Obesity risk is associated with altered cerebral glucose metabolism and decreased µ-opioid and CB1 receptor availability.

BACKGROUND: Obesity is a pressing public health concern worldwide. Novel pharmacological means are urgently needed to combat the increase of obesity and accompanying type 2 diabetes (T2D). Although fully established obesity is associated with neuromolecular alterations and insulin resistance in the brain, potential obesity-promoting mechanisms in the central nervous system have remained elusive. In this triple-tracer positron emission tomography study, we investigated whether brain insulin signaling, µ-opioid receptors (MORs) and cannabinoid CB1 receptors (CB1Rs) are associated with risk for developing obesity. METHODS: Subjects were 41 young non-obese males with variable obesity risk profiles. Obesity risk was assessed by subjects' physical exercise habits, body mass index and familial risk factors, including parental obesity and T2D. Brain glucose uptake was quantified with [18F]FDG during hyperinsulinemic euglycemic clamp, MORs were quantified with [11C]carfentanil and CB1Rs with [18F]FMPEP-d2. RESULTS: Subjects with higher obesity risk had globally increased insulin-stimulated brain glucose uptake (19 high-risk subjects versus 19 low-risk subjects), and familial obesity risk factors were associated with increased brain glucose uptake (38 subjects) but decreased availability of MORs (41 subjects) and CB1Rs (36 subjects). CONCLUSIONS: These results suggest that the hereditary mechanisms promoting obesity may be partly mediated via insulin, opioid and endocannabinoid messaging systems in the brain.

Sex difference in brain CB1 receptor availability in man.

The endocannabinoid system (ECS) has a widespread neuromodulatory function in the central nervous system and is involved in important aspects of brain function including brain development, cortical rhythms, plasticity, reward, and stress sensitivity. Many of these effects are mediated via the cannabinoid CB1 receptor (CB1R) subtype. Animal studies convincingly show an interaction between the ECS and sex hormones, as well as a sex difference of higher brain CB1R in males. Human in vivo studies of sex difference have yielded discrepant findings. Gender differences in CB1R availability were investigated in vivo in 11 male and 11 female healthy volunteers using a specific CB1R tracer [18F]FMPEP-d2 and positron emission tomography (PET). Regional [18F]FMPEP-d2 distribution volume was used as a proxy for CB1R availability. In addition, we explored whether CB1R availability is linked to neuropsychological functioning. Relative to females, CB1R availability was on average 41% higher in males (p = 0.002) with a regionally specific effect larger in the posterior cingulate and retrosplenial cortices (p = 0.001). Inter-subject variability in CB1R availability was similar in both groups. Voxel-based analyses revealed an inverse association between CB1R availability and visuospatial working memory task performance in both groups (p < 0.001). A CB1R sex difference with a large effect size was observed and should be considered in the design of CB1R-related studies on neuropsychiatric disorders. The behavioural correlates and clinical significance of this difference remain to be further elucidated, but our studies suggest an association between CB1R availability and working memory.

Multifunctional liposomes reduce brain ß-amyloid burden and ameliorate memory impairment in Alzheimer's disease mouse models.

Alzheimer's disease is characterized by the accumulation and deposition of plaques of ß-amyloid (Aß) peptide in the brain. Given its pivotal role, new therapies targeting Aß are in demand. We rationally designed liposomes targeting the brain and promoting the disaggregation of Aß assemblies and evaluated their efficiency in reducing the Aß burden in Alzheimer's disease mouse models. Liposomes were bifunctionalized with a peptide derived from the apolipoprotein-E receptor-binding domain for blood-brain barrier targeting and with phosphatidic acid for Aß binding. Bifunctionalized liposomes display the unique ability to hinder the formation of, and disaggregate, Aß assemblies in vitro (EM experiments). Administration of bifunctionalized liposomes to APP/presenilin 1 transgenic mice (aged 10 months) for 3 weeks (three injections per week) decreased total brain-insoluble Aß1-42 (-33%), assessed by ELISA, and the number and total area of plaques (-34%) detected histologically. Also, brain Aß oligomers were reduced (-70.5%), as assessed by SDS-PAGE. Plaque reduction was confirmed in APP23 transgenic mice (aged 15 months) either histologically or by PET imaging with [(11)C]Pittsburgh compound B (PIB). The reduction of brain Aß was associated with its increase in liver (+18%) and spleen (+20%). Notably, the novel-object recognition test showed that the treatment ameliorated mouse impaired memory. Finally, liposomes reached the brain in an intact form, as determined by confocal microscopy experiments with fluorescently labeled liposomes. These data suggest that bifunctionalized liposomes destabilize brain Aß aggregates and promote peptide removal across the blood-brain barrier and its peripheral clearance. This all-in-one multitask therapeutic device can be considered as a candidate for the treatment of Alzheimer's disease.

