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Medical Mycology, 2014, 00, 1–12
doi: 10.1093/mmy/myu070
Advance Access Publication Date: 0 2014
Original Paper
Original Paper
Toxicity and efficacy differences between
liposomal amphotericin B formulations in
uninfected and Aspergillus fumigatus
infected mice
J. A. Olson1,J.A.Schwartz
2, D. Hahka1, N. Nguyen1,T.Bunch
3,
G. M. Jensen3and J. P. Adler-Moore1
1Department of Biological Sciences, California State Polytechnic University, Pomona, California, USA,
2Charles River Laboratories, Pathology Associates, Frederick, Maryland, USA and 3Gilead Sciences, Inc.,
San Dimas, California, USA
Received 21 June 2014; Revised 26 August 2014; Accepted 4 October 2014
Abstract
Because of the reduced toxicity associated with liposomal amphotericin B preparations,
different amphotericin B liposome products have been made. In the present study, we
compared the amphotericin B liposomal formulations, AmBisome R
(AmBi) and Lambin R
(Lbn), in uninfected and Aspergillus fumigatus infected mice, using several in vitro and
in vivo toxicity and efficacy assays. The results showed that the formulations were sig-
nificantly different, with Lbn 1.6-fold larger than AmBi. Lbn was also more toxic than
AmBi based on the RBC potassium release assay and intravenous dosing in uninfected
mice given a single 50 mg/kg dose (80% mortality for Lbn vs. 0% for AmBi). Renal tubular
changes after intravenous daily dosing for 14 days were seen in uninfected mice given
5 mg/kg Lbn but not with AmBi. Survival following A. fumigatus challenge was 30% for
10 mg/kg Lbn and 60% for 10 mg/kg AmBi. When the BAL and lungs were collected
24 h after the second treatment, AmBi at 10 or 15 mg/kg or 15 mg/kg Lbn lowered the
BAL fungal burden significantly vs. the controls (P≤0.05), while there was no difference
in lung fungal burden amongst the groups. In contrast, lung histopathology at this same
early timepoint showed that AmBi was associated with markedly fewer fungal elements
and less lung tissue damage than Lbn. In conclusion, given the differences in size, toxic-
ity, and efficacy, AmBi and Lbn were not physically or functionally comparable, and these
differences underscore the need for adequate testing when comparing amphotericin B
liposome formulations.
Key words: aspergillosis, intravenous toxicity, liposomal amphotericin B, nephrotoxicity, RBC toxicity.
Introduction
Liposomes as drug carriers have a long history of being
able to reduce the toxic side effects of anti-cancer drugs
such as doxorubicin [1], daunorubicin [2,3], cytarabine
[4,5], and vincristine [6]. Liposomes and other lipid for-
mulations have also been used to reduce the toxicity of the
antifungal drug amphotericin B. These amphotericin B lipid
C
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2Medical Mycology, 2014, Vol. 00, No. 0
formulations vary widely in their composition. The com-
mercial lipid formulations of amphotericin B used most ex-
tensively include Amphotec R
, Abelcet R
, and AmBisome R
(AmBi) [7,8]. Amphotec is a colloidal disc-like complex
of amphotericin B and cholesterol sulfate [9]. Abelcet
is a ribbon-like complex of amphotericin B, dimyris-
toyl phosphatidylcholine (DMPC), and dimyristoyl phos-
phatidylglycerol (DMPG) [10]. AmBi is a unilamellar li-
posome composed of amphotericin B, hydrogenated soy
phosphatidylcholine (HSPC), cholesterol, and distearoyl
phosphatidylglycerol (DSPG) [11]. FungisomeTM ,whichis
another liposomal amphotericin B product is composed of
amphotericin B, soy phosphatidylcholine, and cholesterol
and is marketed in India. The manufacturing process for
Fungisome results in multilamellar vesicles that require ul-
trasonication for 45 min prior to their infusion in order to
produce the small unilamellar vesicles suitable for therapy
[12].
Given the sensitivity of the manufacturing conditions
for these amphotericin B drug carriers, even formulations
made up of the same lipid components but produced using
different methods, can vary in their physical, pharmacoki-
netic and pharmacodynamic properties [13]. For example, a
liposome formulation of amphotericin B (Anfogen R
) previ-
ously licensed in Argentina, was reported to have the same
chemical composition as AmBisome R
but was manufac-
tured differently and subsequently found to have very dif-
ferent physical properties and was much more toxic than
AmBisome [14].
There have been several published studies on the an-
imal and human pharmacokinetics of some of these am-
photericin B lipid formulations including AmBisome [15–
23], Abelcet [24,25], and Amphotec [26]. What is clear
from these previous amphotericin B lipid publications is
that the presence of infection, the fungal species causing
the infection, the extent of spread of the infection (localized
or disseminated), as well as the immune status of the host
can play a role in determining dose tolerance [20,27–30].
