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Antimicrobial Agents and Chemotherapy, February 2001, p. 464-470, Vol. 45, No. 2
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.2.464-470.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Therapeutic Efficacy of Liposome-Encapsulated
Gentamicin in Rat Klebsiella pneumoniae Pneumonia in
Relation to Impaired Host Defense and Low Bacterial Susceptibility
to Gentamicin
Raymond M.
Schiffelers,1,2
Gert
Storm,2
Marian T.
ten
Kate,1 and
Irma A. J. M.
Bakker-Woudenberg1,*
Department of Medical Microbiology & Infectious Diseases, Erasmus University Medical Center Rotterdam,
Rotterdam,1 and Department of
Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences,
Utrecht,2 The Netherlands
Received 5 June 2000/Returned for modification 28 August
2000/Accepted 27 October 2000
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ABSTRACT |
Long-circulating liposomes (LCL) may be used as targeted
antimicrobial drug carriers as they localize at sites of infection. As
a result, LCL-encapsulated gentamicin (LE-GEN) has demonstrated superior antibacterial activity over the free drug in a single-dose study of immunocompetent rats with Klebsiella pneumoniae
pneumonia. In the present study, the therapeutic efficacy of LE-GEN was
evaluated by monitoring rat survival and bacterial counts in blood and
lung tissue in clinically relevant models, addressing the issue of impaired host defense and low bacterial antibiotic susceptibility. The
results show that in immunocompetent rats infected with the high-GEN-susceptibility K. pneumoniae strain, a single dose
of LE-GEN is clearly superior to an equivalent dose of free GEN. Yet
complete survival can also be obtained with multiple doses of free GEN.
In leukopenic rats infected with the high-GEN-susceptible K. pneumoniae strain, free GEN at the maximum tolerated dose (MTD) was needed to obtain survival. However, with the addition of a single
dose of LE-GEN to free-GEN treatment, complete survival can be obtained
using a sevenfold-lower cumulative amount of GEN than with free-GEN
treatment alone. In leukopenic rats infected with low-GEN-susceptible
K. pneumoniae cells, free GEN at the MTD did not result in
survival. The use of LE-GEN is needed for therapeutic success.
Increasing LE-GEN bilayer fluidity resulted in an increased GEN release
from the liposomes and hence improved rat survival, thus showing the
importance of the liposome lipid composition for therapeutic efficacy.
These results warrant further clinical studies of liposomal
formulations of aminoglycosides in immunocompromised patients with
severe infections.
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INTRODUCTION |
Clinical practice shows that failure
of antimicrobial treatment is not uncommon. Two major risk factors can
be identified: an impaired host defense and a moderate-to-low
antibiotic susceptibility of the infectious organism(s) (4, 6, 8,
18). An impaired host defense increases the patients'
susceptibility to infections. In addition, a limited ability of the
host defense to support antimicrobial treatment increases the chance of
treatment failure (15). A low antibiotic susceptibility of
bacteria can result in subeffective drug concentrations at the site of
infection despite high drug doses (8).
Targeted antibacterial-drug delivery may increase drug concentrations
at the infectious focus and therefore help to reduce the treatment
failure risks imposed by the impaired host defense or low bacterial
susceptibility. Besides, in the case of potentially toxic antibiotics,
toxicity to nontarget tissues may be reduced, which allows the use of
higher doses. Liposomes have attracted considerable interest as
targeted drug carriers in infectious diseases. A number of studies have
convincingly demonstrated that so-called long-circulating liposomes
tend to localize preferentially at foci of infection or inflammation
after intravenous administration (1, 9, 11, 17, 19). The
preferential localization appears to be the result of the inflammatory
response provoking a locally increased capillary permeability, allowing
liposome extravasation. Generally, intravenously administered liposomes that are rapidly opsonized and taken up by the mononuclear phagocyte system (MPS) hardly localize at foci of infection outside the MPS. By
coating the liposomal surface with polyethylene glycol (PEG),
opsonization and subsequent MPS uptake is reduced, thus prolonging
circulation time and interaction with the infectious target site. As a
result, these PEG-coated long-circulating liposomes (LCL) show superior
target localization characteristics (19). Therefore, LCL
have attractive prospects for the site-specific delivery of
antimicrobial agents. Previous research has shown that LCL-encapsulated
gentamicin (LE-GEN) demonstrates superior antibacterial efficacy
compared to free GEN in a single-dose study of rats with intact host
defenses and with a unilateral Klebsiella pneumoniae
pneumonia (3).
