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Emulsion Admixture Interactions: An Approach Concerning the Reduction of Amphotericin B Toxicity
Amphotericin B/Emulsion Admixture Interactions: An Approach Concerning the Reduction of Amphotericin B Toxicity ´ CRATES T. EGITO,1,2 IVONETE B. ARAU ´ JO,1 BOLIVAR P.G.L. DAMASCENO,1 JAMES C. PRICE2 E. SO 1
Programa de Po´s-graduac¸a˜o em Cieˆncias Farmaceˆuticas (PPCF), Universidade Federal do Rio Grande do Norte (UFRN), Rua Praia de Areia Branca, 8948–Natal-RN, 59094-450, Brazil 2
Department of Pharmaceutical and Biomedical Sciences, The University of Georgia, Athens, Georgia
Received 24 October 2001; revised 5 March 2002; accepted 24 April 2002
ABSTRACT: Mixing FungizoneTM with a fat emulsion used for nutritional purpose (IntralipidTM or LipofundinTM) was reported to decrease Amphotericin B (AmB) toxicity in clinical use. In an effort to understand the reason for this phenomenon, spectral and morphological analyses were done for the FungizoneTM and FungizoneTM/LipofundinTM admixture (FLmix). The absorption spectra analyses showed that not only FungizoneTM but also FLmix presented spectra that were concentration dependent. Moreover, the spectra of FLmix remained stable until the concentration of 5 107 M, and only at 5 108 M did they become similar in shape to the FungizoneTM spectra. Morphological studies revealed that even though emulsion droplets with or without FungizoneTM presented the same particle size, the former was less electron dense compared with LipofundinTM alone. These results suggest a kind of association between FungizoneTM and LipofundinTM that remains over the whole range of concentrations. This hypothesis was confirmed by in vitro studies in which FLmix presented an important selectivity against human and fungal cells compared with FungizoneTM. These findings suggest that parenteral emulsions should be able to reduce the AmB toxicity probably by changing the AmB self-association state by binding it with emulsion droplets. ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:2354–2366, 2002
Keywords: amphotericin B; red blood cells; Candida tropicalis; absorption spectra; parenteral emulsion; transmission electron microscopy (TEM); toxicity
INTRODUCTION Acquired immune deficiency syndrome, cancer, and organ transplants are the three principal reasons for the increase in the number of immunocompromised patients who easily become hosts of fungal infection.1 Although discovered more than 50 years ago, Amphotericin B (AmB) remains the
Correspondence to: Dr. E. So´crates T. Egito (at Universidade Federal do Rio Grande do Norte) Telephone: 84-94318816; Fax: 84-219-2836; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 91, 2354–2366 (2002) ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association
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drug of choice for the treatment of life-threatening fungal infections, primarily because of its broad spectrum of activity.2 However, the outstanding efficacy of its commercially available form, FungizoneTM, in which the drug is solubilized by sodium deoxycholate, is tempered by sometimes severe and frequent adverse reactions.3 To reduce its toxicity, several studies have suggested formulating AmB, which is lipophilic, in lipid carriers such as small unilamellar liposomes (AmBisomeTM), colloidal dispersion (ABCD, AmphocilTM, or AmphotecTM), and lipid complex (ABLC or AbelcetTM).4 All of these lipid-based AmB preparations are reported to have better
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therapeutic indices than FungizoneTM, but their use is limited by their high cost and difficult preparation.5–7 An alternative delivery system, based on the lipid emulsions used for parenteral nutrition in clinical practice (IntralipidTM or LipofundinTM), has been shown to reduce the AmB toxicity with little compromise in efficacy.8–16 In vitro studies revealed that although FungizoneTM/IntralipidTM admixtures (FImix) were at least as active as FungizoneTM against Candida, no hemolytic activity was observed against red blood cells at AmB concentrations up to 80 mg/L. In contrast, FungizoneTM was hemolytic at an AmB concentration of 0.1 mg/L. Moreover, concerning potassium ion (Kþ) leakage, the FImix was reported to be half as toxic to red blood cells as FungizoneTM.15 In contrast, a recent study that also examined the relative toxicity of these formulations as measured by Kþ release from red blood cells during incubation at 378C,16 showed that FImix seems to be slightly more toxic than FungizoneTM. However, Joly et al.12 found that FImix presented a reduced toxicity against renal tubular cells compared with FungizoneTM. In vivo studies showed that although the toxicity of FungizoneTM was 1.7- to 2.5-fold lower in the presence of IntralipidTM, the antifungal activity of FungizoneTM was not reduced.12 Therefore, it was concluded that FImix was more effective than FungizoneTM when both drugs were given at the maximum tolerated dose. Indeed, IntralipidTM improved the therapeutic index of AmB by allowing larger doses of FungizoneTM to be infused12 rather than by altering its antifungal effect of a given dose or by targeting the infected site. As found for the in vitro studies, there are conflicting results from clinical studies. At least three reports have shown that FImix was responsible for a reduction in fever and chills in neutropenic patients,9,17,18 whereas its effectiveness in the treatment of systemic candidiasis was maintained.10 In a pharmacokinetic study, Heinemann et al.14 found an important decrease in both maximum concentration of AmB in serum (Cmax) value and area under the concentrationtime curve value when FImix was used. In addition, a recent study with critically ill patients showed that FImix compared with FungizoneTM was not only effective in eradicating Candida infections but also better tolerated in that the frequency rate and severity of immediate side effects (hypotension, chills, fever, anaphylaxis,
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renal impairment) were diminished.13 Contrary to these authors, two other clinical trials revealed no significant advantage with respect to safety and tolerance in the administration of FImix compared with FungizoneTM.19,20 The reasons for poor or good therapeutic efficacy of AmB in FImix are unclear, and there is a lack of understanding regarding how this mixture works when administered to patients with established infections. The goal of this report was to use spectral and morphological analysis of the FungizoneTM/emulsion admixture (FLmix) and to correlate the results with a study of the effects of FungizoneTM and FLmix on toxicity to red blood cell (RBC) membranes and effectiveness against Candida tropicalis. The effects of concentration and temperature on the spectra shape were evaluated. In addition, in vitro effects of FungizoneTM and FLmix on the membranes of RBCs and C. tropicalis were studied. Afterward, a correlation with the spectra and morphological studies was made.
MATERIALS AND METHODS Materials FungizoneTM and LipofundinTM (MCT/LCT 20%) were obtained from Bristol-Myers Squibb (Sa˜o Paulo, SP, Brazil) and B. Braun (Sa˜o Gonc¸alo, RJ, Brazil), respectively. Sodium deoxycholate, and water for injection were from Sigma (St. Louis, MO). Methods AmB Formulations FungizoneTM solution at the concentration of 5 103 M (5 mg/mL) was obtained by dissolving the components of a vial with 10 mL of water for injection. Each vial of FungizoneTM contains 50 mg of AmB, approximately 41 mg of sodium deoxycholate, and appropriate phosphate buffer to maintain a pH 7.4. Control vials were prepared, containing equivalent amounts of each of the above ingredients except AmB, to yield doses of sodium deoxycholate in the controls equivalent to the FungizoneTM. FLmix was achieved by mixing (Vortex Genie 2, model G-560; Scientific Industries, Inc., Bohemia, NY) 10 mL of LipofundinTM with 10 mL of FungizoneTM solution for 15 min at 258C. This mixture was used within 2 h after its preparation.
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Spectral Studies To determine absorption spectra (Olis-Cary 118 Spectrophotometer; Olis, Inc., Bogart, GA), dispersions of FungizoneTM and FLmix were prepared in phosphate buffer solution, pH 7.4, at various AmB concentrations. FLmix was recorded against a blank consisting of LipofundinTM in phosphate buffer solution, in the reference beam. The optical path of the quartz cuvettes used was 0.1, 1, and 10 cm for the concentrations of 5 105 M (50 mg/L), 5 106 M (5 mg/L), and 5 107 M (0.5 mg/L) and 5 108 M (0.05 mg/L), respectively. These paths were chosen to obtain spectra with absorbance values less than 1.2. Their molar extinction coefficients (e) were calculated using the Beer-Lambert equation. All spectra were recorded at 258C or 378C.
