Comparative in vitro studies on liposomal formulations of amphotericin B and its derivative,N-methyl-N-D-fructosyl amphotericin B methyl ester (MFAME)

N-Methyl-N-D-fructosyl amphotericin B methyl ester (MFAME) is a semisynthetic derivative of the antifungal antibiotic amphotericin B (AMB). In contrast to the parent antibiotic, the derivative is characterised by low toxicity to mammalian cells and good solubility in water of its salts. Comparative studies on biological properties of free MFAME, AMB and their liposomal formulations were performed. To obtain liposomal forms, the antibiotics were incorporated into small unilamellar vesicles composed of dimyristoyl phosphatidylcholine (DMPC) and DMPC:cholesterol or ergosterol, 8:2 molar ratio. The effectivity of the liposomal and free forms of AMB and MFAME were compared by determination of fungistatic and fungicidal activity against Candida albicans ATCC 10261, potassium release from erythrocytes, and haemolysis. The results obtained indicate that in contrast to AMB, incorporation of MFAME into liposomes did not further improve its selective toxicity. Studies on the antagonistic effect of ergosterol and cholesterol on the antifungal activity of the antibiotics indicated that sterol interference was definitely less pronounced in the case of MFAME than in the case of AMB.     

N-Methyl-N-D-fructosyl amphotericin B methyl ester (MF-AME), a novel antifungal agent of low toxicity: monomer/micelle control over selective toxicity

Rational chemical modification of amphotericin B (AMB) led to the synthesis of sterically hindered AMB derivatives. The selected optimal compound, N-methyl-N-D-fructosyl amphotericin B methyl ester (MF-AME) retains the broad spectrum of antifungal activity of the parent antibiotic, and exhibits a two orders of magnitude lower toxicity in vivo and in vitro against mammalian cells. Comparative studies of MF-AME and AMB comprising the determination of the spectroscopic properties of monomeric and self-associated forms of the antibiotics, the investigation of the influence of self-association on toxicity to human red blood cells, and of the antibiotic-sterol interaction were performed. On the basis of the results obtained it can be assumed that the improvement of the selective toxicity of MF-AME could in part be a consequence of the diminished concentration of water soluble oligomers in aqueous medium, and the better ability to differentiate between cholesterol and ergosterol.     

Comparative studies on cell stimulatory, permeabilizing and toxic effects induced in sensitive and multidrug resistant fungal strains by amphotericin B (AMB) and N-methyl-N-D-fructosyl amphotericin B methyl ester (MFAME)

N-Methyl-N-D-fructosyl-amphotericin B methyl ester (MFAME) is a new derivative of amphotericin B, which is characterised by low toxicity to mammalian cells and good solubility in water of its salts. The antifungal activity and effects of MFAME towards Candida albicans and Saccharomyces cerevisiae multidrug resistant MDR(+) and sensitive MDR(-) strains was compared with those of parent compound. The results obtained indicate that MDR(+) S. cerevisiae was sensitive to MFAME as well as to AMB. MFAME exhibited the same effects on fungal cells studied as parent antibiotic. The two antibiotics, depending on the dose applied induced cell stimulation, K+ efflux, and/or had a toxic effect.     

Effect of amphotericin B on membranes: a spin probe study

The effect of the polyene antibiotic amphotericin B on the electron paramagnetic resonance spectra of lipid probes intercalated in model membranes was examined. When the antibiotic was added to the aqueous phase, no spectral effects occurred. However, when the antibiotic was incorporated during membrane preparation, changes in spectral parameters suggested the appearance of a new phase. The spectral changes do not necessarily corroborate the pore models proposed previously for amphotericin B in membranes. With a spin probe that partitions between water and membrane, an interaction between the amphotericin B and probe is observed. This interaction does not occur in the membrane, but in the aqueous phase, between the probe and the aggregated antibiotic. Some of the equilibria involving the antibiotic appear to be achieved slowly.     

Aggregation of amphotericin B in the presence of gamma-cyclodextrin

The macrolide antibiotic amphotericin B (AmB) forms an inclusion complex with gamma-cyclodextrin (gamma-CDx), resulting in a molecularly dispersed state of the drug. The state of aggregation of AmB in different solvents has been studied by absorption (uv-vis) and CD spectroscopy. While in aqueous solutions AmB forms colloid-like multimolecular aggregates, in the presence of gamma-CDx true solutions can be prepared, which show similar spectral properties as AmB dissolved in organic solvents. The AmB-gamma-CDx complex can be isolated as an amorphous, stable, water-soluble powder, indicating that gamma-CDx is a good carrier for the solubilization of this antibiotic. Using gamma-CDx as a carrier, the danger of precipitation of the drug during parenteral or intravenous administration can be largely reduced.     

