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Synthesis and biological evaluation in rat and cat of [18F]12ST05 as a potential 5-HT6 PET radioligand
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Nuclear Medicine and Biology 34 (2007) 995 – 1002 www.elsevier.com/locate/nucmedbio
Synthesis and biological evaluation in rat and cat of [ 18 F]12ST05 as a potential 5-HT6 PET radioligand☆ Sandrine Tang a,1 , Mathieu Verdurand b,c,1 , Benoît Joseph a , Laëtitia Lemoine b,c , Alexia Daoust c , Thierry Billard a , Guy Fournet a , Didier Le Bars a,c , Luc Zimmer b,c,⁎ a
Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, UMR CNRS 5246, Université Lyon 1, Université de Lyon, Lyon, 69677 (Cermep), France b Laboratoire de Neuropharmacologie, FRE CNRS 3006, Université Lyon 1, Université de Lyon, Lyon, 69373 (FRE CNRS 3006), France c CERMEP-Imagerie du Vivant, PET Department, Lyon, 69622 (ICBMS), France Received 6 April 2007; received in revised form 18 June 2007; accepted 2 July 2007
Abstract Introduction: 5-hydroxytryptamine (5-HT)6 receptors represent one of the more recently discovered serotoninergic receptor family. However, no 5-HT6 positron emission tomography (PET) radiotracer is currently used in clinical imaging studies. The purpose of this study was to propose the first fluorinated PET radiotracer for this brain receptor. Methods: A new compound presenting in vitro high affinity towards the serotoninergic 5-HT6 receptor, N-[2-(1-[(4-fluorophenyl)sulfonyl]1H-indol-4-yloxy)ethyl]-N,N-dimethylamine, was labelled with fluorine 18 via a nitro-/fluoronucleophilic substitution. Biological evaluations included (i) in vitro and ex vivo autoradiographies in rat brain and (ii) a PET scan on anaesthetized cat. Results and Conclusion: Although the radioligand showed excellent brain penetration, it did not reveal any specific binding to the 5-HT6 receptors indicating that this radiotracer is not suitable for mapping 5-HT6 receptors using PET. © 2007 Elsevier Inc. All rights reserved. Keywords: 5-HT6 receptors; Radioligand; Positron emission tomography; Rat; Cat
1. Introduction Serotonin [5-hydroxytryptamine (5-HT)] is a central neurotransmitter involved in a great variety of physiological functions, as well as neurological and pathological disorders such as depression, eating disorders and Alzheimer's disease [1]. Pharmacological studies allowed identification of numerous 5-HT receptor families and subtypes. These receptors have been classified by structural, functional and pharmacological criteria into seven distinct receptor classes (5-HT1-7) [2,3].
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Sandrine Tang was supported by a grant from Région Rhône-Alpes “Bourse Prospective.” Mathieu Verdurand was supported by a CIFRE grant with Advanced Accelerator Applications. ⁎ Corresponding author. CERMEP-Imagerie du Vivant, PET Department, F-69006 Lyon, France. E-mail address: [email protected] (L. Zimmer). 1 S.T. and M.V. contributed equally to this article. 0969-8051/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.nucmedbio.2007.07.002
5-HT6 receptors represent one of the more recently discovered serotoninergic receptor family. The 5-HT6 receptor mRNA appears to be almost exclusively present in the brain with little evidence for its presence in peripheral tissues [4,5]. Immunocytochemistry studies in rats agreed well with that seen with 5-HT6 mRNA and have shown that 5-HT6 receptors are localized in striatum, olfactory tubercles, nucleus accumbens, cerebral cortex, hippocampus, hypothalamus and amygdala [6,7]. These data are largely supported by in vitro autoradiographic studies with the selective 5-HT6 radioligands [125I]SB-258585 and [3H]Ro 63-0563 [8–10]. The 5-HT6 receptor messenger RNA is mainly located in the 5-HT projection fields, suggesting that the receptor is located postsynaptically [11]. According to its brain distribution, the 5-HT6 receptors have been implicated in diverse central nervous system pathophysiologies, and several atypical antipsychotics have high affinity for it [12,13]. Although few human postmortem studies are available, the distribution of 5-HT6 receptors appears to be similar to that in rats [14,15].
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Fig. 1. Radiosynthesis of the [18F]12ST05. (A) N,N-dimethylaminoethanol, DIAD, PPh3, THF, rt, 24 h, 100%. (B) NaH, 4-fluorophenylsulfonylchloride 2, THF, rt, 3 h, 50%. (C) NaH, 4-nitrophenylsulfonylchloride 4, THF, rt, 16 h, 60%.
Recently, a team described the carbon 11-radiolabelling of 5-HT6 receptor antagonists, based on the 3-benzenesulfonyl8-piperazin-1-yl-quinoline scaffold, and preclinical positron emission tomography (PET) experiments in porcine brain [16,17]. However, to our knowledge, no clinical studies have been performed yet with PET 5-HT6 radiotracers. We report here the synthesis, the radiolabelling and the first biological evaluations of [18F]-N-[2-(1-[(4-fluorophenyl)sulfonyl]-1H-indol-4-yloxy)ethyl]-N,N-dimethylamine. This molecule belongs to a novel class of 4-(2aminoethoxy)-N-(phenylsulfonyl)indoles recently synthesised and characterised in vitro as selective 5-HT6 ligand [18]. 2. Methods 2.1. Precursor synthesis 4-Hydroxyindole, 4-fluorobenzenesulfonyl chloride (2), 4-nitrobenzenesulfonyl chloride (4) and chemicals were purchased from Sigma-Aldrich, TCI and Alfa Aesar. Melting points were determined using a Büchi melting point apparatus. 1H, 13C and 19F were recorded at 300 K on a Bruker ALS 300 spectrometer. All chemical shifts are reported in parts per million. Coupling constants are expressed in Hertz. Infra-red absorptions were recorded on a Perkin Elmer 681, and values are reported in cm−1. ESI-MS spectra were performed on a ThermoFinnigan LCQ Advantage. Monitoring of reactions was performed using silica gel TLC plates (silica Merck 60 F254). Spots were visualized by ultraviolet light at 254 nm. Column chromatography was performed using silica gel 60 (0.040–0.063 μm, Merck). 2.1.1. N-[2-(1H-Indol-4-yloxy)ethyl]-N,N-dimethylamine (1) To a solution of triphenylphosphine (591 mg, 2.25 mmol) in tetrahydrofurane (12 ml) was added DIAD (diethylazodicarboxylate, 0.45 ml, 2.25 mmol). The solution precipitated few minutes later. After 1 h at room temperature, a
