MXPA01002434A - Methods of treating osteoarthritis with inducible nitric oxide synthase inhibitors - Google Patents

Abstract

Methods of treating osteoarthritis by administering a therapeutically effective amount of NOS inhibitor are provided.

Description

METHODS FOR TREATING OSTEOARTHRITIS WITH INDUCED NITRIC SYNTHUSA INHIBITORS FIELD OF THE INVENTION This invention is generally related to inhibitors of nitric oxide synthase and more specifically relates to the treatment of patients who have osteoarthritis with nitric oxide synthase inhibitors.

BACKGROUND OF THE INVENTION Nitric oxide (NO) is a reactive inorganic gaseous molecule, important in many physiological and pathological processes where it is synthesized by cells that mediate vital biological functions. Nitric oxide serves as a neurotransmitter in the brain, produced in small amounts on an intermittent basis in response to several endogenous molecular signals. The endothelial cells lining the blood vessels also produce nitric oxide in small amounts, which relax the smooth muscle and regulate blood pressure. In fact, the production of nitric oxide has an important effect on the function of circulating blood cells such as platelets and neutrophils as well as on the smooth muscle that includes the blood vessels and other organs. Nitric oxide is also synthesized in the immune system. Endotoxins and cytokines induce the production of large amounts of nitric oxide in response to infection and inflammatory stimuli, which contribute both to the host defense process such as killing bacteria and viruses, and to the pathology associated with acute and chronic inflammation. in a wide variety of diseases. Nitric oxide is formed from the oxidation of L-arginine by at least three different nitric oxide synthases (NOS) isoforms in mammalian cells that are divided into two distinct, constitutive and inducible classes. The three NOS sophormas have been identified as: (i) Endothelial nitric oxide synthase (eNOS); (NOS type III), a constitutive enzyme, dependent on Ca ++ / calmodulin, located in the endothelium that releases nitric oxide in response to a physical stimulus or a receptor; (ii) Neural nitric oxide synthase (nNOS); (NOS type I), a constitutive enzyme dependent on Ca ++ / calmodulin, located in the brain that releases nitric oxide in response to physical stimulation or to a receptor; Y (iii) Nitric oxide synthase inducible (NOS); (NOS type II), an enzyme independent of Ca ++ that is induced after activation of vascular smooth muscle, macrophages, endothelial cells, and a number of other cells by endotoxins and cytokines. Once expressed, this inducible NOS synthesizes large amounts of nitric oxide (NO) for long periods. -w¡ »Éi *% &Nitric oxide generated by the constitutive enzymes acts as a transduociéTi¡ll UDyacente mechanism of several physiological responses. For example, eNOS is < It is used for the production of nitric oxide originally identified as a relaxation factor derived from the endothelium (ERF). Nitric oxide generated by eNOS regulates blood pressure in animals, blood flow in humans and prevents leukocyte adhesion. On the other hand, the nitric oxide produced in large quantities by the inducible enzymes is a cytotoxic effector molecule. As described in the patent of E.U.A. No. 5,629,322, incorporated herein by reference, has been cloned from the human liver and identified in more than a dozen cells and tissues including the smooth muscle cell, the kidney, and numerous epithelial cells in a variety of tissues including the lung and the colon. This enzyme is induced after exposure to lipopolysaccharide (LPS) and to cytokines such as interferon gamma (IFN-α), interleukin-1β (IL-1β), and tumor necrosis factor (TNF). Once induced, the production of nitric acid by NOS continues for a prolonged period of time and the activity of iNOS is relatively independent of intracellular concentrations of calcium. NOS is involved in conditions that lead to cytokine-induced hypotension that includes septic shock, hemodialysis and IL-2 therapy in cancer patients. The excess production of nitric oxide generated by the inducible forms of nitric oxide synthases also seems to contribute to cytokine-mediated inflammation, cytotoxicity and tissue damage. Accordingly, certain conditions have been identified where inhibition in the production of nitric oxide is advantageous. These conditions include arthritis, inflammatory bowel disease, cardiovascular ischemia, diabetes, congestive heart failure, myocarditis, arteriosclerosis, migraine, esophageal reflux, diarrhea, irritable bowel syndrome, cystic fibrosis, emphysema, asthma, bronchiectasis, hyperalgesia (allodynia) , cerebral ischemia (both focal ischemia, thrombotic shock and global ischemia (secondary to cardiac arrest), multiple sclerosis and other disorders of the central nervous system, for example Parkinson's disease and Alzheimer's disease, and other NO-mediated disorders that include opioid tolerance in patients who need opioid analgesics for a long time, and tolerance to benzodiazepine in patients taking benzodiazepines, and other behaviors addictive, for example, nicotine and eating disorders.

Other conditions in which there is an advantage in the inhibition of NO production from L-arginine include systemic hypotension associated with septic and / or toxic shock induced by a variety of agents; therapy with cytokines such as FNT, IL-1 and IL-2; as an adjuvant for short-term immunosuppression in transplant therapies; Y as quimopreventivo. Although the potential uses of NOS inhibitors have been implicated in numerous diseases, the efficacy and the result of using NOS inhibitors to prevent, treat and cure many diseases never ? .ji sS ^ í ^ l's ^ iS ti. '^^?! is & ^; - i.,.

They have been identified. For example, U.S. Patent No. 5,629,322 in column 15 starting at line 60 lists an enormity of types of diseases wherein NOS inhibitors can be used to treat a disease. However, the types of diseases are named as a result of speculation, without examples or analysis. Examples of other compounds that inhibit the production of nitric oxide can be found in the patents of E.U.A. Nos. 5,684,008 and WO 93, 13055, each incorporated herein by reference. The effect and efficacy of inhibitors to NOS and inhibitors specifically selective to iNOS in vivo on the progression of the disease for many diseases has not been applied. Therefore, the success and consequence of the use of inhibitors in the progression of the disease in vivo in many cases remain unknown. Although some information has been generated in vivo in inflammatory arthritis, whose human model of rheumatoid arthritis suggests that either non-selective inhibitors or in a few cases selective inhibitors of NOS reduce the severity of the disease, there are no reports of the use of inhibitors to NOS to modulate the experimental models of osteoarthritis. (18, 19, 21-24) Therefore, there is a need to determine the effects of NOS inhibitors on the progression of osteoarthritis, and to direct new uses for NOS inhibitors and methods for the treatment of osteoarthritis.

