64Cu-Labeled Lys40(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid)NH2–conjugated exendin-4


Chopra A.

Publication Details



In vitro Rodents



Autoimmune processes and other environmental factors that destroy β-cells located in the pancreatic islet cells are known to promote the development of insulin-dependent diabetes mellitus (type 1 diabetes) in individuals genetically predisposed to the disease (1). Due to this destruction, the net mass of the β-cells in the islet cells of the pancreas is reduced, which leads to decreased production of insulin in the individual, and the maintenance of blood glucose at proper physiological levels is impaired. Type 2 diabetes is the most common form of the disease and is primarily caused by insulin resistance as a consequence of low insulin secretion by the β-cells. This form of diabetes can often be corrected with exercise, diet control, and/or medication (2). Upon diagnosis of diabetes, it is important to determine the individual's β-cell mass (BCM) or volume to devise a successful treatment regimen for the condition (3). Changes in the BCM during the onset of diabetes is poorly understood, and only an indirect method that measures the amount of stimulated insulin secretion by the pancreas is used to quantify the BCM in humans (4). However, the β-cells appear to have a reserved capacity to produce insulin, so the use of insulin secretion as a determinant of BCM is of limited value (4).

As an alternative to determine insulin secretion to quantify BCM, investigators have evaluated the use of noninvasive positron emission tomography (PET) imaging techniques to determine the BCM in rats with the use of 11C- or 18F-labeled dihydrotetrabenazine, which is an antagonist of the vesicular monoamine transporter type 2 (VMAT2) in the islet cells (5). Careful evaluation of results obtained with the VMAT2 antagonists has revealed that a large proportion of these radiolabeled compounds tend to reside in the exocrine pancreas, indicating that these radiotracers are not suitable for the determination of BCM with PET imaging (6). Recently, some G-protein–coupled receptors (e.g., the glucagon-like peptide 1 (GLP 1) receptor (GLP-1R)), which show a selective location in the β-cells compared to the surrounding exocrine pancreatic cells, were identified by database mining and immunohistochemical staining of pancreatic tissue (6-8). On the basis of these observations and the known involvement of GLP-1R in β-cell function and biology (for details, see Baggio and Drucker (9)), this receptor was identified as a possible target of radiolabeled probes that can be used to quantify the BCM. Although GLP-1 is the natural ligand for the GLP-1R, a major drawback of using this peptide to measure the BCM is that it is rapidly inactivated (half-life, ~2 min) by proteolytic enzymes while in circulation. As a consequence, investigators identified and have used radiolabeled Exendin-4 or its analogs (Exendin is a peptide of 39 amino acids that has a 54% homology with GLP-1, acts as an agonist of the GLP-1R, and is not inactivated by proteolytic enzymes) for the measurement of BCM with noninvasive molecular imaging techniques (6, 8). Connolly et al. evaluated the use of 64Cu-labeled Lys40(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid)NH2–conjugated exendin-4 ([64Cu](Lys40(DOTA)NH2)Exendin-4) for the in vivo imaging and measurement of the BCM in rats and investigated its biodistribution in these animals (6).



(Lys40(DOTA)NH2)Exendin-4 was obtained from a commercial source and labeled with 64Cu as described by Connolly et al. (6). The labeled peptide was purified on a C18 light SepPak column with high-performance liquid chromatography. The radiochemical purity of the labeled peptide was determined to be 94.2 ± 0.9% with a specific activity of 13.22 ± 6.62 TBq/mmol (357 ± 179 Ci/mmol; n = 5 experiments). The stability of [64Cu](Lys40(DOTA)NH2)Exendin-4 was not reported.

In Vitro Studies: Testing in Cells and Tissues


A commercially available kit that quantifies the amount of cyclic AMP produced by the activation of a receptor was used with Chinese hamster ovary cells that express the human GLP-1R to determine the agonistic potency of (Lys40(DOTA)NH2)Exendin-4 (used as a proof of concept molecule for the study) (6). The EC50 of this molecule was determined to be 0.41 nM; however, the EC50 values of GLP-1 and Exendin-4 were not reported.

In another study, normal and diabetic human and non-human primate pancreas sections were subjected to immunohistochemical analysis with appropriate antibodies to detect the presence of the GLP-1R in the organ (6). The fluorescent signal was detected only in islet cells of the normal specimens, and sections obtained from the diabetic organ generated a very low signal in these cells. This indicated that there was a low level of the GLP-1R in the diabetic specimens and suggested that the receptor could be a suitable target for the noninvasive imaging of β-cells.

