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Keywords:

  • Human 5-HT6 receptor;
  • SB-271046;
  • [3H]-LSD;
  • SB-258585;
  • adenylyl cyclase;
  • rat maximal electroshock seizure threshold (MEST) test;
  • pharmacokinetics

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  • SB-271046, potently displaced [3H]-LSD and [125I]-SB-258585 from human 5-HT6 receptors recombinantly expressed in HeLa cells in vitro (pKi 8.92 and 9.09 respectively). SB-271046 also displaced [125I]-SB-258585 from human caudate putamen and rat and pig striatum membranes (pKi 8.81, 9.02 and 8.55 respectively).

  • SB-271046 was over 200 fold selective for the 5-HT6 receptor vs 55 other receptors, binding sites and ion channels.

  • In functional studies on human 5-HT6 receptors SB-271046 competitively antagonized 5-HT-induced stimulation of adenylyl cyclase activity with a pA2 of 8.71.

  • SB-271046 produced an increase in seizure threshold over a wide-dose range in the rat maximal electroshock seizure threshold (MEST) test, with a minimum effective dose of leqslant R: less-than-or-eq, slant0.1 mg kg−1 p.o. and maximum effect at 4 h post-dose. The level of anticonvulsant activity achieved correlated well with the blood concentrations of SB-271046 (EC50 of 0.16 μM) and brain concentrations of 0.01–0.04 μM at Cmax.

  • These data, together with the observed anticonvulsant activity of other selective 5-HT6 receptor antagonists, SB-258510 (10 mg kg−1, 2–6 h pre-test) and Ro 04-6790 (1–30 mg kg−1, 1 h pre-test), in the rat MEST test, suggest that the anticonvulsant properties of SB-271046 are likely to be mediated by 5-HT6 receptors.

  • Overall, these studies demonstrate that SB-271046 is a potent and selective 5-HT6 receptor antagonist and is orally active in the rat MEST test. SB-271046 represents a valuable tool for evaluating the in vivo central function of 5-HT6 receptors.

British Journal of Pharmacology (2000) 130, 1606–1612; doi:10.1038/sj.bjp.0703457


Abbreviations:
5-HT

5-hydroxytryptamine creatinine sulphate

LSD

D-lysergic acid diethylamide

MEST

maximal electroshock seizure threshold

Ro 04-6790

4-amino-N-(2,6 bis-methylamino-pyrimidin-4-yl)-benzene sulphonamide

SB-258510

N-[4-Methoxy-3-(4-methyl-1-piperazinyl)-phenyl]-5-chloro-3-methylbenzo-thiophene-2-yl sulphonamide monohydrochloride

SB-258585

4-Iodo-N-[4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-benzene sulphonamide

SB-271046

5-Chloro-3-methyl-benzo[b]thiophene-2-sulphonic acid (4-methoxy-3-piperazin-1-yl-phenyl)-amide monohydrochloride

Introduction

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

The rat 5-HT6 receptor was first identified in 1993 by Ruat et al. (1993) and Monsma et al. (1993) and more recently the human 5-HT6 receptor gene was cloned and characterized (Kohen et al., 1996). Since then, a number of publications have suggested a role for 5-HT6 receptor antagonists in the treatment of schizophrenia, depression and cognitive impairment. A role for this receptor in the treatment of schizophrenia and depression is supported by its distribution in the brain and the high affinity of therapeutic atypical antipsychotics, particularly clozapine, and antidepressants for the 5-HT6 receptor (Monsma et al., 1993, Ruat et al., 1993; Roth et al., 1994). Other evidence pointing towards a role for this receptor in anxiety/stress/depression came from a study using antisense oligonucleotides directed at 5-HT6 receptor mRNA. Central administration of 5-HT6 specific antisense oligonucleotides abolished the conditioned fear stress-induced increase in 5-HT release (Yoshioka et al., 1998). In addition, blockade of endogenous corticosterone synthesis modulates 5-HT6 receptor mRNA in specific areas of the rat hippocampus (Yau et al., 1997). However, data from the majority of in vivo studies point to a role for the 5-HT6 receptor in the modulation of cholinergic neuronal activity and, perhaps as a consequence of this, cognitive function. Thus, central administration of 5-HT6 specific antisense oligonucleotides induced a behavioural syndrome which was dose-dependently blocked by the muscarinic receptor antagonist atropine (Bourson et al., 1995), suggesting that an increase in central cholinergic neuronal activity may be a consequence of 5-HT6 receptor blockade. These data were supported by a study using the selective 5-HT6 receptor antagonist Ro 04-6790 (Sleight et al., 1998), where Ro 04-6790 elicited some of the components of the above behavioural syndrome (Sleight et al., 1998; Bentley et al., 1999). Ro 04-6790 has also been shown to block scopolamine-induced rotations in 6-OH-DA-lesioned rats, an effect suggested to support the modulation of the cholinergic system by 5-HT6 receptor antagonists (Bourson et al., 1998). In addition, central administration of the same antisense oligonucleotide was shown to increase cognitive performance in the Morris water maze (Bentley et al., 1997). Despite the first identification of 5-HT6 receptors in 1993, there are relatively few studies demonstrating the in vivo function of this receptor, which is probably due to the lack of suitable pharmacological tools. The development of the selective 5-HT6 receptor antagonist, Ro 04-6790, was a significant improvement over antisense treatment, however, whilst this compound was highly selective for the 5-HT6 receptor, it had moderate affinity (pKi 7.4) and <1% CNS penetration (Sleight et al., 1998).

