Imaging the impact of cyclosporin A and dipyridamole on P-glycoprotein (ABCB1) function at the blood-brain barrier: A [(11)C]-N-desmethyl-loperamide PET study in nonhuman primates.

Cyclosporin A (CsA) and dipyridamole (DPy) are potent inhibitors of the P-glycoprotein (P-gp; ABCB1) in vitro. Their efficacy at inhibiting P-gp at the blood-brain barrier (BBB) is difficult to predict. Efficient and readily available (i.e. marketed) P-gp inhibitors are needed as probes to investigate the role of P-gp at the human BBB. In this study, the P-gp inhibition potency at the BBB of therapeutic doses of CsA or DPy was evaluated in baboons using Positron Emission Tomography (PET) imaging with [(11)C]-N-desmethyl-loperamide ([(11)C]dLop), a radiolabeled P-gp substrate. The preparation of dLop as authentic standard and [(11)C]dLop as radiotracer were revisited so as to improve their production yields. [(11)C]dLop PET imaging was performed in the absence (n=3, baseline condition) and the presence of CsA (15mg/kg/h i.v., n=3). Three animals were injected with i.v. DPy at either 0.56 or 0.96 or 2mg/kg (n=1), corresponding to the usual, maximal and twice the maximal dose in patients, respectively, administered immediately before PET. [(11)C]dLop brain kinetics as well as [(11)C]dLop kinetics and radiometabolites in arterial plasma were measured to calculate [(11)C]dLop area-under the time-activity curve from 10 to 30min in the brain (AUCbrain) and in plasma (AUCplasma). [(11)C]dLop brain uptake was described by AUCR=AUCbrain/AUCplasma. CsA as well as DPy did not measurably influence [(11)C]dLop plasma kinetics and metabolism. Baseline AUCR (0.85±0.29) was significantly enhanced in the presence of CsA (AUCR=10.8±3.6). Injection of pharmacologic dose of DPy did not enhance [(11)C]dLop brain distribution with AUCR being 1.2, 0.9 and 1.1 after administration of 0.56, 0.96 and 2mg/kg DPy doses, respectively. We used [(11)C]dLop PET imaging in baboons, a relevant in vivo model of P-gp function at the BBB, to show the P-gp inhibition potency of therapeutic dose CsA. Despite in vitro P-gp inhibition potency, usual doses DPy are not likely to inhibit P-gp function at the BBB.

Differential influence of propofol and isoflurane anesthesia in a non-human primate on the brain kinetics and binding of [(18)F]DPA-714, a positron emission tomography imaging marker of glial activation.

Translocator protein 18 kDa (TSPO) expression at the mitochondrial membrane of glial cells is related to glial activation. TSPO radioligands such as [(18)F]DPA-714 are useful for the non-invasive study of neuroimmune processes using positron emission tomography (PET). Anesthetic agents were shown to impact mitochondrial function and may influence [(18)F]DPA-714 binding parameters and PET kinetics. [(18) F]DPA-714 PET imaging was performed in Papio anubis baboons anesthetized using either intravenous propofol (n = 3) or inhaled isoflurane (n = 3). Brain kinetics and metabolite-corrected input function were measured to estimate [(18) F]DPA-714 brain distribution (VT). Displacement experiments were performed using PK11195 (1.5 mg/kg). In vitro [(18)F]DPA-714 binding experiments were performed using baboon brain tissue in the absence and presence of tested anesthetics. Brain radioactivity peaked higher in isoflurane-anesthetized animals compared with propofol (SUVmax = 2.7 ± 0.5 vs. 1.3 ± 0.2, respectively) but was not different after 30 min. Brain VT was not different under propofol and isoflurane. Displacement resulted in a 35.8 ± 8.4% decrease of brain radioactivity under propofol but not under isoflurane (0.1 ± 7.0%). In vitro, the presence of propofol increased TSPO density and dramatically reduced its affinity for [(18)F]DPA-714 compared with control. This in vitro effect was not significant with isoflurane. Exposure to propofol and isoflurane differentially influences TSPO interaction with its specific radioligand [(18)F]DPA-714 with subsequent impact on its tissue kinetics and specific binding estimated in vivo using PET. Therefore, the choice of anesthetics and their potential influence on PET data should be considered for the design of imaging studies using TSPO radioligands, especially in a translational research context.

Influence of P-Glycoprotein Inhibition or Deficiency at the Blood-Brain Barrier on (18)F-2-Fluoro-2-Deoxy-D-glucose ( (18)F-FDG) Brain Kinetics.

