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In vivo imaging with fluorescent smart probes to assess treatment strategies for acute pancreatitis.

Agarwal A, Boettcher A, Kneuer R, Sari-Sarraf F, Donovan A, Woelcke J, Simic O, Brandl T, Krucker T - PLoS ONE (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Novartis Institute of BioMedical Research, Cambridge, Massachusetts, USA.

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.

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MPEG-PL-Cy5.5 probe characterization.Activation of 0.2 µM of the mPEG-PL-Cy5.5 probe by 100 nM of various pancreatic enzymes in the presence or absence of 1 µM of SPINK1. Probe is specifically activated in the presence of trypsin. Trypsin activity is diminished in the presence of SPINK1. Data represented mean ± SD.
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pone-0055959-g001: MPEG-PL-Cy5.5 probe characterization.Activation of 0.2 µM of the mPEG-PL-Cy5.5 probe by 100 nM of various pancreatic enzymes in the presence or absence of 1 µM of SPINK1. Probe is specifically activated in the presence of trypsin. Trypsin activity is diminished in the presence of SPINK1. Data represented mean ± SD.

Mentions: When rat anionic trypsin was added to 1 µM of mPEG-PL-Cy5.5 probe, increase in fluorescence intensity was observed which was positively correlated to the concentration of trypsin enzyme and the time of incubation. To establish trypsin selectivity, mPEG-PL-Cy5.5 probe activation was tested by addition to a panel of endopeptidases that are described to be expressed in the pancreas, including human and rat pancreatic elastase, kallikrein 1, kallikrein 5, chymotrypsin, cathepsin L, and cathepsin B [22]. As shown in figure 1, mPEG-PL-Cy5.5 probe was activated in the presence of trypsin in contrast to other pancreatic enzymes. In the presence of the highly specific trypsin inhibiting protein SPINK1, the protease activation was suppressed to background levels (figure 1). Therefore, it can be concluded that mPEG-PL-Cy5.5 probe was highly trypsin selective.


In vivo imaging with fluorescent smart probes to assess treatment strategies for acute pancreatitis.

Agarwal A, Boettcher A, Kneuer R, Sari-Sarraf F, Donovan A, Woelcke J, Simic O, Brandl T, Krucker T - PLoS ONE (2013)

MPEG-PL-Cy5.5 probe characterization.Activation of 0.2 µM of the mPEG-PL-Cy5.5 probe by 100 nM of various pancreatic enzymes in the presence or absence of 1 µM of SPINK1. Probe is specifically activated in the presence of trypsin. Trypsin activity is diminished in the presence of SPINK1. Data represented mean ± SD.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3569412&req=5

pone-0055959-g001: MPEG-PL-Cy5.5 probe characterization.Activation of 0.2 µM of the mPEG-PL-Cy5.5 probe by 100 nM of various pancreatic enzymes in the presence or absence of 1 µM of SPINK1. Probe is specifically activated in the presence of trypsin. Trypsin activity is diminished in the presence of SPINK1. Data represented mean ± SD.
Mentions: When rat anionic trypsin was added to 1 µM of mPEG-PL-Cy5.5 probe, increase in fluorescence intensity was observed which was positively correlated to the concentration of trypsin enzyme and the time of incubation. To establish trypsin selectivity, mPEG-PL-Cy5.5 probe activation was tested by addition to a panel of endopeptidases that are described to be expressed in the pancreas, including human and rat pancreatic elastase, kallikrein 1, kallikrein 5, chymotrypsin, cathepsin L, and cathepsin B [22]. As shown in figure 1, mPEG-PL-Cy5.5 probe was activated in the presence of trypsin in contrast to other pancreatic enzymes. In the presence of the highly specific trypsin inhibiting protein SPINK1, the protease activation was suppressed to background levels (figure 1). Therefore, it can be concluded that mPEG-PL-Cy5.5 probe was highly trypsin selective.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Novartis Institute of BioMedical Research, Cambridge, Massachusetts, USA.

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.

Show MeSH
Related in: MedlinePlus