Gary W. Price

SB-656104-A, a novel selective 5-HT7 receptor antagonist, modulates REM sleep in rats

(6‐((R)‐2‐{2‐[4‐(4‐Chloro‐phenoxy)‐piperidin‐1‐yl]‐ethyl}‐pyrrolidine‐1‐sulphonyl)‐1H‐indole hydrochloride) (SB‐656104‐A), a novel 5‐hydroxytryptamine (5‐HT7) receptor antagonist, potently inhibited [3H]‐SB‐269970 binding to the human cloned 5‐HT7(a) (pKi 8.7±0.1) and 5‐HT7(b) (pKi 8.5±0.2) receptor variants and the rat native receptor (pKi 8.8±0.2). The compound displayed at least 30‐fold selectivity for the human 5‐HT7(a) receptor versus other human cloned 5‐HT receptors apart from the 5‐HT1D receptor (∼10‐fold selective). SB‐656104‐A antagonised competitively the 5‐carboxamidotryptamine (5‐CT)‐induced accumulation of cyclic AMP in h5‐HT7(a)/HEK293 cells with a pA2 of 8.5. Following a constant rate iv infusion to steady state in rats, SB‐656104 had a blood clearance (CLb) of 58±6 ml min−1 kg−1 and was CNS penetrant with a steady‐state brain : blood ratio of 0.9 : 1. Following i.p. administration to rats (10 mg kg−1), the compound displayed a t1/2 of 1.4 h with mean brain and blood concentrations (at 1 h after dosing) of 0.80 and 1.0 μM, respectively. SB‐656104‐A produced a significant reversal of the 5‐CT‐induced hypothermic effect in guinea pigs, a pharmacodynamic model of 5‐HT7 receptor interaction in vivo (ED50 2 mg kg−1). SB‐656104‐A, administered to rats at the beginning of the sleep period (CT 0), significantly increased the latency to onset of rapid eye movement (REM) sleep at 30 mg kg−1 i.p. (+93%) and reduced the total amount of REM sleep at 10 and 30 mg kg−1 i.p. with no significant effect on the latency to, or amount of, non‐REM sleep. SB‐269970‐A produced qualitatively similar effects in the same study. In summary, SB‐656104‐A is a novel 5‐HT7 receptor antagonist which has been utilised in the present study to provide further evidence for a role for 5‐HT7 receptors in the modulation of REM sleep.

SB-224289–a novel selective (human) 5-HT1B receptor antagonist with negative intrinsic activity

1 Human 5‐HT1B (h5‐HT1B) and human 5‐HT1D (h5‐HT1D) receptors show remarkably similar pharmacology with few compounds discriminating the receptors. We report here on a novel compound, SB‐224289 (1′‐Methyl‐5‐[[2′‐methyl‐4′‐(5‐methyl‐1,2,4‐oxadiazol‐3‐yl)biphenyl‐4‐yl]carbonyl]‐2,3,6,7‐tetrahydrospiro [furo [2,3‐f]indole‐3,4′‐piperidine] oxalate), which has high affinity for h5‐HT1B receptors (pK1=8.16±0.06) and displays over 75u2003fold selectivity for the h5‐HT1B receptor over all other 5‐HT receptors including the h5‐HT1D receptor and all other receptors tested thus far. 2 Functional activity of SB‐224289 was measured in a [35S]GTPγS binding assay on recombinant h5‐HT1B and h5‐HT1D receptors expressed in Chinese Hamster Ovary (CHO) cells. SB‐224289 displayed negative intrinsic activity at both receptors with higher potency at h5‐HT1B receptors. SB‐224289 caused a rightward shift of agonist concentration response curves consistent with competitive antagonism and generated affinities comparable with those obtained from competition radioligand receptor binding studies. 3 SB‐224289 potentiated [3H]5‐HT release from electrically stimulated guinea‐pig cerebral cortical slices to the same extent as as the non‐selective 5‐HT1 antagonist methiothepin. SB‐224289 also fully reversed the inhibitory effect of exogenously superfused 5‐HT on electrically stimulated release. 4 Using SB‐224289 as a tool compound, we confirm that in guinea‐pig cerebral cortex the terminal 5‐HT autoreceptor is of the 5‐HT1B subtype.

Serotonergic targets in depression

Serotonin reuptake inhibitors have proved to be a very effective treatment for depression and have strengthened the hypothesis that impaired 5-hydroxytryptamine (5-HT) neurotransmission may contribute to the underlying cause of depressive disorders. Extensive research has been carried out to investigate other 5-HT targets associated with the disease and studies involving combination treatments with selective serotonin reuptake inhibitors and 5-HT(1A) receptor ligands are currently being carried out in the clinic. Whether other 5-HT receptor subtypes are involved in the aetiology of depression remains to be seen.

