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.

Obesity risk is associated with brain glucose uptake and insulin resistance.

Objective: To investigate whether alterations in brain glucose uptake (BGU), insulin action in the brain-liver axis and whole-body insulin sensitivity occur in young adults in pre-obese state. Methods: Healthy males with either high risk (HR; n = 19) or low risk (LR; n = 22) for developing obesity were studied with [18F]fluoro-d-glucose ([18F]FDG)-positron emission tomography during hyperinsulinemic-euglycemic clamp. Obesity risk was assessed according to BMI, physical activity and parental overweight/obesity and type 2 diabetes. Brain, skeletal muscle, brown adipose tissue (BAT), visceral adipose tissue (VAT) and abdominal and femoral s.c. adipose tissue (SAT) glucose uptake (GU) rates were measured. Endogenous glucose production (EGP) was calculated by subtracting the exogenous glucose infusion rate from the rate of disappearance of [18F]FDG. BGU was analyzed using statistical parametric mapping, and peripheral tissue activity was determined using Carimas Software imaging processing platform. Results: BGU was higher in the HR vs LR group and correlated inversely with whole-body insulin sensitivity (M value) in the HR group but not in the LR group. Insulin-suppressed EGP did not differ between the groups but correlated positively with BGU in the whole population, and the correlation was driven by the HR group. Skeletal muscle, BAT, VAT, abdominal and femoral SAT GU were lower in the HR group as compared to the LR group. Muscle GU correlated negatively with BGU in the HR group but not in the LR group. Conclusion: Increased BGU, alterations in insulin action in the brain-liver axis and decreased whole-body insulin sensitivity occur early in pre-obese state.

Gata2, Nkx2-2 and Skor2 form a transcription factor network regulating development of a midbrain GABAergic neuron subtype with characteristics of REM-sleep regulatory neurons.

The midbrain reticular formation (MRF) is a mosaic of diverse GABAergic and glutamatergic neurons that have been associated with a variety of functions, including sleep regulation. However, the molecular characteristics and development of MRF neurons are poorly understood. As the transcription factor, Gata2 is required for the development of all GABAergic neurons derived from the embryonic mouse midbrain, we hypothesized that the genes expressed downstream of Gata2 could contribute to the diversification of GABAergic neuron subtypes in this brain region. Here, we show that Gata2 is required for the expression of several GABAergic lineage-specific transcription factors, including Nkx2-2 and Skor2, which are co-expressed in a restricted group of post-mitotic GABAergic precursors in the MRF. Both Gata2 and Nkx2-2 function is required for Skor2 expression in GABAergic precursors. In the adult mouse and rat midbrain, Nkx2-2-and Skor2-expressing GABAergic neurons locate at the boundary of the ventrolateral periaqueductal gray and the MRF, an area containing REM-off neurons regulating REM sleep. In addition to the characteristic localization, Skor2+ cells increase their activity upon REM-sleep inhibition, send projections to the dorsolateral pons, a region associated with sleep control, and are responsive to orexins, consistent with the known properties of midbrain REM-off neurons.

Cool-Water Immersion Reduces Postexercise Quadriceps Femoris Muscle Perfusion More Than Cold-Water Immersion.

PURPOSE: The muscle perfusion response to postexercise cold-water immersion (CWI) is not well understood. We examined the effects of graded postexercise CWI upon global and regional quadriceps femoris muscle perfusion using positron emission tomography and [15O]H2O. METHODS: Using a matched-group design, 30 healthy men performed cycle ergometer exercise at 70% V̇O2peak to a core body temperature of 38°C, followed by either 10 min of CWI at 8°C, 22°C, or seated rest (control). Quadriceps muscle perfusion; thigh and calf cutaneous vascular conductance; intestinal, muscle, and local skin temperatures; thermal comfort; mean arterial pressure; and heart rate were assessed at preexercise, postexercise, and after CWI. RESULTS: Global quadriceps perfusion was reduced beyond the predefined minimal clinically relevant threshold (0.75 mL per 100 g·min-1) in 22°C water versus control (difference (95% confidence interval (CI)), -2.5 (-3.9 to -1.1) mL per 100 g·min-1). Clinically relevant decreases in muscle perfusion were observed in the rectus femoris (-2.0 (-3.0 to -1.0) mL per 100 g·min-1) and vastus lateralis (-3.5 (-4.9 to -2.0) mL per 100 g·min-1) in 8°C water, and in the vastus lateralis (-3.3 (-4.8 to -1.9) mL per 100 g·min-1) in 22°C water versus control. The mean effects for vastus intermedius and vastus medialis perfusion were not clinically relevant. Clinically relevant decreases in thigh and calf cutaneous vascular conductance were observed in both cooling conditions. CONCLUSIONS: The present findings revealed that less noxious CWI (22°C) promoted clinically relevant postexercise decreases in global quadriceps muscle perfusion, whereas noxious cooling (8°C) elicited no effect.

