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Antimicrobial Agents and Chemotherapy, March 1998, p. 528-533, Vol. 42, No. 3
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Amphotericin B Colloidal Dispersion Combined with
Flucytosine with or without Fluconazole for Treatment of Murine
Cryptococcal Meningitis
DeAnn M.
Diamond,1
Madeline
Bauer,2
Barbra E.
Daniel,3
Mary Ann E.
Leal,1
Debra
Johnson,1
Byron K.
Williams,1
Ann M.
Thomas,4
James C.
Ding,1
Laura
Najvar,5
J. Richard
Graybill,5 and
Robert
A.
Larsen1,*
Departments of Medicine (Infectious
Diseases)1 and
Preventive Medicine
(Biostatistics),2 University of Southern
California, Los Angeles, California;
Preclinical Research
Department, SEQUUS Pharmaceuticals, Inc., Menlo Park,
California3;
Department of Statistics,
University of Northern Colorado, Greeley,
Colorado4; and
Division of Infectious
Diseases, University of Texas Health Sciences Center, San Antonio,
Texas5
Received 24 June 1997/Returned for modification 20 October
1997/Accepted 7 December 1997
 |
ABSTRACT |
Studies with animals and in vitro studies have demonstrated that
flucytosine plus amphotericin B or fluconazole has significantly improved mycologic activity against meningitis caused by
Cryptococcus neoformans compared to the activity of
amphotericin B or fluconazole used alone. However, few doses have been
tested in combination. This study evaluated the antifungal efficacy of
amphotericin B colloidal dispersion (ABCD) combined with flucytosine
with and without fluconazole in a murine model of cryptococcal
meningitis. The following dosages were tested: ABCD at 0 to 12.5 mg/kg
of body weight given intravenously 3 days/week, flucytosine at 0 to 110 mg/kg/day, and fluconazole at 0 to 50 mg/kg/day. Meningitis was
established in male BALB/c mice by intracerebral injection of C. neoformans. Treatment with flucytosine with or without
fluconazole dissolved in the sole source of drinking water was started
on day 2; animals were sacrificed at 16 days, and the numbers of fungal
colonies in the brain were quantified. A survival rate of 100% was
achieved with ABCD plus flucytosine without fluconazole; however, the
addition of fluconazole was required to prevent weight loss
(P < 0.00001) and to achieve the maximum antifungal
effect (P < 0.00001). The only region of dose
combinations for which the 99% confidence intervals were less than 100 CFU/g of brain was defined by ABCD at 5.0 to 7.5 mg/kg combined with
flucytosine at 20 to 60 mg/kg/day and fluconazole at 30 to 40 mg/kg/day. The triple combination of ABCD plus flucytosine and
fluconazole was necessary to achieve the greatest antifungal activity.
| |
INTRODUCTION |
The use of combinations of
antifungal agents has been necessary to achieve the greatest level of
clinical success when treating meningitis caused by Cryptococcus
neoformans. In those clinical studies that have pitted a single
antifungal agent against combination therapy, the combination regimen
has resulted in high rates of success, usually in the range of 55 to
65% for patients with and without human immunodeficiency virus
coinfection compared to rates of success of 35 to 40% when fluconazole
or amphotericin B are used alone (3, 13-15). However, even
these modest improvements in the rates of success are associated with
significant drug toxicity (15, 17, 19). Nevertheless,
treatment with combinations of antifungal agents appears to be
necessary and offers the most immediate opportunity for significant
improvement in outcome. Higher doses of fluconazole alone and in
combination with flucytosine have been evaluated in clinical studies
(4, 17) and in experimental models of cryptococcal
meningitis (1, 8, 12). In vitro studies have evaluated
amphotericin B plus flucytosine (20). However, to date, no
studies have evaluated the potential for improved antifungal activity
by combining these three most commonly used agents, amphotericin B,
fluconazole, and flucytosine, for the treatment of cryptococcal
meningitis. The present study was designed to evaluate the fungicidal
activity of amphotericin B (herein given by the preparation
amphotericin B colloidal dispersion [ABCD]) alone and in combination
with flucytosine with or without fluconazole over a wide dose range in
a murine model of cryptococcal meningitis.
| |
MATERIALS AND METHODS |
Animal protocol.
