Ella Hirani

Imaging the GABA-Benzodiazepine Receptor Subtype Containing the α5-Subunit In Vivo with [11C]Ro15 4513 Positron Emission Tomography:

There is evidence of marked variation in the brain distribution of specific subtypes of the GABA-benzodiazepine receptor and that particular subtypes mediate different functions. The α5-containing subtype is highly expressed in the hippocampus, and selective α5 inverse agonists (which decrease tonic GABA inhibition) are being developed as potential memory-enhancing agents. Evidence for such receptor localization and specialization in humans in vivo is lacking because the widely used probes for imaging the GABA-benzodiazepine receptors, [11C]flumazenil and [123I]iomazenil, appear to reflect binding to the α1 subtype, based on its distribution and affinity of flumazenil for this subtype. The authors characterized for positron emission tomography (PET) a radioligand from Ro15 4513, the binding of which has a marked limbic distribution in the rat and human brain in vivo. Competition studies in vivo in the rat revealed that radiolabeled Ro15 4513 uptake was reduced to nonspecific levels only by drugs that have affinity for the α5 subtype (flunitrazepam, RY80, Ro15 4513, L655,708), but not by the α1 selective agonist, zolpidem. Quantification of [11C]Ro15 4513 PET was performed in humans using a metabolite-corrected plasma input function. [11C]Ro15 4513 uptake was relatively greater in limbic areas compared with [11C]flumazenil, but lower in the occipital cortex and cerebellum. The authors conclude that [11C]Ro15 4513 PET labels in vivo the GABA-benzodiazepine receptor containing the α5 subtype in limbic structures and can be used to further explore the functional role of this subtype in humans.

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‐11C]WAY 100635, was used to image central 5‐HT1A 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 [11C]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 ED50 (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 ∼0.1 mg/kg, resulted in a dose‐dependent decrease in BP, and ED50 values in cortex and hippocampus were estimated as 0.44 ± 0.13 and 0.48 ± 0.12 mg/kg, respectively. The increase in [11C]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. Synapse 36:330–341, 2000.

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

Adenosine A2A receptors are found on striatal neurones projecting to the external pallidum. KW‐6002 (istradefylline) is a potent and selective antagonist for the adenosine A2A 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 [11C]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 [11C]KW‐6002 uptake in the rat brain was seen in striatum and lower levels in cortex and cerebellum. Brain [11C]KW‐6002 uptake was well characterized in humans by a two‐tissue compartmental model with a blood volume term, and the ED50 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 A2A receptors. MRS 1745, an A2B receptor selective antagonist, had no effect on the cerebellar binding of [11C]KW‐6002 in rats, suggesting that this blockable signal is unlikely to result from an affinity for adenosine A2B receptors. Synapse 62:671–681, 2008.

Occupancy of agonist drugs at the 5-HT1A receptor

Drugs acting on the 5-HT1A receptor are used in the treatment of depression, generalized anxiety disorder, and schizophrenia. This study investigated 5-HT1A receptor occupancy by the 5-HT1A agonist drugs flesinoxan (a highly selective probe for the 5-HT1A receptor) and ziprasidone (a novel atypical antipsychotic drug). Using a within-subject design, 14 healthy volunteers each received two positron emission tomography scans using the selective 5-HT1A antagonist radiotracer [11C]WAY-100635. One scan constituted a baseline, while the other followed either 1 mg flesinoxan or 40 mg ziprasidone orally. In addition, rats were pretreated with intravenous flesinoxan at doses ranging from 0.001 to 5 mg/kg then [11C]WAY-100635 binding measured ex vivo. Cerebral cortical and hippocampal regions of interest, and cerebellar reference regions were sampled to estimate 5-HT1A receptor occupancy (inferred from reductions in specific radioligand binding). In man, occupancy was not significant despite volunteers experiencing side effects consistent with central serotonergic activity. The mean cerebral cortex occupancy (±1 SD) for flesinoxan was 8.7% (±13%), and for ziprasidone 4.6% (±17%). However, in rats, flesinoxan achieved significant and dose-related occupancy (17–57%) at 0.25 mg/kg and above. We conclude that 5-HT1A receptor agonists produce detectable occupancy only at higher doses that would produce unacceptable levels of side effects in man, although lower doses are sufficient to produce pharmacological effects. The development of agonist radiotracers may increase the sensitivity of detecting agonist binding, as 5-HT1A antagonists bind equally to low- and high-affinity receptor states, while agonists bind preferentially to the high-affinity state.

In vivo multimodal imaging of stem cell transplantation in a rodent model of Parkinson's disease

Stem cell therapy in the nervous system aims to replace the lost neurons and provide functional recovery. However, it is imperative that we understand the in vivo behaviour of these cells post-implantation. We report visualisation of iron oxide labelled bone marrow-derived stem cells (BMSCs) implanted into the striatum of hemi-parkinsonian rats by magnetic resonance imaging (MRI). Functional efficacy of the donor cells was monitored in vivo using the positron emission tomography (PET) radioligand [11C]raclopride. The cells were visible for 28 days by in vivo MRI. BMSCs provided functional recovery demonstrated by a decreased binding of [11C]raclopride. Although, histology confirmed the persistence of donor cells, no tyrosine hydroxylase positive cells were present. This suggests that BMSCs may have a limited paracrine effect and influence functional recovery. We demonstrate, using multimodal imaging, that we can not only track BMSCs but also establish their effects in a pre-clinical model of Parkinsons disease.

