1 Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, CP 66083, Avenida Lineu Prestes 580 Butantã, CEP 05315-970, São Paulo; 2 Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, CP 26077, Avenida Lineu Prestes 748 Butantã, CEP 05513-970, São Paulo, Brazil
Received 22 November 2004; returned 7 January 2005; revised 12 January 2005; accepted 18 January 2005
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Abstract |
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Methods: Dose-dependent cytotoxicity of DOD/AMB was evaluated in vitro against cultured kidney epithelial cells in culture. For in vivo experiments, Swiss Webster female mice were injected intraperitoneally for 10 consecutive days with 0.4 mg/kg/day AMB in the form of traditional bile salt desoxycholate (DOC)/AMB or DOD/AMB. Body and spleen weight, and biochemical and histopathological data were obtained at days 11 and 180 after injection.
Results: Nephrotoxicity of the novel formulation was lower than that of Fungizone (DOC/AMB), which is the traditional AMB formulation using DOC. Dose-dependent cytotoxicity of DOD/AMB was lower than that exhibited by DOC/AMB. At day 11, DODAB and DOD/AMB caused loss of body weight and increase in spleen weight, which were not observed for DOC/AMB, although the changes were reversible and weights returned to control values at day 180. Ten days after injection, biochemical parameters for hepatic and renal function remained unaltered. At day 180, renal cortex histopathology revealed leucocytic infiltration and moderate hydropic degeneration of the renal tubules in the DODAB and DOD/AMB groups, in contrast to more severe lesions observed for the DOC/AMB group such as tubular cystic degeneration and glomerular injury, which were absent for the former groups.
Conclusions: The DOD/AMB formulation exhibited differential cytotoxicity and low nephrotoxicity, but there were also important aspects of general toxicity that will require evaluation with full-scale toxicity protocols.
Keywords: dioctadecyldimethylammonium bromide , cytotoxicity , renal function , histopathology
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Introduction |
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Dioctadecyldimethylammonium bromide (DODAB) is a synthetic and inexpensive cationic lipid that assembles in water solution forming bilayer vesicles18 or bilayer fragments19 depending on the dispersion method. DODAB bilayer vesicles have been used as interface agents in several instances20 such as vaccine adjuvants interacting with different antigens,2125 biocidal agents against bacteria2629 and fungi,17 and they exhibit differential cytotoxicity.3032 Solubilization of AMB by nanosized, synthetic and charged bilayer fragments electrostatically stabilized in water dispersion has been described; unlike other formulations this did not make use of entire and closed bilayer vesicles.33 These results were also extended to include miconazole, a clinically important hydrophobic drug, which was solubilized and/or colloidally stabilized using synthetic bilayer fragments.34 The AMB solubilization was explained by the very large area of hydrophobic nanosurfaces offered by the bilayer fragments, so that the polyenic moiety of the antibiotic interacted with the hydrophobic borders of the bilayer fragments, while the hydroxylated moiety of the drug remained in contact with the surrounding water from its solubilization site3234 (Figure 1).
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Materials and methods |
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AMB (batch 0008000336) was purchased from Bristol-Myers Squibb (Brazil). DOC/AMB (AMB clinical preparation; Bristol-Myers Squibb) was purchased from a hospital pharmacy. DODAB 99.9% pure was obtained from Sigma Chemical Co. (St Louis, MO, USA). Agar was purchased from Difco. Swiss Webster mice 812 weeks old weighing 3240 g were obtained from the University of São Paulo. The animal ethics committee of the Faculty of Pharmaceutical Sciences at the University of São Paulo approved the study. Candida albicans ATCC 90028 was obtained from the ATCC. A clinical isolate of C. albicans, strain HU168, was obtained from the Microbiology Laboratory Universidade de São Paulo Hospital, Brazil.
