Amphotericin B interactions with a DOPC monolayer. Electrochemical investigations

A model lipid membrane consisting of a monolayer of dioleoyl phosphatidylcholine (DOPC) adsorbed onto a Hg electrode has been used to study the interaction between the lipid and different formulations of Amphotericin B (AmB) [Fungizone® (FZ), Heated Fungizone (HFZ), and Abelcet®]. The lipid organizational order was measured by electrochemical methods [capacitance and metal ion (Tl⁺) reduction], characterizing the change in lipid order due to interaction with the drug. The mean size and number density of pores formed in the monolayer were estimated by fitting the reduction current transients to a random array of microelectrode model. This method was shown sensitive for investigation of the interaction of drugs with the DOPC monolayer. Abelcet was found to have a smaller disruptive effect on lipid order than FZ and HFZ. The formulations used to solubilize the AmB were also studied. Sodium deoxycholate used as a solubilizer in FZ displayed significant influence on lipid order similar to that observed for Abelcet. The lipid complex, used in Abelcet, did not significantly perturb the DOPC monolayer order. The lipid complex used in Abelcet may have an annealing or healing effect that buffers the disruption possible due to AmB.     

Optimizing efficacy of amphotericin B through nanomodification

Fungal infections and leishmaniasis are an important cause of morbidity and mortality in immunocompromised patients. The macrolide polyene antibiotic amphotericin B (AmB) has long been recognized as a powerful fungicidal and leishmanicidal drug. A conventional intravenous dosage form of AmB, AmB- deoxycholate (Fungizone or D-AmB), is the most effective clinically available for treating fungal and parasitic (leishmaniasis) infections. However, the clinical efficacy of AmB is limited by its adverse effects mainly nephrotoxicity. Efforts to lower the toxicity are based on synthesis of AmB analogues such as AmB esters or preparation of AmB-lipid associations in the forms of liposomal AmB (L-AmB or AmBisome), AmB lipid complex (Abelcet or ABLC), AmB colloidal dispersion (Amphocil or ABCD), and intralipid AmB. These newer formulations are substantially more expensive, but allow patients to receive higher doses for longer periods of time with decreased renal toxicity than conventional AmB. Modifications of liposomal surface in order to avoid RES uptake, thus increased targetability has been attempted. Emulsomes and other nanoparticles are special carrier systems for intracellular localization in macrophage rich organs like liver and spleen. Injectable nano-carriers have important potential applications as in site-specific drug delivery.     

Inhibition of the interaction between lipoproteins and amphotericin B by some delivery systems.

Amphotericin B (AmB) is a potent antifungal agent used to treat patients with systemic mycoses. The clinical usefulness of the drug is limited by its high toxicity and several new less toxic formulations of AmB have been recently developed. In order to understand the mechanism of the decreases of toxicity caused by various new delivery systems, we have investigated by uv-visible spectroscopy the interaction of two of these formulations with human blood lipoproteins. The results were compared with those obtained with the commonly used pharmaceutical form of AmB (Fungizone). This study shows that AmB-lipoprotein interaction is hindered when the drug is in a monomeric form and/or when it is included in phospholipid-surfactant micelles. In an in vivo study on mice it is shown here that AmB monomerized by surfactant is less toxic to animals than the same concentration of Fungizone, where the polyene is strongly aggregated. It may be concluded from the present study that the AmB species which is responsible for the in vivo toxicity is a complex of the antibiotic with the low density and the very low density blood lipoproteins and that hindering of this complex formation results in a decrease of AmB toxicity.     

