64Cu- and 68Ga-labelled [Nle(14),Lys(40)(Ahx-NODAGA)NH2]-exendin-4 for pancreatic beta cell imaging in rats.

PURPOSE: Glucagon-like peptide-1 receptor (GLP-1R) is a molecular target for imaging of pancreatic beta cells. We compared the ability of [Nle(14),Lys(40)(Ahx-NODAGA-(64)Cu)NH2]-exendin-4 ([(64)Cu]NODAGA-exendin-4) and [Nle(14),Lys(40)(Ahx-NODAGA-(68)Ga)NH2]-exendin-4 ([(68)Ga]NODAGA-exendin-4) to detect native pancreatic islets in rodents. PROCEDURES: The stability, lipophilicity and affinity of the radiotracers to the GLP-1R were determined in vitro. The biodistribution of the tracers was assessed using autoradiography, ex vivo biodistribution and PET imaging. Estimates for human radiation dosimetry were calculated. RESULTS: We found GLP-1R-specific labelling of pancreatic islets. However, the pancreas could not be visualised in PET images. The highest uptake of the tracers was observed in the kidneys. Effective dose estimates for [(64)Cu]NODAGA-exendin-4 and [(68)Ga]NODAGA-exendin-4 were 0.144 and 0.012 mSv/MBq, respectively. CONCLUSION: [(64)Cu]NODAGA-exendin-4 might be more effective for labelling islets than [(68)Ga]NODAGA-exendin-4. This is probably due to the lower specific radioactivity of [(68)Ga]NODAGA-exendin-4 compared to [(64)Cu]NODAGA-exendin-4. The radiation dose in the kidneys may limit the use of [(64)Cu]NODAGA-exendin-4 as a clinical tracer.

Synthesis and preclinical characterization of [64Cu]NODAGA-MAL-exendin-4 with a Nε-maleoyl-L-lysyl-glycine linkage.

INTRODUCTION: Renal localization of high radioactivity levels during targeted imaging compromises tissue visualization in the kidney region and limits diagnostic accuracy. Radioiodinated antibody fragments with a renal enzyme-cleavable N(ε)-maleoyl-L-lysyl-glycine (MAL) linkage demonstrated low renal radioactivity levels in mice, from early postinjection times. This study tested the hypothesis whether a (64)Cu-labeled NODAGA-exendin-4 peptide with a MAL linkage ([(64)Cu]NODAGA-MAL-exendin-4) could decrease kidney radioactivity levels in rats, compared to a [(64)Cu]NODAGA-exendin-4 reference, without impairing the radioactivity levels in the target tissue. METHODS: NODAGA-MAL-exendin-4 was synthesized in a two-phase approach using solid support to prepare maleoyl-derivatized NODAGA followed by Michael addition to cysteine-derivatized exendin-4 in solution. Radiolabeling was performed in buffered aqua with [(64)Cu]CuCl2, which was produced via the (64)Ni(p,n)(64)Cu nuclear reaction. The in vitro and in vivo stability, lipophilicity, and distribution kinetics in major rat organs for [(64)Cu]NODAGA-MAL-exendin-4 were studied and compared to [(64)Cu]NODAGA-exendin-4. Labeling of pancreatic islets was assessed using autoradiography. RESULTS: NODAGA-MAL-exendin-4 was synthesized, with an overall yield of 9%, and radiolabeled with (64)Cu with high specific radioactivity. Serum incubation studies showed high stability for [(64)Cu]NODAGA-MAL-exendin-4. Similar tissue distribution kinetics was observed for [(64)Cu]NODAGA-MAL-exendin-4 and [(64)Cu]NODAGA-exendin-4, with high kidney radioactivity levels. CONCLUSIONS: The incorporated MAL linkage in [(64)Cu]NODAGA-MAL-exendin-4 was unable to reduce kidney radioactivity levels, compared to [(64)Cu]NODAGA-exendin-4. The applicability of metabolizable linkages in the design of kidney-saving exendin-4 analogs requires further investigation.

