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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, December 1999, p. 2841-2847, Vol. 43, No. 12
Division of Infectious
Diseases1 and Clinical
Pharmacology,3 Department of Medicine, Albany
Medical College, Albany, New York 12208, and Wadsworth Center,
New York State Department of Health, Albany, New York
122012
Received 10 July 1998/Returned for modification 13 December
1998/Accepted 8 September 1999
The interaction between fluconazole (Flu) and amphotericin B (AmB)
was evaluated in a murine model of systemic candidiasis for one
Flu-susceptible strain (MIC, 0.5 µg/ml), two strains with intermediate Flu resistance (Flu mid-resistant strains) (MIC, 64 and
128 µg/ml), and one highly Flu-resistant strain (MIC, 512 µg/ml) of
Candida albicans. Differences in fungal densities in kidneys of infected mice after 24 h of therapy and in survival rates at 62 days of mice treated with an antifungal drug or a combination of antifungal drugs for 4 days were compared. For the
Flu-susceptible and Flu mid-resistant strains, the combination of Flu
and AmB was antagonistic, as shown by both quantitative culture results
and survival. The interaction was additive for the highly Flu-resistant
strain. These results suggest that the combination of Flu and AmB
should be used with caution in infections due to fungi that are usually
susceptible to both antifungal agents and as empirical antifungal drug therapy.
With advances in the treatment of
oncologic malignancies with high-dose antineoplastic chemotherapy and
bone marrow transplantation and improvements in medicine's ability to
support critically ill patients in intensive care units, the incidence
of deep-seated fungal infections is rising (6). Recently, it
was reported that 10% of all nosocomial bloodstream infections were
due to fungi, particularly Candida albicans (6).
In a matched, case-controlled study, Wey et al. (24)
reported that the attributable mortality due to systemic fungal disease
is 38%, despite antifungal drug therapy.
Amphotericin B (AmB) has traditionally been considered the cornerstone
of therapy for deep-seated fungal infections and fungemia. Recently two
blinded, multicenter, randomized controlled trials suggested that
fluconazole (Flu) is as efficacious as AmB in the treatment of C. albicans fungemia in the neutropenic and nonneutropenic host
(3, 20). However, in these studies, treatment failure was
seen in as many as 30% of patients (3).
Because of this high failure rate, there is much interest in using Flu
and AmB as combination therapy in an attempt to improve the outcome.
However, the interaction between Flu and AmB remains poorly defined. In
vitro studies using the checkerboard method and flow cytometry show
that AmB and Flu are antagonistic for C. albicans (4,
16, 18). In contrast, in vivo models of C. albicans
endocarditis in rabbits (22) and systemic candidiasis in
mice (22, 23) reveal additivity. However, the in vivo models were not optimally designed to detect antagonism if it existed.
Yet the characterization of the interaction between Flu and AmB is of
utmost importance. If antagonism is not seen in vivo, this combination
may have practical utility as empirical antifungal therapy in the
febrile, neutropenic patient. Infections due to fungal species that are
resistant to either one of these drugs are being documented for these
immunocompromised patients with greater frequency. Also, the use of
combination therapy may decrease the development of Flu or AmB
resistance by Candida isolates, a problem that may
complicate the treatment of mucosal Candida infections in
the human immunodeficiency virus-infected patient (19, 21).
Furthermore, if the combination is synergistic, it is possible that the
dose of AmB used in combination therapy may be less than the doses used
with AmB monotherapy, thereby decreasing the AmB-related toxicity
experienced by the patient without compromising treatment efficacy.
