A two-compartment description and kinetic procedure for measuring regional cerebral [11C]nomifensine uptake using positron emission tomography.

S-[11C]Nomifensine (S-[11C]NMF) is a positron-emitting tracer suitable for positron emission tomography, which binds to both dopaminergic and noradrenergic reuptake sites in the striatum and the thalamus. Modelling of the cerebral distribution of this drug has been hampered by the rapid appearance of glucuronide metabolites in the plasma, which do not cross the blood--brain barrier. To date, [11C]NMF uptake has simply been expressed as regional versus nonspecific cerebellar activity ratios. We have calculated a "free" NMF input curve from red cell activity curves, using the fact that the free drug rapidly equilibrates between red cells and plasma, while glucuronides do not enter red cells. With this free [11C]NMF input function, all regional cerebral uptake curves could be fitted to a conventional two-compartment model, defining tracer distribution in terms of [11C]NMF regional volume of distribution. Assuming that the cerebellar volume of distribution of [11C]NMF represents the nonspecific volume of distribution of the tracer in striatum and thalamus, we have calculated an equilibrium partition coefficient for [11C]NMF between freely exchanging specific and nonspecific compartments in these regions, representing its "binding potential" to dopaminergic or noradrenergic uptake sites (or complexes). This partition coefficient was lower in the striatum when the racemate rather than the active S-enantiomer of [11C]NMF was administered. In the striatum of patients suffering from Parkinson's disease and multiple-system atrophy, the specific compartmentation of S-[11C]NMF was significantly decreased compared with that of age-matched volunteers.

Regional cerebral glucose transport in insulin-dependent diabetic patients studied using [11C]3-O-methyl-D-glucose and positron emission tomography.

Regional cerebral [11C]3-O-methyl-D-glucose ([11C]MeG) uptake kinetics have been measured in five insulin-dependent diabetic patients and four normal controls using positron emission tomography (PET). Concomitant measurement of regional cerebral blood volume and CBF enabled corrections for the presence of intravascular [11C]MeG signal in cerebral regions of interest to be carried out, and regional cerebral [11C]MeG unidirectional extraction fractions to be computed. Four of the five diabetic subjects were studied with their fasting plasma glucose level clamped at a normoglycaemic level (4 mM), and four were studied at hyperglycaemic plasma glucose levels (mean 13 mM). The four diabetic subjects whose fasting plasma glucose levels were clamped at a normoglycaemic level of 4 mM had mean fasting whole-brain, cortical, and white matter [11C]MeG extraction fractions of 15, 15, and 16%, respectively, values similar to those found for the four normal controls (whole brain, 14%; cortex, 13%; white matter, 17%). Mean regional cerebral [11C]MeG extraction fractions were significantly reduced in diabetic subjects during hyperglycaemia whether their plasma insulin levels were undetectable or whether they were raised by continuous intravenous insulin infusion. Such a reduction in [11C]MeG extraction under hyperglycaemic conditions can be explained entirely in terms of increased competition between [11C]MeG and D-glucose for the passive facilitated transport carrier system for hexoses across the blood-brain barrier (BBB). It is concluded that the number and affinity of D-glucose carriers present in the BBB are within normal limits in treated insulin-dependent diabetic subjects. In addition, insulin appears to have no effect on the transport of D-glucose across the BBB.

In vivo measurement of regional cerebral haematocrit using positron emission tomography.

A method is described for measuring the regional cerebral-to-large vessel haematocrit ratio using inhalation of carbon-11-labelled carbon monoxide and the intravenous injection of carbon-11-labelled methyl-albumin in combination with positron emission tomography. The mean value in a series of nine subjects was 0.69. This is approximately 20% lower than the value of 0.85 previously reported. It is concluded that previous measurements of regional cerebral blood volume using a haematocrit ratio of 0.85 will have underestimated the value of regional cerebral blood volume by 20%.

Glucose transport across the blood-brain barrier in normal human subjects and patients with cerebral tumours studied using [11C]3-O-methyl-D-glucose and positron emission tomography.

