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Journal of Antimicrobial Chemotherapy (2003) 52, 904-910
© 2003 The British Society for Antimicrobial Chemotherapy

Heat-induced reformulation of amphotericin B-deoxycholate favours drug uptake by the macrophage-like cell line J774

Monique Chéron, Caroline Petit, Jacques Bolard and François Gaboriau*

L.P.B.C. (UMR CNRS 7033), Université Pierre et Marie Curie, 4 place Jussieu, F-75252 Cedex 05, France

Received 27 March 2003; returned 23 May 2003; revised 18 August 2003; accepted 21 August 2003


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Aim: Heat treatment of deoxycholate-amphotericin B (AmB-DOC) leads to a therapeutically interesting supramolecular rearrangement (h-AmB-DOC); this reformulation improves the therapeutic index of AmB-DOC by reducing amphotericin B (AmB) toxicity in mammalian cell lines from 3- to 10-fold. Its activity in experimentally induced fungal infection in mice remains unchanged compared with AmB-DOC, whereas its activity is 2.5 times higher in Leishmania donovani-infected mice. This work investigates the in vitro mechanism that allows this improvement.

Methods: In this study, we analysed the role of serum components on the interaction of h-AmB-DOC with two cultured cell lines: murine peritoneal macrophage cells (J774) and kidney epithelial cells (LLCPK1). The methods used were: spectrophotometry for AmB uptake; MTT assay for cell viability; and lactate dehydrogenase release for membrane damage.

Results: In the presence of 10% fetal calf serum (FCS), the toxicity of AmB-DOC or h-AmB-DOC for both cell lines was null or weak. Interestingly, in J774 cells, the uptake of AmB in the form of h-AmB-DOC was much higher. In LLCPK1 cells, AmB uptake was more limited in both cases but remained higher with h-AmB-DOC. In the absence of FCS, no toxicity for either cell line was observed with h-AmB-DOC.

Conclusions: These findings confirm the importance of serum proteins in AmB biodistribution and suggest that, in vivo, the reduced toxicity and the improved antileishmanial activity of AmB-DOC after moderate heating may be the result of its increased uptake by macrophages.

Keywords: amphotericin B, macrophages, serum, lipoproteins, toxicity


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
The deoxycholate salt of amphotericin B (AmB-DOC; Fungizone) is the ‘gold standard’ treatment for systemic fungal infection.1 Unfortunately, amphotericin B (AmB) causes acute side effects following intravenous administration, which limits its more extensive clinical use. Lipid formulations of AmB have recently been developed which drastically reduce the toxicity of the drug in humans.2 However, high dosages of these formulations are needed for treatment to be effective3 and, consequently, make treatment expensive.

In an attempt to reduce the toxicity of AmB aggregates observed in aqueous solution of AmB-DOC, we previously reported that heat treatment leads to an increase in the size of aggregates.4 Heat-induced ‘superaggregates’ were demonstrated to be less toxic than unheated commercial formulations,5 in vitro, in various cultured mammalian cells. Heat treatment of AmB-DOC solutions decreased the acute toxicity of AmB in mice, whereas antifungal activity was retained. In experimentally-induced mycoses in mice, an improvement in antifungal activity was obtained by using heated AmB formulations (h-AmB-DOC) whose decreased toxicity allows administration of drug doses twice those achievable with commercial Fungizone.6 Furthermore, the activity of the h-AmB-DOC formulation against Leishmania donovani was shown to be improved with respect to AmB-DOC.7

The possible practical therapeutic benefits of this new formulation8 deserve further studies to understand the in vitro mechanism that allows this improvement.

