Pharmacological parameters of intravenously administered amphotericin B in rats: comparison of the conventional formulation with amphotericin B associated with a triglyceride-rich emulsion

Liliete Canes Souza and Ana Campa*

Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, S.P., CEP 05508-900, São Paulo, Brazil


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The LD 50 determined in rats for the potent antifungal amphotericin B (AB) increased from 4.2 to 12.0 when the conventional AB-deoxycholate (DOC) was compared with AB associated with a triglyceride-rich emulsion (AB-emulsion). The reduction in amphotericin B toxicity is not due to a modification in plasma clearance, as both formulations seem to be removed from plasma at the same rate. Major differences in amphotericin B tissue distribution were not seen for kidney and liver but were seen for the lung. After 24 h administration of a single amphotericin B dose (2.0 mg/kg body weight) 23.78 ± 11.71 mg/kg tissue was recovered from the lung of animals treated with AB-DOC whereas for AB-emulsion only 5.19 ± 2.50 mg/kg tissue was recovered. The higher lethality of AB-DOC may be related to the higher concentration of amphotericin B in the lung. The therapeutic efficacy of AB-emulsion was similar to that of AB-DOC as attested by survival curves obtained after treatment of mice infected by Candida albicans. This is highly relevant, as the same is not necessarily found for other less toxic proposed vehicles. The equivalent efficacy and the increment in the LD 50 will result in an important improvement in the therapeutic activity of amphotericin B. Furthermore, some data related to storage and stability indicate the clinical utility of this type of drug delivery.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The high potency and the broad spectrum of action of amphotericin B (AB) and the therapeutic experience of almost 40 years with the commercial formulation AB-deoxycholate (DOC) have assured its position as the antibiotic of choice for a great number of systemic mycoses mainly occurring in immunosuppressed patients. 1 ,2 ,3 ,4

The high toxicity of amphotericin B in its conventional formulation with DOC has been successfully prevented in other types of amphotericin B formulations. 5 ,6 ,7 ,8 ,9 ,10 ,11 ,12 ,13 We have studied the reduction of amphotericin B toxicity by its association with a triglyceride-rich emulsion that mimics natural chylomicrons. 14,15 Reduction of toxicity in vitro was observed on erythrocytes 14 and polymorphonuclear leucocytes, 15 and in vivo by the determination of the frequency of acute lethality after iv administration of a high dose (5 mg/kg) of amphotericin B in rats. 14 Importantly, the fungicidal activity in vitro is completely preserved when amphotericin B is associated with the emulsion.

This study addresses two main issues. First, as differences in amphotericin B plasma clearance and tissue distribution could have some role in the reduction of AB-emulsion toxicity when compared with AB-DOC, it is our purpose to define these classical pharmacological parameters, and also the median lethal dose (LD 50), after iv amphotericin B administration in rats. Second, to define whether the antifungal activity of AB-emulsion is maintained in vivo, we evaluated the therapeutic efficiency of the drug in a murine experimental model susceptible to Candida albicans infection. To advance the clinical use of AB-emulsion we also determined K + efflux from erythrocytes in the presence of different concentrations of amphotericin B and the stability of the lipids in the emulsion and the maintenance of amphotericin B antifungal activity on 1-6 months storage.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Reagents

Reagents were high-purity commercial samples from Sigma Chemical Company (St Louis, MO, USA), and Merck-Quimitra (Rio de Janeiro, Brazil). Amphotericin B type I and amphotericin B-deoxycholate (conventional amphotericin B) were supplied by Squibb Bristol-Meyers (São Paulo, Brazil). Stock solutions of amphotericin B were prepared in dimethyl sulphoxide (DMSO) in appropriate concentrations for each assay, protected from light and immediately used. AB-DOC solutions were prepared by resuspension of the commercial deoxycholate preparation (50 mg amphotericin B-41 mg DOC-20.2 mg sodium phosphate) with phosphate-buffered saline (PBS), pH 7.4.

