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To preclinically characterize a mutant form of growth and differentiation factor 5, R399E, with reduced osteogenic properties as a potential disease-modifying osteoarthritis (OA) drug.Cartilage, synovium, and meniscus samples from patients with OA were used to evaluate anabolic and antiinflammatory properties of R399E. In the rabbit joint instability model, 65 rabbits underwent transection of the anterior cruciate ligament plus partial meniscectomy. Three intraarticular (IA) R399E doses were administered biweekly 6 times, and static incapacitance was determined to assess joint pain. OA was evaluated 13 weeks after surgery. In sheep, medial meniscus transection was performed to induce OA, dynamic weight bearing was measured in-life, and OA was assessed after 13 weeks.Intermittent exposure to R399E (1 week per month) was sufficient to induce cell proliferation and release of anabolic markers in 3-dimensional chondrocyte cultures. R399E also inhibited the release of interleukin-1β (IL-1β), IL-6, and prostaglandin ER399E influences several pathologic processes contributing to OA, highlighting its potential as a disease-modifying therapy.

To investigate acute changes in biochemical markers of bone and cartilage turnover in response to moderate intensity exercise with and without joint impact in healthy human subjects. A randomized, cross-over, exploratory, clinical study was conducted. Twenty healthy subjects with no history of joint trauma completed 30 min interventions of standardized moderate intensity cycling and running as well as a resting intervention 1 week apart. Blood samples were taken immediately before, four times after exercise and again the next day. Urine was sampled, before, after and the next day. On the day of rest, samples were taken at timepoints similar to the days of exercise. Markers of type I (CTX-I), II (C2M, CTX-II) and VI (C6M) collagen degradation, cartilage oligomeric matrix protein (COMP) and procollagen C-2 (PRO-C2) was measured. Trial registration number: NCT04542655, 02 September 2020, retrospectively registered. CTX-I was different from cycling (4.2%, 95%CI: 0.4–8.0%, p = 0.03) and resting (6.8%, 95%CI: 2.9–10.7%, p = 0.001) after running and the mean change in COMP was different from cycling (10.3%, 95%CI: 1.1–19.5%, p = 0.03), but not from resting (8.6%, 95%CI: − 0.7–17.8%, p = 0.07) after running. Overall, changes in other biomarkers were not different between interventions. In this exploratory study, running, but not cycling, at a moderate intensity and duration induced acute changes in biomarkers of bone and cartilage extra-cellular matrix turnover.

To examine the effect of dynamic compressive loading applied intermittently on bovine cartilage explants stimulated with proinflammatory cytokines over 21 days.Cartilage explants were cultured for 21 days with Oncostatin M and TNFα (O ​+ ​T) [10/5 ​ng/mL] or in culture medium alone (w/o). The explants were either left free-swelling or subjected to dynamic compressive loading of 20 ​min, at 1 ​Hz, with loads ranging between 0.1 and 1 ​MPa, 5 times weekly. Metabolic activity was measured once weekly using Alamar Blue and cartilage turnover was assessed with biomarkers targeting degradation fragments of aggrecan (AGNx1) and type II collagen (C2M). Glycosaminoglycan degradation was quantified was the DMMB assay. Furthermore, explant weight and histological analysis was used to assess the cartilage degradation.Dynamic compression of cartilage explants attenuated the O ​+ ​T-mediated C2M release on day 21 with 40% (p ​= ​0.0068) compared to the unloaded explants. Additionally, the change in explant weight from day -1 to day 21 showed that O ​+ ​T stimulation alone mediated a cartilage loss of 11%, whereas O ​+ ​T-stimulated explants subjected to compressive loading demonstrated a decreased cartilage weight loss of 6%, which was supported by the histological analysis. However, loading had no effect on aggrecan degradation.In cartilage explants cultured in a proinflammatory milieu, dynamic compressive loading confers anti-catabolic effects, inhibiting type II collagen degradation and reducing explant cartilage loss. These results demonstrate that compressive loading alters cartilage tissue turnover and enforces the need to include mechanical loading in a translation

Objective Currently, there are no disease-modifying osteoarthritis drugs (DMOADs) approved for osteoarthritis. It is hypothesized that a subtype of OA may be driven by inflammation and may benefit from treatment with anti-inflammatory small molecule inhibitors adopted from treatments of rheumatoid arthritis. This study aimed to investigate how small molecule inhibitors of intracellular signaling modulate cartilage degradation and formation as a pre-clinical model for structural effects. Design Bovine cartilage explants were cultured with oncostatin M (OSM) and tumour necrosis factor α (TNF-α) either alone or combined with the small molecule inhibitors: SB203580 (p38 inhibitor), R406 (Spleen tyrosine kinase (Syk) inhibitor), TPCA-1 (Inhibitor of κB kinase (Ikk) inhibitor), or Tofacitinib (Tofa) (Janus kinases (Jak) inhibitor). Cartilage turnover was assessed with the biomarkers of degradation (AGNx1 and C2M), and type II collagen formation (PRO-C2) using ELISA. Explant proteoglycan content was assessed by Safranin O/Fast Green staining. Results R406, TPCA-1 and Tofa reduced the cytokine-induced proteoglycan loss and decreased AGNx1 release 3.7-, 43- and 32-fold, respectively. SB203580 showed no effect. All inhibitors suppressed C2M at a concentration of 3 µM. TPCA-1 and Tofa increased the cytokine reduced PRO-C2 3.5 and 3.7-fold, respectively. Conclusion Using a pre-clinical model we found that the inhibitors TPCA-1 and Tofa inhibited cartilage degradation and rescue formation of type II collagen under inflammatory conditions, while R406 and SB203580 only inhibited cartilage degradation, and SB203580 only partially. These pre-clinical data suggest that TPCA-1 and Tofa preserve and help maintain cartilage ECM under inflammatory conditions and could be investigated further as DMOADs for inflammation-driven osteoarthritis.

Osteoarthritis (OA) and rheumatoid arthritis (RA) are serious and painful diseases. Protease-activated receptor 2 (PAR2) is involved in the pathology of both OA and RA including roles in synovial hyperplasia, cartilage destruction, osteophyogenesis and pain. PAR2 is activated via cleavage of its N-terminus by serine proteases. In this study a competitive ELISA assay was developed targeting the 36-amino acid peptide that is cleaved and released after PAR2 activation. Technical assay parameters including antibody specificity, intra- and inter-assay variation (CV%), linearity, accuracy, analyte stability and interference were evaluated. PAR2 pro-fragment release was confirmed after in vitro cleavage of PAR2 recombinant protein and treatment of human synovial explants with matriptase. Serum levels of 22 healthy individuals, 23 OA patients and 15 RA patients as well as a subset of RA patients treated with tocilizumab were evaluated. The PAR2 antibody was specific for the neo-epitope and intra-inter assay CV% were 6.4% and 5.8% respectively. In vitro cleavage and matriptase treated explants showed increased PAR2 pro-fragment levels compared to controls. In serum, PAR2 pro-fragment levels were increased in RA patients and decreased in RA patients treated with tocilizumab. In conclusion, PAR2 may be a potential biomarker for monitoring RA disease and pharmacodynamics of treatment.

Background Tissue released blood-based biomarkers can provide insight into drug mode of action and response. To understand the changes in extracellular matrix turnover, we analyzed biomarkers associated with joint tissue turnover from a phase 3, randomized, placebo-controlled study of baricitinib in patients with active rheumatoid arthritis (RA). Methods Serum biomarkers associated with synovial inflammation (C1M, C3M, and C4M), cartilage degradation (C2M), bone resorption (CTX-I), and bone formation (osteocalcin) were analyzed at baseline, and weeks 4 and 12, from a subgroup of patients (n = 240) randomized to placebo or 2-mg or 4-mg baricitinib (RA-BUILD, NCT01721057). Mixed-model repeated measure was used to identify biomarkers altered by baricitinib. The relationship between changes in biomarkers and clinical measures was evaluated using correlation analysis. Results Treatment arms were well balanced for baseline biomarkers, demographics, and disease activity. At week 4, baricitinib 4-mg significantly reduced C1M from baseline by 21% compared to placebo (p p p p Conclusion Baricitinib treatment resulted in reduced circulating biomarkers associated with joint tissue destruction as well as concomitant RA clinical improvement. Trial registration ClinicalTrials.gov NCT01721057; date of registration: November 1, 2012

To investigate acute changes in biochemical markers of bone and cartilage turnover in response to moderate intensity exercise with and without joint impact in healthy human subjects.A randomized, cross-over, exploratory, clinical study was conducted. Twenty healthy subjects with no history of joint trauma completed 30 min interventions of standardized moderate intensity cycling and running as well as a resting intervention 1 week apart. Blood samples were taken immediately before, four times after exercise and again the next day. Urine was sampled, before, after and the next day. On the day of rest, samples were taken at timepoints similar to the days of exercise. Markers of type I (CTX-I), II (C2M, CTX-II) and VI (C6M) collagen degradation, cartilage oligomeric matrix protein (COMP) and procollagen C-2 (PRO-C2) was measured.NCT04542655, 02 September 2020, retrospectively registered.CTX-I was different from cycling (4.2%, 95%CI: 0.4-8.0%, p = 0.03) and resting (6.8%, 95%CI: 2.9-10.7%, p = 0.001) after running and the mean change in COMP was different from cycling (10.3%, 95%CI: 1.1-19.5%, p = 0.03), but not from resting (8.6%, 95%CI: - 0.7-17.8%, p = 0.07) after running. Overall, changes in other biomarkers were not different between interventions.In this exploratory study, running, but not cycling, at a moderate intensity and duration induced acute changes in biomarkers of bone and cartilage extra-cellular matrix turnover.

