Automated production of [68Ga]Ga-DOTA-exendin-4 via fractionated radionuclide generator elution on a cassette based synthesis module.

BACKGROUND: PET/CT imaging of glucagon-like peptide receptor 1 has recently filled a gap in reliably diagnosing insulinoma through non-invasive means. 68Ga-labelled derivatives of exendin-4 show high sensitivity as well as sufficient serum stability to enable routine clinical application. Here, we provide data for automated production of [68Ga][Nle14,Lys40(Ahx-DOTA-Ga)NH2]exendin-4 ([68Ga]Ga-DOTA-exendin-4) on a cassette based synthesis module (Modular-Lab PharmTracer, Eckert & Ziegler) using commercially available cassettes in combination with an approved 68Ge/68Ga generator (GalliaPharm, Eckert & Ziegler). This setup ensured high reproducibility as well as low radiation burden for the production team. Quality control including determination of radiochemical purity was performed by RP-HPLC using a water/0.1 % TFA/acetonitrile gradient on a C18 column. A modified TLC system with ammonium acetate & methanol as mobile phase and a novel limit test for determination of polysorbate 80 content in the final formulation are also described in this study. MAIN FINDINGS: Reliable yields as well as high molar activity for patient use were only achieved using a fractionated elution approach. Batch data showed radiochemical purity of >93 % as determined by RP-HPLC and TLC as well as good stability over 2 h post production. Testing for polysorbate 80 confirmed a concentration <1 mg/mL in the final product solution. Specifications for routine production were established based on existing Pharmacopeia monographs for other radiopharmaceuticals and were validated with 5 master batches. CONCLUSION: The described synthesis method enables reproducible, automated in-house production of [68Ga]Ga-DOTA-exendin-4 for routine clinical application.

New Radiolabeled Exendin Analogues Show Reduced Renal Retention.

PET imaging of the glucagon-like peptide-1 receptor (GLP-1R) using radiolabeled exendin is a promising imaging method to detect insulinomas. However, high renal accumulation of radiolabeled exendin could hamper the detection of small insulinomas in proximity to the kidneys and limit its use as a radiotherapeutic agent. Here, we report two new exendin analogues for GLP-1R imaging and therapy, designed to reduce renal retention by incorporating a cleavable methionine-isoleucine (Met-Ile) linker. We examined the renal retention and insulinoma targeting properties of these new exendin analogues in a nude mouse model bearing subcutaneous GLP-1R-expressing insulinomas. NOTA or DOTA was conjugated via a methionine-isoleucine linker to the C-terminus of exendin-4 (NOTA-MI-exendin-4 or DOTA-MI-exendin-4). NOTA- and DOTA-exendin-4 without the linker were used as references. The affinity for GLP-1R was determined in a competitive binding assay using GLP-1R transfected cells. Biodistribution of [68Ga]Ga-NOTA-exendin-4, [68Ga]Ga-NOTA-MI-exendin-4, [177Lu]Lu-DOTA-exendin-4, and [177Lu]Lu-DOTA-MI-exendin-4 was determined in INS-1 tumor-bearing BALB/c nude mice, and PET/CT was acquired to visualize renal retention and tumor targeting. For all tracers, dosimetric calculations were performed to determine the kidney self-dose. The affinity for GLP-1R was in the low nanomolar range (<11 nM) for all peptides. In vivo biodistribution revealed a significantly lower kidney uptake of [68Ga]Ga-NOTA-MI-exendin-4 at 4 h post-injection (p.i.) (34.2 ± 4.2 %IA/g), compared with [68Ga]Ga-NOTA-exendin-4 (128 ± 10 %IA/g). Accumulation of [68Ga]Ga-NOTA-MI-exendin-4 in the tumor was 25.0 ± 8.0 %IA/g 4 h p.i., which was similar to that of [68Ga]Ga-NOTA-exendin-4 (24.9 ± 9.3 %IA/g). This resulted in an improved tumor-to-kidney ratio from 0.2 ± 0.0 to 0.8 ± 0.3. PET/CT confirmed the findings in the biodistribution studies. The kidney uptake of [177Lu]Lu-DOTA-MI-exendin-4 was 39.4 ± 6.3 %IA/g at 24 h p.i. and 13.0 ± 2.5 %IA/g at 72 h p.i., which were significantly lower than those for [177Lu]Lu-DOTA-exendin-4 (99.3 ± 9.2 %IA/g 24 h p.i. and 45.8 ± 3.9 %IA/g 72 h p.i.). The uptake in the tumor was 7.8 ± 1.5 and 11.3 ± 2.0 %IA/g 24 h p.i. for [177Lu]Lu-DOTA-MI-exendin-4 and [177Lu]Lu-DOTA-exendin-4, respectively, resulting in improved tumor-to-kidney ratios for [177Lu]Lu-DOTA-MI-exendin-4. The new exendin analogues with a Met-Ile linker showed 2-3-fold reduced renal retention and improved tumor-to-kidney ratios compared with their reference without the Met-Ile linker. Future studies should demonstrate whether [68Ga]Ga-NOTA-MI-exendin-4 results in improved detection of small insulinomas in close proximity to the kidneys with PET/CT. [177Lu]Lu-DOTA-MI-exendin-4 might open a window of opportunity for exendin-based radionuclide therapy.