Circular dichroism study of interactions of Fungizone or AmBisome forms of amphotericin B with human low density lipoproteins

Amphotericin B (AmB), a potent antifungal agent used to treat invasive fungal infections, is still employed more than 40 years after its introduction in the pharmacopea. When injected into the blood stream, this antibiotic is carried by low density lipoproteins (LDLs) to which it induces the formation of oxidation products responsible in part for some of the severe adverse effects of the drug. However, the oxidative damages induced to LDLs are not yet understood. We present here the effects of the Fungizone and AmBisome forms of AmB on LDLs as compared to those of CuSO(4), a well-known powerful oxidant of LDLs. We use circular dichroism (CD) spectroscopy, which is particularly useful because it allows the investigation of the structural integrity of the proteic moiety of LDL upon interaction with AmB. The CD spectra also yield information on the drug itself because in its oligomer form it presents a strong dichroic signal in a spectral region different from that of the protein. Our results show that neither form of AmB changes the secondary structure of the protein while the helical content of the LDL is increased either in the presence of CuSO(4) alone or in the presence of CuSO(4) and AmBisome or Fungizone. On the other hand, the CD spectra of the antibiotic indicate that Fungizone AmB suffers important oxidative damage in the presence of LDLs and CuSO(4) while this damage is not present with AmBisome AmB. These observations lead us to propose that the structural modifications of the proteic part of LDLs induced by the Cu(2+) ions are involved in the important oxidative damage suffered by Fungizone AmB, which in this form is much more susceptible to interaction with its environment than AmBisome.     

Effect of potassium ions at the different concentration on the interaction between AmB and the lipid monolayer containing cholesterol or ergosterol

AmB is an antifungal drug of polyene. Although it is prone to nephrotoxicity, it is still the gold standard in the clinical treatment of fungal infection. Sterol plays a decisive role in the drug activity of AmB. The antifungal activity of AmB depends on ergosterol in fungal membranes, and its toxicity is related to cholesterol in mammalian membranes. At the same time, AmB interacts with biofilms, leading to a significant loss of potassium ions and affecting the transport of potassium ions across membranes. Meanwhile, metal cation may also affect AmB molecules’ aggregation on the membrane. This paper mainly studied the effects of different concentrations of potassium ions on the interactions between AmB and lipid monolayers containing cholesterol or ergosterol and explored the differences in the impact of varying potassium ions on the drug activity of AmB on monolayers rich in these two kinds of sterols. The results show that potassium ions caused the collapse of lipid monolayer and lipid-AmB monolayer to disappear. The limiting molecular area of these monolayers also increased due to potassium ions. The limiting molecular area of the monolayer in the presence of ergosterol has a great difference in the different concentration of potassium ions, which is different from that in the presence of cholesterol. The presence of potassium ions, regardless of the intensity of K⁺ ions, increased the maximum elastic modulus of the lipid/sterol monolayer with and without AmB. The presence of potassium ions reduced the influence of AmB on the stability of the lipid monolayer containing cholesterol. The impact of AmB on the stability of the lipid monolayer containing ergosterol was related to the concentration of potassium ions. The potassium ions increased the area of the ordered “island” region on the lipid-AmB monolayer containing cholesterol, and the boundary of the microregion produced different degrees of curvature. However, on the lipid/ergosterol monolayer, 5 mM and 10 mM potassium ions made the holes caused by AmB more denser, and the diameter of holes become larger. These results can help to improve the effect of potassium ions on the transmembrane transport of substances affected by AmB. The results will provide a basis for further exploration of the effect mechanism of metal ions on the antifungal activity of polyene drugs.     

