Synthesis and biodistribution of two potential PET radioligands for dopamine reuptake sites: no-carrier-added 4-(2-[18F]fluoroethyl) and 4-[11C]methyl BTCP-piperazine.

Radioligands that specifically target dopamine uptake sites can provide a means of determining dopamine fiber loss at intrastriatal mesencephalic grafts in Parkinsonian patients, using Positron Emission Tomography (PET). The BTCP derivative, 1-[1-(2-benzo(b)thiophenyl)cyclohexyl]-4-(2-hydroxyethyl)-piperazine, shows in vitro high affinity and selectivity for the dopamine transporter. To evaluate the potential of such a compound as a potential dopaminergic PET tracer the positron-emitting analogues, 1-[1-(2-benzo(b)thiophenyl)cyclohexyl]-4-(2-[18F]fluoroethyl)-piperazine and 1-[1-(2-benzo(b)thiophenyl)cyclohexyl]-4-[11C]methylpiperazine, were synthesized. Radiofluorination was carried out by the reaction of 1-[1-(2-benzo(b)thiophenyl)cyclohexyl]-4-(2-chloroethyl)-piperazine with cyclotron-produced n.c.a. 18F-(half life 109.9 min) obtained by the (p,n) reaction on 18O-enriched water. Labelling with carbon-11 (half life 20.4 min) was achieved by 11C methylation of 1-[1-(2-benzo(b)thiophenyl)cyclohexyl]-piperazine with [11C]methyl iodide. After intravenous administration to rats these two compounds enter the brain, but despite their high in vitro affinity they display a high non specific binding in vivo which greatly limits their use as PET radioligands.

Synthesis of [3',5'-3H2]-alpha-fluoromethyl-tyrosine as a radioactive specific label of rat brain tyrosine hydroxylase.

The [3',5']-ditritio-alpha-fluoromethyl-tyrosine 4 (specific activity 15.0 Ci/mmol) has been synthesized and used as a radioactive probe for rat neuronal tyrosine hydroxylase (TH). The route of synthesis for the preparation of 3 and 4 allowed us to not only introduce a fluorine atom into 3/4 using an inorganic source of fluorine (CsF), but also to take advantage of the high-yielding cyclization of (alpha,beta)-acetamido alcohols mediated by diethylaminosulfur trifluoride (DAST) to give the corresponding oxazolines. The distribution and metabolism of 4 have been studied in control conditions within the rat locus caeruleus (LC). Intracisternal injection of 20 microCi of 4 was followed by a rapid disappearance of 4 (t1/2 = 1.5 h) and by a specific accumulation of radioactivity into the LC anatomical limits. This was investigated each 140 microns along the caudo-rostral axis of the noradrenergic nucleus. In each anatomical interval, its distribution correlated nicely with already described caudo-rostral distribution of TH in noradrenergic cells. Thus, 4 may provide a reliable measure of TH activity in such catecholamine structures.

Synthesis of 3-[18F]fluoromethyl-BTCP and evaluation as a potential PET radioligand for the dopamine transporter in baboons.

In an attempt to visualize in vivo the dopamine transporter and evaluate its potential as an imaging tool for monitoring dopamine fiber degeneration by positron emission tomography, the 18F-positron-emitting analogue of 3-fluoromethyl-1-[1-(2-benzothienyl)-cyclohexyl]-piperidine, [18F]BTCP, was synthesized and tested in a primate model of hemiparkinsonism. [18F]BTCP was obtained from cyclotron-produced n.c.a. [18F]fluoride (110 min half-life) and by nucleophilic substitution from 3-bromomethyl-BTCP with a radiochemical yield of 6% (decay-corrected). After intravenous injection, the cerebral distribution of the radioactivity was observed mainly in cortical areas and cerebral structures enriched in catecholamine reuptake sites such as the caudate-putamen complex and the thalamus. The binding ratio, defined with respect to the cerebellum (taken as a region of non-specific binding), was highest in the thalamus (1.42), intermediate in the putamen (1.36) and lowest in the caudate nucleus (1.17), suggesting that some specific binding occurs in these regions. After saturation of dopamine and norepinephrine transporters by nomifensine, the binding ratio in the thalamus, putamen and caudate nucleus striatum remained essentially unchanged in the non-lesioned hemisphere. When comparing binding ratios between the intact and the dopamine-denervated striatum, there was a modest loss of binding in the denervated striatum, suggesting that degeneration of dopaminergic fibers could be detected using 3-[18F]fluoromethyl-BTCP. However due to a high non-specific binding in vivo, the interest of 3-[18F]fluoromethyl-BTCP to image the dopamine reuptake system in vivo appears rather limited.

Binding properties of 3-[125I]iodophencyclidine, a new radioligand for N-methyl-D-aspartate-gated ionic channels.

