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Vilazodone has been reported to be an inhibitor of 5-hydoxytryptamine (5-HT) reuptake and a partial agonist at 5-HT1A receptors. Using [ 35 S]GTPgS binding in rat hippocampal tissue, vilazodone was demonstrated to have an intrinsic activity comparable to the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). Vilazodone (1–10 mg/kg p.o.) dose-dependently displaced in vivo [ 3 H]DASB (N,N-dimethyl-2-(2-amino-4-cyanophenylthio)benzylamine) binding from rat cortex and hippocampus, indicating that vilazodone occupies 5-HT transporters in vivo. Using in vivo microdialysis, vilazodone (10 mg/kg p.o.) was demonstrated to cause a 2-fold increase in extracellular 5-HT but no change in noradrenaline or dopamine levels in frontal cortex of freely moving rats. In contrast, administration of 8OH-DPAT (0.3 mg/kg s.c.), either alone or in combination with a serotonin specific reuptake inhibitor (SSRI; paroxetine, 3 mg/kg p.o.), produced no increase in cortical 5-HT whilst increasing noradrenaline and dopamine 2 and 4 fold, respectively. A 2-fold increase in extracellular 5-HT levels (but no change in noradrenaline or dopamine levels) was observed after combination of the 5-HT1A receptor antagonist, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(pyridinyl)cyclohexanecarboxamide) (WAY-100635; 0.3 mg/kg s.c.) and paroxetine (3 mg/kg p.o.). In summary, vilazodone behaved as a high efficacy partial agonist at the rat hippocampal 5-HT1A receptors in vitro and occupied 5-HT transporters in vivo. In vivo vilazodone induced a selective increase in extracellular levels of 5-HT in the rat frontal cortex. This profile was similar to that seen with a 5-HT1A receptor antagonist plus an SSRI but in contrast to 8-OH-DPAT either alone or in combination with paroxetine. D 2005 Elsevier B.V. All rights reserved.

Starting from a high throughput screening hit, a series of 3,4-dihydro-2H-benzoxazinones has been identified with both high affinity for the 5-HT(1A) receptor and potent 5-HT reuptake inhibitory activity. The 5-(2-methyl)quinolinyloxy derivative combined high 5-HT(1A/1B/1D) receptor affinities with low intrinsic activity and potent inhibition of the 5-HT reuptake site (pK(i)8.2). This compound also had good oral bioavailability and brain penetration in the rat.

At their clinical doses, current antipsychotic agents share the property of both dopamine D(2) and D(3) receptor blockade. However, a major disadvantage of many current medications are the observed extrapyramidal side-effects (EPS), postulated to arise from D(2) receptor antagonism. Consequently, a selective dopamine D(3) receptor antagonist could offer an attractive antipsychotic therapy, devoid of the unwanted EPS. Using SAR information gained in two previously reported series of potent and selective D(3) receptor antagonists, as exemplified by the 2,3,4,5-tetrahydro-1H-3-benzazepine 10 and the 2,3-dihydro-1H-isoindoline 11, a range of 7-sulfonyloxy- and 7-sulfonylbenzazepines has been prepared. Compounds of this type combined a high level of D(3) affinity and selectivity vs D(2) with an excellent pharmacokinetic profile in the rat. Subsequent optimization of this series to improve selectivity over a range of receptors and reduce cytochrome P450 inhibitory potential gave trans-3-(2-(4-((3-(3-(5-methyl-1,2,4-oxidiazolyl))phenyl)carboxamido)cyclohexyl)ethyl)-7-methylsulfonyl-2,3,4,5-tetrahydro-1H-3-benzazepine (58, SB-414796). This compound is a potent and selective dopamine D(3) receptor antagonist with high oral bioavailability and is CNS penetrant in the rat. Subsequent evaluation in the rat has shown that 58 preferentially reduces firing of dopaminergic cells in the ventral tegmental area (A10) compared to the substantia nigra (A9), an observation consistent with a prediction for atypical antipsychotic efficacy. In a separate study, 58 has been shown to block expression of the conditioned place preference (CPP) response to cocaine in male rats, suggesting that it may also have a role in the treatment of cue-induced relapse in drug-free cocaine addicts.

The effects of three structurally related cannabinoids on human and rat recombinant vanilloid VR1 receptors expressed in human embryonic kidney (HEK293) cells and at endogenous vanilloid receptors in the rat isolated mesenteric arterial bed were studied. In the recombinant cells, all three were full agonists, causing concentration-dependent increases in [Ca(2+)](i) (FLIPR), with a rank order of potency relative to the vanilloids capsaicin and olvanil, of olvanil> or =capsaicin>AM404 ((allZ)-N-(4-hydroxyphenyl)-5,8,11,14-eicosatetraenamide)>anandamide>methanandamide. These responses were inhibited by the vanilloid VR1 receptor antagonist, capsazepine. In the mesenteric arterial bed, vasorelaxation was evoked by these ligands with a similar order of potency. The AM404-induced vasorelaxation was virtually abolished by capsaicin pretreatment. AM404 inhibition of capsaicin-sensitive sensory neurotransmission was blocked by ruthenium red, but not by cannabinoid CB(1) and CB(2) receptor antagonists. AM404 had no effect on relaxations to calcitonin gene-related peptide. These data demonstrate that the vasorelaxant and sensory neuromodulator properties of AM404 in the rat isolated mesenteric arterial bed are mediated by vanilloid VR1 receptors.

