Search

Prescription opioids are a mainstay in the treatment of acute moderate to severe pain. However, chronic use leads to a host of adverse consequences including tolerance and opioid-induced hyperalgesia (OIH), leading to more complex treatment regimens and diminished patient compliance. Patients with OIH paradoxically experience exaggerated nociceptive responses instead of pain reduction after chronic opioid usage. The development of OIH and tolerance tend to occur simultaneously and, thus, present a challenge when studying the molecular mechanisms driving each phenomenon. We tested the hypothesis that a G protein-biased µ-opioid peptide receptor (MOPR) agonist would not induce symptoms of OIH, such as mechanical allodynia, following chronic administration. We observed that the development of opioid-induced mechanical allodynia (OIMA), a model of OIH, was absent in β-arrestin1-/- and β-arrestin2-/- mice in response to chronic administration of conventional opioids such as morphine, oxycodone and fentanyl, whereas tolerance developed independent of OIMA. In agreement with the β-arrestin knockout mouse studies, chronic administration of TRV0109101, a G protein-biased MOPR ligand and structural analog of oliceridine, did not promote the development of OIMA but did result in drug tolerance. Interestingly, following induction of OIMA by morphine or fentanyl, TRV0109101 was able to rapidly reverse allodynia. These observations establish a role for β-arrestins in the development of OIH, independent of tolerance, and suggest that the use of G protein-biased MOPR ligands, such as oliceridine and TRV0109101, may be an effective therapeutic avenue for managing chronic pain with reduced propensity for opioid-induced hyperalgesia.

Opioids provide powerful analgesia but also efficacy-limiting adverse effects, including severe nausea, vomiting, and respiratory depression, by activating μ-opioid receptors. Preclinical models suggest that differential activation of signaling pathways downstream of these receptors dissociates analgesia from adverse effects; however, this has not yet translated to a treatment with an improved therapeutic index. Thirty healthy men received single intravenous injections of the biased ligand TRV130 (1.5, 3, or 4.5mg), placebo, or morphine (10mg) in a randomized, double-blind, crossover study. Primary objectives were to measure safety and tolerability (adverse events, vital signs, electrocardiography, clinical laboratory values), and analgesia (cold pain test) versus placebo. Other measures included respiratory drive (minute volume after induced hypercapnia), subjective drug effects, and pharmacokinetics. Compared to morphine, TRV130 (3, 4.5mg) elicited higher peak analgesia (105, 116 seconds latency vs 75 seconds for morphine, P

Drug discovery targeting G protein-coupled receptors (GPCRs) is no longer limited to seeking agonists or antagonists to stimulate or block cellular responses associated with a particular receptor. GPCRs are now known to support a diversity of pharmacological profiles, a concept broadly referred to as functional selectivity. In particular, the concept of ligand bias, whereby a ligand stabilizes subsets of receptor conformations to engender novel pharmacological profiles, has recently gained increasing prominence. This review discusses how biased ligands may deliver safer, better tolerated, and more efficacious drugs, and highlights several biased ligands that are in clinical development. Biased ligands targeting the angiotensin II type 1 receptor and the μ opioid receptor illustrate the translation of the biased ligand concept from basic biology to clinical drug development.

TRV130 is a G protein-biased ligand at the µ-opioid receptor. In preclinical studies it was potently analgesic while causing less respiratory depression and gastrointestinal dysfunction than morphine, suggesting unique benefits in acute pain management. A first-in-human study was conducted with ascending doses of TRV130 to explore its tolerability, pharmacokinetics, and pharmacodynamics in healthy volunteers. TRV130 was well-tolerated over the dose range 0.15 to 7 mg administered intravenously over 1 hour. TRV130 geometric mean exposure and Cmax were dose-linear, with AUC0–inf of 2.52 to 205.97 ng h/mL and Cmax of 1.04 to 102.36 ng/mL across the dose range tested, with half-life of 1.6–2.7 hours. A 1.5 mg dose of TRV130 was also well-tolerated when administered as 30, 15, 5, and 1 minute infusions. TRV130 pharmacokinetics were modestly affected by CYP2D6 phenotype: clearance was reduced by 53% in CYP2D6 poor metabolizers.TRV130 caused dose- and exposure-related pupil constriction, confirming central compartment µ-opioid receptor engagement. Marked pupil constriction was noted at 2.2, 4, and 7 mg doses. Nausea and vomiting observed at the 7 mg dose limited further dose escalation. These findings suggest that TRV130 may have a broad margin between doses causing µ-opioid receptor-mediated pharmacology and doses causing µ-opioid receptor-mediated intolerance.

TRV027 is a novel β-arrestin biased peptide ligand of the angiotensin II type 1 receptor (AT1R). The compound antagonizes G protein coupling while simultaneously stimulating β-arrestin-mediated signaling. In preclinical studies, TRV027 reversibly reduced blood pressure while preserving renal function in a dog tachypaced heart failure model and stimulating cardiomyocyte contractility in vitro. This profile suggests that TRV027 may have unique benefits in acute heart failure, a condition associated with renin-angiotensin system activation. A first-time-in-human study was conducted with ascending doses of TRV027 to explore its tolerability, pharmacokinetics and pharmacodynamics in healthy volunteers. Subjects' salt intake was restricted to stimulate RAS activation. In this study TRV027 was safe and well tolerated with a short-half-life (ranging between 2.4 and 13.2 minutes) and dose-proportional increases in systemic exposure. Consistent with the pre-clinical findings, TRV027 reduced blood pressure to a greater degree in subjects with RAS activation, measured as elevated plasma renin activity, than in those with normal PRA levels. This study in sodium-restricted healthy subjects suggests that TRV027 will successfully target a core mechanism of acute heart failure pathophysiology. Further clinical studies with TRV027 in patients with heart failure are underway.

Background— TRV120027 is a novel β-arrestin biased ligand of the angiotensin II type 1 receptor; it antagonizes canonical G-protein–mediated coupling while, in contrast to classical angiotensin II type 1 receptor antagonists, it engages β-arrestin–mediated signaling. Consequently, TRV120027 inhibits angiotensin II–mediated vasoconstriction while, via β-arrestin coupling, it increases cardiomyocyte contractility. We hypothesized that TRV120027 would elicit beneficial cardiorenal actions when added to furosemide in experimental heart failure. Methods and Results— Two groups of anesthetized dogs (n=6 each) with tachypacing-induced heart failure were studied. After a baseline clearance, 1 group (F+V) received furosemide (1 mg/kg per hour) plus saline for 90 minutes, whereas the other (F+T) received the same dose of furosemide plus TRV120027 (0.3 and 1.5 µg/kg per minute for 45 minutes each); 2 clearances were done during drug infusion. After a washout, a postinfusion clearance was done; * P Conclusions— When added to furosemide, TRV120027, a novel β-arrestin biased angiotensin II type 1 receptor ligand, preserved furosemide-mediated natriuresis and diuresis, while reducing cardiac preload and afterload. These results provide support for TRV120027 as a promising novel therapeutic for the treatment of heart failure.

