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Antimicrobial Agents and Chemotherapy, March 2000, p. 533-539, Vol. 44, No. 3
Biotechnology Laboratory1 and Departments of
Biochemistry & Molecular Biology and Microbiology & Immunology3,
University of British Columbia, Vancouver, British Columbia, Canada V6T
1Z3, and Inex Pharmaceuticals
Corporation, Burnaby, British Columbia, Canada V5J
5J82
Received 26 January 1999/Returned for modification 20 September
1999/Accepted 1 December 1999
Encapsulation of gentamicin in liposomes can be used to achieve
intracellular delivery and broaden the clinical utility of this drug.
We have previously described a novel, rationally designed, pH-sensitive
liposomal carrier for gentamicin that has superior in vitro efficacy
against intracellular infections compared to the efficacies of both
free gentamicin and non-pH-sensitive liposomal controls. This liposomal
carrier demonstrated pH-sensitive fusion that was dependent on the
presence of unsaturated phosphatidylethanolamine (PE) and the
pH-sensitive lipid N-succinyldioleoyl-PE. The
pharmacokinetics and biodistribution of the free and liposomal
gentamicin were examined in mice bearing a systemic Salmonella
enterica serovar Typhimurium infection. Encapsulation of
gentamicin in pH-sensitive liposomes significantly increased the
concentrations of the drug in plasma compared to those of free
gentamicin. Furthermore, the levels of accumulation of drug in the
infected liver and spleen were increased by 153- and 437-fold,
respectively, as a result of liposomal encapsulation. The increased
accumulation of gentamicin in the liver and spleen effected by
liposomal delivery was associated with 104-fold greater
antibacterial activity than that associated with free gentamicin in a
murine salmonellosis model. These pH-sensitive liposomal antibiotic
carriers with enhanced in vitro activity could be used to improve both
in vivo intracellular drug delivery and biological activity.
Gentamicin is an aminoglycoside
antibiotic with broad-spectrum antibacterial activity against several
serious bacterial infections, including typhoid fever. Infections
caused by intracellular pathogens, such as Brucella,
Salmonella, Listeria, Legionella, and
Mycobacteria species, are difficult to treat by conventional
antimicrobial therapies because of the requirement that antibiotics
reach therapeutic levels at the intracellular site of infection. Since
gentamicin is polycationic, it is freely soluble in water and has a low
level of permeation through biological membranes. After intravenous (i.v.) or intramuscular administration the majority of the drug remains
extracellular (7). Therefore, gentamicin and many other antibiotics that are active in vitro are often inactive against intracellular bacteria due to their poor penetration into cells. The
development of new antibacterial formulations or carriers capable of
intracellular delivery will improve therapy for infections that are
presently difficult to treat.
The encapsulation of gentamicin in liposomal delivery systems has been
used to alter the drug's biodistribution, increase its level of
accumulation at the disease site, and reduce its nephrotoxicity that
arises from multiple daily administrations (6). Gentamicin
has been encapsulated into a variety of egg phosphatidylcholine (egg
PC) and egg PC-cholesterol (chol) formulations and has been shown to
have antibacterial activity in both in vitro and in vivo infection
models (for a review, see reference 6). However, the
formulations used by those workers were not specifically designed to
facilitate the intracellular delivery of the antibiotic. Rather, the in
vivo efficacies of these liposomal formulations resulted from the
ability of liposomal carriers to increase the circulation lifetime of
the encapsulated drug and achieve passive accumulation of drug at a
site of infection. Similar effects have been demonstrated previously
for other encapsulated antibacterial (15) and anticancer
(16) agents. It is expected that a liposomal formulation
that can achieve intracellular antibiotic delivery will demonstrate
significantly enhanced antibacterial activity against intracellular
pathogens beyond that conferred by either free drug or drug
encapsulated in nonfusogenic carriers.
In a previous study, we described a novel pH-sensitive liposome
composition that achieved very effective intracellular delivery of
gentamicin into a mouse monocyte/macrophage cell line infected with
Salmonella enterica serovar Typhimurium (8). This
formulation comprises dioleoylphosphatidylethanolamine (DOPE),
N-succinyl-DOPE, and polyethylene glycol (PEG)-ceramide and
delivered drug to the cytoplasm of infected macrophages in a manner
dependent on endosomal acidification (8). The antibacterial
activity achieved by this liposomal carrier was significantly greater
than that achieved by nonfusogenic control carriers (8).
