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Antimicrobial Agents and Chemotherapy, February 1999, p. 390-392, Vol. 43, No. 2
Laboratoire de Physicochimie
Biomoléculaire et Cellulaire (CNRS ESA 7033), Université
Pierre et Marie Curie, 75252 Paris cedex 05, France,1 and
Department of Infectious
and Tropical Diseases, London School of Hygiene and Tropical
Medicine, London WC1E 7HT, United Kingdom2
Received 17 June 1998/Returned for modification 4 September
1998/Accepted 2 November 1998
The heat treatment of amphotericin B deoxycholate (Fungizone),
which was previously shown to induce superaggregation and decrease the
toxicity of the drug to mammalian cells, increased its activity against
Leishmania donovani in BALB/c mice, whereas it reduced its
toxicity. Heat treatment preserved the activity of Fungizone against
L. donovani HU3-infected mouse peritoneal macrophages.
Amphotericin B (AmB) in its
commercial formulation Fungizone is the "gold standard" treatment
for systemic fungal infections and is the recommended second-line
treatment for visceral leshmaniasis (VL) infections when conventional
tetravalent antimony (SbV) therapy is inappropriate or ineffective
(4). Unfortunately, AmB causes acute side effects following
intravenous (i.v.) administration, and these limit its more extensive
clinical use. Recently, lipid formulations of AmB have successfully
been developed to greatly reduce the toxicity of AmB and so enable
higher doses of the drug to be given (see reference
3 for a review). Dose for dose, Fungizone has
greater activity than liposomal AmB against fungal infections
(9); however, against experimental VL infections, lipid
formulations are more active (1, 2, 5, 6, 8, 13, 14, 18, 20-22,
24, 28). Three lipid formulations have been approved for clinical
use: AmBisome (NeXstar, Cambridge, United Kingdom), Abelcet (The
Liposome Co., Princeton, N.J.), and Amphocil (Sequus Pharmaceuticals
Inc., Menlo Park, Calif.). Despite their proven success against
leishmaniasis (7, 10, 23), they are not frequently used
against the disease due to their expense. The developing tropical and
subtropical countries, where leishmaniasis affects 6 million
individuals (27), cannot routinely afford expensive
medication. Simple heating of Fungizone at 70°C for 20 min is an
inexpensive procedure which could be used to improve the therapeutic
index of AmB, as shown for the therapeutic index of AmB against
candidiasis and cryptococcosis (19), and encourage its more
widespread use.
In this study, the in vitro and in vivo antileishmanial activities of
Pentostam (SbV), Fungizone, and heated Fungizone were compared. The
differences in their relative toxicities to mammalian cells and mice
were observed.
Leishmania donovani MHOM/ET/67/L82 amastigotes were
maintained in golden hamsters (Charles Rivers, Margate, United
Kingdom). The parasites were harvested from an infected spleen for in
vitro and in vivo assays.
A stock solution of Fungizone (Bristol Myers Squibb, La Défense,
France) was prepared from a marked bottle by the addition of 10 ml of
sterile 5% dextrose (aqueous). Heated Fungizone was prepared as
described previously (11). Pentostam (100 mg of SbV/ml) and
powdered sodium stibogluconate (NaSbV) were provided by Glaxo Wellcome,
London, United Kingdom. NaSbV powder was dissolved in 0.25%
methylcellulose for in vivo administration. Drug dilutions were made
daily in complete medium for in vitro tests and in 5% dextrose for in
vivo tests.
For in vitro assays, peritoneal macrophages were harvested from female
CD1 mice (Charles Rivers) 24 h after starch (Sigma) induction and
were dispensed into 16-well Lab-tek slides (Nunc Ltd., Chicago, Ill.)
at a concentration of 4 × 104/well (100 µl/well) in
RPMI 1640 medium (Gibco BRL, Paisely, United Kingdom) supplemented with
10% heat-inactivated fetal calf serum (Sera-Lab, Oxon, United
Kingdom). After 24 h, the macrophages were infected with L. donovani amastigotes at a ratio of 10 amastigotes to 1 cell. After
24 h, the infected cells were exposed to drug for 5 days (the cell
overlay and drug were replaced on day 3). Prior to drug administration,
Fungizone solutions were incubated for 15 min at 37°C to allow the
AmB to bind to the proteins in the serum (26). The role of
lipoproteins in the endocytosis of AmB in cells by specific receptors
has already been shown (15, 25). Cells were treated with
both formulations at concentrations ranging from 1 µM to 0.5 nM. The
experiment was terminated on day 5 by methanol fixation and Giemsa
staining. The percentage of infected macrophages was evaluated
microscopically. The 50% effective doses (ED50s) were
determined by linear regression analysis (xlfit; Microsoft
Excel) with 95% confidence limits. P values were calculated
by Student's t test.
