New halogenated [11C]WAY analogues, [11C]6FPWAY and [11C]6BPWAY--radiosynthesis and assessment as radioligands for the study of brain 5-HT1A receptors in living monkey.

[Carbonyl-(11)C]WAY-100635 ([(11)C]WAY) is an established radioligand for the study of brain serotonin(1A) (5-HT(1A)) receptors in living animals and humans with positron emission tomography (PET). There is a recognised need to develop halogenated ligands for 5-HT(1A) receptors, either for labelling with longer-lived fluorine-18 for more widespread application with PET or with iodine-123 for application with single photon emission tomography (SPET). Here we used autoradiography and PET to assess two new halogenated analogues of WAY, namely 6BPWAY and 6FPWAY [N-(2-(1-(4-(2-methoxyphenyl)-piperazinyl)ethyl))-N-(2-(6-bromo-/fluoro-pyridinyl))cyclohexanecarboxamide] as prospective radioligands, initially using carbon-11 as the radiolabel. Labelling of 6BPWAY and 6FPWAY with carbon-11 was accomplished by acylation of the corresponding secondary amine precursors with [carbonyl-(11)C]cyclohexanecarbonyl chloride. After incubation of human brain crysections with [(11)C]6BPWAY or [(11)C]6FPWAY, the highest accumulation of radioactivity was observed in cortical areas and the hippocampal formation. Both radioligands had high nonspecific binding. There was a rapid accumulation of radioactivity in the monkey brain after intravenous injection of [(11)C]6BPWAY and [(11)C]6FPWAY. High accumulation of radioactivity was observed in the frontal and temporal cortex and the raphe nuclei, areas known to contain a high density of 5-HT(1A) receptors. The ratios of radioactivity in receptor-rich temporal cortex to that in receptor-poor cerebellum at peak equilibrium were 1.9 (at 10 min) and 3.0 at (at 20 min) for [(11)C]6BPWAY and [(11)C]6FPWAY, respectively. In pretreatment experiments with high doses of unlabelled WAY, the level of radioactivity in the frontal and temporal cortex and the raphe nuclei was reduced to the same level as in the cerebellum. Radioactive metabolites of [(11)C]6FPWAY appeared at a rate similar to those for [(11)C]WAY, with 17% of the radioactivity in plasma represented by unchanged radioligand after 40 min. Radioactive metabolites of [(11)C]6BPWAY appeared much more slowly. At 40 min after injection 45% of the radioactivity in plasma still represented unchanged radioligand. The results indicate that 6-pyridinyl radiohalogented analogues of WAY are new leads to radioligands for PET or SPET.

SPET imaging of central muscarinic acetylcholine receptors with iodine-123 labelled E-IQNP and Z-IQNP.

1-Azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate (IQNP) is a muscarinic acetylcholine receptor (mAChR) antagonist and the racemic ligand contains eight stereoisomers. In a single-photon emission tomography (SPET) study in monkeys we recently confirmed that [123I]E-(R,R)-IQNP ([123I]E-IQNP) is a radioligand with modest selectivity for the M1 and M4 subtypes, whereas [123I]Z-(R,R)-IQNP ([123I]Z-IQNP) is non-subtype selective. In the present SPET study, E- and Z-IQNP were examined in human subjects. SPET examination was performed on three male subjects after i.v. injection of [123I]E-IQNP and in another three after i.v. injection of [123I]Z-IQNP. The binding potential (BP) for [123I]E-IQNP was calculated using several quantitative approaches with the cerebellum as a reference region. High-performance liquid chromatography was used to measure radioligand metabolism in plasma. Following [123I]E-IQNP, the radioactivity was high in the neocortex and striatum, intermediate in the thalamus and low in the pons and cerebellum, which is consistent with the rank order for the regional density of M1 and M4 subtypes in vitro. For all regions, peak equilibrium was identified within the 48-h data acquisition. The simplified reference tissue approach using SPET data from 0 to 48 h was the most reliable in this limited series of subjects. Following injection of [123I]Z-IQNP, radioactivity was high in the neocortex and striatum, intermediate in the thalamus and pons and low in the cerebellum, which is in agreement with the density of M1, M2 and M4 subtypes as measured in vitro. Quantitative analyses provided indirect support for specific M2 binding of Z-IQNP in the cerebellum. The high selectivity of [123I]E-IQNP for M1 and M4 receptors allowed the use of cerebellum as a reference region devoid of specific binding, and may be advantageous for applied clinical studies of M1 and M4 receptors binding in man. [123I]Z-IQNP has potential for exploration of M2 receptor binding in the cerebellum.

