The effect of aggregation state of amphotericin-B on its interactions with cholesterol- or ergosterol-containing phosphatidylcholine monolayers
Introduction
Since its discovery in 1956 (Gold et al., 1956) Amphotericin-B (AmB) has been, and still is, the major antibiotic used for the treatment of systemic fungi infections. This has been even more so in the past decade as fungi infections have shown an increased frequency in immuno-compromised patients either suffering from cancer (Edwards Jr et al., 1978), AIDS or after organ transplantation.
It is generally accepted that the cellular membrane is the main site of action of AmB (Lampen et al., 1960; Bolard, 1986). In fact, AmB interacts with ergosterol, the major sterol component of fungi membranes (Norman et al., 1972; Kotler-Brajtburg et al., 1974). It is thought that a complex is formed between AmB and ergosterol (Bittman and Fischkoff, 1972; De Kruijff and Demel, 1974; Gruda et al., 1980) which results in the formation of pores that causes an increase in the membrane permeability (Andreoli, 1974; Berges et al., 1990; Cohen, 1992). K+ ions first and, subsequently, other components (e.g. small organites) leave out the cell thus causing its death (Cohen, 1992; Gent and Prestegard, 1976). However, the existence of pores within the cell membrane has not been directly proven experimentally. AmB also interacts, albeit to a lesser extent, with cholesterol, the major sterol of mammal cells and is therefore toxic when administered (Medoff et al., 1983).
Recent experimental evidences, either in vitro or in vivo, have shown that there exists a close correlation between the aggregation state of AmB in the preparations used and its toxicity (Tancrède et al., 1990; Barwicz et al., 1992; Legrand et al., 1992; Schreier and Lamy-Freund, 1993). The selectivity of the drug to ergosterol with respect to cholesterol has indeed been shown to be maximal when monomeric or, preferably, slightly aggregated AmB was used. However, the molecular basis of the interactions of AmB with either ergosterol or cholesterol are not known in details, neither is the relationship between the aggregation states of AmB and its consequences on the better selectivity of AmB towards ergosterol.
In this context, the aim of the present work is to study the interactions of AmB with ergosterol or cholesterol dispersed in a phospholipid monolayer. AmB was dispersed in the subphase at four different concentrations, ranging from a molecular dispersion of the antibiotic in the subphase (10−8 M) to a dispersion of large aggregates (26×10−6 M). Two different concentrations of the sterols were used, 13 and 30%, corresponding to the range of concentrations found in various membranes for these sterols. The lipid used was a phosphatidylcholine (dioleoyl-l-α-phosphatidylcholine (DOPC)), phosphatidylcholines being a major component of the lipid composition of mammal membranes. In addition, DOPC shows a liquid-like phase at the surface pressure studied here, similar to the fluid phase found in the hydrophobic core of a membrane, and is, therefore, extensively used in the literature, specially when studies with sterols are involved (Nag and Keough, 1993; Subczynski et al., 1994; Smaby et al., 1994).
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Materials
AmB, ergosterol and cholesterol were purchased from Sigma Co. (St. Louis, MO). Ergosterol and cholesterol were twice recrystallized from ethanol (Les Alcools de Commerce Inc., Gatineau, Québec). Dioleoyl-l-α-phosphatidylcholine (DOPC, chloroform solution) was purchased from Avanti Polar Lipids (Alabaster, AL) and used without further purification. Ethanol and hexane (Aldrich Chemical Co. Milwaukee, WI) were distilled prior to use. The water used for the monolayer work was demineralized on a
Results and discussion
The molecular structures of the components used in this study are presented in Fig. 1. AmB (Fig. 1A) had its molecular structure elucidated in 1970 by Mechlinski et al. (1970). The molecule has quite a special structure, with one hydrophobic side containing seven conjugated double bonds and the other side, hydrophilic, containing several polar substituents. AmB is also a zwitterionic molecule in solution at neutral pH, the carboxylic acid group being deprotonated while the amino acid group is
Conclusions
In conclusion, therefore, the present work has shown that in its monomeric form (i.e. at concentrations below 10−7 M) AmB interacts selectively with ergosterol-containing and not with cholesterol-containing monolayers. This interaction does not depend on the organization state of the sterol within the monolayer. This work has also shown that micelles represent the active form of AmB when it binds to lipid layers and that the significant selectivity of interaction of the antibiotic with the
Acknowledgements
This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada, to which the authors are grateful.
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