Positron emission tomography analysis of [11C]KW-6002 binding to human and rat adenosine A2A receptors in the brain.

Adenosine A(2A) receptors are found on striatal neurones projecting to the external pallidum. KW-6002 (istradefylline) is a potent and selective antagonist for the adenosine A(2A) receptors in the CNS and acts to inhibit the excessive activity of this pathway in the MPTP marmoset model of PD, thus relieving parkinsonism. The objectives of this study were to investigate the regional binding of the novel positron emission tomography tracer [(11)C]KW-6002 in the healthy human brain and the rat brain, along with receptor occupancy by cold KW-6002 at varying doses in human. The highest [(11)C]KW-6002 uptake in the rat brain was seen in striatum and lower levels in cortex and cerebellum. Brain [(11)C]KW-6002 uptake was well characterized in humans by a two-tissue compartmental model with a blood volume term, and the ED(50) of cold KW-6002 was 0.5 mg in the striatum. Over 90% receptor occupancy was achieved with daily oral doses of greater than 5 mg. In humans, blockable binding was present in all gray matter structures including the cerebellum, which has not been reported to express A(2A) receptors. MRS 1745, an A(2B) receptor selective antagonist, had no effect on the cerebellar binding of [(11)C]KW-6002 in rats, suggesting that this blockable signal is unlikely to result from an affinity for adenosine A(2B) receptors.

In vivo evidence that 5-HT(2C) receptors inhibit 5-HT neuronal activity via a GABAergic mechanism.

BACKGROUND AND PURPOSE: Recent evidence suggests that 5-HT(2C) receptor activation may inhibit midbrain 5-HT neurones by activating neighbouring GABA neurones. This hypothesis was tested using the putative selective 5-HT(2C) receptor agonist, WAY 161503. EXPERIMENTAL APPROACH: The effect of WAY 161503 on 5-HT cell firing in the dorsal raphe nucleus (DRN) was investigated in anaesthetised rats using single unit extracellular recordings. The effect of WAY 161503 on DRN GABA neurones was investigated using double label immunohistochemical measurements of Fos, glutamate decarboxylase (GAD) and 5-HT(2C) receptors. Finally, drug occupancy at 5-HT(2A) receptors was investigated using rat positron emission tomography and ex vivo binding studies with the 5-HT(2A) receptor radioligand [(11)C]MDL 100907. KEY RESULTS: WAY 161503 caused a dose-related inhibition of 5-HT cell firing which was reversed by the 5-HT(2) receptor antagonist ritanserin and the 5-HT(2C) receptor antagonist SB 242084 but not by the 5-HT(1A) receptor antagonist WAY 100635. SB 242084 pretreatment also prevented the response to WAY 161503. The blocking effects of SB 242084 likely involved 5-HT(2C) receptors because the drug did not demonstrate 5-HT(2A) receptor occupancy in vivo or ex vivo. The inhibition of 5-HT cell firing induced by WAY 161503 was partially reversed by the GABA(A) receptor antagonist picrotoxin. Also, WAY 161503 increased Fos expression in GAD positive DRN neurones and DRN GAD positive neurones expressed 5-HT(2C) receptor immunoreactivity. CONCLUSIONS AND IMPLICATIONS: These findings indicate that WAY 161503 inhibits 5-HT cell firing in the DRN in vivo, and support a mechanism involving 5-HT(2C) receptor-mediated activation of DRN GABA neurones.

Evaluation of [4-O-methyl-(11)C]KW-6002 as a potential PET ligand for mapping central adenosine A(2A) receptors in rats.