Identification of 5-hydroxytryptamine receptors positively coupled to adenylyl cyclase in rat cultured astrocytes

1 5‐Hydroxytryptamine (5‐HT) elicited a dose‐dependent stimulation of intracellular adenosine 3′: 5′‐cyclic monophosphate (cyclic AMP) accumulation in cultured astrocytes derived from neonatal rat (Sprague Dawley) thalamic/hypothalamic area with a potency (pEC50) of 6.68 ± 0.08 (mean ± s.e.mean). 2 In order to characterize the 5‐HT receptor responsible for the cyclic AMP accumulation the effects of a variety of compounds were investigated on basal cyclic AMP levels (agonists) and 5‐carboxamidotryptamine (5‐CT) stimulated cyclic AMP levels (antagonists). The rank order of potency for the agonists investigated was 5‐CT (pEC50 = 7.81 ±+ 0.09)>5‐methoxytryptamine (5‐MeOT) (pEC50 = 6.86 ±0.36) > 5‐HT (pEC50 = 6.68± 0.08). The following compounds, at concentrations up to 10 μm, did not affect basal cyclic AMP levels 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (8‐OH‐DPAT), cisapride, sumatriptan, DOI and RU 24969. The rank order of potency of antagonists was meth‐iothepin (p Ki = 7.98 ±0.25)>mesulergine (p Ki = 7.58 ± 0.18)>ritanserin (p Ki = 7.20 ±0.24) >clozapine (p Ki = 7.03±0.19)>mianserin (p Ki= 6.41 ±0.19). The following compounds, at concentrations up to 10 μM, were inactive: ketanserin, WAY100635, GR127935. This pharmacological profile is consistent with that of 5‐HT7 receptor subtype‐mediated effects. 3 The cultured astrocytes exhibited regional heterogeneity in the magnitude of cyclic AMP accumulation (Emax). Cells cultured from the thalamic/hypothalamic area had significantly higher Emax values (588 ± 75% and 572 ± 63% of basal levels for 5‐CT and 5‐HT, respectively) compared to brainstem (274 ± 51% and 318 ± 46%, respectively) and colliculus astrocytes (244 ± 15% and 301±24%, respectively). No significant differences in pEC50 (for either 5‐HT or 5‐CT) values were observed. 4 Reverse transcriptase‐polymerase chain reaction (RT‐PCR) with primers specific for the 5‐HT7 receptor confirmed expression of messenger RNA for this receptor subtype by the cultured astrocytes derived from all regions investigated. Primers specific for the 5‐HT6 receptor also amplified a cDNA fragment from the same samples. 5 From these findings, we conclude that astrocytes cultured from a number of brain regions express functional 5‐HT receptors positively coupled to adenylyl cyclase and that the level of receptor expression or the efficiency of receptor coupling is regionally‐dependent. The pharmacological profile of the receptor on thalamic/hypothalamic astrocytes suggests that the 5‐HT7 receptor is the dominant receptor that is functionally expressed even though astrocyte cultures have the capacity to express both 5‐HT6 and 5‐HT7 receptor messenger RNA.

Cultured astrocytes express messenger RNA for multiple serotonin receptor subtypes, without functional coupling of 5-HT1 receptor subtypes to adenylyl cyclase

The literature describing the expression of 5-HT receptor subtypes by astrocytes is controversial and incomplete. It is clear that primary cultures of astrocytes express receptors of the 5-HT2 family coupled to phospholipase C and of the 5-HT7 receptor family positively coupled to adenylyl cyclase. Cultured astrocytes have also been reported to express receptors of the 5-HT1 family, although the exact subtypes present are unknown. In the present study we have investigated which of the known rat G-protein coupled 5-HT receptor mRNAs are expressed by cultured astrocytes. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed expression of 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT5B, 5-HT6 and 5-HT7 receptor mRNAs in astrocytes derived from 2-day old rats and cultured for 10-12 days. Messenger RNAs for 5-HT4 and 5-HT5A receptors were not detected. The functional expression of 5-HT1 receptor subtypes was investigated by measuring the ability of 5-HT1 receptor agonists: 8-OH-DPAT (5-HT1A receptors), RU24969 (5-HT1A, 5-HT1B, 5-HT1D, and 5-HT1F receptors) or sumatriptan (5-HT1B, 5-HT1D, and 5-HT1F receptors) to modulate forskolin or isoproterenol stimulated cAMP production. These compounds, at concentrations up to 10 microM, did not significantly attenuate cAMP production. These results indicate that although astrocytes express mRNA for each of the five 5-HT1 receptor subtypes which have been isolated from the rat, these receptors are not coupled to the inhibition of adenylyl cyclase.