The experiments reported on here were performed to determine if use of
a pH-sensitive liposomal delivery system would also result in enhanced
antibacterial activity in an in vivo infection model in which S. enterica serovar Typhimurium is an intracellular resident of macrophages.
Materials.
Commercially available lipids were obtained from
Avanti Polar Lipids (Alabaster, Ala.) or Northern Lipids (Vancouver,
British Columbia, Canada). PEG-C20-ceramide (PEG-ceramide)
was manufactured at Inex Pharmaceuticals by Zhao Wang as described
previously (17). Gentamicin sulfate was obtained from Sigma
Chemical Company (St. Louis, Mo.). 14C-cholesterol
hexadecyl ether (14C-CHE) as well as
3H-gentamicin were obtained from Amersham (Oakville,
Ontario, Canada). The murine macrophage cell line J774A.1 was obtained
from the American Type Culture Collection. Dulbecco's modified eagle
medium, fetal bovine serum, and phosphate-buffered saline (PBS) plus
Ca2+ and Mg2+ were obtained from Gibco
(Burlington, Ontario, Canada). Octaethyleneglycol mono-n-dodecyl ether (C12E8) was
obtained from Calbiochem (San Diego, Calif.). All other chemicals used
were of reagent grade and were obtained from major suppliers.
Liposomal gentamicin preparation.
Liposomal gentamicin
formulations were prepared as described previously (8).
Appropriate amounts of lipids were mixed in chloroform, and the solvent
was evaporated by agitation under a nitrogen stream. Residual solvent
was removed from the lipid mixture under high vacuum for at least
1 h. Dried lipid films (representing 25 mg of total lipid) were
rehydrated by the addition of 0.5 ml of 100 mg of gentamicin (as the
gentamicin base) per ml in 20 mM HEPES-150 mM NaCl (HBS; pH 7.4).
Hydration of the lipid was facilitated by extensive vortexing and five
freeze-thaw cycles between
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Antibacterial Efficacy of Gentamicin Encapsulated in pH-Sensitive
Liposomes against an In Vivo Salmonella enterica Serovar
Typhimurium Intracellular Infection Model
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
196°C and room temperature. After
hydration, the samples were diluted so that they contained lipid and
drug concentrations of 25 and 50 mg/ml, respectively. This dispersion
of multilamellar vesicles was converted to large unilamellar vesicles
by 10 extrusions through two stacked 0.1-µm-pore-size filters
(Poretics; AMD Manufacturing, Mississauga, Ontario, Canada) at 25 to
28°C by using a Thermobarrel Extruder (Lipex Biomembranes, Vancouver,
British Columbia, Canada). Liposomes composed of
dipalmitoylphosphatidylcholine (DPPC)-chol were extruded as described
above but at 55°C. Removal of gentamicin from the outside surface of
these anionic liposomes was achieved by adjusting the pH of the
liposome suspension to pH 10 with 500 to 700 µl of glycine-buffered
saline (2 M glycine, 150 mM NaCl [pH 10.0]) or 50 to 75 µl of 3 N
NaOH with rapid vortexing. The liposomes were then passed through a 20- to 25-ml CM-Sepharose column equilibrated to pH 10.0 with 10 mM
glycine-150 mM NaCl. The liposome fractions were collected and
reequilibrated to pH 7.4 with 0.5 M HEPES-150 mM NaCl (pH 7.4).
In vitro lipid mixing assays.
Liposome fusion was determined
by measurement of lipid mixing as described previously (8).
Briefly, donor vesicles containing encapsulated gentamicin were
prepared as described above, but they also contained 0.5 mol% each
N-4-nitrobenzo-2-oxa-1,3-diazole (NBD)
phosphatidylethanolamine (NBD-PE) and N-(lissamine rhodamine B sulfonyl)-dioleoylphosphatidylethanolamine (Rh-PE). Acceptor vesicles
were of the same composition as the donor vesicles but without the
fluorescent molecules. Donor liposomes in 10 mM HBS (pH 7.4) were mixed
in a quartz cuvette with acceptor liposomes (also in 10 mM HBS [pH
7.4]) and were brought to a final volume of 1,900 µl with 150 mM
NaCl. The added volumes of donor and acceptor liposomes were adjusted
to achieve final lipid concentrations of 0.1 and 1.0 mM, respectively.
