In vivo kinetics and displacement study of a carbon-11-labeled hallucinogen, N,N-[11C]dimethyltryptamine

The endogenous hallucinogen, N,N-dimethyltryptamine (DMT), was labeled with carbon-11 and its regional distribution in rat brain studied. [11C]DMT showed higher accumulation in the cerebral cortex, caudate putamen, and amygdaloid nuclei. Studies of the subcellular distribution of [11C]DMT revealed the specific localization in the fractions enriched with serotonin receptors only when a very low dose was injected into rats. The proportions of the radioactivity in receptor-rich fractions were greatly enhanced by pretreatment with the monoamine oxidase inhibitor, pargyline. Specific binding of [11C]DMT to serotonin receptors in dog brain was demonstrated by a positron emission tomographic study in which 5-methoxy-N,N-dimethyltryptamine caused approximately 20% displacement of the radioligand from the receptors.     

An automated SPE-based high-yield synthesis of [11C]acetate and [11C]palmitate: no liquid-liquid extraction, solvent evaporation or distillation required

Introduction

An automated method is described for the rapid and high-yield synthesis of two of the most commonly used radioactive fatty acids: [¹¹C]acetate and [¹¹C]palmitate.

Methods

Reaction of [¹¹C]CO₂ with the respective Grignard reagents in diethyl ether solution proceeded for 2 min at 40°C. Quenching of the reaction and liberation of nonreacted [¹¹C]CO₂ occurred upon addition of a fourfold molar excess of aqueous 0.1 M HCl (acetate) or nonaqueous HCl/Et₂O (palmitate). Labeled products were then purified by adsorption to an Alumina-N Sep-Pak Plus cartridge and eluted with either aqueous NaH₂PO₄ solution (acetate) or 100% ethanol (palmitate).

Results

High-performance liquid chromatography analysis confirmed that the radiochemical purity of each product was >98%, and decay-corrected radiochemical yields averaged 33% for [¹¹C]palmitate and 40% for [¹¹C]acetate.

Conclusion

The method requires no liquid–liquid extraction, solvent evaporation or distillation capabilities and can be readily adapted to existing radiosynthesis modules.     

Development of a modular system for the synthesis of PET [C]labelled radiopharmaceuticals

[¹¹C]labelled radiopharmaceuticals as N-[¹¹C]methyl-choline ([¹¹C]choline), l-(S-methyl-[¹¹C])methionine ([¹¹C]methionine) and [¹¹C]acetate have gained increasing importance in clinical PET and for the routine production of these radiopharmaceuticals, simple and reliable modules are needed to produce clinically relevant radioactivity. On the other hand, flexible devices are needed not only for the routine synthesis but also for more complex applications as the development of new tracers. The aim of this work was the adaptation of an Eckert Ziegler modular system for easy routine synthesis of [¹¹C]choline, [¹¹C]methionine and [¹¹C]acetate using components that account for straightforward scaling up and upgrades.     

Development and evaluation of muscarinic cholinergic receptor ligands N-[11C]ethyl-4-piperidyl benzilate and N-[11C]propyl-4-piperidyl benzilate: a PET study in comparison with N-[11C]methyl-4-piperidyl benzilate in the conscious monkey brain

The muscarinic cholinergic receptor ligands N-[¹¹C]ethyl-4-piperidyl benzilate (4-EPB) and N-[¹¹C]propyl-4-piperidyl benzilate (4-PPB) were developed and evaluated in comparison with N-[¹¹C]methyl-4-piperidyl benzilate (4-MPB) in the conscious monkey brain using positron emission tomography (PET). Time-activity curves of [¹¹C]4-EPB, unlike [¹¹C]4-MPB, showed peaks within 91 min in regions rich in muscarinic receptors. [¹¹C]4-PPB showed no specific binding even in the regions rich in these receptors. These observation demonstrated that increases in [¹¹C]alkyl chain length could alter the kinetic properties of receptor ligands for PET.     

