Cecilie Freja Kjelgaard-Petersen

Osteoarthritis year in review 2015: soluble biomarkers and the BIPED criteria

OBJECTIVE To review and summarize biomarker data published from April 2014 to May 2015 to provide insight to the ongoing work in the field of osteoarthritis (OA). Furthermore, to summarize the BIPED criteria and set it in context of the medical needs of 2015. METHODS PubMed was used as searching machine: Time period 2014/04/01-2015/05/01, MeSH term [Biomarker] AND [Osteoarthritis], Language; English, Full text available. Reviews were excluded. Only papers describing protein based biomarkers measured in human body fluids from OA patients were included. RESULTS Biomarkers of joint tissue turnover, cytokines, chemokines and peptide arrays were measured in different cohorts and studies. Amongst those were previously tested biomarkers such as osteocalcin, Carboxy-terminal cross-linked fragment of type II collagen (CTX-II) and cartilage oligomeric matrix protein (COMP). A majority of the biomarker were classified as I, B or B biomarkers according to the BIPED criteria. Work is continuing on testing biomarkers in OA. There is still a huge, unmet medical need to identify, test, validate and qualify novel and well-known biomarkers. A pre-requisite for this is better characterization and classification of biomarkers to their needs, which may not be reached before higher understanding of OA phenotypes has been gained. In addition, we provide some references to some recent guidelines from Food and Drug Administration (FDA) and European Medicines Agency (EMA) on qualification and usage of biomarkers for drug development and personalized medicine, which may provide value to the field.

Synovitis biomarkers: ex vivo characterization of three biomarkers for identification of inflammatory osteoarthritis

Abstract Objective: Characterize biomarkers measuring extracellular matrix turnover of inflamed osteoarthritis synovium. Methods: Human primary fibroblast-like synoviocytes and synovial membrane explants (SMEs) treated with various cytokines and growth factors were assessed by C1M, C3M, and acMMP3 in the conditioned medium. Results: TNFα significantly increased C1M up to seven-fold (p = 0.0002), C3M up to 24-fold (p = 0.0011), and acMMP3 up to 14-fold (p < 0.0001) in SMEs. IL-1β also significantly increased C1M up to five-fold (p = 0.00094), C3M four-fold (p = 0.007), and acMMP3 18-fold (p < 0.0001) in SMEs. Conclusion: The biomarkers C1M, C3M, and acMMP-3 were synovitis biomarkers ex vivo and provide a translational tool together with the SME model.

Biomarkers of cartilage and surrounding joint tissue

The identification and clinical demonstration of efficacy and safety of osteo- and chondro-protective drugs are met with certain difficulties. During the last few decades, the pharmaceutical industry has, in the field of rheumatology, experienced disappointments associated with the development of disease modification. Today, the vast amount of patients suffering from serious, chronic joint diseases can only be offered treatments aimed at improving symptoms, such as pain and acute inflammation, and are not aimed at protecting the joint tissue. This huge, unmet medical need has been the driver behind the development of improved analytical techniques allowing better and more efficient clinical trial design, implementation and analysis. With this review, we aim to provide a brief and general overview of biochemical markers of joint tissue, with special focus on neoepitopes. Furthermore, we highlight recent studies applying biochemical markers in joint degenerative diseases. These disorders, including osteoarthritis, rheumatoid arthritis and spondyloarthropathies, are the most predominant disorders in Europe and the USA, and have enormous socioeconomical impact.

Translational Biomarkers and Ex Vivo Models of Joint Tissues as a Tool for Drug Development in Rheumatoid Arthritis

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.