Sprifermin, recombinant human fibroblast growth factor 18 (rhFGF18), induces cartilage regeneration in knees of patients with osteoarthritis (OA). We hypothesized that a temporal multiphasic process of extracellular matrix (ECM) degradation and formation underlie this effect. We aimed to characterize the temporal ECM remodeling of human knee OA articular cartilage in response to sprifermin treatment. Articular cartilage explants from patients with knee OA (npatients = 14) were cultured for 70 days, with permanent exposure to sprifermin (900, 450, 225 ng/mL), FGF18 (450 ng/mL), insulin-like growth factor-1 (100 ng/mL, positive control) or vehicle (nreplicates/treatment/patient = 2). Metabolic activity (AlamarBlue) and biomarkers of type IIB collagen (PIIBNP) formation (Pro-C2 enzyme-linked immunosorbent assay [ELISA]) and aggrecanase-mediated aggrecan neo-epitope NITEGE (AGNx1 ELISA) were quantified once a week. At end of culture (day 70), gene expression (quantitative reverse transcription polymerase chain reaction) and proteoglycan content (Safranin O/Fast green staining) were quantified. The cartilage had continuously increased metabolic activity, when treated with sprifermin/FGF18 compared to vehicle. During days 7–28 PIIBNP was decreased and NITEGE was increased, and during days 35–70 PIIBNP was increased. At end of culture, the cartilage had sustained proteoglycan content and relative expression of ACAN

Osteoarthritis (OA) is the most common form of joint disease. This presents the OA research community and pharmaceutical companies developing disease-modifying osteoarthritis drugs (DMOADs) with great opportunities.. The different OA subtypes complicates the traditional approaches for developing new treatments. If we can identify new markers that can distinguish different subtypes this can greatly facilitate drug development from early discovery to late clinical development. Nevertheless, the current approaches result in poorly targeted treatments and the inability to recruit the right patients for clinical trials. Thus, there is an urgent medical need for objective biomarker tools for patient phenotyping.

There is an increasing demand for noninvasive and descriptive biochemical markers (biomarkers) in osteoarthritis; for enabling early drug development (including translational research), evaluating clinical trial at an early stage and for subtyping. Purpose of the review is to review and comment on current availability of such biomarkers.Many different biomarkers have been tested in the last 18 months. The main focus has been on testing whether the biomarkers, whether is reflect joint tissue turnover or inflammatory status, can differentiate osteoarthritis patients from healthy controls or whether the biomarkers are associated with progression. Less than a handful of studies, investigate the role of the biomarkers as response markers. Thus, there is still a great need for developing biomarkers that reflect disease activity and thereby can be used for treatment response or patient phenotyping.Osteoarthritis is the most common form of joint disease. This presents the osteoarthritis research community and pharmaceutical companies developing disease-modifying osteoarthritis drugs (DMOADs) with great opportunities. There are different osteoarthritis subtypes, which complicates the traditional approaches for developing new treatments. If we can identify new markers that can distinguish different subtypes, this can greatly facilitate drug development from early discovery to late clinical development.

Objectives: There is an unmet medical need for biomarkers in OA which can be applied in clinical drug development trials. The present study describes the development of a specific and robust assay measuring type II collagen degradation (T2CM) and discusses its potential as a noninvasive translational biomarker. Methods: A type II collagen specific neoepitope (T2CM) was identified by mass spectrometry and monoclonal antibodies were raised towards the epitope, employed in a chemiluminescence immunoassay. T2CM was assessed in bovine cartilage explants with or without MMP-13 inhibitor, and explant supernatants were analyzed by Western blot. T2CM was measured in plasma samples from one study (n ​= ​48 patients) where OA patients were referred to total knee replacement (TKR). Additionally, T2CM was quantified in serum from OA patients receiving salmon calcitonin treatment (sCT) (n ​= ​50) compared to placebo (n ​= ​57). Results: The T2CM assay was technically robust (13/4 ​% inter/intra-variation) and specific for the type II collagen fragment cleaved by MMP-1 and -13. The MMP-13 inhibitor reduced the T2CM release from bovine cartilage explants receiving catabolic treatment. These results were confirmed by Western blot. In human end-stage OA patients (scheduled for TKR), the T2CM levels were elevated compared to moderate OA (p

Objectives Here we tested the hypothesis that human M-CSF (hM-CSF) overexpressed in CB CD34+ cells would induce differentiation and survival of monocytes and osteoclasts in vitro and in vivo. Methods Human M-CSF was overexpressed in cord blood CD34+ cells using a lentiviral vector. Results We show that LV-hM-CSF-transduced CB CD34+ cells expand 3.6 and 8.5 fold more with one or two exposures to the hM-CSF expressing vector, respectively, when compared to control cells. Likewise, LV-hM-CSF-transduced CB CD34+cells show significantly higher levels of monocytes. In addition, these cells produced high levels of hM-CSF. Furthermore, they are able to differentiate into functional bone resorbing osteoclasts in vitro. However, osteoclast differentiation and bone resorption was blunted compared to control CD34+ cells receiving exogenous hM-CSF. NSG mice engrafted with LV-hM-CSF-transduced CB CD34+ cells have physiological levels of hM-CSF production that result in an increase in the percentage of human monocytes in peripheral blood and bone marrow as well as in the spleen, lung and liver. Conclusion In summary, ectopic production of human M-CSF in CD34+ cells promotes cellular expansion and monocyte differentiation in vitro and in vivo and allows for the formation of functional osteoclasts, albeit at reduced levels, without an exogenous source of M-CSF, in vitro. This article is protected by copyright. All rights reserved.

Purpose: Exercise therapy with a focus on muscle strengthening has proven to be effective for the management of knee osteoarthritis (OA). Yet it is not known whether high-intensity resistance training (RT) is more effective in improving upper leg muscle strength and physical performance than low-intensity RT. Still, there is some controversy regarding the effectiveness of high-intensity RT and whether or not it is harmful, for instance by accelerating cartilage degeneration, osteophyte formation, or increasing synovitis. Any catabolic or anabolic response of musculoskeletal tissue to RT might first be visible on a biochemical level before changes in clinical symptoms are measurable. Serum biomarkers can objectively measure early biochemical changes and assess whether RT leads to a more anabolic or catabolic response. The aim of this study is to assess (i) whether high-intensity RT elicits a different response (e.g. catabolic) on systemic inflammation and musculoskeletal tissues in and surrounding the joint, including bone, cartilage, muscle, and synovial tissue compared to low-intensity RT; and (ii) whether there is an association between changes in serum levels of inflammatory and musculoskeletal tissue-derived biomarkers and improvements in clinical outcomes (performance-based tests and self-reported outcomes on pain and activity limitations).Methods: In a randomized controlled trial, 177 participants with knee OA conducted a high-intensity (70%-80% of the Repetition Maximum (1RM)) or low-intensity (40%-50% 1RM) RT program 3 times a week for 12 weeks. Measures of clinical outcomes and serum samples were collected at the start of RT (pre-intervention), after 3 months at the end of RT (post-intervention), and 6 months after RT (follow-up). As a reflection of systemic inflammation (CRP), synovitis (CRPM, C3M), bone turnover (OC, CTX-I), cartilage turnover (PRO-C2, C2M, huARGS), muscle turnover (PRO-C3, PRO-C6), and cell behaviour (col10neo) a total of eleven serum biomarkers were analysed. With the exception of CRP, which was determined with an immunoturbidimetric assay, ELISA assays were used to quantify serum levels of the other 10 serum biomarkers. The primary outcome measures are the changes in serum biomarker levels. Other outcome measures include upper leg muscle strength, performance-based tests, and self-reported outcomes on pain and activity limitations.Results: High-intensity RT resulted in greater improvements in muscle strength compared to low-intensity RT when measured by the estimated 1RM. No significant differences between groups were found for upper leg muscle strength (Nm/kg) when measured with an isokinetic dynamometer. Both groups showed similar improvements in pain and physical functioning. Although there is no difference between groups in clinical outcomes, except for the estimated 1RM, we expect that participants in the high-intensity RT group are more likely to have enhanced serum levels of catabolic biomarkers than participants in the low-intensity RT group. Since both the high-intensity RT group and low-intensity RT group improved over time, we expect that changes in serum biomarker levels are associated with overall improvements in clinical outcomes. Almost all participants had normal CRP values (Conclusions: The work to date on CRP serum levels suggests that RT did not influence CRP levels. This result may be explained by the high percentage of participants with normal CRP levels (

Ex vivo culture systems cover a broad range of experiments dedicated to studying tissue and cellular function in a native setting. Cartilage is a unique tissue important for proper function of the synovial joint and is constituted by a dense extracellular matrix (ECM), rich in proteoglycan and type II collagen. Chondrocytes are the only cell type present within cartilage and are widespread and relatively low in number. Altered external stimuli and cellular signalling can lead to changes in ECM composition and deterioration, which are important pathological hallmarks in diseases such as osteoarthritis (OA) and rheumatoid arthritis. Ex vivo cartilage models allow 1) profiling of chondrocyte mediated alterations of cartilage tissue turnover, 2) visualizing the cartilage ECM composition, and 3) chondrocyte rearrangement directly in the tissue. Profiling these alterations in response to stimuli or treatments are of high importance in various aspects of cartilage biology, and complement in vitro experiments in isolated chondrocytes, or more complex models in live animals where experimental conditions are more difficult to control. Cartilage explants present a translational and easily accessible method for assessing tissue remodeling in the cartilage ECM in controllable settings. Here, we describe a protocol for isolating and culturing live bovine cartilage explants. The method uses tissue from the bovine knee, which is easily accessible from the local butchery. Both explants and conditioned culture medium can be analyzed to investigate tissue turnover, ECM composition, and chondrocyte function, thus profiling ECM modulation.