Prompted by conflicting literature, this study compared the pharmacology of human 5-hydroxytryptamine2 (5-HT2) receptors expressed in SH-SY5Y cells using a fluorometric imaging plate reader (FLIPR) based Ca2+ assay. 5-Hydroxytryptamine (5-HT) increased intracellular calcium concentration ([Ca2+]i) at 5-HT2A, 5-HT2B and 5-HT2C receptors (pEC(50)=7.73+/-0.03, 8.86+/-0.04 and 7.99+/-0.04, respectively) and these responses were inhibited by mesulergine (pKB=7.42+/-0.06, 8.77+/-0.10 and 9.52+/-0.11). A range of selective agonists and antagonists displayed the expected pharmacology at each receptor subtype. Sodium butyrate pretreatment increased receptor expression in SH-SY5Y/5-HT2B (15-fold) and SH-SY5Y/5-HT2C cells (7-fold) and increased agonist potencies and relative efficacies. In contrast, sodium butyrate pretreatment of SH-SY5Y/5-HT(2A) cells did not affect receptor expression. The present study provides a direct comparison of agonist and antagonist pharmacology at 5-HT(2) receptor subtypes in a homogenous system and confirms that agonist potency and efficacy varies with the level of receptor expression.

SB-258585 (4-Iodo-N-[4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-benzenesulphonamide) is a high affinity ligand at 5-HT6 receptors. It displays over 100 fold selectivity for the 5-HT6 receptor over all other 5-HT receptors tested so far. SB-258585 has been radiolabelled, to high specific activity, for its characterization as a 5-HT6 receptor selective radioligand. [125I]-SB-258585 bound, with high affinity, to a single population of receptors in a cell line expressing human recombinant 5-HT6 receptors. Kinetic and saturation binding experiments gave pKD values of 9.01±0.09 and 9.09±0.02, respectively. In membranes derived from rat or pig striatum and human caudate putamen, [125I]-SB-258585 labelled a single site with high levels (>60%) of specific binding. Saturation analysis revealed pKD values of 8.56±0.07 for rat, 8.60±0.10 for pig and 8.90±0.02 for human. Bmax values for the tissues ranged from 173±23 and 181±25 fmol mg−1 protein in rat and pig striatum, respectively, to 215±41 fmol mg−1 protein in human caudate putamen. The pKi rank order of potency for a number of compounds, determined in competition binding assays with [125I]-SB-258585, at human caudate putamen membranes was: SB-271046>SB-258585>SB-214111>methiothepin>clozapine>5-Me-OT>5-HT>Ro 04-6790>mianserin>ritanserin=amitriptyline>5-CT>mesulergine. Similar profiles were obtained from pig and rat striatal membranes and recombinant 5-HT6 receptors; data from the latter correlated well with [3H]-LSD binding. Thus, [125I]-SB-258585 is a high affinity, selective radioligand which can be used to label both recombinant and native 5-HT6 receptors and will facilitate further characterization of this receptor subtype in animal and human tissues. British Journal of Pharmacology (2000) 130, 1597–1605; doi:10.1038/sj.bjp.0703458

SB-271046, potently displaced [(3)H]-LSD and [(125)I]-SB-258585 from human 5-HT(6) receptors recombinantly expressed in HeLa cells in vitro (pK(i) 8.92 and 9.09 respectively). SB-271046 also displaced [(125)I]-SB-258585 from human caudate putamen and rat and pig striatum membranes (pK(i) 8.81, 9.02 and 8.55 respectively). SB-271046 was over 200 fold selective for the 5-HT(6) receptor vs. 55 other receptors, binding sites and ion channels. In functional studies on human 5-HT(6) receptors SB-271046 competitively antagonized 5-HT-induced stimulation of adenylyl cyclase activity with a pA(2) 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 < or =0.1 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 (EC(50) of 0.16 microM) and brain concentrations of 0.01-0.04 microM at C(max). These data, together with the observed anticonvulsant activity of other selective 5-HT(6) 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-HT(6) receptors. Overall, these studies demonstrate that SB-271046 is a potent and selective 5-HT(6) 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-HT(6) receptors.

The novel 5-HT7 receptor antagonist, SB-269970-A, potently displaced [3H]-5-CT from human 5-HT7(a) (pKi 8.9±0.1) and 5-HT7 receptors in guinea-pig cortex (pKi 8.3±0.2). 5-CT stimulated adenylyl cyclase activity in 5-HT7(a)/HEK293 membranes (pEC50 7.5±0.1) and SB-269970-A (0.03–1 μM) inhibited the 5-CT concentration-response with no significant alteration in the maximal response. The pA2 (8.5±0.2) for SB-269970-A agreed well with the pKi determined from [3H]-5-CT binding studies. 5-CT-stimulated adenylyl cyclase activity in guinea-pig hippocampal membranes (pEC50 of 8.4±0.2) was inhibited by SB-269970-A (0.3 μM) with a pKB (8.3±0.1) in good agreement with its antagonist potency at the human cloned 5-HT7(a) receptor and its binding affinity at guinea-pig cortical membranes. 5-HT7 receptor mRNA was highly expressed in human hypothalamus, amygdala, thalamus, hippocampus and testis. SB-269970-A was CNS penetrant (steady-state brain : blood ratio of ca. 0.83 : 1 in rats) but was rapidly cleared from the blood (CLb=ca. 140 ml min−1 kg−1). Following a single dose (3 mg kg−1) SB-269970 was detectable in rat brain at 30 (87 nM) and 60 min (58 nM). In guinea-pigs, brain levels averaged 31 and 51 nM respectively at 30 and 60 min after dosing, although the compound was undetectable in one of the three animals tested. 5-CT (0.3 mg kg−1 i.p.) induced hypothermia in guinea-pigs was blocked by SB-269970-A (ED50 2.96 mg kg−1 i.p.) and the non-selective 5-HT7 receptor antagonist metergoline (0.3–3 mg kg−1 s.c.), suggesting a role for 5-HT7 receptor stimulation in 5-CT induced hypothermia in guinea-pigs. SB-269970-A (30 mg kg−1) administered at the start of the sleep period, significantly reduced time spent in Paradoxical Sleep (PS) during the first 3 h of EEG recording in conscious rats. British Journal of Pharmacology (2000) 130, 539–548; doi:10.1038/sj.bjp.0703357

