From [11C]CO2 to [11C]amides: a rapid one-pot synthesis via the Mitsunobu reaction.

A novel amide synthesis methodology is described using amines, CO2 and Grignard reagents and Mitsunobu reagents. The method was applied to carbon-11 radiochemistry to label amides using cyclotron-produced [11C]CO2. The synthetic utility of the one-pot labelling methodology was demonstrated by producing [11C]melatonin. The incorporation of [11C]CO2 into [11C]melatonin was 36% - determined by radioHPLC 2 min post [11C]CO2 delivery.

A microfluidic approach for high-throughput droplet interface bilayer (DIB) formation.

We present a simple, automated method for high-throughput formation of droplet interface bilayers (DIBs) in a microfluidic device. We can form complex DIB networks that are able to fill predefined three dimensional architectures. Moreover, we demonstrate the flexibility of the system by using a variety of lipids including 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

FDOPA metabolism in the adult porcine brain: influence of tracer circulation time and VOI selection on estimates of striatal DOPA decarboxylation.

Different methodologies for PET data analysis influence the magnitude of estimates of blood-brain transfer coefficients and rate constants for the metabolism of FDOPA in living striatum. We now test the effects on several kinetic parameters of automatic procedures for volume of interest (VOI) selection. We also tested the sensitivity of the estimates to dynamic frame sequence duration, and produced a standard method for minimizing the variations in physiological estimates for FDOPA kinetics in minipig brain. We used minipigs because our previous work has shown them to provide an appropriate animal model for study normal and pathological cerebral DOPA metabolism using PET. Time-activity curves in striatum of adult minipigs were acquired in VOIs defined manually on MR-images, or alternatively on the basis of the radioactivity concentration based on the most radioactive voxel in the last scan frame. For all frame sequences, the relative decarboxylase activity (k(3)(D)) declined significantly (P < 0.006) as the VOI threshold declined from 95 to 70% of the most radioactive voxel. Irrespective of VOI size, the magnitude of k(3)(D) declined significantly (P < 0.001) from 0.074+/-0.008 to 0.045+/-0.005 per min (mean+/-S.E.M.) as total sequence length increased from 60 to 120 min circulation. The method of VOI selection had no significant effect on the striatum decarboxylation index of FDOPA calculated relative to the radioactivity in cerebellum (k(3)(S)).

[14C]Serotonin uptake and [O-methyl-11C]venlafaxine kinetics in porcine brain.

As part of our program of developing PET tracers for neuroimaging of psychotropic compounds, venlafaxine, an antidepressant drug, was evaluated. First, we measured in vitro rates of serotonin uptake in synaptosomes prepared from selected regions of porcine brain. Then, we determined the pharmacokinetics of venlafaxine, [O-methyl-11C]-labeled for PET. Synaptosomal studies showed that the active uptake of [14C]5-HT differed markedly between brain regions, with highest rates in hypothalamus, raphé region, and thalamus, and lowest rates in cortex and cerebellum. PET studies showed that the unidirectional rate of uptake of [O-methyl-11C]venlafaxine from blood to brain was highest in the hypothalamus, raphé region, thalamus and basal ganglia and lowest in the cortex and cerebellum. Under normal physiological conditions, the capillary permeability-surface area (PS) product for [O-methyl-11C]venlafaxine could not be estimated, because of complete flow-limitation of the cerebral uptake. Nevertheless, a correlation occurred between the apparent partition volume of the radiotracer and the rate of active uptake of 5-HT in selected regions of the porcine brain. During hypercapnia, limitations of blood-brain transfer were observed, giving PS-products for water that were only ca. 50% higher than those of venlafaxine. Thus, under normal physiological conditions, the rate of uptake of venlafaxine from blood into brain is completely flow-limited.

Uptake and distribution of a new SSRI, NS2381, studied by PET in living porcine brain.

This study tests the utility of a new selective serotonin reuptake inhibitor (SSRI), [11C]NS2381 {(+/-)-(8-[11C]methyl-3-(4-trifluoromethyl-phenyl)-8-azabicyclo[3.2.1]oc t-2-ene)}, as positron-emitting radioligand for labelling serotonin (5-HT) reuptake sites in living brain. Studies of monoamine uptake were carried out initially in vitro using rat brain synaptosomes. They showed that NS2381 and its precursor NS2435 are selective inhibitors of serotonin (5-HT) uptake. Then, studies were carried out in vivo on the uptake and distribution of [11C]NS2381 in living porcine brain. They showed that the radiotracer accumulates readily in brain, and binds reversibly in regions rich in serotonin uptake sites (e.g. raphe, basal ganglia and thalamus). In addition, [11C]NS2381 was displaced from brain tissue by the potent SSRI citalopram. The enantiomers of [11C]NS2381 were, in general, found to be similar to the racemate in terms of their uptake and distribution in living pig brain. Thus, [11C]NS2381 fulfilled several criteria of a PET radioligand for studying 5-HT uptake sites in the living brain.

