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Objectives: Based on the assertion that fluorescence spectroscopy detects dimers of the polyene antibiotic amphotericin B (AmB), this technique was recently proposed to analyse the interaction of the drug with cell membranes. However, contradictory results indicate that this 'dimeric' fluorescence might actually originate from polyene impurities. We used a highly purified AmB to challenge this last proposal., Methods: Comparison of the fluorescence of AmB from different origins was made in dimethyl sulphoxide (DMSO); concentration and sodium dodecyl sulphate (SDS) addition dependencies were analysed in water., Results: Excitation of fluorescence in the absorption band of the AmB monomer (around 410 nm) revealed no difference between the different samples, in contrast with what was observed by excitation in the absorption wavelengths of self-associated AmB (around 325 nm). Furthermore, in this latter case, no concentration dependence was observed, in DMSO or in water. SDS addition increased the fluorescence in water., Conclusions: The fluorescence of AmB observed by excitation in the absorption wavelengths of self-associated species (around 325 nm) is explainable by the presence of impurities. Fluorescence is probably not appropriate for characterization of the drug interaction with cell membranes.

Flow cytometry light scattering was used to monitor size increase of Candida albicans (isolate ATCC 10231) cells in the presence or absence of the antifungal drug amphotericin B (AmB). This non-invasive and descriptive method allowed for the differentiation of dead and dormant sub-populations of cells. When inoculated into a growth medium without AmB, a progressive increase in light scattering was observed over a period of approximately 4 h, but without proliferation of the yeast. After this period, the light scattering distribution regressed to baseline level, whereas cell proliferation started. In the presence of AmB, all the cells shrank in size within approximately 4 h and proliferation was temporarily halted. However, in the presence of 0.4 microM AmB, a progressive increase of light scattering occurred after 21 h which was similar to that observed within the first 4 h in the absence of the antifungal. After approximately 24 h of incubation at this concentration of AmB, proliferation resumed. These observations indicate that this renewed cell proliferation was due to the reawakening of dormant cells in the presence of AmB (45% in the presence of 0.4 microM AmB) rather than the result of the development of viable cells that had escaped detection. This simple descriptive approach could be extended to other fungal strains or species, to other antifungal drugs and possibly to bacteria.

Aim: Heat treatment of deoxycholate-amphotericin B (AmB-DOC) leads to a therapeutically interesting supramolecular rearrangement (h-AmB-DOC); this reformulation improves the therapeutic index of AmB-DOC by reducing amphotericin B (AmB) toxicity in mammalian cell lines from 3- to 10-fold. Its activity in experimentally induced fungal infection in mice remains unchanged compared with AmB-DOC, whereas its activity is 2.5 times higher in Leishmania donovani-infected mice. This work investigates the in vitro mechanism that allows this improvement., Methods: In this study, we analysed the role of serum components on the interaction of h-AmB-DOC with two cultured cell lines: murine peritoneal macrophage cells (J774) and kidney epithelial cells (LLCPK1). The methods used were: spectrophotometry for AmB uptake; MTT assay for cell viability; and lactate dehydrogenase release for membrane damage., Results: In the presence of 10% fetal calf serum (FCS), the toxicity of AmB-DOC or h-AmB-DOC for both cell lines was null or weak. Interestingly, in J774 cells, the uptake of AmB in the form of h-AmB-DOC was much higher. In LLCPK1 cells, AmB uptake was more limited in both cases but remained higher with h-AmB-DOC. In the absence of FCS, no toxicity for either cell line was observed with h-AmB-DOC., Conclusions: These findings confirm the importance of serum proteins in AmB biodistribution and suggest that, in vivo, the reduced toxicity and the improved antileishmanial activity of AmB-DOC after moderate heating may be the result of its increased uptake by macrophages.

Heat-induced 'superaggregation' of deoxycholate-amphotericin B (AmB-DOC, Fungizone) was shown previously to reduce the in-vitro toxicity of this antifungal agent. We compared AmB-DOC with the formulation obtained by heating the commercial form (Fungizone, Bristol Myers Squibb, Paris, France) for 20 min at 70 degrees C, in the treatment of murine infections. An improvement of antifungal activity was obtained with heated AmB-DOC formulations due to a lower toxicity which allowed the administration of higher drug doses than those achievable with the commercial preparation. Single intravenous injections of heated AmB-DOC solutions were demonstrated to be two-fold less toxic than unheated ones to healthy mice. For mice infected with Candida albicans, the maximum tolerated dose was higher with heated than with unheated AmB-DOC solutions. In the model of murine candidiasis, following a single dose of heated AmB-DOC 0.5 mg/kg, 85% of mice survived for 3 weeks, whereas at this dose the immediate toxicity of the standard formulation in infected mice restricted the therapeutic efficacy to 25% survival. Both formulations were equally effective in increasing the survival time for murine cryptococcal pneumonia and meningoencephalitis. Injection of heated AmB-DOC solutions at a dose two-fold higher than the maximal tolerated dose observed with the unheated preparation (1.2 mg/kg) increased the survival time by a factor of 1.4 in cryptococcal meningoencephalitis. These results indicate that mild heat treatment of AmB-DOC solutions could provide a simple and economical method to improve the therapeutic index of this antifungal agent by reducing its toxicity on mammalian cells.