With the aim of further elucidating the in vivo function of the 5-HT6 receptor, we have identified a potent, selective and orally active 5-HT6 receptor antagonist, SB-271046 (Bromidge et al., 1999), and report here on its in vitro and in vivo characterization. Recent studies using this compound provide initial supportive evidence for the role of the 5-HT6 receptor in cognitive enhancement (Rogers et al., 1999). A preliminary account of the data presented here has been published in abstract form (Routledge et al., 1999; Stean et al., 1999).

Methods

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

Radioligand binding studies

Cell line

The human 5-HT6 serotonin receptor stably expressed in HeLa cells was obtained from Dr David Sibley of the Molecular Neuropharmacology Section, Experimental Therapeutics Branch, Public Health Service, National Institutes of Health, Bethesda, MA, U.S.A. The cells were grown in Dulbecco' modified Eagle' medium (DMEM) containing 5% foetal bovine serum and were routinely treated with 5 mM sodium butyrate 24 h prior to harvesting.

Radioligand binding

Radioligand binding was carried out as described (Hirst et al., 2000). In brief, radioligand binding was performed on membranes from HeLa cells stably transfected with the human 5-HT6 receptor (see above) and striatal tissue from adult rats (Sprague-Dawley, 200–250 g, Charles River, U.K.), adult pigs (from a local abattoir: Dalehead Foods, Linton, U.K.) and human caudate putamen tissues (from non-identifiable patients aged 64–76 years, whose cause of death was non-neurological, from Resource, Institute of Neurology, London, U.K.). Membranes were incubated with 1 nM [125I]-SB-258585 (Hirst et al., 2000) or 2 nM [3H]-LSD for 45 min at 37°C. Non-specific binding was defined by the inclusion of 10 μM methiothepin and the assay was terminated by rapid filtration through Whatman GF/B filters.

For receptor selectivity studies on other 5-HT receptors, details of the radioligands used and assay conditions are given in Hirst et al. (2000). SB-271046 was also tested in a further 55 binding assays by CEREP (Le Bois l'Eveque, 86600 Celle L'Evescault, France) (CEREP Task Order 882035).

Adenylyl cyclase measurements

Membrane preparation

Cell pellets were stored at −80°C prior to membrane preparation. The cell pellet was thawed at room temperature, and divided into eight volumes, each of which was homogenized in 30 mls ice-cold HEPES buffer (20 mM HEPES, 10 mM MgSO4, pH 7.4) by polytron homogenization. Homogenates were then centrifuged at 18,000 r.p.m. (35,000×g) for 10 min. The supernatant was discarded and the pellet resuspended in 30 mls of HEPES buffer. Homogenates were then incubated at 37°C for 20 min to allow the breakdown of endogenous 5-HT. The suspension was washed a further two times by centrifugation before resuspension at a cell density of 1×108 cell ml−1, and storage at −80°C.