Quantitation of AmB Species AmB in aqueous media is present as a mixture of various species in equilibrium, monomers, and soluble and insoluble aggregates.21,22 The concentration of each species depends on the total AmB concentration,22 on the method of preparing the solutions,22 and on the AmB pharmaceutical delivery system.23 Typically, it is assumed that the spectral bands indicating the existence of monomers and aggregates species of AmB are located around 409 nm, and 327 nm, respectively.23 Previous studies showed that the selectivity against mammalian and fungal cells also depends on the aggregation state of AmB.22,24 – 26 Only self-associated AmB was shown to increase Kþ permeability of cholesterol-containing egg phosphatidyl choline vesicles, whereas both monomer and aggregate species modify Kþ permeability in ergosterolcontaining liposomes.26 Therefore, the equilibrium between monomers and aggregates seems to have a key role in drug activity. Consequently, the structural study and quantitation of the AmB species constitute an essential step in understanding the molecular mechanism of this antifungal agent concerning both selectivity and cytotoxicity.21 To estimate the quantities of each species in aqueous media as a function of the AmB carrier, we propose the following algorithm to process the absorption data. The absorption spectrum of AmB, which results from the spectroscopic contributions of aggregates (b) and monomers (d), depends on the concentration of the forms. Therefore,
absorbances can be expressed according to the Beer-Lambert formulation as: Al ¼ ebl1 ½b l þ edl2 ½d l
ð1Þ
Where l1 ¼ wavelength for aggregates formed (usually around 327 nm); l2 ¼ wavelength for monomers formed (usually around 409 nm); l ¼ cuvette path length; and e ¼ molar extinction coefficients. Assuming that AmB in aqueous media corresponds to a mixture of these two spectroscopic species, the total AmB concentration ([T]) was expressed as the sum of b and d concentrations (eq. 2): ½T ¼ ½b þ ½d
ð2Þ
Molar extinction coefficients (e) for each spectroscopic species were deduced from their typical absorption spectra at l1 and l2 nm for each concentration studied. The mean number of AmB monomeric units in each aggregated form is unknown and probably corresponds to a wide size distribution. Consequently, the e values were estimated considering monomeric AmB. Therefore, the concentration of b and d was expressed in terms of monomeric AmB and calculated according to the expressions: A l1 ½b ¼ b l el1 þ edl2
ð3Þ
½d ¼ ½T ½b
ð4Þ
Mean Particle Size Studies The mean particle size of LipofundinTM and FLmix at two AmB concentrations was estimated by photon correlation spectroscopy using a NICOMP Particle Sizing Systems, Submicron Particle Sizer, Autodilute, model 370A (Santa Barbara, CA). Morphological Analysis Transmission electron microscope (TEM) examination of LipofundinTM and FLmix at two AmB concentrations was performed using a TEM JEOL, model JEM100cx II (Electron Microscope Ltd., Tokyo, Japan) after negative staining with osmium tetroxide solution at 2%(w/v). Preparation of RBC Suspension One healthy adult female donor provided all normal human RBCs for the in vitro experiments;
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this was done to minimize sources of variability. Five milliliters of venous blood was collected in sterile EDTA [1 mg/mL, ethylenediaminetetraacetate at 10%(w/v); Labtest, Lagoa Santa, MG, Brazil] syringes and promptly centrifuged (Refrigerated Centrifuge, ALC, model PK121R, Milan, Italy) in tubes at 1100g for 10 min at 48C. Plasma was aspirated with precautions, and the exposed buffy coat was removed and discarded. The RBCs were washed three times by centrifugation (1100g for 5 min at 48C) and suspension in five volumes of normal saline [NaCl at 0.9% %(w/v); B. Braun]. They were then resuspended in 4 mL of saline, counted in a NeubauerTM chamber, and resuspended again until desired concentration (5 107 cells/mL). They were used within 24 h of collection. Preparation Inocula of C. tropicalis A strain of C. tropicalis isolated from a patient’s urine culture was maintained on SabouraudDextrose-Chloramphenicol agar (SDC; MicroMed, Sa˜o Paulo, SP, Brazil) at room temperature and passaged monthly. Before experiments, an inoculum from the culture was transferred to an SDC agar scope, and incubated at 378C for 16–18 h. The fungal cells were then washed three times with normal saline, resuspended, counted in the central reticule of a NeubauerTM chamber, and resuspended again to obtain the desired concentration (5 107 cfu/mL). Toxicity of AmB Formulations Four milliliters of RBCs (5 107cells/mL) were incubated for 1 h at 378C with the vehicle control or with different concentrations (50, 5, 0.5, and 0.05 mg/L) and formulation of AmB, either FungizoneTM and FLmix. The RBCs were then centrifuged for 5 min at 1100g and washed three times with normal saline. The pellet of RBC was lysed by 4 mL of distilled water, stirred, and centrifuged (1100g for 5 min) to remove membranes. Kþ content of the supernatant was determined using a Flame Photometer 7000 (Tecnow, Sa˜o Paulo, SP, Brazil) calibrated with Kþ reference at 5 mEq/L; hemoglobin was estimated from its absorption at 540 nm recorded with a Coleman Spectrophotometer (model SP395U; Sa˜o Paulo, SP, Brazil). The total potassium and hemoglobin content was estimated from the control RBC tubes. Release was calculated as the difference between control and treated cells and was
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expressed as a percentage of the total hemoglobin or potassium content. At least three different experiments were performed with each formulation and each experimental point was performed in triplicate. Efficacy of AmB Formulations Two-milliliter samples of fungal suspension containing 5 107 cfu/mL were incubated for 1 h at 378C with FungizoneTM and FLmix at AmB concentrations of 50, 5, 0.5, and 0.05 mg/L. Cells were centrifuged for 10 min at 2200g, washed three times in normal saline, and 2 mL of purified water was added to the pellet of fungal cells. An aliquot of this pellet was lysed for 5 min at 1008C and centrifuged to remove membranes, and free potassium was measured. The Kþ leakage was calculated similarly to the RBCs. For the cell viability evaluation, 100-mL aliquots of appropriate dilution of the fungal pellet were seeded, in duplicate, onto agar plates and incubated at 378C. The number of colony-forming units was counted at 24 and 48 h and expressed as a percentage of those obtained from a control inoculum incubated without AmB. Three different experiments were performed with each formulation and each experimental point was performed in duplicate. Statistical Analysis All potassium and hemoglobin release, and cfu viability data were expressed as the mean SE. Statistical analysis was performed using analysis of variance test and significance was defined as p < 0.05.
RESULTS Spectral Studies The absorption spectra of FungizoneTM (Fig. 1) and FLmix (Fig. 2), at 258C are concentration dependent. At low concentration, spectra were similar to those obtained in methanol or other polar organic solvents, exhibiting maxima at 364, 383.5, and 407.5 nm and a shoulder around 347 nm. They are traditionally assigned to monomeric AmB.26 For increasing FungizoneTM concentrations, a new band appeared around 326 nm, and two other ones at 383 and 418 nm. The increase of amplitude of the band at 326 nm, assigned to AmB self-associated species,26 occurred at the expense of those of monomeric AmB. In
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Figure 1. Concentration-induced changes in the FungizoneTM spectra. The spectra were obtained at 258C, in phosphate buffer solution, pH 7.4, at four AmB concentrations. The optical path of the quartz cuvettes used was 0.1, 1, and 10 cm for the concentrations of 5 105 M (50 mg/L), 5 106 M (5 mg/L), and 5 107 M (0.5 mg/L) and 5 108 M (0.05 mg/L), respectively.
It was also noticed that the characteristics of the spectra remained unchanged at 12 h. At 378C, similar changes on concentration were observed (Fig. 3). For FLmix, the band characteristic of self-association was blue-shifted compared with the corresponding band at 258C (319.5 nm). Quantitative changes are described below. Consequently, although variation in temperature did not provoke any important change on the FungizoneTM spectra, the AmB–emulsion complex presented a quite different behavior with a 128C temperature increase. In this case, more monomeric forms are present, probably released from the AmB–emulsion complex, because of the increase of thermodynamic energy on the water/ oil interface. Therefore, it can be concluded from these results that a kind of association between AmB and LipofundinTM remains over the whole range of concentrations. Quantitation of AmB Species
the FLmix spectra, these bands were slightly blue shifted (4 nm). It should be noted that 5 108 M spectra are given to illustrate the described tendency but should not be considered on a quantitative basis in the low wavelength region (they become negative) because of the weakness of the signals (maximum absorbance at 407 nm: 0.05) and the corresponding uncertainty. Beside the small wavelength differences between FungizoneTM and FLmix, the concentration dependency was also different: the formation of self-associated species (monitored by the band around 326 nm) only occurred above 5 107 M whereas it started to occur above 5 108 M for FLmix.
The evaluation of concentration of aggregates [b] and monomers [d] is summarized in Figures 4 and 5. At 258C (Fig. 4), FungizoneTM presented the profile mentioned in the literature.21,22 At low concentration, 5 108 M, 100% of the AmB was in the monomeric form; as the concentration increased, aggregates started to appear and the b/d50%, where sub-exists 50% of aggregates and 50% of monomers, was retrieved at 1.5 106 M. At high concentrations, 5 105 M, almost all the species found were aggregates (4.6 105 M, 92.7%), and the existence of a small quantity of monomer (0.4 105 M, 7.3%) was the result of
Figure 2. Concentration-induced changes in the FungizoneTM/LipofundimTM admixture spectra at 258C.
Figure 3. Concentration-induced changes in the FungizoneTM/LipofundimTM admixture spectra at 378C.
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(at 9 107 M) but also the aggregation structure disappeared at low concentration. Probably, the 128C increase in the temperature was able to release the AmB from the oil/water interface because of the high level of thermodynamic energy there, caused by the surfactants. Particle Size and Morphological Evaluation
Figure 4. Distribution of aggregated [b] and monomeric [d] species as a function of the concentration at 258C. The percentages of both species were calculated from the absorption data at around 326 and 407.5 nm according to eqs. 3 and 4, respectively, described in Material and Methods.
the thermodynamic equilibrium between both species. However, the profile of aggregates and monomers for FLmix was quite distinct. At low concentration, 80% was monomeric and 20% was in the form of aggregates (3.95 108 M and 1.05 108 M, respectively) and the b/d50% was detected at 7 107 M. Also, it was observed that the variation of monomers and aggregates in function of the log of the concentration was quite linear. At 378C, FungizoneTM maintained the same profile as that at 278C (Fig. 5). The only significant variation concerned its b/d50% which was slightly dislocated to the right, at 2.1 106 M. Nevertheless, the FLmix profile presented considerable changes; not only did its b/d50% shift to the right
Figure 5. Distribution of aggregated [b] and monomeric [d] species as a function of the concentration at 378C.