Two routes to [C-carbonyl]organo-isocyanates utilizing [C]phosgene ([C]organo-isocyanates from [C]phosgene)

Two generic radiosynthetic routes for the preparation of [¹¹C-carbonyl]isocyanates have been developed. Reaction of N-organo-sulfinylamines; RNSO, (R = Me, Et, allyl, cyclohexyl and phenyl) with [¹¹C]phosgene gave the corresponding [¹¹C-carbonyl]isocyanates in good radiochemical yield (53–68%) from [¹¹C]phosgene (decay corrected) in ca 16 min from EOB. Alternatively, the reaction of [¹¹C]phosgene with N,N′-organo-ureas; (RNH)₂CO, (R = Me, Et, Pr and phenyl) also gave the corresponding [¹¹C-carbonyl]isocyanates in moderate radiochemical yield (9–37%) from [¹¹C]phosgene (decay corrected) in ca 16 min from EOB. For identification, the [¹¹C-carbonyl]organo-isocyanates were derivatized with 1-(2-methoxyphenyl)piperazine in situ to [¹¹C-carbonyl]carboxamides and the position of radiolabelling in the carbonyl group confirmed by [^11/13C]co-labeling and subsequent carbon-13 NMR spectroscopy.     

Two routes to [11C-carbonyl]organo-isocyanates utilizing [11C]phosgene ([11C]organo-isocyanates from [11C]phosgene).

Two generic radiosynthetic routes for the preparation of [¹¹C-carbonyl]isocyanates have been developed. Reaction of N-organo-sulfinylamines; RNSO, (R = Me, Et, allyl, cyclohexyl and phenyl) with [¹¹C]phosgene gave the corresponding [¹¹C-carbonyl]isocyanates in good radiochemical yield (53–68%) from [¹¹C]phosgene (decay corrected) in ca 16 min from EOB. Alternatively, the reaction of [¹¹C]phosgene with N,N′-organo-ureas; (RNH)₂CO, (R = Me, Et, Pr and phenyl) also gave the corresponding [¹¹C-carbonyl]isocyanates in moderate radiochemical yield (9–37%) from [¹¹C]phosgene (decay corrected) in ca 16 min from EOB. For identification, the [¹¹C-carbonyl]organo-isocyanates were derivatized with 1-(2-methoxyphenyl)piperazine in situ to [¹¹C-carbonyl]carboxamides and the position of radiolabelling in the carbonyl group confirmed by [^11/13C]co-labeling and subsequent carbon-13 NMR spectroscopy.     

Synthesis and in vivo studies of a specific monoamine oxidase B inhibitor: 5-[4-(benzyloxy)phenyl]-3-(2-cyanoethyl)-1,3,4-oxadiazol-[11C]-2(3H)-one

We report the radiochemical synthesis of a specific MAO B inhibitor, namely 5-[4-(benzyloxy)phenyl]-3-(2-cyanoethyl)-1,3,4-oxadiazol-[¹¹C]-2(3H)-one (2b) (in vitro IC₅₀=4nM and selectivity over 71000 for MAO B), by cyclization of its hydrazide precursor1 with [¹¹C]phosgene. The reaction occurred within 2 min. The product obtained after HPLC purification,2b, had a high specific activity (11.1–22.2 GBq/µmol), allowing its use in experiments as a radiotracer in vivo. Biodistribution of2b in the CNS and in the peripheral organs of the rat, and positron emission tomography (PET) studies in the living baboon brain, pretreated or not withl-deprenyl (1 mg/kg, 1 h), an irreversible MAO B-specific inhibitor, were undertaken. The results showed a good uptake of2b in all organs of the rat, with a rapid clearance from the blood (10 min). Metabolite analyses in plasma and in the diencephalon of the rat showed that2b was the only radioactive compound in brain structure whereas in plasma three other radioactive products appeared. PET experiments show that in thel-deprenyl-pretreated baboon brain, specific binding of2b represents around 70% of total radioactivity, whereas in the blood and plasma the radioactivity cleared rapidly (15 min).     

