Comparative pharmacokinetics and safety of a novel lyophilized amphotericin B lecithin-based oil–water microemulsion and amphotericin B deoxycholate in animal models

Begoña Brime1,*, Paloma Frutos1, Pilar Bringas2, Ana Nieto3, M. Paloma Ballesteros1 and Gloria Frutos4

1 Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy; 2 Laboratory of Animal Facilities; 3 Department of Animal Pathology II, Faculty of Veterinary; 4 Department of Statistics and Operational Research, Complutense University of Madrid, 28040 Madrid, Spain

Received 21 June 2002; returned 27 October 2002; revised 24 February 2003; accepted 2 April 2003


    Abstract
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Amphotericin B (AmB) has been a most effective systemic antifungal agent, but its use is circumscribed by the dose-limiting toxicity of the conventional micellar dispersion formulation Fungizone (D-AmB). To lower AmB-associated toxicity, AmB may be integrated into oil-in-water lecithin-based microemulsions. The present study compares the pharmacokinetic characteristics of D-AmB with the alternative formulation of AmB in microemulsion (M-AmB), which has proved effective in a murine candidiasis model. Both formulations were given by intravenous bolus: D-AmB 1 mg/kg, and M-AmB 0.5, 1 or 2 mg/kg. The pharmacokinetics of D-AmB and M-AmB have several differences, specifically with regard to the respective Cmax and AUC0–{infty} values. Elimination of AmB from serum was biphasic for both M-AmB and D-AmB. Single-dose D-AmB (1 mg/kg) achieved a Cmax of 3.89 ± 0.48 mg/L and an AUC0–{infty} of 32.28 ± 7.31 mg·h/L, whereas single-dose M-AmB (1 mg/kg) by comparison achieved a lower Cmax (2.92 ± 0.54 mg/L) and a lower AUC0–{infty} (21.89 ± 5.17 mg·h/L). To evaluate the safety of M-AmB, a multiple-dose toxicity study was performed in groups of 10 mice, each receiving D-AmB 1 mg/kg, or M-AmB 1, 1.5, 2 or 3 mg/kg. The findings suggest that, in comparison with D-AmB, M-AmB produces no histologically demonstrable renal lesions, or changes in clinical chemistry.

Keywords: amphotericin B, microemulsion, safety, pharmacokinetics, rabbits


    Introduction
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Amphotericin B (AmB), and the lipid preparations of this agent, are still the treatment of choice for invasive fungal infections. Since mortality resulting from invasive fungal infections ranges between 30% and 100% in high-risk patients, there is a clinical necessity to administer AmB at high doses in the shortest time possible. Parenteral administration of AmB is limited by toxic side effects that include fever, chills, nausea, vomiting and hypokalaemia, resulting from treatment with desoxycholate preparations.13 In addition, symptoms of nephrotoxicity, including elevated serum creatinine and urea levels, often become apparent upon prolonged administration of AmB. This nephrotoxicity usually becomes more pronounced as therapy continues, and irreversible loss of kidney function has been reported.4

Three novel lipid-containing formulations of AmB have been developed in an attempt to attenuate its nephrotoxicity and increase its therapeutic potential. These formulations include, but are not limited to, AmB lipid complex, AmB colloidal dispersion and small unilamellar vesicle formulations of AmB. However, their higher economic costs and instability are a major limitation in therapeutics.5,6

A new formulation of AmB in an oil–water lecithin-based microemulsion has been developed in our laboratory by Moreno et al.7 This new formulation has proved to be less toxic and more effective than Fungizone, increasing survival and reducing the fungal load in the most representative organs after a single dose; however, little is known about multiple-dose toxicity and the pharmacokinetics in plasma, in comparison with those of conventional desoxycholate AmB after the administration of single doses.

We therefore studied multiple-dose toxicity and the pharmacokinetics in plasma of both conventional AmB (D-AmB) and the microemulsion formulation (M-AmB) in mice and rabbits.


