Imidazoline-I2 PET Tracers in Neuroimaging.

Targeting neuroinflammation, and in particular, microglial activation and astrocytosis, is a current area of the focus of new treatment interventions for a number of neurodegenerative disorders. Probing the roles of microglia and astrocytes in human disease requires the development of useful tools, such as PET imaging tools that are specific for the cell type(s) of interest. This review concentrates on the recent advances in the development of Imidazoline2 binding site (I2BS) PET tracers, which are purported to target astrocytes, and hence could represent key clinical imaging tools for targeting astrocytes in neurodegenerative disease. Five PET tracers for the I2BS are described in this review, with only one (11C-BU99008) being currently validated to GMP for clinical use, and data reported from healthy volunteers, Alzheimer's disease patients, and Parkinson's disease patients. The clinical data utilising 11C-BU99008 have revealed the potential early involvement of astrogliosis in neurodegeneration that might precede the activation of microglia, which, if confirmed, could provide a vital new means for potentially targeting neurodegeneration earlier in the disease course.

Astrogliosis in aging and Parkinson's disease dementia: a new clinical study with 11C-BU99008 PET.

The role of astrogliosis in the pathology of brain aging and neurodegenerative diseases has recently drawn great attention. Imidazoline-2 binding sites represent a possible target to map the distribution of reactive astrocytes. In this study, we use 11C-BU99008, an imidazoline-2 binding sites-specific PET radioligand, to image reactive astrocytes in vivo in healthy controls and patients with established Parkinson's disease dementia. Eighteen healthy controls (age: 45-78 years) and six patients with Parkinson's disease dementia (age: 64-77 years) had one 11C-BU99008 PET-CT scan with arterial input function. All subjects underwent one 3 T MRI brain scan to facilitate the analysis of the PET data and to capture individual cerebral atrophy. Regional 11C-BU99008 volumes of distribution were calculated for each subject by the two-tissue compartmental modelling. Positive correlations between 11C-BU99008 volumes of distribution values and age were found for all tested regions across the brain within healthy controls (P < 0.05); furthermore, multiple regression indicated that aging affects 11C-BU99008 volumes of distribution values in a region-specific manner. Independent samples t-test indicated that there was no significant group difference in 11C-BU99008 volumes of distribution values between Parkinson's disease dementia (n = 6; mean age = 71.97 ± 4.66 years) and older healthy controls (n = 9; mean age = 71.90 ± 5.51 years). Our data set shows that astrogliosis is common with aging in a region-specific manner. However, in this set-up, 11C-BU99008 PET cannot differentiate patients with Parkinson's disease dementia from healthy controls of similar age.

Functional Alternatives to Alcohol.

The consumption of alcohol is associated with well-known health harms and many governments worldwide are actively engaged in devising approaches to reduce them. To this end, a common proposed strategy aims at reducing alcohol consumption. This approach has led to the development of non-alcoholic drinks, which have been especially welcome by younger, wealthier, health-conscious consumers, who have been turning away from alcohol to look toward alternatives. However, a drawback of non-alcoholic drinks is that they do not facilitate social interaction in the way alcohol does, which is the main reason behind social drinking. Therefore, an alternative approach is to develop functional drinks that do not use alcohol yet mimic the positive, pro-social effects of alcohol without the associated harms. This article will discuss (1) current knowledge of how alcohol mediates its effects in the brain, both the desirable, e.g., antistress to facilitate social interactions, and the harmful ones, with a specific focus on the pivotal role played by the gamma-aminobutyric acid (GABA) neurotransmitter system and (2) how this knowledge can be exploited to develop functional safe alternatives to alcohol using either molecules already existing in nature or synthetic ones. This discussion will be complemented by an analysis of the regulatory challenges associated with the novel endeavour of bringing safe, functional alternatives to alcohol from the bench to bars.

Relationship between astrocyte reactivity, using novel 11C-BU99008 PET, and glucose metabolism, grey matter volume and amyloid load in cognitively impaired individuals.