[C]Formaldehyde revisited: considerable concurrent [C]formic acid formation in the low-temperature conversion of [C]carbon dioxide into [C]formaldehyde

The reduction of [¹¹C]carbon dioxide with lithium aluminium hydride in diethyl ether at temperatures ranging from −56°C to 19°C was studied. In contrast to what others have reported, considerable amounts of [¹¹C]formic acid were found at all studied temperatures.     

In vivo imaging of microglial activation using a peripheral benzodiazepine receptor ligand: [11C]PK-11195 and animal PET following ethanol injury in rat striatum

OBJECTIVE: To investigate whether [(11)C]PK-11195, a specific peripheral benzodiazepine receptors (PBRs) ligand for positron emission tomography (PET), can show activated microglia in a rat brain injury model. METHODS: On day 1, ethanol was injected into the rat's right striatum (ST) using a stereotaxic operative procedure. On day 3, head magnetic resonance imaging (MRI) scans for surgically treated rats were performed to evaluate ethanol injury morphologically. On day 4, dynamic PET scans (17 injured rats and 7 non-injured controls) were performed for 60 min with an animal PET scanner under chloral hydrate anesthesia following a bolus injection of [(11)C]PK-11195 through tail vein. Because PBRs are present throughout the brain, there is no suitable receptor-free reference region. The reference tissue model may not be applicable because of low target to background ratio for low affinity of [(11)C]PK-11195 to PBRs. We evaluated the PBRs binding with regions of interest (ROIs)-based approach to estimate total distribution volume (V). We used an integral from 0 min to 60 min (V (60)) as an estimate of V. On the coronal PET image, ROIs were placed on bilateral ST. Differences in right/left ST V (60) ratios between lesioned and unlesioned control rats were compared using unpaired t tests. Immunohistochemical staining was performed for confirming the presence of activated microglia following decapitation on the PET experiment day. RESULTS: The right/left ST V (60) ratios in lesioned rats (1.07 +/- 0.08) were significantly higher than those in unlesioned control rats (1.00 +/- 0.06, P < 0.05). On immunohistochemical staining, activated microglia were exclusively observed in the injured right ST but not in the noninjured left ST of the injury rats and the bilateral ST of the non-injured control rats. CONCLUSIONS: These results suggest that [(11)C]PK-11195 PET imaging would be a useful tool for evaluating microglial activation in a rat brain injury model.     

[1-<Superscript>11</Superscript>C]Acetate uptake is not increased in renal cell carcinoma

Purpose The purpose of this study was to investigate the potential of [1-¹¹C]acetate (AC) as a metabolic tracer for renal cell cancer in human subjects. Methods Twenty-one patients with suspected kidney tumours were investigated with AC and dynamic PET. AC uptake was scored on a five-step scale. Tumour localisation was known from CT/MRI. Histology was available in 18/21 patients. The results in these 18 patients are reported. Results AC uptake by the tumour was less than (n = 11), equal to (n = 5) or higher than (n = 2) uptake in the surrounding renal parenchyma. Histological tumour types showed a typical distribution, with a predominance of clear cell carcinomas (n = 14) and only a small number of papillary cell carcinomas (n = 2) and oncocytomas (n = 2). Only the benign oncocytomas were highly positive with AC. Conclusion In most kidney tumours the AC accumulation was not higher than in normal kidney parenchyma. Therefore, AC PET cannot be recommend for the characterisation of a renal mass.     

In vivo imaging of adenosine A2A receptors in rat and primate brain using [11C]SCH442416

