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The purpose of this study was to evaluate the binding specificity of the radiolabeled glucagon-like peptide 1 receptor (GLP-1R) agonist (Lys40(DOTA)NH2)Exendin-4 in the pancreas using a combination of ex vivo autoradiography and immunohistochemistry. Sprague–Dawley rats were administered [64Cu](Lys40(DOTA)NH2)Exendin-4 i.v. with or without unlabeled Exendin (9-39) to determine binding specificity. Similar experiments were performed using Zucker diabetic fatty (ZDF) and Zucker lean (ZLC) rats. Animals were euthanized and the pancreas was extracted, immediately frozen, and sectioned. The sections were apposed to phosphor imaging plates, scanned, and immunostained for insulin. Co-registration of the autoradiographic and immunohistochemical images revealed that [64Cu] (Lys40(DOTA)NH2)Exendin-4 specific binding was restricted to islet cells. This binding was blocked by the co-administration of Exendin(9-39) indicating that the radiotracer uptake is mediated by GLP-1R. Uptake of [64Cu](Lys40(DOTA)NH2)Exendin-4 was greatly decreased in the pancreas of ZDF rats. Ex vivo autoradiography results using [64Cu](Lys40(DOTA)NH2)Exendin-4 suggest that GLP-1R agonists based on Exendin-4 are attractive PET ligands for the in vivo determination of β-cell mass.

The in vitro and in vivo evaluation of the selective, high affinity (human CB1 IC(50) 0.49 nM) inverse agonist CB1R tracer [(11)C]CB-119, a close analog of the previously disclosed [(18)F]MK-9470, was undertaken.[(11)C]CB-119 was synthesized with high specific activity by alkylation of a phenolic precursor with [(11)C]methyl iodide. In vitro autoradiographic studies using rhesus brain slices were carried out using [(3)H]CB-119, and in vivo imaging studies were carried out using [(11)C]CB-119 in rhesus monkeys under baseline and blocked conditions.Autoradiographic studies in rhesus brain showed the expected distribution pattern for CB1R with highest binding in the cerebral cortex, cerebellum, caudate/putamen, globus pallidus, substantia nigra, and hippocampus. Lower binding was seen in the posterior hypothalamus, ventral tegmental area, and periventricular gray area, and the lowest binding was in the thalamic nuclei. The binding of [(3)H]CB-119 was fully blocked by the addition of 10 microM CB-119. Rhesus positron emission tomography imaging studies showed very good brain uptake and a distribution pattern consistent with that seen in the autoradiographic studies. The kinetics of tracer uptake was slow. The brain uptake was blocked by pretreatment with taranabant, a CB1R inverse agonist. The specific signal (total/nonspecific) in rhesus putamen at 90 min was approximately 6:1.[(11)C]CB-119 is a suitable tracer for imaging central CB1 receptors.

Use of mGluR5 receptor radiotracers to determine whether an in vitro binding assay is able to predict how good a radiotracer is likely to be in imaging receptor in the central nervous system (CNS) via positron emission tomography (PET).Saturation and equilibrium competition studies in rat and rhesus membranes were used to determine receptor concentrations and tracer affinities. In addition, specific binding of metabotropic receptor subtype 5 (mGluR5) radioligands in rhesus and rat brain sections was determined using a "no-wash protocol," and the in vivo binding signal in rats was determined using micro-PET.Affinity values were determined for a series of mGluR5 antagonists (1-5) and ranged from 0.1 to 11 nM in rat. A previously reported "no-wash protocol" was then employed to determine specific binding in tissue sections following a 20-min incubation, and the regional distribution of these mGluR5 radiotracers determined in rat brain via autoradiography. The analogs 1b, 2b, 3b, and 4b, but not 5b, displayed good signal-to-noise ratios under these conditions with high density of binding in caudate, cortex, and hippocampus and lower density in cerebellum. With this information it was predicted that 1c, 2c, 3b, and 4b would display measurable signal-to-noise ratios in vivo, and that the larger in vitro signals for 3b and 4b would translate to 3b and 4b yielding the best in vivo signals. These predictions were investigated using micro-PET imaging in rat. Compound 1c showed a rapid wash-in and rapid wash-out profile in rat brain. Compound 2c showed similar signal-to-noise ratio as 1b, but slower washout. Compounds 3b and 4b showed the best signal-to-noise ratio in vivo, while 5b did not provide a significant signal, as predicted. In vivo occupancy estimates for 2-methyl-6-(phenylethynyl)-pyridine (MPEP) following intravenous administration were determined using radiolabeled compounds 1c, 2c, and 3b; they were essentially the same and were on the order of 1 mg kg(-1) (ID(50)).An in vitro screen of several mGluR5 tracers was used to rapidly predict whether radiolabeled mGluR5 analogs would be useful as PET radiotracers. Results provided an extension to previously reported data. Two of the four radiotracers with the best in vitro "no-wash" results also showed the best potential as measured noninvasively using micro-PET.