Multivalent γ-PGA-Exendin-4 Conjugates to Target Pancreatic ß-Cells.

Targeting of glucagon-like peptide 1 receptor (GLP-1R), expressed on the surface of pancreatic ß-cells, is of great interest for the development of advanced therapies for diabetes and diagnostics for insulinoma. We report the conjugation of exendin-4 (Ex-4), an approved drug to treat type 2 diabetes, to poly-γ-glutamic acid (γ-PGA) to obtain more stable and effective GLP-1R ligands. Exendin-4 modified at Lysine-27 with PEG4-maleimide was conjugated to γ-PGA functionalized with furan, in different molar ratios, exploiting a chemoselective Diels-Alder cycloaddition. The γ-PGA presenting the highest number of conjugated Ex-4 molecules (average 120 per polymeric chain) showed a double affinity towards GLP-1R with respect to exendin per se, paving the way to improved therapeutic and diagnostic applications.

Combined Probe Strategy to Increase the Enzymatic Digestion Rate and Accelerate the Renal Radioactivity Clearance of Peptide Radiotracers.

High and sustained renal radioactivity accumulation is a major challenge in peptide-based radionuclide imaging and therapy. However, neutral endopeptidase (NEP)-based enzymatic hydrolysis to release and excrete the radioactive fragments has been proven to be an effective and promising way to reduce renal accumulation. Despite the improvement, the effect is still far from being satisfactory. To further reduce kidney uptake, we studied the relationship between the enzymatic reaction rate and the substrate concentration and came up with a combined probe strategy. Model compounds Boc-MVK-Dde and Boc-MFK-Dde were used for an in vitro enzymatic digestion study. NOTA-Exendin 4 and NOTA-MVK-Exendin 4 were labeled with 64Cu for in vivo dose-dependent micro-positron emission tomography (PET) studies. Groups 1 and 2 were injected with 0.2 and 0.8 nmol of 64Cu-NOTA-Exendin 4, respectively. Groups 3-6 were injected with 0.2, 0.8, 1.0, and 1.4 nmol of 64Cu-NOTA-MVK-Exendin 4, respectively. Groups 7 and 8 were co-injected with 0.2 nmol of 64Cu-NOTA-MVK-Exendin 4 and NOTA-MVK-PEG5K (1.3 and 2.6 nmol). The radioactivity uptakes were determined and compared within and among the groups. The in vitro cleavage study for both Boc-MVK-Dde and Boc-MFK-Dde indicated that within a certain concentration range, the enzyme digestion rate increased with increasing substrate concentration. The microPET images showed that the renal clearance could be accelerated significantly by increasing the injection dose of 64Cu-NOTA-MVK-Exendin 4, with the kidney uptakes being 60.98, 43.01, and 16.10 % ID/g at 1 h for groups 3, 4 and 5, respectively. Unfortunately, the tumor uptakes were also significantly inhibited as the injected dose of the tracer increased. However, with the co-injection of NOTA-MVK-PEG5K, the renal accumulation was significantly decreased without hampering the tumor uptake. As a result, the tumor-to-kidney ratios were significantly improved, which were 1.93, 3.47, 1.74, and 3.38 times that of group 3 at 1, 4, 24, and 48 h, respectively. The enzymatic reaction rate of NEP is dependent on the concentration of the substrates both in vitro and in vivo. The combined probe strategy developed in this study can dramatically reduce the renal accumulation of a peptide radioligand without affecting the tumor uptake, which shows great potential in peptide-based radiotheranostics.

Development of an 111In-Labeled Glucagon-Like Peptide-1 Receptor-Targeting Exendin-4 Derivative that Exhibits Reduced Renal Uptake.