Low Sensitivity of the Positron Emission Tomography Ligand [11C]Diprenorphine to Agonist Opiates

Previously, we reported minimal opioid receptor occupancy following a clinical dose of the μ-opioid agonist, methadone, measured in vivo using positron emission tomography (PET) with [11C]diprenorphine and subsequently used rats to obtain experimental data in support of a high receptor reserve hypothesis (Melichar et al., 2005). Here, we report on further preclinical studies investigating opioid receptor occupancy with oxycodone (μ- and κ-receptor agonist), morphine (μ-receptor agonist), and buprenorphine (partial agonist at the μ-receptor and antagonist at the δ- and κ-receptors), each given at antinociceptive doses. In vivo binding of [11C]diprenorphine was not significantly reduced after treatment with the full agonists but was reduced by ∼90% by buprenorphine. In addition, given that [11C]diprenorphine is a non-subtype-specific PET tracer, there was no regional variation that might feasibly be interpreted as due to differences in opioid subtype distribution. The data support minimal competition between the high-efficacy agonists and the non-subtype-selective antagonist radioligand and highlight the limitations of [11C]diprenorphine PET to monitor in vivo occupancy. Alternative means may be needed to address clinical issues regarding opioid receptor occupancy that are required to optimize treatment strategies.

Further evaluation of the carbon11-labeled D(2/3) agonist PET radiotracer PHNO: reproducibility in tracer characteristics and characterization of extrastriatal binding.

[11C]‐(+)‐PHNO is a new dopamine D2/3 receptor agonist radiotracer which has been successfully used to measure D2/3 receptor availability in experimental animals and man. Here we report in vivo evaluation in the rat of the biodistribution, metabolism, specificity, selectivity, and dopamine sensitivity of carbon11‐labeled PHNO ([11C]‐3‐PHNO) produced by an alternative radiochemical synthesis method. [11C]‐3‐PHNO showed rapid metabolism and clearance from most peripheral organs and tissues. [11C]‐3‐PHNO, but not its polar metabolite, readily crossed the blood‐brain barrier and showed high levels of uptake in the D2/3‐rich striatum. Pretreatment with unlabeled PHNO and the D2/3 receptor antagonist raclopride indicated that binding in the striatum was specific and selective to D2/3 receptors. PET studies in anesthetized rats revealed significant reductions in [11C]‐3‐PHNO binding in the striatum following amphetamine administration, indicating sensitivity to increases in endogenous dopamine concentrations. D2/3 antagonist pretreatment additionally indicated moderate levels of [11C]‐3‐PHNO specific binding in several extrastriatal brain areas—most notably the olfactory bulbs and tubercles, thalamus, and hypothalamus. Of particular interest, approximately 30% of [11C]‐3‐PHNO signal in the cerebellum—a region often used as a “low‐binding” reference region for PET quantification—was attributable to specific signal. These data demonstrate that [11C]‐3‐PHNO shows similar tracer characteristics to [11C]‐(+)‐PHNO, but additionally indicate that radiolabeled PHNO may be used to estimate D2/3 receptor availability in select extrastriatal brain regions with PET. Synapse 64:301–312, 2010.

Evaluation of [methyl-3H]L655,708 and [ethyl-3H]RY80 as putative PET ligands for central GABAA receptors containing α5 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.

CHAPTER 45 - Preliminary Evaluation of the Glycine Site Antagonists [ 11 C]L 703,717 and [ 3 H]MDL 105,519 as Putative PET Ligands for Central NMDA Receptors: In Vivo Studies in Rats

Two selective antagonists of the glycine site of the N-methyl-D-aspartate (NMDA) receptor—[3H]MDL 105,519, (Z)-2(phenyl)-3- (4,6-dichloroindol-3-yl-2-carboxylic acid) propenoic acid, and [11C]L703,717, 3-substituted 4-hydroxyquinolin-2-(lH)-one—were evaluated in rats as potential in vivo ligands for positron emission tomography (PET). Labeling of L703,717 was carried out by reaction of the phenolic precursor with [11C]iodomethane, giving 98% radiochemical purity and a specific activity of 30–40 GBq/μmol at the end of synthesis. Following intravenous injection of [3H]MDL 105,519 or [11C]L 703,717, radioactivity in plasma was measured and uptake characteristics were assessed in brain within a time period relevant to PET scanning (up to 60 min). Discrete brain regions, such as frontal cortex, entorhinal cortex, striatum, hypothalamus, thalamus, hippocampus, colliculi, medulla oblongata, and cerebellum, were sampled and the temporal distribution of radioactivity analyzed. Additionally, in vitro plasma protein binding of both radioligands was assessed, and the brain uptake index of [3H]MDL 105,519 was measured to evaluate the first-pass extraction. Both [3H]MDL 105,519 and [11C]L 703,717 had a very poor extraction in the rat brain, and consequently, a very low regional biodistribution, in the range of 0.01–0.1% injected dose per gram of tissue, during the time studied. This was consistent with their very high (> 99.8%) in vitro binding to plasma proteins and a pronounced in vivo retention of the radioactivity in plasma. Neither of the glycine site antagonists studied show potential for further development as in vivo PET ligands.