Isomerism of [64Cu-NOTA-Bn]-labeled radiotracers: separation of two complex isomers and determination of their interconversion energy barrier using ion pair chromatography.

The model complex [(64)Cu((S)-p-NH(2)-Bn-NOTA)](-) ([(64)Cu]1) was used to study the isomerism of [(64)Cu-NOTA-Bn]-labeled radiotracers. Two complex isomers [(64)Cu]1a and [(64)Cu]1b, which were formed at a ratio of 1:9 during the complexation of [(64)Cu]Cu(2+) with (S)-p-NH(2)-Bn-NOTA, were separated using ion pair chromatography. To study the interconversion, the nonradioactive complex isomers Cu1a and Cu1b were separated and thermally treated at 90 °C in both ammonium acetate solution and deionized water. A faster interconversion rate was observed for both isomers with lower concentrations of ammonium ions. At the end of reaction, the thermodynamic Cu1a to Cu1b equilibrium ratio was 6:94. The particular energy barriers of the interconversion for Cu1a and Cu1b were 130 kJ mol(-1) and 140 kJ mol(-1). Spectrophotometric measurements with Cu1a and Cu1b revealed two isomers adopting different geometrical configurations.

α2-adrenoceptor regulation of blood glucose homeostasis.

The α(2A)-adrenoceptor has been identified as an important regulator of blood glucose homeostasis. α(2A)-Adrenoceptors on pancreatic ß-cells inhibit insulin secretion, and α(2A)-adrenoceptors on sympathetic nerves and on adrenomedullary chromaffin cells limit sympathoadrenal output. Recently, human α(2A)-adrenoceptor gene polymorphisms that influence α(2A)-adrenoceptor expression and function have been described. Increased α(2A)-adrenoceptor expression has been associated with impaired glucose-stimulated insulin secretion, elevated fasting blood glucose levels and an increased risk of type 2 diabetes. Accordingly, administration of α(2)-adrenoceptor agonists generally increases blood glucose levels, in spite of the ensuing sympatholysis that would be expected to lower blood glucose as a result of diminished α(1)- and ß-adrenoceptor activation. α(2)-Adrenoceptor antagonists increase insulin secretion and reduce blood glucose levels by inhibiting tonically active α(2A)-adrenoceptors on pancreatic ß-cells, but may also enhance sympathoadrenal output. In addition, α(2)-adrenoceptor antagonists potentiate the insulinotropic effect of sulphonylurea drugs, pointing to a potentially serious adverse drug interaction when the two classes of drugs are combined. The α(2)-adrenoceptor antagonist atipamezole is widely used in veterinary medicine, and sulphonylureas are prescribed for the treatment of type 2 diabetes in cats and dogs. Even if no dedicated α(2)-adrenoceptor antagonists are in clinical use in humans, some antipsychotic and antidepressant drugs are relatively potent α(2)-adrenoceptor antagonists. In the treatment of type 2 diabetes, α(2)-adrenoceptor agonists could possibly protect against sulphonylurea-induced hypoglycaemia, and α(2)-adrenoceptor antagonist drugs could improve insulin secretion. The potential usefulness of such drugs may vary between individuals, depending on α(2A)-adrenoceptor genetics, sympathetic tone and concomitant pathological conditions, such as cardiovascular disease and obesity.

Assessment of islet specificity of dihydrotetrabenazine radiotracer binding in rat pancreas and human pancreas.