No significant changes in the LipofundinTM emulsion particle size were observed when the AmB was incorporated to the system (Table 1). Both AmB concentrations studied, 2.5 103 M and 5 105 M, showed a droplet size around 280 70 nm, which was similar to the system without AmB. Only after 12 h of preparation did FLmix at 5 105 M reveal some variation on its distribution profile of droplets (Fig. 6), and its particle size, analyzed in volume by weight, changed from 299.6 81.5 to 347.8 131.8 nm. The morphological analysis of FungizoneTM (Fig. 7) and LipofundinTM with (Fig. 8) or without (Fig. 9) AmB had different aspects. Indeed, the free AmB–FLmix was less electron-dense than AmB-loaded FLmix. Nevertheless, no changes in particle size, which was confirmed to be around 250 nm, were observed (Fig. 10A and B). However, FungizoneTM micelles presented a particle size that were five times lower, around 50 nm, and were high electron-dense because of the unsaturated carbons of the AmB. In Vitro Evaluation of FungizoneTM and FLmix Toxicity against Human RBCs Potassium and hemoglobin leakage after the incubation of 5 107 RBCs (1 h at 378C) with increasing concentrations of FungizoneTM and FLmix are shown in Figures 11 and 12, respectively. No significant potassium leakage was observed below 0.1 mg/L with either FungizoneTM or FLmix. At higher concentrations, 0.5 and 5 mg/L, the permeability of the cell membrane, as reflected by potassium release, was higher with FungizoneTM (46.73 11.33% and 98.96 0.26%, respectively) than with FLmix (9.61 5.99% and 40.01 7.74%, respectively), and only at the highest concentration, 50 mg/L, were the release profiles similar, 99.35 0.16% and 96.13 0.36%, respectively. At AmB concentrations of 5 and 0.5 mg/L, the potassium release induced by FungizoneTM was significantly different from that with FLmix ( p < 0.0001).
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239.5 241.5 245.1 220.5
Blank 1 FLmix at 2.5 103 Blank 2 FLmix at 5 105
64.2 64.5 67.6 83.6
68.6 70.3 66.3 65.0
SD
Mean Diameter 286.4 283.3 279.3 278.3
277.6 279.4 286.5 302.0
CV 0.282 0.299 0.278 0.272
0.268 0.267 0.276 0.379
74.4 74.6 79.1 114.5
0.268 0.267 0.276 0.379
0.282 0.299 0.278 0.272
80.8 84.7 77.7 75.7 After 12 h
CV
SD
Intensity Weighted
298.3 300.0 309.1 347.8
310.1 309.7 301.4 299.6
Mean Diameter
CV, coefficient of variation; SD, Standard deviation. a Blank 1 and Blank 2 samples were prepared from LipofundimTM diluted 1:2 and 1:5 in phosphate buffer solution, pH 7.4, respectively.
243.2 235.1 238.3 239.0
Mean Diameter
Number Weighted
Immediately after Preparation
79.9 80.1 85.3 131.8
87.4 92.6 83.8 81.5
SD
Volume Weighted
Mean Particle Sizes of LipofundinTM and FungizoneTM/LipofundinTM Admixture (FLmix) Immediately and after 12 h of Preparationa
Blank 1 FLmix at 2.5 103 Blank 2 FLmix at 5 105
Sample
Table 1.
0.268 0.267 0.276 0.379
0.282 0.299 0.278 0.272
CV
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Figure 6. Volume weighted particle size distribution of droplets for FungizoneTM/LipofundimTM admixture (FLmix), at 5 105 M, before (A) and after (B) 12 h of preparation. The sample was prepared from FLmix at 2.5 103 M diluted 50 times in phosphate buffer solution, pH 7.4.
No significant hemoglobin leakage was observed with FLmix over the whole range of concentration tested (0.05 to 50 mg/L) and with FungizoneTM below 0.5 mg/L. At this concentration, the increase on hemoglobin leakage started
Figure 7. TEM of FungizoneTM micelles. The sample was stained with osmium tetroxide 2% and analyzed at 36,000 magnification. Bar, 300 nm.
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Figure 8. TEM of FungizoneTM/LipofundimTM admixture showing the incorporation of FungizoneTM on the emulsion droplet, in the oil/water interface. The sample was stained with osmium tetroxide 2% and analyzed at 58,000 magnification. Bar, 170 nm.
(5.36 9.28%) and at 5 mg/L reached 96.99 2.53%. When FLmix was used at 5 and 50 mg/L, the toxicity against RBCs, as reflected by hemoglobin leakage, was significantly reduced compared with FungizoneTM ( p < 0.0001).
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Figure 10. TEM of FungizoneTM/LipofundimTM admixture (A) LipofundimTM emulsion (B) stained with osmium tetroxide 2% and analyzed at 19,000 magnification. Bar, 500 nm.
Potassium leakage after the incubation of 5 107 cfu (1 h at 378C) with increasing concentrations of FungizoneTM and FLmix is shown in Figure 12. Whatever the system tested, a significant potassium release was observed at the lowest concentration (0.05 mg/L) and reached 100% at the 5 mg/L level. However, FungizoneTM at 0.05 mg/L induced a significantly larger potas-
sium release (88.96 3.49%), compared with the FLmix (60.59 21.36%) ( p < 0.0001). The antifungal activity of FungizoneTM and FLmix is shown in Figure 13. Determination of the viability of C. tropicalis cells (ability to form colonies) showed that whereas FungizoneTM was able to kill all seeded fungal cells from a concentration of 5 mg/L (0.36 0.62 cfu%), FLmix was unable to do that. Indeed, after treatment of C. tropicalis cells with FLmix at the highest concentration studied, 50 mg/L, a viability of 11.89 4.65 cfu% was retrieved. Nevertheless,
Figure 9. TEM of LipofundimTM emulsion showing a reduction on the droplet electron density. The sample was stained with osmium tetroxide 2% and analyzed at 58,000 magnification. Bar, 170 nm.
Figure 11. In vitro release of potassium from human RBCs (&, &) and C. tropicalis(*, *) induced by FungizoneTM (*, &) and FungizoneTM/LipofundimTM admixture [FLmix (*, &)]. Each point on the figure is the mean (SD) of three determinations. *Significant difference between the two forms for RBCs ( p < 0.0001).
Activity against C. tropicalis
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Figure 12. In vitro release of hemoglobin from human RBCs induced by FungizoneTM (&) and FungizoneTM/LipofundimTM admixture [FLmix (&)]. Each point on the figure is the mean (SD) of three determinations. *Significant difference between the two formulations ( p < 0.0001).
the difference of activity between both preparations was significantly different only at 0.5 mg/L, when FungizoneTM was more effective than FLmix ( p < 0.0001).
DISCUSSION The use of electronic absorption analysis has been previously reported as a powerful tool to follow the interaction of AmB with a lipidic carrier.23,27 In fact, the strong absorbance that AmB exhibits between 300–450 nm, because of the seven
conjugated double bonds existing on its apolar domain (Fig. 14), is heavily influenced by conformational changes provoked by its self-association in water media or by its interaction with other compounds such as drug carriers. The physicochemical properties of AmB, mainly its amphiphilic and zwitterionic nature, attributed respectively to the apolar and polar sides of the lactone ring and to the presence of ionizable carboxyl and amine groups (Fig. 14), as well as its asymmetrical distribution of hydrophobic and hydrophilic groups, make the drug extremely insoluble in aqueous solvents and in many organic solvents where it generates aggregates beyond a threshold concentration. In fact, beneath 106 M, which is the critical aggregation concentration (CAC), AmB can be present in aqueous media as a soluble monomer, and its absorption spectrum is characterized by a peak at 409 nm. However, above the CAC, AmB molecules are able to selfassociate to form oligomers and then aggregates of oligomers, which are characterized in absorption spectra by a broad intense single band at 340 nm.28–30 It has been reported that the AmB may present various patterns of activity against ergosterol-containing fungal cells with respect to the toxicity against cholesterol-containing mammalian cells, depending on its molecular presentation.22,24,26,31,32 Indeed, monomers were less efficient than aggregates of oligomers at inducing the permeability of a cholesterolcontaining membrane to potassium.33 Self-associated AmB was shown to trigger permeability changes in RBC membranes and to induce cytotoxic events.22 Moreover, the toxic chemotherapeutic effects in mice were previously demonstrated to be correlated to the aggregation state of AmB.25 Binding of the monomeric or oligomeric forms of an amphiphilic drug can lead to an active form either at once or after reorganization in micelles within the lipid bilayer, but only when the
Figure 13. In vitro antifungal activity of FungizoneTM (&) and FungizoneTM/LipofundimTM admixture [FLmix (&)] on C. tropicalis (mean SD of three determinations run in duplicate). *Significant difference between the two formulations ( p < 0.0001). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 11, NOVEMBER 2002
Figure 14.
AmB structure.