Cochleates: New Lipid-Based Drug Delivery System

Abstract

Cochleates are a lipid-based tailored drug delivery system formed by the precipitation of a negatively charged lipid and a cation, for example phosphatidylserine and calcium. Hydrophobic, amphiphilic, negatively or positively charged moieties are suitable candidates to be delivered via cochleates. Various procedures have been developed allowing the control of cochleate particle size, including the trapping and hydrogel methods, which use either a direct addition or a slow diffusion of calcium into the negatively charged liposome/drug suspension. The efficacy of cochleates to encapsulate and deliver drugs was evaluated using amphotericin B as a model. Amphotericin B cochleates (CAMB) were compared to Fungizone® and AmBisome®, two commercially available AmB products. Parenterally, CAMB was given IP to ICR mice infected with Candida albicans. 100% survival was observed with low doses of CAMB (0.5 mg/kg/day, 10 days) compared to 60% for Fungizone, at the same dose. Tissue burden studies were conducted in parallel. Mice were treated daily from day 1 to day 7 post challenge and tissue burden assessed at day 8. In the kidneys, all three formulations were comparable in reducing colony counts. In the spleen, CAMB at 10 mg/kg/day was comparable to AmBisome given IV at the same dose. At 1 mg/kg/day, CAMB was more potent than Fungizone and AmBisome. Oral administration of CAMB in C57BL/6 mice, at 10 mg/kg results in high levels of AmB in target tissues. Multiple daily doses (10) showed accumulation of AmB in key tissues (liver, lungs, spleen, and kidneys) and AmB tissue concentrations are raised to therapeutic levels. Orally administered CAMB are highly effective against fungal infections in mice at very low doses. Balb/C mice were infected with Candida albicans and were given oral CAMB as a daily dose for 15 days. Comparison was done to AmBisome given orally at 10 mg/kg and Fungizone IP. 100% survival was obtained with CAMB at doses as low as 0.5 mg/kg/day (15 days). CAMB eradicate Candida from lungs when given at 2.5 mg/kg/day and was comparable to Fungizone given IP at almost the same dose (2 mg/kg/day). The comparison between CAMB and AmBisome shows that oral CAMB is 10 times more effective than oral AmBisome in reducing colony counts in both kidneys and lungs. Orally administered CAMB were non-toxic even at the highest dose of 50 mg/kg/day (14 days). This was demontrated by 100% survival of the animals and normal histopathology analysis. No lesions in the kidneys, GI tract, lungs, liver and spleen was observed despite the substantial amount of AmB in these organs. AmB cochleate promise to be a safe, broad spectrum, effective and orally available, antifungal formulation.     

Factors Affecting Enhanced Permeation of Amphotericin B Across Cell Membranes and Safety of Formulation

The aim of this study was to determine amphotericin B (AmB) permeation across lipid bilayer membranes mounted on Transwell® and to observe the phagocytosis of the AmB and the AmB-lipid formulations by alveolar macrophage (AM) cell lines using a fluorescence microscope. The lipid bilayer membranes were prepared from phospholipid and ergosterol as well as phospholipid and cholesterol in a ratio (67:33 mol%). AmB-lipid formulations were prepared from AmB incorporated with four lipid derivatives during a lyophilization process. In vitro cytotoxicity studies were carried out on kidney cells by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The levels of nitric oxide production by AMs exposed to these AmB-lipid formulations were determined by the Griess reaction. Phagocytosis of the AmB-lipid formulations was carried out using AM cells. The lipid bilayer membranes and AmB-lipid formulations were successfully prepared. In vitro cytotoxicity results showed less toxicity to kidney cells than pure AmB, and a 1,000-fold less production of nitric oxide by NR8383 cell lines was obtained when compared to lipopolysaccharide. Permeation results were two- to fivefold higher than for pure AmB in the ergosterol containing lipid bilayer and two- to fourfold higher than AmB in the cholesterol containing compositions, both of which were enough to kill the fungi according to their MICs and MFCs. AM phagocytosed the AmB-lipid formulations. We suggest that these products especially the AmB-sodium deoxycholate sulfate are potential candidates for targeting AM cells for the treatment of invasive pulmonary aspergillosis.     

Electrostatic interactions of colicin E1 with the surface of Escherichia coli total lipid

The surface properties of colicin E1, a 522-amino acid protein, and its interaction with monolayers of Escherichia coli (E. coli) total lipid and 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DOPC) were studied using the Langmuir-Blodgett (LB) technique. Colicin E1 is amphiphilic, forming a protein monolayer at the air/buffer interface. The protein is thought to interact with the E. coli total lipid head groups through electrostatic interactions, followed by its insertion into the lipid monolayers. Supported lipid bilayers (SLBs) of E. coli total lipid and DOPC, deposited onto mica at the cell membrane equivalence pressure for E. coli and incubated with colicin E1, were imaged by contact mode atomic force microscopy (CM-AFM). Colicin E1 formed protein aggregates on DOPC SLBs, while E. coli total lipid SLB was deformed following its incubation with colicin E1. Corresponding lateral force images, along with electrostatic surface potentials for colicin E1 P190, imply a direct interaction of colicin E1 with lipid head groups facilitating their charge neutralization.     

