The development of rapidly acting cyanide countermeasures using intramuscular injection (IM) represents an unmet medical need to mitigate toxicant exposures in mass casualty settings. Previous work established that cisplatin and other platinum(II) or platinum(IV)-based agents effectively mitigate cyanide toxicity in zebrafish. Cyanide's in vivo reaction with platinum-containing materials was proposed to reduce the risk of acute toxicities. However, cyanide antidote activity depended on a formulation of platinum-chloride salts with dimethyl sulfoxide (DMSO) followed by dilution in phosphate-buffered saline (PBS). A working hypothesis to explain the DMSO requirement is that the formation of platinum− sulfoxide complexes activates the cyanide scavenging properties of platinum. Preparations of isolated NaPtCl 5 −DMSO and Na (NH 3 ) 2 PtCl−DMSO complexes in the absence of excess DMSO provided agents with enhanced reactivity toward cyanide in vitro and fully recapitulated in vivo cyanide rescue in zebrafish and mouse models. The enhancement of the cyanide scavenging effects of the DMSO ligand could be attributed to the activation of platinum(IV) and (II) with a sulfur ligand. Unfortunately, the efficacy of DMSO complexes was not robust when administered IM. Alternative Pt(II) materials containing sulfide and amine ligands in bidentate complexes show enhanced reactivity toward cyanide addition. The cyanide addition products yielded tetracyanoplatinate(II), translating to a stoichiometry of 1:4 Pt to each cyanide scavenger. These new agents demonstrate a robust and enhanced potency over the DMSO-containing complexes using IM administration in mouse and rabbit models of cyanide toxicity. Using the zebrafish model with these Pt(II) complexes, no acute cardiotoxicity was detected, and dose levels required to reach lethality exceeded 100 times the effective dose. Data are presented to support a general chemical design approach that can expand a new lead candidate series for developing next-generation cyanide countermeasures.

The classification of pancreatic cyst fluids can provide a basis for the early detection of pancreatic cancer while eliminating unnecessary procedures. A candidate biomarker, gastricsin (pepsin C), was found to be present in potentially malignant mucinous pancreatic cyst fluids. A gastricsin activity assay using a magnetic bead-based platform has been developed using immobilized peptide substrates selective for gastricsin bearing a dimeric rhodamine dye. The unique dye structure allows quantitation of enzyme-cleaved product by both fluorescence and surface enhanced Raman spectroscopy (SERS). The performance of this assay was compared with ELISA assays of pepsinogen C and the standard of care, carcinoembryonic antigen (CEA), in the same clinical sample cohort. A retrospective cohort of mucinous (n = 40) and non-mucinous (n = 29) classes of pancreatic cyst fluid samples were analyzed using the new protease activity assay. For both assay detection modes, successful differentiation of mucinous and non-mucinous cyst fluid was achieved using 1 µL clinical samples. The activity-based assays in combination with CEA exhibit optimal sensitivity and specificity of 87% and 93%, respectively. The use of this gastricsin activity assay requires a minimal volume of clinical specimen, offers a rapid assay time, and shows improvements in the differentiation of mucinous and non-mucinous cysts using an accurate standardized readout of product formation, all without interfering with the clinical standard of care.

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4151 Background: One impact of medical imaging technology has been an approximately 3-fold increase in the incidental detection of pancreatic cysts during routine clinical examinations. To reduce burden on the healthcare system and patients, clinicians desire accurate classification of pancreatic cysts into benign non-mucinous or potentially malignant, mucinous populations. Using EUS-FNA, fluid from these cysts can provide molecular biomarkers of predictive value. However, current in vitro diagnostics lack the desired sensitivity and specificity for clinicians to accurately stratify patients for risk of pre-malignant cancers. This situation can be improved by introducing new biomarkers and novel assay platforms. An enzymatic biomarker, pepsin C, has shown high accuracy for diagnosing mucinous cysts. The use of enzymatic activity assays is applicable to clinical workflows without disruption of current standards of care. Methods: A pepsin C activity assay using a magnetic bead-based platform was developed, with both fluorescent and surface-enhanced Raman spectroscopy (SERS) readouts. The assay platform utilizes selective peptide substrates, and a dimeric Rhodamine-6G-based dye, which allows ultrasensitive detection and significantly decreases the sample volume requirement for analysis, down to 1 µL of cyst fluid. The dye-labeled substrate is immobilized on magnetic beads and reacted with enzyme-containing samples to produce a quantitative assay signal that is standardized and expressed in true enzyme activity units. Results: While both readouts were quantitative and produced linear standard curves, SERS-based analysis was more robust against established biological matrix effects than fluorescence. Nevertheless, both assay modes successfully differentiated mucinous and non-mucinous cysts in a retrospective cohort of 69 cyst fluid samples. Compared with the standard of care CEA assay, this activity-based assay displays much improved sensitivity in diagnosing mucinous pancreatic cysts (Table). Conclusions: This pepsin c activity assay differentiates between mucinous and non-mucinous cysts better than the CEA assay and provides a quantifiable standardized readout. This work establishes a path to a true rule-out assay enabling clinicians to better stratify patients into low risk vs. potential malignancy thus impacting treatment and monitoring plans. [Table: see text]