[[(18)F]N-(4'-fluorobenzyl)-4-(3-bromophenyl) acetamide for imaging the sigma receptor status of tumors: comparison with [(18)F]FDG, and [(125)I]IUDR.

A series of biodistribution studies were conducted with the radiotracer, [(18)F]N-(4'-fluorobenzyl)-4-(3-bromophenyl)acetamide, [(18)F]1 in nude mice bearing tumor xenografts of the mouse mammary adenocarcinoma, line 66. This radiotracer has a high affinity for both sigma(1) and sigma(2) receptors. In vivo studies were also conducted in order to assess the effect of blocking sigma(1) receptors on tumor uptake and the tumor:background ratio of this radiotracer. The results of these studies revealed that blocking the sigma(1) receptor so that only the sigma(2) receptors are labeled in vivo, results in a higher tumor:background ratio with only a small reduction in the tumor uptake of the radiotracer relative to the no-carrier-added (i.e., nonselective) conditions. Comparative in vivo studies were also conducted with the anatomic and metabolic imaging agent, [(18)F]FDG, and a radiolabeled DNA precursor, [(125)I]IUdR. Both of these radiolabeled compounds represent classes of agents that have been proposed for imaging the proliferative status of solid tumors. The results of these studies indicated that a sigma(2)-selective imaging agent may be, 1) a better anatomic imaging agent for breast cancer than [(18)F]FDG, and 2) a better functional imaging agent than the radiolabeled DNA precursors, [(123/124)I]IUdR and [(11)C]thymidine, for measuring the proliferative status of breast tumors with PET and SPECT. However, additional studies will be needed to compare sigma(2)-selective imaging agents with [(18)F]FLT in order to determine which is the more appropriate imaging agent for measuring the proliferative status of breast tumors with PET.

Morphine-induced spinal cholinergic activation: in vivo imaging with positron emission tomography.

Positron emission tomography (PET) imaging of spinal cord in monkeys with a cholinergic tracer demonstrates increased spinal cholinergic activity in response to an analgesic dose of morphine, and this PET result correlates with measurement of acetylcholine spillover into spinal cord extracellular space induced by morphine, as measured by microdialysis. Previous studies in rats, mice, and sheep demonstrate activation of spinal cholinergic neurons by systemic opioid administration, and participation of this cholinergic activity in opioid-induced analgesia. Testing the relevance of this observation in humans has been limited to measurement of acetylcholine spillover into lumbar cerebrospinal fluid. The purpose of this study was to apply a recently developed method to image spinal cholinergic terminals non-invasively via PET and to test the hypothesis that the tracer utilized would reflect changes in local cholinergic activity. Following Animal Care and Use Committee approval, seven adult male rhesus monkeys were anesthetized on three separate occasions. On two of the occasions PET scans were performed using [(18)F] (+)-4-fluorobenzyltrozamicol ([(18)F]FBT), which selectively binds to the vesicular acetylcholine (ACh) transporter in the presynaptic cholinergic terminals. PET scans were preceded by injection of either saline or an analgesic dose of IV morphine (10 mg/kg). On the third occasion, microdialysis catheters were inserted in the spinal cord dorsal horn and acetylcholine concentrations in dialysates determined before and after IV morphine injection. Morphine increased cholinergic activity in the spinal cord, as determined by blood flow corrected distribution volume of [(18)F]FBT in the cervical cord compared to the cerebellum. Morphine also increased acetylcholine concentrations in microdialysates from the cervical cord dorsal horn. The one animal which did not show increased spinal cholinergic activity by PET from this dose of morphine also did not show increased acetylcholine from this morphine dose in the microdialysis experiment. These data confirm the ability to use PET to image spinal cholinergic terminals in the monkey spinal cord and suggest that acute changes in cholinergic activity can be imaged with this non-invasive technique. Following preclinical screening, PET scanning with [(18)F]FBT may be useful to investigate mechanisms of analgesic action in normal humans and in those with pain.

