Simon M. Blake

Disease-modifying activity of SB 242235, a selective inhibitor of p38 mitogen-activated protein kinase, in rat adjuvant-induced arthritis

OBJECTIVE To evaluate the effects of SB 242235, a potent and selective inhibitor of p38 mitogen-activated protein (MAP) kinase, on joint integrity in rats with adjuvant-induced arthritis (AIA). METHODS Male Lewis rats with AIA were orally treated either prophylactically (days 0-20) or therapeutically (days 10-20) with SB 242235. Efficacy was determined by measurements of paw inflammation, dual-energy x-ray absorptiometry for bone-mineral density (BMD), magnetic resonance imaging (MRI), microcomputed tomography (CT), and histologic evaluation. Serum tumor necrosis factor alpha (TNFalpha) in normal (non-AIA) rats and serum interleukin-6 (IL-6) levels in rats with AIA were measured as markers of the antiinflammatory effects of the compound. RESULTS SB 242235 inhibited lipopolysaccharide-stimulated serum levels of TNFalpha in normal rats, with a median effective dose of 3.99 mg/kg. When SB 242235 was administered to AIA rats prophylactically on days 0-20, it inhibited paw edema at 30 mg/kg and 10 mg/kg per day by 56% and 33%, respectively. Therapeutic administration on days 10-20 was also effective, and inhibition of paw edema was observed at 60, 30, and 10 mg/kg (73%, 51%, and 19%, respectively). Significant improvement in joint integrity was demonstrated by showing normalization of BMD and also by MRI and micro-CT analysis. Protection of bone, cartilage, and soft tissues was also shown histologically. Serum IL-6 levels were decreased in AIA rats treated with the 60 mg/kg dose of compound. CONCLUSION Symptoms of AIA in rats were significantly reduced by both prophylactic and therapeutic treatment with the p38 MAP kinase inhibitor, SB 242235. Results from measurements of paw inflammation, assessment of BMD, MRI, and micro-CT indicate that this compound exerts a protective effect on joint integrity, and thus appears to have disease-modifying properties.

Potent and Selective Inhibition of Human Cathepsin K Leads to Inhibition of Bone Resorption In Vivo in a Nonhuman Primate

Cathepsin K is a cysteine protease that plays an essential role in osteoclast‐mediated degradation of the organic matrix of bone. Knockout of the enzyme in mice, as well as lack of functional enzyme in the human condition pycnodysostosis, results in osteopetrosis. These results suggests that inhibition of the human enzyme may provide protection from bone loss in states of elevated bone turnover, such as postmenopausal osteoporosis. To test this theory, we have produced a small molecule inhibitor of human cathepsin K, SB‐357114, that potently and selectively inhibits this enzyme (Ki = 0.16 nM). This compound potently inhibited cathepsin activity in situ, in human osteoclasts (inhibitor concentration [IC]50 = 70 nM) as well as bone resorption mediated by human osteoclasts in vitro (IC50 = 29 nM). Using SB‐357114, we evaluated the effect of inhibition of cathepsin K on bone resorption in vivo using a nonhuman primate model of postmenopausal bone loss in which the active form of cathepsin K is identical to the human orthologue. A gonadotropin‐releasing hormone agonist (GnRHa) was used to render cynomolgus monkeys estrogen deficient, which led to an increase in bone turnover. Treatment with SB‐357114 (12 mg/kg subcutaneously) resulted in a significant reduction in serum markers of bone resorption relative to untreated controls. The effect was observed 1.5 h after the first dose and was maintained for 24 h. After 5 days of dosing, the reductions in N‐terminal telopeptides (NTx) and C‐terminal telopeptides (CTx) of type I collagen were 61% and 67%, respectively. A decrease in serum osteocalcin of 22% was also observed. These data show that inhibition of cathepsin K results in a significant reduction of bone resorption in vivo and provide further evidence that this may be a viable approach to the treatment of postmenopausal osteoporosis.

