The interactions of amphotericin B with various sterols in relation to its possible use in anticancer therapy.

Amphotericin B (AmB) is still the most common anti-fungal agent used to treat systemic fungal infections. It is known that this antibiotic acts by forming pores with the ergosterol contained in the membranes of fungi, but it also interacts with the cholesterol contained in the membranes of eukaryotic cells, hence its toxicity. AmB may also interact with the most common oxidation products of cholesterol found in vivo, together with interacting with biosynthetic precursors of cholesterol, namely, lanosterol and 7-dehydrocholesterol (7-DHC). The purpose of the present work was to study the interactions in solution between AmB and these various sterols, the techniques used being UV-Vis spectroscopy and differential scanning calorimetry. The results are globally interpreted in terms of the structural differences between the sterols. We show that AmB selectively interacts with 7-DHC which, according to a recent hypothesis proposed in the literature, has been identified in connexion with a therapeutic strategy against hepatocellular carcinomas. We find that the affinity of AmB towards 7-DHC is even greater than the affinity of the antibiotic towards ergosterol. We also find that AmB selectively interacts with the principal oxidation product of cholesterol, 7-ketocholesterol, a situation that has to be taken into account when AmB is administered.

A kinetic study of the oxidation effects of amphotericin B on human low-density lipoproteins.

The UV-visible results of this kinetic study show that amphothericin B as Fungizone is a much stronger oxidant than CuSO(4), itself a powerful oxidant of low-density lipoprotein (LDL). Amphotericin B as AmBisome alone has no oxidizing effect on LDL while a mixture of both AmBisome and CuSO(4) induces an important potentialization of the LDL oxidation. These results allow us to believe that the high toxicity of amphotericin B is related to its capacity to modify and to weaken the structure of LDL. In addition, differential scanning calorimetry experiments show that the oxidative modifications of LDL by CuSO(4) or by amphotericin B proceed through different mechanisms.

The structuring effects of amphotericin B on pure and ergosterol- or cholesterol-containing dipalmitoylphosphatidylcholine bilayers: a differential scanning calorimetry study.

Amphotericin B (AmB) is the most widely used polyene antibiotic to treat systemic fungal infections which affect an increasing number of immunocompromised patients. It is generally thought that AmB forms pores within the fungi membranes by interacting with ergosterol, the main sterol of fungi. However, it also interacts with the cholesterol contained in mammalian cells, hence its toxicity. In order to have a better understanding of the interactions prevailing between AmB and sterols, differential scanning calorimetry was used to study various mixtures incorporating from 6.5 to 25 mol% of AmB in pure dipalmitoylphosphatidylcholine (DPPC) vesicles and in ergosterol- or cholesterol-containing DPPC vesicles. The sterol concentration was kept constant at 12.5 mol% with respect to the phospholipid. Our results show that three phases co-exist when AmB is dispersed in the pure phospholipid. One corresponds to the phospholipid phase alone. The two others are characterised by a broad transition at temperatures higher than the main transition temperature of the pure phospholipid, corresponding to the drug in interaction with the aliphatic chains of the lipid. The fact that the transition temperatures of these additional components are higher than that of the pure phospholipid suggests that AmB interacts strongly with the aliphatic chains of the lipid, consistent with the idea prevailing in the literature that AmB by itself may form pores in a lipid matrix. When AmB interacts with cholesterol-containing bilayers the thermograms also present three components. Upon increasing the concentration of AmB, though, an important broadening of these components is observed which is explained in terms of destabilisation of the organisation of the aliphatic chains. The situation is strikingly different if ergosterol is present in the lipid matrix. The thermograms remain unmodified as the concentration of AmB is increased and a broad transition, now involving only two components when the thermograms are decomposed, is observed. An analysis of the results shows that various interacting units, e.g. AmB+DPPC and (AmB+ergosterol)+DPPC, are present within the membrane. These units involve the phospholipid and hence contribute to its structurisation. The important differences between the thermograms obtained with the ergosterol- as compared to the cholesterol-containing bilayers, in spite of the structural similarity of these two sterols, provides strong evidence for the selectivity of interaction of AmB with ergosterol as compared to cholesterol. It is thus clear that the action of AmB on cholesterol- as compared to ergosterol-containing membranes results from different mechanisms. Finally, UV-visible spectra of AmB in pure as well as sterol-containing DPPC vesicles show the presence of absorption bands that give support to the interpretation derived from the calorimetric data.

Effect of amphotericin B on dipalmitoylphosphatidylcholine membranes: calorimetry, ultrasound absorption and monolayer technique studies.

Amphotericin B (AmB) is a popular drug frequently applied in the treatment of mycosis. Differential scanning calorimetry (DSC), ultrasound absorption and monomolecular layer technique were applied to study the effect of AmB on organisation of dipalmitoylphosphatidylcholine (DPPC) membranes. DSC-determined enthalpy of the main phase transition of DPPC liposomes was found to be a sensitive parameter to monitor AmB-DPPC interaction. The enthalpy of the phase transition decreases with the increase in molar fraction of AmB incorporated to membranes. The exceptionally sharp decrease in the enthalpy of the transition was observed in the membranes containing 5-7 mol% AmB. Ultrasound absorption-monitored main phase transition of DPPC is very broad under the presence of 5 mol% AmB showing destabilisation and disorganisation of a membrane structure. These findings are discussed in comparison to monomolecular layer study of two-component DPPC-AmB system. Analysis of the surface pressure-molecular area isotherms of compressing DPPC-AmB films at the air-water interface shows pronounced increase in mean molecular area at AmB concentrations corresponding to those found to destabilise DPPC membranes of liposomes. Disorganisation of lipid bilayers due to the presence of AmB in concentrations below 10 mol% with respect to lipid is discussed in terms of toxicity and side effects of this drug.