BRIEF DESCRIPTION OF THE INVENTION Therefore, the present invention provides new methods of treating patients with osteoarthritis by modulating: 1) the amount of synovial fluid, 2) the levels of IL-1β; 3) the development of osteophytes; 4) the amount of cartilage degeneration; 5) the production of metalloproteases; and 6) the acute injury of the joint through the administration of an effective amount of a NOS inhibitor to the patient who needs it. Specifically, the in vivo therapeutic efficacy of a selective inhibitor of inducible NOS, N-iminoethyl-L-lysine (L-NIL), was used to determine the effect of lesion progression in osteoarthritis, on the production and activity of metalloproteases of the joints, and the levels of IL-1ß, prostaglandin E2 (PGE2) and nitrite / nitrate (the stable end products of nitric oxide) in synovial fluid. Treatment with a selective NOS inhibitor reduced the severity of the lesions, demonstrating the effectiveness of the NOS inhibitor in attenuating the progression of the disease. In addition, the inhibitor, L-NIL, reduced the production and activity of metalloproteases in the cartilage, the degradative enzymes known to play a major role in the pathophysiology of osteoarthritic lesions. It was observed that the effect was mediated, partly by the suppressive effect of the inhibitor on the production of IL-1β. "*. & h" * "~ * $ * < In addition, iNOS inhibitors attenuated the improved production of PGE2 associated with an increased production of nitric oxide at sites of inflammation. The proinflammatory action of nitric oxide and PGE2 were suppressed by the selective NOS inhibitor. 5 in vivo treatment with a selective inhibitor of ¡NOS resulted in a marked decrease in the level of nitric oxide and PGE2 in the synovial fluid, and a decrease in the expression of ¡NOS and cyclooxygenase-2 (COX-2), the enzyme responsible for the generation of PGE2. This invention is the first to provide direct evidence that the in vivo suppression of nitric oxide production by selective inhibition of NOS in osteoarthritic tissue (OA) is associated with a reduction in in situ synthesis of interleukin-1 IL -1 ß for the synovium, and metalloproteases for the cartilage, as well as a reduction in the level of NOS, peroxynitrite and COX-2, in OA tissue. This also elucidates the mechanisms responsible for the protective effect of the NOS inhibitor on the structural and biochemical changes seen in experimental osteoarthritis. OA lesions in the cartilage develop as a result of an imbalance in the anabolic and catabolic processes that occur during the development of the disease. (1) The excess production of nitric oxide 20 generated by the inducible forms of nitric oxide synthase contributes to cytokine-mediated inflammation, cytotoxicity and tissue damage. The present invention shows that changes in the metabolism of chondrocytes in this disease are attributed, at least in part, to a é * giá ¡& ^ ¿&¿¿- * * ¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡-fc-Jv a. . , *. - ^ '^^ AÍ ^ s ^^^^ - iitr ^ -! ^^^ i ^ á ^^^ st ^ ^ SÉ¡ ^^ Mi »^^. increase in the synthesis of proinflammatory cytokines such as phyterieucin-1ß (IFN-1ß). This change in the function of the chondrocytes impacts the homeostasis of the cartilage matrix. (2). Proinflammatory cytokines accelerate the degradation of the cartilage matrix. (2,3) By inducing the synthesis of proteolytic enzymes, cytokines interfere with the action of growth factors such as insulin-1 growth factor binding proteins (IGF-1). (4) In addition, inflammatory cytokines also reduce the synthesis of aggrecan, one of the main matrix molecules responsible for the functional properties of articular cartilage. (5,6) An increase in the production of nitric oxide (NO) is one of the main factors by which IL-β reduces the synthesis of aggrecan. (5-7). Nitric oxide is produced in large quantities by chondrocytes once stimulation of proinflammatory cytokines has been carried out. (7-13) In contrast to normal cartilage, osteoarthritic cartilage spontaneously produces nitric oxide. (12-14) High levels of stable end products of nitric oxide have been found in synovial fluid and in the serum of arthritic patients. (15) Similarly, messenger RNA and the protein for inducible NO synthase, an enzyme responsible for the generation of cytotoxic levels of NO, have also been detected in the synovial tissue of OA patients. (14-16). Until the present invention, it was believed that nitric oxide only contributed to the development of arthritic lesions. (17-19). This The hypothesis was based on in vitro data showing that nitric oxide improves the activity of metalloproteases (MMP) and inhibits proteoglycan synthesis. (8,20) Still further, hypotheses have been made that nitric oxide reduces the synthesis of the IL-1 receptor antagonist in chondrocytes, a process that is possibly responsible for the IL-1 enhancing effect on these cells. (13) In the practice of the present invention, 17 dogs were used to test the effects of the NOS inhibitor in vivo on osteoarthritis. The OA dog model was created by sectioning the ligament anterior crusader of the knee joint in the right hind paw of twelve (12) dogs by means of a knife wound. The dogs were divided into three (3) groups. Group I had five (n = 5) dogs and was done with non-operated dogs that did not receive treatment and that were considered normal. Group II consisted of six (6) dogs that had osteoarthritis (OA dogs) without treatment. Group III consisted of six (6) OA dogs that received L-NIL orally (10 mg / kg) twice a day for ten (10) weeks starting immediately after surgery. The knees of dogs treated with L-NIL showed a reduction in the incidence of osteophytes compared to dogs not treated (58% vs. 92%) as well as their size (1.92 ± 0.58 mm vs. 5.08 ± 0.66 mm). Macroscopically, the decrease in L-NIL in the size of the cartilage lesion in both condyles and plateaus compared to untreated dogs was close to fifty percent (50%). On the level Histologically, the severity of cartilage lesions in the condyles and the severity of stnpvial inflammation were statistically lower in dogs treated with L-NIL versus untreated dogs. Treatment with L-NIL also significantly reduced both the overall activity of MMP and 5 of stromelysin in the cartilage and in the levels of IL-β, PGE2 and nitrite / nitrate in synovial fluid. The aim of the present invention is to provide new methods for treating osteoarthritis by administering a therapeutic effective amount of a NOS inhibitor to a patient in need thereof. Many other objects and purposes of the invention will become clear from the following description of the invention. Although the specification concludes with the claims that specifically and specifically claim the material forming the present invention, it is believed that the invention will be better understood from the15 following detailed description of the preferred embodiments.