UNLABELLED: Vesicular monoamine transporter 2 (VMAT2) is a putative molecular target for the quantitative imaging of pancreatic beta-cell mass by PET. The VMAT2 PET tracer (11)C-dihydrotetrabenazine ((11)C-DTBZ) exhibits high pancreatic uptake that is reduced in type 1 diabetes. The aim of this study was to assess the islet and VMAT2 specificity of DTBZ binding in the pancreas. METHODS: The biodistribution of (11)C-DTBZ in rats was determined 10 and 60 min after injection. The localization of DTBZ radioactivity in rat and human pancreatic tissue sections was investigated by autoradiography. Saturation and competition binding assays were performed with (3)H-DTBZ and sections of rat pancreatic and control tissues. The binding of (11)C-DTBZ in pancreatic sections from rats with streptozotocin-induced diabetes was compared with that in control rats. RESULTS: The values for the pancreatic uptake of (11)C-DTBZ (percentage injected dose per gram of tissue) were 3.0 at 10 min and 2.7 at 60 min. At 10 min, pancreatic radioactivity was heterogeneously distributed, with higher levels toward the head of the pancreas (head-to-tail ratio, 1.7). No such gradient was observed in pancreatic sections incubated with (11)C-DTBZ and (3)H-DTBZ in vitro. In rats, (11)C-DTBZ and (3)H-DTBZ binding in pancreatic islets did not exceed binding in the exocrine pancreas. Saturable (3)H-DTBZ binding was observed in the rat brain striatum (dissociation constant [K(d)], 1.3 nM) and the bovine adrenal medulla (K(d), 3.3 nM), whereas in the rat pancreas, (3)H-DTBZ binding was nonsaturable. Competition binding with (3)H-DTBZ and VMAT2 antagonists also indicated that DTBZ binding in the rat pancreas was nonspecific and did not represent binding to VMAT2. Nonspecific pancreatic (11)C-DTBZ binding was lower in rats with streptozotocin-induced diabetes than in control rats. In sections of human pancreas, a subset of pancreatic islets were weakly but VMAT2-specifically labeled with (3)H-DTBZ. CONCLUSION: The results showed that the pancreatic uptake of (11)C-DTBZ is mainly due to nonspecific binding in the exocrine pancreas and suggested that the reduction in pancreatic (11)C-DTBZ binding observed in type 1 diabetes is not specific for the loss of beta-cell mass.

Comparison of 2beta-carbomethoxy-3beta-(4-[18F]fluorophenyl)tropane and N-(3-[18F]fluoropropyl)-2beta-carbomethoxy-3beta-(4-fluorophenyl)nortropane, tracers for imaging dopamine transporter in rat.

PURPOSE: This study compares 2beta-carbomethoxy-3beta-(4-[(18)F]fluorophenyl)tropane ([(18)F]beta-CFT) and N-(3-[(18)F]fluoropropyl)-2beta-carbomethoxy-3beta-(4-fluorophenyl)nortropane ([(18)F]beta-CFT-FP) as radiotracers for imaging the dopamine transporter (DAT) in rat. PROCEDURES: Biodistribution, specificity and selectivity of the radiotracers were studied ex vivo in rats pre-treated with specific antagonists for DAT, serotonin transporter (SERT) and noradrenalin transporter (NET) and in control rats. Positron emission tomography (PET) studies were performed using an HRRT scanner. Radiolabelled metabolites were analyzed with thin-layer chromatography. RESULTS: [(18)F]beta-CFT showed slow kinetics with a maximum striatum/cerebellum uptake ratio of 9.2 at 120 min. [(18)F]beta-CFT-FP showed fast kinetics with a maximum ratio of 3.1 at 5 min. Both tracers bound to DAT. [(18)F]beta-CFT also bound to NET. [(18)F]beta-CFT was more resistant to metabolism than [(18)F]beta-CFT-FP. CONCLUSIONS: Structural modifications of [(18)F]beta-CFT significantly changed its biological properties, as shown by [(18)F]beta-CFT-FP. [(18)F]beta-CFT is a suitable tracer for both preclinical and human PET studies, but [(18)F]beta-CFT-FP is less suitable as a PET tracer.

Autoradiographic characterization of alpha(2C)-adrenoceptors in the human striatum.