The fluorinated D-glucose analog (18)F-2-fluoro-2-deoxy-D-glucose ((18)F-FDG) is the most prevalent radiopharmaceutical for positron emission tomography (PET) imaging. P-Glycoprotein's (P-gp, MDR1, and ABCB1) function in various cancer cell lines and tumors was shown to impact (18)F-FDG incorporation, suggesting that P-gp function at the blood-brain barrier may also modulate (18)F-FDG brain kinetics. We tested the influence of P-gp inhibition using the cyclosporine analog valspodar (PSC833; 5 µM) on the uptake of (18)F-FDG in standardized human P-gp-overexpressing cells (MDCKII-MDR1). Consequences for (18)F-FDG brain kinetics were then assessed using (i) (18)F-FDG PET imaging and suitable kinetic modelling in baboons without or with P-gp inhibition by intravenous cyclosporine infusion (15 mg kg(-1) h(-1)) and (ii) in situ brain perfusion in wild-type and P-gp/Bcrp (breast cancer resistance protein) knockout mice and controlled D-glucose exposure to the brain. In vitro, the time course of (18)F-FDG uptake in MDR1 cells was influenced by the presence of valspodar in the absence of D-glucose but not in the presence of high D-glucose concentration. PET analysis revealed that P-gp inhibition had no significant impact on estimated brain kinetics parameters K 1, k 2, k 3, V T , and CMRGlc. The lack of P-gp effect on in vivo (18)F-FDG brain distribution was confirmed in P-gp/Bcrp-deficient mice. P-gp inhibition indirectly modulates (18)F-FDG uptake into P-gp-overexpressing cells, possibly through differences in the energetic cell level state. (18)F-FDG is not a P-gp substrate at the BBB and (18)F-FDG brain kinetics as well as estimated brain glucose metabolism are influenced by neither P-gp inhibition nor P-gp/Bcrp deficiencies in baboon and mice, respectively.

Effects of selected OATP and/or ABC transporter inhibitors on the brain and whole-body distribution of glyburide.

Glyburide (glibenclamide, GLB) is a widely prescribed antidiabetic with potential beneficial effects in central nervous system injury and diseases. In vitro studies show that GLB is a substrate of organic anion transporting polypeptide (OATP) and ATP-binding cassette (ABC) transporter families, which may influence GLB distribution and pharmacokinetics in vivo. In the present study, we used [(11)C]GLB positron emission tomography (PET) imaging to non-invasively observe the distribution of GLB at a non-saturating tracer dose in baboons. The role of OATP and P-glycoprotein (P-gp) in [(11)C]GLB whole-body distribution, plasma kinetics, and metabolism was assessed using the OATP inhibitor rifampicin and the dual OATP/P-gp inhibitor cyclosporine. Finally, we used in situ brain perfusion in mice to pinpoint the effect of ABC transporters on GLB transport at the blood-brain barrier (BBB). PET revealed the critical role of OATP on liver [(11)C]GLB uptake and its subsequent impact on [(11)C]GLB metabolism and plasma clearance. OATP-mediated uptake also occurred in the myocardium and kidney parenchyma but not the brain. The inhibition of P-gp in addition to OATP did not further influence [(11)C]GLB tissue and plasma kinetics. At the BBB, the inhibition of both P-gp and breast cancer resistance protein (BCRP) was necessary to demonstrate the role of ABC transporters in limiting GLB brain uptake. This study demonstrates that GLB distribution, metabolism, and elimination are greatly dependent on OATP activity, the first step in GLB hepatic clearance. Conversely, P-gp, BCRP, and probably multidrug resistance protein 4 work in synergy to limit GLB brain uptake.

[(18)F]Fallypride: metabolism studies and quantification of the radiotracer and its radiometabolites in plasma using a simple and rapid solid-phase extraction method.