Cerebral 6-[(18)F]fluoro-L-DOPA (FDOPA) metabolism in pig studied by positron emission tomography.

We measured 6-[(18)F]fluoro-L-DOPA (FDOPA) uptake and metabolism in the brain of 4-month-old female pigs (n = 8) using a high-resolution positron emission tomograph (PET) in 3D mode. The mean net blood-brain clearance of FDOPA (K(i)(D)) to striatum was 0.011 ml g(-1) min(-1). Correcting for the elimination of decarboxylated metabolites from striatum (k(loss) = 0.004 min(-1)) increased the apparent magnitude of the estimate of K(i)(D) by 50%, at the expense of doubling the variance of the mean estimate. The mean decarboxylation rate of FDOPA in striatum relative to the cerebellum input (k(3)(s)) was 0.008 min(-1). For multicompartmental analyses, the FDOPA partition volume (V(e)(D)) was constrained to the individual value observed in cerebellum (mean = 0.53 ml g(-1)), with correction for the presence in brain of the plasma metabolite 3-O-methyl-FDOPA (OMFD). Using the first 60 min of the dynamic PET scans, the rate constant of FDOPA decarboxylation (k(3)(D)) was estimated to be 0.037 min(-1 )in striatum, but was not significantly different than zero in frontal cortex. Fitting of a compartmental model correcting for elimination of decarboxylated metabolites to the complete PET frame-sequence (120 min) increased the variance of the estimate of k(3)(D) in striatum. The magnitude of k(3)(D) in striatum of young pig was less than values estimated previously in neonatal piglet, adult monkey, and human. MRI-based simulations predicted that recovery of radioactivity from pig striatum was highly sensitive to the volume of interest. We conclude that the spatial resolution of our tomograph reduces the apparent magnitude of k(3)(D) in striatum. However, anaesthetised pigs are an appropriate experimental model for PET studies of DOPA decarboxylation in striatum.

The synthesis of (R)- and (S)-[N-methyl-11C]beta, beta-difluoromethamphetamine for the investigation of the binding mechanism of biogenic amines in vivo.

In an attempt to elucidate the contribution of the extent of nitrogen protonation on the in vivo binding of methamphetamine in the brain, the enantiomers of [N-methyl-11C]beta, beta-difluoroamphetamine (4) were prepared for use in positron emission tomography (PET) studies. Thus, the enantiomers of beta, beta-difluoroamphetamine were prepared from trans-beta-methylstyrene, via bromination, conversion into the azirine, fluorination and resolution as the tartrate salts. (R)- and (S)-beta, beta-difluoroamphetamine (3) were then each labelled with carbon-11 (tt/2 = 20.4 min) by N-methylation of the corresponding homochiral beta, beta-difluoroamphetamine with [11C]methyl iodide. The labelled products were each synthesised, purified and formulated in 35 min, starting from [11C]carbon dioxide in 15-16% decay-corrected radiochemical yield, with a radiochemical purity of > 99% and specific radioactivity of 50-150 GBq mumol-1 at end of synthesis.

Cerebral blood flow measurements by magnetic resonance imaging bolus tracking: comparison with [(15)O]H2O positron emission tomography in humans.

In six young, healthy volunteers, a novel method to determine cerebral blood flow (CBF) using magnetic resonance (MR) bolus tracking was compared with [(15)O]H2O positron emission tomography (PET). The method yielded parametric CBF images with tissue contrast in good agreement with parametric PET CBF images. Introducing a common conversion factor, MR CBF values could be converted into absolute flow rates, allowing comparison of CBF values among normal subjects.

Absolute cerebral blood flow and blood volume measured by magnetic resonance imaging bolus tracking: comparison with positron emission tomography values.

The authors determined cerebral blood flow (CBF) with magnetic resonance imaging (MRI) of contrast agent bolus passage and compared the results with those obtained by O-15 labeled water (H215O) and positron emission tomography (PET). Six pigs were examined by MRI and PET under normo- and hypercapnic conditions. After dose normalization and introduction of an empirical constant phi Gd, absolute regional CBF was calculated from MRI. The spatial resolution and the signal-to-noise ratio of CBF measurements by MRI were better than by the H215O-PET protocol. Magnetic resonance imaging cerebral blood volume (CBV) estimates obtained using this normalization constant correlated well with values obtained by O-15 labeled carbonmonooxide (C15O) PET. However, PET CBV values were approximately 2.5 times larger than absolute MRI CBV values, supporting the hypothesized sensitivity of MRI to small vessels.

Quantitative PET analysis of regional cerebral blood flow and glucose and oxygen metabolism in response to fenfluramine in living porcine brain.

