A selective cholesterol-dependent induction of H+/OH- currents in phospholipid vesicles by amphotericin B

The effect of amphotericin B on the proton/hydroxide permeability of small unilamellar vesicles has been investigated by using potential-dependent paramagnetic probes. Amphotericin B at 1-10 molecules/vesicle causes a modest 4-8-fold increase in the background H+/OH- permeability of egg phosphatidylcholine (egg PC) vesicles. However, in the presence of cholesterol, amphotericin B promotes a dramatic increase in the H+/OH- permeability of more than 2 orders of magnitude. Surprisingly, this is not observed in vesicle membranes containing ergosterol. In membranes composed of 5-15 mol% ergosterol, amphotericin B is even less effective at promoting H+/OH- currents than in pure egg PC vesicles. The K+ current promoted by amphotericin B in vesicles formed from egg PC and from egg PC plus cholesterol or ergosterol was measured. No significant sterol dependence was found for the K+ current. These results strongly suggest that different mechanisms, or amphotericin B/sterol complexes, are responsible for the induction of H+/OH- and K+ currents. These results have important implications for understanding the therapeutic and toxic effects of amphotericin B.     

Interaction of the polyene antibiotic amphotericin B with model membranes: differences between small and large unilamellar vesicles

The interaction of the polyene antibiotic amphotericin B (AmB) (Fig. 1) with large unilamellar vesicles (LUV) was monitored by circular dichroism (CD) and carboxyfluorescein (CF) release. LUV afford a far better model for biological membranes than small unilamellar vesicles (SUV) which have been used until now. With dimyristoyl phosphatidyl choline (DMPC) LUV (i.e., containing saturated acyl chains), a strong and not saturable binding for AmB/lipid ratios up to 0.5 was observed both above and below the phase transition temperature. Incorporation of cholesterol into the vesicles did not significantly change the interaction. With egg PC (EPC) LUV (i.e., containing unsaturated acyl chains), quite a different picture emerged: the binding reached saturation for AmB/lipid ratios of about 5 x 10(-3), a result not observed with EPC SUV. When sterols were introduced into membranes, the CD spectral features obtained in the presence of ergosterol were different from those obtained in the presence of cholesterol. Such a different behavior was not observed with SUV. We suggest that species whose CD spectrum was observed after 15 min in the presence of ergosterol-containing EPC LUV is the particular one which forms wide channels and induces a Ca2+ release. (H. Ramos, A. Attias, B.E. Cohen and J. Bolard, submitted for publication). The CF release from EPC LUV induced by AmB was very low, even at very high concentrations of the antibiotic (3 x 10(-4)M). In contrast, an important release of the fluorescent dye was observed with DMPC LUV at concentrations of approximately 10(-5)M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dissociation kinetics and equilibrium binding properties of polyene antibiotic complexes with phosphatidylcholine/sterol vesicles

The interactions of sonicated vesicles with the polyene antibiotics amphotericin B, candicidin, mediocidin , and a water-soluble, guanidine derivative of amphotericin B were examined by UV-visible spectroscopy at concentrations below which the polyenes become self-associated. The association constants, Kapp, and the numbers of binding sites per sterol or phospholipid molecule (n) were determined at 30 degrees C and pH 7.4. A single class of binding sites was found, with no evidence of cooperativity. For the binding of mediocidin , amphotericin B, and the guanidine derivative with phosphatidylcholine (PC), PC/cholesterol, and PC/ergosterol vesicles, Kapp was in the range of (1.0-3.0) X 10(6) M-1; Kapp was higher for candicidin-vesicle interaction, reaching 9.0 X 10(6) M-1 with PC/ergosterol vesicles. Binding of the guanidine derivative of amphotericin B to PC vesicles lacking sterol was extensive (n = 0.46); since the other polyenes, which have low aqueous solubilities, had n less than 0.05, positive charges in the mycosamine moiety appear to enhance the extent of polyene antibiotic interaction with the glycerophospholipid head group. Higher values of n (and, therefore, of nKapp ) were found with sterol-containing than with sterol-free vesicles, suggestive of penetration of the polyenes toward the interior of the bilayer when sterol is present. For binding to PC/sterol vesicles, nKapp followed the order of candicidin greater than guanidine derivative of amphotericin B greater than amphotericin B much greater than mediocidin . The values of n and nKapp were appreciably higher for amphotericin B-ergosterol than for amphotericin B-cholesterol interaction in vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic analysis of the interaction between amphotericin B and human serum albumin using surface plasmon resonance and fluorescence spectroscopy.

