Binding characterization of N-(2-chloro-5-thiomethylphenyl)-N'-(3-[3 H]3 methoxy phenyl)-N'-methylguanidine ([3 H]GMOM), a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist.

Labeled with carbon-11, N-(2-chloro-5-thiomethylphenyl)-N'-(3-methoxyphenyl)-N'-methylguanidine ([11 C]GMOM) is currently the only positron emission tomography (PET) tracer that has shown selectivity for the ion-channel site of N-methyl-D-aspartate (NMDA) receptors in human imaging studies. The present study reports on the selectivity profile and in vitro binding properties of GMOM. The compound was screened on a panel of 80 targets, and labeled with tritium ([3 H]GMOM). The binding properties of [3 H]GMOM were compared to those of the reference ion-channel ligand [3 H](+)-dizocilpine maleate ([3 H]MK-801), in a set of concentration-response, homologous and heterologous inhibition, and association kinetics assays, performed with repeatedly washed rat forebrain preparations. GMOM was at least 70-fold more selective for NMDA receptors compared to all other targets examined. In homologous inhibition and concentration-response assays, the binding of [3 H]GMOM was regulated by NMDA receptor agonists, albeit in a less prominent manner compared to [3 H]MK-801. Scatchard transformation of homologous inhibition data produced concave upward curves for [3 H]GMOM and [3 H]MK-801. The radioligands showed bi-exponential association kinetics in the presence of 100 µmol L-1 l-glutamate/30 µmol L-1 glycine. [3 H]GMOM (3 nmol L-1 and 10 nmol L-1 ) was inhibited with dual affinity by (+)-MK-801, (R,S)-ketamine and memantine, in both presence and absence of agonists. [3 H]MK-801 (2 nmol L-1 ) was inhibited in a monophasic manner by GMOM under baseline and combined agonist conditions, with an IC50 value of ~19 nmol L-1 . The non-linear Scatchard plots, biphasic inhibition by open channel blockers, and bi-exponential kinetics of [3 H]GMOM indicate a complex mechanism of interaction with the NMDA receptor ionophore. The implications for quantifying the PET signal of [11 C]GMOM are discussed.

Radiosynthesis and biodistribution of a histamine H3 receptor antagonist 4-[3-(4-piperidin-1-yl-but-1-ynyl)-[11C]benzyl]-morpholine: evaluation of a potential PET ligand.

The potent histamine H(3) receptor antagonist JNJ-10181457 (1) was successfully labeled with (11)C in a novel one-pot reaction sequence, with high chemical yield (decay-corrected yield, 28+/-8%) and high specific radioactivity (56+/-26 GBq/mumol). The binding of [(11)C]1 to H(3) receptors was studied in vitro in rat brain and in vivo in rats and mice. The in vitro binding of [(11)C]1 in rat coronal brain slices showed high binding in the striatum, and this binding was blocked by histamine and by two known H(3) antagonists, JNJ-5207852 (2) and unlabeled Compound (1), in a concentration-dependent manner. The biodistribution of [(11)C]1 in rats was measured at 5, 10, 30 and 60 min. The uptake of [(11)C]1 in regions rich in H(3) receptors was highest at 30 min, giving 0.98%, 1.41%, 1.28% and 1.72% dose/g for the olfactory bulb, hippocampus, striatum and cerebral cortex, respectively. However, the binding of [(11)C]1 in the rat brain could not be blocked by pretreatment with either Compound (2) (30 min or 24 h pretreatment) or cold Compound (1) (30-min pretreatment). The biodistribution of [(11)C]1 in a second species (Balb/c mice) showed a higher overall uptake of the radioligand with an average brain uptake of 8.9% dose/g. In C57BL/6-H(3)(-/-) knockout mice, a higher brain uptake was also observed. Analyses of metabolites and plasma protein binding were also undertaken. It appeared that [(11)C]1 could not specifically label H(3) receptors in rodent brain in vivo. Possible causes are discussed.

Influence of the antipsychotic drug pipamperone on the expression of the dopamine D4 receptor.

