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(S)-5-bromo-N-[(1-cyclopropylmethyl-2-pyrrolidinyl)methyl]-2,3-dimethoxybenzamide (4) has pico-molar in vitro binding affinity to D(2) receptor (K(i) (D(2))=0.003 nM) with lower affinity to D(3) receptor (K(i) (D(3))=0.22 nM). In this study, we describe radiosynthesis of [(11)C]4 and evaluation of its binding characteristics in post-mortem human brain autoradiography and with PET in cynomolgus monkeys. The (11)C labelled 4 was synthesized by using [(11)C]methyltriflate in a methylation reaction with its phenolic precursor with good incorporation yield (64+/-11%, DCY) and high specific radioactivity >370 GBq/micromol (>10,000 Ci/mmol). In post-mortem human brain autoradiography [(11)C]4 exhibited high specific binding in brain regions enriched with dopamine D(2)/D(3) receptors and low level of non-specific binding. In cynomolgus monkeys [(11)C]4 exhibited high brain uptake reaching 4.4% ID at 7.5 min. The binding in the extrastriatal low density D(2)-receptor regions; thalamus and frontal, parietal, temporal, and occipital cortex, was clearly visible. Pre-treatment with raclopride (1 mg/kg as tartrate) caused high reduction of binding in extrastriatal regions, including cerebellum. [(11)C]4 is a promising radioligand for imaging D(2) receptors in low density regions in brain.

[(11)C]FLB 457 is a very high-affinity radiotracer that allows the measurement of dopamine D(2/3) receptor availability in regions of the brain where densities are very low, such as the cerebral cortex. It is not known if [(11)C]FLB 457 binding is sensitive to the concentration of endogenous dopamine in humans in a manner analogous to [(11)C]raclopride and [(123)I]IBZM in the striatum. To test this possibility, extrastriatal [(11)C]FLB 457 binding was measured at baseline and after the oral administration of 40 to 60 mg of the psychostimulant methylphenidate (MP) in 12 healthy volunteers using positron emission tomography (PET) in a balanced-order, double-blind design. The dynamic PET data were quantified using a two-tissue compartment model with a metabolite-corrected arterial plasma input function. Two volunteers were excluded because of excessive head movement. In the remainder, MP caused significant reductions in the volume of distribution (VD) in temporal and frontal cortical regions and thalamus, suggesting that [(11)C]FLB 457 binding is sensitive to endogenous dopamine concentration. Moreover, the change in [(11)C]FLB 457 binding after MP correlated with the dose of MP (in mg/kg body weight) in all regions assessed. We conclude that MP in doses within the therapeutic range for the treatment of attention deficit hyperactivity disorder causes increases in dopamine concentrations in extrastriatal regions and that [(11)C]FLB 457 PET may be a useful tool for the assessment of change in dopamine concentration in these areas in humans.

In positron emission tomography (PET) studies, the detailed mapping of neuroreceptor binding is a trade-off between parametric accuracy and spatial precision. Logan's graphical approach is a straightforward way to quickly obtain binding potential values at the voxel level but it has been shown to have a noise-dependent negative bias. More recently suggested approaches claim to improve parametric accuracy with retained spatial resolution. In the present study, we used PET measurements on regional D2 dopamine and 5-HT1A serotonin receptor binding in man to compare binding potential (BP) estimates of six different parametric imaging approaches to the traditional Logan ROI-based approach which was used as a "gold standard". The parametric imaging approaches included Logan's reference tissue graphical analysis (PILogan), its version recently modified by Varga and Szabo (PIVarga), two versions of the wavelet-based approach, Gunn's basis function method (BFM) and Gunn et al.'s recent compartmental theory-based approach employing basis pursuit strategy for kinetic modeling (called DEPICT). Applicability for practical purposes in basic and clinical research was also considered. The results indicate that the PILogan and PIVarga approaches fail to recover the correct values, the wavelet-based approaches overcome the noise susceptibility of the Logan fit with generally good recovery of BP values, and BFM and DEPICT seem to produce values with a bias dependent on receptor density. Further investigations on this bias and other phenomena revealed fundamental issues regarding the use of BFM and DEPICT on noisy voxel-wise data. In conclusion, the wavelet-based approaches seem to provide the most valid and reliable estimates across regions with a wide range of receptor densities. Furthermore, the results support the use of receptor parametric imaging in applied studies in basic or clinical research.

Dopaminergic neurotransmission in extrastriatal regions may play a crucial role in the pathophysiology and treatment of neuropsychiatric disorders. The high-affinity radioligands [(11)C]FLB 457, [(123)I]epidepride, and [(18)F]fallypride are now used in clinical studies to measure these low-density receptor populations in vivo. However, a single determination of the regional binding potential (BP) does not differentiate receptor density (B(max)) from the apparent affinity (K(D)). In this positron emission tomography (PET) study, we measured extrastriatal dopamine D2 receptor density (B(max)) and apparent affinity (K(D)) in 10 healthy subjects using an in vivo saturation approach. Each subject participated in two to three PET measurements with different specific radioactivity of [(11)C]FLB 457. The commonly used simplified reference tissue model (SRTM) was used in a comparison of BP values with the B(max) values obtained from the saturation analysis. The calculated regional receptor density values were of the same magnitude (0.33-1.68 nM) and showed the same rank order as reported from postmortem studies, that is, in descending order thalamus, lateral temporal cortex, anterior cinguli, and frontal cortex. The affinity ranged from 0.27 to 0.43 nM, that is, approximately 10-20 times the value found in vitro (20 pM). The area under the cerebellar time activity curve (TAC) was slightly lower (11 +/- 8%, mean +/- SD, P = 0.004, n = 10) after injection of low as compared with high specific radioactivity, indicating sensitivity to the minute density of dopamine D2 receptors in the this region. The results of the present study support that dopamine D2 receptor density and affinity can be differentiated in low-density regions using a saturation approach. There was a significant (P < 0.001) correlation between the binding potential calculated with SRTM and the receptor density (B(max)), which supports the use of BP in clinical studies where differentiation of B(max) and K(D) is not required. In such studies, the mass of FLB 457 has to be less than 0.5 microg injected to avoid a mass effect of the radioligand itself.

