ANTIMICROBIAL AGENTS AND CHEMOTHERAPY,
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
May 2001, p. 1487–1492Vol. 45, No. 5
Improved Efficacy of Ciprofloxacin Administered in Polyethylene
Glycol-Coated Liposomes for Treatment of Klebsiella
pneumoniae Pneumonia in Rats
IRMA A. J. M. BAKKER-WOUDENBERG,1* MARIAN T. TEN KATE,1LUKE GUO,2
PETER WORKING,2AND JOHAN W. MOUTON3
Department of Medical Microbiology & Infectious Diseases, Erasmus University Medical Center Rotterdam,
3000 DR Rotterdam,1and Department of Medical Microbiology, Canisius Wilhelmina Hospital,
Nijmegen,3The Netherlands, and ALZA Corporation, Mountain View, California2
Received 12 July 2000/Returned for modification 12 November 2000/Accepted 7 February 2001
Animal and clinical data show that high ratios of the area under the concentration-time curve and the peak
concentration in blood to the MIC of fluoroquinolones for a given pathogen are associated with a favorable
outcome. The present study investigated whether improvement of the therapeutic potential of ciprofloxacin
could be achieved by encapsulation in polyethylene glycol (PEG)-coated long-circulating sustained-release
liposomes. In a rat model of unilateral Klebsiella pneumoniae pneumonia (MIC ? 0.1 ?g/ml), antibiotic was
administered at 12- or 24-h intervals at twofold-increasing doses. A treatment period of 3 days was started 24 h
after inoculation of the left lung, when the bacterial count had increased 1,000-fold and some rats had positive
blood cultures. The infection was fatal within 5 days in untreated rats. Administration of ciprofloxacin in the
liposomal form resulted in delayed ciprofloxacin clearance and increased and prolonged ciprofloxacin con-
centrations in blood and tissues. The ED50(dosage that results in 50% survival) of liposomal ciprofloxacin was
3.3 mg/kg of body weight/day given once daily, and that of free ciprofloxacin was 18.9 mg/kg/day once daily or
5.1 mg/kg/day twice daily. The ED90of liposomal ciprofloxacin was 15.0 mg/kg/day once daily compared with
36.0 mg/kg/day twice daily for free ciprofloxacin; 90% survival could not be achieved with free ciprofloxacin
given once daily. In summary, the therapeutic efficacy of liposomal ciprofloxacin was superior to that of
ciprofloxacin in the free form. PEG-coated liposomal ciprofloxacin was well tolerated in relatively high doses,
permitting once daily administration with relatively low ciprofloxacin clearance and without compromising
The pharmacodynamic and pharmacokinetic properties of
antimicrobial agents have been intensively investigated in the
recent past, years resulting in the optimization of dosage reg-
imens. In vitro studies and dose-effect studies of experimental
infections in animals have been performed with various classes
of antibiotics to investigate which pharmacodynamic param-
eters determine therapeutic efficacy (9, 24, 26, 28, 42). Com-
parative studies of humans with different doses and dosage
regimens have also been utilized to optimize antimicrobial
treatment (9, 24).
The fluoroquinolones are concentration dependent in their
rates of bacterial killing. The area under the concentration-
time curve (AUC)/MIC ratio is the parameter that best corre-
lates with the therapeutic efficacy of these agents. In addition,
a sufficiently high peak concentration in serum/MIC ratio
seems necessary to suppress the emergence of resistant mu-
tants during treatment. This can be concluded from in vitro
models that simulate human pharmacokinetics to study the
effects of changing the concentrations of fluoroquinolones on a
number of pathogens (6, 12, 13, 15, 23, 29, 32). For fluoro-
quinolones, a 24-h AUC/MIC ratio of ?125 is needed for rapid
bacterial eradication, whereas a peak serum drug concentra-
tion/MIC ratio of ?8 is needed to prevent the selection of
The effect of dose or dose interval on the therapeutic efficacy
of fluoroquinolones has been evaluated in experimental infec-
tions such as pneumonia, sepsis, peritonitis, and thigh infection
caused by gram-negative pathogens in mice, rats, guinea pigs,
and rabbits (11, 19, 20, 27, 33, 41). Dosage schedules resulting
in high AUC/MIC ratios and high peak serum drug concen-
tration/MIC ratios of 10 or 20 effected higher therapeutic ef-
ficacy than did regimens in which a more fractionated schedule
was used at the same daily dose (24, 28).
