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The Effects of Orally Administered Calcium Pentosan Polysulfate on Inflammation and Cartilage Degradation Produced in Rabbit Joints by Intraarticular Injection of a Hyaluronate В ЭPolylysine Complex.

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ARTHRITIS & RHEUMATISM Volume 37
Number 1, January 1994, pp 125-136
0 1994, American College of Rheumatology
125
THE EFFECTS OF ORALLY ADMINISTERED
CALCIUM PENTOSAN POLYSULFATE ON
INFLAMMATION AND CARTILAGE DEGRADATION
PRODUCED IN RABBIT JOINTS BY
INTRAARTICULAR INJECTION OF
A HYALURONATE-POLYLYSINE COMPLEX
MARGARET M. SMITH, PETER GHOSH, YUKIKO NUMATA, and MOHINDER K. BANSAL
Objective. To determine the antiinflammatory
and cartilage-protecting activities of orally administered
calcium pentosan polysulfate (CaPPS) in a rabbit model
of inflammatory arthritis.
Methods. A single intraarticular injection of a
preformed polycation complex (PC) of poly-D-lysine and
hyaluronan was used to induce joint inflammation;
saline was injected into the contralateral joint as a
control. Animals were killed 1,4,7, or 10 days post-PC
injection. CaPPS, at 5 mg/kg, 10 mg/kg, or 75 mgkg,
was given every 48 hours commencing 7 days prior to
PC injection. Serum interleukin-6 (IL-6), synovial fluid
(SF) prostaglandin E,, cell numbers, and cartilage proteoglycan (PG) content, composition, and biosynthesis
were determined for PC- and saline-injected joints.
Results. In PC-injected, non-drug-treated animals, serum IL-6 activity, SF leukocyte numbers, and
prostaglandin E, levels were elevated, while cartilage
PG content and biosynthesis were reduced. CaPPS at 10
mgkg, but not at 5 mg/kg, decreased serum IL-6 levels
but maintained cartilage PG concentration and biosynthesis. However, SF leukocyte counts and prostaglandin
E, levels (except on day 1) were not reduced.
From the Raymond Purves Bone and Joint Research Laboratories (University of Sydney) at the Royal North Shore Hospital,
Sydney, Australia.
Supported in part by Arthropharm Proprietary Limited,
Sydney, Australia and the Henry Langley Foundation, University of
Sydney, Sydney, Australia.
Margaret M. Smith, PhD; Peter Ghosh, PhD, FRSC,
FRACI; Yukiko Numata, PhD; Mohinder K. Bansal, PhD.
Address reprint requests to Peter Ghosh, PhD, FRSC,
FRACI, the Raymond Purves Bone and Joint Research Laboratories, Royal North Shore Hospital of Sydney, St. Leonards, NSW
2065, Australia.
Submitted for publication December 1, 1992; accepted in
revised form June 10, 1993.
Conclusion. The ability of CaPPS to attenuate
serum IL-6 levels and preserve cartilage PGs in inflamed
rabbit joints suggests that this substance could be of
value as an effective orally administered chondroprotective, antiarthritic drug.
One of the earliest pathologic features of osteoarthritis (OA) and chronic rheumatoid arthritis (RA) is
the depletion of proteoglycans (PGs) from articular
cartilage of the affected joints (1,2). Articular cartilage
is an aneural, avascular tissue which covers the ends
of long bones and which, because of its unique composition and structure, allows almost frictionless articulation of the joint together with the ability to recover
from deformation after loading. The cartilage matrix is
composed largely of hydrated PG aggregates entrapped within a meshwork of collagen fibers (3).
Immobilization of polyanionic PG aggregates within
the restraining collagen network causes retention of
water molecules in the cartilage and generates a high
osmotic pressure. It is this high osmotic pressure
which confers resilience to articular cartilage (3,4).
Loss of PGs from cartilage in arthritic joints not only
leads to a decline in resilience of this tissue, but also
contributes to the progression of the disease since PG
breakdown products are antigenic and can stimulate
synovial inflammation (5).
While steroidal and nonsteroidal antiinflammatory drugs (NSAIDs) have been shown to modulate
some of the humoral and cellular aspects of inflammation in arthritic joints (6,7), there is limited evidence to
suggest that they offer any protection to articular
cartilage PGs. Indeed, in vitro studies and investigations using animal models suggest that certain
NSAIDs may impair cartilage and bone repair by
SMITH ET AL
126
inhibiting aspects of chondrocyte and bone cell metabolism, including the biosynthesis of matrix PGs (8-11).