AMPHOTERICIN B AND EMULSION ADMIXTURE
concentration exceeds the critical micellar concentration in the membrane.34,35 Consequently, any factor modifying the equilibrium between the different species of AmB in aqueous media may change its overall activity. In the present study, the spectral evaluation of FungizoneTM and FLmix clearly demonstrate that the behavior of AmB molecules was perturbed from one system to another. In fact, the spectra of AmB in the FungizoneTM (Fig. 1) presented a concentration dependency that was reported by other groups.23,28,36,37 At higher concentrations, the spectra presented an intense band at 326 nm and two small ones at 383 and 418 nm. However, as the concentration decreased, the predominant band disappeared and a new one, at 407.5 nm, was observed when it reached 5 107 M. The fact that the aggregation state band was shifted to a slightly lower wavelength, 326 nm, for the FungizoneTM system compared with the classical AmB self-associated band location, which is at 340 nm, indicates that the AmB molecules are complexed with the sodium deoxycholate. However, because the deoxycholate concentration in FungizoneTM diluted to 5 107 M AmB is very low and below its critical micellar concentration (2.8 103 M),38 we believe that the detergent molecules dissociate from the AmB–micelle complex and dissolve in water. Therefore, at high dilutions, FungizoneTM behaved like an aqueous solution of AmB.39 For FLmix, the results indicate that the AmB– emulsion complex generated is quite different (Fig. 2). Firstly, except at very low AmB concentration (5 108 M), the variation of the absorption spectrum was great, and its behavior was similar to FungizoneTM at the same AmB concentration. The self-associated AmB band (at 324 nm in this preparation) was maintained at high dilutions (5 107 M), whereas the proportion of the monomeric form remained low (Fig. 5). This suggests that the AmB molecules were strongly associated with the emulsion droplet. Indeed, it was observed that despite the high dilution of FLmix at AmB concentrations of 5 107 M and 5 108 M, the emulsion system remained stable and presented a milky appearance. Thus, we believe that, when FungizoneTM was mixed with LipofundinTM, the AmB–micelle complex existing in the former dissociates, permitted the detergent to go to the external aqueous phase and the AmB molecules to be incorporated into the oil/water phospholipid monolayer interface that composes the emulsion system.
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Theoretically, AmB could be located either in the oily core of emulsion droplets or at the interface. However, because AmB is not soluble either in soybean oil or in medium-chain fatty acids,40 we assume that it is located in the phospholipid monolayer, as already suggested by Washington et al.41 This hypothesis was partially confirmed by the variation found in the FLmix spectra measured at 378C (Fig. 3). In fact, the behavior of AmB molecules located at the phospholipid monolayer was severely perturbed by the temperature increase of 128C: the aggregate’s band was red shifted from 324 to 319.5 nm; the intensity of the spectrum aggregate’s band at 5 106 M and 5 107 M was reduced with a consequent increase in the monomeric one; the presence of monomeric species was increased to 100% instead of 80% that was found at 258C (Figs. 4 and 5); and the b/d50% was strongly altered. All these events could have happened because of the increase on thermodynamic energy on the oil/water interface caused by the increase in temperature that provokes a complete change in the thermodynamic equilibrium of aggregate and monomeric species, because of the release of the latter from the AmB–emulsion droplet complex. The particle size analysis and the morphological evaluation once again indicated that an interaction between AmB and emulsion droplets had occurred. First, a variation in the distribution profile of droplets charged with AmB was found (Fig. 6). Second, a small variation of the particle size after 12 h was detected (Table 1). Finally, the TEM pictures revealed that not only FungizoneTM micelles (Fig. 7), which present a granulometric size fivefold lower than FungizoneTM/LipofundinTM complex, disappeared from the FLmix external phase (Fig. 10), but also FLmix droplets were more electron-dense (Fig. 8) than LipofundinTM droplets (Figs. 9 and 10). This indicates that FungizoneTM micelles were incorporated into LipofundinTM. The in vitro FLmix evaluation permitted us to go forward on the AmB phospholipid monolayer localization hypothesis. In fact, these studies showed that FLmix presented an important selectivity against human and fungal cells and was less toxic than FungizoneTM. When the target cells were human RBCs, whereas RBC did not present hemoglobin leakage over the whole range of doses tested, the C. tropicalis viability started to decrease at 5 107 M (85.32 10.19% cfu) and became 11.89 4.65% cfu at 5 105 M. No
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significant Kþ leakage, which has often been used as an index of polyene toxicity,22,42 –45 from RBC was observed below 5 107 M (2.97 2.11% at 5 108 M), but for C. tropicalis, the lowest concentration studied caused 60.59 21.36% leakage, reached at 87.77 6.81% as early as the 5 107 M level. For RBCs, this degree of toxicity was observed only at 5 105 M. The results for FungizoneTM were in agreement with those published recently.46 The FLmix presented nearly the same efficacy against C. tropicalis and a lower toxicity against RBCs. However, the ensemble of activity results of FLmix point out that at a particular AmB concentration (0.5 mg/L), the activity toward C. tropicalis is slightly decreased, probably because of the variation of AmB species in equilibrium. In fact, some authors have found that the association of AmB with lipids reduced its activity against fungal cells.47–49 Nevertheless, the high protective effect on RBCs induced by FLmix allows the use of larger doses of FungizoneTM, thus improving its therapeutic index. All the results together suggest that LipofundinTM should be able to reduce the FungizoneTM toxicity probably by changing the AmB selfassociation state by binding it with the emulsion droplet. Indeed, FLmix may be considered as a reservoir of the monomeric form of AmB that releases only limited amounts of free AmB in the aqueous media. As a consequence, the level of free AmB might be below its CAC and the drug could be in its monomeric form. This form would be able to bind to the ergosterol of fungal cells but would be inactive against the cholesterol of mammalian cells. Liposomal and other lipid-based forms of AmB have been extensively studied, and some of them are already marketed.4 However, the FLmix form may have some practical advantages in that it does not include expensive semi-synthetic lipids and is even now in use on clinical trials. Nevertheless, as shown by our results, the mixture of two approved pharmaceutical entities induces large physicochemical changes in both of them. In this specific case, FLmix should be considered as a new delivery system and its use should be made with caution.
ACKNOWLEDGMENTS We thank Elayne C. M. T. Egito, for her drawing of the AmB structure, and Glenn Hawes, for editing the manuscript. The authors are indebted
to one of the referees for his constructive remarks. Dr. Egito is grateful for the financial support from CAPES (Brazilian Department of Education), Brası´lia/Brazil. This work was funded in part by the FMC Foundation.
REFERENCES 1. de Pauw BE, Meunier F. 1999. The challenge of invasive fungal infection. Chemotherapy 45:1–14. 2. Hartsel S, Bolard J. 1996. Amphotericin B: New life for an old drug. Trends Pharmacol Sci 17:445–449. 3. Gallis HA. 1996. Amphotericin B: A commentary on its role as an antifungal agent and as a comparative agent in clinical trials. Clin Infect Dis 22:S145– S147. 4. Groll AH, Muller FMC, Piscitelli SC, Walsh TJ. 1998. Lipid formulations of amphotericin B: Clinical perspectives for the management of invasive fungal infections in children with cancer. Klin Padiatr 210:264–273. 5. Hiemenz JW, Walsh TJ. 1996. Lipid formulations of amphotericin B: Recent progress and future directions. Clin Infect Dis 22:S133–S144. 6. Joly V, Geoffray C, Reynes J, Goujard C, Mechali D, Maslo C, Raffi F, Yeni P. 1996. Amphotericin B in a lipid emulsion for the treatment of cryptococcal meningitis in AIDS patients. J Antimicrob Chemother 38:117–126. 7. Wong-Beringer A, Jacobs RA, Guglielmo BJ. 1998. Lipid formulations of amphotericin B: Clinical efficacy and toxicities. Clin Infect Dis 27:603– 618. 8. Kirsh R, Goldstein R, Tarloff J, Parris D, Hook J, Hanna N, Bugelski P, Poste G. 1988. An emulsion formulation of amphotericin B improves the therapeutic index when treating systemic murine candidiasis. J Infect Dis 158:1065–1070. 9. Moreau P, Milpied N, Fayette N, Ramee JF, Harousseau JL. 1992. Reduced renal toxicity and improved clinical tolerance of amphotericin B mixed with intralipid compared with conventional amphotericin B in neutropenic patients. J Antimicrob Chemother 30:535–541. 10. Caillot D, Casasnovas O, Solary E, Chavanet P, Bonnotte B, Reny G, Entezam F, Lopez J, Bonnin A, Guy H. 1993. Efficacy and tolerance of an amphotericin B lipid (Intralipid) emulsion in the treatment of candidaemia in neutropenic patients. J Antimicrob Chemother 31:161–169. 11. Thakur CP. 1994. Comparison of glucose versus fat emulsion in the preparation of amphotericin B for use in kala-azar. Trans R Soc Trop Med Hyg 88: 698–699. 12. Joly V, Farinotti R, Saintjulien L, Cheron M, Carbon C, Yeni P. 1994. In-vitro renal toxicity and in-vivo therapeutic efficacy in experimental
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study of an amphotericin B emulsion-based delivery system. Int J Pharm 145:17–27. Lambing HE, Wolf BD, Hartsel SC. 1993. Temperature effects on the aggregation state and activity of amphotericin B. Biochim Biophys Acta 1152:185– 188. Barwicz J, Christian S, Gruda I. 1992. Effects of the aggregation state of amphotericin B on its toxicity to mice. Antimicrob Agents Chemother 36:2310– 2315. Bolard J, Legrand P, Heitz F, Cybulska B. 1991. One-sided action of amphotericin B on cholesterolcontaining membranes is determined by its selfassociation in the medium. Biochemistry 30:5707– 5715. Espuelas MS, Legrand P, Cheron M, Barratt G, Puisieux F, Devissaguet JP, Irache JM. 1998. Interaction of amphotericin B with polymeric colloids: A spectroscopic study. Colloids Surf B Biointerfaces 11:141–151. Mazerski J, Bolard J, Borowski E. 1982. Selfassociation of some polyene macrolide antibiotics in aqueous media. Biochim Biophys Acta 719:11–17. Mazerski J, Grzybowska J, Borowski E. 1990. Influence of net charge on the aggregation and solubility behavior of amphotericin B and its derivatives in aqueous media. Eur Biophys J 18:159–164. Bolard J, Seigneuret M, Boudet G. 1980. Interaction between phospholipid-bilayer membranes and the polyene antibiotic amphotericin B: Lipid state and cholesterol content dependence. Biochim Biophys Acta 599:280–293. Barwicz J, Gruszecki WI, Gruda I. 1993. Spontaneous organization of amphotericin B in aqueousmedium. J Colloid Interface Sci 158:71–76. Maher P, Singer SJ. 1984. Structural changes in membranes produced by the binding of small amphipathic molecules. Biochemistry 23:232–240. Hartsel SC, Benz SK, Ayenew W, Bolard J. 1994. Naþ, Kþ and Cl selectivity of the permeability pathways induced through sterol-containing membrane-vesicles by amphotericin B and other polyene antibiotics. Eur Biophys J 23:125–132. Milhaud J, Hartmann MA, Bolard J. 1989. Interaction of the polyene antibiotic amphotericin-B with model membranes: Differences between small and large unilamellar vesicles. Biochimie 71:49–56. Oneill LJ, Miller JG, Petersen NO. 1986. Evidence for nystatin micelles in L-cell membranes from fluorescence photobleaching measurements of diffusion. Biochemistry 25:177–181. Strauss G, Kral F. 1982. Borate complexes of amphotericin B: Polymeric species and aggregates in aqueous-solutions. Biopolymers 21:459–470. Ernst C, Grange J, Rinnert H, Dupont G. 1981. Structure of amphotericin B aggregates as revealed by UV and CD spectroscopies. Biopolymers 20: 1575–1588.