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.

Novel effects of the gastrointestinal hormone secretin on cardiac metabolism and renal function.

The cardiac benefits of gastrointestinal hormones have been of interest in recent years. The aim of this study was to explore the myocardial and renal effects of the gastrointestinal hormone secretin in the GUTBAT trial (NCT03290846). A placebo-controlled crossover study was conducted on 15 healthy males in fasting conditions, where subjects were blinded to the intervention. Myocardial glucose uptake was measured with [18F]2-fluoro-2-deoxy-d-glucose ([18F]FDG) positron emission tomography. Kidney function was measured with [18F]FDG renal clearance and estimated glomerular filtration rate (eGFR). Secretin increased myocardial glucose uptake compared with placebo (secretin vs. placebo, means ± SD, 15.5 ± 7.4 vs. 9.7 ± 4.9 µmol/100 g/min, 95% confidence interval (CI) [2.2, 9.4], P = 0.004). Secretin also increased [18F]FDG renal clearance (44.5 ± 5.4 vs. 39.5 ± 8.5 mL/min, 95%CI [1.9, 8.1], P = 0.004), and eGFR was significantly increased from baseline after secretin, compared with placebo (17.8 ± 9.8 vs. 6.0 ± 5.2 ΔmL/min/1.73 m2, 95%CI [6.0, 17.6], P = 0.001). Our results implicate that secretin increases heart work and renal filtration, making it an interesting drug candidate for future studies in heart and kidney failure.NEW & NOTEWORTHY Secretin increases myocardial glucose uptake compared with placebo, supporting a previously proposed inotropic effect. Secretin also increased renal filtration rate.

Assessment of a digital and an analog PET/CT system for accurate myocardial perfusion imaging with a flow phantom.

In Myocardial Perfusion Imaging (MPI) with Positron Emission Tomography/Computed Tomography (PET/CT) systems, accurate quantification is essential. We assessed flow quantification accuracy over various injected activities using a flow phantom. METHODS: The study was performed on the digital 4-ring Discovery MI (DMI-20) and analog Discovery 690 (D690) PET/CT systems, using 325-1257 MBq of [15O]H2O. PET performance and flow quantification accuracy were assessed in terms of count-rates, dead-time factors (DTF), scatter fractions (SF), time-activity curves (TACs), areas-under-the-curves (AUCs) and flow values. RESULTS: On DMI-20, prompts of 12.8 Mcps, DTF of 2.06 and SF of 46.1% were measured with 1257 MBq of activity. On the D690, prompts of 6.85 Mcps, DTF of 1.57 and SF of 32.5% were measured with 1230 MBq of activity. AUC values were linear over all activities. Mean wash-in flow error was - 9% for both systems whereas wash-out flow error was - 5% and - 6% for DMI-20 and D690. With the highest activity, wash-out flow error was - 12% and - 7% for the DMI-20 and D690. CONCLUSION: DMI-20 and D690 preserved accurate flow quantification over all injected activities, with maximum error of - 12%. In the future, flow quantification accuracy over the activities and count-rates evaluated in this study should be assessed.

Secretin activates brown fat and induces satiation.

