Microwaving in F-18 chemistry: quirks and tweaks.

Since the late 1980s, microwave dielectric heating has been used to speed up chemical transformations, also in radiolabeling tracers for positron emission tomography. In addition to shorter reaction times, higher yields, cleaner product mixtures and improved reproducibility have also been obtained for reactions involving polar components that require heating at elevated temperatures. The conditions used in microwave chemistry can differ considerably from those in conventional heating. Understanding the factors that influence the interaction of the electromagnetic field with the sample is critical for the successful implementation of microwave heating. These parameters are discussed here and exemplified with radiolabelings with fluorine-18.

Effect of acute hyperketonemia on the cerebral uptake of ketone bodies in nondiabetic subjects and IDDM patients.

Using R-beta-[1-(11)C]hydroxybutyrate and positron emission tomography, we studied the effect of acute hyperketonemia (range 0.7-1.7 micromol/ml) on cerebral ketone body utilization in six nondiabetic subjects and six insulin-dependent diabetes mellitus (IDDM) patients with average metabolic control (HbA(1c) = 8.1 +/- 1.7%). An infusion of unlabeled R-beta-hydroxybutyrate was started 1 h before the bolus injection of R-beta-[1-(11)C]hydroxybutyrate. The time course of the radioactivity in the brain was measured during 10 min. For both groups, the utilization rate of ketone bodies was found to increase nearly proportionally with the plasma concentration of ketone bodies (1.0 +/- 0.3 micromol/ml for nondiabetic subjects and 1.3 +/- 0.3 micromol/ml for IDDM patients). No transport of ketone bodies from the brain could be detected. This result, together with a recent study of the tissue concentration of R-beta-hydroxybutyrate in the brain by magnetic resonance spectroscopy, indicate that, also at acute hyperketonemia, the rate-limiting step for ketone body utilization is the transport into the brain. No significant difference in transport and utilization of ketone bodies could be detected between the nondiabetic subjects and the IDDM patients.

In vivo evaluation of the biodistribution of 11C-labeled PD153035 in rats without and with neuroblastoma implants.

The biodistribution of 11C-labeled 4-(3-bromoanilino)-6,7-dimethoxyquinazoline, an inhibitor of the epidermal growth factor (EGF) receptor tyrosine kinase, has been evaluated in vivo in rats using positron emission tomography (PET). Time-activity data obtained after i.v. administration in one rat revealed that the radiotracer rapidly cleared from plasma with subsequent uptake in major organs of the body (brain, heart, liver, gastrointestinal tract and bladder). Uptake in proliferating tissue in rats with human neuroblastoma xenografts indicate that [O-11C-methyl]PD153035 shows promise as a new agent for in vivo imaging of tumors with PET.

N-methylquipazine: carbon-11 labelling of the 5-HT3 agonist and in vivo evaluation of its biodistribution using PET.

N-Methylquipazine (2-[1-(4-methyl)-piperazinyl)quinoline)) was labelled with carbon-11 by reacting [11C]methyl iodide with the nor-compound, quipazine. Radiochemical conversions were 79 +/- 7%, based on the alkylating agent. The total synthesis time including purification was 40 to 45 min. N-[Methyl-11C]methylquipazine thus synthesized was >99% radiochemically pure, and the specific activity ranged between 12-37 GBq/mumol. Dynamic imaging with PET was used to examine in vivo its distribution in rat and monkey. In rat the organ uptake at intermediate times was: liver > heart > whole brain > or = lung > extracerebral tissue. Brain uptake and wash-out were rapid: A maximum was reached in 2 to 3 min with subsequent decrease to approximately equal to 50% the peak value by 13 min. In monkey the tracer uptake was heterogeneous and high in regions known to contain 5-HT3 receptors but also in regions devoid of these receptors. Tissue kinetics were similar for all regions (initial rapid accumulation with tmax < or = 7 min, followed by slow decrease with all regions approaching the level of the cerebellum at 30 to 35 min). Pretreating with quipazine significantly decreased only the ratio of uptake in the medulla oblongata compared to the cerebellum. Although the nonspecificity of its binding limits the usefulness of N-[methyl-11C]methylquipazine, both its kinetic behavior and the blocking results indicate that a more selective arylpiperazine might prove to be a more attractive tracer for PET studies of 5-HT3 receptors.

In vivo biodistribution of [N-11C-methyl]KF 17837 using 3-D-PET: evaluation as a ligand for the study of adenosine A2A receptors.

