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Antimicrobial Agents and Chemotherapy, January 1998, p. 45-52, Vol. 42, No. 1
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Antibacterial Efficacy against an In Vivo Salmonella
typhimurium Infection Model and Pharmacokinetics of a Liposomal
Ciprofloxacin Formulation
Murray S.
Webb,1,*
Nancy L.
Boman,1
David J.
Wiseman,1
Dawn
Saxon,1
Kym
Sutton,1
Kim F.
Wong,2
Patricia
Logan,1 and
Michael J.
Hope1,3
Inex Pharmaceuticals Corporation, Burnaby,
British Columbia, Canada V5J 5J81;
Liposome Research Unit, Department of Biochemistry,
University of British Columbia Vancouver, British Columbia, Canada
V6T 4E62; and
Skin Barrier Research
Laboratory, Department of Medicine, University of British Columbia,
Vancouver, British Columbia, Canada V6T 2B53
Received 7 May 1997/Returned for modification 10 October
1997/Accepted 27 October 1997
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ABSTRACT |
The fluoroquinolone antibiotic ciprofloxacin has been encapsulated
into large unilamellar vesicles (LUV) at efficiencies approaching 100%. Drug accumulation proceeded in response to a transmembrane gradient of methylammonium sulfate and occurred
concomitantly with the efflux of methylamine. A mechanism for the
encapsulation process is described. LUV composed of
dipalmitoylphosphatidylcholine-cholesterol (DPPC/chol),
distearoylphosphatidylcholine-cholesterol (DSPC/chol), or
sphingomyelin-cholesterol (SM/chol) increased the circulation lifetime
of ciprofloxacin after intravenous (i.v.) administration by >15-fold.
The retention of ciprofloxacin in liposomes in the circulation
decreased in the sequence SM/chol > DSPC/chol > DPPC/chol. Increased circulation lifetimes were associated with enhanced delivery
of the drug to the livers, spleens, kidneys, and lungs of
mice. Encapsulation of ciprofloxacin also conferred significant increases in the longevity of the drug in the plasma after
intraperitoneal administration and in the lungs after intratracheal
administration in comparison to free ciprofloxacin. The efficacy of a
single i.v. administration of an SM/chol formulation of ciprofloxacin was measured in a Salmonella typhimurium infection model.
At 20 mg of ciprofloxacin per kg of body weight, the encapsulated
formulation resulted in 103- to 104-fold fewer
viable bacteria in the livers and spleens of infected mice than was
observed for animals treated with free ciprofloxacin. These results
show the utility of liposomal encapsulation of ciprofloxacin in
improving the pharmacokinetics, biodistribution, and antibacterial efficacy of the antibiotic. In addition, these formulations are well
suited for i.v., intraperitoneal, and intratracheal or aerosol administration.
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INTRODUCTION |
Ciprofloxacin is a synthetic
bactericidal fluoroquinolone antibiotic which inhibits the activity of
bacterial DNA gyrase, resulting in the degradation of bacterial DNA by
exonuclease activity. Consequently, ciprofloxacin has broad-spectrum
efficacy against a wide variety of bacteria, including
Staphylococcus aureus, streptococci, Pseudomonas
aeruginosa, Klebsiella pneumoniae, Mycobacterium
tuberculosis, and Mycobacterium avium complex (9,
11, 42). For example, a comprehensive study showed excellent in
vitro ciprofloxacin activity against >20,000 clinical isolates of
members of the family Enterobacteriaceae,
non-Enterobacteriaceae gram-negative bacteria, gram-positive
bacteria, anaerobic bacteria, and other types of bacteria
(37). Despite the enormous success with ciprofloxacin, there
are some factors which limit the drug's clinical utility, such as its
poor solubility at physiological pH, bitter taste in solution, and
rapid renal clearance. For example, in order to administer a typical
0.5-g intravenous (i.v.) dose, the drug must first be diluted to <2
mg/ml and infused slowly to avoid precipitation at the site of
injection.
Encapsulation of therapeutic agents in liposomal carriers is known to
be an effective method for reducing drug toxicity, for increasing the
circulation longevity of drugs after parenteral administration, and for
increasing the accumulation of drugs at sites of disease. In
particular, liposomal encapsulation has been shown to improve the
therapeutic index of anticancer agents, such as doxorubicin (23,
46) and vincristine (4, 24, 26, 27, 46, 48), as well
as antibiotics, such as gentamicin (1, 17, 31, 33),
streptomycin (7, 47), vancomycin (36), amikacin
(1, 8, 35), cefoxitin (18), and ofloxacin (10). One explanation for the enhanced efficacy observed for encapsulated antibiotics in animal models is the natural targeting of
lipid carriers to the fixed macrophages of the reticuloendothelial system, which often harbor microorganisms and protect them from free
drug (18).
Ciprofloxacin passively encapsulated in multilamellar vesicles (MLV)
has shown enhanced in vitro efficacy against Mycobacterium avium-Mycobacterium intracellulare complex infection in human peripheral blood mononuclear cells (22). Similarly,
ciprofloxacin loaded into unilamellar liposomes has shown enhanced in
vitro activity against a Mycobacterium avium infection of
murine J774 macrophages (34). Moreover, several reports have
demonstrated enhanced in vivo efficacy against Francisella
tularensis, Brucella melitensis, and Salmonella
dublin infections in mice with ciprofloxacin passively
encapsulated in MLV (6, 21, 49). It should be noted,
however, that the drug pharmacokinetics, accumulation at infection
site, and antibacterial efficacy will be significantly altered both by
the liposome size (16, 50) and by the rate of drug leakage.
In general, a drug that is actively retained inside a carrier by a
transmembrane ion gradient leaks more slowly than a drug that has been
passively encapsulated (5, 15).
This paper describes and characterizes the active loading of
ciprofloxacin into three different unilamellar liposomal carriers with
a transmembrane gradient of methylammonium sulfate. Previously, we have
shown that ciprofloxacin could not be encapsulated by a simple pH
gradient technique similar to that employed to load doxorubicin
(29) or vincristine (24), but was accumulated when an ammonium sulfate transmembrane gradient was applied. For this
study, a gradient of methylammonium sulfate was used to create the
proton gradient responsible for drug accumulation. Other ammonium salts
can be employed as long as the neutral amine formed after complete
ionization can diffuse out of the vesicle (see Discussion for details
concerning the mechanism of drug loading). In addition, we have
evaluated the effect of liposomal encapsulation on drug pharmacokinetics after i.v., intraperitoneal (i.p.), and intratracheal administration and describe the effects of encapsulation on the antibacterial efficacy of ciprofloxacin against an intracellular Salmonella typhimurium infection in mice.
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MATERIALS AND METHODS |
Materials.
Egg sphingomyelin (SM) was obtained from Avanti
Polar Lipids (Alabaster, Ala.), dipalmitoylphosphatidylcholine (DPPC)
and distearoylphosphatidylcholine (DSPC) were obtained from Northern Lipids (Vancouver, British Columbia, Canada), and cholesterol was
obtained from Sigma Chemical Company. Ciprofloxacin hydrochloride and
[14C]ciprofloxacin were kindly supplied by Bayer.