The amount of amphotericin B (AmB) associated with cultured murine macrophage-like J774 cells, after incubation with various AmB lipid formulations, was determined by absorption spectroscopy. Large, negatively charged, AmB-containing, multilamellar vesicles and small cholesteryl sulphate-AmB complexes both enhanced the amount of AmB associated with J774 cells at 37 degrees C (up to 500-fold the extracellular concentration). In contrast, AmB-containing, small, negatively charged vesicles (AmBisome), positively charged, oligolamellar vesicles and mixed micelles showed a lower association of the antibiotic with cells, compared with AmB added from a solution in dimethylsulphoxide or Fungizone. Experiments performed at 40 degrees C showed a large reduction of AmB uptake for AmB preparations and AmB added from a solution in dimethysulphoxide or Fungizone, suggesting a high percentage of internalization of the antibiotic. Experiments in the presence of cytochalasin B resulted in a decrease of AmB uptake mainly for the preparations of large diameter, suggesting that these formulations were taken up by phagocytosis. A comparative study with Chinese hamster ovary cells, a model of non-phagocytic cells, showed a reduction in the take up of AmB. This reduction was always more marked when AmB was incorporated in lipid formulations. On the other hand, accumulation of the antibiotic in J774 cells was shown to be followed by its release from the cells in an unbound form, the extent of release depending on the type of vector used. The results suggest that in some cases macrophages can be considered as reservoirs of antibiotic, releasing free AmB in the medium.

Renal tubular cells are a target of amphotericin B (AmB) toxicity, but the mechanisms involved in the tubular cell-AmB interactions are unknown. Ketoconazole was selected to lower the cholesterol content of rabbit renal tubular cells in primary culture. The consequences of cholesterol depletion on AmB nephrotoxicity was investigated in vitro as the inhibition of Na(+)-dependent phosphate uptake. After 1 h of exposure, AmB decreased phosphate uptake (49%, 77%, and 82% inhibition with 5, 10, and 20 microM of AmB, respectively). Pretreatment of cells with ketoconazole (10 microM for 24 h) reduced by 50% (P less than .01) the phosphate uptake inhibition induced by AmB, decreased cellular cholesterol synthesis (greater than 80% inhibition), and decreased AmB binding to cell membrane by 50%, as measured by the fluorescence extinction of a probe bound to tubular cell membrane. Incubation with exogenous exchangeable cholesterol again increased AmB binding to plasma membrane and restored AmB toxicity. These results demonstrate that the first step of AmB renal tubular toxicity is mediated by cellular cholesterol content and is parallel to the binding of AmB to cell membrane.

Amphotericin B (AmB) binds to the cholesterol in lipoproteins, as determined by comigration in density gradient ultracentrifugation and changes in the circular dichroic spectrum. The saturation curve and Scatchard plots obtained with circular dichroism suggest that four to 10 cholesterol molecules in low-density lipoproteins bind to one molecule of AmB. AmB interacts more rapidly with low- and very-low-density lipoproteins than with high-density lipoproteins, but the circular dichroic spectrum of the complexed species is the same in all three cases. AmB also binds to other proteins in blood, but much higher concentrations of these proteins than of lipoproteins are needed for comparable binding. Interaction with lipoproteins stabilizes the antifungal activity of AmB. Interaction with lipoproteins and with much higher concentrations of other proteins in blood can also inhibit the effects of AmB on red blood cells, which contain cholesterol in their plasma membranes, but not the effects on Candida albicans, whose membranes contain ergosterol. An appropriate inference is that, when used clinically, AmB circulates in blood bound to lipoproteins and other proteins. The toxic and therapeutic effects of AmB in clinical situations are thus contingent on competitive interactions between sterol-containing cellular membranes of the host and the parasite and components of blood, such as lipoproteins and proteins.