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The longitudinal alterations of the pancreatic β-cell and islet mass in the progression of type 1 diabetes (T1D) are still poorly understood. The objective of this study was to repeatedly assess the endocrine volume and the morphology of the pancreas for up to 24 months after T1D diagnosis (n = 16), by 11C-5-hydroxytryptophan (11C-5-HTP) positron emission tomography (PET) and MRI. Study participants were examined four times by PET/MRI: at recruitment and then after 6, 12, and 24 months. Clinical examinations and assessment of β-cell function by a mixed-meal tolerance test and fasting blood samples were performed in connection with the imaging examination. Pancreas volume has a tendency to decrease from 50.2 ± 10.3 mL at T1D debut to 42.2 ± 14.6 mL after 24 months (P < 0.098). Pancreas uptake of 11C-5-HTP (e.g., the volume of the endocrine pancreas) did not decrease from T1D diagnosis (0.23 ± 0.10 % of injected dose) to 24-month follow-up, 0.21 ± 0.14% of injected dose, and exhibited low interindividual changes. Pancreas perfusion was unchanged from diagnosis to 24-month follow-up. The pancreas uptake of 11C-5-HTP correlated with the long-term metabolic control as estimated by HbA1c (P < 0.05). Our findings argue against a major destruction of β-cell or islet mass in the 2-year period after diagnosis of T1D. [ABSTRACT FROM AUTHOR]

GPR44 expression has recently been described as highly β-cell selective in the human pancreas and constitutes a tentative surrogate imaging biomarker in diabetes. A radiolabeled small-molecule GPR44 antagonist, [11C]AZ12204657, was evaluated for visualization of β-cells in pigs and nonhuman primates by positron emission tomography as well as in immunodeficient mice transplanted with human islets under the kidney capsule. In vitro autoradiography of human and animal pancreatic sections from subjects without and with diabetes, in combination with insulin staining, was performed to assess β-cell selectivity of the radiotracer. Proof of principle of in vivo targeting of human islets by [11C]AZ12204657 was shown in the immunodeficient mouse transplantation model. Furthermore, [11C]AZ12204657 bound by a GPR44-mediated mechanism in pancreatic sections from humans and pigs without diabetes, but not those with diabetes. In vivo [11C]AZ12204657 bound specifically to GPR44 in pancreas and spleen and could be competed away dose-dependently in nondiabetic pigs and nonhuman primates. [11C]AZ12204657 is a first-in-class surrogate imaging biomarker for pancreatic β-cells by targeting the protein GPR44. [ABSTRACT FROM AUTHOR]

Aim: The zinc transporter 8 (ZnT8) has been suggested as a suitable target for non-invasive visualization of the functional pancreatic beta cell mass, due to both its pancreatic beta cell restricted expression and tight involvement in insulin secretion.Methods: In order to examine the potential of ZnT8 as a surrogate target for beta cell mass, we performed mRNA transcription analysis in pancreatic compartments. A novel ZnT8 targeting antibody fragment Ab31 was radiolabeled with iodine-125, and evaluated by in vitro autoradiography in insulinoma and pancreas as well as by in vivo biodistribution. The evaluation was performed in a direct comparison with radio-iodinated Exendin-4.Results: Transcription of the ZnT8 mRNA was higher in islets of Langerhans compared to exocrine tissue. Ab31 targeted ZnT8 in the cytosol and on the plasma membrane with 108 nM affinity. Ab31 was successfully radiolabeled with iodine-125 with high yield and > 95% purity. [125I]Ab31 binding to insulinoma and pancreas was higher than for [125I]Exendin-4, but could only by partially competed away by 200 nM Ab31 in excess. The in vivo uptake of [125I]Ab31 was higher than [125I]Exendin-4 in most tissues, mainly due to slower clearance from blood. Conclusions: We report a first-in-class ZnT8 imaging ligand for pancreatic imaging. Development with respect to ligand miniaturization and radionuclide selection is required for further progress. Transcription analysis indicates ZnT8 as a suitable target for visualization of the human endocrine pancreas. [ABSTRACT FROM AUTHOR]

