Interaction Of Amphotericin Research Articles

Influence of amphotericin B on the DPPC/DOPC/sterols mixed monolayer in the presence of calcium ions

Amphotericin B, an acquainted antifungal drug, has reattracted the attention of most scholars due to its one important advantage of making the fungus less resistant. Amphotericin B's antifungal properties are derived from its ability to interact with ergosterols on the fungal cells' membrane to form pores. However, the cholesterol in the human cell membranes is similar in structure to ergosterol, which cause the drug to produce certain toxicity and make the clinical use of amphotericin B limited. The study of the interaction between amphotericin B and lipid monolayer in the presence of cholesterol or ergosterol is crucial to understanding the mechanism of effect of the drug on cell membranes. Langmuir monolayer as a model for half of cell membranes can precisely control the proportion of components and the solution environment, which has been used to do a lot of research about the interaction of amphotericin B with lipids. It is noteworthy that some ions associated with life activities play an important role in it, such as calcium ions. In this work, the surface pressure-mean molecular area isotherms, elastic modulus and the surface pressure-time curves of DPPC/DOPC/sterol mixed monolayer with or without amphotericin B were studied in the different concentration of calcium ions. The morphology of the Langmuir-Blodgett films transferred on the mica were observed by atomic force microscopy. The results shown that AmB changed the elastic modulus and surface morphology of DPPC/DOPC/sterol mxied monolayer, which was significantly different with different types of sterols. Calcium ions can regulate the effect of this drug, which was clearly different due to different types of sterols. This work provides useful information to further understand the influence mechanism of calcium ions on the interaction between AmB and phospholipid/sterol monolayer, which is helpful to find out the effect mechanism of calcium ion on the interaction between AmB and phospholipid monolayer containing ergosterol or cholesterol and to understand the mechanism of AmB influencing on the membrane of fungal or human cells.

Interaction of amphotericin B and saturated or unsaturated phospholipid monolayers containing cholesterol or ergosterol at the air-water interface

The antimicrobial activity of amphotericin B (AmB) depends on its interaction with ergosterol-containing cell membranes of fungus. Cholesterol is a sterol in mammalian cell membrane, and its structure is very similar to ergosterol, which caused to the toxic of amphotericin B to mammalian or human cell membranes. Even so, it is still the gold standard for the treatment of fungal infections. The mechanism of its toxicity to mammalian cell membrane has become a hot topic. The toxicity mechanism of amphotericin B on the cell membrane is also related to the phospholipids on the membrane. The effects of saturated and unsaturated fat chains on the interaction of amphotericin B with phospholipid monolayers containing cholesterol or ergosterol were studied at the molecular level using an air-water interface monolayer model. Both atomic force microscope and Brewster angle microscope were used to observe the surface morphology of the monolayer. The analysis of limiting molecular area suggested that the interaction between AmB and the two kinds of sterol is significantly different on the unsaturated lipid monolayer. According to the elastic modulus, the AmB molecules can increase the compressibility or viscoelasticity of the phospholipid/sterol monolayer. However, this impact of AmB on the DOPC/sterol monolayer containing ergosterol was stronger than that containing cholesterol at 25 ~ 50 mN/m. While this impact of AmB on the DPPC/sterol monolayer containing cholesterol was stronger than that containing ergosterol at 32 ~ 56 mN/m. The excess Gibbs free energy of the monolayer showed that, in the presence of saturated fat chain, amphotericin B could make the molecules of the DPPC/cholesterol monolayer and the DPPC/ergosterol monolayer arrange more closely and make intermolecular interaction stronger. There was no significant difference between DPPC/cholesterol monolayer and DPPC/ergosterol monolayer. However, in the presence of unsaturated chain, the effects of amphotericin B on the DOPC/cholesterol monolayer and the DOPC/ergosterol monolayer were significantly different. Amphotericin B made the molecular arrangement of DOPC/ergosterol monolayer more loosed, and the intermolecular force weakened at 5-35 mN/m. AFM images reflect that AmB can perforate the phospholipid-ergosterol monolayer, which was no significant correlation with saturation of the lipid monolayer. But the areas of dark areas shaped holes on the DPPC/ergosterol monolayer were larger than that on the DOPC/ergosterol monolayer. The adsorption of amphotericin B on lipid/sterol monolayer suggests that the orientation of amphotericin B may be different when it is inserted into the monolayer of phospholipid-sterol in the presence of saturated or unsaturated chains. The results are helpful to understand the complex mechanism of toxicity of amphotericin B to cell membrane.

