In Vivo Imaging with Fluorescent Smart Probes to Assess Treatment Strategies for Acute Pancreatitis Abhiruchi Agarwal 1 , Andreas Boettcher 2 , Rainer Kneuer 2 , Farid Sari-Sarraf 1 , Adriana Donovan 1 , Julian Woelcke 2 , Oliver Simic 2 , Trixi Brandl 2 , Thomas Krucker 1,3 * 1 Novartis Institute of BioMedical Research, Cambridge, Massachusetts, United States of America, 2 Novartis Institutes for BioMedical Research, Basel, Switzerland, 3 Novartis Institutes of BioMedical Research, Emeryville, California, United States of America Abstract Background and Aims: Endoprotease activation is a key step in acute pancreatitis and early inhibition of these enzymes may protect from organ damage. In vivo models commonly used to evaluate protease inhibitors require animal sacrifice and therefore limit the assessment of dynamic processes. Here, we established a non-invasive fluorescence imaging-based biomarker assay to assess real-time protease inhibition and disease progression in a preclinical model of experimental pancreatitis. Methods: Edema development and trypsin activation were imaged in a rat caerulein-injection pancreatitis model. A fluorescent ‘‘smart’’ probe, selectively activated by trypsin, was synthesized by labeling with Cy5.5 of a pegylated poly-L- lysine copolymer. Following injection of the probe, trypsin activation was monitored in the presence or absence of inhibitors by in vivo and ex vivo imaging. Results: We established the trypsin-selectivity of the fluorescent probe in vitro using a panel of endopeptidases and specific inhibitor. In vivo, the probe accumulated in the liver and a region attributed to the pancreas by necropsy. A dose dependent decrease of total pancreatic fluorescence signal occurred upon administration of known trypsin inhibitors. The fluorescence- based method was a better predictor of trypsin inhibition than pancreatic to body weight ratio. Conclusions: We established a fluorescence imaging assay to access trypsin inhibition in real-time in vivo. This method is more sensitive and dynamic than classic tissue sample readouts and could be applied to preclinically optimize trypsin inhibitors towards intrapancreatic target inhibition. Citation: Agarwal A, Boettcher A, Kneuer R, Sari-Sarraf F, Donovan A, et al. (2013) In Vivo Imaging with Fluorescent Smart Probes to Assess Treatment Strategies for Acute Pancreatitis. PLoS ONE 8(2): e55959. doi:10.1371/journal.pone.0055959 Editor: Yong Jiang, Southern Medical University, China Received July 16, 2012; Accepted January 7, 2013; Published February 11, 2013 Copyright: ß 2013 Agarwal et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding of this research was provided solely within the Novartis organization as per standard research activities. The funders (NIBR) had no role in study design, data collection and analysis, or preparation of the manuscript. The manuscript was assessed by the Novartis legal department as well as senior management in chemistry and biology (Infectious diseases and Center for Proteomic Chemistry) to ascertain that release of this manuscript did not contravene Novartis policy regarding release of proprietary information, but this was limited only to approval for release of the material and was not related to the results or interpretations/conclusions contained in the manuscript. Competing Interests: The authors are, at present, or were during the time of their contribution to this manuscript, employed by Novartis Pharmaceuticals, under the umbrella of the Novartis Institutes for Biomedical Research (NIBR). As such, the authors all own stock in Novartis as part of their remuneration for employment. There are no competing interests as regards consultancies, patents or products in development or currently marketed. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: thomas.krucker@novartis.com Introduction Acute pancreatitis is a serious condition characterized by inflammation, fibrosis and endocrine and exocrine dysfunction of the pancreas. It has a high incidence rate [1,2] and a mortality of up to 40% [1,3,4] in the US. Genetic and environmental factors can lead to an inappropriate activation of trypsin proteases, lipases and other zymogens causing direct pancreatic injury, which in turn triggers an inflammatory immune response. There is growing evidence that genetic variants underlie susceptibility to acute pancreatitis. Hereditary pancreatitis is generally described as an autosomal dominant gain-of-function disorder related to mutations in the cationic trypsinogen gene PRSS1, which has an 80% penetrance. Mutations in this gene promote premature cleavage of trypsinogen to active trypsin in the pancreatic acinar cells, which in turn causes pancreatic autodi- gestion [5,6,7]. In addition, trypsinogen copy number variants (duplications and triplications) appear to be associated with idiopathic pancreatitis in some populations [8]. Moreover, loss- of-function mutations in the gene of the endogenous trypsin inhibitor Kazal type 1 (SPINK1) have been reported to be associated with pancreatitis [9]. SPINK1 is important in limiting ongoing trypsin activity in the pancreatic acinar cells after the onset of an acute inflammatory reaction. Studies in SPINK3 (mouse ortholog of human SPINK1) k.o. mice suggest that the Spink gene plays an essential role in the maintenance of acinar cells [10]. Protease activation targeting trypsinogen or other zymogens within the acinar cells of the pancreas are considered to be early events in the onset of acute pancreatitis [11,12]. This strongly PLOS ONE | www.plosone.org 1 February 2013 | Volume 8 | Issue 2 | e55959