solution of 4-hydroxyindole (200 mg, 1.50 mmol) and N,Ndimethylaminoethanol (0.15 ml, 1.50 mmol) in THF (4 ml) was added. The mixture was stirred for 24 h at room temperature and then concentrated under reduce pressure. The residue obtained was purified by flash chromatography (silica gel) using CH2Cl2/MeOH: 95/5 as eluent to give 1 (307 mg, 100%) as an oil (Fig. 1). IR (film) í 3469, 3053, 1588, 1508, 1459, 1266, 1098 cm−1; 1H-NMR (300 MHz, CDCl3): ä 9.07 (broad s, 1H, NH), 6.99-7.10 (m, 3H, H6, H7, H2), 6.62 (d, 1H, J=2.3 Hz, H3) 6.52 (d, 1H, J=7.5 Hz, H5), 4.23 (t, 2 H, J=5.7 Hz, CH2), 2.88 (t, 2 H, J=5.7 Hz, CH2), 2.42 (s, 6H, CH3); 13C-NMR (75 MHz, CDCl3) ä 152.4 (C), 137.5 (C), 123.0 (CH), 122.5 (CH), 118.9 (C), 104.8 (CH), 100.4 (CH), 99.6 (CH), 66.2 (CH2), 58.3 (CH2), 45.9 (2 CH3); ESI-MS: m/z=205.1 [M + H]+. 2.1.2. N-[2-(1-[(4-Fluorophenyl)sulfonyl]-1H-indol4-yloxy)ethyl]-N,N-dimethylamine (3) To a solution of NaH (88 mg, 2.25 mmol) in THF (20 ml) was added 1 (300 mg, 1.47 mmol) in THF (5 ml) and 4-fluorobenzenesulfonyl chloride 2 (286 mg, 1.47 mmol). After 3 h at room temperature, the mixture was cooled to 0°C, and water was added. The solution was partitioned between CH2Cl2 and water. The aqueous phase was separated and extracted by CH2Cl2. The organic phase was dried over MgSO4 and evaporated under reduced pressure. The residue was purified by flash chromatography under silica gel using CH2Cl2/MeOH: 95/5 as eluent to give 3 (273 mg, 50%) as a solid (Fig. 1). Mp 118–120°C (EtOAc); IR (KBr) í 3058, 2939, 1588, 1494, 1474, 1428, 1371, 1157, 1133, 1058 cm−1; 1H-NMR (300 MHz, CDCl3): δ 7.87 (dd, 2H, J=8.6, 5.0 Hz, H2′), 7.56 (d, 1H, J=8.5 Hz, H7), 7.43 (d, 1H, J=3.6 Hz, H2), 7.22 (t, 1H, J=8.2 Hz, H6), 7.08 (t, 2H, J=8.5 Hz, H3′), 6.80 (d, 1H, J=3.4, H3), 6.65 (d, 1H, J=7.9 Hz, H5), 4.16 (t, 2H, J=5.7 Hz, CH2), 2.77 (t, 2H, J=5.8 Hz, CH2), 2.35 (s, 6H, CH3); 13C-NMR (75 MHz, CDCl3) δ 165.8 (d, J=255.3 Hz, C), 152.6 (C), 136.2 (C), 134.3 (d, J=3.3 Hz, C), 129.7 (d, J=9.3 Hz, 2 CH), 126.0 (CH), 124.7 (CH), 121.5 (C), 116.7 (d, J=22.9 Hz, 2 CH), 107.0 (CH), 106.6 (CH), 104.7 (CH), 66.8 (CH2), 58.3 (CH2), 46.2 (2 CH3); 19F-NMR (282 MHz, CDCl3) δ -103.3 (m); ESI-MS: m/z=363.1 [M + H]+. 2.1.3. N,N-Dimethyl-N-[2-(1-[(4-nitrophenyl)sulfonyl]1H-indol-4-yloxy)ethyl]amine (5) To a solution of NaH (88 mg, 2.25 mmol) in THF (20 ml) was added 1 (301 mg, 1.50 mmol) in THF (5 ml) and 4-nitrobenzenesulfonyl chloride 4 (327 mg, 1.50 mmol). After 16 h at room temperature, the mixture was cooled to 0°C, and water was added. The solution was partitioned between CH2Cl2 and water. The aqueous phase was separated and extracted by CH2Cl2. The organic phase was dried over MgSO4 and evaporated under reduced pressure. The residue was purified by flash chromatography using CH2Cl2/MeOH: 95/5 as eluent to give 5 (311 mg, 60%) as a solid (Fig. 1). Mp 89–91°C (MeOH); IR (KBr) í 3104, 3027, 2920, 1607, 1586, 1426, 1379, 1352, 1194, 1125, 1084,
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742 cm ; H-NMR (300 MHz, CD3OD): δ 8.34 (d, 2H, J=8.7 Hz, H2′), 8.19 (d, 2H, J=8.7 Hz, H3′), 7.69 (d, 1H, J=8.7 Hz, H7), 7.65 (d, 1H, J=3.4 Hz, H2), 7.33 (t, 1H, J=8.3 Hz, H6), 6.98 (d, 1H, J=3.8 Hz, H3), 6.86 (d, 1H, J=7.9 Hz, H5), 4.44 (t, 2H, J=4.7 Hz, CH2), 3.61 (t, 2 H, J=4.7 Hz, CH2), 2.97 (s, 6H, CH3); 13C-NMR (75 MHz, CD3OD) δ 153.7 (C), 152.1 (C), 143.9 (C), 137.3 (C), 129.3 (2 CH), 127.2 (CH), 125.9 (CH), 125.5 (2 CH), 122.6 (C), 108.5 (CH), 107.4 (CH), 106.1 (CH), 66.8 (CH2), 58.8 (CH2), 45.7 (2 CH3); ESI-MS: m/z=390.1 [M + H]+. 1
2.2. Radiochemical synthesis Fluorine-18 was obtained via the 18O (p,n) 18F reaction (IBA Cyclone 18/9 cyclotron). The nitro-/fluoroexchange was realized on a standard Coincidence (GE Mx) synthesizer after reprogramming the automation sequence: after initial fluoride preparation (collection, drying and kryptofix activation), 10 mg of nitro precursor 5 were introduced, and the reaction mixture was heated at 150°C for 10 min. After dilution with 15 ml of water, the reaction mixture was passed through an activated C18 cartridge for prepurification, and the crude product was eluted from the cartridge with 2 ml of ethanol. Pure 3 was obtained after separation on a preparative high-performance liquid chromatography (HPLC) (C18 Symmetry Prep Waters 7 μm 7.8×300 mm) eluted with THF/water pH 5/MeOH 4/9/1 at 2.5 ml min−1, with a retention time of 13 min. For biological use, the radiotracer, called [18F]12ST05, was processed manually and formulated via SPE techniques [19]. The dilution of the product was done with 40 ml of sterile water and loaded on a SEP-Pak Light C18 cartridge (Waters, Milford, MA, USA). The loaded cartridge was eluted with 1 ml of ethanol, and the final product was diluted with isotonic saline and sterilized by filtration (sterile filter Millex-GS, 0.22 μm). Finally, a 5% ethanol in saline injectable solution was obtained.
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2.5. Drugs The 5-HT6 receptor antagonist SB-258585 hydrochloride was purchased from Tocris Biosciences (UK). Ro 04-6790 dihydrochloride, another selective 5-HT6 receptor antagonist, was obtained from Sigma–Aldrich (Germany). Urethane was purchased from Acros Organics (UK). 2.6. In vitro lipophilicity evaluation and ex vivo brain penetration Lipophilicity (log P) was measured to evaluate the lipid solubility of [18F]12ST05 by partitioning between n-octanol and 50 mmol/L Tris-HCl (pH 7.4) buffer. Log P was taken as the concentration of [18F]12ST05 in n-octanol over the concentration in buffer. For the ex vivo evaluation of [18F] 12ST05 brain entry, two series of four rats were anesthetized by intraperitoneal injection of urethane (1.7 g/kg), and a catheter was placed into their caudal vein. The rats were killed by decapitation 5, 10, 20 and 60 min after the bolus injection of [18F]12ST05 (55.5 MBq, 1.5 mCi). The brains were rapidly dissected, rinsed with water and counted for radioactivity with an automated gamma-counter (Cobra II; Packard, Meriden, CT, USA). 2.7. In vitro autoradiographies
Quality control consists of the determination of radiochemical purity and specific activity, determined by analytical HPLC assay [UV set at 254 nm and radioactive detection; elution with 60/40 water pH 5/MeOH, THF (60/40) at 2.5 ml min−1; retention time of 7 min] of an aliquot of the labelled product with comparison to standard curve generated from solutions of known concentration.
Adult Sprague–Dawley males (250-350 g) were anesthetized with a short inhalation of isoflurane and rapidly killed by decapitation. Brains were then dissected, frozen in 2-methylbutane cooled to −29°C with carbon dioxide ice. Cryostat sections (30 μm thick) were cut across the hippocampus, the striatum and the cerebellum, thaw-mounted on slides and allowed to air dry for 30 min before storage at −80°C until used. On the day of [18F]12ST05 synthesis, the slides were allowed to come to room temperature, and the sections incubated for 20 min in Tris phosphate-buffered saline buffer (138 mM NaCl, 2.7 mM KCl, pH adjusted to 7.6) containing 1 μCi/ml of [18F]12ST05. Adjacent sections were incubated in the same medium supplemented with 10 μM serotonin for estimation of nonspecific binding. After 20 min of incubation, the slides were rapidly dipped in distilled water, dried and juxtaposed to sensitive plates. The distribution of the radioactivity was then visualized with a Bio-imaging Analyser System (BAS-1800 II, Raytest, Germany) and analysed using the Multigauge software (Fujifilm, Raytest, Germany).
2.4. Animals
2.8. Ex vivo autoradiographies
These studies were performed in accordance with European guidelines for care of laboratory animals (86/609 EEC) and were approved by the ethical animal use committee of the Université de Lyon. Adult Sprague– Dawley males (Harlan, France) weighing 250–350 g were used for in vitro and ex vivo experiments. A European male cat weighing 3.5 kg (Charles River Laboratories, L'Arbresle, France) was used for PET scan acquisition.