Method

Adenylyl cyclase activity in HeLa cells transfected with the human 5-HT6 receptor (as described above) was determined by measuring the conversion of [α-33P]-ATP to [33P]-cAMP. [α-33P]-cAMP production is small (<1%) as a proportion of the [α-33P]-ATP added and so must be efficiently separated from unconverted [α-33P]-ATP by Dowex-Alumina chromatography using a dual column separation method adapted from Salomon (1979). The assay mixture consisted of 50 mM Tris HCl buffer containing: MgCl2 (5 mM), GTP (50 μM), ATP (100 μM), phosphocreatine (20 mM), creatine phosphokinase (40 units ml−1), myokinase (50 units ml−1), and 1-methyl-3-isobutylxanthine (IBMX) (0.5 μM), in a total assay volume of 50 μl. Incubations (37°C, 10 min) were started by addition of 20 ml of membrane suspension (20–25 mg protein) to tubes containing incubation buffer, [α-33P]-ATP (1–1.5 μCi tube−1 sp. act. 2000 Ci mmol−1) and test drugs as appropriate. The incubation was stopped by addition of 100 μl of 0.5 M HCl containing ATP (40 mM), cyclic AMP (10 mM) and [3H]-cAMP (∼10,000 d.p.m. specific activity 27 Ci mmol−1) for calculation of column recovery. Samples were counted in Ultima-XR scintillant using a dual label protocol and the tritium signal was used to correct for per cent column recovery.

Maximal electroshock seizure threshold (MEST) test

All experimental work was conducted in compliance with the Home Office Guidance on the operation of the Animals (Scientific Procedures) Act 1986, and was reviewed and approved by the SmithKline Beecham Procedures Review Panel.

Male Sprague Dawley rats (100–150 g), supplied by Charles River, U.K. were housed in groups of 10 at a room temperature of 20–22°C. Animals were maintained on a 12 h light/dark cycle with lights on between 0600 and 1800 h Food (Combined Rat and Mouse Diet, Special Diet Services, Witham, U.K.) and water were available ad libitum. Drug treatments were evaluated between 1400 and 1800 h alongside time-matched vehicle-treated controls.

The threshold current for electroshock-induced tonic hindlimb extensor seizure was determined using a Hugo Sachs Electronik stimulator (Germany), which delivered an adjustable constant current (1–300 mA) of 0.3 s duration, 50 Hz, sinewave form, via corneal electrodes. The stimulus intensity was varied, from a typical baseline of 25 mA, by an ‘up and down’ method of shock titration (see Upton et al., 1997, for details). Data generated from treatment groups of n=11–14 were used to calculate the seizure threshold (current producing tonic hindlimb extensor seizure in 50% of animals)±s.e. values according to the method of Kimball et al. (1957). Elevation of seizure threshold is indicative of an anticonvulsant effect whereas a reduction in seizure threshold is indicative of proconvulsant activity. The effects of the selective 5-HT6 receptor antagonists, SB-271046 (0.1–30 mg kg−1 p.o., 4 h pre-test), SB-258510 (10 mg kg−1 p.o., 2–6 h pre-test) (Bromidge et al., 1999) and Ro 04-6790 (0.3–30 mg kg−1 i.p., 1 h pre-test) (Sleight et al., 1998), on seizure threshold were determined. The doses of Ro 04-6790 selected for this study cover the range previously reported to evoke a number of behavioural effects in rats (Sleight et al., 1998; Bentley et al., 1999). SB-271046 and SB-258510 were suspended in 1% methyl cellulose in water and Ro 04-6790 was dispersed in saline. A 1 ml kg−1 dose volume was used for all treatments and doses are expressed as free base.

In order to evaluate the relationship between the level of anticonvulsant activity achieved and blood concentration, the duration of action of a high submaximal dose (10 mg kg−1 p.o.) of SB-271046 in the rat MEST test was evaluated in detail over a 24 h period. Following the conclusion of this study, whole brain and blood samples were taken from randomly selected animals (n=5) at 13 different timepoints. Samples were assayed for SB-271046 using a method based on protein precipitation with acetonitrile, followed by LC/MS/MS analysis employing positive-ion electrospray ionization, with a lowest limit of quantification (LLQ) of 0.01 μM.