A Potent Small Molecule, Nonpeptide Inhibitor of Cathepsin K (SB 331750) Prevents Bone Matrix Resorption in the Ovariectomized Rat

Inhibition of the cyteine proteinase, cathepsin K (E.C. 3.4.22.38) has been postulated as a means to control osteoclast-mediated bone resorption. The preferred animal models for evaluation of antiresorptive activity are in the rat. However, the development of compounds that inhibit rat cathepsin K has proven difficult because the human and rat enzymes differ in key residues in the active site. In this study, a potent, nonpeptide inhibitor of rat cathepsin K (K(i) = 4.7 nmol/L), 5-(2-morpholin-4-yl-ethoxy)-benzofuran-2-carboxylic acid ((S)-3-methyl-1-(3-oxo-1-[2-(3-pyridin-2-yl-phenyl)-ethenoyl]-azepan-4-ylcarbanoyl)-butyl)-amide (SB 331750), is described, which is efficacious in rat models of bone resorption. SB 331750 potently inhibited human cathepsin K activity in vitro (K(i) = 0.0048 nmol/L) and was selective for human cathepsin K vs. cathepsins B (K(i) = 100 nmol/L), L (0.48 nmol/L), or S (K(i) = 14.3 nmol/L). In an in situ enzyme assay, SB 331750 inhibited osteoclast-associated cathepsin activity in tissue sections containing human osteoclasts (IC(50) approximately 60 nmol/L) and this translated into potent inhibition of human osteoclast-mediated bone resorption in vitro (IC(50) approximately 30 nmol/L). In vitro, SB 331750 partially, but dose-dependently, prevented the parathyroid hormone-induced hypercalcemia in an acute rat model of bone resorption. To evaluate the ability of SB 331750 to inhibit bone matrix degradation in vivo, it was administered for 4 weeks at 3, 10, or 30 mg/kg, intraperitoneally (i.p.), u.i.d. in the ovariectomized (ovx) rat. Both 10 and 30 mg/kg doses of compound prevented the ovx-induced elevation in urinary deoxypyridinoline and prevented the ovx-induced increase in percent eroded perimeter. Histological evaluation of the bones from compound-treated animals indicated that SB 331750 retarded bone matrix degradation in vivo at all three doses. The inhibition of bone resorption at the 10 and 30 mg/kg doses resulted in prevention of the ovx-induced reduction in percent trabecular area, trabecular number, and increase in trabecular spacing. These effects on bone resorption were also reflected in inhibition of the ovx-induced loss in trabecular bone volume as assessed using microcomputerized tomography (microCT; approximately 60% at 30 mg/kg). Together, these data indicate that the cathepsin K inhibitor, SB 331750, prevented bone resorption in vivo and this inhibition resulted in prevention of ovariectomy-induced loss in trabecular structure.

Disease‐modifying activity of SB 273005, an orally active, nonpeptide αvβ3 (vitronectin receptor) antagonist, in rat adjuvant‐induced arthritis

Objective To evaluate the effects of SB 273005, a potent, orally active nonpeptide antagonist of the integrin αvβ3 vitronectin receptor, on joint integrity in rats with adjuvant-induced arthritis (AIA). Methods Male Lewis rats with AIA were orally dosed either prophylactically (days 0–20) or therapeutically (days 10–20) with SB 273005. Efficacy was determined by measurement of paw inflammation, assessment of bone mineral density using dual-energy x-ray absorptiometry (DEXA), magnetic resonance imaging (MRI), and histologic evaluation. Results SB 273005 is a potent antagonist of the closely related integrins, αvβ3 (Ki = 1.2 nM) and αvβ5 (Ki = 0.3 nM). When SB 273005 was administered prophylactically to AIA rats twice per day, it inhibited paw edema at doses of 10, 30, and 60 mg/kg, by 40%, 50%, and 52%, respectively. Therapeutic administration twice daily was also effective, and a reduction in paw edema was observed at 30 mg/kg and 60 mg/kg of the antagonist (by 36% and 48%, respectively). SB 273005 was also effective when administered once per day, both prophylactically and therapeutically. Significant improvement in joint integrity in treated rats was shown using DEXA and MRI analyses. These findings were confirmed histologically, and significant protection of bone, cartilage, and soft tissue was observed within the joint. Conclusion Symptoms of AIA in rats were significantly reduced by either prophylactic or therapeutic treatment with the αvβ3 antagonist, SB 273005. Measurements of paw inflammation and of bone, cartilage, and soft tissue structure indicated that this compound exerts a protective effect on joint integrity and thus appears to have disease-modifying properties.