Rats were anesthetized by urethane (1.7 g/kg ip) and an intravenous catheter was placed into their caudal vein. For the competition assays, 5-HT6 antagonists (SB-258585 or Ro 04-6790), or unlabeled 12ST05, were intravenously injected at the dose of 5 mg/kg, 30 min before an 55.5 Bq (1.5 mCi) intravenous injection of [18F]12ST05. Rats were killed by decapitation 20 min later. The brains were rapidly extracted, frozen and cut into 30-μm-thick sections using a
2.3. Quality control
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Fig. 2. (A) Preparative chromatogram of the radiosynthesis of [18F]12ST05 [(top) UV absorbance at 254 nm; (bottom) radioactivity]. The retention times of [18F] 12ST05 and its precursor are 13 and 14 min, respectively. The rectangle indicates the sampled fraction. (B) Chromatogram of the final [18F]12ST05 [(top) UV absorbance at 254 nm; (bottom) radioactivity]. The retention time of [18F]12ST05 is 7 min (indicated by an arrow). Note the absence of precursor and other labelled compounds (radiochemical purity N98%).
cryotome (Microm Microtech, France). The sections were air-dried and apposed on a phosphor imaging plate for 60 min. The distribution of the radioactivity was then visualized with a Bio-imaging Analyser System (BAS-1800 II, Raytest, Germany) and analyzed using the Multigauge software (Fujifilm, Raytest, Germany). Regions of interest (striatum, ventral hippocampus and cerebellum) were manually drawn with the help of a stereotaxic atlas of the rat brain [20]. Background noise was subtracted from all
regions of interest and the radioactivity was expressed in photostimulated luminescence per surface unit (psl/mm2). All experiments were performed in duplicate. 2.9. PET scan acquisition The cat PET acquisition was performed on a Siemens CTI Exact ECAT HR+ (Knoxville, TN, USA) used in threedimensional mode. The cat was anesthetized with 5% isoflurane for 5 min, which was then lowered to 2.5%. The
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3.2. In vitro lipophilicity evaluation and in vivo brain penetration The lipophilicity of [18F]12ST05, determined in vitro by the octanol/water partition coefficient (log P), was found to be 2.03±0.03 (n=5 experiments). This value was confirmed by the calculated log P (2.2 at pH 7.4; ACD/Labs software, version 7.0). Ex vivo, the radiotracer [18F]12ST05 readily entered the brain with a maximal access 20 min post injection. The calculated percentages of injected dose were 0.21%, 0.28%, 0.44%, 0.19%, respectively, at 5, 10, 20 and 60 min post injection. 3.3. In vitro autoradiographies in rats [18F]12ST05 binding on control sections was homogenous throughout the brain with regions like hippocampus and cerebellum bound to a semiquantitative comparable level. No significant binding difference was found between control sections and sections preincubated with serotonin (10 μM) (results not shown). 3.4. Ex vivo autoradiographies in rats Fig. 3. Ex vivo autoradiograms of brain sections 30 min after the preinjection of unlabeled 12ST05 (5 mg/kg iv) (lower images) in comparison to their respective controls (upper images). The histograms represent the corresponding [18F]12ST05 regional radioactivity intensity (psl/mm2).
Ex vivo autoradiographies in control rats, performed 20 min after the [18F]12ST05 injection, confirmed a high
head of the animal was immobilized in a stereotaxic Plexiglas frame with ear bars. A catheter was placed into the forearm branch of the brachiocephalic vein continuously perfused with NaCl 0.9%. A 10-min transmission scan was obtained with three rotating 68Ge–68Ga sources followed by a 74-MBq (2-mCi) bolus injection of [18F]12ST05. Radioactivity was thus measured in a series of 31 sequential frames of increasing duration from 30 s to 10 min. The total duration of a scan was 90 min. After acquisition, the images were reconstructed following the frontal plan. Regions of interest (striatum, thalamus, cingulate cortex, hippocampus and cerebellum) were processed manually according to the stereotaxic atlas of the cat brain [21,22]. Regional radioactivities were expressed in Becquerel per volume unit (Bq/cm3). 3. Results 3.1. Chemistry and radiochemistry Using a reprogrammed automated fluorination module, labelling of 3 from its nitro precursor at 150°C was straightforward, with a radiochemical yield of 32% corrected for decay and 50-min radiosynthesis time. No major radioactive by-products were observed, and HPLC conditions chosen ensured good separation of 3 from its nitro precursor, as confirmed by quality control (Fig. 2). Radiochemical purity was better than 95% and specific activity was between 85 and 120 GBq μmol−1, corrected at end of synthesis.
Fig. 4. Ex vivo autoradiograms of brain sections 30 min after the preinjection of 5-HT6 antagonists (SB-258585 and Ro 04-6790, 5 mg/kg iv) in comparison to their respective controls (upper illustrations). The histograms represent the corresponding [18F]12ST05 regional radioactivity intensity (psl/mm2).
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3.5. PET scan acquisition on a cat After intravenous injection of [18F]12ST05, there was a rapid accumulation of the radioligand in the cat brain within the first 10 min. The time activity curves showed a high initial uptake in all brain regions including striatum, hippocampus, thalamus, cingulate cortex and cerebellum (Fig. 5). Regions with high densities of 5-HT6 receptors like the striatum and the hippocampus presented an uptake ratio (relative to cerebellum) of, respectively, 1.39 and 1.40, 10 min after [18F]12ST05 injection. The highest uptake was observed in thalamus with an uptake ratio (thalamus/ cerebellum) of 1.55, remaining constant over the acquisition time. The radioactivity washout rates were comparable, for all structures, studied except cingulate cortex with a higher washout rate (Fig. 5). 4. Discussion
Fig. 5. PET [18F]12ST05 images of the cat brain in sagittal (A), coronal (B) and transversal (C) planes. Corresponding time–radioactivity curves are presented. Note the intense and peculiar thalamus labelling. Str, striatum; Thal, thalamus; Cg, cingulate cortex; Hip, hippocampus; Cer, cerebellum.
brain uptake of the tracer. The time of analysis was chosen according to the previous in vivo brain penetration results. Regions like striatum and hippocampus, known to be rich in 5-HT6 receptors, displayed a high [18F]12ST05 labelling (Fig. 3). It has to be noted that a similar radiotracer uptake was seen in cerebellum. The preinjection of unlabeled 12ST05 suppressed significantly (P b.05, analysis of variance and Student's t test) the binding of [18F] 12ST05 by 92% in all regions of the brain studied (Fig. 3). In other experiments, the preinjection of SB-258585, a 5-HT6 receptors antagonist, reduced [ 18 F]12ST05 fixation by 17% in striatum (P b.05), 11% in hippocampus and 8% in cerebellum (Fig. 4). Preinjection of Ro 04-6790, another potent antagonist of 5-HT6 receptors, was found to increase significantly [18F]12ST05 binding by 31% in striatum, 24% in hippocampus and 25% in cerebellum (P b.05; Fig. 4).