The thalamus is a neuroanatomic structure that has reciprocal connections with several brain regions suggested to be involved in the pathophysiology of schizophrenia. Recent studies have reported structural as well as functional abnormalities of the thalamus in schizophrenia. The aim of the present exploratory study was to examine D2/D3 dopamine receptors in the thalamus as well as the anterior cingulate and the frontal and temporal cortices by using the high-affinity radioligand [11C]FLB 457 and positron emission tomography (3D PET) and to explore the data in relation to disease, age and psychopathology. Nine drug-naive patients with schizophrenia and eight control subjects were examined. Regional binding potential (BP) values were calculated using the simplified reference tissue model. The D2/D3 receptor binding was significantly lower in the right medial thalamus in the schizophrenia patients compared to control subjects. In addition, we found a significant negative age effect on the D2/D3 BP in the frontal and temporal cortex for both groups. There was no significant age effect on the D2/D3 BP in the thalamus or in the anterior cingulate. The result provides additional support to the view that the age effect on D2/D3 receptors differ between brain regions. The preliminary finding of low thalamic D2/D3 BP in patients strengthens the hypothesis that the thalamus is a key region in the pathophysiology of schizophrenia.

The study of human neuroreceptor systems by means of positron emission tomography (PET) and suitable radioligands has proven to be of great importance in research on normal brain functions and the pathophysiology and treatment of neuropsychiatric disorders. A for long identified goal is to produce detailed parametric maps of showing neuroreceptor binding parameters for the entire human brain in vivo. The application of wavelet filters has recently been proposed as a solution to handle the inherently low signal-to-noise ratio of PET images. In the present study we applied the wavelet approach to data obtained from 10 healthy subjects who were examined with [11C]FLB 457. This high affinity dopamine D2-receptor antagonist provides a signal from a range of regions with a hundredfold difference in receptor density and should thus be suitable for evaluation of the wavelet approach. For cross-validation purposes the data were analysed with four methods: a traditional region-of-interest (ROI) based analysis, a pixel-based analysis and two variants of wavelet-aided analyses. In both variants the wavelet filter was spatially applied, but a two-dimensional filter was used in one case and a three-dimensional one in the other. The same linear-graphical binding potential (BP) estimation step was used for all methods and the results of the three parametric mapping techniques were compared to the reference ROI-based method by calculating the average BP of representative ROIs. The pixel-based and the two-dimensional-wavelet-based methods yielded highly correlated but systematically lower values when compared to the reference ROI-based method. The approach utilising three-dimensional wavelet filters yielded BP maps with regional averages closely matching the values of the ROI-based method. The results show that the combination of three-dimensional spatial wavelet filtering with established parameter estimation procedures provides detailed, accurate maps of radioligand binding parameters. Such maps can be used for in inter-individual or multi-condition comparisons of binding parameters at subregional levels.

Extrastriatal D2 dopamine receptors represent an important target of research into the pathophysiology and pharmacotherapy of psychiatric disorders. The high affinity radioligand [11C]FLB 457 makes possible the measurement of low concentrations of D2 receptors in extrastriatal regions using positron emission tomography (PET). The aim of this study was to assess the test/retest variability and reliability of [11C]FLB 457 binding using a reference tissue model. Eight healthy male subjects (aged 20-33 years) underwent two [11C]FLB 457 PET examinations. Radioactivity in the cerebellum was used as the reference. The binding potentials (BPs) for five cortical regions of interest (ROIs) were calculated using the reference tissue model. The BP was also calculated for each pixel in the form of parametric images. Reproducibility was assessed both for the ROI method and for the parametric images. The test/retest reproducibility for [11C]FLB 457 binding was good, with a mean variability ranging from 4.5% for the thalamus to 15.5% for the hippocampus. The parametric images also demonstrated good reproducibility. These results support the suitability of using [11C]FLB 457 for the quantitative evaluation of extrastriatal D2 receptors and for protocols requiring repeated measurements in the same individual.

The D2 dopamine receptor density ranges from 0.2 to 40 nM among human brain regions. For high density regions radioligands like [(11)C]raclopride provide accurate and reliable estimates of the receptor density. In research on neuropsychiatric disorders there is, however, a growing need for quantitative approaches that accurately measure D2 dopamine receptor occupancy induced by drugs or endogenous dopamine in regions with low receptor density. The new high affinity radioligands [(11)C]FLB 457 and [(123)I]epidepride have been shown to provide a signal for extrasriatal D2 dopamine receptor populations in the human brain in vivo. Initial observations indicate, however, that the time required to reach equilibrium is dependent on receptor density. Ratio analyses may thus not be readily used for comparisons among different brain regions. The aim of the present simulation study was to examine commonly used approaches for calculation of drug induced D2 dopamine receptor occupancy among regions with widely different receptor density. The input functions and the rate constants of [(11)C]FLB 457 and the reference ligand [(11)C]raclopride were first used in a simulation estimating the effect of receptor density on equilibrium time. In a second step we examined how errors produced by inaccurate determination of the binding potential parameter propagate to calculations of drug induced receptor occupancy. The simulations showed a marked effect of receptor density on equilibrium time for [(11)C]FLB 457, but not for [(11)C]raclopride. For [(11)C]FLB 457, a receptor density above about 7 nM caused the time of equilibrium to fall beyond time of data acquisition (1 h). The use of preequilibrium data caused the peak equilibrium and the end time ratio approaches but not the simplified reference tissue model (SRTM) approach to underestimate the binding potential and thus also the drug occupancy calculated for high-density regions. The study supports the use of ratio and SRTM analyses in extrastriatal low-density receptor regions for which the high affinity ligand [(11)C]FLB 457 was developed. However, in high-density regions such as the human striatum simple ratio approaches cannot be validly applied, whereas the SRTM approach has higher potential to provide valid estimates. Interestingly, the results suggest that published data on a proposed extrastriatal selectivity for the antipsychotic drugs clozapine and olanzapine may be due to erroneous estimations of the binding potential when using ratio approaches., (Copyright 2001 Academic Press.)