For example, inflammatory cytokines were upregulated in a
disseminated Aspergillus flavus murine model treated with
10 mg/kg AmBi, but no upregulation of the inflammatory
cytokines was detected in uninfected mice similarly treated
[30]. In another study comparing infected and uninfected
mice, 20 mg/kg Abelcet in uninfected mice was associated
with minimal ongoing renal tubular damage and evidence
of tubular repair (regeneration), while in Aspergillus in-
fected mice given the same amount of Abelcet, renal tubu-
lar damage was more severe, characterized by acute tubular
necrosis and accompanied by surrounding interstitial hem-
orrhage. Treatment with 20 mg/kg AmBi in uninfected or
Aspergillus infected animals showed normal renal tubule
morphology [20].
In the present study, we compared two liposomal am-
photericin B formulations, AmBi and Lambin R
(Lbn), hav-
ing similar chemical compositions but manufactured using
different processes. We evaluated their physical properties
as well as their toxicity in uninfected mice and their toxic-
ity, drug distribution, and efficacy in a murine pulmonary
Aspergillus fumigatus model.
Materials and methods
Mice
Female C57Bl/6 mice, 6 weeks old at the start of treat-
ment (Harlan International, Indianapolis, IN), were used
in the toxicity studies of uninfected animals. We selected
this inbred mouse strain because it is highly sensitive to the
toxic effects of amphotericin B [31]. Female Swiss Web-
ster mice, 7 weeks old at the start of the treatment (Harlan
International, Indianapolis, IN), were used for the studies
with Aspergillus fumigatus infected animals since this is an
outbred strain of mice with a heterogeneous genetic back-
ground somewhat more representative of the genetic diver-
sity in the human population. Animals were maintained in
microisolator cages on a standard rodent diet (Teklad Labo-
ratory rodent diet no. 2918 [18% protein]; Harlan/Teklad,
Madison, WI) with water ad libitum. All animal research
procedures were approved by the Institutional Animal Care
and Use Committee of California State Polytechnic Univer-
sity, Pomona.
Test substances
Lyophilized AmBi (AmBisome, Gilead Sciences, Inc. San
Dimas, CA) and lyophilized Lbn (Lambin, Sun Pharmaceu-
ticals Ind. Ltd., Halol, India) were reconstituted according
to the manufacturers’ instructions to provide final concen-
trations of 4 mg/ml of amphotericin B for each prepara-
tion. Lbn was analyzed by HPLC at Gilead Sciences Inc.
and the content of hydrogenated soy phosphatidylcholine,
distearoyl phosphatidyl glycerol, cholesterol, and ampho-
tericin B was consistent with the composition of AmBi (data
not shown). Both AmBi and Lbn were diluted in sterile
5% dextrose (D5W) for intravenous (i.v.) injection. For the
in vitro assay of potassium release from red blood cells
(RBCs), the cells were purchased from Bioreclamation, Inc.
(Hicksville, NY).
Particle size determination
Aliquots from each of four vials from one lot of AmBi
and two vials from one lot of Lbn were analyzed for
volume-weighted median particle size and size distribution
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Olson et al. 3
parameters as determined by controlled reference dynamic
light scattering using a Microtrac ultrafine particle analyzer
(Honeywell, Morris Township, NJ) [32].
In vitro RBC potassium release toxicity assay
The method of Jensen et al. [33] was used for the assay
of potassium release from RBCs. A serial dilution of each
drug was prepared with D5W as the diluent to provide a
range of amphotericin B concentrations from 0.006% (0.24
μg/ml of AmBi or Lbn) to 12.5% (500 μg/ml AmBi or Lbn)
of the original suspension. For each dilution, 50 μlwas
mixed with 450 μl of washed rat RBCs (Bioreclamation
Inc., Hickville, NY), and the mixtures were incubated at
37◦C for 12 h. After incubation, the supernatant from each
assay dilution was collected, diluted four-fold with 0.9%
(w/v) sodium chloride, and measured by a potassium selec-
tive electrode in a Nova-1 clinical analyzer (Nova Biomed-
ical, Waltham, MA). The baseline level (0% release) was
then defined as the amount of potassium released from the
RBCs after incubation with buffer (147 mM NaCl, 3 mM
KCl, 10 mM dibasic sodium phosphate, pH 7.4) (negative
control); 100% release was defined as the amount of potas-
sium released from the RBCs after incubation with 10 μM
valinomycin (CalBiochem, La Jolla, CA) (positive control).
For each agent, the concentration of amphotericin B that
produced 50% release of the potassium from the RBCs (K50
value) was then calculated.
