Suresh Pandey

Evaluation of 18F-nifene binding to α4β2 nicotinic receptors in the rat brain using microPET imaging

MicroPET imaging studies using 18F-nifene, a new positron emission tomography (PET) radiotracer for nicotinic acetylcholinergic receptors (nAChR) α4β2 receptors in rats, have been carried out. Rats were imaged for 90 min after intravenous injection of 18F-nifene (0.8 to 1 mCi), and binding potential (BPND) was measured. 18F-Nifene binding to thalamic and extrathalamic brain regions was consistent with the α4β2 nAChR distribution in the rat brain. Using the cerebellum as a reference, the values for the thalamus varied less than 5% (BPND = 1.30, n = 3), confirming reproducibility of 18F-nifene binding. 18F-Nifene microPET imaging was also used to evaluate effects of nicotine in a group of Sprague-Dawley rats under isoflurane anesthesia. Nicotine challenge postadministration of 18F-nifene demonstrated reversibility of 18F-nifene binding in vivo. For α4β2 nAChR receptor occupancy (nAChROCC), various doses of nicotine (0, 0.02, 0.1, 0.25, and 0.50 mg/kg nicotine free base) 15 min prior to 18F-nifene were administered. Low-dose nicotine (0.02 mg) reached > 80% nAChROCC while at higher doses (0.25 mg) > 90% nAChROCC was measured. The small amount of 18F-nifene binding with reference to the cerebellum affects an accurate evaluation of nAChROCC. Efforts are underway to identify alternate reference regions for 18F-nifene microPET studies in rodents.

Nicotinic acetylcholine receptors in rat forebrain that bind 18F-nifene: Relating PET imaging, autoradiography, and behavior

Nicotinic acetylcholine receptors (nAChRs) in the brain are important for cognitive function; however, their specific role in relevant brain regions remains unclear. In this study, we used the novel compound 18F‐nifene to examine the distribution of nAChRs in the rat forebrain, and for individual animals related the results to behavioral performance on an auditory‐cognitive task. We first show negligible binding of 18F‐nifene in mice lacking the β2 nAChR subunit, consistent with previous findings that 18F‐nifene binds to α4β2* nAChRs. We then examined the distribution of 18F‐nifene in rat using three methods: in vivo PET, ex vivo PET and autoradiography. Generally, 18F‐nifene labeled forebrain regions known to contain nAChRs, and the three methods produced similar relative binding among regions. Importantly, 18F‐nifene also labeled some white matter (myelinated axon) tracts, most prominently in the temporal subcortical region that contains the auditory thalamocortical pathway. Finally, we related 18F‐nifene binding in several forebrain regions to each animals performance on an auditory‐cued, active avoidance task. The strongest correlations with performance after 14 days training were found for 18F‐nifene binding in the temporal subcortical white matter, subiculum, and medial frontal cortex (correlation coefficients, r > 0.8); there was no correlation with binding in the auditory thalamus or auditory cortex. These findings suggest that individual performance is linked to nicotinic functions in specific brain regions, and further support a role for nAChRs in sensory‐cognitive function. Synapse, 2012.

Evaluation of serotonin 5-HT1A receptors in rodent models using [18F]mefway PET

Serotonin 5‐HT1A receptors have been investigated in various CNS disorders, including epilepsy, mood disorders, and neurodegeneration. [18F]Mefway (N‐{2‐[4‐(2′‐methoxyphenyl)piperazinyl]ethyl}‐N‐(2‐pyridyl)‐N‐(cis/trans−4′‐[18F]fluoromethylcyclohexane)‐carboxamide) has been developed as a suitable positron emission tomography (PET) imaging agent for these receptors. We have now evaluated the suitability of [18F]trans‐mefway in rat and mouse models using PET and computerized tomography (CT) imaging and corroborated with ex vivo and in vitro autoradiographic studies.

Multimodality Imaging Probe for Positron Emission Tomography and Fluorescence Imaging Studies.

Our goal is to develop multimodality imaging agents for use in cell tracking studies by positron emission tomography (PET) and optical imaging (OI). For this purpose, bovine serum albumin (BSA) was complexed with biotin (histologic studies), 5(6)- carboxyfluorescein, succinimidyl ester (FAM SE) (OI studies), and diethylenetriamine pentaacetic acid (DTPA) for chelating gallium 68 (PET studies). For synthesis of BSA-biotin-FAM-DTPA, BSA was coupled to (+)-biotin N-hydroxysuccinimide ester (biotin-NHSI). BSA- biotin was treated with DTPA-anhydride and biotin-BSA-DTPA was reacted with FAM. The biotin-BSA-DTPA-FAM was reacted with gallium chloride 3 to 5 mCi eluted from the generator using 0.1 N HCl and was passed through basic resin (AG 11 A8) and 150 mCi (100 μL, pH 7–8) was incubated with 0.1 mg of FAM conjugate (100 μL) at room temperature for 15 minutes to give 66Ga-BSA-biotin-DTPA-FAM. A shaved C57 black mouse was injected with FAM conjugate (50 μL) at one flank and FAM-68Ga (50 μL, 30 mCi) at the other. Immediately after injection, the mouse was placed in a fluorescence imaging system (Kodak In-Vivo F, Bruker Biospin Co., Woodbridge, CT) and imaged (Λex: 465 nm, Λem: 535 nm, time: 8 seconds, Xenon Light Source, Kodak). The same mouse was then placed under an Inveon microPET scanner (Siemens Medical Solutions, Knoxville, TN) injected (intravenously) with 25 μCi of 18F and after a half-hour (to allow sufficient bone uptake) was imaged for 30 minutes. Molecular weight determined using matrix-associated laser desorption ionization (MALDI) for the BSA sample was 66,485 Da and for biotin-BSA was 67,116 Da, indicating two biotin moieties per BSA molecule; for biotin-BSA-DTPA was 81,584 Da, indicating an average of 30 DTPA moieties per BSA molecule; and for FAM conjugate was 82,383 Da, indicating an average of 1.7 fluorescent moieties per BSA molecule. Fluorescence imaging clearly showed localization of FAM conjugate and FAM-68Ga at respective flanks of the mouse, whereas only a hot spot at the expected flank (FAM-68Ga injection site) was observed in microPET imaging. Our results suggest that BSA-biotin-DTPA-FAM may function as a multiprobe for PET and fluorescence imaging. Experiments are currently in progress to demonstrate cell tracking using both optical and nuclear imaging.

