Nitisinone therapy shows beneficial effects on the chondrocytes and extracellular matrix in the osteoarthropathy of alkaptonuria

Mistry, Jemma and Jackson, D. and Bukhari, Marwan A. S. and Taylor, Adam Michael (2017) Nitisinone therapy shows beneficial effects on the chondrocytes and extracellular matrix in the osteoarthropathy of alkaptonuria. Osteoarthritis and Cartilage, 25 (Suppl.). S152. ISSN 1063-4584

Abstract

Purpose: Alkaptonuria is a rare autosomal recessive form of osteoarthropathy resulting from deficiency of homogentisate 1,2 dioxygenase (HGD) causing inability to metabolise the tyrosine metabolite; homogentisic acid (HGA). HGA levels in the body become elevated and show high affinity for collagenous tissues, primarily load bearing articular cartilages, where it polymerises and deposits as a dark pigment; this process is ochronosis. Over time ochronosis causes early onset joint arthropathy, mimicking osteoarthritis (OA) in presentation but progressing much more rapidly. Recently a therapy; Nitisinone has been shown to be effective in reducing the causative molecule; HGA, therefore potentially halting the arthropathy. This study aims to examine the effect of the drug Nitisinone on cell number, ochronosis and turnover of the extracellular matrix in an in vitro model of this disease whilst also comparing with the effects of IL-1β. Methods: Cultures of immortalised human chondrocytes C20/A4 were cultured in 24 well plates in DMEM containing HGA, IL-1β, Nitisinone or a combination of these reagents for up to 9 days. The rate of pigmentation was assessed using histology and counted as a number of pigment deposits/mm3, degradation and turnover of the matrix was assessed using DMMB assay and CTX-II ELISA on cell culture supernatants. Statistical analysis was performed using Graphpad Prism by ANOVA and Newman Keuls post-hoc analysis. Results: Cultures showed a significance decrease in the number of cells after 3 days of culture when comparing cultures containing HGA, compared to any of those which didn’t (p<0.001). By day 9 of culture similar significance decreases in cell number were seen when comparing cultures containing HGA and those without; Control vs; HGA and vs HGA + IL-1β (p<0.001). The rate of pigmentation in these cultures containing HGA showed no significant differences on day 3 or 6, but by day 9 the cultures containing HGA vs those which contained HGA in combination with Nitisinone showed a significantly higher amount of pigmentation. DMMB assay revealed a significant increase in the amount of GAG in supernatants of culture containing HGA alone vs control, IL-1β, Nitisinone or HGA+IL-1β+NTBC cultures. The cultures containing a combination of HGA+IL-1β vs HGA also showed a significant increase in the amount of GAG released. By day 9 the amount of GAG detectable was not significantly different between treatment groups, although the cultures containing HGA+IL+NTBC did show a decrease in the amount of GAG compared to HGA and HGA+IL-1β. CTX-II showed no significant differences in any treatment groups across 3, 6 or 9 days. Conclusions: This model is the first to test the effect of Nitisinone on the chondrocytes and their extracellular matrix in a model of Alkaptonuria; demonstrating the combined effect on pigmentation rate, cell number and the extracellular matrix. The effect of HGA on cell number shows an early effect on chondrocytes suggesting rapid decrease in chondrocytes at day 3. This is mirrored by loss of GAG from cultures early in the presence of HGA. Nitisinone shows a significant effect in decreasing the pigmentation rate, thus preventing the advancement of disease in these patients. Our results are the first to show the effect of Nitisinone on the release of GAG’s in this in vitro model and provide useful insight into how this drug may protect cartilage in these patients; this also shows some effect of protection of GAG in the presence of IL-1β alone. It also demonstrates that when HGA, the causative molecule in AKU is compared to one of the key mediators of OA; IL-1β, the effect on the cells and their matrix is more severe - giving potential insight into why the arthritis in AKU is more severe and rapidly progressing than that in OA.