In the present study, the antibacterial efficacy of LE-GEN was
evaluated in clinically more relevant models addressing the issue of
impaired host defense and low antibiotic susceptibility. Rat survival
and bacterial counts in lung tissue and blood were monitored in
immunocompetent as well as leukopenic rats with pneumonia caused by
high- or low-GEN-susceptible K. pneumoniae strains.
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MATERIALS AND METHODS |
Liposome preparation and characterization.
Liposomes were
prepared as described previously (3). In brief,
appropriate amounts of partially hydrogenated egg phosphatidylcholine (PHEPC) (Asahi Chemical Industry Co. Ltd., Ibarakiken, Japan), cholesterol (Sigma Chemical Co., St. Louis, Mo.), and
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[polyethylene glycol-2000] (PEG-DSPE) (Avanti Polar Lipids, Alabaster, Ala.) in a
molar ratio of 1.85:1:0.15 were dissolved in a mixture of chloroform
and methanol to obtain "rigid" liposomes. To obtain "fluid"
liposomes, egg phosphatidylcholine (EPC; Asahi Chemical Industry Co.
Ltd.) and PEG-DSPE were dissolved in a molar ratio of 2.85:0.15. After
evaporation of the solvent under constant rotation and reduced
pressure, the lipid mixture was dried under nitrogen, dissolved in
2-methyl-2-propanol (Sigma Chemical Co.), frozen, and freeze-dried
overnight. The resulting lipid film was hydrated for 2 h in 2.5 ml
of aqueous GEN (200 mg/ml) (Duchefa Biochemie b.v., Harlem, The
Netherlands), and subsequently, 7.5 ml of HEPES-NaCl buffer (10 mM
HEPES) (Sigma Chemical Co.) and 135 mM NaCl (Merck, Darmstadt, Germany)
(pH 7.4) were added. The hydrated lipids were sonicated for 8 min with
an amplitude of 8 µm using a 9.5-mm probe in an MSE Soniprep 150 (Sanyo Gallenkamp PLC, Leicester, United Kingdom). Dynamic light
scattering, detected at an angle of 90° to the laser beam on a 4700 system (Malvern Instruments Ltd., Malvern, United Kingdom), was used to
evaluate the particle size distribution. Liposomes with a mean particle size of 100 nm were obtained. In addition to the mean particle size,
the system reports a polydispersity index (a value between 0 and 1). A
polydispersity index of 1 indicates large variations in particle size;
a reported value of 0 means that size variation is apparently absent.
All liposome preparations used had polydispersity indexes below 0.3. Nonencapsulated GEN was removed by ultracentrifugation in two changes
of HEPES-NaCl buffer at 265,000 × g for 2 h at 4°C in an L-70 ultracentrifuge (Beckman, Palo Alto, Calif.). The phosphate concentration was determined spectrophotometrically according
to the method of Bartlett (5). Total (encapsulated and
free) and unencapsulated (free) GEN was measured with a diagnostic sensitivity test agar (Oxoid, Basingstoke, United Kingdom) diffusion test with Staphylococcus aureus Oxford strain (ATCC 9144) as
the indicator organism and standards ranging from 4 to 0.25 µg of GEN/ml, as described previously (3). For total
(unencapsulated and encapsulated) GEN measurements, liposomes were
destroyed by 0.1% (vol/vol) (final concentration) Triton X-100
(Janssen Chimica, Geel, Belgium). Less than 10% of the GEN in the
liposome dispersion was shown to be unencapsulated after
ultracentrifugation. The specific activity was between 70 and 80 µg
of GEN/µmol of total lipid.
Bacterial strains.
The high-GEN-susceptible K. pneumoniae strain (ATCC 43816; capsular serotype 2; MIC = 0.5 µg/ml) was used. The MIC was determined by plating an inoculum of
104 CFU per spot on Mueller-Hinton (MH) agar (Difco
laboratories, Detroit, Mich.) plates containing twofold dilutions of
GEN according to the method of Woods and Washington (20).
The low-GEN-susceptible K. pneumoniae strain (MIC = 4 µg/ml) was obtained by culturing the high-susceptible strain in MH
broth in the presence of increasing concentrations of GEN. The
low-GEN-susceptible strain appeared to be stable in vitro after
repeated culture in antibiotic-free MH broth.
Unilateral pneumonia.