Brown adipose tissue (BAT) thermogenesis is activated by feeding. Recently, we revealed a secretin-mediated gut-BAT-brain axis, which stimulates satiation in mice, but the purpose of meal-induced BAT activation in humans has been unclear. In this placebo-controlled, randomized crossover study, we investigated the effects of intravenous secretin on BAT metabolism (measured with [18F]FDG and [15O]H2O positron emission tomography) and appetite (measured with functional magnetic resonance imaging) in healthy, normal weight men (GUTBAT trial no. NCT03290846). Participants were blinded to the intervention. Secretin increased BAT glucose uptake (primary endpoint) compared to placebo by 57% (median (interquartile range, IQR), 0.82 (0.77) versus 0.59 (0.53) µmol per 100 g per min, 95% confidence interval (CI) (0.09, 0.89), P = 0.002, effect size r = 0.570), while BAT perfusion remained unchanged (mean (s.d.) 4.73 (1.82) versus 6.14 (3.05) ml per 100 g per min, 95%CI (-2.91, 0.07), P = 0.063, effect size d = -0.549) (n = 15). Whole body energy expenditure increased by 2% (P = 0.011) (n = 15). Secretin attenuated blood-oxygen level-dependent activity (primary endpoint) in brain reward circuits during food cue tasks (significance level false discovery rate corrected at P = 0.05) (n = 14). Caloric intake did not significantly change, but motivation to refeed after a meal was delayed by 39 min (P = 0.039) (n = 14). No adverse effects were detected. Here we show in humans that secretin activates BAT, reduces central responses to appetizing food and delays the motivation to refeed after a meal. This suggests that meal-induced, secretin-mediated BAT activation is relevant in the control of food intake in humans. As obesity is increasing worldwide, this appetite regulating axis offers new possibilities for clinical research in treating obesity.

Molecular Fingerprint and Developmental Regulation of the Tegmental GABAergic and Glutamatergic Neurons Derived from the Anterior Hindbrain.

Tegmental nuclei in the ventral midbrain and anterior hindbrain control motivated behavior, mood, memory, and movement. These nuclei contain inhibitory GABAergic and excitatory glutamatergic neurons, whose molecular diversity and development remain largely unraveled. Many tegmental neurons originate in the embryonic ventral rhombomere 1 (r1), where GABAergic fate is regulated by the transcription factor (TF) Tal1. We used single-cell mRNA sequencing of the mouse ventral r1 to characterize the Tal1-dependent and independent neuronal precursors. We describe gene expression dynamics during bifurcation of the GABAergic and glutamatergic lineages and show how active Notch signaling promotes GABAergic fate selection in post-mitotic precursors. We identify GABAergic precursor subtypes that give rise to distinct tegmental nuclei and demonstrate that Sox14 and Zfpm2, two TFs downstream of Tal1, are necessary for the differentiation of specific tegmental GABAergic neurons. Our results provide a framework for understanding the development of cellular diversity in the tegmental nuclei.

Kainate Receptor Auxiliary Subunit NETO2-Related Cued Fear Conditioning Impairments Associate with Defects in Amygdala Development and Excitability.

NETO2 is an auxiliary subunit for kainate-type glutamate receptors that mediate normal cued fear expression and extinction. Since the amygdala is critical for these functions, we asked whether Neto2-/- mice have compromised amygdala function. We measured the abundance of molecular markers of neuronal maturation and plasticity, parvalbumin-positive (PV+), perineuronal net-positive (PNN+), and double positive (PV+PNN+) cells in the Neto2-/- amygdala. We found that Neto2-/- adult, but not postnatal day (P)23, mice had 7.5% reduction in the fraction of PV+PNN+ cells within the total PNN+ population, and 23.1% reduction in PV staining intensity compared with Neto2+/+ mice, suggesting that PV interneurons in the adult Neto2-/- amygdala remain in an immature state. An immature PV inhibitory network would be predicted to lead to stronger amygdalar excitation. In the amygdala of adult Neto2-/- mice, we identified increased glutamatergic and reduced GABAergic transmission using whole-cell patch-clamp recordings. This was accompanied by increased spine density of thin dendrites in the basal amygdala (BA) compared with Neto2+/+ mice, indicating stronger glutamatergic synapses. Moreover, after fear acquisition Neto2-/- mice had a higher number of c-Fos-positive cells than Neto2+/+ mice in the lateral amygdala (LA), BA, and central amygdala (CE). Altogether, our findings indicate that Neto2 is involved in the maturation of the amygdala PV interneuron network. Our data suggest that this defect, together with other processes influencing amygdala principal neurons, contribute to increased amygdalar excitability, higher fear expression, and delayed extinction in cued fear conditioning, phenotypes that are common in fear-related disorders, including the posttraumatic stress disorder (PTSD).

Ruthenium-Mediated 18F-Fluorination and Preclinical Evaluation of a New CB1 Receptor Imaging Agent [18F]FPATPP.