Summary Objective Links between pain and joint degradation are poorly understood. We investigated the role of activation of Toll-like receptors (TLR) by cartilage metabolites in initiating and maintaining the inflammatory loop in OA causing joint destruction. Methods Synovial membrane explants (SMEs) were prepared from OA patients’ synovial biopsies. SMEs were cultured for 10 days under following conditions: culture medium alone, OSM + TNFα, TLR2 agonist - Pam2CSK4, Pam3CSK4 or synthetic aggrecan 32-mer, TLR4 agonist - Lipid A. Release of pro-inflammatory and degradation biomarkers (acMMP3 and C3M) were measured by ELISA in conditioned media along with IL-6. Additionally, human cartilage was digested with ADAMTS-5, with or without the ADAMTS-5 inhibiting nanobody - M6495. Digested cartilage solution (DCS) and synthetic 32-mer were tested for TLR activation in SEAP based TLR reporter assay. Results Western blotting confirmed TLR2 and TLR4 in untreated OA synovial biopsies. TLR agonists showed an increase in release of biomarkers - acMMP3 and C3M in SME. Synthetic 32-mer showed no activation in the TLR reporter assay. ADAMTS-5 degraded cartilage fragments activated TLR2 in vitro. Adding M6495 – an anti-ADAMTS-5 inhibiting nanobody®, blocked ADAMTS-5-mediated DCS TLR2 activation. Conclusion TLR2 is expressed in synovium of OA patients and their activation by synthetic ligands causes increased tissue turnover. ADAMTS-5-mediated cartilage degradation leads to release of aggrecan fragments which activates the TLR2 receptor in vitro. M6495 suppressed cartilage degradation by ADAMTS-5, limiting the activation of TLR2. In conclusion, pain and joint destruction may be linked to generation of ADAMTS-5 cartilage metabolites.

Background: Patients with joint diseases such as osteoarthritis (OA) are believed to have an abundance of endogenous Toll like receptor (TLR) ligands in their joints, which might be responsible for activating TLRs that may ultimately initiate a self-perpetuating inflammatory loop in the disease. TLR ligands may be generated from the breakdown of articular cartilage, which in turn arises from the activity of two key enzymes: A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5) and Matrix metalloproteinases (MMPs). Objectives: In this study, we investigate the effect of M6495, a novel anti ADAMTS-5 inhibiting nanobody® on TLR2 activation by ADAMTS-5 derived cartilage cleaved fragments Methods: Human cartilage biopsies were retrieved from OA patients undergoing total knee replacement. The tissue cartilage was snap frozen in liquid nitrogen to eliminate underlying metabolic activity and then digested with recombinant human ADAMTS-5 (4 μg/ml) for 24, 48 and 72 hours respectively at 37 oC. The undigested remaining cartilage was discarded after each timeframe and the digested cartilage solution (DS) was used for further testing. Tissue cleavage was assessed by measuring the release of aggrecan degradation biomarkers: Aggrecanase mediated aggrecan degradation (AGNx1) and MMP mediated aggrecan degradation (FFGV). DS was tested for TLR activation in a secreted embryonic alkaline phosphatase (SEAP) reporter gene based HEK hTLR2 (human Toll like receptors) cell line. Cartilage tissue in buffer alone was used as control at each time point. Results: Aggrecan degradation in cartilage was confirmed by increased release of AGNx1 (p Conclusion: ADAMTS-5-mediated cartilage degradation leads to release of aggrecan fragments, which activate the TLR2 receptor in vitro in a specialised reporter system. Anti-ADAMTS-5 inhibiting nanobody®, M6495, showed a suppression in release of degradation biomarkers leading to limited activation of TLR2. The data suggest a potential chondro-protective effect by M6495. Disclosure of Interests: Neha Sharma: None declared, Christian Thudium Employee of: I am a full time employee of Nordic Bioscience, Morten Karsdal Shareholder of: I own shares of Nordic Bioscience, Employee of: I am a full-time employee in Nordic Bioscience, Anne-Christine Bay-Jensen Shareholder of: I own shares of Nordic Bioscience, Employee of: I am a full-time employee in Nordic Bioscience, Thorbjorn Gantzel: None declared, Martin Michaelis Employee of: Employee of Merck KGaA, Darmstadt, Germany, Christoph Ladel Employee of: Employee of Merck KGaA, Darmstadt, Germany, Daniela Werkmann Employee of: Employee of Merck KGaA, Darmstadt, Germany, Sven Lindemann Employee of: Employed by Merck KGaA

Summary Objective A hallmark of osteoarthritis (OA) is degradation of articular cartilage proteoglycans. In isolated human OA chondrocytes, the anti-inflammatory cytokine Interleukin-37 (IL-37) lowers the expression of the proteolytic MMP and ADAMTS enzymes, which mediate this degradation. Therefore, we investigated if IL-37 protects against proteoglycan loss in freshly obtained human OA explants. Material and methods Human OA cartilage explants were incubated with IL-37. Release of sulphated proteoglycans (sGAGs) was measured with the dimethylmethylene-blue assay. Production and degradation of newly synthesized proteoglycans was measured using 35S-sulphate. Proteoglycan and proteolytic enzyme expression were analyzed by qPCR and Western Blot. Proteolytic activity was determined by measuring MMP- and ADAMTS-generated aggrecan neo-epitopes with ELISA and by using MMP-3-, MMP-13- or ADAMTS-5-inhibitors. Results Over time, a linear release of sGAGs from OA cartilage was measured. IL-37 reduced this release by 87 μg/ml (24%) 95%CI [21.04–141.4]. IL-37 did not affect 35S-sulphate incorporation or proteoglycan gene expression. In contrast, IL-37 reduced loss of 35S-sulphate labeled GAGs and reduced MMP-3 protein expression, indicating that IL-37 inhibits proteoglycan degradation. Remarkably, we observed two groups of patients; one group in which MMP-3-inhibition lowered sGAG release, and one group in which ADAMTS5-inhibition had this effect. Remarkably, IL-37 was only functional in the group of patients that responded to MMP-3-inhibition. Conclusion We identified a relationship between IL-37 and reduced sGAG loss in OA cartilage. Most likely, this effect is mediated by inhibition of MMP-3 expression. These results suggest that IL-37 could be applied as therapy in a subgroup of OA patients, in which cartilage degradation is mediated by MMP-3.

Background Pain is a debilitating symptom of rheumatoid arthritis (RA), caused by joint inflammation and cartilage and bone destruction. Nonsteroidal anti-inflammatory drugs (NSAIDs) are used to treat pain and inflammation in RA, but are not disease-modifying and do not prevent joint destruction when administered alone. KBPs (Key Bioscience peptides) are synthetic peptides based on salmon calcitonin and are expected to inhibit bone resorption and to be chondroprotective. In this study, we investigated if combining a standard of care NSAID (naproxen) with a KBP resulted in improvement in pain scores, as well as disease activity and structural damage in a rat model of RA. Methods Collagen-induced arthritis (CIA) was induced in 40 female Lewis rats by immunization with porcine type II collagen; 10 rats were given sham injections. CIA rats were treated with KBP and/or naproxen. Health scores and joint scores were evaluated daily. Mechanical and cold allodynia tests and burrowing tests were used to assess pain-like behaviors. Blood samples were collected for biomarker testing, and paws were collected for histology and microcomputed tomography. Results Naproxen monotherapy increased the time until humane endpoints was reached, and improved health score, pain assessments, and trabecular thickness, while KBP monotherapy did not result in improvements. Combination therapy had improved efficacy over naproxen monotherapy; combination therapy resulted in improved health scores, and importantly reduced mechanical and cold allodynia assessment. Furthermore, protection of articular cartilage structure and preservation of bone structure and bone volume were also observed. Conclusions This study demonstrates that combining KBP and naproxen may be a relevant therapeutic strategy for RA, resulting in improvements to the overall health, pain, inflammation, and joint structure. Electronic supplementary material The online version of this article (10.1186/s13075-019-1819-9) contains supplementary material, which is available to authorized users.

