Carrier effects on biological activity of amphotericin B.

Amphotericin B (AmB), the drug of choice for the treatment of most systemic fungal infections, is marketed under the trademark Fungizone, as an AmB-deoxycholate complex suitable for intravenous administration. The association between AmB and deoxycholate is relatively weak; therefore, dissociation occurs in the blood. The drug itself interacts with both mammalian and fungal cell membranes to damage cells, but the greater susceptibility of fungal cells to its effects forms the basis for its clinical usefulness. The ability of the drug to form stable complexes with lipids has allowed the development of new formulations of AmB based on this property. Several lipid-based formulations of the drug which are more selective in damaging fungal or parasitic cells than mammalian cells and some of which also have a better therapeutic index than Fungizone have been developed. In vitro investigations have led to the conclusion that the increase in selectivity observed is due to the selective transfer of AmB from lipid complexes to fungal cells or to the higher thermodynamic stability of lipid formulations. Association with lipids modulates AmB binding to lipoproteins in vivo, thus influencing tissue distribution and toxicity. For example, lipid complexes of AmB can be internalized by macrophages, and the macrophages then serve as a reservoir for the drug. Furthermore, stable AmB-lipid complexes are much less toxic to the host than Fungizone and can therefore be administered in higher doses. Experimentally, the efficacy of AmB-lipid formulations compared with Fungizone depends on the animal model used. Improved therapeutic indices for AmB-lipid formations have been demonstrated in clinical trials, but the definitive trials leading to the selection of an optimal formulation and therapeutic regimen have not been done.

Comparison of the leishmanicidal activity of fungizone, liposomal AmB and amphotericin B incorporated into egg lecithin-bile salt mixed micelles.

We compared leishmanicidal activity of five formulations of amphotericin B (AmB). AmB used as Fungizone immediately caused cell lysis, at lower concentrations partial, and at higher concentrations total; the non-lysed cells reassuming growth. AmB formulated with egg-lecithin and bile salt at doses equivalent to those of Fungizone was not active, whereas at higher doses it resulted in a delayed but complete inhibition of cell growth. The activity of liposomal AmB was comparable to that of Fungizone. The time of initial leishmanicidal activity of the five formulations tested can be ascribed to the rate of free, monomeric AmB delivered. Features that are characteristic of the anticellular effect of AmB formulated with egg lecithin and bile salts, a delay in activity, a requirement for higher concentration and total inhibition of cell growth, were also noted.

Treatment of murine candidiasis and cryptococcosis with amphotericin B incorporated into egg lecithin-bile salt mixed micelles.

Amphotericin B (AmB) with deoxycholate (Fungizone) and AmB incorporated into mixed micelles (AmB-mixMs) composed of egg lecithin with glycocholate, deoxycholate, or taurocholate were compared as treatments for murine infections. For mice infected with Candida albicans, treatment consisted of a single intravenous injection; for mice infected with Cryptococcus neoformans, treatment consisted of two intravenous injections. The maximal tolerated doses of AmB as Fungizone were 1.25 mg/kg of body weight in mice with candidiasis and 2.5 mg/kg of body weight in mice with cryptococcosis. The AmB-mixMs were nontoxic to mice at doses of 80 and 100 mg/kg of body weight and were therapeutically more active than the maximal tolerated dose of Fungizone in both models of infection. However, when Fungizone or AmB-mixMs were administered at equivalent doses of AmB, AmB-mixMs were more active in treating murine candidiasis, whereas Fungizone was more active in treating murine cryptococcosis.

Amphotericin B incorporated into egg lecithin-bile salt mixed micelles: molecular and cellular aspects relevant to therapeutic efficacy in experimental mycoses.

The cellular activities of amphotericin B (AmB) used as Fungizone were compared with those of AmB complexed to either egg lecithin and glycocholate (Egam) or egg lecithin and deoxycholate (Edam). Under conditions in which leakage of K+ from erythrocytes and cultured L cells treated with Fungizone was almost complete, Egam and Edam containing concentrations of AmB severalfold greater than the concentration of AmB in Fungizone had no effect but retained the ability to decrease the level of retention of K+ in fungal cells. Analysis by absorption and circular dichroism spectroscopy demonstrated that when these formulations containing AmB at concentrations of less than 10(-5) M were added to buffer, the AmB dissociated slowly as monomers from Egam or Edam and dissociated rapidly as a mixture of monomers and self-associated species from Fungizone. We propose that in Egam and Edam, the absence of free AmB in the self-associated form reduces the toxicity of AmB to mammalian cells, whereas the presence of monomeric AmB results in the retention of the antifungal activities of these complexes.

