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.     

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.     

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.     

Membrane effects of the polyene antibiotic amphotericin B and of some of its derivatives on lymphocytes

Amphotericin B (AmB) exhibits immunomodulating properties in mice.In vitro studies on lymphocytes, in relation with these properties, are reported here with AmB and two of its derivatives: the N-Fructosyl (N-Fru AmB) and the N-thiopropionyl (AmBSH) derivatives. Interactions of these molecules with thymocytes, a sensitive cell type, demonstrated that the extent of binding is not a toxicity parameter. In contrast, membrane fludity changes have been observed and appeared to be related to toxicity.Experiments performed with normal B lymphocytes have shown that Amphotericin B derivatives were more potent polyclonal B cell activators than the parent compound. To go further in the understanding of these events, we have investigated in a B cell line WEHI 231, the changes in intracellular Ca²⁺ and membrane potential induced by AmB and AmBSH. The two polyenes were shown to induce membrane depolarization but no intracellular Ca²⁺ increase.     

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.     

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.     

Alteration of membrane permeability by amphotericin B

Amphotericin B (AmB) increased unidirectional Na transport and net transcellular sodium movements across the skin of the frog, Rana pipiens, when added to the solution bathing the corium side, but not from the outer epidermal surface. The AmB response was prevented with pretreatment with amiloride, ouabain and mucosal sodium substitution. Alteration in pH markedly reduced the permeability changes induced by AmB. AmB did not interfere with the increase in sodium transport induced by antidiuretic hormone. The present study demonstrates that AmB interacts with the skin of the frog, Rana pipiens, from the corium side specifically increasing transepithelial sodium transport. The increase in transport apparently occurs through the existing sodium pathway.     

Inhibition of the interaction between lipoproteins and amphotericin B by some delivery systems.

Amphotericin B (AmB) is a potent antifungal agent used to treat patients with systemic mycoses. The clinical usefulness of the drug is limited by its high toxicity and several new less toxic formulations of AmB have been recently developed. In order to understand the mechanism of the decreases of toxicity caused by various new delivery systems, we have investigated by uv-visible spectroscopy the interaction of two of these formulations with human blood lipoproteins. The results were compared with those obtained with the commonly used pharmaceutical form of AmB (Fungizone). This study shows that AmB-lipoprotein interaction is hindered when the drug is in a monomeric form and/or when it is included in phospholipid-surfactant micelles. In an in vivo study on mice it is shown here that AmB monomerized by surfactant is less toxic to animals than the same concentration of Fungizone, where the polyene is strongly aggregated. It may be concluded from the present study that the AmB species which is responsible for the in vivo toxicity is a complex of the antibiotic with the low density and the very low density blood lipoproteins and that hindering of this complex formation results in a decrease of AmB toxicity.     

Amphotericin B mediates killing in Cryptococcus neoformans through the induction of a strong oxidative burst

We studied the effects of Amphotericin B (AmB) on Cryptococcus neoformans using different viability methods (CFUs enumeration, XTT assay and propidium iodide permeability). After 1h of incubation, there were no viable colonies when the cells were exposed to AmB concentrations ≥ 1 mg/L. In the same conditions, the cells did not become permeable to propidium iodide, a phenomenon that was not observed until 3h of incubation. When viability was measured in parallel using XTT assay, a result consistent with the CFUs was obtained, although we also observed a paradoxical effect in which at high AmB concentrations, a higher XTT reduction was measured than at intermediate AmB concentrations. This paradoxical effect was not observed after 3h of incubation with AmB, and lack of XTT reduction was observed at AmB concentrations higher than 1mg/L. When stained with dihydrofluorescein, AmB induced a strong intracellular oxidative burst. Consistent with oxidative damage, AmB induced protein carbonylation. Our results indicate that in C. neoformans, Amphotericin B causes intracellular damage mediated through the production of free radicals before damage on the cell membrane, measured by propidium iodide uptake.     

Organization of polyene antibiotic amphotericin B at the argon-water interface

Amphotericin B (AmB) is a life-saving antibiotic, used to treat deep-seated mycotic infections. Both the therapeutic and toxic side effects of AmB are directly dependent on its molecular organization. Organization of AmB was studied in monocomponent monomolecular layers formed at the argon-water interface, by means of polarized and non-polarized electronic absorption spectroscopy and analyzed in terms of the exciton splitting theory. The results provide direct indication that AmB forms spontaneously dimers that can be assembled into molecular structures characterized by homogeneous orientational distribution in the monolayer, interpreted as cylindrical pores. The structures are not stable at surface pressures higher than 20 mN/m and therefore dimers are concluded as abundant molecular organization forms of AmB in biomembranes. Possibility of stabilization of the cylindrical structures, at higher surface pressures, by other molecules, e.g. sterols, is also discussed.     

Comparative theoretical study of the conformations of amphotericin methyl ester and amphotericin B polar heads in the presence of water.