Enhanced fatty acid uptake in visceral adipose tissue is not reversed by weight loss in obese individuals with the metabolic syndrome.

AIMS/HYPOTHESIS: Obesity causes an imbalance in fat mass distribution between visceral and subcutaneous adipose tissue (AT) depots. We tested the hypothesis that this relates to increased NEFA uptake between these depots in obese compared with healthy participants. Second, we hypothesised that a diet very low in energy (very low calorie diet [VLCD]) decreases fat mass in obese participants and that this is associated with the decline in NEFA uptake. METHODS: NEFA uptake in AT depots was measured with [(18)F]-fluoro-6-thia-heptadecanoic acid ((18)F-FTHA) and positron emission tomography (PET) in 18 obese participants with the metabolic syndrome before and after a 6 week VLCD. Whole body fat oxidation was measured using indirect calorimetry and [U-(13)C]palmitate. Sixteen non-obese participants were controls. RESULTS: Obese participants had >100% higher (p < 0.0001) NEFA uptake in the visceral and subcutaneous abdominal AT depots than controls. VLCD decreased AT mass in all regions (12% to 21%), but NEFA uptake was decreased significantly (18%; p < 0.006) only in the femoral AT. Whole body carbohydrate oxidation decreased, while fat oxidation increased. CONCLUSIONS/INTERPRETATION: The data demonstrate that weight loss caused by VLCD does not affect abdominal fasting NEFA uptake rates. We found that visceral fat takes up more NEFAs than subcutaneous AT depots, even after weight loss.

Monoacylglycerol lipase inhibitor JZL184 reduces neuroinflammatory response in APdE9 mice and in adult mouse glial cells.

BACKGROUND: Recently, the role of monoacylglycerol lipase (MAGL) as the principal regulator of simultaneous prostaglandin synthesis and endocannabinoid receptor activation in the CNS was demonstrated. To expand upon previously published research in the field, we observed the effect of the MAGL inhibitor JZL184 during the early-stage proinflammatory response and formation of beta-amyloid (Aß) in the Alzheimer's disease mouse model APdE9. We also investigated its effects in proinflammatory agent - induced astrocytes and microglia isolated from adult mice. FINDINGS: Transgenic APdE9 mice (5 months old) were treated with JZL184 (40 mg/kg) or vehicle every day for 1 month. In vivo binding of the neuroinflammation-related, microglia-specific translocator protein (TSPO) targeting radioligand [(18) F]GE-180 decreased slightly but statistically non-significantly in multiple brain areas compared to vehicle-treated mice. JZL184 treatment induced a significant decrease in expression levels of inflammation-induced, Iba1-immunoreactive microglia in the hippocampus (P < 0.01) and temporal and parietal (P < 0.05) cortices. JZL184 also induced a marked decrease in total Aß burden in the temporal (P < 0.001) and parietal (P < 0.01) cortices and, to some extent, in the hippocampus. Adult microglial and astrocyte cultures pre-treated with JZL184 and then exposed to the neuroinflammation-inducing agents lipopolysaccharide (LPS), interferon-gamma (IFN-γ), and Aß42 had significantly reduced proinflammatory responses compared to cells without JZL184 treatment. CONCLUSIONS: JZL184 decreased the proinflammatory reactions of microglia and reduced the total Aß burden and its precursors in the APdE9 mouse model. It also reduced the proinflammatory responses of microglia and astrocytes isolated from adult mice.

Positron emission tomography imaging of the 18-kDa translocator protein (TSPO) with [18F]FEMPA in Alzheimer's disease patients and control subjects.