The angiotensin II type 1 receptor (AT1R) is an important drug target for hypertension and chronic heart failure. This receptor is also important in the pathophysiology of acute heart failure, stimulating both adverse and beneficial hemodynamic and renal responses. This review will discuss advances in our understanding of the molecular mechanisms of AT1R function, and how these insights have enabled the discovery of β-arrestin-biased AT1R ligands, one of which is now in clinical trials for acute heart failure (AHF) treatment.

Biased G protein-coupled receptor ligands engage subsets of the receptor signals normally stimulated by unbiased agonists. However, it is unclear whether ligand bias can elicit differentiated pharmacology in vivo. Here, we describe the discovery of a potent, selective β-arrestin biased ligand of the angiotensin II type 1 receptor. TRV120027 (Sar-Arg-Val-Tyr-Ile-His-Pro-D-Ala-OH) competitively antagonizes angiotensin II-stimulated G protein signaling, but stimulates β-arrestin recruitment and activates several kinase pathways, including p42/44 mitogen-activated protein kinase, Src, and endothelial nitric-oxide synthase phosphorylation via β-arrestin coupling. Consistent with β-arrestin efficacy, and unlike unbiased antagonists, TRV120027 increased cardiomyocyte contractility in vitro. In rats, TRV120027 reduced mean arterial pressure, as did the unbiased antagonists losartan and telmisartan. However, unlike the unbiased antagonists, which decreased cardiac performance, TRV120027 increased cardiac performance and preserved cardiac stroke volume. These striking differences in vivo between unbiased and β-arrestin biased ligands validate the use of biased ligands to selectively target specific receptor functions in drug discovery.

Periprosthetic osteolysis with or without aseptic loosening is a major clinical problem in total hip arthroplasty. While the macrophage response to prosthetic wear debris and its role in periprosthetic osteolysis has been extensively studied, information regarding other cell types (fibroblasts, osteoblasts) is limited. This study explored the response of fibroblasts to particulate wear debris. Fibroblasts isolated from interfacial membranes of patients with failed total hip replacements and normal synovial tissue, when challenged with small-sized ( < 3 microns) titanium (Ti) particles, responded with significantly enhanced expressions of collagenase, stromelysin and, to a much lesser extent, their tissue inhibitor of metalloproteinases (TIMP). These "regulated" expressions at both mRNA and protein levels were correlated with the size and composition of particles. De novo protein synthesis was required for the regulation of these mRNAs. A similar effect could be induced by the treatment of the cells with particle-free conditioned medium from Ti particle-stimulated fibroblasts. Furthermore, this conditioned medium significantly suppressed the mRNA levels of procollagen alpha 1 (I) and alpha 1 (III) in osteoblast-like MG-63 cells. It is concluded that fibroblasts stimulated with certain particle debris may play an important role in periprosthetic osteolysis by releasing bone-resorbing metalloproteinases and mediator(s) which resulted in suppressed collagen synthesis in osteoblasts.

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 α (TNFα), 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 TNFα. Methods Injured or uninjured cartilage disks were incubated with or without TNFα and/or IL-6/sIL-6R. Additional samples were preincubated with an IL-6–blocking antibody Fab fragment and subjected to injury and TNFα 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 TNFα 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 TNFα 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 TNFα 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 TNFα appeared partly due to endogenous IL-6, since GAG loss was partially abrogated by an IL-6–blocking Fab.

An excess of thyroid hormone results in increased bone turnover and loss of bone mass in humans. Exogenous administration of thyroid hormone to rats has served as a model of human hyperthyroidism in which antiresorptive therapies have been tested. We have further refined this model of thyroxine (T4)-induced turnover in the rat. Daily administration of T4 to aged rats for as short as 1 week resulted in elevated bone resorption determined by significantly higher urinary deoxypyridinoline (Dpd) compared with vehicle controls or animals receiving T4 plus estradiol. Three weeks of daily administration of T4 led to significantly lower bone mineral density compared with untreated controls or animals receiving T4 plus estradiol. In a follow-up study, a depot formulation of T4 caused an increase in Dpd identical to that achieved with a bolus dose. SB-273005 [(4S)-2,3,4,5-tetrahydro-8-[2-[6-(methylamino)-2-pyridinyl] ethoxy]-3-oxo-2-(2,2,2-trifluoroethyl)-1H-2-benzazepine-4- acetic acid] a potent antagonist of the integrins alpha(v)beta(3) and alpha(v)beta(5), has been shown previously to inhibit bone resorption in cultures of human osteoclasts and to protect bone in ovariectomized rats. The effect of SB-273005 by oral administration was evaluated in this thyroxine-induced turnover model. Dose-dependent inhibition of resorption was seen with SB-273005 after 7 days of dosing using Dpd as a measure of bone resorption. In summary, it has been demonstrated that the antiresorptive activity of a vitronectin receptor antagonist can be measured after only 7 days of treatment in this refined rat model of thyroxine-induced bone turnover. These data suggest that SB-273005 may be useful for the treatment of metabolic bone diseases, including those resulting from hyperthyroidism.

Traumatic injury to a joint is known to increase the risk for the development of secondary osteoarthritis, but it is unclear how this process occurs. The existence of such a discrete event that can lead to an increased risk of osteoarthritis has spurred interest in developing in vitro models of traumatic joint injury. The current authors review some of the recent insights gained from these model systems into the pathogenesis of osteoarthritis, including the evidence for an initial, irreversible insult to chondrocytes during mechanical injury, the occurrence of apoptotic chondrocyte death, and attempts to identify the effects of trauma on chondrocyte metabolic response. Results also are presented from the authors' ongoing studies of the degradative pathways initiated by traumatic mechanical loads, the mechanism by which chondrocytes are affected during compression, and possible contributions of the joint capsule to posttraumatic cartilage degradation.

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.

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 situ cytochemical assay (IC(50)1 micrometer) and the osteoclast-mediated bone resorption assay (IC(50)300 nm). Conversely, the cathepsin K selective inhibitor was potently active in both the cytochemical (IC(50) = 63 nm) and resorption (IC(50) = 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 (IC(50) = 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.

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.

The integrin alphavbeta3 is thought to play a key role in the initiation and/or progression of several human diseases, including osteoporosis, restenosis following percutaneous transluminal coronary angioplasty (PTCA), rheumatoid arthritis, cancer and ocular diseases. Antagonism of integrin alphavbeta3 is therefore expected to provide an approach for the treatment and/or prevention of these diseases. A variety of potent, small-molecule alphavbeta3 antagonists have been identified, several of which are active in disease models, thereby demonstrating the therapeutic potential of alphavbeta3 antagonism. This review will focus on recent advances in the identification of small-molecule alphavbeta3 antagonists, with an emphasis on those studies where small-molecule alphavbeta3 antagonists have been used in proof-of-concept studies in vivo.

