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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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Related in: MedlinePlus

Ex vivo analysis of animals receiving the mPEG-PL-Cy5.5 probe in the Camostat study.A) The pancreas from each animal was excised and weighed. Camostat treated animals showed significant reduction in the edema ratio compared to untreated saline animals. B) Total signal intensity of the activated mPEG-PL-Cy5.5 probe in the excised pancreas was quantified. Camostat animals showed a significant reduction in signal intensity compared to untreated saline animals. C) The total signal intensity was plotted against the edema ratio. A positive correlation was observed. D) At the end of the study, pancreas samples were also analyzed for the amount of enzymatically active trypsin. Camostat treated animals showed significant reduction in the amount of active trypsin compared to untreated saline animals. Data represented mean ± SEM.
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pone-0055959-g004: Ex vivo analysis of animals receiving the mPEG-PL-Cy5.5 probe in the Camostat study.A) The pancreas from each animal was excised and weighed. Camostat treated animals showed significant reduction in the edema ratio compared to untreated saline animals. B) Total signal intensity of the activated mPEG-PL-Cy5.5 probe in the excised pancreas was quantified. Camostat animals showed a significant reduction in signal intensity compared to untreated saline animals. C) The total signal intensity was plotted against the edema ratio. A positive correlation was observed. D) At the end of the study, pancreas samples were also analyzed for the amount of enzymatically active trypsin. Camostat treated animals showed significant reduction in the amount of active trypsin compared to untreated saline animals. Data represented mean ± SEM.

Mentions: After the third hour of caerulein, the pancreas was excised from all animals, weighed, and imaged to confirm the in vivo observations. Edema ratio was calculated by dividing the weight of the pancreas by the total body weight of the animal. Upon examination of dissected pancreas, the probe activation was found to be suppressed in the Camostat treated animals when compared to untreated animals (P<0.01 figure 4b). Unlike the edema ratio (figure 4a), the probe activation graph for corresponding pancreas showed that there was a significant difference in Camostat treated animals and controls animals (P<0.05). The sensitivity obtained from the ex vivo imaging is much higher than just the observation of pancreatic weight change.


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)

Ex vivo analysis of animals receiving the mPEG-PL-Cy5.5 probe in the Camostat study.A) The pancreas from each animal was excised and weighed. Camostat treated animals showed significant reduction in the edema ratio compared to untreated saline animals. B) Total signal intensity of the activated mPEG-PL-Cy5.5 probe in the excised pancreas was quantified. Camostat animals showed a significant reduction in signal intensity compared to untreated saline animals. C) The total signal intensity was plotted against the edema ratio. A positive correlation was observed. D) At the end of the study, pancreas samples were also analyzed for the amount of enzymatically active trypsin. Camostat treated animals showed significant reduction in the amount of active trypsin compared to untreated saline animals. Data represented mean ± SEM.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0055959-g004: Ex vivo analysis of animals receiving the mPEG-PL-Cy5.5 probe in the Camostat study.A) The pancreas from each animal was excised and weighed. Camostat treated animals showed significant reduction in the edema ratio compared to untreated saline animals. B) Total signal intensity of the activated mPEG-PL-Cy5.5 probe in the excised pancreas was quantified. Camostat animals showed a significant reduction in signal intensity compared to untreated saline animals. C) The total signal intensity was plotted against the edema ratio. A positive correlation was observed. D) At the end of the study, pancreas samples were also analyzed for the amount of enzymatically active trypsin. Camostat treated animals showed significant reduction in the amount of active trypsin compared to untreated saline animals. Data represented mean ± SEM.
Mentions: After the third hour of caerulein, the pancreas was excised from all animals, weighed, and imaged to confirm the in vivo observations. Edema ratio was calculated by dividing the weight of the pancreas by the total body weight of the animal. Upon examination of dissected pancreas, the probe activation was found to be suppressed in the Camostat treated animals when compared to untreated animals (P<0.01 figure 4b). Unlike the edema ratio (figure 4a), the probe activation graph for corresponding pancreas showed that there was a significant difference in Camostat treated animals and controls animals (P<0.05). The sensitivity obtained from the ex vivo imaging is much higher than just the observation of pancreatic weight change.

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