On the other hand, in vitro and animal studies with
certain polysulfated polysaccharides including glycosaminoglycan polysulfate ester (Arteparon) and sodium pentosan polysulfate (NaPPS; Cartrophen) have
indicated that these agents may be effective in maintaining cartilage PGs in arthritic joints (for reviews,
see refs. 8, 12, 13, and 14). However, these polysulfated polysaccharides are not well absorbed orally,
and their routine use has been limited by the need for
intraarticular or intramuscular administration (12-14).
A new calcium complex of pentosan polysulfate
(CaPPS), in contrast to the corresponding sodium salt,
was shown in rats to be well absorbed orally (15); this
led us to question whether CaPPS was capable of
protecting cartilage PGs in inflammatory arthritis
when the drug was given by the oral route.
The rabbit model of arthritis selected for this
study was originally described by Page-Thomas, who
found that a single intraarticular injection of a preformed polycation complex (PC) of poly-D-lysine and
hyaluronan (HA) into rabbit knee joints rapidly produced a classic inflammatory response (16). In subsequent studies using this model, it was shown that
levels of metalloproteinases and tissue inhibitor of
metalloproteinases (TIMP) were elevated in joint synovium and cartilage (17,18). Dexamethasone, when
incorporated into cultures of synovial and cartilage
explants from PC-treated joints, suppressed collagenase production and increased levels of TIMP (19). In
a more recent study (20), the rapid loss of PGs from
articular cartilage of rabbit joints injected with PC was
shown to be associated with the release, by both the
synovial lining cells and the cartilage, in culture, of
large quantities of latent collagenase. Metalloproteinases including stromelysin and collagenase have been
shown to be present in human RA (21) and OA (for
review, see refs. 21 and 22) cartilage and are known to
degrade matrix components (21-23). The rabbit PC
arthritis model was therefore considered to be an
appropriate model for evaluating the antiinflammatory
and cartilage-preserving activities of CaPPS.
MATERIALS AND METHODS
The rabbit polycation arthritis model. The methods
used in the rabbit polycation model of joint inflammation
have been described in detail elsewhere (17,20). Briefly,
male New Zealand white rabbits of 10 months of age were
anesthetized and given a single intraarticular injection of 7.5
mg of a preformed insoluble complex of poly-D-lysine (MW
150,000-300,000; Sigma, St. Louis, MO) and 7.5 mg of HA
(Sigma) in 1 ml sterile, pyrogen-free isotonic saline. The
-
contralateral knee of each animal was injected with 1 ml
isotonic saline only.
CaPPS (Arthropharm Pty. Ltd., Sydney, Australia)
was administered orally by capsule once every second day
beginning 7 days before the PC injection. The every-otherday dosage of CaPPS was 5 , 10, or 75 mg/kg body weight.
CaPPS is a calcium complex of pentosan polysulfate prepared by ester sulfation of a 1,4-PD-xylopyranose derived
from beechwood hemicellulose. The calcium complex obtained from the sulfated xylopyranose and used for the
present study had an average MW of 5,700 daltons, a degree
of sulfation corresponding to 1.8, and a calcium content of
10.4% (weight/weight).
The doses of CaPPS used in these experiments were
based on previous absorption studies. Klocking et al (15)
demonstrated that oral administration to rats of CaPPS at 5
mgkg led to achievement of peak plasma PPS levels of 3-5
pg/ml within 2 hours. This range of pentosan polysulfate
concentrations has been shown to stimulate PG synthesis by
chondrocytes (24), and HA synthesis by synoviocytes, in
vitro (25). Five mg/kg of CaPPS was therefore selected for
study in the rabbit; however, since the digestive tract of the
rabbit (a herbivore) is different from that of the rat (an
omnivore), the oral dose of 10 mg/kg was also used in order
to accommodate any species differences in drug absorption.
The very high oral dose of 75 mg/kg was included in order to
identify any side effects of the drug. Rabbits (n = 3-6 per
drug-treated group and n = 7 in the non-drug-treated group)
were killed by intravenous injection of pentobarbitone at 1,
4, 7, or 10 days after PC injection.