Interaction of amphotericin B with human and bovine serum albumins: A fluorescence polarization study

Abstract In this study, the interaction of amphotericin B with human and bovine serum albumins has been investigated by fluorescence spectroscopy. Due to the limitations of several analytical methods by the known aggregation of amphotericin B molecules in aqueous solutions, fluorescence polarization technique was employed to determine complex stabilities. Besides the quantitation of reliable stability constants, this method was found to be able determine the stable aggregated amphotericin B fraction in the presence of serum albumins. Our results demonstrate the formation of highly stable albumin-drug complexes, suggesting the significant role of serum albumins regarding the plasma protein binding of amphotericin B.

Interaction of Amphotericin B with Ergosterol/Cholesterol‐Containing POPG Liposomes Studied by Absorption, Fluorescence and Second Harmonic Spectroscopy

AbstractThe membrane permeability induced by the polyene antibiotic Amphotericin B (AmB) against negatively charged L‐α‐phosphatidyl DL‐glycerol (POPG) membranes containing either Ergosterol (Ergo) or Cholesterol (Chol) has been investigated by monitoring the bilayer transport of the LDS‐698 (LDS+) ion using the interfacial selective second harmonic (SH) spectroscopy. The transport of LDS+ becomes faster at very low AmB/Liposome (A/L) ratio whereas Ergo present in the bilayer. As increasing A/L ratio, the average transport time constants (Tav) of the LDS+ ion fall into two kinetic regimes which observed for the different liposomes. In POPG‐Ergo liposomes, the effect of neutralizing either of the two charged functional groups of AmB reveals that interaction of AmB with Ergo is primarily dependent upon the charged amino group present in the mycosamine moiety of AmB. The spectral (absorption and fluorescence) properties of AmB were also studied to supplement the results obtained from SH studies. In summary, at low A/L ratio (< 1), the membrane permeability of the POPG membrane were observed to dependent upon the membrane composition or the pH of the medium, whereas at higher A/L ratio (> 1) the permeability becomes similar for all the cases. The spectroscopic properties of AmB with increasing A/L ratio suggest that the latter phenomena may be due to the aggregated states of AmB in the bilayer which enhance the bilayer permeability irrespective of the bilayer composition and solution pH.

Axial hydrogen at C7 position and bumpy tetracyclic core markedly reduce sterol's affinity to amphotericin B in membrane.

The interaction of amphotericin B (AmB) with fungal ergosterol (Erg) is stronger than its interaction with mammalian cholesterol (Cho), and this property of AmB as an antifungal drug is thought to be responsible for its selective toxicity toward fungi. However, the mechanism by which AmB recognizes the structural differences between sterols, particularly minor difference in the sterol alicyclic portion, is largely unknown. Thus, to investigate the mode of interaction between AmB and the sterol core, we assessed the affinity of AmB to various sterols with different alicyclic structures. Ion flux assays and UV spectral measurements clearly revealed the importance of the Δ7-double bond of the sterol B-ring for interaction with the drug. AmB showed lower affinity for triene sterols, which have double bonds at the Δ5, Δ7, and Δ9 positions. Intermolecular distance measurements by (13)C{(19)F} rotational echo double resonance (REDOR) revealed that the AmB macrolide ring is in closer contact with the steroid core of Erg than it is with the Cho core in the membrane. Conformational analysis suggested that an axial hydrogen atom at C7 of Δ5-sterol (2, 6) and the protruded A-ring of Δ5,7,9-sterol (4, 8) sterically hampered face-to-face contact between the van der Waals surface of the sterol core and the macrolide of AmB. These results further suggest that the α-face of sterol alicycle interacts with the flat macrolide structure of AmB.

