Single photon emission computed tomography experience with (S)-5-[(123)I]iodo-3-(2-azetidinylmethoxy)pyridine in the living human brain of smokers and nonsmokers.

(S)-5-[(123)I]iodo-3-(2-azetidinylmethoxy)pyridine (5-[(123)I]IA), a novel potent radioligand for high-affinity alpha4beta2* neuronal nicotinic acetylcholine receptors (nAChRs), provides a means to evaluate the density and the distribution of nAChRs in the living human brain. We sought in healthy adult smokers and nonsmokers to (1) evaluate the safety, tolerability, and efficacy of 5-[(123)I]IA in an open nonblind trial and (2) to estimate the density and the distribution of alpha(4)beta(2)* nAChRs in the brain. Single photon emission computed tomography (SPECT) was performed for 5 h after the i.v. administration of approximately 0.001 microg/kg ( approximately 10 mCi) 5-[(123)I]IA. Blood pressure, heart rate, and neurobehavioral status were monitored before, during, and after the administration of 5-[(123)I]IA to 12 healthy adults (8 men and 4 women) (6 smokers and 6 nonsmokers) ranging in age from 19 to 46 years (mean = 28.25, standard deviation = 8.20). High plasma-nicotine level was significantly associated with low 5-[(123)I]IA binding in: (1) the caudate head, the cerebellum, the cortex, and the putamen, utilizing both the Sign and Mann-Whitney U-tests; (2) the fusiform gyrus, the hippocampus, the parahippocampus, and the pons utilizing the Mann-Whitney U-test; and (3) the thalamus utilizing the Sign test. We conclude that 5-[(123)I]IA is a safe, well-tolerated, and effective pharmacologic agent for human subjects to estimate high-affinity alpha4/beta2 nAChRs in the living human brain.

Kinetic evaluation in nonhuman primates of two new PET ligands for peripheral benzodiazepine receptors in brain.

Peripheral benzodiazepine receptors (PBRs) are upregulated on activated microglia and are, thereby, biomarkers of cellular inflammation in brain. We recently developed two PET ligands with an aryloxyanilide structure to image PBRs and now evaluate the kinetics of these radiotracers in monkey to determine whether they are suitable to explore in human. Baseline and receptor-blocking scans were performed with [(11)C]PBR01 and [(18)F]PBR06 in conjunction with serial measurements of the arterial plasma concentration of parent radiotracer separated from radiometabolite. We used brain and plasma data with compartmental modeling to calculate regional brain distribution volume, which is equal to the ratio at equilibrium of the concentration of radioligand in brain to that of plasma. The distribution volume of [(11)C]PBR01 was inaccurately estimated in the baseline scans, possibly because of the short half-life of (11)C or the presence of radiometabolite in brain. In contrast, the distribution volume of [(18)F]PBR06 was stably determined within 200 min of scanning, and nondisplaceable uptake was only approximately 10% of total brain uptake. [(18)F]PBR06 is promising for use in human because brain activity could be quantified with standard compartmental models and showed higher ratios ( approximately 10:1) of specific to nonspecific uptake. A critical factor for human use will be whether the tracer has adequately fast wash out from brain relative to the half-life of the radionuclide to obtain stable values of distribution volume.

Identification and regional distribution in rat brain of radiometabolites of the dopamine transporter PET radioligand [11C]PE2I.