In the present study we evaluated the interaction between AmB and Flu
in mice infected with one Flu-susceptible strain (MIC, 0.5 µg/ml),
two strains with intermediate Flu resistance (Flu mid-resistant
strains; MICs, 64 and 128 µg/ml), and one highly Flu-resistant strain
(MIC, 512 µg/ml) of C. albicans. For the two Flu
mid-resistant isolates and one highly Flu-resistant isolate, we also
conducted dose range studies with Flu monotherapy to determine the
highest dose that was associated with no efficacy and correlated these
findings with the MIC breakpoint for Flu that was recently established
by the National Committee for Clinical Laboratory Standards
(17). The pharmacodynamic variable for Flu that best correlates with outcome is the ratio of the area under the
concentration-time curve (AUC) to the MIC (12). Thus, we
used doses of Flu that resulted in AUCs over 24 h that mimicked
the 24-h AUCs measured in humans who were given 100, 200, 400, and 800 mg of Flu per day (10, 13). Since the pharmacodynamic
parameter that predicts the efficacy of AmB is unknown, we used a dose
in mice which resulted in serum peak and trough concentrations and 24-h
AUC values similar to those seen in the serum of humans treated with
0.6 mg of AmB/kg of body weight per day (1). This dose of
AmB is commonly used to treat systemic C. albicans
infections in humans.
C. albicans isolates.
C. albicans ATCC
36082 was purchased from the American Type Culture Collection
(Manassas, Va.). C. albicans 208 and Y-12-99 were gifts from
L. Steele Moore (Christiana Care Health System, Wilmington, Del.), and
strain B59630 was a gift from F. Odds (Janssen Research Foundation,
Beerse, Belgium). The MICs for Flu and AmB were determined on eight
separate occasions by the broth macrodilution method described by the
National Committee for Clinical Laboratory Standards (17).
The median MIC of Flu after 48 h of incubation was 0.5 µg/ml
(range: 0.25 to 0.5 µg/ml) for C. albicans ATCC 36082 (a
Flu-susceptible strain). The median MICs were 64 µg/ml (range: 64 to
128 µg/ml) and 128 µg/ml (range: 64 to 128 µg/ml) for strains 208 and Y-12-99, respectively (both designated Flu mid-resistant strains).
For strain B59630 the median MIC was 512 µg/ml (range: 256 to 1,024 µg/ml; designated a highly Flu-resistant strain). The median MICs for
AmB ranged from 0.125 to 0.25 µg/ml for the various fungal strains.
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Interaction between Fluconazole and Amphotericin B
in Mice with Systemic Infection Due to Fluconazole-Susceptible or
-Resistant Strains of Candida albicans
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Antifungal agents.
Flu powder was supplied by Pfizer Inc.
(New York, N.Y.). AmB-desoxycholate power (Adria Laboratories,
Columbus, Ohio) was purchased from the hospital pharmacy. Flu was
dissolved in sterile, pyrogen-free saline to a stock solution of 4 mg/ml, aliquoted, and stored at 
70°C. For each study, the drug was
thawed and further diluted to the desired concentration(s) with
sterile, pyrogen-free normal saline. AmB was dissolved in sterile water
to the desired concentration and was used immediately.
Animals. Female NYLAR mice (weight 18 to 20 g) were raised at the Animal Research Facility of the Wadsworth Center for Laboratories and Research (Griffin Laboratories, Guilderland, N.Y.). These outbred Swiss mice were housed in plastic boxes at four or five animals per container. They received food and water ad libitum. All animal experimentation procedures were approved by and conducted in accordance with the guidelines of the Institutional Animal Care and Use Committees of the New York State Department of Health, Albany, N.Y., and Albany Medical College.
Dose range pharmacokinetics of fluconazole in infected mice.
Dose range studies were conducted to determine the pharmacokinetics of
Flu and AmB when each drug was administered intraperitoneally (i.p.) as
a single dose. The pharmacokinetic studies with Flu were conducted to
determine the doses to give to mice that resulted in 24-h AUCs in serum
that were similar to the 24-h AUCs that are measured in humans who are
given 100, 200, 400, and 800 mg of Flu per day. Dose range AmB studies
were conducted to determine the dose of AmB that would result in serum
peak and trough concentrations and AUCs similar to those measured in
humans who are given 0.6 mg of AmB/kg per day (1). NYLAR
mice were intravenously infected with 3 × 105 CFU of
blastoconidia of C. albicans ATCC 36082 via a lateral tail
vein. The organism was administered in 0.2 ml of sterile saline. Five
hours later, mice were injected i.p. with one of various doses of Flu
or AmB in 0.2 ml of saline or water, respectively. The doses of Flu
examined were 0, 1, 5, 25, 50, 75, 100, 150, 200, and 250 mg/kg. AmB
doses of 0.5 and 1.0 mg/kg were evaluated. Three or four animals from
each group were humanely sacrificed by CO2 asphyxiation
0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12, 16, and 24 h after
drug administration. Blood was collected by cardiac puncture and
allowed to clot on ice. The serum was separated from the clot by
centrifugation and stored at 70°C.
