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Keywords:

  • Ro 04-6790;
  • Ro 63-0563;
  • 5-HT6 receptor;
  • 5-HT6 receptor antagonists;
  • stretching

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References
  • This study describes the in vitro characterization of two potent and selective 5-HT6 receptor antagonists at the rat and human recombinant 5-HT6 receptor.

  • In binding assays with [3H]-LSD, 4-amino-N-(2,6 bis-methylamino-pyrimidin-4-yl)-benzene sulphonamide (Ro 04-6790) and 4-amino-N-(2,6 bis-methylamino-pyridin-4-yl)-benzene sulphonamide (Ro 63-0563) had mean pKi values ±s.e.mean at the rat 5-HT6 receptor of 7.35±0.04 and 7.83±0.01, respectively and pKi values at the human 5-HT6 receptor of 7.26±0.06 and 7.91±0.02, respectively.

  • Both compounds were found to be over 100 fold selective for the 5-HT6 receptor compared to 23 (Ro 04-6790) and 69 (Ro 63-0563) other receptor binding sites.

  • In functional studies, neither compound had any significant effect on basal levels of cyclicAMP accumulation in Hela cells stably expressing the human 5-HT6 receptor, suggesting that the compounds are neither agonists nor inverse agonists at the 5-HT6 receptor. However, both Ro 04-6790 and Ro 63-0563 behaved as competitive antagonists with mean ±s.e.mean pA2 values of 6.75±0.07 and 7.10±0.09, respectively.

  • In rats habituated to observation cages, Ro 04-6790 produced a behavioural syndrome similar to that seen following treatment with antisense oligonucleotides designed to reduce the expression of 5-HT6 receptors. This behavioural syndrome consisted of stretching, yawning and chewing.

  • Ro 04-6790 and Ro 63-0563 represent valuable pharmacological tools for the identification of 5-HT6 receptors in natural tissues and the study of their physiological function.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

The effects of the neurotransmitter 5-hydroxytryptamine (5-HT) are mediated through at least fourteen distinct receptors. The known 5-HT receptors comprise a ligand gated ion channel (the 5-HT3 receptor) and 13 G-protein coupled receptors (Hoyer et al., 1994; Boess & Martin, 1994).

The 5-HT6 receptor was first isolated from rat striatal mRNA by reverse transcription and polymerase chain reaction, with degenerate oligonucleotide primers derived from conserved regions of known G-protein coupled receptors (Monsma et al., 1993) or by low stringency screening with probes derived from the rat histamine H2 receptor (Ruat et al., 1993). Subsequently, the human 5-HT6 receptor has been isolated (Kohen et al., 1994). In rats the highest levels of 5-HT6 receptor mRNA are present in olfactory tubercle, nucleus accumbens, striatum and hippocampus (Monsma et al., 1993; Ruat et al., 1993; Ward et al., 1995; Gerard et al., 1996). In addition to these regions, 5-HT6 receptor-like immunoreactivity was also identified in frontal and entorhinal cortex and the molecular layer of the cerebellum (Gerard et al., 1997).

The 5-HT6 receptor is positively coupled to adenylyl cyclase and can be radiolabelled with [125I]-lysergic acid diethylamide (LSD), [3H]-LSD and [3H]-5-HT (Monsma et al., 1993; Boess et al., 1997). Many non-selective compounds such as tricyclic antidepressants, antipsychotic agents, tryptamine and ergoline derivatives bind to the 5-HT6 receptor with high affinity (Monsma et al., 1993; Roth et al., 1994; Boess et al., 1997) but to date no selective ligands for the 5-HT6 receptor have been described. Therefore, the identification of functional 5-HT6 receptors in physiological preparations could only be tentative based on the rank order of potency non-selective compounds (Sleight et al., 1997).

The only study exploring the functional significance of the receptor in vivo used antisense-oligonucleotides which should abolish or reduce the expression of the 5-HT6 receptor protein. Intracerebroventricular treatment of rats with 5-HT6 specific antisense-oligonucleotides produced a behavioural syndrome consisting of yawning, stretching and chewing which could be antagonized by atropine but not by haloperidol (Bourson et al., 1995; Sleight et al., 1996).

The further study of 5-HT6 receptors and their physiological function requires potent and selective ligands for the receptor. Here we characterize 2,6-dimethylamino-4-sulphanilamidopyrimidin (Ro 04–6790) and 2,6-dimethylamino-4-sulphanilamido-pyridine (Ro 63–0563) as potent and selective antagonists at the 5-HT6 receptor (Figure 1). These compounds were also used to determine whether 5-HT6 antagonists could produce the same behavioural syndrome as that induced by treatment with antisense oligonucleotides in vivo.

image

Figure 1. Chemical structures of Ro 04–6790 and Ro 63–0563.