Post mortem neuropathology suggests that astrocyte reactivity may play a significant role in neurodegeneration in Alzheimer's disease. We explored this in vivo using multimodal PET and MRI imaging. Twenty subjects (11 older, cognitively impaired patients and 9 age-matched healthy controls) underwent brain scanning using the novel reactive astrocyte PET tracer 11C-BU99008, 18F-FDG and 18F-florbetaben PET, and T1-weighted MRI. Differences between cognitively impaired patients and healthy controls in regional and voxel-wise levels of astrocyte reactivity, glucose metabolism, grey matter volume and amyloid load were explored, and their relationship to each other was assessed using Biological Parametric Mapping (BPM). Amyloid beta (Aß)-positive patients showed greater 11C-BU99008 uptake compared to controls, except in the temporal lobe, whilst further increased 11C-BU99008 uptake was observed in Mild Cognitive Impairment subjects compared to those with Alzheimer's disease in the frontal, temporal and cingulate cortices. BPM correlations revealed that regions which showed reduced 11C-BU99008 uptake in Aß-positive patients compared to controls, such as the temporal lobe, also showed reduced 18F-FDG uptake and grey matter volume, although the correlations with 18F-FDG uptake were not replicated in the ROI analysis. BPM analysis also revealed a regionally-dynamic relationship between astrocyte reactivity and amyloid uptake: increased amyloid load in cortical association areas of the temporal lobe and cingulate cortices was associated with reduced 11C-BU99008 uptake, whilst increased amyloid uptake in primary motor and sensory areas (in which amyloid deposition occurs later) was associated with increased 11C-BU99008 uptake. These novel observations add to the hypothesis that while astrocyte reactivity may be triggered by early Aß-deposition, sustained pro-inflammatory astrocyte reactivity with greater amyloid deposition may lead to astrocyte dystrophy and amyloid-associated neuropathology such as grey matter atrophy and glucose hypometabolism, although the evidence for glucose hypometabolism here is less strong.

Impact of COVID-19 restrictions on alcohol consumption behaviours.

BACKGROUND: We aimed to evaluate how coronavirus (COVID-19) restrictions had altered individual's drinking behaviours, including consumption, hangover experiences, and motivations to drink, and changing levels of depression and anxiety. METHOD: We conducted an online cross-sectional self-report survey. Whole group analysis compared pre- versus post-COVID restrictions. A correlation coefficient matrix evaluated the associations between all outcome scores. Self-report data was compared with Alcohol Use Disorders Identification Test (AUDIT) scores from the 2014 Adult Psychiatric Morbidity Survey. Multiple linear modelling (MLM) was calculated to identify factors associated with increasing AUDIT scores and post-restriction AUDIT scores. RESULTS: In total, 346 individuals completed the survey, of which 336 reported drinking and were therefore analysed. After COVID-19 restrictions 23.2% of respondents reported an increased AUDIT score, and 60.1% a decreased score. AUDIT score change was positively correlated with change in depression (P < 0.01, r = 0.15), anxiety (P < 0.01, r = 0.15) and drinking to cope scores (P < 0.0001, r = 0.35). MLM revealed that higher AUDIT scores were associated with age, mental illness, lack of a garden, self-employed or furloughed individuals, a confirmed COVID-19 diagnosis and smoking status. CONCLUSIONS: COVID-19 restrictions decreased alcohol consumption for the majority of individuals in this study. However, a small proportion increased their consumption; this related to drinking to cope and increased depression and anxiety.

Astrocyte reactivity with late-onset cognitive impairment assessed in vivo using 11C-BU99008 PET and its relationship with amyloid load.