The binding interaction between amphotericin B and human serum albumin (HSA) has been studied using surface plasmon resonance (SPR) spectroscopy combined with a fluorescence quenching method to confirm the binding kinetic results. In this paper, the SPR method used to study the drug-protein interaction has been described in detail. The association rate constant, dissociation rate constant and the equilibrium association constant of amphotericin B binding to HSA were obtained using this method. To confirm the feasibility of the SPR method, a fluorescence quenching method was performed to obtain the equilibrium constant. In order to obtain more accurate results, experiment design was used to optimize the fluorescence quenching process. The two equilibrium association constants obtained using the two methods were 4.017 x 10(4) M(-1) (SPR) and 3.656 x 10(4) M(-1) (fluorescence quenching method) respectively.     

Amphotericin and model membranes. The effect of amphotericin B on cholesterol-containing systems as viewed by 2H-NMR

The interactions between amphotericin B and sterol-containing model membranes were monitored by 2H-NMR of deuterium-labelled dimyristoylphosphatidylcholine (DMPC), cholesterol and epicholesterol. The addition of amphotericin B to a cholesterol/DMPC (3:7) system was perceived differently by the lipid, depending upon the depth in the bilayer: no structural change was manifest in the acyl chain region associated with the plateau in molecular ordering (C4'), whereas the lipid clearly senses two environments near the center of the bilayer (C13', C14'). The amount as well as the ordering properties of the more ordered antibiotic-induced component, sensed at C14', increased with decreasing temperature. The structural parameters of deuterium-labelled cholesterol in cholesterol/DMPC mixtures were unchanged upon addition of amphotericin B, regardless of the bilayer depth. Upon addition of amphotericin B, the lipid T1 values are unchanged, whereas the T2 values are reduced by a factor of 2. The minimum in T1 observed for cholesterol in DMPC at 32-35 degrees C was shifted towards 38-40 degrees C in the presence of amphotericin B. Epicholesterol-containing dispersions of DMPC had properties similar to those of their cholesterol-containing analogs; a noticeable difference between the systems was an approx. 10% increase in the segmental order parameters on the addition of amphotericin B to the system containing the alpha-isomer of cholesterol. The concept of a dynamic complexation between amphotericin B and sterol is discussed.     

Probing amphotericin B single channel activity by membrane dipole modifiers

The effects of dipole modifiers and their structural analogs on the single channel activity of amphotericin B in sterol-containing planar phosphocholine membranes are studied. It is shown that the addition of phloretin in solutions bathing membranes containing cholesterol or ergosterol decreases the conductance of single amphotericin B channels. Quercetin decreases the channel conductance in cholesterol-containing bilayers while it does not affect the channel conductance in ergosterol-containing membranes. It is demonstrated that the insertion of styryl dyes, such as RH 421, RH 237 or RH 160, in bilayers with either cholesterol or ergosterol leads to the increase of the current amplitude of amphotericin B pores. Introduction of 5α-androstan-3β-ol into a membrane-forming solution increases the amphotericin B channel conductance in a concentration-dependent manner. All the effects are likely to be attributed to the influence of the membrane dipole potential on the conductance of single amphotericin B channels. However, specific interactions of some dipole modifiers with polyene-sterol complexes might also contribute to the activity of single amphotericin B pores. It has been shown that the channel dwell time increases with increasing sterol concentration, and it is higher for cholesterol-containing membranes than for bilayers including ergosterol, 6-ketocholestanol, 7-ketocholestanol or 5α-androstan-3β-ol. These findings suggest that the processes of association/dissociation of channel forming molecules depend on the membrane fluidity.     