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Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Animals

Male Sprague-Dawley rats (Füllinsdorf, Switzerland), weighing 250–300 g, were used for the experiment. They were housed in groups of 4 under controlled conditions of temperature (21±1°C) and humidity (55–65%) and under a 12 h light-dark cycle. Rats were allowed free access to food and water.

Receptor binding assays

The affinities of Ro 04–6790 and Ro 63–0563 at rat and human 5-HT6 receptors were measured on membranes obtained from HEK 293 and Hela cells stably expressing the rat and human receptors, respectively. HEK 293 cells were grown in Dulbecco's modified Eagle's medium (DMEM) + 10% foetal bovine serum (FBS) containing penicillin (100 iu ml−1), streptomycin (100 μg ml−1) and 0.5 mg ml−1 geneticin in a humidified atmosphere (5% CO2). Hela cells were grown in exactly the same conditions except that geneticin was omitted from the media. The cells were detached with phosphate buffered saline (PBS) containing 1 mM EDTA, washed with PBS by two centrifugations (10 min, 500 g) and the resulting pellet was resuspended in 50 mM, ice-cold Tris-HCl (pH 7.4), containing 10 mM MgCl2 and 0.5 mM EDTA by use of a polytron homogenizer (15 sec at maximal speed), at a concentration corresponding to 4 × 107 cells ml−1 and aliquots were stored at — 80°C. 5-HT6 receptor binding assays were performed with [3H]-lysergic acid diethylamide ([3H]-LSD; specific activity 86 Ci mmol−1, Amersham). Membranes corresponding to 4 × 105 cells/assay tube were used for the binding assay, resuspended in an assay buffer consisting of Tris-HCl 50 mM, pargyline 10−5 M, MgCl2 5 mM and ascorbic acid 0.1%, pH 7.4. For estimations of the expression levels and the affinity of [3H]-LSD for the receptor binding sites, saturation experiments were performed with 8 concentrations of [3H]-LSD (0.163–20 nM). Competition curves were constructed with 7 concentrations of the displacing agents (1 data point per log unit of concentration: 10−10 M to 10−4 m). Binding assays consisted of 100 μl of the membrane preparation expressing the 5-HT6 receptor, 50 μl of [3H]-LSD and 50 μl of a displacing drug or assay buffer. Non-specific binding was measured in the presence of 10−5 M 5-HT. Incubations were carried out for 1 h at 37°C and reactions were stopped by rapid filtration through Whatmann GF/B filters by use of a Filtermate 196 (Packard Canberra). The filters were washed with 3 × 2 ml Tris HCl (50 mM, pH 7.4) and the radioactivity retained on the filters was measured by scintillation spectroscopy in 50 μl of scintillation fluid. All experiments were performed in triplicate and repeated 3 times. The dissociation constants for [3H]-LSD binding to both human and rat 5-HT6 receptors, IC50 values, Ki values and Hill coefficients were calculated by use of EBDA and LIGAND (Munson & Rodbard, 1980; McPherson, 1985).

In all other receptor binding assays, Ro 04–6790 and Ro 63–0563 were tested at a single concentration (10−5 m) by the methodology described in Table 1. If either compound displaced more than 50% of the specific binding in any of these assays, it was retested at multiple concentrations to estimate pKi values. Ro 63–0563 was also tested in a further 45 binding assays by CEREP (Le Bois l'Eveque, 86600 Celle L'Evescault, France). Here the compound was tested at concentrations of 0.1 and 10 μM at the adenosine receptor subtypes 1 and 2 (rat) and 3 (human); angiotensin receptor subtypes 1 (rat) and 2 (bovine); bradykinin receptor subtypes 1 (rat) and 2 (human); calcitonin gene-related peptide receptors; cholecystokinin receptor subtypes A and B (human); endothelin receptor subtypes A and B (human); GABAA and GABAB receptor subtypes (rat); galanin receptors; glycine receptors (both strychnine sensitive and insensitive); histamine receptor subtypes H1 (CNS, guinea-pig) and H1 (peripheral, guinea-pig), H2 (guinea pig) and H3 (rat); melatonin receptors (chicken); neuropeptide Y receptor 1 and 2 subtypes; neurokinin receptor subtypes 1, 2 and 3 (human); neurotensin receptors; P2X and P2Y purinoceptors (rat); somatostatin receptors. Ro 63–0563 (0.1 μm and 10 μm) was also tested at adenosine (guinea-pig), noradrenaline (rat), dopamine (rat) and 5-HT (rat) uptake sites and at various ion channels, including several subtypes of calcium channels (L, DHP site, L, ditiazem site, L, verapamil site and N), potassium channels (ATP-sensitive, voltage-dependent and Ca2+-dependent) and sodium channels.