Mechanical injury potentiates proteoglycan catabolism induced by interleukin‐6 with soluble interleukin‐6 receptor and tumor necrosis factor α in immature bovine and adult human articular cartilage

OBJECTIVE Traumatic joint injury can damage cartilage and release inflammatory cytokines from adjacent joint tissue. The present study was undertaken to study the combined effects of compression injury, tumor necrosis factor alpha (TNFalpha), and interleukin-6 (IL-6) and its soluble receptor (sIL-6R) on immature bovine and adult human knee and ankle cartilage, using an in vitro model, and to test the hypothesis that endogenous IL-6 plays a role in proteoglycan loss caused by a combination of injury and TNFalpha. METHODS Injured or uninjured cartilage disks were incubated with or without TNFalpha and/or IL-6/sIL-6R. Additional samples were preincubated with an IL-6-blocking antibody Fab fragment and subjected to injury and TNFalpha treatment. Treatment effects were assessed by histologic analysis, measurement of glycosaminoglycan (GAG) loss, Western blot to determine proteoglycan degradation, zymography, radiolabeling to determine chondrocyte biosynthesis, and Western blot and enzyme-linked immunosorbent assay to determine chondrocyte production of IL-6. RESULTS In bovine cartilage samples, injury combined with TNFalpha and IL-6/sIL-6R exposure caused the most severe GAG loss. Findings in human knee and ankle cartilage were strikingly similar to those in bovine samples, although in human ankle tissue, the GAG loss was less severe than that observed in human knee tissue. Without exogenous IL-6/sIL-6R, injury plus TNFalpha exposure up-regulated chondrocyte production of IL-6, but incubation with the IL-6-blocking Fab significantly reduced proteoglycan degradation. CONCLUSION Our findings indicate that mechanical injury potentiates the catabolic effects of TNFalpha and IL-6/sIL-6R in causing proteoglycan degradation in human and bovine cartilage. The temporal and spatial evolution of degradation suggests the importance of transport of biomolecules, which may be altered by overload injury. The catabolic effects of injury plus TNFalpha appeared partly due to endogenous IL-6, since GAG loss was partially abrogated by an IL-6-blocking Fab.

Human articular chondrocytes produce IL-7 and respond to IL-7 with increased production of matrix metalloproteinase-13.

IntroductionFibronectin fragments have been found in the articular cartilage and synovial fluid of patients with osteoarthritis and rheumatoid arthritis. These matrix fragments can stimulate production of multiple mediators of matrix destruction, including various cytokines and metalloproteinases. The purpose of this study was to discover novel mediators of cartilage destruction using fibronectin fragments as a stimulus.MethodsHuman articular cartilage was obtained from tissue donors and from osteoarthritic cartilage removed at the time of knee replacement surgery. Enzymatically isolated chondrocytes in serum-free cultures were stimulated overnight with the 110 kDa α5β1 integrin-binding fibronectin fragment or with IL-1, IL-6, or IL-7. Cytokines and matrix metalloproteinases released into the media were detected using antibody arrays and quantified by ELISA. IL-7 receptor expression was evaluated by flow cytometry, immunocytochemical staining, and PCR.ResultsIL-7 was found to be produced by chondrocytes treated with fibronectin fragments. Compared with cells isolated from normal young adult human articular cartilage, increased IL-7 production was noted in cells isolated from older adult tissue donors and from osteoarthritic cartilage. Chondrocyte IL-7 production was also stimulated by combined treatment with the catabolic cytokines IL-1 and IL-6. Chondrocytes were found to express IL-7 receptors and to respond to IL-7 stimulation with increased production of matrix metalloproteinase-13 and with proteoglycan release from cartilage explants.ConclusionThese novel findings indicate that IL-7 may contribute to cartilage destruction in joint diseases, including osteoarthritis.

Small-molecule inhibitors of NF-κB for the treatment of inflammatory joint disease

Abstract Recent advances in our understanding of the role of cytokine networks in inflammatory processes have led to the development of novel biological agents for the treatment of chronic inflammatory diseases. At the present time, significant efforts are focused on characterizing the complex signal transduction cascades that are activated by these cytokines and, in turn, regulate their expression. The transcription factor NF-κB is a pivotal regulator of the inducible expression of key proinflammatory mediators, and activated NF-κB has been observed in several debilitating inflammatory disorders, including rheumatoid arthritis and osteoarthritis. In light of its central role in inflammation, the identification of inhibitors of NF-κB should provide novel therapeutics for the treatment of chronic joint disease .