Single- and multiple-dose in vivo toxicity studies
Uninfected (nonimmunosuppressed) C57Bl/6 female mice,
6 weeks old (n=5 per group), were given a single i.v. dose
of 20 or 50 mg/kg of AmBi or Lbn in a volume of 0.07–
0.22 ml. The mice were monitored daily over a period of
14 days for survival, weight gain or loss, grooming, and
activity level (general ambulation, laying down, getting up,
and breathing difficulty). If an animal died prior to day 14,
the weight and disease signs of the mouse on the day of
death was included in the average for that group for the re-
mainder of the study. In another study, uninfected C57Bl/6
female mice, 6 weeks old (n=7 per group), were treated
i.v. every day for 14 days with 5.0, 15, or 25 mg/kg of
AmBi or Lbn. The mice were again monitored as described
above. Blood was collected by cardiac puncture from the
uninfected, multidose treated mice 24 h after the last dose
of the drug, and the sera were analyzed for blood urea ni-
trogen (BUN) levels. Histopathological evaluation was also
done on mouse kidneys collected at the same time point. At
necropsy, kidneys were fixed in 10% neutral buffered for-
malin. Fixed tissues were processed routinely and stained
with hematoxylin and eosin (H&E) for evaluation by a li-
censed Board-certified veterinary pathologist (Diplomat of
the American College of Veterinary Pathologists), (Charles
River Laboratories, Davis, CA). Kidney tissues were exam-
ined for evidence of treatment-related changes, and severity
scores were assigned as follows: minimal (fewer than 25%
of tubules affected), mild (25% to 50% of tubules affected),
moderate (50% to 75% of tubules affected), and severe
(more than 75% of tubules affected).
Efficacy and toxicity testing with Aspergillus-
infected mice
Swiss Webster female mice, 7 weeks old, were immunosup-
pressed intraperitoneally with triamcinolone acetonide at
6 mg/kg (Kenalog-10; Bristol-Myers Squibb Co., Prince-
ton, NJ) on day –3, day 0, and day +2 relative to challenge.
The mice were sedated on day 0 with an intraperitoneal in-
jection of 16 mg/kg of xylazine and 80 mg/kg of ketamine
and challenged intranasally with 5.9 ×106A. fumigatus
conidia as previously described [34]. Drug treatment was
initiated 2 h post-challenge, with five groups of mice (n=
22 per group) receiving 10 or 15 mg/kg of i.v. AmBi or Lbn
in a volume of 0.08–0.11 ml. The control group of mice
was given i.v. D5W. Ten mice in each group were moni-
tored for survival, weight gain or loss, and disease signs for
21 days, with additional treatments every 24 h for a total of
six treatments post-challenge. The clinical signs of infection
were scored daily based on weight loss, the level of groom-
ing, and activity level (general ambulation, laying down,
getting up, and breathing difficulty). If an animal died prior
to day 21, the weight and disease signs of the mouse on
the day of death was included in the average for that group
for the rest of the study. The remaining twelve mice in each
group were given a total of only two treatments, one at
2 h and another at 24 h post-challenge and were killed at
48 h post-challenge for fungal burden determination, tis-
sue drug concentration, and serum BUN analysis (n=7
per group); histopathology was done on the tissues of the
other five mice per group. For fungal burden determina-
tion, the lungs were collected aseptically, weighed, homog-
enized in 1 ml of phosphate-buffered saline (PBS), diluted
in PBS, and 200 μl aliquots of each dilution were plated
in duplicate onto Sabouraud agar plates and the plates in-
cubated at 30◦C for 24 h to determine the number of cfu
per gram of lung tissue [20]. Tissue drug concentration was
determined as previously described [34]. Briefly, a 200 μl
aliquot of each lung homogenate was mixed with 200 μl
methanol, heated in a 65◦C water bath for 10 min, cen-
trifuged at 1000 ×g for 8 min, the supernatant collected,
and analyzed in a bioassay using Candida albicans (limit
of sensitivity =0.125 μg/ml). Blood from these same mice
was taken by cardiac puncture, and their sera were analyzed
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Ta b l e 1 . Median particle diameters and 90% passing diameters as determined by dynamic light scattering for individual vials
of AmBi and Lbn.
Vial Diameter (nm) of particles in AmBi Diameter (nm) of particles in Lbn
Median 90% Passing Median 90% Passing
1 75.9 115.3 121.0 174.9
2 76.9 126.5 123.3 165.6
3 80.9 123.1 - -
4 77.5 123.2 - -
Avg (SD) 77.8 (2.2) 122.0 (4.8) 122.2 (1.6) 170.3 (6.6)
for BUN levels. For histopathological analysis, kidneys and
lungs were collected and processed for histopathology as
described above. Infected kidney tissues were examined for
evidence of treatment-related changes and severity scores of
minimal, mild, moderate, and severe were assigned as de-
scribed above. Scoring of the infected lungs reflected both
the severity and the distribution of changes. The lungs were
assessed for the presence of mixed neutrophilic, histiocytic
alveolar infiltrate (alveolar exudates), neutrophilic exudates
in large airways, tissue necrosis, alveolar hemorrhage and
edema, and vascular invasion.
Statistical analysis
Tissue drug concentration (micrograms per gram) and tis-
sue fungal burdens (cfu per gram) were analyzed by using
GraphPad Prism version 5.0 (GraphPad Software, Inc., San
Diego, CA). A Kruskal–Wallis nonparametric analysis of
variance was applied to compare the control to all groups
in each experiment, and where differences occurred, a two-
tailed Mann–Whitney Utest was used for paired-group
comparisons. Survival curves were compared using the log
rank test. A P-value of ≤0.05 was considered significant.