Materials

SB-271046 (5-Chloro-3-methyl-benzo[b]thiophene-2-sulphonic acid (4-methoxy-3-piperazin-1-yl-phenyl)-amide monohydrochloride), SB-258510 (N-[4-Methoxy-3-(4-methyl-1-piperazinyl)-phenyl]-5-chloro-3-methylbenzo-thiophene-2-yl sulphonamide monohydrochloride) and Ro 04-6790 (4-amino-N-(2,6 bis-methylamino-pyrimidin-4-yl)-benzene sulphonamide) were synthesized by SmithKline Beecham Pharmaceuticals (Harlow, U.K.); the chemical structure of SB-271046 is shown in Figure 1. [125I]-SB-258585 was prepared at SmithKline Beecham (Synthetic Isotope Chemistry) by reaction of the tributyltin derivative of SB-258585 with chloramine-T and sodium [125I]-iodide. [3H]-LSD, [α-33P]-ATP and [33P]-cAMP were obtained from NEN Du Pont. 5-hydroxytryptamine creatine sulphate (5-HT), amitriptyline hydrochloride and pargyline were purchased from Sigma (Poole, U.K.). Mesulergine hydrochloride were purchased from Research Biochemicals Inc. (Natick, MA, U.S.A.). Chromatography columns and Dowex resins were obtained from Bio-rad. Cell culture reagents were obtained from Life Technologies Ltd. (Paisley, U.K.) all other reagents were obtained from Sigma or Merck-BDH (Lutterworth, U.K.) and were of analytical grade.

image

Figure 1. Chemical structure of SB-271046.

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Data analysis

The concentration of drug inhibiting specific radioligand by 50% (IC50) was determined by iterative curve fitting (Bowen & Jerman, 1995) and pKi values (negative log10 of the molar Ki) for receptor binding were then calculated from the IC50 values as described by Cheng & Prusoff (1973) using KD values determined in the stauration binding studies. Data are expressed as the mean±s.e.mean of at least three separate experiments.

Drug concentration-response curves from adenylyl cyclase assays were fitted to a 4-parameter logistic equation (GRAFIT, Erithacus Software), constraining the Emax of each curve to 100%. Drug potency was expressed as the pEC50 or pIC50 (−log EC50 or −log IC50) for stimulation or inhibition respectively. Non-enzymic [α-33P]-cAMP production (measured at 4°C) was found to be less than 1% of the basal activity (measured at 37°C). The pA2 for antagonism was determined by Schild analysis of the data where, for a reversible competitive antagonist, provided that the slope is unity, the pA2=pKB. The pKB is −log of the antagonist equilibrium dissociation constant i.e. −log (antagonist concentration/(concentration ratio-1)) where concentration ratio is the ratio of the agonist EC50 in the absence and presence of antagonist. The antagonist concentrations used were chosen to produce a concentration ratio of between 10 and 100. Data represent the mean±s.e.m. of at least three separate experiments and all determinations within an experiment were performed in triplicate.

From the in vivo duration of action study the relationship between the blood concentration of SB-271046 and MEST response (per cent change in seizure threshold from control) was examined in a direct effect Emax pharmacodynamic model. The model produced estimated values for Emax (predicted maximal effect at infinite SB-271046 concentration) and EC50 (SB-271046 concentration producing 50% of maximal increase in seizure threshold).

Statistical analysis.

Significant differences between drug and vehicle-treated animals on seizure threshold were determined according to the method of Litchfield & Wilcoxon (1949).

Results

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

Radioligand binding studies

[3H]-LSD and [125I]-SB-258585 binding to recombinant human 5-HT6 receptors displayed a single saturable binding component with a Kd of 1.5±0.1 nM and 0.80±0.05 nM respectively and Bmax of 3.9±0.8 pmoles mg−1 protein and 6.1±0.95 pmoles mg−1 protein respectively. In rat and pig stiatum and human caudate putamen tissue, [125I]-SB-258585 again displayed single saturable binding components with Kd of 2.8±0.4, 2.8±0.7, and 1.3±0.04, respectively (see Hirst et al., 2000).