    Material and methods
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Chemicals

AmB was supplied by Dumex (Denmark). Fungizone (D-AmB) was a gift from Squibb Pharmaceutical Industry (Barcelona, Spain). The microemulsion oil consisted of isopropyl myristate (IPM) and was obtained from Merck Chemicals (Madrid, Spain). The lipophilic surfactant, soybean lecithin (20% phosphatidylcholine), and the hydrophilic surfactant, a polyoxyethylene sorbitan fatty acid ester derivative, Polysorbate 80 (Tween 80), were purchased from Sigma Chemical Co. (Madrid, Spain). Deionized ultra-pure distilled water, used for the external phase of the microemulsions, was obtained from Milli-Q Plus Equipment, Millipore (Barcelona, Spain). Mannitol, used as a bulking agent for microemulsion lyophilization, was supplied by Merck Chemicals.

Preparation of microemulsion

Lyophilized AmB lecithin-based oil–water microemulsions were prepared using the method described previously by Moreno et al.7 and modified by Brime et al.8 Briefly, a polysorbate 80/water solution (1:3, w/w) was added to a lecithin/IPM dispersion (1:1, w/w), and the mixture stirred at 16 000 rpm for 2 min at 25°C until equilibrium was reached. The resulting system was analysed for homogeneity, isotropy (with cross polarizers) and visual transparency, in order to be considered a true microemulsion. Microemulsions had a polysorbate 80/lecithin relationship of 2:1, w/w.

Addition of AmB

AmB was incorporated at a concentration of 2 mg/mL to the previously obtained microemulsion. Briefly, AmB was dissolved (20 mg/mL) in sodium hydroxide solution (0.1 M) and added to the microemulsion at a temperature of 80°C, which is above the phase transition temperature of the emulsifiers. pH was adjusted to 8.5–9.5, with 1 M orthophosphoric acid. AmB microemulsions were finally passed through cellulose filters with a pore size of 0.45 µm (Millipore) to eliminate suspended AmB. To assess the actual amount of drug incorporated into the dispersal system, recovery of AmB from the microemulsion was determined at the end of the addition process, following the extraction and HPLC procedures validated and described by Moreno et al.9

Lyophilization of AmB microemulsion

AmB microemulsions were lyophilized in order to avoid reaction to hydrolysis by the lecithin phosphatide groups,10 and hence protect the dispersal systems from any decomposition phenomena related to such processes. Five percent (w/v) of mannitol was added to the external phase of the formulation as a bulking agent, because of the fluid nature of most of the excipients. Vials filled with 10 mL of microemulsion were placed inside the lyophilizer chamber. Lyophilization of the samples was performed with Telstar (Tarrasa, Spain) equipment. The resulting product was evaluated by the Karl–Fischer method (Metrohm 658 Processor with 665 Dossimat; Metrohm, Barcelona, Spain), for its macroscopic appearance, ability to reconstitute and water content.

Drug administration

D-AmB was reconstituted with 10 mL distilled water, maintained at 4°C and diluted with sterile 5% dextrose in water immediately before use at a concentration of 1 mg/kg of body weight. AmB microemulsions were prepared from lyophilized powder. The powder was reconstituted initially with 10 mL of sterile water to a 2 mg/mL solution. This solution was diluted with sterile 5% dextrose-in-water to 0.5, 1, 1.5, 2 and 3 mg/kg of body weight concentrations, passed through a filter with a pore size of 0.2 µm and administered at ambient temperature. Neither dilution nor filtration had an effect on size distribution or particle size.7

Extraction and analysis

AmB was extracted from serum by the addition of 2 vols of methanol to one of serum. After centrifugation, the clear supernatant containing the total amount of AmB was subjected to HPLC analysis. The chromatographic method employed was a modification of the method described by Brassinne et al.11 A Luna C18 column (150 x 4.6 mm, 3 µm), a Phenomenex SecurityGuard C18 precolumn (4 x 3 mm) and Hewlett-Packard 1050 series HPLC equipment, with an ultraviolet-visible detector set at a wavelength of 405 nm, were employed. The flow rate was 1 mL/min and the mobile phase was a methanol–acetonitrile–0.0025 M EDTA (40:30:30) (v/v/v) mixture. The retention time of AmB was found to be 5.9 min ± 5%, and the method was successfully validated with an RSD value of 4.7% and a determination coefficient (r2) of 0.9991. As happens with other lipid formulations, only the total AmB (both free and microemulsion-associated) concentration was determined,12 and as AmB microemulsions are supposed to be taken up rapidly by the mononuclear phagocyte system, the percentage of recovery measured experimentally in ex vivo studies cannot be extrapolated to in vivo studies.