Insulinomas are neuroendocrine tumors that are mainly found in the pancreas. Surgical resection is currently the first-line treatment for insulinomas; thus, it is vital to preoperatively determine their locations. The marked expression of the glucagon-like peptide-1 receptor (GLP-1R) is seen in pancreatic ß-cells and almost all insulinomas. Radiolabeled derivatives of exendin-4, a GLP-1R agonist, have been used with nuclear medicine imaging techniques for the in vivo detection of the GLP-1R; however, their marked renal accumulation can hinder the imaging of pancreatic tail lesions. To develop a GLP-1R imaging probe that exhibits reduced renal accumulation, we designed and synthesized a straight-chain GLP-1R-targeting radioligand, [111In]In-E4DA1, which consisted of exendin-4, DOTADG (a chelator), and an (iodophenyl)butyric acid derivative (an albumin binder [ALB]). We performed preclinical evaluations of [111In]In-E4DA1 to investigate its utility as a GLP-1R imaging probe. [111In]In-E4DA1 and [111In]In-E4D (a control compound lacking the ALB moiety) were prepared by reacting the corresponding precursors with [111In]InCl3 in buffer. Cell-binding and human serum albumin (HSA)-binding assays were performed to assess the in vitro affinity of the molecules for INS-1 (GLP-1R-positive) cells and albumin, respectively. A biodistribution assay and single-photon emission computed tomography imaging were carried out using INS-1 tumor-bearing mice. In the cell-binding assay, [111In]In-E4DA1 and [111In]In-E4D exhibited in vitro binding to INS-1 cells. In the HSA-binding assay, [111In]In-E4DA1 bound to HSA, while [111In]In-E4D showed little HSA binding. The in vivo experiments involving INS-1 tumor-bearing mice revealed that the introduction of an ALB moiety into the DOTADG-based exendin-4 derivative markedly increased the molecule's tumor accumulation while decreasing its renal accumulation. In addition, [111In]In-E4DA1 exhibited greater tumor accumulation than renal accumulation, whereas previously reported radiolabeled exendin-4 derivatives demonstrated much higher accumulation in the kidneys than in tumors. These results indicate that [111In]In-E4DA1 may be a useful GLP-1R imaging probe, as it demonstrates only slight renal accumulation.

Development and Validation of an Analytical HPLC Method to Assess Chemical and Radiochemical Purity of [68Ga]Ga-NODAGA-Exendin-4 Produced by a Fully Automated Method.

Background: Glucagon-like peptide 1 receptor (GLP-1R) is preferentially expressed in pancreatic islets, especially in ß-cells, and highly expressed in human insulinomas and gastrinomas. In recent years several GLP-1R-avid radioligands have been developed to image insulin-secreting tumors or to provide a tentative quantitative in vivo biomarker of pancreatic ß-cell mass. Exendin-4, a 39-amino acid peptide with high binding affinity to GLP-1R, has been labeled with Ga-68 for imaging with positron emission tomography (PET). Preparation conditions may influence the quality and in vivo behavior of tracers. Starting from a published synthesis and quality controls (QCs) procedure, we have developed and validated a new rapid and simple UV-Radio-HPLC method to test the chemical and radiochemical purity of [68Ga]Ga-NODAGA-exendin-4, to be used in the clinical routine. Methods: Ga-68 was obtained from a 68Ge/68Ga Generator (GalliaPharma®) and purified using a cationic-exchange cartridge on an automated synthesis module (Scintomics GRP®). NODAGA-exendin-4 contained in the reactor (10 µg) was reconstituted with HEPES and ascorbic acid. The reaction mixture was incubated at 100 °C. The product was purified through HLB cartridge, diluted, and sterilized. To validate the proposed UV-Radio-HPLC method, a stepwise approach was used, as defined in the guidance document released by the International Conference on Harmonization of Technical Requirements of Pharmaceuticals for Human Use (ICH), adopted by the European Medicines Agency (CMP/ICH/381/95 2014). The assessed parameters are specificity, linearity, precision (repeatability), accuracy, and limit of quantification. Therefore, a range of concentrations of Ga-NODAGA-exendin-4, NODAGA-exendin-4 (5, 4, 3.125, 1.25, 1, and 0.75 µg/mL) and [68Ga]Ga-NODAGA-exendin-4 were analyzed. To validate the entire production process, three consecutive batches of [68Ga]Ga-NODAGA-exendin-4 were tested. Results: Excellent linearity was found between 5-0.75 µg/mL for both the analytes (NODAGA-exendin-4 and 68Ga-NODAGA-exendin-4), with a correlation coefficient (R2) for calibration curves equal to 0.999, average coefficients of variation (CV%) < 2% (0.45% and 0.39%) and average per cent deviation value of bias from 100%, of 0.06% and 0.04%, respectively. The calibration curve for the determination of [68Ga]Ga-NODAGA-exendin-4 was linear with a R2 of 0.993 and CV% < 2% (1.97%), in accordance to acceptance criteria. The intra-day and inter-day precision of our method was statistically confirmed using 10 µg of peptide. The mean radiochemical yield was 45 ± 2.4% in all the three validation batches of [68Ga]Ga-NODAGA-exendin-4. The radiochemical purity of [68Ga]Ga-NODAGA-exendin-4 was >95% (97.05%, 95.75% and 96.15%) in all the three batches. Conclusions: The developed UV-Radio-HPLC method to assess the radiochemical and chemical purity of [68Ga]Ga-NODAGA-exendin-4 is rapid, accurate and reproducible like its fully automated production. It allows the routine use of this PET tracer as a diagnostic tool for PET imaging of GLP-1R expression in vivo, ensuring patient safety.

