1 Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo; 2 Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, CP 26077, CEP 05513-970 São Paulo, Brazil
Received 6 September 2002; returned 25 January 2003; revised 12 May 2003; accepted 15 June 2003
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Abstract |
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Keywords: drug delivery, amphotericin B, in vivo
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Introduction |
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Only a limited spectrum of antimycotic agents is available for the treatment of disseminated mycotic infections. Amphotericin B (AMB) is the therapy of choice for most invasive Candida infections. This broad-spectrum fungicide is a polyene antibiotic that exerts its toxic effect on fungal cells by forming complexes with membrane sterols5 that act as transmembrane channels allowing leakage of ions and other vital components from the cell.6 Unfortunately, AMB nephrotoxicity is high7,8 and has often been related to the occurrence of AMB as large aggregates both in aqueous solution and in lipid formulations.9,10 The low solubility of AMB in water and many organic solvents is a problem that is not easily solved. In order to formulate AMB, various strategies including the use of liposomes,11 surfact-ants,12 oil-in-water emulsions13,14 and cochleates15,16 have been employed, and have certainly improved the therapeutic index of the drug. However, DOC/AMB, the very first formulation for the drug, using the bile salt desoxycholate, is still the most frequently used formulation in clinics despite the formation of a very unstable detergentmicelle complex, with considerable toxicity.17 The reason for this seems to be related to two major drawbacks of lipid-based formulations: (i) AMB is required at higher doses than those in DOC/AMB (due to competition between carrier lipids and fungus membrane for drug solubilization);10,18 and (ii) lipids in the formulations are expensive.17
Dioctadecyldimethylammonium bromide (DODAB) is a synthetic and inexpensive cationic lipid that assembles in water solution, forming bilayer vesicles19 or bilayer fragments20 depending on the dispersion method. DODAB bilayer vesicles have been used as interface agents in several settings,21 such as vaccine adjuvants interacting with different antigens,2224 and biocidal agents against bacteria2528 and fungi,18 and they exhibit differential cytotoxicity.29 Recently, solubilization of AMB by nanosized, synthetic and charged bilayer fragments electrostatically stabilized in water dispersion was described; unlike other formulations this did not make use of entire and closed bilayer vesicles.30 These results were also extended to include micon-azole, a clinically important hydrophobic drug that was solubilized and/or colloidally stabilized using synthetic bilayer fragments.31 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 site (see Figure 1).30,31
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Materials and methods |
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AMB (batch 0008000336) was purchased from Bristol-Myers Squibb, São Paulo, 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. Female Swiss Webster mice 68 weeks old weighing 2530 g were obtained from the University of São Paulo. Candida albicans ATCC 90028 was obtained from the ATCC. A clinical isolate of C. albicans, strain HU168, was obtained from the Microbiology Laboratory of the Universidade de São Paulo Hospital, Brazil.
Preparation of DOD/AMB formulation
The DODAB synthetic bilayer fragments (SBF) were obtained by sonication with a titanium macrotip probe.19 The final DODAB concentration was determined by microtitration,32 and adjusted to 10 g/L. AMB solubilization in the 10 g/L DODAB SBF 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 yellow AMB aggregates could be detected in the pellet of centrifuged samples. As a control for complete AMB solubilization in the DODAB SBF, increasing concentrations of AMB and 10 g/L DODAB SBF were mixed, incubated (25°C, 24 h) and centrifuged (15 000g for 50 min). No yellow pellet was observed for AMB concen-trations <0.1 g/L; all AMB molecules were solubilized at the hydrophobic rim of the DODAB bilayer fragments,30 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.33
Physicochemical characterization of DOD/AMB formulation
Prior to parenteral administration, UVvisible optical spectra were obtained in the double-beam mode of a Hitachi spectrophotometer against a blank of lipid dispersions only (without drug), to subtract light scattered by the lipid dispersions from light absorption spectra for the drug. Routinely, the concentration of AMB in the DOD/AMB formulation was determined by means of spectrophotometry at 412 nm against a proper blank composed of DODAB only. The size distribution for DOD/AMB formulations (mean zeta-average diameter, Dz), was determined by using a ZetaPlus Zeta-Potential Analyzer (Brookhaven Instruments Corporation, Holtsville, NY, USA) equipped with a 570 nm laser and dynamic light scattering at 90° for particle sizing.34 All spectra and size distributions were obtained at room temperature (25°C) just after mixing AMB and DODAB SBF or at 24 h, 72 h, 7 days and 15 days thereafter.