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38. Pramauro E, Pelezetti E. 1996. Surfactants in analytical chemistry: Applications of organized amphiphilic media, Vol. 31. New York: Elsevier. 39. Lamyfreund MT, Schreier S, Peitzsch RM, Reed WF. 1991. Characterization and time-dependence of amphotericin B: Deoxycholate aggregation by quasielastic light-scattering. J Pharm Sci 80:262–266. 40. Asher IM, Schwartzman G, USASRG (U.S. Antibiotics Standards Research Group). 1972. Amphotericin B In: Florey K, editor. Analytical profiles of drug substances, Vol. 6. New York: Academic Press, pp 1–42. 41. Washington C, Taylor SJ, Davis SS. 1988. The structure of colloidal formulations of amphotericin B. Int J Pharm 46:25–30. 42. Butler WT, Cotlove E. 1971. Increased permeability of human erythrocytes induced by amphotericin B. J Infect Dis 123:341–350. 43. Davis SS, Washington C, West P, Illum L, Liversidge G, Sternson L, Kirsh R. 1987. Lipid emulsions as drug delivery systems. Ann NY Acad Sci 507: 75–88. 44. Forster D, Washington C, Davis SS. 1988. Toxicity of solubilized and colloidal amphotericin B formulations to human erythrocytes. J Pharm Pharmacol 40:325–328.
45. Souza LC, Maranhao RC, Schreier S, Campa A. 1993. In-vitro and in-vivo studies of the decrease of amphotericin B toxicity upon association with a triglyceride-rich emulsion. J Antimicrob Chemother 32:123–132. 46. Egito EST, Appel M, Fessi H, Barratt G, Puisieux F, Devissaguet JP. 1996. In-vitro and in-vivo evaluation of a new amphotericin B emulsion-based delivery system. J Antimicrob Chemother 38:485– 497. 47. Janoff AS, Perkins WR, Saletan SL, Swenson CE. 1993. Amphotericin B lipid complex (ABLCTM): A molecular rationale for the attenuation of amphotericin B related toxicities. J Liposome Res 3:451–471. 48. Jullien S, Contrepois A, Sligh JE, Domart Y, Yeni P, Brajtburg J, Medoff G, Bolard J. 1989. Study of the effects of liposomal amphotericin B on Candida albicans, Cryptococcus neoformans, and erythrocytes by using small unilamellar vesicles prepared from saturated phospholipids. Antimicrob Agents Chemother 33:345–349. 49. Clark JM, Whitney RR, Olsen SJ, George RJ, Swerdel MR, Kunselman L, Bonner DP. 1991. Amphotericin-B lipid complex therapy of experimental fungal infections in mice. Antimicrob Agents Chemother 35:615–621.
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Programa de Po´s-graduac¸a˜o em Cieˆncias Farmaceˆuticas (PPCF), Universidade Federal do Rio Grande do Norte (UFRN), Rua Praia de Areia Branca, 8948–Natal-RN, 59094-450, Brazil 2
Department of Pharmaceutical and Biomedical Sciences, The University of Georgia, Athens, Georgia
Received 24 October 2001; revised 5 March 2002; accepted 24 April 2002
ABSTRACT: Mixing FungizoneTM with a fat emulsion used for nutritional purpose (IntralipidTM or LipofundinTM) was reported to decrease Amphotericin B (AmB) toxicity in clinical use. In an effort to understand the reason for this phenomenon, spectral and morphological analyses were done for the FungizoneTM and FungizoneTM/LipofundinTM admixture (FLmix). The absorption spectra analyses showed that not only FungizoneTM but also FLmix presented spectra that were concentration dependent. Moreover, the spectra of FLmix remained stable until the concentration of 5 107 M, and only at 5 108 M did they become similar in shape to the FungizoneTM spectra. Morphological studies revealed that even though emulsion droplets with or without FungizoneTM presented the same particle size, the former was less electron dense compared with LipofundinTM alone. These results suggest a kind of association between FungizoneTM and LipofundinTM that remains over the whole range of concentrations. This hypothesis was confirmed by in vitro studies in which FLmix presented an important selectivity against human and fungal cells compared with FungizoneTM. These findings suggest that parenteral emulsions should be able to reduce the AmB toxicity probably by changing the AmB self-association state by binding it with emulsion droplets. ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:2354–2366, 2002
Keywords: amphotericin B; red blood cells; Candida tropicalis; absorption spectra; parenteral emulsion; transmission electron microscopy (TEM); toxicity
INTRODUCTION Acquired immune deficiency syndrome, cancer, and organ transplants are the three principal reasons for the increase in the number of immunocompromised patients who easily become hosts of fungal infection.1 Although discovered more than 50 years ago, Amphotericin B (AmB) remains the
Correspondence to: Dr. E. So´crates T. Egito (at Universidade Federal do Rio Grande do Norte) Telephone: 84-94318816; Fax: 84-219-2836; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 91, 2354–2366 (2002) ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association
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drug of choice for the treatment of life-threatening fungal infections, primarily because of its broad spectrum of activity.2 However, the outstanding efficacy of its commercially available form, FungizoneTM, in which the drug is solubilized by sodium deoxycholate, is tempered by sometimes severe and frequent adverse reactions.3 To reduce its toxicity, several studies have suggested formulating AmB, which is lipophilic, in lipid carriers such as small unilamellar liposomes (AmBisomeTM), colloidal dispersion (ABCD, AmphocilTM, or AmphotecTM), and lipid complex (ABLC or AbelcetTM).4 All of these lipid-based AmB preparations are reported to have better
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therapeutic indices than FungizoneTM, but their use is limited by their high cost and difficult preparation.5–7 An alternative delivery system, based on the lipid emulsions used for parenteral nutrition in clinical practice (IntralipidTM or LipofundinTM), has been shown to reduce the AmB toxicity with little compromise in efficacy.8–16 In vitro studies revealed that although FungizoneTM/IntralipidTM admixtures (FImix) were at least as active as FungizoneTM against Candida, no hemolytic activity was observed against red blood cells at AmB concentrations up to 80 mg/L. In contrast, FungizoneTM was hemolytic at an AmB concentration of 0.1 mg/L. Moreover, concerning potassium ion (Kþ) leakage, the FImix was reported to be half as toxic to red blood cells as FungizoneTM.15 In contrast, a recent study that also examined the relative toxicity of these formulations as measured by Kþ release from red blood cells during incubation at 378C,16 showed that FImix seems to be slightly more toxic than FungizoneTM. However, Joly et al.12 found that FImix presented a reduced toxicity against renal tubular cells compared with FungizoneTM. In vivo studies showed that although the toxicity of FungizoneTM was 1.7- to 2.5-fold lower in the presence of IntralipidTM, the antifungal activity of FungizoneTM was not reduced.12 Therefore, it was concluded that FImix was more effective than FungizoneTM when both drugs were given at the maximum tolerated dose. Indeed, IntralipidTM improved the therapeutic index of AmB by allowing larger doses of FungizoneTM to be infused12 rather than by altering its antifungal effect of a given dose or by targeting the infected site. As found for the in vitro studies, there are conflicting results from clinical studies. At least three reports have shown that FImix was responsible for a reduction in fever and chills in neutropenic patients,9,17,18 whereas its effectiveness in the treatment of systemic candidiasis was maintained.10 In a pharmacokinetic study, Heinemann et al.14 found an important decrease in both maximum concentration of AmB in serum (Cmax) value and area under the concentrationtime curve value when FImix was used. In addition, a recent study with critically ill patients showed that FImix compared with FungizoneTM was not only effective in eradicating Candida infections but also better tolerated in that the frequency rate and severity of immediate side effects (hypotension, chills, fever, anaphylaxis,
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renal impairment) were diminished.13 Contrary to these authors, two other clinical trials revealed no significant advantage with respect to safety and tolerance in the administration of FImix compared with FungizoneTM.19,20 The reasons for poor or good therapeutic efficacy of AmB in FImix are unclear, and there is a lack of understanding regarding how this mixture works when administered to patients with established infections. The goal of this report was to use spectral and morphological analysis of the FungizoneTM/emulsion admixture (FLmix) and to correlate the results with a study of the effects of FungizoneTM and FLmix on toxicity to red blood cell (RBC) membranes and effectiveness against Candida tropicalis. The effects of concentration and temperature on the spectra shape were evaluated. In addition, in vitro effects of FungizoneTM and FLmix on the membranes of RBCs and C. tropicalis were studied. Afterward, a correlation with the spectra and morphological studies was made.