PURPOSE: Imaging of the 18-kDa translocator protein (TSPO) is a potential tool for examining microglial activation and neuroinflammation in early Alzheimer's disease (AD). [(18)F]FEMPA is a novel high-affinity second-generation TSPO radioligand that has displayed suitable pharmacokinetic properties in preclinical studies. The aims of this study were to quantify the binding of [(18)F]FEMPA to TSPO in AD patients and controls and to investigate whether higher [(18)F]FEMPA binding in AD patients than in controls could be detected in vivo. METHODS: Ten AD patients (five men, five women; age 66.9 ± 7.3 years; MMSE score 25.5 ± 2.5) and seven controls (three men, four women; age 63.7 ± 7.2 years, MMSE score 29.3 ± 1.0) were studied using [(18)F]FEMPA at Turku (13 subjects) and at Karolinska Institutet (4 subjects). The in vitro binding affinity for TSPO was assessed using PBR28 in a competition assay with [(3)H]PK11195 in seven controls and eight AD patients. Cortical and subcortical regions of interest were examined. Quantification was performed using a two-tissue compartment model (2TCM) and Logan graphical analysis (GA). The outcome measure was the total distribution volume (V T). Repeated measures analysis of variance was used to assess the effect of group and TSPO binding status on V T. RESULTS: Five AD patients and four controls were high-affinity binders (HABs). Three AD patients and three controls were mixed-affinity binders. V T estimated with Logan GA was significantly correlated with V T estimated with the 2TCM in both controls (r = 0.97) and AD patients (r = 0.98) and was selected for the final analysis. Significantly higher V T was found in the medial temporal cortex in AD patients than in controls (p = 0.044) if the TSPO binding status was entered as a covariate. If only HABs were included, significantly higher V T was found in the medial and lateral temporal cortex, posterior cingulate, caudate, putamen, thalamus and cerebellum in AD patients than in controls (p < 0.05). CONCLUSION: [(18)F]FEMPA seems to be a suitable radioligand for detecting increased TSPO binding in AD patients if their binding status is taken into account.

Amphetamine decreases α2C-adrenoceptor binding of [11C]ORM-13070: a PET study in the primate brain.

BACKGROUND: The neurotransmitter norepinephrine has been implicated in psychiatric and neurodegenerative disorders. Examination of synaptic norepinephrine concentrations in the living brain may be possible with positron emission tomography (PET), but has been hampered by the lack of suitable radioligands. METHODS: We explored the use of the novel α2C-adrenoceptor antagonist PET tracer [(11)C]ORM-13070 for measurement of amphetamine-induced changes in synaptic norepinephrine. The effect of amphetamine on [(11)C]ORM-13070 binding was evaluated ex vivo in rat brain sections and in vivo with PET imaging in monkeys. RESULTS: Microdialysis experiments confirmed amphetamine-induced elevations in rat striatal norepinephrine and dopamine concentrations. Regional [(11)C]ORM-13070 receptor binding was high in the striatum and low in the cerebellum. After injection of [(11)C]ORM-13070 in rats, mean striatal specific binding ratios, determined using cerebellum as a reference region, were 1.4±0.3 after vehicle pretreatment and 1.2±0.2 after amphetamine administration (0.3mg/kg, subcutaneous). Injection of [(11)C]ORM-13070 in non-human primates resulted in mean striatal binding potential (BP ND) estimates of 0.65±0.12 at baseline. Intravenous administration of amphetamine (0.5 and 1.0mg/kg, i.v.) reduced BP ND values by 31-50%. Amphetamine (0.3mg/kg, subcutaneous) increased extracellular norepinephrine (by 400%) and dopamine (by 270%) in rat striata. CONCLUSIONS: Together, these results indicate that [(11)C]ORM-13070 may be a useful tool for evaluation of synaptic norepinephrine concentrations in vivo. Future studies are required to further understand a potential contribution of dopamine to the amphetamine-induced effect.

Synthesis and evaluation of a (18)F-curcumin derivate for ß-amyloid plaque imaging.

INTRODUCTION: Curcumin is a neuroprotective compound that inhibits the formation of amyloid oligomers and fibrils and binds to ß-amyloid plaques in Alzheimer's disease (AD). We aimed to synthesize an (18)F-labeled curcumin derivate ([(18)F]4) and to characterize its positron emission tomography (PET) tracer-binding properties to ß-amyloid plaques in a transgenic APP23 mouse model of AD. METHODS: We utilized facile one-pot synthesis of [(18)F]4 using nucleophilic (18)F-fluorination and click chemistry. Binding of [(18)F]4 to ß-amyloid plaques in the transgenic APP23 mouse brain cryosections was studied in vitro using heterologous competitive binding against PIB. [(18)F]4 uptake was studied ex vivo in rodents and in vivo using PET/computed tomography of transgenic APP23 and wild-type control mice. RESULTS: The radiochemical yield of [(18)F]4 was 21 ± 11%, the specific activity exceeded 1TBq/µmol, and the radiochemical purity exceeded 99.3% at the end of synthesis. In vitro studies of [(18)F]4 with the transgenic APP23 mouse revealed high ß-amyloid plaque binding. In vivo and ex vivo studies demonstrated that [(18)F]4 has fast clearance from the blood, moderate metabolism but low blood-brain barrier (BBB) penetration. CONCLUSIONS: [(18)F]4 was synthesized in high yield and excellent quality. In vitro studies, metabolite profile, and fast clearance from the blood indicated a promising tracer for Aß imaging. However, [(18)F]4 has low in vivo BBB penetration and thus further studies are needed to reveal the reason for this and to possibly overcome this issue.

