Liposomal formulations of inflammatory bowel disease drugs: Local versus systemic drug delivery in a rat model

Article abstract of DOI:10.1007/s11095-005-5376-3

Purpose. Based on adherence to intestinal mucosa, intralumenally administered liposomal formulations of 5-aminosalicylate (5-ASA) and 6-mercaptopurine (6-MP) were studied for their potential to enhance local drug delivery to intestinal tissue for the treatment of inflammatory bowel disease. Methods. 5-ASA was encapsulated in standard phospholipid liposomes while 6-MP required encapsulation in nonphospholipid liposomes to obtain equivalent drug loading. Encapsulation efficiency was measured by size-exclusion chromatography/high-performance liquid chromatogtaphy (HPLC). Liposomal formulations or solution of the drugs were injected into unligated jejunum to compare pharmacokinetics and into ligated loops of rat ileum and colon to evaluate local delivery. Dextran sulfate and acetic acid induced colitis were used as models of lower intestinal inflammation. Plasma, tissue and luminal drug and metabolite levels were measured by liquid scintillation counting or HPLC. Results. Encapsulation efficiency of 6-MP was dependent on lipid content and composition. While liposomal encapsulation significantly reduced systemic absorption of 5-ASA this was not the case for 6-MP. Liposomal adherence to intestinal tissue resulted in increased tissue levels for 5-ASA; however, 6-MP local tissue levels were not improved compared to solution drug. Conclusions. Nonphospholipid liposomes optimize encapsulation of 6-MP. While liposomal formulations show potential for local drug delivery to diseased bowel, drug physicochemical properties, absorption, and metabolic profiles dictate tissue-targeting potential. Liposomes reduce systemic availability from paracellular absorption of hydrophilic 5-ASA, but fail to improve local tissue delivery of 6-MP, a molecule absorbed by passive membrane permeation that undergoes extensive first- pass metabolism.

Full text of DOI:10.1007/s11095-005-5376-3

#
Pharmaceutical Research, Vol. 22, No. 8, August 2005 ( 2005)
DOI: 10.1007/s11095-005-5376-3
Research Paper
Liposomal Formulations of Inflammatory Bowel Disease Drugs: Local versus
Systemic Drug Delivery in a Rat Model
Filippos Kesisoglou,1,2 Simon Yuji Zhou,1 Susan Niemiec,3 Jordan Wing Lee,1 Ellen M. Zimmermann,2
and David Fleisher1,4
Received December 19, 2004; accepted March 15, 2005
Purpose. Based on adherence to intestinal mucosa, intralumenally administered liposomal formulations
of 5-aminosalicylate (5-ASA) and 6-mercaptopurine (6-MP) were studied for their potential to enhance
local drug delivery to intestinal tissue for the treatment of inflammatory bowel disease.
Methods. 5-ASA was encapsulated in standard phospholipid liposomes while 6-MP required
encapsulation in nonphospholipid liposomes to obtain equivalent drug loading. Encapsulation efficiency
was measured by size-exclusion chromatography/high-performance liquid chromatogtaphy (HPLC).
Liposomal formulations or solution of the drugs were injected into unligated jejunum to compare
pharmacokinetics and into ligated loops of rat ileum and colon to evaluate local delivery. Dextran
sulfate and acetic acid induced colitis were used as models of lower intestinal inflammation. Plasma,
tissue and luminal drug and metabolite levels were measured by liquid scintillation counting or HPLC.
Results. Encapsulation efficiency of 6-MP was dependent on lipid content and composition. While
liposomal encapsulation significantly reduced systemic absorption of 5-ASA this was not the case for
6-MP. Liposomal adherence to intestinal tissue resulted in increased tissue levels for 5-ASA; however,
6-MP local tissue levels were not improved compared to solution drug.
Conclusions. Nonphospholipid liposomes optimize encapsulation of 6-MP. While liposomal formula-
tions show potential for local drug delivery to diseased bowel, drug physicochemical properties,
absorption, and metabolic profiles dictate tissue-targeting potential. Liposomes reduce systemic
availability from paracellular absorption of hydrophilic 5-ASA, but fail to improve local tissue delivery
of 6-MP, a molecule absorbed by passive membrane permeation that undergoes extensive first- pass
metabolism.
KEY WORDS: absorption; drug delivery; inflammatory bowel disease; intestine; liposomes.
INTRODUCTION
regimens utilizing oral administration, systemic absorption
is an undesirable delivery feature for these drugs. Disease
Local delivery of therapeutics to diseased bowel tissue localization dictates the need for maximal intestinal tissue
through oral administration is a challenging problem but a drug exposure while systemic delivery should be minimized
desirable goal to treat gastrointestinal diseases such as to avoid unwanted side effects. In the case of IBD, which
inflammatory bowel disease (IBD), irritable bowel syndrome primarily involves pathology in the large intestine and lower
(IBS), or colon cancer. Contrary to most therapeutic small intestine (1), delayed or controlled release oral dosage
forms have proven beneficial to negate upper intestinal
1 Department of Pharmaceutical Sciences, College of Pharmacy, The
University of Michigan, Ann Arbor, Michigan 48109-1065, USA.
2 Division of Gastroenterology, Department of Internal Medicine,
The University of Michigan, Ann Arbor, Michigan 48109-0682,
USA.
absorption and release the drug at the sites of disease (2Y5).
The potential to increase drug residence time in regions of
diseased tissue would serve to further optimize this therapy.