Synovial fluid analysis. Synovial fluid (SF) was collected for total and differential white cell counts and determination of prostaglandin E, levels. S F was obtained by first
injecting 1 ml sterile 50 mM EDTA in isotonic saline into the
joint cavity, and then opening the joints with a sterile scalpel
by slicing across the patellar tendon and collecting the
exudate into a tube by joint manipulation. SF was frozen at
-20°C after centrifugation to remove cells. The cells were
resuspended in 50 mM EDTA for total and differential cell
counts (using a hemocytometer and a Cytospin centrifuge
followed by staining with Daquick, respectively). At a later
stage, S F prostaglandin E, levels were determined using a
commercial radioimmunoassay (enzyme immunoassay kit;
Amersham International, Amersham, UK) after eicosanoid
extraction of the SF washes. Washes were applied to 3 ml
SPE C18 columns (John Moms Scientific Pty. Ltd., Sydney,
Australia) and eicosanoids were eluted with methanol after
each column-bound sample was first washed with water,
hexane, and dichloromethane.
Determination of interleukin-6 levels in serum. Blood
was obtained from the femoral artery immediately after
death and serum was stored frozen at -20°C prior to
analysis. The activity of interleukin-6 (IL-6) in the thawed
(4°C) rabbit serum was measured by a previously described
bioassay employing an IL-6-dependent hybridoma cell line,
7TD1 (26). Cells were cultured in the presence of diluted
serum samples for 3 days. Cell proliferation was measured
by incorporation of 3H-thymidine (0.5 +Ci/well; ICN Biomedicals, Sydney, Australia).
Biochemical analysis of cartilage. Weighed aliquots of
lyophilized cartilage dissected from each joint under sterile
conditions (see below) were assayed for cartilage hexuronate
content using the borosulfuric acid method (27); D-
ORAL CaPPS IN A RABBIT ARTHRITIS MODEL
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Figure 1. Total white cell numbers in the synovial cavity wash of preformed polycation complex (PCb
injected and saline-injectedjoints from control (no drug) and calcium pentosan polysulfate (CaPPS; 5 , 10, or
75 mgikg body weightbtreated rabbits killed 1,4,7, or 10 days after intraarticular PC injection. Hatched bars
indicate saline-injected joints; solid bars indicate PC-injected joints. * = P < 0.05, ** = P < 0.005,
PC-injected versus saline-injected joints; # = P < 0.05, ## = P < 0.005, drug-treated versus non-drugtreated animals. Values are the mean and SEM, logarithmic scale.
glucuronolactone was used as standard after papain digestion of the tissue (24). Hexuronate was used as an index of
PG content (28). Aliquots of papain-digested cartilage were
subjected to ethanol precipitation and the precipitates were
then digested with chondroitinase AC or ABC (Seikagaku,
Tokyo, Japan), as described previously (29). The resulting
unsaturated disaccharides were fractionated by high performance liquid chromatography (HPLC) on a Dynamax aminobonded column (29). The absolute and relative amounts of
HA, chondroitin-4-sulfate (C4S), and chondroitin-6-sulfate
(C6S) were calculated by integration and comparison with
unsaturated disaccharide standards of these sulfated glycosaminoglycans (GAGS) (Seikagaku). Dermatan sulfate
(DS) content was determined by the difference between the
C4S disaccharide peak areas obtained by chondroitinase AC
and ABC digestion.
Cartilage biosynthesis of 35S-proteoglycans. Using
aseptic techniques in a downflow laminar flow hood, fullthickness slices of cartilage were dissected from the femoral
condyles, trochlear grooves, and tibia1 plateaux of salinetreated and PC-treated joints and these were pooled. Eight
explants (1-5 mg) randomly selected from each joint were
blotted on sterile gauze, weighed, and placed in separate
wells of 48-well tissue culture plates in 0.5 ml Ham’s F-12
medium supplemented with 10% (volume/volume) fetal calf
serum (FCS). The remaining cartilage was diced, lyophilized, and used for the biochemical studies (see above).
Cultured cartilage was allowed to normalize in this
medium for 24 hours before the addition of radiolabeled
sulfate (35S0,, specific activity 1,200-1,400 CUmmole; Du
Pont [Australia] Ltd., Sydney, Australia) (5 pCi/well in 0.5
ml Ham’s F-12, 1% [v/v] FCS) to measure 35S-PG biosynthesis by the cartilage chondrocytes. After a further 72 hours
in culture, the medium was removed and the cartilage
washed with phosphate buffered saline. The cartilage and
medium samples (after washings) were subjected to papain
digestion (24) and stored at 4°C until use. Newly synthesized
PG content, as assessed by incorporation of 3 5 S 0 , into GAG
chains, was determined by the method of Collier and Ghosh
(24,30) after selective precipitation of unincorporated radioactivity with Ba++ from papain digests.