Results
Particle size determination
The reconstituted materials in each vial were examined for
median particle size and for the upper limit particle diame-
ter of 90% of the particles. The latter measure is referred to
as the 90% passing diameter and is an indicator of the pres-
ence of particles larger than 100 nm in a small, unilamellar
vesicle dispersion [32]. For AmBi, the median particle size
for the four vials examined was 77.8 +/−2.2 nm (SD)
within the validated method precision of 3.5% [32]. For
Lbn, two vials were examined and the median particle size
was 122.2 +/−1.6 nm, which was 1.6 fold larger than the
particle size for AmBi. The 90% passing diameter values
for all four AmBi vials averaged 122.0 +/−4.8 nm, but
the 90% passing diameter of Lbn was 170.3 +/−6.6 nm,
which was 28.4% larger than AmBi, reflective of a substan-
tially shifted size distribution for Lbn indicating the pres-
ence of substantial amounts of particles larger than 100 nm
(Table 1).
In vitro RBC potassium release toxicity assay
The propensity for amphotericin B in each formulation to
partition into the cell membrane of RBCs during incuba-
tion can be used as one measure of toxicity by determining
the concentration of amphotericin B required to achieve
50% potassium release (K50) from washed rat RBCs af-
ter 12 h of incubation [33]. The concentrations leading to
50% potassium release (K50) in this study were 1.4 μg/ml
for Lbn and 7.4 μg/ml for AmBi (Fig. 1). No vial-to-vial
variability was noted for either product in terms of the re-
sulting K50. Fungizone (deoxycholate amphotericin B) is
normally evaluated in this assay after 4 h of incubation and
would be off scale (fully partitioned) with a 12-h incuba-
tion time. The K50 results indicate a substantially greater
propensity for amphotericin B to partition out of Lbn rel-
ative to AmBi, and a prediction of enhanced toxicity on
infusion and/or at the site of accumulation of the liposomes
in vivo.
0.01 0.1 1 10 100 1000
0
20
40
60
80
100
Amphotericin B (µg/ml)
% Potassium Release
AmBi
Lbn
Figure 1. Potassium (K50) Release from rat red blood cells (RBCs) mea-
sured after 12 h of incubation at 37◦C with dilutions of Lbn or AmBi. K50
values: Lbn (1.4 μg/ml); AmBi (7.3 μg/ml).
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Olson et al. 5
0246810121416
0
20
40
60
80
100
20 Lbn
50 Lbn
20 AmBi
50 AmBi
Stud y Day
Percent survival
A
0246810121416
-30
-20
-10
0
10
20
20 Lbn
50 Lbn
20 AmBi
50 AmBi
Study Day
Percent Weight Change
B
Figure 2. Single-dose i.v. toxicity of Lbn or AmBi in uninfected female
C57Bl/6 mice (n=5/group). (A) Survival, 50 mg/kg Lbn vs. 20 or 50 mg/kg
AmBi or 20 mg/kg Lbn (P<0.001). (B) Mean percent weight change,
50 mg/kg Lbn vs. 20 or 50 mg/kg AmBi or 20 mg/kg Lbn (P<0.001).
Uninfected mice: single-dose in vivo toxicity
studies
To determine if the increased toxicity of Lbn observed in
the in vitro K50 RBC assay was also seen in vivo, we tested
the formulations in vivo by intravenously administering sin-
gle increasing doses of AmBi or Lbn. There were no deaths
in the mice given a single dose of 20 mg/kg of AmBi or
Lbn with no weight loss during the 14 days of monitor-
ing (Fig. 2A, B). However, a single dose of 50 mg/kg Lbn
produced severe weight loss and 80% mortality. In com-
parison, all the mice given a single dose of 50 mg/kg AmBi
survived causing only a slight drop in weight the first day
after injection. This in vivo toxicity study confirmed the
toxicity differences in the formulations predicted by the in
vitro RBC assay (Fig. 1).
Uninfected mice: multiple-dose in vivo toxicity
studies
Since the recommended clinical treatment for liposo-
mal amphotericin B is dosing for a period of 2 to 4
weeks, depending upon the patient and the type of fungal
infection [17,35,36], we compared the toxicities of these
two liposome products in a multiple-dose setting in unin-
fected mice. Doses of each drug were selected based on
previous experience with AmBi [14] and the in vivo tox-
icity single dose study reported above. The multiple-dose
testing included 14 daily i.v. injections of 5.0, 15, or 25
mg/kg AmBi or Lbn. None of the animals died at these
doses, and all test groups showed a consistent weight gain
throughout the 14-day study, similar to that of the control
D5W mice (data not shown). However, toxicity differences
were observed between the formulations at the tissue level
when examined histologically. Renal tubular changes were
not identified in any of the D5W control group kidneys
(Fig. 3A). In comparison, tubular changes with Lbn treat-
ment, noted as minimal or mild degeneration/regeneration,
were identified at 5.0 mg/kg Lbn (Fig. 3C), while treat-
ment with 5.0 mg/kg AmBi was not associated with renal
tubular changes in any of the mice in this AmBi dose group
(Fig. 3B). Renal tubular changes were increased in incidence
in a dose dependent manner between the 5.0 and 15 mg/kg
Lbn groups, and there was no appreciable difference in the
incidence or severity of renal tubular changes between the
15 and 25 mg/kg Lbn groups. Tubular changes in the 15
and 25 mg/kg AmBi treatment groups were predominantly
minimal in severity. Only one 25 mg/kg AmBi dose group
mouse had mild tubular changes.