A Conserved Hydrophobic Moiety and Helix-Helix Interactions Drive the Self-Assembly of the Incretin Analog Exendin-4.

Exendin-4 is a pharmaceutical peptide used in the control of insulin secretion. Structural information on exendin-4 and related peptides especially on the level of quaternary structure is scarce. We present the first published association equilibria of exendin-4 directly measured by static and dynamic light scattering. We show that exendin-4 oligomerization is pH dependent and that these oligomers are of low compactness. We relate our experimental results to a structural hypothesis to describe molecular details of exendin-4 oligomers. Discussion of the validity of this hypothesis is based on NMR, circular dichroism and fluorescence spectroscopy, and light scattering data on exendin-4 and a set of exendin-4 derived peptides. The essential forces driving oligomerization of exendin-4 are helix-helix interactions and interactions of a conserved hydrophobic moiety. Our structural hypothesis suggests that key interactions of exendin-4 monomers in the experimentally supported trimer take place between a defined helical segment and a hydrophobic triangle constituted by the Phe22 residues of the three monomeric subunits. Our data rationalize that Val19 might function as an anchor in the N-terminus of the interacting helix-region and that Trp25 is partially shielded in the oligomer by C-terminal amino acids of the same monomer. Our structural hypothesis suggests that the Trp25 residues do not interact with each other, but with C-terminal Pro residues of their own monomers.

NMR Spectroscopy for Protein Higher Order Structure Similarity Assessment in Formulated Drug Products.

Peptide and protein drug molecules fold into higher order structures (HOS) in formulation and these folded structures are often critical for drug efficacy and safety. Generic or biosimilar drug products (DPs) need to show similar HOS to the reference product. The solution NMR spectroscopy is a non-invasive, chemically and structurally specific analytical method that is ideal for characterizing protein therapeutics in formulation. However, only limited NMR studies have been performed directly on marketed DPs and questions remain on how to quantitively define similarity. Here, NMR spectra were collected on marketed peptide and protein DPs, including calcitonin-salmon, liraglutide, teriparatide, exenatide, insulin glargine and rituximab. The 1D 1H spectral pattern readily revealed protein HOS heterogeneity, exchange and oligomerization in the different formulations. Principal component analysis (PCA) applied to two rituximab DPs showed consistent results with the previously demonstrated similarity metrics of Mahalanobis distance (DM) of 3.3. The 2D 1H-13C HSQC spectral comparison of insulin glargine DPs provided similarity metrics for chemical shift difference (Δδ) and methyl peak profile, i.e., 4 ppb for 1H, 15 ppb for 13C and 98% peaks with equivalent peak height. Finally, 2D 1H-15N sofast HMQC was demonstrated as a sensitive method for comparison of small protein HOS. The application of NMR procedures and chemometric analysis on therapeutic proteins offer quantitative similarity assessments of DPs with practically achievable similarity metrics.

Prolonged activity of exenatide: Detailed comparison of Site-specific linear polyglycerol- and poly(ethylene glycol)-conjugates.

Exenatide is a small therapeutic peptide being currently used in clinic for the treatment of diabetes mellitus type II, however, displaying a short blood circulation time which makes two daily injections necessary. Covalent polymer modification of a protein is a well-known approach to overcome this limitation, resulting in steric shielding, an increased size and therefore a longer circulation half-life. In this study, we employed site-selective C-terminal polymer ligation of exenatide via copper-catalyzed azide-alkyne-cycloaddition (CuAAC) to yield 1:1-conjugates of either poly(ethylene glycol) (PEG) or linear polyglycerol (LPG) of different molecular weights. Our goal was to compare the impact of the two polymers on size, structure and activity of exenatide on the in vitro and in vivo level. Both polymers did not alter the secondary structure of exenatide and expectedly increased its hydrodynamic size, where the LPG-versions of exenatide showed slightly smaller values than their PEG-analogs of same molecular weight. Upon conjugation, GLP-1 receptor activation was diminished, however, still enabled maximum receptor response at slightly higher concentrations. Exenatide modified with a 40 kDa LPG (Ex-40-LPG) showed significant reduction of the blood glucose level in diabetic mice for up to 72 h, which was comparable to its PEG-analog, but 9-fold longer than native exenatide (8 h).

Optoacoustic Imaging of Glucagon-like Peptide-1 Receptor with a Near-Infrared Exendin-4 Analog.