The kinetics of the regional cerebral uptake of [11C]3-O-methyl-D-glucose ([11C]MeG), a competitive inhibitor of D-glucose transport, have been studied in normal human subjects and patients with cerebral tumours using positron emission tomography (PET). Concomitant measurement of regional cerebral blood volume and blood flow enabled corrections for the contribution of intravascular tracer signal in PET scans to be carried out and regional unidirectional cerebral [11C]MeG extractions to be determined. A three-compartment model containing an arterial plasma and two cerebral compartments was required to produce satisfactory fits to experimental regional cerebral [11C]MeG uptake data. Under fasting, resting conditions, normal controls had mean unidirectional whole-brain, cortical, and white matter [11C]MeG extractions of 14, 13, and 17%, respectively. Mean values of k1 and k2, first-order rate constants describing forward and back transport, respectively, of tracer into the first cerebral compartment, were similar for [11C]MeG and [18F]2-fluoro-2-deoxy-D-glucose (18FDG), a second competitive inhibitor of D-glucose transport. k3, a rate constant describing FDG phosphorylation, was 20 times higher for cortical FDG uptake than the k3 fitted for [11C]MeG cortical uptake. Glioma [11C]MeG extractions ranged from normal levels of 12% to raised levels of 30%. Transport of [11C]MeG in and out of contralateral cortical tissue was significantly depressed in patients with gliomas. It is concluded that under fasting, resting conditions, regional cerebral glucose extraction remains relatively uniform throughout normal brain tissue. Gliomas, however, may have raised levels of glucose extraction. The nature of the second cerebral compartment required to describe [11C]MeG uptake is unclear, but it could represent either a useless phosphorylation-dephosphorylation cycle or nonspecific tracer uptake by a cerebral subcompartment.

Benzodiazepine receptor quantification in vivo in humans using [11C]flumazenil and PET: application of the steady-state principle.

Carbon-11-labeled flumazenil combined with positron emission tomography (PET) was used to measure the concentration (Bmax) of the benzodiazepine (Bz) receptor in the brain and its equilibrium dissociation constant (KD) for flumazenil in five normal subjects. The steady-state approach was used injecting the tracer as a bolus of high specific activity. In each subject two studies were carried out. The first study was performed at essentially zero receptor occupancy, the tracer alone study. The second study was performed at a steady-state receptor occupancy of about 50%, achieved by a prolonged constant infusion of nonlabeled ("cold") flumazenil starting 2h before the bolus tracer injection and continuing until the end of scanning period. In this second study the free concentration of unmetabolized flumazenil in plasma water was measured in multiple blood samples. The observed tissue and plasma tracer curves, calibrated in the same units of radioactivity per millimeter, were analyzed in two ways: (a) by the noncompartmental (stochastic) approach making no assumptions regarding number of compartments in the tissue, and (b) by the single-compartment approach assuming rapid exchange (mixing) of tracer between all tissue compartments. The noncompartmental and the compartmental analyses gave essentially the same values for the distribution volume of the tracer, the parameter used for quantitation of the Bz receptor. As the compartmental approach could be applied to a shorter observation period (60 min instead of 120 min) it was preferred. The five subjects had a mean KD value of 12 nM/L of water and Bmax values of the grey matter ranging from 39 +/- 11 in thalamus to 120 +/- 14 nM/L of brain in occipital cortex. Most previous studies have been based on the pseudoequilibrium approach using the brain stem as a receptor-free reference region. This yields practically the same KD but lower Bmax values than the steady-state approach presented here.

Regional lung hematocrit in humans using positron emission tomography.

