ABSTRACT
Studies have shown that the dose-limiting toxicity of amphotericin B (AmB), a key drug for systemic mycoses, depends on its self-aggregation state. In a step toward understanding the various factors in blood mediating the toxicity of AmB, we have investigated the effect of serum albumin, the most abundant plasma protein, on the aggregation state of AmB using absorption spectroscopy. The critical aggregation concentration (CAC) of AmB, which coincides with its concentration at the onset of toxicity (hemolysis), was 1.1 microM, but rose in proportion to the level of serum albumin (1.0 to 4.0% w/v). The CAC of AmB was 8.0 microM at 4.0% w/v serum albumin, which is considerably higher than peak therapeutic levels of AmB in plasma (i.e., 2.0 microM). Serum albumin (4.0% w/v) lowered the degree of aggregation of AmB (size of aggregates) above the CAC and increased its solubility. The results suggest that serum albumin attenuates the toxicity of AmB at a membrane level by affecting its aggregation state. In this way, serum albumin in blood may balance deleterious effects of AmB mediated by serum low-density lipoproteins.
Subject(s)
Amphotericin B/chemistry , Amphotericin B/toxicity , Antifungal Agents/chemistry , Antifungal Agents/toxicity , Serum Albumin/chemistry , Serum Albumin/pharmacology , Absorption , Animals , Cattle , Dimethyl Sulfoxide , Hemolysis , In Vitro Techniques , Male , Solvents , Spectrophotometry, UltravioletABSTRACT
Amphotericin B (AmB) is a membrane-active drug used frequently for the treatment of systemic fungal diseases. Limitations for the use of AmB include poor water solubility and potential for serious systemic toxicities. Recently, it has been demonstrated that the aggregation state of AmB is a determinant factor for toxicity. To increase its therapeutic index, AmB has been solubilized in micelles based on poly(ethylene oxide)-block-poly(beta-benzyl-l-aspartate) (PEO-block-PBLA), using a dialysis method of drug loading. The aggregation state of AmB has been investigated by electronic absorption spectroscopy. AmB loaded in PEO-block-PBLA micelles is non-hemolytic for concentrations up to 15 microgram/ml. AmB as Fungizone(R) initiates hemolysis at 1.0 microgram/ml. The onset of hemolysis correlates with the respective critical aggregation concentrations (CACs) of AmB. The antifungal activity of the AmB-loaded PEO-block-PBLA micelles is four to eight times higher than Fungizone(R) in terms of minimal inhibitory concentrations (MICs). PEO-block-PBLA has no antifungal activity for concentrations up to 200 microgram/ml. The basis for the increase in antifungal activity of AmB-loaded PEO-block-PBLA micelles is unclear, but may be related to a stabilizing effect of the polymeric micelles against auto-oxidation of the AmB heptaene moiety or alternatively, an enhancement in membrane perturbation of fungal cells.
Subject(s)
Amphotericin B/administration & dosage , Amphotericin B/chemistry , Antifungal Agents/administration & dosage , Antifungal Agents/chemistry , Excipients/administration & dosage , Excipients/chemistry , Peptides/administration & dosage , Peptides/chemistry , Polyethylene Glycols/administration & dosage , Polyethylene Glycols/chemistry , Animals , Delayed-Action Preparations , Erythrocytes/drug effects , Male , Micelles , Microbial Sensitivity Tests , Polymers , Rats , Rats, Sprague-Dawley , Spectrophotometry, UltravioletABSTRACT
Polymeric micelles may serve as nanoscopic, long-circulating carriers of hydrophobic drugs. In this study, we have researched the solubilization of amphotericin B (AmB), an antifungal drug, by micelles of poly(ethylene oxide)-block-poly(beta benzyl-L-aspartate) (PEO-PBLA), the properties of the AmB-loaded PEO-PBLA micelles and the resultant haemolytic activity of AmB. AmB loading takes place during self assembly of PEO-PBLA micelles, and this occurs through a dialysis procedure as an alkaline aqueous solution replaces the selective solvent for the polymer and the drug. In this way, AmB reaches levels of 57 to 141 microg/ml, corresponding to a loading efficiency of 27-30% (loaded AmB/initial amount of AmB). The molar ratio of AmB to PEO-PBLA is 0.40 to 1.0. Pictures by transmission electron microscopy reveal spherical AmB-loaded PEO-PBLA micelles with a mean diameter of 25.8+/-4.2 nm. AmB-loaded PEO-PBLA micelles are nonhaemolytic at an AmB level of 10 microg/ml as assessed by release of haemoglobin, measured by UV-Vis spectroscopy. AmB as Fungizone, its standard formulation, completely lyses red blood cells at a level of 3.0 microg/ml in 30 min. In contrast, there is no haemolysis at 5.5 h for AmB-loaded PEO-PBLA micelles at 3.0 microg/ml of AmB, indicating the gradual release of AmB from PEO-PBLA micelles. PEO-PBLA itself is nonhaemolytic even at a level of 0.70 mg/ml. Most amphiphiles, e.g. sodium deoxycholate, present in Fungizone, are haemolytic. Finally, AmB-loaded PEO-PBLA micelles can be freeze-dried and easily reconstituted in water. Afterwards, AmB is present in the intact PEO-PBLA micelles and remains nonhaemolytic.