The cuvette was placed in an SLM Aminco-Bowman Series II luminescence
spectroscope equipped with a stirring mechanism and maintained at
37°C. The fluorescence emission was measured at 535 nm (NBD moiety
fluorescence) by using an excitation wavelength of 445 nm, with 4-nm
slit widths and with the initial fluorescence set at between 50 and
70% of full scale. After a stable baseline was verified, 50 µl of an
acid solution composed of 5% (vol/vol) acetic acid in saline or
dilutions thereof was added. After a 2-min stabilization period to
verify that no fusion had occurred, CaCl2 was added
directly to the cuvette to a final concentration of 20 mM. The
fluorescence was measured continuously until a plateau was achieved,
and then 100% dequenching of the NBD moiety was determined by the
addition of 50 µl of 0.2 M C12E8 detergent.
The final pHs of the solutions were measured with a pH meter. Data were
processed with software provided by Aminco-Bowman. Percent NBD
dequenching, equivalent to percent fusion, was determined as (final
fluorescence initial
fluorescence)/(C12E8 fluorescence initial fluorescence) × 100.
Pharmacokinetics and biodistribution. Gentamicin (labeled with 3H-gentamicin at approximately 2.0 µCi/mg of drug) was encapsulated in liposomes as described above. The liposomes were composed of DPPC-chol (55/45 mol%) or DOPE-N-succinyl-DOPE-chol-PEG-ceramide (35/30/30/5 mol%) that were labeled at approximately 7 µCi/100 mg of lipid with 14C-CHE, a nonexchangeable and nonmetabolizable radioactive tracer lipid (12). These specific activities provided levels in plasma greater than or equal to 880 and 620 dpm/ml for 3H-gentamicin and 14C-CHE, respectively, at their nadirs in the pharmacokinetic studies. For gentamicin, this represents a lowest measured concentration of 0.2 µg/ml of plasma in the pharmacokinetic studies. Pharmacokinetic studies were performed by injecting liposomal gentamicin formulations or free gentamicin i.v. via the lateral tail vein. In preliminary experiments, free and liposomal forms of the drug were administered to uninfected ICR mice at a dose of 10 mg of gentamicin/kg of body weight. In subsequent determinations, free and liposomal forms of the drug were administered to BALB/c mice at a dose of 4.5 mg of gentamicin/kg 24 h after i.v. infection with S. enterica serovar Typhimurium as described below. That is, the pharmacokinetics and biodistribution were determined under in vivo conditions identical to those used for the efficacy experiments described in the next section.
At various time points after drug administration (0.25, 0.5, 1, and 3 h for free gentamicin; 1, 3, 6, and 24 h for liposomal gentamicin), the mice were anesthetized and blood was recovered via cardiac puncture. Subsequently, the animals were killed with an overdose of CO2; and the liver, spleen, lung, kidney, and thigh muscle were recovered. Plasma and tissue homogenates were assayed for gentamicin and lipid levels by LSC. The concentrations of lipid and drug in tissues were corrected for the concentration contribution from the blood as described previously (9). Pharmacokinetic parameters were calculated by using WinNonlin pharmacokinetic software (version 3.0; Pharsight, Mountain View, Calif.). Data were analyzed by using noncompartmental, one- and two-compartment models, and the best-fit results reported. The observed area-under-the-curve (AUC) values for lipid and drug accumulation in tissues were obtained from the linear-trapezoidal calculation in the noncompartmental model by using WinNonlin.In vivo intracellular killing and antibacterial efficacy. S. enterica serovar Typhimurium SL1344 was grown overnight in Luria-Bertani broth in a 37°C shaking incubator. The culture was centrifuged and washed in PBS and was then diluted to achieve a final suspension containing 1,000 CFU/ml. Female BALB/c mice (age, 6 to 8 weeks) were injected i.v. in the tail vein with inocula containing 60 to 90 CFU per mouse.