INTRODUCTION: [(18)F]Fallypride, a fluorinated and substituted benzamide with high affinity for D2/D3 receptors, is a useful PET radioligand for the study of striatal/extrastriatal areas. Since [(18)F]fallypride is extensively metabolized in vivo and since PET examinations are long lasting in humans, the rapid measurement of the unchanged radiotracer in plasma is essential for the quantification of images. The present study aims: i) to evaluate if the radiometabolites of [(18)F]fallypride cross the blood-brain barrier in rodents, ii) to identify these radiometabolites in baboon plasma and iii) to develop a rapid solid phase extraction method (SPE) suitable for human applications to quantify both [(18)F]fallypride and its radiometabolites in plasma. METHODS: The metabolites P450-dependant in rat and human liver microsomes were characterized by LC-MS-MS and compared to those detected in vivo. Sequential solvent elution on Oasis®-MCX-SPE cartridges was used to quantify [(18)F]fallypride and its radiometabolites. RESULT: In rat microsomal incubations, five metabolites generated upon N/O-dealkylation or hydroxylation at the pyrrolidine and/or at the benzamide moiety were identified. No radiometabolite was detected in the rat brain. N-dealkylated and hydroxylated derivatives were detected in human microsomal incubations as well as in baboon plasma. The use of SPE (total recovery 100.2%± 2.8%, extraction yield 95.5%± 0.3%) allowed a complete separation of [(18)F]fallypride from its radiometabolites in plasma and evaluate [(18)F]fallypride at 150 min pi to be 22%± 5% of plasma radioactivity. CONCLUSIONS: The major in vivo radiometabolites of [(18)F]fallypride were produced by N-dealkylation and hydroxylation. Allowing the rapid analysis of multiple plasma samples, SPE is a method of choice for the determination of [(18)F]fallypride until late images required for quantitative PET imaging in humans.

Metabolism and quantification of [(18)F]DPA-714, a new TSPO positron emission tomography radioligand.

[(18)F]DPA-714 [N,N-diethyl-2-(2-(4-(2[(18)F]-fluoroethoxy)phenyl)5,7dimethylpyrazolo[1,5a]pyrimidin-3-yl)acetamide] is a new radioligand currently used for imaging the 18-kDa translocator protein in animal models of neuroinflammation and recently in humans. The biodistribution by positron emission tomography (PET) in baboons and the in vitro and in vivo metabolism of [(18)F]DPA-714 were investigated in rats, baboons, and humans. Whole-body PET experiments showed a high uptake of radioactivity in the kidneys, heart, liver, and gallbladder. The liver was a major route of elimination of [(18)F]DPA-714, and urine was a route of excretion for radiometabolites. In rat and baboon plasma, high-performance liquid chromatography (HPLC) metabolic profiles showed three major radiometabolites accounting for 85% and 89% of total radioactivity at 120 minutes after injection, respectively. Rat microsomal incubations and analyses by liquid chromatography-mass spectrometry (LC-MS) identified seven metabolites, characterized as O-deethyl, hydroxyl, and N-deethyl derivatives of nonradioactive DPA-714, two of them having the same retention times than those detected in rat and baboon plasma. The third plasma radiometabolite was suggested to be a carboxylic acid compound that accounted for 15% of the rat brain radioactivity. O-deethylation led to a nonradioactive compound and [(18)F]fluoroacetic acid. Human CYP3A4 and CYP2D6 were shown to be involved in the oxidation of the radioligand. Finally an easy, rapid, and accurate method--indispensable for PET quantitative clinical studies--for quantifying [(18)F]DPA-714 by solid-phase extraction was developed. In vivo, an extensive metabolism of [(18)F]DPA-714 was observed in rats and baboons, identified as [(18)F]deethyl, [(18)F]hydroxyl, and [(18)F]carboxylic acid derivatives of [(18)F]DPA-714. The main route of excretion of the unchanged radioligand in baboons was hepatobiliary while that of radiometabolites was the urinary system.

Discrepancies in the P-glycoprotein-mediated transport of (18)F-MPPF: a pharmacokinetic study in mice and non-human primates.

PURPOSE: Several in vivo studies have found that the 5-HT(1A) PET radioligand (18)F-MPPF is a substrate of rodent P-glycoprotein (P-gp). However, in vitro assays suggest that MPPF is not a substrate of human P-gp. We have now tested the influence of inhibiting P-gp on the brain kinetics of (18)F-MPPF in mice and non-human primates. METHODS: We measured the peripheral kinetics (arterial input function, metabolism, free fraction in plasma (f(P))) during (18)F-MPPF brain PET scanning in baboons with or without cyclosporine A (CsA) infusion. We measured (3)H-MPPF transport at the mouse BBB using in situ brain perfusion in P-gp/Bcrp deficient mice and after inhibiting P-gp with PSC833. RESULTS: There was an unexpected 1.9-fold increase in brain area under the curve in CsA-treated baboons (n = 4), with no change in radiometabolite-corrected arterial input. However, total volume of distribution corrected for f(P) (V(T)/f(P)) remained unchanged. In situ brain perfusion showed that P-gp restricted the permeability of the mouse BBB to (3)H-MPPF while Bcrp did not. CONCLUSION: These and previous in vitro results suggest that P-gp may not influence the permeability of human BBB to (18)F-MPPF. However, CsA treatment increased (18)F-MPPF free fraction, which is responsible for a misleading, P-gp unrelated enhanced brain uptake.

