Synthesis and evaluation of [18F]exendin (9–39) as a potential biomarker to measure pancreatic β-cell mass

doi:10.1016/j.nucmedbio.2011.07.011

Nuclear Medicine and Biology

Volume 39, Issue 2, February 2012, Pages 167-176

https://doi.org/10.1016/j.nucmedbio.2011.07.011 Get rights and content

Abstract

Introduction

Glucagon-like peptide 1 (GLP-1) is released in response to food intake and plays an important role in maintaining blood glucose homeostasis. Exendin (9–39), a potent glucagon-like peptide 1 receptor antagonist, has been labeled with In-111 for SPECT imaging. We report here the first radiosynthesis of [¹⁸F]exendin (9–39) ([¹⁸F]Ex(9–39)) and an evaluation of its potential as a biomarker for in vivo positron emission tomography (PET) imaging of pancreatic β-cell mass (BCM) in rats.

Methods

F-18 label was introduced by conjugation of [¹⁸F]4-fluorobenzaldehyde with an Ex(9–39) derivative containing a 6-hydrazinonicotinyl group on the ɛ-amine of Lys27. Positron emission tomography imaging was carried out in Sprague–Dawley rats (five control and five streptozotocin-induced diabetic) and BioBreeding diabetes-prone rats (three at 7 weeks and three at 12 weeks) using the high-resolution research tomograph (HRRT) after 0.187±0.084 mCi [¹⁸F]Ex(9–39) administration. Time–activity curves were obtained from pancreas, liver and kidney. Pancreases were assayed for insulin content after the imaging study.

Results

Site-specifically labeled [¹⁸F]Ex(9–39) was purified on a G15 open column with radiochemical and chemical purities >98%. Positron emission tomography imaging showed pancreatic standardized uptake value (SUV) peaked at 10 min and plateaued by 50 min to the end of scan (240 min). No correlations of pancreatic SUV with postmortem measures of insulin content were seen.

Conclusions

[¹⁸F]Ex(9–39) was successfully prepared and used for PET imaging for the first time to measure pancreatic BCM. The results suggest that derivatization of the Lys27 residue might reduce binding affinity, as evidenced by the absence of specific binding. Exendin analogues radiolabeled at other sites may elucidate the active site required for binding.

Introduction

Glucagon-like peptide 1 receptor (GLP-1R) has recently been shown to be overexpressed in insulinomas, gastrinomas and lung neuroendocrine tumors [1], [2], which makes it a promising target for diagnostic and therapeutic purposes, particularly for insulinoma treatment. The endogenous ligand, GLP-1, is ineffective both as an imaging and therapeutic agent because it has a very short half-life (1–2 min) in vivo [3] due to fast degradation by the enzyme dipeptidyl peptidase 4 [4]. Exendin-4, a 39-amino acid peptide hormone found in the saliva of the Gila monster [5], is an agonist of GLP-1R. Exendin-4 has only 53% homology with GLP-1, which increases its resistance to degradation by dipeptidyl peptidase 4 and therefore extends its half-life in vivo to about 2.4 h [6], [7]. Exendin-4 has a high affinity to GLP-1R, with a K_d value of approximately 1.4×10⁻¹⁰ M [8]. Exendin-4 has been radiolabeled with SPECT imaging radionuclides, such as In-111, and was used to image insulinomas in animals [9], [10] as well as in humans [11], [12]. Exendin-4 was also radiolabeled with Ga-68 for positron emission tomography (PET) imaging studies [13]. Exendin (9–39) is a truncated version of exendin-4 and also has a high affinity to GLP-1R, with a K_d value of around 3×10⁻⁹ M [8]. However, unlike exendin-4, truncated exendin (9–39) does not promote the secretion of insulin; instead, it acts as antagonist of GLP-1R and is potentially a competitive inhibitor of exendin-4 [14]. Based on its high affinity to GLP-1R, exendin (9–39) was also labeled with a radionuclide to image the GLP-1R [15].