Cannabinoid receptor 1 (CB1R) controls various physiological and pathological conditions, including memory, motivation, and inflammation, and is thus an interesting target for positron emission tomography (PET). Herein, we report a ruthenium-mediated radiolabeling synthesis and preclinical evaluation of a new CB1R specific radiotracer, [18F]FPATPP. [18F]FPATPP was produced with 16.7 ± 5.7% decay-corrected radiochemical yield and >95 GBq/µmol molar activity. The tracer showed high stability, low defluorination, and high specific binding to CB1Rs in mouse brain.

Automated GMP production and long-term experience in radiosynthesis of CB1 tracer [18 F]FMPEP-d2.

Here, we describe the development of an in-house-built device for the fully automated multistep synthesis of the cannabinoid CB1 receptor imaging tracer (3R,5R)-5-(3-([18 F]fluoromethoxy-d2 )phenyl)-3-(((R)-1-phenylethyl)amino)-1-(4-(trifluoromethyl)phenyl)pyrrolidin-2-one ([18 F]FMPEP-d2 ), following good manufacturing practices. The device is interfaced to a HPLC and a sterile filtration unit in a clean room hot cell. The synthesis involves the nucleophilic 18 F-fluorination of an alkylating agent and its GC purification, the subsequent 18 F-fluoroalkylation of a precursor molecule, the semipreparative HPLC purification of the 18 F-fluoroalkylated product, and its formulation for injection. We have optimized the duration and temperature of the 18 F-fluoroalkylation reaction and addressed the radiochemical stability of the formulated product. During the past 5 years (2013-2018), we have performed a total of 149 syntheses for clinical use with a 90% success rate. The activity yield of the formulated product has been 1.0 ± 0.4 GBq starting from 11 ± 2 GBq and the molar activity 600 ± 300 GBq/µmol at the end of synthesis.

Changes in quadriceps femoris muscle perfusion following different degrees of cold-water immersion.

We examined the influence of graded cold-water immersion (CWI) on global and regional quadriceps muscle perfusion with positron emission tomography (PET) and [15O]H2O. In 30 healthy men [33 ± 8 yr; 81 ± 10 kg; 184 ± 5 cm; percentage body fat: 13 ± 5%; peak oxygen uptake (V̇o2peak): 47 ± 8 mL·kg-1·min-1] quadriceps perfusion, thigh and calf cutaneous vascular conductance (CVC), intestinal, muscle, and local skin temperatures, thermal comfort, mean arterial pressure, and heart rate were assessed before and after 10 min of CWI at 8°C, 15°C, or 22°C. Global quadriceps perfusion did not change beyond a clinically relevant threshold (0.75 mL·100 g·min-1) in any condition and was similar between conditions {range of differences [95% confidence interval (CI)]: 0.1 mL·100 g·min-1 (-0.9 to 1.2 mL·100 g·min-1) to 0.9 mL·100 g·min-1 (-0.2 to 1.9 mL·100 g·min-1)}. Muscle perfusion was greater in vastus intermedius (VI) compared with vastus lateralis (VL) (2.2 mL·100 g·min-1; 95% CI 1.5 to 3.0 mL·100 g·min-1) and rectus femoris (RF) (2.2 mL·100 g·min-1; 1.4 to 2.9 mL·100 g·min-1). A clinically relevant increase in VI muscle perfusion after immersion at 8°C and a decrease in RF muscle perfusion at 15°C were observed. A clinically relevant increase in perfusion was observed in VI in 8°C compared with 22°C water (2.3 mL·100 g·min-1; 1.1 to 3.5 mL·100 g·min-1). There were no clinically relevant between-condition differences in thigh CVC. Our findings suggest that CWI (8-22°C) does not reduce global quadriceps muscle perfusion to a clinically relevant extent; however, colder water increases (8°C) deep muscle perfusion and reduces (15°C) superficial muscle (RF) perfusion in the quadriceps muscle.NEW & NOTEWORTHY Using positron emission tomography, we report for the first time muscle perfusion heterogeneity in the quadriceps femoris in response to different degrees of cold-water immersion (CWI). Noxious CWI temperatures (8°C) increase perfusion in the deep quadriceps muscle, whereas superficial quadriceps muscle perfusion is reduced in cooler (15°C) water. Therefore, these data have important implications for the selection of CWI approaches used in the treatment of soft tissue injury, while also increasing our understanding of the potential mechanisms underpinning CWI.

Fast and efficient copper-mediated 18F-fluorination of arylstannanes, aryl boronic acids, and aryl boronic esters without azeotropic drying.