Difficulties in dopamine transporter radioligand PET analysis: the example of LBT-999 using [18F] and [11C] labelling Part I: PET studies.

BACKGROUND: LBT-999 (E)-N-(4-fluorobut-2-enyl)-2ß-carbomethoxy-3ß-(4'-tolyl)nortropane is a dopamine transporter (DAT) ligand. [(18)F]LBT-999 was first labelled with carbon-11; we will now describe its in vivo behaviour in comparison to that of [(11)C]LBT-999. METHODS/RESULTS: Positron emission tomography (PET) experiments (baboons) confirmed the high affinity/specificity of [(18)F]LBT-999 for DAT. The brain regional distribution was in accordance with that of DAT. Pre-treatment with LBT-999 (1 mg/kg iv), but not with desipramine, a norepinephrine (NET) antagonist, reduced the striatum-to-cerebellum ratio by 96%, confirming the specificity for DAT vs. NET. The parent compound decreased rapidly and represented 24.3 ± 5.0% of plasma radioactivity at 30 min pi. Whole-body scans showed an important bone uptake of free fluorine following metabolism of [(18)F]LBT-999. In the cerebellum and striatum, distribution volumes increased by 30-40% between 80 and 230 min, suggesting the polluting role of a radiometabolite(s). [(11)C]LBT-999 exhibited a 40% higher standardized uptake value in the striata. This difference is likely due to N-dealkylation followed by [(18)F]fluoride release. 2ß-Carbomethoxy-3ß-(4'-tolyl) nortropane is then formed, while [(11)C]2ß-carbomethoxy-3ß-(4'-tolyl) nortropane is formed following injection of [(11)C]LBT-999. This metabolite has high affinity for the DAT. In one specific PET experiment, intravenous injection of this metabolite induced a strong displacement of [(18)F]LBT-999 in the striata, confirming that this metabolite readily crosses the blood-brain barrier (BBB) and binds to DAT. CONCLUSIONS: [(18)F]LBT-999 is N-dealkylated in vivo to yield (1) a nonradioactive metabolite that crosses the BBB and has a high affinity for the DAT and (2) a [(18)F]fluoro-alkyl chain which is further defluorinated. The temporal changes in distribution volumes are consistent with the accumulation of a radiometabolite(s) in the brain. Therefore, the quantification of DAT density with [(18)F]LBT-999 is rather difficult.

Difficulties in dopamine transporter radioligand PET analysis: the example of LBT-999 using [18F] and [11C] labelling: part II: Metabolism studies.

INTRODUCTION: LBT-999, (E)-N-(4-fluorobut-2-enyl)-2ß-carbomethoxy-3ß-(4'-tolyl)nortropane, has been developed for PET imaging of the dopamine transporter. [(18)F]LBT-999 PET studies in baboons showed a lower brain uptake than [(11)C]LBT-999 and a high bone uptake, suggesting the presence of interfering metabolites. Therefore, in vitro and in vivo metabolism of these radiotracers was investigated. METHODS: Rat and human liver microsomal incubations, baboon plasma and rat brain extracts were analyzed by radio-HPLC and LC-MS-MS. RESULTS: In vitro experiments demonstrated the formation by P450s of five polar metabolites. The main routes of LBT-999 metabolism proposed were N-dealkylation, tolyl-hydroxylation and dealkylation plus tolyl-hydroxylation. In vivo in baboons, [(18)F]LBT-999 was rapidly converted into a [(18)F]hydroxylated metabolite likely oxidized in plasma into a [(18)F]carboxylic acid and into unlabeled N-dealkyl-LBT-999. The latter was detected in baboon plasma and in rat brain by LC-MS-MS. The time course of unchanged [(18)F]LBT-999 decreased rapidly in plasma and was higher than that of [(11)C]LBT-999 due to the formation of unlabeled N-dealkyl-LBT-999. In rats, striatum-to-cerebellum ratios of [(18)F]LBT-999, [(18)F]hydroxylated and [(18)F]acidic metabolite were 20, 4.2 and 1.65, respectively, suggesting a possible accumulation of the hydroxylated compound in the striatum. CONCLUSION: P450s catalyzed the formation of dealkylated and hydroxylated metabolites of LBT-999. In baboons, an extensive metabolism of [(18)F]LBT-999, with formation of unlabeled N-dealkyl-LBT-999, [(18)F]fluorobutenaldehyde (or its oxidation product) and [(18)F]hydroxy-LBT-999 able to penetrate the brain, prevented an easy and accurate estimation of the input function of the radiotracer. CYP3A4 being the main P450 involved in the metabolism of LBT-999, a similar pathway may occur in humans and confound PET quantification.

