Structure-Activity Relationship of Nerve-Highlighting Fluorophores

Abstract

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.

Optimization of fluorescent imaging in the operating room through pulsed acquisition and gating to ambient background cycling

“…Fluorescence cannot only be used to mark sites of pathology for resection but also to mark sensitive areas for the surgeon to avoid [5][6][7][8][9]. Increased availability of specific fluorescent probes which delineate ducts, tissues and/or molecular expression will continue, and these will gradually find successful applications in surgical procedures.…”

Nile Red derivatives enable improved ratiometric imaging for nerve-specific contrast

“…However, there are currently few clinically approved fluorophores, none of which are nerve specific. 7,13 The oxazine fluorophore scaffold presents a unique chemical scaffold for tissue-specific Fig. 9 In vivo ratiometric tissue-specific staining after direct administration.…”

“…[5][6][7][8][9] A limited number of small molecule fluorescent contrast agents exist that stain nerve tissue in vivo, with six known classes of small molecule fluorophores that have been shown to have nerve or brain specificity. These include a handful of stilbene derivatives, 10 a coumarin analog, 11 a library of distyrylbenzene derivatives, [12][13][14][15] 8 styryl pyridinium (FM) fluorophores, 16,17 an oxazine fluorophore, 2,18 and a tricarbocyanine fluorophore. 19 Unfortunately, adipose specificity often accompanies nerve specificity for these small molecule fluorophores, 2,13,14 which is largely by necessity since the molecular composition of nerve and adipose tissues is similar, 20,21 making specific accumulation in the nerve without adipose accumulation a substantial challenge.…”

Fluorescence phenomena in nerve-labeling styryl-type dyes

“…The affinity for MBP was maintained provided certain structural features were conserved. 22 We have shown that both the absorption and the emission maxima can be red-shifted significantly by employing through-π system conjugated donor-acceptor (D-A) group, producing dyes with large Stokes shifts, thus minimizing the impact of autofluorescence in the visible region.…”