DETAILED DESCRIPTION OF THE INVENTION For the first time, a selective NOS inhibitor has been observed which reduces in vivo the progression of osteoarthritic lesions. In vivo treatment with a selective NOS inhibitor also reduced the level of proinflammatory mediators, IL-1β and PGE2 and the nitrite / nitrate levels in the synovial fluid, showed a marked reduction in activity and ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ joint. It also resulted in a decrease in the expression of ¡NOS, peroxynitrile and COX-2 in the cartilage. In the first models, osteophyte formation was observed as one of the predominant structural changes of osteoarthritis, particularly in the femoral condyles. (25-28) The formation and growth of osteophytes was thought to be associated with inflamed synovium, mechanical factors, and a variety of growth factors and cytokines. (33.34). The present invention shows that in vivo treatment with a selective inhibitor of NOS reduces the growth of osteophytes and also decreases the production of IL-β. Although in previous models of dogs, intra-articular injections of the IL-1 antagonist receptor showed a decrease in the formation of osteophytes, suggesting a role for IL-1β in the genesis of the osteophyte, the effect of an inhibitor of iNOS on the Osteophyte formation was unknown. (33) The OA dog model used to test the methods of the present invention has previously been used to test the effects of antiarthritic drugs in vivo. (25-28) This model is relevant to human OA and exhibits the development of progressive cartilage lesions. (32) To test the method of the present invention, cartilage lesions in both the condyles and the plateau of untreated dogs at 10 weeks post-surgery were significant in size and degree, as they waited in this model and according to our previous reports (25-28). The treatment with selective inhibitor of ÍNOS markedly reduced the size of the lesions of the caí ago by more than 50%. L-NIL had an effect on the classification of the lesion more specifically on the condyles, which was also reflected by a significantly high reduction in the histological severity of the lesions. The protective effect of the selective NOS inhibitor was mainly attributed to the reduction in the severity of the structural changes (depth of erosion) as indicated by the loss of safranin-O staining. 10 Our findings demonstrate that selective NOS inhibitors mediate the reduction in cartilage matrix breakdown. This may be due to a direct action of the NOS inhibitor, as reflected by the reduction of the nitrite / nitrate level in the synovial fluid, but it is also likely to be secondary to the inhibition of the activity of the synovial fluid. preoteolytic enzymes. Recent reports suggest that NO can stimulate MMP synthesis and / or activity in chondrocytes in vivo. (8,20) Our findings provide evidence that cartilage MMP activity in vivo can be reduced by inhibiting ¡NOS activity and that the reduction in MMP activity in cartilage is associated with the decrease in the expression and synthesis of these proteases by chondrocytes. Therefore, the suppression in the production of NO by selective NOS inhibitors preserved the proteoglycan content of the cartilage by reducing the level of synthesis and MMP activity in the cartilage.

Similarly, the decrease in the severity of structural changes simultaneously with the inhibition of collagenase activity explain how selective NOS inhibitors provide protection to the collagen network that underlies cartilage structure. Another possible mechanism by which inhibition of NOS can ultimately result in a decrease in cartilage destruction includes a reduction in apoptotic cell death of chondrocytes and a reduction in the formation of highly reactive oxidant., peroxynitrite. The reduction in the degree of synovial inflation in OA dogs treated with L-NIL compared to the synovio of untreated OA dogs was associated with a reduction in the level of IL-1β mediated by L-NIL that occurs either directly or through indirect mechanisms. In any case, this reduction in IL-1ß contributes, at least in part, to the mechanisms in which the selective inhibitor reduces the synthesis of MMP in vivo. In the treated dogs, the lower level of synovial inflammation was very remarkable, both macroscopically and microscopically, and was observed mainly as a very significant reduction in hairy hyperplasia and in the number of mononuclear cells infiltrated in the tissue. These findings explain how treatment with a selective inhibitor reduces the amount of effusion of synovial fluid. Similar anti-inflammatory effects of NOS inhibitors were previously reported in an air sac stimulated by carrageenan and in rat leg inflammation models. Oral administration '> v «& m * fl. -M? ßfcé * > * < . - -. "t- ^ j - j-a.

Previous L-NIL exerted other anti-inflammatory effects such as reduction in nitrite / nitrate accumulation and cellular infiltration in the fluid of the sac and in the inflamed leg. In contrast, the in vivo treatment of OA dogs with selective inhibitors reduced the levels of PGE2 or PG isomerase activity as well as its expression. (37.38) Experimental groups Seventeen mestizo adult dogs (2-3 years old), weighing 20 to 25 kg each, were used in this study. The surgical sectioning of the ACL of the right knee, using a knife, was carried out in 12 dogs as previously described (25-28). Before surgery, the animals were anesthetized intravenously with sodium pentobarbital (25 mg / kg) and kept in an incubator. Following the surgery, the dogs were kept in animal care facilities for a week, then sent to housing farms where they were free to exercise in large cages. The dogs were randomly separated into three treatment groups: Group I (n = 5) was formed with non-operated dogs that did not receive treatment (normal); Group II (n = 6) were OA dogs that did not receive treatment; Group III (n = 6) were OA dogs who were given 10 mg / kg L-NIL twice a day.

Drug administration and drug circulation levels L-NIL was administered twice a day in a dose of 10 mg / kg at 8 and 16 hours. The drug was orally given as a liquid solution. Treatment with the drug was started immediately after the surgery with a duration of 10 weeks, after which the animals were sacrificed. The concentration of L-NIL was measured in the serum in the middle of the treatment and at the end of the study following a treatment of 10 weeks, and in the synovial fluid only at the end of the study. L-NIL was measured using MS / MS electrospray (Sciex API III) in the multiple-selection monitoring mode using the fragmented ion (m / z 84) originating from the compound.

Macroscopic classification of the lesions Immediately after sacrifice, the right knees of the dogs were dissected, placed on ice, and the synovial fluid was aspirated and the volume was measured. Each knee was examined to classify morphological changes, including the presence of osteophyte formation and cartilage lesions, as previously described (25-28). The examinations were carried out by two independent observers through blind evaluation. The degree of formation of osteophytes was classified by the measurement of the maximum thickness (mm) of the abutment in each femoral condyle. The changes of the cartilage in the medial and lateral femoral condyles and the tibial plateau were further classified under dissection microscopy (Stereozoom, Bausch &Lomb, Rochester, NY). The depth and erosion was classified on a scale from 0 to 4 as follows: 0 = the surface appears normal, 1 = minimal fibrillation with a slightly yellow discoloration on the surface, 2 = erosion extends within the surface or middle layers , 3 = erosion extends into deep layers, 4 = erosion extends to subchondral bone. The surface area of the changes of the articular surface was measured and expressed in mm2.

Histological classification Histological evaluation was carried out on sagittal sections of cartilage of the injured areas of each femoral condyle and tibial plateau as described (25-28). The specimens were dissected, fixed in 10% buffered formalin and embedded in paraffin for histological evaluation. The serial sections (5 μm) were stained with safranin-O. The severity of the OA lesions was classified on a scale of 0 to 14, using two independent observers, using the histological / histochemical scale of Mankin et al (29). This scale evaluates the severity of OA lesions based on the loss of safranin-0 staining (scale 0-4), cellular changes (scale 0-3), invasion of the advance mark by blood vessels (scale 0-1) and structural changes (scale 0-6). In this last scale, 0 indicates the structure of normal cartilage and 6 section of cartilage. Representative specimens of the synovial membrane of the medial and lateral knee compartments were also dissected from the underlying tissues. The specimens were fixed in 10% buffered formalin, embedded in paraffin, sectioned (5 μm) and stained with hematoxylin-eosin. Two specimens of the synovial membrane were examined for each compartment, with the highest evaluation retained for each compartment. The average was calculated and considered as a unit for the entire knee. The severity of the synovitis was classified on a scale of 0 to 10 (14) by two independent observers, to which was added the labeling for 3 histological criteria: the hyperplasia of cells bordering the synovium (scale 0-2), the hairy hyperplasia (scale 0-3), and the degree of cellular infiltration by mononuclear and polymorphonuclear cells (scale 0-5).