Mice

A total of 140 male albino CR1 mice (25–30 g) were housed in groups of 10 in plastic cages in a 12 h dark–light cycle animal facility, with controlled temperature (25°C) and humidity (70%). Water and food were unrestricted throughout the study.

All animals were housed and maintained in the Laboratory of Animal Facilities, University Complutense, Madrid, according to European Institutes of Health and Spanish guidelines for laboratory animal care (D. C. 86/609/CEE; R. D. 223/1988; O. M. 13/X/1989).

Multiple-dose toxicity study. Six groups of 10 mice each received treatment via the tail vein. M-AmB at 1, 1.5, 2 and the maximum tolerated dose (MTD) of 3 mg/kg of body weight (LD50 for M-AmB is 4 mg/kg), or o/w lecithin-based microemulsion without AmB at 2 mg/kg of body weight, or D-AmB at the MTD of 1 mg/kg of body weight were administered for 5 consecutive days. Mice were observed for toxic effects immediately after the last injection, 1 h after injection and then daily for 14 days. Finally, animals were humanely sacrificed and a post-mortem examination was performed. After the administration of the fifth dose, and 1 week after cessation of treatment, five animals from each group were humanely sacrificed, and blood samples were taken for analysis. A complete necropsy was performed in each animal. Kidneys were taken, fixed in 10% neutral-buffered formalin, embedded in paraffin, sectioned at 4 µm and stained with haematoxylin–eosin. Samples were evaluated microscopically, and the lesion scores assigned objectively, according to the lesion scores published previously by Fielding et al.,13 without knowledge of the treatment group.

Methods of measurement. Haemoglobin values were determined using a haemoglobin Drabkin colorimetric test (SPINREACT, SA, Spain), to detect the possible occurrence of normocytic anaemia, or haemolytic anaemia resulting from bone marrow depression, which can develop as a side effect of AmB treatment. Serum creatinine values, as a parameter associated with renal toxicity, were measured with a Technicon RA creatinine reagent on an RA-500 autoanalizer (Bayer, Germany).

Rabbits

A total of 15 male New Zealand white rabbits weighing between 2.5 and 3.5 kg were used for the study. Animals were housed individually, and provided with food and water ad libitum. All rabbits were allowed 3 days to acclimatize to their environment prior to experimentation.

Single-dose study. A bolus of D-AmB 1 mg/kg, or M-AmB 0.5, 1 or 2 mg/kg was administered via the marginal vein to groups of three rabbits. Heparinized blood samples (2 mL) were obtained at 5 min, 0.5, 1, 2, 3, 4, 7 and 24 h. At the end of the study, animals were humanely sacrificed following pentobarbital anaesthesia.

Pharmacokinetic analyses

Non-compartmental analysis was performed. The AUC0–{infty} and mean residence time (MRT) were estimated by the log-linear trapezoidal rule with extrapolation to infinity, recommended by Chiou.14 CL was calculated as {lambda} x Vc = dose/AUC0–{infty}. The volume of distribution at steady state (Vss) was estimated by using the area under the moment curve (AUMC), such that Vss = CL x (AUMC/AUC0–{infty}). The terminal elimination rate constant (kel) was determined from the slope of the terminal portion of the log concentration–time curve. t1/2 was calculated as t1/2 = ln2/kel. Compartmental analysis was also performed. According to Hutchaleelaha et al.15 the data were fitted to bi- and triexponential functions. Model discrimination was based on Akaike information and Schwartz criterion (SC),16 and the two-compartmental model was selected to define the kinetics of the formulations. The WIN NONLIN 2.1 program was used to perform non-linear regression.


    Results
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
The results obtained in the multiple-dose toxicity study in mice are described below. In an attempt to detect any transient effects, histopathological analysis and creatinine and haemoglobin values were obtained 1 week after the end of treatment with both formulations.

Renal histopathological analysis

Control animals receiving 2 mg/kg of plain microemulsion, without the incorporation of AmB, did not present any nephropathological changes.

Mice sacrificed after five daily doses of D-AmB 1 mg/kg demonstrated kidney lesions of moderate severity (Table 1). These lesions consisted of a generalized vascular congestion, thickened basement membrane and hyalinization of the glomerulus. Tubular lesions included a focal necrosis of epithelial cells, involving principally the ascending limb of the Henle loop and distal convoluted tubules (Figure 1b). Tubular cells of these areas presented large cytoplasmic vacuoles and pyknosis of nuclei. In addition, thickened basement membranes of tubules and tubular calcification were observed (Figure 2). Mice receiving the same formulation at 1 mg/kg for 5 days, but sacrificed 1 week after the end of treatment, presented numerous areas of tubular regeneration, manifest in epithelial cells by a basophilic cytoplasm, large nuclei and the presence of mitosis (Figure 3).


