Method of treatment of osteoarthritis with interleuken-1 receptor antagonist

A method and a composition for the preventative treatment of osteoarthritis comprising the periodic administration to a mammal suffering of this disease of a composition comprising an amount of Human recombinant Interleukin-1 receptor antagonist effective for reducing the progression of lesions and cartilage degradation.

FIELD OF THE INVENTION 
The invention relates to a method and a composition for the preventive 
treatment of osteoarthritis. More particularly the invention relates to a 
method and a composition for reducing the progression of lesion and 
cartilage degradation in osteoarthritis. 
BACKGROUND OF THE INVENTION 
Osteoarthritis, which is also called "degenerative joint disease", is the 
most common rheumatic disease and is characterized by a chronic 
inflammation of the articulation and a progressive depletion of articular 
cartilage matrix macromolecules. Together with the cartilage degeneracy, 
osteophytes (small abnormal body outgrowths) occur and develop on the 
stripped part of the articular bones. Symptoms of osteoarthritis occur in 
many people over the age of 65, and women are affected twice as often as 
men. These symptoms are pain, swelling and stiffness of the articulation. 
In a further stage of the disease, movement of articulations is limited 
and becomes painfil. 
The most commonly used drugs for the treatment of osteoarthritis are the 
nonsteroidal anti-inflammatory agents (NSAID). Even though these drugs 
have proved effectiveness in relieving the symptoms of osteoarthritis and 
in decreasing osteoarthritis cartilage catabolism, some of them, like 
sodium salicylate, have shown inhibiting properties of the proteoglycan 
synthesis which may jeopardize the cartilage repair process. Other drugs, 
such as tiaprofenic acid, which do not inhibit the proteoglycan synthesis 
and have shown in vitro that they are able to decrease osteoarthritis 
cartilage catabolism, (Jean-Pierre Pelletier et al. The Journal of 
Rheumatology 1989;16:5, 646-655), have been unable to provide any 
preventing effect in development of osteoarthritis when administrated to 
patients suffering from the latter, (Edward C. Huskisson et al. The 
Journal of Rheumatology 1995; 22:10-1941-1946). Doxycycline, a member of 
the tetracycline family, was also shown to reduce, in vivo, the severity 
of osteoarthritis lesions in the dog ACL model while reducing 
metalloprotease activity, (Yu LP Jr et al. Arthritis Rheum 35:1150-1159, 
1992). Recent data suggests that the action of corticosteroids is 
associated with a reduction in the synthesis of stromelysin-1 by 
chondrocytes. (see: Pelletier et al., J Arthritis Rheum 37:414-423, 1994; 
and Pelletier et al., J Lab Invest 72:578-586, 1995). 
Accumulating evidence suggests that an important component of the matrix 
loss process is related to proteolytic enzyme activity which degrades the 
principal matrix macromolecules such as collagens and proteoglycans 
(aggrecans). Several matrix metalioproteases including stromelysin, 
collagenase, and gelatinase are believed to play an important role in 
matrix degradation. Support for the role of these enzymes in the arthritic 
process is found in observations showing that these proteases can be 
synthesized by chondrocytes and are present in increased amounts in 
pathological cartilage. Another important factor in osteoarthritis is the 
occurrence of synovial inflammation. There is compelling evidence that 
soluble inflammatory mediators such as cytokines, interleukin-1 (IL-1) and 
tumor necrosis factor-.alpha. a (TNF-(.alpha.), are involved in the 
osteoarthritis process. See for example: 
Pelletier JP et al. A textbook of Rheumatology. Twelfih edition. Edited by 
DJ McCarthy, WJ Koopman. Philadelphia, Lea & Febiger, 1993; 
Pelletier JP, et al. J. In, Osteoarthritis, Edition of Rheumatic Disease 
Clinics of North Ameria. Edited by R W. Moskowitz. Philadelphia, WB 
Saunders, 1993; 
Pelletier JP et al. J. Rheumatol 22:109-114, 1995; and 
Lanick JW et al. Kunkel SL. Pharm Res 5:129-139, 1988. 
While cytokines and other mediators have been implicated in the core of the 
synthesis and release of matrix metalloproteases, IL-1 has also shown 
other deleterious effects on cartilage matrix metabolism. This cytokine, a 
product not only of mononuclear cells but also of synoviocytes and 
chondrocytes, has the ability to suppress the synthesis of collagen type 
II, characteristic of hyaline cartilage, while augmenting the synthesis of 
collagen type I collagen, characteristic of fibroblast cells (Goldring MB 
et al., J Clin Invest 30 82:2026-2037, 1988). In addition, IL-1 reduces 
aggrecan synthesis (Tyler JA. Biochem J 227:869-878, 1985. and Dingle JT 
et al., Cell Biochem Funct 9:99-102, 1991), the macromolecule largely 
responsible for the mechanical properties of articular cartilage. 