Physico-chemical studies (light scattering, circular dichroism, absorbance, cryo-transmission, electron microscopy)4,5,9,10 have shown that the moderate heating of AmB-DOC induces its ‘superaggregation’; they have also shown that rate constants and amplitudes of dissociation of AmB from aggregates or superaggregates to monomers are greater for AmB-DOC than for h-AmB-DOC, respectively. The higher chemical stability of the heat-treated samples (mainly as superaggregates) with respect to the unheated ones (mainly aggregates) has been previously reported for AmB4 as well as for h-AmB-DOC5 solutions. Furthermore, because serum components have been shown to play a determining role in the development of AmB activity and toxicity,1 the influence of moderate heating on their interaction with AmB-DOC was analysed.11 AmB-DOC aggregates appeared far less stable in the presence of albumin than h-AmB-DOC; interaction with serum albumin appeared to be a dominant feature for both drug preparations.

In this study, we have examined two other aspects of the effect of mild heating on AmB-DOC: the consequence on drug uptake by macrophages and the development of toxicity in renal cells. Uptake of AmB or AmB lipid formulations seems to be an important parameter for an increase in AmB therapeutic index.12,13 For renal cells, LLCPK1 cells derived from primary cultures of pig proximal tubular cells were used as a model system to study, in vitro, AmB renal cytotoxicity,14,15 which is considered to be at the origin of important AmB side effects. The incorporation of AmB into these cells was measured by spectrophotometry and their viability in the presence of increasing concentrations of AmB-DOC or h-AmB-DOC was measured by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) test. The AmB-DOC- or h-AmB-DOC-induced membrane damage was measured by the release of the enzyme lactate dehydrogenase (LDH). The presence and absence of fetal calf serum (FCS) and low-density lipoproteins (LDL) were varied in the studies.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Chemicals

AmB-DOC was a gift from Bristol-Myers Squibb (Neuilly, France) and was dissolved in sterile water (Fresenius Pharma, Sèvres, France) at a concentration of 5 x 10–3 M. h-AmB-DOC was obtained by heating the solution at 70°C for 20 min. Further dilutions of these stock solutions were made in PBS. AmB concentrations were determined in a 100-fold dilution in methanol by measurement of absorbance at 407 nm ({varepsilon}407 in MeOH = 150–000 L·mol–1·cm–1) with a Varian Cary 219 UV-visible spectrophotometer.

Unless stated otherwise, other chemicals were purchased from Sigma Chemical Co. (La Verpillière, France). They were of first-grade purity and were used without further purification, except for low-density lipoproteins (LDL), which were dialysed in PBS for one night, at 4°C, in dialysis tubes (Sigma) for molecules with a molecular weight greater than 12 000 Da. The LDL stock solution had a concentration of 5 mg/mL.

Cell treatment

Murine peritoneal macrophages from the cell line J774 and pig kidney tubular cells from the cell line LLCPK1 were grown in monolayers at 37°C in 5% humidified CO2 in RPMI 1640 for J774 cells and DMEM/ L-glutamine for LLCPK1 cells, supplemented with 10% heat-inactivated fetal calf serum (FCS; Gibco, France).

For the experiments, cells were harvested with trypsin and aliquotted 24 h before the Fungizone treatment in 96-well microplates (Nunc Ltd, Chicago, IL, USA) at a density of 2 x 104 cells per well for LLCPK1 cells and 5 x 104 cells per well for J774 cells. Cells were treated at 4 or 37°C in their respective culture media, with the required concentrations of AmB-DOC (10–6 to 2 x 10–4 M) for 4 or 18 h, in the presence or absence of 10% FCS, h-AmB-DOC and/or various LDL concentrations. Each condition was tested in triplicate. When necessary, AmB-DOC and h-AmB-DOC were equilibrated for 15 min with FCS or LDL, at room temperature, before cell treatment.

Cell viability measurements

Supernatants were then collected for lactate dehydrogenase (LDH) release and cells were rinsed twice with 100 µL of culture medium before the viability assay, which was based on formazan formation from MTT.16 MTT measurements were carried out by measuring absorbance at 540 nm with a Multiscan RC microreader (Labsystem, Les Ulis, France). Data were expressed as percentages of the control (untreated cells) and were the mean ± S.D. of three independent measurements.