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Table 1.  Lesion scores from microscopic pathology in groups of five mice examined after five daily injections (A) and 1 week after daily injections (B) of D-AmB and M-AmB
 


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Figure 1. (a) Kidney of mouse exposed to M-AmB 2 mg/kg/day for 5 days, and sacrificed at the end of the fifth dose. Architecture is essentially normal. (b) Kidney of mice at the end of 5 days exposure to D-AmB 1 mg/kg/day. Note the tubular cell necrosis and the thickened basal membrane. Bar, 25 µm.

 


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Figure 2. Kidney of mouse exposed to D-AmB 1 mg/kg/day for 5 days, and sacrificed at the end of the fifth dose. Note the nephrocalcinosis (arrow), tubular cell necrosis (arrow head) and glomerulus hyalinization (asterisk). Bar, 60 µm.

 


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Figure 3. Kidney of mouse exposed to D-AmB 1 mg/kg/day for 5 days, and sacrificed 1 week after the end of the fifth dose. Note the basophilic tubules corresponding with tubular regeneration (arrows). Bar, 60 µm.

 
No significant histopathological changes were observed in animals treated with M-AmB at lower doses of 0.5, 1, 1.5 and 2 mg/kg, after the administration of the fifth dose, or after the recovery period (Figure 1a). Only minimal and focal tubular necrosis was observed in some animals from different groups.

No significant histological lesions were found with a dose of M-AmB 3 mg/kg administered daily for 5 days. A minimal and nonspecific area of hyperplasia was observed in the Henle loop in two of the animals studied after the administration of the fifth dose. We also detected minimal renal tubular regeneration in some mice 1 week after the end of the treatment.

Creatinine values

Creatinine values after the administration of the fifth dose were considered normal for all dosage regimens. Lower values were obtained following administration of M-AmB 1 and 2 mg/kg, where creatinine values were 0.3 mg/dL. Values became higher after the recovery period, but remained within the normal range (0.3–1 mg/dL; Table 2).17


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Table 2.  Group mean (±S.D.) values of creatinine and haemoglobin in mice receiving D-AmB or M-AmB after five daily injections (A) and 1 week after daily injections (B)
 
Haemoglobin values

The haemoglobin value for M-AmB 1.5 mg/kg was slightly under 10 g/dL (9.68 ± 0.74 g/dL), but 1 week after the end of treatment values reached physiological levels. Animals receiving a dose of 1 mg/kg had a value of 9.21 ± 1.13 g/dL. The haemoglobin value following the fifth dose of the 2 mg/kg plain microemulsion was 10.25 g/dL, which is slightly under the physiological value that appears in the literature.17 One week after administration of the fifth dose, the value reached the normal range (10–17 g/dL; Table 2).

Pharmacokinetics of D-AmB

Mean (n = 3) experimental values of D-AmB plasma concentrations, as well as the biexponential fitted curve, are shown in Figure 4. The corresponding pharmacokinetic parameters (taking non-compartmental and compartmental approaches into consideration) are given in Table 3. D-AmB was well tolerated at 1 mg/kg, despite the rapid administration; the Cmax achievable with D-AmB was <5 mg/L, and levels in plasma declined slowly with a t1/2 of 19.840 ± 3.140 h. The AUC0–{infty} was 32.280 ± 7.310 mg·h/L and the MRT 27.100 ± 4.800 h. The distribution of AmB was better described by a two- than a three-compartmental model,18 according to values of Akaike’s information criterion (AIC) of –14.61 and 11.18, and to an SC of –14.29 and 11.65 for the two- and three-compartmental models, respectively. We can see that the predictions of the two-compartmental model are consistent with the data (Figure 4), and CL and Vss parameters calculated by biexponential fitting were 0.082 ± 0.010 L/h and 2.035 ± 0.170 L, respectively.



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Figure 4. Concentrations of AmB in serum after intravenous doses of D-AmB 1 mg/kg (triangles) and M-AmB 0.5 (circles), 1 (diamonds) or 2 mg/kg (squares) in rabbits. Lines represent fitting of data to a two-compartmental model. Each symbol represents the mean ± S.D. of measured serum levels from three rabbits.