[14C]methylammonium sulfate was synthesized by the
method described below for the synthesis of methylammonium sulfate, but
with [14C]methylamine obtained from Amersham.
Tritiated cholesterylhexadecyl ether (3H-CHE) was also
obtained from Amersham. Methylammonium chloride was obtained from Sigma
Chemical Company, and methylamine was obtained from BDH (Vancouver,
British Columbia, Canada). The animals used in this study were 6- to
8-week-old female ICR and BALB/c mice, both obtained from Charles River
Laboratories. Staphylococcus aureus RN450 was obtained from
J. Davies, Department of Microbiology and Immunology, University of
British Columbia. Salmonella typhimurium SL1344 was obtained
from B. Finlay, Department of Biochemistry & Molecular Biology and
Department of Microbiology & Immunology, University of British
Columbia, Vancouver, British Columbia, Canada.
Methods. (i) Ciprofloxacin assay.
Liposomal ciprofloxacin,
in samples not containing [14C]ciprofloxacin, was
assayed by alkalinization of the sample to pH 12 with NaOH and then
extraction from the lipid by a two-phase Bligh and Dyer extraction
procedure (2). The aqueous upper phase was removed after
centrifugation and was assayed for A275 and compared to an aqueous upper-phase blank. This procedure recovered >95% of ciprofloxacin present in the samples and was linear in the
range between 0 and at least 65 µM ciprofloxacin
(r2 for this regression was 0.9998).
(ii) Synthesis of methylammonium sulfate.
Methylammonium
sulfate was prepared from methylamine (40% [wt/vol]in
H2O), and concentrated sulfuric acid was prepared by the
dropwise addition of 34.5 ml of concentrated sulfuric acid to 100 ml of
methylamine solution with continuous stirring in the cold (ice bath).
The pH of the final solution was adjusted to 6.0 to 7.0 with dilute
sulfuric acid or dilute methylamine. The methylammonium sulfate was
dried by rotary evaporation at 80°C. The methylammonium sulfate
slurry was resuspended with 200 ml of absolute ethanol and then dried
and resuspended once with 200 ml and twice with 100 ml of ethanol. The
resulting methylammonium sulfate slurry was taken up with 100 ml of
anhydrous ether, filtered, and then dried extensively under vacuum.
Confirmation of chemical identity and purity was performed by
comparison with methylammonium chloride by 13C-nuclear
magnetic resonance (13C-NMR).
(iii) Formulation of ciprofloxacin into liposomes.
Liposomes
were prepared by dissolving a total of 100 mg of lipid comprised of
phospholipids (DPPC, DSPC, or SM) and cholesterol (DPPC/chol,
DSPC/chol, and SM/chol, respectively) at phospholipid/cholesterol molar
ratios of 55/45 in CHCl3 or a mixture of CHCl3
and CH3OH. Bulk solvent was removed under a stream of
nitrogen gas, and then trace solvent was removed by holding the lipid
film under high vacuum overnight. The lipid films were hydrated by the
addition of 1.0 ml of 0.3 M methylammonium sulfate and then vortexed
and subjected to brief heating (50°C for DPPC/chol, 65°C for
DSPC/chol and SM/chol) to produce MLV. The MLV suspensions were
subjected to five freeze-thaw cycles between 
196°C and the
temperatures described above. Large unilamellar vesicles (LUV) were
produced by 10 passages of the MLV suspensions through two stacked
0.1-µm-pore-diameter filters with an extruder (Lipex Biomembranes,
Vancouver, British Columbia, Canada) maintained at 65°C as
characterized previously (14, 32). Vesicle diameters were
confirmed by quasielastic light scattering with a Nicomp model 270 submicron particle sizer.
Ciprofloxacin was loaded into liposomes in response to a transmembrane
gradient of the methylammonium that was established by overnight
dialysis of liposomes against 1,000 volumes of 150 mM NaCl.
Ciprofloxacin loading was initiated by the addition of the liposomal
suspension to the appropriate quantity of the ciprofloxacin (HCl
· H2O), either as a dry powder or as a 25-mg/ml solution (in H2O), so as to achieve a final ciprofloxacin/lipid
molar ratio of 0.25 or 0.3. Loading was allowed to proceed for 60 min
at 50°C (DPPC/chol) or 65°C (DSPC/chol and SM/chol).
(iv) Measurement of ciprofloxacin loading and transmembrane pH
gradient.
The encapsulation of ciprofloxacin into liposomes was
assessed by column chromatography. At various times during the uptake of ciprofloxacin into liposomes, aliquots were diluted in saline, and
then 50 or 100 µl was loaded onto 1-ml columns of Sephadex G-50 that
had been equilibrated in saline and precentrifuged before use
(38). The loaded columns were then centrifuged for 1 to 1.5 min at 2,000 rpm, and the eluate, containing the void volume, was
recovered for analysis of lipid (liquid scintillation counting [LSC]) and ciprofloxacin (LSC or A275).
The transmembrane pH gradient across the liposome membrane during the
uptake of ciprofloxacin was determined with
[
14C]methylamine as characterized previously
(
12,
41). Liposomes
of DPPC/chol (labeled with
3H-CHE) were equilibrated in the presence of 1 µCi of
[
14C]methylamine. Ciprofloxacin was added to the
sample, which was
then heated to 60°C. At various times, the external
pH was determined
with a pH probe, and the transmembrane pH gradient
and ciprofloxacin
loading were determined by passing the samples over
the 1-ml Sephadex
G-50 columns as described above. The column eluates
were analyzed
for [
14C]methylamine by LSC and for
ciprofloxacin by
A275. The intraliposomal
pH was
calculated assuming a liposome trap volume of 1.15 µl/µmol
of lipid
(
32).
(v) Pharmacokinetics and biodistribution of ciprofloxacin.
Ciprofloxacin (labeled with [14C]ciprofloxacin at 0.5 µCi/mg of ciprofloxacin) was encapsulated in liposomes in response to a methylammonium sulfate gradient as described above. The liposomes were composed of DPPC/chol, DSPC/chol, or SM/chol (55/45
[mol/mol]) labeled at 20 µCi/100 mg of lipid with
3H-CHE (a nonexchangable and nonmetabolizable lipid
radioactive tracer [43]). Pharmacokinetic studies were
performed by injecting liposomal ciprofloxacin formulations or free
ciprofloxacin into ICR mice via the lateral tail vein at a dose of 15 mg of ciprofloxacin/kg of body weight. In other experiments, free
ciprofloxacin and liposomal ciprofloxacin were administered by i.p.
injection or by intratracheal instillation after halothane
anesthetization. At various time points after administration, the mice
were anesthetized, and blood was recovered via cardiac puncture.
Subsequently, the animals were terminated by cervical dislocation, and
the liver, spleen, lung, kidney, and leg muscle were recovered. Plasma
and tissue homogenates were assayed for ciprofloxacin and lipid by LSC.
Concentrations of lipid and ciprofloxacin in tissue were corrected for
the contribution from the blood.
(vi) Determination of in vivo efficacy versus Salmonella
typhimurium.