Amphotericin B still in the headlines

Invited Commentary on key findings in the literature Antifungal drugs currently used for the treatment of Candida infections include polyenes, azoles, echinocandins, allyamines, and flucytosine. These drugs exert either fungicidal or fungistatic activities by interfering with essential processes.1 Intensive prophylactic and therapeutic uses of antifungal agents have selected for drug-resistant strains.2 Moreover, the limited arsenal of antifungal drugs is further compromised by severe side effects in patients and the emergence of species refractory to conventionally used agents. There is a need to develop new antifungals and to explore novel therapeutic approaches to treat Candida infections. To widen the repertoire of antifungal drugs, targets that differ from those of conventional drugs have to be identified. For over forty years, the polyene antibiotic amphotericin B (AmB) has been one of the most important agents used to combat systemic fungal infections.1 In spite of side effects such as nephrotoxicity, anemia, and cardiac arrhythmia, AmB remains the drug of choice for treatment of immunosuppressed patients, such as cancer patients in intensive chemotherapy, solid organ transplant recipients, and AIDS patients. The interaction of amphotericin B with ergosterol, the fungal-specific sterol, and other membrane sterols, results in the production of aqueous pores. As sterols are responsible for the membrane fluidity, sensitive target organisms lose their cellular integrity. The exact molecular architecture of the AmB channel is under debate; different models for the formation and structure of the AmB channel have been proposed.3 The sterol-dependent membrane activity of AmB suggests that the observed therapeutic efficacy of AmB might be related to a differential preference between sterols found in cell membranes. In mammalian cells, cholesterol is the major membrane sterol, whereas in fungi it is ergosterol. It has long been known that lipid bilayers containing sterols are uniquely vulnerable to permeabilization by AmB, but it is still not clear whether the therapeutic effect of AmB is caused by the preferential formation and stability of a complex of polyene and ergosterol over cholesterol, or whether the observed effects result from direct sterol binding. Efforts to improve the therapeutic index of this drug would benefit substantially from a more complete molecular understanding of its mode of action. Enabled by the iterative cross-coupling-based synthesis of a functional group deficient derivative of AmB, Gray et al.4 have discovered that channel formation is not required for potent fungicidal activity of AmB but that AmB primarily kills yeast by simply binding ergosterol. Membrane permeabilization via channel formation represents a second complementary mechanism that further increases drug potency and the rate of yeast killing. This finding clearly indicates that the capacity for AmB to form protein-like ion channels might be separable from its cytocidal effects, sharing this activity with another antifungal polyene macrolide natural product, natamycin, recently found to directly bind ergosterol but not to cause membrane permeabilization.5 The discovery that sterol binding is actually paramount to the antifungal action of AmB has both conceptual and practically important implications. From a conceptual point of view, the findings lend support to the view that antimicrobial mechanisms that can evade resistance not only exist but are ancient, and the mode of action(s) of AmB and perhaps other molecules is one of them. Moreover, although best studied in mammalian cells, lipid signalling is also now appreciated in microbial cells, particularly in yeasts and moulds. The study of Gray et al. accelerates advances in lipid biology and points to lipids as promising new targets in the search for superior antimicrobials that may be less vulnerable to resistance. This study is an illuminating example of how strategies of antifungal drug discovery and therapeutic index implementation benefit substantially from a more complete molecular understanding of the mode of action of old and novel drugs.

Spectral Study of the Amphotericin B - cholesteryl Linoleate Interaction

The interaction of amphotericin B with cholesteryl linoleate has investigated by UV-VIS spectroscopy. The binding results have rationalized in terms of methods Benesi-Hildebrand, Scott and Scatchard, taking into account 1:1 amphotericin B - cholesteryl linoleate system.