Thus, this cytokine contributes both to reduce anabolic and enhance 
catabolic activities in affected joints. 
A substance having inhibitory effects on the activity of IL-1 was found in 
conditioned monocyte medium: Arend WP et al., J Immunol 134:3868-3875, 
1985, and in the urine of febrile patients : Balavoine JF et al., J Clin 
Invest 78:1120-1124, 1986. Characterizations of this molecule has revealed 
a 22 Kd protein and a specific competitive inhibitor of IL-1 known as IL-1 
receptor antagonist or IL-Ira: Carter DB et al., Nature 344:633-638, 1990; 
and Hannum CH et al., Nature 343:336-340, 1990. This antagonist protein is 
a product of several cell types including monocytes, synoviocytes and 
chondrocytes and acts as a competitive inhibitor of IL-1 at the receptor 
level. In addition, IL 1ra binds with a greater affinity to the type 1 as 
compared to the type II IL-1 receptor. It has been shown that IL-1ra is 
capable of blocking some of the effects of IL-1, including the induction 
of matrix metalloproteins, nitric oxide, PGE.sub.2 synthesis, as well as 
the expression of other cytokines (Smith RJ et al., Adv Immunol 
54:167-227, 1993; Arend VP etal., J Clin Invest 85:1694-1697, 1990; and 
Evens CH et al. Receptor 4:9-15, 1994). Most of the above mentionned 
studies have demonstrated a relative deficit in the synthesis of IL-1ra 
vis-a-vis IL-1 in osteoarthritis and rheumatoid arthritis (RA) synovium. 
To date, the majority of the studies exploring the effects of IL-1ra have 
been in vitro. Its in vivo effects have not been studied yet. Moreover 
some studies carried out in vivo have failed to demonstrate a therapeutic 
potential for rhIL-1ra in the treatment of arthritis. For instance, Arner, 
et al in J Rheum 22:1338-1346, 1995, reported that rhIL-1ra administrated 
intravenously fails to inhibit cartilage proteoglycan breakdown in 
polycation induced arthritis in the rabbit. Similarly it has been reported 
that intraperitoneal injections of rhIL-1ra does not affect the 
pathogenesis of antigen induced arthritis in mice (Wooley, et al, 
Arthritis Rheum. 36:1305-1314, 1993). 
While the before mentioned drugs have met with limited success in the 
preventative treatment of osteoarthritis, new and improved method and 
pharmaceutical compositions are constantly being sought which may 
effectively reduce the progression of lesion and cartilage degradation in 
a mammal suffering from osteoarthritis. It is to such a method and a 
composition that the present invention is directed. 
Surprisingly, the inventors have found that the periodic administration to 
a mammal of a composition comprising an amount of human recombinant 
Interleukin-1 receptor antagonist (rhIL-1ra) is effective for reducing the 
progression of lesions and cartilage degradation in a mammal suffering of 
osteoarthritis. 
SUMMARY OF THE INVENTION 
Therefor one of the objects of the invention is to provide a method for the 
preventative treatment of osteoarthritis comprising the periodic 
administration to a mammal of a composition comprising an amount of human 
recombinant Interleukin-1 receptor antagonist (rhIL-1ra) effective for 
reducing the progression of lesions and cartilage degradation. It is, of 
course, much preferred that the method be applied to patients suffering of 
this disease. Advantageously, the period of administration may range from 
a week to a month. It is also very much preferred that the administration 
of the composition be performed by intraarticular injection, even if 
subcutaneous injection or other known methods may also be contemplated. 
Preferably, Human recombinant Interleukin-1 receptor antagonist is 
injected intraarticularly in an amount ranging from 10 to 100 mg per 
injection. 
Another object of the invention is to provide a composition for the 
preventative treatment of osteoarthritis comprising an amount of human 
recombinant Interleuldn-1receptor antagonist (rhIL-1ra) effective for 
reducing the progression of lesions and cartilage degradation and a 
pharmaceutically acceptable carrier. Advantageously, the carrier of such a 
composition is of a type suitable for the formulation of the composition 
for an intraarticular or subcutaneous injection, like a physiological 
saline solution. The amount of human recombinant Interleukin-1 receptor 
antagonist present in each dosage form may range from 10 to 100 mg per 
dosage. 
