Separating dopamine D2 and D3 receptor sources of [11C]-(+)-PHNO binding potential: Independent component analysis of competitive binding.

Development of medications selective for dopamine D2 or D3 receptors is an active area of research in numerous neuropsychiatric disorders including addiction and Parkinson's disease. The positron emission tomography (PET) radiotracer [11C]-(+)-PHNO, an agonist that binds with high affinity to both D2 and D3 receptors, has been used to estimate relative receptor subtype occupancy by drugs based on a priori knowledge of regional variation in the expression of D2 and D3 receptors. The objective of this work was to use a data-driven independent component analysis (ICA) of receptor blocking scans to separate D2-and D3-related signal in [11C]-(+)-PHNO binding data in order to improve the precision of subtype specific measurements of binding and occupancy. Eight healthy volunteers underwent [11C]-(+)-PHNO PET scans at baseline and at two time points following administration of the D3-preferring antagonist ABT-728 (150-1000 â€‹mg). Parametric binding potential (BPND) images were analyzed as four-dimensional image series using ICA to extract two independent sources of variation in [11C]-(+)-PHNO BPND. Spatial source maps for each component were consistent with respective regional patterns of D2-and D3-related binding. ICA-derived occupancy estimates from each component were similar to D2-and D3-specific occupancy estimated from a region-based approach (intraclass correlation coefficients â€‹> â€‹0.95). ICA-derived estimates of D3 receptor occupancy improved quality of fit to a single site binding model. Furthermore, ICA-derived estimates of the regional fraction of [11C]-(+)-PHNO binding related to D3 receptors was generated for each subject and values showed good agreement with region-based model estimates and prior literature values. In summary, ICA successfully separated D2-and D3-related components of the [11C]-(+)-PHNO binding signal, establishing this approach as a powerful data-driven method to quantify distinct biological features from PET data composed of mixed data sources.

Use of Electronic Cigarettes Leads to Significant Beta2-Nicotinic Acetylcholine Receptor Occupancy: Evidence From a PET Imaging Study.

Background: Electronic cigarettes (ECs) can influence nicotine addiction by delivering aerosolized nicotine. We investigated if nicotine from ECs is delivered to the brain ß2*-nicotinic acetylcholine receptors (ß2*-nAChR) and how this relates to the behavioral effects and nicotine delivery from cigarettes. Methods: Seven nicotine users participated in positron emission tomography (PET) studies with (-)-[18F]Flubatine before and after nicotine challenge with 0, 8, and 36 mg/ml nicotine in a 3.3 Volt, 1.5 Ohm EC or a standard tobacco cigarette. Craving was evaluated before and after product use. Results: Average ß2*-nAChR occupancy was higher after 36 mg/ml EC challenge compared to 8 mg/ml EC at trend level. Average ß2*-nAChR occupancy after tobacco cigarette smoking was 68 ± 18% and was not different compared with 8 mg/ml (64 ± 17%,) or 36 mg/ml (84 ± 3%) nicotine in EC users. Area under the curve (AUC) of blood nicotine level was higher in the cigarette smoking group compared with the 8mg/ml group (p = 0.03), but similar compared with the 36 mg/ml EC (p = 0.29). Drug craving was reduced after use of the tobacco cigarette, 8 mg/ml EC, and 36 mg/ml EC. Conclusions: In this novel investigation of EC effects at ß2*-nAChRs, we show that average ß2*-nAChR occupancy was higher after 36 mg/ml EC challenge compared with 8 mg/ml EC. Receptor occupancy and arterial blood nicotine levels after cigarette smoking were similar to 36 mg/ml EC use under controlled conditions. These findings suggest that the ECs studied here have abuse liability and may provide an adequate alternative nicotine delivery system for cigarette smokers. Implications: This is the first study to directly determine the neurologic effects of electronic cigarettes on human brain beta-2 nicotinic acetylcholine receptors using PET neuroimaging with (-)-[18F]Flubatine, a novel radiotracer. Our findings suggest that the e-cigarettes studied here have abuse liability and may provide an adequate alternative nicotine delivery system for cigarette smokers.

PET imaging of α7 nicotinic acetylcholine receptors: a comparative study of [18F]ASEM and [18F]DBT-10 in nonhuman primates, and further evaluation of [18F]ASEM in humans.

