB-AG and more strongly by Fungizone. The increased production of TNF-alpha by cells treated with AmB-AG and the lower inhibitory effect of AmB-AG on lymphocyte stimulation and allocytotoxicity, as compared with Fungizone, explain the better therapeutic efficacy of the AmB-polysaccharide conjugate. AmB is active because of its preferential binding to ergosterol rather than cholesterol, the former sterol preferentially present in parasite surface membranes. This is also valid for the axenic amastigotes, which were sensitive to the AmB-AG. Overall, our results suggest that the antileishmanial activity of AmB-AG is mediated both directly and via modulation of immune functions.     

Evaluation of antifungal pharmacodynamic characteristics of AmBisome against Candida albicans

A liposomal formulation of Amphotericin B (AmBisome), with small unilamellar vesicles containing amphotericin B, shows characteristic pharmacokinetics as liposomes, and in consequence, has different pharmacological activity and toxicity from amphotericin B deoxycholate (Fungizone). In this study, we evaluated the antifungal pharmacodynamic characteristics of AmBisome against Candida albicans using the in vitro time-kill method and murine systemic infection model. A time-kill study indicated that the in vitro fungicidal activities of AmBisome and Fungizone against C. albicans ATCC 90029 increased with increasing drug concentration. For in vivo experiments, leucopenic mice were infected intravenously with the isolate 4 hr prior to the start of therapy. The infected mice were treated for 24 hr with twelve dosing regimens of AmBisome administered at 8-, 12-, 24-hr dosing intervals. Correlation analysis between the fungal burden in the kidney after 24 hr of therapy and each pharmacokinetic/pharmacodynamic parameter showed that the peak level/MIC ratio was the best predictive parameter of the in vivo outcome of AmBisome. These results suggest that AmBisome, as well as Fungizone, has concentration-dependent antifungal activity. Furthermore, since AmBisome can safely achieve higher concentrations in serum than Fungizone, AmBisome is thought to have superior potency to Fungizone against fungal infections.     

Impact of aldehyde content on amphotericin B-dextran imine conjugate toxicity

The biocompatibility of oxidized dextran (40 kDa) was investigated in vitro. The contribution of aldehyde groups to the toxicity of polymer-drug conjugates, such as dextran-amphotericin B (AmB) was evaluated. Oxidized dextran was proved to be toxic against the RAW 264.7 cell line with an IC50 of 3 micromol/mL aldehydes. Modification of aldehyde groups and their reaction with ethanolamine reduced the toxicity at least 15-fold. Accordingly, the antifungal and antileishmanial dextran-AmB imine conjugate, which contains unreacted aldehyde groups, was modified with ethanolamine and compared to dextran-AmB amine and imine conjugates. Modification of the imine conjugate with ethanolamine reduced its toxicity toward the RAW cell line by 100%. The effect on Leishmania major parasites was 5 times higher than that of the dextran-AmB amine conjugate. The dextran-AmB-ethanolamine conjugate was at least 15 times less hemolytic than free AmB. Stability and drug release profiles in buffer solution were investigated. The imine conjugates released free AmB while the amine conjugate did not. It is concluded that aldehyde groups may contribute to cell toxicity. This toxicity is reduced by converting the aldehyde groups into imine conjugates with ethanolamine. The results have direct implications toward the safety of AmB-polysaccharide conjugates used against fungal and leishmanial infections.     

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.     

The effects of amphotericin B on pure and ergosterol- or cholesterol-containing dipalmitoylphosphatidylcholine bilayers as viewed by 2H NMR

Amphotericin B (AmB) is a widely used polyene antibiotic to treat systemic fungal infections. This drug is known to be lethal to fungal cells but it has also side effect toxicity on mammalian cells. The mechanism of action of AmB is thought to be related to the difference of the main sterol present in the mammalian and the fungal cells, namely cholesterol and ergosterol, respectively. The effect of AmB has been investigated on pure dipalmitoylphosphatidylcholine (DPPC) and on cholesterol- and ergosterol-containing DPPC bilayers by 2H NMR spectroscopy. The 2H NMR results first confirm that AmB forms a complex with sterol-free DPPC bilayers, the interaction causing the structurization of the lipids and the increase of the gel-to-lamellar fluid DPPC phase transition temperature with increasing concentration of the antibiotic. The results also show that the effects of AmB on cholesterol- and ergosterol-containing DPPC bilayers are remarkably different. On one hand, the drug causes an increase of the orientational order of the lipid acyl chains in cholesterol-containing membranes, mostly in high cholesterol content membranes. On the other hand, the addition of AmB disorders the DPPC acyl chains when ergosterol is present. This is thought to be due to the direct complexation of the ergosterol by AmB, causing the sterol ordering effect to be weaker on the lipids.