Purpose The aim of this study was to evaluate the suitability of [¹¹C]SCH442416 for the in vivo imaging of adenosine A_2A receptors.Methods In rats and Macaca nemestrina, we evaluated the time course of the cerebral distribution of [¹¹C]SCH442416. Furthermore, in rats we investigated the rate of metabolic degradation, the inhibitory effects of different drugs acting on adenosine or dopamine receptors and the modification induced by the intrastriatal administration of quinolinic acid (QA).Results The rate of metabolic degradation of [¹¹C]SCH442416 in rats was slow; 60 min after tracer injection, more than 40% of total plasma activity was due to unmetabolised [¹¹C]SCH442416. At the time of maximum uptake, radioactive metabolites represented only 6% of total extractable activity in the cerebellum and less than 1% in the striatum. In the striatum, the region with the highest expression of A_2A receptors, the in vivo uptake of [¹¹C]SCH442416 was significantly reduced only by drugs acting on A_2A receptors or by QA, a neurotoxin that selectively reduces the number of intrastriatal GABAergic neurons. Position emission tomography (PET) studies in monkeys indicated that the tracer rapidly accumulates in brain, reaching maximum uptake between 5 and 10 min. Twenty minutes after the injection, radioactivity concentration in the striatum was two times that in the cerebellum.Conclusion The specificity of binding, the rank order of regional distribution in the brain of rats and M. nemestrina, the good signal to noise ratios and the low amount of radioactive metabolites in brain and periphery indicate that [¹¹C]SCH442416 is a promising tracer for the in vivo imaging of A_2A adenosine receptors using PET.     

Biodistribution and radiation dosimetry of [11C]raclopride in healthy volunteers

Purpose This study reports on the whole-body biodistribution and radiation dosimetry of [¹¹C]raclopride, a dopamine D₂ receptor antagonist.Methods In three healthy male volunteers, whole-body scans were performed up to 2 h following i.v. injection of 320±65 MBq [¹¹C]raclopride. Transmission scans (3 min per step, eight or nine steps according to the height of the subject) in 2D mode were used for subsequent attenuation correction of emission scans. Emission scans (1 min per step, eight or nine steps) were acquired over 2 h. Venous blood samples and urine were collected up to 2 h after injection of the radiotracer. For each subject, the percentage of injected activity measured in regions of interest over brain, intestine, lungs, kidneys and liver was fitted to a mono-exponential model, as an uptake phase followed by a mono-exponential washout, for urinary bladder to generate time–activity curves. Using the MIRD method, several source organs were considered in estimating residence time and mean effective radiation absorbed doses.Results Blood pressure and ECG findings remained unchanged after tracer injection. The analysed blood and urine pharmacological parameters did not change significantly after [¹¹C]raclopride injection. The primary routes of clearance were renal and intestinal. Ten minutes after injection, high activities were observed in the gall-bladder, kidneys and liver. High activity was observed in the gall-bladder during the whole study. The kidneys, urinary bladder wall, liver and gall-bladder received the highest absorbed doses. The average effective dose of [¹¹C]raclopride was estimated to be 6.7±0.4 Sv/MBq.Conclusion The amount of [¹¹C]raclopride required for adequate dopamine D₂ receptor imaging results in an acceptable effective dose equivalent, permitting two or three repeated clinical PET imaging studies, with the injection of 222 MBq for each study.     

Metabolic fate of L-[methyl-11C]methionine in human plasma

The metabolites of L-[methyl-¹¹C]methionine in the plasma of 8 patients with tumor were measured for 60 min after injection. In the plasma, after a rapid clearance, the total radioactivity remained constant, and protein-bound radioactivity increased rapidly. Non protein metabolites detected by HPLC as at least two components besides methionine, increased with time. Significant individual variations for the metabolism were observed. AT 60 min after injection, 36.5% (range: 16%–72%) and 45.3% (range: 13%–74%) of the ¹¹C was measured as methionine and labeled proteins, respectively.     

Characteristics of specific in vivo labeling of neuroleptic binding sites with 3-N-[11C]methylspiperone

In vivo binding of 3-N-[¹¹C]methylspiperone ([¹¹C]NMSP) was saturable in the rat forebrain, but not in the cerebellum. Nonspecific binding was almost equivalent in all brain regions except for the white matter. [¹¹C]NMSP binding was localized to receptor-rich fractions when low doses were administered (less than 20 nmol/kg body weight). The striatum-to-cerebellum ratio was a function of time after injection and administered dose. This radio remained constant in low doses of under 30 nmol/kg. The radioactivity curve of the cerebellum in a control positron-emission tomographic study almost equaled that of the striatum in the dog pretreated with spiperone (2 mg). This indicates that the amount of binding in the cerebellum might be considered a nonspecific binding and unbound pool. The data obtained by the pretreatment study was different from that of displacement, which suggested that displaceable [¹¹C]NMSP in the specific binding sites of the striatum was not completely cleared from the brain tissue by a large amount of unlabeled spiperone.     