Integrins, especially α(v)β(3) and α(v)β(5), are upregulated in tumor cells and activated endothelial cells and as such, serve as cancer biomarkers. We developed a novel near-infrared-labeled optical agent for the in vivo detection and quantification of α(v)β(3)/α(v)β(5).A small peptidomimetic α(v)β(3) antagonist was synthesized, coupled to a near-infrared fluorescent (NIRF) dye, and tested for binding specificity using integrin-overexpressing cells, inhibition of vitronectin-mediated cell attachment, binding to tumor and endothelial cells in vitro, and competition studies. Pharmacokinetics, biodistribution, specificity of tumor targeting, and the effect of an antiangiogenic treatment were assessed in vivo.The integrin NIRF agent showed strong selectivity towards α(v)β(3/)α(v)β(5) in vitro and predominant tumor distribution in vivo, allowing noninvasive and real-time quantification of integrin signal in tumors. Antiangiogenic treatment significantly inhibited integrin signal in vivo but had no effect on a cathepsin-cleavable NIR agent. Simultaneous imaging revealed different patterns of distribution reflecting the underlying differences in integrin and cathepsin biology during tumor progression.NIRF-labeled integrin antagonists allow noninvasive molecular fluorescent imaging and quantification of tumors in vivo, improving and providing more refined approaches for cancer detection and treatment monitoring.

This study was conducted to develop a new positron emission tomography (PET) method to visualize neurokinin-1 (NK(1)) receptor systems in the human brain in vivo in order to examine their neuroanatomical distribution and facilitate investigations of the role of substance P, NK(1) receptors, and NK(1) receptor antagonists in central nervous system (CNS) function and dysfunction.PET studies were conducted in 10 healthy male volunteers using a novel selective, high-affinity NK(1) receptor antagonist labeled with fluorine-18 to very high specific radioactivity (up to 2000 GBq/micromol) [F-18]SPA-RQ. Data were collected in 3D mode for greatest sensitivity. Different modeling methods were compared and regional receptor distributions determined for comparison with in vitro autoradiographic studies using postmortem human brain slices with [F-18]SPA-RQ.The studies showed that the highest uptake of [F-18]SPA-RQ was observed in the caudate and putamen. Lower binding was found in globus pallidus and substantia nigra. [F-18]SPA-RQ uptake was also widespread throughout the neocortex and limbic cortex including amygdala and hippocampus. There was very low specific uptake of the tracer in the cerebellar cortex. The distribution pattern was confirmed using in vitro receptor autoradiography with [F-18]SPA-RQ on postmortem human brain slices. Kinetic modeling of the [F-18]SPA-RQ uptake data indicated a binding potential between 4 and 5 in the basal ganglia and between 1.5 and 2.5 in the cortical regions.[F-18]SPA-RQ is a novel tool for exploration of the functions of NK(1) receptors in man. [F-18]SPA-RQ can be used to define receptor pharmacodynamics and focus dose selection of novel NK(1) receptor antagonists in clinical trials thereby ensuring adequate proof of concept testing particularly in therapeutic applications related to CNS dysfunction.

To develop and characterize a radiolabelled Substance-P antagonist useful for quantitation of neurokinin-1 receptors in the brain via PET imaging.[18F]SPA-RQ (Substance-P antagonist - receptor quantifier) was synthesized in good yield and high specific activity by alkylation of a BOC protected phenolate anion using [18F]bromofluoromethane. Removal of the BOC protecting group with trifluoroacetic acid gave [18F]SPA-RQ.SPA-RQ has high affinity for human, rhesus monkey and guinea pig NK1 receptors (h-IC50=67 pM) and has a log P value of 1.8. Biodistribution studies in guinea pig showed that this tracer penetrates the blood-brain barrier and selectively labels NK1 receptors in the striatum and cortex.[18F]SPA-RQ is a potent, high affinity Substance-P antagonist that can be conveniently labeled with high specific activity using [18F]fluoromethylbromide. This tracer is a useful tool for noninvasive imaging of central NK1 receptors.