Preparation of the triglyceride-rich emulsion

Phosphatidylcholine was prepared from egg yolk according to the method of Singleton et al., 16 as modified by Kamp et al. 17 and stored at -80°C under nitrogen, except for the experiments on amphotericin B therapeutic efficiency, K+ efflux from erythrocytes and stability on storage, when phosphatidylcholine was purchased from Lipid Products (Redhill, UK). Triolein and cholesterol were purchased from Nu-Chek Prep (Elysian, MN, USA). Lipids were at least 99% pure as tested by thin-layer chromatography (TLC). Emulsions were prepared from lipid mixtures consisting of 70% triolein, 27% phosphatidylcholine and 3% cholesterol dissolved in chloroform- methanol (2:1). 18 Tracer amounts of labelled [3H]cholesterol, [14C]phosphatidylcholine and [3H]triolein (Amersham International, Amersham, UK) were added in the experiment evaluating lipid stability upon storage. The mixtures were dried under nitrogen and kept in a vacuum desiccator overnight. PBS (pH 7.4) was then added to the dried lipids to give a final concentration of 25 g/L. Emulsions were obtained by sonication in a Branson Cell Disruptor (Branson Ultrasonics, Danbury, CT, USA) for 30 min at 50°C, with an output of 70-80 W. The emulsions were centrifuged at 800g for 12 min to precipitate titanium and filtered through 0.22 µm filters (Durapore-HV, Millipore Ind. e Com. Ltda., São Paulo, Brazil).

Association of amphotericin B with the emulsion

Amphotericin B was associated with emulsion by vortexing a DMSO stock solution of amphotericin B with an appropriate volume of emulsion for 30 s. This solution was prepared just before experiment. During use it was kept at room temperature and protected from light.

LD50 determination

Male Wistar rats (150-200 g) received AB-DOC (amphotericin B 2-10 mg/kg body weight) or AB-emulsion (amphotericin B 2-18 mg/kg body weight) iv via a lateral tail vein as a single dose. The volume of DMSO injected when AB-emulsion was administered varied from 14 to126 µL/200 g body weight. The volume of emulsion was 500 µL/200 g animal body weight. Animals were observed for 4 days. Median lethal doses (LD 50) were calculated by the method of Reed and Muench. 19

Amphotericin B plasma clearance

Male Wistar rats (150-200 g) were injected via the tail vein with amphotericin B (2 mg/kg body weight) as a single dose of AB-DOC or AB-emulsion (the volume of DMSO injected was 14 µL and the volume of the emulsion was 500 µL, both for 200 g body weight). Heparinized (10 IU/mL) blood samples were collected by retro-orbital exsanguination at 3, 30 or 60 min. The blood was centrifuged at 1500g for 10 min at 4°C, and the resulting plasma was stored frozen at -20°C protected from light. Amphotericin B was extracted from plasma samples (200 µL) by mixing with 800 µL of DMSO- methanol-water (10:9:1) for 30 s, incubated at 4°C for 20 min, and clarified by centrifugation at 2000g for 15 min at 4°C, and the supernatant subjected to amphotericin B measurement by UV-visible absorbance (E1%1 cm (411 nm) = 1860) 20 in a Hitachi Spectrophotometer Model U-3210 (Tokyo, Japan). A standard curve was constructed for amphotericin B extracted from plasma to which known (2.0-21.0 mg/L) amphotericin B concentrations were added. Plasma clearance of amphotericin B was plotted and a simple linear regression model was fitted for each animal under study. 21 An analysis of variance was used to compare the slope means. 22

Amphotericin B tissue distribution

Two different protocols were used to evaluate amphotericin B tissue distribution. In the first, six doses of amphotericin B (2 mg/kg body weight), as AB-DOC or as AB-emulsion were iv administered as described above to male Wistar rats (150-200 g) at 48 h intervals. Liver, kidneys and lungs were removed 24 h after the last injection. In the second protocol a single dose of amphotericin B (2 mg/kg) was iv administered to rats. The animals were killed after 30 min, 4 or 24 h and liver, kidneys, heart, spleen and lungs were collected and stored at -20°C. Samples weighing 0.2 g were homogenized with 1 mL DMSO-methanol- water (10:9:1) and placed on ice. The suspension was clarified by centrifugation at 2000g for 15 min at 4°C, and amphotericin B concentration in the supernatants was determined by UV-visible absorbance. Standard curves were prepared with tissues from control rats homogenized with amphotericin B (0.0-21.0 µg). Statistical analysis was by analysis of variance and, when necessary, the Geisser and Greenhouse procedure 22 and the Tukey method of multiple comparisons. 21