Transport of selected PET radiotracers by human P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2): an in vitro screening.

UNLABELLED: Radiolabeled compounds used for brain imaging with PET must readily cross the blood-brain barrier (BBB) to reach their target. Efflux transporters at the BBB-P-glycoprotein (P-gp) and the breast cancer resistance protein (BCRP)-could limit their uptake by the brain. METHODS: We developed and validated an in vitro model using MDCKII cells transfected with human multidrug resistance (MDR1) or BCRP genes and assessed the transport of selected PET ligands by the concentration equilibrium technique. The tested compounds included befloxatone, (R,S)-CGP-12177, clorgyline, R-(-)-deprenyl, diprenorphine, DPA-714, fallypride, flumazenil, 2-fluoro-A-85380, LBT-999, loperamide, p-MPPF, PE2I, Pittsburgh compound B (PIB), (R,S)-PK11195, raclopride, R-(+)-verapamil, and WAY-100635. The assays were performed using the nonradioactive form of each compound (ultraviolet high-performance liquid chromatography analysis) and, when available, the (18)F-labeled analogs (γ-counting). RESULTS: Befloxatone appeared to be transported solely by BCRP. Loperamide, verapamil, and diprenorphine were the only P-gp substrates. Other ligands were transported by neither P-gp nor BCRP. CONCLUSION: The present method can readily be used to screen new-compound transport by P-gp or BCRP, even before any radiolabeling. Compounds that were previously thought to be transported by P-gp in rodents, such as p-MPPF, WAY-100635, and flumazenil, cannot be considered substrates of human P-gp. The impact of BCRP and P-gp at the BBB on the transport of befloxatone and diprenorphine in vivo remains to be evaluated with PET.

In vivo quantification of monoamine oxidase A in baboon brain: a PET study using [(11)C]befloxatone and the multi-injection approach.

[(11)C]befloxatone is a high-affinity, reversible, and selective radioligand for the in vivo visualization of the monoamine oxidase A (MAO-A) binding sites using positron emission tomography (PET). The multi-injection approach was used to study in baboons the interactions between the MAO-A binding sites and [(11)C]befloxatone. The model included four compartments and seven parameters. The arterial plasma concentration, corrected for metabolites, was used as input function. The experimental protocol-three injections of labeled and/or unlabeled befloxatone-allowed the evaluation of all the model parameters from a single PET experiment. In particular, the brain regional concentrations of the MAO-A binding sites (B'(max)) and the apparent in vivo befloxatone affinity (K(d)) were estimated in vivo for the first time. A high binding site density was found in almost all the brain structures (170+/-39 and 194+/-26 pmol/mL in the frontal cortex and striata, respectively, n=5). The cerebellum presented the lowest binding site density (66+/-13 pmol/mL). Apparent affinity was found to be similar in all structures (K(d)V(R)=6.4+/-1.5 nmol/L). This study is the first PET-based estimation of the B(max) of an enzyme.

[(11)C]SL25.1188, a new reversible radioligand to study the monoamine oxidase type B with PET: preclinical characterisation in nonhuman primate.

[(11)C]SL-25.1188 [(S)-5-methoxymethyl-3-[6-(4,4,4-trifluorobutoxy)-benzo[d]isoxazol-3-yl]-oxazolidin-2-one], an oxazolidinone derivative, was characterized in baboons as a radioligand for the in vivo visualization of MAO-B using positron emission tomography (PET). After i.v. injection, [(11)C]SL25.1188 presented a rapid phase of distribution in blood (about 5 min), followed by a T(1/2) elimination of 85 +/- 14 min. Plasma metabolism analysis showed that [(11)C]SL25.1188 is stable in vivo at least for 30 min. Brain uptake was rapid with the highest one observed in the striatum and thalamus, and the lowest in the pons. Calculated distribution volumes (V(T)) were as follows: striatum = 10.3, thalamus = 10.9, hippocampus = 8.9, temporal cortex = 7.7, occipital cortex = 7.2, parietal cortex = 7.4, frontal cortex = 7.4, white matter = 7.4, and pons = 6.1. Pretreatment with deprenyl (2 mg/kg, i.v.) or lazabemide (0.5 mg/kg, i.v.) reduced V(T) values in all brain areas up to 50%. In displacement experiments, injection of SL25.1188 or deprenyl (1 and 2 mg/kg, i.v., respectively) strongly reduced the specific uptake of [(11)C]SL25.1188 in all brain areas (85-100%), while a lesser displacement was observed with lazabemide (0.5 mg/kg, i.v.) (55-70% of specific binding depending on the brain area). Therefore, [(11)C]SL25.1188 is characterized in vivo by reversible binding, high brain uptake and very slow plasma metabolism, strongly suggesting that this radioligand is a potent tool for the in vivo study of brain MAO-B.