The dopamine D4 receptor is a G protein-coupled receptor that binds with high affinity various antipsychotics. The receptor may be involved in attention/cognition, and in genetic studies a polymorphic repeat sequence in its coding sequence has been associated with attention deficit/hyperactivity disorder. We developed an inducible episomal expression system based on the reverse tetracycline transactivator and Epstein-Barr viral sequences. In HEK293rtTA cells expressing the dopamine D4 receptor from this episomal expression vector, addition of doxycycline in combination with sodium butyrate and trichostatin A induces high levels of receptor expression, resulting in 1970 +/- 20 fmol/mg membrane protein. Addition of the dopamine D4 receptor and serotonin 5-HT2A receptor antagonist pipamperone to these cells further increased the expression of the dopamine receptor, reaching 3800 +/- 60 fmol/mg membrane protein. This up-regulation was not restricted to the dopamine D4 receptor but was also found for the serotonin 5-HT2A receptor. We further provide evidence that the increase in receptor expression is not due to increased mRNA synthesis. As pipamperone could rescue the expression of a folding mutant of the dopamine D4 receptor (M345), we propose that pipamperone acts as a pharmacological chaperone for correct receptor folding thereby resulting in an increased dopamine D4 receptor expression. This study describes a strong and inducible expression system for proteins, difficult to express in other heterologous expression systems. This study also demonstrates that pipamperone, an antipsychotic, acts as a pharmacological chaperone and by doing so, increases the expression level of the dopamine D4 receptor. The fact that ligands can also act as pharmacological chaperones is a fairly new additional element in the regulation of receptor expression levels with potential great impact in drug treatment.

Lipophilic nalmefene prodrugs to achieve a one-month sustained release.

Nalmefene is an opioid antagonist which as a once-a-day tablet formulation has recently been approved for reducing ethanol intake in alcoholic subjects. In order to address the compliance issue in this patient population, a number of potential nalmefene prodrugs were synthesized with the aim of providing a formulation that could provide plasma drug concentrations in the region of 0.5-1.0ng/mL for a one-month period when dosed intramuscular to dogs or minipigs. In an initial series of studies, three different lipophilic nalmefene derivatives were evaluated: the palmitate (C16), the octadecyl glutarate diester (C18-C5) and the decyl carbamate (CB10). They were administered intramuscularly to dogs in a sesame oil solution at a dose of 1mg-eq. nalmefene/kg. The decyl carbamate was released relatively quickly from the oil depot and its carbamate bond was too stable to be used as a prodrug. The other two derivatives delivered a fairly constant level of 0.2-0.3ng nalmefene/mL plasma for one month and since there was no significant difference between these two, the less complex palmitate monoester was chosen to demonstrate that dog plasma nalmefene concentrations were dose-dependent at 1, 5 and 20mg-eq. nalmefene/kg. In a second set of experiments, the effect of the chain length of the fatty acid monoester promoieties was examined. The increasingly lipophilic octanoate (C8), decanoate (C10) and dodecanoate (C12) derivatives were evaluated in dogs and in minipigs, at a dose of 5mg-eq. nalmefene/kg and plasma nalmefene concentrations were measured over a four-week period. The pharmacokinetic profiles were very similar in both species with Cmax decreasing and Tmax increasing with increasing fatty acid chain length and the target plasma concentrations (0.5-1.0ng/mL over a month-long period) were achieved with the dodecanoate (C12) prodrug. These data therefore demonstrate that sustained plasma nalmefene concentrations can be achieved in both dog and minipig using nalmefene prodrugs and that the pharmacokinetic profile of nalmefene can be tuned by varying the length of the alkyl group.

MK-801 alters RGS2 levels and adenylyl cyclase sensitivity in the rat striatum.