KW-6002, a xanthine-based adenosine A(2A) antagonist, was labelled with the positron emitter carbon-11 by O-methylation of its precursor, KF23325, using [(11)C]iodomethane and was evaluated in rats as a putative in vivo radioligand for positron emission tomography (PET). Following intravenous injection of [(11)C]KW-6002, radioactivity was measured in blood, plasma, peripheral tissues, and in discrete brain tissues over a 2-h time period commensurate with PET scanning. In brain, [(11)C]KW-6002 showed highest retention in striata, with evidence of saturable binding, and lowest retention in frontal cortex (a tissue low in adenosine A(2A) receptors). PET scanning with [(11)C]KW-6002 demonstrated a specific signal in the striata which could be described using compartmental modelling. Specific binding was, however, also detected in extrastriatal regions, including brain areas reported to have low adenosine A(2A) receptor density. Blocking studies with the A(1) selective antagonist KF15372 and the non xanthine-type A(2A) antagonist ZM 241385 failed to elucidate the nature of this binding. Thus, although [(11)C]KW-6002 shows some potential for development as a PET ligand for quantifying striatal adenosine A(2A) receptor function, its in vivo selectivity requires further investigation.

Effect of 5-HT on binding of [(11)C] WAY 100635 to 5-HT(IA) receptors in rat brain, assessed using in vivo microdialysis nd PET after fenfluramine.

By using a combination of positron emission tomography (PET) and postmortem tissue dissection, the effect of increased endogenous serotonin on specific binding of [(11)C]WAY 100635 to the 5-HT(1A) receptor was investigated in rat brain in vivo. The binding studies were complemented by in vivo microdialysis to monitor 5-HT levels in similarly treated isoflurane-anaesthetised rats, with the dialysis probe locations corresponding to two of the tissues sampled for specific binding of the radioligand. Fenfluramine treatment (10 mg/kg i.p.) resulted in a approximately 5-fold increase in extracellular 5-HT in medial prefrontal cortex and a approximately 15-fold increase in lateral hippocampus, maximal at approximately 40 min after injection. PET scan duration was either 60 or 90 min, beginning 30 min after fenfluramine injection. The specific binding of [(11)C]WAY 100635 was reduced by 10-20% in hippocampus, which showed highest binding in control animals. Specific binding, however, was unaffected in both prefrontal cortex and midbrain raphe, each additional high binding regions. The minimal effects are consistent with a low baseline occupancy of the 5-HT(1A) receptor by 5-HT in vivo, so that only a large change in endogenous agonist concentration will affect radioligand binding. This implies that utilisation of [(11)C]WAY 100635 in human PET to quantify 5-HT(1A) receptor expression can be extended to pathology where synaptic 5-HT levels are altered as a consequence of the disease state.

Evaluation of [O-methyl-11C]RS-15385-197 as a positron emission tomography radioligand for central alpha2-adrenoceptors.

Carbon-11 labelled RS-15385-197 and its ethylsulphonyl analogue, RS-79948-197, were evaluated in rats as potential radioligands to image central alpha2-adrenoceptors in vivo. The biodistributions of both compounds were comparable with that obtained in an earlier study using tritiated RS-79948-197 and were consistent with the known localisation of alpha2-adrenoceptors. The maximal signals (total to non-specific binding) were, however, reduced, in the order [11C]RS-79948-197 < [11C]RS-15385-197 < [3H]RS-79948-197, primarily due to the difference in radiolabel position (O-methyl for carbon- 11 compared with S-ethyl for tritium). This resulted in the in-growth of radiolabelled metabolites in plasma, which, in turn, contributed to the non-specific component of brain radioactivity. Nonetheless, the signal ratio of approximately 5 for a receptor-dense tissue compared with the receptor-sparse cerebellum, at 90-120 min after radioligand injection, encouraged the development of [O-methyl-11C]RS-15385-197 for human positron emission tomography (PET). Unfortunately, in two human PET scans (each of 90 min), brain extraction of the radioligand was minimal, with volumes of distribution more than an order of magnitude lower than that measured in rats. Following intravenous injection, radioactivity was retained in plasma and metabolism of the radiolabelled compound was very low. Retrospective measurements of in vitro plasma protein binding and in vivo brain uptake index (BUI) in rats demonstrated a higher protein binding of the radioligand in human compared with rat plasma and a lower BUI in the presence of human plasma. It is feasible that a higher affinity of RS-15385-197 for human plasma protein compared with receptor limited the transport of the radioligand. Although one of the PET scans showed a slight heterogeneity in biodistribution of radioactivity which was consistent with the known localisation of alpha2-adrenoceptors in human brain, it was concluded that [O-methyl-11C]RS-15385-197 showed little promise for routine quantification of alpha2-adrenoceptors in man.

