Stefanie Willekens

Strain Differences Determine the Suitability of Animal Models for Noninvasive In Vivo Beta Cell Mass Determination with Radiolabeled Exendin.

PurposeNoninvasive beta cell mass (BCM) quantification is a crucial tool to understand diabetes development and progression. [111In]exendin is a promising agent for in vivo beta cell imaging, but tracer testing has been hampered by the lack of well-defined rodent models.ProceduresBiodistribution and pancreatic uptake of [111In]exendin were compared in rats and mice. In selected models, the amount of [111In]exendin accumulation in the pancreas and other organs was determined using a model of alloxan-induced beta cell loss. GLP-1R expression levels were analyzed by RT-PCR and immunohistochemistry.ResultsNamely Brown Norway rats showed beta-cell-specific tracer accumulation and favorable pancreas-to-background ratios for noninvasive BCM determination. Mice displayed receptor-mediated [111In]exendin uptake in endocrine and exocrine pancreas, in spite of very low GLP-1R expression in exocrine tissue.ConclusionsRats display better characteristics for in vivo BCM determination than mice and are suggested as a more adequate model for humans.

SPECT of Transplanted Islets of Langerhans by Dopamine 2 Receptor Targeting in a Rat Model.

Pancreatic islet transplantation can be a more permanent treatment for type 1 diabetes compared to daily insulin administration. Quantitative and longitudinal noninvasive imaging of viable transplanted islets might help to further improve this novel therapy. Since islets express dopamine 2 (D2) receptors, they could be visualized by targeting this receptor. Therefore, the D2 receptor antagonist based tracer [(125/123)I][IBZM] was selected to visualize transplanted islets in a rat model. BZM was radioiodinated, and the labeling was optimized for position 3 of the aromatic ring. [(125)I]-3-IBZM was characterized in vitro using INS-1 cells and isolated islets. Subsequently, 1,000 islets were transplanted in the calf muscle of WAG/Rij rats and SPECT/CT images were acquired 6 weeks after transplantation. Finally, the graft containing muscle was dissected and analyzed immunohistochemically. Oxidative radioiodination resulted in 3 IBZM isomers with different receptor affinities. The use of 0.6 mg/mL chloramine-T hydrate resulted in high yield formation of predominantly [(125)I]-3-IBZM, the isomer harboring the highest receptor affinity. The tracer showed D2 receptor mediated binding to isolated islets in vitro. The transplant could be visualized by SPECT 6 weeks after transplantation. The transplants could be localized in the calf muscle and showed insulin and glucagon expression, indicating targeting of viable and functional islets in the transplant. Radioiodination was optimized to produce high yields of [(125)I]-3-IBZM, the isomer showing optimal D2R binding. Furthermore, [(123)I]IBZM specifically targets the D2 receptors on transplanted islets. In conclusion, this tracer shows potential for noninvasive in vivo detection of islets grafted in the muscle by D2 receptor targeting.

Noninvasive Imaging of Islet Transplants with 111In-Exendin-3 SPECT/CT

Islet transplantation is a promising treatment for type 1 diabetic patients. However, there is acute as well as chronic loss of islets after transplantation. A noninvasive imaging method that could monitor islet mass might help to improve transplantation outcomes. In this study, islets were visualized after transplantation in a rat model with a dedicated small-animal SPECT scanner by targeting the glucagonlike peptide-1 receptor (GLP-1R), specifically expressed on β-cells, with 111In-labeled exendin-3. Methods: Targeting of 111In-exendin-3 to GLP-1R was tested in vitro on isolated islets of WAG/Rij rats. For in vivo evaluation, 400 or 800 islets were transplanted into the calf muscle of WAG/Rij rats (6–8 wk old). Four weeks after transplantation, SPECT/CT images were acquired 1 h after injection of 111In-labeled exendin-3. After SPECT acquisition, the muscles containing the transplant were analyzed immunohistochemically and autoradiographically. Results: The binding assay, performed on isolated islets, showed a linear correlation between the number of islets and 111In-exendin-3 accumulation (Pearson r = 0.98). In vivo, a 1.70 ± 0.44-fold difference in tracer uptake between 400 and 800 transplanted islets was observed. Ex vivo analysis of the islet transplant showed colocalization of tracer accumulation on autoradiography, with insulin-positive cells and GLP-1R expression on immunohistochemistry. Conclusion: 111In-exendin-3 accumulates specifically in the β-cells after islet transplantation and is a promising tracer for noninvasive monitoring of the islet mass.

Non-invasive in vivo determination of viable islet graft volume by 111In-exendin-3

Pancreatic islet transplantation is a promising therapy for patients with type 1 diabetes. However, the duration of long-term graft survival is limited due to inflammatory as well as non-inflammatory processes and routine clinical tests are not suitable to monitor islet survival. 111In-exendin-SPECT (single photon emission computed tomography) is a promising method to non-invasively image islets after transplantation and has the potential to help improve the clinical outcome. Whether 111In-exendin-SPECT allows detecting small differences in beta-cell mass (BCM) and measuring the actual volume of islets that were successfully engrafted has yet to be demonstrated. Here, we evaluated the performance of 111In-exendin-SPECT using an intramuscular islet transplantation model in C3H mice. In vivo imaging of animals transplanted with 50, 100, 200, 400 and 800 islets revealed an excellent linear correlation between SPECT quantification of 111In-exendin uptake and insulin-positive area of islet transplants, demonstrating that 111In-exendin-SPECT specifically and accurately measures BCM. The high sensitivity of the method allowed measuring small differences in graft volumes, including grafts that contained less than 50 islets. The presented method is reliable, convenient and holds great potential for non-invasive monitoring of BCM after islet transplantation in humans.