The animal experiments ethical
committee of the Erasmus University Medical Center Rotterdam approved
the experiments described in this study. Female RP/AEur/RijHsd strain
albino rats (18 to 25 weeks of age; body weight, 185 to 225 g;
Harlan, Horst, The Netherlands) with a specified pathogen-free status
were used. When indicated, rat host defense was impaired by
intraperitoneal injection of 60 mg of cyclophosphamide (Sigma)/kg of
body weight every 4 days, starting at 5 days before bacterial
inoculation. The leukopenic status of cyclophosphamide-treated animals
was ascertained in separate experiments by measuring leukocyte counts in fresh blood samples, obtained by retro-orbital bleeding in EDTA-coated tubes. White blood cells were counted on a Cobas Minos Stex
(Roche Haematology, Montpellier, France) using Minotrol 16 standards
(Roche Haematology) to verify proper functioning of the instrument. As
a result of the cyclophosphamide treatment, the number of leukocytes in
the circulation on the day of bacterial inoculation was reduced sixfold
from approximately 6 × 109 ± 1 × 109 (buffer-treated controls) to 1 × 109 ± 8 × 108 (means ± standard deviations [SD]; n = 3; P < 0.01). Leukocyte counts remained reduced throughout the study period.
From 5 days to 1 day before bacterial inoculation, the drinking water
of the leukopenic rats was supplemented with 1 g of cephalexin
monohydrate (Dopharma, Raamsdonksveer, The Netherlands)/liter to
prevent superinfections. The drinking water in the remaining study
period and the drinking water of immunocompetent rats was autoclaved
water, pH 3. At the time of bacterial inoculation, cephalexin
concentrations were less than 1 µg/ml in the blood and lung tissue of
rats that had received the drinking water supplemented with cephalexin
(n = 3 rats), as measured by an agar diffusion test
using Escherichia coli as the indicator organism, as
described previously (3).
A left-sided unilateral pneumonia was induced as described in detail
elsewhere (
2). In brief, rats were anesthetized, and
the
left primary bronchus was intubated. Through the tube, 0.02
ml of a
saline suspension of
K. pneumoniae was inoculated in the
left lung lobe. The inoculated bacteria were in the logarithmic
phase
of growth. The inoculum was adjusted such that the median
survival
rates of the rats were comparable in the models. The
rats with intact
host defenses were inoculated with 10
6 high-GEN-susceptible
K. pneumoniae (ATCC 43816; capsular serotype
2) cells, and
the leukopenic rats were inoculated with 10
5
high-GEN-susceptible or 10
7 low-GEN-susceptible
K. pneumoniae cells. The cephalexin-containing
water did not have an
effect on rat survival or bacterial outgrowth.
The rats were housed
individually. The in vivo stability of the
phenotype of the
low-susceptibility
K. pneumoniae strain was checked
by
culturing dilutions of homogenized left-lung tissue obtained
24 h
after bacterial inoculation (the starting point of treatment)
on MH
plates. Colonies were isolated, and the MIC was determined
on MH plates
as described above (
20). All 100 tested colonies
had a
stable low-GEN-susceptible phenotype after inoculation in
vivo. The
same procedure was applied to bacteria isolated at the
end of the study
period (after the death of the rats or after
14 days). None of the
treatments in this study resulted in a change
of the MIC of the
K. pneumoniae strains pre- and
postexposure.
Antimicrobial treatment.
The treatments were started at
24 h after bacterial inoculation. GEN was administered twice daily
every 12 h (q12h), and LE-GEN was administered once daily every
24 h (q24h). The formulations were injected intravenously in the
tail vein.
Survival.
Ten rats were used per experimental group. The
survival of the rats was examined every day until 14 days after
bacterial inoculation. The blood of the dead rats was cultured on
Columbia III agar supplemented with 5% sheep blood (Beckton-Dickinson)
overnight at 37°C. Substantial numbers of exclusively K. pneumoniae cells were recovered in the blood samples from the dead rats.
Quantification of bacterial numbers in blood.
At various
time points after bacterial inoculation, blood samples were taken via
retro-orbital bleeding in heparinized tubes on ice. Serial dilutions
were prepared on ice, and 0.2 ml of each dilution was applied to
tryptone-soy-agar plates. The plates were incubated overnight at
37°C, and colonies were counted.
Quantification of bacterial numbers in left-lung tissue.
