A question relevant to nicotine addiction is how nicotine and other nicotinic receptor membrane-permeant ligands, such as the anti-smoking drug varenicline (Chantix), distribute in brain. Ligands, like varenicline, with high pKa and high affinity for α4β2-type nicotinic receptors (α4β2Rs) are trapped in intracellular acidic vesicles containing α4β2Rs in vitro. Nicotine, with lower pKa and α4β2R affinity, is not trapped. Here, we extend our results by imaging nicotinic PET ligands in vivo in male and female mouse brain and identifying the trapping brain organelle in vitro as Golgi satellites (GSats). Two PET 18F-labeled imaging ligands were chosen: [18F]2-FA85380 (2-FA) with varenicline-like pKa and affinity and [18F]Nifene with nicotine-like pKa and affinity. [18F]2-FA PET-imaging kinetics were very slow consistent with 2-FA trapping in α4β2R-containing GSats. In contrast, [18F]Nifene kinetics were rapid, consistent with its binding to α4β2Rs but no trapping. Specific [18F]2-FA and [18F]Nifene signals were eliminated in β2 subunit knock-out (KO) mice or by acute nicotine (AN) injections demonstrating binding to sites on β2-containing receptors. Chloroquine (CQ), which dissipates GSat pH gradients, reduced [18F]2-FA distributions while having little effect on [18F]Nifene distributions in vivo consistent with only [18F]2-FA trapping in GSats. These results are further supported by in vitro findings where dissipation of GSat pH gradients blocks 2-FA trapping in GSats without affecting Nifene. By combining in vitro and in vivo imaging, we mapped both the brain-wide and subcellular distributions of weak-base nicotinic receptor ligands. We conclude that ligands, such as varenicline, are trapped in neurons in α4β2R-containing GSats, which results in very slow release long after nicotine is gone after smoking. SIGNIFICANCE STATEMENT Mechanisms of nicotine addiction remain poorly understood. An earlier study using in vitro methods found that the anti-smoking nicotinic ligand, varenicline (Chantix) was trapped in α4β2R-containing acidic vesicles. Using a fluorescent-labeled high-affinity nicotinic ligand, this study provided evidence that these intracellular acidic vesicles were α4β2R-containing Golgi satellites (GSats). In vivo PET imaging with F-18-labeled nicotinic ligands provided additional evidence that differences in PET ligand trapping in acidic vesicles were the cause of differences in PET ligand kinetics and subcellular distributions. These findings combining in vitro and in vivo imaging revealed new mechanistic insights into the kinetics of weak base PET imaging ligands and the subcellular mechanisms underlying nicotine addiction.

Positron emission tomography (PET) radioligands that bind with high-affinity to α4β2-type nicotinic receptors (α4β2Rs) allow for in vivo investigations of the mechanisms underlying nicotine addiction and smoking cessation. Here, we investigate the use of an image-derived arterial input function and the cerebellum for kinetic analysis of radioligand binding in mice. Two radioligands were explored: 2-[18F]FA85380 (2-FA), displaying similar pKa and binding affinity to the smoking cessation drug varenicline (Chantix), and [18F]Nifene, displaying similar pKa and binding affinity to nicotine. Time–activity curves of the left ventricle of the heart displayed similar distribution across wild type mice, mice lacking the β2-subunit for ligand binding, and acute nicotine-treated mice, whereas reference tissue binding displayed high variation between groups. Binding potential estimated from a two-tissue compartment model fit of the data with the image-derived input function were higher than estimates from reference tissue-based estimations. Rate constants of radioligand dissociation were very slow for 2-FA and very fast for Nifene. We conclude that using an image-derived input function for kinetic modeling of nicotinic PET ligands provides suitable results compared to reference tissue-based methods and that the chemical properties of 2-FA and Nifene are suitable to study receptor response to nicotine addiction and smoking cessation therapies.

