Camilla Lundkvist

Autoradiographic localization of 5-HT1A receptors in the post-mortem human brain using [3H]WAY-100635 and [11C]WAY-100635

The distribution of 5-HT1A receptors was examined in the post-mortem human brain using whole hemisphere autoradiography and the selective 5-HT1A receptor antagonist [3H]WAY-100635 ([O-methyl-3H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2- pyridinyl)cyclohexanecarboxamide trihydrochloride). The autoradiograms showed very dense binding to hippocampus, raphe nuclei and neocortex. The labeling in neocortex was slightly lower than in the hippocampus and was mainly at superficial layers, although a faintly labeled band could be seen in deeper neocortical layers. Other regions, such as the amygdala, septum and claustrum, showed low densities caudatus and putamen, in cerebellum or in structures of the brain stem except in the raphe nuclei. The labeling of human 5-HT1A receptors with [3H]WAY-100635 was antagonised by the addition of 5-HT1A receptor ligands, 5-HT, buspirone, pindolol or 8-OH-DPAT (10 microM), leaving a very low background of non-specific binding. Saturation analysis of semiquantitative data from several human regions indicated that [3H]WAY-100635 has a Kd of approximately 2.5 nM. The selective labeling of 5-HT1A receptors with [3H]WAY-100635 clearly show that this compound is useful for further studies of the human 5-HT1a receptor subtype in vitro [11C]WAY-100635 is used for the characterization of 5-HT1A receptors with positron emission tomography (PET). WAY-100635 was also radiolabeled with the short-lived positron-emitting radionuclide carbon-11 (t1/2 = 20 min) and used for in vitro autoradiography on human whole hemisphere cryosections. [11C]WAY-100635 gave images qualitatively similar to those of [3H]WAY-100635, although with a lower resolution. Thus, the hippocampal formation was densely labeled, with lower density in the neocortex. Buspirone, pindolol or 8-OH-DPAT (10 microM), blocked all binding of [11C]WAY-100635. The in vitro autoradiography of the distribution of 5-HT1A receptors obtained with radiolabeled WAY-100635 provide detailed qualitative and quantitative information on the distribution of 5-HT1A-receptors in the human brain. Moreover, the studies give reference information for the interpretation of previous initial results at much lower resolution in humans with PET and [11C]Way-100635. These data provide a strong basis for expecting [11C]WAY-100635 to behave as a highly selective radioligand in vivo.

Precursor synthesis and radiolabelling of the dopamine D2 receptor ligand [11C]raclopride from [11C]methyl triflate

Desmethyl-raclopride was synthesized via a straightforward, three-step synthetic approach and used for the preparation of [11C]raclopride from [11C]methyl triflate. Conditions for the radiolabelling were optimized to obtain a simple and reproducible procedure suitable for automation. [11C]Raclopride was prepared with an average radiochemical yield of 55–65% (decay corrected, based on starting [11C]methyl triflate) in a total synthesis time (including purification and formulation of product) of 35 min. The radiolabelling procedure used significantly less precursor, avoided the use of DMSO, and was shorter compared to the standard radiolabelling procedure with [11C]methyl iodide. Copyright

Autoradiographic localization of 5-HT2A receptors in the human brain using [3H]M100907 and [11C]M100907

M100907 (MDL 100907, R‐(+)‐α‐(2,3‐dimethoxyphenyl)‐1‐[2‐(4‐fluorophenyl)ethyl]‐4‐piperidinemethanol) is a new selective antagonist of 5‐HT2A receptors. The compound has been labeled with 11C and proved useful for in vivo studies of 5‐HT2A receptors using positron emission tomography (PET). In the present study the distribution of 5‐HT2A receptors was examined in the postmortem human brain using whole hemisphere autoradiography and [3H]M100907 and [11C]M100907. The autoradiograms showed very dense binding to all neocortical regions, whereas the hippocampus was only weakly labeled with [3H]M100907. Other central brain regions, such as the basal ganglia and thalamus, showed low [3H]M100907 binding, reflecting low densities of 5‐HT2A receptors. The cerebellum or structures of the brain stem were virtually devoid of 5‐HT2A receptors. [11C]M100907 gave images qualitatively similar to those of [3H]M100907, although with lower spatial resolution. The labeling of human 5‐HT2A receptors with [3H]M100907 was inhibited by the addition of the 5‐HT2A receptor blockers ketanserin or SCH 23390 (10 μM), leaving a very low background of nonspecific binding. The 5‐HT1A receptor antagonist WAY‐100635 and the D2‐dopamine receptor antagonist raclopride had no effect on the binding of [3H]M100907. The selective labeling of 5‐HT2A receptors with [3H]M100907 clearly shows that this compound is suitable for further studies of the human 5‐HT2A receptor subtype in vitro. The in vitro autoradiography of the distribution of 5‐HT2A receptors obtained with radiolabeled M100907 provides detailed qualitative and quantitative information on the distribution of 5‐HT2A‐receptors in the human brain as well as reference information for the interpretation of previous initial results at much lower resolution in humans in vivo with PET and [11C]M100907. Synapse 38:421–431, 2000.