Other objects, features and advantages of the present invention will be 
apparent from the following detailed description when read in conjunction 
with the accompanying figures and appended claims.

DETAILED DESCRIPTION OF THE INVENTION 
As indicated hereinabove, the present invention is directed to both a 
method and a composition for the treatment of osteoarthritis in a mammal 
and more especially in a human being, by the use of a therapeutic dosage 
of rhIL-ra. The invention is based on the discovery that intraarticular 
injections of rhIL-1ra reduces the osteophyte formation and severity of 
cartilage lesions in osteoarthritis in a dose-dependent fashion. 
At the opposite of several drugs already known in the art for having 
preventing effect for treating osteoarthritis only in vitro, use of 
rhIl-1ra has shown effectiveness in-vivo to reduce the progression of 
lesions and cartilage degradation in mammal suffering of osteoarthritis. 
Although the specific mechanism(s) responsible for this effect remains 
unknown, it is believed that growth factors and cytokines are involved in 
the development of osteophytes in osteoarthritis. It is possible that the 
reduction in incidence and size of osteophytes in this model result from 
the inhibition of direct or indirect effects of IL-1 on osteoid deposition 
in treated animals (Rickard DJ et al., Calcif Tissue Int. 52:227-233, 
1993.; and Taichman RS et al., Inflammation 16:587-601, 1992). Along with 
mechanical factors, growth factors and cytokines may be involved in the 
formation and growth of osteophytes, since these molecules can induce 
growth and differentiation of mesenchymal cells (Van Beuningen HM et al., 
Lab Invest 71:279-290, 1994.; Arend WP. In, Primer on the Rheumatic 
Diseases. Tenth edition. Edited by HR Schumacher, JH Klippel, WJ Koopman. 
The Arthritis Foundation, Georgia, 1993). Injections of TGF-.beta. in the 
murine knee joint induces the outgrowth of chondroid tissue at the femoral 
ridges. Additionally, inhibition of IL-1 effect would decrease cell 
mitosis rate and hence modulate the action of TGF-.beta. by reducing the 
biological effect of this growth factor (Vivien D et al., J Cell Physol 
143:534-545, 1990). Thus, it is conceivable that the increase in the local 
synthesis of growth factors or proinflammatory cytokines by the inflamed 
synovium may be an important factor in osteophyte formation. 
The present invention will be illustrated in detail in the following 
example. This example is include for illustrative purposes and should not 
be considered to limit the present invention. 
EXAMPLE 
A canine ACL model of osteoarthritis has been used in the present example 
to examine the action of intraarticular injections of rhIL-1ra on the 
development of osteoarthritic lesions as well as the expression of 
metalloproteases, collagenase-1 and stromelysin-1. Osteoarthritis has been 
artificially induced in dogs by transection of the anterior cruciate 
ligament (ACL) of the dog's knee which leads to articular changes 
resembling the morphological and biochemical changes observed in human 
osteoarthritis (Brandt KD et al. Arthritis Rheum 34:1560-1570, 1991). 
Similar to naturally-occurring disease in man metalloproteases known as 
collagenase-1, stromelysin-1 and cytokines such as IL-1 are significantly 
increased in the osteoarthritis cartilage and synovial membrane of the 
experimental dog model (Yu LP Jr et al., Arthritis Rheum 35:1150-1159, 
1992). This model has proven to be useful for testing the effects of drugs 
on disease progression and the study of the main pathophysiological 
mechanisms involved in cartilage degradation (Pelletier JP et al., 
Arthritis Rheum 37:414-423, 1994). For example, corticosteroids have 
demonstrated, both under therapeutic and prophylactic conditions, a 
reduction in the progression of cartilage erosion and osteophyte formation 
(Pelletier JP et al., J Arthritis Rheum 37:414-423, 1994). Therefore, the 
canine ACL model of osteoarthritis has been used in the present example to 
be extended to mammal and more particularly to human being, as this model 
is one of the more suitable for human. 