The monomeric form of the drug was stabilized by the DODAB bilayer fragments and remained as such in water solution over at least 15 days at 4°C in contrast to DOC/AMB, a formulation where AMB is preferentially found as large aggregates of the hydrophobic drug.
Determination of in vitro activity of DOD/AMB 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.35 The medium used for diluting cells was culture medium in the case of DOC/AMB, and pure water for DOD/AMB. 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.18 Counts for cell suspension were confirmed by plating on SDA plates after adjusting to 2.5 x 103 cfu/mL. Minimum lethal concentrations (MLC) for DOD/AMB formulations were determined by cfu counts on a volume of 0.1 mL after a 48 h incubation at 35°C. The endpoint was the lowest drug concentration that killed 99.9% of the inoculum.36
Development of systemic candidiasis in mouse models
Mouse model systemic candidiasis was developed in accordance with standard procedures, and ethics committee approval was obtained.37,38 C. albicans isolate HU168 was freshly subcultured (24 h, SDA plates); cells were scraped off, transferred to Sabouraud dextrose broth (SDB) and grown at 37°C overnight. Thereafter, cells were washed three times in saline and adjusted to 70% transmittance at 530 nm yielding a 2.5 x 106 cfu/mL inoculum. Mice were infected by injecting 0.1 mL of the inoculum intravenously (iv) through the tail vein.
Determination of in vivo activity of DOD/AMB against systemic candidiasis in mouse models
The in vivo activity was evaluated by survival and tissue burden experiments for each group under test.
(i) Survival experiments. At 24 h post-infection (p.i.), groups of 10 female Swiss Webster mice were treated with DOD/AMB, DODAB, DOC/AMB or placebo, either iv or intraperitoneally. Animals were given two different treatments with AMB formulations. In the first, 0.2 mg/kg/day AMB was administered intravenously for 4 days. In the second, 0.4 mg/kg/day AMB was given intraperitoneally for (ip) 10 days. Con-trol group mice received DODAB alone or remained untreated. Animal groups were observed twice daily for death or abnormal behaviour over 35 days p.i.
(ii) Tissue burden study. Seven mice per group were infected as described above and treated 24 h p.i. with AMB 0.2, 0.4, 1.0 and 4.0 mg/kg/day over either 4 or 7 days administered as DOC/AMB or DOD/AMB formulations either iv or ip and compared with control groups of untreated or DODAB-treated mice. The tissue burden was assessed at day 4 and 8, for iv- and ip-treated groups, respectively. Kidneys and spleens were aseptically removed, weighed and each was homogenized in 1 mL of sterile saline. Ten-fold dilutions of the homogenate were plated, incubated (48 h, 37°C) and counted for cfu determinations. Results were expressed as cfu/g tissue.
Statistics
Differences in survival after 35 days of observation were assessed by KaplanMeier analysis followed by the Wilcoxon test. Comparisons of colony counts among different treatment groups were performed by the KruskalWallis test. P < 0.05 was considered statistically significant. All statistical analysis was performed with the software STATISTICA for Windows (StatSoft, Inc., Tulsa, OK, USA).
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Results |
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Drug, DODAB SBF and mixtures of both were characterized by light absorption spectroscopy and dynamic light scattering.
The UVvisible spectra of polyenes are very sensitive to conform-ational changes induced by different molecular interactions, including aggregation. Figure 2(a) illustrates the optical spectra for the drug in its monomeric form [dimethyl sulphoxide (DMSO):methanol, 1:1] and as aggregates (in water). Figure 2(b) shows the optical spectra for DOD/AMB and DOC/AMB formulations. For DOC/AMB, the spectrum resembles the one exhibited by AMB in pure water (compare Figure 2a and b). For DOD/AMB, the AMB spectrum became very similar to the one exhibited by AMB in its best organic solvent (compare Figure 2a and b). In DOC/AMB, the drug was present in the form of large aggregates rather than small mixed micelles with deoxycholate, as previously reported in the literature.39 In contrast to DOC/AMB, AMB addition (in minute volumes of its best solvent) to DODAB SBF solubilized the drug in its monomeric form30 as confirmed in this work also for solvent-free AMB/DODAB SBF mixtures.