A question relevant to nicotine addiction is how nicotine and other nicotinic receptor membrane-permeant ligands, such as the anti-smoking drug varenicline (Chantix), distribute in brain. Ligands, like varenicline, with high pKaand high affinity for α4β2-type nicotinic receptors (α4β2Rs) are trapped in intracellular acidic vesicles containing α4β2Rsin vitro. Nicotine, with lower pKaand α4β2R affinity, is not trapped. Here, we extend our results by imaging nicotinic PET ligandsin vivoin male and female mouse brain and identifying the trapping brain organellein vitroas Golgi satellites (GSats). Two PET18F-labeled imaging ligands were chosen: [18F]2-FA85380 (2-FA) with varenicline-like pKaand affinity and [18F]Nifene with nicotine-like pKaand affinity. [18F]2-FA PET-imaging kinetics were very slow consistent with 2-FA trapping in α4β2R-containing GSats. In contrast, [18F]Nifene kinetics were rapid, consistent with its binding to α4β2Rs but no trapping. Specific [18F]2-FA and [18F]Nifene signals were eliminated in β2 subunit knock-out (KO) mice or by acute nicotine (AN) injections demonstrating binding to sites on β2-containing receptors. Chloroquine (CQ), which dissipates GSat pH gradients, reduced [18F]2-FA distributions while having little effect on [18F]Nifene distributionsin vivoconsistent with only [18F]2-FA trapping in GSats. These results are further supported byin vitrofindings where dissipation of GSat pH gradients blocks 2-FA trapping in GSats without affecting Nifene. By combiningin vitroandin vivoimaging, we mapped both the brain-wide and subcellular distributions of weak-base nicotinic receptor ligands. We conclude that ligands, such as varenicline, are trapped in neurons in α4β2R-containing GSats, which results in very slow release long after nicotine is gone after smoking.SIGNIFICANCE STATEMENTMechanisms of nicotine addiction remain poorly understood. An earlier study usingin vitromethods found that the anti-smoking nicotinic ligand, varenicline (Chantix) was trapped in α4β2R-containing acidic vesicles. Using a fluorescent-labeled high-affinity nicotinic ligand, this study provided evidence that these intracellular acidic vesicles were α4β2R-containing Golgi satellites (GSats).In vivoPET imaging with F-18-labeled nicotinic ligands provided additional evidence that differences in PET ligand trapping in acidic vesicles were the cause of differences in PET ligand kinetics and subcellular distributions. These findings combiningin vitroandin vivoimaging revealed new mechanistic insights into the kinetics of weak base PET imaging ligands and the subcellular mechanisms underlying nicotine addiction.

82 Objectives: Although men generally have higher smoking rates than women, nicotine addiction is a public health issue that concerns both genders. Despite its widespread epidemiological relevance, the precise mechanisms underlying nicotine dependence—as well as these gender differences that exist—remains poorly understood. Addiction to nicotine, the substance responsible for tobacco dependence, is predicated on the upregulation of the nicotinic acetylcholine receptor (nAChR) in the brain. Currently, serval positron emission tomography (PET) radiotracers, all of which are weak bases, are available for studies of the binding kinetics of nAChRs. They may be classified according to their differential kinetics based on their ligand pKas and receptor affinity. This study used one of high affinity radiotracer, 2-[18F]FA-85380, to examine the gender differences in binding kinetics of nAChRs. Methods: The 2-[18F]FA-85380 radiotracer was synthesized from the commercially available precursor, 2-TMA-A85380, using an IBA Synthera V2 synthesis module equipped with Synthera preparative HPLC. The tracer (average injected activity = 143.6 uCi, specific activity >3000 mCi/μmole, 99% radiochemical purity) was injected into male and female C57BL/6J mice (3-6 months old) via intraperitoneal catheter within 30 seconds following the start of PET imaging. Dynamic PET acquisition was done for a continuous 180-minute scan and analyzed in 18x10-minute frames. The nAChR binding by 2-[18F]-A85380 in brain regions was assessed using the VivoQuant imaging analysis software with the 3D brain atlas and reported in standardized uptake value (SUV). Results: The dynamic nAChR binding was first analyzed for the whole brain over 180-minute imaging period. The maximum binding capacity of the female is greater than that of males by 11.2% (0.632 vs. 0.564, SUVmax for females and males, respectively) while the binding peaks at 70 minutes in females compared to 50 minutes in males. The elevated binding capacity of the females remains throughout the rest of scanning time after the peak binding, demonstrating a slower dissociation rate of 2-[18F]-A85380 to nAChR. The brain was then segmented into 13 different functional regions. Assessment of regional nAChR binding capacities revealed highest binding activity in the thalamus (0.864 vs. 0.715, SUVmax for females and males, respectively) and lowest within the cerebellum (0.571 and 0.557, SUVmax for females and males, respectively) for both males and females. Consistently, females have higher nAChR binding than the males within all 13 brain regions. Mice with β2 subunit-knockout nAChR were additionally imaged with 2-[18F]-A85380. This gender difference was also observed in the knockout animals with female knockout demonstrating higher nAChR binding than the male knockout. However, regional differences were not observed in the β2-knockout mice. Conclusions: The brain regional distribution of nAChR binding by 2-[18F]-A85380, specifically highest uptake within thalamus in contrast to the weakest binding within the cerebellum, is consistent with findings from human studies. The elevated maximum binding of 2-[18F]FA-85380 in females may suggest a higher density of nAChR in the female brain. In addition, one of the hypotheses of the nicotine binding mechanisms is that the ligand/receptor complex is trapped in the intracellular acidic vesicles. The slower dissociation rate observed in females may be indicative of stronger trapping. Together, these observed gender differences in mice may reflect gender differences in the response to nicotine exposure as well as to nicotine cessation treatments in humans. Therefore, more investigation into the binding kinetics of 2-[18F]FA-85380 and other nAChR ligands is necessary. The differences in kinetics and displaceable binding should provide insight into the cellular mechanism of nicotine induced nAChR upregulation and how nicotine agonists alter this process.