The effects of amphotericin B on pure and ergosterol- or cholesterol-containing dipalmitoylphosphatidylcholine bilayers as viewed by 2H NMR

Amphotericin B (AmB) is a widely used polyene antibiotic to treat systemic fungal infections. This drug is known to be lethal to fungal cells but it has also side effect toxicity on mammalian cells. The mechanism of action of AmB is thought to be related to the difference of the main sterol present in the mammalian and the fungal cells, namely cholesterol and ergosterol, respectively. The effect of AmB has been investigated on pure dipalmitoylphosphatidylcholine (DPPC) and on cholesterol- and ergosterol-containing DPPC bilayers by 2H NMR spectroscopy. The 2H NMR results first confirm that AmB forms a complex with sterol-free DPPC bilayers, the interaction causing the structurization of the lipids and the increase of the gel-to-lamellar fluid DPPC phase transition temperature with increasing concentration of the antibiotic. The results also show that the effects of AmB on cholesterol- and ergosterol-containing DPPC bilayers are remarkably different. On one hand, the drug causes an increase of the orientational order of the lipid acyl chains in cholesterol-containing membranes, mostly in high cholesterol content membranes. On the other hand, the addition of AmB disorders the DPPC acyl chains when ergosterol is present. This is thought to be due to the direct complexation of the ergosterol by AmB, causing the sterol ordering effect to be weaker on the lipids.     

Effects of lipids and detergents on the conformation of the nicotinic acetylcholine receptor from Torpedo californica.

The hydrophobic, photoreactive probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID) was used to characterize the effects of lipids and detergents on acetylcholine receptor (AChR) conformation. Affinity purified AChR reconstituted into dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidic acid (DOPA), and cholesterol showed the same pattern of [125I]TID-labeling and demonstrated the same reduction in labeling of all four subunits upon desensitization by the agonist carbamylcholine, as partially purified AChR in native lipids. On the basis of the patterns of [125I]TID incorporation, reconstitution into DOPC/DOPA also appeared to stabilize the resting (functional) conformation of the AChR, while reconstitution in DOPC/cholesterol or DOPC alone largely desensitized the AChR. The effects of lipids on the functional state of the AChR was determined independently by measuring the ability of AChR reconstituted into different lipid combinations to undergo the change in affinity for agonist diagnostic of desensitization. The dramatic reduction in the apparent levels of [125I]TID associated with the subunits of the AChR observed upon agonist-induced desensitization was shown not to be due to a change in affinity for tightly bound lipid. Solubilization of affinity purified AChR reconstituted into DOPC/DOPA/cholesterol by the non-ionic detergents octyl glucoside, Triton X-100, and Tween 20 (final detergent concentration = 1%) was shown to produce the same pattern of [125I]TID-labeling as desensitization by agonist, while solubilization in 1% sodium cholate appeared to stabilize a conformation of the AChR more similar to the resting state.     

New lipid formulation of amphotericin B: spectral and microscopic analysis

UV-visible and dichroic spectrum analysis and electron microscopy have been used to characterize a new amphotericin B (AmB) lipid formulation prepared by a solvent displacement process. The composition was dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) in molar ratio DMPC/DMPG/AmB 7:3:5, a similar composition to that of Abelcet®. Although the latter has a “ribbon-like” structure, our process gave a thin disc-like structure. Analysis of circular dichroism (CD) and UV-visible spectra of formulations containing different percentages of AmB revealed that a minimum of AmB self-association was observed with 7:3:5 molar ratio. Varying the lipid ratio (DMPC/DMPG) while maintaining the fixed ratio of AmB yielded similar results when DMPC was in excess (DMPC/DMPG from 10:0 to 6:4). However, when the ratio was between 5:5 to 3:7, AmB self-aggregation increased. For compositions rich in DMPG (2:8 and 0:10), inversion of the CD spectrum was observed. The influence of the lipid composition on the morphology of the complex was also evident in electron microscopy. DMPC/DMPG/AmB (10:0:5) gave large unfracturable lamellae. The presence of DMPG shortened the lamellae, which often appeared as disc-like structures. AmB content, the presence of DMPG and the preparation process all contribute to generating these original structures with particular CD spectra.     