The feasibility of imaging the 5-HT6 receptors in the brain by PET studies represents an interesting field for PET imaging. To our knowledge, only a few studies have been done in this new area. In preliminary reports [16,17], the 5-HT6 receptor antagonists GSK215083 and GSK224558 have been radiolabelled with carbon-11 and were evaluated in pigs. However, to our knowledge, no clinical PET studies were recently performed with both radiotracers. In this context, we have recently been interested by a novel class of 4-(2-aminoethoxy)-N-(phenylsulfonyl)indoles with high affinities towards 5-HT6 receptors [18]. We selected one of these structures (3) exhibiting a high affinity towards the 5-HT6 receptors (Ki=4 nM) as well as an excellent selectivity against the closely related 5-HT7 receptors (N2000-fold). The precursor was synthesized, with a chemical structure slightly modified from the one described by Zhou et al. [18] since an NO2-group had to be added for the fluorination. After a classical fluorination via a nitro-/fluoronucleophilic substitution realized on a reprogrammed Coincidence (GE Mx) synthesizer, we obtained [18F]12ST05 in an amount suitable for the biological evaluations. The labelling was straightforward — not surprising given the presence of the parasubstituted sulfonamide. Although the lipophilicity experimentally determined is not systematically predictive of the blood-brain-barrier penetration [23], the [18F]12ST05 log P was encouraging. This in vitro result was confirmed by the radioactive brain distribution of [18F]12ST05 showing an excellent crossing of the blood-brain barrier in comparison to the known brain radiotracers [24]. However, the in vitro autoradiographies were disappointing, suggesting that [18F]12ST05 lacks sufficient affinity or selectivity. Since in vitro studies cannot be directly extrapolated to in vivo studies, it was crucial to perform ex vivo and in vitro studies. Ex vivo autoradiographic experiments in rats were conducted with the aim to evaluate the cerebral distribution of [18F]12ST05 in living animals. In control rats, striatum
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and hippocampus, rich in 5-HT6 receptors [10], showed a high labelling. Unexpectedly, the cerebellum, a region devoid of 5-HT6 receptors, presented a comparable high radioactive level, suggesting a high level of nonspecific binding. The preinjection with unlabelled 12ST05 blocked the binding of [18F]12ST05, showing that its fixation is saturable. In order to determine the specificity of the binding of [18F]12ST05, competition experiments were performed with a well-described 5-HT6 receptor antagonist, SB-258585 [9,10]. The slight reduction of [18F]12ST05 binding in striatum and hippocampus after SB-258585 injection is not sufficient to demonstrate that [18F]12ST05 binds partially to 5-HT6 receptors, since the cerebellum was also reduced in uptake (although not significantly). To further assess the specificity of [18F]12ST05, rats were pretreated with another 5-HT6 antagonist, Ro 04-6790, which crosses the bloodbrain barrier more easily [25]. Unexpectedly, the preinjection of this 5-HT6 antagonist increased [18F]12ST05 binding in striatum, hippocampus and cerebellum. The more plausible explanation could be that Ro 04-6790 acutely increases the cerebral blood flow and modifies the brain penetration of the radiotracer. Finally, we performed a PET scan acquisition on an anaesthetized cat. The cat brain is of sufficient size to allow the use of human-oriented PET cameras in the perspective of a clinical use of the new PET tracer. The [18F]12ST05 rapid brain penetration was consistent with the previous ex vivo studies in rats. Although the thalamus does not present a high density of 5-HT6 receptors, this region was more highly marked than striatum and hippocampus in the first 10 min of acquisition. This PET scan assay demonstrated a homogenous fixation of [18F]12ST05, consistent with a good brain penetration but confirming the lack of ability of this radiotracer to image 5-HT6 receptors. The failure of [18F]12ST05 to label in vivo 5-HT6 receptors can be explained by two factors. The first factor is that the addition of a fluorine atom during the radiolabelling decreased significantly the affinity of 12ST05 for the 5-HT6 receptors. We confirmed recently this hypothesis by the in vitro determination of the affinity of the nonradioactive fluorinated 12ST05 (binding assays on 5-HT6 receptors expressed in Chinese hamster ovary cells vs. [3H]LSD). The 4-fluoro substitution caused an apparent fivefold lowering of affinity for the 5-HT6 receptors (Ki=20 nM, to be compared with the initial value of 4 nM; Cerep France, personal communication). In this same context, a structure–activity relationship study reported previously that the fluorine addition to a phenylsulfonyl derivate (giving the 4-fluorophenylsulfonyl derivate) decreases its affinity toward 5-HT6 receptor [26]. These results highlight the influence of fluorine addition during radiolabeling on the electronic configuration of the 5-HT6 ligand pharmacophore. The second factor explaining this failure is that [18F]12ST05 appeared to have a high affinity to a binding site, blocked by the addition of unlabelled 12ST05. Our results showed that this saturable site is not the 5-HT6 receptor, nor probably
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another serotoninergic receptor (serotonin preincubations did not modify the binding). PET scan obtained in the anaesthetized cat showed that this unknown site is more concentrated in the thalamus. A Scatchard plot could be performed to define if this site is unique and to determine its density. In conclusion, we reported in this study the radiolabelling of a 5-HT6 receptor antagonist. Our preliminary biological results, obtained with complementary ex vivo and in vivo approaches in two different species, demonstrate the inability of [18F]12ST05 to visualize 5-HT6 receptors in vivo. Because the discovery of a potent 5-HT6 radiotracer is crucial for the exploration of brain serotoninergic function, other 5-HT6 leads will be evaluated for future PET investigations. References [1] Cross AJ. Serotonin in Alzheimer-type dementia and other dementing illnesses. Ann NY Acad Sci 1990;600:405–17. [2] Hoyer D, Clarke DE, Fozard JR, Hartig PR, Martin GR, Mylecharane EJ, et al. International Union of Pharmacology classification of receptors for 5-hydroxytryptamine (Serotonin). Pharmacol Rev 1994;446:157–203. [3] Hoyer D, Martin GR. Classification and nomenclature of 5-HT receptors: a comment on current issues. Behav Brain Res 1996;73: 263–8. [4] Monsma FJ, Shen Y, Ward RP, Hamblin MW, Sibley DR. Cloning and expression of a novel serotonin receptor with high affinity for tricyclic psychotropic drugs. Mol Pharmacol 1993;43:320–7. [5] Ruat M, Traiffort E, Arrang JM, Tardivel-Lacombe J, Diaz J, Leurs R, et al. A novel rat serotonin (5-HT6) receptor: molecular cloning, localization and stimulation of cAMP accumulation. Biochem Biophys Res Commun 1993;193:268–76. [6] Gérard C, Martres NP, Lefèvre K, Miquel MC, Vergé D, Lanfumey L, et al. Immuno-localisation of serotonin 5-HT6 receptor-like material in the rat central nervous system. Brain Res 1997;746:207–19. [7] Hamon M, Doucet E, Lefevre K, Miquel MC, Lanfumey L, Insausti R, et al. Antibodies and antisense oligonucleotide for probing the distribution and putative functions of central 5-HT6 receptors. Neuropsychopharmacology 1998;21:68–76. [8] Boess FG, Riemer C, Bos M, Bentley J, Bourson A, Sleight AJ. The 5-hydroxytryptamine6 receptor-selective radioligand [3H]Ro 63-0563 labels 5-hydroxytryptamine receptor binding sites in rat and porcine striatum. Mol Pharmacol 1998;54:577–83. [9] Hirst WD, Minton JAL, Bromidge SM, Moss SF, Latter AJ, Riley G, et al. Characterization of [125I]-SB-258585 binding to human recombinant and native 5-HT6 receptors in rat, pig and human brain tissue. Br J Pharmacol 2000;130:1597–605. [10] Roberts JC, Reavill C, East SZ, Harrisson PJ, Patel S, Routledge C, et al. The distribution of 5-HT6 receptors in rat brain: an autoradiographic binding study using the radiolabelled 5-HT6 receptor antagonist [125I]SB-258585. Brain Res 2002;934:49–57. [11] Gérard C, El Mestikawy S, Lebrand C, Adrien J, Ruat M, Traiffort E, et al. Quantitative RT-PCR distribution of serotonin 5-HT6 receptor mRNA in the central nervous system of control or 5,7-dihydroxytryptamine-treated rats. Synapse 1996;23:164–73. [12] Woolley ML, Marsden CA, Fone KCF. 5-HT6 receptors. Curr Drug Targets CNS Neurol Disord 2004;3:59–79. [13] Mitchell ES, Neumaier JF. 5-HT6 receptors: a novel target for cognitive enhancement. Pharmacol Ther 2005;108:320–33. [14] East SZ, Burnet PWJ, Leslie RA, Roberts JC, Harrison PJ. 5-HT6 receptor binding sites in schizophrenia and following antipsychotic drug administration: autoradiographic studies with [125I]SB-258585. Synapse 2002;45:191–9.
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[15] Lorke DE, Lu G, Cho E, Yew DT. Serotonin 5-HT2A and 5-HT6 receptors in the prefrontal cortex of Alzheimer and normal aging patients. BMC Neurosci 2006;7:36. [16] Martarello L, Ahmed M, Chuang AT, Cunningham VJ, Jakobsen S, Johnson CN, et al. Radiolabelling and in vivo evaluation of [11C] GSK215083 as a potential 5-HT6 pet radioligand in the porcine brain. J Label Compd Radiopharm 2005;48:S7. [17] Huiban M, Passchier J, Martarello L, Cunningham VJ, Jacobson S, Gee AD. [11C]GSK224558 as a potential PET ligand for the delineation of 5-HT6 receptors. Neuroimage 2006;31:T13. [18] Zhou P, Yan Y, Bernotas R, Harrison BL, Huryn D, Robichaud AJ, et al. 4-(2-aminoethoxy)-N-(phenylsulfonyl)indoles as novel 5-HT6 receptor ligands. Bioorg Med Chem Lett 2005;15:1393–6. [19] Lemaire C, Plenevaux A, Aerts J, Del Fiore G, Brihaye C, Le Bars D, et al. Solid Phase Extraction: an alternative to the use of rotary evaporators for solvent removal in the rapid formulation of PET radiopharmaceuticals. J Labelled Compd Radiopharm 1999;42:63–75. [20] Paxinos G, Watson C. The rat brain in stereotaxic coordinates. New York: Academic Press; 1986.
