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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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In vivo study with trypsin and serine protease activity inhibitor Camostat.A) Animals were administered Camostat at 300 mg/kg orally 24 h and 2 h prior to imaging study. Blood pool fluorescent contrast agent Angiosense was administered intravenously and animals were imaged at different times during a 3 h study with Caerulein induced pancreatitis to assess the development of edema. B) (n = 3) The graph is a quantification of the fluorescent intensity corresponding to the time points in A) and normalized to image obtained prior to caerulein administration. C) Another set of animals was administered the mPEG-PL-Cy5.5 probe to monitor the activity of trypsin in caerulein induced pancreatitis animals. D) (n = 6) The signal in the pancreas was quantified and plotted. Data represented mean ± SEM. E) Untreated saline animal average normalized fluorescent intensity from the Angiosense animals and mPEG-PL-Cy5.5 probe was plotted.
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pone-0055959-g003: In vivo study with trypsin and serine protease activity inhibitor Camostat.A) Animals were administered Camostat at 300 mg/kg orally 24 h and 2 h prior to imaging study. Blood pool fluorescent contrast agent Angiosense was administered intravenously and animals were imaged at different times during a 3 h study with Caerulein induced pancreatitis to assess the development of edema. B) (n = 3) The graph is a quantification of the fluorescent intensity corresponding to the time points in A) and normalized to image obtained prior to caerulein administration. C) Another set of animals was administered the mPEG-PL-Cy5.5 probe to monitor the activity of trypsin in caerulein induced pancreatitis animals. D) (n = 6) The signal in the pancreas was quantified and plotted. Data represented mean ± SEM. E) Untreated saline animal average normalized fluorescent intensity from the Angiosense animals and mPEG-PL-Cy5.5 probe was plotted.

Mentions: Edema reduction is a prominent outcome of a trypsin inhibitor study in a caerulein model. However, edema reduction gives inadequate information about trypsin inhibition in the pancreas because it is a biomarker not specific to the target. We compared animals pretreated with Camostat (see Table 1 for protease selectivity) at a dose of 300 mg/kg orally to untreated animals. Both sets of animals received three subsequent caerulein doses SC. Animals that did not receive caerulein were used as baseline controls. When imaged using Angiosense 680, there was no signal enhancement as noticed by reduced intensity (figure 3a, b). Healthy control animals did not show any increase in fluorescence in the region of the pancreas. In another study, the same group of animals was imaged using the mPEG-PL-Cy5.5 smart probe. Animals treated with Camostat showed reduced probe activation (figure 3c, d) in comparison to untreated caerulein animals (P<0.001). Camostat treated animals and control animals did not show any significant difference in probe activation after 3 subsequent caerulein administration. All animals indicated high signal intensity in the liver suggesting that the mPEG-PL-Cy5.5 probe is activated by enzymatic activity in disease pancreas. A correlation plot of untreated animals between Angiosense 680 and mPEG-PL-Cy5.5 probe signal intensity in pancreas showed a good correlation between trypsin mediated probe activation and edema accumulation. We were unable to combine the administration of Angiosense 680 and mPEG-PL-Cy5.5 probe in the same animal due to peak broadening of the activated probe signal that bled over into the Angiosense 680 channel.


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)

In vivo study with trypsin and serine protease activity inhibitor Camostat.A) Animals were administered Camostat at 300 mg/kg orally 24 h and 2 h prior to imaging study. Blood pool fluorescent contrast agent Angiosense was administered intravenously and animals were imaged at different times during a 3 h study with Caerulein induced pancreatitis to assess the development of edema. B) (n = 3) The graph is a quantification of the fluorescent intensity corresponding to the time points in A) and normalized to image obtained prior to caerulein administration. C) Another set of animals was administered the mPEG-PL-Cy5.5 probe to monitor the activity of trypsin in caerulein induced pancreatitis animals. D) (n = 6) The signal in the pancreas was quantified and plotted. Data represented mean ± SEM. E) Untreated saline animal average normalized fluorescent intensity from the Angiosense animals and mPEG-PL-Cy5.5 probe was plotted.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0055959-g003: In vivo study with trypsin and serine protease activity inhibitor Camostat.A) Animals were administered Camostat at 300 mg/kg orally 24 h and 2 h prior to imaging study. Blood pool fluorescent contrast agent Angiosense was administered intravenously and animals were imaged at different times during a 3 h study with Caerulein induced pancreatitis to assess the development of edema. B) (n = 3) The graph is a quantification of the fluorescent intensity corresponding to the time points in A) and normalized to image obtained prior to caerulein administration. C) Another set of animals was administered the mPEG-PL-Cy5.5 probe to monitor the activity of trypsin in caerulein induced pancreatitis animals. D) (n = 6) The signal in the pancreas was quantified and plotted. Data represented mean ± SEM. E) Untreated saline animal average normalized fluorescent intensity from the Angiosense animals and mPEG-PL-Cy5.5 probe was plotted.
Mentions: Edema reduction is a prominent outcome of a trypsin inhibitor study in a caerulein model. However, edema reduction gives inadequate information about trypsin inhibition in the pancreas because it is a biomarker not specific to the target. We compared animals pretreated with Camostat (see Table 1 for protease selectivity) at a dose of 300 mg/kg orally to untreated animals. Both sets of animals received three subsequent caerulein doses SC. Animals that did not receive caerulein were used as baseline controls. When imaged using Angiosense 680, there was no signal enhancement as noticed by reduced intensity (figure 3a, b). Healthy control animals did not show any increase in fluorescence in the region of the pancreas. In another study, the same group of animals was imaged using the mPEG-PL-Cy5.5 smart probe. Animals treated with Camostat showed reduced probe activation (figure 3c, d) in comparison to untreated caerulein animals (P<0.001). Camostat treated animals and control animals did not show any significant difference in probe activation after 3 subsequent caerulein administration. All animals indicated high signal intensity in the liver suggesting that the mPEG-PL-Cy5.5 probe is activated by enzymatic activity in disease pancreas. A correlation plot of untreated animals between Angiosense 680 and mPEG-PL-Cy5.5 probe signal intensity in pancreas showed a good correlation between trypsin mediated probe activation and edema accumulation. We were unable to combine the administration of Angiosense 680 and mPEG-PL-Cy5.5 probe in the same animal due to peak broadening of the activated probe signal that bled over into the Angiosense 680 channel.

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