LDH release constituted the permeability assay, which is a non-invasive means of documenting membrane damage and cell integrity. The LDH activity released by supernatants was determined by measuring the NADH disappearance rate during the LDH-catalysed conversion of pyruvate into lactate.17 AmB-DOC in supernatants was previously shown not to affect NADH measurements. Kinetics data, which were carried out in a microplate reader, were deduced from the linear part of kinetics for a period less than 3 min. Data were expressed as µmoles of NADH consumed per minute and were the mean of three independent measurements.

Uptake of Fungizone by cells

The dosage of cellular AmB was carried out according to the method of Legrand et al.13 After treatment, cells were rapidly washed with cold medium, in order to eliminate free AmB while minimizing the release of internalized AmB. Cells were lysed for 20 min at room temperature with shaking, and with 100 µL of Triton 0.1%. An aliquot of 50 µL of each cell lysate was then mixed with 150 µL of methanol, which solubilized AmB as the monomeric form. The AmB absorbance was measured at 407 nm and converted into AmB concentration by using a calibration curve obtained in the absence of cells, under the same experimental conditions.

Data were fitted iteratively using sigmoid curves (MTT test, release of LDH or uptake by cells) according to the four-parameter equation:


In this equation, c1 and c4 correspond, respectively, to the minimal and maximal values of the sigmoid; c2 is the AmB concentration inducing 50% effect and c3 is the shape parameter of the curve related to the cooperativity of the phenomenon.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Macrophage-like cell line: J774

After incubation for 4 h at 37°C in the absence of FCS, AmB-DOC and h-AmB-DOC developed toxicity above 10 µM but to a lesser degree for h-AmB-DOC (Figure 1). Measured by the MTT test, a 75% survival rate with h-AmB-DOC was observed at 100 µM as opposed to a 15% survival rate with AmB-DOC. The release of LDH was maximal with AmB-DOC above 30 µM, whereas 100 µM h-AmB-DOC was necessary to obtain the same result (Figure 2a). In contrast, in the presence of 10% FCS, no membrane damage was observed with either preparation. The release of LDH was very low, almost independent of the presence of AmB (Figure 2b).



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  		Figure 1. Toxicity of AmB-DOC (open circles) and h-AmB-DOC (filled circles) in J774 cells as measured by the MTT test after a 4 h incubation period at 37°C in the absence of FCS.

 


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  		Figure 2. Toxicity of AmB-DOC and h-AmB-DOC in J774 cells as measured by the release of LDH after a 4 h incubation period at 37°C in the absence (a) or the presence (b) of 10% FCS.

 
After incubation for 4 h with 50 µM AmB in the presence of FCS, the AmB uptake was 2.5-fold more efficient at 37°C with h-AmB-DOC than with AmB-DOC (Figure 3a). In contrast, when incuba- tion was carried out in the presence of FCS at 4°C, the AmB uptake was much smaller for h-AmB-DOC and even null for AmB-DOC (Figure 3b).



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  		Figure 3. Uptake of AmB-DOC and h-AmB-DOC by J774 cells after a 4 h incubation period, in the presence of 10% FCS, at 37°C (a) or 4°C (b).

 
Pig kidney tubular cell line: LLCPK1

After incubation for 18 h at 37°C, in the absence of FCS, AmB-DOC toxicity in LLCPK1 cells was observed at concentrations higher than 20 µM, as shown by the MTT test (Figure 4a) and LDH leakage (Figure 4b). In striking contrast, h-AmB-DOC was not cytotoxic in this range of AmB concentrations. The presence of 10% FCS suppressed the difference in toxicity between AmB-DOC and h-AmB-DOC: no significant toxicity was observed up to 120 µM with both formulations (Figure 5a and b). In the presence of FCS, the AmB uptake was greater by J774 cells than by LLCPK1 cells. Surprisingly, with or without FCS, more drug was incorporated into LLCPK1 cells when they were incubated with the h-AmB-DOC formulation than with conventional AmB-DOC, although toxicity was lower in the first case (Figure 6).



