Rheumatoid Arthritis as a New Disease-Target for Bortezomib.

Bortezomib (Velcade) is the first proteasome inhibitor that has been introduced into the clinic for the treatment of multiple myeloma. Bortezomib, by inhibiting the NFkB function, results in increased tumor cell sensitivity to chemotherapy and induces apoptosis of alloreactive T-cells. Since NF-kB activation results also in transcription of pro-inflammatory molecules, it is speculated that NF-kB inhibition may also ameliorate inflammatory and autoimmune conditions. Rheumatoid arthritis may be a possible target disease for proteasome inhibitors because the most important pro-inflammatory mediators (TNF-a, IL-1, IL-6, V-CAM-1) are regulated by NF-kB. We investigated the effect of Bortezomib in a rat model of Adjuvant Arthritis (AA) which closely resembles human rheumatoid arthritis. The presence of 5,10,100 nM bortezomib for 72h or the presence of 10 nM bortezomib for 48 and 72 h in culture with ConA–activated T-cells from the spleen of rats after AA induction, significanlty inhibited their proliferation as measured by radioactive thymidine incorporation (p<0,00005 in all concentrations and culture times tested). Likewise, the presence of 10 nM Bortezomib for 48 and 72 h in fibroblast-like synoviocytes (FLS) culture from rats after AA induction, significanlty inhibited proliferation (p<0,00005). Bortezomib’s effect in AA was also explored in vivo by intraperitoneal injections at 0,25mg/kg×2days×2weeks in rats after AA induction by FCA injection in the tail base. Adjuvant artrhitis usually starts to develop at day 11–13 post induction. Bortezomib was administered

• before the onset of arthritis

• at the onset of arthritis and

• in established arthritis.

The total arthritic index represented a score based on redness, swelling and deformity of the joint. Arthritis grading was assessed on a 4-point scale per limb with a maximum arthritic score of 16 per rat. The histologic severity of arthritis was also assessed by a histopathological score based on the presence of synovial hyperplasia, inflammatory infiltration, chondroplasia, bone destruction and pannus formation (max score 12 per animal). A significant delay in arthritis onset accompanied by a milder clinical picture was observed in the group which received Bortezomib before the onset of arthritis (mean day21 arthritic score; 1). More importantly, there was a dramatic reduction in the disease score in the group treated with Bortezomib both at the onset of arthritis (mean day26 arthritic score;4,1) and at advanced disease status (mean day30 arthritic score; 3,3) as compared to the untreated AA control group (mean day21, day26 and day30 arthritic score 9,6, 9,9 and 11,3 respectively/p=0,03, p=0,02 and p=0,02, respectively). There was a correlation between the clinical and histopathological assessment of disease severity in Bortezomib-treated animals vs control rats (histological score 3,75 vs 9,8 respectively). A marked reduction in the number of inflammatory cells with little or no bone erosion and limited pannus formation could be seen in the joints of bortezomib-treated rats. A significant reduction of CD3+ cells in the joints and inhibition of NF-kB in spleen sections of Bortezomib-treated rats was detected by immunohistochemistry. Flow cytometry analysis demonstrated a significant increase of CD4+ cells with reduction of CD8+, CD11b+ cells in the blood (p=0,003 and p=0,0002, respectively) and the spleen (p=0,01 and p=0,017, respectively). T-cells from Bortezomib-treated animals exhibited a higher degree of apoptosis measured as Annexin-V + cells, when compared to T-cells from AA control rats. In CT scans before and after treatment with Bortezomib of animals that received bortezomib in advanced disease phase, a significant reduction of soft tissue swelling around the affected joints with restoration of focal osteopenia and bone erosion could be detected. Currently, we are testing the combination of Bortezomib with Mesenchymal Stem Cells in the same disease model, as bortezomib has been shown to enhance the osteogenic potential of MSCs. Our data suggest that Bortezomib exhibits significant anti-inflammatory and proapoptotic activity in a rat model of adjuvant arthritis and human rheumatoid arthritis may represent an important clinical opportunity for this drug