The effect of amphotericin b derivatives on Leishmania and immune functions

The effects of a water-soluble amphotericin B (AmB)-arabinogalactan (AG) conjugate on several immune functions were investigated. The experiments measured the effects of AmB-AG on (1) release of tumor necrosis factor-alpha (TNF-alpha), nitric oxide (NO), and interferon-gamma (IFN-gamma) from phagocytic cells and (2) cell-mediated immune responses. AmB-AG increased TNF-alpha release from mouse peritoneal macrophages and human monocytes but had no effect on IFN-gamma and NO release. A commercial preparation of nonconjugated AmB (Fungizone) also increased TNF-alpha production, but to a lesser extent than AmB-AG. AG alone had no effect on TNF-alpha production, proving that AmB caused the increased TNF-alpha production. AmB-AG and Fungizone were also tested for their effect on B- and T-cell proliferation. Neither compound altered T-lymphocyte responses to concanavalin A, but both inhibited the stimulation of B lymphocytes by lipopolysaccharides. However, Fungizone showed a stronger inhibitory effect on B cells. Allocytotoxicity was also inhibited by AmB-AG and more strongly by Fungizone. The increased production of TNF-alpha by cells treated with AmB-AG and the lower inhibitory effect of AmB-AG on lymphocyte stimulation and allocytotoxicity, as compared with Fungizone, explain the better therapeutic efficacy of the AmB-polysaccharide conjugate. AmB is active because of its preferential binding to ergosterol rather than cholesterol, the former sterol preferentially present in parasite surface membranes. This is also valid for the axenic amastigotes, which were sensitive to the AmB-AG. Overall, our results suggest that the antileishmanial activity of AmB-AG is mediated both directly and via modulation of immune functions.     

Investigations on feasibility of in situ development of amphotericin B liposomes for industrial applications

Amphotericin B (AmB) liposome formulations are very successful in the treatment of fungal infections and leishmaniasis. But higher cost limits its widespread use among people in developing countries. Therefore, we have developed a modified ethanol-injection method for the preparation of AmB liposomes. Two liposomal formulations were developed with dimyristoyl phosphatidylcholine [F-1a] and soya phosphatidylcholine [F-2a], along with egg phosphatidyl glycerol and cholesterol. AmB was dissolved in acidified dimethyl acetamide and mixed with ethanolic lipid solution and rapidly injected in 5% dextrose to prepare liposomes. Liposomes were characterized on the basis of size (~100?nm), zeta (-43.3?±?2.8 mV) and percent entrapment efficiency (>95%). The in vitro release study showed an insignificant difference (P?≥?0.05) for 24-hour release between marketed AmB liposomes (AmBisome) and F-1a and F-2a. Proliposome concentrate, used for the preparation of in situ liposomes, was physically stable for more than 3 months at experimental conditions. Similarly, AmB showed no sign of degradation in reconstituted liposomes stored at 2-8°C for more than 3 months. IC(50) value of Ambisome (0.18 μg/mL) was comparatively similar to F-1a (0.17 μg/mL) and F-2a (0.16 μg/mL) against intramacrophagic amastigotes. Under experimental conditions, a novel modified method for AmB liposomes is a great success and generates interest for development as a platform technology for many therapeutic drug products.     