1060 Objectives: Previous studies utilizing 48V labeled vanadyl (VO2+) chelate bis(acetylacetonato) oxovanadium(IV) [VO(acac)2] in PET and MR [1,2] study of xenograft Caco-2, HCT-116, and HCA-7 colorectal cancer cell line tumors in mice showed relatively low uptakes, despite research showing that VO(acac)2 is an effective contrast agent in MRI for early detection and staging of cancer [3]. To improve compound purity and enhance tumor uptake, the synthesis procedure was revisited following analysis of the cold synthesis [2] and an additional filtration step incorporated [4]. The improved compound was used for cell studies to assess the viability of the HCA-7 cell line as a xenograft model and in-vivo animal studies. We report on the improved synthesis procedure and resulting yields and demonstrate elevated tracer uptake in HCA-7 cells and animal models. Methods: Two thin natural titanium foils were irradiated via the 48Ti(p,n)48V reaction at 40 µA in an 18 MeV IBA cyclotron and left to decay to mitigate short-lived isotopes. The target was dissolved and 48V was isolated via a series of radiochemical steps [5] before being complexed with acetyl acetone under reflux to form 48V-VO(acac)2.While still hot, the solution was passed through a heated fritted glass filter. The filtrate was left to dry to allow VO(acac)2 to crystalize [4]. The VO(acac)2 was then reconstituted in DMSO to prepare for cell and animal studies. To evaluate the compound, half-life analysis and energy spectrum analysis were conducted. HCA-7 cells were grown in McCoy’s 54 medium with 10% FBS (fetal bovine serum). For cell studies, 28 dishes were plated with 5x105 cells each and incubated in 0 to 8 μCi of 48V-VO(acac)2solution at 37°C for 2 hours, washed, and assayed via gamma-ray spectrometry or Molecubes PET. Athymic nude mice were inoculated with 1 - 5 x106 HCA-7 cells and tumors allowed to grow to the appropriate size. Mice were injected with 50-85 µCi of tracer and imaged for four hours via Molecubes PET. After imaging, animals were sacrificed and tumor, muscle, and brain excised for biodistribution studies. Results: Foil irradiation for 87 hours yielded 31.6 mCi. Foils were dissolved in 30 µL HF and 400 µL H2SO4. The 25.2 mCi mixture was transferred to a platinum crucible, neutralized and oxidized at 790°C with a mixture of Na2CO3 and NaNO3 (43:1) for four hours. Several days later to ease radiation shielding, 10.5 mCi was reconstituted with water and centrifuged for 10 minutes at 5000 rpm. The 9.5 mCi supernatant was pipetted off and pH adjusted to 3-4 with HCl, then passed through a Chelex-100 column and eluted with NH3, yielding 5.6 mCi as NH4VO3. The solvent was dried at 300°C before 4.85 mCi was combined with acetyl acetonate and heated under reflux. The solution was passed through a heated, fritted glass filter, yielding 1.49 mCi, a 17% yield with decay corrections. The cell study showed that uptake is linear from 0 - 4 µCi, but plateaus between 4 and 8 µCi. After imaging, cells incubated in up to 4 µCi were still attached to the cell plate, while the cells that had incubated in 8 µCi for this time had died. Animal studies showed 1-2 %ID/mL uptake in tumor, typically higher than the 0.5-1.5 %ID/mL uptake in muscle and the 0.5-1% uptake shown in the brain, but lower than the 3-7% uptake shown in liver and 2-4.5% uptake shown in the kidneys. Conclusions: The yield of this synthesis (17%) was comparable to other runs [1,2] despite an additional filtration, indicating the compound has higher purity with similar yields. Further steps may be implemented in the future to allow the VO(acac)2 to better crystalize and separate, leading to a higher compound purity. The cell uptake curve plateaus after 4 µCi, indicating the maximum activity that can enter the cell is below 4 µCi. Finer sampling between 2 and 4 µCi is needed to determine the threshold. Animal studies demonstrated elevated uptake in tumor compared to other tissues, but high uptake in liver and kidney demonstrate that compound purity could still be improved.