11C-BU99008 is a novel positron emission tomography (PET) tracer that enables selective imaging of astrocyte reactivity in vivo. To explore astrocyte reactivity associated with Alzheimer's disease, 11 older, cognitively impaired (CI) subjects and 9 age-matched healthy controls (HC) underwent 3T magnetic resonance imaging (MRI), 18F-florbetaben and 11C-BU99008 PET. The 8 amyloid (Aß)-positive CI subjects had higher 11C-BU99008 uptake relative to HC across the whole brain, but particularly in frontal, temporal, medial temporal and occipital lobes. Biological parametric mapping demonstrated a positive voxel-wise neuroanatomical correlation between 11C-BU99008 and 18F-florbetaben. Autoradiography using 3H-BU99008 with post-mortem Alzheimer's brains confirmed through visual assessment that increased 3H-BU99008 binding localised with the astrocyte protein glial fibrillary acid protein and was not displaced by PiB or florbetaben. This proof-of-concept study provides direct evidence that 11C-BU99008 can measure in vivo astrocyte reactivity in people with late-life cognitive impairment and Alzheimer's disease. Our results confirm that increased astrocyte reactivity is found particularly in cortical regions with high Aß load. Future studies now can explore how clinical expression of disease varies with astrocyte reactivity.

Acute effects of MDMA on trust, cooperative behaviour and empathy: A double-blind, placebo-controlled experiment.

BACKGROUND: 3,4-Methylenedioxymethamphetamine (MDMA) is being actively researched as an adjunct to psychotherapy. It may be beneficial to trust, empathy and cooperative behaviour due to its acute prosocial effects. AIM: To test (a) the acute effects of MDMA on measures of empathy, trust and cooperative behaviour, and (b) subacute changes in mood three days after MDMA administration. METHODS: Twenty-five participants (n=7 female), participated in this double-blind, repeated-measures, placebo-controlled experiment. Participants attended two acute sessions, one week apart. Each acute session was followed by a subacute session three days later. Participants received placebo (100 mg ascorbic acid) during one acute session, and MDMA (100 mg MDMA-HCl) at the other, with order counterbalanced. Participants completed the following tasks assessing prosocial behaviour: a trust investment task, a trustworthy face rating task, an empathic stories task, a public project game, a dictator game and an ultimatum game. Participants reported subjective effects. Blood was taken pre-drug, 2 and 4 hours post-drug, and tested for plasma MDMA levels. RESULTS: MDMA acutely increased self-reported 'closeness to others' and 'euphoria' and increased plasma concentrations of MDMA. MDMA did not significantly change task-based empathy, trust or cooperative behaviour. Using Bayesian analyses, we found evidence that MDMA and placebo did not differ in their effects on empathy and cooperative behaviour. MDMA did not significantly change subacute mood and this was supported by our Bayesian analyses. CONCLUSION: Despite augmentation in plasma MDMA levels and subjective drug effects, we found no increase in prosocial behaviour in a laboratory setting.

Imidazoline ligand BU224 reverses cognitive deficits, reduces microgliosis and enhances synaptic connectivity in a mouse model of Alzheimer's disease.

BACKGROUND AND PURPOSE: Activation of type 2 imidazoline receptors has been shown to exhibit neuroprotective properties including anti-apoptotic and anti-inflammatory effects, suggesting a potential therapeutic value in Alzheimer's disease (AD). Here, we explored the effects of the imidazoline-2 ligand BU224 in a model of amyloidosis. EXPERIMENTAL APPROACH: Six-month-old female transgenic 5XFAD and wild-type (WT) mice were treated intraperitoneally with 5-mg·kg-1 BU224 or vehicle twice a day for 10 days. Behavioural tests were performed for cognitive functions and neuropathological changes were investigated by immunohistochemistry, Western blot, elisa and qPCR. Effects of BU224 on amyloid precursor protein (APP) processing, spine density and calcium imaging were analysed in brain organotypic cultures and N2a cells. KEY RESULTS: BU224 treatment attenuated spatial and perirhinal cortex-dependent recognition memory deficits in 5XFAD mice. Fear-conditioning testing revealed that BU224 also improved both associative learning and hippocampal- and amygdala-dependent memory in transgenic but not in WT mice. In the brain, BU224 reduced levels of the microglial marker Iba1 and pro-inflammatory cytokines IL-1ß and TNF-α and increased the expression of astrocytic marker GFAP in 5XFAD mice. These beneficial effects were not associated with changes in amyloid pathology, neuronal apoptosis, mitochondrial density, oxidative stress or autophagy markers. Interestingly, ex vivo and in vitro studies suggested that BU224 treatment increased the size of dendritic spines and induced a threefold reduction in amyloid-ß (Aß)-induced functional changes in NMDA receptors. CONCLUSION AND IMPLICATIONS: Sub-chronic treatment with BU224 restores memory and reduces inflammation in transgenic AD mice, at stages when animals display severe pathology.

