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Antimicrobial Agents and Chemotherapy, September 1998, p. 2431-2433, Vol. 42, No. 9
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Superior Efficacy of Liposomal Amphotericin B with
Prolonged Circulation in Blood in the Treatment of Severe
Candidiasis in Leukopenic Mice
Els W. M.
van
Etten,*
Susan V.
Snijders,
Wim
van Vianen, and
Irma
A. J. M.
Bakker-Woudenberg
Department of Medical Microbiology and
Infectious Diseases, Erasmus University Rotterdam, 3000 DR
Rotterdam, The Netherlands
Received 26 January 1998/Returned for modification 17 March
1998/Accepted 10 June 1998
 |
ABSTRACT |
In leukopenic mice with severe systemic candidiasis, single-dose
treatment (5 mg of amphotericin B [AMB]/kg of body weight) with
long-circulating polyethylene glycol-coated AMB liposomes (PEG-AMB-LIP) resulted in zero mortality and a significant
reduction in the number of viable Candida albicans in the
kidney, whereas 70% mortality was seen in mice treated with
five daily doses of AmBisome (5 mg of AMB/kg · day). When the
first of five daily doses of AmBisome was combined with a single low
dose of Fungizone (0.1 mg of AMB/kg), the efficacy was equal to that of
PEG-AMB-LIP.
| |
TEXT |
Fungizone (amphotericin B [AMB]
deoxycholate) remains the therapy of choice for most invasive fungal
infections, but the use of this drug is significantly limited by its
toxic side effects. Lipid formulations of AMB have been developed
by the pharmaceutical industry, with the primary aim of a reduction of
AMB's toxicity. Strikingly, it is not well recognized that the
reduction of AMB's toxicity following lipid formulation seems to be
associated with a substantial reduction of the drug's direct
antifungal activity. It has been previously reported that the in
vitro activity of AmBisome during short-term exposure of Candida
albicans was significantly less than that of Fungizone (4, 5,
7). Furthermore, various in vivo studies have clearly
demonstrated that high dosages of AMB-lipid formulations are often
needed for a treatment to be effective (3, 5, 7).
Recently, we were able to show that it is now possible, by using a
lipid formulation, to substantially reduce the toxicity of AMB without
reducing its direct antifungal activity (7). A new type of
liposomal AMB in which AMB is complexed to a hydrophilic phospholipid
derivative of polyethylene glycol (PEG-AMB-LIP) was prepared in our
laboratory. The PEG-AMB-LIP formulation shows three characteristics
that are expected to be important for improved antifungal efficacy
(6, 7): low toxicity, high direct antifungal activity, and
prolonged circulation time of intact liposomes in blood. Prolonged
blood residence of intact liposomes may be important for increased
accumulation of liposomal AMB at sites of fungal infection outside
the mononuclear phagocyte system, such as the kidney and lung (1,
2).
In a previous study with our model of severe invasive C. albicans infection, it was shown that treatment with a single dose of PEG-AMB-LIP (5 mg of AMB/kg of body weight) resulted in decreasing numbers of viable C. albicans in the kidney within a
short period of time after inoculation with this organism. This effect
could not be achieved with AmBisome at the same dosage; however, a much higher dosage of AmBisome (29 mg of AMB/kg) was as effective as PEG-AMB-LIP. In the present study, the significance of the high direct
antifungal activity of PEG-AMB-LIP for the treatment of acute severe
systemic candidiasis is further explored. The effect of early or
delayed treatment with a single dose of PEG-AMB-LIP was compared with
that of single- or multidose treatment with AmBisome. Furthermore, the
potential benefit of adding a single low dose of Fungizone to the first
dose of AmBisome was investigated.
Sabouraud dextrose agar was from Unipath Ltd. (Basingstoke, England).
AMB and Fungizone were kindly provided by Bristol Myers-Squibb (Woerden, The Netherlands). AmBisome was from NeXstar
Pharmaceuticals, Inc. (San Dimas, Calif.). Hydrogenated
soybean phosphatidylcholine (HSPC) and a polyethylene glycol 1900 derivative of distearoylphosphatidylethanolamine (PEG-DSPE)
were obtained from Avanti Polar Lipids, Inc. (Alabaster, Ala.).
Dimethyl sulfoxide was from Janssen Chimica (Tilburg, The Netherlands). Cyclophosphamide and cholesterol (Chol) were from Sigma (St. Louis, Mo.). Chloroform and methanol were from Merck (Darmstadt, Germany). C. albicans ATCC 44858 was used
(5). Specific-pathogen-free 12- to 20-week-old female
BALB/c mice were obtained from Iffa Credo (L'Arbresle, France).
PEG-DSPE-HSPC-Chol-AMB in a molar ratio of
0.21:1.79:1:0.32 (PEG-AMB-LIP) and placebo liposomes (devoid
of AMB) were prepared as described previously (6, 7).
AmBisome, consisting of HSPC-Chol-distearoylphosphatidylglycerol-AMB in a molar ratio of
2:1:0.8:0.4, was provided as a lyophilized preparation. The powder was
reconstituted according to the manufacturer's instructions.
Leukopenic mice were infected with C. albicans as
previously described (7). The treatment regimens studied
were as follows: (i) single-dose PEG-AMB-LIP, (ii) single- or
multidose AmBisome, (iii) multidose AmBisome combined with a single
dose of Fungizone, and (iv) single-dose Fungizone.