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  		Figure 4. Toxicity of AmB-DOC and h-AmB-DOC in LLCPK1 cells in the absence of FCS, as measured by the MTT test (a) or LDH release in supernatants (b) after an 18 h incubation period at 37°C.

 


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  		Figure 5. Toxicity of AmB-DOC and h-AmB-DOC in LLCPK1 cells in the presence of 10% FCS, as measured by the MTT test (a) or the release of LDH in supernatants (b) after an 18 h incubation period at 37°C.

 


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  		Figure 6. Uptake of AmB-DOC and h-AmB-DOC by LLCPK1 cells after an 18 h incubation period at 37°C, in the absence (a) or in the presence of 10% FCS (b).

 
The same studies were carried out in the presence of increasing concentrations of LDL (from 0 to 0.5 mg/mL) at 50 µM AmB. At this concentration, in the absence of FCS, the drug induced 60–70% of toxicity, measured by the MTT test. The presence of LDL at a concentration as low as 0.2 mg/mL increased the survival of LLCPK1 cells after incubation with AmB-DOC but decreased survival after incubation with h-AmB-DOC (Figure 7). In other words, in the presence of LDL, the toxicities of AmB-DOC and h-AmB-DOC were similar, in contrast to what was observed in the absence of LDL. The presence of LDL also decreased the uptake of AmB-DOC and h-AmB-DOC (Figure 8).



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  		Figure 7. Toxicity of 50 µM AmB-DOC and h-AmB-DOC in LLCPK1 cells after an 18 h incubation period at 37°C in the absence of FCS and with increasing concentrations of LDL, as measured by the MTT test (a) or LDH release (b).

 


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  		Figure 8. Uptake of 50 µM AmB-DOC and h-AmB-DOC by LLCPK1 cells after an 18 h incubation period at 37°C in the absence of FCS.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The purpose of this study was to give an indication of the mechanism by which mild heating of AmB-DOC reduces the toxicity of the drug and allows higher dosages to be administered to mice infected with Candida albicans6,9 or Leishmania donovani,7 resulting in significantly improved therapeutic efficacy. We have looked at two mechanisms that have already been suggested as possible origins of the reduced toxicity of the lipid-AmB formulation:12 (i) increased uptake of the drug into macrophages connected to its slow release from these cells;13 and (ii) decreased toxicity as a result of a lowering of the amount of free drug in serum. Our studies were carried out in the presence of FCS, the role of which has been shown to be determinant in the development of AmB activity.1,2 As expected, we observed a much higher uptake of AmB by macrophages in the presence of FCS when the deoxycholate formulation had been heated than when it had not, although the toxicity was mild for both formulations. Compared with the conventional AmB-DOC formulation, a similar increase in AmB uptake by J774 cells was observed with a preparation similar to the commercial Abelcet (L-Amp B 33; Liposome Company, Princeton, NJ, USA).13 Furthermore, the AmB uptake was significantly reduced with both formulations, AmB-DOC and h-AmB-DOC, by incubation at 4°C. This has been considered13 as an indication that AmB is internalized in J774 cells and not simply bound to the membrane. In the absence of FCS, the relatively high cytotoxicity of the AmB-DOC formulation was strongly reduced by moderate heating.

Similar results were obtained with the LLCPK1 renal cells. We observed a weak toxicity of both formulations in the presence of FCS, compared with the toxicity of AmB-DOC in the absence of FCS and a higher uptake of AmB in cells treated with h-AmB-DOC than with AmB-DOC. This is probably because of the large size of the h-AmB-DOC ‘superaggregates’ (600 nm),6 which contain a high number of AmB molecules and probably allows them to be more efficiently captured by the LLCPK1 cells than smaller aggregates from the unheated AmB-DOC formulation (Figure 9). In such a hypothesis, the h-AmB-DOC formulation could give rise to higher concentrations in tissue, for longer periods of time. This formulation could have similar properties to liposomal AmB, i.e. act as a reservoir of monomeric AmB. The cytotoxicity of the commercial Ambisome formulation (NeXstar, Cambridge, UK) on the LLCPK1 cells was found to be slightly higher than that of h-AmB-DOC (data not shown).