PURPOSE: The α7 nicotinic acetylcholine receptor (nAChR) is implicated in many neuropsychiatric disorders, making it an important target for positron emission tomography (PET) imaging. The first aim of this work was to compare two α7 nAChRs PET radioligands, [18F]ASEM (3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-([18F]fluorodibenzo[b,d]thiophene 5,5-dioxide) and [18F]DBT-10 (7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-2-([18F]fluorodibenzo[b,d]thiophene 5,5-dioxide), in nonhuman primates. The second aim was to assess further the quantification and test-retest variability of [18F]ASEM in humans. METHODS: PET scans with high specific activity [18F]ASEM or [18F]DBT-10 were acquired in three rhesus monkeys (one male, two female), and the kinetic properties of these radiotracers were compared. Additional [18F]ASEM PET scans with blocking doses of nicotine, varenicline, and cold ASEM were acquired separately in two animals. Next, six human subjects (five male, one female) were imaged with [18F]ASEM PET for 180 min, and arterial sampling was used to measure the parent input function. Different modeling approaches were compared to identify the optimal analysis method and scan duration for quantification of [18F]ASEM distribution volume (V T). In addition, retest scans were acquired in four subjects (three male, one female), and the test-retest variability of V T was assessed. RESULTS: In the rhesus monkey brain [18F]ASEM and [18F]DBT-10 exhibited highly similar kinetic profiles. Dose-dependent blockade of [18F]ASEM binding was observed, while administration of either nicotine or varenicline did not change [18F]ASEM V T. [18F]ASEM was selected for further validation because it has been used in humans. Accurate quantification of [18F]ASEM V T in humans was achieved using multilinear analysis with at least 90 min of data acquisition, resulting in V T values ranging from 19.6 ± 2.5 mL/cm3 in cerebellum to 25.9 ± 2.9 mL/cm3 in thalamus. Test-retest variability of V T was 11.7 ± 9.8%. CONCLUSIONS: These results confirm [18F]ASEM as a suitable radiotracer for the imaging and quantification of α7 nAChRs in humans.

Quantitative projection of human brain penetration of the H3 antagonist PF-03654746 by integrating rat-derived brain partitioning and PET receptor occupancy.

1. Unbound brain drug concentration (Cb,u), a valid surrogate of interstitial fluid drug concentration (CISF), cannot be directly determined in humans, which limits accurately defining the human Cb,u:Cp,u of investigational molecules. 2. For the H3R antagonist (1R,3R)-N-ethyl-3-fluoro-3-[3-fluoro-4-(pyrrolidin-1-lmethyl)phenyl]cyclobutane-1-carboxamide (PF-03654746), we interrogated Cb,u:Cp,u in humans and nonhuman primate (NHP). 3. In rat, PF-03654746 achieved net blood-brain barrier (BBB) equilibrium (Cb,u:Cp,u of 2.11). 4. In NHP and humans, the PET receptor occupancy-based Cp,u IC50 of PF-03654746 was 0.99 nM and 0.31 nM, respectively, which were 2.1- and 7.4-fold lower than its in vitro human H3 Ki (2.3 nM). 5. In an attempt to understand this higher-than-expected potency in humans and NHP, rat-derived Cb,u:Cp,u of PF-03654746 was integrated with Cp,u IC50 to identify unbound (neuro) potency of PF-03654746, nIC50. 6. The nIC50 of PF-03654746 was 2.1 nM in NHP and 0.66 nM in human which better correlated (1.1- and 3.49-fold lower) with in vitro human H3 Ki (2.3 nM). 7. This correlation of the nIC50 and in vitro hH3 Ki suggested the translation of net BBB equilibrium of PF-03654746 from rat to NHP and humans, and confirmed the use of Cp,u as a reliable surrogate of Cb,u. 8. Thus, nIC50 quantitatively informed the human Cb,u:Cp,u of PF-03654746.

A modification to improve the reliability of [11 C]CN- production in the GE radiochemistry system.

We report herein a modification to a component of the GE radiochemistry system that increases the yield and reliability of [11 C]CN- production from [11 C]carbon dioxide.

PET imaging evaluation of [(18)F]DBT-10, a novel radioligand specific to α7 nicotinic acetylcholine receptors, in nonhuman primates.