Oral dosage forms that adhere to diseased bowel tissue
could provide sustained exposure of a therapeutic agent to
sites of pathology and improve its therapeutic effect. In this
direction, particulate systems such as nanoparticles and
microspheres have attracted attention due to their mucoad-
hesive properties (6Y11). In a recent study (12), colonic
adhesion properties of liposomal formulations were reported
in studies on a potential delivery vehicle for antioxidant
enzymes to treat intestinal inflammation. Liposomal dosage
forms for local delivery of small drug molecules used for
treatment of IBD were the focus of this research.
3 Pfizer Global Research and Development, Ann Arbor Laborato-
ries, Ann Arbor, Michigan 48105, USA.
4 To whom correspondence should be addressed. (e-mail: fleisher@
umich.edu)
ABBREVIATIONS: 5-ASA, 5-aminosalicylate; FBS, fetal bovine
serum; GDS, glyceryl distearate; HPLC, high-performance liquid
chromatography; IBD, inflammatory bowel disease; IBS, irritable
bowel system; 6-MP, 6-mercaptopurine; NEAA, nonessential amino
acids; NMR, nuclear magnetic resonance; PBS, phosphate-buffered
saline; POE, polyoxyethylene stearyl ether.
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0724-8741/05/0800-1320/0
2005 Springer Science + Business Media, Inc.
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Intralumenal Liposomal Drug Delivery for IBD
1321
Two low molecular weight IBD drugs with very different Liposomal Preparation and Characterization
physicochemical and biological delivery properties, 5-amino-
salicylate (5-ASA) and 6-mercaptopurine (6-MP), were
Phospholipid-based 5-ASA liposomes were prepared by
selected for study in a rat model. Marketed controlled and a solvent evaporation method. Briefly stated, a mixture of
delayed release dosage forms of 5-ASA (3) reduce the rapid saturated egg lecithin, sodium phosphatidylglycerol, and
and complete upper intestinal absorption of this drug (13) to cholesterol (2:1:2 ratio) and 5-ASA solution in phosphate-
achieve delivery to the lower bowel. Systemic absorption of buffered saline (PBS), pH 7.4, was mixed in methyl alcohol
the more potent 6-MP, associated with bone marrow and chloroform to form a clear homogeneous solution. The
suppression, is an important concern in IBD therapy that chloroform and methanol were removed together with a
requires close patient monitoring, can be dose limiting, and fraction of water under vacuum at 65Y70-C, leaving a
further supports the case for local delivery. Local delivery for concentrated liposomal solution. Water was added to obtain
IBD therapy is typically assessed by pharmacodynamic a final lipid concentration of 50 mg/ml. The final 5-ASA
evaluation in animal models by utilizing biomarkers of concentration of the liposomal formulations was 7.5 mg/ml.
inflammation and histological scores (8Y10). In this report,
Nonphospholipid-based 6-MP liposomes were prepared
systemic drug and metabolite measurements are employed to following the procedures described by Niemiec et al. (14).
discern the drug delivery limitations for therapy. Liposomal Glyceryl distearate (GDS), cholesterol (CH), and polyoxy-
formulations of these two drugs were tested for their capacity ethylene (10) stearyl ether (POE) were used as the lipid
to reduce systemic drug levels and increase intestinal tissue components. Lipid composition was varied to maximize 6-
levels in normal and colitic rats. While differing drug MP loading. 6-MP solution in PBS was used as the aqueous
physicochemical properties dictated different liposomal for- phase. 6-MP solutions in PBS were prepared on the day of
mulations, biological factors such as absorption character- the experiment from a stock solution of 10 mM 6-MP in 0.1 N
istics and intestinal metabolic profiles of these drugs also NaOH, with pH adjusted with HCl to 7.4. Appropriate
influence systemic versus local delivery potential.
amounts of the lipids were accurately weighed in small
beakers. The lipids were heated with stirring to 70Y75-C
until the lipid melt became a clear solution. The lipid melt
was then drawn into a preheated syringe. 6-MP solution,
preheated to 60Y65-C, was drawn to a second preheated
syringe. The two syringes were connected via a three-way
Teflon stopcock. The aqueous solution was then injected into
the lipid phase syringe. The mixture was repeatedly mixed
back and forth between the two syringes while being cooled
under tap water, until the mixture reached room temperature.
For both formulations, drug encapsulation efficiency was
measured by size exclusion chromatography followed by
HPLC analysis of both free and liposome-encapsulated drug,
using established methods (15,16). Encapsulation efficiency
(E.E.) was calculated as:
MATERIALS AND METHODS
Materials
6-MP monohydrate and 5-ASA were purchased from
ICN Biochemicals (Costa Mesa, CA) and Sigma-Aldrich (St.
Louis, MO), respectively. [14C]6-mercaptopurine was
obtained from Moravek (Brea, CA). Saturated egg lecithin,
sodium phosphatidylglycerol, and Brij-76 were from Sigma-
Aldrich (St. Louis, MO); glyceryl distearate was purchased
from Pfaltz and Bauer (Waterbury, CT) and cholesterol was
purchased from Fisher Scientific (Pittsburgh, PA). The
scintillation cocktail Ultima-Gold and tissue solubilizer
Solvable were purchased from Perkin-Elmer (Boston, MA).