Pindolol occupancy of 5-HT(1A) receptors measured in vivo using small animal positron emission tomography with carbon-11 labeled WAY 100635.

Positron emission tomography (PET), following an intravenous injection of [carbonyl-(11)C]WAY 100635, was used to image central 5-HT(1A) receptors in rat following pretreatment with graded doses of (-)-pindolol (0.001-3 mg/kg, i.v.). The use of PET had advantages over ex vivo radioligand binding methods in that it produced parametric image volumes and reduced errors due to inter-rat variability. Time-radioactivity curves from regions of interest (ROI) acquired from individual rats enabled the estimation of specific binding of the radioligand using a compartmental model with reference tissue input. Binding potential (BP) of [(11)C]WAY 100635 was estimated for frontal cortex and hippocampus (postsynaptic), and midbrain raphe nuclei (presynaptic). In the latter ROI, pindolol dose-dependently decreased BP. The saturation curve could be fitted to a single-site model up to the lowest dose of pindolol used, giving an ED(50) (dose to cause 50% occupancy) value of 0.26 +/- 0. 05 mg/kg, and inclusion of control (nonpindolol-treated) rats did not affect the fit. In contrast, in cortex and hippocampus ROI, low doses of pindolol caused an increase in BP compared with controls. Pindolol doses greater than approximately 0.1 mg/kg, resulted in a dose-dependent decrease in BP, and ED(50) values in cortex and hippocampus were estimated as 0.44 +/- 0.13 and 0.48 +/- 0.12 mg/kg, respectively. The increase in [(11)C]WAY 100635 binding at low pindolol doses is feasibly related to a decrease in basal receptor occupancy following reduced release of endogenous 5-HT. Considering the apparently greater potency of pindolol at the midbrain raphe ROI, this effect could be mediated via agonist activity at the autoreceptor.

Evaluation of [methyl-3H]L655,708 and [ethyl-3H]RY80 as putative PET ligands for central GABA(A) receptors containing alpha5 subunit.

Two selective radioligands of gamma aminobutyric acid (GABA)A receptors containing the alpha5 subunit, [3H]L655,708 and [3H]RY80, were evaluated in rats as potential in vivo tracers for positron emission tomography (PET). Brain uptake index (BUI), a measure of first pass extraction, was moderate for [3H]L655,708 (BUI of 59%) and good for [3H]RY80 (BUI of 96%). This finding was consistent with their in vitro binding to plasma proteins of approximately 76% and 50%, respectively. Following intravenous injection of either radioligand, radioactivity in plasma was measured and uptake characteristics were assessed in brain within a time period relevant to PET scanning (up to 90 min). Discrete brain regions, such as frontal cortex, striatum, hypothalamus, thalamus, hippocampus, colliculi, medulla, and cerebellum, were sampled and the temporal distribution of radioactivity analysed. Despite the reasonable delivery to the brain, neither of the radioligands had sufficient retention in the tissues rich in alpha5-containing GABA(A) receptors to achieve a good selective signal. For both radioligands, a maximal tissue:cerebellum ratio of 1.5 was seen in hippocampus at 10 min after injection. Thus, neither of the compounds studied shows potential for further development as an in vivo PET ligand.

[carbonyl-11C]Desmethyl-WAY-100635 (DWAY) is a potent and selective radioligand for central 5-HT1A receptors in vitro and in vivo.