Metalloprotease activity assay (MMP) After the macroscopic examination that was carried out, the sections were taken for histological evaluation, the remaining cartilage of the femoral condyles, the tibial plateau and the synovial membranes were carefully dissected. t ^ jfa 5 * ^, f. - MMP extraction was carried out as described (28). Briefly, the tissue was sectioned and homogenized in the extraction buffer (50 mM Tris HCl, 10 mM CaCl2, 2 M guanidine HCl, 0.05% Brij-35, pH 7.5). The mixture was stirred overnight at 4 ° C, and then centrifuged (18,000 g, 30 minutes, 4 ° C). The supernatant was extensively dialyzed (48 hours at 4 ° C) against a test buffer (50 mM Tris HCl, 10 mM CaCl 2, 0.2 M NaCl, 0.05% Brij-35, pH 7.5) using a Spectrapor dialysis tube. with 12,000 Da as a limit value (Spectrum Medical Industries, Los Angeles, CA). The collagenase activity in the tissue extract was measured using a type I collagenase free telopeptide of the rat tail tendon acetylated with I14 acetic anhydride-IC (30). One hundred microliters of the I14 Cl collagenase suspension (12,000 DPM) was incubated under the following conditions: 1) with an aliquot of 100 μl of tissue extract, in the presence of 1 mM of aminophenylmercuric acid (APMA), and 2) with an aliquot of 100 μl of tissue extract, containing 1 mM APMA and 25 mM of ethylenediamine tetraacetic acid (EDTA) which served as target. Each solution was incubated for 28 hours at 30 ° C, after which time each was centrifuged at 12,000 g for 15 minutes at 4 ° C. The radioactivity contained in the supernatant was determined using a beta scintillation counter (Beta Rack, Model 1218; LKB, Stockholm, Sweden). The total enzymatic activity was expressed in units per gram of wet weight tissue (p.h.), with Ü £ »• &» a unit corresponding to the hydrolysis of 1 μg of substrate / hour at 30 ° C. More than 90% of the collagenase activity was inhibited by EDTA. The general MMP activity in the cartilage extract was measured by the method of Chavia et al (31) using azocol (Calbiochem-Novabiochem International, San Diego, CA) as a substrate. One hundred microliters of the azocol suspension were incubated in a manner similar to the collagenase assay, but with 1 mM of 1, 10-phenanthroline serving as a blank. After incubation (48 hours at 37 ° C), this solution was centrifuged (12,000 g for 15 minutes at 4 ° C), and the optical density of the supernatant was determined by spectrophotometric analysis, the overall MMP activity was expressed in units per gram of ph of the tissue, in which one unit corresponds to the hydrolysis of 1 μg of substrate / hour at 37 ° C. The digestion activity of azocol in the extracts was inhibited by more than 90% by 1, 10-phenanthroline.

Enzyme immunoassay The level of IL-1β in the synovial fluid was determined using a douantibody ELISA, in a specific solid phase. PGE2 was measured by enzyme immunoassay. The IL-1ß team was from BioSource International (Camarillo, CA), and the PGE2 team from Cayman Chemical Co. (Ann Arbor, MI). The sensitivity of the assay was 0.3 pa / ml for IL-1 225 and 29 pg / ml for PGE2. The measurements were carried out in duplicate, and according to the manufacturer's specifications. The reaction was measured in a Vmax photometer for micro-ELISA (MQl fülar Devices Corp. Merífo Park, CA, ~ * t USA). They were expressed as total pg or ng in the synovial fluid of the joint.

Nitric Oxide Assay * W Nitrite and nitrate in synovial fluid were determined using a fluorometric assay as described (21). Briefly, the synovial fluid was filtered through an Ultrafree microcentrifuge filter unit with a limit value of 10,000 Da (Millipore, Bedford, MA) at 14,000 rpm for 15 minutes. To convert nitrate to nitrite, 5-10 μl of liquid Synovial filtrate was incubated with 20 M Tris, pH 7.6 containing 40 μM NADPH and 14 mU of nitrate reductase in a final volume of 100 μl. After of the incubation for 5 minutes at 20 ° C, the fluorometric reaction was initiated by adding 10 μl of 0.05 mg / ml 2,3-diaminonaphthalene dissolved in 0. 62 M HCl. After 10 minutes at 20 ° C, the reaction was stopped with 10μl 1. 4 N NaOH. Fluorescence was measured at the wavelength of 365/450 (excitation / emission) using a fluorescence reader (IDEXX Laboratories, Westbrook, ME). Nitrite was expressed as total nmol in the synovial fluid of the joint.

Immunohistochemical studies The specimens of the cartilage of the condyle and the plateau, as well as the synovial membrane, were processed by immunohistochemical analysis as previously described. Briefly, the specimens were fixed in 4% paraformaldehyde for 24 hours, then embedded in paraffin. Sections (76 μm) of specimens embedded in paraffin were placed on slides (Fisher Scientific, Nepean, Ontario, Canada), deparaffinized in toluene, hydrated in a gradual series of ethanol, and pre-incubated with chondroitin ABC (0.25 units / ml) in phosphate buffered saline (PBS; Sigma Aldrich Canada Ltd., Oakville, Ontario, Canada) for 60 minutes at 37 ° C. Following this, the specimens were washed in PBS, then washed again in 0.3% hydrogen peroxide / methanol for 30 minutes. The slides were incubated with 2% normal serum (Dimension: Laboratories, Mississauga, Ontario, Canada) for 20 minutes, blocked and re-incubated with: i) a rabbit polyclonal antibody (IgG) against IOS (100 μg / ml , 1/100 dilution, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) for 2 hours at room temperature in a humid chamber; ii) an anti-nitrotyrosine rabbit polyclonal antibody (IgG) (dilution 1/100, Dr. P. Manning, Searle / Monsanto R &D, St. Louis, MO, USA) for 1 hour at room temperature, iii) with a mouse monoclonal antibody (IgG) against rhlL-13 (1 mg / ml, 1/25 dilution, BioSource International, Montreal, Quebec, Canada); iv) a rabbit polyclonal antibody against stromelysin-1 (IgG) (MMP-3, 500 μg / ml, 1/1000 dilution, Oncogene Science, Cambridge, MA, USA); v) a mouse monoclonal antibody against collagenase (MMP.1, 100 μg / ml, 1/500 dilution, Oncogene Science); or vi) a rabbit polyclonal antibody (IgG) against rhCOX-2 (1/500 dilution, Oxford Biomedical Research Inc., Oxford, MI, USA). With the exception of the antibody against MMP-3, which was incubated for 18 hours at 4 ° C, all the antibodies were incubated for 1 hour at room temperature. Each slide was washed three times in PBS (pH 7.4) and stained using the avidin-biotin complex method (Vectastain ABCkit; Dimension Laboratories). This method links the incubation in the presence of a secondary antibody conjugated to biotin for 30 minutes at room temperature, followed by the addition of an avidin-biotin peroxidase complex for 45 minutes. All incubations were carried out in a humid chamber, and the color was developed with 3,3'-diaminobenzidine (Dimension Laboratories) containing hydrogen peroxide. To determine the specificity of the staining, three control procedures were used according to the experimental protocol: I) the use of an absorbed immune serum (1 hour, 37 ° C) with a molar excess of 10 to 20 times the purified or recombinant antibody; 2) the omission of the primary antibody; and 3) replacement of the primary antibody with an autologous preimmune serum. The purified antigens used in this study were rhlL-1β (Genzyme, Cambridge, MA, USA), rhMMP-1, rhMMP-3, rhiNOS and rhCOX-2 (Monsanto / Searle, St. Louis, MO, USA, and nitrotyrosine (Sigma-Aldrich, St. Louis, MO, USA).