Aggrecanase degradation of type III collagen is associated with clinical knee pain

There is a lack of biochemical markers for non-invasive and objective assessment of symptomatic osteoarthritis (OA). Aggrecanase activity has been shown to be associated with joint deterioration and symptomatic disease through the degradation of extracellular matrix proteins, such as type III collagen. Our study aimed to identify and develop a novel biomarker by measuring an aggrecanase-mediated type III collagen neoepitope, and correlate levels of this biomarker with OA joint pain. Mass spectrometric analysis of purified type III collagen, degraded by the aggrecanase A Disintigrin and Metalloproteinase with Thrombospondin motif (ADAMTS), revealed a fragment generated by ADAMTS-1, -4 and -8. A monoclonal antibody was raised against the neoepitope of this fragment (COL3-ADAMTS) and a competitive ELISA was developed and tested; using serum samples from a cross-sectional cohort of patients with different degrees of knee OA (n = 261). The COL3/ADAMTS ELISA was technically robust and specific for the ADAMTS-1, -4 and -8 generated neoepitope. COL3/ADAMTS was released form cytokine stimulated synovial cultures, indicating a biologic link between the marker and synovium. In OA patients, serum COL3/ADAMTS was independently associated with pain scores (rho = -0.13-0.17, p < 0.05). This association was associated significantly with the presence of radiographic OA. Together, these data indicate that COL3/ADAMTS could be a marker of early osteoarthritis and the underlining pathology.

Tofacitinib and TPCA-1 exert chondroprotective effects on extracellular matrix turnover in bovine articular cartilage ex vivo

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.

THU0024 Anti-Inflammatory Inhibitors Targeting Jak and Ikk Have An Anabolic Effect on Type II Collagen Turnover ex Vivo

Background Rheumatoid arthritis (RA) and a subpopulation of osteoarthritis, inflammatory OA (iOA) are degenerative joint diseases with an inflammatory component. However, the degree of inflammation is much higher in RA than iOA. There are many signaling pathways involved with the inflammation-driven extracellular matrix (ECM) degradation in RA and iOA cartilage that have been suggested as anti-inflammatory targets. However, the results on joint structure in clinical trials have been varying. A better understanding of the intracellular signaling pathways and the downstream effect on ECM turnover could be beneficial for the selection of novel anti-inflammatory treatments for RA and iOA. Objectives The aim of this study was to investigate the direct effect of the anti-inflammatory inhibitors R406 (the active metabolite of Fostamatinib), Tofacitinib, TPCA-1 and SB203580 on the cartilage ECM turnover. Methods Full depth bovine cartilage ex vivo cultures were cultured for 3 weeks with OSM [10 ng/mL] and TNFα [2 ng/mL] (O+T) or together with R406, Tofacitinib or TPCA-1 at 10 μM and a two-fold dilution to 0.16 μM. SB203580 was tested at 3 μM, 1 μM and 0.3 μM. As negative control, untreated explants were included. The ECM turnover of the cartilage was assessed with the biomarkers; C2M, ProC2, AGNx1 and/or ARGS. Additionally, histology of the explants was examined with Safranin O and fast green staining. Results Aggrecanase mediated degradation of aggrecan was assessed with ARGS or AGNx1. The Syk inhibitor R406, the Jak inhibitor Tofacitinib, and the IKK inhibitor TPCA-1 inhibited the release of ARGS or AGNx1, while the p38 inhibitor, SB203580, had no effect. The turnover of type II collagen was measured by the formation of type II collagen (ProC2) and MMP-mediated degradation of type II collagen (C2M). The ratio between ProC2 and C2M was calculated for week 1–3. Tofacitinib and TPCA-1 increased the area under the curve (AUC) of ProC2/C2M significantly compared to O+T (p<0.001). SB203580 and R406 had no effect at 10 μM, 5 μM, 0.31 μM and 0.16 μM, but tended to increase ProC2/C2M at 2.5–0.625 μM compared to O+T (Figure 1). Safranin O and fast green staining of the explants showed that O+T, SB203580 and 0.16 μM of R406, Tofacitinib, and TPCA-1 lead to loss of proteoglycans from the cartilage explants compared to the untreated explants. R406 at 10 μM retained the proteoglycans in the deep and middle zone of the cartilage, while the proteoglycans of the superficial layer was lost. 10 μM of Tofacitinib and TPCA-1 retained the proteoglycans in all layers of the cartilage explants. Conclusions The four inhibitors tested had a positive effect on the degradation of aggrecan and type II collagen. However, only Tofacitinib and TPCA-1 had an increased anabolic effect on type II collagen turnover. The anabolic effect from Tofacitinib and TPCA-1 on top of the anti-catabolic effect indicates that the chondrocytes can repair the cartilage during treatment opposite to the p38 inhibitor that inhibits the catabolic and the anabolic response. Disclosure of Interest C. Kjelgaard-Petersen: None declared, A.-C. Bay-Jensen Shareholder of: Nordic Bioscience A/S, Employee of: Nordic Bioscience A/s, M. Karsdal Shareholder of: Nordic Bioscience A/S, Employee of: Nordic Bioscience A/S, P. Hägglund: None declared, C. Thudium Employee of: Nordic Bioscience A/S