To estimate receptor binding of ligand by positron emission tomography (PET) without an arterial input function, several quantitative approaches based on the use of a reference region have been proposed. We compared three approaches for quantifying extrastriatal D(2) dopamine receptors using [(11)C]FLB 457. The PET measurements were performed on seven healthy men. Binding potential (BP) of [(11)C]FLB 457 was calculated by the reference tissue model method, transient equilibrium method, and late time method. The reference tissue model describes the time-activity curve in a brain region in terms of that in the reference region, assuming that the levels of nondisplaceable radioligand binding in both regions are the same. The transient equilibrium theoretically occurs when the derivative for specific binding is zero. With the late time method, BP is calculated by integrating a late part of the time-activity curve. BP values obtained by all methods were in good agreement with those obtained by the kinetic approach, and the highest coefficient of correlation was observed in the reference tissue model method. In the simulation study, the error of BP calculated by the reference tissue model method was smallest. Moreover, the effect of the difference in the influx rate constant K(1) between the brain and the reference regions on BP was nearly avoided as theoretically predicted. We concluded that the reference tissue model method is most suitable for calculating BP of extrastriatal D(2) dopamine receptors with [(11)C]FLB 457., (Copyright 2001 Academic Press.)

[(11)C]raclopride binding to D2 dopamine receptors in the striatum is sensitive to drug-induced changes of endogenous dopamine concentration. We recently developed the new radioligand [(11)C]FLB 457, which is suitable for positron emission tomography (PET) studies of extrastriatal D2 dopamine receptors. The purpose of this PET study was to examine the effect of amphetamine on [(11)C]FLB 457 binding in extrastriatal regions. Each of three cynomolgus monkeys was examined at baseline conditions, 15 min and 3 h after I.V. injection of amphetamine (2 mg/kg). The effect of amphetamine was calculated from the ratio of specific [(11)C]FLB 457 binding to the binding in the cerebellum, a region which was used as reference for free and nonspecific binding in the brain. The changes of the ratio in the striatum, the thalamus, and the neocortex were between -1.2% and -15.5% at 15 min and -2.1% and -16.3% at 3 h, respectively, after amphetamine administration. The reductions of the binding ratios in the extrastriatal regions are similar to those reported for [(11)C]raclopride binding in the striatum. These data in a limited series of monkeys suggest that [(11)C]FLB 457 binding to D2 dopamine receptors in extrastriatal regions is sensitive to changes in the concentration of endogenous dopamine., (Copyright 2000 Wiley-Liss, Inc.)

The Computerized Brain Atlas (CBA) transforms PET images of individual subjects into a standard brain anatomy. We have previously applied this to PET images with [(11)C]raclopride and confirmed that the D2 dopamine receptors in the striatum can be evaluated accurately with a standard brain anatomy. There is growing evidence that extrastriatal D2 receptors, in spite of their low density, have pathophysiological significance for schizophrenia. We used the CBA to explore the extrastriatal distribution of D2 receptors in 13 healthy subjects using [11C]FLB 457, a substituted benzamide with very high affinity for D2 and D3 receptors. There was good agreement between the specific binding ratios from CBA quantification of standardized images and those from region-of-interest analyses of original images. The highest levels of binding were observed in the putamen and caudate nucleus, followed by the globus pallidus and nucleus accumbens. Besides the basal ganglia, the hypothalamus and nucleus ruber also showed high levels of binding. Intermediate levels were found in the substantia nigra, nucleus subthalami, amygdala, and thalamus. Interestingly, there was very heterogeneous binding among the thalamic nuclei. The anterior and mediodorsal nuclei showed relatively high binding. The cerebral cortices showed lower levels with significant regional differences. Binding was highest in the temporal cortex and hippocampus followed by the anterior cingulate gyrus, and the parietal and frontal cortices, but was lowest in the occipital cortex. The use of CBA for analysis of [11C]FLB 457 binding makes it possible to build a normal database for the extrastriatal D2 receptors in the living human brain. The heterogeneous distribution of D2 receptors provides an attractive opportunity for new research on the pathophysiology and drug treatment of schizophrenia., (Copyright 1999 Academic Press.)

Positron emission tomography (PET) has hitherto been used to examine D2 dopamine receptor binding in the striatum, a region with a high density of receptors. Research has been hampered by the lack of suitable radioligands for detection of the low-density D2 dopamine receptor populations in the limbic and cortical dopamine systems that are implicated in the pathophysiology of schizophrenia. [11C]FLB 457 is a new radioligand with the very high affinity of 20 pmol/L (K(i)) for the D2 and D3 dopamine receptor subtypes. This study in eight healthy subjects was designed to evaluate the suitability of [11C]FLB 457 for quantification of extrastriatal D2/D3 dopamine receptors. PET-data were acquired in the three-dimensional mode and the arterial input function was corrected for labeled metabolites. The standard three-compartment model and four derived approaches were applied to calculate and compare the binding potentials. Besides the striatum, conspicuous radioactivity was found in extrastriatal regions such as the thalamus, the anterior cinguli, and the temporal and frontal cortices. The time activity curves could be described by the three compartment model. The different approaches gave similar binding potential values and the rank order between regions was consistent with that found in vitro. The short time of a PET measurement using [11C]FLB 457 (63 minutes) seemed not to be sufficient for reliable determination of the high binding potential in the striatum. These results are of principal importance because they show the potential for PET quantification of minute receptor populations in the human brain.