Statistical analysis. Differences between PC- and saline-injected joints and differences in serum IL-6 activity
were assessed using paired t-tests. Data from PC- or salineinjected joints from CaPPS-treated versus non-drug-treated
animals were compared overall using 2-factor analysis of
variance (ANOVA) and on each day using unpaired t-tests.
Analyses were performed using Microsoft Excel version 4.0
on an Apple Macintosh personal computer. P values less
than 0.05 were considered significant.
RESULTS
General observations. In the non-drug-treated
control group, rabbits appeared to favor the saline-
128
SMITH ET AL
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Figure 2. Total synovial fluid (SF) prostaglandin E, (PGEZ) levels (mean 2 SEM, logarithmic scale)
expressed as the total amount in the synovial cavity wash of preformed polycation complex (PCtinjected and
saline-injected joints from control (no drug) and calcium pentosan polysulfate (CaPPS; 5 , 10, or 75 mg/kg
body weightttreated rabbits killed 1,4, 7, or 10 days after intraarticular PC injection. Hatched bars indicate
saline-injectedjoints; solid bars indicate PC-injected joints. * = P < 0.05, PC-injected versus saline-injected
joints; # = P < 0.05, drug-treated versus non-drug-treated animals. By analysis of variance, the difference
between 10 mdkg CaPPS-treated and non-drug-treated animals on day 1 was significant at P < 0.005 for
saline-treated joints and at P < 0.05 for PC-treated joints. Values are the mean and SEM, logarithmic scale.
injected joint for the first 24 hours. The PC-injected
joints were swollen and less mobile than the salineinjected joints or joints of non-PC-injected rabbits for
the first 7 days. The animals’ weight, appetite, excretions, and general behavior remained normal throughout
the course of the experiment. Animals treated with the
5-mg/kg and lO-mgkg CaPPS dosage regimens were
more active than the control group even on day 1.
However, rabbits in the high-dose (75 mgkgbtreated
group were not distinguishable from the non-drugtreated PC-injected group, by gross observation. Side
effects of CaPPS treatment were not observed, even
among the animals receiving the 75-mgkg regimen.
Synovial cavity wash. Analysis of the cellular
content of the synovial cavity wash of PC-injected
joints revealed the presence of significantly elevated
numbers of white cells on day 1 (P < 0.02 versus
saline-treated joints), which rapidly declined on subsequent days (Figure 1). Rabbits treated with CaPPS at
400 I
c.
E
2 200
Y
f
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4
7
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Time after injection (days)
Figure 3. Interleukind (IL-6) levels in the sera of control rabbits (no
drug) (solid bars) and 10 mg/kg calcium pentosan polysulfate (CaPPSt
treated rabbits (stippled bars) killed 1,4, 7, or 10 days after intraarticular injection of preformed polycation complex. # = P < 0.05,
drug-treated versus non-drug-treated animals. Values are the mean
and SEM.
1
129
ORAL CaPPS IN A RABBIT ARTHRITIS MODEL
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Time after injection (days)
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Time after injection (days)
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Time after injection (days)
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10
Time after injection (days)
Figure 4. Proteoglycan content, expressed as Fg hexuronic acid per mg cartilage dry weight (cart dry wt), in
articular cartilage from preformed polycation complex (PCkinjected PC and saline-injected joints from
control (no drug) and calcium pentosan polysulfate (CaPPS; 5 , 10, or 75 mgikg body weightbtreated rabbits
killed 1, 4, 7, or 10 days after intraarticular PC injection. Hatched bars indicate saline-injectedjoints; solid
bars indicate PC-injected joints. * = P < 0.05, ** = P < 0.005, PC-injected versus saline-injectedjoints; #
= P < 0.05, ## = P < 0.005,drug-treated versus non-drug-treated animals. By analysis of variance, the
difference between 10 mg/kg CaPPS-treated and non-drug-treated animals was significant at P < O.ooOo5 for
saline-treated joints and P = 0.00 for PC-treated joints, and over time at P < 0.01 for saline-treatedjoints and
P = 0.00 for PC-treated joints. Values are the mean and SEM.
75 mg/kg, but not at 5 or 10 mg/kg, showed reduced
cell numbers on day 1, relative to the non-drug-treated
controls (P< 0.05).
The percentage of white cells present in the
synovial cavity washes that were polymorphonuclear
leukocytes (PMNs) was determined by microscopic
examination of differentially stained slides. PC injection caused a massive influx of PMNs, which accounted for virtually the entire cell population (98%)
on day 1. This percentage was significantly higher than
the proportion of PMNs in the saline-treated joints on
days 1, 4, and 10 (P< 0.05) (data not shown). CaPPS
treatment significantly reduced the percentage of
PMNs on day 1 when given at the 75-mg/kg dose (P<
0.05), but not at the other 2 doses (data not shown).