18F-ZW-104: a new radioligand for imaging neuronal nicotinic acetylcholine receptors--in vitro binding properties and PET studies in baboons.

UNLABELLED: An extensive series of radioligands has been developed for imaging central nicotinic acetylcholine receptors (nAChRs) with PET. Two halogeno-derivatives of A-85380 are being used in humans. Nevertheless, these derivatives still display too-slow brain kinetics and low signal-to-noise ratio. METHODS: A novel nAChR radioligand, 5-(6-fluorohexyn-1-yl)-3-[2(S)-2-azetidinylmethoxy]pyridine (ZW-104), was characterized in vitro using competition binding assays (nAChR subtypes heterologously expressed in HEK 293 cells and in native alpha4beta2 nAChRs from rat brain). (18)F-ZW-104 was prepared as follows: no-carrier-added nucleophilic aliphatic radiofluorination of the corresponding N-Boc-protected tosyloxy derivative 5-(6-tosyloxyhexyn-1-yl)-3-[2(S)-(N-(tert-butoxycarbonyl))-2-azetidinylmethoxy] pyridine) with the activated 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo-[8,8,8]hexacosane (K-(18)F-F-Kryptofix 222 [K(222)] complex), followed by quantitative trifluoroacetic acid-induced removal of the N-Boc protective group. (18)F-ZW-104 was then studied in baboons using PET. RESULTS: ZW-104 showed high binding affinities for rat alpha4beta2 nAChRs (K(i), 0.2 nM) and other subtypes containing the beta2 subunit but much lower affinities for rat alpha3beta4 nAChRs (K(i), 5,500 nM) and other subtypes containing the beta4 subunit. The regional radioactivity distribution in the baboon brain matched that of the alpha4beta2 nAChR, which was similar to that of 2-(18)F-fluoro-3-(2(S)-azetidinylmethoxy)pyridine (2-(18)F-A-85380), a radioligand used in humans. Comparison between (18)F-ZW-104 and 2-(18)F-A-85380 demonstrated better in vivo binding properties of the new radioligand: a substantially greater amount of radioactivity accumulated in the brain, and the occurrence of peak uptake in the thalamus was earlier than that of 2-(18)F-A-85380 and was followed by washout. Distribution volume values in different brain regions were 2-fold higher for (18)F-ZW-104 than for 2-(18)F-A-85380. Displacement by nicotine or unlabeled ZW-104 demonstrated a lower nonspecific binding than that of 2-F-A-85380. CONCLUSION: These results suggest that (18)F-ZW-104 is a promising PET radioligand for studying nAChRs containing the beta2 subunits in humans.

Decrease of nicotinic receptors in the nigrostriatal system in Parkinson's disease.

Smoking is associated with a lower incidence of Parkinson's disease (PD), which might be related to a neuroprotective action of nicotine. Postmortem studies have shown a decrease of cerebral nicotinic acetylcholine receptors (nAChRs) in PD. In this study, we evaluated the decrease of nAChRs in PD in vivo using positron emission tomography (PET), and we explored the relationship between nAChRs density and PD severity using both clinical scores and the measurement of striatal dopaminergic function. Thirteen nondemented patients with PD underwent two PET scans, one with 6-[(18)F]fluoro-3,4-dihydroxy-L-phenylalanine (6-[(18)F]fluoro-L-DOPA) to measure the dopaminergic function and another with 2-[(18)F]fluoro-3-[2(S)-2-azetidinylmethoxy]pyridine (2-[(18)F]fluoro-A-85380), a radiotracer with high affinity for the nAChRs. Distribution volumes (DVs) of 2-[(18)F]fluoro-A-85380 measured in the PD group were compared with those obtained from six nonsmoking healthy controls, with regions-of-interest and voxel-based approaches. Both analyses showed a significant (P <0.05) decrease of 2-[(18)F]fluoro-A-85380 DV in the striatum (-10%) and substantia nigra (-14.9%) in PD patients. Despite the wide range of PD stages, no correlation was found between DV and the clinical and PET markers of PD severity.

Quantification of cerebral nicotinic acetylcholine receptors by PET using 2-[18F]fluoro-A-85380 and the multiinjection approach.