Imidazoline 2 binding sites reflecting astroglia pathology in Parkinson's disease: an in vivo11C-BU99008 PET study.

Astroglia are multifunctional cells that regulate neuroinflammation and maintain homeostasis within the brain. Astroglial α-synuclein-positive cytoplasmic accumulations have been shown post-mortem in patients with Parkinson's disease and therefore astroglia may play an important role in the initiation and progression of Parkinson's disease. Imidazoline 2 binding sites are expressed on activated astroglia in the cortex, hippocampus, basal ganglia and brainstem; therefore, by measuring imidazoline 2 binding site levels we can indirectly evaluate astrogliosis in patients with Parkinson's disease. Here, we aimed to evaluate the role of astroglia activation in vivo in patients with Parkinson's disease using 11C-BU99008 PET, a novel radioligand with high specificity and selectivity for imidazoline 2 binding sites. Twenty-two patients with Parkinson's disease and 14 healthy control subjects underwent 3 T MRI and a 120-min 11C-BU99008 PET scan with volume of distribution (VT) estimated using a two-tissue compartmental model with a metabolite corrected arterial plasma input function. Parkinson's disease patients were stratified into early (n = 8) and moderate/advanced (n = 14) groups according to disease stage. In early Parkinson's disease, increased 11C-BU99008 VT uptake was observed in frontal (P = 0.022), temporal (P = 0.02), parietal (P = 0.026) and occipital (P = 0.047) cortical regions compared with healthy controls. The greatest 11C-BU99008 VT increase in patients with early Parkinson's disease was observed in the brainstem (52%; P = 0.018). In patients with moderate/advanced Parkinson's disease, loss of 11C-BU99008 VT was observed across frontal (P = 0.002), temporal (P < 0.001), parietal (P = 0.039), occipital (P = 0.024), and insula (P < 0.001) cortices; and in the subcortical regions of caudate (P < 0.001), putamen (P < 0.001) and thalamus (P < 0.001); and in the brainstem (P = 0.018) compared with healthy controls. In patients with Parkinson's disease, loss of 11C-BU99008 VT in cortical regions, striatum, thalamus and brainstem correlated with longer disease duration (P < 0.05) and higher disease burden scores, measured with Movement Disorder Society Unified Parkinson's Disease Rating Scale (P < 0.05). In the subgroup of patients with moderate/advanced Parkinson's disease, loss of 11C-BU99008 VT in the frontal (r = 0.79; P = 0.001), temporal (r = 0.74; P = 0.002) and parietal (r = 0.89; P < 0.001) cortex correlated with global cognitive impairment. This study demonstrates in vivo the role of astroglia in the initiation and progression of Parkinson's disease. Reactive astroglia observed early in Parkinson's disease could reflect a neuroprotective compensatory mechanisms and pro-inflammatory upregulation in response to α-synuclein accumulation. However, as the disease progresses and significant neurodegeneration occurs, astroglia lose their reactive function and such loss in the cortex has clinical relevance in the development of cognitive impairment.

Alcohol Hangover: Underlying Biochemical, Inflammatory and Neurochemical Mechanisms.