Twenty-four hours after infection, the mice were treated by a single i.v. administration of free gentamicin (in saline) or liposomal gentamicin at doses between 0.2 and 20 mg of gentamicin/kg. The solutions of both free and liposomal gentamicin were sterilized by passage through a 0.2-µm-pore-size filter prior to administration. At 5 days postinfection, the mice were killed and the spleens and/or livers were removed and placed in sterile 6-ml WhirlPak bags on ice. Individual organs were aseptically homogenized in 3.0 ml of cold PBS, and then aliquots (100 µl) of the homogenates were serially diluted in PBS to a maximum of a 105-fold dilution and plated on duplicate MacConkey agar plates. The plates were incubated overnight at 37°C, and the resulting light red (lac mutant) colonies on plates containing between 30 and 300 colonies were counted. The results were analyzed by the nonparametric Kruskal-Wallis one-way analysis of variance on ranks method by Dunn's pairwise multiple comparison technique on SigmaStat statistical software (version 2.0; SPSS Science, Chicago, Ill.).| |
RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Pharmacokinetics and biodistribution studies with uninfected mice. In a previous publication (8) we demonstrated that a formulation that comprised DOPE-N-succinyl-DOPE-PEG-ceramide had significantly greater efficacy against intracellular S. enterica serovar Typhimurium infections than various nonfusogenic (i.e., DPPC-chol) liposomes by an in vitro killing assay. Therefore, preliminary in vivo screening experiments were performed with healthy (i.e., uninfected) mice to optimize the pharmacokinetics of liposome-encapsulated gentamicin for subsequent in vivo antibacterial efficacy studies. DPPC-chol (55/45), DOPE-N-succinyl-DOPE-PEG-ceramide (69.5/30/0.5 and 65/30/5; mol/mol/mol), and DOPE-N-succinyl-DOPE-chol-PEG-ceramide (35/30/30/5, 39.5/30/30/0.5, 49.5/30/20/0.5, and 45/30/20/5; mol/mol/mol/mol) preparations of liposomal gentamicin were compared for drug and lipid circulation lifetimes after i.v. administration (data not shown).
Free gentamicin was rapidly eliminated from the circulation (data not shown). In contrast, encapsulation of gentamicin in all of the DOPE-N-succinyl-DOPE- and DOPE-N-succinyl-DOPE-chol-PEG-ceramide-based carriers substantially increased the concentrations of gentamicin in the circulation after i.v. administration (data not shown). In some formulations, rapid in vivo leakage of drug from the liposomes was observed, but this could be ameliorated by the addition of chol to the formulation. Overall, the two formulations that provided maximal gentamicin concentrations in the plasma after i.v. administration for in vivo efficacy testing were DPPC-chol (55/45; mol/mol) and DOPE-N-succinyl-DOPE-chol-PEG-ceramide (35/30/30/5; mol/mol/mol/mol).Pharmacokinetics and biodistributions of free and liposomal
gentamicin in infected mice.
In the preceding section, optimized
liposomal formulations of gentamicin were identified on the basis of
the pharmacokinetics of gentamicin and lipid in healthy mice. As a
prelude to evaluating their antibacterial efficacies in a murine
infection model, the pharmacokinetics and biodistributions of
these liposomal gentamicin formulations in animals bearing an
S. enterica serovar Typhimurium infection were characterized
(Fig. 1A to C).
|
) and
for gentamicin encapsulated in liposomes composed of DPPC-chol (55/45;
) or DOPE-N-succinyl-DOPE-chol-PEG-ceramide
(35/30/30/5; 
). Data are means ± standard deviations for three
animals. Error bars are plotted for all datum points; where error bars
are not visible, they are smaller than the size of the symbol.
) of 8.49 h.
In contrast, the elimination of the
DOPE-N-succinyl-DOPE-chol-PEG-ceramide (35/30/30/5)
formulation best fit a two-compartment model (r = 0.9998), with calculated values for the distribution phase
half-life (t1/2
) of 0.22 h and for the
elimination phase half-life (t1/2
) of
8.69 h. For both liposomal formulations of gentamicin, there was
negligible leakage of drug, as indicated by the drug/lipid ratio (Fig.