In clinical studies with fluoroquinolones, the AUC/MIC ra-
tio and the peak serum drug concentration/MIC ratio are im-
portant predictors of both clinical and microbiological cure
(16, 24, 28, 36). Forrest et al. investigated the pharmacody-
namics of intravenous ciprofloxacin in seriously ill patients and
found that a 24-h AUC/MIC ratio of ?125 was significant for
a satisfactory clinical and microbiological outcome (16). The
data from this study show that most treatment failures with
ciprofloxacin are consequences of high MIC, low AUC, or
both. More recent clinical studies with grepafloxacin and levo-
floxacin showed that a peak serum drug concentration/MIC
ratio of 10 or greater was associated with successful outcome;
when the ratio was less than 10 the AUC/MIC ratio was most
closely linked to outcome (17, 38).
Based on the pharmacodynamic properties of the fluoro-
quinolones, as shown in in vitro studies and in dose-response
* Corresponding author. Mailing address: Department of Medical
Microbiology & Infectious Diseases, Erasmus University Medical Cen-
ter Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands.
Phone: 31 10 4087666. Fax: 31 10 4089454. E-mail: firstname.lastname@example.org
studies in animals and patients, regimens of high doses at
infrequent intervals might be most efficacious in terms of erad-
ication time, killing bacteria, and reducing the selection of
drug-resistant bacteria. Relatively infrequent dosing (possibly
once daily) may be superior. However, very high doses of some
quinolones might prove to be toxic when given once daily.
The present study was undertaken to investigate whether
improvement of the therapeutic potential of fluoroquinolones
could be obtained by liposomal encapsulation of the drugs. In
a rat model of left-sided Klebsiella pneumoniae pneumonia, the
therapeutic efficacy of liposome-encapsulated ciprofloxacin
versus free ciprofloxacin was determined. The liposome type
used has shown relatively long blood circulation times, as a
result of coating of the liposome surface with polyethylene
glycol (PEG), and the PEG-coated liposomes are engineered
to release ciprofloxacin slowly. Liposomal encapsulation re-
sults in a decrease in toxic side effects of the drug, permitting
the use of relatively high doses. The aim of using liposomes as
carriers of ciprofloxacin in the present study was, first, to delay
ciprofloxacin clearance and achieve sustained liposomal re-
lease of ciprofloxacin in the blood over time, thus extending
ciprofloxacin activity in the blood (increased AUC) and tissues.
The second objective was to permit relatively high doses and
decreased dose frequency and, as a result, a once-daily treat-
MATERIALS AND METHODS
Animals. Specific-pathogen-free female RP/AEur/RijHsd albino rats (Harlan,
Horst, The Netherlands) were used in all experiments. Animals were 18 to 25
weeks old and weighed 185 to 225 g.
Bacteria. K. pneumoniae ATCC 43816, capsular serotype 2, was used to infect
the rats. The minimum bactericidal concentration of ciprofloxacin for this strain
was 0.1 ?g/ml as determined by the tube dilution test.