A. fumigatus infected mice: survival and disease
signs
To examine the effect of daily dosing for six days in in-
fected animals, we tested these drugs in a murine model
of pulmonary A. fumigatus infection. Mice in the control
D5W group succumbed readily to the infection. All deaths
occurred between 24 h and 72 h post-challenge (Fig. 4A)
with a survival rate of 10% by the end of the study and
maximum weight loss on day 6 of 18% (Fig. 4B) (survival,
P≤0.02 for D5W vs. 10 or 15 mg/kg AmBi or 15 mg/kg
Lamb). The number of deaths in the Lbn and AmBi treat-
ment groups given four doses was comparable up to day 3,
but even with two additional drug doses for the surviving
mice, animals in the 10 mg/kg Lbn group continued to die,
with 30% survival by the end of the study. The mice in the
10 mg/kg Lbn group had a greater percent weight loss than
the control D5W mice, and disease sign scores that were
more severe than the other drug treatment groups (Fig. 4B,
C). In comparison, survival of mice treated with 10 or
15 mg/kg AmBi was 60% and 50%, respectively, with the
least overall weight loss and mild disease signs for the an-
imals in the 10 mg/kg AmBi group. In the 15 mg/kg Lbn
treated mice, survival was 60%, with weight loss and dis-
ease sign scores similar to that observed with 10 mg/kg
AmBi.
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6Medical Mycology, 2014, Vol. 00, No. 0
Figure 3. Kidney histopathology (n=7/group) in uninfected female
C57Bl/6 mice. Representative mice from each group are depicted. (A)
Normal renal tubules in hematoxylin and eosin (HE)-stained kidney from
mouse treated i.v. daily for 14 days with 5% dextrose (D5W, control). Bar,
100 μm. (B) HE-stained kidney from mouse treated i.v. for 14 days with
5 mg/kg AmBi, with normal renal tubular morphology similar to D5W
mice. (C) Renal tubular changes seen in all mice, except one, treated
i.v. for 14 days with 5 mg/kg Lbn with tubular dilation, protein casts
(arrows) and/or tubular regeneration ranging from minimal to mild.
A. fumigatus infected mice: fungal burden in the
BAL and lungs
To obtain more information about the efficacy of these two
drugs, we examined how effective these drugs were in reduc-
0 2 4 6 8 10121416182022
0
20
40
60
80
100
10 Lbn
15 Lbn
10 AmBi
15 AmBi
D5W
A
Stud y Day
Percent survival
0 2 4 6 8 10121416182022
-25
-20
-15
-10
-5
0
5
10 Lbn
15 Lbn
10 AmBi
15 AmBi
D5W
B
Stud y Da y
Percen t Weigh t Ch ang e
0 2 4 6 8 10121416182022
0
2
4
6
8
10 Lbn
15 Lbn
10 AmBi
15 AmBi
D5W
C
Day of Stud y
Disease Sign Score
Figure 4. Efficacy following six daily i.v. treatments with 10 or
15 mg/kg Lbn or AmBi or 5% dextrose (D5W) in triamcinolone-
immunosuppressed Swiss Webster female mice (n=10/group) chal-
lenged intranasally with A. fumigatus (5.6 ×106conidia/mouse).
(A) Survival: D5W vs. 10 or 15 mg/kg AmBi or 15 mg/kg Lbn (P≤0.02);
(B) Weight change: D5W vs. 10 mg/kg AmBi or 15 mg/kg Lbn (P≤0.001);
10 mg/kg Lbn vs. D5W (P=0.0001); (C) Disease Sign Scores: D5W vs.
10 mg/kg or 15 mg/kg AmBi or 15 mg/kg Lbn (P≤0.0001); 10 mg/kg Lbn
vs. 10 mg/kg AmBi (P<0.001), 15 mg/kg Lbn (P<0.0001); 15 mg/kg Lbn
vs. 15 mg/kg AmBi (P=0.006).
