Patent Abstract:
treating or preventing degeneration or destruction of articular cartilage and / or subchondral bone in the affected joint of a mammal is accomplished by administering a compound of formula , wherein the variables have the meanings given in the present description . a preferred compound of formula is formula . this treatment ameliorates , diminishes , actively treats , reverses or prevents any injury , damage or loss of articular cartilage or subchondral bone subsequent to said early stage of said degeneration

Detailed Description:
in order to further demonstrate the methods and compositions of the present invention , there are presented in the paragraphs which follow specific descriptive examples of typical procedures which may be employed in carrying out said methods . however , said examples are intended to be illustrative only and should not be taken as in any way a limitation of the present invention , for which purpose the present claims are appended hereto . the therapeutic effectiveness of ml3000 on the development of lesions in the experimental osteoarthritis ( oa ) dog model was studied . in particular , the action of ml3000 on the synthesis of collagenase - 1 ( mmp - 1 ) in cartilage and interleukin - 1β ( il - 1β ) in synovial membrane as well as on alkaline phophatase activity , igf - 1 production , osteocalcin release , peg 2 and upa activity in or by primary osteoblasts was determined . twenty - one adult crossbred dogs ( 2 - 3 years old ), weighing 20 - 25 kg each , were used in this study . surgical sectioning of the anterior cruciate ligament ( acl ) of the right knee through a stab wound was performed on all dogs as previously described ( 5 , 6 ). prior to surgery , the animals were anesthetized intravenously with pentobarbital sodium ( 25 mg / kg ) and intubated . following surgery , the dogs were kept at a housing farm where they were free to exercise in a large pen . the dogs were randomly separated into 3 treatment groups . group 1 ( n = 7 dogs ) was made up of dogs that received placebo ( encapsulated methylcellulose ) treatment ( oa dogs ); groups 2 and 3 ( n = 7 dogs per group ) received encapsulated ml3000 twice daily for 8 weeks at total doses of 2 . 5 and 5 mg / kg , respectively , beginning the day following surgery . the medication was administered 7 days / week throughout the duration of the study . all dogs were killed 8 weeks after surgery . all dogs in each experimental group completed the study . no clinical signs of drug toxicity , including those related to the gastrointestinal tract , were noted in the group of dogs treated with ml3000 . the level of daily activity was similar in all dogs from the 3 experimental groups , and there was no change in the body weight of the dogs during the study period . values are expressed as mean ± sem . statistical analysis was done using the mann - whitney u test . p values less than 0 . 05 were considered significant . immediately after killing , the right knee of each dog was dissected , placed on ice , and the synovial fluid aspirated . each knee was examined by 2 independent , blinded observers ( dvj , jcf ) for gross morphologic changes including the presence of osteophyte formation and cartilage lesions as previously described ( 5 , 6 ). the degree of osteophyte formation was graded by measuring the maximal width ( mm ) of the spur of each femoral condyle . the cartilage changes on the medial and lateral femoral condyles and tibial plateaus were graded separately under a dissecting microscope ( stereozoom ; bausch & amp ; lomb , rochester , n . y .). the area of the articular surface changes was measured and expressed in mm 2 . the depth of erosion was graded on a scale of 0 - 4 , as follows : 0 = surface appears normal , 1 = minimal fibrillation or a slight yellowish discoloration of the surface , 2 = erosion extended into superficial or middle layers , 3 = erosion extended into deep , layers , and 4 = erosion extended to the subchondral bone . osteophytes . in placebo - treated oa dogs , osteophytes were present in 93 % of the condyles , and their widths measured 4 . 50 ± 0 . 66 mm . in dogs treated with 2 . 5 mg / kg / day and 5 mg / kg / day ml3000 , osteophytes were present in 93 % and 86 % respectively . the width measurements of osteophytes in the two treated groups were marginally smaller compared with the placebo - treated oa group ( 3 . 57 ± 0 . 56 and 3 . 86 ± 0 . 