Radioligands for the study of brain 5-HT(1A) receptors in vivo--development of some new analogues of way.

[Carbonyl-(11)C]WAY-100635 (WAY) has proved to be a very useful radioligand for the imaging of brain 5-HT(1A) receptors in human brain in vivo with positron emission tomography (PET). WAY is now being applied widely for clinical research and drug development. However, WAY is rapidly cleared from plasma and is also rapidly metabolised. A comparable radioligand, with a higher and more sustained delivery to brain, is desirable since these properties might lead to better biomathematical modelling of acquired PET data. There are also needs for other types of 5-HT(1A) receptor radioligands, for example, ligands sensitive to elevated serotonin levels, ligands labelled with longer-lived fluorine-18 for distribution to "satellite" PET centres, and ligands labelled with iodine-123 for single photon emission computerised tomography (SPECT) imaging. Here we describe our progress toward these aims through the exploration of WAY analogues, including the development of [carbonyl-(11)C]desmethyl-WAY (DWAY) as a promising, more brain-penetrant radioligand for PET imaging of human 5-HT(1A) receptors, and (pyridinyl-6-halo)-analogues as promising leads for the development of radiohalogenated ligands.

Z-IQNP: a potential radioligand for SPECT imaging of muscarinic acetylcholine receptors in Alzheimer's disease.

RATIONALE: The density of the M2 subtype of muscarinic acetylcholine receptors (mAChR) has been shown to be reduced in the brain of patients with Alzheimer's disease (AD). It is therefore of interest to develop a brain imaging method for diagnostic purposes. Z-(R,R)-1-azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo1-propen-3-yl)-alpha-phenylacetat e (Z-IQNP) is a muscarinic antagonist with high affinity for the M2 subtype. OBJECTIVE: The pharmacological characteristics and topographic distribution of radiolabelled Z-IQNP as a radioligand for the M2 mAChR subtype were examined in vitro and in vivo. METHODS: Z-IQNP was labelled with 1251 and 123I. Autoradiography was performed on whole-hemisphere cryosections from human post mortem brains. SPECT was performed in a cynomolgus monkey. RESULTS: Autoradiography showed binding of [125I]Z-IQNP in all brain regions, which was inhibited by the non-selective muscarinic antagonist scopolamine. The addition of BIBN 99, a compound with high affinity for the M2 subtype, inhibited [125I]Z-IQNP binding particularly in the cerebellum, which has a high density of the M2 subtype. SPECT demonstrated high uptake of [123I]Z-IQNP in all brain regions. The binding was markedly reduced in all brain regions after pretreatment with the non-selective muscarinic antagonist dexetimide and also the M1 antagonist biperiden. Dexetimide markedly inhibited [123I]Z-IQNP binding in the cerebellum, which is consistent with a high density of M2-receptors in this region. The sigma receptor binding compound DuP 734 had no effect on Z-IQNP binding either in vitro or in vivo. CONCLUSIONS: This study indicates that radiolabelled Z-IQNP has high specificity for mAChR with higher affinity for the M2 than the M1 subtype and negligible affinity for sigma recognition sites both in vitro and in vivo. [123I]Z-IQNP should be useful for future SPECT studies in AD for examination of the density of M2 receptors particularly in the cerebellum.

Dopamine D(2) receptor quantification in extrastriatal brain regions using [(123)I]epidepride with bolus/infusion.

The iodinated benzamide epidepride, which shows a picomolar affinity binding to dopamine D(2) receptors, has been designed for in vivo studies using SPECT. The aim of the present study was to apply a steady-state condition by the bolus/infusion approach with [(123)I]epidepride for the quantification of striatal and extrastriatal dopamine D(2) receptors in humans. In this way the distribution volume of the tracer can be determined from a single SPECT image and one blood sample. Based on bolus experiments, an algorithm using conventional convolution arguments for prediction of the outcome of a bolus/infusion (B/I) experiment was applied. It was predicted that a B/I protocol with infusion of one-third of the initial bolus per hour would be appropriate. Steady-state conditions were attained in extrastriatal regions within 3-4 h but the infusion continued up to 7 h in order to minimize the significance of individual differences in plasma clearance and binding parameters. A steady-state condition, however, could not be attained in striatal brain regions using a B/I protocol of 20 h, even after 11 h. Under near steady-state conditions a striatal:cerebellar ratio of 23 was demonstrated. Epidepride has a unique signal-to-noise ratio compared to [(123)I]IBZM but present difficulties for steady-state measurements of striatal regions. The bolus/infusion approach is particularly feasible for quantification of the binding potential in extrastriatal regions.

