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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 6 h and bioavailability of 23%.

Mass spectrometry-based proteomic analyses performed on cartilage tissue extracts identified the serine protease HtrA1/PRSS11 as a major protein component of human articular cartilage, with elevated levels occurring in association with osteoarthritis. Overexpression of a catalytically active form of HtrA1, but not an active site mutant (S328A), caused a marked reduction in proteoglycan content in chondrocyte-seeded alginate cultures. Aggrecan degradation fragments were detected in conditioned media from the alginate cultures overexpressing active HtrA1. Incubation of native or recombinant aggrecan with wild type HtrA1 resulted in distinct cleavage of these substrates. Cleavage of aggrecan by HtrA1 was strongly enhanced by HtrA1 agonists such as CPII, a C-terminal hexapeptide derived from the C-propeptide of procollagen IIα1 (i.e. chondrocalcin). A novel HtrA1-susceptible cleavage site within the interglobular domain (IGD) of aggrecan was identified, and an antibody that specifically recognizes the neoepitope sequence (VQTV356) generated at the HtrA1 cleavage site was developed. Western blot analysis demonstrated that HtrA1-generated aggrecan fragments containing the VQTV356 neoepitope were significantly more abundant in osteoarthritic cartilage compared with cartilage from healthy joints, implicating HtrA1 as a critical protease involved in proteoglycan turnover and cartilage degradation during degenerative joint disease.

Summary Objective Compare the expression and regulation of nuclear receptors (NRs) in osteoarthritic and normal human articular cartilage. Method The transcriptional levels of 48 NRs and additional related proteins were measured in mRNA from human articular cartilage from subjects with osteoarthritis (OA) and compared to samples from subjects without OA, using microarrays, individual quantitative reverse transcriptase polymerase chain reaction assays, and a custom human NR TaqMan ® Low Density Array (TLDA). The functional effect of liver X receptor (LXR) activity in cartilage was studied by measuring proteoglycan (PG) synthesis and degradation in articular cartilage explant cultures following treatment with the synthetic LXR agonist T0901317. Results Thirty-one of 48 NRs analyzed by TLDA were found to be measurably expressed in human articular cartilage; 23 of these 31 NRs showed significantly altered expression in OA vs unaffected cartilage. Among these, LXRα and LXRβ, and their heterodimeric partners retinoid X receptor (RXR)α and RXRβ were all expressed at significantly lower levels in OA cartilage, as were LXR target genes ABCG1 and apolipoproteins D and E. Addition of LXR agonist to human OA articular chondrocytes and to cartilage explant cultures resulted in activation of LXR-mediated transcription and significant reduction of both basal and interleukin (IL)-1-mediated PG degradation. Conclusions Articular cartilage expresses a substantial number of NRs, and a large proportion of the expressed NRs are dysregulated in OA. In particular, LXR signaling in OA articular cartilage is impaired, and stimulation of LXR transcriptional activity can counteract the catabolic effects of IL-1. We conclude that LXR agonism may be a possible therapeutic option for OA.

The prevention of aggrecan (a key component of cartilage) cleavage via the inhibition of aggrecanase-1 may provide a unique opportunity to stop the progression of cartilage degradation in osteoarthritis. The evaluation of a series of biphenylsulfonamides resulted in the identification of the ((4-keto)-phenoxy)methyl biphenyl-4-sulfonamides analogs (19-21 and 24) with improved Agg-1 inhibition and MMP-2, MMP-13 activity.

5'-Phenyl-3'H-spiro[indoline-3,2'-[1,3,4]thiadiazol]-2-one inhibitors of ADAMTS-5 (Aggrecanase-2) have been prepared via commercially available starting materials. Selected compounds 23, 33-35 show sub-micromolar ADAMTS-5 potency and strong SAR trends with selectivity over the related metalloproteases ADAMTS-4 (Aggrecanase-1), MMP12, and MMP13. This series of compounds represents progress toward a selective ADAMTS-5 inhibitor as a disease modifying osteoarthritis drug.

Summary Objective To evaluate anterior cruciate ligament transection (ACLT) and destabilization of the medial meniscus (DMM) surgical instability models of osteoarthritis (OA) in the 129/SvEv mouse knee joint. Design Micro-surgical techniques were used to perform ACLT or DMM under direct visualization. Histological scoring was performed on multiple sections to assess cartilage damage across the entire joint. Results The ACLT model gave severe OA, chondrogenesis of the joint capsule and, in some cases, severe subchondral erosion of the posterior tibial plateau. Surgical DMM was less invasive than the ACLT procedure and resulted in lesions primarily on the central weight-bearing region of the medial tibial plateau and medial femoral condyles. Lesions in the DMM model progressed from mild-to-moderate OA at 4 weeks, to moderate-to-severe OA at 8 weeks post-surgery. Destruction of the subchondral bone was never observed in the DMM model. Conclusions ACLT is not recommended in the mouse due to the high surgical proficiency required and the development of severe OA that may involve subchondral bone erosion. The severity and location of lesions following DMM are consistent with lesions observed in aged spontaneous mouse models of OA. The DMM model has sufficient sensitivity to show disease modification, as observed with the ADAMTS-5 knock out (KO) mouse. The DMM model should be a first choice to challenge mice with gene deletions of potential targets in OA.