The present report examines the effects of acute NMDA antagonism on Regulator of G Protein Signaling 2 (RGS2) expression and adenylyl cyclase sensitivity in the rat striatum. MK-801 and phencyclidine rapidly down-regulate RGS2 mRNA. The down-regulation of RGS2 by MK-801 was dose dependent and transient. Because previous reports showed that RGS2 attenuates activity of adenylyl cyclase, RGS2 protein level and sensitivity of adenylyl cyclase to forskolin was tested 2 h after administration of MK-801 (1 mg/kg). In striatal membranes of these rats, RGS2 protein level was 17% lower and forskolin-stimulated cAMP production 38% higher than in controls. These findings reveal a cross-talk between NMDA receptors and adenylyl cyclase and suggest a general cross-talk mechanism by which RGS proteins transcriptionally regulated by ionotropic receptors can alter signaling properties of metabotropic receptors.

The dopamine stabilizer (-)-OSU6162 occupies a subpopulation of striatal dopamine D2/D3 receptors: an [(11)C]raclopride PET study in healthy human subjects.

(-)-OSU6162 is a dopamine stabilizer that can counteract both hyperdopaminergic and hypodopaminergic states. In this study, D2/D3 receptor occupancy of (-)-OSU6162 in the human brain was investigated using positron emission tomography (PET). Twelve male healthy volunteers underwent [(11)C]raclopride PET scanning before and 1 h after a single oral dose of (-)-OSU6162 (15-90 mg). Blood samples for determination of (-)-OSU6162 and prolactin plasma levels were collected at Tmax. Parametric images of [(11)C]raclopride binding potential relative to nondisplaceable tissue (cerebellar grey matter) uptake (BPND) at baseline and after (-)-OSU6162 administration were generated using the simplified reference tissue model. MRI-based regions of interest were defined for the striatum, composed of caudate nucleus and putamen, and projected onto the co-registered parametric [(11)C]raclopride BPND image. Furthermore, three striatal subregions, ie, anterior dorsal caudate, anterior dorsal putamen, and ventral striatum, were defined manually and additionally analyzed. Plasma concentrations of (-)-OSU6162, ranging from 0.01 to 0.9 µM, showed a linear relationship with prolactin levels, reflecting blockade of pituitary D2 receptors. A concentration-dependent increase in striatal D2/D3 receptor occupancy was observed, reaching a value of about 20% at an (-)-OSU6162 plasma level of 0.2 µM, and which for higher concentrations leveled off to a maximal occupancy of about 40%. Findings were similar in the striatal subregions. The present data corroborate the notion that (-)-OSU6162 binds preferentially to a subpopulation of D2/D3 receptors, possibly predominantly extrasynaptic, and this may form the basis for the dopamine-stabilizing properties of (-)-OSU6162.

Preclinical evaluation of [(18)F]PK-209, a new PET ligand for imaging the ion-channel site of NMDA receptors.

INTRODUCTION: The present study was designed to assess whether [(18)F]PK-209 (3-(2-chloro-5-(methylthio)phenyl)-1-(3-([(18)F]fluoromethoxy)phenyl)-1-methylguanidine) is a suitable ligand for imaging the ion-channel site of N-methyl-D-aspartate receptors (NMDArs) using positron emission tomography (PET). METHODS: Dynamic PET scans were acquired from male rhesus monkeys over 120min, at baseline and after the acute administration of dizocilpine (MK-801, 0.3mg/kg; n=3/condition). Continuous and discrete arterial blood samples were manually obtained, to generate metabolite-corrected input functions. Parametric volume-of-distribution (VT) images were obtained using Logan analysis. The selectivity profile of PK-209 was assessed in vitro, on a broad screen of 79 targets. RESULTS: PK-209 was at least 50-fold more selective for NMDArs over all other targets examined. At baseline, prolonged retention of radioactivity was observed in NMDAr-rich cortical regions relative to the cerebellum. Pretreatment with MK-801 reduced the VT of [(18)F]PK-209 compared with baseline in two of three subjects. The rate of radioligand metabolism was high, both at baseline and after MK-801 administration. CONCLUSIONS: PK-209 targets the intrachannel site with high selectivity. Imaging of the NMDAr is feasible with [(18)F]PK-209, despite its fast metabolism. Further in vivo evaluation in humans is warranted.

Metabotropic glutamate 1 receptor distribution and occupancy in the rat brain: a quantitative autoradiographic study using [3H]R214127.

