Abstract CN04-02: A novel approach to biomolecular imaging of COX-2
Authors: Uddin Md. Jashim , Crews Brenda C. , Ghebreselasie Kebreab , Blobaum Anna L. , Duggan Kelsey C. , Xu Shu , Kingsley Philip , Cekanova Maria , Marnett Lawrence J. 
Published: January 2014

Abstract
Molecular imaging emerged in the early 21st century as a discipline that enables the visualization of cellular functions, molecular biology of diseases, and diagnosis of disease states in living beings. COX-2 is an attractive target for molecular imaging of cancer, because it is an inducible enzyme that is expressed in inflammation, premalignant and malignant lesions. We designed fluorescent COX-2 inhibitors by tethering bulky fluorescent functional groups onto non-steroidal anti-inflammatory drugs (NSAIDs), and COX-2 inhibitors (COXIBs). To identify the lead compounds, we synthesized a library of 200 hundred fluorescent conjugates, and evaluated them for selective inhibition of purified COX-2, COX-2 in mouse macrophages and COX-2 in human cancer cells. Although a significant number of compounds showed COX-2 inhibitory activity against purified protein, only a fraction of these compounds inhibited COX-2 activity in intact cells, and of those, most did not possess fluorescence properties suitable for in vivo imaging. Optimized lead compounds were then tested for their ability to image COX-2 in intact cells. Among the compounds that emerged from our development pathway, only 5-carboxy-X-rhodamine (5-ROX) and 6-carboxy-X-rhodamine (6-ROX) based conjugates, Fluorocoxib A and Fluorocoxib B, exhibited sufficient metabolic stability to distribute to target tissues in rodents and canines. A detailed kinetic analysis indicated that these compounds are slow, tight-binding inhibitors of COX-2 with very low rates of association and dissociation. These compounds displayed a very high degree of selectivity of uptake in inflammatory tissues or human tumor xenografts in rodents, and in naturally occurring oral or bladder cancer in dogs relative to surrounding normal tissue or muscle as determined by either optical imaging or mass spectrometry. Uptake of these compounds required the expression of COX-2 at the target site and declined as the level of COX-2 decreased. Experiments with COX-2 (-/-) animals or with animals pretreated with indomethacin or celecoxib verified that selective accumulation into inflamed tissue or tumors was due to binding to COX-2. Pharmacokinetic analysis revealed that only the intact parent compound is found in the region of interest. Site-directed mutagenesis and analysis of X-ray co-crystal structure of fluorocoxib A with COX-2 revealed that the n-butyl linker group allows the indomethacin part of fluorocoxib A to fully insert into the binding pocket of COX-2, while the bulky fluorescent 5-ROX group binds in the lobby of the enzyme. Thus, fluorocoxibs represent the first feasible optical imaging reagents for clinical detection of tissues containing high levels of COX-2 in settings amenable to fluorescent excitation and analysis by surface measurement or endoscopy (e.g., skin, esophagus, intestine, and bladder).
(This work has been supported by grants from the National Cancer Institute and National Institutes of Health [CA136465 and CA128323])
Citation Format: Md. Jashim Uddin, Brenda C. Crews, Kebreab Ghebreselasie, Anna L. Blobaum, Kelsey C. Duggan, Shu Xu, Philip Kingsley, Maria Cekanova, Lawrence J. Marnett. A novel approach to biomolecular imaging of COX-2. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr CN04-02.