Regional lung hematocrit ratio (R) was measured in five normal subjects and five patients (2 with pneumonia, 2 with nephrotic syndrome with anemia, and 1 with pancreatitis) using positron emission tomography, a red cell marker 11CO, and a plasma marker [methyl-11C]albumin). The measurements were made in a transaxial thoracic section at midheart level with the subject in supine posture and with a spatial resolution of 1.7 cm. The normal regional hematocrit ratio (means +/- SE) calculated for the lung was 0.90 +/- 0.014, 0.94 +/- 0.023 for the thoracic wall, and 1.00 +/- 0.003 for the heart chambers. The regional lung hematocrit ratio in the patients ranged between 0.81 and 0.86. No correlation was found among the regional lung hematocrit ratio and regional blood volume, lung extravascular density, and the peripheral hematocrit (obtained from venous blood samples). To the extent that 70% of the pulmonary blood in the field of view is in larger vessels with normal hematocrit, the hematocrit in the capillary bed is approximately two-thirds that of the peripheral venous value. Blood volume measurements on the basis of single vascular tracers need to take account of these results.

Radioligands for PET studies of central benzodiazepine receptors and PK (peripheral benzodiazepine) binding sites--current status.

The status of the radiochemical development and biological evaluation of radioligands for PET studies of central benzodiazepine (BZ) receptors and the so-called peripheral benzodiazepine binding sites, here discriminated and referred to as PK binding sites, is reviewed against current pharmacological knowledge, indicating those agents with present value and those with future potential. Practical recommendations are given for the preparation of two useful radioligands for PET studies, [N-methyl-11C]flumazenil for central BZ receptors, and [N-methyl-11C]PK 11195 for PK binding sites. Quality assurance and plasma metabolite analysis are also reviewed for these radioligands and practical recommendations are given on methodology for their performance.

Automated chemoenzymatic synthesis of no-carrier-added [carbonyl-11C]propionyl L-carnitine for pharmacokinetic studies.

Propionyl-L-carnitine (PLC) is under development as a therapeutic for the treatment of peripheral artery disease, coronary heart disease and chronic heart failure. Three methods were examined for labelling PLC in its propionyl group with positron-emitting carbon-11 (t12 = 20.3 min), one chemical and two chemoenzymatic. The former was based on the preparation of [11C]propionyl chloride as labelling agent via 11C-carboxylation of ethylmagnesium bromide with cyclotron-produced [11C]carbon dioxide and subsequent chlorination. Reaction of carrier-added [11C]propionyl chloride with L-carnitine in trifluoroacetic acid gave [11C]PLC in 12% radiochemical yield (decay-corrected) from cyclotron-produced [11C]carbon dioxide. However, the radiosynthesis was unsuccessful at the no-carrier-added (NCA) level of specific radioactivity. [11C]Propionate, as a radioactive precursor for chemoenzymatic routes, was prepared via carboxylation of ethylmagnesium bromide with [11C]carbon dioxide and hydrolysis. NCA [11C]PLC was prepared in 68 min in 14% radiochemical yield (decay-corrected) from [11C]propionate via sequential conversions catalysed by acetate kinase, phosphotransacetylase and carnitine acetyltransferase. A superior chemoenzymatic synthesis of NCA [11C]PLC was developed, based on the use of a novel supported Grignard reagent for the synthesis of [11C]propionate and conversions by S-acetyl-CoA synthetase and carnitine acetyltransferase. This gave an overall radiochemical yield of 30-48% (decay-corrected). This synthesis was automated for radiation safety and provides pure NCA [11C]PLC in high radioactivities ready for intravenous administration within 25 min from radionuclide production. The [11C]PLC is suitable for pharmacokinetic studies in human subjects with PET and the elucidation of the fate of the propionyl group of PLC in vivo.

The radiosynthesis of [N-methyl-11C]-sertraline.

[N-methyl-11C]Sertraline, a potential agent for the study of the serotonergic system in vivo with positron emission tomography, was prepared by N-methylation of the corresponding norcompound with [11C]iodomethane, which was itself prepared from cyclotron-produced [11C]carbon dioxide. Under the best conditions found [norsertraline free base (20 mM) in DMF (0.70 mL), 120 degrees C for 8 min] [N-methyl-11C]-sertraline can be prepared in 43% radiochemical yield from [11C]iodomethane (decay-corrected), corresponding to 20% overall radiochemical yield from [11C]carbon dioxide (decay-corrected), with high specific radioactivity. Preparations can be ready for i.v. injection 50 min from the end of radionuclide production.

