Nerve damage is the major morbidity of many surgeries, resulting in chronic pain, loss of function, or both. The sparing of nerves during surgical procedures is a vexing problem because surrounding tissue often obscures them. To date, systemically administered nerve-highlighting contrast agents that can be used for nerve-sparing image-guided surgery have not been reported. In the current study, physicochemical and optical properties of 4,4’-[(2-methoxy-1,4-phenylene)di-(1E)-2,1-ethenediyl]bis-benzenamine (BMB) and a newly synthesized, red-shifted derivative 4-[(1E)-2-[4-[(1E)-2-[4-aminophenyl]ethenyl]-3-methoxyphenyl]ethenyl]-benzonitrile (GE3082) were characterized in vitro and in vivo. Both agents crossed the blood-nerve barrier and blood-brain barrier, and rendered myelinated nerves fluorescent after a single systemic injection. Although both BMB and GE3082 also exhibited significant uptake in white adipose tissue, GE3082 underwent a hypsochromic shift in adipose tissue that provided a means to eliminate the unwanted signal using hyperspectral deconvolution. Dose and kinetic studies were performed in mice to determine the optimal dose and drug-imaging interval. Results were confirmed in rat and pig, with the latter used to demonstrate, for the first time, simultaneous fluorescence imaging of blood vessels and nerves during surgery using the FLARE™ (Fluorescence-Assisted Resection and Exploration) imaging system. These results lay the foundation for the development of ideal nerve-highlighting fluorophores for image-guided surgery.

The cystine transporter (system xC−) is an antiporter of cystine and glutamate. It has relatively low basal expression in most tissues and becomes upregulated in cells under oxidative stress (OS) as one of the genes expressed in response to the antioxidant response element (ARE) promoter. We have developed 18F-5-fluoro-aminosuberic acid (FASu), a Positron Emission Tomography (PET) tracer that targets system xC−. The goal of this study was to evaluate 18F-FASu as a specific gauge for system xC− activity in vivo and its potential for breast cancer imaging. Methods 18F-FASu specificity towards system xC− was studied by cell inhibition assay, cellular uptake following OS induction with diethyl maleate (DEM), with and without anti-xCT siRNA knockdown, in vitro uptake studies and in vivo uptake in a system xC− transduced xenograft model. In addition, radiotracer uptake was evaluated in three separate breast cancer models MDA-MB-231, MCF-7 and ZR-75-1. Results Reactive oxygen species (ROS)-inducing DEM increased glutathione levels and 18F-FASu uptake, while gene knockdown with anti-xCT siRNA led to decreased tracer uptake. 18F-FASu uptake was robustly inhibited by system xC− inhibitors or substrates, while the uptake was significantly higher in transduced cells and tumors expressing xCT compared to the wild type HEK293T cells and tumors (p<0.0001 for cells, p=0.0086 for tumors). 18F-FASu demonstrated tumor uptake in all three breast cancer cell lines studied. Among them, triple negative breast cancer MDA-MB-231 had the highest tracer uptake (p=0.0058 when compared with MCF-7; p<0.0001 when compared with ZR-75-1), which also has the highest xCT mRNA level. Conclusions 18F-FASu as a system xC− substrate is a specific PET tracer for functional monitoring of system xC− and OS imaging. By enabling non-invasive analysis of xC− responses in vivo, this biomarker may serve as a valuable target for the diagnosis and treatment monitoring of certain breast cancers.