Infection model. A left-sided pneumonia was produced as previously described
(2). In brief, rats were anesthetized with fluanisone and fentanyl citrate (Hyp-
norm) (Janssen, Animal Health, Saunderton, United Kingdom), followed by
pentobarbital (Nembutal) (Sanofi Sante ´ b.v., Maassluis, The Netherlands). The
left primary bronchus was intubated, and the left lung was inoculated with 0.02
ml of a saline suspension containing 106viable K. pneumoniae bacteria in the
logarithmic phase of growth. After bacterial inoculation, the narcotic antagonist
nalorphine bromide (Onderlinge Pharmaceutische Groothandel, Utrecht, The
Netherlands) was injected. Inoculation of the lung resulted in an acute unilateral
pneumonia. The course of the infection was assessed by measuring the number
of viable bacteria in the infected left lung, the right lung, and the blood. Animals
were sacrificed by CO2inhalation, a blood sample was taken, and the left and
right lungs were removed and homogenized (VirtTis, Gardiner, N.Y.) in 20 ml of
phosphate-buffered saline for 30 s at 10,000 rpm. Lung homogenate suspensions
and blood were serially diluted and plated on tryptone soy agar (Unipath Ltd.,
Basingstoke, United Kingdom). At 24 h after bacterial inoculation, the bacterial
numbers in the left lung had increased 103-fold, up to 109(range, 4 ? 108to 5 ?
109; n ? 10). Untreated rats developed septicemia and pleuritis; at 24 h after
inoculation some rats had positive blood cultures. Untreated rats died between
day 3 and day 6 after bacterial inoculation.
Liposomes. PEG-coated liposome preparations of ciprofloxacin (PL Cipro)
consisted of the PEG 2000 derivative of distearoylphosphatidylethanolamine,
hydrogenated soybean phosphatidylcholine, and cholesterol in a molar ratio of
5:50:45. Ciprofloxacin-containing liposomes and placebo liposomes were kindly
supplied by ALZA Corporation (Mountain View, Calif.). Mean particle size was
determined by dynamic light scattering (4700 system; Malvern Instruments,
Malvern, United Kingdom). The ciprofloxacin-containing liposomes had a mean
particle size of 107 ? 7 nm and contained 256 ? 51 ?g of ciprofloxacin/?mol of
total lipid (mean ? standard deviation [SD] of 10 preparations). The mean
particle size of the placebo liposomes was 105 ? 6 nm (mean ? SD of two
Radiolabeling of liposomes. Pharmacokinetics and biodistribution of intact
liposomes were determined by the use of a high-affinity67Ga-deferoxamine-
mesylate (67Ga-DF) complex as an aqueous liposomal marker (44). As shown by
Gabizon et al. (18), this complex is appropriate for in vivo tracing of intact
liposomes because of the advantages of minimal translocation of radioactive
labels to plasma proteins and the rapid renal clearance rate when the label is
released from the liposomes.67Ga was obtained as67Ga-citrate from Mallinck-
rodt Medical b.v., Petten, The Netherlands. The labeling was performed as
described by Gabizon et al. (18). The radiolabeling resulted in the formation of
a67Ga-DF complex in the aqueous interior of the liposomes. Nonentrapped DF
and radiolabels were removed by gel filtration on a Sephadex G-50 column
eluted with HEPES buffer. The circulation times of liposomes in the blood and
localization in the infected left lung, right lung, liver, spleen, and kidneys were
determined using the67Ga-DF complex as a marker for intact liposomes.
Pharmacokinetics and biodistribution. The pharmacokinetics and biodistri-
bution of antimicrobially active ciprofloxacin after administration in the free or
liposome-encapsulated form were determined in rats at 24 h after bacterial
inoculation. At different intervals after intravenous (i.v.) administration, blood
samples were obtained by retro-orbital bleeding under CO2anesthesia. Then the
rats were sacrificed, and the left lung, right lung, spleen, liver, and kidneys were
removed.67Ga served as a marker for intact liposomes and was quantitated in a
Minaxi Autogamma 5000 gamma counter (Packard Instrument Company, Me-
riden, Conn.). Correction for the blood content of the tissues was done using
111In-oxine-labeled syngeneic erythrocytes, injected i.v. 10 min before dissection.