Characterization of the Interaction of Amphotericin B with Cholesteryl Trifluoromethylphenyl-Carbamate by UV-Visible Spectroscopy

Amphotericin B, a polyene antibiotic, self-associates in ethanol. The self-association of antibiotic has characterized by UV-visible spectroscopy. Starting from a simple dimerization model and using different methods described in literature, one determined the molar absorption coefficient of monomer, the molar absorption coefficient of dimer and the dimerization constant. The binding of amphotericin B to cholesteryl trifluoromethylphenyl-carbamate has investigated using absorbance measurements and the results have rationalized in terms of literature models, taking into account both 1:1 drug-sterol system and cooperativity effects. The binding constant of amphotericin B to cholesteryl trifluoromethylphenyl-carbamate has determined using Benesi-Hildebrand, Scott and Scatchard methods.

Interaction of amphotericin B lipid formulations and triazoles with human polymorphonuclear leucocytes for antifungal activity against Zygomycetes

The frequency of zygomycosis has increased considerably over recent years mainly in immunocompromised and diabetic patients. Little is known about the effects of host innate immunity against different Zygomycetes especially under the influence of antifungal agents. The antifungal activity of human polymorphonuclear leucocytes (PMN) in combination with liposomal amphotericin B (LAMB), amphotericin B lipid complex (ABLC), voriconazole (VRC) and posaconazole (PSC) against Rhizopus oryzae and Rhizopus microsporus, frequently isolated Zygomycetes, were studied and compared with Absidia corymbifera, a less pathogenic Zygomycete. Antifungal activity was evaluated as per cent of hyphal damage using the XTT metabolic assay. While A. corymbifera was more susceptible to PMN than the other two Zygomycetes, R. microsporus appeared to be the most susceptible to combined effects of amphotericin B formulations and VRC with PMN. LAMB exhibited synergistic activity with PMN in inducing hyphal damage to R. microsporus but not to the other fungi. In contrast, ABLC exhibited synergistic or additive activity with PMN against all three fungi. Among triazoles, only VRC exhibited additive effect with PMN against R. microsporus. Lipid formulations of amphotericin B and particularly ABLC interact with PMN predominantly in inducing augmented hyphal damage to three different species of Zygomycetes.

Interactions of amphotericin B derivatives with lipid membranes—A molecular dynamics study

Amphotericin B (AmB) is a well-known polyene macrolide antibiotic used to treat systemic fungal infections. AmB targets more efficiently fungal than animal membranes. However, there are only minor differences in the mode of action of AmB against both types of membranes, which is a source of AmB toxicity. In this work, we analyzed interactions of two low toxic derivatives of AmB (SAmE and PAmE), synthesized in our laboratory, with lipid membranes. Molecular dynamics simulations of the lipid bilayers containing ergosterol (fungal cells) or cholesterol (animal cells) and the studied antibiotic molecules were performed to compare the structural and dynamic properties of AmB derivatives and the parent drug inside the membrane. A number of differences was found for AmB and its derivatives' behavior in cholesterol- and ergosterol-containing membranes. We found that PAmE and SAmE can penetrate deeper into the hydrophobic region of the membrane compared to AmB. Modification of the amino and carboxyl group of AmB also resulted in the conformational transition within the antibiotic's polar head. Wobbling dynamics differentiation, depending on the sterol present, was discovered for the AmB derivatives. These differences may be interpreted as molecular factors responsible for the improved selectivity observed macroscopically for the studied AmB derivatives.

In vitro activity and synergism of amphotericin B, azoles and cationic antimicrobials against the emerging pathogen Trichoderma spp.