Comparative assessment of (18) F-Mefway as a serotonin 5-HT1A receptor PET imaging agent across species: Rodents, nonhuman primates, and humans.

We have developed 18F‐trans‐Mefway (18F‐Mefway) for positron emission tomography (PET) imaging studies of serotonin 5‐HT1A receptors which are implicated in various brain functions. Translation of imaging the 5‐HT1A receptor in animal models to humans will facilitate an understanding of the role of the receptor in human brain disorders. We report comparative brain distribution of 18F‐Mefway in normal mice, rats, monkeys, and healthy human volunteers. Mefway was found to be very selective, with subnanomolar affinity for the 5‐HT1A receptor. Affinities of >55 nM were found for all other human‐cloned receptor subtypes tested. Mefway was found to be a poor substrate (>30 μM) for the multidrug resistance 1 protein, suggesting low likelihood of brain uptake being affected by P‐glycoprotein. Cerebellum was used as a reference region in all imaging studies across all species due to the low levels of 18F‐Mefway binding. Consistent binding of 18F‐Mefway in cortical regions, hippocampus, and raphe was observed across all species. 18F‐Mefway in the human brain regions correlated with the known postmortem distribution of 5‐HT1A receptors. Quantitation of raphe was affected by the resolution of the PET scanners in rodents, whereas monkeys and humans showed a raphe to cerebellum ratio of approximately 3. 18F‐Mefway appears to be an effective 5‐HT1A receptor imaging agent in all models, including humans. 18F‐Mefway therefore may be used to quantify 5‐HT1A receptor distribution in brain regions for the study of various CNS disorders. J. Comp. Neurol. 524:1457–1471, 2016.

Synthesis and evaluation of 3-123I-iodo-5-[2-(S)-3-pyrrolinylmethoxy]-pyridine (niodene) as a potential nicotinic α4β2 receptor imaging agent

Nicotinic acetylcholine receptors (nAChRs) are downregulated in disease conditions such as Alzheimers and substance abuse. Presently, (123)I-5-IA-85380 is used in human studies and requires over 6h of scanning time, thus increases patient discomfort. We have designed and synthesized 3-iodo-5-[2-(S)-3-pyrrolinylmethoxy]pyridine (niodene) with the aim to have faster binding kinetics compared to (123)I-5-IA-85380, which may reduce scanning time and help in imaging studies. Binding affinity K(i) of niodene for rat brain α4β2 receptors in brain homogenate assays using (3)H-cytisine was 0.27 nM. Niodene, 10nM displaced >95% of (18)F-nifene bound to α4β2 receptors in rat brain slices. By using the iododestannylation method, (123)I-niodene was obtained in high radiochemical purity (>95%) but with low radiochemical yield (<5%) and low specific activity (∼100 Ci/mmol). Autoradiograms show (123)I-niodene localized in the thalamus and cortex, which was displaced by nicotine (thalamus to cerebellum ratio=4; cortex to cerebellum ratio=1.6). Methods of radioiodination need to be further evaluated in order to obtain (123)I-niodene in higher radiochemical yields and higher specific activity of this potentially useful new SPECT imaging agent.

124I-Epidepride: A PET radiotracer for extended imaging of dopamine D2/D3 receptors

OBJECTIVES A new radiotracer, ¹²⁴I-epidepride, has been developed for the imaging of dopamine D2/3 receptors (D2/3Rs). ¹²⁴I-Epidepride (half-life of ¹²⁴I=4.2 days) allows imaging over extended periods compared to (18)F-fallypride (half-life of ¹⁸F=0.076 days) and may maximize visualization of D2/3Rs in the brain and pancreas (allowing clearance from adjacent organs). D2/3 Rs are also present in pancreatic islets where they co-localize with insulin to produce granules and may serve as a surrogate marker for imaging diabetes. METHODS ¹²⁴I-Epidepride was synthesized using N-[[(2S)-1-ethylpyrrolidin-2-yl]methyl]-5-tributyltin-2,3-dimethoxybenzamide and ¹²⁴I-iodide under no carrier added condition. Rats were used for in vitro and in vivo imaging. Brain slices were incubated with (124)I-epidepride (0.75 μCi/cc) and nonspecific binding measured with 10 μM haloperidol. Autoradiograms were analyzed by OptiQuant. ¹²⁴I-Epidepride (0.2 to 0.3 mCi, iv) was administered to rats and brain uptake at 3 hours, 24 hours, and 48 hours post injection was evaluated. RESULTS ¹²⁴I-Epidepride was obtained with 50% radiochemical yield and high radiochemical purity (>95%). (124)I-Epidepride localized in the striatum with a striatum to cerebellum ratio of 10. Binding was displaced by dopamine and haloperidol. Brain slices demonstrated localization of ¹²⁴I-epidepride up until 48 hours in the striatum. However, the extent of binding was reduced significantly. CONCLUSIONS ¹²⁴I-Epidepride is a new radiotracer suitable for extended imaging of dopamine D2/3 receptors and may have applications in imaging of receptors in the brain and monitoring pancreatic islet cell grafting.