At
various time points after bacterial inoculation, rats were sacrificed
by CO2 inhalation. The infected left lung was dissected and
homogenized in 20 ml of phosphate-buffered saline (4°C), supplemented with aminoglycoside acetylating enzyme, and 2 mM acetyl coenzyme A
(sodium salt) (Sigma Chemical Co.) to inactivate residual GEN present
in the tissues, according to Den Hollander et al. (13). Serial dilutions were prepared, and 0.2 ml of each dilution was applied
on tryptone-soy-agar plates. The plates were incubated overnight at
37°C, and colonies were counted.
Pharmacokinetics and tissue concentrations of free GEN or
LE-GEN.
Free GEN or LE-GEN was injected intravenously in healthy
rats at various doses, used in the survival experiments, via the tail
vein. Blood samples were taken at various time points after injection
from alternate groups of three rats via retro-orbital bleeding under
CO2 anaesthesia. The blood samples were collected in
heparinized tubes, and after centrifugation, the plasma was collected.
Drug concentrations were analyzed using the agar diffusion test as
described above. The sample was divided into two portions. One was
analyzed directly to determine the free (i.e., nonliposomal) drug
concentration. The other was incubated with Triton X-100, as described
above, to disrupt the liposomes in order to determine total (free plus
encapsulated) drug concentrations. Neither liposome type used in the
study showed substantial drug leakage when mixed with plasma and
subsequently placed in agar wells and incubated overnight at 37°C. As
a result, free-GEN concentrations could be accurately determined
separately from the total GEN concentrations.
Total GEN concentrations in lung tissue at different time points after
injection were analyzed by dissection of the infected
left lung and
uninfected right lung, homogenization of the tissue
in
phosphate-buffered saline, and subsequent incubation with 0.1%
Triton
X-100 (final concentration) followed by the agar diffusion
test as
described
above.
Statistical analysis.
Survival in different experimental
groups and controls was compared by the log rank test (Graph Pad
software Inc., San Diego, Calif.).
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RESULTS |
Rats with intact host defenses infected with high-GEN-susceptible
K. pneumoniae.
In the first model, rats with intact host
defenses were infected with the high-GEN-susceptible K. pneumoniae strain (MIC = 0.5 µg/ml). Treatment with either
free GEN or LE-GEN was started 24 h after bacterial inoculation.
The survival rates are shown in Fig. 1.
Untreated control animals died between day 3 and day 5 after bacterial
inoculation. A single dose of free GEN (5 mg/kg) slightly prolonged
survival (P < 0.01 compared to controls), but all
animals still died before day 8. An equivalent dose of LE-GEN yielded
70% survival after 2 weeks (P < 0.001 compared to a
single dose of free GEN [5 mg/kg]). The difference in efficacy
between these two treatments is paralleled by the differences in GEN
concentrations in the lung tissue after injection. Already at 5 h
after a single dose of GEN (5 mg/kg), GEN levels are below 1.5 µg/lung in either the infected left lung or the uninfected right
lung. In contrast, an equivalent dose of LE-GEN results in total GEN
concentrations of 7.9 ± 0.8 µg/left lung and 4.0 ± 2.1 µg/right lung at 5 h after injection. At 24 h,
concentrations of 12.9 ± 4.3 µg/left lung versus 5.5 ± 1.2 µg/right lung were noted, whereas after 48 h, the
concentrations were 16.2 ± 3.9 µg/left lung versus 3.3 ± 2.8 µg/right lung (mean ± SD; n = 5 to 9 rats
per time point).
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FIG. 1.
Effect of free GEN and LE-GEN on survival of rats with
intact host defenses infected with a high-GEN-susceptible K. pneumoniae strain. The treatments were control (no drug treatment)
( ), free GEN at 5 mg/kg (single dose) ( ), free GEN at 5 mg/kg/day
q12h for 3 days ( ), and LE-GEN at 5 mg/kg (single dose) ( ).
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Treatment of rats with free GEN (5 mg/kg/day q12h for 3 days) resulted
in 100%
survival.
Leukopenic rats infected with high-GEN-susceptible K. pneumoniae.
Rat host defense was impaired in the second model by
cyclophosphamide injections, resulting in a sixfold reduction in the number of circulating white blood cells. These leukopenic rats were
infected with the high-GEN-susceptible K. pneumoniae strain (MIC = 0.5 µg/ml). Treatment with either free GEN, LE-GEN, or a
combination of both was started 24 h after bacterial inoculation. The survival rates are shown in Fig. 2
and 3.
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FIG. 2.
Effect of free GEN on survival of leukopenic rats
infected with a high-GEN-susceptible K. pneumoniae strain.