(KF 17837, (E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine, was 11C-labelled by methylation at N-7 of the nor-compound, KF 17440, using [11C]methyl iodide. Radiochemical conversions of 50% or 70-80% were obtained using sodium hydride or potassium carbonate, respectively, as base. Total synthesis time was 40-45 min, including isolation by semipreparative liquid chromatography. Cerebral uptake of [N-11C-methyl]KF 17837 in Cynomolgus monkeys, evaluated using positron emission tomography (PET), was so low that regional differences in distribution kinetics were revealed first after increasing injected dose 3-fold and using 3-D mode of data acquisition. At all times, the relative regional retention (maximum striatum:cerebellum: cortex approximately 1.1:1:0.8 at 20 min) was considerably different from the known relative density of A2A receptors in these regions. Radioactivity decreased more rapidly in the cortex than in the striatum and cerebellum (by 20% vs. 3-7%, respectively, between 5 and 50 min). Addition of carrier to [N-11C-methyl]KF 17837 only marginally affected the cerebral radiotracer uptake. By contrast, in the heart the initial tracer uptake was high and the elimination kinetics was enhanced by adding unlabelled carrier. We have thus shown that KF 17837 passes the blood-brain barrier, though to a very low extent. This fact and the apparently high nonspecific binding in vivo of [N-11C-methyl]KF 17837 in regions with low receptor densities limits its usefulness as a ligand for quantification of the adenosine A2A receptors in the primate brain.

Localized cerebellar reductions in benzodiazepine receptor density in human partial epilepsy.

BACKGROUND: Previous studies suggest that the morphological substrate for cerebellar dysfunction is destruction of Purkinje cells, but disagree on whether this is caused by seizure- or drug-related toxicity. The benzodiazepine (BZ) receptor antagonist flumazenil tagged with carbon 11 is a sensitive marker of Purkinje cells. OBJECTIVE: To investigate whether cerebellar dysfunction in partial epilepsy is related to seizures through cerebrocerebellar connections. DESIGN: Positron emission tomography with [11C] flumazenil was conducted in 5 patients with frontal lobe seizures, 12 patients with mesial temporal lobe seizures, and 7 healthy men. Eight patients also had [18F]-fluorodeoxyglucose positron emission tomography. Cerebellar regions of interest were delineated using magnetic resonance imaging and a computerized anatomical brain atlas, and the epileptogenic regions were determined with a multimethod assessment. RESULTS: Patients with frontal lobe seizures had a significantly reduced BZ receptor density in the anterior cerebellum contralateral to the seizure onset region (P < or = .001), 2-way repeated-measure analysis of variance). Patients with mesial temporal lobe seizures had reductions in the ipsilateral (posterior and anterior) cerebellum (P < or = .001 for both). No significant asymmetries were found in regional glucose metabolism. CONCLUSIONS: The observed distribution of BZ receptor reductions is congruent with animal experiments showing tht frontal lobe projections to the cerebellum are crossed, whereas projections from mesial temporal loe are predominantly ipsilateral. The results thus indicate a functional relation with seizures and may reflect excitotoxic lesions or specific changes in the gamma-aminobutyric BZ system.

Cortical benzodiazepine receptor changes are related to frequency of partial seizures: a positron emission tomography study.

Measurements of benzodiazepine (BZD) receptor density with positron emission tomography (PET) are a promising method of identifying and localizing epileptogenic regions. We investigated whether the pattern of BZD receptor changes depends on seizure frequency, studying 19 patients with matching seizure semiology but different rates of seizure occurrence, using [11C]flumazenil as the ligand. All patients had partial epilepsy and normal magnetic resonance imaging (MRI) of the brain. The visually determined PET focus, characterized by reduced BZD receptor density, corresponded to the epileptogenic focus/seizure onset region in all patients. The degree of BZD receptor reduction showed a positive correlation with seizure frequency. Patients with daily seizures differed from those with fewer seizures in two aspects: (a) the degree and extent of BZD receptor reduction was more pronounced, and (b) BZD receptors were also reduced in the primary projection areas of the focus. Flumazenil-PET reliably identifies epileptogenic brain regions in patients with partial seizures. In addition, flumazenil-PET can distinguish patients with frequent seizures. The method therefore is not only suitable for noninvasive localization of the seizure focus, but also may provide a biochemical marker of epileptogenicity.

[11C]flumazenil positron emission tomography visualizes frontal epileptogenic regions.