PURPOSE: Positron emission tomography (PET) radioligands specific to α7 nicotinic acetylcholine receptors (nAChRs) afford in vivo imaging of this receptor for neuropathologies such as Alzheimer's disease, schizophrenia, and substance abuse. This work aims to characterize the kinetic properties of an α7-nAChR-specific radioligand, 7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-2-[(18)F]-fluorodibenzo[b,d]thiophene 5,5-dioxide ([(18)F]DBT-10), in nonhuman primates. METHODS: [(18)F]DBT-10 was produced via nucleophilic substitution of the nitro-precursor. Four Macaca mulatta subjects were imaged with [(18)F]DBT-10 PET, with measurement of [(18)F]DBT-10 parent concentrations and metabolism in arterial plasma. Baseline PET scans were acquired for all subjects. Following one scan, ex vivo analysis of brain tissue was performed to inspect for radiolabeled metabolites in brain. Three blocking scans with 0.69 and 1.24 mg/kg of the α7-nAChR-specific ligand ASEM were also acquired to assess dose-dependent blockade of [(18)F]DBT-10 binding. Kinetic analysis of PET data was performed using the metabolite-corrected input function to calculate the parent fraction corrected total distribution volume (V T/f P). RESULTS: [(18)F]DBT-10 was produced within 90 min at high specific activities of 428 ± 436 GBq/µmol at end of synthesis. Metabolism of [(18)F]DBT-10 varied across subjects, stabilizing by 120 min post-injection at parent fractions of 15-55%. Uptake of [(18)F]DBT-10 in brain occurred rapidly, reaching peak standardized uptake values (SUVs) of 2.9-3.7 within 30 min. The plasma-free fraction was 18.8 ± 3.4%. No evidence for radiolabeled [(18)F]DBT-10 metabolites was found in ex vivo brain tissue samples. Kinetic analysis of PET data was best described by the two-tissue compartment model. Estimated V T/f P values were 193-376 ml/cm(3) across regions, with regional rank order of thalamus > frontal cortex > striatum > hippocampus > occipital cortex > cerebellum > pons. Dose-dependent blockade of [(18)F]DBT-10 binding by structural analog ASEM was observed throughout the brain, and occupancy plots yielded a V ND/f P estimate of 20 ± 16 ml/cm(3). CONCLUSION: These results demonstrate suitable kinetic properties of [(18)F]DBT-10 for in vivo quantification of α7-nAChR binding in nonhuman primates.

Nicotine and Nicotine Abstinence Do Not Interfere with GABAA Receptor Neuroadaptations During Alcohol Abstinence.

BACKGROUND: Alcohol dependence and tobacco smoking are highly comorbid, and treating both conditions simultaneously is controversial. Previously, we showed that tobacco smoking interferes with GABAA receptor neuroadaptations during alcohol withdrawal in humans, while this effect did not occur with continued nicotine use during alcohol abstinence in nonhuman primates. Here, we extend our previous work by measuring GABAA receptor availability with positron emission tomography (PET) during drug abstinence in nonhuman primates exposed to alcohol alone, nicotine and alcohol together, and alcohol abstinence with continued nicotine exposure. METHODS: Twenty-four adolescent male rhesus macaques orally self-administered alcohol and nicotine, available separately in water and saccharin, over 20 weeks. The groups included alcohol alone (n = 8); nicotine and alcohol with simultaneous abstinence (n = 8); nicotine and alcohol with alcohol abstinence while nicotine was still available (n = 8); and a pilot group of animals consuming nicotine alone (n = 6). Animals were imaged with [(11)C]flumazenil PET to measure binding potential (BPND), an index of GABAA receptor availability. Imaging occurred at baseline (drug-naíve), and following alcohol and/or nicotine cessation at 1 day, 8 days, and 12 weeks of abstinence. Generalized linear mixed models were used to examine the time course of [(11)C]flumazenil BPND during alcohol abstinence across groups. RESULTS: Animals consumed 3.95 ± 1.22 g/kg/d alcohol and 55.4 ± 35.1 mg/kg/d nicotine. No significant group effects were observed in [(11)C]flumazenil BPND during alcohol abstinence; however, a main effect of time was detected. Post hoc analyses indicated that all groups abstaining from alcohol exhibited significantly increased GABAA receptor availability at 1 day and 8 days (but not 12 weeks) of abstinence relative to baseline, while no changes in [(11)C]flumazenil BPND during nicotine abstinence alone were observed. CONCLUSIONS: These data indicate that neither nicotine nor nicotine abstinence interferes with GABAA receptor neuroadaptations during alcohol withdrawal. This conclusion is consistent with our previous study and does not contradict the use of nicotine replacement therapies or non-nicotinic-acting pharmaceuticals to quit smoking during alcohol withdrawal from a GABAergic perspective.

