Tissue distribution of [14C]sucrose octasulfate following oral administration to rats.

Article abstract of DOI:10.1023/A:1016161001013

PURPOSE: Aluminum sucrose octasulfate (SOS) is used clinically to prevent ulcers. Under physiologic conditions, the sodium salt of this drug can be formed. Our objective was to determine whether sodium SOS was absorbed when administered orally. In addition to furthering our understanding of aluminum SOS, this study also aimed to clarify how other polyanionic drugs, such as heparin and low-molecular-weight heparins, are absorbed. METHODS: [14C]-labeled and cold sodium SOS (60 mg/kg) were given to rats by stomach tube. Radioactivity was counted in gut tissue, gut washes, and nongut tissue (i.e., lung, liver, kidney, spleen, endothelial, and plasma samples) at 3 min, 6 min, 15 min, 30 min, 60 min, 4 h, and 24 h, and in urine and feces accumulated over 4 h and 24 h. RESULTS: Peak radioactivity was found in the tissue and washes of the stomach, ileum, and colon at 6 min, 60 min, and 4 h, respectively, showing progression through the gut. Gut recovery accounted for 84% of the dose at 6 min but only 12% of the dose at 24 h, including counts from feces. Radioactivity was recovered from nongut tissue (averaging 8.6% of the dose) and accumulated urine (18% of the dose at 24 h). When total body distribution was considered, the recovery of radioactivity was greater for the endothelium than for plasma (peak percentage of the dose was 65% at 15 min, 20% at 3 min, 5% from 20 to 240 min for the vena cava, aortic endothelium, and plasma, respectively). CONCLUSIONS: Results indicate that sodium SOS is absorbed, agreeing with previous studies demonstrating the oral absorption of other sulfated polyanions. Endothelial concentrations must be considered when assessing the pharmacokinetics of these compounds. The measured plasma drug concentrations reflect the much greater amounts of drug residing with the endothelium.

Full text of DOI:10.1023/A:1016161001013

Pharmaceutical Research, Vol. 19, No. 6, June 2002 (© 2002)
Research Paper
Tissue Distribution of [14C]Sucrose
Octasulfate following Oral
Administration to Rats
City, MO) is used clinically to treat and prevent gastrointes-
tinal ulcers (1,2). Its mechanism of gastroprotection is be-
lieved to be due to the stimulation of wound healing at the
site of the ulcer through binding, protection, and activation of
basic fibroblast growth factor (3,4).
SOS is a negatively charged polyanion, and its aluminum
salt is insoluble, forming a gel in the gastrointestinal tract, and
thus is believed not to be absorbed. Under physiologic con-
ditions, however, aluminum ions can exchange with sodium
ions to form the water-soluble sodium salt of SOS. Despite its
high water solubility, sodium SOS is also not believed to be
absorbed from the gastrointestinal tract because of its highly
charged nature. One goal of this study was to assess whether
sodium SOS is orally bioavailable.
Linda M. Hiebert,1,3 Sandra M. Wice,1 Tilly Ping,1
Ronald E. Hileman,2 Tülay Polat,2 and
Robert J. Linhardt2
Received February 19, 2002; accepted February 28, 2002
Purpose. Aluminum sucrose octasulfate (SOS) is used clinically to
prevent ulcers. Under physiologic conditions, the sodium salt of this
drug can be formed. Our objective was to determine whether sodium
SOS was absorbed when administered orally. In addition to further-
ing our understanding of aluminum SOS, this study also aimed to
clarify how other polyanionic drugs, such as heparin and low-
molecular-weight heparins, are absorbed.
A number of studies have shown that negative poly-
anions are minimally absorbed following oral administration
(5–7). Orally administered heparins show little evidence of
bioavailability when the activated partial thromboplastin time
or anti-factor Xa activity is used as a pharmacodynamic
marker (8). Contrary to current opinion, recent studies by our
group and others have suggested that negative polyanions,
such as unfractionated and low-molecular-weight heparins,
sulodexide, pentosan polysulfate, and dextran sulfate (aver-
age molecular weight, 8000 Da) are absorbed following oral
administration (9–13). Orally administered heparin and dex-
tran sulfate have been demonstrated to be effective anti-
thrombotic agents in a rat jugular vein thrombosis model.
Considerable distribution to the endothelium occurs follow-
ing oral administration, although only low levels (less than
1% of the administered dose) are found in plasma (9,14,15).
This study was undertaken to better understand enteral
absorption of the water-soluble sodium salt of SOS and to
determine whether this compound showed the same distribu-
tion as that reported for other sulfated polyanionic com-
pounds following oral administration. Furthermore, sodium
SOS is monodisperse and homogeneous, making it an excel-
lent model for the study of more complex, polydisperse, and
heterogenous drugs, such as unfractionated and low-
molecular-weight heparins.