Erythrocytes were labeled as previously described (25). Ciprofloxacin concentra-
tions in the blood and tissues were determined as previously described (5). In
short, with diagnostic sensitivity test agar (Oxoid, Basingstoke, United Kingdom)
and an Escherichia coli test strain susceptible to 0.025 ?g of ciprofloxacin per ml,
all tests were done by a standard large-plate agar diffusion procedure. Samples of
100 ?l were assayed. Twofold-increasing standard concentrations ranging from
0.1 to 1.6 ?g of ciprofloxacin per ml were used. The assay system was sensitive to
0.1 ?g of ciprofloxacin per ml. The coefficient of variation of 15 determinations
of solutions containing 0.1 to 1.6 ?g of ciprofloxacin per ml was 1 to 3%.
Blood and tissue homogenates from rats that received PL Cipro were incu-
bated in 0.1% Triton X-100 for 30 min at 25°C to disintegrate intact liposomes
before determination of the presence of antimicrobially active ciprofloxacin.
After centrifugation of the samples for 5 min at 12,000 ? g, ciprofloxacin
concentrations in the supernatant were determined. To exclude the possibility
that the presence of Triton X-100 in the unknown samples may have had an
effect on the bacterial growth inhibition zone on the agar plates, the standard
concentrations were also incubated with Triton X-100.
Toxicity. The maximum tolerated dose (MTD) was assessed using various
parameters. Acute toxicity was characterized in terms of seizures, irritability, and
an apparent dazed state. Long-term toxicity was assessed in terms of a significant
change in renal or hepatic function. Renal function abnormalities were deter-
mined by measuring blood urea nitrogen and serum creatinine; hepatic function
abnormalities were detected by measuring the serum aspartate aminotransferase
and alanine aminotransferase by established tests (Merck Diagnostica, Darm-
Antimicrobial treatment. Ciprofloxacin in the free form (CIP) or PL Cipro was
administered at 24 h after bacterial inoculation of the left lung. The doses were
escalated by twofold increases (n, 8 to 10 per dosage), ranging from 0.3 to 80
mg/kg of body weight/day. The injection frequency was 12 or 24 h for CIP and
24 h for PL Cipro. The duration of treatment was 3 days in all cases. Therapeutic
efficacy was assessed by the survival of rats at day 21 after bacterial inoculation.
Estimates of ED50(dosage that effects 50% survival of rats) and ED90were
obtained using the PROBIT procedure from the SAS program (SAS user’s
guide, SAS Institute Inc., Cary, N.C.), assuming a logistic distribution of data.
Postmortem cultures of the left lung and blood from rats were performed to
check for the presence of K. pneumoniae only, as well as for susceptibility to
ciprofloxacin. Cultures were also done for rats that survived at day 21 postin-
Blood circulation time of liposomes and ciprofloxacin. After
i.v. administration, intact liposomes demonstrated a relatively
long blood residence time, with a half-life of approximately
18 h (Fig. 1). This long-term circulation in blood was not
altered when the liposomes contained encapsulated ciprofloxa-
cin. Figure 2 shows that ciprofloxacin is slowly released from
the intact liposomes in blood. Within 6 h after i.v. administra-
tion about 90% of the encapsulated ciprofloxacin is released,
while the liposomes remain intact (Fig. 2). The concentrations
1488BAKKER-WOUDENBERG ET AL.ANTIMICROB. AGENTS CHEMOTHER.
of ciprofloxacin indicated in Fig. 2 represent primarily PL
Cipro, since it is known that CIP rapidly diffuses from intra-
vascular to extravascular space.
Concentration of ciprofloxacin in blood after administra-
tion in the free or liposome-encapsulated form. Concentra-
tions of ciprofloxacin in the blood were determined at various
intervals after i.v. administration of 20 mg/kg as a single dose.
Total (CIP plus PL Cipro) concentrations of ciprofloxacin are
presented in Table 1. At 3 min after administration of CIP only
5% of the injected dose was present in blood, and at 30 min
only 1% was present, corresponding with 5 ?g of ciprofloxa-
cin/ml. In contrast, after treatment with PL Cipro, 77% of the
injected dose was still circulating in the blood 30 min after
administration. Administration of ciprofloxacin in the lipo-
some-encapsulated form resulted in a substantial increase in
AUC0–6 h, 800 ?g ? h/ml for PL Cipro compared to 15.8 ?g ?
h/ml for CIP.