SB-271046 displayed high affinity for human recombinant 5-HT6 receptors and 5-HT6 receptors in native brain tissues. At the human cloned receptor, using [3H]-LSD and [125I]-SB-258585 as radioligands, SB-271046 gave pKis of 8.92±0.04 and 9.09±0.07 respectively (see Table 1.). SB-271046 also potently displaced [125I]-SB-258585 from rat and pig striatal and human caudate putamen membranes with pKis of 9.02±0.14, 8.55±0.1 and 8.81±0.1 respectively (for inhibition profiles see Hirst et al., 2000). SB-271046 had greater than 200 fold selectivity compared to all other 5-HT receptors tested (see Table 1). Moreover, in a comprehensive selectivity screen, SB-271046 was shown to be more than 200 fold selective for the human 5-HT6 receptor as compared to 55 other receptors, enzymes and ion channels (CEREP Task Order 882035).

Table 1. Selectivity profiles of SB-271046 at recombinant human receptors and native tisue receptorsThumbnail image of

Adenylyl cyclase measurements

Functional studies on the human cloned 5-HT6 receptor showed a 5-HT concentration-dependent increase in cyclic AMP levels, with a pEC50=6.74 (Figure 2a). SB-271046 inhibited the 5-HT stimulation, shifting the 5-HT concentration response curve to the right in a concentration dependent manner with no suppression of the maximal response, consistent with competitive antagonism. Linear regression analysis of Schild plot data (Figure 2b), revealed a correlation coefficient of 0.99, a slope of 1.04 and a pA2=8.71±0.3 for SB-271046, which correlated closely with its 5-HT6 receptor binding affinity. SB-271046 showed no evidence of intrinsic activity in this system.

image

Figure 2. (a) Stimulation of adenylyl cyclase activity in HeLa cells stably expressing human 5-HT6 receptors by 5-HT alone and in the presence of increasing concentrations of SB-271046 (10, 30, 100 and 300 nM). Data points represent the mean and s.e. mean (error bars) of three independent experiments each performed using triplicate determinations. Results are expressed as per cent of the maximal 5-HT response in each assay. (b) Linear regression analysis of this same data displayed as a Schild plot.

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Maximal electroshock seizure threshold (MEST) test

As shown in Table 2, SB-271046 produced an increase in seizure threshold over a wide-dose range in the rat MEST test, with a minimum significantly effective dose of leqslant R: less-than-or-eq, slant0.1 mg kg−1 p.o. At 10 mg kg−1 p.o., the compound had a rapid onset of action (leqslant R: less-than-or-eq, slant30 min); reaching a maximum effect at 4 h post-dose (elevating seizure threshold by 166% from a control level of 23.5±2.1 up to 62.5±1.3 mA) and maintaining biological activity for at least 21 h (P<0.001 30 min–15 h, P<0.05 18–21 h) (Figure 3). In addition, the level of anticonvulsant activity achieved correlated well with the blood concentrations of SB-271046 observed (Figure 3), with a calculated Emax of 155% and EC50 of 0.16 μM (Figure 4). Brain samples had low concentrations of SB-271046 with the majority of values below LLQ, although measurable concentrations (0.01–0.04 μM, 4.5–18.1 ng ml−1) were observed between 2 and 6 h (Cmax), with the highest levels corresponding to the time of maximum plasma concentration and peak anticonvulsant activity (Figure 3). These determined effective brain levels of SB-271046 are in keeping with its affinity at rat 5-HT6 receptors (pKi 9.09).

Table 2. Effect of SB-271046, SB-258510 and Ro 04-6790 on the threshold for electroshock-induced generalized seizures in ratsThumbnail image of
image

Figure 3. Time course profile of anticonvulsant activity in the MEST test and blood concentration of SB-271046 in rats. SB-271046 was administered at a single dose of 10 mg kg−1 p.o. Data represents increase in control EC50±s.d. values for groups of 11–12 rats. Blood concentrations represent mean±s.d. values for groups of five rats.

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image

Figure 4. Relationship between SB-271046 blood concentration and seizure threshold. Data represent a direct effect Emax pharmacodynamic model plotted using ‘WinNonlin Professional Version 1.5’ software.

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In all studies, the anticonvulsant properties of SB-271046 were evident in the absence of any observed overt behavioural depressant effects (data not shown). Furthermore, even at doses up to 100 mg kg−1 p.o., SB-271046 did not impair motor coordination (rotarod test) or spontaneous locomotion (activity counts) in rats (data not shown), suggesting that the anticonvulsant properties of this agent are not secondary to a generalized depressant action.