The uncommon fungal pathogen Trichoderma shows increasing medical importance particularly in immunocompromised patients. Despite systemic antifungal therapy, prognosis of Trichoderma infection is poor regardless of the type of infection and the therapy used. The aim of the present study was to evaluate the in vitro activity and synergism of double antifungal combinations including amphotericin B, voriconazole, fluconazole, chlorhexidine digluconate and Akacid plus against 15 isolates of Trichoderma longibrachiatum and 1 isolate of Trichoderma harzianum. Individual MICs were determined by using broth microdilution method following the NCCLS M38-A guidelines with standard RPMI 1640 broth. Synergy tests were performed using the chequerboard method. All clinical Trichoderma strains showed reduced susceptibility to fluconazole (MICs>or=64 mg/L) and amphotericin B (MICs=2 mg/L), whereas lower MICs of 0.5-1 mg/L were detected for voriconazole. Akacid plus reached the lowest MIC values in a range of 0.06-0.5 mg/L, 4- to 32-fold higher MICs were found for chlorhexidine. No antagonism was observed for any of the antifungal combinations tested. Interaction of amphotericin B and azoles was indifferent (fractional inhibitory concentration index, FICI 2-4). The combination of one azole and one cationic biocide showed different degree of synergism (FICI 0.07-2.03). Interaction of Akacid plus and chlorhexidine resulted in synergism for each Trichoderma isolate (FICI-range 0.05-0.5). These results demonstrate no interaction between antifungals and some degree of synergism between azoles and cationic antimicrobials against Trichoderma spp.

Interaction of amphotericin B and its selected derivatives with membranes: molecular modeling studies

Amphotericin B (AmB) is a well-known antifungal antibiotic that has been used in the clinic for about five decades. Despite its chemotherapeutic importance, AmB is quite toxic and many efforts have been made to improve its pharmacological properties, e.g., by chemical modifications. The lipid membrane is a molecular target for AmB, however, due to heterogeneity of its components, the molecular mechanism of AmB action is still unclear. The lack of this knowledge hinders rational designing of new and less toxic AmB derivatives. Our review is a critical presentation of the current understanding of AmB molecular mechanism of action at the membrane level. Except the experimental approach, the extensive overview of molecular modeling studies, performed mostly in our lab, is presented. The results of interactions between AmB or some of its derivatives and lipid model membranes are discussed. In our studies, different biomembrane models and different associate states of the antibiotic were included. Presented molecular modeling approach is especially valuable with regard to a new paradigm of the structure of lipid membrane containing liquid-ordered domains. Hopefully, all these complementary experimental/computational approaches are going to reach the point at which a new hypothesis about molecular mechanism of AmB activity and selectivity will be put forward.

Interactions of amphotericin B derivative of low toxicity with biological membrane components—the Langmuir monolayer approach

Amphotericin B (AmB)–a polyene macrolide antibiotic–exhibits strong antifungal activity, however, is known to be very toxic to mammalian cells. In order to decrease AmB toxicity, a number of its derivatives have been synthesized. Basing on in vitro and in vivo research, it was evidenced that one of AmB derivatives, namely N-methyl- N- d-fructopyranosylamphotericin B methyl ester (in short MF-AME) retained most of the antifungal activity of the parent antibiotic, however, exhibited dramatically lower animal toxicity. Therefore, MF-AME seems to be a very promising modification product of AmB. However, further development of this derivative as potential new antifungal drug requires the elucidation of its molecular mechanism of reduced toxicity, which was the aim of the present investigations. Our studies were based on examining the binding energies by determining the strength of interaction between MF-AME and membrane sterols (ergosterol-fungi sterol, and cholesterol-mammalian sterol) and DPPC (model membrane phospholipid) using the Langmuir monolayer technique, which serves as a model of cellular membrane. Our results revealed that at low concentration the affinity of MF-AME to ergosterol is considerably stronger as compared to cholesterol, which correlates with the improved selective toxicity of this drug. It is of importance that the presence of phospholipids is essential since–due to very strong interactions between MF-AME and DPPC–the antibiotic used in higher concentration is “immobilized” by DPPC molecules, which reduces the concentration of free antibiotic, thus enabling it to selectively interact with both sterols.

Amphotericin B nephrotoxicity in children.