Intracellular signaling pathways as a target for the treatment of rheumatoid arthritis

Recent advances in our understanding of cellular and molecular mechanisms of rheumatoid arthritis have highlighted a critical role for interleukin-1 and tumor necrosis factor alpha. The quest for chemically amenable targets has recently led to the identification and characterization of the intracellular signaling pathways associated with these inflammatory cytokines. In particular the mitogen-activated protein kinase pathway, the nuclear factor kappaB pathway and the cross-talk between these offer several potential therapeutic opportunities for rheumatoid arthritis.

Potent and selective cathepsin L inhibitors do not inhibit human osteoclast resorption in vitro.

Cathepsins K and L are related cysteine proteases that have been proposed to play important roles in osteoclast-mediated bone resorption. To further examine the putative role of cathepsin L in bone resorption, we have evaluated selective and potent inhibitors of human cathepsin L and cathepsin K in an in vitro assay of human osteoclastic resorption and an in situ assay of osteoclast cathepsin activity. The potent selective cathepsin L inhibitors (K i = 0.0099, 0.034, and 0.27 nm) were inactive in both the in situcytochemical assay (IC50 > 1 μm) and the osteoclast-mediated bone resorption assay (IC50 > 300 nm). Conversely, the cathepsin K selective inhibitor was potently active in both the cytochemical (IC50 = 63 nm) and resorption (IC50 = 71 nm) assays. A recently reported dipeptide aldehyde with activity against cathepsins L (K i = 0.052 nm) and K (K i = 1.57 nm) was also active in both assays (IC50 = 110 and 115 nm, respectively) These data confirm that cathepsin K and not cathepsin L is the major protease responsible for human osteoclastic bone resorption.

A53 MECHANICAL INJURY POTENTIATES THE COMBINED EFFECTS OF TNF-α AND IL-6/sIL-6R ON PROTEOGLYCAN CATABOLISM IN BOVINE CARTILAGE