Salmonella typhimurium SL1344 was grown
overnight in Luria-Bertani broth and then centrifuged and washed in
phosphate-buffered saline (PBS) and subsequently diluted to achieve a
final suspension containing 1,000 CFU/ml. Female BALB/c mice (6 to 8 weeks old) were injected i.v. into the tail vein with inocula
containing 60 to 90 CFU per mouse.
Twenty-four hours after infection, the mice were treated with free
ciprofloxacin (in saline) or liposomal ciprofloxacin by
i.v.
administration at doses of 1 or 20 mg of ciprofloxacin/kg.
The
solutions of both free and liposomal ciprofloxacin were sterilized
by
passage through a 0.2-µm-pore-diameter filter prior to
administration.
The liposomal ciprofloxacin formulations used in these
experiments
were 130-nm SM/chol (55/45 [mol/mol]) liposomes
containing ciprofloxacin
at a ciprofloxacin/lipid molar ratio of 0.239. (For these experiments,
the ciprofloxacin was loaded at a drug/lipid
ratio of 0.25, compared
to 0.30, to improve drug retention
[
30].) At 4 or 5 days postinfection,
the mice were sacrificed,
and the spleens and livers were removed
to sterile 6-ml tubes on ice.
Individual organs were transferred
to sterile plastic Stomacher bags
(Seward Medical, London, England),
and the organs were crushed and
homogenized for 90 s in the presence
of PBS with the Stomacher
apparatus. Aliquots (100 µl) of the
organ suspensions were serially
diluted in PBS to a maximum of
10
5-fold dilution and then
were plated on duplicate MacConkey agar
plates. Plates were incubated
overnight at 37°C, and the resulting
whitish, light-red
(Lac
) colonies were counted for plates containing between
30 and 300
colonies.
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RESULTS |
Characterization of ciprofloxacin loading.
The effect of
solution pH, in the range between 4 and 11, on the solubility of
aqueous ciprofloxacin is shown in Fig. 1.
At pH values below pK1= 6.0 and above pK2= 8.8, ciprofloxacin has a net charge and is highly soluble. However, in the
pH range between these pK values, the compound is zwitterionic or
neutral and is practically insoluble (Fig. 1). Throughout this study,
we employed ciprofloxacin hydrochloride. Dissolved in water at a
concentration of 25 mg/ml, this solution has a pH of 3.5.
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FIG. 1.
Solubility of ciprofloxacin in aqueous solutions
buffered in the pH range between 4 and 11. Ciprofloxacin hydrochloride
was dissolved to saturation in 50 mM HEPES at various pHs. Excess drug
was removed by centrifugation, and the supernatant was assayed for
ciprofloxacin content by A275.
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The kinetics of ciprofloxacin uptake into LUV in response to a
transmembrane gradient of methylammonium sulfate are shown
in Fig.
2A. The amount of drug encapsulated and
retained inside
the vesicles was determined directly from the
ciprofloxacin/lipid
ratio (
20,
25). Using 120-nm LUV and an
initial ciprofloxacin/lipid
molar ratio of approximately 0.3, typical
entrapment efficiencies
of 95 to 100% were observed in formulations
comprised of DPPC/chol,
DSPC/chol, and SM/chol (data not shown).
Complete uptake of ciprofloxacin
within 1 h required that the
loading was performed at temperatures
above the
L
-to-L
lipid phase transition temperature
for the phospholipid employed (i.e., at 50°C for DPPC/chol and
65°C
for DSPC/chol and SM/chol), despite the presence of sufficient
cholesterol to eliminate the L
-to-L
lipid
phase transition.
DPPC/chol LUV possessing a transmembrane gradient of
methylammonium
sulfate retained >96% of the encapsulated methylamine
within the
LUV during 42 h of dialysis at 21°C (data not shown).
However,
upon the addition of external ciprofloxacin and subsequent
ciprofloxacin
loading, methylamine rapidly effluxed from the LUV (Fig.
2A).
The molar stoichiometry of methylamine to ciprofloxacin varied
in
the range between 0.88 and 0.95 during drug accumulation (Fig.
2B),
suggesting that a one-to-one exchange of ciprofloxacin for
methylamine
occurred during drug loading. Analysis of ciprofloxacin
uptake and the
simultaneous changes of the internal and external
pH of 100-nm
DPPC/Chol LUV showed that the internal vesicle pH
increased from
approximately 2.8 prior to the addition of drug
to the external
solution to approximately 3.1 during the encapsulation
process (data
not shown). These results are consistent with the
data (Fig.
2A)
showing that a significant amount of methylamine
remained inside the
vesicles after ciprofloxacin loading. This
is sufficient to maintain
the internal pH and retain the antibiotic
(see Discussion). It should
be noted that all three liposomal
ciprofloxacin formulations retained
100% of the encapsulated drug
during storage for 18 weeks at 4°C, 12 weeks at 21°C, or 8 weeks
at 37°C (data not shown).
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FIG. 2.
Loading of ciprofloxacin into liposomes. (A) Uptake of
ciprofloxacin, expressed as the ciprofloxacin/lipid ratio ( ) and
release of [14C]methylamine, expressed as the
methylamine/lipid ratio ( ). Ciprofloxacin uptake was initiated by
the addition of ciprofloxacin to 100-nm unilamellar liposomes composed
of DPPC/chol (55/45 [mol/mol]) and possessing a transmembrane 0.3 M methylammonium sulfate gradient. (B) Calculated stoichiometry of
moles of methylamine released/moles of ciprofloxacin loaded.
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In vivo pharmacokinetics and biodistribution of free and liposomal
ciprofloxacin.
After i.v. administration, free ciprofloxacin was
removed from the circulation of mice with a half-life of approximately
0.2 h (Fig. 3A). Encapsulation of
ciprofloxacin in all of the carriers increased the circulation
half-life of ciprofloxacin from 0.2 h to >3 h (Fig. 3A),
representing 43- to 105-fold increases in concentrations of
ciprofloxacin in plasma occurring as a consequence of encapsulation.
For example, at 1 h after i.v. administration, the ciprofloxacin
concentrations were 0.132 µg/100 µl of plasma for free
ciprofloxacin and between 5.62 and 13.8 µg/100 µl of plasma for
the liposomal ciprofloxacin formulations. Ciprofloxacin that
leaked from the liposomes would be expected to be removed from
the circulation at rates identical to that for free drug administered
i.v. (Fig. 3A) (28). Higher levels of ciprofloxacin in
plasma were observed in the SM/chol formulation and were a consequence
of the higher ciprofloxacin/lipid ratio in the plasma at various times
after i.v. administration in this formulation compared to those of the
DPPC/chol and DSPC/chol formulations (Fig. 3B). The SM/chol carrier
retained significantly greater proportions of the encapsulated
ciprofloxacin than either the DSPC/chol or DPPC/chol carriers. For
example, at 4 and 6 h after i.v. administration of the
ciprofloxacin formulations, the ciprofloxacin/lipid ratio in the
SM/chol vesicles was 5.2- to 5.8-fold greater than that measured for
the DPPC/chol vesicles and 2.8- to 3.6-fold greater than that in
DSPC/chol LUV.
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FIG. 3.