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Glutathione is the predominant endogenous cellular antioxidant, playing a critical role in the cellular defensive response to oxidative stress by neutralizing free radicals and reactive oxygen species. With cysteine as the rate-limiting substrate in glutathione biosynthesis, the cystine/glutamate transporter (system xc−) represents a potentially attractive PET biomarker to enable in vivo quantification of xc− activity in response to oxidative stress associated with disease. We have developed a system xc− substrate that incorporates characteristics of both natural substrates, l-cystine and l-glutamate (l-Glu). l-aminosuberic acid (l-ASu) has been identified as a more efficient system xc− substrate than l-Glu, leading to an assessment of a series of anionic amino acids as prospective PET tracers. Herein, we report the synthesis and in vitro and in vivo validation of a lead candidate, 18F-5-fluoro-aminosuberic acid (18F-FASu), as a PET tracer for functional imaging of a cellular response to oxidative stress with remarkable tumor uptake and retention. Methods 18F-FASu was identified as a potential PET tracer based on an in vitro screening of compounds similar to l-cystine and l-Glu. Affinity toward system xc− was determined via in vitro uptake and inhibition studies using oxidative stress–induced EL4 and SKOV-3 cells. In vivo biodistribution and PET imaging studies were performed in mice bearing xenograft tumors (EL4 and SKOV-3). Results In vitro assay results determined that l-ASu inhibited system xc− as well as or better than l-Glu. The direct comparison of uptake of tritiated compounds demonstrated more efficient system xc− uptake of l-ASu than l-Glu. Radiosynthesis of 18F-FASu allowed the validation of uptake for the fluorine-bearing derivative in vitro. Evaluation in vivo demonstrated primarily renal clearance and uptake of approximately 8 percentage injected dose per gram in SKOV-3 tumors, with tumor-to-blood and tumor-to-muscle ratios of approximately 12 and approximately 28, respectively. 18F-FASu uptake was approximately 5 times greater than 18F-FDG uptake in SKOV-3 tumors. Dynamic PET imaging demonstrated uptake in EL4 tumor xenografts of approximately 6 percentage injected dose per gram and good tumor retention for at least 2 h after injection. Conclusion 18F-FASu is a potentially useful metabolic tracer for PET imaging of a functional cellular response to oxidative stress. 18F-FASu may provide more sensitive detection than 18F-FDG in certain tumors.

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Contrast-enhanced magnetic resonance imaging is an essential tool for disease diagnosis and management; all marketed clinical magnetic resonance imaging (MRI) contrast agents (CAs) are gadolinium (Gd) chelates and most are extracellular fluid (ECF) agents. After intravenous injection, these agents rapidly distribute to the extracellular space and are also characterized by low serum protein binding and predominant renal clearance. Gd is an abiotic element with no biological recycling processes; low levels of Gd have been detected in the central nervous system and bone long after administration. These observations have prompted interest in the development of new MRI contrast agents based on biotic elements such as iron (Fe); Fe-HBED (HBED = N,N′-bis(2-hydroxyphenyl)ethylenediamine-N,N′-diacetic acid), a coordinatively saturated iron chelate, is an attractive MRI CA platform suitable for modification to adjust relaxivity and biodistribution. Compared to the parent Fe-HBED, the Fe-HBED analogs reported here have lower serum protein binding and higher relaxivity as well as lower relative liver enhancement in mice, comparable to that of a representative gadolinium-based contrast agent (GBCA). Fe-HBED analogs are therefore a promising class of non-Gd ECF MRI CA.

IL-6 was frequently secreted by renal cancer cell lines but it was only present in the serum of approximately half of the patients with metastatic renal cancer. Elevations of serum IL-6 were associated with paraneoplastic manifestations frequently seen in patients with renal cancer, including anemia, thrombocytosis, decreased albumin and elevations of alkaline phosphatase (Stauffer's syndrome). A weak relationship was noted between serum IL-6 level and fever but none was noted between that and survival or response to IL-2.

The enantioselective synthesis of (+)-tetrabenazine (TBZ) and (+)-dihydrotetrabenazine (DTBZ), agents of significant interest for therapeutic and molecular imaging applications, has been completed in 21% (TBZ) and 16% (DTBZ) overall yield and in >97% ee from the starting dihydroisoquinoline. The synthesis utilizes Sodeoka's palladium-catalyzed asymmetric malonate addition to set the initial stereocenter followed by a number of diastereoselective transformations to incorporate the remaining asymmetric centers.