METHODS 
Experimental Groups 
A total of 16 cross-bred dogs, each weighing 20 to 25 kg each were used in 
the study. The ACL of the right knee of each dog was transected via a stab 
incision under general anaesthesia: Pelletier JP et al., J Arthritis Rheum 
37:414-423, 1994. Dogs were randomly assigned to 3 groups. The first group 
(n=5) received intraarticular injections of sterile physiological saline 
solution (1 ml) twice weekly beginning at the time of surgery. The second 
group (n=6) received injections of rhIL-1ra (2 mg) (Amgen/Synergen, 
Boulder, Colo.) in 1 ml of sterile physiological saline solution using the 
same schedule as the first group. The third group of dogs (n=5) received 
intraarticular injections ofrhIL-1ra (4 mg) in 1 ml of physiological 
saline solution at the same frequency as the first two groups. All dogs 
were sacrificed 4 weeks post-surgery. The saline control group was 
included in the protocol in order to evaluate the effect of the IL-1ra 
vehicle solely on the disease progression. The dogs were kept in animal 
care facilities within for 1 week after surgery and then sent to a housing 
farm where they were left free to exercise in a large field for 4 to 6 
hours every day. 
Dissection and Macroscopic Grading 
Immediately after the sacrifice, the right knees of the dogs were removed, 
the synovial fluid aspirated and dissection was performed aseptically on 
ice. Each knee was examined blindly by two independent observers for gross 
morphologic changes, including the presence of osteophyte formation and 
cartilage lesions: Pelletier JP et al., J Arthritis Rheun 37:414-423, 
1994. The degree of osteophyte formation was graded by measuring the 
maxmal width (mm) of the spur on each femoral condyle. The cartilage 
changes of the medial and lateral femoral condyles and tibial plateaus 
were each graded separately under a dissecting microscope 
(Stereozoom.RTM., Bausch & Lomb, Rochester, N.Y.). The depth of the 
erosion was graded on a scale of 0 to 4, with 0 representing a normal 
surface appearance, and 4 a cartilage erosion extending to the subchondral 
bone. The surface area (size) of articular surface lesions was measured 
and expressed in mm.sup.2. These results are shown on table I. 
TABLE I 
______________________________________ 
MACROSCOPIC LESIONS ON FEMORAL 
CONDYLES AND TIBIAL PLATEAUS IN OA DOGS. 
Tibial plateaus 
Osteo- 
No of Femoral condyles Grade, 
arthritic 
ani- Grade, Size, (mm.sup.2) 
(0-4 scale) 
Group* 
mals Size, (mm.sup.2) 
(0-4 scale) 
(p).circleincircle. 
(p).circleincircle. 
______________________________________ 
Saline 
5 4.70 .+-. 2.80 
1.20 .+-. 0.29 
24.40 .+-. 8.17 
1.20 .+-. 0.29 
rhIL-ra 
6 
3.42 .+-. 0.97 
0.75 .+-. 0.22 
20.90 .+-. 8.01 
1.00 .+-. 0.26 
(2 mg) 
rhIL-ra 
5 
1.40 .+-. 0.71 
0.40 .+-. 0.22 
7.70 .+-. 5.16 
0.30 .+-. 0.21 
(4 mg) 
(p &lt; 0.04) 
(p &lt; 0.04) 
______________________________________ 
*After surgery, the dogs were treated twice a week with intraarticular 
injections of either saline, 2 mg or 4mg rhIL1ra, for 4 weeks and 
sacrified. 
.circleincircle.Statistical analysis was done by Mann Whitney Utest; p 
values as compared to OAsaline group. 
Histopathology 
Histologic evaluation was performed on full thickness sagittal sections of 
cartilage from the entire lesional surfaces of each femoral condyle and 
tibial plateau: Pelletier et al., J Arthritis Rheum 37:414-423, 1994. Each 
specimen was dissected and fixed in 10% buffered formalin and embedded in 
paraffin for histologic study. Serial sections (5 .mu.m) were prepared and 
stained with safranin-0. The severity of the osteoarthritis lesions was 
graded on a scale of 0 to 14 by two independent observers using the 
histologic-histochemical scale ofMankin, et al., J Bone Joint Surg Am 
53:523-537, 1971., and the results are shown in FIG. 1. The remaining 
cartilage specimens were rinsed with cold saline and immediately frozen at 
-80.degree. C. 