Thiouric acid was a gift from GlaxoSmithKline. Dextran
sodium sulfate was from Spectrum (Gardena, CA). N-Acetyl
5-ASA was synthesized by reacting 5-ASA with acetic acid
anhydride under catalysis of triethylamine in ethyl ester. The
N-acetyl 5-ASA product was extracted with ethyl ether and
recrystallized. Its structure was verified by nuclear magnetic
resonance (NMR) and its purity was tested using both NMR
and high-performance liquid chromatography (HPLC) with
fluorescence detection. Ketamine-HCl was from Fort Dodge
Lab (Fort Dodge, IA), and Rompum (xylazine) from Bayer
(Shawnee Mission, KS). HPLC solvents were purchased from
Fisher Scientific (Pittsburgh, PA). All other chemicals used
were of analytical grade and purchased from Sigma (St.
Louis, MO) or Fisher Scientific (Pittsburgh, PA).
ðliposome associated drugÞ
E:E:ð%Þ ¼
ꢀ 100
ð free drugÞ þ ðliposome associated drugÞ
Liposomal size was determined using a Nicomp 380 ZLS zeta
potential/particle sizer. Liposomes were used in animal
studies as prepared, without separation of free drug, to
maintain the original total drug concentration. Thus, liposo-
mal formulations provide a 30Y35% reduction in drug
solution compared to pure drug solutions.
Animal Procedures
Male Sprague-Dawley rats weighing approximately 300
g were fasted overnight with water ad libitum prior to
experiment. Anesthesia was induced with an intramuscular
injection of a mixture of ketamine (50 mg/kg) and xylazine
(20 mg/kg) and maintained by an intraperitoneal injection of
sodium pentobarbital (40 mg/kg). A midline longitudinal
incision was made to expose the intestine for further
manipulation.
Animals
Pathogen-free, male Sprague-Dawley rats (Charles
River Laboratories) weighing 300Y400 g were used in accor-
dance with a protocol approved by the Institutional Review
BoardVCommittee on Use and Care of Animals (University
of Michigan).
For in situ intestinal perfusion studies, procedures were
modified from a method previously reported (17). These

1322
Kesisoglou et al.
studies were carried out to assess regional dependence of open longitudinally and the mucosal layer was separated
intestinal absorption and metabolism of 5-ASA and 6-MP as from the rest of the intestinal tissue with a glass cover slip.
limitations for treating lower bowel inflammation with Mucosal or tissue samples were transferred to preweighed
standard oral dosage forms. Liposomal formulations were vials and were homogenized prior to analysis.
not evaluated in the perfusion system since large volumes of
5-ASA and N-acetyl-5-ASA levels in plasma and intes-
formulation would be required to obtain steady-state data tinal tissue were analyzed by HPLC with fluorescent
which would be controlled by unencapsulated drug. Briefly detection as previously described (15). 6-MP and metabolite
stated, after the desired intestinal region (10 cm portion of plasma and tissue levels were assayed by liquid scintillation
upper jejunum or terminal ileum or 5 cm of proximal colon) counting. 6-MP and thiouric acid in the lumenal contents was
was identified, an inlet glass cannula (0.3 cm outer diameter) measured by HPLC using published established methods
was inserted and secured by ligation with silk suture. An (18,19).
outlet glass cannula was inserted 5 or 10 cm distal to the inlet
cannulation site. The inlet tubing was connected to a 30 mL
syringe that was placed in an infusion pump (Harvard Ap-
paratus Company, South Natick, MA). All animals, perfusion
solutions and the pumps were enclosed in a Plexiglas ther-
mostatically controlled chamber set at 30-C. Intestinal
effluent samples were collected into pre-weighed vials every
15 min for up to 120 min. Each effluent fraction was re-
weighed after collection. Steady-state water and solute trans-
port rates were established within 30 min after initiation of
perfusion at the flow rate studied. Water transport was cor-
rected by the gravimetric method. Relative drug loss from
the perfusate was measured by HPLC. The perfusion buffer
consisted of MES (10 mM), NaCl (135 mM), KCl (5 mM),
CaCl2 (1.1 mM), glucose (5 mM), and mannitol (5 mM). The
pH was adjusted to 6.5 with NaOH. All perfusion solutions
were isotonic. The effective permeability (Peff) measured by
intestinal perfusion was based on the loss of drug from the
perfusate according to the following equation:
Partition Coefficient of 5-ASA
5-ASA was dissolved in aqueous buffers with pH ranging
from 0 to 9 (0, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 8, 9). Four
milliliters of these solutions were then equilibrated with an
equal volume of octanol. This was done by first vortex-
mixinging the mixtures vigorously and then letting the phases
stand in a 37-C water bath to reform for 36 h. The 5-ASA
concentrations in each equilibrated phase were then mea-
sured by HPLC.
IBD Animal Model
Colitis was induced using established protocols (20).
Administration of dextran sodium sulfate (DSS) (5% w/v) in
the drinking water of animals for 7 days resulted in
development of colitis within 7 days, confirmed by presence
of blood in the stools with a Hemoccult testing kit (Beckman
Coulter, Fullerton, CA). Alternatively, a 3% v/v solution of
acetic acid in normal saline was instilled in the colon of
anesthetized rats, by inserting a polyethylene tube 10 cm
deep through the anus. Acute colitis developed within 24 h.
The surgical procedure followed is similar to the one
described previously, with liposome encapsulated drug dosed
simultaneously to two separate loops in distal ileum and
proximal colon. For 6-MP, sample collection and preparation
was similar to the described procedures for healthy animals.