Sex differences in the brain's dopamine signature of cigarette smoking.

Cigarette smoking is a major public health danger. Women and men smoke for different reasons and cessation treatments, such as the nicotine patch, are preferentially beneficial to men. The biological substrates of these sex differences are unknown. Earlier PET studies reported conflicting findings but were each hampered by experimental and/or analytical limitations. Our new image analysis technique, lp-ntPET (Normandin et al., 2012; Morris et al., 2013; Kim et al., 2014), has been optimized for capturing brief (lasting only minutes) and highly localized dopaminergic events in dynamic PET data. We coupled our analysis technique with high-resolution brain scanning and high-frequency motion correction to create the optimal experiment for capturing and characterizing the effects of smoking on the mesolimbic dopamine system in humans. Our main finding is that male smokers smoking in the PET scanner activate dopamine in the right ventral striatum during smoking but female smokers do not. This finding-men activating more ventrally than women-is consistent with the established notion that men smoke for the reinforcing drug effect of cigarettes whereas women smoke for other reasons, such as mood regulation and cue reactivity. lp-ntPET analysis produces a novel multidimensional endpoint: voxel-level temporal patterns of neurotransmitter release ("DA movies") in individual subjects. By examining these endpoints quantitatively, we demonstrate that the timing of dopaminergic responses to cigarette smoking differs between men and women. Men respond consistently and rapidly in the ventral striatum whereas women respond faster in a discrete subregion of the dorsal putamen.

Test-retest reliability of the novel 5-HT1B receptor PET radioligand [11C]P943.

PURPOSE: [(11)C]P943 is a novel, highly selective 5-HT1B PET radioligand. The aim of this study was to determine the test-retest reliability of [(11)C]P943 using two different modeling methods and to perform a power analysis with each quantification technique. METHODS: Seven healthy volunteers underwent two PET scans on the same day. Regions of interest (ROIs) were the amygdala, hippocampus, pallidum, putamen, insula, frontal, anterior cingulate, parietal, temporal and occipital cortices, and cerebellum. Two multilinear radioligand quantification techniques were used to estimate binding potential: MA1, using arterial input function data, and the second version of the multilinear reference tissue model analysis (MRTM2), using the cerebellum as the reference region. Between-scan percent variability and intraclass correlation coefficients (ICC) were used to assess test-retest reliability. We also performed power analyses to determine the method that would allow the least number of subjects using within-subject or between-subject study designs. A voxel-wise ICC analysis for MRTM2 BPND was performed for the whole brain and all the ROIs studied. RESULTS: Mean percent variability between two scans across regions ranged between 0.4 % and 12.4 % for MA1 BPND, 0.5 % and 11.5 % for MA1 BPP, 16.7 % and 28.3 % for MA1 BPF, and between 0.2 % and 5.4 % for MRTM2 BPND. The power analyses showed a greater number of subjects were required using MA1 BPF compared with other outcome measures for both within-subject and between-subject study designs. ICC values were the highest using MRTM2 BPND and the lowest with MA1 BPF in ten ROIs. Small regions and regions with low binding had lower ICC values than large regions and regions with high binding. CONCLUSION: Reliable measures of 5-HT1B receptor binding can be obtained using the novel PET radioligand [(11)C]P943. Quantification of 5-HT1B receptor binding with MRTM2 BPND and with MA1 BPP provided the least variability and optimal power for within-subject and between-subject designs.

Further evaluation of [11C]MP-10 as a radiotracer for phosphodiesterase 10A: PET imaging study in rhesus monkeys and brain tissue metabolite analysis.