Our research focuses on identification and evaluation of radiolabeled ligands that bind specifically to molecular targets, allowing for noninvasive in vivo PET imaging of pancreatic islet β-cell mass (BCM). A challenge for imaging BCM is the accumulation of the radioligands by the surrounding organs that can compromise the accuracy of quantitative imaging. Based on database and immunohistochemistry screening, we identified G-protein-coupled receptors (GPCRs), including GLP-1R, which are expressed with a high degree of specificity to islet β-cells for further in vitro and in vivo evaluation for PET imaging of BCM. Immunohistochemical staining of GLP-1R with GLP-1R and insulin antibodies showed colocalization of insulin with GLP-1R. We have also assessed binding and uptake of ¹²⁵I-exendin (9–39) (PerkinElmer, Boston, MA) to a rat insulinoma β-cell line (INS-1 832/13) and a human pancreatic exocrine cell line (PANC-1) and found a preferential binding of exendin (9–39) to rat insulinoma cells (INS-1) and rat islets in comparison to exocrine cells (PANC-1) [16] (see Sections 2 and 3 for brief experimental procedures and results, respectively). The high expression and specificity of GLP-1Rs on islet β-cells makes it an attractive target for molecular imaging using radiolabeled analogues of exendin-4 [10], [17]. Recently, we have shown the feasibility of fluorescent analogues of exendin-4 to image pancreatic islet BCM in vivo. In this study, exendin-4 was conjugated to the monofunctional dye Cy5.5 and the saturation binding was assessed using the INS-1 rat insulinoma cell line. The preferential localization of exendin-4-Cy5.5 to the pancreas was confirmed both in vivo and ex vivo [18] (see Sections 2 and 3 for brief experimental procedures and results, respectively). Furthermore, an ¹¹¹In-labeled exendin-4 analogue, [Lys40(Ahx-DTPA-¹¹¹In)NH₂]exendin-4, has been synthesized and showed high specificity and affinity in targeting GLP-1R with negligible specific and nonspecific binding to surrounding tissue (i.e., liver, stomach, intestines) [10], [17]. We hypothesize that ¹⁸F-labeled exendin (9–39) can be used to specifically target GLP-1R in β-cells with low surrounding tissue accumulations, allowing for quantitative PET imaging of pancreatic BCM.

To the best of our knowledge, Kimura et al. [19] reported the synthesis of [¹⁸F]Ex(9–39) via N-succinimidyl 4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB). [¹⁸F]SFB is an excellent reagent to label peptides with F-18; however, it is not site-specific since the active succinimidyl ester can react with any primary amine group within the peptide molecule. That is, the labeled peptide is a mixture of peptide molecules tagged with [¹⁸F]fluorobenzyl groups ([¹⁸F]FB) on different amino acid residues possessing primary amine groups. Usually, the amine groups in a peptide molecule are critical to its biological function; thus, an improper introduction of a labeling tag to a critical amine group may result in a loss of the biological activity of the labeled peptide. The modification on the ɛ-amine of Lys27 has been reported to have minor influence on the binding affinity of the peptide to GLP-1R [20], [21], [22]; therefore, in the current work, F-18 label was introduced in a site-specific manner by conjugation of [¹⁸F]4-fluorobenzaldehyde ([¹⁸F]FBA)with an exendin derivative containing a 6-hydrazinonicotinyl (HYNIC) group on the ɛ-amine of Lys27 through the formation of a hydrazone. Small-animal PET imaging was carried out in Sprague–Dawley (SD) rats and BioBreeding diabetes-prone (BB-DP) rats following administration of [¹⁸F]Ex(9–39). Time–activity curves (TACs) were obtained for pancreas, liver and kidney. The pancreases were assayed for insulin content after sacrificing the animals at the end of PET scans. Data were examined for correlations between uptake of tracer in pancreas measured by PET and islet insulin expression measured by postmortem histology.