D2 dopamine receptors are of interest in the pathophysiology of schizophrenia. For group comparisons of neuroreceptor distribution measured by PET on a pixel-by-pixel basis, an anatomic standardization technique is required. The aim of the present study is to build a database of normal D2 dopamine receptor distribution using [11C]raclopride and an anatomic standardization technique. In each subject, two PET measurements were performed with rapid bolus injection and with continuous infusion of [11C]raclopride. The radioactivity of the PET images were integrated in the time interval. Integrated images were normalized by the radioactivity of the cerebellum, providing a measure of the binding potential (BP) in each pixel. Each PET image was transformed into a standard brain anatomy using a Computerized Brain Atlas system. From the standardized PET images, the sample mean and the SD of the BP were calculated in each pixel. On the anatomically standardized average images for the both rapid bolus injection and continuous infusion, high BP was observed in the putamen and the caudate nucleus, whereas low BP was observed in the cerebral cortices. The BP for the thalamus and the substantia nigra were slightly higher than those for the cerebral cortices. This regional distribution is in good agreement with the distribution of D2 dopamine receptors known from in vitro studies. The anatomic standardization technique permits to build a database of the normal D2 dopamine receptor distribution in the living human brain. This technique can be applied for group comparisons on a pixel-by-pixel basis., (Copyright 1999 Academic Press.)

Several approaches have been applied for quantification of D2 dopamine receptors in positron emission tomography studies using [11C]raclopride. Initial approaches were based on analyses of data obtained after rapid bolus injection of [11C]raclopride. A continuous infusion paradigm has more recently been applied. The current study compares these approaches in healthy men. Two positron emission tomography measurements were performed in each of six healthy men, the first with rapid bolus injection and the second with continuous infusion of [11C]raclopride. In rapid bolus injection, the binding potential was calculated by the following methods. One approach is the kinetic analysis using the standard three-compartment model. Another is to define a transient equilibrium at the moment when the specific binding reaches its maximum. In continuous infusion, binding potential was calculated by using time-activity data at equilibrium condition. All methods gave almost identical binding potential, representing cross-validation of these methods. The continuous infusion method can provide "true" equilibrium condition. The kinetic analysis is a sophisticated approach but requires determination of an arterial input function. The transient equilibrium method thus is suitable for routine clinical research, since it does not require determination of an arterial input function.

Previous studies of the relationship between plasma prolactin and clinical effects in patients treated with antipsychotic drugs have yielded inconsistent results. A possible explanation may be that most studies have not included subtherapeutic or low doses of antipsychotics. In this exploratory, double-blind study, the relationship between plasma prolactin concentration and central D2 receptor occupancy was examined in 13 schizophrenic patients treated with the experimental antipsychotic drug raclopride (2, 6, or 12 mg daily). D2 receptor occupancy was determined by positron emission tomography and was related to antipsychotic effect as measured by the Brief Psychiatric Rating Scale. Plasma prolactin concentration was increased in eight of nine patients with a D2 receptor occupancy greater than 50%, whereas it was normal among patients with a D2 receptor occupancy less than 50% (p < 0.01). Plasma prolactin concentration measured 4 hours after the morning dose of raclopride correlated significantly with plasma raclopride concentration (r = 0.92, p < 0.01), the degree of D2 receptor occupancy (r = 0.81,p < 0.01), and the antipsychotic effect (r = 0.79, p < 0.01). Further controlled studies that include low doses of antipsychotic drugs may warrant a reconciliation of plasma prolactin as a useful tool in clinical monitoring of antipsychotic drug treatment.

We recently developed [11C]FLB 457 a substituted benzamide with the very high affinity of 20 pM for D2-dopamine receptors in vitro. The aim of the present exploratory study was to examine the anatomical distribution of [11C]FLB 457 binding in the human brain and to determine extrastriatal D2-receptor occupancy in antipsychotic drug-treated patients. [11C]raclopride was used to obtain reference values for D2-dopamine receptor occupancy in the putamen. After IV injection of [11C]FLB 457 there was a high concentration of radioactivity, not only in the caudate putamen but also in the thalamus and the temporal cortex. The concentration of radioactivity in the frontal cortex, the substantia nigra and the colliculi was slightly higher than in the cerebellum. Pretreatment with haloperidol and fluphenazine indicated that [11C]FLB 457 binding in extrastriatal regions to a high degree represent specific binding to D2-dopamine receptors. The D2-occupancy in antipsychotic drug-treated patients was on the same level in the thalamus and the temporal cortex as that determined with [11C]raclopride in the putamen. The study shows that [11C]FLB 457 has potential for quantitative PET-examination of D2-dopamine receptors in man.

Extrastriatal D2-dopamine receptors can be visualised in the monkey and human brain using the benzamides [11C]- and [76Br]FLB 457 in PET and [123I]epidepride in SPECT but not with the salicylamide analogues [76Br]FLB 463 and [123I]NCQ 298. To clarify the background for the differences in binding seen in vivo, we have compared the in vitro binding of [125I]epidepride and [123I]NCQ 298, using human whole hemisphere autoradiography. The images obtained with any radioligand showed detailed distribution with very dense binding in the putamen and the caudate nucleus and with the same detailed extrastriatal distribution. Thus, the divergent results obtained in vivo cannot be explained by different binding properties of the extrastriatal receptors.

Positron emission tomography was used to examine the in vivo binding of [11C]raclopride to D2-dopamine (DA) receptors in the striatum of two Cynomolgus monkeys after a single dose of reserpine (1 mg/kg, i.v.). A Scatchard procedure was repeated five times to follow D2 receptor density and apparent affinity for 7 weeks after reserpine. Reserpine-induced depletion of DA lead to a marked increase in [11C]raclopride binding, which was still detectable 20 days after treatment. Scatchard analyses indicated that the measured increase in [11C]raclopride binding reflected an increase in receptor affinity but no evident change in receptor density (Bmax). Thus, the increase in [11C]raclopride binding after reserpine should correspond to a reduced competition with endogenous DA for binding to D2 receptors. The results were used to estimate the DA-induced D2 occupancy to be about 40% at physiological conditions.