AIM: To review current alcohol hangover research in animals and humans and evaluate key evidence for contributing biological factors. METHOD: Narrative review with alcohol hangover defined as the state the day after a single episode of heavy drinking, when the alcohol concentration in the blood approaches zero. RESULTS: Many of the human studies of hangover are not well controlled, with subjects consuming different concentrations of alcohol over variable time periods and evaluation not blinded. Also, studies have measured different symptoms and use varying methods of measurement. Animal studies show variations with respect to the route of administration (intragastric or intraperitoneal), the behavioural tests utilised and discrepancy in the timepoint used for hangover onset. Human studies have the advantage over animal models of being able to assess subjective hangover severity and its correlation with specific behaviours and/or biochemical markers. However, animal models provide valuable insight into the neural mechanisms of hangover. Despite such limitations, several hangover models have identified pathological changes which correlate with the hangover state. We review studies examining the contribution of alcohol's metabolites, neurotransmitter changes with particular reference to glutamate, neuroinflammation and ingested congeners to hangover severity. CONCLUSION: Alcohol metabolites, neurotransmitter alterations, inflammatory factors and mitochondrial dysfunction are the most likely factors in hangover pathology. Future research should aim to investigate the relationship between these factors and their causal role.

First evaluation of PET-based human biodistribution and radiation dosimetry of 11C-BU99008, a tracer for imaging the imidazoline2 binding site.

BACKGROUND: We measured whole body distribution of 11C-BU99008, a new PET biomarker for non-invasive identification of the imidazoline2 binding site. The purpose of this phase I study was to evaluate the biodistribution and radiation dosimetry of 11C-BU99008 in healthy human subjects. METHODS: A single bolus injection of 11C-BU99008 (296 ± 10.5 MBq) was administered to four healthy subjects who underwent whole-body PET/CT over 120 min from the cranial vertex to the mid-thigh. Volumes of interest were drawn around visually identifiable source organs to generate time-activity curves (TAC). Residence times were determined from time-activity curves. Absorbed doses to individual organs and the whole body effective dose were calculated using OLINDA/EXM 1.1 for each subject. RESULTS: The highest measured activity concentration was in the kidney and spleen. The longest residence time was in the muscle at 0.100 ± 0.023 h, followed by the liver at 0.067 ± 0.015 h and lungs at 0.052 ± 0.010 h. The highest mean organ absorbed dose was within the heart wall (0.028 ± 0.002 mGy/MBq), followed by the kidneys (0.026 ± 0.005 mGy/MBq). The critical organ was the heart wall. The total mean effective dose averaged over subjects was estimated to be 0.0056 ± 0.0004 mSv/MBq for an injection of 11C-BU99008. CONCLUSIONS: The biodistribution of 11C-BU99008 has been shown here for the first time in humans. Our dosimetry data showed the total mean effective dose over all subjects was 0.0056 ± 0.0004 mSv/MBq, which would result in a total effective dose of 1.96 mSv for a typical injection of 350 MBq of 11C-BU99008. The effective dose is not appreciably different from those obtained with other 11C tracers.

Evaluation of 11C-BU99008, a PET Ligand for the Imidazoline2 Binding Site in Human Brain.

The imidazoline2 binding site (I2BS) is thought to be expressed in glia and implicated in the regulation of glial fibrillary acidic protein. A PET ligand for this target would be important for the investigation of neurodegenerative and neuroinflammatory diseases. 11C-BU99008 has previously been identified as a putative PET radioligand. Here, we present the first in vivo characterization of this PET radioligand in humans and assess its test-retest reproducibility. Methods: Fourteen healthy male volunteers underwent dynamic PET imaging with 11C-BU99008 and arterial sampling. Six subjects were used in a test-retest assessment, and 8 were used in a pharmacologic evaluation, undergoing a second or third heterologous competition scan with the mixed I2BS/α2-adrenoceptor drug idazoxan (n = 8; 20, 40, 60, and 80 mg) and the mixed irreversible monoamine oxidase type A/B inhibitor isocarboxazid (n = 4; 50 mg). Regional time-activity data were generated from arterial plasma input functions corrected for metabolites using the most appropriate model to derive the outcome measure VT (regional distribution volume). All image processing and kinetic analyses were performed in MIAKAT. Results: Brain uptake of 11C-BU99008 was good, with reversible kinetics and a heterogeneous distribution consistent with known I2BS expression. Model selection criteria indicated that the 2-tissue-compartment model was preferred. VT estimates were high in the striatum (105 ± 21 mL⋅cm-3), medium in the cingulate cortex (62 ± 10 mL⋅cm-3), and low in the cerebellum (41 ± 7 mL⋅cm-3). Test-retest reliability was reasonable. The uptake was dose-dependently reduced throughout the brain by pretreatment with idazoxan, with an average block across all regions of about 60% (VT, ∼30 mL⋅cm-3) at the highest dose (80 mg). The median effective dose for idazoxan was 28 mg. Uptake was not blocked by pretreatment with the monoamine oxidase inhibitor isocarboxazid. Conclusion:11C-BU99008 in human PET studies demonstrates good brain delivery, reversible kinetics, heterogeneous distribution, specific binding signal consistent with I2BS distribution, and good test-retest reliability.