ing the fungal burden. The Aspergillus infected mice were
evaluated for cfu/ml in the BAL and cfu/g in the lungs at
48 h post-challenge when the animals had only received two
drug treatments (Fig. 5). We had to collect the tissues at this
early timepoint because of the severity of the infection. We
could not wait until after six treatments, as was done in
the mice that were followed for survival. Although Lbn at
10 mg/kg had a significantly higher fungal burden in the
BALcomparedto10or15mg/kgAmBi(P≤0.05)
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Olson et al. 7
D5W Lbn AmBi Lbn AmBi
3
4
5
6
10 mg/kg 15 mg/kg
A
Treatment Group
Log
10
CFU/ml BAL
D5W Lbn AmBi Lbn AmBi
3
4
5
610 mg/kg 15 mg/kg
B
Treatment Group
Log
10
CFU/g lung
Figure 5. Fungal burden (cfu/g lung and cfu/ml BAL) of female Swiss
Webster mice (n=7/group), suppressed with triamcinolone, and chal-
lenged intranasally with A. fumigatus (5.6 ×106conidia/mouse) 24
h after the second i.v. treatment with 5% dextrose (D5W), or 10 or
15 mg/kg Lbn or AmBi. (A) In BAL, 10 mg/kg Lbn vs. 10 or 15 mg/kg
AmBi and 15 mg/kg Lbn (P≤0.05). (B) In lungs, D5W vs. 10 or 15 mg/kg
Lbn and 15 mg/kg AmBi (P≤0.01). Bar =mean/group.
(Fig. 5A), the fungal burden in the BAL with all the liposome
treatments was the same as that seen in the D5W control
group. In the lungs, two treatments with either 10 or 15
mg/kg Lbn or 15 mg/kg AmBi produced significantly lower
fungal burdens compared to the D5W control (P≤0.01),
but there were no significant differences in reduction of lung
fungal burden amongst any of the drug treatment groups
(P>0.05) (Fig. 5B). A better correlation between survival
and reduction of lung fungal burden would probably have
been observed if the lung samples had been collected after
six treatments instead of only two treatments.
A. fumigatus infected mice: drug concentrations
in the BAL and lungs
Because we observed that the 10 mg/kg AmBi produced
more prolonged survival at day 21 compared to Lbn at
10 mg/kg, we investigated whether this difference was re-
Lbn AmBi Lbn AmBi
0
10
20
30
40 10 mg/kg 15 mg/kg
A
Treatment Group
Concentration (µg/g lung)
Lbn AmBi Lbn AmBi
0.0
2.0
4.0
6.0
8.0
10.0
10 mg/kg 15 mg/kg
B
Treatment Group
Concentration (µg/ml BAL)
Figure 6. Drug concentration (μg/ml BAL and μg/g lung) of Swiss Web-
ster mice (n=7/group), suppressed with triamcinolone, and challenged
intranasally with A. fumigatus (5.6 ×106conidia/mouse) determined
24 h after the second i.v. treatment with 5% dextrose (D5W), or 10 or
15 mg/kg Lbn or AmBi. (A) In lung, mean drug concentrations were
lower for 10 or 15 mg/kg AmBi vs. 10 or 15 mg/kg Lbn although not
statistically significantly different. (B) In BAL, mean drug concentrations
were lower for 15 mg/kg Lbn vs. 15 mg/kg AmBi but not statistically
significantly different (P=0.06). Bar =mean/group.
lated to the drug concentration in these tissues collected at
an early timepoint. In the lung tissue, there was no statis-
tically significant difference in drug concentration between
Lbn and AmBi at either dose (Fig. 6A). In the BAL, there
was also no significant difference in drug concentration be-
tween the liposome formulations (Fig. 6B). However, with
15 mg/kg dosing, six out of seven animals had no detectable
drug in the BAL following 15 mg/kg Lbn treatment, while
five out of seven animals given 15 mg/kg AmBi had drug lev-
els that ranged from 2.6 to 8.0 μg/ml. Despite these results,
the difference in BAL concentration between the two drugs
at this dose did not reach significance (P=0.06). These
results indicated that prolonged survival could not be cor-
related with the lung or BAL drug concentrations when the
analysis was done following only two drug treatments.
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8Medical Mycology, 2014, Vol. 00, No. 0
A. fumigatus infected mice: lung histopathological
evaluation of toxicity and efficacy
We also examined the lung tissues after two drug treatments
to evaluate tissue toxicity and fungal penetration. Hema-
toxylin and eosin stained lung sections were evaluated for
efficacy and drug toxicity, with Gridley stained sections
aiding in the identification of fungi. In the lung tissue, there
was severe bronchopneumonia in all mice (Fig. 7). In the
control D5W treated mice, abundant intralesional conidia
and hyphae were associated with acute, severe, necrotizing
bronchoalveolar inflammation, vasculitis, vascular conges-
tion and pulmonary edema (Fig. 7A, B). In the 10 mg/kg
Lbn-treated mice, there was ongoing, bronchiolar necrosis
in some areas, as well as bronchiolar epithelial hyperplasia
in others, indicative of a regenerative response (Fig. 7C),
which was not present in the controls. There was also a lot
of intralesional fungi, but they were less abundant than that
found in the controls, with more conidial than hyphal forms
(Fig. 7D). In the 10 mg/kg AmBi group, lungs displayed a
similar degree of pulmonary inflammation as that of mice
treated with 10 mg/kg Lbn or D5W, but the extent of pul-
monary edema and hemorrhage was less (Fig. 7E). Some
conidia were seen, but the occurrence of hyphae was rare in
the 10 mg/kg AmBi group (Fig. 7F). Infected mice treated
with 15 mg/kg Lbn presented with inflammation compara-
ble to that found in mice treated with 10 mg/kg Lbn with
hyperplastic epithelium lining the bronchiole but without
evidence of vascular invasion (Fig. 7G) and the presence of
some fungal elements (Fig. 7H). Although the severity of
the pulmonary inflammation seen in infected mice treated
with 15 mg/kg AmBi was comparable to that seen in the
other groups, there was no evidence of microabscess forma-
tion or vascular necrosis and hyphal forms were less preva-
lent (Fig. 7I, J). Overall, AmBi was associated with less
lung tissue damage and reduced fungal penetration com-
pared to Lbn when the tissues were examined microscopi-
cally. This difference in lung efficacy indicates that in this
model at an early timepoint, long-term survival correlated
better with the lung histopathologic observations as com-
pared to evaluation of the lung fungal burden using cfu/g
tissue analysis.
