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
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Abstract |
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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
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Introduction |
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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.
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Materials and methods |
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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 103 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 (407 in MeOH = 150000 L·mol1·cm1) 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 (106 to 2 x 104 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.
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Results |
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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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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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Discussion |
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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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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 AmBLDL 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-) 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.
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Acknowledgements |
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Footnotes |
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References |
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