Comparison of AmB and Flu serum concentrations in animals treated
with AmB, Flu, and AmB in combination with Flu.
To define the
effect of AmB on the concentration of Flu in serum, we compared levels
of Flu and/or AmB in serum from infected animals that received
once-daily doses of Flu, AmB, or Flu in combination with AmB on the
second and fourth days of treatment. The doses of Flu and AmB examined
were determined after analysis of the pharmacokinetic studies described
above. Serum was collected from euthanized animals at 1, 4, and 23.45 h
after the drug administration and stored at 70°C until assayed.
Antifungal drug assays. The concentration of Flu in each serum sample was determined by a well diffusion microbiological assay developed by Jorgensen et al. (11) with modifications described by Madu et al. (15). Candida pseudotropicalis (ATCC 46764) was used as the assay organism. Pour plates of the fungus were prepared with molten SAAMF (synthetic amino acid medium fungal) agar and allowed to solidify at room temperature. Four-millimeter-diameter wells were made in the agar. Twenty-microliter aliquots of serum collected from mice or standards were pipetted into wells and kept at 4°C for 1 h and then incubated overnight for 16 h at 30°C in an ambient air incubator. The diameters of inhibition for each serum sample and standards were measured with a vernier caliper to the nearest 0.1 mm. Antifungal drug concentrations in serum samples were calculated with the curves derived from Flu standards. The standard curve was linear for concentrations of Flu between 0.5 and 100 µg/ml of serum. For serum samples that resulted in diameters of inhibition that were greater than those associated with the linear portion of the standard curve, the serum samples were diluted 1:4 with saline and retested. The calculation of the concentration of the drug in serum accounted for this. The intraday and interday coefficients of variation of the microbiological assay were 4.9 and 6.8%, respectively.
Concentrations of AmB in serum were determined by a microbiological assay described by Bannatyne et al. (5) and Granich et al. (9) with modifications. Paecilomyces variotii (ATCC 22319) was used as the assay organism. The fungus was grown on Sabouraud dextrose agar slants for 5 to 7 days at 35°C. Mature spores were harvested with sterile cotton-tipped applicators and placed in normal saline. The total concentration of spores was determined by hemocytometry. Spores were added to molten SAAMF agar to a final concentration of 106 spores/ml. Pour plates of the fungal spores were made and allowed to solidify at room temperature. Ten-millimeter-diameter wells were made in the agar, and 100 µl of serum or standards was pipetted into wells. After incubation for 24 h at 35°C, the diameters of zones of inhibited growth were measured to the nearest 0.1 mm with a vernier caliper. Antifungal drug concentrations in samples were calculated with the curves derived from AmB standards. The standard curve was linear from concentrations of 0.1 to 20 µg/ml. The intraday and interday coefficients of variation of the microbiological assay were 4.3 and 6.2%, respectively. For animals that received AmB and Flu in combination, AmB concentrations in serum were measured with a Flu-resistant C. albicans strain (B59630; gift from F. Odds) in the biological assay. Otherwise the bioassay was conducted as described above. Initial studies demonstrated that serum that contained 0.1 to 4.0 µg of AmB/ml together with 1 to 150 µg of Flu/ml produced zones of growth inhibition whose diameters were the same as those of zones produced by serum containing only AmB. The lower limit of sensitivity of the assay was 0.125 µg/ml. Flu concentrations in sera of animals that received AmB plus Flu were determined by a high-pressure liquid chromatography (HPLC) method described elsewhere (15). Previously, we demonstrated that Flu concentrations measured by the bioassay and HPLC were equivalent (15). The intraday and interday coefficients of variation of the HPLC at 1 µg/ml were 4.5 and 5.6%, respectively.Pharmacokinetic analysis. Pharmacokinetic analysis of the serum samples for Flu and AmB concentration-time relationships were performed with the nonlinear least-squares regression program RSTRIP II (Micromath Scientific Software, Salt Lake City, Utah). The most appropriate pharmacokinetic models were determined by using model selection criteria based on a modified form of Akaike's information criterion (2). Cmax and Cmin (i.e., trough) were defined as the highest and lowest concentrations, respectively, of drug measured in serum after the drug was administered. To determine the AUC in serum, the trapezoidal method was used for the data obtained from time zero to the last time point.