[(11)C]MP-10 is a potent and specific PET tracer previously shown to be suitable for imaging the phosphodiesterase 10A (PDE10A) in baboons with reversible kinetics and high specific binding. However, another report indicated that [(11)C]MP-10 displayed seemingly irreversible kinetics in rhesus monkeys, potentially due to the presence of a radiolabeled metabolite capable of penetrating the blood-brain-barrier (BBB) into the brain. This study was designed to address the discrepancies between the species by re-evaluating [(11)C]MP-10 in vivo in rhesus monkey with baseline scans to assess tissue uptake kinetics and self-blocking scans with unlabeled MP-10 to determine binding specificity. Ex vivo studies with one rhesus monkey and 4 Sprague-Dawley rats were also performed to investigate the presence of radiolabeled metabolites in the brain. Our results indicated that [(11)C]MP-10 displayed reversible uptake kinetics in rhesus monkeys, albeit slower than in baboons. Administration of unlabeled MP-10 reduced the binding of [(11)C]MP-10 in a dose-dependent manner in all brain regions including the cerebellum. Consequently, the cerebellum appeared not to be a suitable reference tissue in rhesus monkeys. Regional volume of distribution (VT) was mostly reliably derived with the multilinear analysis (MA1) method. In ex vivo studies in the monkey and rats only negligible amount of radiometabolites was seen in the brain of either species. In summary, results from the present study strongly support the suitability of [(11)C]MP-10 as a radiotracer for PET imaging and quantification of PDE10A in nonhuman primates.

Decreased norepinephrine transporter availability in obesity: Positron Emission Tomography imaging with (S,S)-[(11)C]O-methylreboxetine.

OBJECTIVES: Noradrenergic dysfunction is implicated in obesity. The norepinephrine transporter (NET) regulates the synaptic availability of norepinephrine. However, NET availability has not been previously characterized in vivo in obese people using Positron Emission Tomography (PET) imaging. Here we report findings evaluating NET availability in individuals with obesity and matched lean (i.e., normal weight) comparison subjects. METHODS: Seventeen obese but otherwise healthy individuals with a mean±SD body mass index (BMI) of 34.7±2.6 and 17 lean individuals with a mean±SD BMI of 23.1±1.4 were studied using a high-resolution research tomograph (HRRT) and (S,S)-[(11)C]O-methylreboxetine ([(11)C]-MRB), a radioligand selective for the NET. The regional brain NET binding potential (BPND) was estimated by the multilinear reference tissue model 2 (MRTM2) with the occipital cortex as a reference region. BPND for regions of interest were obtained with the Automated Anatomic Labeling (AAL) template registered to individual's structural MR scans. RESULTS: Obese individuals had lower NET BPND values in the thalamus (p<0.038, 27% reduction) including within the pulvinar (p<0.083, 30% reduction), but not in the hypothalamus, locus coeruleus or the raphe nuclei, compared to lean individuals. When age was included as a covariate, the difference in NET BPND values remained significant in the thalamus (p<0.025) and pulvinar (p<0.042). CONCLUSIONS: These results indicate that NET availability is decreased in the thalamus, including the pulvinar, in obese individuals. These findings further support data indicating noradrenergic dysfunction in obesity and suggest impaired NE clearance in obesity.

First-in-Human Study of 18F-SynVesT-2: An SV2A PET Imaging Probe with Fast Brain Kinetics and High Specific Binding.