Amphotericin B is the treatment of choice for severe systemic fungal infections. Nephrotoxicity is the most clinically significant adverse effect, but studies examining nephrotoxicity in children are scarce. Nephrotoxicity includes decreased glomerular filtration rate and distal tubulopathy with urinary loss of potassium and magnesium, renal tubular acidosis, loss of urine concentrating ability, and sometimes Fanconi's syndrome. The mechanisms involved in nephrotoxicity include the use of deoxycholate, the vehicle for amphotericin, reduction in renal blood flow and glomerular filtration rate, increased salt concentrations at the macula densa, interaction of amphotericin with ergosterol in the cell membrane, and apoptosis in proximal tubular cells and medullary interstitial cells. Some risk factors for amphotericin nephrotoxicity have been determined over the years. Cumulative dosage, treatment duration, and dosing schedule as well as the combination of amphotericin with other nephrotoxic drugs, such as diuretics and cyclosporine, are important risk factors. Mechanisms to prevent nephrotoxicity include the use of lipid formulations such as amphotericin B lipid complex, amphotericin B colloidal dispersion, and liposomal amphotericin B and the concurrent use of volume repletion. Amiloride can be considered in serious potassium loss.

Interaction of amphotericin B and its low toxic derivative, N-methyl- N-D-fructosyl amphotericin B methyl ester, with fungal, mammalian and bacterial cells measured by the energy transfer method

Amphotericin B (AMB) derivative, N-methyl- N- D-fructosyl amphotericin B methyl ester (MFAME) retains the broad antifungal spectrum and potency of the parent antibiotic, whereas its toxicity towards mammalian cells is reduced by about two orders of magnitude. The purpose of this work was to find out whether the differences observed in the toxicity of MFAME and native AMB are due to the differential drugs affinity to fungal and mammalian cell membranes. Comparative studies on AMB and MFAME biological activity and their affinity to fungal, mammalian and bacterial cells were performed. The interaction of AMB and MFAME with cells have been studied by fluorescence method based on the energy transfer between membrane fluorescent probe (donor) and the polyenic chromophore of the antibiotic (acceptor) simultaneously present in the cell membrane. The amount of the antibiotic bound to cells was indicated by the extent of fluorescence quenching of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) or 1,6-diphenyl-1,3,5-hexatriene (DPH) by polyenic chromophore of the antibiotic. The results obtained indicate that binding extent and characteristics for both antibiotics are comparable in the three types of cells studied. Dramatically lower toxicity of MFAME as compared to AMB towards mammalian cells is not related to the antibiotic-cell affinity, but rather to different consequences of these interactions for cells, reflected in membrane permeabilization. MFAME is definitely less effective than parent AMB in the permeabilizing species formation in mammalian cell membrane.

Interactions of amphotericin B with saturated and unsaturated phosphatidylcholines at the air/water interface

Three synthetic phospholipids, differing in the degree of saturation of phosphatidylcholine (PC) acyl chains, namely: dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylcholine (DOPC) and palmitoyl-oleoyl phosphatidyl choline (POPC) were mixed with the antimycotic polyene antibiotic, amphotericin B (AmB), and investigated as monolayers at the air/water interface. The mixed films were spread on water (pH 6) at room temperature, and the surface pressure–area (π/A) isotherms were recorded upon compression. The interactions were examined by analysing the phase transitions of the monolayers and calculating the films compressibility. The obtained results indicate the existence of stronger interaction between AmB and the saturated phospholipid as compared with unsaturated ones, and reveal the influence of apolar structure of PC chains on the stoichiometry of AmB–PC complexes.

Study of penetration of amphotericin B into cholesterol or ergosterol containing dipalmitoyl phosphatidylcholine Langmuir monolayers

The interactions of amphotericin B (AmB) with sterols and phospholipids have been studied by adsorption of AmB from aqueous solutions into Langmuir monolayers from dipalmitoyl phosphatidylcholine (DPPC), ergosterol, cholesterol and their mixtures. The results show that AmB exhibits stronger interaction with cholesterol than ergosterol in one-component monolayers. However, for DPPC–sterol monolayers, the effectiveness of AmB penetration depends on the proportion of both film components in the mixed film as well as on the strength of interaction between DPPC and particular sterol.

