Studies of Novel Immunosuppressive Agents in Experimental Arthritis

Rheumatoid Arthritis (RA) is a chronic inflammatory disease of the joints. The hallmark of RA is persisting inflammation in diarthrodial joints with infiltration of leukocytes, thickening of the synovial lining layer and synovial pannus formation. The therapeutic aim for RA patients is to alleviate the symptoms, slow the disease progression and optimise the quality of life. In recent years the measures to achieve this goal have improved with the development of new drugs. Despite recent advances in therapies for RA, there is still no drug available that induces remission in all patients. Thus, there is still a need to screen and develop new anti-rheumatic treatments. Because of this we investigated the effects of treatment with compounds which were extracted from three different sources in collagen-induced arthritis (CIA) in Dark Agouti (DA) rats. The constituent of the first compound is dominated by flavonoids together with other extractable compounds derived from Artocarpus leaves. The second compound is Glucurononxyclomannan (GXM) isolated from culture filtrates of Cryptoccocus neoformans. GXM is a polyssacharide with an unbranched mannose backbone. The third compound, code named Rob 803, is 9-chloro-2,3 dimethyl-6-(N,N-dimetylamino-2-oxoethyl)-6H-indolo [2,3-b] quinoxaline, an analogue of a previously reported substance, B220. B220 was originally investigated as an anti-viral drug. Results: 1. Treatment of rats with the Artocarpus extract decreased arthritis incidence and severity and delayed disease onset. In vitro, the Artocarpus extract acted as a T cell modulator, inhibiting mitogen-induced T cell proliferation and inducing apoptosis of activated cells. A new flavonoid glucoside named artonkin-4’-βD-O-glucoside with an average molecular mass of 514.49 Da was isolated together with three other previously known flavonoid molecules. All four compounds were found to have anti-inflammatory effects. These effects correlated well with the inhibition of mitogen-induced T cell proliferation. Furthermore, the compounds inhibited TNF and IFNγ production of ConA stimulated T cells in a concentration-dependent manner. 2. Treatment of CIA with GXM at a dose of 50 mg/kg suppressed disease development both prophylactically and therapeutically. The effect of GXM was associated with a significant decrease of the titers of anti-CII antibodies. GXM therapy diminished MMP-2 activity but had no effect on apoptosis. 3. The anti-arthritic effect of the synthetic compound Rob803 was evaluated in the CIA model. Daily subcutaneous treatment with 40mg/kg/day of Rob803 significantly suppressed arthritis severity and delayed the onset of clinical arthritis. In vitro analysis of the anti-inflammatory effects of Rob803 revealed that Rob803 suppressed NO2 production. Interestingly, T cell proliferation was suppressed at a 1000-fold lower concentration than was NO production. Thus we report that early subcutaneous administration of the synthetic substance Rob803 has antirheumatic effects which are likely to be mediated via T cell suppression. Conclusion: All of the investigated new compounds have anti-arthritic properties when tested in CIA. They have anti-T cell proliferative features which can be of benefit in modulating arthritis pathogenesis. Some of the investigated compounds also have cytokine-inhibiting properties. All compounds have low toxicity. They have the potential to be further investigated in experimental as well as clinical trials of RA and in other inflammatory diseases. LIST OF PUBLICATIONS