Section snippets

Methods and materials

The β-cell selectivity of exendin and its analogues were evaluated via in vitro and in vivo tests before the PET imaging studies with F-18-labeled exendin.

β-Cell selectivity: In vitro cell binding assays using ¹²⁵I-exendin (9–39)

Normalized uptake and retention in the INS-1 cells (34.1±8.0 and 3.0±1.3) was significantly (P<.001) higher than in the PANC-1 cells (1.4±0.2 and 0.1±0.0). Thus, the specificity of ¹²⁵I-exendin (9–39) for the INS-1 cells was ∼24 to 1 for uptake and ∼30 to 1 for retention.

Molecular targeted fluorescent imaging of pancreatic BCM with exendin-4-Cy.5.5

The results from the binding assay of exendin-4-Cy.5.5 to the INS-1 (832/13) rat insulinoma cell line are shown in Fig. 2, indicating a specific and saturable binding of exendin to β-cell. The preferential localization of

Conclusion

Exendin (9–39) was readily radiolabeled with F-18 in a site-specific manner by using the formation of a hydrazone between HYNIC tagged Lys27 and [¹⁸F]FBA. Aniline acted as a catalyst for the formation of the hydrazone and was required for efficient labeling. Introduction of [¹⁸F]FBA to the HYNIC-modified Lys27 residue might lead to a change in binding affinity, as seen by the absence of specific binding to the GLP-1R in pancreas. Preparation of [¹⁸F]exendin analogues with the F-18 label at

Acknowledgments

The authors thank the staff of the Yale University PET Center for their technical expertise and support. Funding for this study was provided by the Juvenile Diabetes Research Foundation (JDRF) and Yale Pfizer Bioimaging Alliance. This publication was also made possible by CTSA Grant Number UL1 RR024139 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH) and NIH roadmap for Medical Research. Its contents are solely the responsibility of