Amphotericin methyl ester (AmE) is an interesting derivative of amphotericin B (AmB) because of its enhancement of selectivity against the fungicells. Both AmB and AmE molecules differ by the structure of their polar heads. This work deals with a theoretical study of conformations of the polar head of AmE in the presence of hydration water molecules. The results will be compared with our previous work concerning AmB.     

Influence of a lipid bilayer on the conformational behavior of amphotericin B derivatives — A molecular dynamics study

Amphotericin B (AmB) is an effective but very toxic antifungal antibiotic. In our laboratory a series of AmB derivatives of improved selectivity of action was synthesized and tested. To understand molecular basis of this improvement, comparative conformational studies of amphotericin B and its two more selective derivatives were carried out in an aqueous solution and in a lipid membrane. These molecular simulation studies revealed that within a membrane environment the conformational behavior of the derivatives differs significantly from the one observed for the parent molecule. Possible reasons for such a difference are analyzed. Furthermore, we hypothesize that the observed conformational transition within the polar head of AmB derivatives may lead to destabilization of antibiotic-induced transmembrane channels. Consequently, the selective toxicity of the derivatives should increase as ergosterol-rich liquid-ordered domains are more rigid and conformationally ordered than their cholesterol-containing counterparts, and as such may better support less stable channel structure.     

The polyene antibiotic amphotericin B acts as a Ca2+ ionophore in sterol-containing liposomes

Amphotericin B (AmB) was shown to induce a Ca2+ influx across ergosterol- and cholesterol-containing large unilamellar liposomes, by following spectrophotometrically the formation of the Arsenazo III-Ca2+ complex. At equivalent antibiotic concentrations the Ca2+ influx was much more extensive through ergosterol-containing membranes (almost 100% with 1 microM AmB, 160 microM lipid) than through cholesterol-containing membranes (below 0.5 microM the influx of Ca2+ was negligible). In the presence of ergosterol-containing membranes the initial rate of Ca2+ influx had the same linear dependence on the ratio antibiotic/lipid whatever the lipid concentration, which was not the case in cholesterol-containing membranes. These results suggest that the channels responsible for the AmB-induced Ca2+ permeability across cholesterol- and ergosterol-containing liposomes have different structures.     

Investigations on feasibility of in situ development of amphotericin B liposomes for industrial applications

Amphotericin B (AmB) liposome formulations are very successful in the treatment of fungal infections and leishmaniasis. But higher cost limits its widespread use among people in developing countries. Therefore, we have developed a modified ethanol-injection method for the preparation of AmB liposomes. Two liposomal formulations were developed with dimyristoyl phosphatidylcholine [F-1a] and soya phosphatidylcholine [F-2a], along with egg phosphatidyl glycerol and cholesterol. AmB was dissolved in acidified dimethyl acetamide and mixed with ethanolic lipid solution and rapidly injected in 5% dextrose to prepare liposomes. Liposomes were characterized on the basis of size (~100?nm), zeta (-43.3?±?2.8 mV) and percent entrapment efficiency (>95%). The in vitro release study showed an insignificant difference (P?≥?0.05) for 24-hour release between marketed AmB liposomes (AmBisome) and F-1a and F-2a. Proliposome concentrate, used for the preparation of in situ liposomes, was physically stable for more than 3 months at experimental conditions. Similarly, AmB showed no sign of degradation in reconstituted liposomes stored at 2-8°C for more than 3 months. IC(50) value of Ambisome (0.18 μg/mL) was comparatively similar to F-1a (0.17 μg/mL) and F-2a (0.16 μg/mL) against intramacrophagic amastigotes. Under experimental conditions, a novel modified method for AmB liposomes is a great success and generates interest for development as a platform technology for many therapeutic drug products.     

Antimycotic-antibiotic amphotericin B promotes influenza virus replication in cell culture

In general, antibiotics are not rated as substances that inhibit or support influenza virus replication. We describe here the enhancing effect of the polyene antibiotic amphotericin B (AmB) on influenza virus growth in Vero cells. We show that isolation rates of influenza A and B viruses from clinical samples can be dramatically enhanced by adding AmB to the culture medium. We demonstrate that AmB promotes the viral uptake and endocytic processing of the virus particles. This effect is specific for Vero and human nasal epithelial cells and was not observed in Madin-Darby canine kidney cells. The effect of AmB was subtype specific and more prominent for human seasonal influenza strains but absent for H5N1 human viruses. The AmB-enhancing effect seemed to be solely due to the viral hemagglutinin function. Our results indicate that the use of AmB may facilitate influenza virus isolation and production in Vero cells.     

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.     