Modulation of polyene antibiotics self-association by ions from the Hofmeister series

The toxicity of the antifungal polyene antibiotic amphotericin B (AMB) has been related to its low solubility, more specifically to a self-associated form termed toxic aggregate. In addition, AMB in aqueous medium gives rise to concentration, ionic strength, and time-dependent polydisperse systems. For this reason different approaches, including the use of several lipid aggregates, have been used in attempts to improve the drug's solubility and increase its therapeutic index. In this context, understanding AMB's self-association properties should help in the preparation of less toxic formulations. Ions from the Hofmeister series alter water properties: while kosmotropes (water structure makers-sulfate, citrate, phosphate) decrease solute solubility, chaotropes (water structure breakers-perchlorate, thiocyanate, trichloroacetate, and the neutral molecule urea) have opposite effects. This work reports a study of the effect of Hofmeister ions and urea on the self-aggregation of AMB and some of its derivatives. Optical absorption and circular dichroism spectra were used to monitor monomeric and aggregated antibiotic. While kosmotropes increased aggregation in a concentration-dependent manner, the opposite was observed for chaotropes. It is shown, for the first time, that thiocyanate and trichloroacetate can induce complete AMB monomerization. The understanding of these processes at the physicochemical and molecular levels and the possibility of modulating the aggregation state of AMB and its derivatives should contribute to elucidate the mechanisms of action and toxicity of this widely used antibiotic and to develop more efficient and less toxic preparations.     

Borate complexes of amphotericin B: Polymeric species and aggregates in aqueous solutions

Amphotericin B, a polyene macrolide antibiotic, exists in aqueous solution as a poorly soluble, high-molecular-weight aggregate. A borate complex of this polyene was prepared that has greater solubility and is less aggregated. In aqueous solution this borate complex exists as a mixture of several molecular species differing in borate content, molecular weight, and molecular conformation. The solubility varied with pH and was minimal at neutrality. Throughout the pH range it was one to two orders of magnitude higher than that of the parent compound. The molecular size distribution, as determined by differential ultrafiltration, showed a progressive increase in the weight fraction of aggregates going from acid to alkaline solutions. The sizes of aggregates ranged from under 25 to over 100 molecules. The borate content of the complexes increased with increasing pH. No borate was complexed in acid solutions. This indicated that amphotericin B and borate ions can complex to form copolymer chains of varying length in which these species alternate, since both are bifunctional. The complexation equilibrium is favored by high pH. Absorption and CD spectra indicated that the polyene molecules can stack reversibly to form dimers. Dimerization constants calculated from the spectra were highest in neutral solution and declined with increasing acidity or alkalinity. In alkaline solutions the polymer chains are long and extended, with minimal stacking. In neutral solution the chains are shorter and extensively stacked. In acid solutions no borate complexes are formed, and the polyenes are stacked to an intermediate degree. The very different effects of pH and concentration on the degree of complexation with borate and on the degree of dimerization of the polyenes shows that these equilibria are independent of each other.     

Structure of amphotericin B aggregates as revealed by UV and CD spectroscopies

The aqueous and hydroalcoholic solutions of the heptenic macrolide amphotericin B display strong and variable signals in CD and absorption spectroscopies in the range of the π* ← π transition. An interpretation of the spectroscopic changes is proposed based on the equilibrium between two forms of the intermolecular organization: the aggregated one (A) with strong excitonic interaction and the nonaggregative one (B) whose spectra are like those of linear conjugated polyenes in true solution with a well-developed vibrational structure. The intermediate spectra are fitted by linear combination of the A- and B-form spectra. A two-level organization of the aggregates is proposed for the A-form: (1) a close packing of few molecules, which is the origin of the absorption maxima hypsochromic shift; and (2) interaction between the preceding small units inside the aggregates, which is spectroscopically expressed by the intense CD couplet.     

Effect of the aggregation state of amphotericin B on its interaction with ergosterol

The interaction of amphotericin B with ergosterol was studied in aqueous solutions of propanol. The mode of the interaction was found to be related to the aggregation state of amphotericin B. Ergosterol does not react (or reacts extremely slowly) with monomeric amphotericin B. Traces of a small aggregate, probably a dimer, enable a cooperative reaction. At high concentrations of the dimer, the reaction is immediate and the concentration of amphotericin B complexed with ergosterol is twice as high as the amount of added sterol. The interaction with ergosterol is hindered when the antibiotic is in micellar form. The pharmaceutical form, Fungizone, behaves similarly to the pure amphotericin B. Fungizone's greater solubility in water does not modify either the extent or the mode of interaction with ergosterol.     