Representative specimens of the synovial membrane from the medial and 
lateral compartments of the knee were dissected from the underlying 
tissues, Pelletier et al., Jr. Arthritis Rheum 28:554-561, 1985. Briefly, 
the specimens were fixed in 10% buffered formalin, embedded in paraffin, 
sectioned (5 .mu.m) and then stained with hematoxylin-eosin. For each 
compartment, two synovial membrane specimens were examined for scoring 
purposes. The highest score from each compartment was averaged and 
considered as a unit for the whole knee. The severity of synovitis was 
graded on a scale of 0 to 10 by two independent observers (Goldring MB, 
Birkhead J, Sandell LJ, Kimura T; Krane SM. J Clin.Invest 82:2026-2037, 
1988), by adding the scores of three histologic criteria: (i) synovial 
lining cell hyperplasia (0 to 2+); (ii) villous hyperplasia (0 to 3+); 
(iii) the degree of cellular infiltration by mononuclear and 
polymorphonuclear cells (0 to 5). The remaining synovial membrane was 
rinsed in cold saline and immediately frozen at -90.degree. C. 
IL-1ra ELISA Assay 
The concentration of rhIL-1ra in the synovial fluid of dogs treated with 
intraarticular injections of rhIL-1ra was determined by using a commercial 
immunoassay kit (Quantikine.RTM. Human IL-1ra; R & D Systems, Minneapolis, 
Minn.). Two hundred microliters of each synovial fluid sample were tested 
following the manufacturer's instructions. The limit of detection of the 
assay is 6.5 pg/ml. 
RNA Extraction 
Total RNA was isolated from cartilage. Cartilage samples were homogenized 
in 10 volumes of 6M guanidine hydrochloride containing 25 mM sodium 
citrate, pH 7,25 mM EDTA, 0.5% sarkosyl and 100 mM 2-mercaptoethanol, 
followed by addition of 0.1 volume of 3 M sodium acetate buffer, pH 5, 
0.25 volume of saturated phenol, and 0.25 volume of isoamyl 
alcohol/chloroform (1:49). The solution was vigorously shaken and cooled 
at 4.degree. C. for 1 hour. The mixture was centrifuged (12,000 g, 30 
minutes, 4.degree. C.), the aqueous phase removed, mixed with 1 volume of 
isopropanol and allowed to stand at -20.degree. C. for 18 hours. After a 
second centrifugation (12,000 g, 20 minutes, 4.degree. C.), the pellet was 
resuspended in 10 ml of 4M guanidine isothiocyanate (GIT) buffer 
containing 3.3 ml of cesium trifluoroacetate (2.01 gm/ml; Pharmacia 
Biotech, Baie d'Urfe, Quebec) and centrifuged for 24 hours (13eckman.RTM. 
SW 40 Ti rotor, 100,000 g, 4.degree. C.). The resultant pellet was 
dissolved in 20 mM sodium acetate buffer, pH 5, 0.5% sodium dodecyl 
sulfate (SDS), 1 mM EDTA and extracted once with preheated (60.degree. C.) 
saturated phenol. The RNA was precipitated with 3 volumes of absolute 
ethanol and maintained at -20.degree. C. for 18 hours. After being 
centrifuged (13,000 g, 20 minutes, 4.degree. C.), the RNA pellet was 
solubilized in DEPC-treated water, and the RNA quantitated 
spectro-photometrically. 
Total RNA was extracted from the synovial membrane as described for 
cartilage, with the following modifications; the initial buffer used was 4 
M GTT, after isopropanol precipitation the pellet was directly extracted 
using a 20 mM sodium acetate buffer. 
Northern Blotting 
Total RNA were resolved on 1.2% agarose-formaldehyde gels and 3 .mu.g of 
RNA were used for specimens from cartilage and 10 .mu.g for those from the 
synovium. Following transfer to nylon membranes (Hybond.RTM. N, Amersham 
Corp., Oakville, Ontario) overnight at 4.degree. C. in 10 mM sodium 
acetate buffer, pH 7.8, containing 20 mM Tris and 0.5 mM EDTA, the RNA was 
cross-linked to the membranes by exposure to ultraviolet light. 
Specific sets of primers for collagenase-1, stromelysin-1 and GAPDH were 
developed. A 460 bp, a 274 bp, and a 272 bp primer were constructed, 
respectively, from the ligation of a DNA polymerase chain reaction 
fragment amplified from canine synovial fibroblasts (collagenase-1, 
stromelysin-1) or chondrocyte (GAPDH) RNA to Bluescript vector. The 
primers were subsequently sequenced, in order to verify the identity of 
the genes. 