Several sections were made? E each block of cartilage, and three slides of each specimen were processed for immunohistochemical analysis. Each section was examined under light microscopy (Leitz Orthoplan, Wilo Leitz, Ville St-OLaurent, Quebec, Canada), and photographed with Kodak Edíachojom film ASA 64 (Rochester, NY, USA).

Morphometric analysis Cartilage The cartilage samples of all the lateral and medial femoral condyles and the tibial plateau were processed. Of each cartilage specimen, three slides were processed for immunohistochemical studies. The quantification of the different antigens in the cartilage and synovium were made using our method previously published with slight modifications. The level of protein synthesis was estimated by determining the number of chondrocytes stained positively within the different layers of the cartilage. Each section of the cartilage was divided into six microscopic fields (X 40, Leitz Diaplan), three fields in each section of the following areas: the surface layers and upper intermediates, and the lower and deeper intermediate layers. The superficial layers include the chondrocytes of the immediate surface characterized by discoidal cells that follow the line pattern of the dominant fibrillar orientation, and the upper 50% of the transition zone. & "? *. **. ^? &) t > i &i &tli? ii characterized by round cells, spheroids.The deep layers include cells of less than 50% of the transitional zone and those aligned in columns For each arthritic specimen, it was ascertained before evaluation that the surface of the intact cartilage could be detected, and used as a marker for the validation of the morphometric analysis, the total number of chondrocytes, and the number of chondrocytes stained positively. for the specific antigen, were estimated for the total thickness of the cartilage.The final results were expressed as the percentage of chondrocytes stained positively for the antigen (cell count), with the maximum count of 100% .Each slide was subjected to an evaluation double blind, resulting in a variation of <5% between the scores The data obtained from the lateral middle condyles and the tibial plateau were considered as independent for the purpose of statistical analysis.

Synovial membrane For the analysis of the synovial membrane, the cell count of the different specimens was determined for each section using our previously published method. Each specimen was divided into 10 distinct areas: five microscopic fields (X 40) at the level of underlying synovial cells, and five in the underlying area of the synovial membrane. The percentage of cells that positively bound for specific antigens was evaluated in each field as described above for cartilage. He percentage of cells with positive staining was averaged for all fields in each area of the synovium (adjacent synovial cells and mononuclear infiltrate). Each slide was blindly evaluated by two independent observers, resulting in a variation < 5% between the two 5 counts. The cell count was given separately for the underlying synovial cells and for the mononuclear cell infiltrate, with the maximum for each area of 100%.

Statistical analysis 10 Mean values and standard errors of the mean were calculated. Statistical analyzes were made using the Mann-Whitney U-test. P values less than 0.05 were considered significant.

Results 15 Levels of Circulating Drug The concentration of L-NIL was measured in serum at half treatment and in serum and synovial fluid after 10 weeks of treatment. The samples were obtained 2 hours after the administration of the drug. The means of the concentration + SEM of L-NIL in the serum at the middle of the treatment and at the end of the treatment were 201 ± 33 and 119 ± 24 μM, respectively. The concentration of L-NIL in the Synovial fluid at the end of the treatment was 96 ± 18 μM, demonstrating the access of the compound to the joint.

Macroscopic findings. 'Osteophytes: In OA dogs, osteophytes were present in 92% of the condyles. Its mean ± SEM width was 5.08 ± 0.66. In dogs treated with L-NIL, osteophytes were present only in 58% of the condyles, and their size was smaller (1.92 ± 0.58 mm, P <0.002) compared to untreated OA dogs. The condyles of the non-operated (normal) dogs were of normal appearance, and no osteophyte could be seen. Cartilage: The cartilage on the condyles and the plateau of normal dogs were microscopically normal in appearance. In untreated OA dogs, cartilage lesions of moderate size and degree were present on both condyles and plateaus, with slightly more severe lesions on the plateaus. Dogs treated with L-NIL showed a marked reduction in the severity of condylar lesions, with a decrease of approximately 50% in size and 20% in grade. Synovial membrane: The synovia of normal dogs had a white shine and were transparent in appearance. The synovia of OA dogs were hypertrophic and showed a reddish yellow discoloration and contained a large number of blood vessels. In OA dogs treated with L-NIL, the synovia was thinner and contained fewer blood vessels and the Discoloration was less intense in coiripation with untreated OA dogs.

Microscopic findings Cartilage: The cartilage of non-operated dogs had a normal histological appearance with the exception of a few dogs that showed either a minimal loss of coloration with safranin-0 in the surface layer or minimal irregularities of the cartilage surface. Specimens from untreated OA dogs had morphological changes that included cartilage fibrillation and fissures, hypercellularity and cloning, and loss of safranin-O staining. The histological count of the condylar lesions was similar to that of the plateaus. In L-NIL dogs treated with OA, condylar lesions were significantly less severe (P <0.005) compared to untreated OA dogs. This difference was largely due to a decrease in the severity of structural changes and to a loss of safranin-O staining. According to the microscopic observation, no difference was observed between the two OA groups (untreated and treated with L-NIL) in the severity of the histological lesions on the plateaus. Synovial membrane: Synovial histology of non-operated dogs was within normal limits, with the exception of a few specimens that had minimal and focal hairy hyperplasia (count = 0.50 ± 0.34). The synovium of the untreated OA dogs was thick, had numerous villi, and showed hyperplasia of the underlying cells - tsft-synovial and a moderate infiltration of the mononuclear cells. The synovial inflammation in the OA bitches with L-NIL was less severe than in the untreated OA dogs (count = 2.50 ± 0.18 vs A ± 0.47, P <0.004). The reduction in the inflammation count that followed L-NIL treatment resulted mainly in the reduction of the intensity of the villous hyperplasia and in the infiltration of mononuclear cells.