Methods. [14C]-labeled and cold sodium SOS (60 mg/kg) were given
to rats by stomach tube. Radioactivity was counted in gut tissue, gut
washes, and nongut tissue (i.e., lung, liver, kidney, spleen, endothe-
lial, and plasma samples) at 3 min, 6 min, 15 min, 30 min, 60 min, 4 h,
and 24 h, and in urine and feces accumulated over 4 h and 24 h.
Results. Peak radioactivity was found in the tissue and washes of the
stomach, ileum, and colon at 6 min, 60 min, and 4 h, respectively,
showing progression through the gut. Gut recovery accounted for
84% of the dose at 6 min but only 12% of the dose at 24 h, including
counts from feces. Radioactivity was recovered from nongut tissue
(averaging 8.6% of the dose) and accumulated urine (18% of the dose
at 24 h). When total body distribution was considered, the recovery of
radioactivity was greater for the endothelium than for plasma (peak
percentage of the dose was 65% at 15 min, 20% at 3 min, 5% from 20
to 240 min for the vena cava, aortic endothelium, and plasma, respec-
tively).
Conclusions. Results indicate that sodium SOS is absorbed, agreeing
with previous studies demonstrating the oral absorption of other sul-
fated polyanions. Endothelial concentrations must be considered
when assessing the pharmacokinetics of these compounds. The mea-
sured plasma drug concentrations reflect the much greater amounts
of drug residing with the endothelium.
MATERIALS AND METHODS
Materials
KEY WORDS: gastrointestinal absorption; sucrose octasulfate; oral;
endothelium; rats.
Sodium SOS standard was a generous gift from Bukh
Meditec (Copenhagen Denmark). Uniformly [14C]-labeled
sucrose was from Amersham Life Sciences (Buckingham-
shire, United Kingdom). Dowex resin was from Aldrich
Chemical Company (Milwaukee, Wisconsin). Bio-Gel resin
was from Bio-Rad Laboratories (Hercules, California). Cel-
lulose acetate membrane was from Gelman Sciences Inc.
(Ann Arbor, Michigan). Enzeco (R) alkaline protease “L”
was from Biddle Sawyer Corp. (New York, New York). All
other reagents, chemicals, and solvents were from Aldrich
Chemical Co.
INTRODUCTION
The water-insoluble aluminum salt of sucrose octasulfate
(SOS) sucralfate (Carafate, Aventis Pharmaceuticals, Kansas
1 Department of Veterinary Biomedical Sciences, Western College of
Veterinary Medicine, University of Saskatchewan, Saskatoon, SK,
S7N 5B4, Canada.
2 Department of Chemistry, Division of Medicinal and Natural Prod-
ucts Chemistry and Chemical and Biochemical Engineering, Uni-
versity of Iowa, Iowa City, Iowa 52242.
3 To whom correspondence should be addressed. (e-mail: linda.
hiebert@usask.ca)
Preparation of Unlabeled and [14C]-Labeled SOS
ABBREVIATIONS: DMF, dimethylformamide; dpm, disintegra-
tions per minute; EtOH, ethanol; NEt3· SO3, triethylamine sulfur
trioxide complex; PAGE, polyacrylamide gel electrophoresis; SOS,
sucrose octasulfate.
Sulfonation of Sucrose
SOS was prepared through the sulfonation of sucrose by
the procedure shown in Scheme 1. Briefly, sucrose (1.0 mg;
0724-8741/02/0600-0838/0 © 2002 Plenum Publishing Corporation
838

Tissue Distribution of [14C]Sucrose Octasulfate following Oral Administration
839
treatment and were anesthetized with barbital and methoxy-
flourane for experimental procedures.
Oral Administration
The drug preparation was introduced into the stomach
using a stomach tube in a measured quantity averaging 0.16
ml to give 60 mg/kg of unlabeled SOS plus 1978 nCi/kg [14C]-
labeled SOS. This was followed by 0.2 ml of saline solution to
give a total administered volume of approximately 0.4 ml.
Three control animals were given 0.4 ml of saline solution.
Rats were killed at 3 min, 6 min, 15 min, 30 min, 60 min, 4 h,
and 24 h following oral SOS administration with two animals
being killed at each time period. To insure that none of the
observed absorption was due to aspiration in the lung, three
additional rats that had been given similar amounts of cold
and [14C]-labeled SOS in gelatin capsules, which were swal-
lowed, were killed at 6 min, 15 min, and 24 h after the drug
administration.
0.003 mmol) was dissolved in an ethanol (EtOH)/H2O mix-
ture (1.25 ml H2O containing 3% EtOH). The solution was
evaporated to dryness in vacuo. At 50°C and under anaerobic
conditions (nitrogen), the residue was dissolved in anhydrous
dimethylformamide (DMF; 0.5 ml), sucrose (9.0 mg; 0.026
mmol) was added followed by the addition of a triethylamine
sulfur trioxide anaerobic (NEt3·SO3) complex (1.5 eq/OH)
slowly over a 10-min period. This reaction mixture was stirred
under conditions (nitrogen) at 50°C for 12 h. An additional
portion of DMF (0.5 ml) and NEt3·SO3 complex (1.5 eq/OH)
was added, and the reaction mixture was stirred at 50°C under
nitrogen for another 12 h. The reaction mixture was concen-
trated in vacuo, and the residue was dissolved in water and
eluted using Dowex-1-chloride (Dowex 1 × 2-200) column.