MATERIALS AND METHODS Materials FungizoneTM and LipofundinTM (MCT/LCT 20%) were obtained from Bristol-Myers Squibb (Sa˜o Paulo, SP, Brazil) and B. Braun (Sa˜o Gonc¸alo, RJ, Brazil), respectively. Sodium deoxycholate, and water for injection were from Sigma (St. Louis, MO). Methods AmB Formulations FungizoneTM solution at the concentration of 5 103 M (5 mg/mL) was obtained by dissolving the components of a vial with 10 mL of water for injection. Each vial of FungizoneTM contains 50 mg of AmB, approximately 41 mg of sodium deoxycholate, and appropriate phosphate buffer to maintain a pH 7.4. Control vials were prepared, containing equivalent amounts of each of the above ingredients except AmB, to yield doses of sodium deoxycholate in the controls equivalent to the FungizoneTM. FLmix was achieved by mixing (Vortex Genie 2, model G-560; Scientific Industries, Inc., Bohemia, NY) 10 mL of LipofundinTM with 10 mL of FungizoneTM solution for 15 min at 258C. This mixture was used within 2 h after its preparation.
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Spectral Studies To determine absorption spectra (Olis-Cary 118 Spectrophotometer; Olis, Inc., Bogart, GA), dispersions of FungizoneTM and FLmix were prepared in phosphate buffer solution, pH 7.4, at various AmB concentrations. FLmix was recorded against a blank consisting of LipofundinTM in phosphate buffer solution, in the reference beam. The optical path of the quartz cuvettes used was 0.1, 1, and 10 cm for the concentrations of 5 105 M (50 mg/L), 5 106 M (5 mg/L), and 5 107 M (0.5 mg/L) and 5 108 M (0.05 mg/L), respectively. These paths were chosen to obtain spectra with absorbance values less than 1.2. Their molar extinction coefficients (e) were calculated using the Beer-Lambert equation. All spectra were recorded at 258C or 378C.
Quantitation of AmB Species AmB in aqueous media is present as a mixture of various species in equilibrium, monomers, and soluble and insoluble aggregates.21,22 The concentration of each species depends on the total AmB concentration,22 on the method of preparing the solutions,22 and on the AmB pharmaceutical delivery system.23 Typically, it is assumed that the spectral bands indicating the existence of monomers and aggregates species of AmB are located around 409 nm, and 327 nm, respectively.23 Previous studies showed that the selectivity against mammalian and fungal cells also depends on the aggregation state of AmB.22,24 – 26 Only self-associated AmB was shown to increase Kþ permeability of cholesterol-containing egg phosphatidyl choline vesicles, whereas both monomer and aggregate species modify Kþ permeability in ergosterolcontaining liposomes.26 Therefore, the equilibrium between monomers and aggregates seems to have a key role in drug activity. Consequently, the structural study and quantitation of the AmB species constitute an essential step in understanding the molecular mechanism of this antifungal agent concerning both selectivity and cytotoxicity.21 To estimate the quantities of each species in aqueous media as a function of the AmB carrier, we propose the following algorithm to process the absorption data. The absorption spectrum of AmB, which results from the spectroscopic contributions of aggregates (b) and monomers (d), depends on the concentration of the forms. Therefore,
absorbances can be expressed according to the Beer-Lambert formulation as: Al ¼ ebl1 ½b l þ edl2 ½d l
ð1Þ
Where l1 ¼ wavelength for aggregates formed (usually around 327 nm); l2 ¼ wavelength for monomers formed (usually around 409 nm); l ¼ cuvette path length; and e ¼ molar extinction coefficients. Assuming that AmB in aqueous media corresponds to a mixture of these two spectroscopic species, the total AmB concentration ([T]) was expressed as the sum of b and d concentrations (eq. 2): ½T ¼ ½b þ ½d
ð2Þ
Molar extinction coefficients (e) for each spectroscopic species were deduced from their typical absorption spectra at l1 and l2 nm for each concentration studied. The mean number of AmB monomeric units in each aggregated form is unknown and probably corresponds to a wide size distribution. Consequently, the e values were estimated considering monomeric AmB. Therefore, the concentration of b and d was expressed in terms of monomeric AmB and calculated according to the expressions: A l1 ½b ¼ b l el1 þ edl2
ð3Þ
½d ¼ ½T ½b
ð4Þ
Mean Particle Size Studies The mean particle size of LipofundinTM and FLmix at two AmB concentrations was estimated by photon correlation spectroscopy using a NICOMP Particle Sizing Systems, Submicron Particle Sizer, Autodilute, model 370A (Santa Barbara, CA). Morphological Analysis Transmission electron microscope (TEM) examination of LipofundinTM and FLmix at two AmB concentrations was performed using a TEM JEOL, model JEM100cx II (Electron Microscope Ltd., Tokyo, Japan) after negative staining with osmium tetroxide solution at 2%(w/v). Preparation of RBC Suspension One healthy adult female donor provided all normal human RBCs for the in vitro experiments;
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this was done to minimize sources of variability. Five milliliters of venous blood was collected in sterile EDTA [1 mg/mL, ethylenediaminetetraacetate at 10%(w/v); Labtest, Lagoa Santa, MG, Brazil] syringes and promptly centrifuged (Refrigerated Centrifuge, ALC, model PK121R, Milan, Italy) in tubes at 1100g for 10 min at 48C. Plasma was aspirated with precautions, and the exposed buffy coat was removed and discarded. The RBCs were washed three times by centrifugation (1100g for 5 min at 48C) and suspension in five volumes of normal saline [NaCl at 0.9% %(w/v); B. Braun]. They were then resuspended in 4 mL of saline, counted in a NeubauerTM chamber, and resuspended again until desired concentration (5 107 cells/mL). They were used within 24 h of collection. Preparation Inocula of C. tropicalis A strain of C. tropicalis isolated from a patient’s urine culture was maintained on SabouraudDextrose-Chloramphenicol agar (SDC; MicroMed, Sa˜o Paulo, SP, Brazil) at room temperature and passaged monthly. Before experiments, an inoculum from the culture was transferred to an SDC agar scope, and incubated at 378C for 16–18 h. The fungal cells were then washed three times with normal saline, resuspended, counted in the central reticule of a NeubauerTM chamber, and resuspended again to obtain the desired concentration (5 107 cfu/mL). Toxicity of AmB Formulations Four milliliters of RBCs (5 107cells/mL) were incubated for 1 h at 378C with the vehicle control or with different concentrations (50, 5, 0.5, and 0.05 mg/L) and formulation of AmB, either FungizoneTM and FLmix. The RBCs were then centrifuged for 5 min at 1100g and washed three times with normal saline. The pellet of RBC was lysed by 4 mL of distilled water, stirred, and centrifuged (1100g for 5 min) to remove membranes. Kþ content of the supernatant was determined using a Flame Photometer 7000 (Tecnow, Sa˜o Paulo, SP, Brazil) calibrated with Kþ reference at 5 mEq/L; hemoglobin was estimated from its absorption at 540 nm recorded with a Coleman Spectrophotometer (model SP395U; Sa˜o Paulo, SP, Brazil). The total potassium and hemoglobin content was estimated from the control RBC tubes. Release was calculated as the difference between control and treated cells and was
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expressed as a percentage of the total hemoglobin or potassium content. At least three different experiments were performed with each formulation and each experimental point was performed in triplicate. Efficacy of AmB Formulations Two-milliliter samples of fungal suspension containing 5 107 cfu/mL were incubated for 1 h at 378C with FungizoneTM and FLmix at AmB concentrations of 50, 5, 0.5, and 0.05 mg/L. Cells were centrifuged for 10 min at 2200g, washed three times in normal saline, and 2 mL of purified water was added to the pellet of fungal cells. An aliquot of this pellet was lysed for 5 min at 1008C and centrifuged to remove membranes, and free potassium was measured. The Kþ leakage was calculated similarly to the RBCs. For the cell viability evaluation, 100-mL aliquots of appropriate dilution of the fungal pellet were seeded, in duplicate, onto agar plates and incubated at 378C. The number of colony-forming units was counted at 24 and 48 h and expressed as a percentage of those obtained from a control inoculum incubated without AmB. Three different experiments were performed with each formulation and each experimental point was performed in duplicate. Statistical Analysis All potassium and hemoglobin release, and cfu viability data were expressed as the mean SE. Statistical analysis was performed using analysis of variance test and significance was defined as p < 0.05.
RESULTS Spectral Studies The absorption spectra of FungizoneTM (Fig. 1) and FLmix (Fig. 2), at 258C are concentration dependent. At low concentration, spectra were similar to those obtained in methanol or other polar organic solvents, exhibiting maxima at 364, 383.5, and 407.5 nm and a shoulder around 347 nm. They are traditionally assigned to monomeric AmB.26 For increasing FungizoneTM concentrations, a new band appeared around 326 nm, and two other ones at 383 and 418 nm. The increase of amplitude of the band at 326 nm, assigned to AmB self-associated species,26 occurred at the expense of those of monomeric AmB. In
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Figure 1. Concentration-induced changes in the FungizoneTM spectra. The spectra were obtained at 258C, in phosphate buffer solution, pH 7.4, at four AmB concentrations. The optical path of the quartz cuvettes used was 0.1, 1, and 10 cm for the concentrations of 5 105 M (50 mg/L), 5 106 M (5 mg/L), and 5 107 M (0.5 mg/L) and 5 108 M (0.05 mg/L), respectively.