Preparation of DOD/AMB formulation
The DODAB synthetic bilayer fragments were obtained by sonication with a titanium macrotip probe.18
The method for obtaining large and closed DODAB vesicles was previously described in 1983,18
and consisted of injecting a chloroformic DODAB solution in a water solution kept at 70°C (above chloroform boiling point and above the mean phase transition temperature for the DODAB bilayer). The method for obtaining open DODAB bilayer fragments based on sonication with a tip was also previously described18
and discussed in comparison to others.19
The final DODAB concentration was determined by microtitration39
and adjusted to 10 g/L. AMB solubilization in the 10 g/L DODAB synthetic bilayer fragments dispersion was performed in the absence of any organic solvent, and the final AMB concentration was 0.05 g/L (DOD/AMB formulation). At final concentrations of AMB 0.1 g/L (10 g/L DODAB), no AMB yellow aggregates could be detected in the pellet of centrifuged samples. As a control for complete AMB solubilization in the DODAB synthetic bilayer fragments, increasing concentrations of AMB and 10 g/L DODAB synthetic bilayer fragments were mixed, incubated (25°C, 24 h) and centrifuged (15 000 g for 50 min). No yellow pellet was observed for AMB concentrations < 0.1 g/L; all AMB molecules were solubilized at the hydrophobic rim of the DODAB bilayer fragments,33
whereas the control mixtures (without DODAB) displayed yellow pellets at the bottom of the glass assay tubes. The bilayer fragments, as usual, remained in the supernatant. DOC/AMB was diluted in 5% glucose for parenteral administration, as recommended.40
Determination of in vitro activity of DODAB against C. albicans
C. albicans ATCC 90028 and HU168 strains were subcultured on Sabouraud dextrose agar (SDA) plates and grown at 35°C for 24 h prior to testing. The macrodilution method was performed according to the recommendations of the NCCLS.41 The medium used for diluting cells was 0.264 M D-glucose. The interaction between DODAB and components of the culture medium would completely prevent targeting of the cationic DODABdrug complex to the oppositely charged Candida cells.17 Counts for cell suspension were confirmed by plating on SDA plates after adjusting to 2.5 x 103 cfu/mL. C. albicans viability in the presence of DODAB formulations was determined by cfu counts in a volume of 0.1 mL after 48 h of incubation at 35°C.42
Culture of kidney epithelial cells (Vero cells from African green monkey)
The kidney epithelial cells were grown as a subconfluent monolayer in 250 mL flasks containing complete minimum essential medium (MEM), 20 mM glutamine and 1% non-essential amino acids, supplemented with 10% bovine fetal serum, penicillin (100 IU/mL) and streptomycin (100 g/L) under a humidified atmosphere containing 5% CO2 at 37°C. Before harvesting and plating cells for the experiments, cells were treated with trypsin and cell concentration (cells/mL) was determined using a Neubauer chamber.
Determination of in vitro cytotoxicity against cultured kidney epithelial cells
The in vitro cytotoxicity against kidney epithelial cells was evaluated by means of a Neutral Red uptake assay.43,44 Neutral Red is a vital dye that is endocytosed by viable cells and internalized inside lysosomes; in this respect it is considered an indicator of lysosome (and cell) integrity.43,44 Cells (1 x 105) in 0.2 mL of culture medium were plated per microplate well. Twenty-four hours after cell seeding, the medium was aspirated from the monolayer and replaced with 0.2 mL of AMB formulation over a range of AMB and DODAB concentrations (0.320 g/L and 0.064.0 g/L, respectively). Two controls for 0 or 100% viability were performed by incubating cells in 0.1% (w/v) Triton X-100 or in culture medium only, respectively. Samples and controls were tested in triplicate, and two independent experiments were carried out. The plates were incubated for 1 h at 37°C in a humidified 5% CO2 incubator. Thereafter, the medium was aspirated from the monolayer, the cell layer was washed twice with phosphate-buffered saline (PBS) pH 7.4, and the medium was replaced by 0.1 mL of MEM containing 6.7 µg of Neutral Red dye. After 2 h of incubation at 37°C, the dye-containing medium was discarded, the wells were washed twice with PBS pH 7.4 and the dye incorporated by the cells was extracted with 0.1 mL of an extractant solution (50% ethanol in acetic acid, 1%). Microplates were shaken gently on a microplate rotator for 2 min before determining absorbance at 540 nm by means of a microplate reader (Ultramark, Model 550; Bio-Rad, Hercules, CA, USA). Percentage viability was calculated by (A540 treated cells/A540 untreated cells)x100 and expressed as mean ± S.D. Percentage cytotoxicity was calculated from: 100percentage viability.