The column was initially washed with water to remove the
unreacted sucrose. The sulfated products then were eluted
with aqueous 2 M sodium chloride. This fraction was concen-
trated by evaporation in vacuo and was desalted by elution
with water on a P-2 column. Then, the residue was dissolved
in water and eluted from a Dowex 50 (Na+) ion exchange
resin to yield the final product. The yield of the sodium salt of
SOS was 65% from sucrose. Purity was assured by ion ex-
change chromatography, thin layer chromatography, gradient
polyacrylamide gel electrophoresis (PAGE), and proton and
carbon nuclear magnetic resonance spectroscopy.
Tissue Collection
Animals to be killed at 4 h and 24 h after drug adminis-
tration were placed in metabolic cages, and urine and feces
were collected. At the designated times, animals were deeply
anesthetized, blood samples of approximately 8 ml (9 parts
blood to 1 part 3.8% sodium citrate) were taken from the
abdominal aorta, and plasma was prepared. As a source of
endothelium, the thoracic aorta and superior vena cava were
removed and placed in saline solution. Liver, lung, spleen,
and kidney were collected. Stomach, duodenum, jejunum, il-
eum, and colon were removed and washed with distilled wa-
ter, and the contents were collected. In the three rats in which
drugs had been administered by gelatin capsules, additional
tissue was harvested including tongue, esophagus, thymus,
heart, brain, bladder, ureters, trachea, scrotum, bile ducts,
thoracic aorta and superior vena cava (minus endothelium),
bone marrow from the tibia of the right hind limb, four areas
of muscle, including the diaphragm, the cleidobrachialis in the
neck, vastus medialis of the left hind limb, and the biceps of
the right forelimb, three areas of skin, including the ear, the
center and caudal areas of the back, and hair samples near the
mouth. A measured quantity of approximately 100 mg of each
tissue, 250 ␮l of the lumenal contents, and 100 ␮l of plasma
were counted for radioactivity. The remaining gut wash fluid
and tissues were frozen for later extraction and analysis of
polyanions. For counting the tissue, samples were treated
with 1 ml of tissue solubilizer NCSII prior to the addition of
5 ml of cocktail for counting aqueous samples. Samples were
counted on a Beckman Scintillation Counter (Beckman
Coulter, Inc., Fullerton, California).
Sulfonation of [14C]-Labeled Sucrose
For the preparation of [14C]-labeled SOS, the identical
procedure as that used to prepare unlabeled SOS was used. In
this reaction, 250 ␮Ci of uniformly [14C]-labeled sucrose (1.7
mCi /mg dissolved in EtOH/H2O mixture, 1.25 ml H2O con-
taining 3% EtOH) and 9 mg of unlabeled sucrose were com-
bined and persulfonated, and the product was purified (as
above). The sample purity was confirmed by ion-exchange
chromatography, thin layer chromatography, and gradient
PAGE analysis.
The [14C]-labeled SOS (21 mg) had a total radioactivity
of 59.5 ␮Ci (specific activity of 6.5 × 106 disintegrations per
minute (dpm) /mg). Cold SOS was dissolved in water at a
concentration of 100 mg/ml to which was added 33 nCi/mg
[14C]-labeled SOS for use in the animal experiments. The
[14C] label is directly incorporated into the carbon backbone
of the sucrose rings, making it stable to all but complete cata-
bolic transformation of the sodium [14C]-labeled SOS.
SOS Extraction from Endothelium
The endothelium was removed from blood vessels ac-
cording to the method of Hiebert and Jaques (16). Vessels
were slit open, pinned to dental wax lumen side up, and rinsed
in Locke’s solution. Cellulose acetate paper was applied to
the lumenal surface, and, when lifted, the endothelium was
Pharmacokinetic Studies
Animals
Twenty male Wistar rats, with a mean ( SD) weight of removed. The length and width of the imprint was measured
284 24 g, were handled and housed according to the Prin- to the nearest millimeter. The mean ( SD) areas for aortic
ciples of Animal Care set by the Canadian Federation of and vena caval endothelium were 2.6 0.3 and 0.8 0.2 cm2,
Biological Societies. Animals were fasted overnight prior to respectively. Endothelium derived from the vena cava was

840
Hiebert et al.
counted immediately following removal by cellulose acetate
paper, whereas the aortic endothelium was further processed.
Cellulose acetate paper was removed from the aortic endo-
thelium by dissolving it in cold acetone followed by centrifu-
gation and the discarding of the supernatant. The process was
repeated to give a dry tissue powder.