Affibodymolecules, small (7 kDa) scaffold proteins, are a promising class of probes for radionuclidemolecular imaging. Radiolabeling of Affibody molecules with the positron-emitting nuclide 68Ga would permit the use of positron emission tomography (PET), providing better resolution, sensitivity, and quantification accuracy than single-photon emission computed tomography (SPECT). The synthetic anti-HER2 ZHER2:S1 Affibodymolecule was conjugated with DOTA at the N-terminus, in themiddle of helix 3, or at the C-terminus. The biodistribution of 68Ga- and 111In-labeled Affibody molecules was directly compared in NMRI nu/nu mice bearing SKOV3 xenografts. The position of the chelator strongly influenced the biodistribution of the tracers, and the influence was more pronounced for 68Ga-labeled Affibody molecules than for the 68In-labeled counterparts. The best 68Ga-labeled variant was 68Ga-[DOTA-A1]-ZHER2:S1, which provided a tumor uptake of 13 ± 1 %ID/g and a tumor to blood ratio of 39 ± 12 at 2 hours after injection. 111In-[DOTA-A1]-ZHER2:S1 and 111In-[DOTA-K58]-ZHER2:S1 were equally good at this time point, providing a tumor uptake of 15 to 16 %ID/g and a tumor to blood ratio in the range of 60 to 80. In conclusion, the selection of the best position for a chelator in Affibody molecules can be used for optimization of their imaging properties. This may be important for the development of Affibody-based and other protein-based imaging probes. [ABSTRACT FROM AUTHOR]

In humans, a well-developed serotonin system is localized to the pancreatic islets while being absent in exocrine pancreas. Assessment of pancreatic serotonin biosynthesis could therefore be used to estimate the human endocrine pancreas. Proof of concept was tested in a prospective clinical trial by comparisons of type 1 diabetic (T1D) patients, with extensive reduction of β-cells, with healthy volunteers (HVs). C-peptide-negative (i.e., insulin-deficient) T1D subjects (n = 10) and HVs (n = 9) underwent dynamic positron emission tomography with the radiolabeled serotonin precursor [11C]5-hydroxy-tryptophan ([11C]5-HTP). A significant accumulation of [11C]5-HTP was obtained in the pancreas of the HVs, with large interindividual variation. A substantial and highly significant reduction (66%) in the pancreatic uptake of [11C]5-HTP in T1D subjects was observed, and this was most evident in the corpus and caudal regions of the pancreas where β-cells normally are the major constituent of the islets. [11C]5-HTP retention in the pancreas was reduced in T1D compared with nondiabetic subjects. Accumulation of [11C]5-HTP in the pancreas of both HVs and subjects with T1D was in agreement with previously reported morphological observations on the β-cell volume, implying that [11C]5-HTP retention is a useful noninvasive surrogate marker for the human endocrine pancreas. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Insulinoma is the most common form of pancreatic endocrine tumors responsible for hyperinsulinism in adults. These tumors overexpress glucagon like peptide-1 (GLP-1) receptor, and biologically stable GLP-1 analogs have therefore been proposed as potential imaging agents. Here, we evaluate the potential of a positron emission tomography (PET) tracer, [68Ga]Ga-DO3A-VS-Cys40-Exendin-4, for imaging and quantification of GLP-1 receptors (GLP-1R) in insulinoma. Methods: [68Ga]Ga-DO3A-VS-Cys40-Exendin-4 was evaluated for binding to GLP-1R by in vitro autoradiography binding studies in INS-1 tumor from xenografts. In vivo biodistribution was investigated in healthy control mice, INS-1 xenografted and PANC1 xenografted immunodeficient mice at two different doses of peptide: 2.5μg/kg (baseline) and 100μg/kg (block). In vivo imaging of [68Ga]Ga-DO3A-VS-Cys40-Exendin-4 in xenografted mice was evaluated by small animal PET/CT using a direct comparison with the clinically established insulinoma marker [11C]5-hydroxy-tryptophan ([11C]5-HTP). Results: GLP-1 receptor density could be quantified in INS-1 tumor biopsies. [68Ga]Ga-DO3A-VS-Cys40-Exendin-4 showed significant uptake (p≤0.05) in GLP1-R positive tissues such as INS-1 tumor, lungs and pancreas upon comparison between baseline and blocking studies. In vivo imaging showed concordant results with higher tumor-to-muscle ratio in INS-1 xenografted mice compared with [11C]5-HTP. Conclusion: [68Ga]Ga-DO3A-VS-Cys40-Exendin-4 has high affinity and specificity for GLP-1R expressed on insulinoma in vitro and in vivo. [Copyright &y& Elsevier]