Little is known about local and systemic biomarkers in relation to synovitis and pain in end-stage osteoarthritis (OA) patients. We investigated the associations between the novel extracellular matrix biomarker, C1M, and local and systemic interleukin 6 (IL-6) with synovitis and pain. Serum C1M, plasma and synovial fluid IL-6 (p-IL-6, sf-IL-6) were measured in 104 end-stage knee OA patients. Contrast-enhanced magnetic resonance imaging (MRI) was used to semi-quantitatively assess an 11-point synovitis score; pain was assessed by the Western Ontario & McMaster Universities Osteoarthritis Index (WOMAC) and the Neuropathic Pain Questionnaire (NPQ). Linear regression was used to investigate associations between biomarkers and synovitis, and biomarkers and pain while controlling for age, sex and body mass index. We also tested whether associations between biomarkers and pain were confounded by synovitis. We found sf-IL-6 was associated with synovitis in the parapatellar subregion (B=0.006; 95% CI 0.003-0.010), and no association between p-IL-6 and synovitis. We also observed an association between C1M and synovitis in the peri-ligamentous subregion (B=0.013; 95% CI 0.003-0.023). Further, sf-IL-6, but not p-IL-6, was significantly associated with pain, WOMAC (B=0.022; 95% CI 0.004-0.040) and NPQ (B=0.043;95% CI 0.005-0.082). There was no association between C1M and WOMAC pain but we did find an association between C1M and NPQ (B=0.229; 95% CI 0.036-0.422). Lastly, synovitis explained both biomarker-NPQ associations, but not the biomarker-WOMAC association. These results suggest C1M and IL-6 are associated with synovitis and pain, and synovitis is an important confounding variable when studying biomarkers and neuropathic features in OA patients., 19 pages, 2 figures, 5 tables and 2 supplementary figures

Spondyloarthritis (SpA) is an umbrella term describing a family of chronic inflammatory rheumatic diseases. These diseases are characterised by inflammation of the axial skeleton, peripheral joints, and entheseal insertion sites throughout the body which can lead to structural joint damage including formation of axial syndesmophytes and peripheral osteophytes. Genetic evidence, preclinical and clinical studies indicate a clear role of interleukin (IL)- 23 and IL-17 as mediators in SpA pathogenesis. Targeting the IL-23/−17 pathways seems an efficient strategy for treatment of SpA patients, and despite the remaining challenges the pathway holds great promise for further advances and improved therapeutic opportunities. Much research is focusing on serological markers and imaging strategies to correctly diagnose patients in the early stages of SpA. Biomarkers may facilitate personalised medicine tailored to each patient's specific disease to optimise treatment efficacy and to monitor therapeutic response. This narrative review focuses on the IL-17 pathway in SpA-related diseases with emphasis on its role in pathogenesis, current approved IL-17 inhibitors, and the need for biomarkers reflecting core disease pathways for early diagnosis and measurement of disease activity, prognosis, and response to therapy.

Background:Altered extracellular matrix (ECM) remodelling is an important part of the pathology seen in joint degenerative diseases. Type II collagen is the most abundant ECM protein in the cartilage and provides the tissue with essential tensile strength in order to withstand high compressive loading. During cartilage erosion, type II collagen is cleaved by matrix metallopeptidases (MMPs) which generates new protein fragments called neo-epitopes. These fragments are released into circulation and may potentially serve as biomarkers by indicating the degree of cartilage destruction.Objectives:The aim of this study is to develop a highly specific immunoassay targeting a neo-epitope fragment of type II collagen cleaved, named T2CM. Moreover, we investigated the assays potential to evaluate type II collagen degradation in anex vivobovine full-depth cartilage explants model (BEX) with catabolic treatment and in healthy controls and osteoarthritis (OA) patients.Methods:A monoclonal antibody was raised in mouse against the C-terminus from protease cleavage site of type II collagen and a direct competitive ELISA was developed and technically validated. The assay specificity was evaluated for the standard peptide excluding cross-reactivity with elongated and truncated peptides, and a non-sense coating peptide. Human OA cartilage was cleaved with MMP-1, -2, -9 and -13 and measured with the T2CM-assay to investigate which MMPs generated the neo-epitope. T2CM levels were measured in supernatant from BEX explants cultured for 21 days in serum free DMEM/F12 medium with six different doses of OSM+TNF-α (O+T) treatment (20/10, 20/20, 20/40, 10/10, 10/20, 10/40 ng/mL) including a control group without (w/o) treatment. The supernatant was harvested 3 times weekly and replaced with new culture medium with O+T treatment. Biomarker results were confirmed by western blot, where T2CM was measured in supernatant from explants with O+T treatment 20/20 ng/mL and 20/40 ng/mL harvested on day 14 and day 21. To confirm the preclinical data, serum samples from 23 healthy controls (age range from 44-59 years with mean 51.4 ± SD 5.1, gender distribution was 56% female and 44% male, and 100% Caucasian) and 23 OA patients (age range from 41-77 years with mean 57.7 ± SD 13.7, gender distribution was 61% female and 39% male, and 100% Caucasian) were measured by T2CM.Results:A technically robust and T2CM-specific assay was developed. The assay linearity and spike-recovery were accepted with percentage of 99.69% and 93.15%. The assay showed no cross-reaction with the elongated, truncated or non-sense coating peptide. In addition, it was demonstrated that the T2CM neo-epitope was derived from MMP-1 and MMP-13 cleavage of type II collagen. O+T treatment induced the T2CM release in BEX compared to the untreated (Figure 1-2). Moreover, the western blot confirmed the T2CM results by the presence of two T2CM bands on day 21 from O+T treated explant compared to day 14 where no bands appeared. T2CM showed to be significantly elevated in patients with OA compared to controls (p=0.036; mean 3.262 ng/mL ± SD 1.065 vs 2.698 ng/mL ± SD 1.118).Conclusion:The newly developed assay was specific for the T2CM neo-epitope and was determined to be generated by MMP-1 and MMP-13. Additionally, the assay detected elevated levels of T2CM in supernatant from explants treated with O+T after 19 days of treatment compared to untreated. This was further confirmed in human OA patients, where the level of T2CM was elevated compared to healthy controls. This suggests that T2CM may have potential as biomarker for type II collagen degradation. Future preclinical and clinical studies are needed to validate these findings.Figure 1-2.T2CM measurements in BEX model. OSM + TNF-a (O+T) ng/mL.Disclosure of Interests:Solveig Skovlund Groen Employee of: Nordic Bioscience, Dovile Sinkeviciute Grant/research support from: Industrial PhD Student, Employee of: Industrial PhD Student, Christian Thudium Employee of: Employee at Nordic Bioscience A/S., Patrik Önnerfjord: None declared, Morten Karsdal Shareholder of: Nordic Bioscience A/S., Employee of: Full time employee at Nordic Bioscience A/S., Anne-Christine Bay-Jensen Shareholder of: Nordic Bioscience A/S, Employee of: Full time employee at Nordic Bioscience A/S., Signe Holm Nielsen Employee of: Full time employee at Nordic Bioscience

Summary Objectives Pain and disability are the main clinical manifestations of osteoarthritis, for which only symptomatic therapies are available. Hence, there is a need for therapies that can simultaneously alter disease progression and provide pain relief. KBP is a dual amylin- and calcitonin-receptor agonist with antiresorptive and chondroprotective properties. In this study we investigated the effect of KBP in a rat model of osteoarthritis. Methods Medial meniscectomy (MNX) was performed in 39 rats, while 10 underwent sham surgery. Rats were treated with KBP and/or naproxen. Nociception was assessed by mechanical and cold allodynia, weight bearing asymmetry, and burrowing behavior. Blood samples were collected for biomarker measurements, and knees for histology. Cartilage histopathology was evaluated according to the advanced Osteoarthritis Research International (OARSI) score and KBPs in vitro antiresorptive effects were assessed using human osteoclasts cultured on bone. Results The MNX animals displayed an increased nociceptive behavior. Treatment with KBP attenuated the MNX-induced osteoarthritis-associated joint pain. The cartilage histopathology was significantly lower in rats treated with KBP than in MNX animals. Bone and cartilage degradation, assessed by CTX-I and CTX-II plasma levels, were decreased in all KBP-treated groups and KBP potently inhibited bone resorption in vitro. Conclusions Our study demonstrates the effectiveness of KBP in ameliorating osteoarthritis-associated joint pain and in protecting the articular cartilage, suggesting KBP as a potential drug candidate for osteoarthritis.

Knee osteoarthritis (KOA) is commonly defined by radiographic changes and knee pain. KOA has a multifactorial aetiology, but there are several well-recognised strong risk factors for the incidence of KOA. These include age, sex, body mass index (BMI), previous injuries, bone density, smoking and pre-existing hand osteoarthritis (HOA). Factors associated with mild systemic and synovial inflammation are potential markers for KOA incidence. The hypothesis for this research was that selected markers associated with synovial inflammation and/or variables linked with metabolic syndrome (MetS) are prognostic for KOA incidence. Self-reported knee stiffness, serum markers associated with knee synovitis, and variables associated with MetS were selected as potential risk factors for KOA incident.