Central D2 dopamine receptor occupancy may be a useful measure to establish clinical guidelines for optimal antipsychotic drug treatment. The use of positron emission tomography (PET) to explore quantitative relationships among D2 receptor occupancy and clinical effects depends on the reliability of such measurements. The calculation of D2 receptor occupancy using [11C]raclopride is routinely based on a ratio-equilibrium analysis, in which the ratio of radioactivity concentration in the striatum to that in the cerebellum is determined. To examine the reliability of such ratios, a test-retest analysis was performed in four schizophrenic patients treated with haloperidol decanoate. PET experiments with [11C]raclopride were repeated in each subject during the same day. The putamen to cerebellum ratio (P/C ratio) ranged from 1.44 to 1.07 among the four patients, corresponding to a D2 receptor occupancy of 62 to 71%. In each subject, the P/C ratios remained highly similar, with quotients 0.98, 1.01, 1.04 and 1.06 between the two experiments. The high test-retest reproducibility of the P/C ratios indicates that measurements of D2 receptor occupancy with the present methods are highly reliable, and support the further use of PET to optimize the drug treatment of schizophrenia.

Both FLB 457 and FLB 463, two substituted benzamides with high affinity for the dopamine D-2 receptors, were labeled with bromine-76 for PET investigations. [76Br]FLB 457 was prepared by electrophilic substitution of the tributyltin precursor. The radiochemical yield was 80%. [76Br]FLB 463 was prepared by a direct electrophilic substitution enhanced by the hydroxyl group of the debromo analogue, with a total radiochemical yield of 50%. Radiochemical and chemical purity values of the radioligands as analyzed by radio-TLC and HPLC were > 99%, and the specific radioactivity was -40 GBq/mumol. During PET examinations of [76Br]FLB 457 and [76Br]FLB 463 binding in baboons there was a rapid and high uptake in the striatum. The striatal radioactivity concentration reached a plateau 1 h postinjection (p.i.). The striatum-to-cerebellum radioactivity concentration ratio increased from 11 at 1 h p.i., to 28 at 4 h p.i. for [76Br]FLB 457, owing to a continuous wash-out from the cerebellum. For [76Br]FLB 463 the corresponding value increased from 10 to 19.5. [76Br]FLB 457 has in contrast to [76Br]FLB 463 a high uptake in thalamic structures and has therefore an additional potential as a radioligand for PET examination of extrastriatal dopamine D-2 receptors in the living human brain.

A large problem in drug discovery is to find relevant in vitro or in vivo animal models and to be able to extrapolate the results obtained to humans. Drug research now benefits from the fast development of imaging technologies that trace radiolabelled molecules directly in the human brain. Positron emission tomography (PET) and allied techniques use molecules that are labelled with short-lived radioisotopes and injected intravenously. The most straightforward approach is to radiolabel a new potential drug and then to trace its anatomical distribution and binding in the brain. An indirect approach is to study how the unlabelled drug inhibits specific radioligand binding. The demonstration of quantitative relationships between drug binding in vivo and drug effects in patients is used to validate targets for drug action and to optimize clinical treatment.

The variability of D2-dopamine receptor binding parameters in man was determined using Positron Emission Tomography (PET) and [11C]raclopride. A saturation analysis based on five PET-experiments was performed in each of ten men and ten women. The mean density of D2-dopamine receptors (Bmax) was 28 +/- 6.9 pmol/ml (mean +/- S.D.) and the apparent affinity (Kdapp) 9.1 +/- 1.9 pmol/ml. The Hill coefficient was in all subjects close to unity (nH: 0.999 +/- 0.020), thereby indicating binding to a homogeneous class of receptors. No significant differences between males and females were found in Bmax or Kdapp. The interindividual difference in Bmax was statistically significant (alpha = 0.01). The difference in Kdapp was not significant. Upregulation of the receptor density (Bmax) has been widely discussed as a mechanism for increased dopaminergic neurotransmission in schizophrenia. This study indicates that receptor density varies considerably in a group of healthy subjects.

Unlabelled: D2 dopamine receptors in extrastriatal brain regions are of central interest for research in schizophrenia and antipsychotic drugs. This article reports the development of [11C]FLB 457 for PET examination of extrastriatal D2 dopamine receptors., Methods: Carbon-11-FLB 457 was prepared by O-methylation of FLB 604 (2-hydroxy precursor) with [11C]methyl iodide. Total radiochemical yield was 25%-35% within a total synthesis time of 30 min. The specific radioactivity at the end of synthesis was about 1300 Ci/mmole (48 GBq/mumole)., Results: FLB 457 bound with high affinity to D2 and D3 dopamine receptors, whereas binding to other putative central receptors was negligible. PET studies in Cynomolgus monkeys demonstrated 15 times higher accumulation of radioactivity in the striatum than in the cerebellum after 60 min. Uptake in the thalamus and neocortex, extrastriatal regions with a low density of D2 dopamine receptors, was, respectively, 4 and 2.5 times higher than in the cerebellum. Radioactivity was displaced by raclopride and haloperidol which confirms the selectivity and reversibility of [11C]FLB 457 binding to D2 dopamine receptors in vivo in the striatum, thalamus and neocortex., Conclusion: Carbon-11-FLB 457 should be a useful PET ligand for quantitative examination of D2 dopamine receptors in extrastriatal regions in the human brain.

The D2-dopamine receptor antagonist raclopride was administered to eight healthy male subjects, who had previously experienced akathisia following antipsychotic drugs. The influence of administration rate on onset, severity and duration of akathisia and on prolactin response was studied. Raclopride 3, 5 or 9 mg or placebo (P) was administered as single IV infusions during 10 min (R10 min/3 mg), 1 h (R1h/5 mg) or 4 h (R4h/9 mg) according to a randomized double-blind design. Despite a 24-fold difference in administration rate a similar peak raclopride concentration of about 350 nmol/l was obtained after all three infusions. Three of the eight subjects experienced akathisia following R10 min/3 mg and R1h/5 mg, respectively. After R4h/9 mg seven subjects experienced akathisia of longer duration but not more severe than after the short infusions. The incidence and duration of akathisia seem to be mainly related to the plasma raclopride concentrations over time, whereas the rate of administration might be more important for the severity. A maximal prolactin response was induced which was not markedly affected by the rate of administration.