Table 1. Methodologies for the binding assays used to determine the selectivity of Ro 04–6790 and R0 63–0563
ReceptorTissue/expression systemRadioligand (concentration)Non-specific ligand (concentration)Incubation conditions
  1. The compounds were tested at a single concentration of 10−5 m and if more than 50% of the specific binding was displaced at this concentration, full displacement curves could be constructed to determine pKi values.

h5-HT1A 5-HT1BHuman recombinant /3T3 cells Rat striatum[3H]-5-HT (1 nm) [3H]-5-HT (1 nm) 8-OH-DPAT (300 nm) Mesulergine (300 nm)5-HT (10 μM) 5-HT (10 μm)60 min: Room temp. 60 min: Room temp.
h5-HT1DHuman recombinant /HEK 293 cells[3H]-LSD (2 nm)5-HT (10 μm)60 min: Room temp.
h5-HT2AHuman recombinant /3T3 cells[3H]-DOB (1 nm)Methysergide (10 μm)60 min: Room temp.
h5-HT2CHuman recombinant /3T3 cells[3H]-5-HT (1 nm)5HT (10 μM60 min: Room temp.
5-HT3N18 cells[3H]-GR 65630 (0.2 nm)Metoclopramide (10 μm)60 min: 4°C
5-HT4Guinea-pig striatum[3H]-GR 113808 (0.1 nm)5-HT (30 μM)30 min: 37°C
h5-HT7Human recombinant /CHO cells[3H]-LSD (2 nm)Methiothepin (10 μm)60 min: 37°C
hD1Human recombinant /GH4 cells[3H]-SCH 23390 (0.5 nM)(+)-Butaclamol (10 μm)45 min: Room temp.
hD2Human recombinant /CHO cells[3H]-spiperone (1.6 nM)(+)-Butaclamol (10 μm)60 min: Room temp.
hD3Human recombinant /CHO cells[3H]-YM-09151–2 (0.1 nM)(+)-Spiperone (10 μm)60 min: Room temp.
hD4Human recombinant /CHO cells[3H]-spiperone (1.6 nM)(+)-Butaclamol (10 μm)60 min: Room temp.
hD5Human recombinant /CHO cells[3H]-SCH 23390 (1 nM)(+)-Butaclamol (10 μm)60 min: Room temp.
α1-AdrenoceptorRat cortex[H]-prazocin (1 nM)Phentolamine (10 μm)60 min: Room temp.
α2-AdrenoceptorRat cortex[3H]-clonidine (1 nM)Yohimbine (10 μm)60 min: Room temp.
β-AdrenoceptorRat cortex[3H]-DHA (0.2 nM)Propranolol (10 μm)60 min: Room temp.
hM1Human recombinant /CHO cells[3H]-pirenzepine (2 nM)Atropine (1 μm)60 min: Room temp.
hM2Human recombinant /CHO cells[3H]-AF-DX 384 (3 nM)Atropine (1 μm)60 min: Room temp.
hM3Human recombinant /CHO cells[3H]-4-DAMP (0.2 nM)Atropine (1 μm)60 min: Room temp.
hM4Human recombinant /CHO[3H]-4-DAMP (0.2 nM)Atropine (1 μm)60 min: Room temp.
hM5Human recombinant /CHO[3H]-4-DAMP (0.2 nM)Atropine (1 μm)60 min: Room temp.
μ-OpioidRat recombinant /CHO[3H]-naloxone (3 nM)Naloxone (10 μm)30 min: Room temp.
k-OpioidRat recombinant /CHO[3H]-naloxoneNaloxone (10 μm)30 min: Room temp.
BenzodiazepineRat cortex[3H]-flumazenil (1 nM)Diazepam (10 μm)30 min: 4°C