Pharmacokinetics of free and liposomal ciprofloxacin
after i.v. administration. (A) Concentrations of ciprofloxacin in
plasma after i.v. administration of free ciprofloxacin () or
ciprofloxacin encapsulated in liposomes comprised of DPPC/chol ( ),
DSPC/chol ( ), or SM/chol ( ). The dose of ciprofloxacin injected
for all treatments was 15 mg/kg. (B) Ciprofloxacin/lipid ratios in
plasma after i.v. administration of the DPPC/chol (), DSPC/chol
(), or SM/chol () formulation of liposomal ciprofloxacin. Data
represent means ± standard errors from three mice.
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The altered pharmacokinetics of ciprofloxacin that occurred as a
consequence of encapsulation in liposomes (Fig.
3) also significantly
altered the biodistribution of ciprofloxacin in liver, lung, spleen,
kidney, and muscle after i.v. administration (Fig.
4). The quantities
of ciprofloxacin that
accumulated in these tissues in mice treated
with free antibiotic were
maximal at 5 min and then decreased
with half-lives in the range of 15 to 30 min (not shown). In contrast,
quantities of ciprofloxacin in
tissue were substantially higher
after administration of all liposomal
formulations of the drug
(not shown), and the times required for levels
of ciprofloxacin
in tissue to decrease to 50% of their highest values
were in the
range of 2 to 4 h in the liver and lung and 3 to
12 h in the spleen
and kidney. Consequently, the amounts of
ciprofloxacin that accumulated
in these tissues during the 24 h
after administration were increased
by 1.5- to 73-fold (Fig.
4A). An
example of the effect of encapsulation
on the quantities of
ciprofloxacin in the lung is shown in Fig.
4B. The increased drug
retention properties of SM/chol vesicles
(Fig.
3) were reflected in
higher quantities of ciprofloxacin
in tissue following i.v.
administration of this formulation compared
to those with the DPPC/chol
and DSPC/chol formulations (Fig.
4A).
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FIG. 4.
Summary of the biodistribution of free and liposomal
ciprofloxacin (cipro) after i.v. administration. Area under the
concentration-time curve (AUC) values over 24 h for the different
liposomal formulations are expressed relative to that for free
ciprofloxacin in liver, lung, spleen, kidney, and muscle (A).
Ciprofloxacin concentrations in the lungs of mice after i.v.
administration of free ciprofloxacin () or ciprofloxacin
encapsulated in SM/chol liposomes () are shown as an example (B).
The dose of ciprofloxacin injected for all treatments was 15 mg/kg.
Data represent means ± standard errors from three mice.
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Pharmacokinetics after i.p. and intratracheal administrations.
The substantial improvements in the pharmacokinetics and
biodistribution of liposomal ciprofloxacin, compared to free
ciprofloxacin, after i.v. administration suggested that similar
advantages might be conferred by other routes of administration.
Consequently, the pharmacokinetics of free and liposomal (SM/chol)
formulations of ciprofloxacin after i.p. and intratracheal
administration were also examined.
Quantities of ciprofloxacin in plasma decreased rapidly after the
administration of free ciprofloxacin by either the i.v.
or i.p. route
(Fig.
5A). As described above (Fig.
3A),
the half-life
of free ciprofloxacin after both i.v. administration and
i.p.
administration was approximately 0.25 h. In contrast, the
administration
of liposomal ciprofloxacin by both the i.v. and i.p.
routes resulted
in significantly higher concentrations of ciprofloxacin
in plasma
than after administration of the free antibiotic. As in Fig.
3,
the half-life of the SM/chol formulation of liposomal ciprofloxacin
after i.v. administration was increased to 2.7 h, representing
a
greater than 10-fold increase in the half-life as a consequence
of
encapsulation. After i.p. administration of liposomal ciprofloxacin,
the concentrations of drug in plasma gradually increased during
the
first 4 h and then exhibited pharmacokinetics identical to
those
of the i.v.-administered formulations between 4 and 24 h
(Fig.
5A). The amounts of ciprofloxacin retained within SM/chol
LUV were
identical after either i.v. or i.p. administration (Fig.
5B).
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FIG. 5.
Comparison of ciprofloxacin pharmacokinetics after i.v.
and i.p. administration. (A) Levels of free (,) or liposomal
(,) ciprofloxacin in plasma after i.v. (,) or i.p.
(,) administration. Liposomes were composed of SM/chol, and the
dose of ciprofloxacin injected for all groups was 15 mg/kg. (B)
Ciprofloxacin/lipid ratios in plasma after i.v. () or i.p. ()
administration of ciprofloxacin encapsulated in SM/chol liposomes. Data
represent means ± standard errors from three mice.
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Free ciprofloxacin was rapidly cleared from the lungs after
intratracheal administration, with a half-life of 0.21 h (Fig.
6), whereas encapsulation dramatically
increased the retention
of drug at this site. Specifically, the
half-lives for ciprofloxacin
in the lungs were increased to 6.9 h
(DPPC/chol), 14.6 h (DSPC/chol),
and 23.6 h (SM/chol),
representing increases of 33-, 69-, and
112-fold, respectively (Fig.
6
and data not shown). Ciprofloxacin
longevity in lung tissue was a
direct consequence of the retention
of the vesicles at this site and
the retention of the drug in
the vesicles (Fig.
6 and data not shown).
At 24 h, the quantities
of lipid remaining in the lungs ranged
from 76 to 106% of the
initial dose, indicating that the carriers did
not extravasate
to the circulation, but remained in the lung tissue and
acted
as slow-release reservoirs of ciprofloxacin. The superior
retention
of ciprofloxacin by SM/chol LUV resulted in a significant
increase
in drug quantities in the lungs over 24 h compared to
those of
free drug and the more leaky DPPC/chol and DSPC/chol
formulations.
The levels of lipid in the plasma after intratracheal
administration
of liposomal ciprofloxacin were negligible (data not
shown), confirming
that the liposomes were not able to extravasate from
the lung
tissue to the circulation. As expected, levels of
ciprofloxacin
in the plasma after intratracheal administration of
liposomal
ciprofloxacin were also negligible, consistent with the short
half-life of free drug that diffuses into the circulation from
the
vesicles trapped in the airways.
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FIG. 6.
Pharmacokinetics after intratracheal administration.
Quantities of ciprofloxacin (,) and lipid ( ) in the lungs of
ICR mice after the intratracheal administration of either free
ciprofloxacin () or ciprofloxacin encapsulated in liposomes
comprised of DPPC/chol (,) are shown. Data represent means ± standard errors from three mice.
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In vivo antibacterial efficacy of free and liposomal
ciprofloxacin.
The antibacterial activities of free and liposomal
ciprofloxacin have been compared in a Salmonella typhimurium
infection model. The SM/chol formulation of liposomal ciprofloxacin was chosen for the efficacy studies because of its superior drug retention properties in vivo (Fig. 3). In these experiments, mice were infected with 66 to 88 CFU of Salmonella typhimurium, and then
24 h later, they were given an i.v. bolus of free or liposomal
(SM/chol) ciprofloxacin or free ciprofloxacin plus empty SM/chol LUV.