One-sided action of amphotericin B on cholesterol-containing membranes is determined by its self-association in the medium

The inducement of K+ permeability through membranes by the polyene antibiotic amphotericin B (AmB) has been analyzed as a measure of the antibiotic activity. Dose-response curves have been obtained with cholesterol- and ergosterol-containing egg yolk phosphatidylcholine large unilamellar vesicles (LUVs), human erythrocytes, and Saccharomyces cerevisiae cells. Conductance changes induced by AmB in sterol-containing planar bilayer membranes have also been studied. AmB self-association in aqueous buffer was determined by circular dichroism (CD) as a function of the antibiotic concentration. Electronic absorption and CD spectra of AmB were recorded in the presence of LUVs. For given AmB concentrations, the extent of permeability inducement is dependent on the lipid concentration. On the other hand, for cholesterol-containing LUVs or erythrocytes, a critical AmB concentration had to be reached before any permeability is observed. Independent of lipid concentration, this concentration was directly related to antibiotic self-association in the aqueous buffer. The same observation was made for erythrocytes and nystatin. The AmB absorption and CD spectra were totally different for ergosterol- and cholesterol-containing LUVs. Formation of single channels by one-sided addition of AmB could be observed only in ergosterol-containing membranes. These data lead us to propose that the permeability pathways induced by amphotericin B or nystatin, in ergosterol- and in cholesterol-containing membranes, are of different natures. In the latter case the antibiotics are only active, by single-sided addition, in the self-associated form. These findings offer important clues for the design of less toxic derivatives of AmB: they should have a low degree of self-association in water.     

Spectroscopic studies of amphotericin B solubilized in nanoscale bilayer membranes

Nanodisks (ND) are discrete nanometer scale phospholipid bilayers whose perimeter is circumscribed by amphipathic apolipoproteins. The membranous environment of ND serves as a matrix for solubilizing the polyene antibiotic amphotericin B (AMB). The spectral properties of AMB in ND are dependent upon AMB concentration. Whereas AMB-ND prepared at a concentration of 2.5 mg AMB per 10 mg phospholipid are consistent with AMB self association in the ND membrane environment, AMB-ND prepared at 0.25 or 0.025 mg AMB per 10 mg phospholipid give rise to spectra reminiscent of AMB in organic solvent. Incubation of ND prepared at a phospholipid/AMB ratio of 400:1 (w/w) at 37 degrees C for 1 h induced a shift in absorbance and near UV circular dichroism spectra consistent with antibiotic self-association. The kinetics of this spectral transition were investigated as a function of incubation temperature. While no change in A388 nm occurred in incubations at 20 degrees C, a time-dependent decrease in A388 nm was observed at 25, 30 and 37 degrees C. Inclusion of ergosterol in the ND membrane attenuated temperature-induced AMB spectral changes. In Saccharomyces cerevisiae growth inhibition assays, ND containing self associated AMB were somewhat less effective than ND possessing a greater proportion of monomeric AMB. On the other hand, inclusion of ergosterol or cholesterol in the ND particle did not alter the growth inhibition properties of AMB-ND. The miniature membrane environment of ND provides a novel milieu for solubilization and characterization of lipophilic biomolecules.     

Spectroscopic studies of amphotericin B solubilized in nanoscale bilayer membranes

Nanodisks (ND) are discrete nanometer scale phospholipid bilayers whose perimeter is circumscribed by amphipathic apolipoproteins. The membranous environment of ND serves as a matrix for solubilizing the polyene antibiotic amphotericin B (AMB). The spectral properties of AMB in ND are dependent upon AMB concentration. Whereas AMB-ND prepared at a concentration of 2.5 mg AMB per 10 mg phospholipid are consistent with AMB self association in the ND membrane environment, AMB-ND prepared at 0.25 or 0.025 mg AMB per 10 mg phospholipid give rise to spectra reminiscent of AMB in organic solvent. Incubation of ND prepared at a phospholipid/AMB ratio of 400:1 (w/w) at 37 °C for 1 h induced a shift in absorbance and near UV circular dichroism spectra consistent with antibiotic self-association. The kinetics of this spectral transition were investigated as a function of incubation temperature. While no change in A₃₈₈ nm occurred in incubations at 20 °C, a time-dependent decrease in A₃₈₈ nm was observed at 25, 30 and 37 °C. Inclusion of ergosterol in the ND membrane attenuated temperature-induced AMB spectral changes. In Saccharomyces cerevisiae growth inhibition assays, ND containing self associated AMB were somewhat less effective than ND possessing a greater proportion of monomeric AMB. On the other hand, inclusion of ergosterol or cholesterol in the ND particle did not alter the growth inhibition properties of AMB-ND. The miniature membrane environment of ND provides a novel milieu for solubilization and characterization of lipophilic biomolecules.     