[21] Jasper HH, Ajmone-Marsan C. A stereotaxic atlas of the diencephalon of the cat. Ottawa: The National Research Council of Canada editors; 1954. [22] Snider RS, Niemer WT. A stereotaxic atlas of the cat brain. Chicago: University of Chicago Press; 1961. [23] Wilson AA, Jin L, Garcia A, DaSilva JN, Houle S. An admonition when measuring the lipophilicity of radiotracers using counting techniques. Appl Radiat Isot 2001;54:203–8. [24] Waterhouse RN. Determination of lipophilicity and its use as a predictor of blood-brain-barrier penetration of molecular imaging agents. Mol Imaging Biol 2003;5:376–89. [25] Bös M, Sleight AJ, Godel T, Martin JR, Riemer C, Stadler H. 5-HT6 receptor antagonists: lead optimisation and biological evaluation of N-aryl and N-heteroaryl 4-amino-benzene sulfonamides. Eur J Med Chem 2001;36:165–78. [26] Cole DC, Lennox WJ, Stock JR, Ellingboe JW, Mazandarani H, Smith DL, et al. Conformationally constrained N1-arylsulfonyltryptamine derivatives as 5-HT6 receptor antagonists. Bioorg Med Chem 2005;15:4780–5.
Nuclear Medicine and Biology 34 (2007) 995 – 1002 www.elsevier.com/locate/nucmedbio
Synthesis and biological evaluation in rat and cat of [ 18 F]12ST05 as a potential 5-HT6 PET radioligand☆ Sandrine Tang a,1 , Mathieu Verdurand b,c,1 , Benoît Joseph a , Laëtitia Lemoine b,c , Alexia Daoust c , Thierry Billard a , Guy Fournet a , Didier Le Bars a,c , Luc Zimmer b,c,⁎ a
Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, UMR CNRS 5246, Université Lyon 1, Université de Lyon, Lyon, 69677 (Cermep), France b Laboratoire de Neuropharmacologie, FRE CNRS 3006, Université Lyon 1, Université de Lyon, Lyon, 69373 (FRE CNRS 3006), France c CERMEP-Imagerie du Vivant, PET Department, Lyon, 69622 (ICBMS), France Received 6 April 2007; received in revised form 18 June 2007; accepted 2 July 2007
Abstract Introduction: 5-hydroxytryptamine (5-HT)6 receptors represent one of the more recently discovered serotoninergic receptor family. However, no 5-HT6 positron emission tomography (PET) radiotracer is currently used in clinical imaging studies. The purpose of this study was to propose the first fluorinated PET radiotracer for this brain receptor. Methods: A new compound presenting in vitro high affinity towards the serotoninergic 5-HT6 receptor, N-[2-(1-[(4-fluorophenyl)sulfonyl]1H-indol-4-yloxy)ethyl]-N,N-dimethylamine, was labelled with fluorine 18 via a nitro-/fluoronucleophilic substitution. Biological evaluations included (i) in vitro and ex vivo autoradiographies in rat brain and (ii) a PET scan on anaesthetized cat. Results and Conclusion: Although the radioligand showed excellent brain penetration, it did not reveal any specific binding to the 5-HT6 receptors indicating that this radiotracer is not suitable for mapping 5-HT6 receptors using PET. © 2007 Elsevier Inc. All rights reserved. Keywords: 5-HT6 receptors; Radioligand; Positron emission tomography; Rat; Cat
1. Introduction Serotonin [5-hydroxytryptamine (5-HT)] is a central neurotransmitter involved in a great variety of physiological functions, as well as neurological and pathological disorders such as depression, eating disorders and Alzheimer's disease [1]. Pharmacological studies allowed identification of numerous 5-HT receptor families and subtypes. These receptors have been classified by structural, functional and pharmacological criteria into seven distinct receptor classes (5-HT1-7) [2,3].
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Sandrine Tang was supported by a grant from Région Rhône-Alpes “Bourse Prospective.” Mathieu Verdurand was supported by a CIFRE grant with Advanced Accelerator Applications. ⁎ Corresponding author. CERMEP-Imagerie du Vivant, PET Department, F-69006 Lyon, France. E-mail address: [email protected] (L. Zimmer). 1 S.T. and M.V. contributed equally to this article. 0969-8051/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.nucmedbio.2007.07.002
5-HT6 receptors represent one of the more recently discovered serotoninergic receptor family. The 5-HT6 receptor mRNA appears to be almost exclusively present in the brain with little evidence for its presence in peripheral tissues [4,5]. Immunocytochemistry studies in rats agreed well with that seen with 5-HT6 mRNA and have shown that 5-HT6 receptors are localized in striatum, olfactory tubercles, nucleus accumbens, cerebral cortex, hippocampus, hypothalamus and amygdala [6,7]. These data are largely supported by in vitro autoradiographic studies with the selective 5-HT6 radioligands [125I]SB-258585 and [3H]Ro 63-0563 [8–10]. The 5-HT6 receptor messenger RNA is mainly located in the 5-HT projection fields, suggesting that the receptor is located postsynaptically [11]. According to its brain distribution, the 5-HT6 receptors have been implicated in diverse central nervous system pathophysiologies, and several atypical antipsychotics have high affinity for it [12,13]. Although few human postmortem studies are available, the distribution of 5-HT6 receptors appears to be similar to that in rats [14,15].
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Fig. 1. Radiosynthesis of the [18F]12ST05. (A) N,N-dimethylaminoethanol, DIAD, PPh3, THF, rt, 24 h, 100%. (B) NaH, 4-fluorophenylsulfonylchloride 2, THF, rt, 3 h, 50%. (C) NaH, 4-nitrophenylsulfonylchloride 4, THF, rt, 16 h, 60%.
Recently, a team described the carbon 11-radiolabelling of 5-HT6 receptor antagonists, based on the 3-benzenesulfonyl8-piperazin-1-yl-quinoline scaffold, and preclinical positron emission tomography (PET) experiments in porcine brain [16,17]. However, to our knowledge, no clinical studies have been performed yet with PET 5-HT6 radiotracers. We report here the synthesis, the radiolabelling and the first biological evaluations of [18F]-N-[2-(1-[(4-fluorophenyl)sulfonyl]-1H-indol-4-yloxy)ethyl]-N,N-dimethylamine. This molecule belongs to a novel class of 4-(2aminoethoxy)-N-(phenylsulfonyl)indoles recently synthesised and characterised in vitro as selective 5-HT6 ligand [18]. 2. Methods 2.1. Precursor synthesis 4-Hydroxyindole, 4-fluorobenzenesulfonyl chloride (2), 4-nitrobenzenesulfonyl chloride (4) and chemicals were purchased from Sigma-Aldrich, TCI and Alfa Aesar. Melting points were determined using a Büchi melting point apparatus. 1H, 13C and 19F were recorded at 300 K on a Bruker ALS 300 spectrometer. All chemical shifts are reported in parts per million. Coupling constants are expressed in Hertz. Infra-red absorptions were recorded on a Perkin Elmer 681, and values are reported in cm−1. ESI-MS spectra were performed on a ThermoFinnigan LCQ Advantage. Monitoring of reactions was performed using silica gel TLC plates (silica Merck 60 F254). Spots were visualized by ultraviolet light at 254 nm. Column chromatography was performed using silica gel 60 (0.040–0.063 μm, Merck). 2.1.1. N-[2-(1H-Indol-4-yloxy)ethyl]-N,N-dimethylamine (1) To a solution of triphenylphosphine (591 mg, 2.25 mmol) in tetrahydrofurane (12 ml) was added DIAD (diethylazodicarboxylate, 0.45 ml, 2.25 mmol). The solution precipitated few minutes later. After 1 h at room temperature, a