Background Cardiac arrest (CA) patients who survived by cardiopulmonary resuscitation (CPR) can present different levels of neurological deficits ranging from minor cognitive impairments to persistent vegetative state and brain death. The pathophysiology of the resulting brain injury is poorly understood and whether changes in post-CA brain metabolism contribute to the injury are unknown. Here we utilized [18F]FDG-PET to study in vivo cerebral glucose metabolism 72 hours following CA in a murine cardiac arrest model. Methods Anesthetized and ventilated adult C57BL/6 mice underwent 12-minute KCl-induced CA followed by CPR. Seventy-two hours following cardiac arrest, surviving mice were intraperitoneally injected with [18F]FDG (~186 μCi/200 μL) and imaged on Molecubes preclinical micro PET/CT imaging systems after a 30-minute awake uptake period. Brain [18F]FDG uptake was determined by the VivoQuant software on fused PET/CT images with the 3D brain atlas. Upon completion of PET imaging, remaining [18F]FDG radioactivity in the brain, heart, and liver was determined using a gamma counter. Results Global increases in brain [18F]FDG uptake in post-CA mice were observed compared to shams and controls. The median standardized uptake value (SUV) of [18F]FDG for CA animals was 1.79 vs. sham 1.25 (p<0.05) and control animals 0.78 (p<0.01). This increased uptake was consistent throughout the 60-minute imaging period and across all brain regions reaching statistical significance in the midbrain, pons, and medulla. Biodistribution analyses of various key organs yielded similar observations that the median [18F]FDG uptake for brain were 7.04%ID/g tissue for CA mice vs 5.537%ID/g tissue for sham animals, p<0.05). Conclusions This study has successfully applied [18F]FDG-PET/CT to measure changes in brain metabolism in a murine model of asystolic CA. Our results demonstrate increased [18F]FDG uptake in the brain 72 hours following CA, suggesting increased metabolic demand in the case of severe neurological injury. Further study is warranted to determine the etiology of these changes.

111 Objectives: Cardiopulmonary Resuscitation (CPR) following cardiac arrest (CA) can lead to neurological deficits ranging from minor cognitive impairments to persistent vegetative state and brain death. Although the resulting brain injury has been well documented in clinical cases and preclinical models, its pathophysiology remains poorly understood. Previous studies have focused primarily on post-arrest changes in in cerebral blood flow or neuroanatomy and few have investigated post arrest changes in brain metabolism. Large fluctuations in cerebral glucose metabolism (CGM) have been reported during and immediately following cerebral ischemia but how these patterns are affected in the context of post-cardiac arrest resuscitation are unknown. In this study we utilized 18F-fluorodeoxyglucose-positron emission tomography (18F-FDG-PET) to study in vivo cerebral glucose metabolism in the post-CA setting. Methods: Anesthetized and ventilated adult C57BL/6 mice were undergone 8-minute KCl-induced CA followed by 90 second cardiopulmonary resuscitation and administration of epinephrine. Surviving mice were intraperitoneally injected with 18F-FDG (~250 uCi/250 ul) 72-hours after the CA and imaged thirty minutes later by high resolution CT imaging on a Molecube’s preclinical imaging system. CT imaging was used as anatomic templates. Brain FDG uptake was determined by Invicro VivoQuant software on fused PET/CT images with the 3D brain atlas. Upon completion of PET imaging, remaining FDG radioactivity in the brain, heart, and liver was determined by isolating organs and measuring 18F activity using a gamma counter. The FDG uptake was expressed as percent of injected dose per gram of tissue (%ID/g). Results: A global increase in FDG uptake in the brains of CA mice was observed compared to the sham group. The PET imaging shows that on average, the normalized uptake of FDG (%ID/g) for CA animals was 7.9%, which is statistically higher than the sham animals (4.89%) (p<0.05). In addition, this increased uptake was consistent throughout 60-min imaging period and across all the brain regions. Biodistribution analyses of various key organs yielded similar observations for brain FDG uptake (7.58% for CA mice vs 5.80% for sham animals). Conclusions: This study demonstrates for the first time, successful application of using PET/CT imaging to measure changes in brain metabolism, utilizing brain FDG uptake, in a murine model of CA. Our results suggest increased FDG uptake in the brain following cardiac arrest indicating altered patterns of cellular metabolism. Further studies are necessary to determine the implications of this altered metabolic pattern.