Cerebral glucose utilization measured with high resolution positron emission tomography in epileptic Finnish Spitz dogs and healthy dogs.

In human epileptic patients, changes in cerebral glucose utilization can be detected 2-deoxy-2-[(18) F] fluoro-D-glucose positron emission tomography (FDG-PET). The purpose of this prospective study was to determine whether epileptic dogs might show similar findings. Eleven Finnish Spitz dogs with focal idiopathic epilepsy and six healthy dogs were included. Dogs were examined using electroencephalography (EEG) and FDG-PET, with epileptic dogs being evaluated during the interictal period. Visual and semi-quantitative assessment methods of FDG-PET were compared and contrasted with EEG findings. Three independent observers, unaware of dog clinical status, detected FDG-PET uptake abnormalities in 9/11 epileptic (82%), and 4/8 healthy dogs (50%). Occipital cortex findings were significantly associated with epileptic status (P = 0.013). Epileptic dogs had significantly lower standardized uptake values (SUVs) in numerous cortical regions, the cerebellum, and the hippocampus compared to the control dogs. The lowest SUVs were found in the occipital lobe. White matter normalized and left-right asymmetry index values for all pairs of homologous regions did not differ between groups. Visual evaluation of the EEGs was less sensitive (36%) than FDG-PET. Both diagnostic tests were consensual and specific (100%) for occipital findings, but EEG had a lower sensitivity for detecting lateralized foci than FDG-PET. Findings supported the use of FDG-PET as a diagnostic test for dogs with suspected idiopathic epilepsy. Visual and semiquantitative analyses of FDG-PET scans provided complementary information. Findings also supported the theory that epileptogenesis may occur in multiple brain regions in Finnish Spitz dogs with idiopathic epilepsy.

FDG-PET in healthy and epileptic Lagotto Romagnolo dogs and changes in brain glucose uptake with age.

Regional cerebral metabolism and blood flow can be measured noninvasively with positron emission tomography (PET). 2-[(18) F]fluoro-2-deoxy-D-glucose (FDG) widely serves as a PET tracer in human patients with epilepsy to identify the seizure focus. The goal of this prospective study was to determine whether juvenile or adult dogs with focal-onset epilepsy exhibit abnormal cerebral glucose uptake interictally and whether glucose uptake changes with age. We used FDG-PET to examine six Lagotto Romagnolo dogs with juvenile epilepsy, two dogs with adult-onset epilepsy, and five control dogs of the same breed at different ages. Three researchers unaware of dog clinical status visually analyzed co-registered PET and magnetic resonance imaging (MRI) images. Results of the visual PET analyses were compared with electroencephalography (EEG) results. In semiquantitative analysis, relative standard uptake values (SUV) of regions of interest (ROI) drawn to different brain regions were compared between epileptic and control dogs. Visual analysis revealed areas of hypometabolism interictally in five out of six dogs with juvenile epilepsy in the occipital, temporal, and parietal cortex. Changes in EEG occurred in three of these dogs in the same areas where PET showed cortical hypometabolism. Visual analysis showed no abnormalities in cerebral glucose uptake in dogs with adult-onset epilepsy. Semiquantitative analysis detected no differences between epileptic and control dogs. This result emphasizes the importance of visual analysis in FDG-PET studies of epileptic dogs. A change in glucose uptake was also detected with age. Glucose uptake values increased between dog ages of 8 and 28 weeks and then remained constant.

New improved radiometabolite analysis method for [18F]FTHA from human plasma: a test-retest study with postprandial and fasting state.