Brain uptake and plasma metabolism of [11C]vinpocetine: a preliminary PET study in a cynomolgus monkey.

Vinpocetine, a vinca alkaloid, is a widely used therapeutic agent in patients with acute and chronic stroke. To reveal the mechanisms of vinpocetine action in the brain, vinpocetine was labeled with 11C. Positron emission tomography (PET) was used to determine the uptake and distribution of [11C]vinpocetine in brain regions and the trunk of a cynomolgous monkey in two independent measurements. The concentration of vinpocetine and its labeled metabolites was determined in blood and plasma using high-performance liquid chromatography (HPLC). Almost identical measurements were obtained in the two independent studies. After intravenous administration, following an initial peak, the total concentration of radioactivity in blood was relatively stable with time, whereas the concentration of the unchanged compound decreased with time in an exponential manner. The uptake of [11C]vinpocetine in brain was rapid, and 5% of the radioactivity totally injected was present in the brain 2 minutes after drug administration, indicating that the compound entered the brain readily. The radioactivity uptake was heterogeneously distributed among brain regions and was highest in the thalamus, the basal ganglia, and certain neocortical regions. The high brain uptake and the heterogeneous regional distribution indicate that direct central nervous system (CNS) effects of vinpocetine must be considered as explanation for the therapeutic effects. The detailed exploration of this suggestion requires further studies.

Iodine-123 labelled Z-(R,R)-IQNP: a potential radioligand for visualization of M(1 )and M(2) muscarinic acetylcholine receptors in Alzheimer's disease.

Z-(R)-1-Azabicyclo[2.2.2]oct-3-yl (R)-alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate (Z-IQNP) has high affinity to the M(1 )and M(2) muscarinic acetylcholine receptor (mAChR) subtypes according to previous in vitro and in vivo studies in rats. In the present study iodine-123 labelled Z-IQNP was prepared for in vivo single-photon emission tomography (SPET) studies in cynomolgus monkeys. SPET studies with Z-[(123)I]IQNP demonstrated high accumulation in monkey brain (>5% of injected dose at 70 min p.i.) and marked accumulation in brain regions such as the thalamus, the neocortex, the striatum and the cerebellum. Pretreatment with the non-selective mAChR antagonist scopolamine (0.2 mg/kg) inhibited Z-[(123)I]IQNP binding in all these regions. The percentage of unchanged Z-[(123)I]IQNP measured in plasma was less than 10% at 10 min after injection, which may be due to rapid hydrolysis, as has been demonstrated previously with the E-isomer of IQNP. Z-[(123)I]IQNP showed higher uptake in M(2)-rich regions, compared with previously obtained results with E-[(123)I]IQNP. In conclusion, the radioactivity distribution from Z-[(123)I]IQNP in monkey brain indicates that Z-[(123)I]IQNP binds to the M(1)- and M(2)-rich areas and provides a high signal for specific binding, and is thus a potential ligand for mAChR imaging with SPET.

Quantification of [11C]FLB 457 binding to extrastriatal dopamine receptors in the human brain.

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.

PET examination of three potent cocaine derivatives as specific radioligands for the serotonin transporter.

Several positron emission tomography (PET) radioligands based on the aryl tropane structure have been used for studies on monoamine reuptake sites. RTI-364, RTI-330, and RTI-357 (3-beta-(4'-n-propyl-,4'-iso-propyl-, and 4'-iso-propenyl-phenyl)nortropane-2-beta-carboxylic acid methyl ester) are three recently synthesized cocaine analogues with higher affinity for the serotonin (5-HTT) than the dopamine transporter (DAT). Unlabelled RTI-364 and RTI-330 were prepared in a two-step synthesis. The key step was the addition of the appropriate propyl Grignard reagent to anhydroecgonine methyl ester. RTI-357 was prepared in a three-step synthesis with a palladium-catalyzed coupling reaction of beta-CIT and isopropenylzinc bromide as key step. Hydrolysis of the ester functions gave the carboxylic acid analogues of RTI-364, RTI-330, and RTI-357, which were labelled with 11C using [11C]methyl iodide in dimethyl formamide (DMF) and tetrabutylammonium hydroxide (TBAH) as base. All three compounds entered the monkey brain in a high degree (approximately 5-10%). There was a low uptake of [11C]RTI-364 in serotonin-rich brain areas, whereas [11C]RTI-330 and [11C]RTI-357 showed a marked uptake of radioactivity in the thalamus and the brainstem, regions known to contain serotonin transporters. Transient equilibrium was reached at 15 and 40 min for [11C]RTI-330 and [11C]RTI-357, respectively. After pretreatment with citalopram, the ratio of radioactivity in the thalamus and the brainstem to the cerebellum were markedly reduced for [11C]RTI-357 but not for [11C]RTI-330. The results indicate that [11C]RTI-357 is a potential PET radioligand for quantitation of the serotonin reuptake site.