The interactions of amphotericin B with various sterols in relation to its possible use in anticancer therapy

Amphotericin B (AmB) is still the most common anti-fungal agent used to treat systemic fungal infections. It is known that this antibiotic acts by forming pores with the ergosterol contained in the membranes of fungi, but it also interacts with the cholesterol contained in the membranes of eukaryotic cells, hence its toxicity. AmB may also interact with the most common oxidation products of cholesterol found in vivo, together with interacting with biosynthetic precursors of cholesterol, namely, lanosterol and 7-dehydrocholesterol (7-DHC). The purpose of the present work was to study the interactions in solution between AmB and these various sterols, the techniques used being UV-Vis spectroscopy and differential scanning calorimetry. The results are globally interpreted in terms of the structural differences between the sterols. We show that AmB selectively interacts with 7-DHC which, according to a recent hypothesis proposed in the literature, has been identified in connexion with a therapeutic strategy against hepatocellular carcinomas. We find that the affinity of AmB towards 7-DHC is even greater than the affinity of the antibiotic towards ergosterol. We also find that AmB selectively interacts with the principal oxidation product of cholesterol, 7-ketocholesterol, a situation that has to be taken into account when AmB is administered.

Interaction of amphotericin B with polymeric colloids: A spectroscopic study

The self-association of the amphiphilic antifungal agent amphotericin B (AmB) in water has been shown to depend on numerous parameters which produce spectroscopic changes and is directly related to modifications in AmB toxicity. We have taken advantage of these changes to study the interactions of AmB with two different polymeric materials: poly(ε-caprolactone) and poloxamer 188. Both materials are components of a recently developed AmB formulation based on polymeric nanospheres, which have demonstrated reduced acute toxicity in mice as compared with free AmB. Poly(ε-caprolactone) composes the core of nanospheres, and poloxamer 188 is the stabilizer used to coat the particles. AmB dispersions with poly(ε-caprolactone) nanospheres led to dramatic spectral changes as compared with free AmB dispersions, indicating an extensive reduction of the aggregation state of AmB and an increase in the threshold of its aggregation. These changes seem to be due to the binding of the drug onto the nanosphere surface. In contrast, dispersions of AmB with poloxamer led to an increase of AmB aggregation as a function of poloxamer concentration due to the formation of mixed micelles. Modification of the ionic charge of AmB molecules during the preparation of nanospheres did not have a significant effect on AmB self-organization. However, interaction of AmB with high concentration of poloxamer containing a larger hydrophobic region (polypropylene oxide, PPO) changed this organization by reducing the number of AmB aggregates as compared to the effect of more hydrophilic poloxamers. These results suggest that ionic and/or hydrogen bond interactions only play a minor role and that predominantly hydrophobic forces drive the interaction between AmB and these compounds. Both types of association were easily dissociated upon dilution (AmB concentration<5 μg/ml), indicating that the interactions of AmB with both polymeric materials are relatively weak. This low affinity between the drug and these compounds has been shown by the fact that such interactions were only achieved with formulations of precise composition obtained with a particular preparative process such as a solvent displacement method. This process forced AmB monomers solubilized in organic solvent — on evaporation of this solvent — to bind to nanospheres or to associate with poloxamer micelles instead of self-aggregating in water.

Interactions of itraconazole with amphotericin B in the treatment of murine invasive candidiasis.

The interactions of amphotericin B and itraconazole were studied in murine invasive candidiasis. Candida albicans-infected mice were treated for 10 consecutive days, 24 h after infection. Survival was monitored over 30 days and kidney cultures were done. Mice treated with amphotericin B (0.2 mg/kg/day intraperitoneally) or itraconazole (100 mg/kg/day by oral gavage in two divided doses/ day) had a 30-day survival of 20% or 40%. Concomitant administration of both drugs resulted in 100% mortality; 90% of mice treated with amphotericin B (1 mg/kg/day) survived. With the combination, 100% were dead by day 28 (P < or = .001 vs. amphotericin B). With sequential therapy (i.e., 5 days with one drug and then 5 days with the other), survival was inferior to that with amphotericin B alone but similar to that with itraconazole alone. Kidney culture results confirmed the antagonism of the combination compared with amphotericin B alone. In treatment of murine invasive candidiasis, the concomitant or sequential use of amphotericin B and itraconazole results in a negative interaction.