SOS Extraction from Tissues and Plasma
Tissues were minced and defatted with acetone and iso-
propanol/petroleum ether. Tissue and plasma were digested
with ENZECO (R) alkaline protease ЈLЈ (Biddle Sawyer
Corp.) in 100 mM Tris-Cl buffer (pH 9.5) at 60°C. Extracts
then were dialyzed against water using 1000 molecular weight
cut-off (MWCO) dialysis tubing. [14C]-labeled SOS and that
derived from extracted tissue were run on an agarose gel
electrophoresis system with toluidine blue staining (17),
where the presence of chemical SOS in tissue extracts was
determined by the migration and staining properties of
samples by comparison to an SOS standard.
Analysis
Results are expressed as mean SEM where three or
more data points are available. A one-way analysis of vari-
ance, followed by the Tukey multiple comparison test, was
used to compare the percentage of the dose recovered in gut
tissue and gut washes between various levels of the gut as well
as that recovered from the endothelium and plasma. A loga-
rithmic transformation was used prior to analysis to ensure
similar variances between groups. To determine the percent-
age of the dose present in the endothelium, the total surface
area of the endothelium was estimated based on the reported
figure of 7000 cm2 per 100 g of muscle (18). Accounting for
less vascular tissue in the body, the corrected value used was
6360 cm2 of endothelial surface per 100 g of tissue. To deter-
mine the percentage of the dose in plasma, the plasma volume
was assumed to be 40.4 ml/kg body weight, as previously re-
ported for rats (19). A paired t test was used to determine
differences in recovery between gut tissues and washes.
Fig. 1. Concentration and percentage recovery of radioactivity from
tissue and washes of different parts of the gut following the admin-
istration of [14C]-labeled SOS (4.5 0.02 × 106 dpm/kg) and cold SOS
(60 mg/kg) by stomach tube or gelatin capsule. Shown are the mean
SEM of three rats per group at 6 min, 15 min, and 24 h, and the
mean of two rats at all other times from different areas of the gut,
stomach (circle), duodenum (square), jejunum (upward triangle), il-
eum (downward triangle), and colon (hexagon). To have equal vari-
ances between groups, a logarithmic transformation was used prior to
analysis using a one-way analysis of variance. a, significantly greater
than all other tissues and washes; b, significantly greater than the
ileum and colon; c, significantly greater than the colon; and d, signifi-
cantly greater than the stomach. The percentage of radioactivity re-
covered in all tissues and washes was significantly greater than that
from untreated control tissues or washes (P < 0.05).
RESULTS
[14C]-Labeled SOS in Gastrointestinal Tissue and Washes
The recovery of radioactivity as percentage of the dose
administered is shown in Fig. 1. Peak radioactivity was found
in stomach tissue at 6 min, in ileum tissue at 60 min, and in 15 min and from colon washes at 30 and 60 min. Recovery
colon tissue at 4 h. Stomach tissue radioactivity dropped to from jejunum washes at 30 and 60 min was greater than that
one third of the original value by 30 min. Recovery from from the colon. Recovery from colon washes was greater than
stomach tissue was significantly greater than all other gut that from stomach washes at 24 h. In general, recovery from
tissue at 3 and 6 min, and was greater than that in the ileum gut washes was slightly higher than that from tissues (Fig. 1;
and colon at 15 min. Recovery from duodenal tissue was con- Table 1) and approached significance when the stomach was
stant from 15 min to 24 h, and in jejunal tissue from 15 min to considered (P ס 0.07). The total radioactivity recovered from
24 h. Radioactivity recovered from the duodenum at 15 min, gastrointestinal tissues and washes decreased with time, with
the jejunum at 30 min, and the ileum at 60 min was signifi- approximately 10% of radioactivity present in washes and
cantly different than that recovered from colon tissue at these tissue at 24 h after drug administration (Table 1). Interest-
times. The peak radioactivity recovered from washes was ob- ingly, concentrations were lower when the drug was admin-
tained at 6 min, 30 min, 15 min, 60 min, and 4 h, respectively, istered by gelatin capsule in the 24-h group. Chemical SOS
for the stomach, duodenum, jejunum, ileum and colon. Ra- also was found in gut washes and tissue following extraction
dioactivity recovered from stomach washes was significantly and analysis with agarose gel electrophoresis, as shown in Fig.
greater than that from all washes at 3 and 6 min, and was 2, A and B. Only trace amounts were found in duodenal
greater than that recovered from ileum and colon washes at tissue.