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  		Figure 9. Model of AmB uptake and cytotoxicity of h-AmB-DOC and AmB-DOC formulations. Heating for 20 min at 70°C leads to the superaggregation of amphotericin B (AmB). The dissociation of the superaggregates (h-AmB-DOC) into the monomeric form occurs at concentrations lower than those for the aggregates (AmB-DOC). This higher thermodynamic stability of the superaggregated form could influence its association with lipoproteins such as LDL and modulate the AmB uptake. Higher AmB uptake is observed with h-AmB-DOC whereas this formulation appears to be less toxic than AmB-DOC in many cell lines. The more efficient endocytosis of AmB deoxycholate aggregates in h-AmB-DOC is probably the result of their larger size (higher number of AmB molecules in the ‘superaggregates’). The size of the cross symbolizes the relative cytotoxicity of the formulation. The size of the AmB circle in the cell is related to AmB uptake.

 
To improve understanding of the role of serum components, we analysed the effects of LDL addition to the serum-depleted medium on the interaction of LLCPK1 cells with AmB-DOC and h-AmB-DOC. We observed that in the presence of LDL, the toxicity and the AmB uptake into this cell line were similar with both formulations. The interaction with LDL of AmB, dissociated from the h-AmB-DOC ‘superaggregates’, reduced the AmB uptake and slightly increased the cytotoxicity of this formulation. In contrast, the cytotoxicity of the AmB-DOC aggregates was markedly decreased in the presence of LDL (Figure 9). For both formulations, the AmB uptake in the presence of LDL could involve the internalization of the AmB-LDL complexes associated with the LDL receptors and the number of these membrane receptors could be the limiting factor. As a matter of fact, AmB-DOC is highly bound in human plasma,18,19 and serum albumin dominates the interaction with the whole serum11 although binding to lipoproteins is also present.20–22 In particular, AmB-DOC binding to LDL may be important because of the high expression of LDL receptors at the surface of LLCPK1 cells (96–000 sites per cell) with high affinity constants.15 The influence of AmB mild heating on the interaction of the drug with serum components has been further determined.11 A significantly lower percentage of AmB from h-AmB-DOC was recovered in high-density lipoprotein (HDL), LDL and triglyceride-rich lipoprotein fractions and a greater percentage recovered in the lipoprotein-deficient plasma. Furthermore, AmB-DOC aggregates were shown to be far less stable in the presence of HDL, LDL and serum albumin than h-AmB-DOC. We also deduced from a spectroscopic study that binding of free AmB to LDL was reduced with the h-AmB-DOC formulation in comparison with the unheated AmB-DOC (data not shown). This probably results from the higher thermodynamic stability of the h-AmB-DOC ‘superaggregates’, which dissociate into AmB monomers more slowly than the AmB-DOC aggregates (Figure 9).

In the presence of FCS, h-AmB-DOC is more internalized into cells, possibly in correlation with the higher binding of h-AmB-DOC to serum albumin. It should be noted that, concerning AmB-DOC our results and those of Krause & Juliano14 differ from those described by Wasan et al.,15 who reported no effect of LDL on AmB-DOC cytotoxicity. The different experimental conditions could be at the origin of this difference (Professor K. M. Wasan, University of British Columbia, personal communication). In our experiments, the LLCKP1 cells were pre-incubated for 24 h before treatment in complete medium, supplemented with 10% FCS. This pre-incubation could have induced a down-regulation of LDL receptors because of the abundance of LDL apolipoprotein in the medium, leading to a reduced internalization of AmB–LDL complex and to a reduced toxicity.

