Imaging disease activity of rheumatoid arthritis by macrophage targeting using second generation translocator protein positron emission tomography tracers.

BACKGROUND: Positron emission tomography (PET) imaging of macrophages using the translocator protein (TSPO) tracer (R)-[11C]PK11195 has shown the promise to image rheumatoid arthritis (RA). To further improve TSPO PET for RA imaging, second generation TSPO tracers [11C]DPA-713 and [18F]DPA-714 have recently been evaluated pre-clinically showing better imaging characteristics. OBJECTIVE: A clinical proof of concept study to evaluate [11C]DPA-713 and [18F]DPA-714 to visualize arthritis in RA patients. METHODS: RA patients (n = 13) with at least two active hand joints were included. PET/CT scans of the hands were obtained after injection of [18F]DPA-714, [11C]DPA-713 and/or (R)-[11C]PK11195 (max. 2 tracers pp). Standardized uptake values (SUVs) and target-to-background (T/B) ratios were determined. Imaging data of the 3 different tracers were compared by pooled post-hoc testing, and by a head to head comparison. RESULTS: Clinically active arthritis was present in 110 hand joints (2-17 pp). Arthritic joints were visualized with both [11C]DPA-713 and [18F]DPA-714. Visual tracer uptake corresponded with clinical signs of arthritis in 80% of the joints. Mean absolute uptake in PET-positive joints was significantly higher for [11C]DPA-713 than for [18F]DPA-714, the latter being not significantly different from (R)-[11C]PK11195 uptake. Background uptake was lower for both DPA tracers compared with that of (R)-[11C]PK11195. Higher absolute uptake and lower background resulted in two-fold higher T/B ratios for [11C]DPA-713. CONCLUSIONS: [11C]DPA-713 and [18F]DPA-714 visualize arthritic joints in active RA patients and most optimal arthritis imaging results were obtained for [11C]DPA-713. Second generation TSPO macrophage PET provides new opportunities for both early diagnosis and therapy monitoring of RA.

Translational evaluation of translocator protein as a marker of neuroinflammation in schizophrenia.

Positron emission tomography (PET) imaging with radiotracers that target translocator protein 18 kDa (TSPO) has become a popular approach to assess putative neuroinflammatory processes and associated microglia activation in psychotic illnesses. It remains unclear, however, whether TSPO imaging can accurately capture low-grade inflammatory processes such as those present in schizophrenia and related disorders. Therefore, we evaluated the validity of TSPO as a disease-relevant marker of inflammation using a translational approach, which combined neurodevelopmental and neurodegenerative mouse models with PET imaging in patients with recent-onset schizophrenia and matched controls. Using an infection-mediated neurodevelopmental mouse model, we show that schizophrenia-relevant behavioral abnormalities and increased inflammatory cytokine expression are associated with reduced prefrontal TSPO levels. On the other hand, TSPO was markedly upregulated in a mouse model of acute neurodegeneration and reactive gliosis, which was induced by intrahippocampal injection of kainic acid. In both models, the changes in TSPO levels were not restricted to microglia but emerged in various cell types, including microglia, astrocytes and vascular endothelial cells. Human PET imaging using the second-generation TSPO radiotracer [11C]DPA-713 revealed a strong trend towards reduced TSPO binding in the middle frontal gyrus of patients with recent-onset schizophrenia, who were previously shown to display increased levels of inflammatory cytokines in peripheral and central tissues. Together, our findings challenge the common assumption that central low-grade inflammation in schizophrenia is mirrored by increased TSPO expression or ligand binding. Our study further underscores the need to interpret altered TSPO binding in schizophrenia with caution, especially when measures of TSPO are not complemented with other markers of inflammation. Unless more selective microglial markers are available for PET imaging, quantification of cytokines and other inflammatory biomarkers, along with their molecular signaling pathways, may be more accurate in attempts to characterize inflammatory profiles in schizophrenia and other mental disorders that lack robust reactive gliosis.

Ring-opened aminothienopyridazines as novel tau aggregation inhibitors.

Aminothienopyridazines (ATPZs) have demonstrated efficacy, in vitro, as tau protein aggregation inhibitors. Modifications were made to the ATPZ scaffold to determine the importance of certain structural features for activity. More specifically, ring-opened analogues detached at the nitrogen-nitrogen bond of the pyridazine, were synthesized and their inhibitory activity evaluated. Preliminary data suggests that the ring-opened structures retain inhibitory activity, independent of tau oxidation. The structures detailed represent the beginnings of a deconstruction-reconstruction-elaboration study, with the aim of identifying simpler scaffolds, which retain activity and can be optimized in terms of physiochemical properties.