For 5-ASA, sampling was performed 90 min after dose
administration.
Q
C0out
Cin
Peff ¼ ꢁ
ꢀ ln
2ꢀrl
where Q is the perfusate flow rate through the segment, r is
the radius of the segment (0.2 cm), L is the length of the
perfused segment, and Cin is the drug concentration of the
perfusate entering the intestinal segment. C0out is the drug
concentration in the exiting perfusate (Cout) corrected for
water transport according to the gravimetric method as
shown below:
ꢀ
ꢁ
V
C0out ¼ Cout
Q ꢂ $t
Caco-2 Cell Studies
where V is the volume of effluent and Dt is the collection
interval.
Caco-2 cells (ATCC HTB37) of passage 44 were seeded
The anesthesia and surgical procedures for intestinal at 4 ꢀ 105 cells/cm2 on six-well snapwell tissue culture insets
delivery and absorption studies to compare drug solution and (growth area = 1.13 cm2) and maintained in Dulbecco’s
liposomal formulations are similar to the ones described modified Eagle’s medium (DMEM) supplemented with 20%
above for the intestinal perfusion studies. After the intestine heat-inactivated fetal bovine serum (FBS), 0.1 mM nones-
was exposed, the desired intestinal segment (terminal ileum sential amino acids (NEAA), 45 nM vitamin E, and 100 units/
or proximal colon) was washed with saline and air dried, ml of penicillin and streptomycin until confluent (3 days).
prior to ligation with silk suture. From 1 to 1.5 ml of Medium was changed to DMEM with 5% FBS, 0.1 mM
formulation were injected with a syringe into the closed NEAA, 45 nM vitamin E, 0.1 mM selenium (Na2SeO3), 0.003
compartment. In some cases, formulations were injected in nM zinc (ZnSO4 I 7H2O), and 100 units/ml of penicillin and
the unligated upper jejunum to allow unhindered intestinal streptomycin, until cells were fully differentiated (2 weeks in
transit. Blood samples were obtained at defined time culture). Cells were grown at 37-C in an atmosphere of 5%
intervals for 2 h from the tail vein or by cardiac puncture. CO2 and 90% relative humidity.
Plasma was separated from erythrocytes by centrifugation.
On the day of the experiment, medium was removed and
At the end of the experiment, animals were euthanized and cells were preincubated for 30 min with HBSS transport
intestine was removed. The removed intestine was washed to medium pH 7.4 at 37-C. Subsequently, 6-MP solution or
collect lumenal contents and nonadherent formulation, cut liposomes were added on the apical chamber and cells

Intralumenal Liposomal Drug Delivery for IBD
1323
aqueous phase pH to 9, which leads to ionization of 6-MP
and therefore higher solubility, did not have a significant
effect on encapsulation. This indicates that ionization of 6-
MP does not produce a significant effect on the lipid-to-
aqueous phase partition coefficient in the formulation.
Particle size was measured to be between 0.5 and 2 mm in
diameter. Size distribution was bimodal with the majority of
the liposomes around 1Y1.5 mm and a smaller population
around 200 nm.
Partition Coefficients
The partition coefficients of 5-ASA between both
octanol and water was measured over a nine log order acidity
range (Fig. 1). The partitioning values were small and
remained unchanged over the pH range of study. The
Fig. 1. Octanol/water partition coefficient of 5-ASA as a function of
pH. 5-ASA was dissolved in aqueous buffers with varying pH and
equilibrated with an equal volume of octanol at 37-C for 36 h. 5-ASA
contents in each phase were assayed by HPLC.
partition coefficient (Ko/w) averaged about 0.03 (log Ko/w
=
j1.52). 6-MP has been reported to exhibit slightly better
octanol partitioning from aqueous pH 6.5 compared to pH
7.4 (22).
returned to the incubator. After 2 h of incubation, aliquots
from the donor and receiver solutions were analyzed by
liquid scintillation counting. Insert membranes were excised,
cells were lysed with 0.1 N NaOHY0.1% sodium dodecyl
sulfate, and radioactivity assayed by liquid scintillation counting.
Intestinal Permeability of 6-MP in Different Intestinal
Regions
Statistical Analysis
The in situ intestinal perfusion studies suggest that 6-MP
does not exhibit region specific intestinal absorption. At the
1 mM (170.2 mg/ml) concentration, which is close to the
reported solubility of 6-MP and the intestinal concentration
expected from a 50 mg oral dose in humans, the effective
Data were analyzed by Student t-test or one-way
ANOVA where applicable. Statistical significance was set
at p < 0.05.
RESULTS
Liposomal Encapsulation of 6-MP and 5-ASA
Encapsulation efficiency of 5-ASA phospholipid lip-
osomes prepared by a reverse phase evaporation method
was 25Y35%. Lower encapsulation (10Y15%) was achieved
by a thin film hydration method. For the liposomes used in
the experiments, the size was narrowly distributed between
0.5 and 1.5 mm.