The relationship between central D2-dopamine receptor occupancy and antipsychotic drug effects was examined in a double-blind study. Raclopride was the compound used to induce a selective occupancy of the D2-dopamine receptors. In addition, 11C-labeled raclopride was the radioligand used to measure occupancy by positron emission tomography (PET). Seventeen schizophrenic patients were randomly assigned to one of three parallel groups treated for 4 weeks with daily doses of 2, 6, or 12 mg of raclopride. D2-receptor occupancy was determined by PET at steady-state conditions in 13 patients who completed the study. A statistically significant relationship was demonstrated between antipsychotic effect and degree of D2-receptor occupancy (p < 0.05). Patients with extrapyramidal side effects had significantly higher D2-receptor occupancy than those without (p = 0.02). The finding of a relationship between selective occupancy of the D2-dopamine receptors and clinical effects in schizophrenic patients principally provides new support for the dopamine hypothesis of antipsychotic drug action.

The pharmacokinetic and pharmacodynamic properties of raclopride, a new antipsychotic, were investigated in 16 healthy men. Single 4 mg doses were administered as intravenous infusion, oral solution and 2 extended release (ER) formulations. Total plasma clearance was about 100 ml/min (6.0 L/h), of which renal clearance accounted for 0.2 ml/min, indicating extensive metabolism. The volume of distribution was 1.5 L/kg; mean absolute bioavailability was 65 to 67% following the oral solution and the ER formulations. A transient increase in plasma prolactin levels followed both the intravenous infusion and the oral solution. The ER formulations resulted in a lower increase, which appeared later. However, the area under the prolactin level curve was similar after administration of all dosage forms. The frequency and severity of the most commonly reported side effects (tiredness and restlessness) were higher after the intravenous infusion than after the ER capsules. These findings indicate that such capsules may be advantageous for clinical antipsychotic treatment with raclopride.

Pharmacological effects were recorded and time course for receptor binding in brain was followed by positron emission tomography after IV injection of the selective D1-dopamine receptor antagonist SCH 23390 in four healthy subjects in doses of 310-810 micrograms. Akathisia, the syndrome of motor restlessness, appeared after the three highest doses. The akathisia was transient and occurred only when [11C]SCH 23390 binding in the basal ganglia was at a high level with a central D1-dopamine receptor occupancy of 45-59%. The D2-dopamine receptor antagonist [11C]raclopride was injected IV into 20 healthy subjects and 13 schizophrenic patients. Akathisia appeared in 14 healthy subjects and 7 patients and coincided with maximal [11C]raclopride binding in the basal ganglia. The findings for [11C]raclopride and [11C]SCH 23390 are the first demonstration of a relationship between time courses for radioligand binding in the human brain and simultaneously induced pharmacological effects.

Five 11C- or 18F-labelled salicylamides ([11C]raclopride (I), [11C]eticlopride (II), [18F]NCQ 258 (III), [18F]NCQ 134 (IV) and [18F]NCQ 135 (V] were prepared. The total radiochemical yields of I-V from EOB were 3-30% (decay-corrected) with an overall synthesis time of 40-110 min. All compounds were isolated by semi-preparative HPLC and the radiochemical purity was greater than 99%. I-V were in separate experiments injected into Cynomolgus monkeys for PET-examination of ligand distribution in brain in vivo. I-V passed rapidly across the blood-brain barrier. With both analogs I and II there was a high uptake in the striatum, a region with a high density of dopamine D-2 receptors. With the 18F-labelled analogs III and IV, the uptake in the striatum was almost identical to that in the dopamine receptor poor cerebellum whereas the striatal uptake of V was clearly higher than in the cerebellum. Unlabelled I-V (raclopride, eticlopride, NCQ 258 (VII), NCQ 134 (VIII) and NCQ 135 (IX)) were also prepared and examined in vitro using [3H]raclopride and [3H]spiperone binding to rat striatal dopamine D-2 receptors. A significantly lower affinity was shown for NCQ 258 and NCQ 134 (5 times) compared to that of raclopride and eticlopride, respectively, whereas the affinity of NCQ 135 was similar to that of eticlopride.

NCQ 298 ((S)-3-iodo-N-[(l-ethyl-2-pyrrolidinyl)methyl]-5,6- dimethoxysalicylamide) has an iodine substituent. We have labelled NCQ 298 with 123I and 125I, and used the radioligands as tracers in receptor studies in vitro, in vivo in autoradiography and in SPECT studies on Cynomolgus monkeys. [125I]NCQ 298 bound in vitro to a single binding site with a KD = 19 pM. NCQ 298 has thus a 10-fold higher affinity for the dopamine D2 receptors than the corresponding des-5-methoxy compound FLA 961 (IBZM), previously used in SPECT studies. The binding of [125I]NCQ 298 was entirely reversible (T1/2 = 17.5 min at 37 degrees C). Autoradiographical studies in vitro on rat and monkey brain tissue sections showed a distinct binding in caudate-putamen, nucleus accumbens, substantia nigra, and in layer 5 of the cerebral cortex. In vivo binding studies in mice showed a ratio of 10 between [125I]NCQ 298 binding in striatum and cerebellum. Binding was displaced by the selective dopamine D2 receptor antagonist raclopride. In SPECT studies with [123I]NCQ 298 in two Cynomolgus monkeys, radioactivity accumulated in the basal ganglia. The measured striatum to cerebellum ratio was about 15 after 3 h. A monkey brain phantom was constructed for the determination of conversion factors from pixel events to actual radioactivity. The resulting, corrected striatum to cerebellum ratio obtained was 30. After administration of 12 mg raclo-pride to one of the monkeys there was a substantial decrease in striatal radioactivity. [125I]NCQ 298 is a suitable ligand for the labelling of dopamine D2 receptors in vitro and in vivo. The specific properties of [123I]NCQ 298 suggest that this compound is a useful ligand for quantitative SPECT studies of dopamine D2 receptors in man.