Indirect experimental evidence suggests that drugs acting on the alpha(2C)-adrenoceptor could be useful in the treatment of neuropsychiatric disorders such as depression and schizophrenia. In rodent brain, the highest levels of alpha(2C)-adrenoceptors are found in the striatum, with lower levels in cerebral cortex and hippocampus. In human brain, because of the poor subtype-selectivity of the available alpha(2)-adrenoceptor ligands, the localization of alpha(2C)-adrenoceptors has remained unknown. Recently, a selective alpha(2C)-adrenoceptor antagonist, JP-1302, was characterized, and to assess the presence of alpha(2C)-adrenoceptors in human brain, we performed competition binding in vitro receptor autoradiography with JP-1302 and the alpha(2)-adrenoceptor subtype nonselective antagonist [ethyl-(3)H]RS79948-197 on rat and human postmortem brain sections. In striatum of both species, JP-1302 vs. [ethyl-(3)H]RS79948-197 competition binding was biphasic, identifying high- and low-affinity binding sites, whereas in cortex and cerebellum, only low-affinity binding sites were detected. The results indicate that a significant portion of the alpha(2)-adrenoceptors in striatum is of the alpha(2C) subtype, whereas non-alpha(2C)-adreocneptors predominate in cortex and cerebellum. Because the alpha(2C)-adrenoceptor subtype distribution pattern appears to be conserved between rodents and humans, results obtained from studies on the role of the alpha(2C)-adrenoceptor in rodent models of neuropsychiatric disorders may be relevant also for human diseases.

Ex vivo evaluation of N-(3-[18F]fluoropropyl)-2 beta-carbomethoxy-3 beta-(4-fluorophenyl)nortropane in rats.

INTRODUCTION: The dopamine transporter (DAT) ligand N-(3-fluoropropyl)-2 beta-carbomethoxy-3beta-(4-fluorophenyl)nortropane (beta-CFT-FP) was labeled with fluorine-18, and its biodistribution was evaluated in rats ex vivo. METHODS: The distribution of 18F radioactivity in the brain and peripheral organs and tissues was determined at several time points 5-120 min after intravenous injection of [18F]beta-CFT-FP. RESULTS: The highest brain uptake of [18F]beta-CFT-FP was localized in the striatum; limbic structures also exhibited high uptake. Low uptake was found in the cerebellum. The highest ratio of striatum-to-cerebellum uptake, already reached within 5 min, was 3.1. Pretreatment with the selective DAT inhibitor GBR12909 significantly decreased [18F]beta-CFT-FP uptake in the striatum. In most peripheral tissues, the highest uptake was found at 5 min, indicating fast washout of the radioligand. Some accumulation of (18)F radioactivity was seen in bone as a function of time, reflecting defluorination of the radioligand. CONCLUSION: The results indicate that [18F]beta-CFT-FP is a potential radioligand for studying DAT in vivo with positron emission tomography.

Reduced blood glucose levels, increased insulin levels and improved glucose tolerance in alpha2A-adrenoceptor knockout mice.

Alpha(2)-Adrenoceptors regulate insulin secretion and sympathetic output. In the present study, alpha(2A)-adrenoceptor knockout (alpha(2A)-KO) mice and their C57BL/6J wild-type (WT) controls were used to assess the glucoregulatory role of the alpha(2A)-adrenoceptor subtype in vivo. Fasting and glucose-stimulated blood glucose and plasma insulin levels were determined with or without (+/-)-propranolol (5 mg/kg) or atropine (10 mg/kg) pre-treatment. Intraperitoneal glucose (1 g/kg) and insulin (0.5 and 1.0 IU/kg) tolerance tests were performed. Fasting plasma glucagon and corticosterone levels were measured. Blood glucose levels (mean+/-S.E.M.) were lower in alpha(2A)-KO males (7.2+/-0.6 mM) and females (7.2+/-0.2 mM) than in WT males (9.8+/-0.3 mM) and females (9.1+/-0.3 mM). Plasma insulin levels were higher in alpha(2A)-KO males (2.2+/-0.5 microg/l) and females (1.7+/-0.3 microg/l) than in WT males (0.7+/-0.1 microg/l) and females (0.8+/-0.2 microg/l). These differences remained after pharmacological beta-adrenoceptor and muscarinic acetylcholine receptor inhibition. In spite of a tendency for slightly decreased insulin sensitivity in alpha(2A)-KO mice, glucose tolerance in alpha(2A)-KO mice was significantly better than in WT mice. However, glucose-stimulated insulin secretion was not increased in alpha(2A)-KO mice compared to WT controls. Plasma glucagon levels, but not corticosterone levels, were elevated in alpha(2A)-KO mice. These results suggest that lack of inhibitory pancreatic beta-cell alpha(2A)-adrenoceptor function results in hyperinsulinaemia, reduced blood glucose levels and improved glucose tolerance in alpha(2A)-KO mice, and demonstrate a key role for the alpha(2A)-adrenoceptor in adrenergic regulation of blood glucose and insulin homeostasis.