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Using beta cell-specific reliable bioimaging probes appears to be an appropriate approach for in vivo beta-cell imaging [20]. GLP-1R is considered as a crucial target in T2DM therapy and a promising target for such probes owing to its high expression in pancreatic beta cells (Table 1) [21–35]. It is a G-protein-coupled receptor [36] expressed physiologically in various organs, such as pancreas and kidney, and overexpressed in various pathological situations, such as insulinomas and pheochromocytomas [36,37].
Loss or dysfunction of the pancreatic beta cells or insulin receptors leads to diabetes mellitus (DM). This usually occurs over many years; therefore, the development of methods for the timely detection and clinical intervention are vital to prevent the development of this disease. Glucagon-like peptide-1 receptor (GLP-1R) is the receptor of GLP-1, an incretin hormone that causes insulin secretion in a glucose-dependent manner. GLP-1R is highly expressed on the surface of pancreatic beta cells, providing a potential target for bioimaging. In this review, we provide an overview of various strategies, such as the development of GLP-1R agonists (e.g., exendin-4), and GLP-1 sequence modifications for GLP-1R targeting for the diagnosis and treatment of pancreatic beta cell disorders. We also discuss the challenges of targeting pancreatic beta cells and strategies to address such challenges.
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Beta cells assume a fundamental role in maintaining blood glucose homeostasis through the secretion of insulin, which is contingent on both beta cell mass and function, in response to elevated blood glucose levels or secretagogues. For this reason, evaluating beta cell mass and function, as well as scrutinizing how they change over time in a diabetic state, are essential prerequisites in elucidating diabetes pathophysiology. Current clinical methods to measure human beta cell mass and/or function are largely lacking, indirect and sub-optimal, highlighting the continued need for noninvasive in vivo beta cell imaging technologies such as optical imaging techniques. While numerous probes have been developed and evaluated for their specificity to beta cells, most of them are more suited to visualize beta cell mass rather than function. In this review, we highlight the distinction between beta cell mass and function, and the importance of developing more probes to measure beta cell function. Additionally, we also explore various existing probes that can be employed to measure beta cell mass and function in vivo, as well as the caveats in probe development for in vivo beta cell imaging.
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This has handicapped efforts to develop radiolabeled Exendin-4 analogues with short half-life isotopes. A lack of purity in other Exendin-derived labels, such as N-2-(4-18F-fluorobenzamido) ethylmaleimide (18F-FBEM) [11] and 18F-fluorobenzaldehyde [12] has rendered them untenable in clinical applications. Previously, we reported on our efforts to develop 18F-TTCO-Cys40-Exendin-4 [13] as a PET-sensitive GLP-1R tracer.
A major limitation in the development of radiolabeled Exendin-4 analogues (short half-life isotopes) is an inability to efficiently and rapidly separate final products from precursors. This is important as lack of purity in the final product decreases probe efficiency. The purpose of this study was to develop a method to prepare the high-purity imaging reagent [¹⁸F] PTTCO-Cys⁴⁰-Exendin-4. To accomplish this, magnetic TCO-beads were incubated with the crude product to remove unlabeled Exendin-4. In rodents pre-treatment with purified [¹⁸F] PTTCO-Cys⁴⁰-Exendin-4 (~1.85 MBq) allowed precise microPET imaging of ectopic insulinomas. Moreover, analogue uptake was successfully blocked by administering non-labelled “cold” Exendin-4. Biodistribution data revealed that [¹⁸F] PTTCO-Cys⁴⁰-Exendin-4 accumulated specifically in GLP-1R-enriched insulinomas in mice, confirming results obtained using miroPET. Investigation of [¹⁸F] PTTCO-Cys⁴⁰-Exendin-4 as a tracer to image portal vein-transplanted pancreatic islets is proceeding in animals.
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In one possible solution, three-dimensionally printed phantom images were used to quantify pancreatic 111In-exendin uptake and initial results showed that this technique is reliable in the quantification of pancreatic uptake [114]. Several groups have used different labeling strategies and developed 18F-labeled exendin (9-39) derivatives [115,116], or exendin-4 analogs [117], for quantitative analysis of BCM. Connolly et al. labeled Lys40(DOTA)NH2Exendin-4 with 64Cu and ex vivo autoradiographic and immunohistochemical studies showed the specific binding of 64Cu-(Lys40(DOTA)NH2)exendin-4 to islet β-cells [118].
Since diabetes is becoming a global epidemic, there is a great need to develop early β-cell specific diagnostic techniques for this disorder. There are two types of diabetes (i.e., type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM)). In T1DM, the destruction of pancreatic β-cells leads to reduced insulin production or even absolute insulin deficiency, which consequently results in hyperglycemia. Actually, a central issue in the pathophysiology of all types of diabetes is the relative reduction of β-cell mass (BCM) and/or impairment of the function of individual β-cells. In the past two decades, scientists have been trying to develop imaging techniques for noninvasive measurement of the viability and mass of pancreatic β-cells. Despite intense scientific efforts, only two tracers for positron emission tomography (PET) and one contrast agent for magnetic resonance (MR) imaging are currently under clinical evaluation. β-cell specific imaging probes may also allow us to precisely and specifically visualize transplanted β-cells and to improve transplantation outcomes, as transplantation of pancreatic islets has shown promise in treating T1DM. In addition, some of these probes can be applied to the preoperative detection of hidden insulinomas as well. In the present review, we primarily summarize potential tracers under development for imaging β-cells with a focus on tracers for PET, SPECT, MRI, and optical imaging. We will discuss the advantages and limitations of the various imaging probes and extend an outlook on future developments in the field.
Evaluation of 18F-labeled exendin(9-39) derivatives targeting glucagon-like peptide-1 receptor for pancreatic β-cell imaging
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β-cell mass (BCM) is known to be decreased in subjects with type-2 diabetes (T2D). Quantitative analysis for BCM would be useful for understanding how T2D progresses and how BCM affects treatment efficacy and for earlier diagnosis of T2D and development of new therapeutic strategies. However, a noninvasive method to measure BCM has not yet been developed.
We developed four ¹⁸F-labeled exendin(9-39) derivatives for β-cell imaging by PET: [¹⁸F]FB9-Ex(9-39), [¹⁸F]FB12-Ex(9-39), [¹⁸F]FB27-Ex(9-39), and [¹⁸F]FB40-Ex(9-39). Affinity to the glucagon-like peptide-1 receptor (GLP-1R) was evaluated with dispersed islet cells of ddY mice. Uptake of exendin(9-39) derivatives in the pancreas as well as in other organs was evaluated by a biodistribution study. Small-animal PET study was performed after injecting [¹⁸F]FB40-Ex(9-39).
FB40-Ex(9-39) showed moderate affinity to the GLP-1R. Among all of the derivatives, [¹⁸F]FB40-Ex(9-39) resulted in the highest uptake of radioactivity in the pancreas 30 min after injection. Moreover, it showed significantly less radioactivity accumulated in the liver and kidney, resulting in an overall increase in the pancreas-to-organ ratio. In the PET imaging study, pancreas was visualized at 30 min after injection of [¹⁸F]FB40-Ex(9-39).
[¹⁸F]FB40-Ex(9-39) met the basic requirements for an imaging probe for GLP-1R in pancreatic β-cells. Further enhancement of pancreatic uptake and specific binding to GLP-1R will lead to a clear visualization of pancreatic β-cells.