The aim of the present investigation was to study and compare the in vitro binding properties of the two radioligands N-[3H]methylspiperone ([3H]NMSP) and [3H]raclopride. These compounds, labeled with 11C, have been extensively used in positron emission tomography studies on central dopamine D2 receptors in schizophrenic patients, although with diverging results. One study (using [11C]NMSP) showed an increased dopamine receptor density in drug-naive schizophrenic patients, whereas in another study (using [11C]raclopride) the density in schizophrenic patients was no different from that in healthy controls. In the present study, using in vitro binding techniques, the density of the binding sites was found to be similar irrespective of which of the two radioligands was used (20 fmol/mg wet weight in rat striatum and 10 fmol/mg in human putamen; the 5-hydroxytryptamine 2 receptors were blocked with 40 nM ketanserin). [3H]NMSP had a 10-fold higher affinity (KD, 0.3 nM in rat striatum and 0.2 nM in human putamen) than [3H]raclopride (KD, 2.1 nM in rat striatum and 3.9 nM in human putamen), which was consistent with the longer dissociation half-life of [3H]NMSP compared with [3H]raclopride (14.8 and 1.19 min, respectively). There was an approximate overall similarity between the inhibition constants for five dopamine antagonists, chlorpromazine, haloperidol, raclopride, remoxipride, and NMSP, when using either radioligand. The Ki values were, however, two- to four-fold higher when using [3H]NMSP as the radioligand, irrespective of inhibiting compound, except for chlorpromazine (and haloperidol in human putamen). NMSP was found to inhibit the binding of [3H]raclopride competitively, whereas raclopride inhibited the binding of [3H]NMSP both competitively and noncompetitively. This difference suggests that part of the binding site is exclusively used by NMSP and can only be allosterically interfered with by raclopride. It is proposed that [3H]NMSP binds to an additional set of accessory binding sites, presumably located more distantly from the agonist binding active site than the sites to which [3H]raclopride binds.

The salicylamide NCQ 298, (S)-3-iodo-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5,6-dimethoxysalicylami de (4), binds with a high affinity and selectivity to central dopamine D-2 receptors. In the present paper the synthesis of NCQ 298 and the efficient labelling both with 123I and 125I are described. The unlabelled NCQ 298 was synthesized by iodination of (S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2,5,6-trimethoxybenzamide followed by demethylation with boron tribromide, which produced 4 and the regioisomer 5 in a ratio of 82:18. The two isomeric salicylamides were separated by radial chromatography and tested for their abilities to inhibit the binding of [3H]raclopride to rat striatal membranes in vitro in relation to some representative salicylamides. The 5,6-dimethoxysalicylamide 4 (NCQ 298) was found to be considerably more active than the corresponding 6-methoxysalicylamide 10 (FLA 961 or IBZM). The radioligands [123I]NCQ 298 and [125I]NCQ 298 were prepared in a carrier-free form from the corresponding desiodo compound by the chloramine-T protocol and isolated by semipreparative HPLC. The total radiochemical yields of [123I]NCQ 298 and [125I]NCQ 298 (based on [123I]- and [125I]iodide and decay-corrected) were 88% and 93%, respectively, with a radiochemical purity of greater than 99%. [125I]NCQ 298 will be studied to evaluate its potential as a radioligand in studies requiring a high specific activity, selectivity and high potency to label dopamine D-2 receptors. [123I]NCQ 298 has potential as a radioligand for the in vivo examination of central dopamine D-2 receptors in human brain by Single Photon Emission Computed Tomography (SPECT).

A new highly selective high-affinity dopamine D-2 receptor antagonist, eticlopride ((-)-(S)-5-chloro-3-ethyl-N-((1-ethyl-2-pyrrolidinyl) methyl)-6-methoxysalicylamide), was labelled with 11C in two different positions ([N-ethyl-11C]eticlopride (I)) and ([methyl-11C]eticlopride (II)). Product I was prepared by N-alkylation of the N-desethyl compound with [11C]ethyl iodide. II was prepared by O-alkylation of the diphenolic precursor with [11C]methyl iodide followed by separation of the two methylated products. The radiochemical yields were 15-20% (EOB) with an overall synthesis time of 45-60 min. Both compounds were isolated by semi-preparative HPLC and the radiochemical purity was in both cases greater than 99%. I was injected i.v. in a Cynomolgus monkey and brain radioactivity was measured by positron emission tomography (PET). The specific activity was 70 Ci/mmol at time of injection. There was a marked accumulation of radioactivity in the basal ganglia, regions known to have a high density of dopamine D-2 receptors.

The selective D2-dopamine receptor antagonist raclopride and its pharmacologically inactive (R)-enantiomer FLB472 were labelled with 11C and used in a study with positron emission tomography to examine the stereoselectivity of 11C-raclopride binding to central D2-dopamine receptors in three healthy men. After the injection of 11C-raclopride, there was a high accumulation of radioactivity in the dopamine-rich basal ganglia, whereas after the injection of 11C-FLB472 there was no such accumulation of radioactivity. Thus, the binding of 11C-raclopride is stereoselective. Distribution ratios [radioactivity in a brain region/"free" (not protein-bound) radioactivity in plasma] were calculated for the two enantiomers to study regional differences in the accumulation of radioactivity. The distribution ratios in white matter were similar for the two enantiomers. In the putamen, a three to four-fold higher distribution ratio was found for 11C-raclopride than for 11C-FLB472, reflecting the presence of specific binding of 11C-raclopride binding to D2-dopamine receptors in the basal ganglia. In the temporal and frontal cortices the distribution ratios were, however, only a few per cent higher for 11C-raclopride than for 11C-FLB472, indicating that if D2-dopamine receptors are present in the human neocortex, then their density is indeed very low.