The multiinjection approach was used to study in vivo interactions between alpha4beta2(*) nicotinic acetylcholine receptors and 2-[(18)F]fluoro-A-85380 in baboons. The ligand kinetics was modeled by the usual nonlinear compartment model composed of three compartments (arterial plasma, free and specifically bound ligand in tissue). Arterial blood samples were collected to generate a metabolite-corrected plasma input function. The experimental protocol, which consisted of three injections of labeled or unlabeled ligand, was aiming at identifying all parameters in one experiment. Various parameters, including B'(max) (the binding sites density) and K(d)V(R) (the apparent in vivo affinity of 2-[(18)F]fluoro-A-85380) could then be estimated in thalamus and in several receptor-poor regions. B'(max) estimate was 3.0+/-0.3 pmol/mL in thalamus, and ranged from 0.25 to 1.58 pmol/mL in extrathalamic regions. Although K(d)V(R) could be precisely estimated, the association and dissociation rate constants k(on)/V(R) and k(off) could not be identified separately. A second protocol was then used to estimate k(off) more precisely in the thalamus. Having estimated all model parameters, we performed simulations of 2-[(18)F]fluoro-A-85380 kinetics to test equilibrium hypotheses underlying simplified approaches. These showed that a pseudo-equilibrium is quickly reached between the free and bound compartments, a favorable situation to apply Logan graphical analysis. In contrast, the pseudo-equilibrium between the plasma and free compartments is only reached after several hours. The ratio of radioligand concentration in these two compartments then overestimates the true equilibrium value, an unfavorable situation to estimate distribution volumes from late images after a bolus injection.

[18F]FPhEP and [18F]F2PhEP, two new epibatidine-based radioligands: evaluation for imaging nicotinic acetylcholine receptors in baboon brain.

The radioligand 2-[(18)F]fluoro-A-85380 has been developed for imaging alpha(4)beta(2) nAChRs with PET. However, it has slow kinetics and a large fraction of bound activity is nondisplaceable. In an attempt to address these problems, two epibatidine-based alpha(4)beta(2) nicotinic antagonists, coded FPhEP and F(2)PhEP, were evaluated in vivo in baboons. They were radiolabeled with fluorine-18 from the corresponding N-Boc-protected bromo-derivatives and the no-carrier-added K[(18)F]F-Kryptofix(222) complex. Radiochemically pure [(18)F]FPhEP or [(18)F]F(2)PhEP was obtained in 80 min in amounts of 1.11-2.22 GBq (111-185 GBq/micromol). After injection of 215 MBq of [(18)F]FPhEP or [(18)F]F(2)PhEP, dynamic PET data were acquired. Thalamic radioactivity peaked at 20 min (4.9% +/- 0.2% ID/100 mL tissue) for [(18)F]FPhEP. For [(18)F]F(2)PhEP, the peak was at 45 min (3.3% +/- 0.1% ID/100 mL tissue). Regional distribution of both radiotracers was in accordance with the known distribution of nAChRs. In presaturation experiments, nicotine, cytosine, or FPhEP reduced brain radioactivity of [(18)F]FPhEP. In a displacement experiment with nicotine only a small amount of [(18)F]F(2)PhEP was dislodged. In spite of a moderate to high in vitro affinity, both ligands do not fulfill the widely adopted criteria for a PET radioligand.

[11C]LBT-999: a suitable radioligand for investigation of extra-striatal dopamine transporter with PET.

A new tropane derivative, (E)-N-(4-fluorobut-2-enyl)-2beta-carbomethoxy-3beta-(4'-tolyl)nortropane (LBT-999), was evaluated in baboons as a carbon-11 radioligand for studies of the dopamine transporter (DAT) using positron emission tomography (PET). Brain uptake was high in the striatum (17 and 13% ID/100 mL tissue in the putamen and the caudate, respectively), moderate in the midbrain and thalamus (5 and 3% ID/100 mL tissue, respectively), and low in the cortex and cerebellum (2% ID/100 mL tissue) at 30 min post injection. The striatum-to-cerebellum ratio was high (30 at 110 min post injection). Specific binding was completely blocked following pretreatment with the DAT antagonists GBR12909 (5 mg/kg i.v.) or PE2I (1 mg/kg i.v.). The [(11)C]LBT-999 uptake was decreased by these antagonists in the putamen (-79 and -92%, respectively), caudate (-80 and -91%, respectively), midbrain (-73 and -78%, respectively), and thalamus (-34 and -46%, respectively). The serotonin transporter (SERT) antagonist citalopram (5 mg/kg i.v.) or the norepinephrine transporter antagonist maprotiline (5 mg/kg i.v.) had no effect on LBT specific binding. Pharmacological challenge with PE2I (1 mg/kg i.v.) induced a rapid and almost complete decrease of the specific binding in the putamen (-97%), caudate (-96%), midbrain (-96%), and thalamus (-81%), confirming the reversibility of [(11)C]LBT-999 binding. The high brain uptake of [(11)C]LBT-999 together with its low nonspecific binding (reflected by the very high brain structure-to-cerebellum ratio) indicate that this radiotracer is an excellent candidate for in vivo quantification of the DAT, especially in extrastriatal structures, such as the midbrain.