PURPOSE: We aimed to determine the composition of radioactivity in rat brain after intravenous administration of the dopamine transporter radioligand, [(11)C]PE2I. METHODS: PET time-activity curves (TACs) and regional brain distribution ex vivo were measured using no-carrier-added [(11)C]PE2I. Carrier-added [(11)C]PE2I was administered to identify metabolites with high-performance liquid radiochromatography (RC) or RC with mass spectrometry (LC-MS and MS-MS). The stability of [(11)C]PE2I was assessed in rat brain homogenates. RESULTS: After peak brain uptake of no-carrier-added [(11)C]PE2I, there was differential washout rate from striata and cerebellum. Thirty minutes after injection, [(11)C]PE2I represented 10.9 +/- 2.9% of the radioactivity in plasma, 67.1 +/- 11.0% in cerebellum, and 92.5 +/- 3.2% in striata, and was accompanied by two less lipophilic radiometabolites. [(11)C]PE2I was stable in rat brain homogenate for at least 1 h at 37 degrees C. LC-MS identified hydroxylated PE2I (1) (m/z 442) and carboxyl-desmethyl-PE2I (2) (m/z 456) in brain. MS-MS of 1 gave an m/z 442-->424 transition due to H(2)O elimination, so verifying the presence of a benzyl alcohol group. Metabolite 2 was the benzoic acid derivative. Ratios of ex vivo measurements of [(11)C]PE2I, [(11)C]1, and [(11)C]2 in striata to their cognates in cerebellum were 6.1 +/- 3.4, 3.7 +/- 2.2 and 1.33 +/- 0.38, respectively, showing binding selectivity of metabolite [(11)C]1 to striata. CONCLUSION: Radiometabolites [(11)C]1 and [(11)C]2 were characterized as the 4-hydroxymethyl and 4-carboxyl analogs of [(11)C]PE2I, respectively. The presence of the pharmacologically active [(11)C]1 and the inactive [(11)C]2 is a serious impediment to successful biomathematical analysis.

Persistent dopamine functions of neurons derived from embryonic stem cells in a rodent model of Parkinson disease.

The derivation of dopamine neurons is one of the best examples of the clinical potential of embryonic stem (ES) cells, but the long-term function of the grafted neurons has not been established. Here, we show that, after transplantation into an animal model, neurons derived from mouse ES cells survived for over 32 weeks, maintained midbrain markers, and had sustained behavioral effects. Microdialysis in grafted animals showed that dopamine (DA) release was induced by depolarization and pharmacological stimulants. Positron emission tomography measured the expression of presynaptic dopamine transporters in the graft and also showed that the number of postsynaptic DA D(2) receptors was normalized in the host striatum. These data suggest that ES cell-derived neurons show DA release and reuptake and stimulate appropriate postsynaptic responses for long periods after implantation. This work supports continued interest in ES cells as a source of functional DA neurons.

In vivo and in vitro measurement of brain phosphodiesterase 4 in rats after antidepressant administration.

Based largely on in vitro measurements, the mechanism of several antidepressant treatments is thought to involve upregulation of 3'-5'-cyclic adenosine monophosphate (cAMP) signal transduction cascade and a corresponding increase in phosphodiesterase (PDE) 4, the enzyme that metabolizes cAMP. To assess the in vivo status of PDE4, rats were chronically treated with imipramine and then studied with: (1) in vivo positron emission tomography (PET) measurement of (R)-[(11)C]rolipram binding, (2) in vitro measurement of [(3)H]rolipram binding in brain homogenates, and (3) Western blotting for protein levels of PDE4 isoforms. Imipramine administration caused no significant change in B(max)/K(d), for both in vivo measurements with (R)-[(11)C]rolipram and in vitro measurements with [(3)H]rolipram in frontal cortex, hippocampus, and diencephalon. None of 10 isoforms of PDE4A, B, and D measured with immunoblots of frontal cortex and hippocampus showed a significant change. In summary, using relatively large brain regions for both in vivo imaging and in vitro measures of radiolabeled ligand binding and protein levels, chronic imipramine treatment via continuous mini-pump administration caused no significant change in PDE4 levels. Most, but not all, prior in vitro studies have found increased PDE4 levels after antidepressant administration. The current results raise questions about the in vivo effects of antidepressant treatment on PDE4 and on other potentially important experimental factors (e.g., continuous infusion vs. intermittent injection of antidepressant) in large brain areas. However, the results do not deny possibility of changes in discrete areas, which were not studied in the current study applying PET.

Effect of a P-glycoprotein inhibitor, Cyclosporin A, on the disposition in rodent brain and blood of the 5-HT1A receptor radioligand, [11C](R)-(-)-RWAY.