Ex vivo autoradiography of pancreas sections obtained from a rat at 60 min after tail vein injection of [64Cu](Lys40(DOTA)NH2)Exendin-4 showed a distinct signal pattern in multiple areas of the tissue, and this was presumed to be generated by radioactivity bound to the islet cells (6). The same section was immunostained to detect insulin (insulin is stained only where the β-cells are present), and a staining pattern similar to that observed with [64Cu](Lys40(DOTA)NH2)Exendin-4 was apparent. The two images were merged, and the autoradiographic and the immunostaining signals were reported to originate from the same location in the specimen, indicating that the distribution of radioactivity corresponded to the distribution of the islet cells. Autoradiography of pancreas sections obtained from a rat administered with unlabeled Exendin(9-39) 10 min before the injection of [64Cu](Lys40(DOTA)NH2)Exendin-4 showed a complete absence of signal, suggesting that the uptake of radioactivity was blocked in this specimen and that the radiolabeled peptide had binding specificity for the GLP-1R. It was also reported that autoradiography of pancreas sections from Zucker ZDF rats (these rodents are the animal model for human type 2 diabetes and show loss of β-cells with progression of the disease) injected with [64Cu](Lys40(DOTA)NH2)Exendin-4 showed very little uptake of radioactivity compared with pancreas sections obtained from control Zucker ZCL rat (6). These results indicated that the tracer was suitable for the determination of any changes in the BCM of the animals.

Animal Studies



The biodistribution of [64Cu](Lys40(DOTA)NH2)Exendin-4 was studied in normal and streptozotocine (STZ)-treated Sprague-Dawley rats (n = 3 animals/group) (6). Immediately prior to initiation of the study, the blood glucose levels of animals from both groups were determined. The rodents were injected with 1.1 MBq (30 μCi) of the tracer through the tail vein and euthanized 60 min later to determine the amount of radioactivity accumulated in the organs of interest. Data were reported as standard uptake values (SUV) corresponding to the injected dose and weight of each animal. The blood glucose level of the normal animals was significantly lower (98.0 ± 12.2 mg/dL; P = 0.02) than that of the STZ-treated rats (442.3 ± 42.7 mg/dL), indicating that the latter group of rodents had a complete onset of diabetes. Both groups of rats showed high accumulation of radioactivity in the kidneys (48.3 ± 5.5 SUV and 27.6 ± 5.1 SUV for the control and STZ-treated animals, respectively), indicating that the tracer was excreted primarily through the urinary system. Uptake of radioactivity in the pancreas of the STZ-treated animals (0.1 ± 0.01 SUV) was significantly lower (P = 0.001) than that observed in the organ of the control rats (0.3 ± 0.02 SUV). All other organs obtained from the control animals showed uptake values between 0.03 ± 0.02 SUV (blood) and 0.7 ± 0.2 SUV (liver), and the STZ-treated group showed a similar trend with tracer accumulation values between 0.2 ± 0.04 SUV (blood) and 0.6 ± 0.05 SUV (liver).

To confirm the uptake of [64Cu](Lys40(DOTA)NH2)Exendin-4 in the pancreas of the rats, dynamic PET images were acquired from two anesthetized animals (6). The rodents were first injected with [11C]methionine to visualize the pancreatic region in the abdomen of the animals (this tracer is known to accumulate primarily in the pancreas (6)). PET scans were performed for 45 min, and images obtained with the 11C probe clearly showed the outline of the pancreas of the animals. Subsequently, the same animals were injected with [64Cu](Lys40(DOTA)NH2)Exendin-4, and PET scans were acquired for 60 min post injection. To confirm the accumulation of the Exendin tracer in the pancreas, images obtained from the two separate scans were merged using appropriate software as described by Connolly et al. (6). In the merged image, however, the radioactivity taken up by the kidneys from the 64Cu-labeled peptide interfered with the image of the pancreas due to the close proximity of the organs in the abdominal cavity of the rats. As a result, the investigators could not clearly visualize the uptake of label in the pancreas.

From these studies, the investigators concluded that [64Cu](Lys40(DOTA)NH2)Exendin-4 bound specifically to the GLP-1R under in vitro conditions. To confirm this observation in vivo and to quantify the BCM in the pancreas, however, similar studies will have to be performed in large animals (6).

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.

Supplemental Information


No information is currently available.


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