Nonphospholipid liposomes were used to overcome the
low encapsulation efficiency of 6-MP in traditional phospho-
lipid liposomes as reported in the literature (16,21). The
encapsulation efficiencies of different nonphospholipid-based
formulations composed of glyceryl distearate, cholesterol and
Brij 76 are shown in Table II. The two major parameters that
appeared to affect encapsulation efficiency were the lipid
composition and the amount of lipid incorporated into the
formulation. Generally, increasing the percentage of lipid,
which favors the formation of multilamellar liposomes, led to
an increase in encapsulation efficiency. In most cases use of
15% lipid resulted in the highest encapsulation. Formulations
with 20% or more lipid were too viscous to be used for
intralumenal administration. Lipid composition also
appeared to significantly affect encapsulation. Highest encap-
sulations were achieved with lipid compositions that con-
sisted of about 40% GDS, 20Y30% cholesterol and 30Y40%
POE. Of all the formulations prepared, the 40:20:40
GDS:CH:POE, 15% (v/v) lipid liposomes exhibited the
highest encapsulation efficiency (31.77% T 5.37%, mean T
SD, n = 4; similar to 5-ASA phospholipids liposomes) and
Fig. 2. Effective permeability (cm/sec) of 6-MP in healthy rat
jejunum, ileum and colon at (A) 1 mM luminal concentration and
(B) 0.1 mM luminal concentration (mean T SD, n = 4Y5) assessed by
in situ rat intestinal perfusion with 6-MP solutions in MES buffer,
were used for most of the in vivo studies. Increasing the pH 6.5.

1324
Kesisoglou et al.
permeability values (cm/s) for jejunum, ileum and colon upper small intestine compared to the lower small intestine
were 5.02 T 0.65 ꢀ 10j5, 4.33 T 0.86 ꢀ 10j5, 3.77 T 1.68 ꢀ (15). Given the fact that liposomal formulations provide only
10j5 respectively (mean T SD, n = 3Y5) (Fig. 2A). At the a 30Y35% reduction in drug solution and that substantial
lower concentration of 0.1 mM effective permeability amounts of liposomal lipid are required for 120-min steady-
exhibited a small, however not statistically significant, state perfusions, regional studies with liposomal formulations
increase to 6.70 T 0.27 ꢀ 10j5, 4.52 T 0.51 ꢀ 10j5, 6.28 T were not carried out.
2.09 ꢀ 10j5 for jejunum, ileum and colon, respectively (mean
T SD, n = 3Y5) (Fig. 2B). At both concentrations, the effective Intralumenal Administration Studies for 5-ASA
permeabilities of intestinal segments for each region were
not statistically significantly different. In contrast, 5-ASA
5-ASA formulations (11.25 mg as 7.5 mg/ml solution) were
permeability is strongly region dependent with higher ab- injected into the unligated upper jejunum of anesthetized rats
sorption through the leakier paracellular pathway of the to study systemic availability of 5-ASA and its major
Fig. 3. Pharmacokinetics of 5-ASA and N-Ace-5-ASA after intralumenal administration of 5-ASA
solution, liposomes or solution with empty liposomes (7.5 mg/ml 5-ASA) to healthy Sprague-Dawley
rats. (A) Plasma levels of 5-ASA (left) and N-Ace-5-ASA (right) and (B) AUC values of 5-ASA,
after direct injection of the formulations in the unligated upper rat jejunum (mg/ml of plasma, mean T
SD, n = 4, ***p < 0.005 compared to solution, **p < 0.01 compared to solution, ++p < 0.01 compared
to liposomes). (C) Intestinal tissue levels of 5-ASA and N-Ace-5-ASA after injection of the
formulations in ligated intestinal loops (mg/mg of tissue, mean T SD, n = 4, ***p < 0.005).

Intralumenal Liposomal Drug Delivery for IBD
1325
metabolite, N-Ace-5-ASA. As shown in Fig. 3A (left graph), graph). To assess tissue delivery of 5-ASA from the different
systemic 5-ASA levels were significantly lower after liposo- formulations, 5-ASA or liposomes were injected into ligated
mal formulation administration, with the AUC value exhibit- ileal intestinal loops. Liposomal encapsulation of 5-ASA
ing a 63.4% reduction (273 T 51 vs. 100 T 18 min mg/ml, mean resulted in significantly higher 5-ASA levels in the intestinal
T SD, n = 4, p < 0.001) (Fig. 3B). Plasma 5-ASA levels also tissue (19.84 T 2.54 vs. 8.56 T 1.03 mg/mg, mean T SD, p <
decreased after administration of 5-ASA solution with empty 0.001, n = 4) (Fig. 3C).
liposomes (Fig. 3A, left graph). In that case a 30.8%
reduction in AUC was observed and the difference was Intralumenal Administration Studies for 6-MP
statistically significant from both solution and liposomal
formulation administration (n = 4, p < 0.01). A similar trend 6-MP solution or liposomal formulation (212.74 mg in
was observed for plasma N-Ace-5-ASA levels (Fig. 3A, right 1 mM solution) were injected in ligated intestinal loops or
Fig. 4. Pharmacokinetics of 6-MP after intralumenal administration of 6-MP solution, liposomes or
solution with empty liposomes (1 mM 6-MP traced with [14C]6-MP) to healthy Sprague-Dawley
rats. (A) Total radioactivity in plasma after injection of 6-MP formulations in ligated intestinal
loops (right) or the unligated upper jejunum (left) (DPM/ml of plasma, mean T SD, n = 3Y5). (B)
Total radioactivity in the intestinal loop tissue after administration of 6-MP formulations (DPM/mg
tissue, mean T SD, n = 4Y5). (C) Lumenal thiouric acid levels after injection of 6-MP formulations in
ligated intestinal loops (mg, mean T SD, n = 4). All DPM values are normalized for initial
radioactivity dose.