Background: Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by progressive inflammation systemically and local joint deterioration. Chronic inflammation leads to elevated levels of tissue remodeling and release of extracellular matrix (ECM) metabolites into circulation. Type VI collagen (Col-VI) is found at the interface between the interstitial- and the basement membrane where it binds other matrix proteins and support cell-cell interactions. Blood levels of Col-VI metabolites (C6M) have been associated with increased tissue turnover and disease activity in animal models and clinical studies of lung 1 and liver2 fibrosis and ankylosing spondylitis3. These studies indicate that systemic levels of C6M are released ubiquitously from many organs and may be modulated by anti-inflammatory treatments. Treatment with the αIL-6 receptor agent Tocilizumab (TCZ) results in decreased ECM remodeling Objectives: Investigate the modulation of Col-VI metabolites in circulation by TCZ treatment and its association with treatment response. Methods: Systemic Col-VI metabolism was measured in the LITHE study (n=740), a one year double blind, placebo controlled phase III parallel group study in patients receiving placebo, 4 mg/kg or 8 mg/kg TCZ in combination with MTX. Col-VI degradation was measured using the C6M assay, measuring a specific MMP generated metabolite at bl, w4 and w16. The odds ratio (OR) of treatment response (ACR20, 50 or 70) at >median decrease was calculated using logistic regression, adjusting for age, sex, BMI, dis. duration (model 1) and treatment (model 2). Results: TCZ treatment dose dependently reduced of Col-VI metabolites in serum at w4 in both 4 and 8 mg/kg (9% ns and 46% p Conclusions: Suppression of Col-VI turnover was significantly associated with a higher level of response to TCZ treatment in RA patients after already 4 weeks. We hypothesize that there is an increased systemic connective tissue turnover in RA and connective tissue metabolites may be used as indicators of treatment response. References 1. Jenkins RG, Simpson JK, Saini G, et al. Lancet Respir Med. 2015;2600(15):1–11. 2. Leeming DJ, Byrjalsen I, Jimenez W, Christiansen C, Karsdal MA, Liver Int. 2013;33(3):439–447. 3. Bay-Jensen AC, Leeming DJ, Kleyer A, Veidal SS, Schett G, Karsdal MA., Rheumatol Int. 2012;32(11):3565–3572. Disclosure of Interest: C. Thudium Employee of: Nordic Bioscience, N. Gudmann Employee of: Nordic Bioscience, M. Karsdal Shareholder of: Nordic Bioscience, Employee of: Nordic Bioscience, A.-C. Bay-Jensen: None declared

Background Glycosaminoglycans (GAGs) are essential for the pressure-resistant function of cartilage. During osteoarthritis (OA), GAGs are lost from cartilage. This loss impairs the functional and structural integrity of cartilage, thereby accelerating further cartilage damage. Proteoglycan degradation is mediated by enzymes such as MMP3, MMP13 and ADAMTS5. Objectives Recently we discovered that interleukin 37 (IL37) lowers the expression of these enzymes in human OA chondrocytes. The goal of this study was to investigate if IL37 protects against GAG loss in freshly obtained human OA explants. Methods Human cartilage was obtained from ten OA patients undergoing total knee or hip arthroplasty. Per condition 6 cartilage explants of 4 mm in diameter were used. Explants were incubated with recombinant IL37 (rhIL37) for up to 6 days. Every other day new rhIL37 was added. Additionally, an MMP3-inhibitor, or MMP13-inhibitor or ADAMTS5-inhibitor was added in the same protocol. Sulfated GAGs (sGAGs) were visualised by histology, and sGAG release in culture medium was quantified using 1,9-dimethylmethylene blue. To study sGAG synthesis, explants were incubated with rhIL37 followed by 4 hour labelling with 35SO4. Alternatively, explants were pre-labelled for 4 hour with 35SO4 followed by incubation with rhIL37 to study sGAG degradation. Additionally, expression levels of proteoglycans and cartilage matrix degrading enzymes were measured by qPCR and Western Blot. Activity of MMP and ADAMTS enzymes was determined by measuring FFGV, ARGS and NITEGE neo-epitope levels in the supernatant of the cultures, using ELISA. Results Culturing human OA explants for up to 6 days, caused a reduction in GAG content as observed by loss of Safranin O staining. In addition, a release of sGAGs was measured in the supernatant of on average 17,5 µg/ml per mg cartilage. Incubation of cartilage explants with rhIL37 significantly reduced the release of GAGs. A maximal reduction of 24% was already observed with the lowest dose of rhIL37 (1 ng/ml). No effect of rhIL37 on the amount of incorporated 35SO4 was observed, indicating that rhIL37 does not alter the synthesis of GAGs. This is supported by the observation that rhIL37 does not affect the mRNA expression of the large proteoglycans and SLRPS. In contrast, we did find that rhIL37 significantly reduced the loss of 35SO4-labelled GAGs from cartilage explants. In addition, rhIL37 reduced MMP3 and MMP13 protein expression and lowered both MMP and ADAMTS mediated degradation of proteoglycan fragments: FFGV neo-epitope and ARGS and NITEGE neo-epitopes, confirming that rhIL37 reduces active degradation of sGAGs. Lastly, to investigate which proteolytic enzyme contributes to the sGAG release in our culture system, a MMP3-, MMP13- or ADAMTS5-inhibitor was added to the explants. Strikingly, we found that only MMP3 inhibition mimicked IL37 function, suggesting that the effects of rhIL37 run via MMP3. Conclusions Our data show that rhIL37 reduces sGAG release of cartilage explants, indicating that IL37 supports cartilage matrix integrity. To our knowledge this is the first report demonstrating this anti-destructive effect of IL37 on freshly obtained human OA cartilage explants. Possible these effects run via MMP3 because IL37 reduced MMP3 expression and only MMP3 inhibition results in similar effects as rhIL37 addition. Disclosure of Interest None declared

Background The innate immune system is important for the initiation and development of joint diseases such as rheumatoid arthritis (RA) and Osteoarthritis (OA). Exogenous TLR (Toll like receptor) ligands are abundant in the synovial joints of RA and OA patients and induce the first immune response. Stimulation of TLRs results in an increased immune response and is characterised by induction of cytokines expression which in turn causes tissue damage and results in generation of more DAMPs. Hence, resulting in a self-perpetuating loop of TLR activation and TLR mediated tissue damage. Objectives The aim of this study was to investigate the effect of degradation fragments of human cartilage generated ex vivo or in vitro, on TLR-2 activation in a TLR-2 reporter cell system. Methods Human cartilage biopsies retrieved from OA patients undergoing total knee replacement at Gentofte hospital were used to generate human explants (HEX). HEX were cultured for 21 days either without (w/o) treatment or in the presence of IL-1α (10 ng/ml, 5 ng/ml and 2.5 ng/ml). Unconditioned media incubated without cartilage tissue was used as control. Tissue degradation was verified by measuring the release of aggrecan neo-epitope biomarkers AGNx1 and FFGV into the conditioned media (CM). Correspondingly, human cartilage was digested with ADAMTS5 for different time intervals (16 hour, 24 hour and 88 hour) at 37°C. The digested cartilage was removed by centrifugation and the supernatant was stored. Crushed cartilage in buffer alone was used as a control set up for each time point and buffer alone was subtracted as background. The CM and digested media (DM) was tested in the SEAP (secreted embryonic alkaline phosphatase) reporter gene based HEK hTLR2 cell line. The HEK null 1 parent cell line was used as a control. Pam3CSK4 was used as a positive control for the hTLR2 cell line. Results IL-1α induced aggrecan degradation was confirmed by increased exAGNx1 and exFFGV release in the conditioned media compared to w/o (p Conclusions IL-1α induced cartilage degradation leads to the release of aggrecan fragments into the CM, and this CM as well as in vitro cleavage products from ADAMTS-5 digestion of human cartilage was able to activate the TLR2 receptor in vitro in a specialised reporter cell system. These data indicate that DAMPs may be released from human cartilage in the presence of pro-inflammatory cytokines and proteolytic enzymes. The released fragments can lead to TLR2 activation and cause further inflammation. These data suggest that DAMPs may play a role in the onset and maintenance of inflammation in diseases such as OA and RA. Disclosure of Interest None declared