However, examination of histologic sections of the
synovial lining from the 75 mg/kg-treated rabbits
showed elevated numbers of PMNs in this tissue
compared with PC-injected joints (results not shown).
Prostaglandin E, levels in the synovial cavity
were expressed as the total amount recovered in the
cavity wash (-1 ml) (Figure 2). The normal volume of
SF in a rabbit knee joint was -200 pl; however, this
increased during joint inflammation. Thus, the absolute concentration of prostaglandin E, within the joints
may be up to 5 times the amounts reported here.
Nonetheless, these results provide data on the relative
amount of prostaglandin E, present in the synovial
cavity with the various treatments (control versus drug
treatment). By this means it was shown that there
were greater amounts of prostaglandin E, in the PCinjected joints than in the saline-injected joints, with
minimal levels achieved by day 10 (Figure 2). Treatment with CaPPS at 10 mg/kg significantly reduced
SMITH ET AL
130
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Time after injection (days)
Figure 5. Chondroitin-6-sulfate(C6S) levels expressed as a percentage of total glycosaminoglycancontent in
articular cartilage from preformed polycation complex (PCbinjected and saline-injectedjoints from control
(no drug) and calcium pentosan polysulfate (CaPPS; 5 , 10, or 75 mg/kg body weight)-treated rabbits killed I ,
4, 7, or 10 days after intraarticular PC injection. Hatched bars indicate saline-injected joints; solid bars
indicate PC-injected joints. * = P < 0.05, PC-injected versus saline-injected joints; # = P < 0.05,
drug-treated versus non-drug-treated animals. By analysis of variance, the difference between 10 mg/kg
CaPPS-treated and non-drug-treated animals was significant at P < 0.0005 for saline-treated joints and P =
0.005 for PC-treated joints, and over time at P < 0.005 for saline-treated joints. Values are the mean and
SEM.
prostaglandin E, levels relative to levels in the nondrug-treated PC-injected joints on day 1 (P < 0.05),
but not thereafter. No statistically significant effects
on synovial prostaglandin E, were demonstrable with
CaPPS dosages of 5 or 75 mg/kg (Figure 2).
Serum IL-6 levels. Levels of IL-6 in the rabbit
sera, determined by bioassay (26), were elevated at all
time periods after PC injection. However, IL-6 activity in the sera of animals treated with 10 mg/kg of
CaPPS was significantly reduced relative to the nondrug-treated PC group on days f , 7 , and I0 (P < 0.05).
The reduction by CaPPS was -80% on day 1 and
-70% on day 10 (Figure 3).
In animals treated with CaPPS at 5 mglkg,
serum IL-6 levels were not statistically different from
those in the non-drug-treated, PC-injected group (data
not shown). Serum from the 75 mg/kg CaPPS group
was not available for analysis of IL-6 activity.
Articular cartilage analysis. The total PG content of cartilage as determined by hexuronate analysis
declined progressively over 10 days in the non-drugtreated PC- and saline-injected joints (Figure 4). However, the cartilage PG content in the PC-treated joints
was significantly lower (P < 0.001) than that in the
saline-injected joints from day 4 to day 10 (Figure 4).
CaPPS treatment at 10 mg/kg, but not at 5 mg/kg,
significantly prevented both the loss of PGs caused by
PC injection (P < 0.02) and that sustained by saline
injection (P< 0.005) at days 7 and 10. However, with
the 75-mgikg dose, cartilage hexuronate levels were
significantly lower than those in non-drug-treated controls on day 1 (P< 0.05), but higher on days 7 and 10
131
ORAL CaPPS IN A RABBIT ARTHRITIS MODEL
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Control no drug
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Time after injection (days)
Figure 6. Dermatan sulfate (DS) levels expressed as a percentage of total glycosaminoglycan content in
articular cartilage from preformed polycation complex (PChinjected and saline-injectedjoints from control
(no drug) and calcium pentosan polysulfate (CaPPS; 5 , 10, or 75 mg/kg body weight)-treated rabbits killed 1,
4, 7, or 10 days after intraarticular PC injection. Hatched bars indicate saline-injected joints; solid bars
indicate PC-injected joints. * = P < 0.05, PC-injected versus saline-injected joints; # = P < 0.05,
drug-treated versus non-drug-treated animals. By analysis of variance, the difference between 10 mg/kg
CaPPS-treated and non-drug-treated animals was significant at P < 0.005 for saline-treated joints, and over
time at P < 0.005 for saline-treated joints and P = 0.05 for PC-treated joints. Values are the mean and SEM.
