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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

Application of the mPEG-PL-Cy5.5 probe to evaluate the efficacy of trypsin inhibitor Novartis166.Animals were administered PBS, Vehicle ( = “control”), or varying concentration of Novartis166 IP prior to caerulein induction of pancreatitis. Pancreas were excised and in vivo fluorescent images were acquired at the end of a 3 h caerulein induced pancreatitis study. A) The graph shows the fluorescent intensity of the mPEG-PL-Cy5.5 probe. Novartis166 at 30 mg/kg significantly reduced the activation of the trypsin mPEG-PL-Cy5.5 probe compared to other the Vehicle group. B) The corresponding edema ratio also indicated that Novartis166 at 30 mg/kg significantly abrogated the development of edema compared to the Vehicle group. C) The amount of Novartis166 present in the pancreas was also assessed. The amount of Novartis166 present correlated to the dose administered. D) At 30 mg/kg of Novartis166, in vivo probe activation time course was also evaluated. With increasing time, the activation of the mPEG-PL-Cy5.5 probe was significantly reduced. Data represented mean ± SEM.
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pone-0055959-g005: Application of the mPEG-PL-Cy5.5 probe to evaluate the efficacy of trypsin inhibitor Novartis166.Animals were administered PBS, Vehicle ( = “control”), or varying concentration of Novartis166 IP prior to caerulein induction of pancreatitis. Pancreas were excised and in vivo fluorescent images were acquired at the end of a 3 h caerulein induced pancreatitis study. A) The graph shows the fluorescent intensity of the mPEG-PL-Cy5.5 probe. Novartis166 at 30 mg/kg significantly reduced the activation of the trypsin mPEG-PL-Cy5.5 probe compared to other the Vehicle group. B) The corresponding edema ratio also indicated that Novartis166 at 30 mg/kg significantly abrogated the development of edema compared to the Vehicle group. C) The amount of Novartis166 present in the pancreas was also assessed. The amount of Novartis166 present correlated to the dose administered. D) At 30 mg/kg of Novartis166, in vivo probe activation time course was also evaluated. With increasing time, the activation of the mPEG-PL-Cy5.5 probe was significantly reduced. Data represented mean ± SEM.

Mentions: We validated our capability to obtain a dose response relationship using in vivo imaging. An in-house trypsin inhibitor (Novartis166) was used. Novartis166 was administered intra peritonealy (IP) using a vehicle, one hour prior to caerulein administration, at doses (3, 10, or 30 mg/kg). Control animals that did not receive any caerulein were used for baseline comparison. As seen in figure 5a, at 30 mg/kg dose, there was a significant difference between vehicle+caerulein-treated baseline controls and trypsin inhibitor treated animals (P<0.01). However, the edema graph (figure 5b) indicated no significant difference between baseline controls and treated animals. In addition, we were able to gain a dose response relationship. At 3 and 10 mg/kg, there was no significant difference between untreated and treated animals (figure 5a). We further validated our capability to use in vivo imaging for observing the effect of a trypsin inhibitor in real time. As shown in figure 5d, at 30 mg/kg dose, the animals were not significantly different from baseline control animals in terms of the concentration of active trypsin in the pancreas. These results indicate that in vivo imaging may be used to predict the outcome of a trypsin inhibitor dynamically.


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)

Application of the mPEG-PL-Cy5.5 probe to evaluate the efficacy of trypsin inhibitor Novartis166.Animals were administered PBS, Vehicle ( = “control”), or varying concentration of Novartis166 IP prior to caerulein induction of pancreatitis. Pancreas were excised and in vivo fluorescent images were acquired at the end of a 3 h caerulein induced pancreatitis study. A) The graph shows the fluorescent intensity of the mPEG-PL-Cy5.5 probe. Novartis166 at 30 mg/kg significantly reduced the activation of the trypsin mPEG-PL-Cy5.5 probe compared to other the Vehicle group. B) The corresponding edema ratio also indicated that Novartis166 at 30 mg/kg significantly abrogated the development of edema compared to the Vehicle group. C) The amount of Novartis166 present in the pancreas was also assessed. The amount of Novartis166 present correlated to the dose administered. D) At 30 mg/kg of Novartis166, in vivo probe activation time course was also evaluated. With increasing time, the activation of the mPEG-PL-Cy5.5 probe was significantly reduced. Data represented mean ± SEM.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0055959-g005: Application of the mPEG-PL-Cy5.5 probe to evaluate the efficacy of trypsin inhibitor Novartis166.Animals were administered PBS, Vehicle ( = “control”), or varying concentration of Novartis166 IP prior to caerulein induction of pancreatitis. Pancreas were excised and in vivo fluorescent images were acquired at the end of a 3 h caerulein induced pancreatitis study. A) The graph shows the fluorescent intensity of the mPEG-PL-Cy5.5 probe. Novartis166 at 30 mg/kg significantly reduced the activation of the trypsin mPEG-PL-Cy5.5 probe compared to other the Vehicle group. B) The corresponding edema ratio also indicated that Novartis166 at 30 mg/kg significantly abrogated the development of edema compared to the Vehicle group. C) The amount of Novartis166 present in the pancreas was also assessed. The amount of Novartis166 present correlated to the dose administered. D) At 30 mg/kg of Novartis166, in vivo probe activation time course was also evaluated. With increasing time, the activation of the mPEG-PL-Cy5.5 probe was significantly reduced. Data represented mean ± SEM.
Mentions: We validated our capability to obtain a dose response relationship using in vivo imaging. An in-house trypsin inhibitor (Novartis166) was used. Novartis166 was administered intra peritonealy (IP) using a vehicle, one hour prior to caerulein administration, at doses (3, 10, or 30 mg/kg). Control animals that did not receive any caerulein were used for baseline comparison. As seen in figure 5a, at 30 mg/kg dose, there was a significant difference between vehicle+caerulein-treated baseline controls and trypsin inhibitor treated animals (P<0.01). However, the edema graph (figure 5b) indicated no significant difference between baseline controls and treated animals. In addition, we were able to gain a dose response relationship. At 3 and 10 mg/kg, there was no significant difference between untreated and treated animals (figure 5a). We further validated our capability to use in vivo imaging for observing the effect of a trypsin inhibitor in real time. As shown in figure 5d, at 30 mg/kg dose, the animals were not significantly different from baseline control animals in terms of the concentration of active trypsin in the pancreas. These results indicate that in vivo imaging may be used to predict the outcome of a trypsin inhibitor dynamically.

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