Background Osteoarthritis (OA) patients suffer from progressive degradation of articular cartilage, to which there is no available treatment to block or reverse the catabolic mechanisms. Articular cartilage confers a biomechanical function in the joints, and in concordance chondrocytes have shown to be mechanosensitive. The effect of dynamic compression on cartilage extracellular matrix (ECM) could prove to be crucial for translational research in development and screening of new OA drug candidates. Objectives This study investigates the effect of long-term dynamic compression of a bovine articular cartilage ex vivo model through quantification of cartilage associated biomarkers. Methods Full depth bovine cartilage explants were cultured for 2 weeks. The explants were treated 3 times a week with either OSM [10 ng/mL] and TNF-a [2 ng/mL] (O+T), or TGF-B1 [50 ng/mL]. Untreated samples were included as negative controls (w/o). For each condition two groups were established; an unloaded group and a group compressed 3 times a week. Compression was applied using Electroforce 5500 (TA Instruments), in a sine wave with a maximum load per cycle of 1 MPa, at 1 Hz frequency for 1200 cycles. Metabolic activity was measured once a week using AlamarBlue. Biomarkers released to the supernatant were assessed using the following well-described ELISAs: formation of type II collagen was measured by ProC2, and MMP-degradation of type II collagen was measured by C2M. Sulfated glycosaminoglycans (sGAG) released to media, and sGAG extracted from the explants on the last day using a 4 mM GuHCl solution were quantified using a 1.9-dimethylmethylene blue (DMB) assay. Results Compression of bovine explants significantly decreased release of C2M compared to unloaded samples in O-T and w/o groups with 42% and 48% respectively. ProC2 release was not affected by compression. Compression of w/o or TGF-b treated explants increased sGAG release with 195% and 152% relative to their unloaded controls. However, the total amount of sGAG extracted from the explants on the last day remained unchanged. Compression of the explants did not affect the metabolic activity. Conclusions Compression significantly reduced the degradation of type II collagen, which in combination with unaltered formation as measured by ProC2, may suggest suppression of the involved catabolic processes and increased deposition of type II collagen in the cartilage matrix. Furthermore, compression elevated the sGAG release without reduction in the explant GAG content, indicating an increased turnover of GAG. In conclusion, the cartilage ECM turnover is significantly modulated by dynamic compression. This method adds essential translational value to the continuous research addressing OA. Disclosure of Interest None declared

Background Osteoarthritis (OA) is a heterogeneous disease described by a combination of joint pain, physical disability and radiographic alterations leading to joint failure and total joint replacement (TJR). Commonly used endpoints in OA trials are worsening of pain and joint space narrowing. TJR is normally not considered an endpoint. Age and female gender are considered as major risk factors for developing OA. Objectives We hypothesise that TJR can be used as an endpoint in OA outcome studies within reasonable time frame. To investigate the basis for this hypothesis, we explored the prevalence and incidence of TJR as a reflection of joint failure in the Prospective Epidemiologic Risk Factor (PERF I) study. Methods A total of 5,855 Danish postmenopausal women aged 49–88 enrolled in the Prospective Epidemiologic Risk Factor (PERF I) study during 1999–2001 (baseline). Three, six and twelve year follow-up data from the Danish National registry was collected in end of 2014, including occurrence of TJR, OA and other relevant diagnosis. Also, women where at baseline and in 2014 asked whether they had a TJR or OA. The biomarker C1M was measured in baseline serum samples. The PERF I study was carried out in accordance with ICH-GCP and the study protocol was approved by the local ethics committees. Results There were 798 women that had their first TJR between baseline and 12 year follow-up; giving an incidence proportion of 13.6%. The TJR women were on average 1 year older (p=0.010) and heavier (1.7 kg/cm2, p 40 ng/mL, median) that after baseline underwent there first ever TJR (841). Group 1 is as described above. The 3, 6 and 12 incidence rates were 9.0, 17.3% and 29.7% for the prior TJR group, 6.2, 12.1% and 23.3% for the OA group, and 7.6, 13.6% and 25.1% for the OA +C1M group. The age-dependent prevalence and incidence for the first TJR. The prevalence was insignificant for the age group younger than 60 years old ( Conclusions Within a timeframe of 3, 6 or 12 years TJR incidence for women with an OA diagnosis reached 6, 12% and 23%, which was a doubling compared to the All population. The incidence increased by adding a single diagnostic measure. This reflects that TJRs are frequent amongst elderly women and that if designed minutiously, such as including specific diagnostic criteria (e.g. biomarker, OA diagnose), it may be feasible to conduct clinical studies with TJR as an endpoint. However, special attention much be directed to the objectiveness of the criteria for TJR. This may build a case for design of outcome studies (joint failure) for developing drugs in OA. Disclosure of Interest A. Bay-Jensen Shareholder of: Nordic Bioscience, Grant/research support from: ApproacH (IMI support), Employee of: Nordic Bioscience, C. Bager Employee of: Proscion, A. Bihlet Shareholder of: Nordic Bioscience, Employee of: Nordic Bioscience, C. Thudium Employee of: Nordic Bioscience, I. Byrjalsen Employee of: Nordic Bioscience, H. Nielsen Employee of: Nordic Bioscience, J. Andersen Shareholder of: Nordic Bioscience, Employee of: Nordic Bioscience, B. Riis Shareholder of: Nordic Bioscience, C. Christiansen Shareholder of: Nordic Bioscience, M. Karsdal Shareholder of: Nordic Bioscience, Grant/research support from: ApproacH (IMI support), Employee of: Nordic Bioscience

Background Response to any treatment in rheumatoid arthritis (RA) is assessed by symptomatic changes, such as swollen joint count. Such assessments does not provide information regarding the effect of the treatment at tissue level. Chronic inflammation has a detrimental effect resulting in elevated levels of tissue remodelling and the release of extracellular matrix (ECM) metabolites into the circulation. The tissues consist mainly of interstitial matrix, basement membrane and cells, which are all affected by auto-immune disorders. Tissue metabolites can be measured in serum as biomarkers of tissue remodelling. Objectives The purpose was to investigate if tissue remodelling is differently modulated by tocilizumab (TCZ) and methotrexate (MTX). Methods The AMBITION study, a phase III RCT with tocilizumab vs. Methotrexate in which TCZ monotherapy (8 mg/kg every 4 weeks) was compared to methotrexate monotherapy over 24 weeks in patients with moderate-severe RA (AMBITION, NCT00109408). TCZ is a compound that inhibits the IL-6 receptor. Tissue metabolites were measured in baseline and 8 weeks sera (n=319) by ECM specific ELISAs: Connective tissue remodelling was measured by C3M (type III collagen degradation), basement membrane remodelling by C4M (type IV collagen), inflammation by C-reactive protein (CRP) and its metabolite CRPM. Comparison between groups were done by ANCOVA adjusting for age, gender, BMI and disease duration. Results Tissue remodelling was increased by 10% in the placebo group and significantly (p Conclusions Chronic inflammation results in an increased amount of tissue remodelling. There was a significant difference in the magnitude of effect MTX and TCZ on tissue remodelling. In addition there was a disconnect between tissue remodelling and change in disease activity, which was treatment dependent. Disclosure of Interest A. Bay-Jensen Shareholder of: Nordic Bioscience, Employee of: Nordic Bioscience, A. S. Siebuhr Employee of: Nordic Bioscience, C. Thudium Employee of: Nordic Bioscience, M. Karsdal Shareholder of: Nordic Bioscience, Employee of: Nordic Bioscience

Background: Osteoarthritis (OA) leads to joint failure and total joint replacement (TJR, either hip (H) or knee (K)). Worsening of pain and joint space narrowing are believed to be surrogates for joint failure; however, we hypothesize that TJR, as a reflection of joint failure, can be used as an endpoint in event-driven clinical trials within a reasonable duration. We explored the incidence of TJR in the Prospective Epidemiologic Risk Factor (PERF I) study. Methods: A total of 5,855 Danish postmenopausal women aged 49-88 enrolled in the PERF I study during 1999-2001 (baseline). Three-, six- and twelve-year follow-up data from the Danish National Patient Registry was collected, including occurrence of TJR and OA diagnosis. At baseline the women were asked whether they had OA. Findings: The TJR women were on average 1 year older (p

Osteoporosis is a major health care problem, and whereas efficacious treatments for vertebral fracture reduction are available for osteoporosis patients, these therapies are still limited with respect to capacity for restoration of bone loss, as well as efficacy on non-vertebral fractures, such as hip fractures, which are the source of morbidity and mortality.Studies of rare bone diseases in humans, such as osteopetrosis, sclerosteosis, pycnodysostosis and more, have shed light on a series of drug targets in bone that have the potential to result in therapies for osteoporosis with novel mechanisms of action, and the potential to improve the standard of care substantially. We focus on how they are separated from classic treatments for osteoporosis, in terms of novel modes of action, additional beneficial effects on bone turnover and importantly also safety. We focus on the status of anti-sclerostin antibodies, novel parathyroid hormone-related protein analogs, inhibitors of cathepsin K and ClC-7 in osteoclasts, all of which are currently in development.There is a good possibility that the treatment of osteoporosis will be greatly improved within the coming years; however, with numerous effective and safe drugs already available careful attention to the safety of these novel candidates is crucial.

Osteoarthritis (OA) is the most common arthritic disease in the world, leading to debilitating pain and destruction of joint tissues. While pain is the hallmark symptom of osteoarthritis, clear associations between pain and disease processes involved in joint deterioration are lacking. OA pain is multifactorial and may arise from multiple distinct or concurrent mechanisms, and may thus present as different pain sub-types. Several biomarkers developed to reflect important pathological processes are available, and associations between such biomarkers and OA pain may give hints to important pathological features, which have not been possible to assess using clinical, radiographic or magnetic resonance imaging techniques. This review highlights a selection of important, protein-derived biomarkers measured in body fluids from OA patients, which have been associated with different types and aspects of OA pain, and discusses the potential mechanisms behind the associations. SIGNIFICANCE: Osteoarthritis (OA) is a heterogenous disease affecting the entire joint, including cartilage, bone and synovium. While pain is the hallmark symptom of osteoarthritis, clear associations between pain and disease processes involved in joint deterioration are lacking. Thus, there is clear need for biomarkers that can accurately describe the underlying processes and distinguish between different disease and pain pathologies. In this review we discuss a selected number of biomarkers which have been directly or indirectly associated with pain mechanisms and development of pain in OA either via structural correlates or as molecular sensitizing agents. We further evaluate the challenges that the OA field faces in the development and application of biomarkers for OA pain.

