Richard Melton

Inhibition of interleukin‐1α‐induced cartilage oligomeric matrix protein degradation in bovine articular cartilage by matrix metalloproteinase inhibitors: Potential role for matrix metalloproteinases in the generation of cartilage oligomeric matrix protein fragments in arthritic synovial fluid

OBJECTIVE To determine whether matrix metalloproteinases (MMPs) degrade cartilage oligomeric matrix protein (COMP) to produce fragments similar to those found in synovial fluid (SF) from patients with arthritis. METHODS COMP fragments were generated in vitro by treating (a) bovine articular cartilage with interleukin-1alpha (IL-1alpha), (b) purified bovine COMP with MMPs, and (c) articular cartilage with MMPs. The fragments generated in each case were analyzed by Western blot, using an antibody to the C-terminal heptadecapeptide of COMP. RESULTS IL-1alpha stimulation of cartilage resulted in a fragmentation of COMP, which was inhibited by MMP inhibitors CGS 27023A and BB-94. Isolated, recombinant MMPs rapidly degraded purified COMP, as well as COMP residing in cartilage. Several COMP fragments produced in vitro had similar electrophoretic mobility to those in SF of patients with arthritis. CONCLUSION MMPs may contribute to the COMP fragments found in vivo. Quantitation of MMP-specific fragments may be useful in the evaluation of MMP inhibitors in patients with arthritis.

Potent and selective 2-naphthylsulfonamide substituted hydroxamic acid inhibitors of matrix metalloproteinase-13.

The matrix metalloproteinase enzyme MMP-13 plays a key role in the degradation of type II collagen in cartilage and bone in osteoarthritis (OA). An effective MMP-13 inhibitor would provide a disease modifying therapy for the treatment of arthritis, although this goal still continues to elude the pharmaceutical industry due to issues with safety. Our efforts have resulted in the discovery of a series of hydroxamic acid inhibitors of MMP-13 that do not significantly inhibit MMP-2 (gelatinase-1). MMP-2 has been implicated in the musculoskeletal side effects resulting from pan-MMP inhibition due to findings from spontaneously occurring human MMP-2 deletions. Analysis of the SAR of hundreds of previously prepared hydroxamate based MMP inhibitors lead us to 2-naphthylsulfonamide substituted hydroxamates which exhibited modest selectivity for MMP-13 versus MMP-2. This Letter describes the lead optimization of 1 and identification of inhibitors exhibiting >100-fold selectivity for MMP-13 over MMP-2.

Design and synthesis of thiol containing inhibitors of matrix metalloproteinases

A series of thiol containing derivatives was prepared. Several of these compounds were found to inhibit matrix metalloproteinases 1, 3, and 9 with selectivity towards 3 and 9. Compounds 15, 20, and 22 were administered to rats orally at 75 mumol/kg. Drug levels of compounds 20 and 22 in the plasma were found to exceed the IC50 values for MMP 3 and 9 four hours after administration.

Matrix Metalloproteinase Inhibitor CGS 27023A Protects COMP and Proteoglycan in the Bovine Articular Cartilage but not the Release of Their Fragments from Cartilage after Prolonged Stimulation in Vitro with IL‐1α

In arthritis, the metalloproteinases (MP) such as stromelysin-1, collagenase-1 and -3, MT1-MMP, 92-kDa gelatinase, and as yet unidentified MP aggrecanase are thought to play a role in the degradation of proteoglycans (PG), cartilage oligomeric matrix protein (COMP), and type II collagen, the components of articular cartilage. It is not yet clear which of these MP plays a dominant role in the degradation of cartilage. The matrix metalloproteinase inhibitors CGS 27023A 1 and BB-94 2 are potent inhibitors of several MPs, but only BB-94 inhibits aggrecanase activity. 3 Here, we investigated whether inhibition of these two activities is needed in protecting the cartilage components PG and COMP. We used them in an IL-1 α –induced bovine cartilage degradation assay 4 and determined their effect on the release of these cartilage components from cartilage and also their retention in the cartilage after prolonged IL-1 stimulation of cartilage.

A stromelysin assay for the assessment of metalloprotease inhibitors on human aggregated proteoglycan

Human proteoglycan was aggregated to an immobilized hyaluronan solid phase on a 96-well ELISA plate. This complex was then degraded by recombinant human stromelysin. The remaining proteoglycan fragments were detected using a monoclonal antibody probe directed against the chondroitin sulfate (CS) region of the core protein. Stromelysin degraded the aggregate in a time and dose dependent manner as reflected by the loss of the CS epitope. Assay sensitivity was 0.125 U/well with total loss of the CS epitope occurring at 4 U/well.o-phenanthroline (IC50=52 μM) and U24522 (IC50=9 μM) inhibted degradation, while phosphoramidon did not. Serine and cysteine protease inhibitors had no effect. A comparative analysis of this assay with a reference method, substance P assay, gave similar inhibitor profiles. The use of aggregated human proteoglycan (native conformation) as a substrate, may better reflect how stromelysin inhibitors behave in the presence of complex substrates such as cartilage matrix.

Intra-articular injection of stromelysin into rabbit knees as a model to evaluate matrix metalloprotease inhibitors

Mat r ix me ta l lopro teases ( M M P s ) such as s t romelysin , col lagenase and gela t inase are e levated in the synovial fluids o f pa t ien ts wi th arthri t is . The ext racel lu lar ma t r ix o f car t i lage is suscept ible to deg rada t ion by these proteases . A desi rable p r o p e r t y o f a ma t r ix me ta l lop ro tease inh ib i to r ( M M P I s ) is one tha t inhibi ts ma t r ix me ta l lopro teases f rom degrad ing car t i lage in the joint . W e repor t here our f indings of a mode l tha t tests the d u r a t i o n o f ac t ion and oral b ioava i lab i l i ty o f M M P I s to inhibi t M M P s in the synovia l fluid.

Elevation of synovial plasminogen activator activity after injection of interleukin-1α into rabbit knee joint

We investigated production of plasminogen activator (PA) and cartilage degradation induced by injection of recombinant human interleukin-1 (rhIL-1α) in rabbit knees. Rabbits were injected intra-articularly (i.a.) with 100 ng rhIL-1α and necropsied at 0, 3, 6, 18 and 54 h and synovial lavage and articular cartilage were collected. PA activity in the joint lavage was measured using Z-Lys-thiobenzyl ester as a substrate. Cartilage degradation was assessed by quantitating sulfated glycosaminoglycan (S-GAG) to hydroxyproline (Hyp) and appearance of keratan sulfate (KS) in synovial lavage by and ELISA. The PA activity in the lavage of IL-1 injected knees at 3, 6, and 18 h was elevated 8 to 10 fold compared to vehicle controls. At 54 h the activity declined to approximately one third of that seen at the earlier time points. KS in the joint lavage was highest at 18 h, suggesting proteoglycan degradation. The maximal loss of cartilage proteoglycan (S-GAG/Hyp) occurred by 54 h. These observations demonstrate that i.a. injection of IL-1 stimulated the production of PA activity within the rabbit joint. Since elevation of PA activity is followed by cartilage degradation, we investigated effect of anti-inflammatroy agents on PA activity and cartilage degradation in this model. We found that triamcinolone, indomethacin and dexamethasone were able to suppress PA activity but not the cartilage degradation. These observations suggest that in this model of cartilage degradation suppression of PA is not sufficient to inhibit cartilage degradation.