PET imaging of synaptic vesicle glycoprotein 2A allows for noninvasive quantification of synapses. This first-in-human study aimed to evaluate the kinetics, test-retest reproducibility, and extent of specific binding of a recently developed synaptic vesicle glycoprotein 2A PET ligand, (R)-4-(3-(18F-fluoro)phenyl)-1-((3-methylpyridin-4-yl)methyl)pyrrolidine-2-one (18F-SynVesT-2), with fast brain kinetics. Methods: Nine healthy volunteers participated in this study and were scanned on a High Resolution Research Tomograph scanner with 18F-SynVesT-2. Five volunteers were scanned twice on 2 different days. Five volunteers were rescanned with preinjected levetiracetam (20 mg/kg, intravenously). Arterial blood was collected to calculate the plasma free fraction and generate the arterial input function. Individual MR images were coregistered to a brain atlas to define regions of interest for generating time-activity curves, which were fitted with 1- and 2-tissue-compartment (1TC and 2TC) models to derive the regional distribution volume (V T). The regional nondisplaceable binding potential (BP ND) was calculated from 1TC V T, using the centrum semiovale (CS) as the reference region. Results: 18F-SynVesT-2 was synthesized with high molar activity (187 ± 69 MBq/nmol, n = 19). The parent fraction of 18F-SynVesT-2 in plasma was 28% ± 8% at 30 min after injection, and the plasma free fraction was high (0.29 ± 0.04). 18F-SynVesT-2 entered the brain quickly, with an SUVpeak of 8 within 10 min after injection. Regional time-activity curves fitted well with both the 1TC and the 2TC models; however, V T was estimated more reliably using the 1TC model. The 1TC V T ranged from 1.9 ± 0.2 mL/cm3 in CS to 7.6 ± 0.8 mL/cm3 in the putamen, with low absolute test-retest variability (6.0% ± 3.6%). Regional BP ND ranged from 1.76 ± 0.21 in the hippocampus to 3.06 ± 0.29 in the putamen. A 20-min scan was sufficient to provide reliable V T and BP ND Conclusion: 18F-SynVesT-2 has fast kinetics, high specific uptake, and low nonspecific uptake in the brain. Consistent with the nonhuman primate results, the kinetics of 18F-SynVesT-2 is faster than the kinetics of 11C-UCB-J and 18F-SynVesT-1 in the human brain and enables a shorter dynamic scan to derive physiologic information on cerebral blood flow and synapse density.

Studies of the metabotropic glutamate receptor 5 radioligand [¹¹C]ABP688 with N-acetylcysteine challenge in rhesus monkeys.

Detecting changes in receptor binding at the metabotropic glutamate receptor 5 (mGluR5) with the PET allosteric antagonist, [¹¹C]ABP688, may be valuable for studying dysfunctional glutamate transmission associated with psychiatric illnesses. This study was designed to validate the findings of a recent pilot study in baboons which reported a significant global decrease from baseline [¹¹C]ABP688 binding after increasing endogenous glutamate with 50 mg/kg N-acetylcysteine (NAC), with no change from test to retest. In rhesus monkeys (n = 5), paired [¹¹C]ABP688 scans were performed on the same day on the Focus-220 as follows (n = 3 per group): test-retest, baseline-NAC (50 mg/kg), and baseline-NAC (100 mg/kg). Multiple modeling methods were evaluated for kinetic analysis to estimate the total volume of distribution (VT ) and non-displaceable binding potential (BP(ND)) in regions-of-interest (ROIs), with the cerebellum gray matter (CGM) as the reference region. There was an increasing trend from test to retest BP(ND) across ROIs (13%). NAC (50 mg/kg and 100 mg/kg) increased VT (5% and 19%) and decreased BP(ND) (3% and 10%), respectively, significant only for VT in ROIs at the 100 mg/kg dose. High intersubject variability in BP(ND) was comparable to that reported in the baboon study. However, interpretability of BP(ND) is difficult with increases in VT in the CGM reference region at the higher NAC dose. Additionally, the net reduction in BP(ND) from the baseline-NAC scans may be obscured due to observed increases in test-retest BP(ND). Thus, we did not strictly replicate the findings in the baboon study based on BP(ND).

Synthesis and evaluation of 4-(substituted styryl/alkenyl)-3,5-bis(4-hydroxyphenyl)-isoxazoles as ligands for the estrogen receptor.

A series of 3,5-bis (4-hydroxyphenyl) isoxazoles bearing a styryl/alkyl vinyl group at the 4-position were prepared and evaluated as ligands for the estrogen receptor-alpha (ERα). The target compounds were prepared using the Suzuki reaction to couple an iodo-isoxazole intermediate with a series of styryl/alkenyl boronic acids, followed by O-demethylation. The products were evaluated for their estrogen receptor-α ligand binding domain (ERα-LBD) binding affinity using a competitive binding assay. The 4-(4-hydroxystyryl) derivative 4h displays binding properties similar to those of the previously described pyrazole class of ER ligands, indicating that the ERα-LBD tolerates the presence of the added vinyl group at the 4-position of the isoxazole ring.

Targeting the estrogen receptor with metal-carbonyl derivatives of estradiol.