We used the selective metabotropic glutamate (mGlu) 1 receptor antagonist [3H]1-(3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-2-phenyl-1-ethanone ([3H]R214127) to investigate the distribution of mGlu1 receptor binding sites in rat brain. We found high mGlu1 receptor binding in the cerebellum, thalamus, dentate gyrus and medial central gray, moderate binding within the CA3 of the hippocampus and hypothalamus, and low mGlu1 receptor binding in the basal ganglia and cortex. The mGlu1 receptor is also present in variable degree in the dorsal lateral septal nucleus, amygdala, interpeduncular nucleus and median raphe nucleus. Additionally, we employed [3H]R214127 autoradiography as a means of investigating the occupancy of central mGlu1 receptors following in vivo administration of mGlu1 receptor antagonists that prevent binding of this radioligand. We found that the mGlu1 receptor antagonist (3aS,6aS)-6a-naphtalan-2-ylmethyl-5-methyliden-hexahydro-cyclopenta[c]furan-1-on (BAY 36-7620), administered subcutaneously (s.c.) at 10 mg/kg, only occupied about 30% of cerebellar and thalamic mGlu1 receptors. The mGlu1/5 receptor antagonist 2-quinoxaline-carboxamide-N-adamantan-1-yl (NPS 2390) exhibited a relatively high potency in occupying mGlu1 receptors in rat cerebellum (ED50 = 0.75 mg/kg, s.c.) and thalamus (ED50 = 0.63 mg/kg, s.c). In the future, this method can be employed to gain more insight into the in vivo profile and central activity of potential therapeutic agents that act upon the mGlu1 receptor.

Does phenylethylamine act as an endogenous amphetamine in some patients?

In brain capillary endothelium and catecholaminergic terminals a single decarboxylation step effected by aromatic amino-acid decarboxylase converts phenylalanine to phenylethylamine, at a rate comparable to that of the central synthesis of dopamine. Phenylethylamine, however, is not stored in intra-neuronal vesicles and is rapidly degraded by monoamine oxidase-B. Despite its short half-life, phenylethylamine attracts attention as an endogenous amphetamine since it can potentiate catecholaminergic neurotransmission and induce striatal hyperreactivity. Subnormal phenylethylamine levels have been linked to disorders such as attention deficit and depression; the use of selegiline (Deprenyl) in Parkinson's disease may conceivably favour recovery from deficient dopaminergic neurotransmission by a monoamine oxidase-B inhibitory action that increases central phenylethylamine. Excess phenylethylamine has been invoked particularly in paranoid schizophrenia, in which it is thought to act as an endogenous amphetamine and, therefore, would be antagonized by neuroleptics. The importance of phenylethylamine in mental disorders is far from fully elucidated but the evolution of phenylethylamine concentrations in relation to symptoms remains a worthwhile investigation for individual psychotic patients.

Daphnane-type diterpene orthoesters and their biological activities.

Daphnane orthoesters are the active ingredients of plant remedies from the Western, Chinese and African traditional medicine, and have provided important tools to investigate medicinally relevant processes like tumour promotion, apoptosis, neurotrophism, and VR1 activation. The occurrence, biological activity, and molecular pharmacology of these compounds will be reviewed.

Nuclei and subnuclei gene expression profiling in mammalian brain.

Information on the neuroanatomical expression of a given gene is critical to understanding its function in the central nervous system. The integration of laser capture microdissection (LCM), T7-based RNA amplification and cDNA microarrays allows for this information to be simultaneously generated for thousands of genes. To validate this integrative approach, we catalogued the gene expression profiles of seven rat brain nuclei or subnuclei. A hundred cells from the following seven brain nuclei were analyzed: locus coeruleus (LC), dorsal raphe nucleus (DR), parvocellular division (PA) and magnocellular division (MG) of the hypothalamic paraventricular nucleus (PVN) and CA1, CA3 and dentate gyrus (DG) divisions of the hippocampal formation. Of the 2145 genes investigated, 1402 genes (65%) gave a hybridization signal statistically different from the background level that was defined by non-specific hybridizations to 15 different plant genes. Validation of our microarray data on four arbitrarily selected genes was confirmed by Real-Time PCR. Previous research showing expression patterns of 'signature' genes (n=17) for specific brain nuclei are consistent with our findings. For example, as previously shown, enriched mRNA expression encoding the serotonin transporter or tyrosine hydroxylase was found in DR and LC cells, respectively. Interestingly, expression of the serotonin 5-HT(2B) receptor mRNA was also found in DR cells. We confirmed this new finding by in-situ hybridization. The hierarchical clustering analysis of gene expression shows that the two divisions of the PVN (PA and MG) are closely related to each other, as well as the three regions of the hippocampal formation (CA1, CA3 and DG), which also showed similar gene expression profiles. This study demonstrates the importance, feasibility and utility of cellular brain nuclei profiling.