It was also noticed that the characteristics of the spectra remained unchanged at 12 h. At 378C, similar changes on concentration were observed (Fig. 3). For FLmix, the band characteristic of self-association was blue-shifted compared with the corresponding band at 258C (319.5 nm). Quantitative changes are described below. Consequently, although variation in temperature did not provoke any important change on the FungizoneTM spectra, the AmB–emulsion complex presented a quite different behavior with a 128C temperature increase. In this case, more monomeric forms are present, probably released from the AmB–emulsion complex, because of the increase of thermodynamic energy on the water/ oil interface. Therefore, it can be concluded from these results that a kind of association between AmB and LipofundinTM remains over the whole range of concentrations. Quantitation of AmB Species
the FLmix spectra, these bands were slightly blue shifted (4 nm). It should be noted that 5 108 M spectra are given to illustrate the described tendency but should not be considered on a quantitative basis in the low wavelength region (they become negative) because of the weakness of the signals (maximum absorbance at 407 nm: 0.05) and the corresponding uncertainty. Beside the small wavelength differences between FungizoneTM and FLmix, the concentration dependency was also different: the formation of self-associated species (monitored by the band around 326 nm) only occurred above 5 107 M whereas it started to occur above 5 108 M for FLmix.
The evaluation of concentration of aggregates [b] and monomers [d] is summarized in Figures 4 and 5. At 258C (Fig. 4), FungizoneTM presented the profile mentioned in the literature.21,22 At low concentration, 5 108 M, 100% of the AmB was in the monomeric form; as the concentration increased, aggregates started to appear and the b/d50%, where sub-exists 50% of aggregates and 50% of monomers, was retrieved at 1.5 106 M. At high concentrations, 5 105 M, almost all the species found were aggregates (4.6 105 M, 92.7%), and the existence of a small quantity of monomer (0.4 105 M, 7.3%) was the result of
Figure 2. Concentration-induced changes in the FungizoneTM/LipofundimTM admixture spectra at 258C.
Figure 3. Concentration-induced changes in the FungizoneTM/LipofundimTM admixture spectra at 378C.
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(at 9 107 M) but also the aggregation structure disappeared at low concentration. Probably, the 128C increase in the temperature was able to release the AmB from the oil/water interface because of the high level of thermodynamic energy there, caused by the surfactants. Particle Size and Morphological Evaluation
Figure 4. Distribution of aggregated [b] and monomeric [d] species as a function of the concentration at 258C. The percentages of both species were calculated from the absorption data at around 326 and 407.5 nm according to eqs. 3 and 4, respectively, described in Material and Methods.
the thermodynamic equilibrium between both species. However, the profile of aggregates and monomers for FLmix was quite distinct. At low concentration, 80% was monomeric and 20% was in the form of aggregates (3.95 108 M and 1.05 108 M, respectively) and the b/d50% was detected at 7 107 M. Also, it was observed that the variation of monomers and aggregates in function of the log of the concentration was quite linear. At 378C, FungizoneTM maintained the same profile as that at 278C (Fig. 5). The only significant variation concerned its b/d50% which was slightly dislocated to the right, at 2.1 106 M. Nevertheless, the FLmix profile presented considerable changes; not only did its b/d50% shift to the right
Figure 5. Distribution of aggregated [b] and monomeric [d] species as a function of the concentration at 378C.
No significant changes in the LipofundinTM emulsion particle size were observed when the AmB was incorporated to the system (Table 1). Both AmB concentrations studied, 2.5 103 M and 5 105 M, showed a droplet size around 280 70 nm, which was similar to the system without AmB. Only after 12 h of preparation did FLmix at 5 105 M reveal some variation on its distribution profile of droplets (Fig. 6), and its particle size, analyzed in volume by weight, changed from 299.6 81.5 to 347.8 131.8 nm. The morphological analysis of FungizoneTM (Fig. 7) and LipofundinTM with (Fig. 8) or without (Fig. 9) AmB had different aspects. Indeed, the free AmB–FLmix was less electron-dense than AmB-loaded FLmix. Nevertheless, no changes in particle size, which was confirmed to be around 250 nm, were observed (Fig. 10A and B). However, FungizoneTM micelles presented a particle size that were five times lower, around 50 nm, and were high electron-dense because of the unsaturated carbons of the AmB. In Vitro Evaluation of FungizoneTM and FLmix Toxicity against Human RBCs Potassium and hemoglobin leakage after the incubation of 5 107 RBCs (1 h at 378C) with increasing concentrations of FungizoneTM and FLmix are shown in Figures 11 and 12, respectively. No significant potassium leakage was observed below 0.1 mg/L with either FungizoneTM or FLmix. At higher concentrations, 0.5 and 5 mg/L, the permeability of the cell membrane, as reflected by potassium release, was higher with FungizoneTM (46.73 11.33% and 98.96 0.26%, respectively) than with FLmix (9.61 5.99% and 40.01 7.74%, respectively), and only at the highest concentration, 50 mg/L, were the release profiles similar, 99.35 0.16% and 96.13 0.36%, respectively. At AmB concentrations of 5 and 0.5 mg/L, the potassium release induced by FungizoneTM was significantly different from that with FLmix ( p < 0.0001).
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239.5 241.5 245.1 220.5
Blank 1 FLmix at 2.5 103 Blank 2 FLmix at 5 105
64.2 64.5 67.6 83.6
68.6 70.3 66.3 65.0
SD
Mean Diameter 286.4 283.3 279.3 278.3
277.6 279.4 286.5 302.0
CV 0.282 0.299 0.278 0.272
0.268 0.267 0.276 0.379
74.4 74.6 79.1 114.5
0.268 0.267 0.276 0.379
0.282 0.299 0.278 0.272
80.8 84.7 77.7 75.7 After 12 h
CV
SD
Intensity Weighted
298.3 300.0 309.1 347.8
310.1 309.7 301.4 299.6
Mean Diameter
CV, coefficient of variation; SD, Standard deviation. a Blank 1 and Blank 2 samples were prepared from LipofundimTM diluted 1:2 and 1:5 in phosphate buffer solution, pH 7.4, respectively.
243.2 235.1 238.3 239.0
Mean Diameter
Number Weighted
Immediately after Preparation
79.9 80.1 85.3 131.8
87.4 92.6 83.8 81.5
SD
Volume Weighted
Mean Particle Sizes of LipofundinTM and FungizoneTM/LipofundinTM Admixture (FLmix) Immediately and after 12 h of Preparationa
Blank 1 FLmix at 2.5 103 Blank 2 FLmix at 5 105
Sample
Table 1.
0.268 0.267 0.276 0.379
0.282 0.299 0.278 0.272
CV
2360 EGITO ET AL.
Figure 6. Volume weighted particle size distribution of droplets for FungizoneTM/LipofundimTM admixture (FLmix), at 5 105 M, before (A) and after (B) 12 h of preparation. The sample was prepared from FLmix at 2.5 103 M diluted 50 times in phosphate buffer solution, pH 7.4.
No significant hemoglobin leakage was observed with FLmix over the whole range of concentration tested (0.05 to 50 mg/L) and with FungizoneTM below 0.5 mg/L. At this concentration, the increase on hemoglobin leakage started
Figure 7. TEM of FungizoneTM micelles. The sample was stained with osmium tetroxide 2% and analyzed at 36,000 magnification. Bar, 300 nm.
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Figure 8. TEM of FungizoneTM/LipofundimTM admixture showing the incorporation of FungizoneTM on the emulsion droplet, in the oil/water interface. The sample was stained with osmium tetroxide 2% and analyzed at 58,000 magnification. Bar, 170 nm.
(5.36 9.28%) and at 5 mg/L reached 96.99 2.53%. When FLmix was used at 5 and 50 mg/L, the toxicity against RBCs, as reflected by hemoglobin leakage, was significantly reduced compared with FungizoneTM ( p < 0.0001).
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Figure 10. TEM of FungizoneTM/LipofundimTM admixture (A) LipofundimTM emulsion (B) stained with osmium tetroxide 2% and analyzed at 19,000 magnification. Bar, 500 nm.
Potassium leakage after the incubation of 5 107 cfu (1 h at 378C) with increasing concentrations of FungizoneTM and FLmix is shown in Figure 12. Whatever the system tested, a significant potassium release was observed at the lowest concentration (0.05 mg/L) and reached 100% at the 5 mg/L level. However, FungizoneTM at 0.05 mg/L induced a significantly larger potas-
sium release (88.96 3.49%), compared with the FLmix (60.59 21.36%) ( p < 0.0001). The antifungal activity of FungizoneTM and FLmix is shown in Figure 13. Determination of the viability of C. tropicalis cells (ability to form colonies) showed that whereas FungizoneTM was able to kill all seeded fungal cells from a concentration of 5 mg/L (0.36 0.62 cfu%), FLmix was unable to do that. Indeed, after treatment of C. tropicalis cells with FLmix at the highest concentration studied, 50 mg/L, a viability of 11.89 4.65 cfu% was retrieved. Nevertheless,
Figure 9. TEM of LipofundimTM emulsion showing a reduction on the droplet electron density. The sample was stained with osmium tetroxide 2% and analyzed at 58,000 magnification. Bar, 170 nm.