Amphotericin B therapeutic efficacy

An inoculum of C. albicans, a clinical isolate from sinovial fluid, was transferred to 10 mL of liquid Sabouraud's medium and incubated overnight at 37°C. Cells were washed twice with PBS and counted in a Neubauer chamber. Male BALB/c mice (weight 30 ± 2 g) were injected with 100 µL of a suspension containing 3 x 105 cfu of fungal cells via the lateral tail vein for the establishment of experimental candidosis. After 72 h the infected mice were randomly divided into three groups, which received PBS alone (n = 26) or amphotericin B 0.5 mg/kg body weight as AB-DOC (n = 25) or AB-emulsion (n = 25). The volume of injection was 100 µL. Survivors were recorded daily until day 45. The efficacy of the drug was monitored in two ways: (i) by observing survival of the mice; (ii) by evaluating the presence of viable C. albicans in the kidneys after drug therapy. The survival curves were constructed using the Kaplan-Meier method, and for the comparison of curves a generalization of the Kruskal-Wallis method for censured data was used. 23 The fungal viability was determined by plating slices of kidney on Sabouraud's dextrose agar for 48 h at 30°C.

An identical experiment was also performed with a higher amphotericin B dose (1 mg/kg body weight). The groups PBS, AB-DOC and AB-emulsion had seven animals each and were observed for 30 days.

Amphotericin B induction of K+ efflux from red blood cells (RBC)

RBC were obtained from EDTA-treated blood of healthy donors, after centrifugation at 1000g for 2 min. The cells were washed twice with PBS before being dispersed in the same buffer and counted in a haemocytometer. Intracellular K+ loss was measured after incubation of the cells (1.5 x 108) with amphotericin B 1-15 mg/L as AB-DOC or AB-emulsion (0.5 mL) in PBS (incubation final volume was 1.0 mL). After 10 or 60 min of incubation an aliquot of 100 µL was collected and centrifuged at 2800g for 2 min. The pellets were washed twice with saline solution and then cells were lysed by addition of 15 mM LiNO 3. The concentration of K+ was determined in the supernatant obtained after centrifugation of the lysate at 11,200g for 10 min by flame photometry (Corning Flame Photometer Model 400, Acton, MA, USA). Data obtained in this study were analysed by Student's t-test.

Stability of AB-emulsion solution on storage

Amphotericin B (72 µg) was added to 12 mL of lipid emulsion and sterilized by filtration (0.45 µm Millex-HV, Millipore Ind. e Com. Ltda., São Paulo, Brazil). The resulting filtered AB-emulsion (amphotericin B 4.4 mg/L) was distributed in sterilized flasks (2.0 mL) and stored protected from light in a refrigerator. The stability of the lipids in the AB-emulsion was assessed by the appearance of hydrolysis products and lipid peroxidation, and the stability of amphotericin B activity was assessed by determination of K + efflux in C. albicans.

To measure lipid hydrolysis, tracer amounts of [3H]cholesterol (2381 Bq), [14C]phosphatidylcholine (1755 Bq) and [3H]triolein (2381 Bq) were added to the total lipids. After 1, 2, 4, 6 and 8 months, 100 µL of the emulsion were extracted in cloroform-methanol-water (5.0:2.5:2.5) overnight as described by Kobayashi and Maudsley. 24 The organic phase was evaporated by N 2 flux and the solid was resuspended in 200 µL of a chloroform-methanol (2:1) standard carrier solution (40 µg of triolein, cholesterol ester and free cholesterol) and separated by TLC with silica gel 60H and hexane-diethyl ether-acetic acid (70:30:1) as solvent system. The separated fraction was visualized by iodine vapour, and the bands corresponding to triglycerides, phosphatidylcholine and cholesterol were scraped into scintillation vials and 7 mL of scintillation solution were added. Radioactivity was measured in the 3H and 14C region in a Tri-Carb Model 1900 TR scintillator (Packard Instrument Co., Meriden, CT, USA). The hydrolysis was evaluated as a percentage of the fresh preparation.

Thiobarbituric acid reactive substances (TBARS) were used as a lipid peroxidation index. Two hundred µL of the AB-emulsion solution were used and the conditions were as described by Winterbourn et al. 25 Measurements were carried out in a Hitachi Spectrophotometer Model U-3210 (Tokyo, Japan) in the 532-535 nm region, where the thiobarbituric acid-malonaldehyde adduct absorption is maximal (pink-absorbing chromogen).

