Args neoepitope, presumably derived from adamts5 activity, is elevated in serum and urine from osteoarthritis patients undergoing knee replacemenT

Abstract

Purpose: Proteolytic degradation of aggrecan in articular cartilage is a characteristic feature of osteoarthritis (OA). ADAMTS5-mediated cleavage of aggrecan generates the ARGS neoepitope. The objective of this study was to evaluate ARGS levels in variousmatrices from different OA populations in contrast to human subjects and assess performance of a sensitive target-specific immunoassay to facilitate the clinical development of an ADAMTS5 inhibitor for the treatment of OA. Methods:Matched sera and urine were collected from 20 healthy subjects (13 women, 7 men) aged from 26 to 60 years (mean SD: 43.2 8.4) and from 20 subjects with knee OA (12women, 8 men aged from 49 to 77 years (mean SD: 66.1 7.36). Matched serum, urine and synovial fluid was obtained from 19 subjects undergoing joint replacement for knee OA (15 women, 9 men) aged from 47 to 85 years (mean SD: 69.1 11.30). Diurnal and inter-day variation (over 3 visits) was evaluated in the non-surgical OA group. Serum and urine samples were collected on two visits for the patients undergoing knee replacement with synovial fluid taken only at the time of surgery. Correlation to additional biomarkers including OSM, COMP, PIIANP, CTX-II, CTX-1, OC, hsCRP, CPII, CIIM, AGNx-1 was assessed. ARGS neoepitope was quantitated using a chemiluminescent Meso-ScaleDiscovery (MSD) immunoassaywhich had been optimised and validated in serum, plasma, urine and synovial fluid. In brief, a commercial antiaggrecan capture antibody was coupled with a sulfo TAG labelled GSK antibody specific to the ARGS neoepitope (OA-1). ARGS standard was generated by digestion of aggrecan with ADAMTS5 which was diluted in appropriate matrices pre-depleted of ARGS. The lower limit of quantification was 1.37, 0.46, and 4.12 ng/mL in serum, urine, and synovial fluid respectively. Results: The mean concentration of ARGS neoepitope in serum from healthy subjects was 4.3 2.2ng/mL (range 0.9-8.9 ng/mL), 5.9 2.7ng/mL (range 1.1-10.0) in non-surgical OA subjects, and 9.3 4.6ng/mL (range 2.016.5) in the surgical OA patients. Synovial fluid concentrations of ARGS in the surgical OA patients were similar to serum levels in those patients (mean SD: 8.28 5.29). ARGS levels were lower in urine than serum in all subjects but showed a similar trend of elevation in the surgical OA patients (mean SD 5.9 4.5 range 0.5-13.9) compared to 3.31 2.28 (mean SD) in non-surgical OA patients (range 0.5-8.7) and 2.7 3.3 (mean SD) in healthy subjects (range 0-10.8). Compared to healthy subjects, both serum (p1⁄40.004) and urine levels (p1⁄40.008) of ARGS were significantly elevated in the surgical OA patients. No significant difference was seen between healthy and non-surgical OA subjects. No significant diurnal effect or inter-day variance was observed in urine or serum. Serum levels of ARGS correlated positively with COMP and CTXII (p<0.05) but not with other markers (OSM, PIIANP, CTX-1, OC, hsCRP, CPII, CIIM, AGNx-1). Conclusions: This study utilized a highly sensitive and robust assay to provide an initial evaluation of ARGS neoepitope concentrations in various matrices in OA patients and healthy volunteers. There was a general trend for increasing ARGS neoepitope concentration in serum going from healthy subjects to non-end stage knee OA patients to end stage (knee replacement) knee OA. Additional studies are planned to evaluate ARGS as a diagnostic tool to facilitate patient selection as well as provide an early indication of the pharmacodynamic effects of an ADAMTS5 inhibitor in clinical trials.