Tissue Distribution of [14C]Sucrose Octasulfate following Oral Administration
841
Table I. Recovery of Radioactivity from Nongut Rat Tissue at Various Times following Administration of
[
14C]-Labeled SOS (4.5 0.02 × 106 Dpm/kg) and Cold SOS (60 mg/kg) by Stomach Tube or Gelatin Capsulea
Time
Spleen
Liver
Lung
Kidney
Plasma
Urine
min (dpm × 103/g) (dpm × 103/g)
(dpm × 103/g)
(dpm × 103/g) (dpm × 103/ml) (dpm × 103/ml)
3
6
0.0
0.0, 0.0
0.5
0.0
0.0, 0.0
0.9
89.0
172.2, 5.7
74.3
1.1
0.7, 1.5
2.6
3.9
3.7, 4.2
2.8
0.0
0.0b
44.7
44.7b
0.4, 0.0, 1.0b
1.1
0.6, 0.4, 1.8b
4.6
34.9, 149.1, 39.0b
87.8
1.1, 4.1, 2.5b
5.0
1.3, 4.7, 2.4b
8.3
15
0.6, 1.9, 0.8b
0.9
2.6, 9.7, 1.5b
3.7
5.1, 256.8, 1.4b
1.9
6.5, 6.1, 2.4b
5.6
6.9, 16.2, 1.8b
5.6
30
1.4, 0.5
1.2
4.7, 2.7
7.0
1.5, 2.3
22.3
4.8, 6.3
7.2
6.7, 4.6
7.6
60
0.9, 1.5
1.9
7.9, 6.0
4.7
5.0, 39.7
3.3
7.3, 7.1
4.9
9.4, 5.9
5.5
240
1440
12.7
7.5, 17.8
40.8
2.7, 1.1
1.2
6.4, 3.1
3.5
2.8, 3.8
2.9
5.6, 4.3
3.1
5.9, 5.2
2.7
1.4, 1.8, 0.3b
4.6, 3.9, 2.0b
5.7, 2.3, 0.8b
3.7, 4.1, 1.5b
3.2, 4.0, 0.9b
42.6, 74.3, 5.6b
a Bold numbers show averages. Numbers below these in standard type show individual values being averaged.
b Doses administered by gelatin capsule; all others administered by stomach tube.
[14C]-Labeled SOS in Nongut Tissue
the trachea, the 0.62%, 0.05%, and 0.23% found in the brain,
and the 0.06%, 0.04%, and 0.46% found in bone marrow
samples (25 mg) at 6 min, 15 min, and 24 h, respectively.
Counts also were obtained from the thymus, scrotum, and
aortic and vena caval walls minus the endothelium and in
samples of muscle, skin, and hair. The amounts of radioactiv-
ity found in urine increased with time and accounted for
18.3% of the administered dose at 24 h.
Endothelial samples also were harvested from the aortic
and vena caval endothelium. Results reported in terms of
dpm per square centimeter are also expressed as the percent-
age of the dose that is estimated from the total body endo-
thelium (Fig. 3). Counts were recovered from both the aortic
and vena caval samples beginning at 3 min following oral
administration. Peak amounts were observed in the aortic and
vena caval endothelium at 3 and 15 min after drug adminis-
tration, respectively. The label remained in the endothelium
throughout the study period but dropped to near 0 at 24 h.
Radioactivity in plasma, expressed as a concentration and as
the percentage of the dose by considering total blood volume,
never rose above 10% of the dose. The percentage of the dose
recovered from endothelium vs. plasma was significantly
greater when calculations were based on venal caval concen-
trations at 15, 30, 60, and 240 min after administration, and
when they were based on aortic endothelial concentrations at
240 min. Significantly more radioactivity was associated with
vena caval vs. aortic endothelium at 240 min.
Radioactivity was found in lung, kidney, and plasma as
early as 3 min following drug administration, and was found
in spleen and liver at 6 min (Table 2). Concentrations were
similar in plasma throughout the 24-h time period. In spleen,
liver and kidney, concentrations reached a plateau at 15 min,
which was maintained for 24 h. Radioactivity was found in
urine at 15 min, 4 h, and 24 h, but not at 6 min, after drug
administration. The highest concentrations were observed in
the lung at 3, 6, and 15 min after drug administration. Recov-
ery from the lung was quite variable and was evident even
when the drug was administered by gelatin capsule. Average
lung radioactivity concentrations dropped significantly by 4 h
but were still found at 24 h after drug administration. Con-
siderable chemical SOS was observed in lung and urine, as
shown in Fig. 2C. Only trace amounts were recovered from
kidney and liver.
Total [14C] Recovery
The amount of radioactivity recovered averaged 74%
(Table 1). Up to 4 h, 80.4% of the total administered amount
of the drug was accounted for, whereas at 24 h only 34% of
the drug was recovered. Only trace amounts were recovered
in the feces, which were accumulated up to 24 h (0.001%). An
average of 8.6% was recovered from the lung, liver, kidney,
spleen, and plasma, with less recovered at 24 h than at pre-
vious times. A gelatin capsule was used to preclude the pos-
sibility that any of the observed absorption was due to aspi-
ration into the lung when the stomach tube was used.
Amounts recovered at 24 h were less when given by gel cap-
DISCUSSION
Highly sulfated polyanionic compounds are considered
sule vs. stomach tube, suggesting a decreased absorption to be poorly absorbed following oral drug administration.
when a gelatin capsule was used.