SAT0045 The Activation of the Synovial Membrane Results in Release of Tissue Specific Biomarkers, Which in Turn can Act as Soluble Disease Activity Measures in Rheumatoid Arthritis

Background Swollen and tender joints in rheumatoid arthritis (RA) are associated with increased turnover of the connective tissue of the joint. The synovial membrane, one of those tissues, contains type III collagen, whose breakdown and formation is highly regulated in response to changes in the inflammatory burden. Matrix metalloproteinases (MMPs) are believed to play a major role in this regulation. Previous studies have shown that type III collagen turnover, measured by serum C3M, is elevated in RA as compared to controls, and can by modulated by treatment. Objectives 1) to confirm that C3M and MMP3 are directly released from the synovial membrane in inflammatory conditions using an ex vivo model, and 2) to investigate whether these biomarkers are associated with disease activity pain in RA patients. Methods Human synovial membrane isolated from arthritis patients undergoing total knee replacement at Gentofte Hospital, Denmark, were cultured as explants (SME) for 3 wks with either media (w/o), 10ng/mL Tumor Necrosis Factor α (TNFα), Interleukin (IL) 1β or Transforming Growth Factor β (TGFβ)-2. Gelatin zymography and western blotting were used to detect MMP3. C3M and MMP3 were assessed in the supernatant by ELISA. Biomarker marker measures were acquired from previously established biomarker database from the LITHE study. The biomarker study included 569 patients with moderate to severe RA. The data extraction for cross-sectional analysis included age, gender, BMI, disease duration, DAS28, VAS pain, and the log-transformed biomarkers C3M and MMP3. Results 1) Ex vivo model: Increases of 12-fold (p<0.01) and 8-fold (p<0.05) in the release of C3M were seem when the SME were treated for 10 days with the pro-inflammatory cytokines TNFα or IL1β, respectively. The increased release of C3M was in line with the release of acMMP3, with an 11-fold increase from day 7 to 21 (p<0.05) in response to TNFα and 4-5 fold increase from day 4 to 7 (p<0.01) in response to IL1β. As assessed by western blotting and gelatin zymography secretion of MMP3 was also increased by TNFα and IL1β. TGFβ2 did not increase MMP secretion of the release of C3M or acMMP3. 2) RA patients: Serum logC3M was correlated to both DAS28 (p<0.00001) and VASpain (p=0.0016) and remained highly statistical significant for both clinical outputs (p<0.001) after adjusting for age, sex, BMI and disease duration. Serum logMMP3 was also correlated to both DAS28 (p<0.00001) and VASpain (p=0.0010), which also remained highly statistical significant for both clinical outputs (p<0.00001) after adjusting for age, sex, BMI and disease duration. Conclusions This study confirms that C3M and MMP3 are released directly from the synovial tissue and thus may serve as biomarkers of synovial turnover. Furthermore there was a strong association between DAS28, VASpain, and the biomarkers, which may indicate that these are soluble markers of disease activity. Disclosure of Interest C. Kjelgaard-Petersen: None declared, A. Siebuhr Grant/research support from: EC FP7 programs, Employee of: Nordic Bioscience, I. Byrjalsen Employee of: Nordic Bioscience, K. Musa: None declared, T. Christiansen: None declared, M. Karsdal Shareholder of: Nordic Bioscience, Grant/research support from: EC FP7 programs, Employee of: Nordic Bioscience, A. Bay-Jensen Shareholder of: Nordic Bioscience, Grant/research support from: EC FP7 programs, Employee of: Nordic Bioscience