 

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Table 3.  Pharmacokinetic parameters of AmB in rabbits receiving D-AmB or M-AmBa
 
Pharmacokinetics of M-AmB

Mean (n = 3) experimental values of M-AmB plasma concentrations, as well as the biexponential fitted curve, are shown in Figure 4. The corresponding pharmacokinetic parameters (taking into consideration both non-compartmental and compartmental approaches) are given in Table 3. Groups of three rabbits each received a single intravenous bolus dose of M-AmB 0.5, 1 or 2 mg/kg. At M-AmB 1 mg/kg, mean Cmax and AUC0–{infty} values amounted to 2.920 ± 0.540 mg/L and 21.890 ± 5.170 mg·h/L, respectively. The Cmax and AUC0–{infty} following administration of M-AmB rose proportionately with increasing dose; at a dose of 0.5 mg/kg they were 2.673 ± 0.450 mg/L and 14.457 ± 2.702 mg·h/L, whereas at a dose of 2 mg/kg they were 14.995 ± 1.175 mg/L and 39.359 ± 8.896 mg·h/L. These data show the almost linear rise in M-AmB levels in plasma with increasing dose, mainly with the AUC0–{infty} values. We observed similar values of CL and t1/2 of ~0.12 L/h and 12.00 h, respectively, for M-AmB at the three doses administered. The elimination of AmB from serum was biphasic for AmB microemulsions presenting an AIC of –20.07 and 5.86, and an SC of –19.75 and 6.34, for the two- and three-compartmental models, respectively.

Comparison of D-AmB and M-AmB

Table 3 illustrates several differences between the pharmacokinetic parameters of M-AmB and D-AmB. At a dose of 1 mg/kg, D-AmB had a greater Cmax and AUC0–{infty} than M-AmB. The Vss for D-AmB was also greater and CL slightly greater for M-AmB. This could demonstrate a faster elimination of AmB microemulsions from plasma via reticuloendothelial organ uptake. The intervals at the 95% confidence level for mean values of AUC and CL parameters, corresponding to D-AmB and M-AmB formulations administered at 1 mg/kg, were (20.36; 40.48) and (18.64; 25.24), and (0.05; 0.10) and (0.09; 0.13), respectively. For the two parameters, the confidence intervals partially overlap, so the statistical differences are not definitively proved due to the poor potency of proof (n = 3). Both formulations presented a biexponential elimination phase, which has also been observed in previous studies of AmB.1820


    Discussion
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
In a report recently submitted for publication, an infectious model for candidiasis was developed in immunocompetent and neutropenic mice. The non-treated mice and mice receiving D-AmB showed high mortality with heavy fungal load, unlike mice treated with M-AmB that were able to overcome the infection and showed an improved survival rate.

This study demonstrates that AmB incorporated into a lecithin-based oil–water microemulsion is safe and well tolerated at a dose of 3 mg/kg of body weight, when administered to healthy mice and rabbits; no acute adverse effects occurred and haematological parameters were all normal.

The highest D-AmB dose that could be administered during five daily doses to mice without any deaths was 1 mg/kg, in comparison with a dose of 3 mg/kg of M-AmB, confirming that M-AmB was considerably safer than the conventional micellar formulation of D-AmB.

AmB belongs to the class of polyene antibiotics characterized by a structure comprising a hydrophilic and a lipophilic region, enabling it to become incorporated in the cell membrane.21 The mechanisms of nephrotoxicity are not completely clear. Rhoades et al.4 were the first to suggest that they resulted from direct AmB action on the renal tubules, as well as from drug-induced renal vasoconstriction, with a consequent reduction in renal blood flow and glomerular filtration rate.22 As AmB is insoluble in saline at neutral pH, it is formulated with sodium deoxycholate, forming ribbon-like aggregates (Fungizone), which, when reconstituted, produce a colloidal suspension. However, these aggregates are relatively unstable and dissociate rapidly after infusion, releasing AmB into the bloodstream. It has also been demonstrated in vitro that some nephrotoxicity may be the result of the vehicle sodium deoxycholate.23