Carbon-11 epidepride: a suitable radioligand for PET investigation of striatal and extrastriatal dopamine D2 receptors.

Epidepride [(S)-(-)-N-([1-ethyl-2-pyrrolidinyl]methyl)-5-iodo-2,3-dimethoxybenza mide] binds with a picomolar affinity (Ki = 24 pM) to the dopamine D2 receptor. Iodine-123-labeled epidepride has been used previously to study striatal and extrastriatal dopamine D2 receptors with single photon emission computed tomography (SPECT). Our aim was to label epidepride with carbon-11 for comparative quantitative studies between positron emission tomography (PET) and SPECT. Epidepride was synthesized from its bromo-analogue FLB 457 via the corresponding trimethyl-tin derivative. In an alternative synthetic pathway, the corresponding substituted benzoic acid was reacted with the optically pure aminomethylpyrrolidine-derivative. Demethylation of epidepride gave the desmethyl-derivative, which was reacted with [11C]methyl triflate. Total radiochemical yield was 40-50% within a total synthesis time of 30 min. The specific radioactivity at the end of synthesis was 37-111 GBq/micromol (1,000-3,000 Ci/mmol). Human postmortem whole-hemisphere autoradiography demonstrated dense binding in the caudate putamen, and also in extrastriatal areas such as the thalamus and the neocortex. The binding was inhibited by unlabeled raclopride. PET studies in a cynomolgus monkey demonstrated high uptake in the striatum and in several extrastriatal regions. At 90 min after injection, uptake in the striatum, thalamus and neocortex was about 11, 4, and 2 times higher than in the cerebellum, respectively. Pretreatment experiment with unlabeled raclopride (1 mg/kg) inhibited 50-70% of [11C]epidepride binding. The fraction of unchanged [11C]epidepride in monkey plasma determined by a gradient high performance liquid chromatography (HPLC) method was about 30% of the total radioactivity at 30 min after injection of [11C]epidepride. The availability of [11C]epidepride allows the PET-verification of the data obtained from quantitation studies with SPECT.

[Cerebral uptake and metabolism of (11C) Vinpocetine in monkeys: PET studies]. / [11C] Vinpocetin agyi felvétele és metabolizmusa majomban: PET vizsgálatok.

Vinpocetine, a vinca alkaloid, is a therapeutic agent widely used in the treatment of acute and chronic stroke patients. To explore the uptake and distribution of vinpocetine in the primate brain, vinpocetine was labelled with 11C and positron emission tomography (PET) was used to measure the uptake and distribution of 11C-vinpocetine in the brain and the trunk of a cynomolgous monkey. HPLC was used to determine the concentration of vinpocetine and its labelled metabolites in blood and plasma. Following the radioligand's intravenous administration, after an initial peak, the total concentration of radioactivity in blood was relatively stable with time. The uptake of 11C-vinpocetine into the brain was rapid and about 5% of the total injected radioactivity was present in the brain two minutes after drug administration. These facts indicate that the compound passes the blood-brain barrier readily and enters the brain. The radioactivity uptake was heterogeneously distributed among brain regions. The highest concentrations were found in the thalamus, the basal ganglia and certain neocortical regions. In an earlier PET investigation on chronic stroke patients the highest increases in cerebral blood flow and glucose metabolism after intravenous vinpocetine treatment occurred in these anatomical structures. The heterogenous regional distribution of vinpocetine and the observation that the highest uptake values in brain structures go parallel with the greatest regional blood flow and glucose metabolic rate increases indicate that direct CNS effects of vinpocetine should be considered as an explanation for the therapeutic effects. The confirmation of this suggestion requires further investigations.