It has already been shown that the heat-induced superaggregation of AmB-DOC provokes a smaller release of tumour necrosis factor (TNF-{alpha}) from THP-1 human monocytes, and this cytokine is possibly associated with the unpleasant side effects of AmB.11,23

In conclusion, it appears that in the presence of FCS, that is under conditions relevant to the in vivo situation, the consequence of AmB-DOC mild heating is to strongly increase the uptake of AmB by macrophages. A low AmB toxicity for renal cells is observed, but it is the same as conventional AmB-DOC. Under these conditions, it can be suggested that the increased therapeutic index of the heated formulation, observed in mice, compared with AmB-DOC, may result from greater phagocytosis of AmB deoxycholate aggregates in h-AmB-DOC, owing to their larger size (higher number of AmB molecules in the ‘superaggregates’). It has often been suggested that macrophages act as reservoirs of AmB and slowly release it as monomeric AmB. Another point is that the large size of the h-AmB-DOC ‘superaggregates’ (600 nm) probably allows them to be efficiently captured by the macrophage and to be transferred to the site of infection with L. donovani amastigotes. Studies in the absence of FCS would have led to different conclusions, stressing instead decreased toxicity of h-AmB-DOC in cells. Such a decrease is not observed in the presence of FCS, which already has low AmB-DOC toxicity.

Further experiments are under way to compare the biological efficiency and the mechanism of action on various fungal and parasite cells of the heated deoxycholate AmB formulation with that of other commercial AmB formulations.


   Acknowledgements
 
This work was supported by the Ministère de la Recherche et de la Technologie and the Fondation pour la Recherche Médicale.


   Footnotes
 
* Corresponding author. Present address. Régulation des équilibres fonctionnels du foie normal et pathologique, INSERM U522, Centre Hospitalier Pontchaillou, F-35033, Rennes Cedex, France. Tel: +33-2-99-54-74-02; Fax: +33-2-99-54-01-37; E-mail: francois.gaboriau{at}univ-rennes1.fr Back


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Hartsel, S. & Bolard, J. (1996). Amphotericin B: new life for an old drug. Trends in Pharmacological Sciences 17, 445–9.[CrossRef][Medline]

2 . Dupont, B. (2002). Overview of the lipid formulations of amphotericin B. Journal of Antimicrobial Chemotherapy 49, 31–6.[Abstract/Free Full Text]

3 . Wongberinger, A., Jacobs, R. A. & Guglielmo, B. J. (1998). Lipid formulations of amphotericin B: clinical efficacy and toxicities. Clinical Infectious Diseases 27, 603–18.[Web of Science][Medline]

4 . Gaboriau, F., Chéron, M., Leroy, L. et al. (1997). Physicochemical properties of the heat-induced superaggregation of amphotericin B. Biophysical Chemistry 66, 1–12.[Medline]

5 . Gaboriau, F., Chéron, M., Petit, C. et al. (1997). Heat-induced superaggregation of amphotericin B reduces its in vitro toxicity: a new way to improve its therapeutic index. Antimicrobial Agents and Chemotherapy 41, 2345–51.[Abstract]

6 . Petit, C., Chéron, M., Joly, V. et al. (1998). In-vivo therapeutic efficacy in experimental murine mycoses of a new formulation of deoxycholate-amphotericin B obtained by mild heating. Journal of Antimicrobial Chemotherapy 42, 779–85.[Abstract/Free Full Text]

7 . Petit, C., Yardley, V., Gaboriau, F. et al. (1999). Activity of a heat-induced reformulation of amphotericin B deoxycholate (Fungizone) against Leishmania donovani. Antimicrobial Agents and Chemotherapy 43, 390–2.[Abstract/Free Full Text]

8 . Bau, P., Bolard, J. & Dupouy-Camet, J. (2003). Heated amphotericin to treat leishmaniasis. Lancet Infectious Diseases 3, 188.[CrossRef][Web of Science][Medline]

9 . van Etten, E. W., van Vianen, W., Roovers, P. et al. (2000). Mild heating of amphotericin B-desoxycholate: effects on ultrastructure, in vitro activity and toxicity, and therapeutic efficacy in severe candidiasis in leukopenic mice. Antimicrobial Agents and Chemotherapy 44, 1589–603.