Synthesis and radiolabeling of N-[4-[4-(2-[11C]methoxyphenyl)piperazin-1-yl]butyl]benzo[b]thiophene-2-carboxamide -- a potential radiotracer for D3 receptor imaging with PET.

FAUC346 (N-[4-[4-(2-methoxyphenyl)piperazin-1-yl]butyl]benzo[b]thiophene-2-carboxamide), an in vitro D(3)-selective ligand, and its normethyl derivative have been synthesized from commercially available 1-(2-substituted-phenyl)piperazines. FAUC346 has been labeled using [(11)C]methyl triflate in acetone containing aqueous NaOH (5 Eq) at -10 degrees C for 1 min, purified on semipreparative reverse-phase high-performance liquid chromatography (HPLC) and formulated as an intravenous injectable solution using a Sep-Pak Plus C(18) device. Up to 5.5 GBq of [(11)C]FAUC346 (N-[4-[4-(2-[methyl-(11)C]methoxyphenyl)piperazin-1-yl]butyl]benzo[b]thiophene-2-carboxamide), with a specific radioactivity of 45-75 GBq/micromol, could be obtained in 30-35 min, including HPLC purification and formulation starting from 44.4 GBq of [(11)C]carbon dioxide. Preliminary pharmacological evaluation of [(11)C]FAUC346 in rat brain clearly demonstrated in vivo selectivity for D(3) receptors and the absence of radiolabeled metabolite within the brain. These encouraging results, however, could not be confirmed in nonhuman primates; therefore, this radioligand does not appear to have the required pharmacological profile for a positron emission tomography probe for imaging D(3) receptors.

Synthesis and radiosynthesis of [(18)F]FPhEP, a novel alpha(4)beta(2)-selective, epibatidine-based antagonist for PET imaging of nicotinic acetylcholine receptors.

FPhEP (1, (+/-)-2-exo-(2'-fluoro-3'-phenyl-pyridin-5'-yl)-7-azabicyclo[2.2.1]heptane) belongs to a recently described novel series of 3'-phenyl analogues of epibatidine, which not only possess subnanomolar affinity and high selectivity for brain alpha4beta2 neuronal nicotinic acetylcholine receptors (nAChRs), but also were reported as functional antagonists of low toxicity (up to 15 mg/kg in mice). FPhEP (1, K(i) of 0.24 nM against [(3)H]epibatidine) as reference as well as the corresponding N-Boc-protected chloro- and bromo derivatives (3a,b) as precursors for labelling with fluorine-18 were synthesized in eight and nine steps, respectively, from commercially available N-Boc-pyrrole (overall yields=17% for 1, 9% for 3a and 8% for 3b). FPhEP (1) was labelled with fluorine-18 using the following two-step radiochemical process: (1) no-carrier-added nucleophilic heteroaromatic ortho-radiofluorination from the corresponding N-Boc-protected chloro- or bromo derivatives (3 a,b-1mg) and the activated K[(18)F]F-Kryptofix(222) complex in DMSO using microwave activation at 250 W for 1.5 min, followed by (2) quantitative TFA-induced removal of the N-Boc-protective group. Radiochemically pure (>99%) [(18)F]FPhEP ([(18)F]-1, 2.22-3.33 GBq, 66-137 GBq/micromol) was obtained after semi-preparative HPLC (Symmetry C18, eluent aq 0.05 M NaH(2)PO(4)/CH(3)CN, 80:20 (v:v)) in 75-80 min starting from a 18.5 GBq aliquot of a cyclotron-produced [(18)F]fluoride production batch (10-20% nondecay-corrected overall yield). In vitro binding studies on rat whole-brain membranes demonstrated a subnanomolar affinity (K(D) 660 pM) of [(18)F]FPhEP ([(18)F]-1) for nAChRs. In vitro autoradiographic studies also showed a good contrast between nAChR-rich and -poor regions with a low non-specific binding. Comparison of in vivo Positron Emission Tomography (PET) kinetics of [(18)F]FPhEP ([(18)F]-1) and [(18)F]F-A-85380 in baboons demonstrated faster brain kinetics of the former compound (with a peak uptake at 20 min post injection only). Taken together, the preliminary data obtained confirm that [(18)F]FPhEP ([(18)F]-1) has potential for in vivo imaging nAChRs in the brain with PET.