Different brain radioactivity curves in a PET study with [11C]beta-CIT labelled in two different positions.

The cocaine congener 2beta-carbomethoxy-3beta-(4'-iodophenyl)tropane (beta-CIT) has a chemical structure that enables labelling with carbon-11 either by N-methylation or by O-methylation. The regional brain uptake of [N-methyl-11C]beta-CIT and [O-methyl-11C]beta-CIT was compared in cynomolgus monkeys using positron emission tomography (PET). The striatal uptake of radioactivity after intravenous injection of [O-methyl-11C]beta-CIT reached a plateau at 30-40 min, whereas the uptake of [N-methyl-11C]beta-CIT increased continuously during the time of the PET measurement. Two of the putative labelled metabolites, [N-methyl-11C]beta-CIT-acid and [O-methyl-11C]nor-beta-CIT were prepared and examined with PET to investigate if they may enter the brain and thus add to the radioactivity uptake obtained with [11C]beta-CIT. Less than 0.4% of injected [N-methyl-11C]beta-CIT-acid entered the brain whereas 5-6% of [O-methyl-11C]nor-beta-CIT entered the brain and accumulated in the striatum and in the thalamus. The fraction of [O-methyl-11C]nor-beta-CIT obtained in plasma after intravenous injection of [O-methyl-11C]nor-beta-CIT, however, never exceeded 3%. Consequently, the formation of [N-methyl-11C]beta-CIT-acid and [O-methyl-11C]nor-beta-CIT cannot be the explanation for the different time-activity curves in the monkey brain demonstrated with [11C]beta-CIT labelled in two different positions. An unidentified labelled lipophilic metabolite, detected in monkey plasma after injection of [O-methyl-11C]beta-CIT, remains as the only possible explanation for the differences between [N-methyl-11C]beta-CIT and [O-methyl-11C]beta-CIT.

Changes in striatal D2-receptor density following chronic treatment with amphetamine as assessed with PET in nonhuman primates.

Recent brain imaging studies suggest that schizophrenia may be related to abnormally high amphetamine-induced dopamine release. It is known that repeated use of amphetamine may cause paranoid psychosis and persisting stereotypies. The biochemical background for these signs and symptoms has not been clarified. In this study, positron emission tomography and [11C]raclopride were used to determine central D2-dopamine receptor density (Bmax) and apparent affinity (K(D)app) in Cynomolgus monkeys before and after 14 days of treatment with d-amphetamine sulphate (2 mg/kg/day; s.c.). One day after withdrawal from amphetamine, K(D)app was increased, suggesting [11C]raclopride competition with elevated concentration of dopamine. At 7 and 14 days after withdrawal, there was a 19-26% decrease in Bmax but no change in K(D)app as compared to baseline. Although this study was performed on two monkeys only, there was thus no support for the view that chronic intermittent hyperactivity of the dopamine system may be related to an upregulation of striatal D2-dopamine receptors. Repeated administration of amphetamine may, rather, cause a long-lasting downregulation of the D2-receptor density, which may be a neurochemical correlate to the abnormal movements, anhedonia, anxiety, and depression seen in psychostimulant abusers.

Carbon-11-NNC 112: a radioligand for PET examination of striatal and neocortical D1-dopamine receptors.

UNLABELLED: The aim of this work was to explore the potential of a selective D1-dopamine receptor antagonist as a new radioligand for PET examination of striatal and neocortical D1-dopamine receptors. METHODS: The active (+)- and inactive (-)-enantiomers of [11C]NNC 112 were radiolabeled using the N-methylation approach and were examined by PET in cynomolgus monkeys and healthy men. Metabolite levels in plasma were measured by gradient high-performance liquid chromatography (HPLC). RESULTS: N-methylation of the corresponding desmethyl precursors with [11C]methyl triflate gave high total radiochemical yield (50%-60%) and specific radioactivity (110 GBq/micromol). (+)-[11C]NNC 112 binding in cynomolgus monkeys was 5.77+/-0.31 and 2.36+/-0.14 times higher in the striatum and neocortex, respectively, than in the cerebellum at a transient equilibrium that appeared 40-50 min after injection. The binding of (+)-[11C]NNC 112 is stereoselective, because the brain distribution of the inactive (-)-enantiomer was on an equally low level for all brain regions. Displacement and pretreatment experiments using unlabeled SCH 23390 and ketanserin confirms that (+)-[11C]NNC 112 binds specifically and reversibly to D1-dopamine receptors. The radioactivity ratios of the striatum, frontal cortex and nucleus accumbens to the cerebellum were 3.8-4.0, 1.7-2.0 and 2.8-3.1, respectively, at a transient equilibrium that appeared 40-50 min after injection in four healthy human subjects. Linear graphical analysis gave distribution volume ratios of 3.9 and 1.5 in the putamen and frontal cortex, respectively. The fraction of the total radioactivity in human plasma representing unchanged (+)-[11C]NNC 112 was 85% at 5 min and 25% at 75 min after injection. CONCLUSION: (+)-[11C]NNC 112 should be a useful PET radioligand for quantitative examination of not only striatal but neocortical D1-dopamine receptors in man.