A. fumigatus infected mice: kidney histopathology
and BUN evaluation of toxicity
As renal tubular changes had been noted in uninfected mice
given 5 mg/kg Lbn every day for 14 days, the blood and kid-
neys of infected mice were collected at 24 h after only two
drug doses to determine whether any toxicity was evident
in the infected kidneys at this early timepoint. As mentioned
above, we had to collect the tissues at 48 h post-challenge
due to several deaths that occurred in some of the groups,
particularly the D5W group. The kidneys of D5W treated
mice had normal tubular structure. However, mice given
either Lbn or AmBi had some evidence of minimal renal
changes, which increased in a dose dependent manner for
both drugs. There was tubular dilation, protein casts, and
single cell necrosis, and these effects were slightly more ev-
ident in the AmBi treatment groups. After only two drug
treatments, BUN levels in all mice were within normal limits
(12 to 39 mg/dl serum) [37].
Discussion
The toxic effects of the antifungal drug, deoxycholate am-
photericin B, have been markedly reduced by its formu-
lation into liposomes. However, as reported previously in
a study of Anfogen and AmBi, if the amphotericin B li-
posomes are not manufactured under the same conditions,
even though the liposomes have the same chemical composi-
tion, their physical properties as well as toxicity and efficacy
profiles can be very different [14]. In the present study, the
liposomal amphotericin B products, Lbn and AmBi, also
seem to have the same chemical components but they have
different particle sizes, with Lbn being 1.6-fold larger than
AmBi (122.2 nm Lbn vs. 77.8 nm AmBi). This difference in
size can affect how the liposomes distribute in the host and
their rate of cellular uptake [38].
The conventional in vitro toxicity assay for amphotericin
B is the RBC lytic assay [39]. RBCs are used because they
have high levels of cholesterol in their membranes to which
the amphotericin B will bind resulting in pore formation
and lysis of the cells. A more sensitive RBC assay measures
the leakage of potassium from the RBCs when they are ini-
tially damaged by the amphotericin B and has been used
successfully to compare the in vitro toxicity of different
amphotericin B lipid formulations [33]. When the potas-
sium release assay was used in this study, AmBi required
5×more drug than Lbn to produce 50% potassium leak-
age of the RBCs (K50 =1.4 μg/ml Lbn vs. 7.4 μg/ml AmBi)
demonstrating the reduced in vitro toxicity of AmBi relative
to Lbn.
In vitro assays are helpful in identifying toxicity at the
cellular level, and in the case of lipid amphotericin B for-
mulations, indicative of the binding affinity of the ampho-
tericin B for the liposome bilayer vs. the mammalian cell
membrane. However, in vivo toxicity testing is needed to
evaluate how the pharmacodynamics of the drug will affect
its toxicity. In the present study, we evaluated both lipo-
somal amphotericin B drugs in uninfected C57Bl/6 mice
following either a single injection or daily treatment for
14 days. We used this extended therapy since amphotericin
B treatment of fungal infections requires repeated dosing
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Olson et al. 9
Figure 7. Lung histopathology (HE or Gridley stain) of Swiss Webster mice, suppressed with triamcinolone and challenged with A. fumigatus (5.6 ×
106conidia/mouse), with lung collection 24 h after the second i.v. treatment with 5% dextrose (D5W), or 10 or 15 mg/kg Lbn or AmBi. Representative
mice from each group are depicted (n=5/group). (D5W group: A, B) Inflammation and abundant intralesional conidia and hyphae, necrosis, vascular
congestion, and edema. (10 mg/kg Lbn group: C, D) Severe bronchopneumonia, with inflammation, pulmonary edema, and/or hemorrhage similar to
that in the control D5W group, with large amounts of conidia and hyphae in all mice treated. (10 mg/kg AmBi group: E, F) Inflammation comparable
to that with 10 mg/kg Lbn or D5W but less pulmonary edema and hemorrhage, along with some conidia and rare fragmented hyphae. (15 mg/kg
Lbn group: G, H) Inflammation similar to that in mice given 10 mg/kg Lbn but with reduced amounts of conidia and hyphae. (15 mg/kg AmBi group:
I, J) Similar inflammation to that with 10 mg/kg AmBi but with mild epithelial necrosis, no microabscess formation, and few hyphal elements. B =
bronchial epithelium, BV =blood vessel, TA =terminal airway, C =conidia, H =hyphae. Bar for HE-stained lungs (A, C, E, G, I) =1 mm. Bar for
Gridley-stained lungs (B, D, F, H, J) =50 μm.