As a measurement of renal function, creatinine values were studied during and after treatment with D-AmB and M-AmB, and no prompt changes were observed after administration of the fifth dose. The serum creatinine value is a parameter used to detect abnormalities of renal function when it exceeds 1 mg/dL.6,17 Serum creatinine values were low at doses of M-AmB 1 and 2 mg/kg after the administration of the fifth dose, although there was no correlation between the decrease in the serum creatinine value and the total dose of AmB received. Serum creatinine values for the plain microemulsion were at normal levels in both determinations, indicating the safety of the formulation. For both formulations, and in all dosing regimens for M-AmB, we also observed that after the administration of the fifth dose serum creatinine values were lower than values obtained 1 week after the end of treatment. For D-AmB, creatinine values were at physiological levels and no nephrotoxic side effects could be related to use of this formulation according to this parameter. These results may suggest that, for the treatment regimens selected, the creatinine parameter is not as sensitive a marker as histopathological analysis; this may be due to the short exposure of mice to the drug (5 days).

Haemoglobin was not significantly changed in any of the dosing regimens (Table 2). Haemoglobin values under 8 g/dL would indicate the existence of anaemia; however, since values remained within the physiological range, we can conclude that AmB did not damage or depress the bone marrow, thereby producing haemolytic anaemia as a side effect (which has been described in 5% of patients treated with AmB in a clinical trial with 556 patients).24

Blood samples obtained were highly haemolysed, owing to the severe mechanism of blood extraction by heart puncture, and the impossibility of employing a better mechanism. This obstructed the detection of other clinical chemistry parameters, such as alkaline phosphatase, alanine aminotransferase or aspartate aminotransferase.

Mice receiving D-AmB 0.125 mg showed tubular damage and renal glomerulus degeneration, in comparison with mice receiving M-AmB 0.375 mg, which did not present a seriously damaged renal structure despite the three-fold higher dose of the antifungal agent (Figures 13). The mechanism by which AmB microemulsions attenuate mammalian cellular toxicity has not been elucidated fully as yet. However, we think it could be very similar to AmB lipid complex, not only because both formulations present similar pharmacokinetics, but because their composition is similar as both contain phosphatidylcholine in their structures.25

AmB is highly bound to the lipoproteins, erythrocytes and cholesterol present in plasma and in tissues throughout the body.20 The primary storage site appears to be the liver. Liposomal forms of the drug are believed to be taken up rapidly by the reticuloendothelial system, thereby reducing binding to cholesterol and plasma membranes.26 The M-AmB formulation should achieve a similar decrease in plasma membrane lipoprotein binding, and consequently reduce associated toxicity, as has been observed in animal studies carried out in our laboratory.

Linear kinetics at the doses studied were observed, along with a biphasic elimination pattern, but we would expect the use of higher doses of M-AmB to result in non-linear kinetics, as happens with other lipid formulations.27 This pattern suggests uptake of the drug by the reticuloendothelial system cells, followed by redistribution, as has been observed in other lipid-based AmB formulations with similar pharmacokinetic behaviour.28 As described in Figure 4, there is an important difference in the slope of elimination for the D-AmB and M-AmB formulations: it is faster for M-AmB. This may be one of the reasons why this new formulation is less toxic, as shown by the histopathological analysis.

There may be large differences in intra-individual distribution, owing to the nature of the microemulsion and its unknown mechanism of action, as seen with other lipid-based formulations;29 however, these are not reflected in a study of this size. Moreover, there is uncertain extrapolation to the effects in humans.

The instability of lipid formulations of AmB and very high cost of treatment have been a cause of concern in its use as a therapeutic agent, and constitute a hindrance to widespread use in poor countries. The results of this study show that the AmB lecithin-based oil–water microemulsion can be prepared simply and at a very low cost. Its low toxicity and good efficacy in animals make it a valuable system for the delivery of AmB; however, a multiple-dose pharmacokinetic study with larger numbers of rabbits, and toxicity studies employing different animal models, are warranted.


    Acknowledgements
 
We thank Dr W. J. Jusko, Department of Pharmaceutics, School of Pharmacy, State University of New York, Buffalo, for consultation on the pharmacokinetic analysis. The work was supported by a grant from the Science and Education Ministry and by a grant number 99/0853 from the National Funds for Health Investigation (FIS).


    Footnotes
 
* Corresponding author. Tel: +34-91-394-17-22; Fax: +34-91-394-17-05; E-mail: begonab{at}farm.ucm.es Back


    References
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
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