This study, however, supports the hypothesis that [14C]-
Additional tissues were examined for the presence of the labeled SOS and cold SOS are absorbed in rats when admin-
drug at 6 min, 15 min, and 24 h after drug administration by istered by stomach tube. Evidence of this is radioactive or
gelatin capsules. These additional tissues accounted for 4%, chemical SOS was found in urine, plasma, and nongut tissue.
4.5%, and 1.2% of the recovered radioactivity at 6 min, 15 Radioactivity recovered from urine accounted for 18.3% of
min, and 24 h, respectively. Notable were the 1.3%, 0.75%, the administered dose at 24 h, whereas that from the lung,
and 0.01% found in the esophagus, the 1%, 3.1%, and 0.07% liver, kidney, and spleen accounted for an average of 8.6% of
found in the tongue, the 0.25%, 0.18%, and 0.01% found in the dose. Radioactivity was found in plasma at all times up to

842
Hiebert et al.
tissue harvested from the duodenum relative to other gut
tissue, thus making chemical extraction more difficult. We
were also able to recover chemical SOS readily from lung
tissue and urine, supporting the hypothesis that SOS is ab-
sorbed orally. Only trace chemical amounts were recovered
from the liver and kidney, although this tissue was examined
when peak radioactivity was found. This suggests that me-
tabolizing of SOS may be occurring. This finding agrees with
previous studies using [14C]-labeled and unlabeled heparin in
which the amount of chemical heparin recovered from the
liver was much less than the amount of radioactivity, suggest-
ing that the liver may be a site of metabolism (11).
Previous studies with sulfated polyanions have shown
that these compounds readily bind to the endothelium. This
binding, when all the body endothelium is considered, can
account for a high percentage of the administered dose, as
demonstrated when using unfractionated heparin or dextran
sulfate (9). This study demonstrates the same effect with SOS,
where the amounts recovered (reported as the percentage of
the dose administered) in the vena caval and aortic endothe-
lium were higher than those observed in plasma. The higher
amounts found in vena caval vs. aortic endothelium agree
with a similar trend observed with the low-molecular-weight
heparin reviparin, in which concentrations in the vena caval
endothelium were significantly higher than those from the
aorta (10).
Results from gut tissue and gut washes showed the ex-
pected transit through the gastrointestinal tract with peak re-
coveries from both gut tissue and gut washes at 6, 60, and 240
min, respectively, for the stomach, ileum, and colon. At 24 h,
only 35.3% of the total radioactivity could be accounted for,
with 12.1% found in the gut and gut washes, including that
recovered from the feces accumulated over the 24-h period.
This suggests that SOS is widely distributed in the tissues.
This is supported by observations that radioactivity could be
found in other tissue throughout the body including muscle
and bone marrow.
These results support those of previous studies with sul-
fated polyanionic compounds in which the effects of oral ad-
ministration have been demonstrated. Dextran sulfate, un-
fractionated heparin, and low-molecular-weight heparin ad-
ministered by the oral route have shown antithrombotic
activity in a rat jugular vein model (9,10,20). Oral sulodexide,
a mixture of dermatan sulfate and fast-migrating heparin, de-
Fig. 2. Agarose gel electrophoresis of gut washes, tissues, and urine
extracts from rats given [14C]-labeled SOS and unlabeled SOS by
stomach tube. Tissue and urine extracts were obtained from samples
obtained near the times when radioactivity peaked; specifically at 3 creased fibrinogen concentrations following oral administra-
min, 15 min, 1 h, 1 h, and 4 h, respectively, for stomach (S), duodenum
(D), jejunum (J), ileum (I) and colon (Co) tissue and washes (A and
B), and at 30 min, 15 min, 15 min, 30 min, and 24 h, respectively, for
spleen (Sp), lung (Lu), liver (Li), kidney (K), and urine (Ur) (C).
Radioactivity in tissue samples was counted and dissolved in water
such that 2 ␮l contained 0.05 nCi. Following the application of
samples (2 ␮l) where indicated by arrows, slides were subjected to
electrophoresis, were fixed, and then were stained with toluidine blue
and destained with 0.1% glacial acetic acid.
tion to patients with peripheral vascular disease (21). Oral
pentosan polysulfate has been shown to be effective for the
treatment of interstitial cystitis and prostatitis (12), whereas
oral chondroitin sulfate has been suggested to be efficacious
in the treatment of arthritis (22,23).
