Sharon Ashworth

The design and physical characteristics of a small animal positron emission tomograph

A small diameter positron emission tomography, designed specifically for small animal studies, was constructed from existing, commercially available, bismuth germanate (BGO) detectors and electronics. The scanner consists of 16 BGO detector blocks arranged to give a tomograph with a diameter of 115 mm and an axial field of view (FOV) of 50 mm. Each block is cut to produce eight (axial) by seven (radial) individual detector elements. The absence of interplane septa enables the acquisition of 3D data sets consisting of 64 sinograms. A 2D data set of 15 sinograms, consisting of eight direct and seven adjacent cross planes, can be extracted from the 3D data set. Images are reconstructed from the 2D sinograms using a conventional filtered backprojection algorithm. Two methods of normalization were investigated, based on either a rotating 68Ge rod source, or a uniform 68Ge plane source, with a uniform cylindrical 18F phantom. Attenuation of the emitted photons was estimated using a rotating 68Ge rod source. The transaxial resolution of the tomograph was measured as 2.3 mm full width at half maximum (FWHM) and 5.6 mm full width at tenth maximum (FWTM) at the centre of the FOV, degrading to 6.6 mm (radial) and 4.4 mm (tangential) FWHM and 10.4 mm (radial) and 14.4 mm (tangential) FWTM at 40.0 mm from the centre of the FOV. The axial slice width was 4.3 mm FWHM, 10.3 mm FWTM at the centre of the transaxial field of view and 4.4 mm FWHM, 10.6 mm FWTM at 20.0 mm from the centre of the FOV. A scatter fraction of 31.0% was measured at 250-850 keV, for an 18F line source centred in a 60 mm diameter, water-filled phantom, reducing to 20.4% and 13.8% as the lower energy discrimination was increased to 380 keV and 450 keV, respectively. The count rate performance was measured using a noise equivalent count rate method, and the linearity of the dead time correction was confirmed over the count rates encountered during routine scanning. In 2D mode, the absolute sensitivity of the tomograph was measured as 9948 counts s-1 MBq-1 at 250-850 keV, 8284 counts s-1 MBq-1 at 380-850 keV and 6280 counts s-1 MBq-1 at 450-850 keV.

Synthesis of the enantiomers of [N-methyl-11C]PK 11195 and comparison of their behaviours as radioligands for PK binding sites in rats

The enantiomers of [N-methyl-11C]PK 11195, a radioligand for PET studies of PK (peripheral benzodiazepine) binding sites, have been prepared from the newly synthesized N-desmethyl-enantiomers by 11C-methylation with N.C.A. [11C]iodomethane. The brain uptake and retention of each enantiomer was compared with that of the racemic radioligand after i.v. administration into normal rats and into rats with focal cortical lesions. No significant differences in the uptakes of the enantiomers were observed in regions devoid of PK binding sites. However, the R-enantiomer was retained to a significantly greater extent than the S-enantiomer in olfactory bulbs-tubercles, which contain some PK binding sites, and also in 9-day-old focal cortical lesions, which are greatly enriched in PK binding sites associated with macrophage infiltration. The observed differences are consistent with the approximately 2-fold greater affinity of the R-enantiomer for PK binding sites reported in vitro and imply that the use of this enantiomer would have advantages over the use of the racemate currently used for PET studies.

The potential of high-resolution positron emission tomography to monitor striatal dopaminergic function in rat models of disease

The use of a recently commissioned small-diameter, high-resolution positron emission tomography (PET) to obtain a measure of specific binding of 3 carbon-11 labelled ligands in rat striatum is described. Using cerebellum as a reference tissue, compartmental modelling was used to obtain individual estimates of striatal binding potential (defined as the ratio of rate constants to and from the specifically bound compartment) for [11C]raclopride (D2 receptors), [11C]SCH 23390 (D1 receptors) and [11C]RTI-121 (dopamine transporter). The coefficients of variation in control, anaesthetized rats were of the order of 10%. Using two models of human disease, namely striatal injection of ibotenic acid to produce postsynaptic cell loss as in Huntingtons disease, and 6-hydroxydopamine injection into substantia nigra pars compacta to mimic dopaminergic terminal loss in Parkinsons disease, marked reductions in binding potential were observed for the corresponding pre- or postsynaptic markers. When the regions of interest are so small as to be of the order of the spatial resolution of the system, factor such as spill over and partial volume negate absolute quantification of tissue radioactivity. Nevertheless, the use of PET to monitor relative changes in dopaminergic integrity should be considered as a viable complement to established in vivo microdialysis and post mortem techniques.

GDNF protects against 6-OHDA nigrostriatal lesion: in vivo study with microdialysis and PET.