On the existence of an amphotericin B--sterol complex in lipid vesicles and in propanol-water systems

The amphotericin B (AmB) - ergosterol complex, formed by interaction of the antibiotic with ergosterol-containing phospholipid vesicles, is associated with the lipid bilayer. It has been shown by circular dichroism studies that the AmB-ergosterol complex formed in water-propanol binary mixtures has a similar structure to that observed in phospholipid vesicles. A positive cooperativity is found for the interaction of AmB with ergosterol. The similar AmB-cholesterol complex is much less stable and rearranges rapidly to a different conformation.     

Temperature dependence of the structural dimensions of the inverted hexagonal (HII) phase of phosphatidylethanolamine-containing membranes

The characteristic temperature dependence of the lattice basis vector length d of phospholipid-water systems in the inverted hexagonal (HII) phase has been investigated with X-ray diffraction. For 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), d falls sharply from 78.1 A at 10 degrees C to 62.5 A at 90 degrees C. When used in conjunction with the volume fractions of the constituents, d can be used to determine the dimensions within the lipid and water regions. These data showed that a reduction in the radius of the HII-phase water cylinders Rw accounted for most of the reduction in d. From geometrical relationships between the dimensions in the HII phase, it was shown that both d and Rw are sensitive functions of the thickness of the lipid monolayer dHII. The characteristic shape of d(T) could be parameterized with the small temperature dependence of dHII along with the ratio v/a, which is the ratio of the specific volume to the area per lipid molecule at the polar interface. The ratio v/a was found to be independent of temperature for the fully hydrated HII system. Additional measurements made with a mixture of DOPE and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), mole ratio 5.07:1, produced a similar parameterization of d(T). The larger basis vector lengths for this mixture compared to those for DOPE can be attributed to a smaller ratio of v/a, which was also found to be temperature independent for this mixture. The smaller value of v/a is due to the larger effective headgroup area of DOPC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Investigations on feasibility of in situ development of amphotericin B liposomes for industrial applications

Amphotericin B (AmB) liposome formulations are very successful in the treatment of fungal infections and leishmaniasis. But higher cost limits its widespread use among people in developing countries. Therefore, we have developed a modified ethanol-injection method for the preparation of AmB liposomes. Two liposomal formulations were developed with dimyristoyl phosphatidylcholine [F-1a] and soya phosphatidylcholine [F-2a], along with egg phosphatidyl glycerol and cholesterol. AmB was dissolved in acidified dimethyl acetamide and mixed with ethanolic lipid solution and rapidly injected in 5% dextrose to prepare liposomes. Liposomes were characterized on the basis of size (~100?nm), zeta (–43.3?±?2.8 mV) and percent entrapment efficiency (>95%). The in vitro release study showed an insignificant difference (P?≥?0.05) for 24-hour release between marketed AmB liposomes (AmBisome) and F-1a and F-2a. Proliposome concentrate, used for the preparation of in situ liposomes, was physically stable for more than 3 months at experimental conditions. Similarly, AmB showed no sign of degradation in reconstituted liposomes stored at 2–8°C for more than 3 months. IC₅₀ value of Ambisome (0.18 µg/mL) was comparatively similar to F-1a (0.17 µg/mL) and F-2a (0.16 µg/mL) against intramacrophagic amastigotes. Under experimental conditions, a novel modified method for AmB liposomes is a great success and generates interest for development as a platform technology for many therapeutic drug products.     

Electrostatic interactions of colicin E1 with the surface of Escherichia coli total lipid

The surface properties of colicin E1, a 522-amino acid protein, and its interaction with monolayers of Escherichia coli (E. coli) total lipid and 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DOPC) were studied using the Langmuir-Blodgett (LB) technique. Colicin E1 is amphiphilic, forming a protein monolayer at the air/buffer interface. The protein is thought to interact with the E. coli total lipid head groups through electrostatic interactions, followed by its insertion into the lipid monolayers. Supported lipid bilayers (SLBs) of E. coli total lipid and DOPC, deposited onto mica at the cell membrane equivalence pressure for E. coli and incubated with colicin E1, were imaged by contact mode atomic force microscopy (CM-AFM). Colicin E1 formed protein aggregates on DOPC SLBs, while E. coli total lipid SLB was deformed following its incubation with colicin E1. Corresponding lateral force images, along with electrostatic surface potentials for colicin E1 P190, imply a direct interaction of colicin E1 with lipid head groups facilitating their charge neutralization.     