As part of our program to develop new probes for the estrogen receptor binding domain, we prepared and evaluated a novel 17α-(rhenium tricarbonyl bipyridyl) vinyl estradiol complex. Preparation of the final compound was achieved using the Stille coupling between the preformed brominated rhenium tricarbonyl bipyridine complex and the tributylstannyl vinyl estradiol. Competitive receptor binding assays and stimulatory assays demonstrated that the final complex retained affinity and efficacy comparable to the corresponding pyridyl vinyl estradiol analog, but lower than that of the phenyl vinyl estradiol analog.

Synthesis and evaluation of 17α-E-20-(heteroaryl)norpregn-1,3,5(10),20 tetraene-3,17ß-diols [17α-(heteroaryl)vinyl estradiols] as ligands for the estrogen receptor-α ligand binding domain (ERα-LBD).

A series of 17α-(heteroaryl)vinyl estradiols was prepared to evaluate the influence of heteroatom on the affinity and efficacy of estrogenic ligands for the estrogen receptor-alpha ligand binding domain (ERα-LBD). The products demonstrated reduced binding affinity compared to the parent 17α-E-phenyl vinyl estradiol, but the binding was relatively independent of the heteroatom. The greatest influence of the heteroatom was evident in the efficacy of the compounds as the thienyl derivatives 2f,g were more potent than either the pyridyl 2b-d or pyrimidinyl 2e analogs. The results suggest that a subtle interplay of interactions between the ligands and the receptor influences the biological response.

Convergent synthesis of a steroidal antiestrogen-mitomycin C hybrid using "click" chemistry.

A convergent synthesis of a novel estrogen receptor-targeted drug hybrid was developed based on structures of the potent anti-proliferative mitomycin C and the steroidal anti-estrogen RU 39411. The steroidal antiestrogen was prepared with an azido-triethylene glycoloxy linker while the mitomycin C derivative (porfirimycin) incorporated a complementary 7-N-terminal alkyne. The two components were ligated using the Huisgen [3 + 2] cycloaddition ("click") reaction. Preliminary biological assays demonstrated that the final hybrid compound retained both potent anti-estrogenic and anti-proliferative activities.

Synthesis and evaluation of 11ß-(4-substituted phenyl) estradiol analogs: transition from estrogen receptor agonists to antagonists.

INTRODUCTION: As part of our program to develop estrogen receptor (ER) targeted imaging and therapeutic agents we chose to evaluate 11ß-substituted estradiol analogs as a representative scaffold. Previous synthetic studies provided an entry into this class of compounds and other work indicated that 11ß-(substituted aryl) estradiol analogs were potent antagonists of the ER. Little information existed about the specific structural features involved in the transition from agonism to antagonism for the 11ß-aryl estradiol analogs or their potential as scaffolds for drug conjugation. METHODS: We prepared and characterized a series of 11ß-(4-Substituted phenyl) estradiol analogs using modifications of existing synthetic methods. The new compounds, as well as standard steroidal agonists and antagonists, were evaluated as competitive ligands for the ERß-LBD. Functional assays used the induction of alkaline phosphatase in Ishikawa cells to determine potency of the compounds as ER agonists or antagonists. RESULTS: The synthetic strategy successfully generated a series of compounds in which the 4-substituent was sequentially modified from hydroxyl to methoxy to azidoethoxy/N,N-dimethylaminoethoxy and eventually to a prototypical 1,4-naphthoquinone-containing moiety. The new compounds all retained high relative binding affinity (RBA) for the ERα-LBD, ranging from 13-83% that of estradiol. No subtype selectivity was observed. More importantly, the transition from agonist to antagonist activity occurs at the 4-methoxy stage where the compound is a mixed antagonist. More notably, antagonism appeared to be more dependent upon the size of the 11ß-substituent than upon the nature of the terminal group CONCLUSIONS: We have developed a synthetic strategy that provides facile access to potent 11ß-(4-substituted phenyl) estradiol analogs. The resultant compounds retain high affinity for the ERα-LBD and, more importantly, demonstrate potent antagonist activity in cells. Large functionalities distal to the 11ß-phenyl ring had little additional effect on either affinity or efficacy, suggesting the incorporation of diverse imaging or biologically active groups can be attached without significantly compromising the ER-binding capacity. Future studies are in progress to exploit the 11ß-aryl estradiol analogs as potential drug delivery systems and imaging agents.