Amphotericin B channel conductance inactivation

Effects induced in bilayer lipid membranes by amphotericin B and its alkyl derivatives was analysed. Inactivation of the antibiotic-dependent multichannel membrane conductance was discovered. Kinetics of membrane conductivity was shown to depend on the antibiotic concentration in the membrane. At concentrations between 10(-8) and 10(-7) M, the resulting conductance appeared to the transient. We suggest that the phenomenon of biphasic kinetics of membrane conductance is the result of a consecutive transformation of polyene channels in the membrane: half-pores are assembled on either side of membrane-nonconducting 1; two half-pores combine to build up a conducting channels-conducting 2, and the conducting channels are disassemled to monomers and nonconducting self-associated forms inside the membrane-disassembled state (nonconducting 3). To explain the transient characteristics of the induced conductance, it is proposed that the antibiotic, present in the solution under self-associated form, binds the membrane and forms pores, then dissociates in the bilayer in a non-active monomeric form. The existence of definite monomers and nonconducting self-associated forms of amphotericin B molecules inside the membrane was estimated from the dependence of kinetic conductance of lipid membranes of amphotericin B and its alkyl derivatives, when the antibiotics are washed out from aqueous medium. Equilibrium between different antibiotic assemblies inside the membrane was demonstrated by the kinetics of conductance decrease following washing the antibiotic. Using circular dichroism measurements, we observed that amphotericin B alkyl derivatives were in self-associated form being susceptible to form pores across cholesterol-containing membranes. The phenomenon of biophasic kinetics was observed only in the cholesterol-containing membrane. The substitution of membrane cholesterol for ergosterol provides monotonic kinetics of membrane conductance at any antibiotic concentration.     

Nitroxide spin-probe study of amphotericin B-ergosterol interaction in egg phosphatidylcholine multilayers

The interaction between the polyene macrolide antibiotic, amphotericin B, and ergosterol in egg phosphatidylcholine multilayers was investigated using head group and acyl chain nitroxide spin-labelled phosphatidylcholine as probes. At physiological concentrations of less than 15 mol% sterol in egg phosphatidylcholine multilayers amphotericin B accumulates near the head group region until an amphotericin B : ergosterol ratio of approximately 0.7 is achieved. As the proportion of amphotericin B is increased above this value, formation of an acyl chain disordering complex occurs which has an approximate antibiotic:sterol ratio of unity. Dicetyl phosphate was used to increase the solubility of ergosterol past its normal limit in pure egg phosphatidylcholine (approximately 15 mol%). At concentrations of ergosterol higher than 15 mol% a complex of two ergosterol molecules and one amphotericin B was postulated when there was insufficient antibiotic to form a 1:1 complex.     

Interaction between phospholipid bilayer membranes and the polyene antibiotic amphotericin B: Lipid state and cholesterol content dependence

The interaction between amphotericin B and egg yolk phosphatidylcholine, dimyristoyl (DMPC) and dipalmitoyl phosphatidylcholine (DPPC) phospholipid bilayer vesicles has been monitored by the circular dichroism (CD) spectra of amphotericin B at a 1 · 10^?5 M concentration. This method has revealed that amphotericin B may be present in a number of different forms depending on the time elapsed after the mixing, the cholesterol content of the vesicles and the vesicles' physical state. Some striking features of these CD detected species are the following: with egg yolk phosphatidylcholine and a molar cholesterol percentage lower than 25, at 25°C several forms are coexistent, their amount is time-dependent; with dipalmitoyl or dimyristoyl phosphatidylcholines without cholesterol or with a cholesterol molar percentage lower than 25, in the gel state, a form different from the former appears very rapidly; with egg yolk phosphatidylcholine, DMPC and DPPC at a molar cholesterol percentage between 25 and 50 a new form is monitored, identical in the three cases and observed in the liquid crystalline state as well as in the gel state. In the case of the three phospholipids without cholesterol a definite interaction with the antibiotic is observed but with different characteristics according to the nature of lipid.With amphotericin B ‘Fungizone’ the same species are monitored but their appearance is much slower.Two explanations are proposed for the origin of the discrepancies between CD and electronic absorption.     

Rates of amphotericin B and filipin association with sterols. A study of changes in sterol structure and phospholipid composition of vesicles

The influence of structural modifications in sterols and phospholipids on the rate of polyene antibiotic-sterol interaction was studied. For filipin and amphotericin B association with sterols in vesicles, a preferential interaction was found with sterols whose side chain length is close to that of cholesterol. Introduction of trans double bonds into the sterol side chain did not alter the rate of interaction in vesicles. The delta 7-bond of the sterol appears to be of critical importance in amphotericin B-sterol interaction, whereas the delta 5-bond is not essential. These observations are relevant to the well-known effects of amphotericin B on cell membranes containing ergosterol compared with those containing cholesterol. The dependence of the rates of sterol-polyene antibiotic interaction on the phospholipid composition of the vesicles indicates that phospholipid vesicles may be an inadequate model for reaching a comprehensive understanding of the effects exerted on biological membranes by these agents.     