In vivo imaging of microglial activation by positron emission tomography with [(11)C]PBR28 in the 5XFAD model of Alzheimer's disease.

Microglial activation has been linked with deficits in neuronal function and synaptic plasticity in Alzheimer's disease (AD). The mitochondrial translocator protein (TSPO) is known to be upregulated in reactive microglia. Accurate visualization and quantification of microglial density by PET imaging using the TSPO tracer [(11)C]-R-PK11195 has been challenging due to the limitations of the ligand. In this study, it was aimed to evaluate the new TSPO tracer [(11)C]PBR28 as a marker for microglial activation in the 5XFAD transgenic mouse model of AD. Dynamic PET scans were acquired following intravenous administration of [(11)C]PBR28 in 6-month-old 5XFAD mice and in wild-type controls. Autoradiography with [(3)H]PBR28 was carried out in the same brains to further confirm the distribution of the radioligand. In addition, immunohistochemistry was performed on adjacent brain sections of the same mice to evaluate the co-localization of TSPO with microglia. PET imaging revealed that brain uptake of [(11)C]PBR28 in 5XFAD mice was increased compared with control mice. Moreover, binding of [(3)H]PBR28, measured by autoradiography, was enriched in cortical and hippocampal brain regions, coinciding with the positive staining of the microglial marker Iba-1 and amyloid deposits in the same areas. Furthermore, double-staining using antibodies against TSPO demonstrated co-localization of TSPO with microglia and not with astrocytes in 5XFAD mice and human post-mortem AD brains. The data provided support of the suitability of [(11)C]PBR28 as a tool for in vivo monitoring of microglial activation and assessment of treatment response in future studies using animal models of AD.

Optimising PET approaches to measuring 5-HT release in human brain.

A major goal in neuroscience is the measurement of neurotransmitters in living human brain. To date this has only been done reliably with dopamine using certain PET and SPECT radiotracers. The use of this technique has greatly advanced our understanding of dopamine and the dopaminergic system in normal and abnormal brain function. Transferring this technology to other neurotransmitter systems has proved less fruitful. The serotonergic system (5-HT) is one such system. 5-HT has been implicated in a wide range of brain functions and their disorders. The ability to measure 5-HT using this technique would be invaluable. In this article, we explore the key pharmacological features of current radiotracers for 5-HT receptors that might be sensitive to endogenous 5-HT. We also estimate the likely brain concentrations of the current available tranche of agents that might be used to enhance synaptic 5-HT concentration, so taking into account the potential for these to interact with the receptors directly and produce a spurious displacement signal.

Influence of agonist induced internalization on [3H]Ro15-4513 binding-an application to imaging fluctuations in endogenous GABA with positron emission tomography.

Low affinity α1/α2 containing GABAA receptors are significantly less able to bind [(11) C]/[(3) H]Ro15-4513 following translocation to the endosomal environment. The alterations in [(11) C]Ro15-4513 binding observed in vivo following perturbations in endogenous GABA are likely driven by both alterations in receptor binding parameters following agonist induced internalisation and the GABA Shift.

The Effects of Acutely Administered 3,4-Methylenedioxymethamphetamine on Spontaneous Brain Function in Healthy Volunteers Measured with Arterial Spin Labeling and Blood Oxygen Level-Dependent Resting State Functional Connectivity.