+Sui, Y; **Song, X-Y; +**Lee, J H; *DiMicco, M; **Blake, S M; *Hung, H; **James, I; ** Lark, M W; +§¶*Grodzinsky, A J +Biological, §Mechanical, ¶Electrical Engineering, *Center for Biomedical Engineering, MIT, Cambridge, MA; **Centocor R&D, Inc, Radnor, PA ysui@mit.edu INTRODUCTION: Acute traumatic joint injury increases the risk of developing osteoarthritis (OA) [1]. The mechanisms by which injury causes chronic cartilage degradation in vivo are not fully understood, but elevated levels of injury-induced pro-inflammatory cytokines [2], including TNF-α and IL-6 [3], may play pivotal roles in the pathogenesis of OA. TNF-α causes a synergistic loss of PG from mechanically-injured cartilage in vitro [4], but the pathways regulating this synergy are unclear. The objectives of this study were to (1) examine the combined effect of TNF-α and IL-6/sIL6R on proteoglycan degradation in mechanically-injured cartilage, and (2) to determine the role of endogenous IL-6 in the cartilage catabolism induced by both TNF-α and mechanical injury. METHODS: Cartilage disks (3 mm diam., 1 mm thick) were harvested from the middle zone of the femoropatellar grooves of 1-2-week old calves, and equilibrated for 2 days in normal medium (DMEM + 1% ITS) prior to treatment. Cytokine and Mechanical Injury Treatments: Location-matched disks were either injuriously compressed (50% strain, 100%/second strain rate), cultured in medium with rhTNF-α (25 ng/ml), treated with rhIL-6 (50 ng/ml) plus soluble IL-6 receptor (sIL-6R, 250ng/ml), or treated with combinations of these three conditions (Fig.1). Culture was terminated after 6 days of treatment. In a separate experiment, half the cartilage disks (Fig. 3) were pre-equilibrated for 6 days with an IL-6 blocking Fab fragment (50 ug/ml, Centocor, J&J) prior to treatment; the remaining disks were incubated in normal medium during this period. Afterward, disks were either injuriously compressed, incubated with rhTNF-α (25 ng/ml), or treated with combined injury + TNF-α. Disks that were pre-treated with the IL-6 blocking Fab fragment continued to receive Fab fragments until the termination of the experiment. Aggrecan Western Blotting, GAG Content and Histology: Culture medium from each condition was collected on day 2, 4 and 6 after the initial injury and/or cytokine treatments. Concentrated medium was used to perform Western blot analysis using a monoclonal Ab specific to the G1NITEGE fragment of aggrecan (kindly provided by C. Flannery, Wyeth). DMMB dye was used to quantify sGAG released into the medium. Selected cartilage samples were fixed in gluteraldehyde with RHT, paraffin-embedded, sectioned, and stained with Toluidine Blue. Additional disks were radiolabeled during days 4-6 with 5 μCi/ml SO4 to assess proteoglycan synthesis. RESULTS: 3-way ANOVA analyses followed by post-hoc Tukey’s pairwise comparisons showed that TNF-α formed interactions with both IL-6/sIL-6R (p<0.001) and injurious compression (p<0.001) causing increased sGAG release (Fig. 1(i)) and decreased proteoglycan synthesis (data not shown). While IL-6/sIL-6R significantly augmented TNF-αinduced proteoglycan degradation (Fig.1(i)B,G), the largest amount of GAG loss was caused by the combination of injury+TNF-α + IL-6/sIL6R (Fig.1(i)D). Histology showed that GAG loss was not uniform across the disk cross-section, but instead was initiated at the disk periphery and progressed towards the disk center with time (Fig. 2). The most rapid, severe progression of GAG loss was observed in disks treated with the combination of injury + TNF-α + IL-6/sIL-6R (Fig. 2e,f). Analysis of conditioned medium for aggrecan fragments by Western blotting demonstrated that the most dramatic release of aggrecanase-generated cleavage products occurred in response to treatment with TNF-α + IL6/sIL-6R, both with and without injurious compression (Fig. 1(ii)B,D). The IL-6 blocking Fab fragment was effective in neutralizing exogenous rhIL-6 in the medium, and was not toxic to cells (data not shown). In separate studies, TNF-α + mechanical injury caused greater GAG loss than either treatment alone (Fig.3G,H,I). Importantly, the IL-6 blocking Fab fragment significantly reduced the combined catabolic effects of TNF-α + mechanical injury on GAG loss, with no exogenous IL-6 present (Fig.3D,I). DISCUSSION: We found that the combined treatment with TNF-α and IL-6/sIL-6R induced significantly more GAG loss than either cytokine alone did, consistent with previous studies of TNF-α/IL-6 treatment [3], and suggesting this catabolic response was associated with aggrecanase (but not MMP) activity. Additionally, we now report that the catabolic effect of TNF-α, and the combined effect of TNF-α + IL-6/sIL-6R are both highly potentiated by mechanical injury. The degradative effects of injury + TNF-α appear to be due, in part, to the action of endogenous IL-6, as sGAG loss was partly abrogated by the IL-6 blocking Fab fragment. This result is also consistent with the increased loss of sGAG upon addition of exogenous IL-6 to the combination of TNF-α and mechanical injury. Histology observations (Fig. 2) suggest that the kinetics of cartilage degradation is not merely a consequence of the activities of proteolytic enzymes, but it also depends strongly on the transport of cytokines, proteases, anti-IL-6 Fab and other cartilage biomolecules, which may be altered by overload injury. In conclusion, our study suggests that pro-inflammatory cytokines, whose productions are elevated by traumatic joint injury, can interact to potentiate cartilage catabolism. The mechanobiological (cell-mediated) responses to overload [5], as well as altered transport of cytokines and proteases in the damaged matrix, may both be affected by joint injury, making the damaged cartilage tissue more susceptible to further degradation by biochemical mediators. REFERENCES:[1] Gelber+, Ann Intern Med 133:3211, ‘00; [2] Irie+, Knee 10:93, ‘03; [3] Flannery+, Matrix Biol, 19:549, ‘00. [4] Patwari+, Arth Rheum, 48:1292, ‘03 [5] Lee+, Arth Rheum, 52:2386, ‘05. Acknowledgements: Supported by Centocor and NIH Grant AR45779