Osteonecrosis of the femoral head often results in secondary osteoarthritis of the hip joint; however, the pathologic processes underlying the destruction of articular cartilage are not fully understood. Molecular markers in the hip joint fluids were measured to examine the changes in turnover of cartilage and other joint tissues. Marker data were related to clinical, radiological, and histopathological changes in the articular cartilage of the hip. Forty-five patients (median age: 43 years) were studied. The median time between the onset of symptoms and sampling of hip synovial fluid was 6 months. Aggrecan fragments, C-propeptide of type-II collagen, matrix metalloproteinase-3, and tissue inhibitor of metalloproteinases-1 levels in joint fluid were determined by immunoassay. Osteonecrosis of the femoral head was graded by radiology as minimal collapse of the femoral head (stage 2: 26 patients), severe collapse (stage 3: 15 patients), or severe collapse with osteoarthritis (stage 4: four patients). Histological changes of the articular cartilage, consistent with early-stage osteoarthritis, were evident at stage 3 and were more advanced at stage 4. The average concentrations of proteoglycan fragments and C-propeptide of type-II collagen were 207 (SD 182) μg/ml and 19.6 (SD 19.3) ng/ml, respectively. The average concentrations of matrix metalloproteinase-3 and tissue inhibitor of metalloproteinases-1 were 177 (SD 291) nM and 23.0 (SD 9.9) nM, respectively. Measurable levels for all markers assayed were noted in the earliest stage of the disease, only a few months after the onset of symptoms and well before the appearance of radiological changes. Levels of matrix metalloproteinase-3 and molar ratios of matrix metalloproteinase-3/tissue inhibitor of metalloproteinases-1 were higher in early stage disease than in later stage disease. (Less)

We have previously demonstrated that a tartrate-resistant acid phosphatase (TRAP)-positive subpopulation of mononuclear cells isolated from collagenase digests of human osteoclastoma tissue exhibits an osteoclast phenotype and can be induced to resorb bone. Using these osteoclast precursors as a model system, we have assessed the chemotactic potential of 16 chemokines. Three CC chemokines, the recently described CKbeta-8, RANTES, and MIP-1alpha elicited significant chemotactic responses. In contrast, 10 other CC chemokines (MIP-1beta, MCP-1, MCP-2, MCP-3, MCP-4, HCC-1, eotaxin-2, PARC, SLC, ELC) and 3 CXC chemokines (IL-8, GROalpha, SDF-1) were inactive. None of these chemokines showed any chemotactic activity for either primary osteoblasts derived from human bone explants or the osteoblastic MG-63 cell line. The identity of the osteoclast receptor that mediates the chemotactic response remains to be established. However, all three active chemokines have been reported to bind to CCR1 and cross-desensitization studies demonstrate that RANTES and MIP-1alpha can partially inhibit the chemotactic response elicited by CKbeta-8. CKbeta-8, the most potent of the active CC chemokines (EC(max) 0.1-0.3 nM), was further characterized with regard to expression in human bone and cartilage. Although expression is not restricted to these tissues, CKbeta-8 mRNA was shown to be highly expressed in osteoblasts and chondrocytes in human fetal bone by in situ hybridization. In addition, CKbeta-8 protein was shown to be present in human osteophytic tissue by immunolocalization. These observations suggest that CKbeta-8, and perhaps other chemokines, may play a role in the recruitment of osteoclast precursors to sites of bone resorption.

Caspases have been strongly implicated to play an essential role in apoptosis. A critical question regarding the role(s) of these proteases is whether selective inhibition of an effector caspase(s) will prevent cell death. We have identified potent and selective non-peptide inhibitors of the effector caspases 3 and 7. The inhibition of apoptosis and maintenance of cell functionality with a caspase 3/7-selective inhibitor is demonstrated for the first time, and suggests that targeting these two caspases alone is sufficient for blocking apoptosis. Furthermore, an x-ray co-crystal structure of the complex between recombinant human caspase 3 and an isatin sulfonamide inhibitor has been solved to 2.8-A resolution. In contrast to previously reported peptide-based caspase inhibitors, the isatin sulfonamides derive their selectivity for caspases 3 and 7 by interacting primarily with the S(2) subsite, and do not bind in the caspase primary aspartic acid binding pocket (S(1)). These inhibitors blocked apoptosis in murine bone marrow neutrophils and human chondrocytes. Furthermore, in camptothecin-induced chondrocyte apoptosis, cell functionality as measured by type II collagen promoter activity is maintained, an activity considered essential for cartilage homeostasis. These data suggest that inhibiting chondrocyte cell death with a caspase 3/7-selective inhibitor may provide a novel therapeutic approach for the prevention and treatment of osteoarthritis, or other disease states characterized by excessive apoptosis.

A new series of potent nonpeptide vitronectin receptor antagonists, based on a novel carbocyclic Gly-Asp mimetic, has been discovered. A representative of this series, SB 265123 (4), has 100% oral bioavailability in rats, and is orally active in vivo in the ovariectomized rat model of osteoporosis.

There is a tremendous unmet therapeutic need for the treatment of osteoporosis and osteoarthritis. The ovariectomized rat and the guinea pig are widely used animal models for the evaluation of new therapeutics for osteoporosis and osteoarthritis, respectively. We have utilized X-ray micro-CT techniques to quantitatively evaluate the differences in trabecular bone in the rat proximal tibia following ovariectomy and treatment with estrogen (17-B-estradiol). Results demonstrate a loss of trabecular bone and architecture following ovariectomy (p

The preservation of the structure of articular cartilage depends on the availability of inhibitors of matrix-degrading enzymes. Tissue inhibitor of metalloproteinases-1 is thought to be an important contributor to the integrity of the matrix of articular cartilage, but the mechanisms that regulate its availability within the tissue are poorly understood. These studies elucidate the contributions of diffusion, fluid flow, and electrical migration to the transport of iodinated recombinant human tissue inhibitor of metalloproteinases-1 through explants of adult bovine articular cartilage under conditions relevant to the loading of cartilage. With use of measured partition coefficients of the cartilage explants, the diffusivity of the inhibitor was 0.5-1.6 x 10(-7) cm2/sec. Fluid velocities that were induced by applying an electrical current across the cartilage disks increased the flux of the inhibitor by approximately 20 to more than 150-fold compared with the effect of diffusion alone for the range of current densities that were applied. We examined the contribution of electrophoretic migration by titrating the charge on the inhibitor during measurements of flux and found that flux in the presence of the applied current decreased as the inhibitor became more negatively charged. Enhancements in the flux of the inhibitor were observed relative to the flux during diffusion alone even under conditions in which electrophoretic migration opposed the flux due to fluid flow, suggesting that of the transport mechanisms tested, fluid flow was dominant. These results suggest that the physical phenomena present during physiologic loading conditions (e.g., fluid flows and streaming currents) can affect the transport of tissue inhibitor of metalloproteinases-1 through the matrix of cartilage.