5-HT2A and 5-HT2C receptors and their atypical regulation properties.

The 5-HT(2A) and 5-HT(2C) receptors belong to the G-protein-coupled receptor (GPCR) superfamily. GPCRs transduce extracellular signals to the interior of cells through their interaction with G-proteins. The 5-HT(2A) and 5-HT(2C) receptors mediate effects of a large variety of compounds affecting depression, schizophrenia, anxiety, hallucinations, dysthymia, sleep patterns, feeding behaviour and neuro-endocrine functions. Binding of such compounds to either 5-HT(2) receptor subtype induces processes that regulate receptor sensitivity. In contrast to most other receptors, chronic blockade of 5-HT(2A) and 5-HT(2C) receptors leads not to an up- but to a (paradoxical) down-regulation. This review deals with published data involving such non-classical regulation of 5-HT(2A) and 5-HT(2C) receptors obtained from in vivo and in vitro studies. The underlying regulatory processes of the agonist-induced regulation of 5-HT(2A) and 5-HT(2C) receptors, commonly thought to be desensitisation and resensitisation, are discussed. The atypical down-regulation of both 5-HT(2) receptor subtypes by antidepressants, antipsychotics and 5-HT(2) antagonists is reviewed. The possible mechanisms of this paradoxical down-regulation are discussed, and a new hypothesis on possible heterologous regulation of 5-HT(2A) receptors is proposed.

Internalization of human 5-HT4a and 5-HT4b receptors is splice variant dependent.

The family of 5-HT4 receptors comprises 16 putative splice variants. We have previously shown that there are differences in signal transduction of the h5-HT(4a) and h5-HT(4b) receptors. In the present study, the internalization of these two splice variants following receptor stimulation was investigated with confocal microscopy on living cells. Chimeric receptors, h5-HT(4a)-GFP and h5-HT(4b)-GFP were generated by fusing the coding sequence of the 5-HT4 receptor with the coding sequence of the GFP. The agonist stimulation of fluorescent receptors resulted in a time-dependent internalization of the h5-HT(4b)-GFP receptor, but not of the h5-HT(4a)-GFP receptor. The h5-HT(4b) receptor displays a dual coupling to G(alpha)i,o and G(alpha)s proteins, in contrast to the h5-HT4a receptor, which couples to Galphas proteins only. We investigated whether the difference in internalization of the two splice variant receptors was related to their differential coupling. Therefore, we performed agonist-stimulation of the receptor following inhibition of the G(alpha)i,o protein coupling using PTX. The h5-HT(4b) receptor internalization is PTX insensitive. We co-transfected the fluorescent chimeric receptors with other wild-type variants, which did not produce an alteration of the receptor trafficking. These findings provide the first evidence of differential internalization between the two splice variants, 5-HT(4a) and 5-HT(4b) receptors.

Synthesis and preclinical evaluation of carbon-11 labelled N-((5-(4-fluoro-2-[(11)C]methoxyphenyl)pyridin-3-yl)methyl)cyclopentanamine as a PET tracer for NR2B subunit-containing NMDA receptors.