1326
Kesisoglou et al.
the unligated upper jejunum of anesthetized rats to study
systemic availability and local tissue delivery of 6-MP. Prior
to analysis of drug in mucus and tissue, adherence of the
liposomal formulation to the mucosal border was visually
apparent. Because of the complex nature of 6-MP metabo-
lism, total radioactivity was assayed by liquid scintillation
counting to assess total drug availability. Significant differ-
ences in plasma radioactivity levels between solution and
liposomal formulations (Fig. 4A) were not observed for
either method of intestinal administration. In contrast to
observations for 5-ASA, the presence of empty liposomes,
did not appear to alter absorption of 6-MP from solution
(Fig. 4A, left graph). Although intestinal tissue radioactivity
contents (Fig. 4B) were generally higher after liposomal
6-MP administration compared to solution, no statistically
significant differences were observed between the two formu-
lations. The Bobserved^ difference in tissue 6-MP radioactiv-
ity was attributed to the mucus containing tissue fraction
where it has been suggested that the majority of the particles
are localized (23). Consistent with these findings, no statis-
tically significant difference was observed for concentrations
of luminal thiouric acid, the predominant intestinal metabo-
lite of 6-MP (Fig. 4C).
Studies in Animal Models of IBD
Similarly to the observations made with healthy animals,
encapsulation of 6-MP in nonphospholipid liposomes failed
to alter either intestinal tissue levels (Fig. 5A, B) or the
systemic absorption (Fig. 5C) of 6-MP dosed in DSS-colitic
loops. While plasma levels of 5-ASA and its metabolite were
not significantly different for lower intestinal administration,
tissue associated drug levels were higher in IBD rats
compared to normal rats (Fig. 6). The intestinal tissue levels
from the two 6-MP liposomal formulations were identical in
the ileal loops (Fig. 5A). In the colon, where more
inflammation is expected after DSS treatment, liposomes
resulted in higher intestinal mucosal 6-MP levels than from
solution, but the levels were not statistically significantly
different (Fig. 5B).
Fig. 5. Pharmacokinetics of 6-MP after intralumenal administration
of 6-MP solution, or (1 mM 6-MP traced with [14C]6-MP) to DSS-
colitic Sprague-Dawley rats. (A) Total radioactivity in ileal intestinal
loop tissue after administration of 6-MP formulations (DPM/mg
tissue, mean T SD, n = 4). (B) Total radioactivity in colonic intestinal
loop tissue after administration of 6-MP formulations (DPM/mg
tissue, mean T SD, n = 4). (C) Lumenal thiouric acid levels after
injection of 6-MP formulations in ligated intestinal loops (DPM/ml of
plasma, mean T SD, n = 4). All DPM values are normalized for initial
radioactivity dose.
6-MP Caco-2 Transport Study
The results from the 6-MP transport studies in Caco-2
monolayers are shown in Fig. 7. No significant differences
were observed in disappearance of 6-MP from the apical
chamber or appearance in the basolateral chamber. Pres-
ence of liposomes does not appear to inhibit 6-MP ab-
sorption. Monolayer radioactivity content was significantly
higher after application of liposomes compared to solution
at the same concentration (graph C). At a concentration of
1 mM, a substantial amount of liposomal formulation ad-
hered to the mucus-free Caco-2 surface, which most likely
accounts for the increase in radioactivity within the mono-
layer. When a 10-fold diluted formulation was used fewer
liposomes adhered to the monolayer surface resulting in
smaller differences in cell monolayer radioactivity. The lack
of detectable xanthine oxidase activity in this system, lim-
ited assessment of intracellular drug delivery by measuring
6-MP metabolite.
DISCUSSION
Particulate formulations, such as polymeric micro-
spheres, nanoparticles, and liposomes, have attracted atten-
tion for oral delivery of drugs to diseased intestinal tissue for
the treatment of alimentary tract disorders. These formula-
tions were reported to exhibit increased binding to the
intestinal wall in healthy intestinal tissue (6,24). Further,
Lamprecht et al. (7) reported that microsphere and nano-

Intralumenal Liposomal Drug Delivery for IBD
1327
Fig. 6. Tissue (A) and plasma levels (B) of 5-ASA and N-Ace-5-
ASA after administration of a 5-ASA liposomal formulation (7.5 mg/
ml of 5-ASA) in isolated colonic loops of healthy and acetic acid-
induced colitic animals.
sphere gut wall attachment significantly increases in inflamed
intestinal segments of colitic rats. More recently, Jubeh et al.
(12) reported that binding of liposomes to healthy and colitic
intestinal tissue is dependent on liposomal charge and size.
Attachment of the formulation to the intestinal wall could
provide sustained exposure of a therapeutic agent to local
sites of pathology and improve therapy.
In this study, we compared the efficiency of liposomes to
locally deliver two commonly used IBD drugs, 5-ASA and 6-
MP, to intestinal tissue. While 5-ASA was readily encapsu-
lated in phospholipid liposomes, non-phospholipid based
liposomes were required to achieve equivalent encapsulation
of 6-MP. Based on encapsulation efficiency, liposomal for-
mulation reduced equilibrium drug solution availability by
30Y35%. Intralumenal administration of the formulation
resulted in increased tissue levels and reduced systemic
distribution compared to solution for 5-ASA, but no signif-
icant differences were observed for 6-MP. The differences in
drug physicochemical properties not only dictate differences
in liposomal formulations but also generate differences in
intestinal drug absorption pathways and metabolic profiles
underlying local delivery results for these two drugs.