Purpose: Human epidermal growth factor receptor type 3 (HER3) is a transmembrane receptor tyrosine kinase belonging to the HER (ErbB) receptor family. Membranous expression of HER3 is associated with trastuzumab resistance in breast cancer and the transition to androgen independence in prostate cancer. Imaging of HER3 expression in malignant tumors may provide important diagnostic information that can influence patient management. Affibody molecules with low picomolar affinity to HER3 were recently selected. The aim of this study was to investigate the feasibility of HER3 imaging using radiolabeled Affibody molecules. Methods: A HER3-binding Affibody molecule, Z, with a HEHEHE-tag on N-terminus was labeled with Tc(CO) using an IsoLink kit. In vitro and in vivo binding specificity and the cellular processing of the labeled binder were evaluated. Biodistribution of Tc(CO)-HEHEHE-Z was studied over time in mice bearing HER3-expressing xenografts. Results: HEHEHE-Z was labeled with Tc(CO) with an isolated yield of >80 % and a purity of >99 %. Binding of Tc(CO)-HEHEHE-Z was specific to BT474 and MCF7 (breast cancer), and LS174T (colon cancer) cells. Cellular processing showed rapid binding and relatively quick internalization of the receptor/Affibody molecule complex (70 % of cell-associated radioactivity was internalized after 24 h). The tumor targeting was receptor mediated and the excretion was predominantly renal. Receptor-mediated uptake was also found in the liver, lung, stomach, intestine, and salivary glands. At 4 h pi, tumor-to-blood ratios were 7 ± 3 for BT474, and 6 ± 2 for LS174T xenografts. LS174T tumors were visualized by microSPECT 4 h pi. Conclusions: The results of this study suggest the feasibility of HER3-imaging in malignant tumors using Affibody molecules. [ABSTRACT FROM AUTHOR]

Affibody molecules are a class of small (7 kDa) non-immunoglobulin scaffold-based affinity proteins, which have demonstrated substantial potential as probes for radionuclide molecular imaging. The use of positron emission tomography (PET) would further increase the resolution and quantification accuracy of Affibody-based imaging. The rapid in vivo kinetics of Affibody molecules permit the use of the generator-produced radionuclide 68Ga (T1/2 = 67.6 min). Earlier studies have demonstrated that the chemical nature of chelators has a substantial influence on the biodistribution properties of Affibody molecules. To determine an optimal labeling approach, the macrocyclic chelators 1,4,7,10-tetraazacylododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-N,N,N-triacetic acid (NOTA) and 1-(1,3-carboxypropyl)-1,4,7- triazacyclononane-4,7-diacetic acid (NODAGA) were conjugated to the N-terminus of the synthetic Affibody molecule ZHER2:S1 targeting HER2. Affibody molecules were labeled with 68Ga, and their binding specificity and cellular processing were evaluated. The biodistribution of 68Ga-DOTA-ZHER2:S1,68Ga-NOTA-ZHER2:S1 and 68Ga-NODAGA-ZHER2:S1, as well as that of their 111In-labeled counterparts, was evaluated in BALB/C nu/nu mice bearing HER2-expressing SKOV3 xenografts. The tumor uptake for 68Ga-DOTA-ZHER2:S1 (17.9±0.7%IA/g) was significantly higher than for both 68Ga-NODAGA-ZHER2:S1(16.13±0.67%IA/g) and 68Ga-NOTA-ZHER2:S1 (13±3%IA/g) at 2 h after injection. 68Ga-NODAGA-ZHER2:S1 had the highest tumor-to-blood ratio (60±10) in comparison with both 68Ga-DOTA-ZHER2:S1 (28±4) and 68Ga-NOTA-ZHER2:S1 (42±11). The tumor-to-liver ratio was also higher for 68Ga-NODAGA-ZHER2:S1 (7±2) than the DOTA and NOTA conjugates (5.5±0.6 vs.3.3±0.6). The influence of chelator on the biodistribution and targeting properties was less pronounced for 68Ga than for 111In. The results of this study demonstrate that macrocyclic chelators conjugated to the N-terminus have a substantial influence on the biodistribution of HER2-targeting Affibody molecules labeled with 68Ga.This can be utilized to enhance the imaging contrast of PET imaging using Affibody molecules and improve the sensitivity of molecular imaging. The study demonstrated an appreciable difference of chelator influence for 68Ga and 111In. [ABSTRACT FROM AUTHOR]