Limitations in current imaging tools have long challenged the imaging of small pancreatic islets in animal models. Here, we report the first development and in vivo validation testing of a broad-spectrum and high-absorbance near-infrared optoacoustic contrast agent, E4x12-Cy7. Our near-infrared tracer is based on the amino acid sequence of exendin-4 and targets the glucagon-like peptide-1 receptor (GLP-1R). Cell assays confirmed that E4x12-Cy7 has a high-binding affinity (dissociation constant, Kd, 4.6 ± 0.8 nM). Using the multispectral optoacoustic tomography, we imaged E4x12-Cy7 and optoacoustically visualized ß-cell insulinoma xenografts in vivo for the first time. In the future, similar optoacoustic tracers that are specific for ß-cells and combines optoacoustic and fluorescence imaging modalities could prove to be important tools for monitoring the pancreas for the progression of diabetes.

Indirectly radioiodinated exendin-4 as an analytical tool for in vivo detection of glucagon-like peptide-1 receptor in a disease setting.

OBJECTIVE: Glucagon-like peptide-1 receptor agonist (GLP-1RA) has been reported to have therapeutic effects on diabetes and various diseases. Precise detection of GLP-1 receptor (GLP-1R) can be useful to diagnose and elucidate the mechanism of such diseases. Here we aimed to develop an imaging probe based on GLP-1RA that has high molar activity and sensitivity for detection of low-level GLP-1R expression in non-pancreatic diseases. METHODS: We selected the agonist exenatide (Ex4) as the parent peptide of a GLP-1R targeting probe and prepared Cys-Ex4 by addition of an N-terminal Cys residue and labeling with the prosthetic agent N-(3-[125I]iodophenyl)maleimide ([125I]IPM) to generate [125I]Ex4ipm. We evaluated the affinity of [125I]Ex4ipm for GLP-1R, as well as cellular binding profiles in insulinoma and prostate cancer cell lines, and in vivo biodistributions in normal and tumor-bearing mice to assess GLP-1R-dependent accumulation of radioactivity in tissues. RESULTS: [125I]Ex4ipm was easily synthesized with high radiochemical yield (73%), radiochemical purity (> 99%), and molar activity (81 GBq/µmol) via a thiol/maleimide reaction. Following administration to mice, [125I]Ex4ipm accumulated to high levels in the pancreas (23.3% ID/g), with radioactivity co-localizing in areas having insulin-positive ß cells. High amounts of radioactivity also accumulated in insulinomas that overexpressed GLP-1R (27.5% ID/g). In contrast, low amounts of [125I]Ex4ipm accumulation, corresponding to low expression levels of GLP-1R, were observed in prostate cancer cells and xenografts used as a model of non-pancreatic applications. CONCLUSION: Our results suggested that [123I]Ex4ipm could be valuable for GLP-1R imaging in diabetes, insulinomas, and various diseases related to GLP-1R.

Beta Cell Imaging-From Pre-Clinical Validation to First in Man Testing.

There are presently no reliable ways to quantify human pancreatic beta cell mass (BCM) in vivo, which prevents an accurate understanding of the progressive beta cell loss in diabetes or following islet transplantation. Furthermore, the lack of beta cell imaging hampers the evaluation of the impact of new drugs aiming to prevent beta cell loss or to restore BCM in diabetes. We presently discuss the potential value of BCM determination as a cornerstone for individualized therapies in diabetes, describe the presently available probes for human BCM evaluation, and discuss our approach for the discovery of novel beta cell biomarkers, based on the determination of specific splice variants present in human beta cells. This has already led to the identification of DPP6 and FXYD2ga as two promising targets for human BCM imaging, and is followed by a discussion of potential safety issues, the role for radiochemistry in the improvement of BCM imaging, and concludes with an overview of the different steps from pre-clinical validation to a first-in-man trial for novel tracers.

Nasal absorption enhancement of protein drugs independent to their chemical properties in the presence of hyaluronic acid modified with tetraglycine-L-octaarginine.