Adenylyl cyclase measurements

Hela cells expressing human recombinant 5-HT6 receptors were grown to 90% confluency in DMEM +10% FBS (dialyzed), washed once with DMEM without phenol red (DMEM-), detached with PBS +1 mM EDTA and washed 2 × with DMEM (450 g, 5 min). The final cell density was adjusted to approximately 1.25 × 106 cells ml−1. Aliquots of 80 μl were transferred to 96 well plates (approximately 105 cells/well) and incubated at 37°C in a humidified atmosphere for 30 min. 5-HT, combined with pargyline and the phosphodiesterase inhibitor Ro 20–1724, was added in a volume of 20 μl/well (final incubation volume 100 μl/well, final concentration of pargyline and Ro 20–1724: 20 μM and 100 μM, respectively). In order to determine the agonist potential of the two compounds, full concentration-response curves were constructed with Ro 04–6790 and Ro 63–0563 at the h5-HT6 receptor and compared to 5-HT as a positive control. To determine the antagonist affinities of the 2 compounds full dose-response curves to 5-HT were constructed in the presence of either 1, 3, 10 and 30 μM Ro 04–6790 or 0.1, 0.3, 1 and 3 μM Ro 63–0563. After a period of 20 min at 37°C in a humidified atmosphere (5% CO2), the incubation was terminated by the addition of 200 μl ethanol/well. All experiments were performed in triplicate and repeated at least 3 times. After at least 2 h at — 20°C, the plates were centrifuged for 5 min at 470 g (4°C) and 75 μl aliquots of the supernatant were transferred to Packard OptiPlates, evaporated under vacuum, and resuspended in 0.05 m acetate buffer. The concentration of adenosine 3′:5′-cyclic monophosphate (cyclicAMP) was determined by use of the BIOTRAK cyclicAMP [125I] scintillation proximity assay (SPA) system (Amersham) adapted to 96-well plates. Agonist dose-response curves were analysed from the equation; E = B + Emax × x/(EC50 + x), where × is the concentration of agonist, E and Emax the measured and the maximum effect (cyclicAMP/well), EC50 is the concentration of agonist producing 50% of the response and B the basal cyclicAMP level. pA2 values were calculated as described by Arunlakshana & Schild (1959) by calculating the ratio of the EC50 for 5-HT in the presence or absence of various concentrations of each antagonist. A pA2 value was calculated for each separate experiment together with the gradient of the Schild regression. The experiment was performed 3 times and the mean pA2 values ± s.e.mean and mean slopes ± s.e.mean were calculated for both Ro 04–6790 and Ro 63–0563.

Measurement of Ro 04–6790 and Ro 63–0563 in CSF and plasma

Rats were injected with Ro 04–6790 (30 mg kg−1, i.p.) or Ro 63–0563 (10 mg kg−1, i.v.) and 0.5, 1, 2, 3 and 6 h later the rats were anaesthetized with 5% isoflurane 95% O2. CSF samples were taken from the cisterna magna by inserting a 22 G needle between the 1st and 2nd cervical vertebra. A sample of blood was then removed from the aorta and stored in tubes each containing 7 μg EDTA and 5 μg NaF to prevent coagulation. Blood and CSF samples were taken from 3 rats 0.5 h following administration of the 5-HT6 antagonists, from 2 rats 1 h after administration and from 1 rat at 2, 3 and 6 h after. Blood samples were centrifuged for 5 min at 1200 r.p.m. and CSF and plasma samples were stored at — 20°C until analysis.

Samples were analysed by high performance liquid chromatography with ultra violet detection (h.p.l.c.-u.v.) with gradient elution and a 150 × 4 mm analytical column packed with Inertsil ODS-3.5 μm (GL Sciences Inc. U.S.A.). The two mobile phases contained a mixture of acetonitrile and 50 mM NaHPO4 (pH 5.5). U.V. detection was performed at 290 nm for Ro 04–6790 and 270 nm for Ro 63–0563. Plasma samples of 100 μl were deproteinated by adding 100 μl HC104 (1 n). Following centrifugation for 5 min at 1200 r.p.m., 100 μl of the supernatant were injected onto the column. CSF samples (50 μl) were diluted with 50 μl H2O and injected directly onto the column. The limits of detection of Ro 04–6790 in CSF and plasma were 6 ng ml−1 and 80 ng ml−1, respectively, and those for Ro 63–0563 were 8 ng ml−1 and 80 ng ml−1, respectively.

