Effect of acetylcholinesterase inhibitors on the binding of nicotinic alpha4beta2 receptor PET radiotracer, (18)F-nifene: A measure of acetylcholine competition.

Acetylcholinesterase inhibitors (AChEI's) are used to treat Alzheimer's disease (AD), and the putative mode of action is to increase acetylcholine (ACh) levels. Our goal is to evaluate competition of ACh with nicotinic alpha4beta2 receptor PET agonist radiotracer, 2-[(18)F]fluoro-3-[2-((S)-3-pyrrolinyl)methoxy]pyridine ((18)F-nifene). This ability to measure ACh-(18)F-nifene competition may have potential to assess efficacy of AChEI's in vivo. In vitro studies in rat brain slices used two AChEI's, physostigmine (PHY) and galanthamine (GAL). Brain slices were incubated with (18)F-nifene and various concentrations of PHY (0.2-20 microM) or GAL (0.4-4 microM) prior to (18)F-nifene treatment. For ACh competition, slices were also incubated with PHY + 100 nM ACh or GAL + 100 nM ACh or 100 nM ACh alone. Nonspecific binding of (18)F-nifene was determined using 300 microM nicotine. In the in vitro rat brain homogenate binding assay, ACh inhibited (3)H-cytisine binding to alpha4beta2 receptors with K(i) values of 19.2 nM (with PHY) and 34.7 microM (no PHY) indicating approximately 1.8 x 10(3) weaker binding of ACh in the absence of AChEI. Binding of (18)F-nifene was not affected by PHY (0.2-20 microM) or ACh 100 nM alone but decreased substantially by PHY + ACh 100 nM in all brain regions (down by >40% of control in thalamus). Similarly, for GAL (4 microM) no effect on (18)F-nifene binding occurred but GAL (0.4-4 microM) + ACh 100 nM showed a reduction of (18)F-nifene binding in all brain regions (down by approximately 15%). The reduction in both cases is a result of ACh competition with (18)F-nifene in the presence of AChEI. These preliminary in vitro results suggest that ACh is able to compete with (18)F-nifene at the alpha4beta2 receptors in the presence of PHY or GAL. The effect is AChEI-concentration dependent and is greater for PHY than GAL. Thus (18)F-nifene has promise for assessing ACh levels and AChEI effects in vivo.

Synthesis and biologic evaluation of a novel serotonin 5-HT1A receptor radioligand, 18F-labeled mefway, in rodents and imaging by PET in a nonhuman primate.

UNLABELLED: Serotonin 5-HT1A receptors have been implicated in disorders of the central nervous system and, therefore, are being studied by PET. Efforts are under way to improve in vivo stability of 5-HT1A agents currently in human use (11C-labeled N-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl-N-(2-pyridinyl)cyclohexanecarboxamide [11C-WAY-100635], 4-(2'-methoxyphenyl)-1-[2'-(N-2''-pyridinyl)-p-18F-fluorobenzamido]ethylpiperazine [18F-MPPF], and 18F-labeled trans-4-fluoro-N-(2-[4-(2-methoxyphenyl)piperazin-1-yl)ethyl]-N-(2-pyridyl)cyclohexanecarboxamide [18F-FCWAY]). We have synthesized N-{2-[4-(2-methoxyphenyl)piperazinyl]ethyl}-N-(2-pyridyl)-N-(4-18F-fluoromethylcyclohexane)carboxamide (18F-mefway), which contains a 18F on a primary carbon to make the compound more stable to defluorination. METHODS: Radiosynthesis of 18F-mefway was performed in a single tosylate for 18F-fluoride exchange. In vitro binding studies on rat brain slices using 18F-mefway were read on a phosphor imager. Monkey PET studies were performed on a whole-body PET scanner. RESULTS: Binding affinity (inhibitory concentration of 50% [IC50]) of mefway was 26 nmol/L and was comparable to that of WAY-100635, 23 nmol/L. Yields of 18F-mefway were 20%-30% in specific activities of 74-111 GBq/micromol at the end of synthesis. In vitro binding of 18F-mefway in the hippocampus (Hp), colliculus (Co), cortex (Ctx), and other brain regions-with limited binding in the cerebellum (Cer)--was observed, with ratios of Hp/Cer = 82.3, Co/Cer = 45.8, and Ctx/Cer = 40. Serotonin displaced 18F-mefway from various brain regions with IC50 values in the range of 169-243 nmol/L. PET studies in a rhesus monkey showed 18F-mefway binding in the fontal cortex (FC), temporal cortex (TC) including hippocampus, raphe (Rp), and other brain regions, with ratios of FC/Cer = 9.0, TC/Cer = 10, and Rp/Cer = 3.3. Plasma analysis indicated the presence of approximately 30% of 18F-mefway at 150-180 min after injection. CONCLUSION: The high ratios in specific brain regions such as the hippocampus suggest that 18F-mefway has potential as a PET agent for 5HT1A receptors.