1B), after administration to infected mice. Consequently, the
pharmacokinetics of drug elimination (Fig. 1C) from the plasma of mice
bearing an S. enterica serovar Typhimurium infection were very similar to those described above for liposome elimination rates
(contrast Fig. 1A with C). That is, the elimination of gentamicin removal from the circulation of infected mice treated with the DPPC-chol form of liposomal gentamicin was best described by a noncompartmental model (r = 0.9991) with a calculated
t1/2 of 8.13 h. The elimination of drug
administered as the
DOPE-N-succinyl-DOPE-chol-PEG-ceramide (35/30/30/5) form
of liposomal gentamicin best fit a two-compartment model (r = 0.9998) with calculated values of t1/2
of 0.24 h and t1/2 of 8.88 h. While
the elimination of free gentamicin was best described by a
two-compartment model (r = 1.000), the calculated
t1/2s were significantly shorter, with a
t1/2 of 0.07 h and a
t1/2 of 0.49 h. These elimination rates
were associated with observed AUC values of 17.23, 129.3, and 1,195.4 µg · h/ml for free gentamicin and for the DPPC-chol and
DOPE-N-succinyl-DOPE-chol-PEG-ceramide (35/30/30/5)
liposomal gentamicin formulations, respectively. These represent 7.5- and 69.4-fold increases in the plasma AUCs for the DPPC-chol and
DOPE-N-succinyl-DOPE-chol-PEG-ceramide liposomal formulations of gentamicin, respectively, compared to that for the free drug.
The effects of liposomal encapsulation of gentamicin on drug
biodistribution are shown in Fig. 2A to C
and are summarized in Table 1.
Encapsulation of gentamicin in either liposome formulation redirected
gentamicin to both the liver and the spleen (Fig. 2A and B; Table 1)
and substantially reduced the level of drug accumulation in the kidneys
(Fig. 2C and Table 1). The increase in drug accumulation conferred by
liposomal encapsulation was most pronounced in spleens, in which
DPPC-chol liposomes increased drug quantities by 497-fold and
DOPE-N-succinyl-DOPE-chol-PEG-ceramide formulations
increased drug quantities by 437-fold (Table 1). The two formulations
increased the quantities delivered to the liver by factors of 80- and
153-fold, respectively (Table 1), whereas delivery of increased
quantities to the lungs was observed only for the DPPC-chol liposomal
formulation of gentamicin (5.9- versus 1.05-fold for
DOPE-N-succinyl-DOPE-chol-PEG-ceramide; data not
shown). The gentamicin/lipid ratios in the livers and spleens of mice
receiving liposomal gentamicin were very similar to the
gentamicin/lipid ratios of the injected formulations (Table 1). This
result suggests that the liposomes with the encapsulated gentamicin
were accumulating intact in the liver and spleen. In contrast, the
gentamicin/lipid ratios were significantly higher in the kidneys of
these animals, by 2.4- and 13.3-fold for the DPPC-chol and
DOPE-N-succinyl-DOPE-chol-PEG-ceramide formulations, respectively (Table 1). This result suggests that gentamicin leaking
from these liposomal delivery formulations rapidly accumulated in the
kidneys in a fashion identical to that observed after the administration of the free drug (Table 1).
|
) and lipid (
,
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In vivo antibacterial efficacy. To evaluate the therapeutic benefit conferred by encapsulation of gentamicin in a pH-sensitive liposome, we have chosen an in vivo S. enterica serovar Typhimurium infection model (11). This model was used because it is a well-characterized model in which the infecting bacteria reside primarily in the macrophages of the liver and the spleen (11). Since the quantities of drug that accumulated in the spleens were similar for these nonfusogenic (DPPC-chol) and pH-sensitive (DOPE-N-succinyl-DOPE-chol-PEG-ceramide) formulations (Fig. 2; Table 1), this represents an opportunity to distinguish between liposomal drug delivery to a diseased tissue and additional intracellular delivery to the infecting intracellular pathogen.
By administration of free gentamicin as single i.v. doses between 0.2 and 5.0 mg/kg 24 h after bacterial infection, the free gentamicin had no statistically significant antibacterial activity (P = 0.05) (Fig. 3). In contrast, the DOPE-N-succinyl-DOPE-chol-PEG-ceramide liposomal formulation of gentamicin was very effective at killing the intracellular pathogen (Fig. 3). That is, at doses between 0.2 and 5.0 mg/kg, the number of bacteria surviving in the spleens was decreased by approximately 103 (Fig. 3). At doses between 0.2 and 1.0 mg/kg, the DOPE-N-succinyl-DOPE-chol-PEG-ceramide formulation of gentamicin had a good dose-response relationship (r2 = 0.84; Fig. 3). At all doses tested, the number of bacteria observed in the spleens of animals treated with the liposomal form of the antibiotic were significantly lower (P < 0.05) than those in the spleens of control and free gentamicin-treated mice (with the exception of the numbers after treatment with liposomal gentamicin at 5.0 mg/kg compared to those in no-treatment controls; P = 0.081).
|
) or for mice
receiving either free gentamicin (
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) or the free gentamicin control (*) for
which P was <0.05 are indicated by matching symbols. For
all other pairwise comparisons P was >0.05.