Quantitative analyses of carbonyl-carbon-11-WAY-100635 binding to central 5-hydroxytryptamine-1A receptors in man.

UNLABELLED: The serotonin 5-hydroxytryptamine-1A (5-HT1A) receptor subtype is of central interest in research on the pathophysiology and treatment of psychiatric disorders. Carbonyl-11 C-WAY-100635 is a new radioligand that, in PET experiments, provides high-contrast delineation of brain regions that are rich in 5-HT1A receptors. The aim of this PET study was to examine the prospects for quantitation of carbonyl-11C-WAY-100635 binding to 5-HT1A receptors in the human brain. METHODS: A PET examination was performed in each of six healthy male subjects after intravenous injection of carbonyl-11C-WAY-100635. Radioactive metabolites in plasma were determined with high-performance liquid chromatography. The metabolite-corrected arterial input function was used in a kinetic three-compartment analysis, and the cerebellum was used as reference region in linear graphical and transient equilibrium analyses. RESULTS: The highest radioactivity concentration was observed in the neocortex and the raphe nuclei, whereas radioactivity was low in the cerebellum. The time-activity curves were well-described by a three-compartment model for all regions. Uptake in the cerebellum could not be described by a two-compartment model. The transient equilibrium and linear graphical analyses, which are both dependent on the cerebellum as the reference region, gave lower binding potential values than did the kinetic analysis. The metabolism was rapid, and the fraction of unchanged carbonyl-11C-WAY-100635 was <10% 10 min after injection in all human subjects. The major radioactive metabolites were unidentified polar components. One metabolite comigrated with reference cyclohexanecarboxylic acid, and another comigrated with reference desmethyl-WAY-100635. CONCLUSION: The suitability of carbonyl-11C-WAY-100635 for research on central 5-HT1A receptors in neuropsychiatric disorders was supported by the observation that the high signals in the neocortex and raphe nuclei can be described using a kinetic analysis with a metabolite-corrected arterial input function. It cannot be excluded that kinetically distinguishable nonspecific binding or the formation of a metabolite that passes the blood-brain barrier may represent measurable components of the low radioactivity in the cerebellum. Simplified quantitative methods, using the relatively low radioactivity in the cerebellum as reference, should accordingly be applied with some caution until the biochemical nature of the radioactivity is better understood and the reliability of these approaches has been confirmed in larger samples.

[11C]NNC 22-0215, a metabolically stable dopamine D1 radioligand for PET.

NNC 22-0215 has been found to be a metabolically stable dopamine D1 antagonist with high affinity and selectivity for D1 receptors in vitro. We prepared [11C]NNC 22-0215 with a specific radioactivity of about 50 GBq/micromol at time of administration. In PET experiments with [11C]NNC 22-0215 there was a rapid uptake of radioactivity in the cynomolgus monkey brain (1.8% of total radioactivity injected). Radioactivity accumulated most markedly in the striatum and the neocortex. The striatum to cerebellum ratio was about 4, with specific binding that remained at a plateau level from 50 min to 100 min after injection. Binding in the striatum and neocortex was markedly displaced by SCH 23390, whereas binding in the cerebellum was not reduced. Metabolite studies showed that about 80% of the radioactivity in the monkey plasma represented unchanged radioligand 30 min after injection. The rate of metabolism in monkey plasma in vivo was also determined for a series of structurally related 11C-labelled benzazepines, previously used as dopamine D1 receptor ligands for PET. Results indicate a significantly slower rate of metabolism for [11C]NNC 22-0215 than for any of the previously labelled benzazepines. Thus [11C]NNC 22-0215 has potential for imaging of selective binding to the dopamine D1 receptors in the human brain with high count rates at time of equilibrium.