Behavioural observations

Every day for 4 consecutive days, rats were injected intraperitoneally with saline (1 ml kg−1) and immediately placed in transparent plexiglass cages (55 × 34 × 18 cm) in groups of 4 for 1 h. A mirror was positioned behind the cages to allow all-round observation of the rats. On the 5th day, rats received either Ro 04–6790 (3, 10, 30 mg kg−1, i.p.) or saline and were again immediately placed in the observation boxes (4 groups of 4 per cage) into which they had been habituated with one animal per treatment group in each box. The number of yawns and stretches was counted by direct observation for a period of 60 min from the time that the rats were treated with the 5-HT6 antagonist. The experiments were performed in this manner so that the results obtained could be compared with those obtained in animals treated with antisense oligonucleotides designed to block the translation of 5-HT6 receptors (Bourson et al., 1995). In these published experiments, the animals were injected daily with the oligonucleotides and then placed in observation cages each day. These experiments were carried out between 8 h 30 min and 12 h 30 min; all behavioural studies were performed on a‘blind’ basis.

Analysis of behavioural data

The results are expressed as the mean number of yawns or stretches per hour per group. Although these data were not normally distributed, in Figure 7, s.e.mean values are also given for an indication of variability. A Kruskall-Wallis analysis of variance was first carried out, and if significant, a Mann-Whitney U test was used to compare differences between treatment groups.

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Figure 7. Effect of treatment with either saline, Ro 04–6790 (3 mg kg−1, i.p.), Ro 04–6790 (10 mg kg−1, i.p.) or Ro 04–6790 (30 mg kg−1, i.p.) on the number of stretches (mean ± s.e.mean) counted for a period of 1 h immediately after treatment with the 5-HT6 antagonist. Eight animals were used per treatment group and animals were habituated to the observation cages for a 1 h period on each of the 4 days preceding treatment. In order to minimize the stress effects of injections on the test day, the animals were injected with saline (i.p.) on each of the habituation days. * P < 0.05.

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Materials

[3H]-5-HT, [3H]-LSD, [3H]-GR 113808 ([1[2-(methylsuphonyl)-aminoethyl] −4- piperidinyl]methyl — 1-methyl-1H-indole-3-carboxylate), [3H]-SCH 23390 (7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride), [3H]-spiperone, [3H]-prazocin, [3H]-DHA (dihydroalprenolol) and [3H]-flumazenil were purchased from Amersham (U.K.) and [3H]-DOB ([3H]4-bromo-2,5-dimethoxyphenylisopropylamine), [3H]-GR 65630 ([3H]3-(5-methyl-1H-imidazol-4-yl)-l-(1 — methyl −1H — indol — 3 — yl) −1 — propanone), [3H] — YM-09151–2 ([3H](μ)-cis-N-l-benyl-2-methylpyrrolidin-3-yl)-5-chloro-2-methyoxy-4-methylamiono benzamide), [3H]-clonidine, [3H]-pirenzepine, [3H]-AF-DX 384 ([3H](±)-5,11-dihydro-11-[2–2-[(dipropylamino)methyl] — 1 — piperidinylethylamino]carbonyl-6H — pyrido(2,3-b)(1,4) — benzodiazepine — 6 — one), [3H] — DAMP and [3H]-naloxone were purchased from New England Nuclear, (U.S.A.). 5-HT was purchased from Fluka (Switzerland). Atropine, (+)-butaclamol, metoclopramide, methiothepin, methysergide, naloxone, pargyline phentolamine, propranolol, spiperone and yohimbine were purchased from Research Biochemicals International U.S.A. DMEM, FBS, penicillin, streptomycin and geneticin were obtained from GIBCO Life Technologies and Ro 20–1724 (4-(3-butoxy-4-methylbenzyl)-2-inidazolidinone), Ro 04–6790, Ro 63–0563 and diazepam were synthesized at F. Hoffmann-La Roche.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

[3H]-LSD labelled recombinant rat 5-HT6 receptors with a dissociation constant (Kd) of 1.9 ± 0.1 nM and recombinant human 5-HT6 receptors with a Kd of 1.6 ± 0.1 nM. As can be seen in Figure 2, both Ro 04–6790 and Ro 63–0563 displaced [3H]-LSD from both the human and the rat 5-HT6 receptor with high affinity. The affinity values (mean ± s.e.mean pKi) of Ro 04–6790 for the rat and human 5-HT6 receptor were 7.35 ± 0.04 and 7.26 ± 0.06, respectively (Table 2). Ro 63–0563 had higher affinity than Ro 04–6790 for the rat and human 5-HT6 receptors with pKi values of 7.83 ± 0.01 and 7.91 ± 0.02, respectively (Table 2). The curves shown in Figure 2 represent the means of 3 experiments for both compounds.