The treatments were control (no drug treatment) (), free GEN at 5 mg/kg/day q12h for 3 days (), free GEN at 5 mg/kg/day q12h for 5 days (), free GEN at 20 mg/kg/day q12h for 5 days (), and free
GEN at 40 mg/kg/day q12h for 5 days ( ).
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FIG. 3.
Effect of GEN and LE-GEN on survival of leukopenic rats
infected with a high-GEN-susceptible K. pneumoniae strain.
The treatments were control (no drug treatment) (), free GEN at 5 mg/kg/day q12h for 5 days (), free GEN at 5 mg/kg/day q12h for 5 days plus a single dose of LE-GEN at 5 mg/kg on day 1 (), free GEN
at 5 mg/kg/day q12h for 5 days plus a single dose of free GEN at 5 mg/kg on day 1 (), and LE-GEN at 5 mg/kg/day q24h for 5 days
().
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Figure
2 shows that, similar to the previous model, untreated control
animals died between days 2 and 4 after bacterial inoculation.
The
therapeutic regimen that resulted in complete survival in
the previous
model (free GEN at 5 mg/kg/day q12h for 3 days) hardly
prolonged
survival in leukopenic rats. Free GEN at 5 mg/kg/day
q12h for 5 days
prolonged survival during treatment, but after
termination of treatment
only 10% of the rats survived up to 14
days (
P < 0.0001 compared to control animals). Increasing the
dose of free GEN to
20 mg/kg/day q12h for 5 days showed 70% survival
on day 14. A further
increase to 40 mg/kg/day q12h for 5 days,
which is the maximum
tolerated dose (MTD), resulted in complete
survival.
Figure
3 shows that addition of a single dose of LE-GEN at 5 mg/kg on
day 1 to a 5-day treatment with free GEN at 5 mg/kg/day
q12h also
resulted in 100% survival. The complete survival obtained
with that
therapeutic regimen is the result of the addition of
liposomal GEN, as
the addition of free GEN on day 1 to the 5-day
treatment with free GEN
showed only 30% survival on day 14 (
P < 0.001 for the
addition of free GEN versus the addition of LE-GEN).
Treatment with
LE-GEN only at 5 mg/kg/day q24h for 5 days was
not effective, as the
majority of rats died during
treatment.
The median numbers of bacteria (± range) recovered from the left lung
and blood on day 0, 1, 6, 10, and 14 after bacterial
inoculation of
leukopenic rats infected with the high-GEN-susceptible
K. pneumoniae strain are shown in Table
1. The rats received
a 5-day treatment
with free GEN at 5 mg/kg/day q12h with the addition
of a single dose of
either LE-GEN (5 mg/kg) or free GEN (5 mg/kg)
on day 1. Median bacterial counts 24 h after the last dose (day
6) in the
left lungs of rats treated with the combination of free
GEN plus LE-GEN
were 10-fold lower than those in rats treated
with equivalent doses of
free GEN alone. In the following days,
complete bacterial killing was
achieved in rats treated with free
GEN plus LE-GEN whereas the majority
of rats treated with free
GEN alone died. Bacterial counts in blood on
day 6 were approximately
100-fold lower for free GEN plus
LE-GEN-treated animals than for
rats treated with free GEN alone.
Median bacterial blood counts
stabilized in the following days in the
rats that received free
GEN plus LE-GEN. Examination of bacterial
counts in consecutive
blood samples of individual rats revealed that
the bacteremia
was episodic in nature. The GEN susceptibility of
K. pneumoniae recovered from dead or surviving animals was
not changed compared
to the inoculated bacteria.
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TABLE 1.
Number of bacteria in left lung and blood in leukopenic
rats infected with the high-GEN-susceptible K. pneumoniae strainsa
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Leukopenic rats infected with low-GEN-susceptible K. pneumoniae.
The third model combines the clinically encountered
problems of an impaired host defense and low bacterial antibiotic
susceptibility. Leukopenic rats were infected with the
low-GEN-susceptible K. pneumoniae strain (MIC = 4 µg/ml). Treatment consisted of either free GEN or a combination of
free GEN and LE-GEN and was started 24 h after bacterial
inoculation. The survival rates are shown in Fig.
4.
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FIG. 4.
Effect of GEN and rigid LE-GEN or fluid LE-GEN on
survival of leukopenic rats infected with a low-GEN-susceptible
K. pneumoniae strain. The treatments were control (no drug
treatment) (), free GEN at 40 mg/kg/day q12h for 5 days (), free
GEN at 40 mg/kg/day q12h for 5 days plus rigid LE-GEN at 20 mg/kg/day
q24h for 5 days (), and free GEN at 40 mg/kg/day q12h for 5 days
plus fluid LE-GEN at 20 mg/kg/day q24h for 5 days ().