Pathogen-free BALB/c male mice (age,
approximately 6 weeks; weight, 21 to 26 g) were used in all
experiments. The animals were weighed individually, housed in isolation
cages at four or five mice per cage, and given free access to food and
water. The mice were briefly anesthetized (CO2 narcosis)
and were challenged intracerebrally with approximately 500 CFU of
C. neoformans 1597. The inoculum was delivered in a volume
of 0.06 ml through a 27-gauge needle by direct puncture through the
cranial vault approximately 6 mm posterior to the orbit. The animal
protocol was approved by the University of Southern California
Institutional Animal Care and Use Committee.
Chemotherapy.
The mice were randomly assigned to treatment
groups 2 days after intracerebral challenge, and treatment was
initiated with the assigned concentrations of flucytosine or
fluconazole, or both, dissolved in the sole source of drinking water.
ABCD was administered intravenously via the lateral tail vein starting on day 3 and then thrice weekly for 2 weeks. ABCD was reconstituted in
sterile water and was diluted with sterile 5% dextrose on the day of
administration of the concentration calculated to deliver the assigned
dose in a constant volume of 10 ml/kg of body weight. The water intake
by the mice in each cage was recorded daily. Water containing the
treatment was replaced every 3 to 4 days; the concentrations of
fluconazole and flucytosine were recalculated on the basis of the
weights of the animals in each cage, measured water intake during the
preceding days, and assigned drug doses. Treatment was continued for 14 days. ABCD was tested at a dosage of 0 to 7.5 mg/kg of body weight 3 days/week in combination with flucytosine at 0 to 110 mg/kg/day without
fluconazole. When fluconazole (10 to 50 mg/kg/day) was added,
flucytosine was tested at dosages of 20 to 110 mg/kg/day. In addition,
ABCD was tested alone at 2.5 to 12.5 mg/kg. Four to five animals in one
cage were treated with each dosage combination tested.
Mycologic procedures.
The C. neoformans isolate
(isolate 1597) was obtained from a patient with AIDS-associated
cryptococcal meningitis who responded promptly to treatment with
fluconazole and flucytosine. Two days prior to use, the isolate was
plated onto Sabouraud dextrose agar. Twenty-four hours prior to
infection of the mice, 1 CFU was placed in brain heart infusion broth
and the broth was incubated at 35°C overnight. The organisms were
washed twice with normal pyrogen-free saline before suspension in
saline. The concentration of the organisms injected into the mice was
confirmed by making serial 10-fold dilutions of the initial suspension
and by counting the numbers of CFU on plates prepared from 0.06 ml of
the suspension ejected from the inoculation syringe just before and
just after inoculation of the mice in each cage.
For measurement of the brain fungal burden, the animals were killed and
the brains were removed, weighed, and homogenized in 1.0 ml of normal
saline. Serial dilutions of the whole-brain homogenate were prepared
for quantitative counts of CFU. A 0.01-ml aliquot from each dilution
was plated onto Sabouraud dextrose agar, the agar plate was incubated
at 35°C for 72 to 96 h, and the numbers of CFU were recorded. In
addition, the remaining original whole-brain homogenate was plated on a
large agar plate to assess the sample for low colony counts or
sterility.
Measurements of efficacy.
Treatment activity was determined
by measuring or evaluating the following endpoints: measuring the
survival rate and weight change relative to the initial weight and
evaluating the brain tissue mycologically. Survival was considered in
two forms: first, as the duration of the survival time and, second, as
the proportion of animals in each cage which were alive at the end of
the 16-day experiment. The weight change for each animal was based on
the weight at the time of killing relative to the initial weight
recorded at the time of infection. Groups of four to five control mice were killed on day 2 and at additional time points following infection to estimate the growth curve for the numbers of CFU of C. neoformans. Animals exhibiting signs of distress were killed, and
the day following the date of killing was considered the date of death. Survival times for animals alive on the scheduled day of killing were
censored at that day. Any animal that died prior to the scheduled day
of killing was considered a death, with survival time equal to the day
of death.
Statistical analysis.
