Background/Purpose: Osteoarthritis
(OA) and rheumatoid arthritis are degenerative diseases of the whole joint.  The
extracellular matrix (ECM) turnover is highly regulated in response to the
inflammatory status. This leads to an increased degradation of collagen type
III in the synovium and an increased degradation of collagen type II and
aggrecan in cartilage. The spleen tyrosine kinase (Syk) is involved in
pro-inflammatory signaling and R406, the active metabolite of the
anti-inflammatory drug, Fostamatinib inhibits Syk. Fostamatinib has been tested
in both phase II and phase III clinical trials with mixed results. The aim of this
study was to investigate R406 effects on the ECM
turnover of the synovium and cartilage under inflammatory conditions ex vivo.

Methods: Cartilage
turnover was investigated using a bovine cartilage explant (BEX) model. Synovial
tissue turnover was investigated using a human synovial membrane OA explant
(SME) model.  BEX: BEX was cultured 3 weeks without (w/o) treatment, or
in the presence of 2 ng/mL TNFα + 10 ng/mL OSM  (O+T), or O+T with R406 in
7 different concentrations from 10 μM to 0.156 μM. SME: The
explants were cultured for 2 weeks w/o treatment, or in the presence of 10
ng/mL TNFα, or TNFα with 5 μM, 0.5 μM or 0.05 μM R406.
Both models were cultured with O+T or TNFα with the p38 inhibitor 10
μM SB203580. The tissue turnover was assessed with the biomarkers; C2M, C3M,
acMMP3 the ARGS neo-epitope in the supernatant with ELISA.

Results: BEX)
O+T increased the MMP-mediated degradation of collagen type II (C2M) after 14 days
and ARGS after 7 days compared to w/o (p<0.0001). All concentrations of R406
inhibited the release of C2M in a dose depended manner. R406 at 10 μM (p<0.0001), 5 μM (p<0.0001) and 2.5 μM (p
= 0.0281) decreased the release of ARGS compared to O+T, whereas lower
concentrations of R406 did not affect ARGS (Figure 1A).  SB202580 at 10 μM
decreased C2M (p<0.0001), but did not affect the ARGS release compared to
O+T. SME) TNFα increased the released of the MMP-mediated
degradation of collagen type III (C3M) (p=0.0400) and activated MMP3 (acMMP3)
(p=0.0371) compared to w/o. R406 at 5 μM tended to decrease the release of
C3M, while R406 at 0.5 μM (p=0.0219) increased and 0.05 μM tended to increased
C3M compared to TNFα. (Figure 1B). R406 tended to decrease the release of acMMP3
at 5 μM and had no effect at 0.5 μM and 0.05 μM compared to TNFα.
SB203580 did not affect C3M or acMMP3 compared to TNFα.

Conclusion: These
data show that R406 can inhibit pro-inflammatory ECM joint degradation at
concentrations higher than 0.5 μM, but at 0.5 μM or lower R406
increased the inflammatory degradation of collagen type III and had no effect
on aggrecan degradation. In contrast, the p38 inhibitor SB203580 had no effect on
synovial ECM turnover or aggrecan degradation, underlining the importance of
understanding the differences between inhibitors of pro-inflammatory mediators
and their effect on the joint tissue.