BACKGROUND: Fatty acid uptake can be measured using PET and 14-(R,S)-[18F]fluoro-6-thia-heptadecanoic acid ([18F]FTHA). However, the relatively rapid rate of [18F]FTHA metabolism significantly affects kinetic modeling of tissue uptake. Thus, there is a need for accurate chromatographic methods to analyze the unmetabolized [18F]FTHA (parent fraction). Here we present a new radiometabolite analysis (RMA) method, with comparison to a previous method for parent fraction analysis, and its use in a test-retest clinical study under fasting and postprandial conditions. We developed a new thin-layer chromatography (TLC) RMA method for analysis of [18F]FTHA parent fraction and its radiometabolites from plasma, by testing stationary phases and eluent combinations. Next, we analyzed [18F]FTHA, its radiometabolites, and plasma radioactivity from subjects participating in a clinical study. A total of 17 obese or overweight participants were dosed with [18F]FTHA twice under fasting, and twice under postprandial conditions and plasma samples were obtained between 14 min (mean of first sample) and 72 min (mean of last sample) post-injection. Aliquots of 70 plasma samples were analyzed using both methods, enabling head-to-head comparisons. We performed test-retest and group comparisons of the parent fraction and plasma radioactivity. RESULTS: The new TLC method separated seven [18F]FTHA radiometabolite peaks, while the previous method separated three. The new method revealed at least one radiometabolite that was not previously separable from [18F]FTHA. From the plasma samples, the mean parent fraction value was on average 7.2 percentage points lower with the new method, compared to the previous method. Repeated [18F]FTHA investigations on the same subject revealed reproducible plasma SUV and parent fractions, with different kinetics between the fasted and postprandial conditions. CONCLUSIONS: The newly developed improved radio-TLC method for [18F]FTHA RMA enables accurate parent fraction correction, which is required to obtain quantitative data for modelling [18F]FTHA PET data. Our test-retest study of fasted and postprandial conditions showed robust reproducibility, and revealed clear differences in the [18F]FTHA metabolic rate under different study settings. TRIAL REGISTRATION: EudraCT No: 2020-005211-48, 04Feb2021; and Clinical Trials registry NCT05132335, 29Oct2021, URL: https://classic. CLINICALTRIALS: gov/ct2/show/NCT05132335 .

Glial reactivity in a mouse model of beta-amyloid deposition assessed by PET imaging of P2X7 receptor and TSPO using [11C]SMW139 and [18F]F-DPA.

BACKGROUND: P2X7 receptor has emerged as a potentially superior PET imaging marker to TSPO, the gold standard for imaging glial reactivity. [11C]SMW139 is the most recently developed radiotracer to image P2X7 receptor. The aim of this study was to image reactive glia in the APP/PS1-21 transgenic (TG) mouse model of Aß deposition longitudinally using [11C]SMW139 targeting P2X7 receptor and to compare tracer uptake to that of [18F]F-DPA targeting TSPO at the final imaging time point. TG and wild type (WT) mice underwent longitudinal in vivo PET imaging using [11C]SMW139 at 5, 8, 11, and 14 months, followed by [18F]F-DPA PET scan only at 14 months. In vivo imaging results were verified by ex vivo brain autoradiography, immunohistochemical staining, and analysis of [11C]SMW139 unmetabolized fraction in TG and WT mice. RESULTS: Longitudinal change in [11C]SMW139 standardized uptake values (SUVs) showed no statistically significant increase in the neocortex and hippocampus of TG or WT mice, which was consistent with findings from ex vivo brain autoradiography. Significantly higher [18F]F-DPA SUVs were observed in brain regions of TG compared to WT mice. Quantified P2X7-positive staining in the cortex and thalamus of TG mice showed a minor increase in receptor expression with ageing, while TSPO-positive staining in the same regions showed a more robust increase in expression in TG mice as they aged. [11C]SMW139 was rapidly metabolized in mice, with 33% of unmetabolized fraction in plasma and 29% in brain homogenates 30 min after injection. CONCLUSIONS: [11C]SMW139, which has a lower affinity for the rodent P2X7 receptor than the human version of the receptor, was unable to image the low expression of P2X7 receptor in the APP/PS1-21 mouse model. Additionally, the rapid metabolism of [11C]SMW139 in mice and the presence of several brain-penetrating radiometabolites significantly impacted the analysis of in vivo PET signal of the tracer. Finally, [18F]F-DPA targeting TSPO was more suitable for imaging reactive glia and neuroinflammatory processes in the APP/PS1-21 mouse model, based on the findings presented in this study and previous studies with this mouse model.

Evaluation of [18F]FMTEB in Sprague Dawley rats as a PET tracer for metabotropic glutamate receptor 5.