Objective: To determine the within- and between-patient variability in the concentrations of synovial fluid, serum and urine markers of joint tissue metabolism in a cohort of patients with knee pain and cartilage changes consistent with early-stage knee osteoarthritis. Design: Samples of synovial fluid, serum, and urine were obtained from 52 patients on eight different occasions during 1 year, as part of a clinical trial in patients with cartilage abnormalities and knee pain. In joint fluid, aggrecan fragments were quantified by dye precipitation and enzyme-linked immunosorbent assay (ELISA), and matrix metalloproteinases-1 and -3, and tissue inhibitor of metalloproteinases-1 by sandwich ELISAs. In serum, keratan sulfate was quantified by ELISA. Type I collagen N-telopeptide cross-links in urine were determined by ELISA. Results, The degree of cross-sectional variability in marker concentrations did not vary between the different sampling occasions, and did not differ between the periods of weeks 0 (baseline), 1–4 (treatment) and 13–26 (follow-up). Both between-patient and within-patient coefficients of variation varied for markers in different body fluid compartments, with the lowest variability for serum keratan sulfate, followed by urine type I collagen N-telopeptide crosslinks, and the highest for synovial fluid markers. For synovial fluid, aggrecan fragments showed the least variability, and matrix metalloproteinases the highest. One patients with septic arthritis showed a fivefold peak increase in joint fluid aggrecan fragment concentrations, while the concentration of matrix metalloproteinase-3 increased 100-fold. Conclusions: Molecular markers of joint tissue metabolism have been suggested as, for example, outcome measures for clinical trials of disease-modifying drugs in osteoarthritis. This report is the first to present data on between- and within-patient variability for such molecular markers in three different body fluid compartments in stable cohort of patients. The availability of such data enables calculations to determine the number of patients needed in prospective studies using these markers as outcome measures.

Nitric oxide (NO) is implicated in a number of inflammatory processes and is an important mediator in animal models of rheumatoid arthritis and in in vitro models of cartilage degradation. The pyridinyl imidazole SB 203580 inhibits p38 mitogen-activated protein (MAP) kinase in vitro, blocks proinflammatory cytokine production in vitro and in vivo, and is effective in animal models of arthritis. The purpose of this study was to determine whether SB 203580 could inhibit p38 MAP kinase activity, NO production, and inducible NO synthase (iNOS) in IL-1 stimulated bovine articular cartilage/chondrocyte cultures. The results indicated that SB 203580 inhibited both IL-1 stimulated p38 MAP kinase activity in isolated chondrocytes and NO production in bovine chondrocytes and cartilage explants with an IC50 value of approximately 1 μM. To inhibit NO production, SB 203580 had to be present in cartilage explant cultures during the first 8 h of IL-1 stimulation, and activity was lost when it was added 24 h following IL-1. SB 203580 did not inhibit iNOS activity, as measured by the conversion of arginine to citrulline, when added directly to cultures where the enzyme had already been induced, but had to be present during the induction period. Using a 372-bp probe for bovine iNOS we demonstrated inhibition of IL-1-induced mRNA by SB 203580 at both 4 and 24 h following IL-1 treatment. The iNOS mRNA levels were consistent with NO levels in 24-h cell culture supernatants of the IL-1-stimulated bovine chondrocytes used to obtain the RNA.

Bovine cartilage explants were treated with 100 ng/ml recombinant human interleukin-1beta (IL-1beta) or 1 microM all-trans retinoic acid (RA) and changes in biochemical, biomechanical, and physicochemical properties were assessed. Additionally, samples cultured with IL-1beta or RA were treated with 4 microM recombinant human tissue inhibitor of metalloproteinases-1 (TIMP-1) or a synthetic metalloproteinase inhibitor (L-758,354) to inhibit this degradation. Treatment with IL-1beta or RA each resulted in90% GAG loss after 8 days in culture. Addition of TIMP or L-758,354 to the culture media inhibited IL-1beta-induced loss of tissue GAG by 40 and 65%, respectively, and inhibited RA-induced GAG loss by 35 and 65%, respectively. Analysis of degradation products in the culture media using a G1 antibody indicated that IL-1beta- and RA-treated plugs released 68-kDa fragments of aggrecan, corresponding to a segment of the aggrecan core protein from the G1 domain to the C-terminus NITEGE, consistent with "aggrecanase" activity. Release of the G1 fragment was inhibited by treatment with L-758,354. Both IL-1beta and RA induced significant loss of hyaluronan from cartilage explants after 8 days of exposure and HA loss was also inhibited by addition of L-756,354 to the culture media. IL-1beta, but not RA, induced a significant increase in swelling ratio (wet weight in 0.01 M NaCl normalized to wet weight in DMEM) after 8 days in culture, consistent with degradation of the collagen network, and the increase in tissue swelling was inhibited by treatment with TIMP-1 or L-758,354. Exposure to IL-1beta or RA resulted in significant changes in cartilage physical properties including streaming potential, equilibrium modulus, hydraulic permeability, and electrokinetic coupling coefficient after 8 days in culture, and these changes were inhibited by 40-90% by exposure to TIMP and by 50-90% by exposure to L-758,354. Measurement of dynamic streaming potential showed that changes due to treatment with IL-1beta alone were highly dependent in compression frequency, with dramatic changes seen at high frequency prior to changes in mechanical properties, and little initial change seen at low frequency. Streaming potential and equilibrium modulus of explants treated with RA decreased to 10% of their initial values after 8 days in culture, but decreased to only 40 and 90%, respectively, when treated with RA plus TIMP-1.

To examine the activity of matrix metalloproteinases (MMPs) and aggrecanase in control and diseased human articular cartilage, metabolic fragments of aggrecan were detected with monospecific antipeptide antibodies. The distribution and quantity of MMP-generated aggrecan G1 fragments terminating in VDIPEN341 were compared with the distribution of aggrecanase-generated G1 fragments terminating in NITEGE373. Both types of G1 fragments were isolated from osteoarthritic cartilage. The sizes were consistent with a single enzymatic cleavage in the interglobular domain region, with no further proteolytic processing of these fragments. Both neoepitopes were also detected by immunohistochemistry in articular cartilage from patients undergoing joint replacement for osteoarthritis (OA), rheumatoid arthritis (RA), and in cartilage from adults with no known joint disease. In control specimens, the staining intensity for both G1 fragments increased with age, with little staining in cartilage from 22-wk-old fetal samples. There was also an increase with age in the extracted amount of MMP-generated neoepitope in relation to both aggrecan and collagen content, confirming the immunohistochemical results. After the age of 20-30 yr this relationship remained at a steady state. The staining for the MMP-generated epitope was most marked in control cartilage exhibiting histological signs of damage, whereas intense staining for the aggrecanase-generated fragment was often noted in adult cartilage lacking overt histological damage. Intense staining for both neoepitopes appeared in the more severely fibrillated, superficial region of the tissue. Intense immunostaining for both VDIPEN- and NITEGE- neoepitopes was also detected in joint cartilage from patients with OA or RA. Cartilage in these specimens was significantly more degraded and high levels of staining for both epitopes was always seen in areas with extensive cartilage damage. The levels of extracted VDIPEN neoepitope relative to collagen or aggrecan in both OA and RA samples were similar to those seen in age-matched control specimens. Immunostaining for both types of aggrecan fragments was seen surrounding the cells but also further removed in the interterritorial matrix. In some regions of the tissue, both neoepitopes were found while in others only one was detected. Thus, generation and/or turnover of these specific catabolic aggrecan fragments is not necessarily coordinated. Our results are consistent with the presence in both normal and arthritic joint cartilage of proteolytic activity against aggrecan based on both classical MMPs and "aggrecanase."