The binding properties of the 125I-labeled phencyclidine derivative N-[1-(3-[125I]iodophenyl)cyclohexyl]piperidine (3-[125I]iodo-PCP), a new ligand of the N-methyl-D-aspartate (NMDA)-gated ionic channel, were investigated. Association and dissociation kinetic curves of 3-[125I]iodo-PCP with rat brain homogenates were well described by two components. About 32% of the binding was of fast association and fast dissociation, and the remaining binding was of slow association and slow dissociation. Saturation curves of 3-[125I]iodo-PCP also were well described using two binding sites: one of a high affinity (KDH = 15.8 +/- 2.3 nM) and the other of a low affinity (KDL = 250 +/- 40 nM). 3-Iodo-PCP inhibited the binding of 3-[125I]iodo-PCP with inhibition curves that were well fitted by a two-site model. The binding constants (KiH, BmaxH; KiL, BmaxL) so obtained were close to those obtained in saturation experiments. Ligands of NMDA-gated ionic channels also inhibited the binding of 3-[125I]iodo-PCP with two constants, KiH and KiL. There was a very good correlation (r = 0.987) between the affinities of these ligands to bind to NMDA-gated ionic channels and their potencies to inhibit the binding of 3-[125I]iodo-PCP with a high affinity. Moreover, the regional distribution of the high-affinity binding of 3-[125I]-iodo-PCP paralleled that of tritiated N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP). In contrast to that of [3H] TCP, the binding of 3-[125I]iodo-PCP to well-washed rat brain membranes was fast and insensitive to glutamate and glycine.(ABSTRACT TRUNCATED AT 250 WORDS)

[3H]RP 62203, a ligand of choice to label in vivo brain 5-HT2 receptors.

There are only a few ligands available for labelling brain receptors simply because in vivo binding requires more severe experimental conditions than in vitro binding. We now describe the in vivo binding properties of [3H]RP 62203, a new potent and selective 5-HT2 antagonist. After intravenous injection into rats, [3H]RP 62203 accumulated predominantly in brain regions containing 5-HT2 receptors, with a frontal cortex/cerebellum ratio of 6 to 7. A good correlation was obtained between the regional distribution of [3H]RP 62203 in the brain and the density of 5-HT2 receptors measured in vitro. In vivo binding of [3H] RP 62203 was saturable in the frontal cortex but not in the cerebellum. The Bmax in the frontal cortex was equal to 42.5 fmol/mg, thus in the same range as was found in vitro. The 5-HT2 selectivity was ascertained by displacement (prevention) experiments; 5-HT2 antagonists or the agonist 2,5-dimethoxy- 4-iodophenylisopropylamine could prevent specific labelling of [3H]RP 62203 only in brain regions containing 5-HT2 receptors. Interestingly, the radioactivity remaining in various brain regions after displacement with pipamperone corresponded exactly to that measured in the cerebellum, with or without pipamperone. In conclusion, [3H]RP 62203 possesses striking properties of in vivo binding which make it a suitable candidate for examining 5-HT2 receptors in human brain by positron emission tomography scanning.

Quantitative autoradiographic mapping of 5-HT3 receptors in the rat CNS using [125I]iodo-zacopride and [3H]zacopride as radioligands.

Substitution of the chlorine atom by a radio-iodine in position 5 in the zacopride molecule yielded [125I]iodo-zacopride that bound with high affinity (Kd = 4.3 nM) to 5-HT3 receptors in the rat central nervous system. Assays with membranes from the posterior (mainly entorhinal) cortex confirmed that the pharmacological properties and regional distribution of [125I]iodo-zacopride-specific binding sites were identical with those of 5-HT3 sites labelled by the reference radioligand [3H]zacopride. Autoradiographic investigations for the visualization and quantification of 5-HT3 receptors yielded similar results with both radioligands, but autoradiograms could be obtained after only 1-3 days of exposure of sections labelled with [125I]iodo-zacopride, instead of 4-6 months using [3H]zacopride. The highest density of 5-HT3 sites was found in the nucleus tractus solitarius followed by, in decreasing order, the dorsal motor nucleus of the vagus nerve, the superficial layers of the dorsal horn in the spinal cord, the nucleus of the spinal tract of the trigeminal nerve, and the area postrema. Significant labelling of 5-HT3 receptors was also observed in limbic areas (amygdala, hippocampus, frontal and entorhinal cortex), and to a much lower extent in the dorsal raphe nucleus, striatum, and substantia nigra. These multiple locations further support the idea that 5-HT3 receptors are probably involved in several 5-HT-mediated functions in the central nervous system.

[(125I)iodo-zacopride: new ligand for the study by autoradiography of central 5-HT3 receptors]. / [125I]iodo-zacopride: un nouveau ligand pour l'étude par autoradiographie des récepteurs 5-HT3 centraux.