Limited brain uptake of radioligands with otherwise optimal properties for imaging brain receptors can be improved by blocking the effect of P-glycoprotein (P-gp), an efflux pump at the blood-brain barrier. Using small animal positron emission tomography (PET), we investigated how P-gp and its blockade with Cyclosporin A (CsA) affect rodent brain uptake of [(11)C](R)-(-)-RWAY, a radioligand for brain 5-HT(1A) receptors. Brain uptake of radioactivity was compared in control and CsA-treated rats as well as P-gp knockout and wild type mice. Parent radioligand and radiometabolite in plasma and brain samples were determined at 30 min after injection of radioligand. PET binding potential (BP) was calculated with a reference tissue model. P-gp knockout mice had 2.5- and 2.8-fold greater brain uptake of [(11)C](R)-(-)-RWAY than wild type ones, measured by in vivo PET and ex vivo tissue sampling, respectively. Similarly, CsA increased rat brain uptake 4.9- and 5.3-fold, in vivo and ex vivo. In addition, CsA increased the plasma free fraction of [(11)C](R)-(-)-RWAY in rats by 2.7-fold. Although CsA increased brain uptake in wild type mice by 2.5-fold, it had no effect on plasma free fraction in knockout animals. Three radiometabolites of [(11)C](R)-(-)-RWAY were uniformly distributed in rat brain, suggesting they were inactive. CsA treatment increased brain uptake of [(11)C](R)-(-)-RWAY and only one of its radiometabolites. Regional rat brain BP increased 27-70% in the CsA-treated rats and the P-gp knockout mice. [(11)C](R)-(-)-RWAY is a P-gp substrate in rat and mouse. The effects of CsA in rats are mediated by both P-gp blockade and displacement of the radiotracer from plasma proteins. Studies with wild type and knockout mice showed that CsA affected only P-gp in this species.

PET imaging with [11C]PBR28 can localize and quantify upregulated peripheral benzodiazepine receptors associated with cerebral ischemia in rat.

Peripheral benzodiazepine receptors (PBRs) are upregulated on activated microglia. We recently developed a promising positron emission tomography (PET) ligand, [11C]PBR28, with high affinity and excellent ratio of specific to nonspecific binding. We assessed the ability of [11C]PBR28 PET to localize PBRs in a rat permanent middle cerebral artery occlusion (MCAO) model of neuroinflammation. [11C]PBR28 was intravenously administered to rats at 4 and 7 days after permanent MCAO. In all experiments, arterial blood was sampled for compartmental modeling of regional distribution volumes, and rat brains were sampled after imaging for in vitro [3H]PK 11195 autoradiography and histological evaluation. [11C]PBR28 PET and [3H]PK 11195 autoradiography showed similar areas of increased PBRs, especially in the peri-ischemic core. Results from these in vivo and in vitro methods were strongly correlated. In this first study to demonstrate neuroinflammation in vivo with small animal PET, [11C]PBR28 had adequate sensitivity to localize and quantify the associated increase in PBRs.

Sazetidine-A, a novel ligand that desensitizes alpha4beta2 nicotinic acetylcholine receptors without activating them.

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels found throughout the central and peripheral nervous systems. They are crucial to normal physiology and have been clearly implicated in nicotine addiction. In addition, they are possible therapeutic targets in a wide range of pathological conditions, including cognitive disorders, Parkinson's disease, and neuropathic pain. Nicotinic ligands are usually classified as agonists (or partial agonists), competitive antagonists, or noncompetitive antagonists. Sazetidine-A is a new nicotinic ligand that shows a different pharmacological profile from any of these known classes of ligands. Sazetidine-A competes with very high binding affinity (Ki approximately 0.5 nM) and selectivity for the alpha4beta2 nAChR subtype (Ki ratio alpha3beta4/alpha4beta2 approximately 24,000). Despite its high affinity, sazetidine-A neither activates nAChR channel function nor prevents channel activation when it is applied simultaneously with nicotine. However, when it is pre-incubated for 10 min with the receptors, it potently blocks nicotine-stimulated alpha4beta2 nAChR function (IC50 approximately 30 nM). The action of sazetidine-A may be explained by its very low affinity for the resting conformation of the alpha4beta2 nAChRs, and its very high affinity for the desensitized state of the receptor. We propose that sazetidine-A is a "silent desensitizer" of nAChRs, meaning that it desensitizes the receptor without first activating it. Furthermore, comparison of the effects of sazetidine-A and nicotine at alpha4beta2 nAChRs suggests that the predominant effects of nicotine and other nicotinic agonists are related to desensitization of the receptors and that sazetidine-A potently mimics these effects.

Development of new brain imaging agents based upon nocaine-modafinil hybrid monoamine transporter inhibitors.