Cholinergic activity of aged rhesus monkeys revealed by positron emission tomography.

In the present study, the radiotracer [(18)F] (+)-4-fluorobenzyltrozamicol ((+)-[(18)F]FBT) and positron emission tomography (PET) were used to examine the vesicular acetylcholine transporter and determine if presynaptic cholinergic activity was altered with age in 23 rhesus monkeys that varied in age from 10 to 37 years. Binding of (+)-[(18)F]FBT in the basal ganglia was reduced significantly with increasing age of the monkeys. However, there were individual differences noted in that some middle-aged and aged monkeys demonstrated levels of (+)-[(18)F]FBT binding that were comparable to the binding measured in adult monkeys. These data indicate that presynaptic cholinergic function may decrease with age, but that there may be a differential susceptibility of the cholinergic system to the aging process in different individuals.

Fluorine-18-labeled tropane analogs for PET imaging studies of the dopamine transporter.

A series of PET imaging studies were conducted with two fluorine-18-labeled tropane analoges, [(18)F](+)-FTT and [(18)F](+)-FCT. Both compounds possessed a high affinity and selectivity for the dopamine transporter and had a higher accumulation in the basal ganglia, a brain region having a high density of the dopamine transporter (DAT) than the cerebellum, a reference region devoid of dopaminergic terminals. [(18)F](+)-FCT had a higher brain uptake and more suitable basal ganglia:cerebellum (BG:Cb) ratio than [(18)F](+)-FTT. [(18)F](+)-FCT also displayed reversible binding kinetics in vivo, indicating that the measurement of DAT density in vivo with PET will be relatively insensitive to changes in cerebral blood flow that can occur as a consequence of disease or prolonged cocaine abuse. The uptake of [(18)F](+)-FCT was also displaced by an intravenous injection of cocaine (1.0 mg/kg), which is consistent with the labeling of the DAT in vivo by this radiotracer. These data suggest that [(18)F](+)-FCT may be a suitable radiotracer for studying DAT function in vivo with PET.

Reproducibility of repeated measures of cholinergic terminal density using.

UNLABELLED: [18F](+)-4-fluorobenzyltrozamicol (FBT), which selectively binds to the vesicular acetylcholine transporter in the presynaptic cholinergic neuron, has previously been shown to be a useful ligand for the study of cholinergic terminal density in the basal ganglia with PET. The goal of this study was to assess the test-retest variability of [18F]FBT and PET measurements under baseline conditions in the basal ganglia. METHODS: After approval from the Animal Care and Use Committee, 6 rhesus monkeys underwent a series of 2 [18F]FBT PET scans (time between scans, 32-301 d) under isoflurane anesthesia. Each scan was initiated on the bolus injection of the radiotracer and consisted of 26 frames acquired during 180 min. Arterial blood samples were collected over the course of each scan to determine the metabolite-corrected arterial input function. Tissue time-activity curves were obtained from the scan data by drawing regions of interest over the basal ganglia and cerebellum. The distribution volume ratio for the basal ganglia was then determined for each scan by taking the ratio of the basal ganglia (specific binding) to cerebellum (nonspecific binding) distribution volume. Distribution volumes were derived using the Logan graphic analysis technique as well as a standard 3-compartment model. Additionally, the radioactivity concentration ratio was calculated as the ratio of the average [18F]FBT concentration in the basal ganglia to that in the cerebellum during the last half of the study (85-170 min). The constant K1, determined using the standard 3-compartment model, was used as an index of blood flow changes between studies. RESULTS: For all subjects, the test-retest variability was less than 15% for the distribution volume ratio and 12% for the radioactivity concentration ratio. Good agreement was found between the distribution volume ratio calculated using the graphic technique and the standard 3-compartment model. Using K1 as an index, the variability in blood flow seen in both the basal ganglia and the cerebellum was significantly reduced in their ratio. CONCLUSION: These results show the reproducibility of [18F]FBT and PET measurements in the basal ganglia.