A number of compounds, including the selective 5-HT7 receptor antagonist SB-258719, were investigated for their effect on [3H]-5-carboxamidotryptamine (5-CT) radioligand binding and 5-CT-stimulated adenylyl cyclase activity in guinea-pig hippocampal membranes, in order to confirm the presence of functionally coupled 5-HT7 receptors in this tissue. The [3H]-5-CT radioligand binding profile was consistent with binding predominantly to 5-HT7 receptors. The affinity of SB-258719 (pKi 7.2±0.1) was similar to its reported human 5-HT7 receptor affinity. In the adenylyl cyclase functional assay, 5-CT was a potent and full agonist compared to 5-HT, whereas 8-hydroxy-dipropylaminotetralin (8-OH-DPAT) was a partial agonist (intrinsic activity 0.4±0.1). The rank order of potency for agonists (5-CT>5-HT∼8-OH-DPAT) was consistent with activation of 5-HT7 receptors. SB-258719 (5 μM) and methiothepin (1 μM) surmountably antagonized the response to 5-CT, consistent with competitive antagonism. The pKB for SB-258719 (7.2±0.1) was in good agreement with its reported antagonist potency at the human cloned 5-HT7 receptor. In the functional assay, WAY-100635 (100 nM) and cyanopindolol (1 μM) induced a biphasic 5-CT response curve, consistent with selective antagonism of a component of the response to 5-CT. The estimated pKB values for WAY-100635 and cyanopindolol (9.6 and 8.4 respectively) were in good agreement with their reported 5-HT1A receptor affinities. The data are consistent with the presence of 5-HT7 receptors in guinea-pig hippocampus which are positively coupled to adenylyl cyclase. In addition, 5-HT7 receptor-mediated stimulation of adenylyl cyclase activity in this tissue appears to be augmented by a mechanism involving 5-HT1A receptor activation.

1. This study describes the pharmacological comparison of the muscarinic partial agonists sabcomeline, xanomeline and milameline at human cloned muscarinic receptor subtypes (hM1-5). 2. Radioligand binding studies at the hM1-5 muscarinic receptor subtypes were compared with functional studies using microphysiometry using carbachol as the standard full agonist. 3. In binding assays none of the compounds studied displayed preferential affinity for the M1,3,4 or M5 subtypes although carbachol was less potent at hM1 than hM3,4,5. 4. In functional studies, all of the compounds studied displayed similar levels of efficacy across the muscarinic receptors with the exception of M3, where there was a large apparent receptor reserve and the compounds behaved essentially as full agonists. 5. Sabcomeline was the most potent agonist in functional studies but also showed the lowest efficacy. In terms of potency, xanomeline showed some selectivity for M1 over M2 receptors and milameline showed some selectivity for M2 over M1 receptors. 6. These results show the value of microphysiometry in being able to compare receptor pharmacology across subtypes irrespective of the signal transduction pathway. 7. None of the partial agonists showed functional selectivity for M1 receptors, or indeed any muscarinic receptor, in the present study.

We have investigated the pharmacology of the functionally selective muscarinic M1 receptor partial agonist, sabcomeline [SB-202026 (R-(Z)-(+)-alpha-(methoxyamino)-1-azabicyclo[2.2.2] octane-3-acetonitrile)], in rat cortex and heart using radioligand binding and functional studies. The Quinuclidinyl benzilate/Oxotremorine-M acetate ratio from radioligand binding studies suggested that sabcomeline and xanomeline [3(3-hexyloxy-1,25-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-met hylpyridine] are muscarinic receptor partial agonists in cortical and heart membranes. In [35S]GTPgammaS binding studies in rat cortex, carbachol stimulated binding via muscarinic M2/M4 receptors which could be blocked by sabcomeline with a pA2 of 7.2. In rat heart membranes, carbachol also stimulated [35S]GTPgammaS binding studies through muscarinic M2 receptors. Sabcomeline caused a small stimulation of basal [35S]GTPgammaS binding in both rat and heart tissues. Sabcomeline did not stimulate phosphoinositide hydrolysis in rat cortical slices, but did block the muscarinic M1 receptor-mediated response caused by carbachol with apparent pKb of 6.9. Xanomeline and milameline also had no effect on phosphoinositide hydrolysis up to 100 microM. In adenylyl cyclase studies in rat atria, sabcomeline inhibited forskolin-stimulated adenylyl cyclase activity to a similar extent to that of carbachol, xanomeline and milameline. The present study, using the techniques of radioligand binding, supports previous publications which have claimed that sabcomeline is a muscarinic receptor partial agonist. As expected, this study shows that the functional actions of this compound at muscarinic receptor subtypes and in different tissues will depend on receptor reserve.

