As shown in figure 6a, at 10 except 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, ,3,3, 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. Figure 6 Evaluation of trypsin inhibitor Novartis848 on intrapancreatic trypsin activity and edema formation using mPEG-PL-Cy5.5 probe. Discussion In this report, we successfully demonstrated the use of an activatable fluorescent smart probe to image the trypsin-dependent development of experimental pancreatitis and treatment response to protease inhibitors in a caerulein-injection animal model.
The development of optical fluorescence imaging in the near infrared has extended the field of in vitro fluorescence imaging at the molecular level to in vivo disease diagnosis and measurement of treatment response in animal models. Optical probes that emit in the near infrared can be employed successfully for various biomedical applications since hemoglobin and water have low coefficient of absorption in this region [23]. Therefore, deeper tissue can be accessed using fluorescent optical signal. Highly specific activatable, or ��smart�� [13] fluorescent probes have further enabled the real time visualization of the desired molecular function combined with specific localization.
In pancreatitis, Carfilzomib the pre-mature activation of trypsinogen to trypsin leads to autodigestion of the pancreatic tissue [24], [25]. The use of a fluorescent probe activated upon cleavage by trypsin was attractive for the characterization of intrapancreatic trypsin inhibitors in an in vivo model of experimental pancreatitis. Our in-house developed mPEG-PL-Cy5.5 probe was very selectively cleaved by trypsin as confirmed by an assay panel of pancreatic proteases.