66 respectively ), and these differences did not reach statistical significance . cartilage . in the placebo - treated oa dogs , cartilage lesions of moderately severe grade and size were present on both condyles and plateaus with more severe lesions on the plateaus ( fig1 ). there was a significant reduction in the size of lesions on the condyles and plateaus of both groups treated with ml3000 . in the 2 . 5 mg / kg / day group , the size of lesions on femoral condyles was lower by 39 % ( p & lt ; 0 . 05 ) and by 45 % ( p & lt ; 0 . 01 ) on tibial plateaus . in the group that received 5 mg / kg / day , the size of the lesions on femoral condyles was also reduced ( 61 %, p & lt ; 0 . 04 ), while the effect on tibial plateaus was about the same as those found in the 2 . 5 mg / kg / day group ( 54 %, p & lt ; 0 . 01 ). both concentrations of ml3000 significantly reduced the grade of lesions on tibial plateaus to about the same extent . although the lesions on femoral condyles of treated dogs showed a tendency towards a reduction in their grade , it did not reach statistical significance . synovial membrane . synovium from placebo - treated oa dogs was hypertrophic , demonstrated a red and yellowish discoloration and contained a large number of blood vessels . in dogs treated with ml3000 , the synovia was thinner , contained fewer blood vessels , and the discoloration was less intense compared with placebo - treated oa dogs . histologic evaluation was performed on sagittal sections of cartilage from the lesioned areas of each femoral condyle and tibial plateau as described ( 7 , 8 ). specimens were dissected , fixed in tissufix # 2 ( laboratoires gilles chaput , montréal , québec , canada ), and embedded in paraffin for histologic evaluation . serial sections ( 5 μm ) were stained with safranin o . the severity of the oa lesions was graded on scale of 0 - 14 , by 2 independent observers ( dj , jf ), using the histologic / histochemical scale of mankin et al ( 12 ). this scale evaluates the severity of oa lesions based on the loss of staining with safranin o ( scale 0 - 4 ), cellular changes ( scale 0 - 3 ), invasion of the tidemark by blood vessels ( scale 0 - 1 ), and structural changes ( scale 0 - 6 , where 0 = normal cartilage structure and 6 = erosion of the cartilage down to the subchondral bone ). this scoring system was based on the most severe histologic changes within each cartilage section . representative specimens of synovial membrane from the gutters of the medial and lateral knee compartments were also dissected from underlying tissue . the specimens were fixed in tissufix # 2 , embedded in paraffin , sectioned ( 5 μm ), and stained with hematoxylin and eosin . two synovial membrane specimens from each compartment were examined , serial selections were made throughout the specimens , and each one scored separately . the highest score from each specimen was recorded . the average was calculated and considered as a unit for the whole knee . the severity of synovitis was graded on a scale of 0 - 10 ( 13 ) by 2 independent observers ( dvj , jcf ) adding the scores for 3 histologic criteria : synovial lining cell hyperplasia ( scale 0 - 2 ), villous hyperplasia ( scale 0 - 3 ), and degree of cellular infiltration by mononuclear and polymorphonuclear cells ( scale 0 - 5 ). cartilage . cartilage from placebo - treated oa dogs presented morphologic changes including fibrillation and fissures , hypercellularity and cloning , and a loss of safranin o staining . histologic scores for the lesions on the plateaus were slightly more severe than for those on the condyles ( fig4 ). in the ml3000 - treated dogs , the lesions on the condyles were less severe compared with the placebo - treated oa dogs , and statistical significance was obtained in the group treated with the highest dosage of the drug ( 5 mg / kg / day ). this reduction in the histologic score was largely due to a decrease in severity of structural changes and loss of safranin o staining . on tibial plateaus , a statistically significant decrease in the severity of lesions ( p & lt ; 0 . 002 and p & lt ; 0 . 02 , respectively ) was obtained with both doses of ml3000 ( 2 . 5 and 5 mg / kg / day ). this results from a decrease in the loss of safranin o staining and severity of structural changes in addition to a reduction in cell cloning . synovial membrane . synovia from placebo - treated oa dogs was thick , had numerous villi , and showed synovial lining cell hyperplasia . in the ml3000 - treated dogs , there was a significant reduction in villous hyperplasia in the 5 mg / kg / day group . synovial fluid taken at the time of sacrifice was centrifuged ( 14000 g , 15 minutes , 4 ° c . ), and supernatants used for pge 2 determination . the level of pge 2 was determined using a specific enzyme immunoassay ( eia ) ( cayman chemical , ann arbor , mich .). synovial fluids from placebo - treated oa dogs contained a high level of pge 2 ( 652 . 