E.A. Morris

Prevention of Cartilage Degeneration in a Rat Model of Osteoarthritis by Intraarticular Treatment With Recombinant Lubricin

OBJECTIVE Lubricin, also referred to as superficial zone protein and PRG4, is a synovial glycoprotein that supplies a friction-resistant, antiadhesive coating to the surfaces of articular cartilage, thereby protecting against arthritis-associated tissue wear and degradation. This study was undertaken to generate and characterize a novel recombinant lubricin protein construct, LUB:1, and to evaluate its therapeutic efficacy following intraarticular delivery in a rat model of osteoarthritis (OA). METHODS Binding and localization of LUB:1 to cartilage surfaces was assessed by immunohistochemistry. The cartilage-lubricating properties of LUB:1 were determined using a custom friction testing apparatus. A cell-binding assay was performed to quantify the ability of LUB:1 to prevent cell adhesion. Efficacy studies were conducted in a rat meniscal tear model of OA. One week after the surgical induction of OA, LUB:1 or phosphate buffered saline vehicle was administered by intraarticular injection for 4 weeks, with dosing intervals of either once per week or 3 times per week. OA pathology scores were determined by histologic analysis. RESULTS LUB:1 was shown to bind effectively to cartilage surfaces, and facilitated both cartilage boundary lubrication and inhibition of synovial cell adhesion. Treatment of rat knee joints with LUB:1 resulted in significant disease-modifying, chondroprotective effects during the progression of OA, by markedly reducing cartilage degeneration and structural damage. CONCLUSION Our findings demonstrate the potential use of recombinant lubricin molecules in novel biotherapeutic approaches to the treatment of OA and associated cartilage abnormalities.

Elevated aggrecanase activity in a rat model of joint injury is attenuated by an aggrecanase specific inhibitor

OBJECTIVE To evaluate aggrecanase activity after traumatic knee injury in a rat model by measuring the level of aggrecanase-generated Ala-Arg-Gly-aggrecan (ARG-aggrecan) fragments in synovial fluid, and compare with ARG-aggrecan release into joint fluid following human knee injury. To evaluate the effect of small molecule inhibitors on induced aggrecanase activity in the rat model. METHOD An enzyme-linked immunosorbent assay (ELISA) was developed to measure ARG-aggrecan levels in animal and human joint fluids. A rat model of meniscal tear (MT)-induced joint instability was used to assess ARG-aggrecan release into joint fluid and the effects of aggrecanase inhibition. Synovial fluids were also obtained from patients with acute joint injury or osteoarthritis and assayed for ARG-aggrecan. RESULTS Joint fluids from human patients after knee injury showed significantly enhanced levels of ARG-aggrecan compared to uninjured reference subjects. Similarly, synovial fluid ARG-aggrecan levels increased following surgically-induced joint instability in the rat MT model, which was significantly attenuated by orally dosing the animals with AGG-523, an aggrecanase specific inhibitor. CONCLUSIONS Aggrecanase-generated aggrecan fragments were rapidly released into human and rat joint fluids after injury to the knee and remained elevated over a prolonged period. Our findings in human and preclinical models strengthen the connection between aggrecanase activity in joints and knee injury and disease. The ability of a small molecule aggrecanase inhibitor to reduce the release of aggrecanase-generated aggrecan fragments into rat joints suggests that pharmacologic inhibition of aggrecanase activity in humans may be an effective treatment for slowing cartilage degradation following joint injury.

Tenascin-C induces inflammatory mediators and matrix degradation in osteoarthritic cartilage

BackgroundTenascin-C (TN-C) is an extracellular matrix glycoprotein that is involved in tissue injury and repair processes. We analyzed TN-C expression in normal and osteoarthritic (OA) human cartilage, and evaluated its capacity to induce inflammatory and catabolic mediators in chondrocytes in vitro. The effect of TN-C on proteoglycan loss from articular cartilage in culture was also assessed.MethodsTN-C in culture media, cartilage extracts, and synovial fluid of human and animal joints was quantified using a sandwich ELISA and/or analyzed by Western immunoblotting. mRNA expression of TN-C and aggrecanases were analyzed by Taqman assays. Human and bovine primary chondrocytes and/or explant culture systems were utilized to study TN-C induced inflammatory or catabolic mediators and proteoglycan loss. Total proteoglycan and aggrecanase -generated ARG-aggrecan fragments were quantified in human and rat synovial fluids by ELISA.ResultsTN-C protein and mRNA expression were significantly upregulated in OA cartilage with a concomitant elevation of TN-C levels in the synovial fluid of OA patients. IL-1 enhanced TN-C expression in articular cartilage. Addition of TN-C induced IL-6, PGE2, and nitrate release and upregulated ADAMTS4 mRNA in cultured primary human and bovine chondrocytes. TN-C treatment resulted in an increased loss of proteoglycan from cartilage explants in culture. A correlation was observed between TN-C and aggrecanase generated ARG-aggrecan fragment levels in the synovial fluid of human OA joints and in the lavage of rat joints that underwent surgical induction of OA.ConclusionsTN-C expression in the knee cartilage and TN-C levels measured in the synovial fluid are significantly enhanced in OA patients. Our findings suggest that the elevated levels of TN-C could induce inflammatory mediators and promote matrix degradation in OA joints.