INTRODUCTION: [18F]FMTEB, along with other tracers, was developed as a promising PET radioligand for imaging metabotropic glutamate receptor subtype 5 (mGluR5). Despite favorable preliminary results, it has not been used further for studies of mGluR5. This paper presents an in-depth preclinical evaluation of [18F]FMTEB in healthy Sprague Dawley rats. METHODS: [18F]FMTEB was synthesized from a boronic ester precursor using copper-mediated fluorination. In vivo PET imaging was performed on six rats, of which three were pre-treated with a high affinity mGluR5 receptor antagonist. An additional 18 rats were used for ex vivo experiments for metabolite analyses in plasma, brain and urine, and for biodistribution and ex vivo brain autoradiography at different time points. RESULTS: [18F]FMTEB was synthesized in adequate radiochemical yield and a molar activity of 154 ± 64 GBq/µmol. Both in vivo imaging and ex vivo brain autoradiography showed high specificity for mGluR5, and the blocking experiments showed a clear decrease in radioactivity in mGluR5-rich brain areas. Metabolite analyses confirmed fast metabolism of the tracer in plasma. The percentage of parent compound in brain tissue exceeded 90 % up to 90 min after injection. CONCLUSION: [18F]FMTEB produced via copper-mediated 18F-fluorination fulfilled the requirements for preclinical evaluation in rats. The absence of specific uptake in cerebellum and absence of defluorination of the tracer allowed cerebellum to be used as a reference tissue. Due to the fast kinetics in rats, the region-to-cerebellum ratios equilibrated within 30 min. These results prove [18F]FMTEB to be a good candidate for mapping mGluR5 in rat brain and a suitable alternative to [18F]FPEB.

[18F]Fluoride uptake in various bone types and soft tissues in rat.

BACKGROUND: In the development of new 18F-labelled tracers, it is important to assess the amount of released [18F]fluoride taken up in the bones of experimental animals because all 18F-labelled PET-tracers are prone, to lesser or higher degree, to undergo defluorination, with subsequent release of [18F]fluoride during scanning. However, the pharmacokinetics of [18F]fluoride in bones and other organs of healthy rats have not been well documented in a comprehensive manner. We aimed to study pharmacokinetics of [18F]NaF in rats in order to increase our understanding of the biodistribution of [18F]fluoride originating from defluorination of 18F-labelled tracers. We studied [18F]fluoride uptake in Sprague Dawley rat bones, including the epiphyseal parts of the tibia and radius, the mandible, ilium, lumbar vertebrae, costochondral joints, tibia, radius, and ribs, with 60-min in vivo PET/CT imaging. Kinetic parameters, K1, Ki, Ki/K1, and k3 were calculated with a three-compartment model. In addition, separate groups of male and female rats were studied with ex vivo bone and soft tissue harvesting and gamma counting over a 6-h period. RESULTS: [18F]fluoride perfusion and uptake varied among the different bones. [18F]fluoride uptake was higher in trabecular bones, due to high perfusion and osteoblastic activity, compared to cortical bones. In soft tissues, the organ-to-blood uptake ratios increased over time in the eyes, lungs, brain, testes, and ovaries during the 6 h study period. CONCLUSION: Understanding the pharmacokinetics of [18F]fluoride in various bones and soft tissues is highly useful for assessing 18F-labelled radiotracers that release [18F]fluoride.

Quantification of the purinergic P2X7 receptor with [11C]SMW139 improves through correction for brain-penetrating radiometabolites.

The membrane-based purinergic 7 receptor (P2X7R) is expressed on activated microglia and the target of the radioligand [11C]SMW139 for in vivo assessment of neuroinflammation. This study investigated the contribution of radiolabelled metabolites which potentially affect its quantification. Ex vivo high-performance liquid chromatography with a radio detector (radioHPLC) was used to evaluate the parent and radiometabolite fractions of [11C]SMW139 in the brain and plasma of eleven mice. Twelve healthy humans underwent 90-min [11C]SMW139 brain PET with arterial blood sampling and radiometabolite analysis. The volume of distribution was estimated by using one- and two- tissue compartment (TCM) modeling with single (VT) and dual (VTp) input functions. RadioHPLC showed three major groups of radiometabolite peaks with increasing concentrations in the plasma of all mice and humans. Two radiometabolite peaks were also visible in mice brain homogenates and therefore considered for dual input modeling in humans. 2TCM with single input function provided VT estimates with a wide range (0.10-10.74) and high coefficient of variation (COV: 159.9%), whereas dual input function model showed a narrow range of VTp estimates (0.04-0.24; COV: 33.3%). In conclusion, compartment modeling with correction for brain-penetrant radiometabolites improves the in vivo quantification of [11C]SMW139 binding to P2X7R in the human brain.

Lower abdominal adipose tissue cannabinoid type 1 receptor availability in young men with overweight.