(i) Single-dose PEG-AMB-LIP.
PEG-AMB-LIP was administered
intravenously (i.v.) as a single dose of the maximum tolerated
dosage of 5 mg of AMB/kg (7). Treatment was started at
either 6, 16, or 20 h after C. albicans inoculation.
(ii) Single- or multidose AmBisome.
AmBisome was administered
i.v. either as a single dose of 5 mg of AMB/kg or daily at 5 mg
of AMB/kg · day for three or five consecutive days.
Treatment was started at either 6, 16, or 20 h after
C. albicans inoculation.
(iii) Multidose AmBisome combined with a single dose of
Fungizone.
The first of five daily doses of AmBisome (5 mg of
AMB/kg · day) was administered at 20 h after inoculation
with C. albicans and combined with a single i.v. dose
of the maximum tolerated dosage (0.1 mg of AMB/kg) of Fungizone
(5). The two agents were administered separately, with the
first dose of AmBisome being given directly after Fungizone.
(iv) Single-dose Fungizone.
Fungizone, at 0.1 mg of AMB/kg,
was administered i.v. 20 h after C. albicans
inoculation.
The efficacy of treatment was assessed as previously described
(7). In the present study, only the numbers of viable
C. albicans in the kidneys were determined, since it
was previously shown (5, 7) that in this animal model the
kidney is the most severely infected organ. Differences in
C. albicans CFU between the various treatment groups
were analyzed by the Mann-Whitney test.
By increasing the delay of the start of treatment, the efficacy of
treatment in relation to the severity of infection could
be
investigated. This was reflected in increasing numbers
(
P 
0.01) of viable
C. albicans in
the kidneys at the start of treatment
(Table
1). The effect of early or delayed
treatment with a single
dose of PEG-AMB-LIP versus single- or
multidose AmBisome treatment
is presented in Table
1. When
AmBisome treatment was started
6 h after
C. albicans inoculation, five daily doses (5 mg/kg
·
day) were needed to significantly reduce the number of viable
C. albicans at 7 days after inoculation compared to the
number
at the start of treatment. The rate of response to this therapy
was, thus, relatively low.
View this table:
[in this window]
[in a new window]
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TABLE 1.
Effect of early or delayed treatment with a single dose
of PEG-AMB-LIP versus single- or multidose AmBisome on survival of
leukopenic micea and growth of C. albicans in the kidney
|
|
In this model of acute severe systemic candidiasis, in which untreated
animals start to die 24 h after
C. albicans
inoculation,
the efficacy of a 5-day AmBisome treatment regimen after a
delay
in the start of treatment was questioned. When treatment was
delayed
until 16 h after inoculation, 10% of the animals died
after five
daily doses of AmBisome. The further delay in treatment to
20
h after inoculation resulted in an increase in mortality to
70%.
In sharp contrast, a single dose of PEG-AMB-LIP administered at
20 h after inoculation still resulted in zero mortality and a
significant reduction in the number of viable
C. albicans in the
kidney. The present study shows that the high
efficacy observed
for PEG-AMB-LIP at a single-dose administration could
not be matched
by a multidose regimen of AmBisome, although the
latter results
in prolonged blood residence. Probably the high
direct antifungal
activity of AMB is of major importance in the
successful treatment
of severely infected animals with PEG-AMB-LIP:
treatment with
PEG-AMB-LIP resulted in a rapid response in terms
of decreasing
numbers of viable
C. albicans in the
kidney, whereas AmBisome
treatment did not.
Apparently, AMB in PEG-AMB-LIP is readily available for immediate
antifungal activity. To gain more insight into the mechanism
by which
PEG-AMB-LIP exerts its rapid antifungal effect, factors
important for
the specific transfer of AMB from PEG-AMB-LIP to
fungal cells are
presently under investigation. The possibility
of a slight release of
AMB from PEG-AMB-LIP in the blood after
i.v. administration cannot be
excluded, although it has been previously
shown that during circulation
AMB is tightly associated with the
liposomes in PEG-AMB-LIP
(
6).
The second part of the study involved investigating whether a lack of
direct availability of AMB in AmBisome might be responsible
for the low
efficacy of that drug in severely infected mice. At
20 h after
C. albicans inoculation, the first of five daily doses
of AmBisome was combined with a single low dose of Fungizone (0.1
mg of
AMB/kg) (Table
2). Strikingly, the
efficacy of this combined
treatment was equal to that of PEG-AMB-LIP
(Tables
1 and
2).
After receiving a single low dose of Fungizone alone,
the animals
died (Table
2). The observation that in severe invasive
fungal
infection the efficacy of treatment with AmBisome can be greatly
improved by addition of a single low dose of Fungizone in the
early
phase of treatment may be of great clinical significance.
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Effect of multidose AmBisome treatment with or
without a single dose of Fungizone on survival of leukopenic
micea and growth of C. albicans in the kidney
|
|
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Medical Microbiology and Infectious Diseases, Erasmus University
Rotterdam, P. O. Box 1738, 3000 DR Rotterdam, The Netherlands.
Phone: 31-10-4088068. Fax: 31-10-4364730. E-mail:
vanetten{at}kmic.fgg.eur.nl.
| |
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Antimicrobial Agents and Chemotherapy, September 1998, p. 2431-2433, Vol. 42, No. 9
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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