Evaluation and metabolite studies of 125I- and 123I-labelled E-(R,R)-IQNP: potential radioligands for visualization of M1 muscarinic acetylcholine receptors in brain.

A new ligand for the M1 muscarinic receptor subtype, E-(R,R)-1-azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate (E-IQNP), was labelled with 125I and 123I for autoradiographic studies on human whole-brain cryosections and SPET studies, respectively, in Cynomolgus monkey. Autoradiography demonstrated E-[125I]IQNP binding in M1 receptor-rich regions such as the neocortex and the striatum. The binding was displaceable by the selective M1 antagonist biperiden. In vivo single photon emission tomography (SPET) studies with E-[123I]IQNP demonstrated a high accumulation of radioactivity in the monkey neocortex. Rapid hydrolysis of the quinuclidinyl ester to the free acid was found to be a major biotransformation route for E-[123I]IQNP. The free acid of E-[123I]IQNP does not pass the blood-brain barrier, but the plasma concentration was high as compared to the total radioactivity in brain. It is thus necessary to correct for the high concentration of radioactive metabolites in parenchymal blood (CBV) to obtain accurate values for E-[123I]IQNP binding in brain.

Characterisation of the appearance of radioactive metabolites in monkey and human plasma from the 5-HT1A receptor radioligand, [carbonyl-11C]WAY-100635--explanation of high signal contrast in PET and an aid to biomathematical modelling.

N-(2-(4-(2-Methoxy-phenyl)-1-piperazin-1-yl)ethyl)-N-(2-pyridyl)++ +cyclohexanecarboxamide (WAY-100635), labelled in its amido carbonyl group with 11C (t1/2 = 20.4 min), is a promising radioligand for the study of brain 5-HT1A receptors with positron emission tomography (PET). Thus, in PET experiments in six cynomolgus monkeys and seven healthy male volunteers, [carbonyl-11C]WAY-100635 was taken up avidly by brain. Radioactivity was retained in regions rich in 5-HT1A receptors, such as occipital cortex, temporal cortex and raphe nuclei, but cleared rapidly from cerebellum, a region almost devoid of 5-HT1A receptors. [Carbonyl-11C]WAY-100635 provides about 3- and 10-fold higher signal contrast (receptor-specific to nonspecific binding) than [O-methyl-11C]WAY-100635 in receptor-rich areas of monkey and human brain, respectively. To elucidate the effect of label position on radioligand behaviour and to aid in the future biomathematical interpretation of the kinetics of regional cerebral radioactivity uptake in terms of receptor-binding parameters, HPLC was used to measure [carbonyl-11C]WAY-100635 and its radioactive metabolites in plasma at various times after intravenous injection. Radioactivity cleared rapidly from monkey and human plasma. Parent radioligand represented 19% of the radioactivity in monkey plasma at 47 min and 8% of the radioactivity in human plasma at 40 min. [Carbonyl-11C]desmethyl-WAY-100635 was below detectable limits in monkey plasma and at most a very minor radioactive metabolite in human plasma. [11C]Cyclohexanecarboxylic acid was identified as a significant radioactive metabolite. In human plasma this maximally represented 21% of the radioactivity at 10 min after radioligand injection. All other major radioactive metabolites in monkey and human plasma were even more polar. No-carrier-added [carbonyl-11C]cyclohexanecarboxylic acid was prepared in the laboratory and after intravenous administration into cynomolgus monkey was shown with PET to give only a low uptake of radioactivity into brain tissue. The acid rapidly gave rise to several radioactive metabolites of higher polarity in plasma. The observed lack of any significant metabolism of [carbonyl-11C]WAY-100635 to highly lipophilic or pharmacologically potent radioactive compounds is consistent with its high signal contrast in primate brain.

[carbonyl-11C]Desmethyl-WAY-100635 (DWAY) is a potent and selective radioligand for central 5-HT1A receptors in vitro and in vivo.