solution of 4-hydroxyindole (200 mg, 1.50 mmol) and N,Ndimethylaminoethanol (0.15 ml, 1.50 mmol) in THF (4 ml) was added. The mixture was stirred for 24 h at room temperature and then concentrated under reduce pressure. The residue obtained was purified by flash chromatography (silica gel) using CH2Cl2/MeOH: 95/5 as eluent to give 1 (307 mg, 100%) as an oil (Fig. 1). IR (film) í 3469, 3053, 1588, 1508, 1459, 1266, 1098 cm−1; 1H-NMR (300 MHz, CDCl3): ä 9.07 (broad s, 1H, NH), 6.99-7.10 (m, 3H, H6, H7, H2), 6.62 (d, 1H, J=2.3 Hz, H3) 6.52 (d, 1H, J=7.5 Hz, H5), 4.23 (t, 2 H, J=5.7 Hz, CH2), 2.88 (t, 2 H, J=5.7 Hz, CH2), 2.42 (s, 6H, CH3); 13C-NMR (75 MHz, CDCl3) ä 152.4 (C), 137.5 (C), 123.0 (CH), 122.5 (CH), 118.9 (C), 104.8 (CH), 100.4 (CH), 99.6 (CH), 66.2 (CH2), 58.3 (CH2), 45.9 (2 CH3); ESI-MS: m/z=205.1 [M + H]+. 2.1.2. N-[2-(1-[(4-Fluorophenyl)sulfonyl]-1H-indol4-yloxy)ethyl]-N,N-dimethylamine (3) To a solution of NaH (88 mg, 2.25 mmol) in THF (20 ml) was added 1 (300 mg, 1.47 mmol) in THF (5 ml) and 4-fluorobenzenesulfonyl chloride 2 (286 mg, 1.47 mmol). After 3 h at room temperature, the mixture was cooled to 0°C, and water was added. The solution was partitioned between CH2Cl2 and water. The aqueous phase was separated and extracted by CH2Cl2. The organic phase was dried over MgSO4 and evaporated under reduced pressure. The residue was purified by flash chromatography under silica gel using CH2Cl2/MeOH: 95/5 as eluent to give 3 (273 mg, 50%) as a solid (Fig. 1). Mp 118–120°C (EtOAc); IR (KBr) í 3058, 2939, 1588, 1494, 1474, 1428, 1371, 1157, 1133, 1058 cm−1; 1H-NMR (300 MHz, CDCl3): δ 7.87 (dd, 2H, J=8.6, 5.0 Hz, H2′), 7.56 (d, 1H, J=8.5 Hz, H7), 7.43 (d, 1H, J=3.6 Hz, H2), 7.22 (t, 1H, J=8.2 Hz, H6), 7.08 (t, 2H, J=8.5 Hz, H3′), 6.80 (d, 1H, J=3.4, H3), 6.65 (d, 1H, J=7.9 Hz, H5), 4.16 (t, 2H, J=5.7 Hz, CH2), 2.77 (t, 2H, J=5.8 Hz, CH2), 2.35 (s, 6H, CH3); 13C-NMR (75 MHz, CDCl3) δ 165.8 (d, J=255.3 Hz, C), 152.6 (C), 136.2 (C), 134.3 (d, J=3.3 Hz, C), 129.7 (d, J=9.3 Hz, 2 CH), 126.0 (CH), 124.7 (CH), 121.5 (C), 116.7 (d, J=22.9 Hz, 2 CH), 107.0 (CH), 106.6 (CH), 104.7 (CH), 66.8 (CH2), 58.3 (CH2), 46.2 (2 CH3); 19F-NMR (282 MHz, CDCl3) δ -103.3 (m); ESI-MS: m/z=363.1 [M + H]+. 2.1.3. N,N-Dimethyl-N-[2-(1-[(4-nitrophenyl)sulfonyl]1H-indol-4-yloxy)ethyl]amine (5) To a solution of NaH (88 mg, 2.25 mmol) in THF (20 ml) was added 1 (301 mg, 1.50 mmol) in THF (5 ml) and 4-nitrobenzenesulfonyl chloride 4 (327 mg, 1.50 mmol). After 16 h at room temperature, the mixture was cooled to 0°C, and water was added. The solution was partitioned between CH2Cl2 and water. The aqueous phase was separated and extracted by CH2Cl2. The organic phase was dried over MgSO4 and evaporated under reduced pressure. The residue was purified by flash chromatography using CH2Cl2/MeOH: 95/5 as eluent to give 5 (311 mg, 60%) as a solid (Fig. 1). Mp 89–91°C (MeOH); IR (KBr) í 3104, 3027, 2920, 1607, 1586, 1426, 1379, 1352, 1194, 1125, 1084,
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742 cm ; H-NMR (300 MHz, CD3OD): δ 8.34 (d, 2H, J=8.7 Hz, H2′), 8.19 (d, 2H, J=8.7 Hz, H3′), 7.69 (d, 1H, J=8.7 Hz, H7), 7.65 (d, 1H, J=3.4 Hz, H2), 7.33 (t, 1H, J=8.3 Hz, H6), 6.98 (d, 1H, J=3.8 Hz, H3), 6.86 (d, 1H, J=7.9 Hz, H5), 4.44 (t, 2H, J=4.7 Hz, CH2), 3.61 (t, 2 H, J=4.7 Hz, CH2), 2.97 (s, 6H, CH3); 13C-NMR (75 MHz, CD3OD) δ 153.7 (C), 152.1 (C), 143.9 (C), 137.3 (C), 129.3 (2 CH), 127.2 (CH), 125.9 (CH), 125.5 (2 CH), 122.6 (C), 108.5 (CH), 107.4 (CH), 106.1 (CH), 66.8 (CH2), 58.8 (CH2), 45.7 (2 CH3); ESI-MS: m/z=390.1 [M + H]+. 1
2.2. Radiochemical synthesis Fluorine-18 was obtained via the 18O (p,n) 18F reaction (IBA Cyclone 18/9 cyclotron). The nitro-/fluoroexchange was realized on a standard Coincidence (GE Mx) synthesizer after reprogramming the automation sequence: after initial fluoride preparation (collection, drying and kryptofix activation), 10 mg of nitro precursor 5 were introduced, and the reaction mixture was heated at 150°C for 10 min. After dilution with 15 ml of water, the reaction mixture was passed through an activated C18 cartridge for prepurification, and the crude product was eluted from the cartridge with 2 ml of ethanol. Pure 3 was obtained after separation on a preparative high-performance liquid chromatography (HPLC) (C18 Symmetry Prep Waters 7 μm 7.8×300 mm) eluted with THF/water pH 5/MeOH 4/9/1 at 2.5 ml min−1, with a retention time of 13 min. For biological use, the radiotracer, called [18F]12ST05, was processed manually and formulated via SPE techniques [19]. The dilution of the product was done with 40 ml of sterile water and loaded on a SEP-Pak Light C18 cartridge (Waters, Milford, MA, USA). The loaded cartridge was eluted with 1 ml of ethanol, and the final product was diluted with isotonic saline and sterilized by filtration (sterile filter Millex-GS, 0.22 μm). Finally, a 5% ethanol in saline injectable solution was obtained.
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2.5. Drugs The 5-HT6 receptor antagonist SB-258585 hydrochloride was purchased from Tocris Biosciences (UK). Ro 04-6790 dihydrochloride, another selective 5-HT6 receptor antagonist, was obtained from Sigma–Aldrich (Germany). Urethane was purchased from Acros Organics (UK). 2.6. In vitro lipophilicity evaluation and ex vivo brain penetration Lipophilicity (log P) was measured to evaluate the lipid solubility of [18F]12ST05 by partitioning between n-octanol and 50 mmol/L Tris-HCl (pH 7.4) buffer. Log P was taken as the concentration of [18F]12ST05 in n-octanol over the concentration in buffer. For the ex vivo evaluation of [18F] 12ST05 brain entry, two series of four rats were anesthetized by intraperitoneal injection of urethane (1.7 g/kg), and a catheter was placed into their caudal vein. The rats were killed by decapitation 5, 10, 20 and 60 min after the bolus injection of [18F]12ST05 (55.5 MBq, 1.5 mCi). The brains were rapidly dissected, rinsed with water and counted for radioactivity with an automated gamma-counter (Cobra II; Packard, Meriden, CT, USA). 2.7. In vitro autoradiographies
Quality control consists of the determination of radiochemical purity and specific activity, determined by analytical HPLC assay [UV set at 254 nm and radioactive detection; elution with 60/40 water pH 5/MeOH, THF (60/40) at 2.5 ml min−1; retention time of 7 min] of an aliquot of the labelled product with comparison to standard curve generated from solutions of known concentration.
Adult Sprague–Dawley males (250-350 g) were anesthetized with a short inhalation of isoflurane and rapidly killed by decapitation. Brains were then dissected, frozen in 2-methylbutane cooled to −29°C with carbon dioxide ice. Cryostat sections (30 μm thick) were cut across the hippocampus, the striatum and the cerebellum, thaw-mounted on slides and allowed to air dry for 30 min before storage at −80°C until used. On the day of [18F]12ST05 synthesis, the slides were allowed to come to room temperature, and the sections incubated for 20 min in Tris phosphate-buffered saline buffer (138 mM NaCl, 2.7 mM KCl, pH adjusted to 7.6) containing 1 μCi/ml of [18F]12ST05. Adjacent sections were incubated in the same medium supplemented with 10 μM serotonin for estimation of nonspecific binding. After 20 min of incubation, the slides were rapidly dipped in distilled water, dried and juxtaposed to sensitive plates. The distribution of the radioactivity was then visualized with a Bio-imaging Analyser System (BAS-1800 II, Raytest, Germany) and analysed using the Multigauge software (Fujifilm, Raytest, Germany).
2.4. Animals
2.8. Ex vivo autoradiographies
These studies were performed in accordance with European guidelines for care of laboratory animals (86/609 EEC) and were approved by the ethical animal use committee of the Université de Lyon. Adult Sprague– Dawley males (Harlan, France) weighing 250–350 g were used for in vitro and ex vivo experiments. A European male cat weighing 3.5 kg (Charles River Laboratories, L'Arbresle, France) was used for PET scan acquisition.
