Intraoperative Fluorescence Imaging of Peripheral and Central Nerves Through a Myelin-Selective Contrast Agent

Victoria E. Cotero 0 Tiberiu Siclovan 0 Rong Zhang 0 Randall L. Carter 0 Anshika Bajaj 0 Nicole E. LaPlante 0 Evgenia Kim 0 Daniel Gray 0 V. Paul Staudinger 0 Siavash Yazdanfar 0 Cristina A. Tan Hehir 0 0 GE Global Research, One Research Circle , Niskayuna, NY , 12309, USA Purpose: Patients suffer from complications as a result of unintentional nerve damage during surgery. We focus on improving intraoperative visualization of nerves through the use of a targeted fluorophore and optical imaging instrumentation. Procedure: A myelin-targeting fluorophore, GE3111, was synthesized, characterized for its optical and myelin-binding properties using purified myelin basic protein, and evaluated in mice. Additionally, a compact instrument was adapted to visualize nerves. Results: GE3111 was synthesized using a versatile methodology. Its optical properties were sensitive to the local environment both in vitro and in vivo. Following intravenous injection, central and peripheral nerves were visualized, with the kinetics of nerve uptake modifiable depending on the formulation. Fluorescence polarization showed specific and strong binding to purified myelin basic protein. Nerves were visualized in vivo using a dedicated compact imaging device requiring less than 2.5 mW/cm2 of illumination at 405 nm. Conclusions: Fluorescence imaging of nerves through myelin showed a potential for use in image-guided surgery. Intraoperative nerve imaging is an example where contrast agent and instrument development come together as a result of clinical need. - I morbidity associated with many surgical procedures, atrogenic damage to peripheral nerves is a major cause of including prostatectomy [14], coronary artery bypass graft [57], thyroidectomy [8, 9], rhytidectomy [10], and breast cancer surgery [1113]. Symptoms associated with nerve damage are dependent upon the location, type of nerve, and the severity of the damage, and may result in loss of function, weakness, muscle atrophy, fasciculation, paralysis, cardiac irregularities, allodynia, and chronic neuropathy [14]. The cause of nerve damage during open and laparoscopic surgical procedures is variable but is often the result of inadvertent surgical damage due to poor visibility of the nerve as compared to surrounding tissues or an unfortunate necessity due to close proximity of the nerve to target structures [15]. Currently, most surgical procedures are performed without image guidance, as available technologies lack the specificity needed to provide nerveselective imaging [16]. Applied nerve-sparing procedures generally rely on anatomical landmark identification and are highly dependent on the surgeon's skill and experience. In addition to visual identification, intraoperative electrical stimulation devices are often employed to verify continued stimulation, via nerve, of the muscle or organ in question [17, 18]. However, there are inherent limitations to relying on these methods alone. Visual identification of nerves can be inconsistent due to the intricacy and size of the individual nerves, and overall variation in the anatomic location across patient populations [19]. Furthermore, intraoperative electrical stimulation fails to prevent nerve damage; rather, it identifies damage that has already occurred. Thus, optical imaging could provide a valuable clinical tool for imageguided surgery by allowing direct and real-time visualization of nerves. We have previously reported the generation of a nervespecific fluorophore, 4-[(1E)-2-[4-[(1E)-2-[4-aminophenyl] ethenyl]-3-methoxyphenyl] ethenyl]-benzonitrile (GE3082), that crosses the bloodnerve and bloodbrain barriers, producing significant fluorescence in myelinated nerves after a single systemic injection [20]. Because of its lipophilic nature, GE3082 requires a specialized intravenous formulation consisting of 65 % serum, 20 % HEPES, 10 % dimethyl sulfoxide (DMSO), and 5 % Cremophor EL to maintain aqueous solubility, and thus, it is non-ideal for clinical intravenous use due to the potential negative physiologic and pharmacologic effects arising from this formulation [21, 22]. The goal of our study is to advance the current understanding of myelin-targeting fluorophores and to demonstrate in vivo imaging of nerves during surgery. We describe here the in vitro and in vivo characterization of a newly synthesized fluorophore, 1-methylsulfonyl-4-[(1E)-2[4-[(1E)-2-[4-aminophenyl] ethenyl]-3-methoxyphenyl] ethenyl]-benzene (GE3111). GE3111 was made using a more versatile synthetic methodology with reduced number of steps and more amenable to creating chemical libraries by parallel synthesis. GE3111 had improved aqueous solubility as well as reduced lipophilicity compared with GE3082, allowing for the development of more clinically relevant formulations for intravenous injection. We also describe advancements in the understanding of the myelin-targeting binding interaction, pharmacodynamics, pharmacokinetics, and environmental influences on the optical properties of this fluorophore. Materials and Methods Synthesis of GE3111 GE3111 was synthesized in a stepwise procedure as shown in Fig. 1. Heck coupling [23] of 4-bromo-3-methoxybenzaldehyde [24] with Boc-protected 4-amino styrene in the presence of the water-soluble TPPTS catalyst proceeded in 70 % yield after purification, to give stylbene aldehyde 2. Subsequent olefination [25] with the phosphonate 3 proceeded in 65 % yield after purification to give the bis-stylbene 4, exclusively in the transtrans configuration [26]. Deprotection with trifluoroacetic acid (TFA) in amylene-containing dichloromethane, gave the desired dye 5 in essentially quantitative yield and better than 95 % purity by nuclear magnetic resonance (NMR) spectroscopy. Removal of traces of fluorescent impurities was achieved through a final purification by reverse phase chromatography, eluting with wateracetonitrile gradient containing 0.1 % v/v TFA. The dye was found to be more stable upon storage as its TFA salt; whenever free base dye was needed, a simple aqueous workup (NaHCO3/dichloromethane) supplied the required dye as 999.9 % purity. Details of the synthetic methodology can be found in the Supplementary Material. Physical and Optical Properties of GE3111 A 10 mM stock solution of GE3111 was prepared in anhydrous dimethylsulfoxide (DMSO) to ensure complete dissolution of the fluorophore. Subsequent aliquots of the stock solution were taken to prepare 10 M solutions of GE3111 in the following solvents: toluene, olive oil, DMSO, water, and a selected intravenous (IV) formulation (58.5 % distilled water, 30 % 2-hydroxypropyl-cyclodextrin, 10 % propylene glycol, 1 % PEG-300, and 0.5 % DMSO). Absorbance spectra were taken using a Lambda 20 UV/ Vis spectrometer (Perkin Elmer, Waltham, MA). The wavelength of maximum absorbance was then used as the excitation wavelength for the collection of the fluorescence emission spectra on a steady-state fluorimeter (Photon Technology I (...truncated)