Metalloprotease activity General MMP activity: The overall average MMP activity in cartilage and synovium was significantly higher (P <0.004 and P <0.02, respectively) in untreated OA dogs compared to normal dogs. In OA dogs treated with L-NIL, the enzymatic activity was reduced both in the synovial membrane and in the cartilage and a statistical difference for the cartilage was reached (P <0.004). Collagenase: The average activity of collagenase found in the cartilage and synovium of dogs OA was significantly higher (P <0.002) compared to the activity found in normal dogs. The enzymatic activity in both tissues was reduced by treatment with L-NIL, and again a statistical difference was reached only for cartilage (P <0.004). 3rd Values of IL-lß. Nitrite-Nitrate, v PGE? in synovial fluid! The synovial fluid volume was increased from 0.3 + 0.04 ml in the joints of normal dogs to 6.8 ± 1.0 ml in the joints of untreated OA dogs. Treatment with L-NIL reduced the volume by 68% to 2.4 ± 0.8 ml (P <0.009). The total amount of IL-lβ in the synovial fluid of normal dogs was very low (0.1 pg). A significantly higher level of IL-lβ (32.9 ± 15.3 pg, P <0.001) was found in untreated OA dogs. In OA dogs treated with L-NIL, the level of IL-lβ decreased significantly to near normal values (0.5 ± 0.2 pg) having a value of P < 0.004 when compared with the untreated OA group. The total amount of nitrite / nitrate was also markedly increased in the synovial fluid of the untreated OA group (96.4 ± 17.9 nmol, P <0.001), compared to the normal (6.5 ± 0.8 nmol). OA dogs treated with L-NIL exhibited a 53% decrease (48.6 ± 12.9 nmol, P <0.04) in the total nitrite / nitrate content of synovial fluid over those of untreated OA dogs. Furthermore, the total amount of PGE2 was also reduced from 9.4 ± 5.4 ng in the synovial fluid of untreated OA dogs to 0.8 ± 0.3 in OA dogs treated with L-NIL (P <0.009). Due to the limitations of the samples, the amount of PGE2 was not measured in normal dogs.

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Synovial membranes In non-operated dogs, IL-1 B was detected only in a few cells in the underlying synovial cells. In OA dogs not treated, OA staining was found in all specimens, both in the underlying synovial cells and in the mononuclear cell infiltrate. The cell count of the OA synovial membrane specimens was significantly higher than that of the non-operated dogs, both for the infiltrated mononuclear cells and for the synovial underlying cells 10. In the dogs treated with L-NIL, although the IL-1 B was expressed in all specimens, the cell count was significantly lower than that of untreated OA dogs, both for the underlying synovial cells (P <0.008) and for the mononuclear cell infiltrate (P <0.008). The 15 controls using absorbed immune serum showed only background staining. b) NOS and 3-nitrotyrosine With the exception of an occasional synovial underlying cell, the The synovial membrane of the non-operated dogs showed no specific staining. In the OA synovial membrane of untreated dogs, a strong staining for both NOS and 3-nitrotyrosine was observed in a small percentage of the mononuclear and underlying infiltrating cells. The cell count for Both mononuclear cells and underlying genes were significantly higher when compared with non-operated dogs. There was a significant decrease in the cell count of both the mononuclear cells and the underlying cells, with the exception of the cellular count for NOS in mononuclear cells, in dogs treated with L-NIL. Controls that used absorbed immune serum showed only background staining. c) COX-2 In non-operated dogs, COX-2 staining was detected in only a few cells of the synovial lining. In untreated dogs, there was a large number of cells, both in the synovial lining and in the mononuclear cell infiltrate, which showed positive staining for COX-2. The osteoarthritis of the dogs treated with L-NIL showed a marked decrease in the number of cells that stained positively for COX-2, both in the mononuclear cell infiltrate and in the lining.

Cartilage a) metalloproteases Only a few cells stained positively for collagenase-1 in the superficial and deep layers of the condyles and cartilage plateau of non-operated dogs. The cell count was substantially higher in the OA cartilage of untreated operated dogs, with a higher proportion of positive cells in the superficial layers. In contrast, operated dogs treated with L-NIL showed a marked and significant decrease in the counting of cells with collagenase-1 both in the condyles and the plateau. The results for stromilicin-1 (General MMP) were similar to those of collagenase-1, with a marked and significant increase in the cellular count in the OA cartilage of untreated operated dogs, with similar changes in both the condyles and in the plateau. Treatment with L-NIL significantly reduced the cell count for stromelysin-1 in both the condyles and the plateau. b) NOS 3-nitrotyrosine Only a few cells in the cartilage specimens of the condyles and the plateau of non-operated dogs showed specific cytoplasmic staining for iNOS. These cells were located in both the deep and superficial layers. There was a large and significant increase in the number of chondrocytes that expressed iNOS in the OA cartilage, both in the condyles and in the plateau, which was significantly higher than in the specimens of non-operated dogs. The cell control was similar in the superficial and deep layers. There was a very significant decrease in the cellular count of NOS in the condyles and in the plateau in operated dogs treated with L-NIL. The coloring pattern for 3-nitrotyrosine was almost identical to that of NOS. Only a few cells stained positively on all the cartilage layers of the condyle and the plateau of non-operated dogs, while found a marked and sÉÉÉpÉcativo increase in the cellular count in the OA cartilage. The number of positive chondrocytes was distributed homogeneously between the superficial and deep layers. The cartilage specimens of the dogs treated with L-NIL showed a significantly low cellular control 5 compared to that of the treated dogs, both in the condyle and in the plateau. c) COX-2 The level of COX-2 was greatly increased in the cartilages OA, where the number of chondrocytes stained positively for this enzyme was five to eight times higher than in normal cartilage. Dogs OA treated with L-NIL presented a very significant reduction in the cell count in the condyles and plateaus. ? * ^ > -a- r and 6-t * "^ - TABLE 1 Expression of IL-18 in dog synovial tissue Groups Synovial Coating Mononuclear Cells (cellular counting) (P) ® (cellular counting) (P) Not operated '5 20 F? 65 0 23 + 0 23 (p < 0 008) (p < 0 008) (n = = 5) OA§ 58 71 ± 3 20 46 36 ± 4 76 = 5) L-NIL, »33 32 + 2 52 18 51 ± 2 84 (n = = 5) (p <0 008) (p <0 008) Values are the measures ± SEM Animals slaughtered neither operated nor treated Animals killed slaughtered and tissue examined at 12 weeks after of surgery Exposed animals and tissue examined at 12 weeks after surgery, were given L-NIL orally (10 mg / kg / day / orally) for 12 weeks starting immediately after surgery ® Statistical analysis made with the Mann-Whitney U-test, the p values were compared with the OA group Í & S & ! NOS 3-Nitrotyrosine Groups Cladding Cells Cladding Synovial cells mononuclear synovial mononuclear (cell count) ® (cell count) (cell count) (cell count) No 0 002 + 0 002 0 002 ± 0 002 1 36 ± 0 2 0 002 ± 0 002 operated * (p <0008) (p <0 06) (p <0 008) (p <0008) (n = 5) OA§ 8 34 ± 0 96 5 26 ± 2 07 14 84 ± 3 57 8 87 ± 1 86 (n = 5) L-NIL @ 1 09 ± 039 2 46 ± 2 15 2 05103 2 36 ± 0 84 (n = 5) (p <0008) NS (p ± 0 008) (p <0 02) The values are the measures ± SEM. Animals slaughtered neither operated nor treated. Sacrificed operated animals and tissue examined at 12 weeks after surgery. Sacrificed operated animals and tissue examined at 12 weeks after surgery; L-NIL was given orally (10 mg / kg / day / orally) for 12 weeks starting immediately after surgery. ® Statistical analysis done with the Mann-Whitney U-test; the p values were compared with the OA group.