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In vitro and in vivo activity of the DOD/AMB formulation
In this work, the MLC of DOD/AMB against C. albicans ATCC 90028 and pathogenic C. albicans HU168 was determined as 0.125 and 0.250 mg/L, respectively (Table 1). The activity of DOD/AMB in vitro was similar to that exhibited by DOC/AMB, since there was no significant difference between the mean values (Table 1). It is important to emphasize that the DOD/AMB formulation did lose its fungicidal activity in the culture medium where cationic DODAB SBF interacted with several anionic medium components.
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Discussion |
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Many studies have characterized DODAB self-assembly in aqueous solution.40 Depending on the DODAB dispersion method, large or small vesicles or bilayer fragments can be obtained. Using ultrasonic disruption with a tip, which introduces a high energy input in the DODAB powder/water system, DODAB bilayer vesicles are not only formed but also disrupted; bilayer fragments conveniently offer a very large area of hydrophobic nanosurfaces suitable for the solubilization of hydrophobic substances or drugs (Figure 1).30,31 Figure 2 confirmed solubilization of AMB using the 10 g/L DODAB dispersion prepared by ultrasonic disruption with a tip. In contrast, aggre-gation of AMB in the presence of deoxycholate (DOC/AMB) was evident from light absorption spectra (Figure 2). In addition to the advantageous monomeric state for AMB in the DOD/AMB formu-lation, Figures 3 and 4 also demonstrated its high colloid stability. Whereas the size of DOD/AMB aggregates decreased as a function of time, the opposite occurred for DOC/AMB at 25°C (Figures 3 and 4). Therefore, colloid stability for the new formulation was also better than the one exhibited by DOC/AMB.
The AMB monomeric state in the DOD/AMB formulation may be responsible for the similar survival percentiles relative to those obtained with DOC/AMB at equivalent AMB doses (Figure 5). Thirty-five days after infection, 0.4 mg/kg/day given intraperitoneally for 10 days resulted in 100% survival for the DOD/AMB formulation; similar survival patterns were obtained with the same dose as DOC/AMB. At this point, since the efficacy of both formulations was similar, one should recognize that there are not yet convincing data to show the real therapeutic advantages of this formulation over Fungizone or other lipid-based formulations that have markedly reduced toxicity relative to Fungizone.41 In fact, the major problem with Fungizone, its toxicity, is well circumvented by using the lipid-based formulations such as liposomal AMB (AmBisome) or AMB lipid complex (Abelcet). The presence of lipids has improved the therapeutic index by markedly reducing AMB nephrotoxicity. As DODAB is also a lipid preparation, a similar reduction in nephrotoxicity for this novel formulation could be anticipated and toxicity evaluations are currently being undertaken.
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 lower than 0.1 g/L. Therefore, DOD/AMB treatment regimens would possibly have to be prolonged to administer small daily AMB doses distributed over a larger number of days.
Regarding administration of the novel formulation, there would be no need to wait for longer than overnight at 37°C to administer the DOD/AMB formulation, since complete AMB solubilization under these conditions has been documented.30
A central question regards the mechanism by which AMB would be delivered to the fungal cell in vivo. The drug is expected to be readily available from the bilayer disc border in order to complex with ergosterol present in the fungus membrane. However, contrary to other lipid carriers such as fusogenic liposomes or cochleates, which require fusion of the bilayer carrier with the target cell, the DODAB SBF bilayer is in the rigid gel state and is not expected to fuse with the target cell membrane. Studies have failed to demonstrate leakage of radioactive water-soluble markers in the inner compartment of large DODAB vesicles upon their interaction with bacterial cells or leakage of phosphorylated cytosol components from the bacterial cells upon interaction with DODAB bilayers.27 In contrast, leakage was observed upon cell interaction with surfactants that form micelles instead of bilayers.27 Possibly, since the affinity of DODAB for serum proteins was high in vitro,41 DODAB SBF complexed with AMB could interact with albumin from serum and the stable complex DOD/AMBalbumin might then freely travel in the blood until the ergosterol- or cholesterol-rich cell membrane of the fungus or of the host, respectively, was found and the drug delivered.
In conclusion, DOD/AMB formulation is a monomeric, colloidally and chemically stable, organic solvent-free, inexpensive lipid-based formulation with in vitro and in vivo activity against C. albicans. DOD/AMB was as effective as DOC/AMB against C. albicans in vivo. Reduced nephrotoxicity relative to Fungizone has been was repeatedly reported in the literature for lipid-based AMB formulations.42 Therefore, it is to be anticipated that the lipid-based DOD/AMB formulation will also exhibit reduced nephrotoxicity.
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Acknowledgements |
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Footnotes |
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References |
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