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Figure 2. Displacement of [3H]-LSD from rat recombinant 5-HT6 receptors by Ro 04–6790 and Ro 63–0563 and from human recombinant 5-HT6 receptors by Ro 04–6790 and Ro 63–0563. Each data point represents the mean of 3 separate competition experiments; vertical lines show s.e.mean.

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Table 2. Affinities of Ro 04–6790 and Ro 63–0563 for various binding sites
ReceptorRo 04–6790Ro 63–0563
  1. Affinities are given as pKi values except where 10 μM of the displacing agent failed to displace 50% of the specific binding. ND = not determined.

h5-HT67.26 ± 0.067.91μ 0.02
r5-HT67.35 ± 0.047.83 ± 0.01
h5-HT1AIC50 > 10 μMIC50 > 10 μM
5-HT1BIC50 > 10 μMIC50 > 10 μM
h5-HT1DIC50 > 10 μMIC50 > 10 μM
h5-HT2AIC50 > 10 μM5.32 ± 0.02
h5-HT2CIC50 > 10 μM5.69 ± 0.01
5-HT3IC50 > 10 μMIC50 > 10 μM
5-HT4NDIC50 > 10 μM
h5-HT7IC50 > 10 μMIC50 > 10 μM
hD12IC50 > 10 μMIC50 > 10 μM
hD2IC50 > 10 μMIC50 > 10 μM
hD3IC50 > 10 μMIC50 > 10 μM
hD4IC50 > 10 μMIC50 > 10 μM
hD5IC50 > 10 μMIC50 > 10 μM
α1 -AdrenoceptorIC50 > 10 μMIC50 > 10 μM
α2-AdrenoceptorIC50 > 10 μMIC50 > 10 μM
β-AdrenoceptorIC50 > 10 μMIC50 > 10 μM
hM1IC50 > 10 μMIC50 > 10 μM
hM2IC50 > 10 μMIC50 > 10 μM
hM3IC50 > 10 μMIC50 > 10 μM
hM4IC50 > 10 μMIC50 > 10 μM
hM5IC50 > 10 μMIC50 > 10 μM
r μ-OpioidIC50 > 10 μMIC50 > 10 μM
r k-OpioidIC50 > 10 μMIC50 > 10 μM
BenzodiazepineIC50 > 10 μMIC50 > 10 μM

Ro 04–6790 has over 100 fold selectivity for the 5-HT6 receptor with respect to the other receptor binding sites examined. We were unable to measure the affinity of Ro 04–6790 for any of the other 23 receptor binding sites studied (Table 2), since it failed to displace more than 50% of the specific binding at a concentration of 10 μM. Indeed, for all of the other 5-HT receptors tested there was no displacement of specific binding by 10 μM Ro 04–6790.

Ro 63–0563 also had over 100 fold selectivity for the 5-HT6 receptor with respect to the other binding sites tested. The only other receptors for which Ro 63–0563 had any measurable affinity were the 5-HT2A and the 5-HT2C receptors with pKi values of 5.32 ± 0.02 and 5.69 ± 0.01, respectively (Table 2). In all other binding assays, Ro 63–0563 failed to displace more than 50% of the specific binding at a concentration of 10 μM. This was also true for all the binding sites tested at CEREP (see Methods section).

At the human 5-HT6 receptor, neither Ro 04–6790 nor Ro 63–0563 had any agonist activity (Figure 3), since they had no effect on the accumulation of cyclicAMP. In addition no inverse agonist activity was seen in that the compounds themselves did not significantly reduce basal accumulation of cyclicAMP (Figure 3). However, both Ro 04–6790 and Ro 63–0563 behaved as competitive antagonists. Ro 04–6790 had a pA2 value of 6.75 ± 0.07 calculated from Schild regressions with a slope of 1.16 ± 0.04. The pA2 value of Ro 63–0563 at the 5-HT6 receptor was 7.10 ± 0.09 calculated from Schild regressions with a slope of 1.14 ± 0.13. All of these values are means ± s.e.mean of 3 separate experiments each with 4 concentrations of each antagonist. Figure 4 shows a typical example of concentration-response curves to 5-HT in the presence of different concentrations of Ro 04–6790 and, likewise, Figure 5 shows a typical example of concentration-response curves to 5-HT in the presence of different concentrations of Ro 63–0563.

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Figure 3. Effect of 5-HT, Ro 04–6790 and Ro 63–0563 on cyclicAMP accumulation in Hela cells stably expressing the recombinant human 5-HT6 receptor. Data points represent the means of at least 3 separate experiments.