Previous studies examining the tissue distribution of
[14C]-labeled heparin and cold unfractionated heparin in the
rat have shown a wide distribution of heparin in the tissues, a
presence in the endothelium, and only small amounts in the
feces (15), agreeing with the pattern of distribution for SOS
24 h, including as early as 3 min after administration, suggest- that was observed in this study. However, radioactive counts
ing stomach absorption. However, the amounts recovered found in accumulated urine were 1.5% of the administered
from plasma never rose above 10% of the administered dose. dose at 24 h, and plasma counts were always less than 1% of
We were able to recover chemical SOS from gut washes the dose over the 24-h period. These amounts were consid-
and tissues when examined at times when radioactivity erably less than the radioactivity observed in this study in
peaked, although only trace amounts were recovered from which 18.3% of the administered dose was observed in accu-
duodenal tissue. This may be due to the small amount of mulated urine and less than 10% of the dose was observed in

Tissue Distribution of [14C]Sucrose Octasulfate following Oral Administration
843
Table II. Total Radioactivity in Gut, Gut Washes, and Nongut Tissue following Oral Adminis-
tration of [14C]-Labeled SOS and Cold SOS (60 mg/kg) by Stomach Tube or Gelatin Capsulea
Nongut
Time
(min)
Gut tissue
(% of total)
Gut washes
(% of total)
tissue & plasma
(% of total)
Urine
(% of total)
Recovery
(%)
3
6
15
30
60
240
1440
Mean
22.3
23.7
31.1
17
33
18.5
5.3
35.6
60.1
50.4
35
55.4
33.5
6.8b
39.5
10.4
13.7
20.4
5.1
10.1
5.3
ND
0
0.06
ND
ND
2.2
68.3
97.5
102
57.1
98.5
59.5
35.3
74
5
8.6
18.3
21.5
a ND, not determined.
b Includes feces.
plasma over the 24-h period. The increased amounts found in throughout the entire gastrointestinal tract with considerable
urine and plasma with oral SOS as compared to unfraction- absorption taking place in the stomach. Once absorbed, the
ated heparin may be due to an enhanced rate of absorption or sodium SOS binds primarily to the endothelium, with a small
to a decreased ability to bind to the endothelium. The en- amount freely circulating in the plasma. Thus, endothelial
hanced rate of absorption of SOS may be due to its much concentrations must be considered when the pharmacokinet-
smaller average molecular weight relative to unfractionated ics of this compound are studied. The amounts found in
heparin. This is supported by our recent observations that plasma likely reflect a much greater concentration associated
showed effectiveness at much lower single doses for oral low- with the endothelium. These results also support the hypoth-
molecular-weight heparin vs. oral unfractionated heparin esis that sulfated polyanions are absorbed following drug ad-
(24).
ministration by the oral route. This is in contrast to the cur-
These results demonstrate that sodium SOS is absorbed rently accepted view that polyanions are not bioavailable
following oral administration. It seems this drug is absorbed when administered orally (5,7,8). The possibility of the oral
absorption of negative polyanions opens avenues for long-
term use when systemic effects are desired. Furthermore, ad-
ditional considerations are likely to be required regarding the
pharmacokinetics and side effects of these polyanionic com-
pounds.
ACKNOWLEDGMENTS
This study was supported by grants from the Heart and
Stroke Foundation of Saskatchewan, by a sabbatical leave
grant to L. Hiebert, and by National Institutes of Health
grants HL62244, HL52622, and GM38060 to R. Linhardt.
REFERENCES
1. J. L. DePriest. Stress ulcer prophylaxis: Do critically ill patients
need it? Postgrad. Med. 98:159–168 (1995).
2. S. A. Ziller and J. C. Netchvolodoff. Uncomplicated peptic ulcer
disease: An overview of formation and treatment principles.
Postgrad. Med. 93:126–140 (1993).
3. J. Folkman, S. Szabo, M. Stovroff, P. Mcneil, W. Li, and Y. Shing.
Fig. 3. The recovery of radioactivity, reported as the percentage of
the administered dose, from plasma and endothelium following the
administration of [14C]-labeled SOS (4.5 0.02 × 106 dpm/kg) and
cold SOS (60 mg/kg) by stomach tube or gelatin capsule. The per-
centage of the dose assumes recovery from total endothelium when
concentrations on vena caval endothelium (circle) and aortic endo-
thelium (triangle) are considered, and from total plasma (square)
when concentrations in plasma samples are considered. The calcula-
tion of the percentage of the dose assumed a total surface area of
6360 cm2 per 100 g of tissue for the endothelium and a total plasma
volume of 40.4 ml/kg body weight. Shown are the mean SEM of
three rats per group at 6 min, 15 min, and 24 h, and mean of two rats
at all other times. When comparing the percentages of the doses
recovered, a logarithmic transformation was used prior to analysis
using a one-way analysis of variance. a, significantly greater than
recovery from plasma; b, significantly greater than recovery from
aortic endothelium (P < 0.05).
Duodenal ulcer: Discovery of a new mechanism and development
of angiogenic therapy that accelerates healing. Ann. Surg. 214:
414–425 (1991).
4. S. Szabo, P. Vattay, E. Scarbrough, and J. Folkman. Role of
vascular factors, including angiogenesis, in the mechanisms of
action of sucralfate. Am. J. Med. 91:158S–160S (1991).