Bovine cartilage explants were cultured with 1 mM 4-aminophenylmercuric acetate (APMA) to activate endogenous matrix metalloproteinases (MMPs) and changes in biochemical, biomechanical, and physicochemical properties were assessed. Additionally, graded levels of either rhTIMP-1 (recombinant human tissue inhibitor of metalloproteinases-1) or L-696-418 (a synthetic metalloproteinase inhibitor) were used to inhibit degradation induced by APMA. Treatment with APMA resulted in as much as 80% loss in tissue GAG content, a greater than threefold increase in denatured type II collagen as determined by the presence of CB11B epitope, and complete loss of biosynthetic activity after 3 days in culture. Physicochemical studies revealed that APMA treatment resulted in a significant increase in tissue swelling response, consistent with damage to the collagen network. Activation of MMPs by APMA also resulted in > 80% decrease in equilibrium modulus, dynamic stiffness, and streaming potential and > 50% decrease in electrokinetic coupling coefficient. The addition of 4 microM, 400 nM, and 40 nM TIMP inhibited PG loss by 95, 50, and 20%, respectively, and all doses effectively inhibited swelling response. The addition of 4 microM and 400 nM L-696-418 inhibited PG loss by 95% while 40 nM L-696-418 inhibited PG loss by 60%, and all doses effectively inhibited swelling response. The inhibition of APMA-induced GAG loss by 4 microM TIMP was accompanied by maintenance of streaming potential, electrokinetic coupling coefficient, dynamic stiffness, and equilibrium modulus.

Atherosclerotic plaque rupture may occur when regions of weakened extracellular matrix are subjected to increased mechanical stresses. Since collagen is a major determinant of extracellular matrix strength, enzymes that degrade collagen may play an important role in destabilizing the atherosclerotic lesion. To test the hypothesis that matrix metalloproteinase 1 (interstitial collagenase, or MMP-1), which initiates degradation of fibrillar collagens, colocalizes with increased stress in the fibrous cap of the atherosclerotic lesion, 12 unruptured human coronary lesions were studied. Finite-element analysis was used to determine the distribution of stress in the lesion, with estimates of material properties from previous measurements of human tissues. A computerized image analysis system was used to determine the distribution of immunoreactive MMP-1 within the fibrous tissue of the lesion. There was a significant correlation between immunoreactive MMP-1 and circumferential tensile stress in the fibrous cap within a given lesion (median Spearman rank correlation coefficient, .36; interquartile range, −.02 to .81; P

The concept of ligand bias at G protein-coupled receptors broadens the possibilities for agonist activities and provides the opportunity to develop safer, more selective therapeutics. Morphine pharmacology in β-arrestin-2 knockout mice suggested that a ligand that promotes coupling of the μ-opioid receptor (MOR) to G proteins, but not β-arrestins, would result in higher analgesic efficacy, less gastrointestinal dysfunction, and less respiratory suppression than morphine. Here we report the discovery of TRV130 ([(3-methoxythiophen-2-yl)methyl]({2-[(9R)-9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]ethyl})amine), a novel MOR G protein-biased ligand. In cell-based assays, TRV130 elicits robust G protein signaling, with potency and efficacy similar to morphine, but with far less β-arrestin recruitment and receptor internalization. In mice and rats, TRV130 is potently analgesic while causing less gastrointestinal dysfunction and respiratory suppression than morphine at equianalgesic doses. TRV130 successfully translates evidence that analgesic and adverse MOR signaling pathways are distinct into a biased ligand with differentiated pharmacology. These preclinical data suggest that TRV130 may be a safer and more tolerable therapeutic for treating severe pain.

Objective. To characterize the effects of intra-articular injection of recombinant human stromelysin (SLN) on the matrix composition and physical properties of cartilage from lapine stifle joints and the modulation of these effects by the systemic administration of an N-carboxyalkyl synthetic matrix metalloproteinase inhibitor, L-696,418. Methods. Female 6–8-week-old New Zealand white rabbits received an intraarticular injection of 100 μg activated SLN in 1 stifle joint and buffer in the contralateral control knee; these animals were killed after 1 hour. A separate group of animals received an intravenous injection of either 30 mg/kg L-696,418 or buffer prior to intraarticular injection of SLN. Joints were dissected and analyzed for proteoglycan (PG) loss into joint fluid, tissue biochemical composition, and histology by toluidine blue or anti-VDIPEN antibody staining, or were frozen for physical property analysis. Disks of femoropatellar groove cartilage were harvested from the stifle joint and tested in uniaxially confined compression for determination of electromechanical and mechanical properties. Results. Lapine stifle joints that received injection of SLN without systemic administration of L-696,418 showed a 13-fold increase in loss of PG into synovial fluid. Cartilage from these joints showed significant decreases in streaming potential at 1 Hz and electrokinetic coupling coefficient, but no change in equilibrium modulus, dynamic stiffness, or hydraulic permeability. Systemic treatment with L-696,418 resulted in a significant decrease in loss of PG into joint fluid and elimination of changes in cartilage highfrequency streaming potential and coupling coefficient in joints that were injected with SLN. Conclusion. The 1-hour exposure to SLN in vivo resulted in loss of PG and exposure of the VDIPEN epitope of the aggrecan core protein in the superficial region of the tissue near the articular surface. This highly localized degradation resulted in electromechanical behavior changes, but little or no change occurred in mechanical properties. Systemic administration of L-696,418 significantly decreased loss of PG from cartilage and prevented the highly localized tissue degradation and the resultant changes in electromechanical behavior caused by intraarticular SLN injection.

