A radioactive and fluorescent dual modality cysteine cathepsin-B activity-based probe for the detection and treatment evaluation in rheumatoid arthritis.

Activated macrophages are key effector cells and specific markers in patients with rheumatoid arthritis (RA). Cysteine cathepsin B (CTS-B) is highly expressed in macrophages and positively associated with RA activity and severity. This study aims to evaluate an activity-based multi-modality diagnostic agent, ⁶⁸Ga-BMX2, which targets CTS-B to visualize the arthritis activity and evaluate the treatment efficacy. A CTS-B activity-based probe, BMX2, was labeled efficiently with ⁶⁸Ga to produce ⁶⁸Ga-BMX2 for fluorescent and positron emission tomography (PET) multi-modality imaging. The affinity and specificity of BMX2 binding with the CTS-B enzyme in macrophages were determined by radioactive experiment using RAW 264.7 cell lines, with CA074 and BMX5 as the inhibitors to test the specificity of the binding. Then, PET and fluorescence imaging were acquired on collagen-induced arthritis (CIA) mice. Additionally, the treatment monitoring capability of ⁶⁸Ga-BMX2 PET/CT imaging was tested with methotrexate (MTX). RAW 264.7 macrophage cells showed significant uptake of ⁶⁸Ga-BMX2. The binding of BMX2 with CTS-B in RAW 264.7 macrophage cells is time-dependent and could be blocked by CA074 and BMX5. In vivo optical and PET imaging showed high signals in the right hind arthritis in CIA mice from ⁶⁸Ga-BMX2 and BMX2 accumulated for at least 120 h. Additionally, ⁶⁸Ga-BMX2 signals were significantly reduced in the MTX-treated CIA mice compared to the control group. The ⁶⁸Ga-BMX2, a radioactive and fluorescent dual-modality diagnostic agent targeting CTS-B, demonstrated a practical approach for CIA PET and fluorescence imaging. The ⁶⁸Ga-BMX2 multimodality imaging could significantly monitor the treatment response in the CIA mice.