8 ± 149 . 9 pg / ml ). treatment with ml3000 at 2 . 5 and 5 mg / kg / day markedly decreased this level ( fig5 ). representative specimens of synovial membrane from all dogs were aseptically dissected from underlying tissue . the specimens were rinsed several times in dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dmem ; gibco - brl life technologies , burlington , ontario , canada ), and 150 mg of tissue incubated ( duplicate ) for 48 hours at 37 ° c . in a humidified atmosphere of 5 % co 2 / 95 % air in dmem medium in the presence of 1 μg / ml lipopolysaccharide ( lps ; sigma - aldrich canada , oakville , ontario , canada ). the ltb 4 was extracted from cultured synovium explants as follows : membranes were homogenized in eia buffer ( 1 m phosphate , ph 7 . 4 , containing 1 % bsa , 4 m nacl , 10 mm edta and 0 . 1 % sodium azide ) and centrifuged ( 30000 g , 20 minutes , 4 ° c .). supernatants were collected and the levels of ltb 4 determined using eia ( cayman chemical ). cultured synovium from oa dogs treated with 1 μg / ml lps contained a significant amount of ltb 4 ( 2 . 48 ± 0 . 69 pg / mg tissue ). both groups treated with ml3000 demonstrated a statistically significant reduction in ltb 4 levels ( fig5 ). cartilage and synovial membrane specimens were processed for immunohistochemical analysis as previously described ( 7 , 14 ). briefly , specimens were fixed in 4 % neutral buffered formalin for 24 hours , then embedded in paraffin . sections ( 5 μm ) of paraffin - embedded specimens were placed on superfrost plus slides ( fisher scientific , nepean , ontario , canada ), deparaffinized in toluene , dehydrated in a graded series of ethanol , and preincubated with chondroitinase abc ( 0 . 25 units / ml ) in phosphate buffered saline ( pbs ; sigma - aldrich canada ) for 60 minutes at 37 ° c . after this , the specimens were washed in pbs , then again in 0 . 3 % hydrogen peroxide / pbs for 30 minutes . slides were further incubated with a blocking solution ( dako diagnostics , mississauga , ontario , canada ) and 5 % skim milk for 60 minutes , blotted and then overlaid with the primary monoclonal antibody against collagenase - 1 ( 100 μg / ml , dilution 1 : 500 , oncogene research products , cambridge , mass .) ( cartilage ) or the primary antibody against human il - 1β ( 1 μg / ml , dilution 1 : 50 ; biosource international , camarillo , calif .) ( synovial membrane ) for 18 hours at room temperature in a humidified chamber . each slide was washed 3 times in pbs ( ph 7 . 4 ) and stained using the avidin - biotin complex method ( vectastain abc kit ; dako diagnostics canada ). this method entails incubation in the presence of the biotin - conjugated secondary antibody for 30 minutes at room temperature followed by the addition of the avidin - biotin - peroxidase complex for 30 minutes . all incubations were carried out in a humidified chamber and the colour developed with a 3 , 3 ′- diaminobenzidine ( dako diagnostics canada ) containing hydroxide peroxide . slides were counterstained with neutral red ( cartilage ) or hematoxylin / eosin ( synovium ). to determine the specificity of staining , 3 control procedures were employed according to the same experimental protocol : 1 ) use of absorbed immune serum ( 1 hour , 37 ° c .) with a 20 - fold molar excess of recombinant collagenase - 1 or il - 1β ; 2 ) omission of the primary antibody ; and 3 ) substitution of the primary antibody with an autologous preimmune serum . the purified antigens used in our study were human recombinant collagenase - 1 ( oncogene research products ) or human recombinant il - 1β ( genzyme , cambridge , mass ., usa ). several sections were made from each block of cartilage , and slides from each specimen , were processed for immunohistochemical analysis . each section was examined under a light microscope ( leitz orthoplan ; wild leitz , st . laurent , quëbec , canada ) and photographed with kodak ektachrome 64 asa film ( kodak , rochester , n . y .). cartilage ( fig3 ). in placebo - treated dogs , a large number of chondrocytes in the superficial layers of cartilage specimens stained positive for collagenase - 1 on both the condyles and plateaus . in the ml3000 - treated oa dogs , both condyles and plateaus presented a significant decrease in the chondrocyte