Victoria E. Cotero

Dual-mode laparoscopic fluorescence image-guided surgery using a single camera

Iatrogenic nerve damage is a leading cause of morbidity associated with many common surgical procedures. Complications arising from these injuries may result in loss of function and/or sensation, muscle atrophy, and chronic neuropathy. Fluorescence image-guided surgery offers a potential solution for avoiding intraoperative nerve damage by highlighting nerves that are otherwise difficult to visualize. In this work we present the development of a single camera, dual-mode laparoscope that provides near simultaneous display of white-light and fluorescence images of nerves. The capability of the instrumentation is demonstrated through imaging several types of in situ rat nerves via a nerve specific contrast agent. Full color white light and high brightness fluorescence images and video of nerves as small as 100 µm in diameter are presented.

Identification of the Protein Target of Myelin-Binding Ligands by Immunohistochemistry and Biochemical Analyses

The ability to visualize myelin is important in the diagnosis of demyelinating disorders and the detection of myelin-containing nerves during surgery. The development of myelin-selective imaging agents requires that a defined target for these agents be identified and that a robust assay against the target be developed to allow for assessment of structure-activity relationships. We describe an immunohistochemical analysis and a fluorescence polarization binding assay using purified myelin basic protein (MBP) that provides quantitative evidence that MBP is the molecular binding partner of previously described myelin-selective fluorescent dyes such as BMB, GE3082, and GE3111.

A compact fluorescence and white light imaging system for intraoperative visualization of nerves

Fluorescence image guided surgery (FIGS) allows intraoperative visualization of critical structures, with applications spanning neurology, cardiology and oncology. An unmet clinical need is prevention of iatrogenic nerve damage, a major cause of post-surgical morbidity. Here we describe the advancement of FIGS imaging hardware, coupled with a custom nerve-labeling fluorophore (GE3082), to bring FIGS nerve imaging closer to clinical translation. The instrument is comprised of a 405nm laser and a white light LED source for excitation and illumination. A single 90 gram color CCD camera is coupled to a 10mm surgical laparoscope for image acquisition. Synchronization of the light source and camera allows for simultaneous visualization of reflected white light and fluorescence using only a single camera. The imaging hardware and contrast agent were evaluated in rats during in situ surgical procedures.

Performance of Redox Active and Chelatable Iron Assays to Determine Labile Iron Release From Intravenous Iron Formulations

Emerging data from global markets outside the United States, where many generic iron sucrose formulations are available, have revealed that non‐US generic intravenous (i.v.) iron formulations may have iron release profiles that differ from the reference listed drug (RLD). The first generic i.v. iron approved in the United States was sodium ferric gluconate complex in 2011. We evaluated chelatable and redox labile iron assay methods to measure the amount of labile iron released from i.v. iron formulations in biorelevant matrices in vitro. The majority of published labile iron assays evaluated were not suitable for use in vitro due to overwhelming interference by the presence of the i.v. iron products. However, an optimized high‐performance liquid chromatography (HPLC)‐based method performed well for use in vitro labile iron detection in a biorelevant matrix. Application of this method may enhance bioequivalence evaluation of generic i.v. iron formulations in the future.

Improved Intraoperative Visualization of Nerves through a Myelin-Binding Fluorophore and Dual-Mode Laparoscopic Imaging

The ability to visualize and spare nerves during surgery is critical for avoiding chronic morbidity, pain, and loss of function. Visualization of such critical anatomic structures is even more challenging during minimal access procedures because the small incisions limit visibility. In this study, we focus on improving imaging of nerves through the use of a new small molecule fluorophore, GE3126, used in conjunction with our dual-mode (color and fluorescence) laparoscopic imaging instrument. GE3126 has higher aqueous solubility, improved pharmacokinetics, and reduced non-specific adipose tissue fluorescence compared to previous myelin-binding fluorophores. Dosing and kinetics were initially optimized in mice. A non-clinical modified Irwin study in rats, performed to assess the potential of GE3126 to induce nervous system injuries, showed the absence of major adverse reactions. Real-time intraoperative imaging was performed in a porcine model. Compared to white light imaging, nerve visibility was enhanced under fluorescence guidance, especially for small diameter nerves obscured by fascia, blood vessels, or adipose tissue. In the porcine model, nerve visualization was observed rapidly, within 5 to 10 minutes post-intravenous injection and the nerve fluorescence signal was maintained for up to 80 minutes. The use of GE3126, coupled with practical implementation of an imaging instrument may be an important step forward in preventing nerve damage in the operating room.