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Untreated control animals died, similar to the previous models, on days
3 and 4. Free GEN given at the MTD of 40 mg/kg/day
q12h for 5 days,
which resulted in complete survival in the previous
model, was
unsuccessful. Higher doses of free GEN produced acute
mortality.
Addition of LE-GEN at 20 mg/kg/day q24h for 5 days
to 5-day treatment
with free GEN at the MTD increased survival
up to 50%
(
P < 0.01 compared to treatment with free GEN alone)
without producing acute
mortality.
Up to this point, only a rigid LE-GEN formulation had been used, as it
proved highly effective. In the present model, the
maximum dose of free
GEN plus rigid LE-GEN was only partially
successful (50% survival).
Therefore, a fluid LE-GEN formulation
was investigated, as it has been
shown that bilayer fluidity can
influence the therapeutic efficacy of
liposome-encapsulated aminoglycosides
(
7). A previous
study had already demonstrated that the fluid
and rigid LE-GEN localize
with the same targeting efficiency at
the target site
(
19). Addition of the fluid LE-GEN at 20 mg/kg/day
for 5 days instead of equivalent doses of rigid LE-GEN to the
5-day free-GEN
treatment at the MTD resulted in complete survival
(
P < 0.05 compared to free GEN plus rigid LE-GEN).
The median number of bacteria (± range) recovered from the left lung
and blood on days 0, 1, 6, 10, and 14 after bacterial
inoculation of
leukopenic rats infected with the low-GEN-susceptible
K. pneumoniae strain is shown in Table
2. Rats received 5-day
treatment with
free GEN at 40 mg/kg/day q12h with the addition
of rigid LE-GEN or
fluid LE-GEN at 20 mg/kg/day q24h for 5 days.
The median bacterial
counts 24 h after the last dose (day 6) in
the left lungs of rats
treated with free GEN plus fluid LE-GEN
were 10-fold lower than those
in rats treated with equivalent
doses of free GEN plus rigid
LE-GEN. In the following days, the
median numbers of bacteria
in the left lungs of rats treated with
free GEN plus fluid LE-GEN were
reduced further to zero. Fifty
percent of the rats treated with the
rigid LE-GEN combination
died during this period. The median numbers of
K. pneumoniae cells
in the blood of rats treated with GEN
plus fluid LE-GEN remained
zero throughout the study period, whereas
bacteria were present
in the blood of rats treated with GEN plus rigid
LE-GEN. The GEN
susceptibility of
K. pneumoniae recovered
from dead or surviving
animals was not changed compared to the
inoculated bacteria.
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TABLE 2.
Number of bacteria in left lung and blood in leukopenic
rats infected with the low-GEN-susceptible K. pneumoniae straina
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Pharmacokinetics.
The time courses of blood concentrations of
therapeutically available GEN or total (therapeutically available plus
liposome-encapsulated) GEN after injection of free GEN, fluid LE-GEN,
or rigid LE-GEN are shown in Fig. 5). The
pharmacokinetics of free GEN are independent of the dose in the range
of 2.5 to 20 mg/kg, as the GEN concentrations in the circulation over
time were approximately proportional to the injected dose.
Disappearance of free GEN from the bloodstream was relatively rapid,
with a half-life of approximately 20 min. At 8 h after injection
of the highest dose (20 mg/kg), GEN levels in the blood were already
below 0.1 µg/ml.
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FIG. 5.
(A) Time course of GEN concentrations in plasma of rats
after injection of a single dose of free GEN at 2.5 (), 5 (), 10 (), or 20 () mg/kg; There were three animals per experimental
group. (B) Time course of GEN concentrations in plasma of rats after
injection of a single dose of rigid LE-GEN or fluid LE-GEN. The open
symbols indicate microbiologically active (i.e., not
liposome-encapsulated) GEN concentrations after a single dose of rigid
LE-GEN at 5 mg/kg (), rigid LE-GEN at 20 mg/kg (), or fluid
LE-GEN at 20 mg/kg (). The solid symbols indicate total (i.e.,
microbiologically active and liposome-encapsulated) GEN concentrations
after a single dose of rigid LE-GEN at 5 mg/kg (), rigid LE-GEN at
20 mg/kg (), or fluid LE-GEN at 20 mg/kg ( ). There were three
animals per experimental group. All points represent means ± SD.