Effect of polymyxin B on the conductivity of negatively charged bilayer lipid membranes

Effect of polymyxin B on the planar bilayer lipid membranes (BLM) formed from synthetic phosphatidic acid has been studied. The addition of cholesterol to phospholipid in molar ratio 1 : 2 was followed by an increase of BLM conductance from 2 x 10(-8) to 3 x 10(-7) Ohm-1 cm-2. It was suggested that the observed increase of conductance was due to the fluidity of the membrane matrix in the presence of cholesterol. It was shown that 10(-6)--10(-5) M polymyxin slightly affected the conductance of BLM from phosphatidic acid. It was found that polymyxin increased conductance of negatively charged BLM modified by palmitic acid from 10(-8) to 10(-6) Ohm-1 cm-2.     

Thermodynamic and spectroscopic features of the behavior of amphotericin B in aqueous medium

The interaction between amphotericin B molecules in aqueous medium solution was studied using absorption and circular dichroism approaches. The results showed that at concentrations below 1 microM of amphotericin B, an equilibrium between the monomer and aggregate occurred with a constant of approximately 0.6x10(6) M(-1). The aggregate formation constant was dependent on the experimental conditions of the medium: its value increased at acidic pH values, while alkaline medium induced the equilibrium displacement to the monomer formation. Either neutral salts or chaotropic agents such as urea prevented the formation of the aggregate. The presence of net electrical charge on the amine and carboxyl groups plays a role in the thermodynamic stability of the aggregate. A hydrophobic effect was also found between the monomer form and the water molecules of neighbours. In the aggregate formation water molecules were released contributing to an increase in the entropic change.     

Correlation between the association and the desmutagenicity of benzo(a)pyrene by hemin: comparison with hemoproteins, transient metals and amines

The mutagenicity of the potent carcinogen benzo(a)pyrene proved to be lost by binding with hemin and other tested compounds. The association constant (Kass) and the desmutation constant (Kmut) of benzo(a)pyrene and hemin were 8.5 x 10(4) M(-1) and 7.0 x 10(-4) M(-1), respectively. The correlation coefficient (gamma) of the association and the desmutation of benzo(a)pyrene and hemin was nearly 1, and the coefficient of regression of association on the desmutation, gamma (sigma ass/sigma mut), was 0.78. The correlation of other compounds, ferric chloride among transient metals, and 3,3'-diaminodipropylamine and p-phenylenediamine, were also almost 1 in both values, but their Kass an Kmut were must smaller than those of hemin: Kass:Kmut, 525 M(-1):520 M(-1) for ferric chloride, 2.2 x 10(3) M(-1):2.3 x 10(3) M(-1) for diaminodipropylamine and 175 M(-1):125 M(-1) for p-phenylenediamine. Good agreement of Kass and Kmut means that the desmutation of benzo(a)pyrene is caused by binding with tested materials.     

Amphotericin B inhibits entry of Leishmania donovani into primary macrophages

Visceral leishmaniasis is a vector-borne disease caused by an obligate intra-macrophage protozoan parasite Leishmania donovani. The molecular mechanisms involved in internalization of Leishmania are still poorly understood. Amphotericin B and its formulations are considered as the best existing drugs against visceral leishmaniasis and are being increasingly used. The reason for its antileishmanial activity is believed to be its ability to bind ergosterol found in parasite membranes. In case of in vivo amphotericin B treatment, both host macrophages and parasites are exposed to amphotericin B. The effect of amphotericin B treatment could therefore be due to a combination of its interaction with both sterols i.e., ergosterol of Leishmania and cholesterol of host macrophages. We report here that cholesterol complexation by amphotericin B markedly inhibits binding of L. donovani promastigotes to macrophages. These results represent one of the first reports on the effect of amphotericin B on the binding of Leishmania parasites to host macrophages. Importantly, these results offer the possibility of reevaluating the mechanism behind the effectiveness of current therapeutic strategies that employ sterol-complexing agents such as amphotericin B to treat leishmaniasis.     

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.     