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Synthesis and evaluation of [18F]exendin (9–39) as a potential biomarker to measure pancreatic β-cell mass

Abstract

Introduction

Methods

Results

Conclusions

Introduction

Section snippets

Methods and materials

β-Cell selectivity: In vitro cell binding assays using 125I-exendin (9–39)

Molecular targeted fluorescent imaging of pancreatic BCM with exendin-4-Cy.5.5

Conclusion

Acknowledgments

J Biol Chem

J Biol Chem

Peptides

Biochem Biophys Res Commun

J Control Release

J Control Release

Biochem Biophys Res Commun

Nucl Med Biol

J Biol Chem

Concomitant expression of several peptide receptors in neuroendocrine tumours: molecular basis for in vivo multireceptor tumour targeting

Eur J Nucl Med Mol Imaging

GLP-1 receptor expression in human tumors and human normal tissues: potential for in vivo targeting

J Nucl Med

Similar elimination rates of glucagon-like peptide-1 in obese type 2 diabetic patients and healthy subjects

J Clin Endocrinol Metab

DPP-4 inhibitors and their potential role in the management of type 2 diabetes

Int J Clin Pract

Exenatide: a review of its use in patients with type 2 diabetes mellitus (as an adjunct to metformin and/or a sulfonylurea)

Drugs

Pharmacology of GLP-1-based therapies

Br J Diabetes Vasc Dis

[Lys40(Ahx-DTPA-111In)NH2]-Exendin-4 is a highly efficient radiotherapeutic for glucagon-like peptide-1 receptor-targeted therapy for insulinoma

Clin Cancer Res

[Lys40(Ahx-DTPA-111In)NH2]Exendin-4, a very promising ligand for glucagon-like peptide-1 (GLP-1) receptor targeting

J Nucl Med

Synthesis and evaluation of [¹⁸F]exendin (9–39) as a potential biomarker to measure pancreatic β-cell mass

β-Cell selectivity: In vitro cell binding assays using ¹²⁵I-exendin (9–39)

[Lys40(Ahx-DTPA-111In)NH₂]-Exendin-4 is a highly efficient radiotherapeutic for glucagon-like peptide-1 receptor-targeted therapy for insulinoma

[Lys40(Ahx-DTPA-111In)NH₂]Exendin-4, a very promising ligand for glucagon-like peptide-1 (GLP-1) receptor targeting