Amphotericin B toxicity as related to the formation of oxidatively modified low-density lipoproteins.

The effect of amphotericin B on the oxidation and degradation of low- and high-density lipoproteins was investigated by UV-vis spectroscopy, electron microscopy, electrophoresis, and size-exclusion chromatography. Two formulations of the drug were used: the commercial Fungizone and a new, less toxic, liposomal formulation, AmBisome. It was shown that Fungizone strongly enhanced the oxidative deformation of low-density lipoprotein structure while AmBisome did not bind to this lipoprotein fraction and did not affect its oxidation. It was shown that amphotericin B contained in Fungizone extracted cholesterol from low-density lipoproteins which sensitized them to oxidation. Both formulations of amphotericin B studied here did not bind to high-density lipoprotein and did not affect the process of its oxidation.

The effect of aggregation state of amphotericin-B on its interactions with cholesterol- or ergosterol-containing phosphatidylcholine monolayers.

Amphotericin B (AmB) is the most effective antibiotic used in the treatment of systemic fungal infections. It is generally thought that the activity of this drug results from its interaction with ergosterol, the main sterol of fungi membranes. However, AmB also interacts with cholesterol, the major sterol of mammal membranes, thus limiting the usefulness of this drug due to its relatively high toxicity. The aim of the present work is to study the molecular basis of the interactions of AmB with these sterols contained in a DOPC film by using the monolayer technique. Two different concentrations of the sterols in the film (13 and 30%) at an initial surface pressure of 30 mN/m were studied, which correspond to conditions found in various biological membranes. Four concentrations of AmB in the subphase, ranging from a molecularly dispersed to a highly aggregated state of the drug were studied. Our results show that the monomeric form of AmB interacts with the ergosterol containing film solely. On the other hand, when AmB is dispersed as a pre-micellar or as a highly aggregated state in the subphase, a very significant selectivity of its interactions between the two sterols is observed which is shown in our experimental results by a difference of 8 mN/m in the surface pressure when AmB interacts with ergosterol as compared to cholesterol. We show that the activity of AmB is most likely related to the micellar form of the antibiotic. In addition, we observe that upon increasing the amount of ergosterol in the film, the insertion of AmB is largely promoted, results that are discussed in terms of the molecular organization of the sterols within the monolayer film. We show that these results provide a better understanding of the action of AmB (activity/toxicity) at the membrane level.

Modulation of amphotericin B activity by association with mannose ester.

The biological and molecular properties of a new formulation of Amphotericin B complexed with the surfactant palmitoyl mannose were studied in in vitro as well as in in vivo situations. The properties analyzed include toxicity towards two types of mammalian cells and four fungi strains, effect on macrophage activity, inflammatory properties, acute toxicity in mice and spectral behavior in presence of foetal calf serum or 6% propanol. The results demonstrate that, in presence of palmitoyl mannose, the cytotoxicity of AmB is decreased towards both, fungal and mammalian cells while its fungistatic potential is increased, its inflammatory properties are conserved and its acute toxicity is significantly diminished. These effects can be potentially explained by the formation of a complex between AmB and the sugar ester that impedes the interaction of the drug with either serum components or cell membrane constituents. The overall properties of AmB in the complex would be expected to favor an increase in the immunoadjuvant properties of the drug, a more localized inflammation during fungal infection and consequently a better therapeutic efficiency.

Effects of the aggregation state of amphotericin B on its toxicity to mice.

Amphotericin B (AmB) is a very effective antifungal agent for most systemic fungal infections. However, the relatively high toxicity of this drug imposes limits on its clinical usefulness. Most of the current work in this field is devoted to the search for less-toxic formulations of the drug. Here we describe the effects of three surfactants, one anionic and the other two nonionic, on the aggregation state of AmB in solutions which were injected intravenously into mice. The degree of aggregation of AmB was monitored spectroscopically and by light scattering. The toxicity was expressed as percentage of survivors. These results were compared with those obtained with doses of AmB the same as those present in a commercial formulation of AmB, Fungizone. Two surfactants, lauryl sucrose and sodium deoxycholate, used at concentrations which induced monomerization of AmB, substantially decreased the acute toxicity of AmB to mice. Conversely, the third surfactant, Tween 80, showed a synergistic potentiation of the toxicity of the antibiotic. A good correlation was found between the in vivo toxicity and the aggregation state of AmB in injected solutions. Solutions in which AmB was almost entirely monomeric were half as toxic after 24 h and about six times less toxic after 1 week than the corresponding solutions of Fungizone.

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.

The effect of surfactants on the aggregation state of amphotericin B.

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