In vitro characteristics of liposomal gentamicin formulations.
The pH dependence of the fusion of the
DOPE-N-succinyl-DOPE-chol-PEG-ceramide (35/30/30/5)
formulation of gentamicin was confirmed by a resonance energy transfer
assay (Fig. 5). In the presence of 20 mM
Ca2+ and at pH values between 6.9 and 4.0, a control
formulation composed of
DOPC-N-succinyl-DOPE-chol-PEG-ceramide
(39.5/30/30/0.5) had no fluorescence dequenching indicative
of membrane fusion. In contrast, the
DOPE-N-succinyl-DOPE-chol-PEG-ceramide formulation (39.5/30/30/0.5) showed significant fusion activity, particularly in
the pH range from 5.5 to 4.5 that is characteristic of late endosomes.
These data are consistent with our previous report that a very similar
liposomal gentamicin composition,
DOPE-N-succinyl-DOPE-PEG-ceramide, undergoes a
pH-dependent destabilization or membrane fusion event that
facilitates intracellular delivery of liposomal contents (8). These results are also consistent with the report
(3) that pH-sensitive liposomes containing up to 40 mol%
cholesterol retain their pH-dependent membrane fusion activity.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Gentamicin is an aminoglycoside antibiotic whose clinical utility against serious pathogens is limited by poor penetration of host cells to the location of the intracellular bacterial infection. In a previous study (8) we described a novel liposomal formulation of gentamicin that is capable of achieving efficient intracellular antibiotic delivery and improved antibacterial therapy. This formulation was designed to achieve intracellular drug delivery via the inclusion of lipids (N-succinyl-DOPE and DOPE) that destabilize the carrier membrane at endosomal pH values. It was the purpose of the present study to evaluate if this pH-sensitive liposomal delivery vehicle that demonstrates significantly enhanced intracellular delivery activity in vitro would also show increased antibacterial activity in vivo.
On the basis of preliminary screening experiments, the formulations
initially identified for in vivo efficacy studies
(DOPE-N-succinyl-DOPE with 0.5 or 5.0% PEG-ceramide) based
on in vitro experiments (8) were modified to include 30 mol% cholesterol. Significant improvements in the liposome circulation
lifetime, drug retention within the liposomes, and drug concentrations
in the circulation were observed with the inclusion of 30 mol%
cholesterol in the DOPE-N-succinyl-DOPE-based vesicles
(data not shown). Despite this improvement, a significant proportion of
the DOPE-N-succinyl-DOPE-chol-PEG-ceramide liposome population showed an initial clearance phase
(t1/2 = 0.22 h) that was associated with
rapid liposome accumulation in the liver (Fig. 2A). It is likely that
the clearance of these anionic vesicles was a result of protein
opsonization and subsequent uptake by both fixed and circulating
macrophages (5). Similarly, opsonization to these anionic
formulations could promote their destabilization and subsequent drug
release. This observation is consistent with previous reports that the
presence of cholesterol increases the circulation longevity of
liposomes and improves drug retention (5, 9). A remaining
proportion of the DOPE-N-succinyl-DOPE-chol-PEG-ceramide liposome population (<10%) had a t1/2 (8.69 h) very similar to the DPPC-chol t1/2 (8.49 h). It is possible that this small proportion of the
DOPE-N-succinyl-DOPE-chol-PEG-ceramide liposome
population may have had a negligible surface charge and consequently
acquired clearance characteristics comparable to those of a neutral
liposomal carrier such as DPPC-chol (5).