089 SELECTIVE AGGRECANASE INHIBITION IS DISEASE MODIFYING AND PAIN ALLEVIATING IN A RAT MENISCAL TEAR MODEL OF OSTEOARTHRITIS

Results: Following ACL section, limb impairment rapidly developed in all dogs, with PVF and GCA values dropping by week 4. After this acute disability phase, the dogs underwent a slow remission phase that was still incomplete by week 26. Prediction of PVF change was best estimated (R2=0.96) from GCA and BMLSPGR (p<0.0001), particularly during the phase of acute disability, whereas cartilage defect was more influential (R2=0.97) during the remission phase (p=0.0051) (week 8 to week 26). The other joint structural damages had insignificant effect on limb impairment and recovery. Both BML-SPGR and cartilage defect adversely affected the recovery in PVF, with mutually independent effects. Similar to ln_PVF, GCA showed an acute drop by week 4, followed afterwards by a remission phase (p<0.0001), which attained baseline values by week 26 (p=0,46). The time-course of GCA recovery was negatively affected by cartilage defect, and was positively affected by joint effusion (p<0.0001 for both variables). Conclusions: In recent human OA studies, pain and limb impairment were related mostly to BML and joint effusion. Our data from dogs with experimental OA confirms the role of BML and joint effusion on limb function. On one hand, BML and cartilage defect hinder the recovery of PVF. On the other hand, joint effusion positively influences GCA, supporting the existence of alleviating mechanisms that oppose to abnormal biomechanics. This study also clarifies the role of cartilage and other joint structural components in OA: cartilage volume and osteophytosis act as confounding factors with negligible role in limb impairment. Such structure/function modeling opens promising avenues for assessing outcome of disease-modifying OA drugs at the preclinical development stage.

Continued exploration of biphenylsulfonamide scaffold as a platform for aggrecanase-1 inhibition.

Design, synthesis and structure-activity relationship of a series of biphenylsulfonamido-3-methylbutanoic acid based aggrecanase-1 inhibitors are described. In addition to robust aggrecanase-1 inhibition, these compounds also exhibit potent MMP-13 activity. In cell-based cartilage explants assay compound 48 produced 87% inhibition of proteoglycan degradation at 10 μg/mL. Good pharmacokinetic properties were demonstrated by 46 with a half-life of 6h and bioavailability of 23%.

A13 INTRAARTICULAR LUBRICIN SUPPLEMENTATION IS CHONDROPROTECTIVE AND PREVENTS CARTILAGE DEGENERATION IN EXPERIMENTAL OSTEOARTHRITIS

12 weeks post-BTM while Group C were sacrificed 12 [n = 6], 24 [n = 6] and 52 [n = 6] weeks post-BTM. At necropsy, both medial compartments of BTM joints were scored by 2 blinded observers for AC lesions and osteophytes (OP) using a 0−4 scale. Synovial tissue and a 5mm wide coronal osteochondral slice were removed from the mid-line of the femur and tibia and processed for histochemical and histomorphometric analyses using published methods. Intact patellae were used for topographical biomechanical AC indentation studies. Results: Gross morphological scores 12 wks post BTM showed a dosedependent effect of MPC on AC integrity and OP formation; 100 mil MPC emerging as the most effective chondroprotective dose relative to HA alone. Total AC score ratios (HA+MPC)/(HA) showed 100>150>25=10 while OP ratios were 100>25>10>150 mil MPC. Statistically significant (SS) lower scores were observed for total femoral & tibial AC (p = 0.019) and total AC and OP (p = 0.009) for Group C MPC joints compared HA alone. Histomorphometric analysis of Group C MPC+HA tibial plateaus revealed that AC were thicker than the corresponding HA-AC in the middle (p = 0.057) and outer regions (p = 0.028); for all regions (p = 0.01). The mean phase lag for the patellae AC of Group C MPC injected joints was significantly lower than the contralateral patella AC (p = 0.002). Mean modified Mankin scores for AC sections from Group C MPC+HA joints were less than corresponding HA sections but were not SS. There was no evidence of synovial histopathology modulation. The chondroprotective effects observed for the 100 mil MPC injected joints diminished with time; the positive effects noted at 12 and 24 weeks BTM being lost by 52 weeks. Conclusions: This is the first report, as far as we are aware, of a beneficial therapeutic effect of allogenic Stro-3+ MPC on cartilage integrity in a model of early OA. MPC/MSC are known to release growth factors and cytokines and also suppress the production of TNF-alpha by other cells, while up-regulating anti-inflammatory cytokines (eg. Il-4, Il-10). These paracrine activities of MPC could stimulate chondrocyte biosynthesis of new matrix but also attenuate local production and activity of catabolic mediators. The finding in this study that 100 million MPC were chondroprotective was consistent with such a mechanism of action.