Fig. 7. 6-MP transport in Caco-2 monolayers. 1 mM or 0.1 mM 6-MP
solution or liposomes (traced with [14C]6-MP) were applied on the
apical chamber of differentiated Caco-2 monolayers. Data are
expressed as % of initial radioactivity remaining in apical chamber
(A), appearance in basolateral chamber (B), and contents of Caco-2
While both 5-ASA and 6-MP are small molecules,
differences in solubility (Table I), partition coefficient, monolayer (C) (mean T SD, n = 3). *p < 0.05 compared to solution.

1328
Kesisoglou et al.
Table I. Physicochemical Properties and Use in IBD Therapy for 6-MP and 5-ASA
6-MP 5-ASA
Physicochemical properties
MW
pKa
152.18
2.5, 7.77, 11.17
0.376Y1.22b
153.14
2.3, 5.69a
0.03c
>18
Poctanol/water
Aqueous solubility
pH 7.4 (mg/ml)
Dose (oral, mg)
0.180Y0.355d
50
800Y1200
Use in IBD therapy
Disease variable
Moderate to
severe
Severe
Mild
Side effects
Generally well
tolerated
(bone marrow
suppression)
a Ref. (33).
b Refs. (22,31).
c Fig. 1.
d Refs. (22,32).
absorption characteristics and first-pass metabolism play a human upper small intestine (13). Thus complete absorption
role in both formulation and biological delivery. The high in the upper bowel reduces the ability to deliver drug locally
solubility of 5-ASA permits 30Y35% entrapment of the drug to inflamed lower bowel and provides a rationale for delayed
in the aqueous interior of a phospholipid unilammelar release dosage forms. In previous rat intestinal perfusion
formulation. Similar formulations of 6-MP produce very low
drug entrapment even when the pH was raised to promote
Table II. Indicative Encapsulation Efficiencies of Nonphospholipid-
Based Liposomal Formulations of 6-MP
drug ionization (16). Drug entrapment equivalent to that of
5-ASA was achieved for 6-MP with a non-phospholipid
multilammelar formulation. Furthermore, nonphospholipid
formulations offer the advantage of lower cost, better
stability and ease of preparation (25). The two major factors
that affected encapsulation were the lipid composition and
the amount of lipid incorporated into the formulations (Table
II). A cholesterol concentration of 20Y30% appeared to
enhance encapsulation and increasing lipid content, which
promotes formation of multilamellar vesicles, resulted in
higher encapsulation efficiency. The biphasic solubility of 6-
MP likely results in drug association with liposomal mem-
brane layers as well as loading in the aqueous layers.
Solubility differences for the two drugs result in
different partition coefficient and permeation behavior.
The low octanol/water partition coefficient of anionic/
zwitterionic 5-ASA shows weak pH dependence consistent
with higher octanol partitioning for the zwitterionic species
(Fig. 1). With a basic and one acidic pKa out of the range of
physiologic pH, only the second acidic pKa results in slightly
better octanol partitioning from aqueous pH 6.5 compared to
pH 7.4 (22).
GDS:CH:POE
Weight Ratio
% Lipid
(v/v)
Aqueous
Phase
% Encapsulation
50:15:35
5
10
15
5
PBS, pH 7.4
PBS, pH 7.4
PBS, pH 7.4
PBS, pH 7.4
PBS, pH 7.4
PBS, pH 7.4
PBS, pH 7.4
PBS, pH 9
<5
5.73
11.33
17.75
18.68
16.94
27.29
21.42
23.00
16.37
20.98
27.34
13.66
26.02
33.66
16.52
31.77 T 5.37
32.29
45:25:30
10
15
20
15
20
5
45:25:30
40:30:30
PBS, pH 9
PBS, pH 7.4
PBS, pH 7.4
PBS, pH 7.4
PBS, pH 7.4
PBS, pH 7.4
PBS, pH 7.4
PBS, pH 7.4
PBS, pH 7.4
PBS, pH 7.4
10
15
5
40:25:35
40:20:40
10
15
10
15
20
The low partition coefficient of 5-ASA is consistent with
high paracellular permeability in the small intestine. In fact,
% Encapsulation is typically the average of two different prepara-
tions. For the GDS:CH:POE 40:20:40 15% lipid (v/v) liposomes that
saturated solutions of 5-ASA are 100% absorbed in the were used in the in vivo studies, mean T SD (n = 4) is provided.