BACKGROUND: The compound 3,4-methylenedioxymethamphetamine (MDMA) is a potent monoamine releaser that produces an acute euphoria in most individuals. METHODS: In a double-blind, placebo-controlled, balanced-order study, MDMA was orally administered to 25 physically and mentally healthy individuals. Arterial spin labeling and seed-based resting state functional connectivity (RSFC) were used to produce spatial maps displaying changes in cerebral blood flow (CBF) and RSFC after MDMA administration. Participants underwent two arterial spin labeling and two blood oxygen level-dependent scans in a 90-minute scan session; MDMA and placebo study days were separated by 1 week. RESULTS: Marked increases in positive mood were produced by MDMA. Decreased CBF only was observed after MDMA, and this was localized to the right medial temporal lobe (MTL), thalamus, inferior visual cortex, and the somatosensory cortex. Decreased CBF in the right amygdala and hippocampus correlated with ratings of the intensity of global subjective effects of MDMA. The RSFC results complemented the CBF results, with decreases in RSFC between midline cortical regions, the medial prefrontal cortex, and MTL regions, and increases between the amygdala and hippocampus. There were trend-level correlations between these effects and ratings of intense and positive subjective effects. CONCLUSIONS: The MTLs appear to be specifically implicated in the mechanism of action of MDMA, but further work is required to elucidate how the drug's characteristic subjective effects arise from its modulation of spontaneous brain activity.

Imaging endogenous opioid peptide release with [11C]carfentanil and [3H]diprenorphine: influence of agonist-induced internalization.

Understanding the cellular processes underpinning the changes in binding observed during positron emission tomography neurotransmitter release studies may aid translation of these methodologies to other neurotransmitter systems. We compared the sensitivities of opioid receptor radioligands, carfentanil, and diprenorphine, to amphetamine-induced endogenous opioid peptide (EOP) release and methadone administration in the rat. We also investigated whether agonist-induced internalization was involved in reductions in observed binding using subcellular fractionation and confocal microscopy. After radioligand administration, significant reductions in [(11)C]carfentanil, but not [(3)H]diprenorphine, uptake were observed after methadone and amphetamine pretreatment. Subcellular fractionation and in vitro radioligand binding studies showed that amphetamine pretreatment only decreased total [(11)C]carfentanil binding. In vitro saturation binding studies conducted in buffers representative of the internalization pathway suggested that µ-receptors are significantly less able to bind the radioligands in endosomal compared with extracellular compartments. Finally, a significant increase in µ-receptor-early endosome co-localization in the hypothalamus was observed after amphetamine and methadone treatment using double-labeling confocal microscopy, with no changes in δ- or κ-receptor co-localization. These data indicate carfentanil may be superior to diprenorphine when imaging EOP release in vivo, and that alterations in the ability to bind internalized receptors may be a predictor of ligand sensitivity to endogenous neurotransmitter release.

The influence of different cellular environments on PET radioligand binding: an application to D2/3-dopamine receptor imaging.

Various D2/3 receptor PET radioligands are sensitive to endogenous dopamine release in vivo. The Occupancy Model is generally used to interpret changes in binding observed in in vivo competition binding studies; an Internalisation Hypothesis may also contribute to these changes in signal. Extension of in vivo competition imaging to other receptor systems has been relatively unsuccessful. A greater understanding of the cellular processes underlying signal changes following endogenous neurotransmitter release may help translate this imaging paradigm to other receptor systems. To investigate the Internalisation Hypothesis we assessed the effects of different cellular environments, representative of those experienced by a receptor following agonist-induced internalisation, on the binding of three D2/3 PET ligands with previously reported sensitivities to endogenous dopamine in vivo, namely [3H]spiperone, [3H]raclopride and [3H]PhNO. Furthermore, we determined the contribution of each cellular compartment to total striatal binding for these D2/3 ligands. These studies suggest that sensitivity to endogenous dopamine release in vivo is related to a decrease in affinity in the endosomal environment compared with those found at the cell surface. In agreement with these findings we also demonstrate that ∼25% of total striatal binding for [3H]spiperone originates from sub-cellular, microsomal receptors, whereas for [3H]raclopride and [3H]PhNO, this fraction is lower, representing ∼14% and 17%, respectively. This pharmacological approach is fully translatable to other receptor systems. Assessment of affinity shifts in different cellular compartments may play a crucial role for understanding if a radioligand is sensitive to endogenous release in vivo, for not just the D2/3, but other receptor systems.