We have investigated whether glial cell line-derived neurotrophic factor (GDNF) protects against a complete unilateral 6-hydroxydopamine (6-OHDA) nigrostriatal lesion, a robust rat model of Parkinsons disease. GDNF or vehicle were administered above the rat substantia nigra and into the lateral ventricle immediately before an ipsilateral 6-OHDA injection into the medial forebrain bundle. In vivo tests were employed to assess the effects of the treatment: microdialysis to measure striatal dopamine release, amphetamine challenge to estimate turning behaviour, and positron emission tomography (PET) to image dopamine reuptake sites. The present results show that GDNF can protect dopaminergic neurones against an acute and irreversible 6-OHDA lesion. They are encouraging for potential use of GDNF in Parkinsons disease.

Evaluation of [O-methyl-3H]WAY-100635 as an in vivo radioligand for 5-HT1A receptors in rat brain

N-(2-(4-(2-Methoxyphenyl)-1-piperazinyl)ethyl)-N-(2- pyridyl)cyclohexanecarboxamide trihydrochloride (WAY-100635) is a new, potent and selective 5-HT1A receptor antagonist. We have evaluated radiolabelled WAY-100635 as a prospective radioligand for positron emission tomography (PET) by studying biodistribution in rat ex vivo. After intravenous injection, [O-methyl-3H]WAY-100635 cleared rapidly from plasma but was retained in brain. Specific binding was quantified from kinetic studies, using a reference-tissue compartment model, fitting for binding potential (k3/k4). The regional variation in binding potential correlated with the known distribution of 5-HT1A receptors. Saturation studies gave Bmax values in vivo that were consistent with those reported in vitro. At 60 min after injection, the ratio of radioactivity in 5-HT1A receptor-rich regions (e.g. septum, entorhinal cortex and hippocampus) to that in cerebellum reached approximately 16. Pre-dosing the rats with WAY-100635 (2 mg/kg) reduced this ratio to one, whereas similar pre-dosing with citalopram (5-HT uptake site inhibitor), prazosin (alpha 1A-adrenoceptor antagonist) or idazoxan (alpha 2-adrenoceptor antagonist) caused little or no reduction. Substantial (77%) blockade of [3H]WAY-100635 binding was achieved with the 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), and the partial agonists, ipsapirone and buspirone. Thus, the properties of WAY-100635 are such that, when labelled with carbon-11, it could provide a radioligand suitable for clinical and pharmacological investigations of central 5-HT1A receptors in man using PET.

Evaluation of 11C-GSK189254 as a Novel Radioligand for the H3 Receptor in Humans Using PET

The histamine H3 receptor is implicated in the pathophysiology of several central nervous system disorders. N-methyl-6-(3-cyclobutyl-2,3,4,5-tetrahydro-1H-benzo[d]azepin-7-yloxy)-nicotamide (GSK189254) is a highly potent, selective, and brain-penetrant H3 receptor antagonist. Previous studies in the pig using PET have shown that 11C-GSK189254 uptake in H3-rich regions of the brain can be blocked by the selective H3 antagonist ciproxifan. The purpose of the present study was to evaluate 11C-GSK189254 as a PET radioligand for human studies and to determine the dose–receptor occupancy relationship of GSK189254 in the human brain. Methods: Dynamic PET scans were obtained in healthy subjects over 90 min after intravenous administration of approximately 370 MBq of 11C-GSK189254. Blood samples were taken throughout the scans to derive the arterial plasma parent input function. Each subject was scanned twice, either with tracer alone (test–retest) or before and after a single oral dose of GSK189254 (10–100 μg). Data were analyzed by compartmental analysis, and regional receptor-occupancy estimates were obtained by graphical analysis of changes in the total volumes of distribution (VT) of the radioligand. Results: 11C-GSK189254 readily entered the brain; its regional brain distribution reflected the known distribution of H3 receptors, with high binding in the caudate and putamen, intermediate binding in cortical regions, and low binding in the cerebellum. GSK189254 displayed a high receptor affinity, and a marked reduction in VT was apparent at all the doses tested. The oral dose equaling 50% occupancy of the available receptor sites (ED50) was estimated as 4.33 μg. Additional data on plasma pharmacokinetics after oral dosing and the plasma free fraction gave a corresponding estimate of the free concentration of GSK189254 required to occupy 50% of the available receptor sites (EC50) (0.011 nM). The test–retest data showed reductions in regional VT on the second scan in all subjects. A nonlinear compartmental analysis of this effect demonstrated that this reduction was consistent with carryover of a tracer mass dose effect with an estimated in vivo apparent dissociation constant of 0.010 nM, close to the independent estimate of the plasma EC50. Conclusion: 11C-GSK189254 can be used to quantify H3 receptor availability in humans in vivo using PET but requires high specific activity; the possibility of tracer mass dose effects should be carefully analyzed.

Assessment of striatal graft viability in the rat in vivo using a small diameter PET scanner.