[carbonyl-11C]Desmethyl-WAY-100635 (DWAY) is possibly a low-level metabolite appearing in plasma after intravenous administration of [carbonyl-11C]WAY-100635 to human subjects for positron emission tomographic (PET) imaging of brain 5-HT1A receptors. In this study we set out to assess the ability of DWAY to enter brain in vivo and to elucidate its possible interaction with 5-HT1A receptors. Desmethyl-WAY-100635 was labelled efficiently with carbon-11 (t1/2 = 20.4 min) in high specific radioactivity by reaction of its descyclohexanecarbonyl analogue with [carbonyl-11C]cyclohexanecarbonyl chloride. The product was separated in high radiochemical purity by high-performance liquid chromatography (HPLC) and formulated for intravenous injection. Rats were injected intravenously with DWAY, sacrificed at known times and dissected to establish radioactivity content in brain tissues. At 60 min after injection, the ratios of radioactivity concentration in each brain region to that in cerebellum correlated with previous in vitro and in vivo measures of 5-HT1A receptor density. The highest ratio was about 22 in hippocampus. Radioactivity cleared rapidly from plasma; HPLC analysis revealed that DWAY represented 55% of the radioactivity in plasma at 5 min and 33% at 30 min. Only polar radioactive metabolites were detected. Subsequently, a cynomolgus monkey was injected intravenously with DWAY and examined by PET. Maximal whole brain uptake of radioactivity was 5.7% of the administered dose at 5 min after injection. The image acquired between 9 and 90 min showed high radioactivity uptake in brain regions rich in 5-HT1A receptors (e.g. frontal cortex and neocortex), moderate uptake in raphe nuclei and low uptake in cerebellum. A transient equilibrium was achieved in cortical regions at about 60 min, when the ratio of radioactivity concentration in frontal cortex to that in cerebellum reached 6. The corresponding ratio for raphe nuclei was about 3. Radioactive metabolites appeared rapidly in plasma, but these were all more polar than DWAY, which represented 52% of the radioactivity in plasma at 4 min and 20% at 55 min. In a second PET experiment, in which a cynomolgus monkey was pretreated with the selective 5-HT1A receptor antagonist, WAY-100635, at 25 min before DWAY injection, radioactivity in all brain regions was reduced to that in cerebellum. Autoradiography of post mortem human brain cryosections after incubation with DWAY successfully delineated 5-HT1A receptor distribution. Receptor-specific binding was eliminated in the presence of the selective 5-HT1A receptor agonist, 8-OH-DPAT [(+/-)-8-hydroxy-2-dipropylaminotetralin]. These findings show that: (a) intravenously administered DWAY is well able to penetrate brain in rat and monkey, (b) DWAY is a highly effective radioligand for brain 5-HT1A receptors in rat and monkey in vivo and for human brain in vitro, and (c) the metabolism and kinetics of DWAY appear favourable to successful biomathematical modelling of acquired PET data. Thus, DWAY warrants further evaluation as a radioligand for PET studies of 5-HT1A receptors in human brain.

In vivo saturation kinetics of two dopamine transporter probes measured using a small animal positron emission tomography scanner.

When estimated in vitro, the parameters which describe the binding of radiolabelled analogues of cocaine to sites on the dopamine transporter are very much influenced by the methodology used. In the present study, a small animal positron emission tomography (PET) scanner was used to estimate in vivo saturation kinetics for two carbon-11 labelled compounds presently used to monitor dopamine terminal function. The binding of [11C]CFT (WIN 35,428) in rat striatum was adequately described by a single-site model, giving an apparent dissociation constant corresponding to an intravenous dose of 242 nmol/kg. In contrast, the binding of [11C]RTI-121 was better described by a two-site model with the 'high-affinity' site or state (dissociation constant = 1 nmol/kg) being significantly occupied at doses routinely used in PET scanning. Such findings cannot readily be predicted from in vitro work, but could aid in both the choice of ligand and the model used in quantification of scan data. While multi-dose in vivo PET studies are difficult in man, rat PET can easily be employed either pre-clinically for putative radioligands, or experimentally, to study drug interactions and receptor occupancy related to functional efficacy.