Rats were anesthetized by urethane (1.7 g/kg ip) and an intravenous catheter was placed into their caudal vein. For the competition assays, 5-HT6 antagonists (SB-258585 or Ro 04-6790), or unlabeled 12ST05, were intravenously injected at the dose of 5 mg/kg, 30 min before an 55.5 Bq (1.5 mCi) intravenous injection of [18F]12ST05. Rats were killed by decapitation 20 min later. The brains were rapidly extracted, frozen and cut into 30-μm-thick sections using a
2.3. Quality control
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Fig. 2. (A) Preparative chromatogram of the radiosynthesis of [18F]12ST05 [(top) UV absorbance at 254 nm; (bottom) radioactivity]. The retention times of [18F] 12ST05 and its precursor are 13 and 14 min, respectively. The rectangle indicates the sampled fraction. (B) Chromatogram of the final [18F]12ST05 [(top) UV absorbance at 254 nm; (bottom) radioactivity]. The retention time of [18F]12ST05 is 7 min (indicated by an arrow). Note the absence of precursor and other labelled compounds (radiochemical purity N98%).
cryotome (Microm Microtech, France). The sections were air-dried and apposed on a phosphor imaging plate for 60 min. The distribution of the radioactivity was then visualized with a Bio-imaging Analyser System (BAS-1800 II, Raytest, Germany) and analyzed using the Multigauge software (Fujifilm, Raytest, Germany). Regions of interest (striatum, ventral hippocampus and cerebellum) were manually drawn with the help of a stereotaxic atlas of the rat brain [20]. Background noise was subtracted from all
regions of interest and the radioactivity was expressed in photostimulated luminescence per surface unit (psl/mm2). All experiments were performed in duplicate. 2.9. PET scan acquisition The cat PET acquisition was performed on a Siemens CTI Exact ECAT HR+ (Knoxville, TN, USA) used in threedimensional mode. The cat was anesthetized with 5% isoflurane for 5 min, which was then lowered to 2.5%. The
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3.2. In vitro lipophilicity evaluation and in vivo brain penetration The lipophilicity of [18F]12ST05, determined in vitro by the octanol/water partition coefficient (log P), was found to be 2.03±0.03 (n=5 experiments). This value was confirmed by the calculated log P (2.2 at pH 7.4; ACD/Labs software, version 7.0). Ex vivo, the radiotracer [18F]12ST05 readily entered the brain with a maximal access 20 min post injection. The calculated percentages of injected dose were 0.21%, 0.28%, 0.44%, 0.19%, respectively, at 5, 10, 20 and 60 min post injection. 3.3. In vitro autoradiographies in rats [18F]12ST05 binding on control sections was homogenous throughout the brain with regions like hippocampus and cerebellum bound to a semiquantitative comparable level. No significant binding difference was found between control sections and sections preincubated with serotonin (10 μM) (results not shown). 3.4. Ex vivo autoradiographies in rats Fig. 3. Ex vivo autoradiograms of brain sections 30 min after the preinjection of unlabeled 12ST05 (5 mg/kg iv) (lower images) in comparison to their respective controls (upper images). The histograms represent the corresponding [18F]12ST05 regional radioactivity intensity (psl/mm2).
Ex vivo autoradiographies in control rats, performed 20 min after the [18F]12ST05 injection, confirmed a high
head of the animal was immobilized in a stereotaxic Plexiglas frame with ear bars. A catheter was placed into the forearm branch of the brachiocephalic vein continuously perfused with NaCl 0.9%. A 10-min transmission scan was obtained with three rotating 68Ge–68Ga sources followed by a 74-MBq (2-mCi) bolus injection of [18F]12ST05. Radioactivity was thus measured in a series of 31 sequential frames of increasing duration from 30 s to 10 min. The total duration of a scan was 90 min. After acquisition, the images were reconstructed following the frontal plan. Regions of interest (striatum, thalamus, cingulate cortex, hippocampus and cerebellum) were processed manually according to the stereotaxic atlas of the cat brain [21,22]. Regional radioactivities were expressed in Becquerel per volume unit (Bq/cm3). 3. Results 3.1. Chemistry and radiochemistry Using a reprogrammed automated fluorination module, labelling of 3 from its nitro precursor at 150°C was straightforward, with a radiochemical yield of 32% corrected for decay and 50-min radiosynthesis time. No major radioactive by-products were observed, and HPLC conditions chosen ensured good separation of 3 from its nitro precursor, as confirmed by quality control (Fig. 2). Radiochemical purity was better than 95% and specific activity was between 85 and 120 GBq μmol−1, corrected at end of synthesis.
Fig. 4. Ex vivo autoradiograms of brain sections 30 min after the preinjection of 5-HT6 antagonists (SB-258585 and Ro 04-6790, 5 mg/kg iv) in comparison to their respective controls (upper illustrations). The histograms represent the corresponding [18F]12ST05 regional radioactivity intensity (psl/mm2).
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3.5. PET scan acquisition on a cat After intravenous injection of [18F]12ST05, there was a rapid accumulation of the radioligand in the cat brain within the first 10 min. The time activity curves showed a high initial uptake in all brain regions including striatum, hippocampus, thalamus, cingulate cortex and cerebellum (Fig. 5). Regions with high densities of 5-HT6 receptors like the striatum and the hippocampus presented an uptake ratio (relative to cerebellum) of, respectively, 1.39 and 1.40, 10 min after [18F]12ST05 injection. The highest uptake was observed in thalamus with an uptake ratio (thalamus/ cerebellum) of 1.55, remaining constant over the acquisition time. The radioactivity washout rates were comparable, for all structures, studied except cingulate cortex with a higher washout rate (Fig. 5). 4. Discussion
Fig. 5. PET [18F]12ST05 images of the cat brain in sagittal (A), coronal (B) and transversal (C) planes. Corresponding time–radioactivity curves are presented. Note the intense and peculiar thalamus labelling. Str, striatum; Thal, thalamus; Cg, cingulate cortex; Hip, hippocampus; Cer, cerebellum.
brain uptake of the tracer. The time of analysis was chosen according to the previous in vivo brain penetration results. Regions like striatum and hippocampus, known to be rich in 5-HT6 receptors, displayed a high [18F]12ST05 labelling (Fig. 3). It has to be noted that a similar radiotracer uptake was seen in cerebellum. The preinjection of unlabeled 12ST05 suppressed significantly (P b.05, analysis of variance and Student's t test) the binding of [18F] 12ST05 by 92% in all regions of the brain studied (Fig. 3). In other experiments, the preinjection of SB-258585, a 5-HT6 receptors antagonist, reduced [ 18 F]12ST05 fixation by 17% in striatum (P b.05), 11% in hippocampus and 8% in cerebellum (Fig. 4). Preinjection of Ro 04-6790, another potent antagonist of 5-HT6 receptors, was found to increase significantly [18F]12ST05 binding by 31% in striatum, 24% in hippocampus and 25% in cerebellum (P b.05; Fig. 4).