The destruction of articular cartilage in immune inflammatory arthritic disease involves the proteolytic degradation of its extracellular matrix. The role of activated matrix metalloproteinases (MMPs) in the chondrodestructive process was studied by identifying a selective cleavage product of aggrecan in murine arthritis models initiated by immunization with either type II collagen or proteoglycan. We conducted semiquantitative immunocytochemical studies of VDIPEN341 using a monospecific polyclonal antibody requiring the free COOH group of the COOH-terminal Asn for epitope detection. This antibody recognizes the aggrecan G1 domain fragment generated by MMP [i.e., stromelysin (SLN) or gelatinase A] cleavage of aggrecan between Asn341-Phe342 but does not recognize intact aggrecan. VDIPEN was undetectable in normal mouse cartilage but was observed in the articular cartilage (AC) of mice with collagen-induced arthritis 10 d after immunization, without histological damage and clinical symptoms. This aggrecan neoepitope was colocalized with high levels of glycosaminoglycans (GAGs) in pericellular matrices of AC chondrocytes but was not seen at the articular surface at this early time. Digestion of normal (VDIPEN negative) mouse paw cryosections with SLN also produced heavy pericellular VDIPEN labeling. Computer-based image analysis showed that the amount of VDIPEN expression increased dramatically by 20 d (70% of the SLN maximum) and was correlated with GAG depletion. Both infiltration of inflammatory cells into the synovial cavity and early AC erosion were also very prominent at this time. Analysis of adjacent sections showed that both induction of VDIPEN and GAG depletion were strikingly codistributed within sites of articular cartilage damage. Similar results occurred in proteoglycan-induced arthritis, a more progressive and chronic model of inflammatory arthritis. These studies demonstrate for the first time the MMP-dependent catabolism of aggrecan at sites of chondrodestruction during inflammatory arthritis.

Objective. To determine the effects of stromelysin treatment on biochemical, histologic, and swelling characteristics of intact cartilage explants and to correlate these effects with changes in the functional physical properties of the tissue. Methods. Bovine articular cartilage explants were cultured for up to 3 days in the presence or absence of recombinant human stromelysin (SLN). Damage to matrix proteoglycans and collagens was assessed and characterized by N-terminal sequencing and Western blot analysis, respectively. Explants were mechanically tested to assess the ability of the tissue to withstand cyclic and static compressive loads. Results. Treatment with SLN resulted in a time-and dose-dependent loss of proteoglycans from cartilage explants, with significant loss seen after 3 days of exposure to 20 nM SLN. Histology indicated that initial loss of proteoglycans occurred in regions near the tissue surface and proceeded inward with increasing time of SLN exposure. SLN treatment resulted in degradation of matrix collagen types IX and II, and a concomitant increase in tissue swelling. This matrix degradation resulted in severe alterations in functional physical properties of the tissue, including compressive stiffness. The initial, focal loss of proteoglycans that resulted from SLN treatment was most accurately detected with highfrequency streaming potential measurements. Conclusion. Exposure of intact cartilage to SLN caused specific, molecular-level degradation of matrix molecules, which resulted in changes in the swelling behavior and marked deterioration of functional physical properties of the tissue.

A rat chondrosarcoma cell line and primary bovine chondrocytes have been used to study cell-mediated aggrecan catabolism. Addition of 1 microM retinoic acid to chondrosarcoma cultures resulted in aggrecan proteolysis with the release of greater than 90% of the cell layer aggrecan into the medium within 4 days. NH2-terminal sequencing of chondroitin sulfate-substituted catabolic products gave a single major NH2-terminal sequence of ARGNVILTXK, initiating at Ala374. This showed that the proteinase, commonly referred to as "aggrecanase," which cleaves the Glu373-Ala374 bond of the interglobular domain of aggrecan (Sandy, J. D., Neame, P. J., Boynton, R. E., and Flannery, C. R. (1990) J. Biol. Chem. 266, 8683-8685), is active in this cell system. Aggrecan G1 domain, generated by cleavage of the interglobular domain, was also liberated during catabolism and this was characterized with three antipeptide antisera. Anti-CDAGWL was used as a general probe for G1 domain. Anti-FVDIPEN was used to specifically detect G1 domain with COOH terminus of Asn341, the form which is readily generated by cleavage of aggrecan by a wide range of matrix metalloproteinases. Anti-NITEGE antiserum was used to specifically detect G1 domain with COOH terminus of Gln373, the form which is the expected product of "aggrecanase"-mediated cleavage of aggrecan. Western blot analysis indicated that a single form of G1 domain of about 60 kDa was formed. G1 domain of this size reacted with both anti-CDAGWL and anti-NITEGE but not with anti-FVDIPEN. Similar experiments with primary bovine chondrocyte cultures, treated with either retinoic acid or interleukin 1, showed that two forms of catabolic G1 domain, of about 62 and 66 kDa, were formed. Both of these forms reacted on Western blots with anti-CDAGWL and also with anti-NITEGE. It is suggested that cell-mediated catabolism of the aggrecan interglobular domain in these culture systems, whether promoted by retinoic acid or interleukin 1, primarily involves cleavage of the Glu373-Ala374 bond by aggrecanase. The accumulation of G1 domain with a COOH-terminal of Glu373 shows that such aggrecanase-mediated cleavage can occur independent of the cleavage of the Asn341-Phe342 bond by matrix metalloproteinases.

Osteoarthritis (OA) involves the loss of the physiological balance between degradation and replenishment of intercellular matrix in the cartilage and other tissues of the joint. This uncoupling eventually results in changes in the structure of the affected joints, which may cause pain and physical disability. OA evolves slowly and is a heterogeneous condition. Concurrent with the slow natural progression of OA, the patient and the joint tissues age, inducing additional and confounding functional changes in the musculoskeletal system. The molecular signals regulating cither the breakdown of the matrix or its replenishment are unknown.

Aggrecan turnover is critically important to maintain extracellular matrix homeostasis in articular cartilage. Cartilage aggrecan metabolism has been studied in chondrocyte cell cultures, cartilage explant cultures, and in whole animal models. In many of these studies, matrix metalloproteinases (MMPs) are proposed to degrade cartilage aggrecan. MMP expression appears elevated in joint tissues from patients with osteoarthritis (OA) and rheumatoid arthritis (RA) (Dean et al. 1989, Wolfe et al. 1993) and inhibitors of both MMPs and thiol proteinases have been shown to block aggrecan (Buttle et al. 1992) and type II collagen (Mort et al. 1993) degradation in cartilage explant cultures. Using antibodies and cDNA probes, elevations in expression and concentration of many of these enzymes in animal models and in OA and RA have been described. Human cartilage aggrecan has now been cloned and sequenced (Doege et al. 1991). This information, in combination with the ability to sequence aggrecan and aggrecan fragment...