BACKGROUND: Copper-mediated radiofluorination is a straightforward method to produce a variety of [18F]fluoroarenes and [18F]fluoroheteroarenes. To minimize the number of steps in the production of 18F-labelled radiopharmaceuticals, we have developed a short and efficient azeotropic drying-free 18F-labelling method using copper-mediated fluorination. Our goal was to improve the copper-mediated method to achieve wide substrate scope with good radiochemical yields with short synthesis time. RESULTS: Solid phase extraction with Cu (OTf)2 in dimethylacetamide is a suitable activation method for [18F]fluoride. Elution efficiency with Cu (OTf)2 is up to 79% and radiochemical yield (RCY) of a variety of model molecules in the crude reaction mixture has reached over 90%. Clinically relevant molecules, norepinephrine transporter tracer [18F]NS12137 and monoamine transporter tracer [18F]CFT were produced with 16.5% RCY in 98 min and 5.3% RCY in 64 min, respectively. CONCLUSIONS: Cu (OTf)2 is a suitable elution agent for releasing [18F]fluoride from an anion exchange cartridge. The method is fast and efficient and the Cu-complex is customizable after the release of [18F]fluoride. Alterations in the [18F]fluoride elution techniques did not have a negative effect on the subsequent labelling reactions. We anticipate this improved [18F]fluoride elution technique to supplant the traditional azeotropic drying of [18F]fluoride in the long run and to concurrently enable the variations of the copper-complex.

Radiosynthesis of the norepinephrine transporter tracer [18 F]NS12137 via copper-mediated 18 F-labelling.

[18 F]NS12137 (exo-3-[(6-[18 F]fluoro-2-pyridyl)oxy]8-azabicyclo[3.2.1]octane) is a highly selective norepinephrine transporter (NET) tracer. NETs are responsible for the reuptake of norepinephrine and dopamine and are linked to several neurodegenerative and neuropsychiatric disorders. The aim of this study was to develop a copper-mediated 18 F-fluorination method for the production of [18 F]NS12137 with straightforward synthesis conditions and high radiochemical yield and molar activity. [18 F]NS12137 was produced in two steps. Radiofluorination of [18 F]NS12137 was performed via a copper-mediated pathway starting with a stannane precursor and using [18 F]F- as the source of the fluorine-18 isotope. Deprotection was performed via acid hydrolysis. The radiofluorination reaction was nearly quantitative as was the deprotection based on HPLC analysis. The radiochemical yield of the synthesis was 15.1 ± 0.5%. Molar activity of [18 F]NS12137 was up to 300 GBq/µmol. The synthesis procedure is straightforward and can easily be automated and adapted for clinical production.

The SGLT2 Inhibitor Dapagliflozin Reduces Liver Fat but Does Not Affect Tissue Insulin Sensitivity: A Randomized, Double-Blind, Placebo-Controlled Study With 8-Week Treatment in Type 2 Diabetes Patients.

OBJECTIVE: The aim of this study was to investigate tissue-specific effects of dapagliflozin on insulin sensitivity and liver and body fat in patients with type 2 diabetes. RESEARCH DESIGN AND METHODS: This randomized, double-blind, parallel group, placebo-controlled study recruited 32 patients with type 2 diabetes. Enrolled patients were to have HbA1c 6.5-10.5% (48-91 mmol/mol) and ≥3 months of stable treatment with metformin, dipeptidyl peptidase 4 inhibitor, or their combination. Patients were randomized 1:1 to receive 10 mg dapagliflozin or placebo daily for 8 weeks. Before and after the intervention, tissue insulin sensitivity was measured using [18F]-fluorodeoxyglucose and positron emission tomography during hyperinsulinemic-euglycemic clamp. Liver proton density fat fraction (PDFF) and adipose tissue volumes were assessed using MRI, and blood biomarkers were analyzed. RESULTS: After 8 weeks, glycemic control was improved by dapagliflozin (placebo-corrected change in HbA1c -0.39%, P < 0.01), but whole-body glucose uptake was not increased (P = 0.90). Tissue-specific insulin-stimulated glucose uptake did not change in skeletal muscle, liver, myocardium, or white and brown adipose tissue, and endogenous glucose production remained unaffected. However, there were significant placebo-corrected decreases in liver PDFF (-3.74%, P < 0.01), liver volume (-0.10 L, P < 0.05), visceral adipose tissue volume (-0.35 L, P < 0.01), interleukin-6 (-1.87 pg/mL, P < 0.05), and N-terminal prohormone of brain natriuretic peptide (-96 ng/L, P = 0.03). CONCLUSIONS: In this study, 8 weeks of treatment with dapagliflozin reduced liver PDFF and the volume of visceral adipose tissue in obese patients with type 2 diabetes. Although glycemic control was improved, no effect on tissue-level insulin sensitivity was observed.