The serotonin agonist fenfluramine has been used widely in humans for studying neuronal activation. We carried out the present study in order to determine whether anesthetized pigs could be used for studying effects of fenfluramine on cerebral functions using positron emission tomography (PET). We obtained quantitative measures of regional cerebral blood flow (rCBF) and of glucose and oxygen utilization (rCMRglc and rCMR(O2)) during intravenous administration of fenfluramine, using [15O]water, [18F]FDG and [15O]oxygen, respectively. Fenfluramine (25 mg/h i.v.) caused a significant rise in rCBF and, to a lesser extent, in rCMR2(O2), but it failed to affect rCMRglc. The findings indicate that quantitative estimation of rCBF by repeated injection of [15O]water was more sensitive than either rCMRO2 or rCMRglc for detecting effects of fenfluramine on serotonin neurotransmission in living porcine brain.

PET neuroimaging with [11C]venlafaxine: serotonin uptake inhibition, biodistribution and binding in living pig brain.

The brain binding kinetics and distribution of the antidepressant venlafaxine, labelled with 11C in the O-methyl position, was studied by PET after intravenous injection in anesthetized pigs. In addition, venlafaxine's action on serotonin (5-HT) uptake was studied in vitro in blood platelets obtain from humans or pigs. Venlafaxine resembled imipramine, paroxetine and citalopram in causing a dose-dependent inhibition of 5-HT uptake in blood platelets from pigs and humans. Venlafaxine-derived radioactivity entered the living brain readily and showed higher binding potentials in diencephalic and telencephalic regions than in cerebellum. Acute administration of an antidepressant drug (i.e. imipramine, citalopram or paroxetine) enhanced the distribution and altered the binding of venlafaxine in certain brain regions. The findings show that [11C]venlafaxine is not an ideal PET radiotracer mainly because of its relatively low binding potentials and its lack of specificity for the 5-HT transporter in living brain.

Synthesis of compounds of interest for positron emission tomography with particular reference to synthetic strategies for 11C labeling.

The future impact of positron emission tomography in clinical and basic sciences will be closely related to further developments of detector systems with regard to sensitivity and resolution, but probably even more with respect to developments in the synthesis of relevant labeled tracer molecules. Among the interesting radionuclides, 11C is of special interest since it is a radionuclide of an element, carbon, frequently occurring in the biosystem. In this paper some of the recent progress within the field of using the short-lived radionuclide 11C with a half-life of 20.4 min is presented. The paper deals with synthetic strategies which have been applied starting from simple one-carbon precursors like 11C carbon dioxide. In these strategies the development of reliable methods for production of one-carbon and multiple-carbon precursors, as well as multiple-carbon difunctional precursors, is important. Some examples are discussed. Labeled precursors can be applied conventional types of organic synthesis, in enzyme catalyzed reactions, or by using a combination of these. The synthesis of interesting 11C labeled receptor ligands and amino acids such as alanine, valine, phenylalanine, DOPA, tryptophan, 5-hydroxytryptophan, 2-methyltyrosine and some neuropeptides are presented.

Synthesis of racemic (+) and (-) N-[methyl-11C]nomifensine, a ligand for evaluation of monoamine re-uptake sites by use of positron emission tomography.

The synthesis of racemic or enantiomeric N-[methyl-11C]nomifensine (1,2,3,4-tetrahydro-2-[11C]methyl-4-phenyl-8-isoquinolinamine), a potential ligand for the evaluation of monoamine re-uptake sites at the presynaptic dopaminergic terminals, using the appropriate N-desmethylcompounds and [11C]methyl iodide is described. The radiochemical conversion of [11C]methyl iodide to [11C]nomifensine was in the order of 85-95%. Radiochemical purity of the LC-purified radiopharmaceutical was in the order of 98-99%. In a typical run, starting with 120 mCi (4.4 GBq) of [11C]carbon dioxide, 380 MBq (8.6% not decay corrected) of a final solution was obtained within 55 min (roughly 20 min of that is related to transport time). The specific radioactivity corresponding to the [11C]methyl iodide was 30-100 mCi/mumol (typical: a total mass of 30 micrograms and 150 MBq was administered in the PET-studies). A procedure for resolving the racemate of N-desmethylnomifensine (1,2,3,4-tetrahydro-4-phenyl-8-isoquinoline) into its enantiomers using triacetylcellulose as the stationary phase and methanol/ethanol as solvents by use of LC is also described.

Rapid decrease in amino acid metabolism in prolactin-secreting pituitary adenomas after bromocriptine treatment: a PET study.

Four patients with prolactin-secreting pituitary adenomas were examined with positron emission tomography using L-[11C]methionine to monitor the effect of dopamine agonist treatment on the amino acid metabolism in the tumors. Within the first few hours after intramuscular injection of bromocriptine retard (50 mg) the amino acid metabolism decreased by 40%. Two of the patients were reexamined 7 and 9 days later and showed a 70% reduction in the metabolism of the adenomas. This metabolic effect was later accompanied by significant tumor shrinkage in all adenomas. It is suggested that bromocriptine has a general and rapid effect on the protein synthesis of the prolactin-secreting pituitary adenoma cells.