OBJECTIVE--To examine if unilateral knee injury affects the synovial fluid concentrations of aggrecan fragments, cartilage oligomeric matrix protein (COMP) fragments, stromelysin-1, and tissue inhibitor of metalloproteinases-1 (TIMP-1) in the contralateral uninjured knee. METHODS--Synovial fluids from the injured and uninjured knees were obtained at different times in a group of patients after unilateral knee trauma. Serum samples were obtained on the same occasion. Concentrations of aggrecan fragments were determined by precipitation with Alcian Blue; those of COMP fragments, stromelysin-1, and TIMP-1 were measured by immunoassay. Concentrations were compared with those in a reference group of 10 healthy volunteers. RESULTS--Immediately after knee injury, concentrations of aggrecan fragments, COMP fragments, stromelysin-1 and TIMP-1 were increased in the synovial fluid of the injured knee. However, concentrations of aggrecan and COMP fragments, and stromelysin-1 increased also in the contralateral uninjured knee immediately after injury, but less than in the injured knee. Subsequently, the concentrations of all markers decreased in the synovial fluid of the injured knee, but remained unchanged in the uninjured knee. The concentration of aggrecan fragments in the injured knee decreased to less than that in the uninjured knee in the chronic phase. Serum concentrations of COMP were much smaller than those in synovial fluid. CONCLUSIONS--The increased concentrations of aggrecan and COMP fragments and stromelysin-1 in the joint fluid of the contralateral, uninjured knee following unilateral knee injury, compared with concentrations in healthy reference knees, suggest changes in joint cartilage metabolism in both knees following unilateral knee injury. The mechanisms for these changes are unclear. The low serum concentration of COMP makes it less likely that there is any significant 'exchange' of molecular makers between the knees. A further consequence of these findings is that the contralateral knee cannot be recommended as the only control joint in studies of matrix metabolism in patients with unilateral knee injury.

Stromelysin-1, tissue inhibitor of metalloproteinases-1 (TIMP-1), and proteoglycan fragments were quantified in knee synovial fluid samples in a cross-sectional study of patients who had injury to the anterior cruciate ligament or the meniscus. The average concentrations of stromelysin-1 and TIMP-1 increased 25-fold and 10-fold within the first day after the trauma, respectively, and the concentration of proteoglycan fragments increased 4-fold. From approximately 1-6 months after injury, the levels of these markers were higher after injury to the cruciate ligament than after injury to the meniscus. From 6 months to 18 years after trauma, however, the levels of stromelysin-1 and TIMP-1 in patients who had an injury to the ligament were the same as the levels in patients who had a meniscal lesion, but the levels were increased compared with those for a reference group of healthy volunteers. The molar balance of stromelysin-1 to TIMP-1 in synovial fluid in both groups of injured joints changed from a balance representing an excess of free inhibitor in the normal joint to one representing an excess of free enzyme in the injured joint. The increased release of these markers to joint fluid both early and late after trauma may be caused by a change in the loading patterns in the knee with an injured ligament or meniscus or by synovitis induced by bleeding. The increased release may be associated with the frequent development of posttraumatic osteoarthritis in patients with these injuries.

Background— The angiotensin II type 1 receptor (AT1R) plays a key role in regulating cardiorenal function. Classic “unbiased” AT1R antagonists block receptor coupling to both G αq and ß-arrestin–mediated signals, which desensitize G-protein signaling as well as transduce G-protein–independent signals. TRV120027 is a novel ß-arrestin–biased AT1R ligand, which engages ß-arrestins while blocking G-protein signaling. At the AT1R, TRV120027 can inhibit angiotensin II–mediated vasoconstriction, whereas, through ß-arrestin coupling, increase cardiomyocyte contractility. We defined for the first time the acute cardiorenal actions of TRV120027 in healthy and heart failure (HF) canines. Methods and Results— Healthy and HF canines (induced by tachypacing) were anesthetized. After instrumentation and equilibration, a 30-minute baseline clearance was performed, followed by further clearance with escalating doses of intravenous TRV120027 (0.01, 0.1, 1, 10, and 100 μg/kg per minute) and a 30-minute washout. In healthy canines, TRV120027 decreased pulmonary capillary wedge pressure and systemic and renal vascular resistances, while increasing cardiac output, renal blood flow, glomerular filtration rate, and urinary sodium excretion. In HF canines, TRV120027 decreased mean arterial pressure, right atrial pressure, and pulmonary capillary wedge pressure, systemic and renal vascular resistances and increased cardiac output and renal blood flow. Glomerular filtration rate and urinary sodium excretion were maintained. Conclusions— We report for the first time the cardiorenal actions of the novel ß-arrestin–biased AT1R ligand TRV120027. In both normal and HF canines, TRV120027 demonstrated cardiac unloading actions while preserving renal function. With this beneficial pharmacological profile, TRV120027 represents a novel strategy for the treatment of HF.

The role of cytokines in the regulation of articular inflammation and cartilage degradation was evaluated in the rabbit model of Haemophilus influenzae type b arthritis. At 6 and 12 h after intraarticular infection, treatment with IB4 monoclonal antibody to the CD18 leukocyte receptor alone or in combination with dexamethasone resulted in significant reduction of synovial fluid (SF) neutrophil concentration. Treatment with dexamethasone alone was associated with lower SF concentrations of interleukin-1 (IL-1), tumor necrosis factor-alpha, and stromelysin than in other groups. At 24 h after infection, increased cartilage degradation was detected in untreated controls and in animals treated with IB4 alone or in combination with dexamethasone compared with those treated with dexamethasone alone. Multiple regression analyses indicated SF concentration of IL-1 and stromelysin as the significant predictors of cartilage degradation. These data suggest that IL-1 mediates cartilage degradation by regulation of metalloproteinases, such as stromelysin, during acute experimental bacterial arthritis.

Objective. To determine the concentrations of human stromelysin-1, collagenase, tissue inhibitor of metalloproteinases (TIMP), and proteoglycan fragments in knee synovial fluid in patients with injury to the meniscus or anterior cruciate ligament, posttraumatic osteoarthritis, primary osteoarthritis, or pyrophosphate arthritis. Methods. Synovial fluid samples were collected from patients with knee disease diagnosed arthroscopically and radiologically. Concentrations of stromelysin-1, collagenase, and TIMP-1 were determined by sandwich immunoassay, using monoclonal and polyclonal antibodies. Fragments of cartilage proteoglycan containing the chondroitin sulfate-binding region were determined by immunoassay with a polyclonal antibody. Results. Average concentrations of metalloproteinases, TIMP, and proteoglycan fragments in joint fluid were significantly elevated in patients from all disease groups as compared with volunteers with healthy knees (reference group). Stromelysin concentrations in disease groups averaged 15-45 times that of the reference group. The molar ratios between stromelysin and collagenase varied between 10 and 150. The molar ratio between total stromelysin and free TIMP was 0.5 in the reference group and between 1.6 and 5.3 in the disease groups. Conclusion. Stromelysin concentration in joint fluid is a parameter that distinguishes diseased joints from healthy joints, with a sensitivity of 84% and a specificity of 90%. The high concentrations of metalloproteinase relative to TIMP in joint fluid from patients with the conditions studied may be associated with cartilage matrix degradation in these arthritides. (Less)

In this study, we evaluated the in vitro and in vivo potency of human leukocyte elastase (HLE) and human cathepsin G (HCG) as proteoglycanases. In vitro evaluation was done using bovine nasal septum aggrecan and aggrecan/hyaluronan aggregate as substrates. Enzyme activity was assessed by the ability of the proteinases to abrogate the ability of aggrecan to aggregate with hyaluronan. In vivo activity of the proteinases was tested by injecting purified HLE and HCG intra-articularly into rabbit stifle joints and quantifying the levels of proteoglycan released into synovial fluids. On a molar basis, HCG was at least tenfold more potent than HLE as a proteoglycanase in vitro. Moreover, HCG was twofold more potent as a proteoglycanase in vivo. In contrast, HLE hydrolyzed elastin approximately 22-fold faster than HCG, but was only slightly more rapid than HCG when [3H]-transferrin was used as substrate. These data indicate that HCG is more potent than HLE as a proteoglycanase both in vitro and in vivo. Thus, HCG could be more important in the pathogenesis of rheumatoid arthritis than previously suspected.