Structure-activity study of inhibition of amphotericin B (Fungizone) binding to sterols, toxicity to cells, and lethality to mice by esters of sucrose.

The effects of four monoesters of sucrose with different acyl chain lengths (palmitate, C16; myristate, C14; laurate, C12; and caprate, C10) on the aggregation state of amphotericin B (AmB), its binding to cholesterol and ergosterol, its toxicity to cells, and its lethality to mice were determined. In solution, all four of these esters inhibited AmB binding to cholesterol more than to ergosterol; this effect correlated with the ester-induced shift from the mainly aggregated form of AmB to the mainly monomeric form. In experiments with cells, the esters inhibited the toxicity of AmB to mouse erythrocytes and cultured mouse fibroblast L-929 cells more than its toxicity to Candida albicans cells. When injected intravenously with AmB, these esters decreased AmB lethality to mice. In all of these assays, the ester with the shortest chain length (caprate) was much less potent than the other three esters. Our results indicate a correlation between in vitro and in vivo assays and suggest that the in vitro and in vivo selectivity of AmB may be enhanced by surface-active agents which modulate the aggregation state of AmB.

Inhibition of amphotericin B (Fungizone) toxicity to cells by egg lecithin-glycocholic acid mixed micelles.

Mixed micelles prepared from egg lecithin and the sodium salt of glycocholic acid markedly inhibited amphotericin B toxicity to mammalian cells without significantly affecting the antifungal effects of the drug.

Lysis of human promyelocytic HL-60 cells by amphotericin B in combination with 2-chloroethyl-1-nitrosoureas: role of the carbamoylating activity of nitrosoureas.

The combinations of amphotericin B (AmB) with 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) or 2-cyclohexyl isocyanate, the carbamoylating decomposition product of CCNU, were more potent in lysing HL-60 cells than the combinations of AmB with 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) or 2-chloroethyl isocyanate, the carbamoylating decomposition product of BCNU. The noncarbamoylating nitrosoureas 1-(2-chlorethyl)-3-(2,6-dioxo-3-piperydyl)-1-nitrosourea and 2-[3-(2-chloroethyl)-3-nitrosoureido]-D-glucopyranose did not alter AmB effects on HL-60 cells. These results indicate that the potentiating action of CCNU and BCNU on the lytic effects of AmB is associated with the carbamoylating activity of these nitrosoureas. It is likely that the greater carbamoylating activity of CCNU, compared to BCNU, is responsible for the differences in potency of the two AmB-drug combinations.

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.

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.

Effects of ascorbic acid on the antifungal action of amphotericin B.

Ascorbic acid enhanced the lethal but not the permeabilizing effects of amphotericin B on Candida albicans and Cryptococcus neoformans cells. Two other ene-diol acids, D-erythorbate and dihydroxyfumarate, also enhanced the lethal action of amphotericin B on Can. albicans. Maleic acid and gulanolactone, compounds structurally related to ascorbic acid but not containing the ene-diol group, had no such effect. It is assumed that ascorbic acid and the two other ene-diol acids acting as pro-oxidants augmented the oxidation-dependent killing of fungal cells induced by amphotericin B.

Study of the effects of liposomal amphotericin B on Candida albicans, Cryptococcus neoformans, and erythrocytes by using small unilamellar vesicles prepared from saturated phospholipids.

We compared the anticellular effects of liposomal amphotericin B (AmB) formed from AmB and small unilamellar vesicles. The small unilamellar vesicles with or without cholesterol were prepared from three L-alpha-phosphatidylcholines with saturated acyl chains of different lengths: distearoyl (C18), dipalmitoyl (C16), and dimyristoyl (C14). We found that the anticellular potency of liposomal AmB, compared with that of free AmB, decreased with decreasing length of the acyl chain of the phospholipid and increased with the addition of cholesterol. In a parallel study (S. Jullien, A. Vertut-Croquin, J. Brajtburg, and J. Bolard, Anal. Biochem. 172:197-202, 1988), we found that binding of AmB to vesicles decreased with increasing length of the acyl chain of the phospholipid and decreased with the addition of cholesterol. We conclude that the anticellular effects of liposomal AmB preparations are due to the levels of AmB remaining free (unbound to the lipids) in these preparations.