Toxic side effects. Toxicity was assessed using various param-
eters in a treatment schedule at twofold-increasing doses for a
period of 3 days, with PL Cipro administered once daily and
CIP administered once or twice daily. The MTD for CIP was
40 mg/kg/dose. At higher doses, acute toxicity was observed
(e.g., seizures, irritability followed by an apparent dazed state)
shortly after the first dose. After administration of PL Cipro,
no signs of acute toxicity were observed at dosages up to 160
mg/kg/dose. Long-term toxicity was investigated at dosage
schedules yielding 100% survival of rats: 40 mg of CIP/kg/dose
twice daily and 20 mg of PL Cipro/kg/day once daily. At 12 or
24 h after the last dose of CIP or PL Cipro, respectively,
significant abnormalities in renal or hepatic functions were not
observed. Thus, PL Cipro was well tolerated at doses above the
MTD of CIP.
Therapeutic efficacy of PL Cipro versus CIP. In untreated
rats, after inoculation of the left lung with 106CFU of K.
pneumoniae, the infection in the left lung developed progres-
sively, whereas no infection developed in the right lung.
Antimicrobial treatment was started at 24 h after bacterial
inoculation, when the bacterial count in the left lung had in-
creased approximately 103-fold, to 3 ? 109CFU (range, 5 ?
108to 8 ? 109; n ? 10), and 7 out of 10 rats had developed
positive blood cultures. All untreated rats died between day 3
and day 6 after bacterial inoculation. The parameter for ther-
apeutic efficacy of antimicrobial treatment was the survival of
rats assessed for a period of 21 days after bacterial inoculation.
Treatment with CIP twice daily was effective in a dose-depen-
dent manner and resulted in 100% survival of rats at doses that
were well tolerated (Table 2). When CIP was administered
once daily, 100% survival of rats could not be achieved at doses
below the MTD. In contrast, the administration of PL Cipro
once daily was fully effective at a dosage of 20 mg/kg/day.
ED50sand ED90scalculated from the survival data demon-
strate that the therapeutic efficacy of PL Cipro was superior to
that of CIP (Table 2). PL Cipro was significantly (P ? 0.05)
more effective than CIP in achieving 50% survival of rats with
once-daily administration and slightly, but not significantly,
more active than CIP given twice daily. The difference between
FIG. 1. Levels of
floxacin or placebo liposomes in blood. Liposomes were injected (inj.)
i.v. as a single dose (ciprofloxacin, 20 mg/kg; total lipid, 83 ?mol/kg) in
rats at 24 h after inoculation of K. pneumoniae in the left lung. Levels
of67Ga-DF label in the blood after injection of ciprofloxacin-contain-
ing liposomes (G) or placebo liposomes (?) were determined. Data
are expressed as mean ? SD for six rats.
67Ga-DF-labeled liposomes containing cipro-
FIG. 2. Levels of
floxacin in blood. Liposomes were injected (inj.) i.v. as a single dose
(ciprofloxacin, 20 mg/kg; total lipid, 83 ?mol/kg) into rats at 24 h after
inoculation of K. pneumoniae in the lung. Levels of67Ga-DF label and
antimicrobially active ciprofloxacin in the blood were determined.
Data are expressed as mean ? SD for six rats.
67Ga-DF-labeled liposomes containing cipro-
TABLE 1. Concentrations of total ciprofloxacin in blood at various
intervals after i.v. administration of PL Cipro or CIP in
rats with K. pneumoniae lung infectiona
Concn (?g/ml) (% of injected dose) of
ciprofloxacin in blood after treatment with:
17.6 ? 1.6 (4.7)
8.6 ? 2.1 (2.3)
5.0 ? 1.2 (1.3)
3.3 ? 1.1 (0.9)
291 ? 32 (77)
212 ? 30 (56)
97 ? 7.5 (26)
35 ? 7.0 (9.2)
aPL Cipro (20 mg/kg; total lipid, 86 ?mol/kg) or CIP (20 mg/kg) was injected
as a single dose into rats at 24 h after inoculation of K. pneumoniae in the left
lung. Total (CIP plus PL Cipro) concentrations of ciprofloxacin are presented.
