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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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Evaluation of trypsin inhibitor Novartis848 on intrapancreatic trypsin activity and edema formation using mPEG-PL-Cy5.5 probe.Animals were administered PBS, vehicle, or varying concentration of Novartis848 IP 1 h prior to caerulein induction of pancreatitis (n = 5∼7). Vehicle-1 = vehicle for Novartis848+ SC PBS, Vehicle-2 = vehicle for Novartis848+ SC caerulein. A) Animals were administered the mPEG-PL-Cy5.5 probe to monitor the activity of trypsin in caerulein induced pancreatitis animals. Animals receiving 30 mg/kg of Novartis848 and caerulein showed marked reduction in the signal intensity in the areas of the pancreas compared to vehicle animals. B), C) & D) Pancreas were excised, weighed and fluorescent images were acquired at the end of a 3 h caerulein induced pancreatitis study. B) Fluorescence heat maps of excised pancreas. C) Excised pancreas fluorescence showed results similar to the in vivo imaging study. At 30 mg/kg of Novartis848 the fluorescence of the probe was significantly reduced in comparison to Vehicle-2 animals. (D) The edema ratio. Data represented mean ± SEM.
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pone-0055959-g006: Evaluation of trypsin inhibitor Novartis848 on intrapancreatic trypsin activity and edema formation using mPEG-PL-Cy5.5 probe.Animals were administered PBS, vehicle, or varying concentration of Novartis848 IP 1 h prior to caerulein induction of pancreatitis (n = 5∼7). Vehicle-1 = vehicle for Novartis848+ SC PBS, Vehicle-2 = vehicle for Novartis848+ SC caerulein. A) Animals were administered the mPEG-PL-Cy5.5 probe to monitor the activity of trypsin in caerulein induced pancreatitis animals. Animals receiving 30 mg/kg of Novartis848 and caerulein showed marked reduction in the signal intensity in the areas of the pancreas compared to vehicle animals. B), C) & D) Pancreas were excised, weighed and fluorescent images were acquired at the end of a 3 h caerulein induced pancreatitis study. B) Fluorescence heat maps of excised pancreas. C) Excised pancreas fluorescence showed results similar to the in vivo imaging study. At 30 mg/kg of Novartis848 the fluorescence of the probe was significantly reduced in comparison to Vehicle-2 animals. (D) The edema ratio. Data represented mean ± SEM.

Mentions: To establish a dose response relationship with real time in vivo imaging, we used another in-house trypsin inhibitor Novartis848. As shown in figure 6a, at 10 and 30 mg/kg doses of Novartis848, there was a significant difference between treated and untreated animals by the third dose of caerulein. Animals receiving 30 mg/kg of Novartis848 and caerulein showed marked reduction in the signal intensity in the areas of the pancreas compared to Vehicle-2 animals (P<0.01 and P<0.001 after Cer2 and Cer3 time points respectively). The difference was not apparent at other concentrations of Novartis848 indicating a dose effect on the activation of the mPEG-PL-Cy5.5 probe. These results validate that fluorescent whole body imaging can be used in an animal like rat to assess the trypsin-dependent edema formation in the absence and presence of a trypsin inhibitor in the pancreas. In vivo results were further validated by ex vivo examination of the pancreas. As shown in figure 6c, 3, 10 and 30 mg/kg of Novartis848 doses were significantly different from untreated Vehicle-2 animals (P<0.05, P<0.001, and P<0.001 respectively). However, the results were not the same on the edema ratio chart (figure 6d). Our studies indicate that in vivo imaging maybe used to assess the trypsin-dependent edema formation in the absence and presence of a trypsin inhibitor in a dose response manner.


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)

Evaluation of trypsin inhibitor Novartis848 on intrapancreatic trypsin activity and edema formation using mPEG-PL-Cy5.5 probe.Animals were administered PBS, vehicle, or varying concentration of Novartis848 IP 1 h prior to caerulein induction of pancreatitis (n = 5∼7). Vehicle-1 = vehicle for Novartis848+ SC PBS, Vehicle-2 = vehicle for Novartis848+ SC caerulein. A) Animals were administered the mPEG-PL-Cy5.5 probe to monitor the activity of trypsin in caerulein induced pancreatitis animals. Animals receiving 30 mg/kg of Novartis848 and caerulein showed marked reduction in the signal intensity in the areas of the pancreas compared to vehicle animals. B), C) & D) Pancreas were excised, weighed and fluorescent images were acquired at the end of a 3 h caerulein induced pancreatitis study. B) Fluorescence heat maps of excised pancreas. C) Excised pancreas fluorescence showed results similar to the in vivo imaging study. At 30 mg/kg of Novartis848 the fluorescence of the probe was significantly reduced in comparison to Vehicle-2 animals. (D) The edema ratio. Data represented mean ± SEM.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569412&req=5

pone-0055959-g006: Evaluation of trypsin inhibitor Novartis848 on intrapancreatic trypsin activity and edema formation using mPEG-PL-Cy5.5 probe.Animals were administered PBS, vehicle, or varying concentration of Novartis848 IP 1 h prior to caerulein induction of pancreatitis (n = 5∼7). Vehicle-1 = vehicle for Novartis848+ SC PBS, Vehicle-2 = vehicle for Novartis848+ SC caerulein. A) Animals were administered the mPEG-PL-Cy5.5 probe to monitor the activity of trypsin in caerulein induced pancreatitis animals. Animals receiving 30 mg/kg of Novartis848 and caerulein showed marked reduction in the signal intensity in the areas of the pancreas compared to vehicle animals. B), C) & D) Pancreas were excised, weighed and fluorescent images were acquired at the end of a 3 h caerulein induced pancreatitis study. B) Fluorescence heat maps of excised pancreas. C) Excised pancreas fluorescence showed results similar to the in vivo imaging study. At 30 mg/kg of Novartis848 the fluorescence of the probe was significantly reduced in comparison to Vehicle-2 animals. (D) The edema ratio. Data represented mean ± SEM.
Mentions: To establish a dose response relationship with real time in vivo imaging, we used another in-house trypsin inhibitor Novartis848. As shown in figure 6a, at 10 and 30 mg/kg doses of Novartis848, there was a significant difference between treated and untreated animals by the third dose of caerulein. Animals receiving 30 mg/kg of Novartis848 and caerulein showed marked reduction in the signal intensity in the areas of the pancreas compared to Vehicle-2 animals (P<0.01 and P<0.001 after Cer2 and Cer3 time points respectively). The difference was not apparent at other concentrations of Novartis848 indicating a dose effect on the activation of the mPEG-PL-Cy5.5 probe. These results validate that fluorescent whole body imaging can be used in an animal like rat to assess the trypsin-dependent edema formation in the absence and presence of a trypsin inhibitor in the pancreas. In vivo results were further validated by ex vivo examination of the pancreas. As shown in figure 6c, 3, 10 and 30 mg/kg of Novartis848 doses were significantly different from untreated Vehicle-2 animals (P<0.05, P<0.001, and P<0.001 respectively). However, the results were not the same on the edema ratio chart (figure 6d). Our studies indicate that in vivo imaging maybe used to assess the trypsin-dependent edema formation in the absence and presence of a trypsin inhibitor in a dose response manner.

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