Our previous mouse studies demonstrated that mean bioavailability of exendin-4, which is an injectable glucagon-like peptide-1 (GLP-1) analogue whose molecular weight (Mw) and isoelectric point (pI) are ca. 4.2 kDa and 4.5, respectively, administered nasally with poly(N-vinylacetamide-co-acrylic acid) (PNVA-co-AA) bearing D-octaarginine, which is a typical cell-penetrating peptide, was 20% relative to subcutaneous administration even though it was less than 1% when exendin-4 alone was given nasally. The studies also revealed that the absorption-enhancing ability of D-octaarginine-linked PNVA-co-AA for exendin-4 was statistically equivalent to that of sodium salcaprozate (SNAC), which is an absorption enhancer formulated in tablets of semaglutide approved recently as an orally available GLP-1 analogue. From a perspective of clinical application of our technology, we have separately developed hyaluronic acid modified with L-octaarginine via a tetraglycine spacer which would be degraded in biological conditions. The present study revealed that tetraglycine-L-octaarginine-linked hyaluronic acid enhanced nasal absorption of exendin-4 in mice, as did D-octaarginine-linked PNVA-co-AA. There was no significant difference in absorption-enhancing abilities between the hyaluronic acid derivative and SNAC when octreotide (Mw: ca. 1.0 kDa, pI: 8.3) and lixisenatide (Mw: ca. 4.9 kDa, pI: 9.5) were used as a model protein drug. On the other hand, SNAC did not significantly enhance nasal absorption of somatropin (Mw: ca. 22.1 kDa, pI: 5.3) when compared with absorption enhancer-free conditions. Substitution of SNAC with tetraglycine-L-octaarginine-linked hyaluronic acid resulted in a 5-fold increase in absolute bioavailability of somatropin with statistical significance. It appeared that pI hardly ever influenced absorption-enhancing abilities of both enhancers. Results indicated that our polysaccharide derivative would be a promising absorption enhancer which delivers biologics applied on the nasal mucosa into systemic circulation and was of greater advantage than SNAC for enhancing nasal absorption of protein drugs with a larger Mw.

Recombinant Expression and Stapling of a Novel Long-Acting GLP-1R Peptide Agonist.

Owing to their pleiotropic metabolic benefits, glucagon-like peptide-1 receptor (GLP-1R) agonists have been successfully utilized for treating metabolic diseases, such as type 2 diabetes and obesity. As part of our efforts in developing long-acting peptide therapeutics, we have previously reported a peptide engineering strategy that combines peptide side chain stapling with covalent integration of a serum protein-binding motif in a single step. Herein, we have used this strategy to develop a second generation extendin-4 analog rigidified with a symmetrical staple, which exhibits an excellent in vivo efficacy in an animal model of diabetes and obesity. To simplify the scale-up manufacturing of the lead GLP-1R agonist, a semisynthesis protocol was successfully developed, which involves recombinant expression of the linear peptide followed by attachment of a polyethylene glycol (PEG)-fatty acid staple in a subsequent chemical reaction step.

Targeted Optical Imaging of the Glucagonlike Peptide 1 Receptor Using Exendin-4-IRDye 800CW.

The treatment of choice for insulinomas and focal lesions in congenital hyperinsulinism (CHI) is surgery. However, intraoperative detection can be challenging. This challenge could be overcome with intraoperative fluorescence imaging, which provides real-time lesion detection with a high spatial resolution. Here, a novel method for targeted near-infrared (NIR) fluorescence imaging of glucagonlike peptide 1 receptor (GLP-1R)-positive lesions, using the GLP-1 agonist exendin-4 labeled with IRDye 800CW, was examined in vitro and in vivo. Methods: A competitive binding assay was performed using Chinese hamster lung (CHL) cells transfected with GLP-1R. Tracer biodistribution was determined in BALB/c nude mice bearing subcutaneous CHL-GLP-1R xenografts. In vivo NIR fluorescence imaging of CHL-GLP-1R xenografts was performed. Localization of the tracer in the pancreatic islets of BALB/c nude mice was examined using fluorescence microscopy. Laparoscopic imaging was performed to detect the fluorescent signal of the tracer in the pancreas of mini pigs. Results: Exendin-4-IRDye 800CW binds GLP-1R with a half-maximal inhibitory concentration of 3.96 nM. The tracer accumulates in CHL-GLP-1R xenografts. Subcutaneous CHL-GLP-1R xenografts were visualized using in vivo NIR fluorescence imaging. The tracer accumulates specifically in the pancreatic islets of mice, and a clear fluorescent signal was detected in the pancreas of mini pigs. Conclusion: These data provide the first in vivo evidence of the feasibility of targeted fluorescence imaging of GLP-1R-positive lesions. Intraoperative lesion delineation using exendin-4-IRDye 800CW could benefit open as well as laparoscopic surgical procedures for removal of insulinomas and focal lesions in CHI.

PET-Based Human Dosimetry of 68Ga-NODAGA-Exendin-4, a Tracer for ß-Cell Imaging.