Type 1 diabetes (T1D) is characterized by the loss of insulin-producing cells and hence insulin secretion and metabolic control. In addition to insulin, there are a number of hormones and cytokines that influence metabolism, and many of these can be secreted from brown adipose tissue (BAT). However, the presence and activity of BAT in T1D have not been studied, despite the fact that preclinical studies have shown that transplantation of BAT in mouse models of T1D can restore metabolic control. The metabolic activity of BAT, white adipose tissue (WAT), and skeletal muscle was investigated in patients with T1D (n = 11) by 2-deoxy-2-(18F)fluoro-D-glucose PET/CT after cold stimulation. Functional BAT was detected in 4 out of 11 individuals with T1D with a prevalence of 36%. The glucose utilization rate in the supraclavicular BAT regions ranged from 0.75–38.7 µmol × min−1 × 100 g−1. The glucose utilization per gram tissue was higher in BAT when compared with both WAT (p = 0.049) and skeletal muscle (p = 0.039). However, no correlation between BAT activity and metabolic control or insulin requirements was found. In conclusion, for the first time, cold-induced BAT was detected in patients with T1D with a wide range in metabolic activity. Contrary to findings in animal models, the metabolic activity of BAT had negligible impact on insulin requirements or metabolic control in T1D under normal physiological conditions. [ABSTRACT FROM AUTHOR]

Background: The G-protein-coupled receptor 44 (GPR44) is a beta cell-restricted target that may serve as a marker for beta cell mass (BCM) given the development of a suitable PET ligand.Methods: The binding characteristics of the selected candidate, AZ12204657, at human GPR44 were determined using in vitro ligand binding assays. AZ12204657 was radiolabeled using 11C- or 3H-labeled methyl iodide ([11C/3H]CH3I) in one step, and the conversion of [11C/3H]CH3I to the radiolabeled product [11C/3H]AZ12204657 was quantitative. The specificity of radioligand binding to GPR44 and the selectivity for beta cells were evaluated by in vitro binding studies on pancreatic sections from human and non-human primates as well as on homogenates from endocrine and exocrine pancreatic compartments.Results: The radiochemical purity of the resulting radioligand [11C]AZ12204657 was > 98%, with high molar activity (MA), 1351 ± 575 GBq/μmol (n = 18). The radiochemical purity of [3H]AZ12204657 was > 99% with MA of 2 GBq/μmol. Pancreatic binding of [11C/3H]AZ12204657 was co-localized with insulin-positive islets of Langerhans in non-diabetic individuals and individuals with type 2 diabetes (T2D). The binding of [11C]AZ12204657 to GPR44 was > 10 times higher in islet homogenates compared to exocrine homogenates. In human islets of Langerhans GPR44 was co-expressed with insulin, but not glucagon as assessed by co-staining and confocal microscopy.Conclusion: We radiolabeled [11C]AZ12204657, a potential PET radioligand for the beta cell-restricted protein GPR44. In vitro evaluation demonstrated that [3H]AZ12204657 and [11C]AZ12204657 selectively target pancreatic beta cells. [11C]AZ12204657 has promising properties as a marker for human BCM. [ABSTRACT FROM AUTHOR]