The ciprofloxacin doses administered were either 1 or 20 mg/kg. Results
are summarized in Fig. 7. Free
ciprofloxacin, administered at either dose, had only minor effects on
the numbers of viable bacteria recovered from the livers and spleens of
infected animals in comparison to those recovered from controls (Fig.
7). Liposomal ciprofloxacin at a drug dose of 1 mg/kg reduced the
number of viable bacteria in the liver and spleen by approximately 10- to 100-fold compared to the number reduced by free ciprofloxacin at the
same dose (data not shown). At 20 mg/kg, the numbers of viable bacteria
remaining in the livers and spleens of infected animals were
103- to 104-fold lower in those treated with
encapsulated ciprofloxacin than those in control animals (untreated or
saline treated) or animals treated with the same dose of free
ciprofloxacin in the presence or absence of empty SM/chol carriers
(Fig. 7 and data not shown). Finally, it should be added that single
i.v. administration of the SM/chol formulation of liposomal
ciprofloxacin resulted in an approximate doubling of the survival time
for mice bearing this Salmonella typhimurium infection.
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|
FIG. 7.
In vivo antibacterial efficacy against Salmonella
typhimurium. The CFU of viable bacteria per milliliter of liver
(open bars) or spleen (shaded bars) homogenates for animals infected
with 66 to 88 CFU of Salmonella typhimurium were either left
untreated, were treated once with saline, or were treated once with
free or liposomal ciprofloxacin at 20 mg/kg. Data represent means ± standard errors from three mice.
|
|
An additional control experiment was performed to ensure that the
reduction in the number of viable bacteria in the organs
of the
infected animals that had been treated with liposomal ciprofloxacin
was
not due to the release of ciprofloxacin from liposomes during
tissue
homogenization. Homogenates of livers obtained from control
animals or
from animals treated with liposomal ciprofloxacin 5
days earlier were
centrifuged to remove particulate material.
The supernatants
(containing any remaining liposomes or ciprofloxacin
released from the
liposomes during homogenization) were filter
sterilized with a
0.22-µm-pore diameter filter, and then 3 · 10
6 CFU
of
Salmonella typhimurium SL1344 in PBS were added to 1 ml
of PBS or to 1 ml of each of the liver suspension filtrates. These
bacterial suspension-organ filtrates were incubated for 1 h at
4°C, and then 100 µl of 10
0- to 10
5-fold
dilutions were assayed for viable bacteria. Neither free
nor liposomal
ciprofloxacin treatments reduced the number of bacteria
in spleen or
liver homogenates (not shown). Therefore, the bactericidal
efficacy of
the liposomal ciprofloxacin occurred postadministration
in the mice and
did not occur as an artifact of organ-associated
ciprofloxacin that was
plated onto the microbiological growth
media.
| |
DISCUSSION |
Ciprofloxacin loading.
The most prominent examples of active
drug loading into liposomes for the purpose of drug delivery are
doxorubicin (44) and vincristine (3). Both are
lipophilic amines and are loaded into vesicles which possess an
acidic interior with respect to the external solution. Accumulation
proceeds because lipophilic weak bases (a characteristic of a
surprising number of drugs) distribute across the liposomal membrane
according to the relationship [drug]IN/[drug]OUT = [H+]IN/[H+]OUT
(5). Consequently, a transmembrane 
pH of 3 U (inside acidic) will result in drug loading until, at equilibrium, there is a
1,000-fold-higher concentration of drug inside the vesicle than
outside.
The practical advantages of the process have enabled the successful
clinical development of several liposome-based drug delivery
systems.
Active loading is simple and efficient. Conditions can
be selected such
that 100% of the drug can be effectively encapsulated
into preformed
LUV, therefore minimizing process development issues.
Moreover, the
presence of a transmembrane ion gradient helps retain
drugs inside
carriers during storage and after administration.
Most drugs for which
active loading has been characterized are
lipophilic cations or anions
(
20). Therefore, given the zwitterionic
nature of
ciprofloxacin, it was not obvious that this drug could
be loaded by an
active process. However, recently we (
15) and
others
(
19,
34) demonstrated that ciprofloxacin accumulation
occurred in response to an ammonium sulfate ion gradient, and
in this
report, we have demonstrated that rapid and complete uptake
is also
achieved with a methylamine sulfate chemical gradient
(Fig.
2). A
scheme is presented in Fig.
8 which
describes the
uptake process and explains the characteristics of
ciprofloxacin
loading we observed. Encapsulated methylamine sulfate
ionizes
into methylammonium and sulfate. The methylammonium ion further
disassociates into methylamine and a proton, both of which can
escape
the vesicle by diffusing through the bilayer down their
concentration
gradients. The bilayer is relatively impermeable
to sulfate ions.
Furthermore, the efflux of protons is limited
by the rapid formation of
a transmembrane potential (negative
inside) caused by movement of the
positive charge out of the vesicles.
As methylamine continues to
diffuse from the vesicle, the internal
concentration of protons
increases until the membrane potential
and
pH are in equilibrium
(Fig.
8).
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|
FIG. 8.
Schematic representation of the active loading method
for ciprofloxacin. Ciprofloxacin exists in cationic, anionic,
zwitterionic, and neutral forms. (Note that this ionization scheme is
identical both inside and outside the liposome, but for clarity, is
shown only on the outside.) Inside the liposome is a low internal pH as
a consequence of methylammonium ionization to methylamine and a proton.
The neutral form of ciprofloxacin is membrane permeable and crosses to
the aqueous lumen of the liposomes, where the low pH favors the
protonated species. Cationic ciprofloxacin cannot diffuse back out of
the vesicle; consequently, the drug accumulates until
[drug]IN/[drug]OUT = [H+]IN/[H+]OUT.
As a proton is consumed by each neutral drug species, equilibrium is
maintained by disassociation of methylammonium into methylamine and a
proton. This accounts for the observed 1:1 molar stoichiometry between
drug uptake and methylamine efflux.
|
|
Ciprofloxacin possesses both a carboxyl function and an amino function.
At a pH of <6, the molecule exhibits a net positive
charge, whereas
for pHs of >9, the charge is net negative (
15,
45). Above
or below these pH extremes, ciprofloxacin is very
soluble, but over the
physiological pH range, the drug is practically
insoluble (Fig.
1). The
four ionization states for ciprofloxacin
are shown in Fig.
8. In
general, charged molecules cannot cross
the bilayer at a significant
rate; consequently, it is reasonable
to assume that it is the uncharged
species that diffuses into
the vesicle down its concentration gradient.
Inside, the low internal
pH favors the protonated ciprofloxacin
species, trapping the drug
which is unable to diffuse back across the
bilayer in the charged
form. Drug accumulates inside the vesicle
(stoichiometrically
with the efflux of methylamine [Fig.
2B])
until an electrochemical
equilibrium is reached such that
[drug]
IN/[drug]
OUT = [H
+]
IN/[H
+]
OUT
(
5,
13). However, it should be noted that it is an
oversimplification
to assume that the transmembrane drug distribution
exactly reflects
the
pH; factors such as the membrane-water
partitioning coefficient
for the drug and precipitation with internal
counterions also
need to be taken into consideration (
5). As
long as the internal
concentration of methylamine sulfate exceeds that
of the drug,
then at equilibrium, a stable, internal acidic pH will be
maintained,
as was observed in this study.