Structure of amphotericin B aggregates based on calculations of optical spectra

The Degenerate ground state approximation was used to calculate the optical absorption and CD spectra for helical polymer models of amphotericin B aggregates in aqueous solution. Comparisons with experimental spectra indicate that a two-molecule/unit cell helical polymer model is a possible structure for aggregates of amphotericin B.     

Spin-labeled amphotericin B: synthesis, characterization, biological and spectroscopic properties

A biologically active spin-labeled derivative of amphotericin B has been synthesized by the nucleophilic addition of amphotericin B to 4-(2-iodoacetamido)-2,2',6,6'-tetramethylpiperadine-N-oxyl in dimethyl-sulphoxide at 40 degrees C. The derivative is a moderately water-soluble compound which displays the same biological activity of the parental compound against the sensitive organism Leishmania mexicana; also, the rates of proton-cation exchange induced by the two compounds in large unilamellar liposomes are indistinguishable. The ESR spectra of spin-labeled amphotericin B in lipid vesicles indicate a high degree of motion, very similar to that encountered for the compound in aqueous solutions at neutral pH and in deoxycholate micelles, and suggest that the structures formed by the antibiotic in membranes are composed by a small number of molecules. In contrast, the spectra of the labeled antibiotic in ethanol, diethyl ether and dimethylformamide indicate restricted motion and exchange interactions, probably resulting from the micellar aggregation induced in these media. Ascorbate at 10 mM is able to reduce completely the nitroxide group of the labeled antibiotic in lipid vesicles in less than 30 s, indicating that an asymmetric disposition of the antibiotic molecules across the membrane is capable of inducing its biological and ionophoric properties. Ni2+ and Cu2+ produce moderate exchange broadening of the ESR signal of spin-labeled amphotericin B in lipid vesicles; the comparison of this phenomenom with the exchange broadening produced by the same ions in the ESR spectrum of 2,2',6,6'-tetramethylpiperidine-N-oxyl in water solution suggests an specific Cu2+-amphotericin B interaction in membranes.     

Transient permeability induced by alkyl derivatives of amphotericin B in lipid membranes

Individual ionic channels were shown to be formed in the brain cholesterol containing phospholipid membranes by two-sided addition of the amphotericin B alkyl derivatives. At concentrations between 10(-8) and 10(-7) M, the resulting conductance appeared to be transient. Existence of different antibiotic assemblies was justified by the kinetic analysis of the membrane conductance decline following the antibiotic washing out. In order to account for the transient characteristics of the induced conductance, it was proposed that the antibiotic oligomers incorporate into the membrane from the aqueous phase, form channels aggregating with cholesterol, and then dissociate in the bilayer into non-active degraded oligomeric or monomeric forms.     

Transient permeability induced by alkyl derivatives of amphotericin B in lipid membranes

Individual ionic channels were shown to be formed in the brain cholesterol containing phospholipid membranes by two-sided addition of the amphotericin B alkyl derivatives. At concentrations between 10⁻⁸ and 10⁻⁷ M, the resulting conductance appeared to be transient. Existence of different antibiotic assemblies was justified by the kinetic analysis of the membrane conductance decline following the antibiotic washing out. In order to account for the transient characteristics of the induced conductance, it was proposed that the antibiotic oligomers incorporate into the membrane from the aqueous phase, form channels aggregating with cholesterol, and then dissociate in the bilayer into non-active degraded oligomeric or monomeric forms.     

Acid-base properties and the thermodynamics of amphotericin B ionization in aqeous solutions]

Acid-base properties of amphotericin B, polyenic antibiotic in aqueous solutions was studied. A special procedure provided the use of potentiometric titration for investigation of ionization of the groups of the water-insoluble substance. The ionization constants of the carboxylic and amine groups of the antibiotic were determined at several temperatures. It was found that ionization of the acid group did not depend on the temperature. At the same time the heat effect of the amine group ionization was significant and amounted to about 10 kcal/mole. Thermodynamic analysis of the ionization process of amphotericin B in aqueous solutions was performed. Integral components defining the process energetics were calculated.     