Data are expressed as mean ? SD six rats.
bAUC0–6hwas 800 ?g ? h/ml.
cAUC0–1hwas 15.8 ?g ? h/ml.
VOL. 45, 2001THERAPEUTIC EFFICACY OF PL Cipro1489
PL Cipro and CIP with once-daily administration is even more
striking at the ED90. Survival of 90% of rats could not be
achieved with once-daily treatment with CIP; twice-daily treat-
ment was required. In contrast, PL Cipro given once daily was
effective. The ED90daily dose for CIP given twice daily was
slightly higher than the ED90daily dose for PL Cipro given
As shown in Fig. 3, the administration of placebo liposomes
had no effect on the mortality rate. In this figure, the mortality
rates of rats treated with PL Cipro at doses of 20 mg/kg, 2.5
mg/kg, or 0.3 mg/kg for 3 days are also represented.
K. pneumoniae was present only in the infected left lung of
rats that had died. In addition, the susceptibility to ciprofloxa-
cin of the bacteria that were recovered from the infected left
lung tissue of treated rats appeared to be unchanged.
Biodistribution of PL Cipro versus CIP. Concentrations of
ciprofloxacin (CIP plus PL Cipro) in different organs and the
blood of infected rats were determined at 1- and 6-h intervals
after administration of a single dose of CIP or PL Cipro at 20
mg/kg (Table 3). When rats were sacrificed 1 h after injection
of CIP, about 1% of the injected dose was still present in the
blood. Rats treated with PL Cipro were sacrificed at 1 or 6 h
after administration. One hour after the administration of PL
Cipro, the total recovery of ciprofloxacin based on its levels in
the blood, liver, spleen, kidney, and lung was 75% of the
injected dose. In contrast, when CIP was administered, recov-
ery was only 3% of the injected dose at the same time point.
Concentrations of ciprofloxacin in tissue were substantially
increased following administration in the liposomal form com-
pared with injected CIP. The degrees of localization of cipro-
floxacin in the infected left lung and in the uninfected right
lung were not different and appeared to decrease with time.
A possible approach for intensifying antibiotic treatment
may be the use of an appropriate delivery system, such as
liposomes, which may enhance antibiotic pharmacokinetics or
penetration to infected sites. Liposomes are considered to be
versatile delivery systems. Depending on the liposomal size
and physicochemical characteristics, which can be manipulated
by changing the lipid composition (surface charge, surface
coating, bilayer rigidity), liposomes can be used in various
ways. One rationale for using liposomes as carriers of antibi-
otics is to reduce the toxicity of potentially toxic antibiotics
such as amphotericin B (21, 22). Liposomes can also be ex-
ploited to achieve high and prolonged intracellular antibiotic
concentrations in infected cells (3, 37). Another application of
liposomes targets the delivery of antibiotics to infected tissues.
In this respect, previous studies in this experimental model of
K. pneumoniae pneumonia in rats have demonstrated a sub-
stantial increase in therapeutic efficacy for gentamicin or cefta-
zidime resulting from administration in long-circulating lipo-
somes (4, 40). Finally, liposomes carrying antibiotics may also
be used as microreservoirs of antibiotics during circulation.
Again, long-circulating liposomes are needed for this purpose.
FIG. 3. Survival of rats with K. pneumoniae lung infection after
administration of PL Cipro at 20 mg/kg (G) (n ? 8), 2.5 mg/kg (f) (n ?
8), or 0.3 mg/kg (?) (n ? 5); placebo liposomes (?) (n ? 10); or buffer
(?) (n ? 10) at 24, 48, and 72 h after bacterial inoculation of the lung.
Total lipid doses were 81 ?mol/kg/dose.