Xanomeline [3(3-hexyloxy-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-me thylpyridine] has been reported to act as a functionally selective muscarinic partial agonist with potential use in the treatment of Alzheimer's disease. This study examined the functional activity of xanomeline at 5-HT1 and 5-HT2 receptors in native tissue and/or human cloned receptors. Xanomeline had affinity for muscarinic receptors in rat cortical membranes where the ratio of the displacement affinity of [3H]-Quinuclidinyl benzilate vs that of [3H]-Oxotremorine-M was 16, indicative of partial agonist activity. Radioligand binding studies on human cloned receptors confirmed that xanomeline had substantial affinity for M1, M2, M3, M4, M5 receptors and also for 5-HT1 and 5-HT2 receptor subtypes. Carbachol and xanomeline stimulated basal [35S]-GTPgammaS binding in rat cortical membranes with micromolar affinity. The response to carbachol was attenuated by himbacine and pirenzepine with pA2 of 8.2, 6.9 respectively consistent with the response being mediated, predominantly, via M2 and M4 receptors. Xanomeline-induced stimulation of [35S]-GTPgammaS binding was inhibited by himbacine with an apparent pKb of 6.3, was not attenuated by pirenzepine up to 3 microM and was inhibited by the selective 5-HT1A antagonist WAY100635 with an apparent pKb of 9.4. These data suggest the agonist effect of xanomeline in this tissue is, in part, via 5-HT1A receptors. Similar studies on human cloned receptors confirmed that xanomeline is an agonist at human cloned 5-HT1A and 5-HT1B receptors. In studies using the fluorescent cytoplasmic Ca2+ indicator FLUO-3AM, xanomeline induced an increase in cytoplasmic Ca2+ concentration in SH-SY5Y cells expressing recombinant human 5-HT2C receptors. Atropine antagonized this response, consistent with mediation via endogenously-expressed muscarinic receptors. In the presence of atropine, xanomeline antagonized 5-HT-induced cytoplasmic changes in Ca2+ concentration in cells expressing h5-HT2A, h5-HT2B and h5-HT2c receptors with potencies similar to its affinity at these receptors. These studies indicate that xanomeline is a potent agonist at 5-HT1A and 5-HT1B receptors and an antagonist at 5-HT2 receptor subtypes.

1 The functional profile of the long form of the human cloned 5-HT7 receptor (designated h5-HT7(a)) was investigated using a number of 5-HT receptor agonists and antagonists and compared with its binding profile. Receptor function was measured using adenylyl cyclase activity in washed membranes from HEK293 cells stably expressing the recombinant h5-HT7(a) receptor. 2 The receptor binding profile, determined by competition with [3H]-5-CT, was consistent with that previously reported for the h5-HT7(a) receptor. The selective 5-HT7 receptor antagonist SB-258719 ((R)-3,N-Dimethyl-N-[1-methyl-3-(4-methylpiperidin-1-yl)propyl]benzene sulfonamide) displayed high affinity (pKi 7.5) for the receptor. 3 In the adenylyl cyclase functional assay, 5-CT and 8-OH-DPAT were both full agonists compared to 5-HT and the rank order of potency for agonists (5-CT>5-HT>8-OH-DPAT) was the same in functional and binding studies. 4 Risperidone, methiothepin, mesulergine, clozapine, olanzapine, ketanserin and SB-258719 antagonised surmountably 5-CT-stimulated adenylyl cyclase activity. Schild analysis of the antagonism by SB-258719 gave a pA2 of 7.2±0.2 and slope not significantly different from 1, consistent with competitive antagonism. 5 The same antagonists also inhibited basal adenylyl cyclase activity with a rank order of potency in agreement with those for antagonist potency and binding affinity. Both SB-258719 and mesulergine displayed apparent partial inverse agonist profiles compared to the other antagonists tested. These inhibitory effects of antagonists appear to be 5-HT7 receptor-mediated and to reflect inverse agonism. 6 It is concluded that in this expression system, the h5-HT7(a) receptor shows the expected binding and functional profile and displays constitutive activity, revealing inverse agonist activity for a range of antagonists. British Journal of Pharmacology (1998) 124, 1300–1306; doi:10.1038/sj.bjp.0701946

5-HT1 receptors are members of the G-protein-coupled receptor superfamily and are negatively linked to adenylyl cyclase activity. The human 5-HT1B and 5-HT1D receptors (previously known as 5-HT1Dbeta and 5-HT1Dalpha, respectively), although encoded by two distinct genes, are structurally very similar. Pharmacologically, these two receptors have been differentiated using nonselective chemical tools such as ketanserin and ritanserin, but the absence of truly selective agents has meant that the precise function of the 5-HT1B and 5-HT1D receptors has not been defined. In this paper we describe how, using computational chemistry models as a guide, the nonselective 5-HT1B/5-HT1D receptor antagonist 4 was structurally modified to produce the selective 5-HT1B receptor inverse agonist 5, 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] (SB-224289). This compound is a potent antagonist of terminal 5-HT autoreceptor function both in vitro and in vivo.