Reconstituted high density lipoprotein enriched with the polyene antibiotic amphotericin B

The polyene antibiotic amphotericin B (AMB) is an effective antifungal agent whose therapeutic potential is limited by poor aqueous solubility and toxicity toward host tissues. Addition of apolipoprotein A-I to a multilamellar phospholipid vesicle dispersion containing 20% (w/w) AMB induces the formation of reconstituted high density lipoprotein (rHDL), with solubilization of the antibiotic. Density gradient ultracentrifugation resulted in flotation of the complexes to a density of 1.16 g/ml, and negative stain electron microscopy revealed a population of disk-shaped particles. Native gradient polyacrylamide gel electrophoresis indicated a particle diameter of approximately 8.5 nm. Absorbance spectroscopy provided evidence for AMB integration into the lipid milieu. AMB-rHDLs were potent inhibitors of Saccharomyces cerevisiae growth, yielding 90% growth inhibition at <1 microg/ml yeast culture. In studies with pathogenic fungal species, similar growth inhibition characteristics were observed. Compared with AMB-deoxycholate micelles, AMB-rHDL displayed greatly attenuated red blood cell hemolytic activity and decreased toxicity toward cultured hepatoma cells. In in vivo studies in immunocompetent mice, AMB-rHDLs were nontoxic at 10 mg/kg, and they showed efficacy in a mouse model of candidiasis at concentrations as low as 0.25 mg/kg. These results indicate that AMB-rHDLs constitute a novel formulation that effectively solubilizes the antibiotic and elicits strong in vitro and in vivo antifungal activity with no observed toxicity at therapeutic doses.     

Kinetic study of interaction between [14C]amphotericin B derivatives and human erythrocytes: relationship between binding and induced K+ leak

The relationship between polyene antibiotic binding to red cells and their membrane permeabilization was studied using two 14C-labelled amphotericin B (AmB) derivatives: N-fructosyl AmB and N-acetyl methyl ester AmB. The binding kinetics of both derivatives were determined on whole red cells and ghosts. The resulting experimental points were well fitted by monoexponential functions, and the characteristic t1/2 for both derivatives were calculated from these functions. At 2 X 10(-5) M, the half time t1/2 for N-acetyl methyl ester AmB (30.2 min) which forms aqueous aggregates was longer than the t1/2 for the more soluble species N-fructosyl AmB (4.5 min). At lower concentrations (10(-7) M), the t1/2 for N-acetyl methyl ester AmB (6.3 min) in a more solubilized form was close to that of N-fructosyl AmB (7.9 min). These results suggest that only solubilized species bound to red cell membranes and that disaggregation of aggregates is the limiting step in the binding process. The permeabilization of red cell membranes by N-fructosyl AmB, measured as intracellular K+ leak, was not instantaneous and at 10 degrees C external K+ was only detected 20 min after antibiotic addition. In contrast, binding occurs without lag time. Consequently, different mecanisms underlie binding and K+ permeability inducement. Absorption spectroscopy data showed that bound antibiotic is located in the hydrophobic membrane interior and that this penetration of the membrane by AmB derivatives occurs without lag time. Consequently, the lag time occurring for K+ permeability inducement would be due to some steps subsequent to binding and probably located in the hydrophobic membrane interior. This statement is further supported by the observation that the lag time is sensitive to changes in membrane fluidity as shown here by the break between 20 and 30 degrees C in the slope of the Arrhenius plot for the lag time, coinciding with the phase transition in red cell membranes.     

Molecular organization of antibiotic amphotericin B in dipalmitoylphosphatidylcholine monolayers induced by K(+) and Na(+) ions: the Langmuir technique study

The effect of potassium (K(+)) and sodium (Na(+)) ions on the self-association of antibiotic amphotericin B (AmB) in the lipid membrane was reported. Mixed Langmuir monolayers of AmB and dipalmitoylphosphatidylcholine (DPPC) were investigated by recording surface pressure-area isotherms spread on aqueous buffers containing physiological concentration of K(+) and Na(+) ions. The analyses of the π-A isotherms and compressional modulus curves indicate the interactions in the AmB-DPPC system. The strength of the AmB-DPPC interactions and the stability of the mixed monolayers were examined on the basis of the excess free energy of mixing values. The obtained results proved a high affinity of AmB towards lipids induced by the presence of K(+) than Na(+) ions. The most stable monolayers in the presence of K(+) and Na(+) ions were formed by AmB and DPPC with the 1:1 and 2:1 stoichiometry. The understanding of the AmB aggregation processes at the molecular level should contribute to elucidate the mechanisms of action and toxicity of this widely used drug. The presented results are potentially valuable in respect to develop more efficient and less toxic AmB formulations.