Anfotericina B liposomal: farmacología clínica,farmacocinética y farmacodinamia

Liposomal amphotericin B is a lipid formulation of the antifungal drug amphotericin B with some distinguishing characteristics in its pharmacological behavior that entail some clinical differences of great interest. The significant improvement in the systemic and renal tolerability is one of them. This fact is related to the great stability of the liposome, promoted by its negative charge, the presence of cholesterol and the remarkable thermo-stability of the remaining lipids that compose it. In this situation, amphotericin B seems to be released from the liposome not spontaneously but when the liposome binds to the ergosterol in the fungal cell membrane. For this reason, there is almost no free amphotericin B in plasma or tissues, although it seems that its availability is greater when there is fungal infection. As a consequence, when the pharmacokinetic behavior is studied, the concentration and availability of liposomal amphotericin B are very high, and its volume of distribution is reduced in comparison with the other formulations.     

Effect of amphotericin B on cholesterol-containing liposomes of egg phosphatidylcholine and didocosenoyl phosphatidylcholine. A refinement of the model for the formation of pores by amphotericin B in membranes

(1) Binding and K+-permeability measurements were performed on egg and 22 : 1c/22 : 1c-phosphatidylcholine liposomes with or without cholesterol. (2) Amphotericin B binds specifically to cholesterol in both types of liposome despite the difference in bilayer thickness. (3) Addition of amphotericin B to one side of the cholesterol-containing egg phosphatidylcholine bilayers induces a fast K+ efflux from the outermost compartment of the liposomes. In contrast, the total K+ content of sonicated unilamellar cholesterol-containing egg phosphatidylcholine vesicles is released by amphotericin B. (4) Amphotericin B addition to one side of the cholesterol-containing 22 : 1c/22 : 1c-phosphatidylcholine liposomes does not cause a change in K+ permeability. The presence of amphotericin B on both sides of the bilayer, however, induces an increase in K+ permeability. (5) A model is proposed which accounts for the effect of bilayer thickness on the amphotericin B-induced permeability changes in membranes.     

Effects of amphotericin B and its methyl ester on plasma membranes of Candida albicans and erythrocytes as examined by freeze-fracture electron microscopy

Freeze-fracture electron microscopy of the plasma membranes of Candida albicans yeast cells and red blood cells treated with amphotericin methyl ester and amphotericin B showed that amphotericin B (50 micrograms ml-1) caused extreme aggregation of intramembranous particles on the protoplasmic fracture face of the C. albicans membrane, and a marked reduction of the density of intramembranous particles. On the other hand, the rearrangement of intramembranous particles induced by amphotericin methyl ester (50 micrograms ml-1) produced elevations of the particle-free membrane domains toward the outside of the cells, so that the particles were aggregated in linear furrows surrounding these elevations on the protoplasmic fracture face, and the corresponding ridges on the exoplasmic fracture face. The density of intramembranous particles was greatly reduced on the protoplasmic fracture face. Both polyenes produced only small changes in the erythrocyte membranes at the same concentration. These results suggest that amphotericin methyl ester affects the ergosterol-containing membranes more than amphotericin B, and that ergosterol has a higher sensitivity for these two polyene antibiotics than cholesterol.     

In Vitro Activity of a New Antifungal Azolyl-substituted Indole Against Aspergillus fumigatus

A new 2- (α -azolylbenzyl)indole derivative exhibited high in vitro activity against 10 strains of Aspergillus fumigatus. This active compound, MT18n, had MIC of 2 μg/mL and is slightly less active than itraconazole and amphotericin B. The mechanism of action of this compound was evaluated through scanning electron microscopy, ergosterol biosynthesis inhibition and phospholipase A 2 -like activity inhibition studies. Scanning electron microscopy allowed observation of the membrane perturbations caused by MT18n and inference of a critical role of MT18n in membrane synthesis inhibition. Like other azole derivatives MT18n inhibits ergosterol biosynthesis, with a minimal inhibitory concentration of 6 μM. On the other hand, MT18n (10 μM) decreased the secreted phospholipase A 2 -like activity of Aspergillus fumigatus, an enzyme involved in the invasion process of the host. These results show the high in vitro activity of MT18n against Aspergillus fumigatus and suggest that this compound disturbs the membrane structure via ergosterol biosynthesis inhibition and exhibits phospholipase activity inhibition.