In vivo markers of inflammatory response in recent-onset schizophrenia: a combined study using [(11)C]DPA-713 PET and analysis of CSF and plasma.

Several lines of evidence suggest aberrant immune response in schizophrenia, including elevated levels of cytokines. These cytokines are thought to be produced by activated microglia, the innate immune cells of the central nervous system. However, increase in translocator protein 18 kDa (TSPO), a marker of activated glia, has not been found in patients with chronic schizophrenia using second-generation radiotracers and positron emission tomography (PET)-based neuroimaging. In this study we focused on patients with recent onset of schizophrenia (within 5 years of diagnosis). Quantified levels of TSPO in the cortical and subcortical brain regions using the PET-based radiotracer [(11)C]DPA-713 were compared between the patients and healthy controls. Markers of inflammation, including interleukin 6 (IL-6), were assessed in the plasma and cerebrospinal fluid (CSF) in these participants. We observed no significant change in the binding of [(11)C]DPA-713 to TSPO in 12 patients with recent onset of schizophrenia compared with 14 controls. Nevertheless, the patients with recent onset of schizophrenia showed a significant increase in IL-6 in both plasma (P<0.001) and CSF (P=0.02). The CSF levels of IL-6 were significantly correlated with the levels of IL-6 in plasma within the total study population (P<0.001) and in patients with recent onset of schizophrenia alone (P=0.03). Our results suggest that increased levels of IL-6 may occur in the absence of changed TSPO PET signal in the brains of medicated patients with recent onset of schizophrenia. Future development of PET-based radiotracers targeting alternative markers of glial activation and immune response may be needed to capture the inflammatory signature present in the brains of patients with early-stage disease.

Body temperature and cardiac changes induced by peripherally administered oxytocin, vasopressin and the non-peptide oxytocin receptor agonist WAY 267,464: a biotelemetry study in rats.

BACKGROUND AND PURPOSE: There is current interest in oxytocin (OT) as a possible therapeutic in psychiatric disorders. However, the usefulness of OT may be constrained by peripheral autonomic effects, which may involve an action at both OT and vasopressin V1A receptors. Here, we characterized the cardiovascular and thermoregulatory effects of OT, vasopressin (AVP) and the non-peptide OT receptor agonist WAY 267,464 in rats, and assessed the relative involvement of the OT and V1A receptors in these effects. EXPERIMENTAL APPROACH: Biotelemetry in freely moving male Wistar rats was used to examine body temperature and heart rate after OT (0.01 - 1 mg kg(-1); i.p.), AVP (0.001 - 0.1 mg kg(-1); i.p.) or WAY 267,464 (10 and 100 mg kg(-1); i.p.). The actions of the OT receptor antagonist Compound 25 (C25, 5 and 10 mg kg(-1)) and V1A receptor antagonist SR49059 (1 and 10 mg kg(-1)) were studied, as well as possible V1A receptor antagonist effects of WAY 267,464. KEY RESULTS: OT and AVP dose-dependently reduced body temperature and heart rate. WAY 267,464 had similar, but more modest, effects. SR49059, but not C25, prevented the hypothermia and bradycardia induced by OT and AVP. WAY 267,464 (100 mg·kg(-1)) prevented the effects of OT, and to some extent AVP. CONCLUSIONS AND IMPLICATIONS: Peripherally administered OT and AVP have profound cardiovascular and thermoregulatory effects that appear to principally involve the V1A receptor rather than the OT receptor. Additionally, WAY 267,464 is not a simple OT receptor agonist, as it has functionally relevant V1A antagonist actions.

The nonpeptide oxytocin receptor agonist WAY 267,464: receptor-binding profile, prosocial effects and distribution of c-Fos expression in adolescent rats.