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Encapsulation of GEN in rigid liposomes resulted in dramatically
increased total GEN concentrations in the circulation after
injection.
Even at 24 h after injection, 15 to 20% of the injected
dose was
still present in the bloodstream. Fluid LE-GEN showed
a completely
different picture. Less than 5% of the injected dose
was present in
the circulation at 24 h after injection. Since
rigid and fluid
liposomes exhibit similar blood clearance kinetics,
as has been
demonstrated previously (
19), the difference between
the
two liposome types is the result of faster drug release from
the fluid
liposomes, as can also be deduced from the higher free
GEN levels after
injection of the fluid liposomes. It is not the
result of renal
impairment, as administration of the highest doses
of free GEN, rigid
LE-GEN, or fluid LE-GEN used in this experiment
did not result in
significantly different blood creatinin or blood
urea nitrogen levels
in these three experimental groups 24 h after
administration (data
not
shown).
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DISCUSSION |
The preferential localization of liposomes at sites of infection
offers an attractive way to selectively increase antibacterial drug
concentrations at the target location, with the intention to increase
the therapeutic efficacy of antimicrobial treatment. Most studies in
this field have been performed in animal models with an intact host
defense and with high-antibiotic-susceptible bacteria. However, in
clinical practice, two important complicating factors should be taken
into account. In particular, patients having impaired host defenses
carry a high risk of treatment failure, which further increases when
the infectious organism has moderate to low susceptibility to the
applied antimicrobial agent. Generally, the studies with animals with
intact host defenses report enhanced therapeutic efficacy of the
liposomal formulation compared to that of the free drug (3, 7,
16, 21). The same conclusion can be drawn from the present study
in the intact host defense model of a high-GEN-susceptible K. pneumoniae pneumonia. As a result of the local delivery of the
antibiotic by LE-GEN, a single dose of the liposome-encapsulated drug
dramatically improves survival compared to an equivalent dose of free
GEN. Increasing the dose of LE-GEN may improve survival to 100%. Yet
the clinical relevance of this approach is limited, as a relatively
short course of treatment for only 3 days with free GEN at 5 mg/kg/day
q12h already yields complete survival. Therefore, the present study was
undertaken to investigate the therapeutic potential of LE-GEN in
clinically more relevant models of infection that are difficult to
treat with conventional antibiotics as a result of impaired host
defense and low antibiotic susceptibility of the inoculated bacteria.
In leukopenic rats inoculated with the high-susceptible K. pneumoniae strain, approximately 10 to 100 bacteria per ml of
blood were already present 24 h after injection. Thus, in these
leukopenic rats, antimicrobial therapy should be directed not only
towards the bacteria at the infectious focus (the left lung) but also towards the rapidly occurring bacteremia. The survival experiments in
the leukopenic model of a high-GEN-susceptible K. pneumoniae pneumonia show that antimicrobial treatment is far less effective as a
result of impaired host defense. Treatment with free GEN at 5 mg/kg/day
q12h for 3 days, which produced complete survival in the rats with
intact host defenses, hardly prolongs survival in the leukopenic
animals. Continuing treatment up to 5 days prolonged survival during
treatment. However, after termination of treatment, only 10% survived
up to 14 days. Doses of free GEN have to be increased up to 40 mg/kg/day for 5 days (the MTD) to obtain complete survival. These data
illustrate why in clinical practice aminoglycosides are used in
combination with other classes of antibiotics to increase the
therapeutic efficacy under these conditions.
Addition of a single dose of LE-GEN at 5 mg/kg on day 1 to the 5-day
treatment with free GEN at 5 mg/kg/day q12h appeared to confer
substantial therapeutic benefit. All rats survived, and the cumulative
amount of GEN administered was sevenfold lower than the amount of free
GEN (40 mg/kg/day for 5 days) needed to obtain complete survival. This
reduction in GEN exposure may reduce the risk of the well-known
toxicity of GEN on the kidney and audiovestibular apparatus. On the
other hand, the altered tissue distribution in general and increased
GEN concentrations at the site of infection in particular as a result
of the liposome encapsulation might change the toxicity profile. Yet
previous studies in beagle dogs with liposome-encapsulated amikacin
suggest a favorable safety profile for liposome-encapsulated
aminoglycosides, as doses of 20 mg/kg/day for 1 month did not result in
adverse effects despite steady-state plasma concentrations of 750 µg/ml (14). Rats treated with only LE-GEN at 5 mg/kg for
5 days already show mortality during treatment. The blood clearance
kinetics of free GEN and LE-GEN offer an explanation for the superior
efficacy of the combination of free GEN and LE-GEN. Free GEN is
therapeutically active in the circulation against the bacteremia but is
rapidly cleared after injection, with a half-life of approximately 20 min. Activity in the infected left lung is expected to be limited. When
encapsulated in liposomes, on the other hand, the drug only slowly
leaves the circulation. LE-GEN hardly releases GEN into the bloodstream
and consequently shows limited activity against bacteremia but
localizes substantially in the infected left lung (3).