Despite only modest homology between h5-HT1B and h5-HT1D receptor amino acid sequences, these receptors display a remarkably similar pharmacology. To date there are few compounds which discriminate between these receptor subtypes and those with some degree of selectivity, such as ketanserin, have greater affinity for other 5-HT receptor subtypes. We now report on two compounds, SB-216641 (N-[3-(2-dimethylamino) ethoxy-4-methoxyphenyl]-2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)-(1,1'-biphenyl)-4-carboxamide) and BRL-15572 3-[4-(3-chlorophenyl) piperazin-1-yl]-1,1-diphenyl-2-propanol), which display high affinity and selectivity for h5-HT1B and h5-HT1D receptors, respectively. In receptor binding studies on human receptors expressed in CHO cells, SB-216641 has high affinity (pKi = 9.0) for h5-HT1B receptors and has 25-fold lower affinity at h5-HT1D receptors. In contrast, BRL-15572 has 60-fold higher affinity for h5-HT1D (pKi = 7.9) than 5-HT1B receptors. Similar affinities for these compounds were determined on native tissue 5-HT1B receptors in guinea-pig striatum. Functional activities of SB-216641 and BRL-15572 were measured in a [35S]GTPgammaS binding assay and in a cAMP accumulation assay on recombinant h5-HT1B and h5-HT1D receptors. Both compounds were partial agonists in these high receptor expression systems, with potencies and selectivities which correlated with their receptor binding affinities. In the cAMP accumulation assay, results from pK(B) measurements on the compounds again correlated with receptor binding affinities (SB-216641, pK(B) = 9.3 and 7.3; BRL-15572, pK(B) =6 and 7.1, for h5-HT1B and h5-HT1D receptors respectively). These compounds will be useful pharmacological agents to characterise 5-HT1B and 5-HT1D receptor mediated responses.

Human cerebral cortical slices and synaptosomes, guinea-pig cerebral cortical slices and human right atrial appendages were used to study the effects of SB-216641, a preferential h5-HT1B receptor ligand, and of BRL-15572, a preferential h5-HT1D receptor ligand, on the presynaptic h5-HT1B and h5-HT1B-like autoreceptors in the human and guinea-pig brain preparations, respectively, and on the presynaptic h5-HT1D heteroreceptors in the human atrium. The brain preparations, preincubated with [3H]serotonin ([3H]5-HT), and the segments of atrial appendages, preincubated with [3H]noradrenaline, were superfused with modified Krebs' solution and tritium overflow was evoked electrically (human and guinea-pig cerebral cortex slices and human atrial appendages) or by high K+ (human cerebral cortex synaptosomes). The electrically evoked tritium overflow from guinea-pig cerebral cortex slices was reduced by the 5-HT receptor agonist 5-carboxamidotryptamine (5-CT). This effect was not modified by BRL-15572 (2 microM; concentration 154 times higher than its Ki at h5-HT1D receptors) but was antagonized by SB-216641 (0.1 microM; concentration 100 times higher than its Ki at h5-HT1B receptors; apparent pA2 8.45). SB-216641 (0.1 microM) by itself facilitated, whereas BRL-15572 (2 microM) did not affect, the evoked overflow. In human cerebral cortex slices SB-216641 (0.1 microM) also facilitated, and BRL-15572 (2 microM) again failed to affect, the electrically evoked tritium overflow. In human cerebral cortical synaptosomes, 5-CT reduced the K+-evoked tritium overflow. This response was unaffected by BRL-15572 (300 nM) but antagonized by SB-216641 (15 nM; drug concentrations 23 and 15 times higher than their Ki at h5-HT1D and h5-HT1B receptors, respectively). Both drugs, given alone, did not modify the K+-evoked tritium overflow. In human atrial appendages, the electrically evoked tritium overflow was inhibited by 5-HT in a manner susceptible to antagonism by BRL-15572 (300 nM; 23 times Ki at h5-HT1D receptors) but not by SB-216641 (30 nM; 30 times Ki at h5-HT1B receptors). Both drugs by themselves did not change the electrically evoked tritium overflow. In conclusion, SB-216641 behaves as a preferential antagonist at native human 5-HT1B receptors and BRL-15572 as a preferential antagonist at native human 5-HT1D receptors. These compounds are clearly useful tools for the differentiation between human 5-HT1B and 5-HT1D receptors in functional studies.