cell score for collagenase - 1 — the effect of which was slightly more pronounced in the group treated with the highest dosage of the drug . synovial membrane ( fig3 ). the examination of the synovium samples from placebo - treated oa dogs showed the presence of a large number of lining cells staining strongly positive for il - 1β . in dogs treated with ml3000 , there was a dose - dependent and significant decrease in the number of cells showing positive staining for il - 1β . cartilage . quantification of the different antigens in cartilage was done using a published method ( 7 , 14 ). the presence of the antigen was estimated by determining the number of chondrocytes staining positive in the upper ( superficial and upper intermediate layers ) zone of cartilage . in this zone , cartilage was divided into 3 microscopic fields ( x 40 ; leitz diaplan ), and averaged . for each arthritic specimen , it was ensured prior to evaluation that an intact cartilage surface could be detected and used as a marker for validation of morphometric analysis . the total number of chondrocytes and the number of chondrocytes staining positive for the specific antigen were determined . the final results were expressed as the percentage of chondrocytes staining positive for the antigen ( cell score ) with the maximum score being 100 %. each slide was subjected to double blind evaluation resulting in a variation of & lt ; 5 %. the data obtained from the medial and lateral condyles and tibial plateaus were considered as independent for the purpose of statistical analysis . synovial membrane . for synovial membrane analysis , a cell score of the different specimens was determined for each section using our published method ( 8 ). each specimen was divided into 5 microscopic fields ( x 40 ) at the synovial lining level . the percentage of cells staining positive for the specific antigen was evaluated in each field as described above for cartilage . cell count scores were given separately for the synovial lining cells and mononuclear cell infiltrate with the maximum for each area being 100 %. the proximal end of the tibia was removed as below - described , rinsed in a cold physiological saline solution , and placed on ice prior to and throughout dissection . medial tibial plateaus was extracted to prepare explants and primary bone cell cultures ; no marginal cortical bone tissue was included . the overlying cartilage was first removed from the tibial plateaus , and plug explants were dissected out exclusively from the midportion of the medial plateau . the trabecular bone tissue was then dissected away from the subchondral bone plate . all manipulations were performed under a magnifying microscope to ensure complete removal of cartilage and trabecular bone . the samples were used to prepare primary cell cultures as described in ( 16 ), with minor modifications . bone samples were cut into small pieces ( 2 mm 2 ) prior to their sequential digestion in the presence of 1 mg / ml type i collagenase ( sigma ) in ham &# 39 ; s f - 12 / dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dmem ; sigma ) without serum , at 37 ° c . for 20 , 20 , and 240 minutes . this treatment removed both adherent and remaining bone marrow cells from the cortical bone pieces . after washing with the same medium , the digested bone pieces were cultured in bgj medium containing 20 % fetal bovine serum ( fbs ; wisent , st . bruno , quebec , canada ). this medium was replaced every 2 days until cells are observed in the petri dishes , at which time the culture medium was replaced with fresh medium containing 10 % fbs . at confluence , cells were passaged once at a ratio of 25 , 000 cells / cm 2 and are grown in 24 - well plates ( falcon , lincoln park , n . j .) for 5 days prior to assay . cells obtained under these culture conditions showed an osteoblast - like cell phenotype , as noted in ( 16 ). conditioning is performed for the last 2 days of culture , in the presence or absence of 50 nm 1 , 25 ( oh ) 2 d 3 ( 1 , 25 - dihydroxyvitamin d ) for maximal stimulation , in ham &# 39 ; s f - 12 / dmem containing 2 % charcoal - stripped fbs , which yields maximal stimulation of alkaline phosphatase activity and osteocalcin secretion , as noted ( 16 ). the medium was collected at the end of the incubation and frozen at − 80 ° c . prior to assay . cells were then washed twice with phosphate buffered saline ( pbs ), ph 7 . 4 , and solubilized in alkaline phosphatase buffer ( 100 mm glycine , 1 mm mgcl 2 , 1 mm zncl 2 , 1 % triton x - 100 ; ph 10 . 