11C-labeled (+)-trans-2-[[(3R,4S)-4-(4-chlorophenyl)-1-methylpiperidin-3-yl]methylsulfanyl]ethanol ([11C]5) and (+)-trans-2-[[(3R,4S)-4-(4-chlorophenyl)-1-methylpiperidin-3-yl]methylsulfanyl]-1-(piperidin-1-yl)ethanone ([11C]6) were synthesized and evaluated as new imaging agents for the norepinephrine transporter (NET). [11C]5 and [11C]6 display high affinity for the NET in vitro (Ki = 0.94 and 0.68 nM, respectively) and significant selectivity over the dopamine (DAT) and serotonin transporters (SERT). Because of their high affinity and favorable transporter selectivities we speculated that these ligands might serve as useful PET agents for imaging NET in vivo. Contrary to our expectations, both of these ligands provided brain images that were more typical of those shown by agents binding to the DAT.

PET imaging of the dopamine transporter with 18F-FECNT: a polar radiometabolite confounds brain radioligand measurements.

UNLABELLED: 18F-2beta-Carbomethoxy-3beta-(4-chlorophenyl)-8-(2-fluoroethyl)nortropane (18F-FECNT), a PET radioligand for the dopamine transporter (DAT), generates a radiometabolite that enters the rat brain. The aims of this study were to characterize this radiometabolite and to determine whether a similar phenomenon occurs in human and nonhuman primate brains by examining the stability of the apparent distribution volume in DAT-rich (striatum) and DAT-poor (cerebellum) regions of the brain. METHODS: Two rats were infused with 18F-FECNT and sacrificed at 60 min. Extracts of brain and plasma were analyzed by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometric (LC-MS) techniques. Two human participants and 3 rhesus monkeys were injected with 18F-FECNT and scanned kinetically, with serial arterial blood analysis. RESULTS: At 60 min after the injection of rats, 18F-FECNT accumulated to levels about 7 times higher in the striatum than in the cortex and cerebellum. The radiometabolite was distributed at equal concentrations in all brain regions. The LC-MS techniques identified N-dealkylated FECNT as a major metabolite in the rat brain, and reverse-phase HPLC detected an equivalent amount of radiometabolite eluting with the void volume. The radiometabolite likely was 18F-fluoroacetaldehyde, the product expected from the N-dealkylation of 18F-FECNT, or its oxidation product, 18F-fluoroacetic acid. The distribution volume in the cerebellum increased up to 1.7-fold in humans between 60 and 300 min after injection and 2.0 +/- 0.1-fold (mean +/- SD; n = 3) in nonhuman primates between 60 and 240 min after injection. CONCLUSION: An 18F-fluoroalkyl metabolite of 18F-FECNT originating in the periphery confounded the measurements of DAT in the rat brain with a reference tissue model. Its uniform distribution across brain regions suggests that it has negligible affinity for DAT (i.e., it is an inactive radiometabolite). Consistent with the rodent data, the apparent distribution volume in the cerebellum of both humans and nonhuman primates showed a continual increase at late times after injection, a result that may be attributed to entry of the radiometabolite into the brain. Thus, reference tissue modeling of 18F-FECNT will be prone to more errors than analysis with a measured arterial input function.

Whole-body biodistribution and estimation of radiation-absorbed doses of the dopamine D1 receptor radioligand 11C-NNC 112 in humans.

UNLABELLED: The present study estimated radiation-absorbed doses of the dopamine D(1) receptor radioligand [(11)C]((+)-8-chloro-5-(7-benzofuranyl)-7-hydroxy-3-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine) (NNC 112) in humans, based on dynamic whole-body PET in healthy subjects. METHODS: Whole-body PET was performed on 7 subjects after injection of 710 +/- 85 MBq of (11)C-NNC 112. Fourteen frames were acquired for a total of 120 min in 7 segments of the body. Regions of interest were drawn on compressed planar images of source organs that could be identified. Radiation dose estimates were calculated from organ residence times using the OLINDA 1.0 program. RESULTS: The organs with the highest radiation-absorbed doses were the gallbladder, liver, lungs, kidneys, and urinary bladder wall. Biexponential fitting of mean bladder activity demonstrated that 15% of activity was excreted via the urine. With a 2.4-h voiding interval, the effective dose was 5.7 microSv/MBq (21.1 mrem/mCi). CONCLUSION: (11)C-NNC 112 displays a favorable radiation dose profile in humans and would allow multiple PET examinations per year to be performed on the same subject.