Infection model for evaluating the interaction between Flu and AmB. NYLAR mice were infected intravenously with 3 × 105 CFU (C. albicans ATCC 36082) or 105 CFU (all other fungal strains) of yeast. With these inocula, mice succumbed of their infections within 2 to 8 days of fungal injection. The blastoconidia were administered via a lateral tail vein in 0.2 ml of pyrogen-free saline.
For the studies that used C. albicans ATCC 36082, infected mice were randomly divided into four groups. Each group consisted of 29 to 31 animals. Group I received Flu once per day at a dose that resulted in a serum 24-h AUC that was equivalent to the 24-h AUC measured in humans that received 400 mg of Flu per day (10). The drug was given 5, 30, 54, and 78 h after fungal inoculation. Group II received AmB at 0.5 mg/kg every 24 h for four doses beginning 5 h after fungal inoculation. Group III was given Flu in combination with AmB at the doses and dosing schedules indicated for the individual drugs. For this group, AmB was given 1 h before Flu. Group IV received saline once every 24 h and served as untreated controls. The animals were observed for 62 days. Preliminary studies demonstrated that the administration of four daily doses of Flu and AmB, either alone or in combination, was not toxic to healthy mice for a 62-day observation period. Therefore, noninfected treatment controls were not studied in subsequent trials. Mortality was assessed at least twice each day for the duration of the study. Moribund animals (defined as mice that were unable to ambulate or to rise from a supine position) were humanely sacrificed by CO2 asphyxiation followed by induction of bilateral pneumothoraces. Euthanized animals were included in the mortality counts of the following day. Previously, we found that 90% of treated animals that demonstrated either of these conditions died within 24 h (mean: 12.6 h; range: 4 to 34 h) (unpublished results). This study was conducted twice. Quantitative cultures were conducted with kidneys of infected animals after 24 h of treatment. Briefly, seven or eight animals from each of the groups described above were randomly chosen to receive just one dose of Flu and/or one dose of AmB. The dose of Flu used was the dose that resulted in a 24-h AUC in mice that was equivalent to the 24-h AUC measured in the serum of humans who are given 400 mg of Flu per day. AmB at 0.5 mg/kg was used. Both drugs were given i.p. in 0.2 ml of solution 5 to 6 h after fungal injection. These animals were humanely sacrificed 24 h after fungal inoculation. The right kidney was removed aseptically. Each kidney was weighed, homogenized, and serially diluted with saline. Two hundred microliters of each dilution was plated onto potato dextrose agar that was supplemented with 100 IU of penicillin and 100 µg of streptomycin per ml of agar. After 48 h of incubation at 35°C, the colonies were counted and the results for different groups were compared. The cultures reproducibly detected
50 organisms/g of tissue. The studies were
conducted at least twice for each fungal strain.
For the two Flu mid-resistant strains and the one highly Flu-resistant
strain of C. albicans, the protocol described above for
C. albicans ATCC 36082 was used with the following changes. First, the fungal inoculum used was 105 CFU/mouse since
higher inocula resulted in the death of untreated animals between 12 and 24 h after the fungus was administered. Second, additional
treatment groups were studied to evaluate the effect of increasing Flu
doses on the interaction between this azole and AmB at 0.5 mg/kg per
dose. The groups consisted of mice treated with Flu at doses that
resulted in 24-h AUCs that were similar to those measured in humans
given 100, 200, 400, and 800 mg of Flu per day (10, 13) and
each of these Flu dosages in combination with AmB at 0.5 mg/kg/day.