A time-related increase in seizure threshold was also observed following administration of SB-258510 (10 mg kg−1 p.o., 2–6 h pre-test). The maximum observed effect (an elevation of seizure threshold of 132% from a baseline of 30±1.2 to 70.8±3.9 mA at 6 h) was comparable to that induced by SB-271046 given at the same dose (Table 2). Similarly, Ro 04-6790 produced a modest (maximum of 46%), but significant, elevation of seizure threshold at doses ranging from 1–30 mg kg−1 i.p., 1 h pretest (Table 2). The apparently reduced level of anticonvulsant efficacy of Ro 04-6790 compared to SB-271046 and SB-258510, may reflect the poor CNS penetration (<1%) of the former compound (Sleight et al., 1998).

Discussion

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

The present study describes the in vitro and in vivo characterization of SB-271046, a potent and selective receptor antagonist for the human recombinant 5-HT6 receptor and for rat striatal, pig striatal and human caudate 5-HT6 receptors. The affinities of SB-271046 in rat (pKi 9.02), pig (pKi 8.55) and human (pKi 8.81) were similar suggesting a lack of species differences in this receptor for this given ligand. In addition, this compound has greater than 200 fold selectivity over 69 other receptor, enzyme and binding sites, including all other 5-HT receptor subtypes tested.

In functional studies on human recombinant 5-HT6 receptors, SB-271046 antagonized 5-HT-induced stimulation of adenylyl cyclase activity, in a surmountable manner with a pA2 of 8.7, which is in good agreement with the radioligand binding affinity of this compound for the 5-HT6 receptor. The inhibition of 5-HT-stimulated adenylyl cyclase activity is competitive, displaying a rightward, concentration-dependent shift of the 5-HT concentration-effect curve with no depression of the maximum response (Figure 2a). Linear regression analysis of Schild plot data revealed a correlation coefficient of 0.99 and a slope of 1.04 (Figure 2b). At concentrations up to 300 nM, SB-271046 displayed no intrinsic activity, having neither stimulatory nor inhibitory activity per se. No demonstration of inhibitory activity in the adenylyl cyclase assay suggests that this compound is either a silent antagonist or that there is no constitutive activity of the 5-HT6 receptor in this system, hence no inverse agonism is observed. Failure to demonstrate any partial agonism in this very high receptor expression cell line suggests that this compound is highly unlikely to display intrinsic activity in any native brain tissues.

Following the studies of Schoeffter & Waeber (1994), we have attempted to duplicate these functional studies in rat and pig striatal tissue but we have consistently failed to produce a 5-HT6 receptor mediated stimulation of adenylyl cyclase activity (data not shown).

SB-271046 represents the most selective and potent 5-HT6 receptor antagonist reported to date and therefore, offers significant advantages over other tools/compounds that have been used to characterize the 5-HT6 receptor, both in vitro and in vivo. For example, whilst SB-271046 has a similar 5-HT6 receptor affinity to those of LSD, a partial agonist at the 5-HT6 receptor, and clozapine and methiothepin, antagonists at the 5-HT6 receptor (Boess et al., 1997), it has a superior selectivity profile with respect to other 5-HT receptor subtypes. The identification of Ro 04-6790 (Sleight et al., 1998) provided the first selective 5-HT6 receptor ligand. Although the affinity of this compound is relatively low (pKi 7.4 compared with 8.9 for SB-271046) and the CNS penetration is very limited (<1%), in vivo effects of this compound were observed following systemic administration (Bourson et al., 1998; Sleight et al., 1998).

In the absence of selective 5-HT6 receptor ligands, intracerebroventricular administration of 5-HT6 specific antisense oligonucleotide was used to characterize 5-HT6 receptor function in vivo. Whilst this approach was shown to selectively reduce CNS 5-HT6 receptors (using [3H]-LSD as the radioligand for receptor number determination; Bourson et al. (1995); Bentley et al. (1997); Yoshioka et al. (1998)) there are both practical and interpretational difficulties with using this technology in vivo. Given these difficulties it is interesting, and perhaps reassuring, to note that there are some similarities between the data obtained using 5-HT6 specific antisense oligonucleotide and selective 5-HT6 receptor antagonists. For example, Bourson et al. (1995) showed that administration of 5-HT6 specific antisense oligonucleotide elicited yawning, stretching and chewing in rats, similarly Ro 04-6790 elicited stretching in rats. A similar pattern is seen in animal models of cognition where the same antisense was shown to alter performance in the Morris water maze task (Bentley et al., 1997). The latter findings on cognitive function have recently been confirmed and expanded using SB-271046, to include different animal models of cognitive performance (Rogers et al., 1999).

