Background/Purpose: A primary challenge for intra-articular delivery of protein and peptide therapies has been their short residence time in the joint tissues. The IL-1 receptor antagonist (IL1RA) could be therapeutic for treating local joint inflammation in monoarticular gout flare, osteoarthritis flare, or post-ACL injury. However, it has been limited by its short half-life in the joint, and previous attempts to prolong its residence time have led to decreased potency. A novel strategy for tissue-targeted local delivery involves fusion of a matrix-binding domain that binds to charged proteoglycans within the extracellular matrix. We describe here the generation of a matrix-binding interleukin-1 receptor antagonist (MB-IL1RA) fusion without loss of activity.

Methods: The matrix binding peptide is 21 amino acid sequence derived from the heparin-binding domain of HB-EGF. The MB-IL1RA fusion protein was expressed in E. coli and purified to homogeneity. Activity was tested in a cell-based assay using IL-1 to stimulate an NF-kB-driven luciferase reporter. Retention in articular cartilage tissue was tested in newborn bovine cartilage tissue explants. After incubation of either anakinra or MB-IL1RA with the tissue, the cartilage was washed into medium with no drug and assayed via Western analysis for drug remaining in the tissue.  

Results: The 50% inhibitory concentrations (IC50s) of MB-IL1RA and anakinra were determined in the NFkB reporter cells in the presence of 5 ng/ml interleukin-1β, yielding IC50s of 175 pM ± 23 pM for anakinra and 152 pM ± 26 pM for MB-IL1RA (mean of 9 experimental repeats; a representative run is shown in Figure 1).

After wash-out of bovine cartilage explant disks for 30 min., IL-1RA (anakinra) was undetectable in the tissue by Western analysis (densitometric analysis shown in Figure 2). In contrast, the MB-IL1RA fusion protein was robustly detectable within the tissue even after two days of wash-out in plain medium.

Conclusion: The novel therapeutic fusion protein MB-IL1RA allows delivery of an IL-1 inhibitor to the articular cartilage and extended residence time within the tissue, while retaining full potency of the inhibitor. MB-IL1RA is a candidate therapeutic for IA administration to treat local inflammation in the joint in acute flares of gout and OA.