PET imaging of dopamine D2 receptors with [18F]fluoroclebopride in monkeys: effects of isoflurane- and ketamine-induced anesthesia.

The purpose of the present study was to determine whether positron emission tomography (PET) studies in monkeys with the dopamine (DA) D2 receptor ligand [18F]fluoroclebopride (FCP) would be significantly influenced by anesthetic induction with isoflurane (approximately 5.0%) compared to induction with 10 mg/kg ketamine. Five experimentally-naive adult male cynomolgus monkeys (Macaca fascicularis) were trained to sit calmly in a primate restraint chair. Before the first PET scan, each monkey was anesthetized, by mask, with isoflurane. After complete sedation, the monkey was intubated and anesthesia was maintained throughout the PET study by isoflurane (approximately 1.5%). At least 1 month later, a second PET study was conducted in which anesthesia was induced with ketamine and maintained by isoflurane (approximately 1.5%). Irrespective of induction anesthetic, there was a high uptake of [18F]FCP and a linear rate of washout from the basal ganglia for all monkeys. There were also no differences in time to peak uptake (approximately 25 min), in clearance half-life (t1/2 = 140-164 min) or in D2 binding (distribution volume ratios of 2.48 vs. 2.50). These results indicate that induction anesthetic did not differentially affect D2 binding of [18F]FCP in monkeys. Furthermore, the low variability between studies indicates that [18F]FCP is an excellent ligand for longitudinal studies of D2 receptors in nonhuman primates.

(+)-p-([18F]fluorobenzyl)spirotrozamicol [(+)-[18F]spiro-FBT]: synthesis and biological evaluation of a high-affinity ligand for the vesicular acetylcholine transporter (VAChT).

(+)-1'-[4-Hydroxy-1-(4-fluorobenzyl)piperidin-3-yl]spiro[1H- indene-1,4'- piperidine] {(+)-Spiro-FBT}, a high-affinity vesicular acetylcholine transporter ligand, was labeled with fluorine-18, and evaluated in the rat and monkey. In the rat brain, (+)-[18F]Spiro-FBT accumulated preferentially in the striatum, hippocampus, and cortex, brains regions containing high-to-moderate densities of cholinergic terminals. However, due to rapid metabolism, no preferential accumulation of the radiotracer was observed in corresponding regions of the monkey brain. Consequently, rapid metabolism renders (+)-[18F]Spiro-FBT unsuitable for studying cholinergic function with positron emission tomography.

In vivo imaging of the spinal cord cholinergic system with PET.

PURPOSE: Our goal was to demonstrate the feasibility of an in vivo noninvasive method for imaging spinal cord cholinergic terminals using (+)-4-[18F]fluorobenzyltrozamicol ([18F]FBT) and PET. METHOD: In vitro and in vivo experiments in rats were conducted to demonstrate the specific binding characteristics, localization, and time course of [3H]FBT binding in the spinal cord. PET imaging was then performed on seven rhesus monkeys. RESULTS: The rat studies demonstrate high specific binding in the spinal cord with a distribution coinciding with the known distribution of cholinergic terminals. In vivo tracer concentrations in the spinal cord and basal ganglia were of the same magnitude. With use of [18F]FBT and PET in the rhesus monkey, the spinal cord was clearly visualized, with tracer concentration in the spinal cord being approximately one-fourth of that seen in the basal ganglia. CONCLUSION: This work demonstrates the feasibility of imaging cholinergic terminals in vivo in the spinal cord using [18F]FBT and PET.

Use of positron emission tomography to study the dynamics of psychostimulant-induced dopamine release.