The present study shows that SB-271046 produces potent and long-lasting anticonvulsant activity in the rat MEST test, a model of previously reported utility for studying the role of serotonergic pathways in seizure regulation (e.g. Upton et al., 1998). Since SB-271046 is a very selective 5-HT6 receptor antagonist (Bromidge et al., 1999; Routledge et al., 1999), the anticonvulsant properties of SB-271046 are likely to be mediated by blockade of 5-HT6 receptors. This is further supported by the demonstrated close correlation between the pharmacokinetic and pharmacodynamic profiles of the compound and the presently observed anticonvulsant activity of other selective 5-HT6 receptor antagonists, SB-258510 (Bromidge et al., 1999; pKi at human 5-HT6 receptor 9.2) and the chemically distinct agent Ro 04-6790 (Sleight et al., 1998). Overall, these data suggest that the MEST test may provide a robust model of in vivo 5-HT6 receptor function, and also illustrate that SB-271046 is a potent and orally active 5-HT6 receptor antagonist. However, the magnitude of these anti-seizure effects was modest in comparison to that of known anti-epileptic drugs For example, using identical test conditions, agents such as carbamazepine can elevate seizure threshold by >1200% (Upton et al., 1997) as compared to the maximum increase of only 132 and 166% produced by SB-258510 and SB-271046, respectively. This low level of anticonvulsant efficacy associated with 5-HT6 receptor blockade probably contributes to the apparent lack of dose-dependency for SB-271046, SB-258510 and Ro 04-6790 in the MEST test, since the anticonvulsant activity of SB-271046 is clearly related to the level of exposure in blood. Therefore, the relevance of this observation to the possible clinical utility of SB-271046 in the treatment of epilepsy is, at this stage, unclear.

The anticonvulsant effects of the 5-HT6 receptor antagonists observed in the present studies are intriguing in view of previous evidence indicating that agents which elevate extracellular serotonin, inhibit generalized (and limbic) seizures, whereas agents which deplete brain serotonin are generally associated with proconvulsant activity, as seen in a range of experimental models of epilepsy (see Upton et al., 1998).

In addition, blockade of 5-HT1A and 5-HT1B receptors with WAY 100635 (0.03–0.3 mg kg−1 s.c., 1 h pre-test) (Fletcher et al., 1996) and SB-224289 (1–10 mg kg−1 p.o., 3 h pre-test) (Gaster et al., 1998), respectively, does not alter electroshock-induced seizure threshold in rats (unpublished observations) suggesting that blockade of 5-HT1A and 5-HT1B receptors is unlikely to affect seizure propagation. It has also been previously demonstrated that blockade of 5-HT3 receptors (Upton et al., 1993) does not induce anticonvulsant activity in the rat intravenous metrazol infusion test. Furthermore, blockade of 5-HT2C and/or 5-HT2B receptors has no effect on electroshock-induced seizure threshold in rats (Upton et al., 1998), which is further supported by the lack of anticonvulsant activity of the mixed 5-HT receptor agonist metergoline in this model (unpublished data). It was therefore unexpected that a 5-HT6 receptor antagonist would be anticonvulsant, albeit a modest effect. This suggests that endogenous 5-HT has both anticonvulsant and proconvulsant activity, but under normal physiological conditions, the former dominates.

Taken together, these data demonstrate that SB-271046 is a potent, selective and orally active 5-HT6 receptor antagonist. This compound provides a useful tool for further elucidating the physiological function of 5-HT6 receptors in vivo.

Acknowledgments

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

We would like to thank Brenda Trail and Tracey Ashmeade for their excellent technical assistance, Phil Jeffrey, Doug Smith, Lisa Bennicosa, Tania Buck, Sarah Murray, and Nigel Deeks for providing pharmacokinetic support and Julia Loudon for her critical evaluation of the manuscript.

References

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