The longitudinal alterations of the pancreatic β-cell and islet mass in the progression of type 1 diabetes (T1D) are still poorly understood. The objective of this study was to repeatedly assess the endocrine volume and the morphology of the pancreas for up to 24 months after T1D diagnosis ( n = 16), by 11 C-5-hydroxytryptophan ( 11 C-5-HTP) positron emission tomography (PET) and MRI. Study participants were examined four times by PET/MRI: at recruitment and then after 6, 12, and 24 months. Clinical examinations and assessment of β-cell function by a mixed-meal tolerance test and fasting blood samples were performed in connection with the imaging examination. Pancreas volume has a tendency to decrease from 50.2 ± 10.3 mL at T1D debut to 42.2 ± 14.6 mL after 24 months ( P < 0.098). Pancreas uptake of 11 C-5-HTP (e.g., the volume of the endocrine pancreas) did not decrease from T1D diagnosis (0.23 ± 0.10 % of injected dose) to 24-month follow-up, 0.21 ± 0.14% of injected dose, and exhibited low interindividual changes. Pancreas perfusion was unchanged from diagnosis to 24-month follow-up. The pancreas uptake of 11 C-5-HTP correlated with the long-term metabolic control as estimated by HbA 1c ( P < 0.05). Our findings argue against a major destruction of β-cell or islet mass in the 2-year period after diagnosis of T1D., (© 2021 by the American Diabetes Association.)

Quantitative PET imaging with [(68)Ga]Ga-DO3A-VS-Cys(40)-Exendin-4 has potential use in diabetes and cancer. However, the radiation dose to the kidneys has been a concern for the possibility of repeated imaging studies in humans. Therefore, we investigated the dosimetry of [(68)Ga]Ga-DO3A-VS-Cys(40)-Exendin-4 based on the biodistribution data in rats, pigs, non-human primates (NHP) and a human.Organ distribution of [(68)Ga]Ga-DO3A-VS-Cys(40)-Exendin-4 in rats (Male Lewis; n=12; 30, 60, and 80 min) was measured ex vivo. The dynamic uptake of [(68)Ga]Ga-DO3A-VS-Cys(40)-Exendin-4 in the abdomen was assessed by PET/CT scanning of pigs (male; n = 4, 0-60 min), NHP (Female; cynomolgus; n=3; 0-90 min), and human (female; n=1; 0-40, 100, 120 min).The organ distribution data in each species were extrapolated to those of a human, assuming similar distribution between the species. Residence times were assessed by trapezoidal approximation of the kinetic data. Organ doses (mGy/MBq) and the whole body effective dose (mSv/MBq), was extrapolated by using the OLINDA/EXM 1.1 software. The extrapolated human whole body effective dose was 0.017 ± 0.004 (rats), 0.014 ± 0.004 (pigs), 0.017 ± 0.004 (NHP), and 0.016 (human) mSv/MBq. The absorbed dose to the kidneys was limiting:0.33 ± 0.06 (rats), 0.28±0.05 (pigs), 0.65 ± 0.11 (NHP), and 0.28 (human) mGy/MBq, which corresponded to the maximum yearly administered amounts of 455 (rat), 536 (pig), 231 (NHP), and 536 (human) MBq before reaching the yearly kidney limiting dose of 150 mGy. More than 200 MBq of [(68)Ga]Ga-DO3A-VS-Cys(40)-Exendin-4 can be administered yearly in a human, allowing for repeated (2-4 times) scanning. This potentially enables longitudinal clinical PET imaging studies of the GLP-1R in the pancreas, transplanted islets, or insulinoma.

Affibody molecules are a class of affinity agents for molecular imaging based on a non-immunoglobulin protein scaffold. Previous studies have demonstrated high contrast for in vivo imaging of cancer-associated molecular abnormalities using Affibody molecules. Using the radionuclide (18)F for labeling and PET as the imaging modality, the sensitivity of molecular imaging using Affibody molecules can be further increased. The use of oxime formation between an aminooxy-functionalized peptide and (18)F-fluorobenzaldehyde ((18)F-FBA) is a promising way of radiolabeling of targeting peptides. However, previous studies demonstrated that application of this method to Affibody molecules is associated with high liver uptake. We hypothesized that incorporation of a triglutamyl spacer between the aminooxy moiety and the N-terminus of a synthetic Affibody molecule would decrease the hepatic uptake of the (18)F-N-(4-fluorobenzylidine)oxime) ((18)F-FBO)-labeled tracer. To verify this, we have produced two variants of the HER2-targeting ZHER2:342 Affibody molecule by peptide synthesis: OA-PEP4313, where aminooxyacetic acid was conjugated directly to the N-terminal alanine, and OA-E3-PEP4313, where a triglutamyl spacer was introduced between the aminooxy moiety and the N-terminus. We have found that the use of the spacer is associated with a minor decrease of affinity, from KD = 49 pM to KD = 180 pM. Radiolabeled (18)F-FBO-E3-PEP4313 demonstrated specific binding to HER2-expressing ovarian carcinoma SKOV-3 cells and slow internalization. Biodistribution studies in mice demonstrated that the use of a triglutamyl linker decreased uptake of radioactivity in liver 2.7-fold at 2 h after injection. Interestingly, radioactivity uptake in kidneys was also reduced (2.4-fold). Experiments in BALB/C nu/nu mice bearing SKOV-3 xenografts demonstrated HER2-specific uptake of (18)F-FBO-E3-PEP4313 in tumors. At 2 h pi, the tumor uptake (20 ± 2% ID/g) exceeded uptake in liver 5-fold and uptake in kidneys 3.6-fold. The tumor-to-blood ratio was 21 ± 3. The microPET/CT imaging experiment confirmed the biodistribution data. In conclusion, the use of a triglutamyl spacer is a convenient way to improve the biodistribution profile of Affibody molecules labeled at the N-terminus using (18)F-FBA. It provides a tracer capable of producing high-contrast images of HER2-expressing tumors.