Objective. To examine the in vivo expression of the matrix metalloproteinase stromelysin in the synovium and articular cartilage of rabbits injected intraarticularly with recombinant human interleukin-1β (IL-1). Methods. The direct isolation of messenger RNA (mRNA) from articular cartilage without the prior isolation of chondrocytes is described. The in vivo expression of stromelysin was examined at the mRNA level by Northern blot analysis, and at the protein level by in situ immunolocalization and by enzyme-linked immunosorbent assay. Results. In the synovium of IL-1–injected joints, stromelysin mRNA levels were highest at 4 hours and declined to background levels within 24 hours. In the cartilage of IL-1–injected joints, stromelysin mRNA was elevated at 4 hours and continued to increase until 8 hours, before declining. Stromelysin mRNA expression preceded a similar increase in stromelysin protein levels in both synovium and cartilage. Conclusion. Intraarticular injection of IL-1 induced the endogenous expression of stromelysin mRNA and protein in both synovium and cartilage. The kinetics of stromelysin expression correlated well with the accumulation of stromelysin and proteoglycan in synovial fluids. Therefore, the de novo synthesis of stromelysin in cartilage may have contributed to the loss of proteoglycan from that tissue.

Osteoarthritis is a major cause of disability and early retirement. Yet we lack the means to diagnose the disease in its early stages or to monitor the effects of treatment on the target tissue, the joint cartilage. Neither can we identify the disease mechanisms at the tissue or cell level. Current research focuses on the use of markers of cartilage matrix metabolism in body fluids as a means to diagnose and monitor osteoarthritis. Cartilage proteoglycan, collagen and glycoprotein fragments, as well as proteinases and their inhibitors, are being suggested for this purpose. Structural information on matrix molecule fragments released into body fluids may also help to identify the enzymes active in the destruction of the cartilage, a central issue in osteoarthritis.

One of the best studied animal models of osteoarthritis is a dog model in which the anterior cruciate ligament of the hind limb stifle joint is transected (Pond-Nuki model). To determine whether stromelysin might play a role in this model, it was necessary to purify the enzyme for production of suitable probes. In the present study, dog synovial fibroblasts were stimulated to express a metalloproteinase that was demonstrated to be canine prostromelysin by Northern blot, protein purification and amino-terminal sequence analyses. Unlike rabbit synoviocytes, passaged dog synoviocytes did not express stromelysin mRNA in response to recombinant human IL-1, but expressed stromelysin mRNA only upon treatment with dog monocyteconditioned medium (dMCM). The aminophenylmercuric acetate (APMA)-activatable metalloproteinase present in the culture supernatants of stimulated dog synoviocytes was purified using a combination of ion-exchange and dye matrix affinity chromatography. The purified canine metalloproteinase co-migrated on reducing SDS-PAGE with recombinant human prostromelysin-1 as a doublet with apparent molecular masses of 54 and 56 kDa. Similar to APMA-activated human prostromelysin-1, the APMA-activated canine metalloproteinase was inhibited by 1,10- phenanthroline or recombinant human tissue inhibitor of metalloproteinases (TIMP). The amino-terminal sequences of the canine pro- and APMA -activated enzymes were compared with those of human, rabbit and rat stromelysin. The striking homologies among the sequences demonstrated that the purified canine metalloproteinase was indeed canine prostromelysin. A rabbit anti-canine prostromelysin polyclonal antiserum was generated and used to localize the enzyme within cultured dog synoviocytes and articular cartilage stimulated with dMCM. The reagents developed in this study should be useful for examining the expression and distribution of prostromelysin in canine models of osteoarthritis.

Products generated by the digestion of human aggrecan with recombinant human stromelysin have been purified and analyzed by N-terminal sequencing and C-terminal peptide isolation. N-terminal analysis of chondroitin sulfate-bearing fragments revealed a clearly identifiable sequence initiating at residue Phe342 of human aggrecan, providing evidence for a cleavage site at the Asn341-Phe342 bond located within the interglobular domain. This cleavage site, which separates the G1 domain from the remainder of the molecule, was confirmed by isolation from the liberated G1 domain of a C-terminal tryptic peptide with the sequence YDAICYTGEDFVDIPEN (in which the C-terminal residue is Asn341). This peptide was also isolated from tryptic digests of hyaluronan-binding proteins (A1D4 samples) prepared by CsCl gradient centrifugation of extracts of mature human articular cartilages. Since these A1D4 samples contain G1 domain which accumulates as a result of aggrecan catabolism in vivo, these results clearly indicate that stromelysin cleaves the Asn341-Phe342 bond of human aggrecan in situ.

Primary and passaged human synovial fibroblasts isolated from rheumatoid pannus were treated with recombinant interleukin-1 (IL-1) alpha or beta, tumor necrosis factor-alpha (TNF), or phorbol myristate acetate (PMA) to determine the effects of these stimuli on the relative expression of stromelysin, collagenase, and tissue inhibitor of metalloproteinases (TIMP). The steady-state mRNA levels for these genes and glyceraldehyde-3-phosphate dehydrogenase were determined on Northern blots. Immunoblot analyses of the conditioned media using monoclonal antibodies generated against recombinant human stromelysin, collagenase, or TIMP showed that protein levels reflected the corresponding steady-state mRNA levels. The results revealed that 1) stromelysin and collagenase were not always coordinately expressed; 2) IL-1 was more potent than TNF or PMA in the induction of stromelysin expression; 3) neither IL-1 nor TNF significantly affected TIMP expression; 4) PMA induced both metalloproteinase and TIMP expression; and 5) the combination of IL-1 plus TNF had a synergistic effect on stromelysin expression. Dose response and time course experiments demonstrated that the synergistic effect of IL-1 plus TNF occurred at saturating concentrations of each cytokine and lasted for 7 days. In summary, the ability of IL-1 and TNF to preferentially induce stromelysin and collagenase expression, versus TIMP, may define a pivotal role for these cytokines in the pathogenesis of rheumatoid arthritis.