1 SB-204269 (trans-(+)-6-acetyl-4S-(4-fluorobenzoylamino)-3,4-dihydro-2,2-dimethyl-2H-benzol[b]pyran-3R-ol, hemihydrate) shows potent anticonvulsant activity in a range of animal seizure models, with a lack of neurological or cardiovascular side-effects. The profile of the compound suggests that it may have a novel mechanism of action. This study describes the characteristics of a binding site for [3H]-SB-204269 in rat forebrain membranes. 2 Specific [3H]-SB-204269 binding was saturable and analysis indicated binding to a homogenoeous population of non-interacting binding sites with a dissociation constant (KD) of 32±1 nM and a maximum binding capacity (Bmax) of 253±18 fmol mg−1 protein. Kinetic studies indicated monophasic association and dissociation. Binding was similar in HEPES or Tris-HCl buffers and was unaffected by Na+, K+, Ca2+ or Mg2+ ions. Specific binding was widely distributed in brain, but was minimal in a range of peripheral tissues. 3 Specific [3H]-SB-204269 binding was highly stereoselective, with a 1000 fold difference between the affinities of SB-204269 and its enantiomer SB-204268 for the binding site. The affinities of analogues of SB-204269 for binding can be related to their activities in the mouse maximal electroshock seizure threshold (MEST) test of anticonvulsant action. 4 None of the standard anticonvulsant drugs, phenobarbitone, phenytoin, sodium valproate, carbamazepine, diazepam and ethosuximide, or the newer anticonvulsants, lamotrigine, vigabatrin, gabapentin and levetiracetam, showed any affinity for the [3H]-SB-204269 binding site. A wide range of drugs active at amino acid receptors, Na+ or K+ channels or various other receptors did not demonstrate any affinity for the binding site. 5 These studies indicate that SB-204269 possesses a specific CNS binding site which may mediate its anticonvulsant activity. This binding site does not appear to be directly related to the sites of action of other known anticonvulsant agents, but may have an important role in regulating neuronal excitability. British Journal of Pharmacology (1997) 121, 1687–1691; doi:10.1038/sj.bjp.0701331

The selective, brain penetrant, 5-HT(1B/D) (formerly 5-HT(1D beta/alpha)) receptor agonist SKF-99101H (3-(2-dimethylaminoethyl)-4-chloro-5-propoxyindole hemifumarate) (30 mg/kg i.p.) causes a dose related fall in rectal temperature in guinea pigs which previous studies have shown to be blocked by the non-selective 5-HT(1B/D) receptor antagonist GR-127935 (N-[4-methoxy-3-(4-methyl-1-piperazinyl) phenyl]-2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl) [1,1'biphenyl]-4-carboxamide oxalate). The present study shows that the hypothermic response to SKF-99101H is dose-dependently blocked by SB-224289G (1'-methyl-5-(2'-methyl-4'-[(5-methyl-1,2,4-oxadiazol-3-yl)bipheny l-4-yl]carbonyl)-2,3,6,7-tetrahydrospiro[furo[2,3-f]indole-3,4'-pi peridone] hemioxalate) (0.3-10.0 mg/kg p.o.) (ED50 3.62 mg/kg), which is the first compound to be described which is more than 60 fold selective for the 5-HT1B receptor over the 5-HT1D receptor. SB-216641A (N-[3-(2-dimethylamino) ethoxy-4-methoxy-phenyl] 2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)-(1,1'-biphenyl)-4-car boxamide hydrochloride) (0.6-20.0 mg/kg i.p.), which is somewhat less selective (30 fold) for the 5-HT1B receptor over the 5-HT1D receptor had a similar effect (ED50 4.43 mg/kg). The brain penetrant 5-HT1D selective receptor antagonist, BRL-15572 (4-(3-chlorophenyl)-alpha-(diphenylmethyl)-1-piperazineethanol+ ++ dihydrochloride) (0.3-100.0 mg/kg i.p.) was inactive. When administered alone neither BRL-15572 (0.1-10 mg/kg i.p.) nor SB-224289G (2.2-22 mg/kg p.o.) had an effect on body temperature. These data demonstrate that 5-HT1B (formerly 5-HT(1D beta)) and not 5-HT1D (formerly 5-HT(1D alpha)) receptors mediate the hypothermic response to SKF-99101H (30 mg/kg i.p.) in guinea pigs. The compounds described are useful pharmacological tools for distinguishing responses to 5-HT1B and 5-HT1D receptors.

This study, carried out in pig caudate membranes, characterises the radioligand binding site labelled with [ 3 H]L-694,247 (2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1 H -indole-3-yl]ethylamine). The affinities of 27 standard compounds are consistent with binding to a 5-HT 1D recognition site. In addition the results indicate that, under the assay conditions described, [ 3 H]L-694,247 specifically labels the 5-HT 1Dβ recognition site since ketanserin and ritanserin display a low affinity consistent with their activities at this subtype of the 5-HT 1D receptor.

1. With radioligand binding techniques, MDL 73005 EF (8-[2-(2,3-dihydro-1,4-benzodioxin-2-yl-methylamino)ethyl]-8-az aspiro[4, 5]decane-7,9-dione methyl sulphonate) shows high affinity (pIC50 8.6) and selectivity (greater than 100 fold compared to other monoamine and benzodiazepine receptor sites) for the 5-hydroxytryptamine (5-HT)1A recognition site; it was both more potent and more selective than buspirone in this respect. 2. In rats pretreated with reserpine, 8-hydroxy-2-(di-n-propyl-amino) tetralin (8-OH-DPAT) induced forepaw treading and flat body posture; in the same model, MDL 73005EF and buspirone showed minimal agonist activity and at high doses MDL 73005EF inhibited responses to 8-OH-DPAT. 3. In rats trained to discriminate 8-OH-DPAT from saline in a drug discrimination paradigm, both MDL 73005EF and buspirone generalized dose-dependently and completely to the 8-OH-DPAT cue. 4. To define the anxiolytic potential of MDL 73005EF, it was examined in the elevated plus-maze test and in the water-lick conflict test in comparison with diazepam and buspirone. In both tests MDL 73005EF induced effects similar to those seen following diazepam. Buspirone had similar effects to both MDL 73005EF and diazepam in the water-lick conflict test but opposite effects in the elevated plus-maze. 8-OH-DPAT also had opposite effects in the elevated plus-maze test to MDL 73005EF and diazepam. 5. The anti-conflict effects of MDL 73005EF were reversed by low doses of the 5-HT1A receptor agonist, 8-OH-DPAT; those of buspirone were neither antagonised nor mimicked by 8-OH-DPAT. 6. These results suggest that an interaction with 5-HTIA receptors is the basis of the anxiolytic-like activity of MDL 73005EF. However, its mechanism of action is clearly different from that of buspirone, possibly reflecting a greater selectivity for the 5-HTlA receptors located presynaptically on central 5- hydroxytryptaminergic neurones.