The results presented in Fig. 2 demonstrate that the DOPE-N-succinyl-DOPE-based formulations effectively precluded drug accumulation in the kidneys and redirected the antibiotic to the liver and spleen. While the accumulation of drugs in the liver and spleen is well known for liposomal formulations, the rate of the DOPE-N-succinyl-DOPE-chol-PEG-ceramide liposomal form of gentamicin accumulation in these organs was substantially faster than that for neutrally charged DPPC-chol liposomes. These data are consistent with those that were reported previously and that demonstrated the extensive accumulation of empty 200-nm egg PC-chol-N-succinyl-DOPE liposomes in the livers of mice (10). pH-sensitive liposomes composed of DOPE-oleic acid have also been observed to be removed rapidly from the circulation and to accumulate to high levels in the livers, lungs, and spleens of mice (2). The data reported by those investigators also suggested rapid leakage of the entrapped marker after i.v. administration and subsequent marker distribution to organs different from those to which the injected carrier was distributed (2). In contrast, the drug/lipid ratios presented in Table 1 demonstrate that the liposomes localized in the liver and spleen contained encapsulated gentamicin, while the kidney appeared to be accumulating gentamicin that had been released from liposomes in the circulation. The blood circulation times for these pH-sensitive formulations are not as long as those reported for sterically stabilized pH-sensitive liposomes composed of cholesterol hemisuccinate (13). However, the rapid accumulation of these carriers in the target organs (the liver and spleen) makes these pH-sensitive carriers ideal for in vivo evaluation in an antibacterial efficacy model.
The in vivo antibacterial activity of the pH-sensitive liposomal gentamicin carriers was excellent, achieving 103- to 104-fold reductions in the numbers of S. enterica serovar Typhimurium organisms residing in the spleen (Fig. 3) and liver (data not shown). Similar decreases in bacterial load have been reported in rats bearing pulmonary Klebsiella pneumoniae infections and treated with liposomal gentamicin (1), and substantial increases in survival have been reported in mice bearing S. enterica serovar Typhimurium infections that were treated with liposomal gentamicin (14). The previous results, achieved with nonfusogenic liposomes based on egg PC or egg PC and chol mixtures, are consistent with the results presented in Fig. 4. That is, the antibacterial effects observed with the pH-sensitive DOPE-N-succinyl-DOPE-based formulations (Fig. 4) were also achieved with two different nonfusogenic control formulations of gentamicin. There are two primary reasons why this might occur: (i) the pH-sensitive formulation is rendered nonfusogenic upon systemic administration, possibly by the adsorption of serum proteins, or (ii) after liposomal drug accumulation at the disease site (Fig. 2), the therapeutic benefits that are achieved by fusogenic carrier-mediated intracellular delivery of gentamicin are outweighed by those benefits that arise from the substantial increases in the AUC for the antibiotic at the infection site (Table 1) for both fusogenic and nonfusogenic carriers. The latter explanation is more likely, given the observation that these liposomal formulations retain their pH-sensitive behavior in the presence of fetal bovine serum (8) (data not shown). It is pertinent that the antibacterial activities of the liposomal gentamicin formulations were poorly correlated to the pharmacokinetic parameters. Rather, antibacterial activity was closely associated with the AUC for drug accumulation in the target organs, the liver and spleen, irrespective of the liposomal formulation used to deliver the antibiotic.
It should be noted that the numbers of bacteria that survived in the spleens of mice treated with liposomal gentamicin were similar to those that were present at the start of gentamicin therapy (11). Therefore, it cannot be excluded that the liposomal formulations may be exerting cytostatic rather than cytotoxic effects. However, since the infection is exclusively present intracellularly, these data demonstrate that the use of these liposomal formulations enhanced the intracellular delivery of the drug to increase either the cytotoxic or the cytostatic activities of the drug compared to those achieved with free gentamicin.
This is the first report of a study that has characterized the in vivo efficacy of an antibiotic encapsulated in a pH-sensitive fusogenic liposome. It is anticipated that the ability of pH-sensitive fusogenic liposomal carriers to significantly improve in vivo therapeutic activity will be seen with the optimum combination of encapsulated drug, disease target, and carrier pharmacodynamics.
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ACKNOWLEDGMENTS |
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We thank Maryse St. Louis for advice and suggestions and Dana Masin for expert technical assistance. Senior authorship is shared equally by B.B.F. and M.S.W.
This work was supported in part by the Technology Partnership Program (Natural Sciences and Engineering Research Council of Canada [NSERC]). P.L. is the recipient of an NSERC Industrial Fellowship.
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FOOTNOTES |
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* Corresponding author. Mailing address: Inex Pharmaceuticals Corporation, 100-8900 Glenlyon Parkway, Burnaby, B.C., Canada V5J 5J8. Phone: (604) 419-3200. Fax: (604) 419-3201. E-mail: mwebb{at}inexpharm.com.
Present address: QLT Phototherapeutics, Vancouver, British
Columbia, Canada V5T 4T5.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Antimicrobial Agents and Chemotherapy, March 2000, p. 533-539, Vol. 44, No. 3
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