Additional groups included untreated controls and mice treated with AmB
at 0.5 mg/kg/day as monotherapy. Animals that received both antifungal
drugs were given AmB 1 h before Flu was administered. There were
21 to 30 infected mice per group. The survival rate in each group was
evaluated at least twice daily for up to 62 days. Seven or eight mice
from groups that received Flu at a dose resulting in a 24-h AUC
equivalent to the 24-h AUC measured in humans who received 400 mg of
Flu per day, this dose of Flu in combination with AmB, AmB monotherapy, or saline were sacrificed 24 h after fungal inoculation. The
kidneys from these animals were assessed for fungal densities.
To monitor for drug carryover, kidneys were collected from uninfected
mice that were given Flu, AmB, or Flu plus AmB at the doses described
previously. The AmB or Flu or both were administered i.p. approximately
18 h before the mice were euthanized. Kidneys collected from
uninfected mice that received saline served as controls. The kidneys
were weighed and homogenized. One hundred microliters of homogenate was
added to 100 µl of saline containing one of the C. albicans strains used in the in vivo studies. The entire volume of
material was cultured onto drug-free potato dextrose agar plates. The
plates were incubated for 48 h at 35°C, and the colonies were
counted. The study was conducted in duplicate on two separate occasions
for each fungal strain. No differences in counts between cultures
containing homogenates of kidneys collected from saline- and antifungal
drug-treated animals were seen, indicating that drug carryover did not
occur for the doses of the drugs examined (data not shown).
Statistical analysis. Comparisons of colony counts among the different treatment groups were performed by the Kruskal-Wallis test with multiple comparisons followed by Newman-Keuls analysis with the software program True Epistat, version 5.3 (Epistat Services, Richardson, Tex.). A difference was considered statistically significant at P < 0.05. Differences in survival after 62 days of observation were assessed by Kaplan-Meier analysis followed by the Wilcoxon test. A P < 0.05 was considered a statistically significant difference. Correction of P values for multiple comparisons was not done. The interaction between AmB and the various doses of Flu was defined as an enhanced effect if combination therapy resulted in a statistically significant improvement in survival compared with the most active monotherapeutic agent. The combination was antagonistic if it resulted in a statistically significant decrease in survival versus the most active monotherapy. If the combination did not meet either criterion it was deemed additive. For the quantitative culture results, the combination of Flu and AmB was defined as producing enhanced effect if it resulted in a statistically significant decrease in fungal density in kidneys versus the most active monotherapeutic agent. The combination was defined as antagonistic if it resulted in a significant increase in counts versus the most active monotherapy. It was additive if neither criterion was met.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Dose range pharmacokinetic studies in infected mice.
The
pharmacokinetics of Flu were determined in mice that received a single
i.p. injection of drug 5 h after they were inoculated intravenously with C. albicans. The
Cmax was observed 1 h after the drug was
administered. Both the Cmax and AUC increased in proportion to the dose of Flu administered. The
Cmax was described by the linear equation
Cmax = 1.2893 × dose 2.3651, with r2 = 0.996. The AUC was described by
the linear equation: AUC = 3.3271 × dose + 7.4691, with
r2 = 0.998. The pharmacokinetics was best
described by a two-compartment model with a terminal half-life of
3.4 h. The terminal half-life did not change with increasing Flu doses.
|
Effect of AmB on serum Flu concentrations with combination
therapy.
Concentrations of Flu and AmB in serum were measured for
infected mice that were treated for 2 or 4 days with Flu at 25, 50, or
100 mg/kg per day, AmB at 0.5 mg/kg per day, or each of these dosages
of Flu in combination with AmB at 0.5 mg/kg/day. Animals were infected
with one of the four C. albicans strains used in this
project. The purpose of these studies was to determine whether AmB
and/or Flu serum concentrations were altered when these drugs were used
together. Table 1 shows the
concentrations of drugs in mice infected with C. albicans
ATCC 36082 at 1 (peak level in serum), 4, and 23.45 h after the animals
received their second and fourth daily doses of Flu (100 mg/kg) and/or
AmB (0.5 mg/kg). On both days, the serum Flu concentrations for groups
that received Flu alone and together with AmB were similar. Also, the
serum AmB concentrations did not differ between groups treated with AmB
alone and those treated with AmB in combination with Flu. Similar
results were seen in mice that were treated with Flu at 25 or 50 mg/kg/day alone and in combination with AmB (data not shown). Also,
these findings were independent of the strain of C. albicans
used to infect the animals (data not shown).