5-HT1A receptor agonist-antagonist binding affinity difference as a measure of intrinsic activity in recombinant and native tissue systems

It has been reported that radiolabelled agonistu2003:u2003antagonist binding affinity ratios can predict functional efficacy at several different receptors. This study investigates whether this prediction is true for recombinant and native tissue 5‐HT1A receptors. Saturation studies using [3H]‐8‐OH‐DPAT and [3H]‐MPPF revealed a single, high affinity site (KD∼1u2003nM) in HEK293 cells expressing human 5‐HT1A receptors and rat cortex. In recombinant cells, [3H]‐MPPF labelled 3–4 fold more sites than [3H]‐8‐OH‐DPAT suggesting the presence of more than one affinity state of the receptor. [3H]‐Spiperone labelled a single, lower affinity site in HEK293 cells expressing h5‐HT1A receptors but did not bind to native tissue 5‐HT1A receptors. These data suggest that, in transfected HEK293 cells, human 5‐HT1A receptors exist in different affinity states but in native rat cortical tissue the majority of receptors appear to exist in the high agonist affinity state. Receptor agonists inhibited [3H]‐MPPF binding from recombinant 5‐HT1A receptors in a biphasic manner, whereas antagonists and partial agonists gave monophasic inhibition curves. All compounds displaced [3H]‐8‐OH‐DPAT and [3H]‐spiperone binding in a monophasic manner. In rat cortex, all compounds displaced [3H]‐MPPF and [3H]‐8‐OH‐DPAT in a monophasic manner. Functional evaluation of compounds, using [35S]‐GTPγS binding, produced a range of intrinsic activities from full agonism, displayed by 5‐HT and 5‐CT to inverse agonism displayed by spiperone. [3H]‐8‐OH‐DPATu2003:u2003[3H]‐MPPF pKi difference correlated well with functional intrinsic activity (r=0.86) as did [3H]‐8‐OH‐DPATu2003:u2003[3H]‐spiperone pKi difference with functional intrinsic activity (r=0.96). Thus agonistu2003:u2003antagonist binding affinity differences may be used to predict functional efficacy at human 5‐HT1A receptors expressed in HEK293 cells where both high and low agonist affinity states are present but not at native rat cortical 5‐HT1A receptors in which only the high agonist affinity state was detectable.

Serotonin transporters in adult rat brain astrocytes revealed by [3H]5-HT uptake into glial plasmalemmal vesicles

Cultured astrocytes derived from neonatal rat brain exhibited high affinity, Na+-dependent, paroxetine and fluoxetine sensitive [3H]5-HT uptake. Reverse transcriptase-PCR demonstrated that astrocytes in culture expressed messenger RNA for the cloned serotonin transporter protein which has been characterised as the neuronal serotonin transporter. Although the serotonin transporter in cultured astrocytes displayed a Km value approximately 10 times greater than found in adult brain synaptosomes, these observations indicated that astrocytes in vitro may express the same serotonin transporter as neurons. Reverse transcriptase-PCR demonstrated the presence of serotonin transporter mRNA in the adult rat cerebral cortex, suggesting that astrocytes in vivo may express low levels of this mRNA. To investigate whether astrocytes in the adult CNS express functional serotonin transporters, glial plasmalemmal vesicles were prepared from cerebral cortex, representing a subcellular fraction composed primarily of vesicles derived from astrocytes. These vesicles were characterised by [3H]-glutamate and [3H]-dopamine uptake and by immunoblot analysis, using glial and synaptic markers: glutamate synthase, SNAP-25 and synaptobrevin. [3H]5-HT was taken up into glial plasmalemmal vesicles in a high affinity (Km approximately 40 nM), Na+ dependent, paroxetine-sensitive manner. The [3H]5-HT uptake capacity (Vmax) in these vesicles was approximately one quarter of that observed in synaptosomes. These data indicate that astrocytes in culture and in vivo are capable of 5-HT uptake via the previously characterised neuronal serotonin transporter.