Ciprofloxacin has also been actively loaded into unilamellar liposomes
in response to a transmembrane gradient of ammonium
sulfate in a
procedure dependent on the pH of the external solution
(
19,
34). These workers concluded that the protonated species
of
ciprofloxacin is the membrane-permeable form of the drug
(
34)
and, once diffused across the membrane, is rendered
impermeable
by precipitation within the liposome interior (
19,
40). However,
analysis of liposome-encapsulated ciprofloxacin by
proton NMR
does not support the conclusion that intraliposomal
ciprofloxacin
exists as a ciprofloxacin sulfate precipitate
(
49a). Rather,
the upfield shift of the aromatic proton
resonances associated
with the encapsulated drug is more consistent
with ciprofloxacin
self-association.
Pharmacokinetics, biodistribution, and efficacy.
The longevity
of ciprofloxacin in plasma was considerably increased by encapsulation
in 100-nm LUV (Fig. 3A). SM/chol LUV had the greatest drug retention in
vivo compared to DSPC/chol or DPPC/chol LUV, and this was reflected in
the drug/lipid ratio measured in the blood over 24 h (Fig. 3B).
The observation that SM/chol vesicles were better able to retain
actively loaded drug than glycerol-based
phospholipid-cholesterol-containing vesicles has been made previously
with formulations of vincristine (48). The data from
the latter study suggested that SM/chol vesicles are more stable in
blood and consequently can maintain the pH required to keep the drug
entrapped.
Levels of ciprofloxacin in tissue following i.v. administration of
encapsulated formulations reflected the biodistribution
of the
liposomal carrier systems. It is well known that phospholipid
vesicles
accumulate in organs of the RES. We analyzed liver, spleen,
lung, and
kidney tissues, and in all cases, encapsulated ciprofloxacin
was
readily measured out to 4 or 6 h postinjection. In comparison,
free drug was cleared quickly, and was undetectable after 1 h
according to our assay protocol (Fig.
4B). Interestingly, i.p.
administration of encapsulated ciprofloxacin gave rise to a profile
for
the drug in blood almost identical to that obtained after
i.v. delivery
(Fig.
5A); the only difference in clearance kinetics
was seen during
the first 4 h as vesicles drained into the circulation
via the
lymphatics. The drug/lipid ratio data (Fig.
5B) indicated
that during
this time, the release of encapsulated ciprofloxacin
proceeded at the
same rate as that for vesicles administered i.v.
In contrast, vesicles
introduced into the lungs via tracheal intubation
were well retained at
the epithelium-air interface. As a result,
a reservoir of ciprofloxacin
can be maintained in the lung for
at least 24 h (Fig.
6A), with
free drug being steadily released
into surrounding tissue. It is worth
making the point that because
the formulations described here are
stable in solution, they are
readily aerosolized and therefore could be
targeted directly to
lung tissue. This would represent a novel route of
administration
for ciprofloxacin. Aerosolized formulations of
ciprofloxacin have
not been developed, in part because of the drug's
poor solubility
and extremely bitter taste. However, the actively
loaded formulations
described here overcome both of these limitations.
Because ciprofloxacin
is 100% encapsulated and is held inside the
delivery system by
a
pH, the drug can be suspended in physiological
media without
precipitation at concentrations that far exceed the free
drug
solubility. Furthermore, encapsulation would be expected to mask
the bitter taste. The potential for aerosolized delivery of
ciprofloxacin
may have clinical significance in the light of recent
data demonstrating
that formulations of liposomal ciprofloxacin were
highly effective,
following intratracheal administration, in treating
mice whose
lungs had been inoculated with
Francisella
tularensis, a virulent
respiratory pathogen (
49).
The systemic
Salmonella typhimurium infection used here is
one in which the infecting bacteria seed primarily in the liver
and
spleen, and, furthermore, at least 88% of the liver-resident
bacteria
are localized within macrophages (
39). We have demonstrated
significantly enhanced efficacy of encapsulated ciprofloxacin
against
these intracellularly localized bacteria in vivo. The
drug was
administered as a single dose i.v. and compared to an
equivalent dose
of free ciprofloxacin. Given the superior drug
retention properties of
the SM/chol vesicles, we chose to test
this formulation in the
infection model. At 20 mg/kg, the free
antibiotic exhibited very little
activity, but an equivalent dose
of encapsulated ciprofloxacin reduced
the viable bacterial count
in both the liver and spleen by 3 to 4 orders of magnitude (Fig.
7). Presumably the increased activity over
that of the free drug
was due to a combination of increased drug
concentrations in the
circulation and the accumulation of a
ciprofloxacin reservoir
in the target organs. Taken in sum, these data
highlight the versatility
and efficacy of the liposomal ciprofloxacin
formulations.
| |
ACKNOWLEDGMENTS |
We thank Sharon Ruschkowski, Agneta Richter-Dahlfors, and Brett
Finlay for cooperation with the in vivo efficacy experiments and
Norbert Maurer for performing the 13C-NMR analyses.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Inex
Pharmaceuticals Corporation, 100-8900 Glenlyon Parkway, Burnaby,
British Columbia, Canada V5J 5J8. Phone: (604) 264-9954. Fax: (604)
264-9690. E-mail: mwebb{at}inexpharm.com.
| |
REFERENCES |
| 1.
|
Bermudez, L. E.,
A. O. Yao-Young,
J.-P. Lin,
J. Cogger, and L. S. Young.
1990.
Treatment of disseminated Mycobacterium avium complex infection of beige mice with liposome-encapsulated aminoglycosides.
J. Infect. Dis.
161:1262-1268[Medline].
|
| 2.
|
Bligh, E. G., and W. J. Dyer.
1959.
A rapid method of total lipid extraction and purification.
Can. J. Biochem. Physiol.
37:911-917.
|
| 3.
|
Boman, N. L.,
M. B. Bally,
P. R. Cullis,
L. D. Mayer, and M. S. Webb.
1995.
Encapsulation of vincristine in liposomes reduces its toxicity and improves its anti-tumor efficacy.
J. Liposome Res.
5:523-541.
|
| 4.
|
Boman, N. L.,
D. Masin,
L. D. Mayer,
P. R. Cullis, and M. B. Bally.
1994.
Liposomal vincristine which exhibits increased drug retention and increased circulation longevity cures mice bearing P388 tumors.
Cancer Res.
54:2830-2833[Abstract/Free Full Text].
|
| 5.
|
Cullis, P. R.,
M. J. Hope,
M. B. Bally,
T. D. Madden,
L. D. Mayer, and D. B. Fenske.
1997.
Influence of pH gradients on the transbilayer transport of drugs, lipids, peptides and metal ions into large unilamellar vesicles.
Biochim. Biophys. Acta
1331:187-211[Medline].
|
| 6.
|
Di Ninno, V. L.,
J. W. Cherwonogrodzky, and J. P. Wong.
1993.
Liposome-encapsulated ciprofloxacin is effective in the protection and treatment of BALB/c mice against Francisella tularensis.