Quantification of brain phosphodiesterase 4 in rat with (R)-[11C]Rolipram-PET.

OBJECTIVE: Phosphodiesterase 4 (PDE4) catabolizes the second messenger 3', 5'-cyclic adenosine monophosphate and may play a critical role in brain diseases. Our aim was to quantify PDE4 in rats with positron emission tomography (PET). METHODS: High (n = 6) and low specific activity (SA) (n = 2) higher affinity ((R)-[(11)C]rolipram) and high SA lower affinity ((S)-[(11)C]rolipram) (n = 2) enantiomers were intravenously administered to Sprague-Dawley rats. Brain data were acquired using the ATLAS PET scanner and reconstructed using the 3D-ordered subset expectation maximization algorithm. Arterial samples were taken to measure unmetabolized [(11)C]rolipram. Total distribution volumes (V(T)') were calculated using a 1-tissue compartment (1C) and an unconstrained 2-tissue compartment (2C) model. RESULTS: High SA R experiments showed later and greater brain uptake, and slower washout than low SA R and S experiments. In all regions and in all experiments, the 2C model gave significantly better fitting than the 1C model. The poor fitting by the latter caused underestimation of V(T)' by 19-31%. The 2C model identified V(T)' reasonably well with coefficients of variation less than 10%. V(T)' values by this model were 16.4-29.2 mL/cm(3) in high SA R, 2.9-3.5 in low SA R, and 3.1-3.7 in S experiments. CONCLUSIONS: Specific binding of (R)-[(11)C]rolipram was accurately measured in living rats. In high SA R experiments, approximately 86% of V(T)' was specific binding. Distribution and changes of PDE4 in animal models can now be studied by measuring V(T)' of high SA (R)-[(11)C]rolipram.

PET imaging of brain with the beta-amyloid probe, [11C]6-OH-BTA-1, in a transgenic mouse model of Alzheimer's disease.

PURPOSE: The purpose of this study was to evaluate the capacity of [11C]6-OH-BTA-1 and positron emission tomography (PET) to quantify beta-amyloid (Abeta) plaques in the Tg2576 mouse model of Alzheimer's disease (AD). METHODS: PET imaging was performed with the NIH ATLAS small animal scanner in six elderly transgenic mice (Tg2576; age 22.0+/-1.8 months; 23.6+/-2.6 g) overexpressing a mutated form of human beta-amyloid precursor protein (APP) known to result in the production of Abeta plaques, and in six elderly wild-type litter mates (age 21.8+/-1.6 months; 29.5+/-4.7 g). Dynamic PET scans were performed for 30 min in each mouse under 1% isoflurane inhalation anesthesia after a bolus injection of 13-46 MBq of [11C]6-OH-BTA-1. PET data were reconstructed with 3D OSEM. On the coronal PET image, irregular regions of interest (ROIs) were placed on frontal cortex (FR), parietal cortex (PA), striatum (ST), thalamus (TH), pons (PO), and cerebellum (CE), guided by a mouse stereotaxic atlas. Time-activity curves (TACs) (expressed as percent injected dose per gram normalized to body weight: % ID-kg/g) were obtained for FR, PA, ST, TH, PO, and CE. ROI-to-CE radioactivity ratios were also calculated. Following PET scans, sections of mouse brain prepared from anesthetized and fixative-perfused mice were stained with thioflavin-S. RESULTS: TACs for [11C]6-OH-BTA-1 in all ROIs peaked early (at 30-55 s), with radioactivity washing out quickly thereafter in both transgenic and wild-type mice. Peak uptake in all regions was significantly lower in transgenic mice than in wild-type mice. During the later part of the washout phase (12-30 min), the mean FR/CE and PA/CE ratios were higher in transgenic than in wild-type mice (1.06+/-0.04 vs 0.98+/-0.07, p=0.04; 1.06+/-0.09 vs 0.93+/-0.08 p=0.02) while ST/CE, TH/CE, and PO/CE ratios were not. Ex vivo staining revealed widespread Abeta plaques in cortex, but not in cerebellum of transgenic mice or in any brain regions of wild-type mice. CONCLUSION: Marked reductions in brain uptake of this radioligand in transgenic mice may be due to reduced cerebral blood flow relative to that in wild-type mice. Specific [11C]6-OH-BTA-1 binding to Abeta plaques, if any, is probably very low, as reflected in the small FR/CE and PA/CE ratio differences. FR/CE and PA/CE ratios are considerably higher in AD patients while Abeta plaque densities in 22-month-old transgenic mice may be expected to show essentially the same density as is observed in the AD brain. This implies that the absence of tracer retention in 22-month-old transgenic mice may be due to the smaller number of Abeta plaque binding sites and/or to lower affinity of the binding sites for [11C]6-OH-BTA-1 as compared with AD patients. [11C]6-OH-BTA-1 shows excellent brain uptake in mice.