[11C]MDL 100907, a radioligand for selective imaging of 5-HT2A receptors with positron emission tomography

The highly selective 5-HT2A receptor antagonist, MDL 100907 ((R)-(+)-4 -(l-hydroxy-1-(2,3-dimethoxyphenyl)methyl)-N -2-(4-fluorophenylethyl)piperidine), was labeled with 11C for Positron Emission Tomography (PET) studies. After i.v. injection of (R)-(+)-[3-OCH3-11C]MDL 100907 [11C]MDL 100907) in Cynomolgus monkeys a marked accumulation in the 5-HT2A receptor rich neocortical regions was obtained with a neocortex to cerebellum ratio of 3.5-4.5 after 60-80 minutes. In the neocortical regions a transient equilibrium occurred within 40-60 minutes. Radioactivity in the neocortex, but not in the cerebellum, was reduced after injection of ketanserin, indicating that neocortical radioactivity following injection of [11C]MDL 100907 represents specific binding to 5-HT2A receptors. There was no evident effect on neocortical binding after pretreatment with raclopride or SCH 23390. [11C]MDL 100907 has potential to become the first selective radioligand for PET-quantitation of 5-HT2A receptors in the human brain in vivo.

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.

[11C]?-CIT-FE, a radioligand for quantitation of the dopamine transporter in the living brain using positron emission tomography

The cocaine analogue β‐CIT‐FE (N‐(2‐fluoroethyl)‐2β‐carbomethoxy‐3β‐(4‐iodophenyl)nortropane) was labeled with 11C for positron emission tomography (PET) studies of the dopamine transporter. After intravenous administration to a cynomolgus monkey, [11C]β‐CIT‐FE accumulated in the striatum with a striatum‐to‐cerebellum ratio of about 9 after 60 min. Pseudoequilibrium of specific [11C]β‐CIT‐FE binding in the striatum was obtained within 30–50 min. The radioactivity ratios of the thalamus to the cerebellum and the neocortex to the cerebellum were about 2 and 1.5, respectively. In displacement and pretreatment experiments, radioactivity in the striatum but not in the cerebellum was reduced after injection of β‐CIT or the dopamine transporter inhibitor GBR 12909, indicating that striatal radioactivity following injection of [11C]β‐CIT‐FE represents reversible binding to dopamine transporter sites. After displacement or pretreatment with cocaine there was a marked effect not only in the striatum but also in the thalamus and neocortex. [11C]β‐CIT‐FE has potential as a useful PET radioligand for quantitation of the dopamine transporter in the primate brain in vivo.

Characterization of the radioactive metabolites of the 5-HT1A receptor radioligand, [O-methl-11C]WAY-100635, in monkey and human plasma by HPLC: Comparison of the behaviour of an identified radioactive metabolite with parent radioligand in monkey using PET

N-(2-(4-(2-Methoxy-phenyl)-1-piperazin-1-yl)ethyl)-N-(2-pyridyl) cyclohexanecarboxamide (WAY-100635), labelled in the O-methyl group with carbon-11 (t1/2 = 20.4 min), is a promising radioligand for application with positron emission tomography (PET) to the study of 5-HT1A receptors in living human brain. An understanding of the metabolism of this new radioligand is crucial to the development of a biomathematical model for the interpretation of the kinetics of radioactivity uptake in brain in terms of receptor-binding parameters. After intravenous injection of [O-methyl-11C]WAY-100635 into humans, radioactivity was found to clear rapidly from blood and plasma. By using established methods for the analysis of radioactivity in plasma, it was found that intravenously injected [O-methyl-11C]WAY-100635 is rapidly metabolised to more polar radioactive compounds in a cynomolgus monkey and in humans. Thus, at 60 min postinjection, parent radioligand represented 40% and 5% of the radioactivity in monkey and human plasma, respectively. In monkey and human, one of the radioactive metabolites was identified as the descyclohexanecarbonyl analogue of the parent radioligand, namely [O-methyl-11C]WAY-100634. This compound is known to have high affinity for 5-HT1A receptors and alpha 1-adrenoceptors. In a PET experiment it was demonstrated that, after IV injection of [O-methyl-11C]WAY-100634 into a cynomolgus monkey, radioactivity was avidly taken up by brain. Uptake of radioactivity was higher in 5-HT1A receptor-rich frontal cortex than in cerebellum, which is devoid of 5-HT1A receptors. Polar radioactive metabolites appeared in plasma. The results suggest that the use of WAY-100635 labelled with carbon-11 in its cyclohexanecarbonyl moiety may provide enhanced signal contrast in PET studies and a possibility to develop a simple biomathematical model for regional brain radioactivity uptake.