Pharmacological profile of SB-357134: a potent, selective, brain penetrant, and orally active 5-HT6 receptor antagonist

N-(2,5-Dibromo-3-fluorophenyl)-4-methoxy-3-piperazin-1-ylbenzenesulfonamide (SB-357134) potently inhibited [125I]SB-258585 and [3H]LSD binding in a HeLa cell line expressing human 5-HT(6) receptors (pK(i)=8.6 and 8.54, respectively). Furthermore, SB-357134 inhibited [125I]SB-258585 binding in human caudate--putamen and in rat and pig striatum membranes (pK(i)=8.82, 8.44, and 8.61, respectively). SB-357134 displayed over 200-fold selectivity for the 5-HT(6) receptor versus 72 other receptors and enzymes. 5-HT-stimulated cyclic AMP (cAMP) accumulation in human 5-HT(6) receptors was competitively antagonised by SB-357134 (pA(2)=7.63). SB-357134 inhibited ex vivo [125I]SB-258585 binding in the rat with an ED(50) of 4.9 +/- 1.3 mg/kg po, 4 h postdose. In the rat maximal electroshock seizure threshold (MEST) test, SB-357134 produced a potent and dose-dependent increase in seizure threshold, with a minimum effective dose of 0.1 mg/kg po. At 10 mg/kg po, maximum activity occurred between 4 and 6 h postdose. Good exposure was observed with SB-357134 at 10 mg/kg po, reaching maximal blood and brain concentrations of 4.3 +/- 0.2 and 1.3 +/- 0.06 microM, respectively, 1 h postdose. In addition, SB-357134 (10 mg/kg po) enhanced memory and learning following chronic administration (twice a day for 7 days) in the rat water maze. Overall, these studies demonstrate that SB-357134 is a potent, selective, brain penetrant, and orally active 5-HT(6) receptor antagonist.

Importance of h5-HT1B Receptor Selectivity for 5-HT Terminal Autoreceptor Activity: an In Vivo Microdialysis Study in the Freely-moving Guinea-pig

The importance of h5-HT1B receptor selectivity for 5-HT terminal autoreceptor activity was investigated with the selective h5-HT1B receptor ligands SB 219085, SB 220272, SB 224289 and SB 216641. The studies employed measurement of compound affinity and efficacy in vitro and the measurement of extracellular 5-HT in the frontal cortex of the freely-moving guinea-pig using in vivo microdialysis. All compounds had high affinity and selectivity for the h5-HT1B receptor, with SB 224289 the most selective for h5-HT1B over h5-HT1D receptors. Compounds exhibited a range of efficacies at both receptors: SB 224289 and SB 219085 were inverse agonists, SB 220272 was an antagonist and SB 216641 was a partial agonist. SB 220272, SB 216641 and SB 224289 had no effect on extracellular 5-HT following systemic administration, however, SB 219085 produced a significant increase. The SB 219085-induced increase in extracellular 5-HT was attributed to the compounds non-specific releasing properties as it was also demonstrated to increase basal release of [3H]5-HT from pre-loaded guinea-pig cortical slices. The lack of effect of the above h5-HT1B receptor selective compounds and the decrease in extracellular 5-HT elicited by the non-selective compounds GR 127935, GR125743 and methiothepin suggest that antagonism of 5-HT1D receptors may mediate this decrease in 5-HT levels. It is plausible that blockade of 5-HT1D receptors increases 5-HT levels in the raphe, this activates 5-HTtA receptors which results in an overall decrease in terminal 5-HT release. Definitive proof now awaits elucidation of the action of a selective 5-HT1D receptor antagonist.

[3H]-SB-269970 radiolabels 5-HT7 receptors in rodent, pig and primate brain tissues.

The selective 5-HT7 receptor antagonist radioligand, [3H]-SB-269970, has been reported to radiolabel the human cloned 5-HT7(a) receptor and 5-HT7 receptors in guinea pig cortex (thomas et al, 2000). Saturation analysis of [3H]-SB-269970 binding to mouse forebrain, rat cortex, pig cortex, marmoset cortex and human thalamus membranes was consistent with labelling a homogenous population of binding sites in each tissue. K(D) values for [3H]-SB-269970 binding in these tissues ranged from 0.9 to 2.3 nM, being similar to those reported for the human cloned and guinea pig cortex 5-HT7 receptors (1.3 and 1.7 nM, respectively). Bmax values for [3H]-SB-269970 binding to the mouse, rat, pig, marmoset and human brain membranes were 20, 30, 31, 14 and 68 fmoles x mg x protein(-1), respectively. For each species the profile of inhibition of [3H]-SB-269970 binding, using a number of 5-HT7 receptor agonists and antagonists, correlated well with that reported for the human cloned 5-HT7(a) receptor (correlation coefficients were 0.95, 0.94, 0.92, 0.95, 0.97 versus the mouse, rat, pig, marmoset and human tissues, respectively). In conclusion, [3H]-SB-269970 has been shown to radiolabel 5-HT7 receptors in rodent, pig and primate brain and represents a valuable tool with which to further characterise the distribution and function of 5-HT7 receptors in native tissues and elucidate their potential role in disease states.