Nicotinic alpha4beta2 receptor imaging agents: part II. Synthesis and biological evaluation of 2-[18F]fluoro-3-[2-((S)-3-pyrrolinyl)methoxy]pyridine (18F-nifene) in rodents and imaging by PET in nonhuman primate.

The alpha4beta2 nicotinic acetylcholine receptor (nAChR) has been implicated in various neurodegenerative diseases. Optimal positron emission tomography (PET) imaging agents are therefore highly desired for this receptor. We report here the development and initial evaluation of 2-fluoro-3-[2-((S)-3-pyrrolinyl)methoxy]pyridine (nifene). In vitro binding affinity of nifene in rat brain homogenate using 3H-cytisine exhibited a K(i) = 0.50 nM for the alpha4beta2 sites. The radiosynthesis of 2-18F-fluoro-3-[2-((S)-3-pyrrolinyl)methoxy]pyridine (18F-nifene) was accomplished in 2.5 h with an overall radiochemical yield of 40-50%, decay corrected. The specific activity was estimated to be approx. 37-185 GBq/micromol. In vitro autoradiography in rat brain slices indicated selective binding of 18F-nifene to anteroventral thalamic (AVT) nucleus, thalamus, subiculum, striata, cortex and other regions consistent with alpha4beta2 receptor distribution. Rat cerebellum showed some binding, whereas regions in the hippocampus had the lowest binding. The highest ratio of >13 between AVT and cerebellum was measured for 18F-nifene in rat brain slices. The specific binding was reduced (>95%) by 300 microM nicotine in these brain regions. Positron emission tomography imaging study of 18F-nifene (130 MBq) in anesthetized rhesus monkey was carried out using an ECAT EXACT HR+ scanner. PET study showed selective maximal uptake in the regions of the anterior medial thalamus, ventro-lateral thalamus, lateral geniculate, cingulate gyrus, temporal cortex including the subiculum. The cerebellum in the monkeys showed lower binding than the other regions. Thalamus-to-cerebellum ratio peaked at 30-35 min postinjection to a value of 2.2 and subsequently reduced. The faster binding profile of 18F-nifene indicates promise as a PET imaging agent and thus needs further evaluation.

Positron autoradiography for intravascular imaging: feasibility evaluation.

Approximately 70% of acute coronary artery disease is caused by unstable (vulnerable) plaques with an inflammation of the overlying cap and high lipid content. A rupturing of the inflamed cap of the plaque results in propagation of the thrombus into the lumen, blockage of the artery and acute ischaemic syndrome or sudden death. Morphological imaging such as angiography or intravascular ultrasound cannot determine inflammation status of the plaque. A radiotracer such as 18F-FDG is accumulated in vulnerable plaques due to higher metabolic activity of the inflamed cap and could be used to detect a vulnerable plaque. However, positron emission tomography (PET) cannot detect the FDG-labelled plaques because of respiratory and heart motions, small size and low activity of the plaques. Plaques can be detected using a miniature particle (positron) detector inserted into the artery. In this work, a new detector concept is investigated for intravascular imaging of the plaques. The detector consists of a storage phosphor tip bound to the end of an intravascular catheter. It can be inserted into an artery, absorb the 18F-FDG positrons from the plaques, withdrawn from the artery and read out. Length and diameter of the storage phosphor tip can be matched to the length and the diameter of the artery. Monte Carlo simulations and experimental evaluations of coronary plaque imaging with the proposed detector were performed. It was shown that the sensitivity of the storage phosphor detector to the positrons of 18F-FDG is sufficient to detect coronary plaques with 1 mm and 2 mm sizes and 590 Bq and 1180 Bq activities in the arteries with 2 mm and 3 mm diameters, respectively. An experimental study was performed using plastic tubes with 2 mm diameter filled with an FDG solution, which simulates blood. FDG spots simulating plaques were placed over the surface of the tube. A phosphor tip was inserted into the tube and imaged the plaques. Exposure time was 1 min in all simulations and experiments. Experiments showed that detecting the coronary plaques using the proposed technique is possible. The proposed technique has the potential for fast and accurate detection of vulnerable coronary and other intravascular plaques.

Entrapment of highly active membrane-bound receptors in macroporous Sol-Gel derived silica.

The immobilization of membrane-associated proteins remains a challenging task. Herein, we report on the entrapment of two classes of membrane-bound receptors into sol-gel derived silica. Both nicotinic acetylcholine receptor (nAChR), a ligand-gated ion channel, and dopamine D(2Short) receptor (D2R), a G-protein coupled receptor, were entrapped into a series of sol-gel derived nanocomposite materials. In cases where the silica had a bimodal pore size distribution wherein both mesopores and macropores were present, the two receptors showed 40-80% of solution activity over periods of at least 1 month. Furthermore, the dissociation constants of entrapped nAChR and D2R for binding to known agonists and antagonists were very close to the values obtained for free receptors in solution. These results indicate that membrane-bound receptors entrapped into bimodal meso/macroporous silica should provide a useful platform for the development of bioanalytical devices such as bioaffinity columns or microarrays, which could aid in diagnosis and high-throughput drug screening.