Infantile malignant osteopetrosis (IMO) is a rare, lethal, autosomal recessive disorder characterized by nonfunctional osteoclasts. More than 50% of the patients have mutations in the TCIRG1 gene, encoding for a subunit of the osteoclast proton pump. The aim of this study was to develop a clinically applicable lentiviral vector expressing TCIRG1 to correct osteoclast function in IMO. Two mammalian promoters were compared: elongation factor 1α short (EFS) promoter and chimeric myeloid promoter (ChimP). EFS promoter was chosen for continued experiments, as it performed better. IMO osteoclasts corrected in vitro by a TCIRG1-expressing lentiviral vector driven by EFS (EFS-T) restored Ca2+ release to 92% and the levels of the bone degradation product CTX-I to 95% in the media compared to control osteoclasts. IMO CD34+ cells from five patients transduced with EFS-T were transplanted into NSG mice. Bone marrow was harvested 9–19 weeks after transplantation, and human CD34+ cells were selected, expanded, and seed...

The objective of this study was to examine the tissue degradation in response to anti-IL6 receptor treatment in rheumatoid arthritis (RA) patients which are anti-TNF-α inadequate responders.RADIATE was a randomised, double-blinded, placebo-controlled, parallel-group, phase III trial. RA patients with previous inadequate response to anti-TNFα therapy (n=299) were randomly assigned to tocilizumab 4 or 8 mg/kg with methotrexate (10-25 mg weekly) or placebo with methotrexate. Type III collagen degradation (C3M) and CRP degradation (CRPM) were analysed in serum samples at baseline and 16 weeks.Treatment with 4 and 8 mg/kg tocilizumab significantly decreased C3M (p=0.0001 and p=0.0007) and CRPM (p0.0001) levels after 16 weeks. Changes in C3M and CRPM levels after 16 weeks correlated well with the changes in disease activity score 28 (DAS28). Change in CRPM levels furthermore correlated moderately with the change in patient pain (VAS) (rpartial of 0.20) and Health assessment questionnaire disability index (HAQ-DI) (rpartial of 0.24). Changes in biomarker levels above median change led to an odds ratio of 1.95 (C3M) and 3.00 (CRPM) for achieving the American College of Rheumatology 20% improvement criteria (ACR20).This study shows that a decrease in inflammation leads to a decrease in excessive extracellular matrix degradation. It furthermore supports earlier shown evidence that tocilizumab works in the treatment of RA patients, although there is a clear need for identifying and selecting patients who are more likely to respond to treatment.

Background It is important to establish translational methods that aid in pre-clinical go/no-go decision points to increase the success rate of approved DMARDs. The Spleen tyrosine kinase (Syk) inhibitor Fostamatinib (Fosta) was terminated for development for RA, due to insufficient effect on joint structure in phase III. Objectives The objective was to use a translational system of ex vivo cultures to back-translate the insufficient effect on joint structure described in clinical studies. Methods Human mature osteoclasts (HOC) seeded on bone, bovine cartilage explants (BEX) and human synovial explants (SME) were treated with R406 (API of Fosta) at 5μM-0.05μM. Osteoclasts were co-stimulated with 25 ng/ml M-CSF and RANKL, while BEX and SME were co-stimulated with TNFα 2 ng/mL and OSM 10 ng/mL (O+T) or TNFα 10 ng/mL, respectively. CTX-1 and Ca2+ were measured in conditioned medium (CM) from HOC. Metabolic activity of HOC was assessed with Alamar Blue. C2M and AGNx1 were measured in CM from BEX, while acMMP3, C1M, and C3M were measured in CM from SME. The biomarkers in BEX and SME CM were measured at 4 time points and the total release were quantified by area under the curve (AUC). CTX-1, C2M, AGNx1, acMMP3, C1M, C2M and C3M were measured with ELISA. Ca2+was measured with ADVIA Chemistry system. Statistical differences of metabolic activity was calculated with One-way ANOVA with Dunnett9s multiple comparison test. Statistical differences between biomarkers levels or AUC were calculated with Kruskall Wallis test with Dunn9s multiple comparision test. Results R406 decreased the release of CTX-1 (Fig 1A) and Ca2+ in a dose-dependent manner, with a significant decrease at 1μM (P Conclusions Serum-based biomarkers of the joint ECM turnover were measured in CM from HOC, cartilage and synovial explant cultures. R406 decreased bone resorption and HOC metabolic activity in a dose-dependent manner, together with the MMP-mediated degradation of type II (C2M) collagen and aggrecanse degradation of aggrecan (AGNx1) in cartilage. However, R406 had limited effect on the inflammation driven MMP-mediated degradation of type I (C1M) and III (C3M) collagen and activation of MMP-3. CTX-1, C2M, and C3M have previously been measured in OSKIRA-1, with a profile identical to the ex vivo measurements here [1]. References Platt A, Bay-Jensen AC, Braddock M, et al. The Effect of Fostamatinib with Methotrexate on Circulating Biomarkers of Synovium, Cartilage and Bone Metabolism: Potential Utility for Clinical Development Decision Making in Rheumatoid Arthriti [abstract]. Arthritis Rheumatol. 2016; 68 (suppl 10). Disclosure of Interest C. Kjelgaard-Petersen: None declared, T. Christensen: None declared, P. Hagglund: None declared, M. Karsdal Shareholder of: Nordic Bioscience A/S, Employee of: Nordic Bioscience A/S, C. Thudium Employee of: Nordic Bioscience A/S, A.-C. Bay-Jensen Shareholder of: Nordic Bioscience A/S, Employee of: Nordic Bioscience A/S

Background There is currently a lack of disease modifying OA drugs approved by the drug administration agencies. One of the obstacles in the development of DMOADs that can effectively halt or reverse cartilage degradation is the lack of robust and reproducible model systems for early drug testing. Objectives To develop inducable anabolic bovine ex vivo models of 1) synovial membrane explants and 2) synovial membrane explants in co-culture with cartilage explants. Methods Synovium (bSME) from healthy bovine ( Results Explants were viable throughout the experiments, albeit the bSME lost some viability with time. bSME treated with O+T showed increased C1M and C3M (>400% and >200%) from day 10, compared to w/o, whereas in bCC O+T increased C1M from day 21 and C3M from day 14 (>800%, >1900%). O+T treatment increased C2M was increased from day 21 (>400% and >1000%) in both BEX and bCC. The release was blocked by the generic MMP inhibitor GM6001 which also decreased the C1M and C3M compared to w/o. O+T treatment increased AGNxI at day 7 and 10 (>600%) and exFFGV from day 21 (>650%) in both BEX and bCC. In bSME, TGF-b1 continuously and dose-dependently increased P1NP from day 7 compared to w/o (250%). O+T pre-treatment for 10 days followed by TGF-b1 stimulation increased P1NP after 7 days of TNF-b1 treatment (150%, figure). IGF-1 did not affect the P1NP level at any time point in bSME. In bCC both TGF-b1 (dose-dependently) and IGF-1 sustained the PRO-C2 level at the level of baseline throughout the study periods (figure), whereas O+T decreased PRO-C2 compared to with w/o. The PRO-C2 level in BEX with TGF-b1 was unaltered compared to w/o. Conclusions We here show that bovine synovium can be anaobolic and catabolic stimulated, both alone and in co-culture with cartilage. Anabolic stimulation was achieved with TGF-b1 in both bSME and bCC, while IGF-1 only showed an anabolic effect in the bCC. Previous human explant models using human tissuehave lacked the anabolic capacity. These translational explants models may be applied in the early development of anabolic drugs for cartilage degenerative diseases. Disclosure of Interest A. S. Siebuhr Employee of: Nordic Bioscience, C. Cabanes: None declared, S. Dahab: None declared, M. Karsdal Shareholder of: Nordic Bioscience, Employee of: Nordic Bioscience, A.-C. Bay-Jensen Employee of: Nordic Bioscience, C. Thudium Employee of: Nordic Bioscience