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Survival of infected animals treated with Flu, AmB, or Flu in combination with AmB. For C. albicans ATCC 36082, a Flu-susceptible strain, the survivals of the various treatment groups are shown in Fig. 2A. The results were similar in two separate trials. Therefore, the results were combined. None of the animals were excluded from analysis. All treatment regimens were better than controls. AmB as monotherapy was the most active regimen. The combination of Flu and AmB was superior to Flu monotherapy (P = 0.000003). However, the combination of Flu and AmB was less active than AmB monotherapy (P < 0.000001). Therefore, this combination was antagonistic.
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0.6; Fig. 2D). This
finding supports the results of the studies using C. albicans 208 and Y-12-99; the interaction between AmB and Flu is
dependent on the doses of Flu used relative to the MIC of Flu for that
organism. Of note, the results of two separate trials were similar.
Therefore, the figure represents the combined outcomes of two trials.
Fungal density in kidneys of mice treated with Flu, AmB, or Flu
plus AmB.
Flu monotherapy was superior to no therapy for the
treatment of systemic fungal infection due to the Flu-susceptible
strain (ATCC 36082). For all three Flu-resistant strains Flu therapy was no better than controls (Table 2).
For the one Flu-susceptible strain and one of the Flu mid-resistant
strains (isolate 208; Flu MIC of 64 µg/ml), the combination of Flu
and AmB demonstrated antagonism relative to AmB monotherapy (Table 2;
P < 0.01). For the two C. albicans strains
for which the Flu MICs were the highest (128 and 512 µg/ml), Flu
monotherapy had little effect in reducing the fungal densities in
kidneys and the efficacies of the AmB-plus-Flu regimens were not
different from that for AmB monotherapy. These results are consistent
with the survival data for each of the strains of C. albicans examined.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In this study we showed that the interaction between Flu and AmB was antagonistic for one Flu-susceptible strain and two Flu mid-resistant (MIC, 64 to 128 µg/ml) strains of C. albicans as determined by both survival and quantitative cultures of the kidneys. The efficacy of Flu plus AmB was similar to that of AmB monotherapy for the highly Flu-resistant isolate (MIC, 512 µg/ml). The results of the present study confirmed our observation, in a rabbit model of aortic valve endocarditis and pyelonephritis, that the combination of Flu and AmB is antagonistic (14). Furthermore, the present study demonstrates that the degree of antagonism between Flu and AmB that is observed is dependent upon the dose of Flu used relative to the Flu MIC of the fungal isolate.
AmB is the more active of the two antifungal agents studied. Based on the known mechanisms of action of Flu and AmB, it is reasonable to expect that the interaction between these drugs would be antagonistic. AmB exerts its fungicidal effect by binding to ergosterol, a lipid constituent of the fungal membrane (7). Ergosterol-bound AmB aggregates to form channels across the fungal membrane through which essential nutrients and electrolytes exit from the fungus, resulting in the death of the organism (7). Flu, an alpha-14 demethylase inhibitor, incompletely inhibits the production of ergosterol by certain fungal species, including C. albicans, and is fungistatic (8, 10). Since this azole decreases ergosterol production, less targets are available in the fungal membrane for AmB to interact with, thus decreasing the activity of the more active of the two agents.
Most in vitro studies report antagonism between Flu and AmB (4, 16, 18). In contrast, on the basis of in vivo studies with a non-neutropenic-mouse model of systemic candidiasis, Sugar et al. (23) reported additivity between these drugs. However, these in vivo studies were not optimally designed to identify an antagonistic interaction since the 100% survival associated with AmB monotherapy places the outcome at the top of the dose-response curve. This makes it extremely difficult to identify antagonism between Flu and AmB unless Flu completely abolishes the activity of AmB.