Determination of in vivo toxicity after repeated administration
Toxicity was evaluated from percentage average weight change, relative spleen weights, determination of hepatic and renal function by biochemical parameters, and renal histopathology. Measurements were performed for 4 groups of 10 female mice (for histopathology and other evaluations) and 4 groups of 15 female mice (for blood parameters). Two groups received DOD/AMB (DODAB/AMB, 80:0.4 mg/kg/day); two groups received DOC/AMB (AMB 0.4 mg/kg/day); two groups received DODAB (80 mg/kg/day); and two control groups received sterile dextrose, 5% in water. For 10 days, 240 µL of each formulation was given daily by intraperitoneal injection. The protocol choice was based on the survival evaluation of in vivo activity of the formulation against systemic candidiasis in a mouse model.35
Percentage average weight change and relative spleen weights
Average weight change (%) was calculated by the following equation: 100x[(Wdaily Winitial)/Winitial], where W is weight. At day 11 and 180, after injection, five mice/group were weighed and euthanized, spleens were excised and weighed, and the relative spleen weight as a percentage of body weight was calculated for each group.
Determination of hepatic and renal function
At day 11 after injection, five mice from each group were weighed and euthanized. Blood was collected without anticoagulant, centrifuged at 2000 g and the serum was frozen at 30°C until analysis. For each experimental condition, serum pools were prepared by mixing equal volumes collected from three mice so that five serum pools were obtained. Alkaline phosphatase (ALP) activity, transaminase activity, and serum urea and creatinine levels were determined in 1 mL of each serum pool using an auto-analyser for clinical chemistry assay (LIASYS Random Access Analyzer; AMS, Roma, Zenica). The auto-analyser was validated for mouse liver enzymes [alanine aminotransferase (ALT), aspartate aminotransferase (AST) and ALP] before analytical determinations.
Histopathology of renal cortex
At day 11 and 180 after injection, five mice from each group were weighed and euthanized. The kidneys were carefully dissected and fixed by immersion in a 10% solution of buffered formalin (pH 7.4). Transverse sections were processed according to usual histological techniques for paraffin embedding. Five micrometre sections were taken and stained with haematoxylin and eosin.
Statistical analysis
All data are expressed as means ± S.D. Differences between groups were analysed by ANOVA one-way multiple comparison tests, and the KruskalWallis non-parametric test was used when needed. A P value of 0.05 was considered significant.
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Results and discussion |
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The DOD/AMB formulation based on a single synthetic lipid which forms bilayer fragments upon sonication with a tip is shown schematically in Figure 1. Bilayer fragments are bilayer discs produced by ultrasonically dispersing certain synthetic lipids in water solution. They have been observed by transmission electron microscopy (TEM)45 and cryo-TEM46 over the last 20 years for single lipid dispersions, such as those of sodium dihexadecylphosphate, or dioctadecyldimethylammonium bromide or chloride. DODAB bilayer fragments33 are stiff and flat due to their rigid gel state at room temperature.47 They are composed of open, charged and electrostatically stabilized bilayer pieces obtained from sonication with a tip. This is a high energy input procedure that not only disperses the lipid powder in water producing bilayer vesicles, but also disrupts them, thereby originating bilayer fragments. The main pieces of evidence for their existence are: (i) osmotic non-responsiveness of the dispersion indicative of absence of inner vesicle compartment; (ii) TEM micrographs with electronic staining;45 (iii) cryo-TEM micrographs;46 (iv) quasi-elastic light scattering and electron paramagnetic resonance spectroscopy; and (v) solubilization of hydrophobic drugs such as AMB at the borders of DODAB bilayer fragments, which does not occur for DODAB closed bilayer vesicles.19,33,45,46 They differ from the closed vesicles in the AmBisome formulation by providing hydrophobic borders at their edges that are absent in closed bilayer systems such as vesicles or liposomes. The inexpensive DODAB synthetic lipid is a potent bactericide2628,32 and exhibits cytotoxicity against mammalian cells in culture30,32 that is much lower than against prokaryotic or yeast cells.17,32 Bacteria are very susceptible to DODAB, with micromolar DODAB concentrations effectively killing four bacteria species of clinical importance.26 Table 1 shows DODAB concentrations for 0% survival of C. albicans HU168 and C. albicans ATCC 90028: 0.8 and 0.4 mM, respectively. Therefore, DODAB concentrations for killing yeast cells are much higher than those required to kill bacteria. Regarding kidney epithelial cells, the DODAB concentration required for 50% survival of cultured cells was 5.4 mM, i.e. these cells were much less susceptible to DODAB than bacteria or yeast cells.