Affibody molecules, small (7 kDa) scaffold proteins, are a promising class of probes for radionuclide molecular imaging. Radiolabeling of Affibody molecules with the positron-emitting nuclide 68Ga would permit the use of positron emission tomography (PET), providing better resolution, sensitivity, and quantification accuracy than single-photon emission computed tomography (SPECT). The synthetic anti-HER2 ZHER2:S1 Affibody molecule was conjugated with DOTA at the N-terminus, in the middle of helix 3, or at the C-terminus. The biodistribution of 68Ga- and 111In-labeled Affibody molecules was directly compared in NMRI nu/nu mice bearing SKOV3 xenografts. The position of the chelator strongly influenced the biodistribution of the tracers, and the influence was more pronounced for 68Ga-labeled Affibody molecules than for the 111In-labeled counterparts. The best 68Ga-labeled variant was 68Ga-[DOTA-A1]-ZHER2:S1, which provided a tumor uptake of 13 ± 1 %ID/g and a tumor to blood ratio of 39 ± 12 at 2 hours after injection. 111In-[DOTA-A1]-ZHER2:S1 and 111In-[DOTA-K58]-ZHER2:S1 were equally good at this time point, providing a tumor uptake of 15 to 16 %ID/g and a tumor to blood ratio in the range of 60 to 80. In conclusion, the selection of the best position for a chelator in Affibody molecules can be used for optimization of their imaging properties. This may be important for the development of Affibody-based and other protein-based imaging probes.

Accurate detection of cancer-associated molecular abnormalities in tumors could make cancer treatment more personalized. Affibody molecules enable high contrast imaging of tumor-associated protein expression shortly after injection. The use of the generator-produced positron-emitting radionuclide (68)Ga should increase sensitivity of HER2 imaging. The chemical nature of radionuclides and chelators influences the biodistribution of Affibody molecules, providing an opportunity to further increase the imaging contrast. The aim of the study was to compare maleimido derivatives of DOTA and NODAGA for site-specific labeling of a recombinant ZHER2:2395 HER2-binding Affibody molecule with (68)Ga. DOTA and NODAGA were site-specifically conjugated to the ZHER2:2395 Affibody molecule having a C-terminal cysteine and labeled with (68)Ga and (111)In. All labeled conjugates retained specificity to HER2 in vitro. Most of the cell-associated activity was membrane-bound with a minor difference in internalization rate. All variants demonstrated specific targeting of xenografts and a high tumor uptake. The xenografts were clearly visualized using all conjugates. The influence of chelator on the biodistribution and targeting properties was much less pronounced for (68)Ga than for (111)In. The tumor uptake of (68)Ga-NODAGA-ZHER2:2395 and (68)Ga-DOTA-ZHER2:2395 and tumor-to-blood ratios at 2 h p.i. did not differ significantly. However, the tumor-to-liver ratio was significantly higher for (68)Ga-NODAGA- ZHER2:2395 (8 ± 2 vs 5.0 ± 0.3) offering the advantage of better liver metastases visualization. In conclusion, influence of chelators on biodistribution of Affibody molecules depends on the radionuclides and reoptimization of labeling chemistry is required when a radionuclide label is changed.