Intralumenal Liposomal Drug Delivery for IBD
1329
experiments, significantly higher jejunal than ileal permeabil- absorption. While 5-ASA tissue levels were higher from
ity for 5-ASA was observed (6.17 T 1.82 ꢀ 10j6 cm/s vs. 0.04 liposomal formulation no differences in drug metabolite were
T 1.36 ꢀ 10j6 cm/s) (15). This is likely due to the fact that the observed (Fig. 3C). This indicates that 5-ASA is likely
paracellular pathway is more available for drug transport in maintained at the mucosal surface by the liposomal formu-
the leaky jejunal tissue as compared to the ileum. This suggests lation as compared to solution but tissue entry, as gauged by
that delayed release dosage forms should reduce total metabolite, is not improved. In addition, 5-ASA intestinal
absorption of 5-ASA although IBD is known to compromise metabolism is easily saturable and could also account for the
lower gut barrier resistance to transport of hydrophilic solutes formulation-independent metabolite levels. Furthermore,
(26). The permeability of 6-MP did not show any dependence when tissue levels following liposomal 5-ASA administration
on intestinal region (Fig. 2). Combined with the better were compared between healthy and colitis animals, signifi-
partitioning behavior of 6-MP compared to 5-ASA, this data cant liposomal accumulation in colitic tissue was observed
suggests that 6-MP absorption is most likely dominated by (Fig. 6A), while no difference was observed in systemic drug
transcellular membrane permeation as has been previously levels (Fig. 6A). In the case of 6-MP, no differences in
suggested (19). Given that the liposomal formulations tested systemic or tissue levels were observed between formulation
in this study include a substantial drug solution component, and solution regardless of the method of intestinal adminis-
liposomal entrapment is observed to more strongly limit tration (Fig. 4A). While there was a trend toward greater 6-
systemic absorption of 5-ASA than of 6-MP. Absorption of MP association with the mucosa from liposomal formulation
solution 5-ASA is greater in the upper than lower intestine than from solution, measurements were not statistically
which is not the case for 6-MP. Although less absorbing significantly different (Fig. 4B). Lumenal thiouric acid levels
surface area would be exposed with a delayed release 6-MP were also slightly elevated after liposomal administration but
dosage form, extent of drug absorption might not necessarily the difference was not statistically significant (Fig. 4C).
be reduced (27) as observed with 5-ASA.
Although it has been previously reported that tissue patho-
Given the liposomal adherence to intestinal tissue and physiological state can affect both binding of liposomal
difficulty in separating the formulation from the mucosa, formulations (12) as well as alter mechanisms of absorption
attempts to monitor tissue delivery of drug utilized intestinal (26), studies with DSS-colitic animals revealed a similar
or luminal drug metabolite levels. The appearance of N- pattern for tissue and systemic 6-MP levels, with no clear
acetyl-5-ASA and thiouric acid in the tissue or perfusate differences between liposomes and solution (Fig. 5). While no
would indicate that 5-ASA and 6-MP, respectively, have differences were observed for 6-MP in the in vivo studies,
entered intestinal tissue from the adherent liposomal formu- application of the formulations on a Bcleaner, mucus-free^
lation. While the dominant paracellular absorption should Caco-2 system clearly exhibited the potential of liposomes to
bypass cellular metabolism for 5-ASA, there is some cellular adhere to intestinal tissue and locally increase drug levels
uptake by a parallel pathway that may be partially carrier- (Fig. 7B). As was the case with the in vivo studies, liposomes
mediated (15). Intestinal and hepatic first-pass metabolism do not appear to significantly influence basolateral (systemic)
does not have a significant impact on systemic availability of drug appearance (Fig. 7C).
5-ASA but significantly limits oral bioavailability of 6-MP
An orally administered local delivery system for 6-MP
(19,28Y30). In fact, intestinal metabolism of 6-MP by would be therapeutically beneficial for IBD to minimize dose-
xanthine oxidase is so extensive that it likely enhances limiting systemic toxicity while targeting the site of pathology.
absorptive 6-MP flux by maintaining a high transmucosal However, these animal studies indicate that 6-MP is a poorer
drug concentration gradient. This would serve to promote the candidate than 5-ASA for such a strategy and this is at least
complete absorption of 6-MP from a typical 50-mg oral dose partially based on differences in absorption pathways and
within the small intestinal residence time. In addition, it intestinal metabolism. The fact that empty phospholipid
would perturb drug formulation equilibrium toward rapid liposomes reduced absorption of 5-ASA from solution sug-
removal of 6-MP and promote absorption. Although 6-MP gests that this lipid formulation hinders paracellular absorp-
aqueous solubility is fairly low, it is sufficient to insure that tion of 5-ASA. This is not the case for 6-MP where
dissolution rate of a 50-mg tablet would not be rate limiting transcellular absorption is effectively coupled to intestinal
to drug absorption. Thus, 6-MP is well absorbed and, while metabolism which minimizes liposomal control of delivery.
first-pass metabolism limits oral bioavailability, systemic One possible advantage of liposomal formulation for IBD
toxicity motivates formulation for local delivery in IBD.
drugs over currently marketed dosage forms is an increased
Subsequent to intralumenal administration of 5-ASA drug residence time at the inflamed intestinal tissue due to
and 6-MP in liposomal formulations vs. equivalent drug mucosal adherence. Nonphospholipid liposomes are worthy
concentration solutions in rats, plasma levels of 5-ASA and of consideration due to greater chemical and physical
N-acetyl-5-ASA were monitored as a function of time by stability, as well as easier large scale manufacturing. While
HPLC while 6-MP total drug and metabolite in the circula- in these studies lumenal liposomal stability was maximized
tion were measured by radiolabel counts. In addition, by direct administration in the intestinal site of interest,
intestinal lumen and tissue levels were assessed via the same stability issues in the stomach and duodenum would certainly
analytical methods. In the case of 5-ASA, drug and metab- require additional formulation modification for oral admin-
olite plasma levels were reduced with liposomal formulation istration of the liposomal formulations. Alternatively, li-
compared to solution (Fig. 3A, B). Furthermore, empty posomes could be incorporated in enema formulations.
liposomes administered with solution drug resulted in plasma Adherent liposomes may gradually release their drug con-
levels intermediate to those of formulation and solution tents in the vicinity of the inflamed bowel over time, pro-
(Fig. 3A), suggesting that liposomes interfere with 5-ASA viding sustained intestinal tissue levels not achievable with

1330
Kesisoglou et al.
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