How does Amphotericin B Work?: Studies on Model Membrane Systems

Abstract

The drug Amphotericin B is a very important antifungal agent as well as one of the first model systems for transmembrane pore structures. The most widely accepted model for the anticellular activity of this drug involves the formation of 1:1 Amphotericin/ sterol aggregates which subsequently associate into a transmembrane barrel with a large -OH lined aqueous pore down the middle. The stronger association of Amphotericin with ergosterol versus cholesterol explains the higher toxicity toward fungi. However, conflicting membrane permeability data concerning Amphotericin channel ion selectivity, sterol requirements, and mode of delivery has accumulated over the past fifteen years and suggests there exists a multiplicity of AmB channel structures and modes of action. Some of these mechanisms of action may be even more relevant clinically than the Amphotericin/sterol pore structure. Some of the anticellular membrane damage caused by Amphotericin may be due to formation of membrane defects and non-bilayer phases, channels without sterol or even induction of oxidative damage. In this article we present a survey of recent observations on AmB's activity on model membrane systems. As such, we are mostly concerned with liposome and planar bilayer studies. Some of the newer models explaining AmB s differential effects on cholesterol versus ergosterol containing membranes are presented along with a brief overview of membrane disruption models based on current research on membrane-active amphiphilic peptides. A synthesis and reconciliation of many of these diverse observations is attempted in a model which can accommodate most aspects of the classical sterol/Amphotericin barrel model and more recent observations as well.     

Effects of new amphotericin analogues on the scrapie isoform of the prion protein

Prion diseases or Transmissible Spongiform Encephalopathies (TSEs) are a group of neurodegenerative disorders associated with the conversion of a normal host prion protein (PrP(C)) into a pathogenic isoform (PrP(Sc)). Despite years of research, there is still no known cure for TSEs. Amphotericin B (AmB), an anti-fungal antibiotic, has antiprion activity but its usage is limited by its toxicity. This study assessed the antiprion properties of new amphotericin analogues in which the exocyclic carboxyl groups were replaced by methyl groups. These analogues reduced levels of the abnormal PrP(Sc) isoform of the mouse prion protein in cultured cells. 16-descarboxyl-16-methyl-amphotericin B (16B) had antiprion activity equivalent to that of amphotericin B and was significantly less toxic to cells as determined by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide dye reduction assay. A non-anti-fungal analogue, 16-descarboxyl-16-methyl-19-O-(6-deoxyhexosyl)-19-O-desmycosaminyl-amphotericin (16-19B) had higher antiprion activity and significantly lower toxicity than AmB. Some of the new amphotericin analogues may have potential as antiprion drugs.     

Head-to-tail interaction between amphotericin B and ergosterol occurs in hydrated phospholipid membrane

Amphotericin B (AmB) is thought to exert its antifungal activity by forming an ion-channel assembly in the presence of ergosterol. In the present study we aimed to elucidate the mode of molecular interactions between AmB and ergosterol in hydrated phospholipid bilayers using the rotational echo double resonance (REDOR) spectra. We first performed (13)C{(19)F}REDOR experiments with C14-(19)F-labeled AmB and biosynthetically (13)C-labeled ergosterol and implied that both "head-to-head" and "head-to-tail" orientations occur for AmB-ergosterol interaction in the bilayers. To further confirm the "head-to-tail" pairing, (13)C-labeled ergosterol at the dimethyl terminus (C26/C27) was synthesized and subjected to the REDOR measurements. The spectra unambiguously demonstrated the presence of a "head-to-tail" orientation for AmB-ergosterol pairing. In order to obtain information on the position of the dimethyl terminus of ergosterol in membrane, (13)C{(31)P}REDOR were carried out using the labeled ergosterol and the phosphorus atom of a POPC headgroup. Significant REDOR dephasing was observed at the C26/C27 signal of ergosterol in the presence of AmB, but not in the absence of AmB, clearly indicating that the side-chain terminus of ergosterol in the AmB complex comes close to the bilayer surface.     

Investigations on feasibility of in situ development of amphotericin B liposomes for industrial applications

Amphotericin B (AmB) liposome formulations are very successful in the treatment of fungal infections and leishmaniasis. But higher cost limits its widespread use among people in developing countries. Therefore, we have developed a modified ethanol-injection method for the preparation of AmB liposomes. Two liposomal formulations were developed with dimyristoyl phosphatidylcholine [F-1a] and soya phosphatidylcholine [F-2a], along with egg phosphatidyl glycerol and cholesterol. AmB was dissolved in acidified dimethyl acetamide and mixed with ethanolic lipid solution and rapidly injected in 5% dextrose to prepare liposomes. Liposomes were characterized on the basis of size (~100?nm), zeta (–43.3?±?2.8 mV) and percent entrapment efficiency (>95%). The in vitro release study showed an insignificant difference (P?≥?0.05) for 24-hour release between marketed AmB liposomes (AmBisome) and F-1a and F-2a. Proliposome concentrate, used for the preparation of in situ liposomes, was physically stable for more than 3 months at experimental conditions. Similarly, AmB showed no sign of degradation in reconstituted liposomes stored at 2–8°C for more than 3 months. IC₅₀ value of Ambisome (0.18 µg/mL) was comparatively similar to F-1a (0.17 µg/mL) and F-2a (0.16 µg/mL) against intramacrophagic amastigotes. Under experimental conditions, a novel modified method for AmB liposomes is a great success and generates interest for development as a platform technology for many therapeutic drug products.