[11C]Raclopride binding to central D2-dopamine receptors in humans has previously been examined by positron emission tomography (PET). Based on the rapid occurrence of binding equilibrium, a saturation analysis has been developed for the determination of receptor density (Bmax) and affinity (Kd). For analysis of PET measurements obtained with other ligands, a kinetic three-compartment model has been used. In the present study, the brain uptake of [11C]raclopride was analyzed further by applying both a kinetic and an equilibrium analysis to data obtained from four PET experiments in each of three healthy subjects. First regional CBV was determined. In the second and third experiment, [11C]-raclopride with high and low specific activity was used. In a fourth experiment, the [11C]raclopride enantiomer [11C]FLB472 was used to examine the concentration of free radioligand and nonspecific binding in brain. Radio-activity in arterial blood was measured using an automated blood sampling system. Bmax and Kd values for [11C]raclopride binding could be determined also with the kinetic analysis. As expected theoretically, those values were similar to those obtained with the equilibrium analysis. In addition, the kinetic analysis allowed separate determination of the association and dissociation rate constants, kon and koff, respectively. Examination of [11C]raclopride and [11C]FLB472 uptake in brain regions devoid of specific D2-dopamine receptor binding indicated a fourth compartment in which uptake was reversible, nonstereoselective, and nonsaturable in the dose range studied.

1. The use of raclopride, a new compound of the salicylamide series, as a ligand for the labelling of dopamine-D2 receptors in vitro and in vivo is described. 2. 3H-Raclopride has a high affinity for the dopamine-D2 receptors (Kd = 1 nM in rat striatum) with much less affinity for any other receptor. 3. 3H-Raclopride enters the brain easily and has therefore also been used in in vivo binding and autoradiography. The nonspecific binding is very low both in vitro and in vivo. 4. Raclopride has been labelled with 11C, and is used as a marker for dopamine-D2 receptors in the living human brain using positron emission tomography.

Tracer doses of 11C-SCH 23390 and 11C-raclopride, selective D1-dopamine and D2-dopamine receptor antagonists, respectively, were injected intravenously into three healthy male volunteers and two drug-treated schizophrenic patients. Regional radioactivity in brain and plasma was followed during 1 h by positron emission tomography (PET). After injection of both ligands a high accumulation of radioactivity was observed in the dopamine-rich caudate putamen. Experiments with 11C-SCH 23390, but not 11C-raclopride, showed a conspicuous accumulation of radioactivity also in the neocortex. None of the ligands accumulated in the dopamine-poor cerebellum. Specific binding of 11C-raclopride in the putamen was reduced by more than 80% in schizophrenic patients treated with antipsychotic doses of sulpiride or cis(Z)-flupentixol decanoate. 11C-SCH 23390 binding was slightly reduced in both the cortex and the putamen after treatment with cis(Z)-flupentixol decanoate but not after sulpiride. The results indicate that clinical antipsychotic drug treatment with sulpiride and cis(Z)-flupentixol decanoate causes a substantial blockade of D2-dopamine receptors in the basal ganglia but has only a minor effect on D1-dopamine receptors.

Raclopride, a highly selective D2-dopamine receptor antagonist, was administered in doses up to 4 mg b.i.d. to ten schizophrenic patients in an open label non-comparative study lasting 4 weeks. Safety, tolerability, potential antipsychotic effect, prolactin response and drug effect on plasma homovanillic acid were evaluated. Central D2-dopamine receptor occupancy was determined by positron emission tomography (PET). No major deviations were found in biochemical and physiological safety parameters. Raclopride was well tolerated. The mean BPRS score was reduced by 55% at endpoint. In the global evaluation seven patients were "very much" or "much" improved. Extrapyramidal side effects were recorded in four patients and disappeared after dose reduction or single doses of biperiden. An increase in plasma prolactin of short duration was observed in both sexes. A significant decrease of plasma HVA was obtained after 4 weeks of treatment. In two of the patients the central D2-dopamine receptors occupancy was measured using PET. The receptor occupancy was 68 and 72% which is the same as that found in patients treated with conventional neuroleptics.

Affinities and regional densities of the D1- and D2-dopamine receptor subtypes were studied in the human post-mortem brain in vitro using the two selective radioligands 3H-SCH 23390 and 3H-raclopride. 3H-Raclopride binding was confined to the caudate nucleus, the putamen and the substantia nigra, while 3H-SCH 23390 bound to cortical regions as well. The binding of 3H-SCH 23390 was reduced by a low concentration of ketanserin, indicating binding to 5-HT2 receptors in addition to the D1-dopamine receptors. The endogenous neurotransmitter dopamine interacted potently both with the D1-dopamine receptor and the D2-dopamine receptor, displaying two affinity states for each subtype. The distribution of the dopamine receptor subtypes obtained in the present in vitro investigation is in agreement with data obtained with 11C-SCH 23390 and 11C-raclopride in positron emission tomographic studies in human volunteers.

Raclopride, a new potential antipsychotic drug, with high selectivity and affinity for central D2-dopamine receptors, was in this first human study administered to 8 healthy male volunteers in single oral doses from 0.1 to 16 mg. Two subjects, known to be slow metabolizers of debrisoquine, were also included. Pharmacokinetics, safety, tolerability, and effect on plasma prolactin levels were evaluated. The maximum plasma concentrations of raclopride (Cmax) and the area under the raclopride curve vs time (AUC) increased proportionally with dose. No deviant kinetic parameters were seen in the slow debrisoquine metabolizers. Only minor deviations in biochemical and physiological safety parameters were found. Raclopride was well tolerated by all subjects at doses up to 8 mg but not at 16 mg because of akathisia. No other extrapyramidal side-effects were recorded. The drug induced a rapid and transient increase of plasma prolactin concentrations.