The oligonucleotide primers were prepared with a DNA synthesizer (Cyclone 
Model (trade name), Miffipore, Bedford, Mass.) and used at a final 
concentration of 200 nM. The sequences for collagenase-1 primers were 
5'-CCAAAAGCGTGTGACAGTAAGC-3' (sense primer) which corresponded to position 
891-912 bp of the sequence of the human gene publkished in Goldberg el 
al., J Biol. Chem 261:6600-6605, 1986. and 5'-CAACTTTGTGGCCAATTCCAGG-3' 
(antisense primer) from position 1326-1347 bp. The sequences for 
stromelysin-1 primers were 5'-GAAAGTCTGGGAAGAGGTGACTCCAC-3' (sense primer) 
and 5'-CAGTGTTGGCTGAGTGAAAGAGACCC-3' (antisense primer), corresponding to 
positions 414-440 bp and 671-697 bp, respectively, of the sequence 
published in Saus et al., J Biol Chem 263:6742-6745, 1988. The sequences 
for GAPDH primers were 5'-CAGAACATCATCCCTGCCTCT-3' (sense primer), which 
corresponded to position 604-624 of the published sequence of the human 
gene, (Tso et al., Nucleic Acids Res. 13:2485-2502, 1985) and 
5'-GCTTGACAAAGTGGTCGTTGA-3' (antisense primer), which corresponded to 
position 901-922 bp. 
Detection was done with a luminescent method using Digoxigenin-11-Uridyl 
Triphosphate (DIF-11dUTP) (Boehringer Mannheim Biochemica, Mannheim, 
Germany) with Lumigen PPD 
[4-Methoxy4-(3-phosphatephenyl)Spiro-(1,2-dioxetane-3,2'-adamant ane) 
disodium salt] as substrate for alkaline phosphatase conjugated to 
anti-DIG antibody Fab-fragments, 
The membranes were then subjected to autoradiography using Kodak XAR5.RTM. 
films (Eastman Kodak LTD, Rochester, N.Y.) at room temperature. Each 
membrane was probed, first for coliagenase-1 or stromelysin- 1, then 
stripped and reprobed with GAPDH. The stripping buffer consisted of 50 mM 
Tris-HCl, pH 8, containing 60% formamide and 1% SDS. The membrane was 
heated at 75.degree. C. for 1 hour, rinsed thoroughly in distilled water 
and probed again starting at the prehybridization step. After exposure, 
all autoradiograph films were subjected to laser scanning densitometry 
(GS-300 (trade name), Hoefer Scientific Instruments, San Francisco, 
Calif.) to determine relative mRNA abundance. Standardization against 
GAPDH MRNA levels enabled the quantitative evaluation of specific mRNA. 
Relative expression of coflagenase-1 and stromelysin-1 were calculated as 
the ratio of the relative intensity of the metalloprotease band to the 
relative intensity of the GAPDH band. 
Statistical Analysis 
The data were expressed as mean.+-. SEM and when appropriate analyzed with 
the Mann-Whitney U-test. A p value equal to or less than 0.05 was 
considered significant. 
RESULTS 
IL-1ra Synovial Fluid Levels 
Human rhIL-1ra was detectable by ELISA assay in the synovial fluid 
collected at time of sacrifice from all dogs treated with the antagonist. 
The level in the dogs treated with 2 mg injections was lower 
(103.8.+-.86.9 ng/ml) than those treated with the 4 mg injections 
(153.3.+-.132.1 ng/ml). 
Macroscopic Grading 
Osteophytes 
Osteophytes were present on 70% of condyles in the saline-treated dogs. The 
mean width of osteophytes in these dogs was 2.3.+-.0.7 mm. In general, 
when present, the osteophytes were observed on both condyles and their 
sizes were similar. Dogs treated with rhIL-1ra presented a dose-dependent 
decrease in the incidence and size of osteophytes on condyles. Dogs 
treated with the 2 mg rhIL-1ra injections has a lower incidence (42%) and 
smaller osteophyte size (0.7.+-.0.3 mm) than the control group. The 
reduction in dogs treated with 4 mg rhIL-1ra injections was even more 
pronounced than the previous group, with a reduction in the incidence of 
osteophytes to 20% (p.ltoreq.0.06) and mean size to 0.5.+-.0.3 mm 
(p.ltoreq.0.04). 
Cartilage Lesions 
In saline-treated dogs, fibrillated lesions usually of a small size and low 
grade were present on both condyles (Table I). Both groups of dogs treated 
with rhIL-1ra presented a reduction in the size and/or the grade of 
condyle lesions. However, the effect was more pronounced in the dogs 
treated with the 4 mg injections (Table I). The tibial plateau lesions in 
the saline-treated dogs were similar on both plateaus and were more severe 
than on the femoral condyles, particularly with regards to their size, 
which was much larger (Table I). Dogs treated with rhIL-1ra injections at 
a dosage of2 mg had lesions that were slightly less severe compared to the 
saline-treated dogs. As for femoral condyles, the dogs treated with 4 mg 
rhIL-1ra injections presented a marked and statistically significant 
reduction (p.ltoreq.0.04) in both size and grade (Table I). 