Microdialysis studies have shown that psychostimulants act through a common neurochemical mechanism of elevating synaptic dopamine content in the mesocorticolimbic dopaminergic system. However, little information is available regarding the dynamics of the interaction between the elevated synaptic dopamine levels induced by a psychostimulant and postsynaptic dopamine receptors. The goal of the current investigation was to determine if positron emission tomography (PET) studies using the dopamine D2-selective radioligand [18F]4'-fluoroclebopride ([18F]FCP) could be used to measure synaptic dopamine levels. Rhesus monkeys were used because our previous studies revealed that [18F]FCP has a low test/retest variability in this species. Under control conditions, [18F]FCP had a high uptake and slow rate of washout from the basal ganglia, a region of brain that expresses a high density of D2 receptors, reaching kinetic equilibrium at approximately 40 min. Challenge studies, each separated by at least 1 month, were conducted by administering an intravenous dose of (-)cocaine, d-amphetamine, methylphenidate, or d-methamphetamine (1.0 mg/kg) at 40 min post-IV injection of a no-carrier-added dose of [18F]FCP. In each case, the psychostimulant caused an increase in the rate of washout of [18F]FCP from the basal ganglia. Methamphetamine and amphetamine had more pronounced effects on the washout kinetics of [18F]FCP relative to cocaine and methylphenidate, a result that is consistent with the ability of each drug to elevate synaptic dopamine levels. Our results indicate that challenge studies with [18F]FCP may be a useful technique for studying the dynamics of the interaction between psychostimulant-induced increases in synaptic dopamine and postsynaptic D2 receptors.

Imaging of cholinergic terminals using the radiotracer [18F](+)-4-fluorobenzyltrozamicol: in vitro binding studies and positron emission tomography studies in nonhuman primates.

The goal of the present set of studies was to characterize the in vitro binding properties and in vivo tissue kinetics for the vesicular acetylcholine transporter (VAcChT) radiotracer, [18F](+)-4-fluorobenzyltrozamicol ([18F](+)-FBT). In vitro binding studies were conducted in order to determine the affinity of the (+)- and (-)-stereoisomers of FBT for the VAcChT as well as sigma (sigma 1 and sigma 2) receptors. (+)-FBT was found to have a high affinity (Ki = 0.22 nM) for the VAcChT and lower affinities for sigma 1 (21.6 nM) and sigma 2 (35.9 nM) receptors, whereas (-)-FBT had similar affinities for the VAcChT and sigma 1 receptors (approximately 20 nM) and a lower affinity for sigma 2 (110 nM) receptors. PET imaging studies were conducted in rhesus monkeys (n = 3) with [18F](+)-FBT. [18F](+)-FBT was found to have a high accumulation and slow rate of washout from the basal ganglia, which is consistent with the labeling of cholinergic interneurons in this brain region. [18F](+)-FBT also displayed reversible binding kinetics during the 3 h time course of PET and produced radiolabeled metabolites that did not cross the blood-brain barrier. The results from the current in vitro and in vivo studies indicate that [18F](+)-FBT is a promising ligand for studying cholinergic terminal density, with PET, via the VAcChT.

Social stress, depression, and brain dopamine in female cynomolgus monkeys.

Socially subordinate adult female cynomolgus monkeys are hypercortisolemic, the targets of aggression, fearful, vigilant, receive little positive affiliative contact, exhibit pathological behaviors indicating anxiety, and are disengaged in the social events around them. Subordinates also have altered dopaminergic activity that may be due to decreased D2 receptor binding. Dopaminergic activity indices were more closely associated with affiliative than agonistic behaviors.

Comparison of two fluorine-18 labeled benzamide derivatives that bind reversibly to dopamine D2 receptors: in vitro binding studies and positron emission tomography.