In this study, we examined the effect of the acute and chronic administration of the selective 5-HT2C receptor antagonist SB-243213 (SB) on the activity of spontaneously active dopamine (DA) cells in the substantia nigra pars compacta (SNC) and ventral tegmental area (VTA) in anesthetized, albino, male Sprague-Dawley rats. This was accomplished using the technique of in vivo extracellular single cell recording. The acute i.v. administration of SB-243213 (0.025–3.2 mg/kg) did not significantly alter the basal firing rate or pattern of either spontaneously active SNC or VTA DA neurons compared to vehicle-treated controls. The acute i.p. administration of either 1 or 10 mg/kg of SB-243213 did not significantly alter the number of spontaneously active DA cells in the SNC or VTA compared to vehicle-treated controls, whereas the 3 mg/kg dose only significantly decreased the number of spontaneously active VTA DA neurons. Overall, the 1 mg/kg dose of SB-243213 did not significantly alter the firing pattern of either SNC or VTA DA neurons compared to vehicle-treated controls. In contrast, the 3 mg/kg dose significantly altered the firing pattern of SNC DA neurons, whereas the 10 mg/kg dose altered the firing pattern of DA neurons in both the SNC and VTA. The repeated i.p. administration (21 days) of 1, 3, and 10 mg/kg of SB-243213 or 20 mg/kg of clozapine produced a significant decrease in the number of spontaneously active DA cells in the VTA compared to vehicle-treated controls. The decrease in the number of spontaneously active VTA DA cells was not reversed by the i.v. administration of (+)-apomorphine (50 μg/kg). The repeated administration of either 1 or 3 mg/kg of SB-243213 had minimal effects on the firing pattern of either SNC or VTA DA neurons. In contrast, the firing pattern of VTA DA neurons was significantly altered by 10 mg/kg dose of SB-243213. Overall, our results indicate that antagonism of the 5-HT2C receptor alters the activity of midbrain DA neurons in anesthetized rats and suggest that SB-243213 has an atypical antipsychotic profile following chronic administration. Synapse 46:129–139, 2002. © 2002 Wiley-Liss, Inc.

5-CT (5-carboxamidotryptamine)-insensitive (5-HT1E/5-HT1F) 5-HT1-like recognition sites have been mapped autoradiographically in rat and guinea pig brain using [3H]5-HT in the presence of 5-CT and mesulergine to mask 5-HT1A, 5-HT1B, 5-HT1D and 5-HT2C binding sites. Binding was more dense in the guinea pig but in both species 5-CT-insensitive 5-HT1-like sites were located in the olfactory tubercle, interpeduncular nucleus, caudate putamen, nucleus accumbens, substantia nigra, frontal cortex and hippocampus. These receptors were particularly marked in the claustrum of the guinea pig but not the rat.

5‐HT(1B) autoreceptors are involved in the control of extracellular 5‐HT levels from both the terminal and cell body regions of serotonergic neurons. In this manuscript we review the pharmacological and pharmacokinetic data available for the selective and potent 5‐HT(1B) receptor inverse agonist, SB‐236057‐A (1′‐ethyl‐5‐(2′‐methyl‐4′‐(5‐methyl‐1,3,4‐oxadiazolyl‐2‐yl)biphenyl‐4‐carbonyl)‐2,3,6,7‐tetrahydrospiro (furo[2,3‐f]indole‐3,4′‐piperidine) hydrochloride). SB 236057‐A has been shown to have high affinity for human 5‐HT(1B) receptors (pK (i)= 8.2) and displays 80 or more fold selectivity for the human 5‐HT(1B) receptor over other 5‐HT receptors and a range of additional receptors, ion channels and enzymes. In functional studies at human 5‐HT(1B) receptors SB‐236057‐A displayed inverse agonism (pA(2)= 8.9) using [(35)S]GTPγS binding, and silent antagonism (pA(2)= 9.2) using cAMP accumulation. SB‐236057‐A also acted as an antagonist at the 5‐HT terminal autoreceptor as measured by [(3)H]5‐HT release from electrically stimulated guinea pig and human cortical slices. In the guinea pig, pharmacokinetic analysis demonstrated that SB‐236057‐A was bioavailable and according to in vivo pharmacodynamic assays it enters brain and has a long duration of action. Importantly no side effect liability was evident at relevant doses from anxiogenic, cardiovascular, sedative or migraine viewpoints. In vivo microdialysis studies demonstrated that SB‐236057‐A is an antagonist in the guinea pig cortex but has no effect on extracellular 5‐HT levels per se. In contrast, SB‐236057‐A increased extracellular 5‐HT levels in the guinea pig dentate gyrus. This increase in 5‐HT release was comparable to that observed after 14 days of paroxetine administration. SB‐236057‐A has been a useful tool in confirming that, in either guinea pigs or humans, the terminal 5‐HT autoreceptor is of the 5‐HT(1B) subtype. It appears that acute 5‐HT(1B) receptor blockade, by virtue of increased 5‐HT release in the dentate gyrus, may provide a rapidly acting antidepressant.