The primary objective of this
experiment was to evaluate the dependence of observed measures of
efficacy (survival, weight loss, and fungicidal activity) on the doses
of ABCD alone and in combination with flucytosine or fluconazole, or
both. Loess regression was used to estimate the dose-response surface
for each measure of response (2, 6, 7). Unlike classical least-squares regression methods, loess regression estimates the response to each dose combination by using locally weighted linear or
quadratic regression on the observed responses to nearby dose combinations. Thus, the patterns of association are not forced to be
the same over the entire range of doses. Loess regression is
nonparametric, in that it does not require the specification of an
explicit equation for the association between response and doses. The
loess method also does not require the assumption of normally
distributed errors. Robust resistant iterative methods are used to
minimize the distortion of the estimated surface by unusually large or
small observations (5, 10).
The relative association of potentially explanatory variables with
response was assessed by robust analysis of variance (
7,
10). Pointwise 99% confidence intervals (CIs) for the estimated
response were used to identify regions of dose combinations with
similar levels of response for each measure of efficacy (
2).
For survival and weight change, dose combinations for which the
lower
limits of the 99% CIs were greater than selected values
were
identified. For fungicidal activity, dose combinations for
which the
upper limits of the 99% CIs were less than selected
values of the
numbers of CFU per gram of brain tissue were identified.
The duration of survival for untreated controls was estimated by the
method of Kaplan and Meier (
11). CIs for the proportion
of
mice surviving up to a time point were estimated by Greenwood's
(
9) formula by the method described by Link (
16).
Analysis
of survival for treated animals was based on the proportion of
animals alive at the end of the 14-day treatment period, determined
separately for the animals in each cage. Analysis of fungicidal
activity for treated animals was based on the numbers of CFU per
gram
of brain tissue. Descriptive statistics were based on medians
and
robust 99% CIs (
10). All statistical analyses were
performed
with S-
PLUS statistical software (
21,
22). Because of the
exploratory nature of these analyses, only
P values less than
0.001 were considered significant.
Since no animals were treated with combinations of ABCD plus
fluconazole without flucytosine, these data cannot be used to
test
whether the addition of flucytosine affected the response
but can be
used to test whether the response was associated with
the dosage of
flucytosine in the range evaluated (20 to 110 mg/kg/day)
in the
three-drug combination.
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RESULTS |
Survival and growth curve of C. neoformans for control
mice.
Figure 1 shows the
Kaplan-Meier survival curve for control animals. Median survival was 7 days. The median inoculum for controls was 933 CFU/g of brain tissue
(99% CI; 645 to 1,260 CFU/g). By day 8, the fungal burden had
reached 108 CFU/g of brain tissue. ice tolerated up to
7 days of untreated infection, resulting in 108 CFU/g of
brain tissue, before exhibiting signs of distress or death.
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FIG. 1.
Kaplan-Meier survival ( ) and numbers of CFU of
C. neoformans per gram of brain tissue ( ) by day
postinfection for untreated control animals.
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Survival for treated mice.
The proportion of animals treated
with ABCD alone surviving to the end of treatment increased from 0%
for mice receiving 2.5 mg/kg to 100% for mice receiving 7.5 mg/kg and
then decreased to 50% for mice receiving 12.5 mg/kg (Fig.
2A). Two of the four animals treated with
12.5 mg of ABCD per kg alone died during injection (days 8 and 13, respectively), with >105 and 103 CFU/g being
recovered from the brains, respectively.
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FIG. 2.
Response surfaces showing the loess fit of the
association between the proportion of animals alive at the end of
treatment and dose combinations of ABCD (A), ABCD plus flucytosine (B),
and fluconazole plus flucytosine (C). (A) ABCD alone. (B) ABCD plus
flucytosine without fluconazole. The loess fit used a neighborhood of
75% with a local regression quadratic for ABCD and linear for
flucytosine. The association between ABCD and survival was highly
significant (P < 0.00001); the additional contribution
of flucytosine was not significant (P = 0.42). (C)
Flucytosine plus fluconazole without ABCD. The loess fit used a
neighborhood of 70% with a local regression quadratic for fluconazole
and linear for flucytosine. The association between fluconazole and
survival was highly significant (P < 0.00001);
flucytosine had no additional contribution (P = 0.6).
 , 99% CIs contain 100% survival.
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For animals treated with ABCD plus flucytosine without fluconazole, the
association between survival and dose of ABCD was
highly
significant (
P < 0.00001); the additional contribution
of flucytosine was not significant (
P = 0.4; Fig.