The purpose of the present set of studies was to characterize, in vitro and in vivo, two benzamide analogues, 2,3-dimethoxy-N-[1-(4-fluorobenzyl)piperidin4yl]benzamide (MBP) and 4'-fluoroclebopride (FCP), for studying dopamine D2 receptors with Positron Emission Tomography (PET). In vitro binding studies were conducted to determine the affinities of MBP and FCP to the three subtypes of dopamine D2 receptors: D2(long), D3, and D4 receptors. MBP was found to have a high affinity (Ki = 1-8 nM) for all three subtypes of the D2 receptor, whereas FCP had nanomolar affinity (Ki approximately 5.5 nM) for D2(long) and D3 receptors, and a lower affinity for D4 receptors (Ki = 144 nM). In vitro binding studies also revealed that MBP had a relatively high affinity for rho1 receptors (Ki = 11 nM) compared to FCP (Ki = 340 nM). PET imaging studies were conducted in rhesus monkeys with the fluorine-18 labeled analogues of each compound. Both [18F]MBP and [18F]FCP displayed reversible binding kinetics during the 3 h time course of PET. [18F]FCP was found to have a higher basal ganglia:cerebellum ratio and lower variability in the rate of washout from D2 receptors in vivo relative to [18F]MBP. Neither radiotracer was found to produce radiolabeled metabolites capable of crossing the blood-brain barrier. The high rho1 binding affinity and low basal ganglia:cerebellum ratio of [18F]MBP indicate that this ligand may not be suitable for quantitative studies of D2 receptors. The results from the in vitro and in vivo studies indicate that [18F]FCP is a promising ligand for studying D2 receptors with PET.

PET imaging studies of dopamine D2 receptors: comparison of [18F]N-methylspiperone and the benzamide analogues [18F]MABN and [18F]MBP in baboon brain.

A series of positron emission tomography (PET) imaging studies was conducted in a baboon with the benzamide derivatives [18F]2,3-dimethoxy-N-[9-(4-fluorobenzyl)-9-azabicyclo[3.3.1]non an-3 beta-yl]benzamide ([18F]MABN) and [18F]2,3-dimethoxy-N-[1-(4-fluorobenzyl)piperidin-4-yl]be nza mide ([18F]MBP). Studies were also conducted with the butyrophenone [18F]N-methylspiperone (NMSP) for comparison. Tissue-time activity curves of [18F]MABN are similar to those of [18F]NMSP since both compounds displayed approximately the same uptake in the basal ganglia and displayed irreversible binding kinetics in vivo. However, the rapid rate of clearance from the cerebellum and high basal ganglia:cerebellum ratio of [18F]MABN indicate that this compound has a much lower amount of nonspecific binding than [18F]NMSP. [18F]MBP displayed a higher uptake in the basal ganglia relative to [18F]NMSP and [18F]MABN and exhibited reversible binding kinetics in vivo. This property of [18F]MBP is desirable since the uptake of radioactivity in D2-rich ligands is less likely to be influenced by changes in cerebral blood flow. The current data suggest that both [18F]MABN and [18F]MBP are promising ligands for studying dopamine D2 receptors with PET.

Synthesis and in vivo evaluation of [18F]-N-(p-nitrobenzyl)spiperone ([18F]PNBS) in rats.

[18F]-N-(p-nitrobenzyl)spiperone, [18F]PNBS, was prepared and its in vivo uptake in rat brain was measured. A temporal increase in striatum:cerebellum ratio and frontal cortex:cerebellum (FC:Cb) ratio was observed. Blocking studies suggested the formation of a radiolabeled metabolite, presumably [18F]spiperone, that is capable of crossing the blood-brain barrier and labeling cortical 5-HT2 receptors. This was confirmed using the ISRP method of metabolite analysis indicating [18F]PNBS is not a suitable tracer for studying D2 receptors in vivo with PET.

[15O]H2O, [15O]O2 and [15O]CO gas production, monitoring and quality control system.

The 15O gas production system selects the gas production mode, monitors gas delivery conditions and performs quality control needed during the production and delivery of continuous flow radioactive gas for equilibrium and bolus PET studies. Target gas switches choose the appropriate valve positioning for [15O]H2O, [15O]O2 or [15O]CO production while flow rate, pressure and radioactivity are constantly monitored. The system also includes an IBM PC interfaced gas chromatograph which provides chemical and radiochemical quality control by on-line, real time, gas sampling of the delivery line.