5. O. Iqbal, S. Aziz, D. Hoppenstead, S. Ahmad, J. Walenga, M.
Bakhos, and J. Fareed. Emerging anticoagulant and thrombolytic
drugs. Emerging Drugs 6:111–135 (2001).
6. K. J. Lorentsen, C. W. Hendrix, J. M. Collins, D. M. Kornhauser,
B. G. Petty, R. W. Klecker, C. Flexner, R. H. Eckel, and P. S.
Lietman. Dextran sulfate is poorly absorbed after oral adminis-
tration. Ann. Intern. Med. 111:561–566 (1989).
7. R. A. Faaij, N. Srivastava, J. M. van-Griensven, R. C. Schoe-
maker, C. Kluft, J. Burggraaf, and A. F. Cohen. The oral bio-

844
Hiebert et al.
availability of pentosan polysulphate sodium in healthy volun-
teers. Eur. J. Clin. Pharmacol. 54:929–935 (1999).
8. K. Salartash, M. Lepore, M. D. Gonze, B. A. Leone, R. Baugh- 16. L. M. Hiebert and L. B. Jaques. Heparin uptake on endothelium.
lowing administration to rats by the oral route. Can. J. Physiol.
Pharmacol. 78:307–320 (2000).
man, S. W. Charles, J. C. Bowen, and S. R. Money. Treatment of
Artery. 2:26–37 (1976).
experimentally induced caval thrombosis with oral low molecular 17. L. B. Jaques, S. M. Wice, and L. M. Hiebert. Determination of
weight heparin and delivery agent in a porcine model of deep
venous thrombosis. Ann. Surg. 231:789–794 (2000).
9. L. B. Jaques, L. M. Hiebert, and S. M. Wice. Evidence from 18. C. A. Keele and E. Neil. Samson Wright’s Applied Physiology,
absolute amounts of heparin and of dextran sulfate in plasma in
microgram quantities. J. Lab. Clin. Med. 115:422–432 (1990).
endothelium of gastric absorption of heparin and of dextran sul-
fates 8000. J. Lab. Clin. Med. 117:122–130 (1991).
10. L. M. Hiebert, S. M. Wice, T. Ping, D. Herr, and V. Laux. An-
tithrombotic efficacy in a rat model of the low molecular weight
heparin, reviparin sodium, administered by the oral route.
Thromb. Haemost. 85:114–118 (2001).
11. F. A. Ofosu. Pharmacological actions of sulodexide. Semin.
Thromb. Hemost. 24:127–138 (1998).
12. J. C. Nickel, B. Johnston, J. Downey, J. Barkin, P. Pommerville,
M. Gregoire, and E. Ramsey. Pentosan polysulfate therapy for
chronic nonbacterial prostatitis (chronic pelvic pain syndrome
Oxford University Press, London, 1971.
19. P. L. Altman and D. S. Ditmer. Biology Data Book, Federation
of American Societies for Experimental Biology, Washington,
DC, 1964.
20. L. M. Hiebert, S. M. Wice, and L. B. Jaques. Antithrombotic
activity of oral unfractionated heparin. J. Cardiovasc. Pharmacol.
28:26–29 (1996).
21. M. Lunetta and T. Salanitri. Lowering of plasma viscosity by the
oral administration of the glycosaminoglycan sulodexide in pa-
tients with peripheral vascular disease. J. Intern. Med. Res. 20:
45–53 (1992).
category IIIA): A prospective multicenter clinical trial. Urology 22. S. O. Canapp, R. M. McLaughlin, J. J. Hoskinson, J. K. Roush,
56:413–417 (2000).
and M. D. Butine. Scintigraphic evaluation of dogs with acute
synovitis after treatment with glucosamine hydrochloride and
chondroitin sulfate. Am. J. Vet. Res. 60:1552–1557 (1999).
23. L. Bucsi and G. Poor. Efficacy and tolerability of oral chondrotin
sulfate as a symtomatic slow-acting drug for osteoarthritis
(SYSADOA) in the treatment of knee osteoarthritis. Osteoar-
thritis Cartilage 6:31–36 (1998).
13. L. M. Hiebert, S. M. Wice, L. B. Jaques, K. E. Williams, and J. M.
Conly. Orally administered dextran sulfate is absorbed in HIV-
positive individuals. J. Lab. Clin. Med. 133:161–170 (1999).
14. L. M. Hiebert, S. M. Wice, N. M. McDuffie, and L. B. Jaques. The
heparin target organ-the endothelium: Studies in a rat model. Q.
J. Med. 86:341–348 (1993).
15. L. M. Hiebert, S. M. Wice, T. Ping, R. E. Hileman, I. Capila, and
R. J. Linhardt. Tissue distribution and antithrombotic activity of
unlabeled or 14C-labeled porcine intestinal mucosal heparin fol-
24. L. M. Hiebert, T. Ping, and S. M. Wice. Antithrombotic activity
of orally administered low molecular weight heparin (logiparin)
in a rat model. Haemostasis 30:196–203 (2000).