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10 Medical Mycology, 2014, Vol. 00, No. 0
over a period of several weeks [40–42]. Although all the
uninfected mice given a single i.v. injection of 20 mg/kg
Lbn or AmBi survived, all but one mouse given a single
dose of 50 mg/kg Lbn died, while there were no deaths in
those mice given a single dose of 50 mg/kg AmBi. When un-
infected animals received multiple drug treatments (5, 15,
or 25 mg/kg/day) for 14 days, evidence of chronic kidney
damage was detected microscopically in those mice given
5 mg/kg Lbn but not in the kidneys of mice treated with
5 mg/kg AmBi. This difference is significant since the clin-
ical dose of AmBi is 3–5 mg/kg in patients being treated
for fungal infections and treatment is often for at least 2
to 4 weeks [17,35,36,43–45]. Furthermore, amphotericin
B treatment in an already immunosuppressed patient has
to be considered carefully, especially if they are being given
other nephrotoxic or hepatoxic drugs [46–48].
Although there were clear toxicity differences between
the two formulations based on our in vitro assays and in
vivo intravenous toxicity testing in uninfected animals, an-
imals infected with A. fumigatus do not always tolerate
repeated dosing with the same high doses of amphotericin
B lipid formulations as the uninfected animals [14,20]. To
address this issue, in the present study we used the steroid
immunosuppressed pulmonary A. fumigatus murine model
for evaluating the efficacy of daily intravenous treatment
with AmBi or Lbn in infected mice. We administered 10
or 15 mg/kg of each drug since those doses have been re-
ported to be most effective in previous mouse aspergillosis
studies [14,25,43]. AmBi at either dose (10 or 15 mg/kg)
given every day for 6 days significantly prolonged survival,
whereas only the higher dose of Lbn (15 mg/kg) increased
survival. Thus, administering Lbn at the required higher
dose could potentially result in increased toxicity since an-
tifungal treatment often requires 2–4 weeks of therapy.
It has been reported that if Aspergillus infected tissue is
collected after at least four drug treatments, there is a cor-
relation between reduction in fungal burden and drug con-
centration in the tissues [20]. However, due to the severity
of the infection in the present study, we had to collect tis-
sues after only 2 days of treatment to obtain enough tissue
samples for each group. We observed significantly lower
cfu/g lung in the 10 or 15 mg/kg Lbn and 15 mg/g AmBi
groups compared to the D5W control mice, while there was
no significant difference in efficacy amongst any of the drug
treatment groups. Similarly, the amount of drug in the lungs
was the same for all drug treatments. This would suggest
a possible correlation between reduction in cfu/g lung and
lung drug concentration. However, histopathologic evalua-
tion of the lung tissues even at this early timepoint showed
that AmBi treatment at either dose was more effective than
Lbn with less lung tissue damage and fewer fungal elements,
particularly at the 10 mg/kg dose. This correlated better
with the prolonged survival achieved with 10 mg/kg AmBi
vs. 10 mg/kg Lbn. As reported in other studies compar-
ing amphotericin B formulations in pulmonary and central
nervous system aspergillosis, important additional informa-
tion about drug efficacy and toxicity was obtained through
histologic examination of infected tissues, which comple-
mented traditional methods of fungal burden assessment
and toxicity testing [49,50]. These studies underscore the
need to include histological examination of target tissues
when evaluating drug efficacy and toxicity of lipid ampho-
tericin B formulations. Furthermore, in subsequent studies,
consideration should be given to using an inhalation mouse
pulmonary aspergillosis challenge model as it would be less
acute and would allow a longer-term follow up with multi-
ple sampling times to evaluate toxicity [51].
The observations from this study re-enforce the conclu-
sion that the process used to formulate a drug is critical
with respect to ensuring that it has the desired physical and
pharmacokinetic/pharmacodynamic properties. This is par-
ticularly important when the drug has to be incorporated
into a carrier system, such as a liposome or other nanopar-
ticle. Consequently, it is essential that supposedly compa-
rable antifungal drugs prepared under different processing
conditions be tested in both uninfected and fungal infected
animals, using a range of in vitro and in vivo toxicity and
efficacy assays. By using this multiple testing approach, we
have shown that there were significant differences between
Lbn and AmBi based on their physical properties as well as
their biological activity in both uninfected and Aspergillus
infected mice. This type of analysis for evaluating similari-
ties and differences among antifungal drugs can be readily
applied to other antimicrobial agents and is of particular
importance when examining drugs incorporated into car-
rier systems.
Declaration of Interest
Support for this research was provided by a research grant
from Gilead Sciences, Inc.
G. M. and T. B. are employees of Gilead Sciences, Inc.
J. A. S. and J. P. A.-M. are consultants for Gilead Sciences,
Inc.
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