Internal-surface reversed-phase chromatography for plasma metabolite analysis of radiopharmaceuticals.

The use of internal-surface reversed-phase (ISRP) chromatography of unprocessed plasma samples was investigated as an alternative method of quantitation of the arterial plasma metabolite time course of [18F]N-methylspiperone. The ISRP method was directly compared to standard solid phase extraction/HPLC (SPE/HPLC) methods currently in wide use. Results indicate that: (1) the ISRP method is rapid and minimizes sample preparation; (2) recovery of radioactivity from the ISRP column is greater than 90%; (3) no radioactivity remains associated with chromatographically size excluded proteins and (4) the quantitative results are well correlated with conventional SPE/HPLC methods.

Optimization of [11C]CO2 trapping efficiencies from nitrogen gas streams.

Three new systems were developed for trapping [11C]CO2 from high flow rate nitrogen gas streams and the trapping efficiency was determined for each trap design. The results of this study indicate that both the stainless steel shot and stainless steel frit systems yield high trapping efficiencies under the range of flow rates evaluated (400-3600 sccm) and may prove to be alternatives to the traditional stainless steel/copper coiled tubing or molecular sieve trapping systems in common use.

Effect of N-alkylation on the affinities of analogues of spiperone for dopamine D2 and serotonin 5-HT2 receptors.

Two series of N-substituted spiperone analogues were prepared and evaluated in vitro to measure their affinities for dopamine D2 and serotonin 5-HT2 receptors. Substitution of the amide nitrogen with an alkyl group of five carbon units or less resulted in analogues displaying a low selectivity for D2 compared to 5-HT2 receptors. However, a moderate improvement in selectivity for D2 receptors was observed with N-benzylspiperone. Substitution at either the ortho or para position of the benzyl group resulted in a further reduction in affinity for 5-HT2 receptors and improvement in the selectivity ratio. Examination of N-substituted analogues of spiperone may provide insights into the topography of the antagonist binding region of the 5-HT2 receptor. The results also suggest that an 18F-labeled analogue of N-(4-nitrobenzyl)spiperone (4p) may be a suitable tracer for studying D2 receptors with positron emission tomography since this compound displays a high selectivity for D2 receptors relative to that of spiperone and N-methylspiperone.

In vivo muscarinic cholinergic receptor imaging in human brain with [11C]scopolamine and positron emission tomography.

Cerebral muscarinic cholinergic receptors were imaged and regionally quantified in vivo in humans with the use of [11C]scopolamine and positron emission tomography. Previous studies in experimental animals have suggested the utility of radiolabeled scopolamine for in vivo measurements, on the bases of its maintained pharmacologic specificity following systemic administration and the exclusion of labeled metabolites from the brain. The present studies describe the cerebral distribution kinetics of [11C]scopolamine in normal subjects following intravenous injection. Scopolamine is initially delivered to brain in a perfusion-directed pattern. After 30 to 60 min, activity is lost preferentially from cerebral structures with low muscarinic receptor density including the cerebellum and thalamus. Activity continues to accumulate throughout a 2 h postinjection period in receptor-rich areas including cerebral cortex and the basal ganglia. The late regional concentration of [11C]scopolamine does not, however, accurately parallel known differences in muscarinic receptor numbers in these receptor-rich areas. Tracer kinetic analysis of the data, performed on the basis of a three-compartment model, provides receptor binding estimates in good agreement with prior in vitro measurements. Kinetic analysis confirms significant contributions of ligand delivery and extraction to the late distribution of [11C]scopolamine, reconciling the discrepancy between receptor levels and tracer concentration. Finally, a novel dual-isotope method for rapid chromatographic processing of arterial blood samples in radiotracer studies is presented. The combination of rapid chromatography and compartmental analysis of tracer distribution should have broad utility in future in vivo studies with short-lived radioligands.