TABLE 2. Therapeutic efficacy of treatment of rats
with K. pneumoniae lung infectiona
No. of surviving rats/total no. of rats receiving treatment
CIP twice daily CIP once dailyPL Cipro once daily
aTreatment was started at 24 h after inoculation of K. pneumoniae in the lung
and continued for 3 days. Survival of rats was assessed for a period of 21 days.
Untreated rats died before day 6 after inoculation.
bED50s and ED90s are expressed as milligrams per kilograms per day. Values
in parentheses are 95% confidence intervals.
cToxic side effects (acute toxicity) were observed in four out of eight rats.
TABLE 3. Biodistribution of total ciprofloxacin at various intervals
after i.v. administration of PL Cipro or CIP in rats
with K. pneumoniae lung infectiona
Concnb(% of injected dose) of ciprofloxacin at time
6 h, PL Cipro
3.3 ? 1.1 (0.9)
33 ? 4.2 (0.8)
7.3 ? 0.5 (0.2)
42 ? 17 (1.0)
3.7 ? 0.4 (0.1)
4.6 ? 0.9 (0.1)
212 ? 32 (56)
552 ? 50 (14)
68 ? 4.1 (1.7)
44 ? 19 (1.1)
54 ? 20 (1.4)
47 ? 21 (1.2)
35 ? 7.0 (9.2)
83 ? 27 (2.0)
134 ? 22 (3.2)
9.5 ? 3.3 (0.2)
12 ? 2.9 (0.3)
7.5 ? 3.4 (0.2)
aPL Cipro (20 mg/kg; total lipid, 86 ?mol/kg) or CIP (20 mg/kg) was injected
as a single dose into rats at 24 h after inoculation of K. pneumoniae in the left
lung. Total (CIP plus PL Cipro) concentrations of ciprofloxacin are presented.
bConcentrations of ciprofloxacin (mean ? SD for six rats) are presented as
micrograms per milliliter of blood or per organ.
1490BAKKER-WOUDENBERG ET AL.ANTIMICROB. AGENTS CHEMOTHER.
Experimental evidence to support the application of liposomes
in this way is provided in the present study.
In the current study, liposomes were used first to influence
the pharmacokinetics of ciprofloxacin, thus extending cipro-
floxacin activity in the blood and tissues, and secondly to pro-
tect encapsulated ciprofloxacin, which facilitates the use of
relatively high doses and possibly once-daily dosing. The lipo-
somes exhibited sustained liposomal release of ciprofloxacin in
the blood over time while remaining intact. In earlier studies
with PEG-coated liposomes containing gentamicin or ceftazi-
dime, the liposomes retained their content during circulation
(4), and as a consequence, substantial targeting of liposomal
antibiotic to the infected left lung tissue was observed (40). In
the present study, as expected, targeting of liposomal cipro-
floxacin to the infected tissue was not achieved, because of the
relatively rapid release of the antibiotic from the liposomes.
Ciprofloxacin concentrations in the infected left lung and un-
infected right lung were similar. However, the administration
of PL Cipro resulted in relatively low ciprofloxacin clearance,
prolonged ciprofloxacin concentrations in the blood, and in-
creased ciprofloxacin concentrations in both infected and un-
infected tissues. Probably as a result of this, the therapeutic
efficacy of PL Cipro was superior to that of CIP. It was also
observed that liposomal ciprofloxacin was well tolerated in
relatively high doses and could be administered once daily
without compromising its therapeutic efficacy.