Novel pyridyl ring C5 substituted analogues of epibatidine and 3-(1-methyl-2(S)-pyrrolidinylmethoxy)pyridine (A-84543) as highly selective agents for neuronal nicotinic acetylcholine receptors containing beta2 subunits.

Introduction of a hydrophobic or hydrogen-bonding alkynyl group into the C5 position of the pyridyl ring of epibatidine and A-84543 significantly increased the selectivity for neuronal nicotinic acetylcholine receptors (nAChRs) containing beta2 subunits over nAChRs containing beta4 subunits (K(i) ratio up to 92000-fold). Our data indicate that the extracellular domains of the nAChRs are sufficiently different to allow for the design of novel ligands with high affinity and selectivity for the nAChR subtypes.

Synthesis and biodistribution of radiolabeled alpha 7 nicotinic acetylcholine receptor ligands.

UNLABELLED: Our objective was to develop an array of alpha(7)-selective nicotinic cholinergic receptor (nAChR)-based imaging agents for PET and SPECT. METHODS: (2'R)-N-(11)C-Methyl-N-(phenylmethyl)-spiro[1-azabicyclo[2.2.2]octane-3,2'(3'H)-furo[2,3-b]pyridin]-5'-amine 1 was synthesized by reaction of the corresponding desmethyl precursor with (11)C-CO(2) and reduction. N-(R)-1-Aza-bicyclo[2.2.2]oct-3-yl-4-(11)C-methylsulfanyl-benzamide 2 was synthesized by reduction of the corresponding disulfide precursor and reaction with (11)C-iodomethane. N-(R)-1-Aza-bicyclo[2.2.2]oct-3-yl-4-(125)I-iodo-benzamide 3 was synthesized by halogen exchange of the corresponding bromide. (2'R)-5'-(2-(125)I-iodo-3-furanyl)spiro[1-azabicyclo[2.2.2]octane]-3,2'(3'H)-furo[2,3-b]pyridine 4 was synthesized by the chloramine-T method. Kinetic biodistribution studies were done in male CD-1 mice by tail vein injection of 3.7 MBq (100 microCi) of the (11)C-labeled radiotracer or 0.67 MBq (2 microCi) of the (125)I-labeled radiotracer followed by brain dissection and tissue counting. Receptor blockade was determined by pretreatment of the mice with an excess of either unlabeled precursor or nicotine. RESULTS: We synthesized 4 radiolabeled, moderate- to high-affinity, alpha(7)-nAChR-based ligands. The compounds were a series of quinuclidine derivatives with an inhibition constant (K(i)) < 6 nmol/L (33 pmol/L for 4) for alpha(7)-nAChR and selectivities of alpha(7)/alpha(4)beta(2) subtypes of > or =14,000. All of the compounds were produced in adequate radiochemical yield and specific radioactivity (>74 GBq/micromol [2,000 Ci/mmol]). No site selectivity or receptor blockade was shown for 1 and 2 (0.91 +/- 0.05 and 0.14 +/- 0.03 %ID/g [percentage injected dose per gram] in the hippocampus [target tissue], respectively). Compound 3 showed low hippocampal uptake (0.25 +/- 0.05 %ID/g) but prolonged retention within that structure. Pretreatment with nicotine decreased its uptake by up to 50% in the hippocampus. Similar reductions were also observed within the cerebellum (nontarget tissue). Compound 4 showed hippocampal uptake of 2.41 +/- 0.03 %ID/g and target-to-nontarget uptake ratios of up to 2. Pretreatment of animals with unlabeled 4 resulted in a decrease of hippocampal uptake to 60% of its preblockade value without a corresponding decrease in cerebellar uptake. CONCLUSION: With further structural optimization, selective imaging of alpha(7)-nAChR may be possible.