2B).
For animals treated with flucytosine plus fluconazole without ABCD,
100% survival was observed when mice received fluconazole
at
20
mg/kg/day (
P < 0.00001; Fig.
2C), regardless of the
dose
of flucytosine (
P = 0.6). For animals treated with
the three-drug
combination, 100% survived to the end of treatment with
all dose
combinations (data not shown).
Weight change.
Untreated control animals lost 20% of their
initial weight; the median weight change for treated animals ranged
from 
20 to +10%. ABCD alone did not protect animals from weight
loss, regardless of the dose (median weight loss, 20% for mice
receiving 2.5 mg/kg and 10 to 15% for mice receiving 12.5 mg/kg; Fig.
3A). The combination of ABCD plus
flucytosine without fluconazole did not protect animals from
significant weight loss (Fig. 3B). Animals treated with flucytosine plus fluconazole at 10 to 45 mg/kg/day without ABCD maintained their
weight, regardless of the flucytosine dose (Fig. 3C). However, weight
loss was seen for mice receiving fluconazole at dosages of 50 alone and
>40 mg/kg/day in combination with flucytosine at >60 mg/kg/day.
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FIG. 3.
Loess fit of the association between percent weight
change and combinations of doses of ABCD (A), ABCD plus flucytosine
(B), fluconazole plus flucytosine (C), and the three-drug combination
(D). (A) ABCD alone. (B) ABCD plus flucytosine without fluconazole. The
loess fit used a neighborhood of 75% with a local regression quadratic
for ABCD (P = 0.002) and linear for flucytosine
(P = 0.01). (C) Flucytosine plus fluconazole without
ABCD. The loess fit used a neighborhood of 75% with a local regression
quadratic for fluconazole (P < 0.00001) and linear for
flucytosine (P = 0.38). (D) Three-drug combination with
20 to 110 mg of flucytosine per kg per day. The loess fit used a
neighborhood of 50% with a local regression quadratic for fluconazole
(P < 0.00001) and linear for ABCD (P < 0.00001). There was no additional association with the flucytosine
dose over the range tested (P = 0.9). Lower limits of
99% CIs for weight change: >0% ( ) and 2.5 to 0% ().
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For animals treated with the three-drug combination, weight change was
strongly associated with the dosages of both ABCD and
fluconazole
(
P < 0.00001); there was no additional association
with flucytosine in the range of dosages tested (
P = 0.9; Fig.
3D). The region of dosage combinations at which weight was
maintained
included fluconazole at
20 mg/kg/day combined with
flucytosine
without ABCD and fluconazole at
10 mg/kg/day combined
with flucytosine
plus ABCD at 5.0 or 7.5 mg/kg. Again, weight loss was
seen in
mice receiving the highest dosages of the three-drug
combination,
fluconazole at
40 mg/kg/day and ABCD at 7.5 mg/kg,
regardless
of the dose of flucytosine.
Fungicidal activity.
ABCD alone at 10 to 12.5 mg/kg reduced
the numbers of CFU per gram of brain tissue from 108 for
untreated controls to 105 for treated animals (Fig.
4A). The combination of ABCD at 7.5 mg/kg
plus flucytosine at 110 mg/kg/day without fluconazole reduced the
numbers of CFU per gram of brain from 108 to
104 (Fig. 4B; for ABCD, P < 0.00001; for
flucytosine, P < 0.0001). For animals treated with
fluconazole plus flucytosine without ABCD, the numbers of CFU per gram
of brain tissue recovered had a strong association with the fluconazole
dosage (P < 0.00001) and a moderate association with
the dosage of flucytosine (P < 0.01; Fig. 4C).
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FIG. 4.
Loess fit of the association between the numbers of CFU
per gram of brain tissue at end of treatment and combinations of doses
of ABCD (A), ABCD plus flucytosine (B), and fluconazole plus
flucytosine (C). (A) ABCD alone. (B) ABCD plus flucytosine without
fluconazole. The loess fit used a neighborhood of 75% with a local
regression quadratic for ABCD (P < 0.00001) and linear
for flucytosine (P < 0.0001). (C) Flucytosine plus
fluconazole without ABCD. The loess fit used a neighborhood of 70%
with a local regression quadratic for fluconazole (P < 0.00001) and linear for flucytosine (P = 0.006). Upper
limit of 99% CI (CFU per gram of brain tissue): <101
(), 101 to 102 ( ), and 102 to
103 ( ).