Synovial Membrane 
Synovium from saline-treated dogs showed definite signs of synovial 
inflammation with a yellowish-red discoloration and a large number of 
blood vessels. Both goups of dogs treated with rhIL-1ra demonstrated 
similar changes as the control group, except that they generally had a 
more pronounced tissue discoloration and thickening. 
Microscopic Grading 
Cartilage 
Specimens from the saline-treated dogs showed morphological changes 
characteristic of osteoarthritis. These included fibrillation and fissures 
of the cartilage surface, loss of safranin-0 staining, as well as an 
increase in tissue cellularity and cloning. There was no tidemark invasion 
by blood vessels in the samples studied. 
The total score of histological lesions on femoral condyles (3.06.+-.0.69) 
was less severe than those on tibial plateaus (FIG. 1). Although no 
significant effect on the total score of femoral condyle lesions was 
observed in both rhIL-1ra treated groups when compared to controls, a 
trend towards less severe structural changes was observed (FIG. 2). The 
score for structural changes (0-6 scale; FIG. 2) was 0.8.+-.0.3 in the 
saline-treated dogs, 0.4.+-.0.1 and 0.5.+-.0.3 for the 2 mg and4 mg 
rIL-1ra-treated groups, respectively. 
Interestingly, the total histological score (FIG. 1) of lesions on tibial 
plateaus was less severe in the dogs treated with rhIL-1ra compared to the 
control group (FIG. 1). The dogs treated with 2 mg rhIL-1ra injections had 
a total lesion score of 3.40.+-.0.79 (p.ltoreq.0.002; FIG. 1) for the 4 mg 
rhIL-1ra treated dogs. The reduction in the severity of lesions was 
particularly noticeable for structural changes (0-6 scale; FIG. 2) and 
tissue cellularity (0-3 scale). With regards to the structure, values of 
1.1.+-.0.4 and 0.9.+-.0.4 (p.ltoreq.0.02) were obtained for the 2 mg and 4 
mg treated groups, and 1.9.+-.0.3 for the control group, whereas 
0.9.+-.0.3, 0.9.+-.0.2 (p.ltoreq.0.005) and 1.6.+-.0.1 were obtained for 
the 2 mg, 4 mg rhIL-1ra treated groups and control group, respectively, 
for the cellularity. 
Synovial Membrane 
A moderate inflammatory reaction was present in specimens from the three 
groups with similar total histological scores. The scores were 
5.10.+-.0.71, 4.58.+-.0.45, an 5.20.+-.0.34 for the saline, 2 mg and 4 mg 
rhIL-1ra-treated groups, respectively. However, qualitative differences in 
synoial inflammation were observed between rhIL-1ra-treated and control 
dogs. Specimens from rhIL-1ra-treated dogs had higher scores for 
mononuclear cell infiltration (0-5 scale) (2 mg, 2.83.+-.0.31, 
p.ltoreq.0.05; 4 mg, 2.50.+-.0.27) compared to thesaline-treated dogs 
(1.80.+-.0.44). 
Metalloprotease Expression 
The level of expression of collagenase-1 and stomelysin-1 in the synovial 
membrane and cartilage were measured. In the synovium, the levels of 
expression for collagenase-1 and stromelysin-1 were similar among the 
three groups. However, the levels of collagenase-1 and stromelysin-1 MRNA 
in cartilage showed differences. The stromelysin-1 expression demonstrated 
a slight decrease only for the 2 g rhIL-1ra-treated-groups when compared 
to the saline-treated group. In contrast, the collagenase-1 mRNA level was 
significanly lower (p.ltoreq.0.005) in both rhIL-1ra-treated groups. 
DISCUSSION 
The above example demonstrates that intraarticular injections of rhIL-1ra 
reduced the osteophyte formation and severity of cartilage lesions in the 
canine ACL model of OA in a dose-dependent fashion. The fact that the 
effect is more pronounced in the group receiving the higher dose of 
rhIL-1ra, is an additional argument for suggesting the role of IL-1 in the 
genesis of osteophytes. 