Previous research suggests that the nonpeptide oxytocin receptor (OTR) agonist WAY 267,464 may only partly mimic the effects of oxytocin in rodents. The present study further explored these differences and related them to OTR and vasopressin 1a receptor (V(1a) R) pharmacology and regional patterns of c-Fos expression. Binding data for WAY 267,464 and oxytocin were obtained by displacement binding assays on cellular membranes, while functional receptor data were generated by luciferase reporter assays. For behavioural testing, adolescent rats were tested in a social preference paradigm, the elevated plus-maze (EPM) and for locomotor activity changes following WAY 267,464 (10 and 100 mg/kg, i.p.) or oxytocin (0.1 and 1 mg/kg, i.p.). The higher doses were also examined for their effects on regional c-Fos expression. Results showed that WAY 267,464 had higher affinity (K(i) ) at the V(1a) R than the OTR (113 versus 978 nm). However, it had no functional response at the V(1a) R and only a weak functional effect (EC(50) ) at the OTR (881 nm). This suggests WAY 267,464 is an OTR agonist with weak affinity and a possible V(1a) R antagonist. Oxytocin showed high binding at the OTR (1.0 nm) and V(1a) R (503 nm), with a functional EC(50) of 9.0 and 59.7 nm, respectively, indicating it is a potent OTR agonist and full V(1a) R agonist. WAY 267,464 (100 mg/kg), but not oxytocin, significantly increased the proportion of time spent with a live rat, over a dummy rat, in the social preference test. Neither compound affected EPM behaviour, whereas the higher doses of WAY 267,464 and oxytocin suppressed locomotor activity. WAY 267,464 and oxytocin produced similar c-Fos expression in the paraventricular hypothalamic nucleus, central amygdala, lateral parabrachial nucleus and nucleus of the solitary tract, suggesting a commonality of action at the OTR with the differential doses employed. However, WAY 267,464 caused greater c-Fos expression in the medial amygdala and the supraoptic nucleus than oxytocin, and lesser effects in the locus coeruleus. Overall, our results confirm the differential effects of WAY 267,464 and oxytocin and suggest that this may reflect contrasting actions of WAY 267,464 and oxytocin at the V(1a) R. Antagonism of the V(1a) R by WAY 267,464 could underlie some of the prosocial effects of this drug either through a direct action or through disinhibition of oxytocin circuitry that is subject to vasopressin inhibitory influences.

Is there any correlation between binding and functional effects at the translocator protein (TSPO) (18 kDa)?

The translocator protein (TSPO) is a potential drug target for the treatment of CNS diseases, with TSPO ligands being able to modulate steroidogenesis, apoptosis, and cell proliferation. While there exist multiple TSPO binding sites, the nature of these sites--either overlapping or allosterically linked--remains largely uncharacterized. Furthermore, while evidence suggests that microglial activation and polymerization result in changes to TSPO binding sites, these changes are poorly understood. While current pharmacophoric models can be used to synthesize TSPO ligands with high affinity and selectivity, these models are unable to predict ligands with desirable functional effects. Better characterization of TSPO binding sites in health and disease may provide insight into particular sites which mediate promising therapeutic profiles, thus refining the TSPO pharmacophore.

Evidence for complex binding profiles and species differences at the translocator protein (TSPO) (18 kDa).

The translocator protein (TSPO) (18 kDa) is an emerging drug target for the treatment of numerous pathologies including cancer and neurodegenerative disease. However, our limited knowledge of TSPO binding site(s) has hindered the development of TSPO ligands with potential therapeutic effects. We have synthesized a series of pyrrolobenzoxazepines (1-10) to better characterize the interaction of ligands with the TSPO across species, and to determine their functional profiles. All ligands 1-10 displaced the binding of [3H]PK 11195 to the TSPO at nanomolar concentrations, with discrepancies in binding affinity between rat and human TSPO. Interestingly, non-linear regression analysis revealed that some ligands bound to the protein with a Hill slope not equal to 1.0, suggesting possible additional TSPO binding sites with allosteric effects. However, this trend was not conserved between rat and human. When tested for their effects on pregnenolone production in rat C6 glioma cells, nitric oxide release in murine microglia, and cell proliferation in human MCF-7 breast cancer cells, the pyrrolobenzoxazepines (40 µM) displayed functional effects which did not correlate to the binding trend observed in competition assays. We propose that consideration of species differences and binding site cooperativity, plus optimization of currently accepted functional assays, will aid in the development of drugs targeting TSPO that can be used as therapeutics for human disease.

Resilience and reduced c-Fos expression in P2X7 receptor knockout mice exposed to repeated forced swim test.