To detemine the K + efflux produced in C. albicans, an inoculum of the fungal culture was transferred to 5 mL of liquid Sabouraud’s medium and incubated overnight at 37°C. Twenty mL of fresh medium were added, and the mixture was incubated at 37°C for 60 min. Cells were washed twice with saline solution and counted in a Neubauer chamber. Intracellular K + loss was measured after incubation of the cells (4 x 10 7) with AB-emulsion (1.5 mL) in PBS (final volume 3.0 mL) for periods varying from 10 to 60 min. Three-hundred microlitre aliquots were centrifuged for 2 min at 2800g and the pellets washed twice with saline solution. Cells were then lysed by addition of 15 mM LiNO 3 and heated at 100°C for 3 min to facilitate lysis. K + concentration in the supernatant obtained after centrifugation of the lysate at 11,200g for 10 min was determined by flame photometry (Corning Model 400, Acton, MA, USA)


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
LD50 determination

Table I shows the mortality rates observed after a single iv dose of AB-DOC or AB-emulsion in rats. The LD 50 values obtained from these data were amphotericin B 4.2 and 12.0 mg/kg body weight for AB-DOC and AB-emulsion respectively. For both formulations, doses <=2 mg/kg did not cause death. Doses of AB-DOC >=6 mg/kg led to 100% mortality, whereas with AB-emulsion 100% mortality occurred only with 18 mg/kg. Deaths occurred 30-120 min after amphotericin B administration.


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Table I. Deaths after iv administration of a single dose of AB-DOC or AB-emulsion in rats
 
No additional protective effect of the emulsion on amphotericin B toxicity seemed to occur on increasing the amount of emulsion. In a preliminary experiment, with a double amount of emulsion, AB-emulsion with amphotericin B at 8 and 10 mg/kg body weight was administered and deaths/number of injected animals were 0/4 and 4/4 respectively. These data were indicative of an LD 50 value close to that seen for AB-emulsion. Conversely, decrease in AB-DOC toxicity was seen on mixing emulsion with AB-DOC before its injection. Administering amphotericin B at 3 and 4 mg/kg body weight, deaths/number of injected animals were, respectively, 1/3 and 3/3 for AB-DOC and 0/4 and 0/4 for AB-DOC plus emulsion.

Amphotericin B plasma clearance

The mean ± S.D. of amphotericin B plasma concentration after 3, 30 and 60 min following the iv administration of amphotericin B at 2 mg/kg body weight was 13.50 ± 7.76, 4.60 ± 1.69 and 1.97 ± 0.94 mg/L for AB-DOC and 8.59 ± 4.99, 1.55 ± 1.43 and 1.21 ± 0.53 mg/L for AB-emulsion. There was no statistical difference (P = 0.7360) between the two groups (each of 27 animals). The average serum concentration of amphotericin B decreased rapidly with time and the clearance of amphotericin B was the same for both treatments.

Amphotericin B tissue distribution

The mean ± S.D. of amphotericin B concentration for lung, liver and kidney of animals treated with six doses of amphotericin B 2 mg/kg body weight were 49.66 ± 13.53, 10.66 ± 7.01 and 6.38 ± 1.52 mg/kg of tissue for animals that received AB-DOC (n = 5) and 10.66 ± 8.79, 13.79 ± 4.41 and 10.75 ± 3.51 mg/kg of tissue for animals that received AB-emulsion (n = 8). In animals treated with AB-DOC, a higher mean concentration was observed for lung (P < 0.00001).

The average and standard deviation of the amphotericin B concentration in tissues at different times following a single dose of amphotericin B of 2 mg/kg body weight is shown in Table II. Amphotericin B was not detectable in the heart. The values for AB-emulsion in kidney at 24 h and in spleen at 30 min refer to the maximum concentration detectable from the constructed standard curve. Comparing tissues, for all treatments and sampling times, with the exception of the AB-emulsion group at 24 h, the organ that showed the highest mean amphotericin B concentration was the lung.


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Table II. Tissue distribution of amphotericin B (mg/kg tissue) after a single iv amphotericin B dose (2 mg/kg body weight) as AB-DOC or AB-emulsion
 
With AB-DOC, the maximum amphotericin B concentration in lung was seen after 4 h, whereas for AB-emulsion the highest concentration was observed at 30 min and decreased with time. For the other organs studied no significant differences were observed between treatments. The administration of multiple amphotericin B doses resulted in accumulation of amphotericin B in all studied organs (data not shown).