The injections of rhIL-1ra induced a reduction in both the macroscopic and 
the microscopic lesion scores in condyles and plateaus, the reduction 
being more pronounced on plateaus, however. The reduction of macroscopic 
lesions was noted for the size as well as the depth of the cartilage 
lesions. A preferential effect of drugs on tibial plateau lesions has 
already been reported and may be explained by the fact that on the 
plateaus, lesions are more severe than on the condyles. The 
chondroprotective effect of rhIL-1ra was dose-dependent and more 
pronounced at 4 mg than at 2 mg. On the plateaus, the reduction in 
histological grades of lesions was essentially the result of a better 
conservation of the cartilage structure, and a reduction in cell cloning. 
In condyles, although the total histological score was not improved, one 
could note a decrease in the severity of structural changes. These 
findings emphasized the possibility that the inhibition of IL-1 activity 
was responsible, at least in part, for these improvements. IL-1 has been 
shown to have some mitogenic effects on articular chondrocytes, in vitro, 
Frazer et al., Biochim Biophys Acta 1226:193-200, 1994 and it is capable 
of modulating the mitogenic activity of certain growth factors, including 
TGF-.beta., (Guerne et al. J Cell Physiol 158:476-484, 1994 and Pujol et 
al. Endocrinol (Paris) 55:109-120, 1994). Alternatively, it is possible 
that the reduced incidence of chondrocytic hypercellularity observed in 
treated dogs was indirectly related to the inhibition of IL-1 activity, 
although it was a secondary effect, since the inhibition of synthesis of 
oncoproteins like c-Myc, c-Fos and c-Jun have mitogenic effects. This 
hypothesis is supported by recent studies showing that in the dog ACL 
model intraarticular corticosteroid injections reduced the severity of 
osteoarthritis lesions and chondrocyte cloning while simultaneously 
suppressing oncoprotein synthesis (Pelletier et al. J Lab Invest 
72:578-586, 1995). 
Northern blotting analysis indicates that there was an important 
collagenase-1 expression suppressive effect of rhIL-1ra in cartilage. This 
in vivo reduction by a known specific inhibitor of IL-1 again strongly 
supports the contention that this cytokine is an important mediator of 
this metalloprotease synthesis. Surprisingly, no dramatic difference was 
observed in the cartilage expression of stromelysin-1 in the 
rhIL-1ra-treated dogs, nor were important differences noted in the 
expression levels of these two metalloproteases in the synovial membrane. 
Either IL-1 is not a pivotal mediator of stromelysin-1 synthesis in this 
model, at least during the early stage, or that uncharacterized 
intraarticular conditions maintained stromelysin-1 expression despite the 
inhibition of IL-1 activity. Alternatively, perhaps even a very low level 
of IL-1 receptor occupancy by IL-1 suffices to induce the expression of 
stromelysin-1. However, the observation of differences in expression 
between collagenase-1 and stromelysin-1 is not totally surprising as 
discoordinate expression of these two metalloprotease genes has been 
previously reported (Nguyen et al., J Biol Chem 265:17238-17245, 1990). 
Moreover, it is possible that the reduction in collagenase-1 expression 
was responsible for the preservation of the collagenous architecture of 
the cartilage. This hypothesis is supported by the histological findings, 
in which the cartilage from the rhIL-1ra-treated dogs demonstrated a much 
lower incidence in structural damage. The histological score from the two 
rhIL-1ra-treated groups was approximately half that of the saline-treated 
dogs, both for the femoral condyles and the tibial plateaus. This finding 
suggests that rhIL-1ra was capable of reducing matrix damage, principally 
collagen type II, possibly by inhibiting IL-1-mediated collagenase 
synthesis. On the other hand, the absence of improvement in the the 
safranin-O staining in rhIL-1ra-treated dogs, suggesting that the 
depletion in cartilage proteoglycan was minimally affected, is not 
surprising in view of the continued expression of stromelysin-1. 
The weaker effect from the higher dosage of rhIL-1ra at suppressing 
collagenase-1 expression is intriguing and contrasts with its greater 
chondroprotective effect. Should one believe that the chondroprotective 
action of rhIL-1ra be directly related to its suppressive effect on 
metalloprotease synthesis, then a dose-dependent suppression would be 
expected. 
While the invention has been described with respect to certain specific 
embodiments, it will be appreciate that many modifications and changes may 
be made by those skilled in the art without departing from the spirit of 
the invention. It is intended, therefore by the appended claims to cover 
all such modification and changes as fall within the true spirit and scope 
of the invention.