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Figure 4. Stimulation of cyclicAMP accumulation in Hela cells stably expressing the human 5-HT6 receptor by either 5-HT alone or 5-HT in the presence of Ro 04–6790 (1, 3, 10 or 30 μM). This figure shows a typical example of 3 separate experiments. Inset, Schild regression of the data.

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Figure 5. Stimulation of cyclicAMP accumulation in Hela cells stably expressing the human 5-HT6 receptor by either 5-HT alone or 5-HT in the presence of Ro 63–0563 (0.1, 0.3, 1 or 3 μm). This figure shows a typical example of 3 separate experiments. Inset, Schild analysis of the data.

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The measurement of Ro 04–6790 and Ro 63–0563 in both plasma and cerebrospinal fluid (CSF) showed that a small but significant fraction of the plasma concentration of Ro 04–6790 could be detected in the CSF (Figure 6). After treatment with Ro 04–6790 (30 mg kg−1, i.p.), its concentration in the CSF was sufficient to occupy more than 70% of the 5-HT6 receptors. Ro 63–0563 was detected in plasma at similar concentrations to Ro 04–6790 but could not be detected in the CSF at any time point (results not shown). Therefore, Ro 04–6790 alone was used for behavioural studies, to determine whether the same behavioural syndrome could be produced as in rats treated with antisense oligonucleotides designed to prevent the translation of 5-HT6 mRNA. Animals were habituated to the observation cages for 4 days. On the 5th day, rats treated with Ro 04–6790 exhibited a behavioural syndrome of yawning, stretching and chewing identical to that produced by antisense oligonucleotides. As can be seen from Figure 7, Ro 04–6790 produced a dose-related and statistically significant increase in the number of stretches observed over a 1 h period immediately after treatment. A maximal effect was observed at a dose of 30 mg kg−1, i.p., which is in agreement with the concentration of Ro 04–6790 in the CSF being sufficient to occupy 5-HT6 receptors. Similar results were observed for yawning. However, this failed to reach significance (P = 0.17, data not shown).

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Figure 6. Mean concentrations of Ro 04–6790 in rat plasma and CSF at various time points following administration of Ro 04–6790 (30 mg kg−1, i.p.). Separate rats were used for measurements at each time point. Data points at 0.5 h are the mean ± s.e.mean of determinations from 3 rats, at the 1 h time point, 2 rats were used and at all other time points 1 rat was used.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

The present study describes two compounds, Ro 04–6790 and Ro 63–0563, which are potent and highly selective antagonists for the cloned 5-HT6 receptor (Monsma et al., 1993; Ruat et al., 1993; Kohen et al., 1994; Boess et al., 1997). Both compounds had similar binding affinities at the rat and human 5-HT6 receptor showing that there are no species differences between the human and the rat receptors for these compounds. Furthermore, both compounds were highly selective for the 5-HT6 receptor, with Ro 04–6790 having over 100 fold selectivity with respect to 23 other binding sites including 8 5-HT receptors. The same was true for Ro 63–0563 which was also tested in a further 45 binding assays by CEREP. In total, Ro 63–0563 was tested in 69 binding assays and was found to have over 100 fold selectivity for the 5-HT6 receptor. When studied in functional assays, neither compound had any significant effect on basal cyclicAMP accumulation, suggesting that they are not either agonists or inverse agonists. However, both compounds behaved as competitive antagonists at the human 5-HT6 receptor with pA2 values of 6.75 ± 0.07 and 7.10 ± 0.09, respectively. These values are in agreement with the binding affinities of the compounds at the human 5-HT6 receptor (7.26 ± 0.06 and 7.91± 0.02, respectively).