In these assays, no toxicity to macrophages was seen with either
formulation at the doses tested. ED50s were found to be
0.035 µg/ml for heated AmB deoxycholate and 0.024 µg/ml for
unheated AmB deoxycholate (Table 1). The
difference in the activities of the two formulations was not
significant (P > 0.05). Both Fungizone formulations
were more active than sodium stibogluconate. In aqueous solution, AmB
exists as a mixture of different species in equilibrium: monomers and
soluble and unsoluble aggregates (17). Under the conditions
of the in vitro experiments, for concentrations of AmB below 1 µM,
both formulations were mainly in the monomeric form (12),
and this could give an explanation for their similar activities.
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Copyright © 1999, American Society for Microbiology. All rights reserved.
Activity of a Heat-Induced Reformulation of
Amphotericin B Deoxycholate (Fungizone) against Leishmania
donovani
ABSTRACT
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TABLE 1.
In vitro activities of Pentostam and unheated and heated
Fungizone against L. donovani MHOM/ET/67/L82 in mouse
peritoneal macrophages
For in vivo assays, 8- to 10-week-old female BALB/c mice (weight, 20 g) were infected i.v. with 1.5 × 107 L. donovani L82 amastigotes and were randomly sorted into groups of five mice. At 7 days postinfection, one mouse was killed to check for the patency of infection and drug administration commenced. Sodium stibogluconate was administered subcutaneously for 5 consecutive days. Both Fungizone formulations were administered i.v. for 3 days (dosed on alternate days): 0.04 and 0.2 mg/kg of body weight for the unheated Fungizone formulation and 0.04, 0.2, and 1 mg/kg for the heated Fungizone formulation. At 1 mg/kg, unheated Fungizone was fatal to the mice, so a reduced dose of 0.5 mg/kg was administered daily. The maximum tolerated dosage of heated Fungizone was 2.5 mg/kg per day. All mice were killed on day 14 postinfection. Their livers were weighed, and impression smears were made, fixed with 100% methanol, and stained with Giemsa stain. Parasite numbers were determined by counting the number of amastigotes per 500 nuclei and multiplying that value by the weight of the liver (in milligrams). The ED50s were also determined by linear regression analysis. In a second experiment a higher inoculum of amastigotes, 2 × 107/mouse, was used.
The acute toxicity of unheated Fungizone prevented a direct comparison
with the heat-treated formulation. Unheated Fungizone was toxic at 1 mg/kg, while it was possible to inject safely 2.5 mg of the heated
formulation per kg. This reduction in toxicity has already been
demonstrated for healthy mice or for mice with candidiasis or
cryptococcosis (19). Possible explanations for this are the
physicochemical properties of the heat-induced superaggregates (11). Both experiments demonstrated that heat-treated
Fungizone had an approximately twofold increased antileishmanial
activity over that of the untreated formulation (Table
2). The elevated ED50s in
experiment 2 reflect the higher level of infection in the mice due to
the larger parasite inoculum given in this assay. Both Fungizone
formulations were 20- to 60-fold more active than sodium
stibogluconate. These formulations were administered by the intravenous
route and were then passively transferred to the liver. In general,
relatively large (diameter, >0.1 µm) structures are cleared from the
blood by the mononuclear phagocyte system (16). The large
size of the heated Fungizone aggregates (600 nm) perhaps allows them to
be efficiently captured by the mononuclear phagocyte system and to be
transferred to the site of infection with amastigotes. This formulation
could also act as a reservoir for monomeric AmB.
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The pharmacokinetics and activity of heated Fungizone remain to be elucidated, but this study suggests that the heat treatment of Fungizone could provide a simple and inexpensive way to increase the therapeutic index of this formulation for the treatment of visceral leishmaniasis.
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ACKNOWLEDGMENTS |
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V. Yardley and S. L. Croft received financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR). C. Petit received financial support from the Fondation pour la Recherche Médicale.
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FOOTNOTES |
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* Corresponding author. Present address: Groupe de Recherche en Thérapeutique Anticancéreuse (CNRS ESA 6027), Université Rennes 1, Faculté de Médecine, 2 Avenue du Professeur Léon Bernard 35043 Rennes cedex, France. Phone: (2) 99 33 62 69. Fax: (2) 99 33 68 99. E-mail: Francois.Gaboriau{at}univ-rennes1.fr.
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Antimicrobial Agents and Chemotherapy, February 1999, p. 390-392, Vol. 43, No. 2
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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