68Ga-NODAGA-exendin-4 is a promising tracer for ß-cell imaging using PET/CT. Possible applications include preoperative visualization of insulinomas and discrimination between focal and diffuse forms of congenital hyperinsulinism. There is also a significant role for this tracer in extending our knowledge on the role of ß-cell mass in the pathophysiology of type 1 and type 2 diabetes by enabling noninvasive quantification of tracer uptake as a measure for ß-cell mass. Calculating radiation doses from this tracer is important to assess its safety for use in patients (including young children) with benign diseases and healthy individuals. Methods: Six patients with hyperinsulinemic hypoglycemia were included. After intravenous injection of 100 MBq of the tracer, 4 successive PET/CT scans were obtained at 30, 60, 120, and 240 min after injection. Tracer activity in the pancreas, kidneys, duodenum, and remainder of the body were determined, and time-integrated activity coefficients for the measured organs were calculated. OLINDA/EXM software, version 1.1, was applied to calculate radiation doses using the reference adult male and female models and to estimate radiation doses to children. Results: The mean total effective dose for adults was very low (0.71 ± 0.07 mSv for a standard injected dose of 100 MBq). The organ with the highest absorbed dose was the kidney (47.3 ± 10.2 mGy/100 MBq). The estimated effective dose was 2.32 ± 0.32 mSv for an injected dose of 20 MBq in newborns. This dose decreased to 0.77 ± 0.11 mSv/20 MBq for 1-y-old children and 0.59 ± 0.05 mSv for an injected dose of 30 MBq in 5-y-old children. Conclusion: Our human PET/CT-based dosimetric calculations show that the effective radiation doses from the novel tracer 68Ga-NODAGA-exendin-4 are very low for adults and children. The doses are lower than reported for other polypeptide tracers such as somatostatin analogs (2.1-2.6 mSv/100 MBq) and are beneficial for application as a research tool, especially when repeated examinations are needed.

Glucagon-like peptide-1 receptor expression after myocardial infarction: Imaging study using 68Ga-NODAGA-exendin-4 positron emission tomography.

BACKGROUND: Activation of glucagon-like peptide-1 receptor (GLP-1R) signaling protects against cardiac dysfunction and remodeling after myocardial infarction (MI). The aim of the study was to evaluate 68Ga-NODAGA-exendin-4 positron emission tomography (PET) for assessment of GLP-1R expression after MI in rats. METHODS AND RESULTS: Rats were studied at 3 days, 1 and 12 weeks after permanent coronary ligation or a sham-operation. Rats were injected with 68Ga-NODAGA-exendin-4 and scanned with PET and contrast-enhanced computed tomography (CT) followed by digital autoradiography and histology of left ventricle tissue sections. 68Ga-NODAGA-exendin-4 PET/CT showed focally increased tracer uptake in the infarcted regions peaking at 3 days and continuing at 1 week after MI. Pre-treatment with an unlabeled exendin-4 peptide significantly reduced 68Ga-NODAGA-exendin-4 uptake. By autoradiography, 68Ga-NODAGA-exendin-4 uptake was 8.6-fold higher in the infarcted region and slightly increased also in the remote, non-infarcted myocardium at 1 week and 12 weeks post-MI compared with sham. Uptake of 68Ga-NODAGA-exendin-4 correlated with the amount of CD68-positive macrophages in the infarcted area and alpha-smooth muscle actin staining in the remote myocardium. CONCLUSIONS: 68Ga-NODAGA-exendin-4 PET detects up-regulation of cardiac GLP-1R expression during healing of MI in rats and may provide information on the activated repair mechanisms after ischemic myocardial injury.

Superior Diagnostic Performance of the GLP-1 Receptor Agonist [Lys40(AhxHYNIC-[99mTc]/EDDA)NH2]-Exendin-4 over Conventional Imaging Modalities for Localization of Insulinoma.

PURPOSE: Insulinomas are the most common functioning neuroendocrine neoplasms of the pancreas, typically diagnosed due to characteristic symptoms. In the vast majority, the treatment is surgical and curative, requiring accurate localization of the tumour; conventional imaging, including somatostatin receptor molecular imaging, is negative in up to 10 % of cases. Recently, labelled glucagon-like peptide receptor (GLP-1R) analogues were introduced as a sensitive diagnostic method for localization of insulinomas. The aim of this study was to assess the diagnostic accuracy of a Tc-99m-labelled GLP-1R agonist [Lys40(AhxHYNIC-[99mTc]EDDA)NH2]-exendin-4 for localization of occult insulinoma. PROCEDURES: Eight patients (all females; age range 35-75 years) with biochemically proven insulinoma and with negative or inconclusive conventional imaging (consisting of somatostatin receptor scintigraphy, computed tomography, endoscopic ultrasound and magnetic resonance imaging) were enrolled. Whole-body single-photon emission tomography/computed tomography (SPECT/CT) imaging was performed 4 h post-injection of 740 MBq of [Lys40(AhxHYNIC-[99mTc]EDDA)NH2]-exendin-4. Surgical treatment was performed based on imaging findings. Histology of the removed lesions and biochemical and clinical symptom resolution was considered as the gold standard for analysis of the imaging results. RESULTS: Focal uptake of [Lys40(AhxHYNIC-[99mTc]EDDA)NH2]-exendin-4 was found in all patients, leading to successful removal of the offending lesion and complete biochemical and symptomatic resolution. Histological analysis confirmed insulinoma in all included patients. CONCLUSIONS: [Lys40(AhxHYNIC-[99mTc]EDDA)NH2]-exendin-4 SPECT/CT appears to be an excellent molecular imaging method for preoperative localization of an occult insulinoma, surpassing conventional imaging methods. If routinely available, it could be considered as a method of choice due to its favorable combination of imaging characteristics.