There is considerable evidence suggesting genetic factors play an important role in the pathophysiology of depression, possibly by increasing susceptibility to repeated environmental stressors. Recent linkage studies have associated a polymorphism of the gene coding for the P2X7 receptor (P2X7R) with both major depressive disorder and bipolar disorder. Here we assessed whether P2X7 deletion affected the behavioural and neural response to repeated stress. P2X7R knockout (P2X7-/-) mice were subjected to the forced swim test for three consecutive days and neuronal activation in response to the third exposure was assessed using c-Fos immunohistochemistry. In addition, anxiety was evaluated in another group of P2X7-/- mice using the elevated plus maze (EPM) and light dark emergence (LDE) tests. Equivalent levels of immobility were observed in P2X7-/- mice and wild-type (WT) mice on the first exposure to forced swim, but much greater immobility was seen in WT mice on second and third exposures. This suggests that P2X7-/- mice exhibit an impaired adaptive coping response to repeated stress. Reinforcing this view, c-Fos expression in the dentate gyrus of the hippocampus and in the basolateral amygdala was seen in WT mice but not P2X7-/- mice following repeated forced swim. In addition, decreased locomotor activity was detected in P2X7-/- mice without any specific effects on anxiety in the LDE test. However, P2X7-/- mice showed greater anxiety-like behaviour in the EPM. These data suggest that the P2X7R may be involved in the adaptive mechanisms elicited by exposure to repeated environmental stressors that leads to the development of depression-like behaviours. This suggests that P2X7R antagonists may be useful therapeutics for the treatment of major depression, possibly by increasing resilience in the face of repeated stress.

Insights into structure-activity relationships and CNS therapeutic applications of NR2B selective antagonists.

Excessive stimulation of NMDA receptors is involved in various CNS pathologies such as Parkinson's disease, acute and chronic pain and cerebral ischaemia. The use of NMDA antagonists as therapeutic agents has been restricted as a result of unwanted side effects including hallucinations and loss of co-ordination. NR2B subtype selective antagonists have previously shown a therapeutic effect without causing the side effects of broad spectrum NMDA antagonists. Considerable research has since been devoted to the development of orally bioavailable, selective NR2B antagonists and their applications in various neurological diseases. The improved therapeutic index of these compounds is expected to be the result of the subtype selectivity and cellular location of the NR2B receptors within the CNS. This review describes recent advances in the development of NR2B antagonists as well as their therapeutic applications.

Attenuation correction for the large non-human primate brain imaging using microPET.

Assessment of the biodistribution and pharmacokinetics of radiopharmaceuticals in vivo is often performed on animal models of human disease prior to their use in humans. The baboon brain is physiologically and neuro-anatomically similar to the human brain and is therefore a suitable model for evaluating novel CNS radioligands. We previously demonstrated the feasibility of performing baboon brain imaging on a dedicated small animal PET scanner provided that the data are accurately corrected for degrading physical effects such as photon attenuation in the body. In this study, we investigated factors affecting the accuracy and reliability of alternative attenuation correction strategies when imaging the brain of a large non-human primate (papio hamadryas) using the microPET Focus 220 animal scanner. For measured attenuation correction, the best bias versus noise performance was achieved using a (57)Co transmission point source with a 4% energy window. The optimal energy window for a (68)Ge transmission source operating in singles acquisition mode was 20%, independent of the source strength, providing bias-noise performance almost as good as for (57)Co. For both transmission sources, doubling the acquisition time had minimal impact on the bias-noise trade-off for corrected emission images, despite observable improvements in reconstructed attenuation values. In a [(18)F]FDG brain scan of a female baboon, both measured attenuation correction strategies achieved good results and similar SNR, while segmented attenuation correction (based on uncorrected emission images) resulted in appreciable regional bias in deep grey matter structures and the skull. We conclude that measured attenuation correction using a single pass (57)Co (4% energy window) or (68)Ge (20% window) transmission scan achieves an excellent trade-off between bias and propagation of noise when imaging the large non-human primate brain with a microPET scanner.

Behavioural effects of a selective NMDA NR1A/2B receptor antagonist in rats with unilateral 6-OHDA+parafascicular lesions.