5-Benzylidene-2-thioxo-thiazolidin-4-one inhibitors of ADAMTS-5 (Aggrecanase-2) have been prepared via commercially available starting materials. The identified compounds show micromolar ADAMTS-5 potency and demonstrate SAR trends for both the aryl group and thioxothiazolidinone zinc chelator. This series of compounds represents steps toward a metalloprotease inhibitor as a disease-modifying osteoarthritis drug.

Aggrecanases are ADAMTS (a disintegrin and metalloproteinase with thrombospondin type I motifs) proteases capable of primary (patho)physiological cleavage at specific Glu-Xaa bonds within the core protein of the hyaluronan-binding proteoglycan aggrecan. Accumulating evidence suggests that regulation of the activity of one such aggrecanase, ADAMTS-4 (or Aggrecanase-1), involves post-translational C-terminal processing (truncation) which modulates both glycosaminoglycan (GAG)-binding affinity and enzymatic activity. In the present study, we compared the effects of C-terminal truncation on the GAG-binding properties and aggrecanase activity of ADAMTS-5 (Aggrecanase-2) relative to three other ADAMTS family members, ADAMTS-9, ADAMTS-16 and ADAMTS-18. Full-length recombinant human ADAMTS-5 (M(r) approximately 85 kDa; ADAMTS-5p85) underwent autolytic cleavage during expression by CHO/A2 cells, and co-purified with C-terminally truncated (tr) isoforms of M(r) approximately 60 kDa (ADAMTS-5p60 and M(r) approximately 45 kDa (ADAMTS-5p45). All three ADAMTS-5 isoforms bound to sulfated GAGs (heparin and chondroitin sulfate (CS)). An ADAMTS-5p45 structural mimetic, terminating at Phe628 and comprising the catalytic domain, disintegrin-like domain and thrombospondin type I repeat (TSR)-1 domain (designated trADAMTS-5F628), also bound to heparin, and exhibited potent aggrecanase activity toward cleavage sites both in the aggrecan CS-2-attachment region (at Glu1771-Ala1772) and in the interglobular domain (at Glu373-Ala374). Further truncation (deletion of the TSR-1 domain) of ADAMTS-5 significantly reduced aggrecanase activity, although appreciable GAG (heparin)-binding affinity was maintained. Other TSR-1 domain-bearing truncated ADAMTS constructs demonstrating either positive GAG-binding ability (trADAMTS-9F649) or negligible GAG-affinity (trADAMTS-16F647 and trADAMTS-18F650) displayed comparably low aggrecanase activities. Thus, the presence of TSR-1 on truncated ADAMTSs appears to be necessary, but not sufficient, for effective aggrecanase-mediated catalysis of target Glu-Xaa bonds. Similarly, GAG-binding ability, irrespective of the presence of a TSR-1 domain, does not necessarily empower truncated ADAMTSs with proficient aggrecanase activity.

Aggrecanases are recently discovered enzymes that cleave aggrecan, a key component of cartilage. Aggrecanase inhibitors may provide a unique means to halt the progression of cartilage destruction in osteoarthritis. The synthesis and evaluation of biphenylsulfonamidocarboxylic acid inhibitors of aggrecanase-1 are reported. Compound 24 demonstrated 89% inhibition of proteoglycan degradation at 10 μg/mL and has an oral bioavailability in rat of 35%.

The structure-based design and synthesis of a series of novel biphenyl sulfonamide carboxylic acids as potent MMP-13 inhibitors with selectivity over MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-14, Aggrecanase 1, and TACE are described.

Maximal exercise testing is a valuable diagnostic tool for the evaluation of exercise intolerance and level of fitness in the equine athlete. A description of the standardized incremental exercise testing protocol and interpretation of the results of the test is followed by a discussion of more specific diagnostic techniques aimed at identifying pulmonary dysfunction as a cause of suboptimal performance.

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.

Equisport is a comprehensive clinical sports medicine program for equine athletes. This program combines the diagnostic capabilities of a full service large animal veterinary hospital with the ability to evaluate physiological parameters during exercise, using a high speed treadmill. The goal of the equisport program is to optimize athletic performance using a series of clinical programs aimed at evaluation of performance potential in young horses, an early training program to improve fitness, a poor performance evaluation for adult horses and a rehabilitation program for horses recovering from musculoskeletal injury or orthopedic surgery. Over 250 horses have been evaluated since the inception of the equisport program 2 years ago. Although we have evaluated a wide variety of equine athletes, the majority of the caseload is made up of Thoroughbred and Standardbred racehorses.