Ilona Gruda

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.

Application of differential spectra in the ultraviolet-visible region to study the formation of amphotericin B-sterol complexes

The extent of complex formation between the polyene antibiotic, amphotericin B, and cholesterol or ergosterol was investigated and a method for a quantitative measurement of the complex formation was developed. The effect of experimental conditions on the magnitude of the amphotericin B-sterol interaction and on the selectivity of this interaction showed that there was only a narrow range of solvent composition in which the differential selectivity of amphotericin B towards these two sterols could be observed.

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.

Effects of the detergent sucrose monolaurate on binding of amphotericin B to sterols and its toxicity for cells.

Amphotericin B (AmB) is a potent antifungal agent used to treat patients with systemic mycoses. The cytotoxicity of AmB is related to its binding to membrane sterols and its clinical usefulness is based on its greater affinity to ergosterol, the fungal sterol, compared to the mammalian cell sterol, cholesterol (1-3). Here we report that sucrose monolaurate (L.S.) decreased the binding of AmB to cholesterol without interfering with its binding to ergosterol. Furthermore, the toxicity of AmB for mouse erythrocytes (RBC) and cultured mouse fibroblasts, L-929, cells was significantly decreased by low concentrations of L.S., whereas under the same conditions, its toxicity for Candida albicans was unaffected. We observed a very good correlation between the spectroscopic and cell studies. The results reported here on the effects of L.S. on the selectivity of AmB toxicity for fungal cells compared to animal cells and the relative nontoxic nature of sugar esters suggest a potential for compounds of this type to enhance the therapeutic index of AmB.

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 CuSO4, 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 CuSO4 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 CuSO4 or by amphotericin B proceed through different mechanisms.

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.

Polarized spectra of a series of stilbazolium merocyanines in polyvinyl alcohol films

Abstract Polarized absorption, fluorescence and photoacoustic spectra of a series of stilbazolium merocyanines in stretched and unstretched polyvinylalcohol films were measured. Protonated dyes and free based are present in PVA films. The degree of orientation of the dye molecules depends on the substituents present in the molecule. The bulky t-butyl causes a decrease in the degree of orientation and prevents the formation of ground state complexes. In the excited state, the steric hindrance is less important and exciplexes are formed. Only ground state complexes and exciplexes fluoresce. The excitation of monomers is followed by effective thermal deactivation. Ground state complexes and exciplexes have different geometries. Axial deformation of PVA films favours excimer formation, but causes a partial dissociation of the ground state complexes.

Separation of overlapping spectra from evolving systems using factor analysis.: 2. Amphotericin B in aqueous propanol and in aqueous lauroyl sucrose

Abstract A method based on factor analysis is used to separate the spectra of individual species from the experimental absorption spectra obtained from a fixed amount of amphotericin B dissolved in aqueous solutions. In aqueous propanol solutions and in aqueous lauroyl sucrose solutions, five species are identified: two monomers and three aggregates. Although the spectra of the different species in the two media are similar, the intensity signatures of the two systems are quite different and serve to characterize them.

Concentration dependent dual effect of the monolauryl ester of sucrose on the antifungal activity and absorption spectra of amphotericin B (Fungizone)

A mild detergent, the monolauryl ester of sucrose (LS), at concentrations which ranged from 0.008 to 0.03%, enhanced amphotericin B (AmB) toxicity against Saccharomyces cerevisiae and Cryptococcus neoformans cells. At higher concentrations, 0.06 to 2.5%, LS inhibited AmB effects on these two fungi. We analyzed changes in the absorption spectrum of AmB induced by LS at these two concentration ranges by comparing ratios (R values) of AmB absorbance at 409 nm, the wavelength characteristic of non-aggregated (monomeric) AmB, to absorbance at 328 nm, the wavelength characteristic of aggregated AmB. Low concentrations of LS caused a decrease in R, whereas the higher LS concentrations increased R. Therefore, LS had concentration-dependent dual effects on the antifungal activity of AmB which correlated with shifts in the physical states of AmB. The concentration range of LS required to inhibit the antifungal effects of AmB was about 1000-fold greater than the previously reported concentrations required to inhibit AmB toxicity to mammalian cells (Gruda, I., Gauthier, E., Elberg, S., Brajtburg, J. and Medoff, G. (1988) Biochem. Biophys. Res. Commun. 154, 954-958). This suggests that LS may be a useful agent to decrease AmB toxicity to host cells without affecting the antifungal effects. Moreover, increase in AmB toxicity induced by low concentrations of LS suggests the possibility that synergistic interaction between fatty acid esters and polyene antibiotics may have therapeutic value.

Analysis of amphoteric surfactants of the alkylaminopropylglycine type by gas chromatography

A procedure was developed for gas chromatographic analysis of glycine-type amphoteric surfactants. The commercial samples contained N-alkylaminopropylglycines, N-alkylamines and N-alkylaminopropylamines. Some di- and triacids were also detected. The method proposed allows a good separation according to both the carbon number and the chemical function.