AB0123 Real-Time in Vivo Apoptosis Imaging with a Histone-H1 Targeting Probe for the Measurement of therapeutic Response in an Experimental Arthritis Model

BackgroundApoptosis is a well-organized process that is an important and active regulatory pathway of cell infiltration within inflammatory tissues. Decision on response after treatment with disease modifying anti-rheumatic drugs in rheumatoid arthritis has been performed by measuring acute phase reactants and clinical parameters such as disease activity score (DAS). Real-time monitoring of apoptosis may improve our decision on the response to therapy.ObjectivesTo examine whether in vivo imaging of apoptosis obtained by uptake of Histone H1-targeting probe labeled with near-infrared fluorescence is able to detect early response and monitor quantitative changes after treatment in a chronic inflammatory arthritis model.MethodsHistone H1-targeting probe (CQRPPRC, ApoPep-1) was synthesized using HPLC and labeled with FPR675 near infrared fluorescence dye. To induce apoptosis, cells were treated with staurosporine (STS) or methotrexate (MTX). Apoptotic cells were measured using flow cytometer and confocal microscopy. For in vivo imaging, collagen-induced arthritis (CIA) model was treated with drugs and optical imaging was performed using OpTix Explore. Total photon count (TPC) was measured by the summation of photon counts from the region of interests of four paws.ResultsTreatment of Jurkat T cells and THP-1 cells with STS and MTX induced apoptosis in a dose-dependent manner, which was detected by binding with ApoPep-1-FITC or annexin V-FITC in combination with propodium iodide in a similar sensitivity. Detection of apoptotic cells using ApoPep-1-FITC within sections of joint tissues from active CIA mice was as effective as that using tunnel assay. In active CIA mice, apoptosis imaging was performed using either annexing V or ApoPep-1 labeled with FPR675 (5 mg/kg, each), which showed a marked enhancement of TPC in ApoPep-1 imaging upto 8 hours after intravenous (i.v.) injection (Figure). According to the stage of disease progression, optical intensity of ApoPep-1-FPR675 was significantly increased compared to that of annexin V-FPR675 from the early stage of arthritis, which showed the most conspicuous difference in active arthritis. After treatment with high dose of MTX (50mg/kg, i.v.), the difference of TPC was greatest after 3 days of treatment compared with control-treated mice. The number of apoptotic cells detected using ApoPep-1-FITC within tissue sections from the joints was significantly increased after treatment with high dose of MTX.ConclusionsThe pre