Radiosynthesis and Preclinical Evaluation of an α2A-Adrenoceptor Tracer Candidate, 6-[18F]Fluoro-marsanidine.

PURPOSE: The α2-adrenoceptors mediate many effects of norepinephrine and epinephrine, and participate in the regulation of neuronal, endocrine, cardiovascular, vegetative, and metabolic functions. Of the three receptor subtypes, only α2A and α2C are found in the brain in significant amounts. Subtype-selective positron emission tomography (PET) imaging of α2-adrenoceptors has been limited to the α2C subtype. Here, we report the synthesis of 6-[18F]fluoro-marsanidine, a subtype-selective PET tracer candidate for α2A-adrenoceptors, and its preclinical evaluation in rats and mice. PROCEDURES: 6-[18F]Fluoro-marsanidine was synthesized using electrophilic F-18 fluorination with [18F]Selectfluor bis(triflate). The tracer was evaluated in Sprague Dawley rats and in α2A-knockout (KO) and wild-type (WT) mice for subtype selectivity. In vivo PET imaging and ex vivo brain autoradiography were performed to determine the tracer distribution in the brain. The specificity of the tracer for the target was determined by pretreatment with the subtype-non-selective α2-agonist medetomidine. The peripheral biodistribution and extent of metabolism of 6-[18F]fluoro-marsanidine were also analyzed. RESULTS: 6-[18F]Fluoro-marsanidine was synthesized with [18F]Selectfluor bis(triflate) in a radiochemical yield of 6.4 ± 1.7 %. The molar activity was 3.1 to 26.6 GBq/µmol, and the radiochemical purity was > 99 %. In vivo studies in mice revealed lower uptake in the brains of α2A-KO mice compared to WT mice. The results for selectivity were confirmed by ex vivo brain autoradiography. Blocking studies revealed reduced uptake in α2A-adrenoceptor-rich brain regions in pretreated animals, demonstrating the specificity of the tracer. Metabolite analyses revealed very rapid metabolism of 6-[18F]fluoro-marsanidine with blood-brain barrier-permeable metabolites in both rats and mice. CONCLUSION: 6-[18F]Fluoro-marsanidine was synthesized and evaluated as a PET tracer candidate for brain α2A-adrenoceptors. However, rapid metabolism, extensive presence of labeled metabolites in the brain, and high non-specific uptake in mouse and rat brain make 6-[18F]fluoro-marsanidine unsuitable for α2A-adrenoceptor targeting in rodents in vivo.

In vivo characterization of a novel norepinephrine transporter PET tracer [18F]NS12137 in adult and immature Sprague-Dawley rats.

Norepinephrine modulates cognitive processes such as working and episodic memory. Pathological changes in norepinephrine and norepinephrine transporter (NET) function and degeneration of the locus coeruleus produce irreversible impairments within the whole norepinephrine system, disrupting cognitive processes. Monitoring these changes could enhance diagnostic accuracy and support development of novel therapeutic components for several neurodegenerative diseases. Thus, we aimed to develop a straightforward nucleophilic fluorination method with high molar activity for the novel NET radiotracer [18F]NS12137 and to demonstrate the ability of [18F]NS12137 to quantify changes in NET expression. Methods: We applied an 18F-radiolabeling method in which a brominated precursor was debrominated by nucleophilic 18F-fluorination in dimethyl sulfoxide. Radiolabeling was followed by a deprotection step, purification, and formulation of the radiotracer. The [18F]NS12137 brain uptake and distribution were studied with in vivo PET/CT and ex vivo autoradiography using both adult and immature Sprague-Dawley rats because postnatal NET expression peaks at 10-20 days post birth. The NET specificity for the tracer was demonstrated by pretreatment of the animals with nisoxetine, which is well-known to have a high affinity for NET. Results: [18F]NS12137 was successfully synthesized with radiochemical yields of 18.6±5.6%, radiochemical purity of >99%, and molar activity of >500 GBq/µmol at the end of synthesis. The in vivo [18F]NS12137 uptake showed peak standard uptake values (SUV) of over 1.5 (adult) and 2.2 (immature) in the different brain regions. Peak SUV/30 min and peak SUV/60 min ratios were calculated for the different brain regions of the adult and immature rats, with a peak SUV/60 min ratio of more than 4.5 in the striatum of adult rats. As expected, in vivo studies demonstrated uptake of the tracer in brain areas rich in NET, particularly thalamus, neocortex, and striatum, and remarkably also in the locus coeruleus, a quite small volume for imaging with PET. The uptake was significantly higher in immature rats compared to the adult animals. Ex vivo studies using autoradiography showed very strong specific binding in NET-rich areas such as the locus coeruleus and the bed nucleus of the stria terminalis, and high binding in larger grey matter areas such as the neocortex and striatum. The uptake of [18F]NS12137 was dramatically reduced both in vivo and ex vivo by pretreatment with nisoxetine, demonstrating the specificity of binding. Conclusions: [18F]NS12137 was synthesized in good yield and high molar activity and demonstrated the characteristics of a good radiotracer, such as good brain penetration, fast washout, and high specific binding to NET.