Syntheses of 11C- and 18F-labeled carboxylic esters within a hydrodynamically-driven micro-reactor.

Carboxylic esters were successfully labeled with one of two short-lived positron-emitters, carbon-11 or fluorine-18, within a hydrodynamically-driven micro-reactor. The non-radioactive methyl ester was obtained at room temperature; its yield increased with higher substrate concentration and with reduced infusion rate. Radioactive methyl ester was obtained from the reaction of (10 mM) with in 56% decay-corrected radiochemical yield (RCY) at an infusion rate of 10 microL min(-1), and when the infusion rate was reduced to 1 microL min(-1), the RCY increased to 88%. The synthesis of the non-radioactive fluoroethyl ester from and required heating of the micro-reactor on a heating block at 80 degrees C (14-17% RCY), whilst the corresponding radioactive from and was obtained in 10% RCY. The radioactive 'peripheral' benzodiazepine receptor ligand was obtained from the reaction of acid with labeling agent in 45% RCY at an infusion rate of 10 microL min(-1). When the infusion rate was reduced to 1 microL min(-1), the RCY increased to 65%. The results exemplify a new methodology for producing radiotracers for imaging with positron emission tomography that has many potential advantages, including a requirement for small quantities of substrates, enhanced reaction, rapid reaction optimisation and easy product purification.

Chronic nicotine administration does not increase nicotinic receptors labeled by [125I]epibatidine in adrenal gland, superior cervical ganglia, pineal or retina.

Neuronal nicotinic acetylcholine receptors (nAChRs) were measured in CNS and peripheral tissues following continuous exposure to saline or nicotine hydrogen tartrate (3.3 or 10 mg/kg/day) for 14 days via osmotic pumps. Initially, binding of [3H](-)nicotine, [3H]cytisine and [3H]epibatidine to nAChRs was compared to determine the suitability of each for these kinds of studies. The predominant nAChR labeled by agonists in the cerebral cortex is an alpha 4 beta 2 subtype, whereas the predominant nicotinic receptors in the adrenal gland, superior cervical ganglia and pineal gland contain an alpha 3 subunit, and they do not bind either [3H](-)nicotine or [3H]cytisine with high affinity. In retina some nAChRs bind all three ligands with high affinity, and others appear to bind only [3H]epibatidine. Thus, only [3H]epibatidine had high enough affinity to be useful for measuring the nAChRs in all of the tissues. The receptors from nicotine-treated rats were then measured using [125I]epibatidine, which has binding characteristics very similar to [3H]epibatidine. Treatment with the two doses of nicotine hydrogen tartrate increased binding sites in the cerebral cortex by 40% and 70%, respectively. In contrast, no significant changes in the density of receptor binding sites were found in the adrenal gland, superior cervical ganglia, pineal gland or retina. These data indicate that chronic administration of nicotine even at high doses does not increase all nicotinic receptor subtypes, and that receptors containing alpha 3 subunits may be particularly resistant to this nicotine-induced change.

Declines in different beta2* nicotinic receptor populations in monkey striatum after nigrostriatal damage.

In the present study we used the nicotinic ligand 5-iodo-A-85380 [5-iodo-3(2(S)-azetidinylmethoxy)pyridine], which selectively binds to beta2-containing nicotinic acetylcholine receptors, to elucidate the nicotinic receptor subtypes affected by nigrostriatal damage in the monkey. Autoradiographic studies in control monkeys showed that 5-[(125)I]A-85380 ([(125)I]A-85380) binds throughout the brain with the characteristics of a nicotinic receptor ligand. Competition experiments with cytisine and nicotine yielded K(i) values of approximately 1 and 10 nM, respectively, with complete inhibition of [(125)I]A-85380 binding at a 10(-6) M concentration of these ligands. In contrast, alpha-conotoxin MII blocked radioligand binding in the striatum by 30% at the highest concentrations, suggesting that a subset of striatal [(125)I]A-85380 sites are alpha-conotoxin MII-sensitive. Monkeys treated with the nigrostriatal neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine showed a selective decrease in striatal [(125)I]A-85380 sites, with a 42% reduction in the caudate and putamen of animals with moderate nigrostriatal lesioning and a 53% decline in the striatum of severely lesioned animals. Our previous work had demonstrated that there were two populations of nicotinic receptors eliminated after nigrostriatal damage, an alpha-conotoxin MII-sensitive and an alpha-conotoxin MII- resistant subtype. Analysis of both striatal [(125)I]A-85380 and [(125)I]epibatidine competition studies extend our earlier studies by demonstrating that the alpha-conotoxin MII-sensitive sites eliminated after moderate nigrostriatal lesioning appear to be composed of two nicotinic receptor subtypes. The data may be important for potential therapeutic approaches because they suggest that there are at least three populations of nicotinic receptors in monkey striatum, of which two are selectively vulnerable to nigrostriatal damage, while the third is more resistant.