1310 Objectives: The α4β2 subtype of nicotinic acetylcholine receptors (nAChRs) is upregulated in response to nicotine exposure. Varenicline, an FDA approved medication for nicotine addiction, is a high affinity-weak base α4β2R ligand. However, the mechanisms of its action are yet to be fully investigated. One of the proposed mechanisms suggests that varenicline is trapped within intracellular acidic vesicles and slowly released, which is thought to be the neurobiological basis for its smoking cessation properties. Currently available PET tracers for α4β2Rs, all of which are weak bases, can be classified according to their differential kinetics based on their ligand pKas and receptor affinity. These differences in kinetics and displaceable binding should provide insight into the cellular mechanism of nicotinic receptor upregulation and how varenicline alters this process. The purpose of this study is to compare the differential kinetics and selective binding in two [18F]labeled α4β2R PET probes, i.e., 2-[18F]A85380 and [18F]Nifene, in mouse models. Methods: 2-[18F]A85380 (2-[18F]Fluoro-3-(2(S)-azetidinylmethoxy)pyridine), a radiotracer known with slow kinetics, was synthesized from the commercially available precursor, 2-TMA-A85380. [18F]Nifene, a radiotracer with relatively fast kinetics, was synthesized from the precursor N-Boc-nitronifene. An IBA Synthera V2 automatic synthesis module equipped with Synthera preparative HPLC was used for the radiolabeling inside a Comecer Hotcell. To establish ligand binding kinetics, wild type C57BL/J6 mice were used for both PET probes, while their littermates of β2-subunit knockout mice were used to confirm the specificity of α4β2Rs. [18F]labeled ligands were injected into anesthetized mice via tail vein (~250 μCi/100 μl), immediately followed by PET/CT imaging using the Molecubes preclinical imaging system. Dynamic PET acquisition was done in a 7 hour continuous scanning for 2-[18F]A85380 and in 2 hour for [18F]Nifene. High resolution CT imaging was performed after the PET scan for the purpose of anatomic co-registration. The binding kinetics of different brain regions were analyzed using Invicro Vivoquant software on CT-fused PET images co-registered with the available 3D brain atlas. Results: Synthesis of both radioligands was carried out at the cyclotron facility of the University of Chicago. Typical yields for 2-[18F]A85380 were 34% (decay corrected) with a specific activity of 8,300 mCi/μmole and higher than 99% radiochemical purity, while representative radiochemical yield was 6.3% (decay corrected) with higher than 99% purity for [18F]Nifene. The dynamic analyses demonstrated a fast binding kinetics of [18F]Nifene, peaking at ~10 minutes after tracer injection and a slow binding kinetics of 2-[18F]A85380, peaking at ~2 hours post injection. In wild type mice, both 2-[18F]A85380 and [18F]Nifene showed the highest binding as measured by %ID/g tissue in the thalamus (2.5% and 11.5%, respectively) and lowest binding in the cerebellum (1.1% & 5.9%). This regional difference of binding was eliminated in β2-subunit knockout mice, further confirming the specificity of the two ligands. Conclusions: Both 2-[18F]A85380 and [18F]Nifene were successfully employed in PET/CT mouse brain imaging for the kinetic study of α4β2Rs nicotine receptor, making this study the first to use 2-[18F]A85380 in mouse models. The selectivity of the synthesized probes is consistent with other publications showing the distribution of the α4β2 nicotine receptor in the brain of human and mice. With an understanding of the differences in probe kinetics, we will be able to better study the precise mechanism of nicotine receptor upregulation and the displaceable binding of nicotine and varenicline, which has implications for not only smoking cessation but other neurological conditions such as dementia, anxiety, and attention deficits.