The 5-HT6 receptor was identified by molecular biological methods and its pharmacology made the study of naturally occurring 5-HT6 receptors very difficult. All compounds previously identified as having high affinity for the 5-HT6 receptor were non-selective ligands. Potent agonists at the receptor are LSD, ω-N-methyl-5-HT, bufotenine, 5-methoxy-tryptamine, 5-HT, 2-methyl-5-HT, 5-benzyloxytryptamine and tryptamine all of which are non-selective, having affinities at other 5-HT receptors (Boess et al., 1997). LSD, lisuride, 2-methyl-5-HT, tryptamine and 5-benzloxytryptamine behaved as partial agonists compared to 5-HT. Potent antagonists at the receptor are also non-selective and include methiothepin, clozapine, mianserin and ritanserin (Boess et al., 1997). Therefore, the identification of physiological responses mediated by the 5-HT6 receptor has not been possible and responses with properties similar to that of the cloned receptor can only be classified as 5-HT6-like (Sleight et al., 1997). Indeed, with respect to receptor nomenclature the fact that no selective 5-HT6 ligands existed excluded the 5-HT6 receptor from being officially recognised as a functional, naturally occurring 5-HT receptor (Hoyer et al., 1994; Hoyer & Martin, 1997). We propose that responses can now be classified as mediated by the 5-HT6 receptor with the use of Ro 04–6790 and Ro 63–0563. For example, in vitro studies have suggested that naturally occurring 5-HT6-like receptors are present in mouse neuroblastoma-derived cell lines N18TG2 and NCB-20 (MacDermot et al., 1979; Berry-Kravis & Dawson, 1983; Conner & Mansour, 1990; Unsworth & Molinoff, 1994). However, this classification was only based on a comparison of the rank order of potency of compounds in the functional models with that at the recombinant receptor. In addition, 5-HT6-like receptors have also been demonstrated in pig caudate membranes and mouse striatal neurones in culture where they stimulate cyclicAMP accumulation (Schoeffter & Waeber, 1994; Sebben et al., 1994).

The lack of selective tools has also meant that the pharmacological evaluation of the function of 5-HT6 receptors in vivo has not been possible. The only study investigating the role of 5-HT6 receptors in vivo used i.c.v. injections of antisense oligonucleotides to reduce the expression of the 5-HT6 receptor protein. This treatment gave rise to a behavioural syndrome of yawning, stretching and chewing and caused a 30% reduction in the number of [3H]-LSD binding sites (measured in the presence of 300 nM spiperone). This behavioural syndrome was dose-dependently antagonized by atropine suggesting a modulatory role for 5-HT6 receptors on cholinergic neurones (Bourson et al., 1995; Sleight et al., 1996). In order to determine whether a 5-HT6 antagonist will produce the same behavioural syndrome, the ability of both Ro 04–6790 and Ro 63–0563 to cross the blood-brain barrier was determined by measuring the CSF and plasma concentrations of the two compounds. Ro 04–6790 but not Ro 63–0563 could be detected in the CSF at concentrations that would occupy more than 70% of the 5-HT6 receptors after administration of 30 mg kg−1, i.p. Interestingly Ro 04–6790 produced a similar behavioural syndrome to that produced by the 5-HT6 antisense consisting of yawning, stretching and chewing (Figure 7). The stretching behaviour was statistically significant while the yawning just failed to reach statistical significance. Since the behavioural syndrome is produced by both antisense oligonucleotide treatment and selective 5-HT6 antagonists, we conclude that it is indeed mediated by 5-HT6 receptor blockade. It should also be noted that the maximal effect of Ro 04–6790 at 30 mg kg−1, i.p. would also agree with its concentration in the CSF being sufficient to occupy 5-HT6 receptors. These data suggest that experiments with antisense oligonucleotides can be of use in the study of newly cloned receptors where no selective compounds are available, provided that correct controls and techniques are used.

Currently, there is no good binding assay to label 5-HT6 receptors in animal tissue although it has been suggested that [3H]-clozapine labels two populations of receptors in the rat brain, one of which may be the 5-HT6 receptor binding site (Glatt et al., 1995). However, 5-HT had no measurable affinity for these binding sites and [3H]-clozapine was not observed in the corpus striatum. The presence of 5-HT6 receptor binding sites in this brain region would have been predicted from in situ hybridization studies (Ward et al., 1995) and from the mapping of 5-HT6-like immunoreactivity with polyclonal antibodies (Gerard et al., 1996). Therefore, the lack of affinity for 5-HT and the localization of [3H]-clozapine binding raises questions as to the validity of this radioligand binding assay. Consequently, it would be interesting to radiolabel a compound such as Ro 63–0563 in order to determine whether there are indeed specific 5-HT6 receptor binding sites in the brain. This work has been started in our group and is now ongoing.

In summary, the present study describes and characterizes two potent and selective 5-HT6 receptor antagonists that can be used to study and characterize the functional role of naturally occurring 5-HT6 receptors. Perhaps, more importantly such compounds may now allow the identification of possible therapeutic uses of 5-HT6 receptor ligands.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

The expert technical assistance of Nadine Petit, Alain Rudler, Daniel Wanner and Catherine Zwingelstein is gratefully acknowledged.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References
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