INTRODUCTION: The N-methyl-D-Aspartate (NMDA) receptor plays an important role in learning and memory. Overactivation is thought to play an important role in neurodegenerative disorders such as Alzheimer's disease. Currently, it is not possible to assess N-methyl-D-aspartate receptor (NMDAr) bio-availability in vivo. The purpose of this study was to develop a positron emission tomography (PET) ligand for the NR2B binding site of the NMDA receptor. METHODS: N-((5-(4-fluoro-2-methoxyphenyl)pyridin-3-yl)methyl)cyclopentanamine was radiolabelled with carbon-11 in the phenyl moiety. Biodistribution and blocking studies were carried out in anaesthetized mice and in non-anaesthetized rats. RESULTS: N-((5-(4-fluoro-2-[(11)C]methoxyphenyl)pyridin-3-yl)methyl)cyclopentanamine was prepared in 49±3% (decay-corrected) yield, affording 4.1±0.3 GBq of formulated product at the end of synthesis with a radiochemical purity of >99% and with a specific activity of 78±10 GBq/µmol. CONCLUSION: A new NR2B PET ligand was developed in high yield. [(11)C]4 readily enters the brain and binds to the NR2B subunit-containing NMDAr in the rodent brain. High sigma-1 receptor binding may, however, limit its future application as a PET probe for imaging the NR2B subunit-containing NMDAr. Anaesthesia has an effect on NMDAr function and therefore can complicate interpretation of preclinical in vivo results. In addition, effects of endogenous compounds cannot be excluded. Despite these potential limitations, further studies are warranted to investigate the values of [(11)C]4 as an NR2B PET ligand.

[11C]AF150(S), an agonist PET ligand for M1 muscarinic acetylcholine receptors.

BACKGROUND: The M1 muscarinic acetylcholine receptor (M1ACh-R) is a G protein-coupled receptor that can occur in interconvertible coupled and uncoupled states. It is enriched in the basal ganglia, hippocampus, olfactory bulb, and cortical areas, and plays a role in motor and cognitive functions. Muscarinic M1 agonists are potential therapeutic agents for cognitive disorders. The aim of this study was to evaluate [11C]AF150(S) as a putative M1ACh-R agonist PET ligand, which, owing to its agonist properties, could provide a tool to explore the active G protein-coupled receptor. METHODS: Regional kinetics of [11C]AF150(S) in rat brain were measured using a high-resolution research tomograph, both under baseline conditions and following pre-treatment with various compounds or co-administration of non-radioactive AF150(S). Data were analysed by calculating standard uptake values and by applying the simplified reference tissue model (SRTM). RESULTS: [11C]AF150(S) was rapidly taken up in the brain, followed by a rapid clearance from all brain regions. Analysis of PET data using SRTM revealed a binding potential (BPND) of 0.25 for the striatum, 0.20 for the hippocampus, 0.16 for the frontal cortical area and 0.15 for the posterior cortical area, all regions rich in M1ACh-R. BPND values were significantly reduced following pre-treatment with M1ACh-R antagonists. BPND values were not affected by pre-treatment with a M3ACh-R antagonist. Moreover, BPND was significantly reduced after pre-treatment with haloperidol, a dopamine D2 receptor blocker that causes an increase in extracellular acetylcholine (ACh). The latter may compete with [11C]AF150(S) for binding to the M1ACh-R; further pharmacological agents were applied to investigate this possibility. Upon injection of the highest dose (49.1 nmol kg-1) of [11C]AF150(S) diluted with non-radioactive AF150(S), brain concentration of AF150(S) reached 100 nmol L-1 at peak level. At this concentration, no sign of saturation in binding to M1ACh-R was observed. CONCLUSIONS: The agonist PET ligand [11C]AF150(S) was rapidly taken up in the brain and showed an apparent specific M1ACh-R-related signal in brain areas that are rich in M1ACh-R. Moreover, binding of the agonist PET ligand [11C]AF150(S) appears to be sensitive to changes in extracellular ACh levels. Further studies are needed to evaluate the full potential of [11C]AF150(S) for imaging the active pool of M1ACh-R in vivo.

Radiosynthesis and preclinical evaluation of [11C]prucalopride as a potential agonist PET ligand for the 5-HT4 receptor.