OBJECTIVE: Cannabinoid type 1 receptors (CB1R) modulate feeding behavior and energy homeostasis, and the CB1R tone is dysgulated in obesity. This study aimed to investigate CB1R availability in peripheral tissue and brain in young men with overweight versus lean men. METHODS: Healthy males with high (HR, n = 16) or low (LR, n = 20) obesity risk were studied with fluoride 18-labeled FMPEP-d2 positron emission tomography to quantify CB1R availability in abdominal adipose tissue, brown adipose tissue, muscle, and brain. Obesity risk was assessed by BMI, physical exercise habits, and familial obesity risk, including parental overweight, obesity, and type 2 diabetes. To assess insulin sensitivity, fluoro-[18 F]-deoxy-2-D-glucose positron emission tomography during hyperinsulinemic-euglycemic clamp was performed. Serum endocannabinoids were analyzed. RESULTS: CB1R availability in abdominal adipose tissue was lower in the HR than in the LR group, whereas no difference was found in other tissues. CB1R availability of abdominal adipose tissue and brain correlated positively with insulin sensitivity and negatively with unfavorable lipid profile, BMI, body adiposity, and inflammatory markers. Serum arachidonoyl glycerol concentration was associated with lower CB1R availability of the whole brain, unfavorable lipid profile, and higher serum inflammatory markers. CONCLUSIONS: The results suggest endocannabinoid dysregulation already in the preobesity state.

Pharmacokinetics of [¹8F]flutemetamol in wild-type rodents and its binding to beta amyloid deposits in a mouse model of Alzheimer's disease.

PURPOSE: The aim of this study was to investigate the potential of [(18)F]flutemetamol as a preclinical PET tracer for imaging ß-amyloid (Aß) deposition by comparing its pharmacokinetics to those of [(11)C]Pittsburgh compound B ([(11)C]PIB) in wild-type Sprague Dawley rats and C57Bl/6N mice. In addition, binding of [(18)F]flutemetamol to Aß deposits was studied in the Tg2576 transgenic mouse model of Alzheimer's disease. METHODS: [(18)F]Flutemetamol biodistribution was evaluated using ex vivo PET methods and in vivo PET imaging in wild-type rats and mice. Metabolism and binding of [(11)C]PIB and [(18)F]flutemetamol to plasma proteins were analysed using thin-layer chromatography and ultrafiltration methods, respectively. Radiation dose estimates were calculated from rat ex vivo biodistribution data. The binding of [(18)F]flutemetamol to Aß deposits was also studied using ex vivo and in vitro autoradiography. The location of Aß deposits in the brain was determined with thioflavine S staining and immunohistochemistry. RESULTS: The pharmacokinetics of [(18)F]flutemetamol resembled that of [(11)C]PIB in rats and mice. In vivo studies showed that both tracers readily entered the brain, and were excreted via the hepatobiliary pathway in both rats and mice. The metabolism of [(18)F]flutemetamol into radioactive metabolites was faster than that of [(11)C]PIB. [(18)F]Flutemetamol cleared more slowly from the brain than [(11)C]PIB, particularly from white matter, in line with its higher lipophilicity. Effective dose estimates for [(11)C]PIB and [(18)F]flutemetamol were 2.28 and 6.65 µSv/MBq, respectively. Autoradiographs showed [(18)F]flutemetamol binding to fibrillar Aß deposits in the brain of Tg2576 mice. CONCLUSION: Based on its pharmacokinetic profile, [(18)F]flutemetamol showed potential as a PET tracer for preclinical imaging. It showed good brain uptake and was bound to Aß deposits in the brain of Tg2576 mice. However, its high lipophilicity might complicate the analysis of PET data, particularly in small-animal imaging.

ImmunoPET imaging of amyloid-beta in a rat model of Alzheimer's disease with a bispecific, brain-penetrating fusion protein.