Measuring nicotinic receptors with characteristics of alpha4beta2, alpha3beta2 and alpha3beta4 subtypes in rat tissues by autoradiography.

Comparison of [125I]epibatidine and 5-[125I]iodo-3-(2-azetidinylmethoxy)pyridine ([125I]A-85380) autoradiography showed evidence for nicotinic receptor heterogeneity. To identify the receptor subtypes, we performed [125I]epibatidine autoradiography in the presence of cytisine or A-85380. By comparing these results with binding data from human embryonic kidney (HEK) 293 cells stably transfected with different combinations of rat nicotinic receptor subunits, we were able to quantify three distinct populations of [125I]epibatidine binding sites with characteristics of alpha4beta2, alpha3beta2 and alpha3beta4 receptors. Although the predominant subtype in rat brain was alpha4beta2, non-alpha4beta2 binding sites were prominent in many regions. In the habenulo-peduncular system, cerebellum, substantia gelatinosa, and many medullary nuclei, alpha3beta4-like binding accounted for more than 40% of [125I]epibatidine binding, and nearly all binding in superior cervical ganglion and pineal gland. Other regions enriched in alpha3beta4-like binding included locus ceruleus, dorsal tegmentum, subiculum and anteroventral thalamic nucleus. Regions enriched in alpha3beta2-like binding included the habenulo-peduncular system, many visual system structures, certain geniculate nuclei, and dopaminergic regions. The combination of autoradiography using a broad spectrum radioligand in the presence of selective competitors, and data from binding to defined receptor subtypes in expression systems, allowed us to quantify the relative populations of these three subtypes.

Pharmacological evaluation of a Br-76 analog of epibatidine: a potent ligand for studying brain nicotinic acetylcholine receptors.

[(76)Br]-Norchlorobromoepibatidine ([(76)Br]BrPH) is a specific and high affinity radioligand for the nicotinic acetylcholine receptors (nAChRs). In vitro, on rat thalamus membranes [(76)Br]BrPH bound to two sites with apparent affinities of 8 pM and 3 nM. The density of binding sites were 1.9 and 70 fmol/mg protein, respectively. In vivo, in biodistribution and autoradiographic studies in rats the regional distribution of [(76)Br]BrPH paralleled the neuroanatomical localization of nAChRs. Two hours postinjection, the highest concentration in the brain was found in thalamus and colliculi (4% ID/g). Competition experiments with specific nicotinic, muscarinic, dopaminergic, and serotoninergic drugs confirmed that the in vivo binding of [(76)Br]BrPH was consistent with neuronal nicotinic receptors. PET imaging of [(76)Br]BrPH in baboon demonstrated a rapid and high uptake in the brain. Peak uptake occurred at 30-40 min for the thalamus. Due to the constant washout in the cerebellum, the thalamus to cerebellum ratio was 5 at 2 h postinjection. Subcutaneous injection of cytisine (1 mg/kg), 3 h postinjection of [(76)Br]BrPH reduced the radioactivity concentration in thalamus and cortex by 58 and 50%, respectively, as observed 1 h later. Cytisine pretreatment (5 mg/kg s.c.) inhibited completely the radioligand accumulation in the thalamus. Chronic MPTP pretreatment resulted in reduction of [(76)Br]BrPH uptake in all brain regions except in cerebellum. These preliminary results suggest that [(76)Br]BrPH has the potential to be a useful radioligand for studying the pharmacology of nicotinic acetylcholine receptors in preclinical experiments.