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At day 11 after injection of each formulation in mice, DODAB, DOD/AMB and DOC/AMB did not affect any of the serum biochemical parameters currently used to evaluate renal and hepatic function (Table 2). Activity of ALT, AST or ALP, as well as creatinine and urea concentrations in serum, were not altered relative to controls after intraperitoneal administration of 80 mg/kg/day DODAB or 80/0.4 mg/kg/day DOD/AMB or 0.4 mg/kg/day AMB in DOC/AMB for 10 consecutive days. In clinics, DOC/AMB is used at doses 1.0 mg/kg/day13
because this is the limit for tolerable nephrotoxicity, meaning no significant changes in creatinine serum level. Above this limit, nephrotoxicity and creatinine serum levels rapidly increase within days of treatment. One should note that the present study was conducted at low AMB doses both for DOC/AMB and DOD/AMB owing to the high efficacy of the DOD/AMB formulation.35
Therefore, increases in creatinine serum levels were not expected even for DOC/AMB. In fact, measured creatinine levels did not increase at day 11 compared with controls for both formulations (Table 2). As a more sensitive marker for nephrotoxicity, renal histopathology examination revealed differences between DOC/AMB and DOD/AMB effects on renal morphology.
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DODAB and DOD/AMB formulations reduced the percentage average weight change, whereas DOC/AMB did not (Figure 3). Nevertheless, this effect was reversible and not significant over the long term (see percentage average weight at day 180 in Figure 3). DODAB has been recognized as an efficient immunostimulator in animals.2124 On the other hand, the normal pathway of drug clearance from the peritoneum is through direct absorption across the peritoneal membrane and by drainage into the lymphatic system.50 Owing to intraperitoneal administration, the formulations were expected to interact with the spleen, the main lymphatic organ connected to systemic circulation. DODAB, DOD/AMB and DOC/AMB affected relative spleen weight as a percentage of body weight as determined at days 11 and 180 after administration. The spleen responded to DODAB formulations by having significantly increased relative weight at day 11 (Table 3). However, this effect receded at day 180, at which point no significant increase in relative spleen weight was seen (Table 3). In contrast, DOC/AMB, which was not expected to act as an immunostimulator, did not affect relative spleen weight.
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The histopathology of the mouse kidneys at day 11 is shown in Figure 4 at two magnifications: x100 and x400. On the left in Figure 4, the control showed cross-sections of intact glomeruli and normal tubuli. In the middle, DOD/AMB treatment caused mild tubular hydropic degeneration, although at x100 magnification, the overall aspect of glomeruli and tubuli appeared normal. On the right, DOC/AMB caused early focal lymphocytic infiltrates (indicated by an asterisk) and tubular cystic degeneration filled with haemorrhagic fluid (indicated by an arrow) plus cellular debris in the lumen (indicated by D).
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The main disadvantage of the DOD/AMB formulation is related to its limited capacity to carry the monomeric form of the drug: 10 g/L DODAB dispersion was able to solubilize AMB over a drug concentration range that had to be < 0.1 g/L. Therefore, the DOD/AMB treatment regimen would possibly have to be prolonged to administer small daily AMB doses distributed over a large number of days. Interestingly, the inherent immunostimulatory effect associated with DODAB carrier could be favourable to neutropenic patients, who suffer severe fungal infections. Consistently, we have been determining a DODAB-induced rise in the count of polymorphonuclear neutrophils cells in peripheral blood (N. Lincopan, P. Borelli, R. A. Fock, E. M. Mamizuka and A. M. Carmona-Ribeiro, unpublished results).
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Acknowledgements |
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