Release of GEN from the liposomes localizing in the infected lung leads
to the efficient bacterial killing seen. As a result, combination of free GEN and LE-GEN reduces the numbers of bacteria in left-lung tissue
and blood 10- and 100-fold more efficiently, respectively, than the
treatment with free GEN alone. These numbers were further reduced to
zero in the remaining study period. In contrast, the majority of rats
treated with free GEN or LE-GEN alone died during this period. The
third model addresses an additional factor complicating clinical
antimicrobial therapy, i.e., low antibiotic susceptibility of the
bacteria. The eightfold increase in the MIC clearly had an effect on
the efficacy of treatment, as free GEN at the MTD was no longer
effective and all rats died. The addition of LE-GEN at 20 mg/kg/day
q24h for 5 days to the 5-day treatment with free GEN at the MTD
resulted in 50% survival on day 14 without producing acute toxicity.
Probably, as a result of the low GEN susceptibility of the K. pneumoniae strain, the therapeutic availability of GEN released
from this rigid liposome formulation in the infected lung is
insufficient to obtain 100% survival. As fluid liposomes have been
reported to release encapsulated aminoglycosides more easily than their
rigid counterparts (7), a fluid LE-GEN formulation was
investigated. Cholesterol was omitted from the liposome formulation, as
cholesterol has a major rigidifying effect on liposomal bilayers (10). Furthermore, the partially hydrogenated phospholipid
PHEPC was replaced by EPC as bilayer rigidity increases with the degree of hydrogenation of the phospholipids in the bilayer. The addition of
fluid LE-GEN instead of rigid LE-GEN to 5-day free-GEN treatment at the
MTD resulted in an increase in therapeutic effect: complete survival
was obtained. The blood clearance kinetics of fluid LE-GEN show that
GEN is released into the circulation to a larger extent from this
liposome formulation than from rigid LE-GEN. As a result, higher
therapeutically active drug concentrations were measured in the
bloodstream. Yet, a sufficient amount of GEN remained liposomally encapsulated to be delivered to the left lung to control the local infection, as is supported by the efficient bacterial killing observed
in the left lung. These results show that lipid composition is an
important determinant of therapeutic efficacy. A careful balance must
be sought between release of antibiotic into the circulation to obtain
sufficiently high drug levels there versus liposomal retention of the
drug in order to achieve sufficiently high levels of (locally released)
antibiotic at the infectious focus.
In conclusion, in rats with intact host defenses infected with a
high-GEN-susceptible K. pneumoniae strain, LE-GEN is clearly superior to free GEN treatment. Yet the clinical relevance is limited,
as complete survival can also be obtained with multiple doses of the
free drug. In leukopenic rats infected with high-GEN-susceptible K. pneumoniae cells, the addition of LE-GEN to free GEN
treatment shows substantial therapeutic benefit. Complete survival can
be obtained by using a sevenfold-lower amount of GEN compared to administration of free GEN alone. In leukopenic rats infected with
low-GEN-susceptible K. pneumoniae cells, free GEN at the MTD
shows 0% survival. The use of LE-GEN is a strict requirement for
achieving therapeutic success. It appears that the increased release of
GEN by fluid LE-GEN compared to rigid LE-GEN is more favorable. These
results warrant further clinical studies of liposomal formulations of
aminoglycosides in immunocompromised patients with severe infections.
| |
ACKNOWLEDGMENTS |
This work was supported by grant 902-21-161 from the Dutch
Organization for Scientific Research (N.W.O.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Ee1751,
Department of Medical Microbiology & Infectious Diseases, Erasmus
University Medical Center Rotterdam, P.O. Box 1738, 3000 DR Rotterdam,
The Netherlands. Phone: 31 10 4087666. Fax: 31 10 4089454. E-mail: bakker{at}kmic.fgg.eur.nl.
| |
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Antimicrobial Agents and Chemotherapy, February 2001, p. 464-470, Vol. 45, No. 2
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.2.464-470.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