Presently available noninvasive methods correctly localize epileptogenic regions in only approximately 50% of patients with frontal lobe epilepsy (FLE). Earlier studies have shown that temporal lobe epileptogenic regions may be identified readily by positron emission tomography (PET) measurements of regional benzodiazepine (BZD) receptor binding. We tested the specific applicability of this method in patients with FLE. Six patients with frontal partial seizures and 7 healthy men were investigated with PET and the BZD receptor ligand [11C]flumazenil. All patients had magnetic resonance (MR) brain scans. The independent assessment of seizure-onset region was based on seizure semiology, intra- and extracranial EEG and, in 4 cases, also on [18F]fluorodeoxyglucose (FDG)-PET. The epileptic focus/seizure-generating region was correctly identified by [11C]flumazenil PET in all patients. This region was characterized by a significant reduction in BZD receptor density. The area with reduced BZD receptor density was better delimited than the corresponding hypometabolic region, which was observed in 50% of patients investigated with [18F]FDG-PET. MRI was normal in 5 patients. Visualization of BZD receptors with [11C]flumazenil PET appears to be a promising approach for noninvasive identification of frontal lobe epileptogenic regions.

Use of R-beta-[1-11C]hydroxybutyrate in PET studies of regional cerebral uptake of ketone bodies in humans.

A method for determining regional cerebral utilization of ketone bodies in humans is described. After a bolus injection of R-beta-[1-11C]hydroxybutyrate, the time course of the tracer in the brain was measured with positron emission tomography in five healthy volunteers. The regional cerebral blood flow was measured separately. The tracer uptake in the brain could be well described by a single rate constant, indicating that the concentration of unmetabolized ketone bodies in the brain is very low and that transport across the blood-brain barrier is the rate-limiting step. At an average plasma concentration of beta-hydroxybutyrate of 0.043 mumol/ml, the utilization rate was estimated to be 0.48 nmol.ml-1.min-1. In accordance with previous animal studies, the utilization rate was found to increase almost linearly with increasing plasma concentration of beta-hydroxybutyrate. Furthermore, the utilization was higher in gray than in white matter. Finally, the ratio between the utilization in the basal ganglia and the brain as a whole was lower for ketone bodies than for glucose.

In vivo demonstration of altered benzodiazepine receptor density in patients with generalised epilepsy.

Electrophysiological data suggest that an abnormal oscillatory pattern of discharge in cortical and thalamic neurons may be the major mechanism underlying primary generalised epilepsy. No neurochemical or anatomical substrate for this theory has hitherto been demonstrated in humans and the pathophysiology of primary generalised epilepsy remains unknown. By means of PET and the benzodiazepine (BZ) ligand [11C]flumazenil it has been previously shown that the BZ receptor density is reduced in the epileptic foci of patients with partial epilepsy. In the present study the method was further developed and used in a comparative analysis of cortical, cerebellar, and subcortical BZ receptor binding in patients with primary generalised tonic and clonic seizures (n = 8), and healthy controls (n = 8). Patients with generalised seizures had an increased BZ receptor density in the cerebellar nuclei (p = 0.006) and decreased density in the thalamus (p = 0.003). No significant changes were seen in the cerebral and cerebellar cortex or in the basal ganglia. The observed alterations suggest that the gamma-amino-butyric acid (GABA)-BZ system may be affected in the cerebello-thalamocortical loop of patients with generalised epilepsy and indicate possible targets for selective pharmacological treatment.

Synthesis of [1-11C]D-glucosamine and evaluation of its in vivo distribution in rat with PET.

D-Glucosamine is a structural unit of many biologically interesting macromolecules. To investigate the feasibility of using labelled D-glucosamine as a tracer for anabolic processes, a two-step synthetic procedure for specifically labelling D-glucosamine in position 1 with carbon-11 was developed. [11C]Cyanide was reacted with an imine precursor, N-benzyl-D-arabinosylamine, to generate the [1-11]alpha-amino nitrile. Reduction to [1-11C]D-glucosamine was accomplished by catalytic hydrogenation using PdCl2 and the N-benzyl group was simultaneously removed. The total synthesis time from end-of-trapping of [11C]cyanide was 40-45 min and the decay-corrected radiochemical yield was 5-10% after HPLC isolation. The biodistribution of [1-11C]D-glucosamine in rat following i.v. bolus injection was investigated using positron emission tomography and showed that the availability of this substance for CNS anabolism is low with the primary limitation being the intact blood-brain barrier.

In vivo distribution of [11C]-busulfan in cynomolgus monkey and in the brain of a human patient.

