G S Kobayashi

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

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.

Regulation of dimorphism in the pathogenic fungus Histoplasma capsulatum

Histoplasma capsulatum is a dimorphic pathogenic fungus which is the causative agent of the disease histoplasmosis. The disease is worldwide in occurrence and approximately 40 million people have been infected in the USA1. In soil the fungus is filamentous, but in the infected host it exists as a budding yeast and it is believed that the phase transition is important in the pathogenicity. In the laboratory, the mycelial form can convert to the yeast phase when the temperature of incubation is shifted from room temperature (25 °C) to body temperature (37 °C). In this report we show that the elevated temperature initiates a series of reactions leading to changes in the intracellular level of 3′, 5′-cyclic adenosine monophosphate (cyclic AMP);the shifts in intracellular cyclic AMP levels are an important determinant of the morphological phase of the organism and therefore of its disease producing potential.

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.

Morphological transition in the human fungal pathogen Histoplasma capulatum

Considerable information has accumulated recently about specific genes of Histoplasma capsulatum that are expressed during the process of adaptation when the organism undergoes morphological transition at the onset of infection. The study of these genes is crucial to identify targets for the development of novel antifungal agents.

Permeability control in animal cells by polyenes: a possibility.

The polyenes amphotericin B and vitamin A enhanced the specific actions of rifampin, fusidic acid, and 1,3-bis(2-chloroethyl)-1-nitrosourea against mouse L cells in tissue culture as measured by macromolecular synthesis and cell survival.

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.

Stability of Amphotericin B in Fungal Culture Media

We have found that amphotericin B is unstable in two commonly employed fungal culture media. This instability leads to inaccuracies in determining the actual level of susceptibility of slow-growing strains that require prolonged incubation for growth. To help compensate for this problem, we have described two rapid methods of susceptibility testing.

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.