(P < 0.05). A notable finding was the marked increase
in hexuronate content in cartilage from the salinetreated joints in animals receiving the 10-mg/kgdosage
relative to the non-drug-treated group ( P < 0.00005 by
ANOVA) (Figure 4).
The GAG composition of the rabbit articular
cartilage PGs revealed no significant changes with
regard to cartilage C4S content (data not shown). The
C6S levels declined progressively over 10 days in the
PC-injected joints of the non-drug-treated animals (P
< 0.05 on days 7 and 10) (Figure 5). While treatment
with 5 mg/kg or 75 mg/kg CaPPS had minimal effects
on the PC-induced decline of cartilage C6S, the level
of this GAG isomer in the PC-injected and salineinjected joints was maintained for up to 10 days in
animals receiving the lO-mg/kg dosage (P < 0.005 and
P < 0.0005, by ANOVA). Cartilage DS levels, in
contrast to C6S levels, were significantly increased in
the PC-injected joints on days 1, 7, and 10 (P < 0.05)
(Figure 6). This response was reduced in the CaPPStreated groups when the drug was administered at 10
mg/kg ( P < 0.005 by ANOVA) and 75 mg/kg (P <
0.001 by ANOVA), but the effect was statistically
significant by t-test for only the lO-mg/kg dose on day
4 and the 75-mg/kg dose on days 1,4, and 7 (P < 0.05).
No significant effects were evident with the 5-mg/kg
CaPPS dose.
HPLC fractionation of unsaturated disaccharides derived from GAG precipitates showed that HA
levels were significantly lower in the articular cartilage
of the PC-injected joints relative to the saline-injected
joints on days 4 and 7 (P < 0.01) and day 10 (P < 0.05)
132
SMITH ET AL
15
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Figure 7. Hyaluronic acid (HA) levels, expressed as pg per mg cartilage dry weight (cart dry wt), in articular
cartilage from preformed polycation complex (PC)-injected and saline-injectedjoints from control (no drug)
and calcium pentosan polysulfate (CaPPS; 5, 10, or 75 mg/kg body weightktreated rabbits killed 1, 4, 7, or
10 days after intraarticular PC injection. Hatched bars indicate saline-injected joints; solid bars indicate
PC-injected joints. * = P < 0.05, PC-injected versus saline-injectedjoints; # = P < 0.05, ## = P < 0.005,
drug-treated versus non-drug-treated animals. By analysis of variance, the difference between 10 mg/kg
CaPPS-treated and non-drug-treated animals was significant at P < 0.0005 for saline-treatedjoints and P =
0.005 for PC-treated joints, and over time at P < 0.005 for PC-treated joints. Values are the mean and SEM.
(Figure 7). Treatment with CaPPS at 10 mg/kg produced higher mean levels of HA in the articular
cartilage of PC-injected joints at all time points (P <
0.005 by ANOVA), but this was significant by t-test on
day 4 only ( P < 0.05). HA levels did not differ between
cartilage from the saline-injected and that from the
PC-injected joints of the 10 mg/kg-treated animals (P
> 0.05). The corresponding HA values obtained for
cartilage from saline-injected joints of animals receiving the 75-mg/kg dose were found to be significantly
lower than those in the non-drug-treated group (P <
0.05) on days 1, 4, and 7.
Synthesis of 35S-proteoglycans.In vitro PG synthesis in cartilage removed from the joints at the time
of death was determined by monitoring the incorporainto newly synthesized macromolecules
tion of 35S04
over a 72-hour incubation period. The results obtained
are presented as the ratio of the incorporation of 35S
into PGs in cartilage from saline-injected joints relative
to PC-injected joints (Figure 8). Ratios are used to
enable comparison between experiments using 35S04
batches of different specific activities. PC injection
into joints caused a significant depression of 35S04
incorporation into PGs on days 1, 4, and 7, but
synthesis had partially recovered by day 10. CaPPS at
75 mg/kg failed to prevent the PC-induced inhibition of
PG synthesis and was found to exacerbate this process, since the ratios at days 4 and 10 were lower than
those in non-drug-treated controls (P < 0.05). In
contrast, CaPPS in doses of 10 mg/kg partially reversed the PC-induced inhibition of PG biosynthesis
on days 1 and 4 (P < 0.05).