On the basis of a neutropenic-mouse model of systemic candidiasis, Sugar et al. (23) reported a "positive effect" between Flu and AmB. In this model, Flu and Flu-plus-AmB recipients received Flu beginning 1 h after fungal inoculation. However, AmB was initiated 2 days after fungal injection in the AmB-plus-Flu group and AmB monotherapy group. If one excludes the deaths that occurred before AmB therapy was initiated in the latter group, the survival rates seen in the Flu-plus-AmB and AmB groups would be similar. Of note, in another trial (23) these investigators reported a 40% reduction in survival rates, from 62.5% for AmB alone to 37.5% for Flu plus AmB. The difference was not statistically significant. However, the number of animals in each group was small.
Sanati et al. (22) evaluated the interaction between Flu and AmB in a neutropenic-mouse model of systemic candidiasis. Eight days after fungal inoculation the survival rates were approximately 15, 43, 60, and 72% for mice that received placebo, Flu, Flu plus AmB, and AmB, respectively. Although differences in survival between groups did not reach statistical significance, the investigators could not discount the possibility that their study lacked sufficient power to identify antagonism (22).
In a rabbit model of C. albicans endocarditis, Sanati (22) reported that the interaction between Flu and AmB was indifferent. In contrast, using the same fungal strain, Louie et al. (14) noted the combination of Flu and AmB to be antagonistic. Both investigators found the fungal densities in cardiac tissues of Flu-plus-AmB recipients to lie between those of the two monotherapies. The discrepancy in results may be explained by the fact that Louie et al. observed a larger difference between the fungal densities in the cardiac vegetations of the Flu and AmB monotherapy groups than that observed by Sanati et al. (a 5-log10 difference versus a 2-log10 difference). Thus, the model of Louie et al. was more sensitive than that of Sanati et al. for identifying statistically significant differences between Flu-plus-AmB and the other treatment groups. Of note, Louie et al. (14) also reported antagonism between Flu and AmB in the clearance of C. albicans from the kidneys of the same infected rabbits. With 5 and 14 days of therapy, the combination of Flu plus AmB was significantly less effective that AmB but more active than Flu. However, by day 21 of therapy Flu, AmB, and Flu plus AmB all sterilized this site. Thus, antagonism in the kidney was manifested by a delay in the sterilization of this organ.
In summary, in the present study we found the interaction between Flu and AmB to be antagonistic in our murine model of systemic candidiasis. This was manifested by a decrease in the clearance of fungi from the kidneys and a worsening of survival with combination therapy relative to the most active regimen, AmB monotherapy. These findings are consistent with the antagonistic interaction that was observed in vitro (4, 16, 18) and in our rabbit model of endocarditis and pyelonephritis (14). However, our study and those of others show that the efficacy of Flu plus AmB is no worse than Flu monotherapy. Thus, for infections in which clinical studies have shown Flu and AmB monotherapies to have equivalent efficacies, such as catheter-related fungemia due to C. albicans (20), outcomes associated with Flu plus AmB, Flu, and AmB should be similar. Of note, in a nonfatal rabbit model of systemic candidiasis, we demonstrated that antagonism between Flu and AmB was manifested as a slower rate of clearance of the fungus from the kidney than that for AmB monotherapy. However, the kidneys of AmB, Flu-plus-AmB, and Flu recipients all were sterilized with 21 days of treatment. Flu resistance in human immunodeficiency virus-infected patients who are receiving long-term Flu for mucocutaneous candidiasis is well documented (19, 21). The results of our nonfatal rabbit model of Candida pyelonephritis (14) suggest that the combination of Flu and AmB should be evaluated in the treatment of non-life-threatening C. albicans infections as a means of treating the infection while, perhaps, reducing the emergence of Flu or AmB resistance during therapy. However, the results of our murine model of fatal systemic candidiasis suggest that this antifungal drug combination should be used with caution in deep-seated fungal infections in which the relative activities of AmB and Flu for the fungus are not known or the activity of AmB for the fungus is greater than that of Flu.
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ACKNOWLEDGMENT |
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This project was supported by an unrestricted educational grant by Pfizer Inc., New York, N.Y.
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FOOTNOTES |
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* Corresponding author. Mailing address: Division of Infectious Diseases, Mail Code-49, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208. Phone: (518) 262-6548. Fax: (518) 262-6727. E-mail: arnold_louie_at_amc01-3{at}ccgateway.amc.edu.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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