In vitro and in vivo DFO-chelatable labile iron release profiles among commercially available intravenous iron nanoparticle formulations

ABSTRACT Intravenous (IV) iron formulations are complex colloidal suspensions of iron oxide nanoparticles. Small changes in formulation can allow more labile iron to be released after injection causing toxicity. Thus, bioequivalence (BE) evaluation of generic IV iron formulations remains challenging. We evaluated labile iron release in vitro and in vivo using a high performance liquid chromatography chelatable iron assay to develop a relational model to support BE. In vitro labile iron release and in vivo labile iron pharmacokinetics were evaluated for Venofer®, Ferrlecit®, generic sodium ferric gluconate complex, InFeD®, Feraheme® and a pre‐clinical formulation GE121333. Labile iron release profiles were studied in vitro in 150mM saline and a biorelevant matrix (rat serum) at 0.952 mgFe/mL. In vivo plasma labile iron concentration‐time profiles (t0–240min) were studied in rats after a 40 mgFe/kg IV dose. In vitro labile iron release in saline was significantly higher compared to rat serum, especially with InFeD®. An in vitro release constant (iKr) was calculated which correlated well with maximal plasma concentrations in the in vivo rat PK model (R2=0.711). These data suggest an in vitro to in vivo correlation model of labile iron release kinetics could be applied to BE. Other generic IV iron formulations need to be studied to validate this model. HIGHLIGHTSIntravenous iron formulations are complex drugs that are colloidal suspensions of iron oxide nanoparticles.Small formulation changes that are not discerned by physicochemical characterization may allow for more labile iron release.These characteristics of IV iron formulations make bioequivalence evaluation challenging.An in vitro to in vivo correlation model for labile iron release could improve bioequivalence evaluation.

Applications of phosphorescent materials for in-vivo imaging of brain structure and function

A number of approaches have been developed for in-vivo imaging of neural function at the time scale of action potentials and at the spatial resolution of individual neurons. Remarkable results have been obtained with optogenetics, although the need for genetic modification is an important limitation of these approaches. Similarly, voltage and ion-sensitive dyes allow for optical imaging of action potentials but toxicity remains a problem. Additionally, optical techniques are often only able to be used up to a limited depth. Our preliminary work has shown that nanoparticles of common phosphorescent materials, believed to be generally non-toxic, specifically lutetium oxide and strontium aluminate, can be utilized for cellular imaging, for tomographic imaging, and that the particles can be designed to adhere to neurons. Additionally, lutetium oxide has been shown to be highly X-ray luminescent, potentially allowing for imaging deep within the brain, if the particles can be targeted properly. In ex vivo experiments, we have shown that the phosphorescence of strontium aluminate particles is significantly affected by electric fields similar in strength to those found in the vicinity of the cellular membrane of a neuron. This phenomenon is consistent with early published reports in the electroluminescence literature, namely the Gudden-Pohl effect. We will show results of the ex vivo imaging and dynamic electrical stimulation experiments. We will also show some preliminary ex vivo cell culture results, and will describe plans for future research, focusing on potential in both cell cultures and in vivo for animal models.

PD13-08 EVALUATION OF NERVE-HIGHLIGHTING CONTRAST AGENT GE3126 FOR IMAGE-GUIDED SURGERY

INTRODUCTION AND OBJECTIVES: Inadvertent injury to nerves is a significant and often inevitable consequence of surgery that can lead post-operative morbidity and loss of function. Nerve-sparing during radical prostatectomy can reduce the incidence of post-surgical erectile dysfunction. In retroperitoneal lymph node dissection for testis cancer, the prospective identification and preservation of nerves in is essential in maintaining ejaculatory function and fertility. Although there is a significant need, there is no widely adopted system for real-time identification of nerves in either the open or laparoscopic surgical settings. We sought to investigate the performance of a myelin-targeted fluorophore and optical imaging instrumentation in the intraoperative visualization of nerves. METHODS: A myelin-targeting small molecule fluorophore, GE3126, was synthesized and characterized for its optical and myelinbinding properties using purified myelin basic protein. 2 Yorkshire pigs were utilized in a non-survival study. Peripheral and retroperitoneal nerves were exposed and control images taken using a dedicated compact imaging device adapted to both open and minimally-invasive approaches. Both white light and 405nm illumination were used. Following intravenous injection of the agent, blood, urine, and bile were drawn at fixed intervals to determine the pharmacokinetics of the agent. Central and peripheral nerves were visualized. Tissues were harvested for ex-vivo analysis and histopathology. 4 blinded observers evaluated captured images. RESULTS: The primary route of excretion was renal. The fluorescence peak was achieved at 60-80 min following injection. Label/nonlabel fluorescence signal ratio was 5:1 at peak. Nerve to muscle signal was 7:1. Fluorescence polarization showed specific and strong binding to purified myelin basic protein. Retroperitoneal nerves from 100mm-10mm were evaluated. Inter-observer disagreement was 22% with white light images, and 0% with fluorescence images, which was confirmed by histology. No adverse effects were noted in the animals. CONCLUSIONS: GE3126 provides a safe and effective means of identifying nerves through fluorescence imaging and is adaptable to both open and minimally invasive surgical procedures.