Experimental lesions involving the parafascicular (Pf) nucleus and medial forebrain bundle (MFB) may model to some extent the pathological loss of glutamatergic neurons from the centromedian-parafascicular (CM-Pf) complex and nigral dopaminergic cell loss observed clinically at post-mortem in Parkinson's disease (PD) cases. Our study investigated whether there were alterations in symptomatology in such rats with unilateral 6-OHDA+Pf lesions after treatment with either a selective NR1A/NR2B NMDA antagonist and/or l-dopa. Rats were given dual surgery to the MFB with 6-hydroxydopamine (6-OHDA) and Pf with N-methyl-d-aspartate (NMDA). (i) An NR1A/NR2B selective NMDA antagonist (BZAD-01; 10mg/kg), (ii) l-dopa (25mg/kg), (iii) BZAD-01+l-dopa (10mg/kg; 25mg/kg) or (iv) vehicle solution were administered for 6 weeks, during which behavioural testing was performed. BZAD-01 improved postural asymmetry in the first month as well as apomorphine-induced rotation. The latter was also improved by l-dopa in this model. These data support the use of selective NR1/NR2B NMDA antagonists in the therapeutics of PD.

Evaluation of behavioural effects of a selective NMDA NR1A/2B receptor antagonist in the unilateral 6-OHDA lesion rat model.

The degeneration of the dopaminergic nigrostriatal pathway in Parkinson's disease (PD) is associated with altered transmission at striatal NMDA receptors containing NR2B subunits. We investigated a potential novel therapeutic compound, 4-trifluoromethoxy-N-(2-trifluoromethyl-benzyl)-benzamidine (BZAD-01), a selective NMDA NR1A/2B receptor antagonist for PD and compared it with levodopa, the standard treatment for PD. This study also evaluated whether combining levodopa and BZAD-01 gave better improvements of parkinsonian symptoms. Parkinsonism was induced by microinjection of the toxin, 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle (MFB) of 40 Sprague-Dawley rats. Parkinsonism and the efficacy of drugs were assessed using a battery of behavioural tests including balance beam, apomorphine-induced rotation, body axis bias or "curling", head position bias and disengage sensorimotor latency test. Immunohistochemistry was performed on post-mortem tissue to estimate the loss of dopaminergic neurons. The main effects were that BZAD-01 co-administration prevented chronic levodopa-induced potentiation of apomorphine rotation. However levodopa-treated rats were slower than either controls or BZAD-01-treated rats in the locomotor test. The improvement in the apomorphine rotation test suggests that BZAD-01 may be a useful adjunct to levodopa monotherapy.

Neuroprotective effects of a selective N-methyl-D-aspartate NR2B receptor antagonist in the 6-hydroxydopamine rat model of Parkinson's disease.

1. Current pharmacotherapies for the treatment of Parkinson's disease (PD) are largely symptomatic and do not attenuate the characteristic nigral (dopamine) cell loss. 2. Using the 6-hydroxydopamine (6-OHDA) rat model of PD, we investigated the novel, potentially neuroprotective compound BZAD-01, which is an N-methyl-D-aspartate (NMDA) glutamate receptor antagonist selective for the NR2B subunit. 3. Forty female Sprague-Dawley rats were pretreated with either 10 mg/kg BZAD-01 or vehicle (5% sucrose and 0.1% ascorbate) in their drinking water for 11 days prior to and for 3 days following 6-OHDA surgery. During surgery, rats received an injection of either a toxic dose of 16 microg 6-OHDA or a non-toxic dose of 1 microg 6-OHDA (sham) into the medial forebrain bundle. A series of behavioural tests, including curling (measuring body axis bias), head position bias and narrow beam, was performed fortnightly for 8 weeks after surgery to assess the effects of BZAD-01 pretreatment on parkinsonism. Drug-induced rotational asymmetry was also assessed just before rats were killed. Post-mortem immunohistochemistry was performed to quantify the degree of nigral dopamine cell loss. 4. Pretreatment of 6-OHDA-lesioned rats with BZAD-01 significantly reduced the amount of dopamine cell loss and significantly improved all behavioural measures. Furthermore, there was no significant difference in any of the behavioural measures between lesioned rats pretreated with BZAD-01 and rats that underwent sham surgery.

High-resolution imaging of the large non-human primate brain using microPET: a feasibility study.