Preparation of [177Lu]Lu-DOTA-Ahx-Lys40-Exendin-4 for radiotherapy of insulinoma: a detailed insight into the radiochemical intricacies.

INTRODUCTION: [177Lu]Lu-DOTA-Ahx-Lys40-Exendin-4 ([177Lu]Lu-DOTA-Exendin-4) is a potential agent for radiotherapy of insulinomas owing to its specificity towards GLP-1 (Glucagon like peptide-1) receptors over-expressed on such cancers. The objective of the present study is to optimize the various radiochemistry parameters for the consistent formulation of the agent with high radiolabeling yield using carrier added [177Lu]LuCl3 and also to evaluate its biological behaviour in small animal model. METHODS: In order to optimize the radiolabeling parameters, DOTA-Exendin-4 was radiolabeled with [177Lu]LuCl3 in two different buffer systems (sodium acetate and HEPES) at three different temperatures (45, 65 and 95 °C) using three different ligand to metal ratios (3:1, 4:1 and 5:1). The radiolabeled peptide was characterized by both paper chromatography and HPLC. The effect of addition of three different radio-protectors on complexation yield was also studied. Bio-distribution studies were carried out in healthy Swiss mice to evaluate the pharmacokinetic behaviour of the radiolabeled peptide as well as to determine the in vivo specificity of the radiotracer towards GLP-1 receptors (blocking studies). Urine and kidney lysate of the animals were analyzed at various post-administration time-points in order to determine the in vivo stability of the radiolabeled peptide. RESULTS: The [177Lu]Lu-DOTA-Exendin-4 complex could be prepared consistently with >95% radiolabeling yield using the optimized reaction conditions. Bio-distribution studies revealed early accumulation of [177Lu]Lu-DOTA-Exendin-4 in pancreas along with fast clearance via renal pathway. Significantly high accumulation of the radiotracer was observed in kidneys. Analyses of urine and kidney lysate of the animals revealed in vivo stability of [177Lu]Lu-DOTA-Exendin-4. Blocking studies showed displacement of significant amount of radiotracer from GLP-1 receptor-positive organs such as, pancreas and lungs (p <0.05) in presence of unlabeled peptide, indicating the specificity of the radiolabeled preparation towards GLP-1 receptors. CONCLUSIONS: Present study shows that [177Lu]Lu-DOTA-Exendin-4 could be formulated for radiotherapeutic application with high radiochemical purity and adequate in vivo stability using [177Lu]LuCl3 produced via direct neutron irradiation. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: Findings of the present study will be helpful in preparing the patient dose of [177Lu]Lu-labeled Exendin for radiotherapy of insulinoma using carrier added [177Lu]LuCl3, produced in a medium flux reactor, without the requirement of post-labeling purification.

Mono-PEGylates of exenatide in branched and dimeric structures can improve in vivo stability and hypoglycemic bioactivity.

Exenatide, a synthetic version of exendin-4, is a glucagon-like peptide-1 receptor agonist (GLP-1RA) used for treating diabetes, but its relatively short half-life is a major disadvantage. In this study, we attempted residue-specific mono-PEGylation to the middle of the amino acid backbone to extend its in vivo half-life. Exenatide was point-mutated from Lys to Cys at the 12th residue to yield a variant (K12C), and PEG-maleimide of varying molecular weights (MW) (5, 10, 20, 40 kD) was site-specifically conjugated to yield a mono-PEGylate with branched T-shape molecular structure. In another approach, we conjugated a bis-maleimide PEG (10 kD) to the middle of two K12Cs to yield an H-shape homodimer PEGylate In vitro bioactivity assays indicated that: (1) PEGylates conjugated with higher MW PEG lead to stronger receptor binding, (2) the branched form was superior to the linear configuration in the binding, and (3) both T-shape and H-shape mono-PEGylates demonstrated better potency than the native exenatide, evidenced by lower EC50. Db/db mouse experiments to evaluate in vivo hypoglycemic activity indicated that: (1) all mono-PEGylates resulted in improved glucose tolerance compared to the native exenatide, (2) the homodimer PEGylate demonstrated much stronger hypoglycemic activity, especially during the initial period, and (3) the H-shape and T-shape mono-PEGylates (40 kD) maintained hypoglycemia for up to ca. 168 and 140 h, representing approximately 12- and 14-fold increase, respectively, compared with the native exenatide. Our findings suggest that the exenatide mono-PEGylates in unclassical molecular structures can improve in vivo pharmacokinetics properties.