J. Infect. Dis.
168:793-794[Medline].
|
| 7.
|
Düzgüne , N.,
D. R. Ashtekar,
D. L. Flasher,
N. Ghori,
R. J. Debs,
D. S. Friend, and P. R. J. Gangadharam.
1991.
Treatment of Mycobacterium avium-intracellulare complex infection in beige mice with free and liposome-encapsulated streptomycin: role of liposome type and duration of treatment.
J. Infect. Dis.
164:143-151[Medline].
|
| 8.
|
Düzgüne, N.,
V. K. Perumal,
L. Kesavalu,
J. A. Goldstein,
R. J. Debs, and P. R. J. Gangadharam.
1988.
Enhanced effect of liposome-encapsulated amikacin on Mycobacterium avium-M. intracellulare complex infection in beige mice.
Antimicrob. Agents Chemother.
32:1404-1411[Abstract/Free Full Text].
|
| 9.
|
Fenlon, C. H., and M. H. Cynamon.
1986.
Comparative in vitro activities of ciprofloxacin and other 4-quinolones against Mycobacterium tuberculosis and Mycobacterium intracellulare.
Antimicrob. Agents Chemother.
29:386-388[Abstract/Free Full Text].
|
| 10.
|
Fresta, M.,
A. Spadaro,
G. Cerniglia,
I. M. Ropero,
G. Puglisi, and P. M. Furneri.
1995.
Intracellular accumulation of ofloxacin-loaded liposomes in human synovial fibroblasts.
Antimicrob. Agents Chemother.
39:1372-1375[Abstract].
|
| 11.
|
Gay, J. D.,
D. R. DeYoung, and G. D. Roberts.
1984.
In vitro activities of norfloxacin and ciprofloxacin against Mycobacterium tuberculosis, M. avium complex, M. chelonei, M. fortuitum, and M. kansasii.
Antimicrob. Agents Chemother.
26:94-96[Abstract/Free Full Text].
|
| 12.
|
Harrigan, P. R.,
M. J. Hope,
T. E. Redelmeier, and P. R. Cullis.
1992.
Determination of transmembrane pH gradients and membrane potentials in liposomes.
Biophys. J.
63:1336-1345[Medline].
|
| 13.
|
Harrigan, P. R.,
K. F. Wong,
T. E. Redelmeier,
J. J. Wheeler, and P. R. Cullis.
1993.
Accumulation of doxorubicin and other lipophilic amines into large unilamellar vesicles in response to transmembrane pH gradients.
Biochim. Biophys. Acta
1149:329-338[Medline].
|
| 14.
|
Hope, M. J.,
M. B. Bally,
G. Webb, and P. R. Cullis.
1985.
Production of large unilamellar vesicles by a rapid extrusion procedure. Characterization of size distribution trapped volume and ability to maintain a membrane potential.
Biochim. Biophys. Acta
812:55-56.
|
| 15.
|
Hope, M. J., and K. F. Wong.
1995.
Liposomal formulation of ciprofloxacin, p. 121-134.
In
P. N. Shek (ed.), Liposomes in biomedical applications. Harwood Academic Publishers, Germany.
|
| 16.
|
Hwang, K. J.
1987.
Liposome pharmacokinetics, p. 109-156.
In
M. J. Ostro (ed.), Liposomes: from biophysics to therapeutics. Marcel Dekker, Inc., New York, N.Y.
|
| 17.
|
Klemens, S. P.,
M. H. Cynamon,
C. E. Swenson, and R. S. Ginsberg.
1990.
Liposome-encapsulated-gentamicin therapy of Mycobacterium avium complex infection in beige mice.
Antimicrob. Agents Chemother.
34:967-970[Abstract/Free Full Text].
|
| 18.
|
Kresta, A.,
P. N. Shek,
J. Odumeru, and J. M. A. Bohnen.
1993.
Distribution of free and liposome-encapsulated cefoxitin in experimental intra-abdominal sepsis in rats.
J. Pharm. Pharmacol.
45:779-783[Medline].
|
| 19.
|
Lasic, D. D.,
B. Ceh,
M. C. A. Stuart,
L. Guo,
P. M. Frederik, and Y. Barenholz.
1995.
Transmembrane gradient driven phase transitions within vesicles: lessons for drug delivery.
Biochim. Biophys. Acta
1239:145-156[Medline].
|
| 20.
|
Madden, T. D.,
P. R. Harrigan,
L. C. L. Tai,
M. B. Bally,
L. D. Mayer,
T. E. Redelmeier,
H. C. Loughrey,
C. P. S. Tilcock,
L. W. Reinish, and P. R. Cullis.
1990.
The accumulation of drugs within large unilamellar vesicles exhibiting a proton gradient: a survey.
Chem. Phys. Lipids
53:37-46[Medline].
|
| 21.
|
Magallanes, M.,
J. Dijkstra, and J. Fierer.
1993.
Liposome-incorporated ciprofloxacin in treatment of murine salmonellosis.
Antimicrob. Agents Chemother.
37:2293-2297[Abstract/Free Full Text].
|
| 22.
|
Majumdar, S.,
D. Flasher,
D. S. Friend,
P. Nassos,
D. Yajko,
W. K. Hadley, and N. Düzgüne.
1992.
Efficacies of liposome-encapsulated streptomycin and ciprofloxacin against Mycobacterium avium-M. intracellulare complex infections in human peripheral blood monocyte/macrophages.
Antimicrob. Agents Chemother.
36:2808-2815[Abstract/Free Full Text].
|
| 23.
|
Mayer, L. D.,
M. B. Bally,
P. R. Cullis,
S. L. Wilson, and J. T. Emerman.
1990.
Comparison of free and liposome encapsulated doxorubicin tumor drug uptake and antitumor efficacy in the SC115 murine mammary tumor.
Cancer Lett.
53:183-190[Medline].
|
| 24.
|
Mayer, L. D.,
M. B. Bally,
H. Loughrey,
D. Masin, and P. R. Cullis.
1990.
Liposomal vincristine preparations which exhibit decreased drug toxicity and increased activity against murine L1210 and P388 tumors.
Cancer Res.
50:575-579[Abstract/Free Full Text].
|
| 25.
|
Mayer, L. D.,
T. D. Madden,
M. B. Bally, and P. R. Cullis.
1993.
pH gradient-mediated drug entrapment in liposomes, p. 27-44.
In
G. Gregoriadis (ed.), Liposome technology, vol. II. Entrapment of drugs and other materials. CRC Press, Boca Raton, Fla.
|
| 26.
|
Mayer, L. D.,
D. Masin,
R. Nayar,
N. L. Boman, and M. B. Bally.
1995.
Pharmacology of liposomal vincristine in mice bearing L1210 ascitic and B16/BL6 solid tumors.
Br. J. Cancer
71:482-488[Medline].
|
| 27.
|
Mayer, L. D.,
R. Nayar,
R. L. Thies,
N. L. Boman,
P. R. Cullis, and M. B. Bally.
1993.
Identification of vesicle properties that enhance the antitumour activity of liposomal vincristine against murine L1210 leukemia.
Cancer Chemother. Pharmacol.
33:17-24[Medline].
|
| 28.
|
Mayer, L. D., and G. St.-Onge.
1995.