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.

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.

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.

Interference with effects of amphotericin B on Candida albicans cells by 2-chloroethyl-1-nitrosoureas.

Two nitrosoureas, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) and 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), with strong carbamoylating and weak alkylating activities, interfered with the induction of potassium leakage and lethal action of amphotericin B (AmB) on Candida albicans. 2-Cyclohexyl isocyanate, the product of decomposition of CCNU, and 2-chloroethyl isocyanate, the product of decomposition of BCNU, also interfered with the anticandidal actions of AmB. In contrast, two nitrosoureas with weak carbamoylating and strong alkylating activities, 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperydyl)-1-nitrosourea and 2-[3-(2-chloroethyl)-3-nitrosoureido]-D-glucopyranose, did not affect AmB action against C. albicans. These results indicate that the inhibitory action of CCNU and BCNU on the anticandidal effects of AmB is associated with the carbamoylating activity of these nitrosoureas.

Two mechanisms of synergism when amphotericin B is used in combination with actinomycin D or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea against the human promyelocytic leukemia cell line HL-60.

The toxic effects of the combinations of amphotericin B (AmB) and actinomycin D or AmB and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea were measured against the human promyelocytic leukemia cells HL-60. The toxicities of both drug combinations were greater than the additive toxicity of each of the drugs used singly, but the exact conditions under which synergism was achieved differed for each combination. The synergism achieved by the AmB-actinomycin combination was accompanied by an AmB-induced increase in uptake of actinomycin D by the HL-60 cells, whereas the synergism of the AmB-1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea combination could be linked to potentiation by 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea of AmB-induced oxidative injury. These results indicate that the synergism of these two drug combinations was caused by different mechanisms.

Toxicity and induction of resistance to Listeria monocytogenes infection by amphotericin B in inbred strains of mice.

Amphotericin B (AmB) treatment before infection with the bacterium Listeria monocytogenes prolonged survival of AKR mice but shortened survival of C57BL/6 mice compared with survival of untreated infected controls. C57BL/6 mice were also more sensitive to the acute toxic effects of AmB than AKR mice, as were (C57BL/6 X AKR)F1 hybrid mice. Spleen cells and erythrocytes (RBCs) from the C57BL/6 and the F1 hybrid mice were both more sensitive to the lytic and lethal effects of AmB than corresponding cells from AKR mice. Biochemical analysis indicated that catalase levels in RBCs from C57BL/6 and F1 hybrid mice were about 60% of those found in RBCs from AKR mice. The lysis by AmB of RBCs from all these strains of mice was inhibited by catalase or incubation in a low-oxygen environment. These findings suggest that (i) the low catalase levels in C57BL/6 and F1 hybrid mice may limit the protection of cells from the oxidant damage involved in AmB action, and (ii) the toxicity which occurs at low concentrations of AmB in the mouse strains with low intracellular catalase levels may interfere with or ablate the AmB-induced increases in mouse resistance to L. monocytogenes infection.

Amphotericin B-induced oxidative damage and killing of Candida albicans.

Amphotericin B (AmB) is known to bind to ergosterol in fungal cell membranes, but the precise mechanism of its toxicity to cells is as yet poorly understood. AmB autooxidizes, and it is possible that its antifungal effects could result from oxidative damage. Exposure of protoplasts of Candida albicans to AmB under hypoxic conditions reduced protoplast lysis by as much as 80% compared with incubations in air. Protoplasts were protected from AmB-induced lysis by exogenous catalase and/or superoxide dismutase (SOD). Whole cells of C. albicans were protected by exogenous catalase from AmB-induced leakage of [3H]leucine and from killing by AmB. Cells grown on medium inducing high levels of endogenous catalase were resistant to AmB-induced growth inhibition. In contrast, AmB-induced K+ leakage was not hindered under hypoxic conditions or in the presence of catalase or SOD. Thus the lethal and lytic effects of AmB on C. albicans cells and protoplasts, but not prelethal AmB-induced K+ leakage, are mediated by oxidative damage.