[carbonyl-11C]Desmethyl-WAY-100635 (DWAY) is possibly a low-level metabolite appearing in plasma after intravenous administration of [carbonyl-11C]WAY-100635 to human subjects for positron emission tomographic (PET) imaging of brain 5-HT1A receptors. In this study we set out to assess the ability of DWAY to enter brain in vivo and to elucidate its possible interaction with 5-HT1A receptors. Desmethyl-WAY-100635 was labelled efficiently with carbon-11 (t1/2 = 20.4 min) in high specific radioactivity by reaction of its descyclohexanecarbonyl analogue with [carbonyl-11C]cyclohexanecarbonyl chloride. The product was separated in high radiochemical purity by high-performance liquid chromatography (HPLC) and formulated for intravenous injection. Rats were injected intravenously with DWAY, sacrificed at known times and dissected to establish radioactivity content in brain tissues. At 60 min after injection, the ratios of radioactivity concentration in each brain region to that in cerebellum correlated with previous in vitro and in vivo measures of 5-HT1A receptor density. The highest ratio was about 22 in hippocampus. Radioactivity cleared rapidly from plasma; HPLC analysis revealed that DWAY represented 55% of the radioactivity in plasma at 5 min and 33% at 30 min. Only polar radioactive metabolites were detected. Subsequently, a cynomolgus monkey was injected intravenously with DWAY and examined by PET. Maximal whole brain uptake of radioactivity was 5.7% of the administered dose at 5 min after injection. The image acquired between 9 and 90 min showed high radioactivity uptake in brain regions rich in 5-HT1A receptors (e.g. frontal cortex and neocortex), moderate uptake in raphe nuclei and low uptake in cerebellum. A transient equilibrium was achieved in cortical regions at about 60 min, when the ratio of radioactivity concentration in frontal cortex to that in cerebellum reached 6. The corresponding ratio for raphe nuclei was about 3. Radioactive metabolites appeared rapidly in plasma, but these were all more polar than DWAY, which represented 52% of the radioactivity in plasma at 4 min and 20% at 55 min. In a second PET experiment, in which a cynomolgus monkey was pretreated with the selective 5-HT1A receptor antagonist, WAY-100635, at 25 min before DWAY injection, radioactivity in all brain regions was reduced to that in cerebellum. Autoradiography of post mortem human brain cryosections after incubation with DWAY successfully delineated 5-HT1A receptor distribution. Receptor-specific binding was eliminated in the presence of the selective 5-HT1A receptor agonist, 8-OH-DPAT [(+/-)-8-hydroxy-2-dipropylaminotetralin]. These findings show that: (a) intravenously administered DWAY is well able to penetrate brain in rat and monkey, (b) DWAY is a highly effective radioligand for brain 5-HT1A receptors in rat and monkey in vivo and for human brain in vitro, and (c) the metabolism and kinetics of DWAY appear favourable to successful biomathematical modelling of acquired PET data. Thus, DWAY warrants further evaluation as a radioligand for PET studies of 5-HT1A receptors in human brain.

[18F] beta-CIT-FP is superior to [11C] beta-CIT-FP for quantitation of the dopamine transporter.

beta-CIT-FP [N-(3-fluoropropyl)-2 beta-carbomethoxy-3 beta-(4-iodophenyl)nortropane] is a cocaine analogue with high affinity for the dopamine transporter. Positron emission tomography (PET) studies with [O-methyl-11C] beta-CIT-FP ([11C] beta-CIT-FP) has shown that equilibrium conditions were approached but, however, not reached at the end of measurement. Moreover, metabolite studies of [11C] beta-CIT-FP in monkey plasma demonstrated a lipophilic-labelled metabolite that may enter the brain. We therefore labelled beta-CIT-FP with fluorine-18 in a position that may avoid the formation of labelled lipophilic metabolites. The more long-lived radionuclide (18F) was used to allow for measurements over longer time. [N-fluoropropyl- 18F] beta-CIT-FP ([18F] beta-CIT-FP) was prepared by N-alkylation of nor-beta-CIT with [18F]fluoropropyl bromide. PET studies were performed in cynomolgus monkeys. [18F] beta-CIT-FP entered the brain rapidly. There was a high concentration of radioactivity in the striatum and much lower in the thalamus, neocortex, and cerebellum. The striatum-to-cerebellum ratio was about 5 at time of transient equilibrium, which occurred after 60 to 100 min. After pretreatment with GBR 12909, radioactivity in the striatum was markedly reduced, thus indicating specific [18F] beta-CIT-FP binding to the dopamine transporter. The fraction of unchanged [18F] beta-CIT-FP determined by HPLC was 10-15% after 140 min. No lipophilic labelled metabolites were detected. The absence of measurable lipophilic labelled metabolites and the occurrence of transient equilibrium within the time of the PET measurement indicate that [18F] beta-CIT-FP is superior to [11C] beta-CIT-FP as a PET radioligand for quantification of the dopamine transporter in the human brain.