Determination of free and liposome-associated doxorubicin and vincristine levels in plasma under equilibrium conditions employing ultrafiltration techniques.
Anal. Biochem.
232:149-157[Medline].
|
| 29.
|
Mayer, L. D.,
L. C. L. Tai,
M. B. Bally,
G. N. Mitilenes,
R. S. Ginsberg, and P. R. Cullis.
1990.
Characterization of liposomal systems containing doxorubicin entrapped in response to pH gradients.
Biochim. Biophys. Acta
1025:143-151[Medline].
|
| 30.
|
Mayer, L. D.,
L. C. L. Tai,
D. S. C. Ko,
D. Masin,
R. S. Ginsberg,
P. R. Cullis, and M. B. Bally.
1989.
Influence of vesicle size, lipid composition, and drug-to-lipid ratio on the biological activity of liposomal doxorubicin in mice.
Cancer Res.
49:5922-5930[Abstract/Free Full Text].
|
| 31.
|
Nacucchio, M. C.,
M. J. Gatto Bellora,
D. O. Sordelli, and M. D'Aquino.
1988.
Enhanced liposome-mediated antibacterial activity of piperacillin and gentamicin against gram-negative bacilli in vitro.
J. Microencapsulation
5:303-309[Medline].
|
| 32.
|
Nayar, R.,
M. J. Hope, and P. R. Cullis.
1989.
Generation of large unilamellar vesicles from long-chain saturated phosphatidylcholines by extrusion technique.
Biochim. Biophys. Acta
986:200-206.
|
| 33.
|
Nightingale, S. D.,
S. L. Saletan,
C. E. Swenson,
A. J. Lawrence,
D. A. Watson,
F. G. Pilkiewicz,
E. G. Silverman, and S. X. Cal.
1993.
Liposome-encapsulated gentamicin treatment of Mycobacterium avium-Mycobacterium intracellulare complex bacteremia in AIDS patients.
Antimicrob. Agents Chemother.
37:1869-1872[Abstract/Free Full Text].
|
| 34.
|
Oh, Y.-K.,
D. E. Nix, and R. M. Straubinger.
1995.
Formulation and efficacy of liposome-encapsulated antibiotics for therapy of intracellular Mycobacterium avium infection.
Antimicrob. Agents Chemother.
39:2104-2111[Abstract].
|
| 35.
|
Omri, A.,
M. Ravaoarinoro, and M. Poisson.
1995.
Incorporation, release and in vitro antibacterial activity of liposomal aminoglycosides against Pseudomonas aeruginosa.
J. Antimicrob. Chemother.
36:631-639[Abstract/Free Full Text].
|
| 36.
|
Onyeji, C. O.,
C. H. Nightingale, and M. N. Marangos.
1994.
Enhanced killing of methicillin-resistant Staphylococcus aureus in human macrophages by liposome-entrapped vancomycin and teicoplanin.
Infection
22:338-342[Medline].
|
| 37.
|
Paganoni, R.,
C. Herzog,
A. Braunsteiner, and P. Hohl.
1988.
Fleroxacin: in-vitro activity worldwide against 20,807 clinical isolates and comparison to ciprofloxacin and norfloxacin.
J. Antimicrob. Chemother.
22:3-17.
|
| 38.
|
Pick, U.
1981.
Liposomes with a large trapping capacity prepared by freezing and thawing of sonicated phospholipid mixtures.
Arch. Biochem. Biophys.
212:186-194[Medline].
|
| 39.
|
Richter-Dahlfors, A.,
A. M. J. Buchan, and B. B. Finlay.
1997.
Murine salmonellosis studies by confocal microscopy: Salmonella typhimurium resides intracellularly inside macrophages and exerts a cytotoxic effect on phagocytes in vivo.
J. Exp. Med.
186:569-580[Abstract/Free Full Text].
|
| 40.
|
Rodriguez, J. L.,
S. Z. Trooskin,
R. S. Greco,
R. A. Herbstman,
A. P. Donetz, and R. A. Harvey.
1986.
Reduced bacterial adherence to surfactant-coated catheters.
Curr. Surg.
43:423-425[Medline].
|
| 41.
|
Rottenberg, H.,
T. Grunwald, and M. Avron.
1972.
Determination of (delta)pH in chloroplasts. 1. Distribution of [14C]methylamine.
Eur. J. Biochem.
25:54-63[Medline].
|
| 42.
|
Sanders, C. C.,
W. E. Sanders, Jr., and R. V. Goering.
1987.
Overview of preclinical studies with ciprofloxacin.
Am. J. Med.
82:2-11[Medline].
|
| 43.
|
Sherphof, G. L.,
F. Kuipers,
J. T. P. Denkser,
H. H. Spanyer, and R. J. Wonk.
1987.
Liposomes in vivo: conversion of liposomal cholesterol to bile salts.
Biochem. Soc. Trans.
15:625-631.
|
| 44.
|
Tardi, P. G.,
N. L. Boman, and P. R. Cullis.
1996.
Liposomal doxorubicin.
J. Drug Targeting
4:129-140[Medline].
|
| 45.
|
Teixeira, M. H. S. F.,
L. F. Vilas-Boas,
V. M. S. Gil, and F. Teixeira.
1995.
Complexes of ciprofloxacin with metal ions contained in antacid drugs.
J. Chemother.
7:126-132[Medline].
|
| 46.
|
Vaage, J.,
D. Donovan,
E. Mayhew,
P. Uster, and M. Woodle.
1993.
Therapy of mouse mammary carcinomas with vincristine and doxorubicin encapsulated in sterically stabilized liposomes.
Int. J. Cancer
54:959-964[Medline].
|
| 47.
|
Vladimirsky, M. A., and G. A. Ladigina.
1982.
Antibacterial activity of liposome-entrapped streptomycin in mice infected with Mycobacterium tuberculosis.
Biomedicine
36:375-377.
|
| 48.
|
Webb, M. S.,
T. O. Harasym,
D. Masin,
M. B. Bally, and L. D. Mayer.
1995.
Sphingomyelin-cholesterol liposomes significantly enhance the pharmacokinetic and therapeutic properties of vincristine in murine and human tumour models.
Br. J. Cancer
72:896-904[Medline].
|
| 49.
|
Wong, J. P.,
J. W. Cherwonogrodzky,
V. L. Di Ninno,
R. De la Cruz, and E. G. Saravolac.
1995.
Liposome-encapsulated ciprofloxacin for the prevention and treatment of infectious diseases caused by intracellular pathogens, p. 105-120.
In
P. N. Shek (ed.), Liposomes in biomedical applications. Harwood Academic Publishers, Germany.
|
| 49a.
| Wong, K. F., et al. Submitted for publication.
|
| 50.
|
Zou, Y. Y.,
Y. H. Ling,
S. Reddy,
W. Priebe, and R. Perez-Soler.
1995.
Effect of vesicle size and lipid composition on the in vivo tumor selectivity and toxicity of the non-cross-resistant anthracycline Anamycin incorporated in liposomes.
Int. J. Cancer
61:666-671[Medline].
|
Antimicrobial Agents and Chemotherapy, January 1998, p. 45-52, Vol. 42, No. 1
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