Our observation that prolonged residence of ciprofloxacin in
the blood is important for therapeutic efficacy agrees with the
findings of others investigating the therapeutic efficacy of fluo-
roquinolones in the free form at various dose schedules in
animal infection models. In models of pneumonitis caused by
K. pneumoniae and thigh infection caused by K. pneumoniae or
Pseudomonas aeruginosa in mice, Leggett et al. investigated
dose-effect relations for ciprofloxacin (27). It was shown that
the AUC/MIC ratio was the variable most closely linked to
outcome, even though the peak serum drug concentration/
MIC ratio was ?10. The dosing interval had little impact. In
contrast, Drusano et al. emphasized the role of the dosing
interval for therapeutic efficacy. They examined the impact of
dose fractionation and altered MICs in a neutropenic rat
model of P. aeruginosa sepsis using lomefloxacin (11). Once-
daily administration of the drug, produced a peak serum drug
concentration/MIC ratio of 20 and resulted in better efficacy
than a more fractionated treatment schedule at the same daily
dose. At lower doses producing peak serum drug concentra-
tion/MIC ratios of ?10, the AUC/MIC ratio appeared to be
most closely linked to outcome. The relative importance of the
peak serum drug concentration/MIC ratio for therapeutic ef-
ficacy has also been demonstrated in a model of P. aeruginosa
pneumonia in neutropenic guinea pigs. Gordin et al. showed
that ciprofloxacin and pefloxacin had the same rate of bacterial
killing when the peak serum drug concentration/MIC ratio was
the same for each agent (19). Similar conclusions can be drawn
from studies by Hackbarth et al. and Shibl et al. that examined
the efficacy of ciprofloxacin and pefloxacin in experimental
meningitis caused by P. aeruginosa or E. coli, respectively, in
rabbits (20, 41). Both studies demonstrated that the peak se-
rum drug concentration/MIC ratio was predictive for bacterial
killing in cerebrospinal fluid.
The PEG-coated liposomes used in the present study show a
relatively long blood residence time due to decreased uptake
by the mononuclear phagocyte system (1). Long-term circula-
tion of liposomes is needed to achieve prolonged ciprofloxacin
activity in blood. Other investigators using liposomes contain-
ing fluoroquinolones focused on the treatment of intracellular
infections, which are difficult to treat due to poor penetration
of antibiotics into the infected cells or decreased intracellular
activity. In these studies, classical non-PEG-coated liposomes
were used, which rapidly accumulate in cells of the mononu-
clear phagocyte system after i.v. administration, resulting in
increased intracellular concentrations. Enhanced efficacy of
ciprofloxacin in the protection and treatment of mice with
intracellular Francisella tularensis infection was demonstrated
when it was administered in the liposomal form i.v. intranasally
(10), or by aerosol delivery (8). Liposomal ciprofloxacin also
appeared effective in the i.v. treatment of intracellular infec-
tions caused by Salmonella enterica serovar Dublin (30) or S.
enterica serovar Typhimurium (43) in mice. In vitro studies in
which monocytes or macrophages in culture were infected with
Mycobacterium avium-M. intracellulare complex and exposed to
ciprofloxacin (31, 34) or ofloxacin (35) in the free or liposome-
encapsulated form also showed the superiority of the lipo-
some-encapsulated drugs. In contrast, free and liposomal spar-
floxacin had similar effects on the growth of intracellular M.
avium-M. intracellulare complex (14).
Sustained drug release of fluoroquinolones from liposomes
has also been demonstrated for enrofloxacin encapsulated in
non-PEG-coated liposomes composed of phosphatidylcholine
and cholesterol. This formulation was administered intramus-
cularly in rabbits and provided therapeutic and prolonged
plasma concentrations (7). In addition, ciprofloxacin liposomes
have been used for external coating of catheters to prevent
catheter-associated urinary tract infections in a rabbit model
For the fluoroquinolones, animal data showing that high
ratios of the AUC and the peak concentration in blood to the
MIC for the pathogen are associated with favorable outcomes
are in agreement with the clinical data. For infections caused
by highly susceptible bacteria, the optimal AUC/MIC ratio and
peak serum drug concentration/MIC ratio criteria can easily be
reached. The difficulty lies in infections caused by bacteria such
as Staphylococcus and Pseudomonas species that are only mar-
ginally susceptible to fluoroquinolones (MICs ? 0.5 ?g/ml). In
future studies the efficacy of PL Cipro will be investigated in a
model of P. aeruginosa pneumonia and septicemia in rats.
The financial support of ALZA Corporation (Mountain View, Cal-
if.) is gratefully acknowledged.
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1492BAKKER-WOUDENBERG ET AL.ANTIMICROB. AGENTS CHEMOTHER.