Structure-Activity Relationship of Nerve-Highlighting Fluorophores

Citation: Gibbs SL, Xie Y, Goodwill HL, Nasr KA, Ashitate Y, et al. ( Structure-Activity Relationship of Nerve-Highlighting Fluorophores Summer L. Gibbs 0 Yang Xie 0 Haley L. Goodwill 0 Khaled A. Nasr 0 Yoshitomo Ashitate 0 Victoria J. Madigan 0 Tiberiu M. Siclovan 0 Maria Zavodszky 0 Cristina A. Tan Hehir 0 John V. Frangioni 0 A Ganesan, University of East Anglia, United Kingdom 0 1 Department of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States of America, 2 Advanced Imaging Research Center, University of Texas Southwestern Medical Center , Dallas, Texas , United States of America, 3 Diagnostics and Biomedical Technologies, GE Global Research, Niskayuna, New York, United States of America, 4 Department of Radiology, Beth Israel Deaconess Medical Center , Boston, Massachusetts , United States of America Nerve damage is a major morbidity associated with numerous surgical interventions. Yet, nerve visualization continues to challenge even the most experienced surgeons. A nerve-specific fluorescent contrast agent, especially one with near-infrared (NIR) absorption and emission, would be of immediate benefit to patients and surgeons. Currently, there are only three classes of small molecule organic fluorophores that penetrate the blood nerve barrier and bind to nerve tissue when administered systemically. Of these three classes, the distyrylbenzenes (DSBs) are particularly attractive for further study. Although not presently in the NIR range, DSB fluorophores highlight all nerve tissue in mice, rats, and pigs after intravenous administration. The purpose of the current study was to define the pharmacophore responsible for nerve-specific uptake and retention, which would enable future molecules to be optimized for NIR optical properties. Structural analogs of the DSB class of small molecules were synthesized using combinatorial solid phase synthesis and commercially available building blocks, which yielded more than 200 unique DSB fluorophores. The nerve-specific properties of all DSB analogs were quantified using an ex vivo nerve-specific fluorescence assay on pig and human sciatic nerve. Results were used to perform quantitative structure-activity relationship (QSAR) modeling and to define the nerve-specific pharmacophore. All DSB analogs with positive ex vivo fluorescence were tested for in vivo nerve specificity in mice to assess the effect of biodistribution and clearance on nerve fluorescence signal. Two new DSB fluorophores with the highest nerve to muscle ratio were tested in pigs to confirm scalability. - Funding: This work was funded by grants from the National Institute of Health, including R01-EB-022872 (CTH), R01- CA-115296 (JVF), and K01EB-010201 (SLG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared the following interests: FLARETM technology is owned by Beth Israel Deaconess Medical Center, a teaching hospital of Harvard Medical School. It has been licensed to the FLARETM Foundation, a non-profit organization focused on promoting the dissemination of medical imaging technology for research and clinical use. Dr. Frangioni is the founder and chairman of the FLARETM Foundation. The Beth Israel Deaconess Medical Center will receive royalties for sale of FLARETM Technology. Dr. Frangioni has elected to surrender post-market royalties to which he would otherwise be entitled as inventor, and has elected to donate pre-market proceeds to the FLARETM Foundation. Dr. Frangioni has started three for-profit companies, Curadel, Curadel ResVet Imaging, and Curadel Surgical Innovations, which may someday be non-exclusive sublicensees of FLARETM technology. Co-authors Tiberiu M. Siclovan, Maria Zavodszky and Cristina A. Tan Hehir are employed by General Electric Company. Drs. Siclovan and Tan Hehir are inventors of patents and patent applications on technologies related to nerve imaging. These include US8169696B2 (Systems for intraoperative nerve imaging); US8114382B2 (Myelin detection using benzofuran derivatives); US20110142759A1 (Agents and methods for the imaging of myelin basic protein); US20100310457A1 and US20100310456A1 (imaging of myelin basic protein). There are no further products in development or marketed products to declare. This does not alter the authors adherence to all the PLOS ONE policies on sharing data and materials. Nerve damage during surgery results in significant morbidity for patients, causing both chronic pain and permanent paralysis [13]. Nerve-sparing surgery can prove difficult, as currently no nerve-specific contrast agents are clinically available to aid in intraoperative visualization. At present, nerve detection during surgery is largely completed through electromyographic (EMG) monitoring in delicate areas, such as surgical procedures near the larynx, thyroid, or spinal cord [46], or direct visualization by the surgeon. Current methods are suboptimal as EMG monitoring is an electrical stimulus detection method rather than an imaging methodology, and direct visualization can be hampered by the nature of the small, translucent nerve structures that are typically protected deep within the tissue. Improved nerve visualization would result from a nerve-specific optical contrast agent that could aid nerve visualization during image-guided surgery. Fluorescence-guided surgery is quickly gaining traction because it provides real-time assessment of normal and diseased tissues. A number of fluorescent image-guided surgery systems are in development, clinical trials, or are commercially available for use [7]. However, targeted fluorophore availability is currently limited, and a clinically viable nerve-specific fluorophore does not exist. Histopathological examination of myelin has been possible for many years using a number of colorimetric stains [810], and fluorophores that specifically label myelin have also been developed [11,12]. However, none of the currently available histopathological contrast for myelin can be administered systemically to stain nerve tissue in vivo, because the contrast agents will not penetrate the blood nerve barrier (BNB). There are only four classes of fluorescent molecules that have been found to penetrate the BNB and stain nerve tissue in vivo following systemic administration, which include nervespecific peptides and three classes of small molecule organic fluorophores. The nerve-specific peptides are a targeting sequence that largely highlights the epineurium with some binding to the endoneurium [13]. The three classes of nervespecific small molecule organic fluorophores include the stilbene derivatives [14], the distyrylbenzene (DSB) derivatives [1417], and the styryl pyridinium (FM) fluorophores [18,19]. The FM dyes have been found to stain only the dorsal root and trigeminal ganglia when administered systemically [18]. The stilbene derivatives (...truncated)