10 . Baas, B., Kindt, K., Scott, A. et al. (1999). Activity and kinetics of dissociation and transfer of amphotericin B from a novel delivery form. AAPS pharmSci 1, E10.[CrossRef][Medline]

11 . Hartsel, S. C., Baas, B., Bauer, E. et al. (2001). Heat-induced superaggregation of amphotericin B modifies its interaction with serum proteins and lipoproteins and stimulation of TNF-alpha. Journal of Pharmaceutical Sciences 90, 124–33.[CrossRef][Web of Science][Medline]

12 . Brajtburg, J. & Bolard, J. (1996). Carrier effects on biological activity of amphotericin B. Clinical Microbiology Reviews 9, 512–31.[Abstract]

13 . Legrand, P., Vertut-Doï, A. & Bolard, J. (1996). Comparative internalization and recycling of different amphotericin B formulations by a macrophage-like cell line. Journal of Antimicrobial Chemotherapy 37, 519–33.[Abstract/Free Full Text]

14 . Krause, H. M. & Juliano, R. L. (1988). Interactions of liposome-incorporated amphotericin B with kidney epithelial cell cultures. Molecular Pharmacology 34, 296–7.

15 . Wasan, K. M., Rosenblum, M. G., Cheung, L. et al. (1994). Influence of lipoproteins on renal cytotoxicity and antifungal activity of amphotericin B. Antimicrobial Agents and Chemotherapy 38, 223–7.[Abstract/Free Full Text]

16 . Mosman, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65, 55–63.[CrossRef][Web of Science][Medline]

17 . Bergmeyer, H. U., Bernt, E. & Hess, B. (1963). Lactic dehydrogenase. In Methods of Enzymatic Analysis (Bergmeyer, H. U., Ed.), pp. 736–41. Academic Press, New York, NY, USA.

18 . Ridente, Y., Aubard, J. & Bolard, J. (1999). Absence in amphotericin B-spiked human plasma of the free monomeric drug, as detected by SERS. FEBS Letters 446, 283–6.[CrossRef][Web of Science][Medline]

19 . Bekersky, I, Fielding, R. M., Dressler, D. E. et al. (2002). Plasma protein binding of amphotericin B and pharmacokinetics of bound versus unbound amphotericin B after administration of intravenous liposomal amphotericin B (AmBisome) and amphotericin B deoxycholate. Antimicrobial Agents and Chemotherapy 46, 834–40.[Abstract/Free Full Text]

20 . Brajtburg, J., Elberg, S., Bolard, J. et al. (1984). Interaction of plasma proteins and lipoproteins with amphotericin B. Journal of Infectious Diseases 149, 986–97.[Web of Science][Medline]

21 . Wasan, K. M., Brazeau, G. A. & Keyhani, A. (1993). Roles of liposome composition and temperature in distribution of amphotericin B in serum lipoproteins. Antimicrobial Agents and Chemotherapy 37, 246–50.[Abstract/Free Full Text]

22 . Bhamra, R., Sa’ad, A., Bolcsak, L. E. et al. (1997). Behavior of amphotericin B lipid complex in plasma in vitro and in the circulation of rats. Antimicrobial Agents and Chemotherapy 41, 886–92.[Abstract]

23 . Rogers, P. D., Barkers, K. S., Herring, V. et al. (2003). Heat-induced superaggregation of amphotericin B attenuates its ability to induce cytokine and chemokine production in the human monocytic cell line THP-1. Journal of Antimicrobial Chemotherapy 51, 405–8.[Abstract/Free Full Text]


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J. A. Sanchez-Brunete, M. A. Dea, S. Rama, F. Bolas, J. M. Alunda, R. Raposo, M. T. Mendez, S. Torrado-Santiago, and J. J. Torrado
Treatment of Experimental Visceral Leishmaniasis with Amphotericin B in Stable Albumin Microspheres
Antimicrob. Agents Chemother., September 1, 2004; 48(9): 3246 - 3252.
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