Labelled agents for PET studies of the dopaminergic system--some quality assurance methods, experience and issues.

Practical methods are described for the quality assurance of three labelled agents (L-6-[18F]fluoro-DOPA, S-[N-methyl-11C]nomifensine and [O-methyl-11C]raclopride) now produced regularly for PET studies of the dopaminergic system in man. These include indirect methods for the initial determination of label position (e.g. 13C-NMR spectroscopy) and also direct methods for the assessment of chiral purity (TLC and HPLC) and the routine determination of radiochemical purity, chemical purity and specific activity (HPLC). Mass spectrometry has been used to identify some impurities. L-6-hydroxy-DOPA (a precursor in vivo of the neurotoxin, L-6-hydroxydopamine) has been detected by HPLC in some preparations of L-6-[18F]fluoro-DOPA. Formulated S-[N-methyl-11C]nomifensine has been found to decrease in radiochemical purity with storage, whereas formulated [O-methyl-11C]raclopride has been found to be stable. Some quality assurance issues are discussed in relation to experience in the application of the described methods and the obtained results.

A comparison between regional cerebral blood flow measurements obtained in human subjects using 11C-methylalbumin microspheres, the C15O2 steady-state method, and positron emission tomography.

Regional cerebral blood flow (rCBF) values were measured in nine normotensive subjects with known previous myocardial infarctions using 15 mu 11C-methylalbumin microspheres and positron emission tomography (PET). Microspheres were injected directly into the left ventricle of each subject during routine cardiac angiography and blood flow calibrated using the reference sample technique. rCBF values were compared with those obtained for a group of fifteen age-matched normal controls using the C15O2 steady-state inhalation technique. Using 1 cm radius circular regions of interest, the 11C-microspheres approach yielded mean blood flow values of 51 ml/100 ml/min and 48 ml/100 ml/min for regions of interest dominated by temporal and frontal cortical grey matter respectively. An rCBF value of 32 ml/100 ml/min was obtained for regions of interest dominated by frontal white matter. Mean rCBF values obtained for these regions using the C15O2 method were not significantly different (52 ml/100 ml/min, 44 ml/100 ml/min, and 28 ml/100 ml/min respectively), but the C15O2 approach gave a significantly lower rCBF value than the 11C-microspheresfor regions of interest dominated by occipital grey matter. Although the two groups of subjects studied were not strictly equivalent, the good agreement between blood flow values obtained using the 11C-microspheres and the C15O2 techniques is of interest, and suggests that the assumptions of the C15O2 steady-state approach do not lead to large errors in practice.

Studies on regional cerebral haematocrit and blood flow in patients with cerebral tumours using positron emission tomography.

Regional cerebral haematocrit has been measured in seven patients with brain tumours, and in one normal subject, using positron emission tomography (PET). Red cell and plasma volumes of distribution were assessed using 11CO and [methyl-11C]albumin, respectively. Haematocrit values were compared with values of regional cerebral blood flow (rCBF) measured using steady-state inhalation of C15O2. Only two of the seven cerebral tumours studied showed any increase in uptake of [methyl-11C]albumin over 45 min. Values of r, the regional ratio of cerebral small-to-large vessel haematocrit, varied from 0.52 to 0.84 for the seven tumours studied. No correlation between r and tumour blood flow was observed. The normal subject yielded an r value of 0.69 for the mean whole brain small-to-large vessel haematocrit ratio. No significant difference between gray and white matter r values was found. The contralateral hemispheres of the seven tumour patients studied yielded an overall mean r value of 0.71 +/- 0.05. We conclude that it is reasonable to assume an r value of 0.7 in tomographic calculations of regional cerebral blood volume (rCBV) from red cell or plasma volumes of distribution in normal brain. Such an assumption for tumours, however, may lead to errors of 35% in estimated rCBV.

Preparation of human serum [methyl-11C]methylalbumin microspheres and human serum [methyl-11C]methylalbumin for clinical use.