Quantitative and longitudinal imaging of intramuscular transplanted islets of Langerhans with SPECT using [ 123 I]IBZM

A non‐invasive imaging method to monitor islet grafts could provide novel and improved insight into the fate of transplanted islets and, potentially, monitor the effect of therapeutic interventions. Therefore, such an imaging method could help improve long‐term transplantation outcome. Here, we investigated the use of [ 123I]IBZM for insulin positive graft volume quantification and longitudinal graft monitoring. SPECT images were acquired 6 weeks after islet transplantation in the calf muscle of rats. For longitudinal graft analysis, rats were monitored by SPECT for 10 weeks. After animals were euthanized, graft containing muscles were dissected for ex vivo analysis and insulin‐positive graft volume determination. Six weeks after transplantation, a clear signal was observed in all grafts by SPECT imaging. Moreover, the intensity of the SPECT signal correlated linearly with insulin‐positive graft volume, as determined histologically. Longitudinal graft follow‐up showed a clear SPECT signal of the transplant from 3 until 10 weeks after transplantation. In this study, we demonstrate for the first time the successful application of a radiotracer, [ 123I]IBZM, for non‐invasive, in vivo graft volume quantification and longitudinal graft monitoring.

Characterization of In-111-labeled Glucose-Dependent Insulinotropic Polypeptide as a Radiotracer for Neuroendocrine Tumors

Somatostatin receptor targeting is considered the standard nuclear medicine technique for visualization of neuroendocrine tumors (NET). Since not all NETs over-express somatostatin receptors, the search for novel targets, visualizing these NETs, is ongoing. Many NETs, expressing low somatostatin receptor levels, express glucose-dependent insulinotropic polypeptide (GIP) receptors (GIPR). Here, we evaluated the performance of [Lys37(DTPA)]N-acetyl-GIP1-42, a newly synthesized GIP analogue to investigate whether NET imaging via GIPR targeting is feasible. Therefore, [Lys37(DTPA)]N-acetyl-GIP1-42 was radiolabeled with 111In with specific activity up to 1.2 TBq/µmol and both in vitro and in vivo receptor targeting properties were examined. In vitro, [Lys37(111In-DTPA)]N-acetyl-GIP1-42 showed receptor-mediated binding to BHK-GIPR positive cells, NES2Y cells and isolated islets. In vivo, both NES2Y and GIPR-transfected BHK tumors were visualized on SPECT/CT. Furthermore, co-administration of an excess unlabeled GIP1-42 lowered tracer uptake from 0.7 ± 0.2%ID/g to 0.6 ± 0.01%ID/g (p = 0.78) in NES2Y tumors and significantly lowered tracer uptake from 3.3 ± 0.8 to 0.8 ± 0.2%ID/g (p = 0.0001) in GIPR-transfected BHK tumors. In conclusion, [Lys37(111In-DTPA)]N-acetyl-GIP1-42 shows receptor-mediated binding in various models. Furthermore, both GIPR-transfected BHK tumors and NES2Y tumors were visible on SPECT/CT using this tracer. Therefore, [Lys37(111In-DTPA)]N-acetyl-GIP1-42 SPECT seems promising for visualization of somatostatin receptor negative NETs.

Beta Cell Imaging as Part of “Imaging on Metabolic Diseases”

Currently, radionuclide imaging after injection of radiolabeled tracers seems to be the most promising approach to noninvasively determine the beta cell mass (BCM) in vivo. Despite the limited resolution of positron emission tomography (PET) and single photon emission computed tomography (SPECT), the very high sensitivity of these imaging modalities allows detection of very low radiotracer concentrations. Since the noninvasive imaging modalities that offer the highest spatial resolution, i.e., computed tomography (CT) and magnetic resonance imaging (MRI), which are not able to resolve single islets in vivo, radionuclide imaging would be the method of choice for beta cell imaging. However, noninvasive visualization of the pancreatic beta cells is a highly challenging endeavor. The beta cells are located in the islets of Langerhans in the pancreas. The islets are small (usually between 50 and 400 μm), only account for 1–2 % of the total pancreatic mass, and are spread throughout the pancreas (Bonner-Weir 1994; Weir et al. 1990). Targeting of these small structures spread through an organ is highly challenging when compared to tumor imaging, where a focal solid mass containing the target cells is visualized. Moreover, the islets consist of several cell types (beta cells, alpha cells, delta cells, and pp cells) with the beta cells representing around 60–80 % of the islet mass, making specific visualization of beta cells even more difficult. Since PET and SPECT cannot resolve single islets, let alone beta cells, a highly beta cell-specific radiotracer is required. Only when the radiotracer specifically accumulates in beta cells and not in other cell types of the endocrine or exocrine pancreas, the accumulation of the radiotracer in the pancreas could be used as a surrogate measure of the beta cell mass. To enable this, a target specifically expressed on beta cells and an optimal respective ligand should be selected for the development of beta cell targeting radiotracers. In contrast to tumor imaging, where several targets are overexpressed, beta cells express endogenous levels of receptors leading to generally lower accumulation of radiotracers when compared to tumor cells. In this book chapter, the challenges regarding tracer development and preclinical and clinical characterization will be discussed in detail.

PS2 - 6. Proof of Principle Study: IBZM based imaging of transplanted islets of Langerhans

Currently, transplantation of islets of Langerhans is a promising experimental treatment for diabetes type 1 patients. Despite the ability of the transplanted islets to normalize blood glucose, the rate of insulin-independency drops to less than 15% after 5 years. The visualization of transplanted islets could gain important insights in the processes underlying islet transplantation and might improve the outcome of islet transplantation.