Further characterization of a CNS adenosine A2a receptor ligand [11C]KF18446 within vitro autoradiography andin vivo tissue uptake

PET assessment of the adenosine A2a receptors localized in the striatum offers us a potential new diagnostic tool for neurological disorders. In the present study, we carried out in vitro receptor autoradiography of a newly developed PET ligand [11C]KF18446 ([7-methyl-11C]-(E)-8-(3,4,5-trimethoxystyryl)-1,3,7-trimethylxanthin e) with rat brain sections. [11C]KF18446 showed a high striatum/cortex binding ratio (5.0) and low nonspecific binding (<10%), suggesting that [11C]KF18446 has characteristics comparable or slightly superior to [3H]CGS 21680 or [3H]SCH 58261, which are currently available representative A2a receptor ligands. Scatchard analysis indicated a Kd of 9.8 nM and a Bmax of 170 fmol/mm3 tissue in the striatum and a Kd of 16.4 nM and a Bmax of 33 fmol/mm3 tissue in the cortex. Seven xanthine-type and four nonxanthine-type adenosine receptor ligands with an affinity for the adenosine A2a receptors significantly reduced the in vitro binding of [11C]KF18446 to the brain section. The blocking effects were much stronger in the striatum than in the cortex, but did not necessarily parallel their affinity. On the other hand, four xanthine-type ligands and one nonxanthine-type ligand (SCH 58261) of the 11 ligands studied reduced the in vivo uptake of [11C]KF18446 in mice, but other ligands, including A1-selective and nonselective ligands and three nonxanthine-type A2a-selective antagonists did not. We conclude that [11C]KF18446 is a promising adenosine A2a receptor ligand for PET study.     

Evaluation of [C]laniquidar as a tracer of P-glycoprotein: radiosynthesis and biodistribution in rats

At present, P-glycoprotein (P-gp) function can be studied using positron emission tomography (PET) together with a labelled P-gp substrate such as (R)-[¹¹C]verapamil. Such a tracer is, however, less suitable for investigating P-gp (over)expression. Laniquidar is a third-generation P-gp inhibitor, which has been used in clinic trials for modulating multidrug resistance transporters. The purpose of the present study was to develop the radiosynthesis of [¹¹C]laniquidar and to assess its suitability as a tracer of P-gp expression.The radiosynthesis of [¹¹C]laniquidar was performed by methylation of the carboxylic acid precursor with [¹¹C]CH₃I. The product was purified by HPLC and reformulated over a tC₁₈ Seppak, yielding a sterile solution of [¹¹C]laniquidar in saline. For evaluating [¹¹C]laniquidar, rats were injected with 20 MBq [¹¹C]laniquidar via a tail vein and sacrificed at 5, 15, 30 and 60 min after injection. Several tissues and distinct brain regions were dissected and counted for radioactivity. In addition, uptake of [¹¹C]laniquidar in rats pretreated with cyclosporine A and valspodar (PSC 833) was determined at 30 min after injection. Finally, the metabolic profile of [¹¹C]laniquidar in plasma was determined.[¹¹C]Laniquidar could be synthesized in moderate yields with high specific activity. Uptake in brain was low, but significantly increased after administration of cyclosporine A. Valspodar did not have any effect on cerebral uptake of [¹¹C]laniquidar. In vivo rate of metabolism was relatively low. Further kinetic studies are needed to investigate the antagonistic behaviour of [¹¹C]laniquidar at tracer level.     