Background There are two major needs in clinical development of DMOADs: 1) Identifying a suitable population with an active and progressive disease in order to demonstrate significant improvements by an efficacious intervention; and 2) Phenotyping patients and linking them to a corresponding treatment mode-of-action (e.g. anti-inflammatory). The biochemical marker CRPM is a neoepitope of C-reactive protein (CRP) and is released from the local inflamed tissue when CRP is degraded by proteases such as matrix metalloproteinases Objectives The purpose of this study was to translate the CRPM, from rheumatoid arthritis (RA), where it has been extensively tested, to OA, and to test whether it is predictive of radiographic OA. Methods The placebo arms of two phase III OA trial (clinicaltrial.gov: NCT00486434 and NCT00704847), a phase III RA study (the LITHE study, N=490), which included patients with active, moderate-severe RA (NCT00106535), as well as in an early RA cohort (N=92) were used. Subjects with symptomatic and radiographic knee OA: WOMAC pain ≥150mm and/or WOMAC function ≥510mm, and Kellgren-Lawrence grade (KLG) 2 or 3. KLG were scored for both knees at baseline and year 2 (Y2). Serum CRPM and hsCRP were measured at baseline. The association between serum CRPM levels and disease activity score (DAS28) and hsCRP was investigated by Spearman9s correlations. Quartile ranges of CRPM in the early RA cohort were used to define the cut-off between inflammatory OA and non-inflammatory OA. OA knees were divided into cases and controls based on a terminology proposed by the FNIH-OAI consortium (1); knees with KLG≥2 at BL were excluded, and incidence OA at Y2 was defined KLG≥2. Logistic regression was used to compare cases and controls. Results There was a significant correlation between disease activity measures and CRPM in both RA studies. Seventy-five percent of the LITHE patients had high or very high levels of CRPM at BL, which was changed to a pattern similar to early RA after treatment. The mean CRPM levels were significantly lower in OA (8.5 [95% CI 8.3–8.8] ng/mL) compared to the RA patients (15.6 [9.5–21.6] ng/mL); however, a significant subset of OA patients (41% and 31% in SMC2301 and SMC2302) had CRPM levels ≥9ng/mL, as 75% of patients with early RA. Patients with BL or Y2 CRPM levels ≥9ng/mL were more likely to develop knee OA than patients with low level of CRPM. Overall, moderate to very high levels of CRPM at BL and Y2 were predictive of incidence OA with odds ratio of 4.6 [1.2–17] and 2.5 [1.2–4.8]. Conclusions A subset of OA patients, up to 41%, appear to have tissue inflammation comparable to that of RA, reflected by the level of CRPM. Furthermore, high CRPM levels was prognostic of incident knee OA. These data suggest that CRPM is blood-based biochemical marker for finding OA patients with an inflammatory phenotype. References Longitudinal validation of periarticular bone area and 3D shape as biomarkers for knee OA progression? Data from the FNIH OA Biomarkers Consortium.Hunter D et al.Ann Rheum Dis. 2016 Sep;75(9):1607–14. Disclosure of Interest A. Bay-Jensen Shareholder of: Nordic Bioscience, Grant/research support from: DBOARD, Employee of: Nordic Bioscience, A. Bihlet Shareholder of: Nordic Bioscience, Employee of: Nordic Bioscience, I. Byrjalsen Employee of: Nordic Bioscience, J. Andersen Shareholder of: Nordic Bioscience, Employee of: Nordic Bioscience, Y. He Employee of: Nordic Bioscience, A. S. Siebuhr Employee of: Nordic Bioscience, C. Thudium Employee of: Nordic Bioscience, B. Riis Shareholder of: Nordic Bioscience, C. Christiansen Shareholder of: Nordic Bioscience, M. Karsdal Shareholder of: Nordic Bioscience, Employee of: Nordic Bioscience

Objective Rheumatoid arthritis (RA) is a chronic and degenerative autoimmune joint disease that leads to disability, reduced quality of life, and increased mortality. Although several synthetic and biologic disease-modifying antirheumatic drugs are available, there is still a medical need for novel drugs that control disease progression. As only 10% of experimental drug candidates for treatment of RA that enter phase I trials are eventually registered by the Food and Drug Administration, there is an immediate need for translational tools to facilitate early decision-making in drug development. In this study, we aimed to determine if the inability of fostamatinib (a small molecule inhibitor of Syk) to demonstrate sufficient efficacy in phase III of a previous clinical study could have been predicted earlier in the development process. Methods Biomarkers of bone, cartilage, and interstitial matrix turnover (C-telopeptide of type I collagen [CTX-I], matrix metalloproteinase-derived types I, II, and III collagen neoepitopes [C1M, C2M, and C3M]) were measured in 450 serum samples from the Oral Syk Inhibition in Rheumatoid Arthritis 1 study (OSKIRA-1, a phase III clinical study of the efficacy of fostamatinib in RA) at baseline and follow-up. Additionally, the same biomarkers were subsequently measured in conditioned media from osteoclast, cartilage, and synovial membrane cultured with the active metabolite of fostamatinib, R406, to assess the level of suppression induced by the drug. Results In OSKIRA-1 serum samples and osteoclast and cartilage cultures, fostamatinib suppressed the levels of CTX-I and C2M. In OSKIRA-1 serum samples and synovial membrane cultures, fostamatinib did not mediate any clinical or preclinical effect on either C1M or C3M, which have previously been associated with disease response and efficacy. Conclusion These data demonstrate that translational biomarkers are a potential tool for early assessment and decision-making in drug development for RA treatment.

Summary Objective Knee osteoarthritis (OA) is among the higher contributors to global disability. Despite its high prevalence, currently, there is no cure for this disease. Furthermore, the available diagnostic approaches have large precision errors and low sensitivity. Therefore, there is a need for new biomarkers to correctly identify early knee OA. Method We have created an analytics pipeline based on machine learning to identify small models (having few variables) that predict the 30-months incidence of knee OA (using multiple clinical and structural OA outcome measures) in overweight middle-aged women without knee OA at baseline. The data included clinical variables, food and pain questionnaires, biochemical markers (BM) and imaging-based information. Results All the models showed high performance (AUC > 0.7) while using only a few variables. We identified both the importance of each variable within the models as well its direction. Finally, we compared the performance of two models with the state-of-the-art approaches available in the literature. Conclusions We showed the potential of applying machine learning to generate predictive models for the knee OA incidence. Imaging-based information were found particularly important in the proposed models. Furthermore, our analysis confirmed the relevance of known BM for knee OA. Overall, we propose five highly predictive small models that can be possibly adopted for an early prediction of knee OA.

Here, we tested the hypothesis that human M-CSF (hM-CSF) overexpressed in cord blood (CB) CD34Human M-CSF was overexpressed in cord blood CD34We show that LV-hM-CSF-transduced CB CD34In summary, ectopic production of human M-CSF in CD34

Osteopetrosis due to lack of acid secretion by osteoclasts is characterized by abolished bone resorption, increased osteoclast numbers, but normal or even increased bone formation. In contrast, osteoclast-poor osteopetrosis appears to have less osteoblasts and reduced bone formation, indicating that osteoclasts are important for regulating osteoblast activity. To illuminate the role of the osteoclast in controlling bone remodeling, we transplanted irradiated skeletally mature 3-month old wild-type mice with hematopoietic stem cells (HSCs) to generate either an osteoclast-rich or osteoclast-poor adult osteopetrosis model. We used fetal liver HSCs from (1) oc/oc mice, (2) RANK KO mice, and (3) compared these to wt control cells. TRAP5b activity, a marker of osteoclast number and size, was increased in the oc/oc recipients, while a significant reduction was seen in the RANK KO recipients. In contrast, the bone resorption marker CTX-I was similarly decreased in both groups. Both oc/oc and Rank KO recipients developed a mild osteopetrotic phenotype. However, the osteoclast-rich oc/oc recipients showed higher trabecular bone volume (40 %), increased bone strength (66 %), and increased bone formation rate (54 %) in trabecular bone, while RANK KO recipients showed only minor trends compared to control recipients. We here show that maintaining non-resorbing osteoclasts, as opposed to reducing the osteoclasts, leads to increased bone formation, bone volume, and ultimately higher bone strength in vivo, which indicates that osteoclasts are sources of anabolic molecules for the osteoblasts. Osteopetrosis due to lack of acid secretion by osteoclasts is characterized by abolished bone resorption, increased osteoclast numbers, but normal or even increased bone formation. In contrast, osteoclast-poor osteopetrosis appears to have less osteoblasts and reduced bone formation, indicating that osteoclasts are important for regulating osteoblast activity. To illuminate the role of the osteoclast in controlling bone remodeling, we transplanted irradiated skeletally mature 3-month old wild-type mice with hematopoietic stem cells (HSCs) to generate either an osteoclast-rich or osteoclast-poor adult osteopetrosis model. We used fetal liver HSCs from (1) oc/oc mice, (2) RANK KO mice, and (3) compared these to wt control cells. TRAP5b activity, a marker of osteoclast number and size, was increased in the oc/oc recipients, while a significant reduction was seen in the RANK KO recipients. In contrast, the bone resorption marker CTX-I was similarly decreased in both groups. Both oc/oc and Rank KO recipients developed a mild osteopetrotic phenotype. However, the osteoclast-rich oc/oc recipients showed higher trabecular bone volume (40 %), increased bone strength (66 %), and increased bone formation rate (54 %) in trabecular bone, while RANK KO recipients showed only minor trends compared to control recipients. We here show that maintaining non-resorbing osteoclasts, as opposed to reducing the osteoclasts, leads to increased bone formation, bone volume, and ultimately higher bone strength in vivo, which indicates that osteoclasts are sources of anabolic molecules for the osteoblasts.