BACKGROUND: Hijacking the transferrin receptor (TfR) is an effective strategy to transport amyloid-beta (Aß) immuno-positron emission tomography (immunoPET) ligands across the blood-brain barrier (BBB). Such ligands are more sensitive and specific than small-molecule ligands at detecting Aß pathology in mouse models of Alzheimer's disease (AD). This study aimed to determine if this strategy would be as sensitive in rats and to assess how TfR affinity affects BBB transport of bispecific immunoPET radioligands. METHODS: Two affinity variants of the rat TfR antibody, OX26, were chemically conjugated to a F(ab')2 fragment of the anti-Aß antibody, bapineuzumab (Bapi), to generate two bispecific fusion proteins: OX265-F(ab')2-Bapi and OX2676-F(ab')2-Bapi. Pharmacokinetic analyses were performed 4 h and 70 h post-injection of radioiodinated fusion proteins in wild-type (WT) rats. [124I]I-OX265-F(ab')2-Bapi was administered to TgF344-AD and WT rats for in vivo PET imaging. Ex vivo distribution of injected [124I]I-OX265-F(ab')2-Bapi and Aß pathology were assessed. RESULTS: More [125I]I-OX265-F(ab')2-Bapi was taken up into the brain 4 h post-administration than [124I]I-OX2676-F(ab')2-Bapi. [124I]I-OX265-F(ab')2-Bapi PET visualized Aß pathology with significantly higher signals in the TgF344-AD rats than in the WT littermates without Aß pathology. The PET signals significantly correlated with Aß levels in AD animals. CONCLUSION: Affinity to TfR affects how efficiently a TfR-targeting bispecific fusion protein will cross the BBB, such that the higher-affinity bispecific fusion protein crossed the BBB more efficiently. Furthermore, bispecific immunoPET imaging of brain Aß pathology using TfR-mediated transport provides good imaging contrast between TgF344-AD and WT rats, suggesting that this immunoPET strategy has the potential to be translated to higher species.

Novel plasma protein binding analysis method for a PET tracer and its radiometabolites: A case study with [11C]SMW139 to explain the high uptake of radiometabolites in mouse brain.

Radiometabolites of PET tracers interfere with imaging and need to be taken into account when modeling PET data. Various tracer and radiometabolite characteristics affect the uptake rate into tissue. In this study, we investigated two such factors, lipophilicity and protein-free fraction. A novel rapid method was developed using thin-layer chromatography with digital autoradiography (radioTLC) and ultrafiltration for analyzing the protein-free fractions of an exemplar PET tracer, [11C]SMW139 (fP, free parent tracer over all radioactivity), and its radiometabolites (fM, free radiometabolites over all radioactivity). Detailed understanding of the uptake of radiometabolites into extravascular cells requires analyzing fM, which has not previously been performed for PET tracers. Mice were injected with [11C]SMW139, and time-activity curves from plasma and brain coupled with the parent fraction and free fraction data were analyzed to demonstrate the true levels of protein-free and protein-bound [11C]SMW139 and its radiometabolites in plasma. The ultrafiltration method included separate membrane correction factors for the parent tracer and its radiometabolites for analysis of unbiased fP and fM. Metabolism of [11C]SMW139 was rapid, and after 45 min, the parent fraction was 0.33 in plasma and 0.28 in brain. Ultrafiltration membrane correction had a significant effect on the fP but not the fM. From 10-45 min, the fP decreased from 0.032 to 0.007, while fM remained between 0.52 and 0.35. The much higher fM in plasma could explain why the less lipophilic radiometabolites enter the brain efficiently. This detailed understanding of fP and fM from rodents can be used in translational studies to explain the behavior of the tracer in humans. Similar parent fraction and plasma protein binding methods can be used for human in vivo analysis.

Direct Comparison of [18F]F-DPA with [18F]DPA-714 and [11C]PBR28 for Neuroinflammation Imaging in the same Alzheimer's Disease Model Mice and Healthy Controls.

PURPOSE: In this study we compared the recently developed TSPO tracer [18F]F-DPA, with [18F]DPA-714 and [11C]PBR28 by performing in vivo PET imaging on the same Alzheimer's disease mouse model APP/PS1-21 (TG) and wild-type (WT) mice with all three radiotracers. PROCEDURES: To compare the radiotracer uptake, percentage of injected dose/mL (%ID/mL), standardized uptake value ratios to cerebellum (SUVRCB), and voxel-wise analyses were performed. RESULTS: The peak uptake of [18F]F-DPA was higher than 4.3% ID/mL, while [18F]DPA-714 reached just over 3% ID/mL, and [11C]PBR28 was over 4% ID/mL in only one brain region in the WT mice. The peak/60-min uptake ratios of [18F]F-DPA were significantly higher (p < 0.001) than those of [18F]DPA-714 and [11C]PBR28. The differences in [18F]F-DPA SUVRCB between WT and TG mice were highly significant (p < 0.001) in the three studied time periods after injection. [18F]DPA-714 uptake was significantly higher in TG mice starting in the 20-40-min timeframe and increased thereafter, whereas [11C]PBR28 uptake became significant at 10-20 min (p < 0.05). The voxel-wise analysis confirmed the differences between the radiotracers. CONCLUSIONS: [18F]F-DPA displays higher brain uptake, higher TG-to-WT SUVRCB ratios, and faster clearance than [18F]DPA-714 and [11C]PBR28, and could prove useful for detecting low levels of inflammation and allow for shorter dynamic PET scans.