A small diameter positron emission tomography (PET) scanner has been used to monitor [11C]raclopride (D2 receptor) binding in vivo in either intact striatum, denervated striatum following an excitotoxic lesion with ibotenic acid, or lesioned and grafted striatum following implantation of cortical or striatal tissue grafts in rats. Binding of [11C]raclopride was localized in the intact striatum within 20 min of injection of the radioligand, and was much reduced within the lesioned striatum. Cortical grafts exhibited a similar low level of binding to the lesioned striatum, whereas striatal grafts showed specific binding at an intermediate level. The [11C]raclopride binding signal in vivo correlated well with the extent of surviving or grafted striatal tissue observed post morten by Nissl staining and acetylcholinesterase histochemistry. Thus, the distribution of dopamine receptors as seen in the PET scanner are consistent with post mortem anatomical observations of striatal, lesion and graft sizes, and suggest that PET can provide a useful tool for monitoring the viability of implanted striatal graft tissues in vivo.

CHAPTER 3 – Quantification of Dopamine Receptors and Transporter in Rat Striatum Using a Small Animal PET Scanner

This chapter reports on the use of a system to quantify specific binding of three positron-emitting tracers in rat striatum in vivo: [ 11 C]SCH 23390 (D 1 receptor antagonist), [ 11 C]raclopride (D 2 receptor antagonist), and the recently described dopamine transporter ligand [ 11 C]RTI-121. A dedicated small-diameter positron emission tomography (PET) scanner has been used to quantify binding of three positron-emitting tracers in rat striatum in vivo: [ 11 C]SCH 23390 (D 1 receptor antagonist), [ 11 C]-raclopride (D 2 receptor antagonist), and the dopamine transporter ligand [ 11 C]RTI-121. As the sizes of the striatal regions of interest used for analysis are of the same order as the dimensions of a detector element (3–4 mm), slight differences in the position of the rat within the scanner significantly affected the final images. Rats therefore were held in a perspex stereotaxic frame during scanning, thus abolishing movement and allowing precise positioning such that the striata were at the center of the field of view. Data are reported from a total of 31 previously untreated rats scanned over a period of 17 months. In addition, for each radioligand, groups of three rats were scanned after pretreatment with a blocking dose of the stable compound. Specific binding was quantified from the kinetic data using a reference tissue compartment model, with the cerebellum as an indirect input function. The results demonstrate that regional time-radioactivity data acquired using small animal positron emission tomography (PET) can provide reproducible and consistent quantitative information on pre- and postsynaptic dopaminergic function in rat striatum and that the system is sufficiently sensitive to allow the study of animal models of disease.

Modulatory effects of L-DOPA on D2 dopamine receptors in rat striatum, measured using in vivo microdialysis and PET

Summary. Putative modulatory effects of L-3,4-dihydroxyphenylalanine (L-DOPA) on D2 dopamine receptor function in the striatum of anaesthetised rats were investigated using both in vivo microdialysis and positron emission tomography (PET) with carbon-11 labelled raclopride as a selective D2 receptor ligand. A single dose of L-DOPA (20 or 100 mg/kg i.p.) resulted in an increase in [11C]raclopride binding potential which was also observed in the presence of the central aromatic decarboxylase inhibitor NSD 1015, confirming that the effect was independent of dopamine. This L-DOPA evoked D2 receptor sensitisation was abolished by a prior, long-term administration of L-DOPA in drinking water (5 weeks, 170 mg/kg/day). In the course of acute L-DOPA treatment (20 mg/kg), extracellular GABA levels were reduced by ∼20% in the globus pallidus. It is likely that L-DOPA sensitising effect on striatal D2 receptors, as confirmed by PET, may implicate striato-pallidal neurones, hence a reduced GABA-ergic output in the projection area. Since the L-DOPA evoked striatal D2 receptor supersensitivity habituates during long-term treatment, the effects reported here may contribute to the fluctuations observed during chronic L-DOPA therapy in Parkinsons disease.

Evaluation in rat of RS-79948-197 as a potential PET ligand for central α2-adrenoceptors

Tritium-labelled RS-79948-197 {(8aR, 12aS, 13aS)-5,8,8a,9,10,11,12,12a,13,13a-decahydro-3-methoxy-12-(ethylsulphonyl)-6H-isoquino[2,1-g][1,6]naphthyridine} was evaluated in rat brain as an in vivo ligand for central α2-adrenoceptors, as a preliminary step in the development of a radioligand for positron-emission tomography (PET) studies. The maximal receptor-specific signal was achieved within 90–120 min after i.v. injection of [ethyl-3H]RS-79948-197 and was selective for the α2-compared with the α1-adrenoceptor, with no detectable binding to the imidazoline-I2 site. Estimates for binding potential (approximating to BmaxKd) ranged between 3.4 in entorhinal cortex and 0.5 in medulla oblongata. The results, which indicate a similarly localised but 2-fold increase in specific binding compared with that previously demonstrated using [3H]RX 821002 (2-methoxy-idazoxan), are sufficiently encouraging as to support further investment in the development of 11C-labelled RS-79948-197, or a close structural analogue, as a ligand for clinical PET.