The neuroanatomy and physiology of the baboon brain closely resembles that of the human brain and is well suited for evaluating promising new radioligands in non-human primates by PET and SPECT prior to their use in humans. These studies are commonly performed on clinical scanners with 5 mm spatial resolution at best, resulting in sub-optimal images for quantitative analysis. This study assessed the feasibility of using a microPET animal scanner to image the brains of large non-human primates, i.e. papio hamadryas (baboon) at high resolution. Factors affecting image accuracy, including scatter, attenuation and spatial resolution, were measured under conditions approximating a baboon brain and using different reconstruction strategies. Scatter fraction measured 32% at the centre of a 10 cm diameter phantom. Scatter correction increased image contrast by up to 21% but reduced the signal-to-noise ratio. Volume resolution was superior and more uniform using maximum a posteriori (MAP) reconstructed images (3.2-3.6 mm(3) FWHM from centre to 4 cm offset) compared to both 3D ordered subsets expectation maximization (OSEM) (5.6-8.3 mm(3)) and 3D reprojection (3DRP) (5.9-9.1 mm(3)). A pilot (18)F-2-fluoro-2-deoxy-d-glucose ([(18)F]FDG) scan was performed on a healthy female adult baboon. The pilot study demonstrated the ability to adequately resolve cortical and sub-cortical grey matter structures in the baboon brain and improved contrast when images were corrected for attenuation and scatter and reconstructed by MAP. We conclude that high resolution imaging of the baboon brain with microPET is feasible with appropriate choices of reconstruction strategy and corrections for degrading physical effects. Further work to develop suitable correction algorithms for high-resolution large primate imaging is warranted.

In vivo evidence for microglial activation in neurodegenerative dementia.

Evidence from numerous neuropathological observations and in vivo clinical imaging studies suggests a prominent role of activated microglia, the main effector cell of the brain's innate immune system, in Alzheimer's disease and other neurodegenerative diseases. Though the comprehensive molecular definition of the microglial activation process is still incomplete, the de novo expression of 'peripheral benzodiazepine-binding sites (PBBS)' by activated but not resting microglia has been established as a useful descriptor of functional state changes in microglia. As microglial transformation to an activated state is closely linked to progressive changes in brain disease, the detection of activated microglia can provide information about disease distribution and rate of disease progression. Positron emission tomography (PET) and [(11)C](R)-PK11195, a specific ligand of the PBBS, have been used to study systematically microglial activation in vivo. Significant microglial activation is present in the brains of patients with neurodegenerative dementia even at early and possibly preclinical stages of the disease with a spatial distribution reflecting different clinical phenotypes. We review some of the posited functions of activated microglia in the pathophysiology of dementia and speculate on the relationship between increased regional [(11)C](R)-PK11195 signals and the ensuing changes in brain volume. Finally, we provide a brief outlook on the development of new radioligands for the PBBS.

Developing a preclinical model of Parkinson's disease: a study of behaviour in rats with graded 6-OHDA lesions.

Injection of increasing concentrations of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle (MFB) can be used to establish a graded model of different clinical stages of Parkinson's disease (PD). We investigated the relationship between behavioural alterations and loss of dopaminergic neurons in the substantia nigra pars compacta (SNc). Forty female Sprague-Dawley rats were injected with either (i) 4 microg (ii) 8 microg or (iii) 16 microg 6-hydroxydopamine (6-OHDA) to mimic the preclinical, mild and advanced clinical stages of PD, respectively. Vehicle was injected in a separate control group. Behaviours analysed included postural asymmetry, balance, locomotion, sensorimotor deficits and apomorphine rotation. At post-mortem the degree of tyrosine immunoreactive dopaminergic cell (TH-ir) loss was then estimated. There was a graded and consistent trend in each of the behaviours studied with respect to cell loss between the different sized lesion groups when examined using correlation analysis (all comparisons, r > 0.8, p < 0.001). Rats with large lesions demonstrated more significant behavioural changes over 8 weeks of testing than those with intermediate and smaller lesions (group comparisons p < 0.001). PD symptomatology became overt when cell loss reached 70%, however some significant changes can be observed with as little as 40% dopaminergic cell loss. Thus, injection with increasing concentrations 6-OHDA into the MFB can produce increasing extents of cell loss and behavioural changes, which were well correlated. This graded model can be useful for testing potential neuroprotective compounds for PD.

Improved synthesis of the peripheral benzodiazepine receptor ligand [11C]DPA-713 using [11C]methyl triflate.