Imaging Pituitary Vasopressin 1B Receptor in Humans with the PET Radiotracer 11C-TASP699.

Arginine vasopressin is a hormone that is synthesized mainly in the hypothalamus and stored in the posterior pituitary. Receptors for vasopressin are categorized into at least 3 subtypes (V1A, V1B, and V2). Among these subtypes, the V1B receptor (V1BR), highly expressed in the pituitary, is a primary regulator of hypothalamic-pituitary-adrenal axis activity and thus a potential target for treatment of neuropsychiatric disorders such as depression and anxiety. N-tert-butyl-2-[2-(6-methoxypyridine-2-yl)-6-[3-(morpholin-4-yl)propoxy]-4-oxopyrido[2,3-d]pyrimidin-3(4H)-yl]acetamide (TASP699) is a novel PET radiotracer with high affinity and selectivity for V1BR. The purpose of this study was to characterize the pharmacokinetic and binding profiles of 11C-TASP699 in humans and determine its utility in an occupancy study of a novel V1BR antagonist, TS-121. Methods: Six healthy subjects were scanned twice with 11C-TASP699 to determine the most appropriate kinetic model for analysis of imaging data and test-retest reproducibility of outcome measures. Nine healthy subjects were scanned before and after administration of TS-121 (active component: THY1773) to assess V1BR occupancy. Metabolite-corrected arterial input functions were obtained. Pituitary time-activity curves were analyzed with 1- and 2-tissue-compartment (1TC and 2TC, respectively) models and multilinear analysis 1 (MA1) to calculate distribution volume (VT). Relative test-retest variability (TRV) and absolute TRV were calculated. Since no brain region could be used as a reference region, percentage change in VT after TS-121 administration was computed to assess its receptor occupancy and correlate with plasma concentrations of the drug. Results:11C-TASP699 showed high uptake in the pituitary and no uptake in any brain region. The 2TC model provided better fits than the 1TC model. Because the MA1 VT estimates were similar to the 2TC VT estimates, MA1 was the model of choice. The TRV of VT was good (TRV, -2% ± 14%; absolute TRV, 11%). THY1773 reduced VT in a dose-dependent fashion, with a half-maximal inhibitory concentration of 177 ± 52 ng/mL in plasma concentration. There were no adverse events resulting in discontinuation from the study. Conclusion:11C-TASP699 was shown to display appropriate kinetics in humans, with substantial specific binding and good reproducibility of VT Therefore, this tracer is suitable for measurement of V1BR in the human pituitary and the V1BR occupancy of TS-121, a novel V1BR antagonist.

Binding of the synaptic vesicle radiotracer [11C]UCB-J is unchanged during functional brain activation using a visual stimulation task.

The positron emission tomography radioligand [11C]UCB-J binds to synaptic vesicle glycoprotein 2 A (SV2A), a regulator of vesicle release. Increased neuronal firing could potentially affect tracer concentrations if binding site availability is altered during vesicle exocytosis. This study assessed whether physiological brain activation induces changes in [11C]UCB-J tissue influx (K1), volume of distribution (VT), or binding potential (BPND). Healthy volunteers (n = 7) underwent 60-min [11C]UCB-J PET scans at baseline and during intermittent presentation of 8-Hz checkerboard visual stimulation. Sensitivity to intermittent changes in kinetic parameters was assessed in simulations, and visual stimulation was repeated using functional magnetic resonance imaging to characterize neural responses. VT and K1 were determined using the one-tissue compartment model and BPND using the simplified reference tissue model. In primary visual cortex, K1 increased 34.3 ± 15.5% (p = 0.001) during stimulation, with no change in other regions (ps > 0.12). K1 change was correlated with fMRI BOLD response (r = 0.77, p = 0.043). There was no change in VT (-3.9 ± 8.8%, p = 0.33) or BPND (-0.2 ± 9.6%, p = 0.94) in visual cortex nor other regions (ps > 0.19). Therefore, despite robust increases in regional tracer influx due to blood flow increases, binding measures were unchanged during stimulation. [11C]UCB-J VT and BPND are likely to be stable in vivo measures of synaptic density.