[18F]F-DPA for the detection of activated microglia in a mouse model of Alzheimer's disease.

INTRODUCTION: Neuroinflammation is associated with several neurological disorders, including Alzheimer's disease (AD). The translocator protein 18 kDa (TSPO), due to its overexpression during microglial activation and relatively low levels in the brain under normal neurophysiological conditions, is commonly used as an in vivo biomarker for neuroinflammation. Reported here is the preclinical evaluation of [18F]F-DPA, a promising new TSPO-specific radioligand, as a tool for the detection of activated microglia at different ages in the APP/PS1-21 mouse model of AD and a blocking study to determine the specificity of the tracer. METHODS: [18F]F-DPA was synthesised by the previously reported electrophilic 18F-fluorination methodology. In vivo PET and ex vivo brain autoradiography were used to observe the tracer distribution in the brains of APP/PS1-21 and wildtype mice at different ages (4.5-24 months). The biodistribution and degree of metabolism of [18F]F-DPA were analysed and the specificity of [18F]F-DPA for its target was determined by pre-treatment with PK11195. RESULTS: The in vivo PET imaging and ex vivo brain autoradiography data showed that [18F]F-DPA uptake in the brains of the transgenic animals increased with age, however, there was a drop in the tracer uptake at 19 mo. Despite the slight increase in [18F]F-DPA uptake with age in healthy animal brains, significant differences between wildtype and transgenic animals were seen in vivo at 9 months and ex vivo already at 4.5 months. The specificity study demonstrated that PK11195 can be used to significantly block [18F]F-DPA uptake in all the brain regions studied. CONCLUSIONS: In vivo time activity curves plateaued at approximately 20-40 min suggesting that this is the optimal imaging time. Significant differences in vivo are seen at 9 and 12 mo. Due to the higher resolution, ex vivo autoradiography with [18F]F-DPA can be used to visualise activated microglia at an early stage of AD pathology.

[18F]FMPEP-d2 PET imaging shows age- and genotype-dependent impairments in the availability of cannabinoid receptor 1 in a mouse model of Alzheimer's disease.

Contradictory findings on the role of the type 1 cannabinoid receptor (CB1R) during the pathogenesis of Alzheimer's disease (AD) have been reported. Here, we evaluated the CB1R brain profile in an AD mouse model using longitudinal positron emission tomography with an inverse agonist for CB1R, [18F]FMPEP-d2. APP/PS1-21 and wild-type (n = 8 in each group) mice were repeatedly imaged between 6 to 15 months of age, accompanied by brain autoradiography, western blot, and CB1R immunohistochemistry with additional mice. [18F]FMPEP-d2 positron emission tomography demonstrated lower (p < 0.05) binding ratios in the parietotemporal cortex and hippocampus of APP/PS1-21 mice compared with age-matched wild-type mice. Western blot demonstrated no differences between APP/PS1-21 and wild-type mice in the CB1R abundance, whereas significantly lower (p < 0.05) receptor expression was observed in male than female mice. The results provide the first demonstration that [18F]FMPEP-d2 is a promising imaging tool for AD research in terms of CB1R availability, but not expression. This finding may further facilitate the development of novel therapeutic approaches based on endocannabinoid regulation.