BACKGROUND: Serotonin 5-HT4 receptor (5-HT4-R) agonists are potential therapeutic agents for enterokinetic and cognitive disorders and are marketed for treatment of constipation. The aim of this study was to develop an agonist positron emission tomography (PET) ligand in order to label the active G-protein coupled 5-HT4-R in peripheral and central tissues. For this purpose prucalopride, a high-affinity selective 5-HT4-R agonist, was selected. METHODS: [11C]Prucalopride was synthesized from [11C]methyl triflate and desmethyl prucalopride, and its LogDoct,pH7.4 was determined. Three distinct studies were performed with administration of IV [11C]prucalopride in male rats: (1) The biodistribution of radioactivity was measured ex vivo; (2) the kinetics of radioactivity levels in brain regions and peripheral organs was assessed in vivo under baseline conditions and following pre-treatment with tariquidar, a P-glycoprotein efflux pump inhibitor; and (3) in vivo stability of [11C]prucalopride was checked ex vivo in plasma and brain extracts using high-performance liquid chromatography. RESULTS: [11C]Prucalopride was synthesized in optimised conditions with a yield of 21% ± 4% (decay corrected) and a radiochemical purity (>99%), its LogDoct,pH7.4 was 0.87. Ex vivo biodistribution studies with [11C]prucalopride in rats showed very low levels of radioactivity in brain (maximal 0.13% ID·g-1) and ten times higher levels in certain peripheral tissues. The PET studies confirmed very low brain levels of radioactivity under baseline conditions; however, it was increased three times after pre-treatment with tariquidar. [11C]Prucalopride was found to be very rapidly metabolised in rats, with no parent compound detectable in plasma and brain extracts at 5 and 30 min following IV administration. Analysis of levels of radioactivity in peripheral tissues revealed a distinct PET signal in the caecum, which was reduced following tariquidar pre-treatment. The latter is in line with the role of the P-glycoprotein pump in the gut. CONCLUSION: [11C]Prucalopride demonstrated low radioactivity levels in rat brain; a combination of reasons may include rapid metabolism in the rat in particular, low passive diffusion and potential P-glycoprotein substrate. In humans, further investigation of [11C]prucalopride for imaging the active state of 5-HT4-R is worthwhile, in view of the therapeutic applications of 5-HT4 agonists for treatment of gastrointestinal motility disorders.

Reproducible Analysis of Rat Brain PET Studies Using an Additional [(18)F]NaF Scan and an MR-Based ROI Template.

Background. An important step in the analysis of positron emission tomography (PET) studies of the brain is the definition of regions of interest (ROI). Image coregistration, ROI analysis, and quantification of brain PET data in small animals can be observer dependent. The purpose of this study was to investigate the feasibility of ROI analysis based on a standard MR template and an additional [(18)F]NaF scan. Methods. [(18)F]NaF scans of 10 Wistar rats were coregistered with a standard MR template by 3 observers and derived transformation matrices were applied to corresponding [(11)C]AF150(S) images. Uptake measures were derived for several brain regions delineated using the MR template. Overall agreement between the 3 observers was assessed by interclass correlation coefficients (ICC) of uptake data. In addition, [(11)C]AF150(S) ROI data were compared with ex vivo biodistribution data. Results. For all brain regions, ICC analysis showed excellent agreement between observers. Reproducibility, estimated by calculation of standard deviation of the between-observer differences, was demonstrated by an average of 17% expressed as coefficient of variation. Uptake of [(11)C]AF150(S) derived from ROI analysis closely matched ex vivo biodistribution data. Conclusions. The proposed method provides a reproducible and tracer-independent method for ROI analysis of rat brain PET data.

Design, synthesis, radiolabeling, and in vitro and in vivo evaluation of bridgehead iodinated analogues of N-{2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl}-N-(pyridin-2-yl)cyclohexanecarboxamide (WAY-100635) as potential SPECT ligands for the 5-HT1A receptor.