5 ) for 60 minutes with agitation at 4 ° c . osteocalcin release was measured in conditioned ham &# 39 ; s f - 12 / dmem ( 1 : 1 ) prepared for the last 2 days of culture of osteoblast - like cells as described in ( 16 ), containing 2 % charcoal - treated fbs , and in the presence of 50 nm 1 , 25 ( oh ) 2 d 3 or vehicle ( 0 . 1 % ethanol ). nascent osteocalcin was determined using a specific enzyme immunoassay ( biomedical technologies , stoughton , mass .). the detection limit of this assay is 0 . 5 ng / ml , and 2 % charcoal - treated fbs contains & lt ; 0 . 1 ng / ml osteocalcin . results are shown in fig6 . cellular alkaline phosphatase activity was determined , on cells used for osteocalcin release , as the release of p - nitrophenol hydrolyzed from p - nitrophenyl phosphate ( 12 . 5 mm final concentration ) at 37 ° c . for 30 minutes after solubilizing the cells in alkaline phosphatase buffer as above - described . alkaline phosphatase was determined immediately on aliquots . protein determination was performed by the bicinchoninic acid method described in smith , p . k . ; krohn , r . i . ; hermanson , g . t . ; mallia , a . k . ; gartner , f . h . ; provenzano , m . d . ; et al . ; “ measurement of protein using bicinchoninic acid ”, anal biochem , 150 , 1985 , 76 - 85 . results are shown in fig7 . for evaluation of upa and igf - 1 , the conditioned media from confluent osteoblast - like cells fed with ham &# 39 ; s f - 12 / dmem , without pbs , but containing 1 % insulin - transferrin - selenium mix ( its , sigma ) for the last 2 days of culture . first , upa levels were determined by specific enzyme - linked immunosorbent assay ( elisa ; american diagnostica , greenwich , conn .). there was then used the procedure described in leprince , p . ; rogister , b . ; moonen , g . a . ; “ colorimetric assay for the simultaneous measurement of plasminogen activators and plasminogen ativator inhibitors in serum - free conditioned media from cultured cells ”, anal biochem , 177 , 1989 , 341 - 346 , to determine the activity of upa via the hydrolysis of the specific substrate dl - val - leu - arg - p - nitroanilide ( sigma ), which releases p - nitroaniline that can be detected at 405 nm . pai - 1 levels are determined by elisa , using materials available from american diagnostica ( greewich , conn .). igf - 1 was determined using a high - sensitivity elisa ( diagnostic systems laboratories , webster , tex .) that does not cross - react with insulin . internal control studies are performed with the media alone containing 1 % its , and any values obtained should be below the limit of detection . for the conditioned medium of cell culture samples , 3 or 4 supernatants are pooled , lyophilized , and then reconstituted in pbs buffer , ph 7 . 4 . samples are then treated according to the method described in mohan , s . ; bautista , c . m . ; herring , s . j . ; linkhart , t . a . ; baylink , d . j . ; “ development of valid methods to measure insulin - like growth factors - i and - ii in bone cell - conditioned medium , endocrinology , 126 , 1990 , 2534 - 42 . results are shown in fig8 and 9 . pge2 in primary osteoblasts was determined essentially in the same manner as described above for synovial fluid . results are shown in fig1 . from the above results it can be concluded that ml3000 , a balanced dual inhibitor of cox / 5 - lo , can significantly reduce the development of early experimental oa at the same time as inhibiting in vivo the production of pge 2 and ltb 4 . the protective effect of the drug is believed largely related to the marked inhibition of major oa pathophysiological pathways — namely the excess synthesis of il - 1β and collagenase - 1 . some of these effects appeared to be linked to the inhibition of the excess production of ltb 4 . 1 . pelletier j p , martel - pelletier j , howell d s . etiopathogenesis of osteoarthritis . in : koopman w j , editor . arthritis & amp ; 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40 : 1653 - 61 . 15 . huskisson e c , berry h , gishen p , jubb r w , whitehead j . effects of antiinflammatory drugs on the progression of osteoarthritis of the knee . link study group . longitudinal investigation of nonsteroidal antiinflammatory drugs in knee osteoarthritis . j rheumatol 1995 ; 22 : 1941 - 6 . 16 . lajeunesse , d . ; busque , l . ; ménard , p . ; brunette , m . g . ; bonny , y . ; “ demonstration of an osteoblast defect in two cases of human malignant osteoporosis : correction of the phenotype after bone marrow transplant ”; j . clin invest , 98 , 1996 , 1835 - 1842 .