Periodic Report Summary 1 - IMAGFIB (Imaging Fibrosis - Chemistry and Optical Confocal Laser Endomicroscopy) Fibrosis, or scarring, is a process which is associated with many diseases of the lung and other organs, which constitute a heavy burden of morbidity and untimely deaths and for which there is no current treatment and fibrotic diseases. Active fibrosis is also associated with many cancers. There are currently no suitable biomarkers to detect active fibrosis in humans. The global aims of the project were to generate and validate, in a biological setting, a series of probes for the detection and analysis of fibrosis in vitro and in vivo. This project targeted the development of ‘smart’ probes to allow the detection of active fibrogenesis in ‘real time’ and brought together the application of organic synthesis and ‘cutting-edge’ technology to perform optical imaging microscopy in diseased organs. This approach is based on the micro-dosing of a ‘tiny’ dose of a fluorogenic compound into the lungs of patients - with the compound designed to specifically detect and respond to key events in the fibrotic process. The biological targets chosen are well recognised as key components/activants of the fibrogenic pathway and were amenable for optical imaging using confocal laser endoscopy and "smart" probes. The "smart" probes developed during this project had various mechanisms of action depending on their enzyme target and were focussed on:(a). Protease Based Probes for MMP-9(b). Protease Based Probes for Thrombin (c). Lysyl oxidase (LOX) activity fluorescent reporters and inhibitorsKey drivers for my project were the generation of "smart" probes that could be readily synthesised, have appropriate water solubility and stability, and target specificity and detectability by the imaging system. This lead to multiple and iterative rounds of synthesis and screening throughout the project, in a dynamic and responsive manner, where the results of the biological analysis fed into the next round of synthesis to allow probe optimisation. The probes were designed to monitor extracellular MMP's and/or thrombin in the lung providing an OFF/ON fluorescent signal, and were tuned for optimal S/N ratio, rate of cleavage, specificity and solubility. Evaluation in vitro against recombinant enzymes showed an increase in fluorescecne following activation, which could be knocked down with inhibitors. When human tissue samples were treated with smart probes a significant increase in fluorescence was observed, with this rise suppressed by known inhibitors. The activation of the LOX probe by the enzyme was confirmed in human lung homogenate models. Furthermore, probe activation was inhibited in the presence of specific LOX inhibitors, thus confirming target specificity. The probe also showed utility in a size-relevant model of lung fibrogenesis. This optical Smartprobe has the potential to image real-time LOX activity within the lungs of patients, since significant signal amplification is detected after 30min, a time-point that is feasible for bedside imaging. The research project aim was to create novel active chemical “smartprobes” as markers of fibrogenesis in situ to rapidly establish early diagnosis and efficacy of much needed new therapies in scarring diseases of the lung and other organs. As a result, one of the lead probes developed in this research is being prepared to GMP standards, and we anticipate 'first-into-man' studies in the next few months.