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The most potent fungicidal effect was seen in the group of animals
receiving the three-drug combination. The numbers of CFU
recovered per
gram of brain tissue had a strong association with
the dosages of both
ABCD and fluconazole (
P < 0.00001) and a moderate
association with the dosage of flucytosine (
P < 0.01).
The loess
fit of the association between the numbers of CFU per gram of
brain tissue and the dosages of the three-drug combinations are
displayed as slices from the four-dimensional surface, which are
dose-response curves for one drug at selected dosages of the other
two
drugs (Fig.
5). The effect of ABCD at
each dosage of fluconazole
was to decrease the overall level of the
dose-response curve for
flucytosine as the dose of ABCD increased.
However, this reduction
in the numbers of CFU per gram of brain tissue
appeared primarily
in the range of 0 to 5 mg of ABCD per kg, with
little additional
reduction for ABCD at 7.5 mg/kg (Fig.
5, two right
panels in each
row). As the fluconazole dosage increased, the level of
the flucytosine
dose-response curve decreased and the slope of the
curve changed
from negative to positive, so that at fluconazole dosages
of
20
mg/kg/day, the numbers of CFU per gram of brain tissue
increased
slightly with increasing dosages of flucytosine. This
interaction
between fluconazole and flucytosine was seen at all doses
of ABCD,
but it was somewhat more pronounced at ABCD doses of
5.0
mg/kg
(Fig.
5, two right panels in each row). The upper limits of the
99% CIs were not below 100 CFU/g until both ABCD was used at
5.0
mg/kg and fluconazole was used at
30 mg/kg/day (Figure
5; two
right
panels in two top rows).
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FIG. 5.
Flucytosine dose-response curves for selected levels of
ABCD (increasing from 0 mg/kg on the left to 7.5 mg/kg on the right)
and fluconazole (increasing from 0 mg/kg/day in the bottom row to 40 mg/kg/day in the top row). The solid black curves represent the loess
estimate of the dose-response curve; the dots show the upper and lower
limits of the pointwise 99% CIs for the estimated response. The gray
horizontal reference lines are 102 CFU/g.
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The maximum fungicidal effect of the three-drug combination was seen at
dosages of fluconazole of
30 mg/kg/day, flucytosine
in the dosage
range of 20 to 60 mg/kg/day, and ABCD at doses of
5.0 and 7.5 mg/kg,
with the exception that the numbers of CFU
per gram of brain tissue
increased slightly at the highest dosages
of the three drugs. Figure
6 shows the regions of dose combinations
with similar fungicidal activities superimposed on the regions
of the
dose combinations resulting in similar weight loss in the
animals
treated with the three-drug combination. The region of
dose
combinations of fluconazole and flucytosine with the greatest
fungicidal activity and no weight loss were similar when they
were used
in combination with ABCD at both 5.0 and 7.5 mg/kg (Fig.
6A and B,
respectively). The three-drug combination with ABCD
at 2.5 mg/kg and
the drug combination without ABCD (Fig.
6C and
D, respectively) had
similar regions where weight was maintained;
however, the level of
fungicidal activity achieved at the two
higher doses of ABCD was not
achieved with ABCD at 2.5 mg/kg or
in its absence.
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FIG. 6.
Regions with the most effective dose combinations. For
each dose of ABCD tested, the regions of combinations of fluconazole
and flucytosine dosages with similar fungicidal activities are
superimposed on the regions where similar weight loss was found. The
size and locations of the regions for weight loss (indicated by gray
shading) were similar for all doses of ABCD tested in the three-drug
combination, with the exception of the slight increase in weight loss
for ABCD at 7.5 mg/kg with higher doses of fluconazole in combination
with low dosages of flucytosine (A). In contrast, the regions with
fungicidal activity in the range 100 to 1,000 CFU per gram of brain
tissue showed a strong shift toward the inclusion of lower doses of
fluconazole as the dose of ABCD in the three-drug combination
increased, from 0 mg/kg (D) to 7.5 mg/kg (A). However, the maximum
fungicidal effect (less than 100 CFU/g of brain tissue) was achieved
only for the three-drug combination with ABCD 5.0 to 7.5 mg/kg (as
indicated by the presence of regions with dotted lines in panels A and
B and their absence in panels C and D).