Here we describe the design, synthesis, and pharmacological profile of 5-HT(1A) receptor ligands related to 1 (WAY-100635). The cyclohexyl moiety in 1 and its O-desmethylated analogue 3 were replaced by the bridgehead iodinated bridge-fused rings: adamantyl, cubyl, bicyclo[2.2.2]octyl, or bicyclo[2.2.1]heptyl. All analogues displayed a (sub)nanomolar affinity for the 5-HT(1A) receptor in vitro. Compounds 6b and 7b appeared to be selective for this receptor over other relevant receptors and could easily be iodinated with radioactive iodine-123. In humane hepatocytes, [(123)I]6b showed a low propensity for amide hydrolysis and a stable carbon-iodine bond. The biodistribution of [(123)I]6b and [(123)I]7b in rats revealed that the carbon-iodine bond was also stable in vivo. Unfortunately, the brain uptake and the specificity for both radioligands were significantly lower than those of the parent molecule 1. In conclusion, the designed tracers are not suitable for SPECT imaging.

Quantification of the neurokinin 1 receptor ligand [¹¹C]R116301.

PURPOSE: Neurokinin 1 (NK1) receptors have been implicated in depression, anxiety, and pain perception. Recently, it was shown that, in the human brain, a specific NK1 receptor-related signal was obtained with the novel radioligand, [¹¹C]R116301, using positron emission tomography. The purpose of this study was to evaluate various methods for quantifying specific [¹¹C]R116301 binding. METHODS: Two dynamic 90-min [¹¹C]R116301 scans, separated by 5 h, were performed in 11 healthy volunteers. In three patients, the second scan was performed after an oral blocking dose of 125 mg of aprepitant, whereas in the other eight, no intervention was performed (test-retest). Whole striatum was used as the tissue of interest, as it has the highest density of NK1 receptors. Cerebellum was used as the reference tissue. RESULTS: Reference tissue models were stable with the simplified reference tissue model (SRTM) performing best. Average (± standard deviation) SRTM-derived mean nondisplaceable binding potential (BP(ND)) of all (first) baseline scans was 0.64±0.31 (n=11), which reduced to -0.01±0.03 (n=3) after aprepitant administration. Test-retest results showed low variability (14.0±10.7%) and excellent reliability, as indicated by the intraclass correlation coefficient (0.93). The ratio of standardized uptake values of striatum and cerebellum minus 1, an approximation of BP(ND), showed very low variability (6.2±3.1%) with excellent reliability (intraclass correlation coefficient=0.98), and correlated well with SRTM-derived BP(ND) (R²=0.96). CONCLUSION: SRTM is the model of choice for quantifying [¹¹C]R116301 binding. Semiquantitative tissue ratios hold promise for routine clinical applications.

Evaluation of [11C]laniquidar as a tracer of P-glycoprotein: radiosynthesis and biodistribution in rats.

At present, P-glycoprotein (P-gp) function can be studied using positron emission tomography (PET) together with a labelled P-gp substrate such as R-[11C]verapamil. Such a tracer is, however, less suitable for investigating P-gp (over)expression. Laniquidar is a third-generation P-gp inhibitor, which has been used in clinic trials for modulating multidrug resistance transporters. The purpose of the present study was to develop the radiosynthesis of [11C]laniquidar and to assess its suitability as a tracer of P-gp expression. The radiosynthesis of [11C]laniquidar was performed by methylation of the carboxylic acid precursor with [11C]CH3I. The product was purified by HPLC and reformulated over a tC18 Seppak, yielding a sterile solution of [11C]laniquidar in saline. For evaluating [11C]laniquidar, rats were injected with 20 MBq [11C]laniquidar via a tail vein and sacrificed at 5, 15, 30 and 60 min after injection. Several tissues and distinct brain regions were dissected and counted for radioactivity. In addition, uptake of [11C]laniquidar in rats pretreated with cyclosporine A and valspodar (PSC 833) was determined at 30 min after injection. Finally, the metabolic profile of [11C]laniquidar in plasma was determined. [11C]Laniquidar could be synthesized in moderate yields with high specific activity. Uptake in brain was low, but significantly increased after administration of cyclosporine A. Valspodar did not have any effect on cerebral uptake of [11C]laniquidar. In vivo rate of metabolism was relatively low. Further kinetic studies are needed to investigate the antagonistic behaviour of [11C]laniquidar at tracer level.