Evaluation of 11C-BU99008, a PET ligand for the imidazoline2 binding sites in rhesus brain.

UNLABELLED: The development of a PET radioligand selective for I2-imidazoline binding sites (I2BS) would enable, for the first time, specific, measurable in vivo imaging of this target protein, along with assessment of alterations in expression patterns of this protein in disease pathophysiology. METHODS: BU99008 was identified as the most promising I2BS radioligand candidate and radiolabeled with (11)C via methylation. The in vivo binding properties of (11)C-BU99008 were assessed in rhesus monkeys to determine brain penetration, brain distribution, binding specificity and selectivity (via the use of the unlabeled blockers), and the most appropriate kinetic model for analyzing data generated with this PET radioligand. RESULTS: (11)C-BU99008 was demonstrated to readily enter the brain, resulting in a heterogeneous distribution (globus pallidus > cortical regions > cerebellum) consistent with the reported regional I2BS densities as determined by human tissue section autoradiography and preclinical in vivo PET studies in the pig. In vivo competition studies revealed that (11)C-BU99008 displayed reversible kinetics specific for the I2BS. The multilinear analysis (MA1) model was the most appropriate analysis method for this PET radioligand in this species. The selective I2BS blocker BU224 was shown to cause a saturable, dose-dependent decrease in (11)C-BU99008 binding in all regions of the brain assessed, further demonstrating the heterogeneous distribution of I2BS protein in the rhesus brain and binding specificity for this radioligand. CONCLUSION: These data demonstrate that (11)C-BU99008 represents a specific and selective PET radioligand for imaging and quantifying the I2BS, in vivo, in the rhesus monkey. Further work is under way to translate the use of (11)C-BU99008 to the clinic.

Imaging imidazoline-I2 binding sites in porcine brain using 11C-BU99008.

UNLABELLED: Changes in the density of imidazoline-I(2) binding sites have been observed in a range of neurologic disorders including Alzheimer's disease, Huntington's chorea, and glial tumor; however, the precise function of these sites remains unclear. A PET probe for I(2) binding sites would further our understanding of the target and may find application as a biomarker for early disease diagnosis. Compound BU99008 has previously been identified as a promising I(2) ligand from autoradiography studies, displaying high affinity and good selectivity toward the target. In this study, BU99008 was radiolabeled with (11)C in order to image the I(2) binding sites in vivo using PET. METHODS: (11)C-BU99008 was radiolabeled by N-alkylation of the desmethyl precursor using (11)C-methyl iodide. A series of PET experiments was performed to investigate the binding of (11)C-BU99008 in porcine brains, in the presence or absence of a nonradiolabeled, competing I(2) ligand, BU224. RESULTS: (11)C-BU99008 was obtained in good yield and specific activity. In vivo, (11)C-BU99008 displayed good brain penetration and gave a heterogeneous distribution with high uptake in the thalamus and low uptake in the cortex and cerebellum. (11)C-BU99008 brain kinetics were well described by the 1-tissue-compartment model, which was used to provide estimates for the total volume of distribution (V(T)) across brain regions of interest. Baseline V(T) values were ranked in the following order: thalamus > striatum > hippocampus > frontal cortex ≥ cerebellum, consistent with the known distribution and concentration of I(2) binding sites. Administration of a selective I(2) binding site ligand, BU224, reduced the V(T) to near-homogeneous levels in all brain regions. CONCLUSION: (11)C-BU99008 appears to be a suitable PET radioligand for imaging the I(2) binding sites in vivo.