BOX HJ - * .- i4? Macroscopic classification of femoral condyles and tibial plateaus Femoral condyles Tibial plateaus Group Size No. Classification Size Animal classification (mm2) (0-4 scale) (mm2) (0-4 scale) Not operated * OAf 15.33 ± 3.52 1.42 + 0.19 17.02 ± 3.49 1.42 ± 0.29 L-NIL§ 7.33 ± 2.32 1.08 + 0.26 9.75 ± 2.45 1.33 ± 0.26 The values are the measures ± SEM. 10 * Animals slaughtered neither operated nor treated. t Animals that were sacrificed and the tissue was examined at 10 weeks after surgery. Animals that were sacrificed and the tissue was examined at 10 weeks after surgery. L-NIL was given orally (10 mg / kg / twice a day) for 10 weeks, starting after surgery.

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CUADROjtV Expression of colaqenase (MMP-1) and stromelysins (MMP-3) in dog cartilage Collagenase-1 Estromelysin-1 (MMP-1) (MMP-3) Femoral condyle groups Tibial platelet Femoral condyle Tibial plateau (cell count) ® (cell count) (cell count) (cell count) No 4.12 ± 0.42 5.43 ± 0.71 4.62 ± 0.61 5.98 ± 0.52 operated (p? .0001) (p < 0.0001) (p < 0.0001) (p < 0.0001) (n = 5) OA§ (n = 5) 30.28 ± 3.94 36.25 ± 3.58 43.48 ± 1.98 44.94 ± 2.17 L-NIL @ 10.02 ± 2.26 18.25 ± 1.97 34.45 ± 2.14 33.21 ± 1.90 (n = 5) (p <0.0002) (p <0 0003) (p <0.0005) (pO.OOOl) values are the means ± SEM. 10 * Animals slaughtered neither operated nor treated. § Sacrificed operated animals and tissue examined at 12 weeks after surgery. @ Sacrificed operated animals and tissue examined at 12 weeks after surgery; L-NIL was given orally (10 mg / kg / day / orally) for 12 weeks starting immediately after surgery. ® Statistical analysis done with the Mann-Whitney U-test; the p values compared to the OA group.

TABLE Expression of inductible nitric oxide synthase (iNOS) and 3-nitrotyrosine in dog cartilage Nitric oxide synthase inducible (iNOS) 3-Nitrotyrosine Groups Condyles femoral Tibial platelet Femoral condyle Tibial plateau (cell count) ® (cell count) (cell count) (cell count) Not operated 5.49 ± 0.30 6.98 ± 0.42 5.36 ± 0.49 6.27 ± 0.36 (n = 5) (p? .0001) (p? .0001) (p? .0001) (p < 0.0001) OA§ (n = 5) 47.11 ± 1.98 45.82 ± 2.13 36.68 ± 2.75 45.50 ± 2.35 L- NIL @ 26.46 ± 2.04 35.82 ± 1.97 21.81 ± 2.14 27.30 ± 1.88 (n = 5) (p? .0001) (p? .001) (p < 0.0004) (p < 0.0001) The values are the means ± SEM . Animals slaughtered neither operated nor treated. 10 Animals operated sacrificed and tissue examined at 12 weeks after surgery. Sacrificed operated animals and tissue examined at 12 weeks after surgery; L-NIL was given orally (10 mg / kg / day / orally) for 12 weeks starting immediately after surgery. ® Statistical analysis done with the Mann-Whitney U-test; the p values compared to the OA group.

Aunt & amp; amp; MS & amp; amp; > & «?» Awg-g TABLE VI Expression of COX-2 in dog cartilage Femoral condyles groups (% positive tibial plateau (p) ® cells (% of positive cells) (p) Not operated 5.02 ± 0.36 7.64 ± 0.52 (n = 5) (p <0.0001) (p? .0001) OA§ (n = 5) 40.99 ± 3.26 39.74 ± 2.70 L-NIL @ (n = 5) 18.20 ± 2.29 27.68 ± 2.12 (p <0.0001) (p? .001) The values are the means ± SEM. * Animals slaughtered neither operated or treated § Sacrificed operated animals and tissue examined at 12 10 weeks after surgery @ Sacrificed operated animals and tissue examined at 12 • weeks ^ after surgery, were given L-NIL orally (10 mg / kg / i every day / orally) for 12 weeks starting immediately after surgery. ® Statistical analysis made with the Mann-Whitney U-test, the p-values compared to the OA group.

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Production of nitric oxide in the synovial membrane of rheumatoid and osteoarthris patients. J. Exp. Med. 184: 1519-1524. 15. Farrell, A.J., D.R. Blake, R.M. Palmer, and S. Moneada. 1992. Increased concentration of nitrite in synovial fluid and serum samples suggest ncreased nitric oxide synthesis n rheumatic diseases. Ann Rheum. Dis. 51: 1219-1222. 16. Sakurai, H., H. Kohsaka, M. Liu H. Higashiyama, Y. Hirata, K. 4 J Kanno, I. Saito, and N. Miyasaka. 1995. Nitric Oxide Production and inducible nitric oxide synthase expression in nflammatory arthritides. J. Clin. Invest. 96: 2357-2363. 15 17. Cannon, G. W., S. J. Openshaw, J. B. Hibbs, Jr., J. R. Hoidal, T. P. Huecksteadt, and M. M. Griffiths. 1996. Nitric oxide production during adjuvant-induced and collagen-induced arthritis. Arthritis Rheum. 39: 1677-1684. 18. Evans, C. H., M. Stefanovic-Racic, and J. Lancaster. 1995. 20 Nitric oxide and its role in orthopaedic disease. Clin Orthop 312: 275-294. «F 19. Stefanovic-Racic, M., J. Stadler, and C. H. Evans. 1993. Nitric oxide and arthritis. Arthritis Rheum. 36: 1036-1044.