Altered glucose homeostasis in alpha2A-adrenoceptor knockout mice.

To elucidate the functions of alpha2-adrenoceptor subtypes in metabolic regulation, we determined plasma glucose and insulin levels and tissue uptake of the glucose analogue 2-[18F]fluoro-2-deoxy-d-glucose ([18F]FDG) in C57Bl/6J wild-type (WT) and alpha2A-adrenoceptor knockout (alpha2A-KO) mice at baseline and following alpha2-adrenoceptor agonist ((+)-4-(S)-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole (dexmedetomidine)) and antagonist (4-[2-ethyl-2,3-dihydro-1H-inden-2-yl]-1H-imidazole (atipamezole)) administration. Basal glucose levels were 30% lower in alpha2A-KO mice than in WT mice. In WT mice, dexmedetomidine lowered insulin and elevated glucose levels, and atipamezole reduced glucose levels. In alpha2A-KO mice, neither drug affected the glucose or insulin levels. [18F]FDG uptake was investigated in plasma, heart, liver, kidney, pancreas, lung, fat, and skeletal muscle. Cardiac [18F]FDG uptake was a sensitive indicator of sympathetic function. Liver [18F]FDG uptake conformed to the plasma glucose levels. In alpha2A-KO mice, drug effects on [18F]FDG tissue uptake were absent. Thus, the alpha2A-adrenoceptor is the alpha2-adrenoceptor subtype primarily involved in the regulation of blood glucose homeostasis in vivo.

[Ethyl-3H]RS-79948-197 alpha2-adrenoceptor autoradiography validation in alpha2-adrenoceptor knockout mice.

[Ethyl-(3)H][8aR,12aS,13aS]-5,8,8a,9,10,11,12,12a,13,13a-decahydro-3-methoxy-12-(ethylsulfonyl)-6H-isoquino[2,1-g][1,6]naphthyridine ([ethyl-(3)H]RS-79948-197) was evaluated for alpha(2)-adrenoceptor autoradiography in brain sections from wild-type mice and alpha(2A)- and alpha(2ABC)-adrenoceptor knockout mice. Receptor numbers were 83% lower in cortex and 28% lower in caudate putamen of alpha(2A)-knockout mice than in wild-type mice. No specific binding was seen in alpha(2ABC)-knockout mice. [Ethyl-(3)H]RS-79948-197 saturation binding parameters were compared to those of [(3)H]2-(2,3-dihydro-2-methoxy-1,4-benzodioxan-2-yl)-4,5-dihydro-1H-imidazoline ([(3)H]RX821002) and [methyl-(3)H]17alpha-hydroxy-20alpha-yohimban-16beta-carboxylic acid methyl ester ([methyl-(3)H]rauwolscine). [Ethyl-(3)H-]RS-79948-197 detected a larger number of both alpha(2A)- and alpha(2B/C)-adrenoceptors than [(3)H]RX821002, while [methyl-(3)H]rauwolscine only underestimated the number of alpha(2A)-adrenoceptors. Oxymetazoline and prazosin competed for [ethyl-(3)H]RS-79948-197 binding with the expected rank order of affinities. Higher than necessary [ethyl-(3)H]RS-79948-197 concentrations resulted in a rapid increase in non-specific binding. Slow dissociation kinetics, high specific radioactivity and high alpha(2)-adrenoceptor affinity (slightly lower for the alpha(2A)-adrenoceptor than for the other subtypes) confer [ethyl-(3)H]RS-79948-197 distinct advantages compared to [(3)H]RX821002 for detection of alpha(2)-adrenoceptor subtypes in a mixed alpha(2)-adrenoceptor population.