Reduced toxicity of amphotericin B methyl ester (AME) vs. amphotericin B and fungizone in tissue culture.

The comparative toxicities of amphotericin B methyl ester (AME), the parent antibiotic amphotericin B (AB), and the deoxycholate solubilized complex of AB, Fungizone (FZ), toward five cell lines has been determined as measured by early membrane damage (51Cr release), 24 hr survival, 72 hr viability, and growth rate. Cells used were of turtle (TH-1), marsupial (PT K2), human MA 160), rabbit (RK-13) and hamster (BHK-21) origin. AME: (a) caused less membrane damage at 1 hr than AB or FZ; (b) was less toxic than AB or FZ as indicated by 24 hr cell survival and 72 hr cell viability; and (c) was required in higher levels than AB or FZ to reduce the growth rate of all five cell lines. Spectrophotometric analysis of residual polyene levels indicated that AME had good stability in tissue culture medium. Previous studies have indicated that AME has the same in vitro antifungal activity as the parent antibiotic AB (1, 2). These findings suggest that AME may prove to be superior to AB and FZ for use as an antifungal agent in tissue culture systems.     

Interaction of the NMDA receptor noncompetitive antagonist MK-801 with model and native membranes.

MK-801, a noncompetitive antagonist of the NMDA (N-methyl-D-aspartate) receptor, has protective effects against excitotoxicity and ethanol withdrawal seizures. We have determined membrane/buffer partition coefficients (Kp[mem]) of MK-801 and its rates of association with and dissociation from membranes. Kp[mem] (+/- SD) = 1137 (+/- 320) in DOPC membranes and 485 (+/- 99) in synaptoneurosomal (SNM) lipid membranes from rat cerebral cortex (unilamellar vesicles). In multilamellar vesicles, Kp[mem] was higher: 3374 (+/- 253) in DOPC and 6879 (+/- 947) in SNM. In cholesterol/DOPC membranes, Kp[mem] decreased as the cholesterol content increased. MK-801 associated with and dissociated from membranes rapidly. Addition of ethanol to SNM did not affect Kp[mem]. MK-801 decreased the cooperative unit size of DMPC membranes. The decrease was smaller than that caused by 1,4-dihydropyridine drugs, indicating a weaker interaction with the hydrocarbon core. Small angle x-ray diffraction, with multilayer autocorrelation difference function modeling, indicated that MK-801 in a cholesterol/DOPC membrane (mole ratio = 0.6) causes a perturbation at approximately 16.0 A from the bilayer center. In bilayers of cholesterol/DOPC = 0.15 (mole ratio) or pure DOPC, the perturbation caused by MK-801 was more complex. The physical chemical interactions of MK-801 with membranes in vitro are consistent with a fast onset and short duration of action in vivo.     

Design of Micelle Nanocontainers Based on PDMAEMA-b-PCL-b-PDMAEMA Triblock Copolymers for the Encapsulation of Amphotericin B

The clinical application of amphotericin B (AmB), a broad spectrum antifungal agent, is limited by its poor solubility in aqueous medium and also by its proven renal toxicity. In this work, AmB was encapsulated in micelles obtained from the self-assembly of PDMAEMA-b-PCL-b-PDMAEMA triblock copolymers. The amount of encapsulated AmB depended on the copolymer composition, and short blocks of polycaprolactone (PCL) and poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) showed better performance. All the studied formulations exhibited a controlled release of AmB along 150 h. The formulations presented reduced hemotoxicity while maintaining antifungal activities against Candida albicans, Candida krusei, and Candida glabrata comparable with free AmB. A reduction on the hemotoxicity was found to be due to the slow release and subsequent low aggregation achieved with the use of polymer micelle nanocontainers.KEY WORDS: amphotericin B, antifungal agent, hemotoxicity, micelles