133
ORAL CaPPS IN A RABBIT ARTHRITIS MODEL
DISCUSSION
The inflammatory response induced in the rabbit knee by PC injection has been previously characterized with respect to joint swelling, the presence of
inflammatory cells, increased levels of prostaglandin
E,, and kinetics of PG loss from cartilage (16-20). In
the present study, the PC-induced inflammatory reaction was assessed not only in terms of prostaglandin E,
levels and leukocyte infiltration into the joint cavity,
but by the increase in IL-6 levels in rabbit sera. IL-6
has been shown to be elevated in sera of patients with
RA and OA (31-33) as well as sera of rats with
adjuvant-induced arthritis (34,35). In the rat studies
(34,35), a positive correlation was found between
mean paw volume and serum IL-6 activities, leading
those authors to suggest that IL-6 levels in serum
could be a useful marker of arthritis disease activity.
Furthermore, it was noted that daily treatment of rats
with 5 mg/kg cyclosporin A or 1.5 mg/kg dexamethasone prevented paw swelling and maintained IL-6
activity to within control levels. However, 1 mg/kg of
oral indomethacin only partially prevented swelling
and reduced IL-6 activity by only 50% relative to
controls (34,35).
In the present study, CaPPS at 10 mg/kg reduced serum IL-6 activity by -80% on day 1 and
-70% on day 10 (Figure 3). This finding was consistent
with the results of in vitro studies (36) which had
shown that CaPPS was a potent inhibitor of IL-6
production by rat peritoneal macrophages and human
peripheral blood monocytes. The reduction by CaPPS
of IL-6 levels in the sera of PC-treated rabbits was
therefore indicative of antiinflammatory activity even
though white cell numbers and prostaglandin E, levels
in S F were not reduced by the drug. In a parallel study
using the rat air pouch model of inflammation (37),
subcutaneous administration of 1-10 mg/kg of CaPPS
stimulated the production of IL-6 but reduced tumor
necrosis factor a (TNFa) levels in pouch fluid. In
contrast, leukocyte numbers, fluid volume, and prostaglandin E, levels remained unaffected by the drug.
Furthermore, since pouch fluid IL-1 activity
remained constant and this cytokine is known to
stimulate the production of IL-6 by synoviocytes (38),
we consider that the pouch lining cells, which exhibit
many similarities to synoviocytes (39), were the
source of the elevated IL-6 activity in pouch fluid. It
would therefore appear that CaPPS may exhibit specificity of action with regard to its effects on the
production of cytokines by different cell types.
'
I
1
4
7
10
Time after injection (days)
Figure 8. Relative ex vivo incorporation of '30,into sulfated
glycosaminoglycans in preformed polycation complex (PC)-injected
and saline-injected joints from control rabbits (no drug) (
and
I)
from rabbits treated with calcium pentosan polysulfate at 5 mg/kg
( Q ), 10 mg/kg ( E Z l ) , or 75 mdkg (m)weight, killed 1 , 4, 7, or
10 days after intraarticular PC injection. Each bar represents the
ratio between the dpdmg cartilage wet weight incorporated into the
PC-injectedjoint and the dpdmg cartilage wet weight incorporated
into the saline-injected joint during 72-hour ex vivo culture of
cartilage explants (see Materials and Methods). * = P < 0.05,
drug-treated versus non-drug-treated animals. Values are the mean
and SEM.
With synoviocytes, stimulation of 1L-6 activity
may occur, but with monocytes, IL-6 production is
reduced. IL-6 has been shown in vitro to both stimulate TIMP (4042), which can block the proteolytic
activities of stromelysin (17,21,22), and promote collagen and GAG synthesis in dermal fibroblasts (43).
Administration of IL-6 in vivo also significantly suppressed the development of rat adjuvant arthritis (44).
Notwithstanding the importance of the anti-TNFa
effect exhibited by CaPPS (37), we propose that the
preservation of cartilage PG levels by this drug in the
present study could be mediated by its ability to
promote production of IL-6 by synovial cells. Additional studies are in progress to evaluate this
hypothesis.
PMNs constituted the largest cell population
accumulating in the SF of PC-injected joints, with
mononuclear cells representing only 2-3%. While
PMNs can degrade cartilage by releasing inflammatory
prostanoids and cytokines (45,46), they are also an
abundant source of oxygen-derived free radicals
(ODFR) and proteinases, particularly elastase and
cathepsin G. Elastase and ODFR have been demon-
134
strated to inhibit PG biosynthesis (47), degrade cartilage in vitro and in vivo (47), and activate kininogen
(48) and complement components (49). Polysulfated
polysaccharides including CaPPS and NaPPS are potent inhibitors of both elastase and cathepsin G (50; for
review, see ref. 8); thus, in addition to its effects on
TNFa and IL-6, CaPPS could also preserve cartilage
PGs directly by inhibiting leukocyte proteinases which
degrade the matrix or which activate latent metalloproteinases (21) produced by chondrocytes in response to IL-1 and prostaglandin E, (21-23).