Amphotericin B therapeutic efficacy

Mice were infected with 3 x 10 5 cfu of C. albicans and after 72 h were treated with amphotericin B 0.5 mg/kg body weight as AB-DOC or AB-emulsion. Most infected animals treated with AB-DOC showed symptoms of acute toxicity, such as respiratory distress, soon after drug administration. The same was not observed after AB-emulsion administration. The animals were observed for 45 days. All animals in the infected control group died during the period of observation, whereas for the AB-DOC and AB-emulsion treated groups 15 (60%) and 12 (48%) animals died, respectively (Figure 1). At the end of the experiment kidney tissue from all surviving animals was cultured and was positive for C. albicans for two animals per treatment. The statistical analysis of the survival curves did not show differences between AB-DOC and AB-emulsion treatments (P > 0.10), but both differed from the untreated control (P < 0.001).



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Figure 1. Survival curves for BALB/c mice infected with C. albicans (3 x 105cfu): {blacksquare}, untreated controls (n = 26); {triangleup}, AB-DOC treated (n = 25); {circ}, AB-emulsion treated (n = 25). Treatment was single doses of amphotericin B 0.5 mg/kg body weight.

 
In a second experiment the infected animals received PBS or amphotericin B 1 mg/kg body weight, as AB-DOC or AB-emulsion, and were observed for 30 days (seven animals per group). After 16 days following infection all animals in the untreated control group had died. All treated animals survived until the end of the experiment at which time cultures of kidney were all negative.

Amphotericin B induction of K+ efflux from RBC

Table III shows the mean and standard deviation obtained for the K + efflux from erythrocytes incubated for 10 and 60 min with AB-DOC (1-10 mg/L) or AB-emulsion (1-15 mg/L). Incubation with AB-DOC resulted in an accelerated loss of intracellular K + in the first 10 min whereas the efflux of K + was markedly reduced with AB-emulsion. Significant differences between AB-DOC and AB-emulsion were found for all concentrations assayed (P < 0.018, P <= 0.003, P < 0.0001, respectively for 1, 5 and 10 mg/L).


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Table III. K+ intracellular content in erythrocytes (1.5 x 108), incubated with amphotericin B as AB-DOC or AB-emulsion
 
Stability of AB-emulsion on storage

Phosphatidylcholine present in the emulsion was hydrolysed by 22, 30, 34 and 50% after 1, 2, 4 and 8 months, respectively. For cholesterol plus triglycerides observed hydrolysis was 12, 14, 17, 25 and 36% after 1, 2, 4, 6 and 8 months, respectively.

Lipid peroxidation was indicated by malonaldehyde concentration, which changed from 0.0577 µM on the first day of preparation to 0.1218, 0.232, 0.320 and 0.365 µM after 1, 2, 4 and 8 months of storage at 4°C.

The stability of amphotericin B activity assessed by the degree of K +efflux it produces in C. albicans did not change after storage for at least 6 months. AB-emulsion caused a C. albicans K + efflux of approximately 62% after 60 min incubation.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The LD 50 experiments clearly show the efficiency of the triglyceride-rich emulsion in reducing amphotericin B lethality in rats, with an increase of approximately three times in the LD 50 (Table I). Increases in LD 50 have been reported for amphotericin B delivered with other carriers, 13 ,26 ,27 and curiously also for AB-DOC in a post-prandial situation in mice. 28 The mechanism operating in the reduction of toxicity in these cases is not known but it is possible that it is similar to that seen with the decrease in toxicity observed in our experiments. The inverse correlation between high values of plasma triglycerides and amphotericin B toxicity suggests that lipoproteins may be involved in the protective effect of feed. Post-prandial serum was also shown to be more protective than normal serum in preventing the K + efflux from erythrocytes caused by AB-DOC. 28 Some other conditions that alter the vascular lipid composition also modify the amphotericin B toxic effect, 11 ,29 ,30 possibly as a result of changes in drug distribution among plasma fractions.

The absence of any change in LD 50 on doubling the volume of the emulsion shows that the improvement obtained with emulsion had reached its limit. It is possible that even smaller lipid quantities would give the same protection. This may be an important factor in prolonged treatment where a reduction in volume and amount of administered lipids diminishes the possibility of associated problems. 31 Additionally, the observed reduction in lethality when AB-DOC is mixed with the emulsion points to a simpler approach to clinical use of amphotericin B. This would be similar to what was observed when AB-DOC was administered with Intralipid. 32,33

Drug clearance usually plays an important role in drug toxicity and efficiency. For other proposed amphotericin B formulations, both a reduced 34,35 and a prolonged time 36 of amphotericin B presence in the circulation were found. No differences in clearance were noted for the formulation studied in this work, hence it follows that this factor does not contribute to the observable reduction in toxicity.