The in vivo distribution of the antileukemic agent busulfan labeled with the positron-emitting radionuclide carbon 11 was investigated in cynomolgus monkeys and in a human patient using positron emission tomography. After i.v. injection of the radiotracer, its regional uptake was monitored for about 1 h in the monkey's body and, in a separate experiment, in the monkey's brain. The concentration of radioactivity in the liver, which showed the highest levels of all the organs scanned, increased throughout the experiment and was 9-fold that in the brain at the end of the experiment. [11C]-Busulfan rapidly crossed the blood-brain barrier. The radioactivity peaked in both the cortex and the white matter showing a ratio of 1.25, at 3 min but declined quickly to yield a ratio of approximately 1 after 30 min. In the human brain, radioactivity in the cerebellum, cortex, and white matter reached a maximum within 5 min showing a cortex:white matter ratio of 1.6. The activity in the cortex declined to yield a ratio of 1 within 30 min. Of the delivered dose, 20% penetrated into the brain.

11C-labeling of busulphan.

Busulphan [1,4-bis(methanesulfonoxy)butane], an alkylating agent used in the treatment of chronic myelocytic leukemia, was labeled with the positron-emitting radionuclide carbon-11 in a four-step synthetic procedure [1-11C]4-Hydroxybutyronitrile was obtained in 60-70% yield by the reaction of [11C]cyanide with 3-bromopropanol. The nitrile was hydrolysed to [1-11C]gamma-butyrolactone (80-90% yield) with sulfuric acid. Solid phase extraction was used to isolate the lactone and change the solvent before reduction to [1-11C]1,4-butanediol. Dimesylation of the diol with methanesulfonyl chloride in dichloromethane/pyridine yielded [1-11C]busulphan with conversions in the order of 30-35%. The total time of synthesis, including HPLC purification, was 65-75 min from the end-of-trapping of [11C]ammonium cyanide. The decay-corrected isolated yield of no-carrier-added [11C]busulphan was 4-7% and the radiochemical purity was better than 99%.

Cortical benzodiazepine receptor binding in patients with generalized and partial epilepsy.

Impaired cortical inhibition may be involved in epileptogenic mechanisms. In a positron-emission tomography (PET) study, we demonstrated a reduction of the cortical benzodiazepine (BZD) receptor density in the epileptic foci of patients with partial epileptic seizures. In the present study, we used the same method in 10 patients with primary generalized epilepsy to determine whether an altered BZD receptor binding could also be demonstrated in this patient group. The [11C]-labeled BZD receptor antagonist Ro 15-1788 was used as ligand. Receptor affinities and densities were calculated in various cortical regions and then compared with the values from corresponding "nonepileptic" regions in the previously examined partial epilepsy patients. Focal alterations of the BZD receptor density or affinity were not demonstrated in patients with generalized epilepsy. This patient group had a slight tendency toward lower mean cortical BZD receptor density, however, as compared with corresponding values from 10 patients with partial epilepsy. Our results strongly suggest that a focal alteration of cortical inhibition is not a prominent feature of human generalized epilepsy. The observed tendency toward lower mean cortical BZD receptor density may be a consequence of diffusely impaired cortical inhibition. Further investigations of this issue are therefore indicated.

11C-labelling of Ro 15-1788 in two different positions, and also 11C-labelling of its main metabolite Ro 15-3890, for PET studies of benzodiazepine receptors.

The benzodiazepine receptor antagonist Ro 15-1788 has been labelled with 11C in two different positions [( methyl-11C]Ro 15-1788 (I) and [ethyl-11C]Ro 15-1788 (II]. Product I was prepared by N-alkylation of the desmethyl compound (Ro 15-5528) with [11C]methyl iodide and product II was prepared by esterification of the desethyl compound (Ro 15-3890) with [11C]ethyl iodide. Ro 15-3890, the main metabolic product of Ro 15-1788, was labelled by two synthetic routes. In route A, [methyl-11C]Ro 15-3890 (III) was prepared by N-alkylation of the corresponding desmethyl compound (Ro 15-6877) with [11C]methyl iodide. In route B, III was prepared by a subsequent hydrolysis of I. The radiochemical yields were on the order of 15-60% (EOB) with an overall synthesis time of 40-50 min. Compounds I, II and III were isolated by semi-preparative HPLC and the radiochemical purity was in all cases greater than 99%.

Peripheral benzodiazepine binding sites in human gliomas evaluated with positron emission tomography.

A specific ligand for peripheral type benzodiazepine receptors, Ro5-4864, was synthesised and labelled with 11C for use in positron emission tomography (PET). Four patients with suspected glioma, two patients with high grade astrocytoma and two with low grade astrocytoma, as well as one patient with meningioma were examined with PET after the administration of 200 MBq 11C-Ro5-4864. The images as well as kinetic analyses revealed lower uptake of the ligand in tumor as compared with cortical tissue. Markedly elevated uptake in the thalamus suggests a functional role of the ligand.