Development and evaluation of muscarinic cholinergic receptor ligands n-[c]ethyl-4-piperidyl benzilate and n-[c]propyl-4-piperidyl benzilate: a pet study in comparison with n-[c]methyl-4-piperidyl benzilate in the conscious monkey brain

The muscarinic cholinergic receptor ligands N-[¹¹C]ethyl-4-piperidyl benzilate (4-EPB) and N-[¹¹C]propyl-4-piperidyl benzilate (4-PPB) were developed and evaluated in comparison with N-[¹¹C]methyl-4-piperidyl benzilate (4-MPB) in the conscious monkey brain using positron emission tomography (PET). Time-activity curves of [¹¹C]4-EPB, unlike [¹¹C]4-MPB, showed peaks within 91 min in regions rich in muscarinic receptors. [¹¹C]4-PPB showed no specific binding even in the regions rich in these receptors. These observation demonstrated that increases in [¹¹C]alkyl chain length could alter the kinetic properties of receptor ligands for PET.     

Automated no-carrier-added synthesis of [1-C]-labeled d- and l-enantiomers of lactic acid

The first purely chemical method for automated no-carrier-added synthesis of [1-(11)C]-labeled d(R)- and l(S)-2-hydroxypropanoic acid (lactic acid) was developed for experimental neurophysiology studies and position emission tomography (PET) diagnosis. Starting from sodium 1-hydroxyethanesulfonate and [(11)C]HCN (trapped as [(11)C]KCN) the intermediate dl-(R,S)-[1-(11)C]-2-hydroxypropanenitrile was prepared. Its rapid acid hydrolysis gave dl-(R,S)-[1-(11)C]lactic acid, which was isolated by preparative reversed phase HPLC and automatically injected on a second preparative C(18) HPLC column coated with a chiral selector, where both [1-(11)C]lactic acid enantiomers were separated by chiral ligand-exchange chromatography. Two novel chiral selectors for HPLC enantiomeric separation of alpha-hydroxy acids, namely d(R)- or l(S)-2-amino-3-methyl-3-(5-phenylpentylsulfanyl)-butanoic acid were utilized for the preparative HPLC separation of the [1-(11)C]lactic acid enantiomers. The preparation of the selectors and the coating procedure for the manufacturing of the preparative chiral HPLC columns are described. A highly efficient trap for [(11)C]HCN is presented. The whole radiosynthesis is automated, takes about 45 min and leads to more than 80% decay corrected overall radiochemical yield of each enantiomer (up to 2.5 GBq) with over 99% radiochemical, chemical and enantiomeric purity. The specific activity at the end of the synthesis is about 400 GBq/micromol.     

[<Superscript>18</Superscript>F]Fluoroacetate is not a functional analogue of [<Superscript>11</Superscript>C]acetate in normal physiology

Purpose  [¹¹C]Acetate (C-AC) is a general PET tracer of cellular carbon flux and useful for clinical imaging in heart disease as well as prostate cancer and other tumours. C-AC has a high (70%) whole-body extraction fraction, proportional to blood flow in many organs. Trapping is related to organ-specific enzymatic activation and formation of [¹¹C]-acetyl-CoA, the fate of which has been well characterized. Due to the logistic challenges with C-AC, 2-[¹⁸F]fluoroacetate (F-AC) has been proposed as a marker for prostate cancer imaging. Method  We evaluated the potential of F-AC as a tracer for imaging blood flow and early enzymatic steps in the intermediary metabolism. C-AC and F-AC were injected serially in three cynomolgus monkeys and one domestic pig and scanned using PET/CT. A dynamic scan covering heart and liver was followed by repeated whole-body imaging. Kinetic patterns were compared for the myocardium, liver, blood and other organs. Results  C-AC kinetics and organ distribution in both species were similar to those previously established in man. In contrast, F-AC showed prolonged blood retention, no detectable trapping in myocardium or salivary glands, rapid clearance from liver and extensive excretion to bile and urine. Massive defluorination was seen in the pig, resulting in intense skeletal activity. Conclusion  2-[¹⁸F]Fluoroacetate cannot be regarded as a functional analogue of 1-[¹¹C]acetate in normal physiology and appears to be of little use for studies of organ blood flow, intermediary metabolism or lipid synthesis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.