S? Ia ^ - 'i 20. Murrell, G. G. C, D. Jang, and R. J. Williams. 1995. Nitric oxide activates metalloprotease enzymes n joint cartilage. Biochem Biophys Res Commun 206: 15-21. 21. Connor, J. R., P. T. Manning, S. Settle, W. M. Moore, G. M. Jerome, R. K. Webber, F. S. Tjoeng, and M. G. Currie, 1995. Suppron of adjuvant-induced arthritis by selective inhibition of inducible nitric oxide synthase. Eur J Pharmacol 273: 15-24. 22. Stefanovic-Racic, M., K. Meyers, C. Meschter, J. W. Coffey, R. A. Hoffman, and C. H. Evans. 1994. N-monomethy arginine, an inhibitor of nitric oxide synthase, supprs the development of adjuvant arthritis n rats. Arthritis Rheum. 37: 1062-1069. 23. Stefanovic-Racid, M., K. Meyers, C. Meschter, J. W. Coffey, R. A. Hoffman, and C. H. Evans. 1995. Comparison of the nitric oxide synthase inhibitors methylarginine and aminoguanidine as prophylactic and therapeutic agents in rat adjuvant arthritis. J. Rheumatol. 22: 1922-1928. 24. McCartney-Francis, N., J. B. Alien, D. E. Mizel, J. E. Albina, Q. Xie. D. F. Nathan, and S. M. Wahl. 1993. Suppron of arthritis by an inhibitor of nitric oxide synthase. J. Exp. Med. 178: 749-754. 25. Moore, W, M., R. K. Webber, G. M. Jerome, F. S. Tjoeng, T. P. Misko, and M. G. Currie. 1994. L -? / 6- (1-lminoethyl) lysine: a selective inhibitor of inducible nitric oxide synthase. J. Med Chem 37: 3886-3888. 26. Pelletier, J. P., J. A. DiBattista, J. P. Raynauld, S. Wilhelm, and J. Martel-Pelletier. 1995. The in vivo effects of intraarticular corticosteroid injections on cartilage lesions, stromelysin, interleukin-1 and oncogene protein synthesis in experimental osteoarthritis. Lab. Invest. 72: 578-586. 27. Pelletier, JP, F. Mineau, JP Raynauld, JF Jr. Woer, Z. Gunja-Smith, and J. Martel-Pelletier, 1994. Intraarticular injections with methylprednisolone acetate reduces osteoarthritic lesions in parallel with chondrocyte stromelynsin synthesis in experimental Osteoarthritis: Arthritis Rheum. 3: 414-423. 28. Fernandes, J. C, J. Martel-Pelletier, I. G. Ottern A. Lopez-Anaya, F. Mineau, G. Tardif, and J. P. Pelletier. 1995. Effects of tenidap on canine experimental osteoarthritis: I. Morphologic and metalloprotease analysis. Arthritis Rheum. 38: 1290-1303. 29. Mankin, H. J., H. Dorfman, L. Lippiello, and a. Zarins, 1971. Biochemical and metabolic abnormalities in articular cartilage from osteoarthritic human hips. II. Correlation of morphology with biochemical and metabolic data. J. Bone Joint Surg. Am. 53: 523-537. 30. Cawson, T. E. and A. J. Barrett. 1979. A rapid and reproducible assay for collagenase using [1-14c] acetylated collagen. Anal Biochem. 99: 340-345. 31. Chavira, R. Jr., t. J. Burnett, and J. H. Hageman. 1984. Assaying proteinases with azocoll. Anal. Biochem. 36: 446-450. 32. Brandt. K. D. 1994. Insights into the natural history of osteoarthritis provided by the cruciate-deficient dog. An animal model of osteoarthritis. [Review] Ann. NYAcad. Sci. 732: 199-205. . - * < < fe- "> ** • * '33. Caron, J. P., J. C. Femandes, J. Martel-Pelletier, G. Tardif, F. Mineau. C. Geng. and J. P. Pelletier. 1996. Chondroprotective effect of intraarticular injections of interleukin-1 receptor antagonist in experimental osteoarthritis: suppron of collagenase-1 expron. Arthritis Rheum 39: 1535-1544. 34. Van beuningen, H. M., P. M. van der Kraan, O. J. Arntz, and W. B. van den Berg. 1994. Transforming growth factor-beta 1 stimulates articular chondrocyte proteoglycan synthesis and induces osteophyte formation in the murine knee joint. Lab. Invest. 71: 279-290. 10 35. Blanco F. J., R. L. Ochs, H. Schwarz and M. Lotz. 1995. Chondrocyte apoptosis induced by nitric oxide. Am J. Pathol. 146: 75-85. i. • 7 36. Beckman, J. S. and Koppenol. 1996. Nitric oxide, superoxide and peroxynitrite: the good, the bad, and the ugly. Am. J. Physiol. 271: C1424- C1437. 15 37. Salvemini, D., P. T. Manning, B. S. Zweifel, K. Siebert, I. Connor, M. G. Currie, P. Needleman, and J. L. Masferrer. 1995. Dual inhibition of nitric oxide and prostanglandin production contributes to the antiflammatory properties of nitric oxide synthase inhibitors. J. Clin. Invest. 96: 301-308. 38. Salvemini, D., Z. -Q. Wang P. P. Wyatt, D. M. Bourdon, M. H.

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DESCRIPTION OF THE FIGURES Figure 1 Levels of general activity of metalloprotease in the articular cartilage and in the synovial membrane of dogs. The values are units / g per wet weight (p.h.) of tissue, the P values are versus the AO group of 10 weeks, by Mann-Whitney U-test. Figure 2 Levels of collagenase activity in the cartilage and in the synovial membrane of dogs. The values are units / g d per wet weight (p.h.) of tissue. The P values are versus the 10-week AO group, using the Mann-Whitney U-test. i

Claims (14)

NOVELTY OF THE INVENTION < ,, ' CLAIMS

1. - The use of an inducible nitric oxide synthase inhibitor, for the manufacture of a medicament for modulating the levels of IL-1β in a patient.

2. The use of an inducible nitric oxide synthase inhibitor, for the manufacture of a drug to reduce the amount of synovial fluid in a patient.

3. The use of an inducible nitric oxide synthase inhibitor for the manufacture of a medicament for modulating the cartilage degeneration of a patient who has osteoarthritis.

4. The use according to claim 3, wherein the severity of said disease is reduced.

5. The use according to claim 3, wherein the size of the lesion is reduced.

6. The use according to claim 3, wherein the disorganization of the disease matrix is modified.

7. The use of an inhibitor of nitric oxide synthase, for the manufacture of a medicine to modulate the formation and development of osteophytes in a patient. «To? * Twata- 8.- The use of an inducible nitric oxide synthase inhibitor for the manufacture of a medicament to modulate the cartilage lesions in a patient. 9. The use according to claim 8, wherein said lesions are in the condyles. 10. The use according to claim 8, wherein said lesions are on the plateaus. 11. The use of an inducible nitric oxide synthase inhibitor for the manufacture of a medicament for treating osteoarthritis in a patient. 12. The use of an inhibitor of inducible nitric oxide synthase, for the manufacture of a medicine to prevent osteoarthritis in a patient. 13. The use of a inducible nitric oxide synthase inhibitor for the manufacture of a medicament for modulating metalloproteases in a patient. 14. The use of a inducible nitric oxide synthase inhibitor for the manufacture of a medicament for treating acute joint damage in a patient. & * & ***, -_ * ^ ™ ~ ™ »« > m ™ í »» - - ^ - ~ # *