Janina Brajtburg

Amphotericin B: current understanding of mechanisms of action.

This minireview describes the problems associated with the use of Amphotericin B and what is known about the mechanism(s) of its action. The efforts to design a more efficient vehicle for AmB are summarized in the following minireview (8)

Amphotericin B: delivery systems.

Because the clinical utility of amphotericin B (AmB) is limited by its toxicity of host cells, an important question is how to best direct it specifically to the fungus or at least to the site of infection or, alternatively, how to keep it away from the host cells. One strategy is to use a vehicle other than deoxycholate. In this minireview, we review some of the relevant studies addressing this issue

Role of cell defense against oxidative damage in the resistance of Candida albicans to the killing effect of amphotericin B.

A laboratory-derived mutant of Candida albicans B311 (L) and a clinical isolate (C) of C. albicans, both lacking membrane ergosterol, were less susceptible to amphotericin B (AmB)-induced cell membrane permeability to K+ and lethality than was the wild-type laboratory strain (B311) which contained ergosterol. The resistance of L and C to AmB-induced killing was much greater than the level of resistance to AmB-induced cell membrane permeability. L and C were also less susceptible to killing by H2O2 than was B311, and when treated with menadione, they each produced less H2O2 than did B311. In addition, their levels of catalase activity were 3.8-fold (L) and 2-fold (C) higher than that of B311. The ergosterol deficiency in L and C probably impaired AmB binding to the cells, thereby lowering AmB effectiveness as measured by both cell membrane permeability and killing. Resistance of strains L and C to oxidation-dependent damage likely contributed to a diminished response to AmB-induced lethality.

Involvement of oxidative damage in erythrocyte lysis induced by amphotericin B.

Lysis of human erythrocytes induced by amphotericin B was retarded when the oxygen tension of the incubation mixture was reduced or when the antioxidant catalase was added; lysis was accelerated when cells were preincubated with the prooxidant ascorbate. In the atmosphere of reduced oxygen tension, the erythrocytes containing carboxyhemoglobin lysed at a slower rate than did the cells containing oxyhemoglobin. Consistent with a role for oxidative damage in lysis, the mixture of erythrocytes and amphotericin B showed an increase in malonyldialdehyde, the product of peroxidation, which paralleled the progression of hemolysis. In contrast, the permeabilizing effect of amphotericin B, measured as a decrease in intracellular K+, was not affected by changes in oxygen tension, catalase, or ascorbate treatment. These results imply that oxidant damage is involved in the lytic, but not in the permeabilizing, action of amphotericin B.

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.

Affinity of amphotericin B for phosphatidylcholine vesicles as a determinant of the in vitro cellular toxicity of liposomal preparations

Candida albicans and human erythrocytes were treated with liposomal amphotericin B (AmB) obtained by incubation of free AmB with small unilamellar vesicles (SUV) composed of unsaturated fatty acyl chains phosphatidylcholine (egg-yolk PC). Cellular effects were determined by changes in the K+ internal content of cells and in the number of colonies formed by fungal cells or as hemolysis, measured as a decrease in haemoglobin retention by erythrocytes. Dose-response curves were obtained by two procedures: either the ratio of AmB to phospholipids was kept constant over the AmB concentration range used (R = 10(-2] or the phospholipid concentration was kept constant (C = 0.2 mM) and the concentration of AmB varied. The liposomal preparations of AmB were as active against fungi as AmB in dimethylsulfoxide but less active (internal K+ changes) or inactive (hemolysis) against erythrocytes. On the other hand the binding of AmB to the SUV, as a function of the AmB concentration, was monitored by circular dichroism, fluorescence and UV absorption, in the two conditions used for the cellular studies. The amount of AmB bound when the total concentration of antibiotic was 2.10(-7) M was very low but increased with concentration and reached 90% at 10(-5) M. In all the assays we used, the anticellular effects could be attributed to the levels of AmB remaining free (unbound to the lipids). The variations of these levels with total AmB concentration could therefore explain the increased selectivity of liposomal AmB in toxicity against fungi and erythrocytes as compared to that of AmB added as a solution in dimethylsulfoxide. Indeed fungal cells are sensitive to low concentrations of AmB in dimethylsulfoxide; at these concentrations, in liposomal preparations, AmB is not bound to phospholipids and therefore as active as if added in dimethylsulfoxide. By contrast erythrocytes are only sensitive to much higher concentrations of AmB in dimethylsulfoxide; at these concentrations AmB is almost totally bound to phospholipids and therefore much less active.

Circular dichroism for the determination of amphotericin B binding to liposomes

Circular dichroism (CD) of the antifungal antibiotic amphotericin B (AmB) can be used to characterize the liposomal preparations of the drug with regard to the levels of drug bound to the lipids. The very intense dichroic doublet centered around 340 nm of free amphotericin B in water or the dichroism observed above 435 nm can be used to determine the percentages of bound AmB and free AmB in preparations containing high antibiotic/lipid ratios (ranging from 10(-2) to 10(-1] used in these carrier systems. Examples are given for AmB in the presence of small unilamellar vesicles prepared from four saturated fatty acyl chain phosphatidylcholines of different chain lengths, with or without cholesterol. The transfer of AmB from vesicles to two blood components, serum albumin, and lipoproteins can also be monitored by CD under particular conditions.

Effects of elevation of serum cholesterol and administration of amphotericin B complexed to lipoproteins on amphotericin B-induced toxicity in rabbits.

Amphotericin B was infused into normal rabbits or rabbits made hypercholesterolemic by diet. There was no difference in amphotericin B-induced toxicity between these two groups. Amphotericin B given in a mixture with human low-density lipoproteins was more toxic than when given without lipoproteins.

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.

Stimulatory, permeabilizing, and toxic effects of amphotericin B on L cells.

High concentrations of amphotericin B (AmB) killed mouse L cells, but low concentrations increased plating efficiency and stimulated the incorporation of labeled precursors into DNA and RNA. Thus, there were two disparate effects of AmB on L cells, stimulatory and toxic, and they occurred in distinct dose-related stages. AmB also affected the permeability of L cells. In dose-response studies, increases in cell membrane permeability, measured as the loss of K+ ions, occurred along with the stimulation of [3H]uridine incorporation into RNA. In contrast, stimulation of [3H]thymidine incorporation into DNA was only observed in cells recuperating from AmB-induced permeability changes. When the K+ concentration in the medium was lowered to 0.5 from 4.5 mM, or when 1 mM ouabain was added to the cultures, cell killing was potentiated, but the stimulatory and permeabilizing effects of subtoxic concentrations of AmB were unaffected. Furthermore, etruscomycin, a polyene antibiotic without any permeabilizing effects, nevertheless induced an enhancement of plating efficiency and of incorporation of [3H]uridine into RNA and [3H]thymidine into DNA. Our results suggest that the dose-related stimulatory, permeabilizing, and toxic effects of AmB most probably have distinct mechanisms of action and may be independent of one another.