Twenty million Americans suffer from peripheral nerve injury caused by trauma and medical disorders, resulting in a broad spectrum of potentially debilitating side effects. In one out of four cases, patients identify surgery as the root cause of their nerve injury. Particularly during tumor resections or after traumatic injuries, tissue distortion and poor visibility can challenge a surgeon's ability to precisely locate and preserve peripheral nerves. Intuitively, surgical outcomes would improve tremendously if nerves could be highlighted using an exogeneous contrast agent. In clinical practice, however, the current standard of care ? visual examination and palpation ? remains unchanged. The inability of surgeons to identify nerves during surgery represents an immense unmet clinical need, which we propose to address within this application. We will develop and validate a translational fluorescent imaging agent for the peripheral nervous system. In preparation for a clinical trial, we will show in mice and large animals that the agent is safe for use in humans. Specifically, this proposal centers on Hsp1a, a peptide which we isolated from the venom of a Peruvian tarantula, Homoeomma Spec. Peru, and which we found to be highly specific for human Nav1.7. It has been shown that Nav1.7 is highly expressed in human peripheral nerve tissue. We believe that a fluorescent Hsp1a will have tremendous value as an injectable, intraoperative guide during surgery, and that the agent will provide surgeons with additional contrast, reducing iatrogenic injury and therefore surgical morbidity. To achieve this goal, we have assembled three specific aims (SAs), each of which will explore a distinct goal toward showing that fluorescent Nav1.7 tracers can be translated to the clinic. In SA1, and supported by our proof-of-principle data, we will synthesize a library of fluorescently labeled Hsp1a derivatives (n = 20), featuring different fluorophores, attachment points and linker lengths. For all derivatives, we will determine the IC50 values against Nav1.7 using patch-clamp electrophysiology; in SA2, pharmacokinetics and in vivo performance will be determined using Nav1.7 expressing, tumor-bearing athymic nude mice and Nav1.7 deficient Scn9atm1Dgen/J mice. For the most promising tracers (n = 3, based on IC50 and signal/noise ratios), we will test the minimum detectable dose in cohorts of pigs. In SA3, we will interrogate the pharmacodynamic properties of the most promising Hsp1a peptides. We will determine the maximum tolerated doses in mice while monitoring cardiovascular and respiratory function. For the lead candidate, we will perform a 2-species GLP toxicity study (mice and dogs). If successful, the current research proposal and generated data will form the foundation of a clinical phase I trial with Hsp1a in oral cancer.
Peripheral nerves are filigree structures that, due to poor visibility, are often damaged during surgery (in up to 75% of cases, depending on tumor location), leading to loss of function, weakness, muscle atrophy, fasciculation, paralysis, cardiac irregularities, allodynia and chronic neuropathy. We have identified a novel targeted peptide, Hsp1a, for intraoperative nerve imaging, and showed that modification with fluorescent dyes retains selectivity and produces exceptional contrast. Now, we propose to develop a fluorescently labeled translational Hsp1a candidate, providing evidence for specificity and the absence of side effects when injected into mice and large animals. 1