Safely-injectable suspensions of human serum [methyl-11 C] methylalbumin microspheres have been prepared via the reaction of human serum albumin microspheres with [11C]methyl iodide, itself prepared in a novel one-pot synthesis from cyclotron-produced [11C]carbon dioxide. The preparation takes only 30 min from the end of radionuclide production and proceeds in 22% radiochemical yield based on the activity of [11C]carbon dioxide used and decay-corrected. It has been shown that such microspheres are highly stable in vivo and may be used as reference blood flow markers in positron emission tomography (PET). Similarly, safely-injectable and radiochemically pure solutions of human serum [methyl-11C] methylalbumin have been prepared in 31% radiochemical yield and in 40 min from the end of [11C]carbon dioxide production via the reaction of [11C]methyl iodide with human serum albumin. This radiopharmaceutical is intended for studies of lung permeability and blood-brain barrier permeability by PET.

Citalopram: labelling with carbon-11 and evaluation in rat as a potential radioligand for in vivo PET studies of 5-HT re-uptake sites.

In vivo autoradiography of [N-methyl-3H]citalopram in rat brain shows a differential regional localization which correlates with the localization of 5-HT re-uptake binding sites defined in vitro. A comparison of the biodistribution of [N-methyl-3H]citalopram over 2 h after i.v. injection in (1) control rats (2) rats pre-dosed with either citalopram or paroxetine and (3) rats chemically-lesioned with p-chloroamphetamine provides an estimate of specific binding relative to total binding in vivo. The ratio of binding in certain regions (e.g. cingulate) to binding in a reference tissue (e.g. cerebellum) at 30-120 min post injection is c. 1.4. In view of these results a method was developed for labelling citalopram with carbon-11 (t1/2 = 20.3 min, beta + = 99.8%) to provide a potential radioligand for studies using positron emission tomography. Thus, reaction of nca [11C]iodomethane, prepared from cyclotron-produced [11C]carbon dioxide, with norcitalopram in ethanol containing 2,2,6,6-tetramethyl-piperidine for 5 min at 95 degrees C gives crude [N-methyl-11C]citalopram in 60% radiochemical yield, decay-corrected. HPLC on silica gel provides radiochemically and chemically pure [N-methyl-11C]citalopram, as assessed by TLC, HPLC and MS. This product (isolated radiochemical yield, 49%) is easily formulated for i.v. injection. Up to 2 GBq of formulated product with a specific activity of c. 15 GBq/mumol have been prepared within 40 min from the end of radionuclide production. The described radiosynthesis has also been applied to give the single biologically active (+)-enantiomer of [N-methyl-11C]citalopram rather than the racemate. This product gives enhanced specific signal in the rat following i.v. injection, the ratio of uptake in regions of interest relative to cerebellum approaching 2 at 90 min.

The distribution of radioactivity in brains of rats given [N-methyl-11C]PK 11195 in vivo after induction of a cortical ischaemic lesion.

PK 11195 is a selective ligand for the peripheral-type benzodiazepine binding site (PTBBS). There are few such sites in normal brain but their number increases in association with tissue necrosis. The time-course of appearance of PTBBS around a focally induced ischaemic lesion in frontal cortex of rat brain was established by autoradiography using [N-methyl-3H]PK 11195. Using this information and the same experimental model of ischaemia, the distribution of radioactivity after injection of carbon-11 (t1/2 = 20.3 min, beta+ = 99.8%) labelled PK 11195 was studied. The purpose was to synthesize [N-methyl-11C]PK 11195 and to test its suitability as a tracer for depicting the presence of PTBBS in ischaemic lesions. The time-profiles of distribution of radioactivity in brain regions after intravenous injection of tracer and the ratio of radioactivity in lesioned compared with unlesioned cortex were determined. Data for the temporal (days after lesion induction) and for the regional retention of radioactivity were consistent with independent evidence (autoradiographic and immunohistochemical) for the occurrence of increased numbers of PTBBS, predominantly in association with macrophages, in areas undergoing necrosis.