A consistent observation in the present study
was the loss of PGs over 10 days from cartilage of the
contralateral (saline-injected) joints of the non-drugtreated, PC-injected animals. Since the white cell
numbers accumulating in the saline-treated joints were
low, it is possible that factor(s), such as TNFa and
IL- 1, produced by the activated inflammatory cells in
the PC-treated joint were transmitted via the circulation to the contralateral joint. The elevated levels of
PGs in the cartilage of saline-injected joints of animals
receiving 10 mg/kg CaPPS orally (Figure 4) might again
be due to the ability of this drug to inhibit TNFa as
well as to stimulate IL-6 activities within the
synovium.
Levels of HA are reported to be lower in OA
cartilage than in non-OA cartilage (51,52). Since this
GAG is required for the formation of PG aggregates
(53), its depletion in OA cartilage may be associated
with the diffusion of PG monomers from the matrix
into the SF. Orally administered CaPPS at 10 mg/kg
caused maintenance of cartilage HA levels (as determined by HPLC as unsulfated GAG) in PC-injected
joints over the 10-day experimental period (Figure 7).
This result was not entirely unexpected since a stimulatory effect by the sodium analog (NaPPS) on HA
biosynthesis by synoviocytes (25) and in cartilage in a
model of rabbit arthropathy induced by chronic hydrocortisone injection (52) has been previously reported,
and both NaPPS and CaPPS promoted HA and PG
synthesis by rabbit chondrocytes in vitro (24). Hence,
apart from its anticatabolic effect, CaPPS could be
supporting chondrocyte anabolic activities. This activity would be of considerable importance in maintaining
cartilage integrity in inflamed joints in which PG
biosynthesis was suppressed (47,5435).
Examination of the chondroitin sulfates present
in cartilage of the rabbit joints revealed some interesting findings. While the percentage of DS present
increased significantly over 10 days in non-drugtreated animals, administration of 10 mg/kg or 75
SMITH ET AL
mg/kg CaPPS attenuated this increase. Two DScontaining small PGs have been isolated from cartilage
(56,57). Biglycan (DS-PGI) consists of a core protein
of M , 42,500 and 2 DS chains, while decorin (DS-PGII)
has a similar core protein but only 1 DS chain (57).
Decorin levels are elevated in cartilage from aged (58),
degenerative (59), and mechanically overloaded joints
(60,61). While the effects of inflammatory mediators
such as IL-1 on the expression of biglycan and decorin
is still the subject of investigation, IL-1 has been
shown to increase DS-PG core protein messenger
RNA levels in human skin fibroblasts (62) and arterial
smooth muscle cells (63).
In the present study the elevation of DS-PG
levels in cartilage of non-drug-treated PC-injected
joints was therefore consistent with mechanical overloading of chondrocytes induced by the depletion of
the large aggregating PG species (aggrecans) from the
extracellular matrix. Our finding that CaPPS attenuated the production of these small DS-containing PGs
in cartilage of the inflamed joints was compatible with
the maintenance of cartilage integrity. However, effects of the drug on cytokines which could also influence chondrocyte biosynthetic activities cannot be
excluded.
The present study has shown that CaPPS not
only preserved PG levels and biosynthesis in cartilage
of inflamed joints, but was also capable of maintaining
chondrocyte phenotypic expression. Since these effects of CaPPS were observed with an oral dosage of
10 mg/kg but not at 75 mg/kg, a dosage which proved
to be antagonistic to chondrocyte PG biosynthesis, it
would appear that the “chondroprotective” activities
of this drug follow a bell-shaped curve representing a
dose-dependent relationship, as has been observed
with other antirheumatic drugs (24,30,64,65).
ACKNOWLEDGMENTS
The authors would like to thank Mr. Colin Boyd and
Mr. Don Ernst of the Gore Hill Research Laboratories for
animal care, Ms. Suzanne Smith and Ms. Anne Wilcox of the
Raymond Purves Bone and Joint Research Laboratories for
technical assistance with animal experimentation, and Mr.
Daniel Burkhardt and Dr. Dechang Sun of the same laboratones for assisting with the HPLC.
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