PET-characterization of [carbonyl-11C]WAY-100635 binding to 5-HT1A, receptors in the primate brain

Abstract [carbonyl-11C]WAY-100635 is a new radioligand which can be used with positron emission tomography (PET) to provide high contrast delineation of human brain regions that are rich in 5-HT1A receptors. In the present PET study, the binding of [carbonyl-11C]WAY-100635 was characterized in the cynomolgus monkey brain. Pretreatment with each of the two reference compounds, WAY-100635 and 8-OH-DPAT, as well as the drugs buspirone and pindolol, induced a marked inhibition of [carbonyl-11C]WAY-100635 binding in the neocortex and the raphe nuclei. A preliminary Scatchard analysis yielded 5-HT1A receptor density values of the same order as those that have been reported in vitro. The study shows that [carbonyl-11C]WAY-100635 binds specifically to 5-HT1A receptors in the primate brain and has potential for determination of 5-HT1A receptor occupancy and density in psychiatric patients.

[O-methyl-11C]β-CIT-FP, a potential radioligand for quantitation of the dopamine transporter: Preparation, autoradiography, metabolite studies, and positron emission tomography examinations

beta-CIT-FP [N-(3-fluoropropyl)-2 beta-carbomethoxy-3 beta-(4-iodophenyl)nortropane] is a cocaine analogue with a high affinity for the dopamine transporter. [O-methyl-11C]beta-CIT-FP ([11C]beta-CIT-FP) was prepared by O-alkylation of the free acid with [11C]methyl iodide. The total radiochemical yield of [11C]beta-CIT-FP was 50 to 60% with an overall synthesis time of 30 min. The radiochemical purity was > 99%, and the specific radioactivity at time of injection was about 37 GBq/mumol (1000 Ci/mmol). Autoradiographic examination of [11C]beta-CIT-FP binding in human brain postmortem demonstrated specific binding in the caudate nucleus and putamen. Positron emission tomography (PET) examination of [11C]beta-CIT-FP in a Cynomolgus monkey demonstrated accumulation in the striatum with a striatum-to-cerebellum ratio of about 8 after 60 min. Equilibrium in the striatum was attained within 70 to 90 min. The radioactivity ratios of thalamus/cerebellum and neocortex/cerebellum were about 2 and 1.5, respectively. In a displacement experiment, radioactivity in the striatum but not in the cerebellum was reduced after injection of beta-CIT, indicating that striatal radioactivity following injection of [11C]beta-CIT-FP is associated with dopamine transporter sites and that the binding is reversible. The fraction of the total radioactivity in plasma representing [11C]beta-CIT-FP determined by high-performance liquid chromatography (HPLC) was 84% at 15 min and 50% at 95 min. [11C]beta-CIT-FP should be a useful PET radioligand for the quantitation of dopamine transporters in the human brain in vivo.

In vitro and in vivo characterisation of nor-β-CIT: a potential radioligand for visualisation of the serotonin transporter in the brain

Radiolabelled 2β-Cabomethoxy-3β-(4-iodophenyl)tropane (β-CIT) has been used in clinical studies for the imaging of dopamine and serotonin transporters with single-photon emission tomography (SPET). 2β-Carbomethoxy-3β-(4-iodophenyl)nortropane (nor-β-CIT) is a des-methyl analogue of β-CIT, which in vitro has tenfold higher affinity (IC50=0.36 nM) to the serotonin transporter than β-CIT (IC50=4.2 nM). Nor-β-CIT may thus be a useful radioligand for imaging of the serotonin transporter. In the present study iodine-125 and carbon-11 labelled nor-β-CIT were prepared for in vitro autoradiographic studies on post-mortem human brain cryosections and for in vivo positron emission tomography (PET) studies in Cynomolgus monkeys. Whole hemisphere autoradiography with [125I]nor-β-CIT demonstrated high binding in the striatum, the thalamus and cortical regions of the human brain. Addition of a high concentration (1 μM) of citalopram inhibited binding in the thalamus and the neocortex, but not in the striatum. In PET studies with [11C]nor-β-CIT there was rapid uptake of radioactivity in the monkey brain (6% of injected dose at 15 min) and high accumulation of radioactivity in the striatum, thalamus and neocortex. Thalamus to cerebellum and cortex to cerebellum ratios were 2.5 and 1.8 at 60 min, respectively. The ratios obtained with [11C]nor-β-CIT were 20%–40% higher than those previously obtained with [11C]β-CIT. Radioactivity in the thalamus and the neocortex but not in the striatum was displaceable with citalopram (5 mg/kg). In conclusion, nor-β-CIT binds to the serotonin transporter in the primate brain in vitro and in vivo and has potential for PET and SPET imaging of the serotonin transporter in human brain.