The feasibility of imaging the 5-HT6 receptors in the brain by PET studies represents an interesting field for PET imaging. To our knowledge, only a few studies have been done in this new area. In preliminary reports [16,17], the 5-HT6 receptor antagonists GSK215083 and GSK224558 have been radiolabelled with carbon-11 and were evaluated in pigs. However, to our knowledge, no clinical PET studies were recently performed with both radiotracers. In this context, we have recently been interested by a novel class of 4-(2-aminoethoxy)-N-(phenylsulfonyl)indoles with high affinities towards 5-HT6 receptors [18]. We selected one of these structures (3) exhibiting a high affinity towards the 5-HT6 receptors (Ki=4 nM) as well as an excellent selectivity against the closely related 5-HT7 receptors (N2000-fold). The precursor was synthesized, with a chemical structure slightly modified from the one described by Zhou et al. [18] since an NO2-group had to be added for the fluorination. After a classical fluorination via a nitro-/fluoronucleophilic substitution realized on a reprogrammed Coincidence (GE Mx) synthesizer, we obtained [18F]12ST05 in an amount suitable for the biological evaluations. The labelling was straightforward — not surprising given the presence of the parasubstituted sulfonamide. Although the lipophilicity experimentally determined is not systematically predictive of the blood-brain-barrier penetration [23], the [18F]12ST05 log P was encouraging. This in vitro result was confirmed by the radioactive brain distribution of [18F]12ST05 showing an excellent crossing of the blood-brain barrier in comparison to the known brain radiotracers [24]. However, the in vitro autoradiographies were disappointing, suggesting that [18F]12ST05 lacks sufficient affinity or selectivity. Since in vitro studies cannot be directly extrapolated to in vivo studies, it was crucial to perform ex vivo and in vitro studies. Ex vivo autoradiographic experiments in rats were conducted with the aim to evaluate the cerebral distribution of [18F]12ST05 in living animals. In control rats, striatum
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and hippocampus, rich in 5-HT6 receptors [10], showed a high labelling. Unexpectedly, the cerebellum, a region devoid of 5-HT6 receptors, presented a comparable high radioactive level, suggesting a high level of nonspecific binding. The preinjection with unlabelled 12ST05 blocked the binding of [18F]12ST05, showing that its fixation is saturable. In order to determine the specificity of the binding of [18F]12ST05, competition experiments were performed with a well-described 5-HT6 receptor antagonist, SB-258585 [9,10]. The slight reduction of [18F]12ST05 binding in striatum and hippocampus after SB-258585 injection is not sufficient to demonstrate that [18F]12ST05 binds partially to 5-HT6 receptors, since the cerebellum was also reduced in uptake (although not significantly). To further assess the specificity of [18F]12ST05, rats were pretreated with another 5-HT6 antagonist, Ro 04-6790, which crosses the bloodbrain barrier more easily [25]. Unexpectedly, the preinjection of this 5-HT6 antagonist increased [18F]12ST05 binding in striatum, hippocampus and cerebellum. The more plausible explanation could be that Ro 04-6790 acutely increases the cerebral blood flow and modifies the brain penetration of the radiotracer. Finally, we performed a PET scan acquisition on an anaesthetized cat. The cat brain is of sufficient size to allow the use of human-oriented PET cameras in the perspective of a clinical use of the new PET tracer. The [18F]12ST05 rapid brain penetration was consistent with the previous ex vivo studies in rats. Although the thalamus does not present a high density of 5-HT6 receptors, this region was more highly marked than striatum and hippocampus in the first 10 min of acquisition. This PET scan assay demonstrated a homogenous fixation of [18F]12ST05, consistent with a good brain penetration but confirming the lack of ability of this radiotracer to image 5-HT6 receptors. The failure of [18F]12ST05 to label in vivo 5-HT6 receptors can be explained by two factors. The first factor is that the addition of a fluorine atom during the radiolabelling decreased significantly the affinity of 12ST05 for the 5-HT6 receptors. We confirmed recently this hypothesis by the in vitro determination of the affinity of the nonradioactive fluorinated 12ST05 (binding assays on 5-HT6 receptors expressed in Chinese hamster ovary cells vs. [3H]LSD). The 4-fluoro substitution caused an apparent fivefold lowering of affinity for the 5-HT6 receptors (Ki=20 nM, to be compared with the initial value of 4 nM; Cerep France, personal communication). In this same context, a structure–activity relationship study reported previously that the fluorine addition to a phenylsulfonyl derivate (giving the 4-fluorophenylsulfonyl derivate) decreases its affinity toward 5-HT6 receptor [26]. These results highlight the influence of fluorine addition during radiolabeling on the electronic configuration of the 5-HT6 ligand pharmacophore. The second factor explaining this failure is that [18F]12ST05 appeared to have a high affinity to a binding site, blocked by the addition of unlabelled 12ST05. Our results showed that this saturable site is not the 5-HT6 receptor, nor probably
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another serotoninergic receptor (serotonin preincubations did not modify the binding). PET scan obtained in the anaesthetized cat showed that this unknown site is more concentrated in the thalamus. A Scatchard plot could be performed to define if this site is unique and to determine its density. In conclusion, we reported in this study the radiolabelling of a 5-HT6 receptor antagonist. Our preliminary biological results, obtained with complementary ex vivo and in vivo approaches in two different species, demonstrate the inability of [18F]12ST05 to visualize 5-HT6 receptors in vivo. Because the discovery of a potent 5-HT6 radiotracer is crucial for the exploration of brain serotoninergic function, other 5-HT6 leads will be evaluated for future PET investigations. References [1] Cross AJ. Serotonin in Alzheimer-type dementia and other dementing illnesses. Ann NY Acad Sci 1990;600:405–17. [2] Hoyer D, Clarke DE, Fozard JR, Hartig PR, Martin GR, Mylecharane EJ, et al. International Union of Pharmacology classification of receptors for 5-hydroxytryptamine (Serotonin). Pharmacol Rev 1994;446:157–203. [3] Hoyer D, Martin GR. Classification and nomenclature of 5-HT receptors: a comment on current issues. Behav Brain Res 1996;73: 263–8. [4] Monsma FJ, Shen Y, Ward RP, Hamblin MW, Sibley DR. Cloning and expression of a novel serotonin receptor with high affinity for tricyclic psychotropic drugs. Mol Pharmacol 1993;43:320–7. [5] Ruat M, Traiffort E, Arrang JM, Tardivel-Lacombe J, Diaz J, Leurs R, et al. A novel rat serotonin (5-HT6) receptor: molecular cloning, localization and stimulation of cAMP accumulation. Biochem Biophys Res Commun 1993;193:268–76. [6] Gérard C, Martres NP, Lefèvre K, Miquel MC, Vergé D, Lanfumey L, et al. Immuno-localisation of serotonin 5-HT6 receptor-like material in the rat central nervous system. Brain Res 1997;746:207–19. [7] Hamon M, Doucet E, Lefevre K, Miquel MC, Lanfumey L, Insausti R, et al. Antibodies and antisense oligonucleotide for probing the distribution and putative functions of central 5-HT6 receptors. Neuropsychopharmacology 1998;21:68–76. [8] Boess FG, Riemer C, Bos M, Bentley J, Bourson A, Sleight AJ. The 5-hydroxytryptamine6 receptor-selective radioligand [3H]Ro 63-0563 labels 5-hydroxytryptamine receptor binding sites in rat and porcine striatum. Mol Pharmacol 1998;54:577–83. [9] Hirst WD, Minton JAL, Bromidge SM, Moss SF, Latter AJ, Riley G, et al. Characterization of [125I]-SB-258585 binding to human recombinant and native 5-HT6 receptors in rat, pig and human brain tissue. Br J Pharmacol 2000;130:1597–605. [10] Roberts JC, Reavill C, East SZ, Harrisson PJ, Patel S, Routledge C, et al. The distribution of 5-HT6 receptors in rat brain: an autoradiographic binding study using the radiolabelled 5-HT6 receptor antagonist [125I]SB-258585. Brain Res 2002;934:49–57. [11] Gérard C, El Mestikawy S, Lebrand C, Adrien J, Ruat M, Traiffort E, et al. Quantitative RT-PCR distribution of serotonin 5-HT6 receptor mRNA in the central nervous system of control or 5,7-dihydroxytryptamine-treated rats. Synapse 1996;23:164–73. [12] Woolley ML, Marsden CA, Fone KCF. 5-HT6 receptors. Curr Drug Targets CNS Neurol Disord 2004;3:59–79. [13] Mitchell ES, Neumaier JF. 5-HT6 receptors: a novel target for cognitive enhancement. Pharmacol Ther 2005;108:320–33. [14] East SZ, Burnet PWJ, Leslie RA, Roberts JC, Harrison PJ. 5-HT6 receptor binding sites in schizophrenia and following antipsychotic drug administration: autoradiographic studies with [125I]SB-258585. Synapse 2002;45:191–9.
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[21] Jasper HH, Ajmone-Marsan C. A stereotaxic atlas of the diencephalon of the cat. Ottawa: The National Research Council of Canada editors; 1954. [22] Snider RS, Niemer WT. A stereotaxic atlas of the cat brain. Chicago: University of Chicago Press; 1961. [23] Wilson AA, Jin L, Garcia A, DaSilva JN, Houle S. An admonition when measuring the lipophilicity of radiotracers using counting techniques. Appl Radiat Isot 2001;54:203–8. [24] Waterhouse RN. Determination of lipophilicity and its use as a predictor of blood-brain-barrier penetration of molecular imaging agents. Mol Imaging Biol 2003;5:376–89. [25] Bös M, Sleight AJ, Godel T, Martin JR, Riemer C, Stadler H. 5-HT6 receptor antagonists: lead optimisation and biological evaluation of N-aryl and N-heteroaryl 4-amino-benzene sulfonamides. Eur J Med Chem 2001;36:165–78. [26] Cole DC, Lennox WJ, Stock JR, Ellingboe JW, Mazandarani H, Smith DL, et al. Conformationally constrained N1-arylsulfonyltryptamine derivatives as 5-HT6 receptor antagonists. Bioorg Med Chem 2005;15:4780–5.