Figure 11. In vitro release of potassium from human RBCs (&, &) and C. tropicalis(*, *) induced by FungizoneTM (*, &) and FungizoneTM/LipofundimTM admixture [FLmix (*, &)]. Each point on the figure is the mean (SD) of three determinations. *Significant difference between the two forms for RBCs ( p < 0.0001).
Activity against C. tropicalis
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Figure 12. In vitro release of hemoglobin from human RBCs induced by FungizoneTM (&) and FungizoneTM/LipofundimTM admixture [FLmix (&)]. Each point on the figure is the mean (SD) of three determinations. *Significant difference between the two formulations ( p < 0.0001).
the difference of activity between both preparations was significantly different only at 0.5 mg/L, when FungizoneTM was more effective than FLmix ( p < 0.0001).
DISCUSSION The use of electronic absorption analysis has been previously reported as a powerful tool to follow the interaction of AmB with a lipidic carrier.23,27 In fact, the strong absorbance that AmB exhibits between 300–450 nm, because of the seven
conjugated double bonds existing on its apolar domain (Fig. 14), is heavily influenced by conformational changes provoked by its self-association in water media or by its interaction with other compounds such as drug carriers. The physicochemical properties of AmB, mainly its amphiphilic and zwitterionic nature, attributed respectively to the apolar and polar sides of the lactone ring and to the presence of ionizable carboxyl and amine groups (Fig. 14), as well as its asymmetrical distribution of hydrophobic and hydrophilic groups, make the drug extremely insoluble in aqueous solvents and in many organic solvents where it generates aggregates beyond a threshold concentration. In fact, beneath 106 M, which is the critical aggregation concentration (CAC), AmB can be present in aqueous media as a soluble monomer, and its absorption spectrum is characterized by a peak at 409 nm. However, above the CAC, AmB molecules are able to selfassociate to form oligomers and then aggregates of oligomers, which are characterized in absorption spectra by a broad intense single band at 340 nm.28–30 It has been reported that the AmB may present various patterns of activity against ergosterol-containing fungal cells with respect to the toxicity against cholesterol-containing mammalian cells, depending on its molecular presentation.22,24,26,31,32 Indeed, monomers were less efficient than aggregates of oligomers at inducing the permeability of a cholesterolcontaining membrane to potassium.33 Self-associated AmB was shown to trigger permeability changes in RBC membranes and to induce cytotoxic events.22 Moreover, the toxic chemotherapeutic effects in mice were previously demonstrated to be correlated to the aggregation state of AmB.25 Binding of the monomeric or oligomeric forms of an amphiphilic drug can lead to an active form either at once or after reorganization in micelles within the lipid bilayer, but only when the
Figure 13. In vitro antifungal activity of FungizoneTM (&) and FungizoneTM/LipofundimTM admixture [FLmix (&)] on C. tropicalis (mean SD of three determinations run in duplicate). *Significant difference between the two formulations ( p < 0.0001). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 11, NOVEMBER 2002
Figure 14.
AmB structure.
AMPHOTERICIN B AND EMULSION ADMIXTURE
concentration exceeds the critical micellar concentration in the membrane.34,35 Consequently, any factor modifying the equilibrium between the different species of AmB in aqueous media may change its overall activity. In the present study, the spectral evaluation of FungizoneTM and FLmix clearly demonstrate that the behavior of AmB molecules was perturbed from one system to another. In fact, the spectra of AmB in the FungizoneTM (Fig. 1) presented a concentration dependency that was reported by other groups.23,28,36,37 At higher concentrations, the spectra presented an intense band at 326 nm and two small ones at 383 and 418 nm. However, as the concentration decreased, the predominant band disappeared and a new one, at 407.5 nm, was observed when it reached 5 107 M. The fact that the aggregation state band was shifted to a slightly lower wavelength, 326 nm, for the FungizoneTM system compared with the classical AmB self-associated band location, which is at 340 nm, indicates that the AmB molecules are complexed with the sodium deoxycholate. However, because the deoxycholate concentration in FungizoneTM diluted to 5 107 M AmB is very low and below its critical micellar concentration (2.8 103 M),38 we believe that the detergent molecules dissociate from the AmB–micelle complex and dissolve in water. Therefore, at high dilutions, FungizoneTM behaved like an aqueous solution of AmB.39 For FLmix, the results indicate that the AmB– emulsion complex generated is quite different (Fig. 2). Firstly, except at very low AmB concentration (5 108 M), the variation of the absorption spectrum was great, and its behavior was similar to FungizoneTM at the same AmB concentration. The self-associated AmB band (at 324 nm in this preparation) was maintained at high dilutions (5 107 M), whereas the proportion of the monomeric form remained low (Fig. 5). This suggests that the AmB molecules were strongly associated with the emulsion droplet. Indeed, it was observed that despite the high dilution of FLmix at AmB concentrations of 5 107 M and 5 108 M, the emulsion system remained stable and presented a milky appearance. Thus, we believe that, when FungizoneTM was mixed with LipofundinTM, the AmB–micelle complex existing in the former dissociates, permitted the detergent to go to the external aqueous phase and the AmB molecules to be incorporated into the oil/water phospholipid monolayer interface that composes the emulsion system.
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Theoretically, AmB could be located either in the oily core of emulsion droplets or at the interface. However, because AmB is not soluble either in soybean oil or in medium-chain fatty acids,40 we assume that it is located in the phospholipid monolayer, as already suggested by Washington et al.41 This hypothesis was partially confirmed by the variation found in the FLmix spectra measured at 378C (Fig. 3). In fact, the behavior of AmB molecules located at the phospholipid monolayer was severely perturbed by the temperature increase of 128C: the aggregate’s band was red shifted from 324 to 319.5 nm; the intensity of the spectrum aggregate’s band at 5 106 M and 5 107 M was reduced with a consequent increase in the monomeric one; the presence of monomeric species was increased to 100% instead of 80% that was found at 258C (Figs. 4 and 5); and the b/d50% was strongly altered. All these events could have happened because of the increase on thermodynamic energy on the oil/water interface caused by the increase in temperature that provokes a complete change in the thermodynamic equilibrium of aggregate and monomeric species, because of the release of the latter from the AmB–emulsion droplet complex. The particle size analysis and the morphological evaluation once again indicated that an interaction between AmB and emulsion droplets had occurred. First, a variation in the distribution profile of droplets charged with AmB was found (Fig. 6). Second, a small variation of the particle size after 12 h was detected (Table 1). Finally, the TEM pictures revealed that not only FungizoneTM micelles (Fig. 7), which present a granulometric size fivefold lower than FungizoneTM/LipofundinTM complex, disappeared from the FLmix external phase (Fig. 10), but also FLmix droplets were more electron-dense (Fig. 8) than LipofundinTM droplets (Figs. 9 and 10). This indicates that FungizoneTM micelles were incorporated into LipofundinTM. The in vitro FLmix evaluation permitted us to go forward on the AmB phospholipid monolayer localization hypothesis. In fact, these studies showed that FLmix presented an important selectivity against human and fungal cells and was less toxic than FungizoneTM. When the target cells were human RBCs, whereas RBC did not present hemoglobin leakage over the whole range of doses tested, the C. tropicalis viability started to decrease at 5 107 M (85.32 10.19% cfu) and became 11.89 4.65% cfu at 5 105 M. No
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significant Kþ leakage, which has often been used as an index of polyene toxicity,22,42 –45 from RBC was observed below 5 107 M (2.97 2.11% at 5 108 M), but for C. tropicalis, the lowest concentration studied caused 60.59 21.36% leakage, reached at 87.77 6.81% as early as the 5 107 M level. For RBCs, this degree of toxicity was observed only at 5 105 M. The results for FungizoneTM were in agreement with those published recently.46 The FLmix presented nearly the same efficacy against C. tropicalis and a lower toxicity against RBCs. However, the ensemble of activity results of FLmix point out that at a particular AmB concentration (0.5 mg/L), the activity toward C. tropicalis is slightly decreased, probably because of the variation of AmB species in equilibrium. In fact, some authors have found that the association of AmB with lipids reduced its activity against fungal cells.47–49 Nevertheless, the high protective effect on RBCs induced by FLmix allows the use of larger doses of FungizoneTM, thus improving its therapeutic index. All the results together suggest that LipofundinTM should be able to reduce the FungizoneTM toxicity probably by changing the AmB selfassociation state by binding it with the emulsion droplet. Indeed, FLmix may be considered as a reservoir of the monomeric form of AmB that releases only limited amounts of free AmB in the aqueous media. As a consequence, the level of free AmB might be below its CAC and the drug could be in its monomeric form. This form would be able to bind to the ergosterol of fungal cells but would be inactive against the cholesterol of mammalian cells. Liposomal and other lipid-based forms of AmB have been extensively studied, and some of them are already marketed.4 However, the FLmix form may have some practical advantages in that it does not include expensive semi-synthetic lipids and is even now in use on clinical trials. Nevertheless, as shown by our results, the mixture of two approved pharmaceutical entities induces large physicochemical changes in both of them. In this specific case, FLmix should be considered as a new delivery system and its use should be made with caution.
ACKNOWLEDGMENTS We thank Elayne C. M. T. Egito, for her drawing of the AmB structure, and Glenn Hawes, for editing the manuscript. The authors are indebted
to one of the referees for his constructive remarks. Dr. Egito is grateful for the financial support from CAPES (Brazilian Department of Education), Brası´lia/Brazil. This work was funded in part by the FMC Foundation.
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