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| |
DISCUSSION |
We studied the fungicidal activity of ABCD alone and in
combination with flucytosine or fluconazole, or both, in a murine model
of cryptococcal meningitis. A 100% survival rate was achieved with
ABCD plus flucytosine without fluconazole; however, in the absence of
fluconazole, neither ABCD nor flucytosine, alone or in combination,
prevented weight loss. ABCD alone at 10 to 12.5 mg/kg decreased the
numbers of CFU per gram of brain tissue from 108 to
105 but proved to have lethal toxicity for some animals.
When ABCD was tested in combination with flucytosine without
fluconazole, the numbers of CFU recovered per gram of brain tissue were
104 for the highest dose combination (ABCD at 7.5 mg/kg
plus flucytosine at 110 mg/kg/day). In contrast, when fluconazole was
added, the numbers of CFU recovered per gram of brain tissue were
between 10 and 100 for animals treated with ABCD at 5 or 7.5 mg/kg
combined with fluconazole at 30 to 40 mg/kg/day and flucytosine at 20 to 60 mg/kg/day. For one animal treated with ABCD at 7.5 mg/kg,
fluconazole at 40 mg/kg/day, and flucytosine at 40 mg/kg/day, cultures
of whole-brain samples were sterile.
In our laboratory, murine models of cryptococcal meningitis
consistently demonstrate that flucytosine has increased antifungal activity when it is combined with fluconazole (8, 12). This dramatic effect of fluconazole on the fungicidal activity of
flucytosine, which has also been demonstrated in in vitro studies
(18), has now been demonstrated for fluconazole in
combination with amphotericin B. In these models, we took advantage of
the availability of new statistical methods for estimating and
visualizing the dose-response surfaces for survival, weight loss, and
fungicidal activity (2, 6, 7, 21, 22). By evaluating these
easily determined endpoints over a wide range of dose combinations, we
were able to identify a range of dosages of fluconazole and flucytosine that, in combination with ABCD, define a region of doses which has
promising fungicidal activity, which has a 100% survival rate, and
where weight is maintained. The reproducibility of the results obtained
by these methods is demonstrated by comparing the regions of
fluconazole and flucytosine dosages with the greatest fungicidal activity obtained in this experiment with the regions obtained in
previous experiments (8).
The identification of a region of dose combinations with the most
promising therapeutic effect provides more useful information than
statistical summaries based on statistically significant differences
between specific dose combinations and controls or estimation of
specific dose combinations, e.g., the 50% lethal dose, for guiding
investigators in designing subsequent experiments and clinical trials
(2). Furthermore, these regions can be used to evaluate the
effect of systematic changes in experimental conditions such as
severity of meningitis, host immune status, and the possible need to
alter therapies based on known clinical conditions that affect the
rates of clinical success.
It is widely recognized that animal models are not likely to provide
the precise doses which will produce the optimum therapeutic effect in
the clinic. However, information which is essential for the design of
clinical trials can be determined from the location and size of the
region of dose combinations which provide the best response in the
animal model. For example, in the present model, the region of dose
combinations with the most promising therapeutic effect is defined by
higher doses of fluconazole combined with low to moderate doses of
flucytosine and ABCD rather than the combination of all three drugs at
their respective maximum tolerated doses.
| |
ACKNOWLEDGMENTS |
This work was supported in part by SEQUUS Pharmaceuticals, Inc.,
grants from the National Institute of Allergy and Infectious Diseases
(N01-AI-15082) and the State of California (California Collaborative
Treatment Group), contributions from Robert B. Henry, Jr., and Edwin G. Snyder, and an anonymous grant in honor of the family of Richard Pitts.
We thank Hoi Pham for expertise in database management and Lucia Noll
for expert technical editing.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Medicine (Infectious Diseases), 2020 Zonal Ave., IRD Room 220, University of Southern California, Los Angeles, CA 90033. Phone: (213)
226-7556. Fax: (213) 226-2775.
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