Strong differences were found for the amphotericin B tissue distribution in lung. The drug concentration in lungs was very high for the group treated with AB-DOC, exceeding by several times the amphotericin B recovered from liver, kidney and spleen. After 4 and 24 h of administration, the concentration of amphotericin B in lungs was around three to four times lower for AB-emulsion than for AB-DOC. The high concentration of amphotericin B in lungs could be one of the reasons for the acute lethality of AB-DOC in rats. The animals injected with AB-DOC showed a respiratory insufficiency with subsequent convulsion, possibly by cerebral oxygen reduction, that culminated in the observed death. For surviving animals there were many lesions microscopically observed (Souza, Saldiva & Campa, unpublished results).

Concentrations of amphotericin B in lung even higher than that obtained by the administration of AB-DOC were found for liposomes 37 and lipid complex. 38 Indeed, treatment with high doses of amphotericin B-liposomes causes severe lung impairment. 39 Thus, the observed decreased distribution of amphotericin B in lung on administration of AB-emulsion may have important consequences when high doses of amphotericin B have to be employed. Except for the lung, there were no other indications of a role for tissue distribution in the reduction of toxicity of AB-emulsion.

The therapeutic efficiency of AB-emulsion was similar to that of AB-DOC, because AB-emulsion maintains its antifungal activity in vivo. This is very important, as the enhancement in LD 50 indicates the possibility of using higher doses of AB-emulsion than of AB-DOC, resulting in increased therapeutic effect. The demonstration that AB-emulsion is as efficient as AB-DOC will not necessarily be found for other lipid formulations, for instance for some liposomic formulations and lipid complexes. 32 ,40 ,41 ,42 AmBisome in the treatment of systemic and localized candidosis in immunocompromised mice showed four to eight times lower antifungal activity than AB-DOC. 43

The toxicity of amphotericin B depends on several factors, some of which are affected by the association of amphotericin B with different fractions of plasma. 44 All these factors operate in our in-vivo experiments. On the other hand, the toxicity of the drug independent of these complex factors can be observed in the in-vitro assays of toxicity. Whereas AB-DOC induced a marked intracellular K + efflux from human erythrocytes, the same is only observed with 15 times more AB-emulsion.

The clinical value of AB-emulsion will depend on stability on storage, ease of handling and costs. The present results show that the AB-emulsion is relatively stable. Partial losses of lipids seem not to interfere with amphotericin B activity for at least 6 months. No special care was taken in the preparation of AB-emulsion for storage, and it is possible that conditions guaranteeing sterility and the addition of an anti-oxidant will give improved stability. In relation to ease of handling and reduction of costs the mixture of AB-DOC plus emulsion also shows promise.

The elevated toxicity associated with amphotericin B therapy is a particular problem in severely compromised patients, in which doses of amphotericin B higher than 0.5 mg/kg/day cannot be tolerated. 45 The results presented provide pharmacological support for the suggested use of AB-emulsion as a lipid formulation highly favourable for use in antifungal therapy. There is strong evidence that the applicability of AB-emulsion could be more successful than other proposed formulations, because the reduction in toxicity is not accompanied by a decrease in therapeutic efficiency. The possible relatively low cost and the stability, as well as the possibility of using AB-DOC plus emulsion, make its use highly attractive.


    Acknowledgments
 
The authors thank Dr Claudette de Paula from the Parasitology Laboratory of the Instituto de Ciências Biomédicas, Universidade de São Paulo, for kindly supplying the C. albicans inoculum, and Squibb Bristol-Meyers (São Paulo, Brazil) for amphotericin B. We also thank the Applied Statistical Centre of the Instituto de Matematica e Estatística, Universidade de São Paulo, and Flavio Bianchi for technical help. This work received financial support from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP).


    Notes
 
* Corresponding author. Tel: +55-11-8183637; Fax: +55-11-8132197; E-mail: anacampa{at}usp.br Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 7 April 1998; returned 8 September 1998; revised 26 October 1998; accepted 26 February 1999