This paper describes the synthesis and the pharmacological characteristics of the first radioiodinated ligand of central 5-HT3 receptors: [125I]iodo-zacopride. Specific sites having a high affinity (Kd = 4.3 nM) for [125I]iodo-zacopride have been found in membranes from the rat entorhinal cortex. In addition, a highly significant correlation (r = 0.995) existed between the Ki of several 5-HT-related drugs for displacing both [125I]iodo-zacopride from its specific binding sites, and [3H]zacopride from 5-HT3 receptors. Finally, [125I]iodo-zacopride was successfully used for the autoradiographic mapping of 5-HT3 receptors in the rat central nervous system.

Chromatographic analyses of the serotonin 5-HT1A receptor solubilized from the rat hippocampus.

Serotonin 5-HT1A receptors in rat hippocampal membranes were solubilized by 10 mM 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) and chromatographed on various gels in an attempt to design a relevant protocol for their (partial) purification. In particular, an affinity gel made of the 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) derivative 8-methoxy-2-[(N-propyl, N-butylamino)amino]tetralin (8-MeO-N-PBAT) coupled to Affigel 202 was specially developed for this purpose. First, studies of the effects of various compounds (detergents, lipids, reducing agents, sugars, etc.) on the specific binding of [3H]8-OH-DPAT and on the rate of heat-induced inactivation of solubilized 5-HT1A sites led to a buffer composed of 50 mM Tris-HCl, 50 microM dithiothreitol, 1 mM CHAPS, 10% glycerol, 0.1 mM MnCl2, and 50 micrograms/ml of cholesteryl hemisuccinate, pH 7.4, ensuring a high degree of stability of solubilized 5-HT1A sites, compatible with chromatographic analyses for 2-4 days at 4 degrees C. Adsorption and subsequent elution of [3H]8-OH-DPAT specific binding sites were found with several chromatographic gels, including wheat germ agglutinin-agarose, phenyl-Sepharose, hydroxylapatite-Ultrogel, diethylaminoethyl (DEAE)-Sepharose, and DEAE-Sephacel. Similarly, 8-MeO-N-PBAT-Affigel 202 allowed the adsorption and subsequent elution (by 1 mM 5-HT) of active 5-HT1A binding sites solubilized from rat hippocampal membranes. The two-step chromatography using 8-MeO-N-PBAT-Affigel 202 followed by wheat germ agglutinin-agarose gave a fraction enriched (by at least 400-fold) in 5-HT1A sites. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this partially purified fraction revealed a major protein band with Mr close to 60,000.

125I-Bolton-Hunter-8-methoxy-2-[N-propyl-N-propylamino]tetralin as a new selective radioligand of 5-HT1A sites in the rat brain. In vitro binding and autoradiographic studies.

In vitro binding assays with 125I-[8-methoxy-2-[N-propyl-N-(3'-iodo-4'-hydroxyphenyl)-propionamido -N'- propylamino] tetralin] (125I-BH-8-MeO-N-PAT), a 125I-labeled derivative of the potent serotonin (5-HT) agonist 8-hydroxy-2-[di-n-propylamino]tetralin [( 3H]-8-OH-DPAT), showed that this compound recognized specific sites with nanomolar affinity for 5-HT and 5-HT1A ligands such as spiroxatrine, ipsapirone, buspirone and gepirone in rat hippocampal membranes. Comparison of the binding characteristics of 125I-BH-8-MeO-N-PAT with those of [3H]-8-OH-DPAT revealed striking similarities: at the hippocampal level, both binding sites exhibited nanomolar affinity for their respective ligands and the same Bmax; their pharmacological profiles defined by the inhibition of each bound ligand by a series of 26 serotonin, dopamine- or norepinephrine-related agonists and antagonists were identical; and their regional distributions examined by membrane binding assays and autoradiography of labeled brain sections were highly correlated. These observations indicate that 125I-BH-8-MeO-N-PAT is the first 125I-reversible ligand for the selective labeling of 5-HT1A sites in the rat central nervous system.

[125I-BH-8-MeO-N-PAT, a new ligand for the study of 5-HT1A receptors in the central nervous system]. / 125I-BH-8-MeO-N-PAT, un nouveau ligand pour l'étude des récepteurs 5-HT1A dans le système nerveux central.

Specific radioactive ligands are needed for studying the pharmacological properties and the regional distribution of the different classes of 5-HT1 receptors within the central nervous system. We describe here the synthesis and some characteristics of the first iodinated specific ligand of 5-HT1A receptors. Like its parent compound, the agonist 8-hydroxy-2-(di-n-propylamino)tetralin or 8-OH-DPAT, [125I]-BH-8-MeO-N-PAT, exhibits a high affinity and excellent selectivity for 5-HT1A sites. Its high specific radioactivity makes this ligand a useful tool for studying 5-HT1A receptors in membranes and sections of the rat brain.