Recently, the pyrazolopyrimidine, [11C] N,N-Diethyl-2-[2-(4-methoxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl]acetamide (DPA-713) has been reported as a new promising marker for the study of peripheral benzodiazepine receptors with positron emission tomography. In the present study, DPA-713 has been labelled from the corresponding nor-analogue using [11C]methyl triflate (CH3OTf). Conditions for HPLC were also modified to include physiological saline (aq. 0.9% NaCl)/ethanol:60/40 as mobile phase making it suitable for injection. The total time of radiosynthesis, including HPLC purification, was 18-20 min. This reported synthesis of [11C]DPA-713, using [11C]CH3OTf, resulted in an improved radiochemical yield (30-38%) compared to [11C]methyl iodide (CH3I) (9) with a simpler purification method. This ultimately enhances the potential of [11C]DPA-713 for further pharmacological and clinical evaluation. These improvements make this radioligand more suitable for automated synthesis which is of benefit where multi-dose preparations and repeated syntheses of radioligand are required.

Radiosynthesis and pharmacological evaluation of [11C]EMD-95885: a high affinity ligand for NR2B-containing NMDA receptors.

EMD-95885, 6-[3-[4-(4-fluorobenzyl)piperidino]propionyl]-3H-benzoxazol-2-one (1) has been described as a selective antagonist for the NMDA receptors containing NR2B subunits, displaying an IC50 of 3.9 nM for this subtype. EMD-95885 (1) has been synthesized in good overall yield and labelled with carbon-11 ( T1/2 : 20.4 min) at its benzoxazolinone moiety using [11C]phosgene. The pharmacological profile of [11C]EMD-95885 ([11C]-1) was evaluated in vivo in rats with biodistribution studies and brain radioactivity monitored with intracerebral radiosensitive beta-microprobes. The brain uptake of [11C]-1 was homogeneous (0.4-0.6%ID/mL) across the different brain structures studied. This in vivo brain regional distribution of [11C]-1 was not consistent with the known distribution of NR2B subunits. Also as a measure of specificity the hippocampus/cerebellum ratio reached 0.8 throughout the time course of the experiment supporting the lack of specificity. Competition studies with the NR2B prototypic ligand ifenprodil and EMD-95885 (1), 30 min before the radioligand injection, displayed homogeneous reduction of [11C]-1 uptake of 40-60%. Pre-treatment of rats with DTG (sigma ligand), MDL105519 (glycine site antagonist) and MK801 (ion channel blocker) had no inhibitory effect on [11C]-1 uptake. Use of haloperidol as a blocking drug also resulted in a homogeneous inhibition of [11C]-1 uptake by 66-60%, which does not reflect binding to dopamine or sigma receptors. Due to the homogeneous radioligand uptake and inhibition and no measure of cerebral blood flow effects during these blocking studies it is uncertain whether any specific binding is observed. In view of these results, [11C]EMD-95885 ([11C]-1) does not have the required properties for imaging NR2B containing NMDA receptors using positron emission tomography.

3-Pyridyl ethers as SPECT radioligands for imaging nicotinic acetylcholine receptors.

To develop a suitable single photon emission computed tomography (SPECT) radioligand for neuronal nicotinic acetylcholine receptors (nAChRs) that displays faster in vivo kinetics than 5-[123I]iodo-A-85380, we synthesised the radioiodinated analogue of A-84543. 5-[123I]Iodo-A-84543 was prepared by electrophilic iododestannylation in a modest yield of 23%. In the baboon brain, 5-[123I]iodo-A-85380 displayed a profile consistent with the known distribution of nAChRs, however, 5-[123I]iodo-A-84543 displayed a homogenous uptake with no preferential localisation in regions known to contain nAChRs. To examine the effect of halogen substitution on the 3-pyridyl ether, A-84543, the 5-chloro, 5-bromo and 5-iodo analogues were synthesised and evaluated with respect to nAChR binding. In vitro binding data revealed that halogen substitution at the 5-position of A-84543 was not well tolerated with an increase in halogen size resulting in lower binding towards nAChRs. The 5-chloro analogue 4 displayed highest affinity, Ki =1.3 nM, compared to the 5-bromo and 5-iodo compounds, 5 Ki =3.3 nM and 3 Ki =40.8 nM, respectively. Taken together, these results clearly indicate that 5-[123I]iodo-A-84543 is not suitable for the study of nAChRs in vivo using SPECT.