Structure-activity relationships of a lead hydroxamic acid inhibitor of recombinant human stromelysin were systematically defined by taking advantage of a concise synthesis that allowed diverse functionality to be explored at each position in a template. An ex vivo rat model and an in vivo rabbit model of stromelysin-induced cartilage degradation were used to further optimize these analogs for oral activity and duration of action. The culmination of these modifications resulted in CGS 27023A, a potent, orally active stromelysin inhibitor that blocks the erosion of cartilage matrix.

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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 therefore be a novel disease modifying therapy for the treatment of arthritis. Our efforts have resulted in the discovery of a series of carboxylic acid inhibitors of MMP-13 that do not significantly inhibit the related MMP-1 (collagenase-1) or tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE). It has previously been suggested (but not proven) that inhibition of the latter two enzymes could lead to side effects. A promising carboxylic acid lead 9 was identified and a convergent synthesis developed. This paper describes the optimization of 9 and the identification of a compound 24f for further development. Compound 24f is a subnanomolar inhibitor of MMP-13 (IC(50) value 0.5 nM and K(i) of 0.19 nM) having no activity against MMP-1 or TACE (IC(50) of >10000 nM). Furthermore, in a rat model of MMP-13-induced cartilage degradation, 24f significantly reduced proteoglycan release following oral dosing at 30 mg/kg (75% inhibition, p < 0.05) and at 10 mg/kg (40% inhibition, p < 0.05).

Cytokines, interleukin-1 (IL-I), tumor necrosis factor a, and the neurotransmitter, substance P, have been implicated in the pathogenesis of arthritis because they stimulate synovial cells to secrete prostaglandin E, and collagenase in vitro. We investigated in vivo changes in intraarticular substance P and the degradation of cartilage proteoglycan in response to intraarticular cytokine injections in rabbits. Twenty-four hours after a single injection of 10 ng, 30 ng, or 100 ng of recombinant human &la (rHuIL-la) per joint, the mean f SEM levels of substance P detected in the cell-free joint lavage fluid were 250 & 67 fmoles, 480 2 60 fmoles, and 530 & 130 fmoles (n = 4-9, respectively. The level of substance P in the contralateral knees injected with diluent was 58 -c 8 fmoles (n = 12). The level of substance P had increased by 2 hours after IL-1 injection and remained elevated in the joint 48 hours after injection. Cytokineinduced proteoglycan depletion was also time-and dosedependent. Proteoglycan concentrations in articular cartilage dissected from the weight-bearing condyles were calculated as the ratio of sulfated glycosaminoglycan measured using 1,9-dimethyImethylene blue:hydroxyproline. After 48 hours, 10 ng, 30 ng, or 100 ng of rHuIL-la per joint decreased proteoglycan levels by 9 f 4%, 14 f 4%, and 21 2 3% (n = a), respectively. Likewise, the injection of recombinant human

Cartilage degeneration in osteoarthritis is initiated by a loss of proteoglycan. Intra-articular injection of papain causes a reversible loss of proteoglycan in rabbit knees. Rabbits were scanned with magnetic resonance imaging (MRI), using a 1.5T Signa superconducting magnet with 3 inch surface coil. Spin echo sequences were performed in the coronal and sagittal planes at 0, 24, 48, and 72 h after intra-articular injection of papain to obtain T1, proton density, and T2-weighted images. Cartilage proteoglycan content was measured biochemically and histochemically. Reduced articular cartilage thickness in the MR images of papain-treated knees corresponded to changes in cartilage proteoglycan content.

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Intra-articular (i.a.) injection of papain causes a reversible loss of proteoglycan in intact rabbit knees. Twelve rabbits were scanned with magnetic resonance imaging (MRI) at 0, 24, 48 and 72 hours after 5 units of papain i.a. in a 1.5 Tesla Signa with a three inch surface coil using spin echo sequence. Total cartilage thickness in proton density images was 1.08 +/- 0.09 mm prior to papain injection. The magnetic resonance images showed a reduction in articular cartilage thickness in papain-treated rabbit femurs at 24 hours to 0.69 +/- 0.18 mm and partial restoration by 72 hours to 0.77 +/- 0.21 mm.

Osteoarthritis was surgically induced in mature male Dutch Belted rabbits by sectioning the fibular collateral and sesamoid ligaments and removal of the anterior horn of the lateral meniscus. The site of surgical intervention was detectable by MRI. Histopathologic analysis revealed severe focal cartilage lesions on opposing surfaces of the tibia and femur. Histology of cartilage adjacent to the osteoarthritic lesions appeared normal. In another animal model, arthritis was induced by immunization against ovalbumin followed by intra-articular injection of ovalbumin. MRI of immune arthritic rabbit knees showed accumulation of synovial fluid and cartilage degradation. Histopathology was characterized by vascular necrosis of the synovium and depletion of cartilage proteoglycan. MRI can be used to non-invasively follow the therapeutic effects of drug treatment on synovial inflammation and cartilage degradation in rabbit knees.