SB-243213 (5-methyl-1-[[-2-[(2-methyl-3-pyridyl)oxy]-5-pyridyl]carbamoyl]-6-trifluoromethylindoline hydrochloride) is a new, selective 5-hydroxytryptamine (5-HT)2C receptor inverse agonist. SB-243213 has high affinity for the human 5-HT2C receptor (pKi 9.37) and greater than a 100-fold selectivity over a wide range of neurotransmitter receptors, enzymes and ion channels. In in vitro functional studies, SB-243213 acted as an inverse agonist at the human 5-HT2C receptor with a pKb of 9.8. In in vivo studies, SB-243213 was a potent inhibitor of central 5-HT2C receptor-mediated function in rats, blocking meta-chlorophenylpiperazine-induced hypolocomotion with an ID50 of 1.1 mg/kg p.o. and a long duration of action (>8 h). In rats, SB-243213 exhibited anxiolytic-like activity in both the social interaction and Geller–Seifter conflict tests. Importantly, unlike diazepam, chronic administration of SB-243213 did not result in the development of either tolerance to the anxiolytic-like effects or withdrawal anxiogenesis. Furthermore, in rodents, SB-243213 did not affect seizure threshold, did not increase body weight or induce catalepsy, but attenuated the haloperidol-induced catalepsy. SB-243213 did not affect amphetamine-, MK-801- or phencyclidine-induced hyperactivity. In conclusion, SB-243213 may possess an improved anxiolytic profile compared to benzodiazepines. SB-243213 also modulates dopaminergic transmission, lacks pro-psychotic properties and may have utility in the treatment of schizophrenia and motor disorders.

The effect of low pH on capsaicin-sensitive sensory neurotransmission in the rat isolated mesenteric arterial bed and at recombinant (rVR1) vanilloid receptors was investigated. Mesenteric sensory neurogenic vasorelaxation elicited by electrical field stimulation was reversibly inhibited by lowering pH from 7.4 to 6.9 and 6.3. Capsaicin-induced vasorelaxation was not different at pH 6.9, but was attenuated at pH 6.3. Vasorelaxation to calcitonin gene-related peptide, the principal sensory motor neurotransmitter in rat mesenteric arteries, was not different at pH 6.9 or pH 6.3. In rVR1-transfected HEK293 cells, acidic conditions enhanced the affinities of capsaicin and capsazepine at rVR1, but did not affect the potency of carbachol at endogenous muscarinic receptors. Following inactivation of endogenous acid-sensitive ion channels, lowering pH (6.0-4.5) directly increased [Ca2+]i in rVR1-HEK293 cells (EC50 5.5). This response was abolished by 1 microM capsazepine. In conclusion, a decrease in pH (to 6.9 and 6.3) enhances the affinity of capsaicin at rVR1, but inhibits sensory neurotransmission in the rat mesenteric arterial bed. This likely explains why there is no evidence of an enhancement of sensitivity to capsaicin at endogenous vanilloid receptors, as observed with rVR1. When pH is reduced still further (6.0-5.5) there is direct activation of rVR1.

Bisarylmethoxyethers have been identified with nanomolar 5-HT2C affinity and selectivity over both 5-HT2A and 5-HT2B receptors. Compounds such as 1, 2, 8, 12, 14 and 18 have potent oral activity in a centrally mediated pharmacodynamic model of 5-HT2C function and their therapeutic potential is currently under further investigation.

Using homology searching of public databases with a metabotropic glutamate receptor sequence from Caenorhabditis elegans, two novel protein sequences (named RAIG-2 (HGMW-approved symbol GPRC5B) and RAIG-3 (HGMW-approved symbol GPRC5C) were identified containing seven putative transmembrane domains characteristic of G-protein-coupled receptors (GPCRs). RAIG-2 and RAIG-3 encode open reading frames of 403 and 442 amino acid polypeptides, respectively, and show 58% similarity to the recently identified retinoic acid-inducible gene-1 (RAIG-1, HGMW-approved symbol RAI3). Analysis of the three protein sequences places them within the type 3 GPCR family, which includes metabotropic glutamate receptors, GABA(B) receptors, calcium-sensing receptors, and pheromone receptors. However, in contrast to other type 3 GPCRs, RAIG-1, RAIG-2, and RAIG-3 have only short N-terminal domains. RAIG-2 and RAIG-3 cDNA sequences were cloned into the mammalian expression vector pcDNA3 with c-myc or HA epitope tags inserted at their N-termini, respectively. Transient transfection experiments in HEK239T cells using these constructs demonstrated RAIG-2 and RAIG-3 expression at the cell surface. Distribution profiles of mRNA expression obtained by semiquantitative Taq-Man PCR analysis showed RAIG-2 to be predominantly expressed in human brain areas and RAIG-3 to be predominantly expressed in peripheral tissues. In addition, expression of RAIG-2 and RAIG-3 mRNA was increased following treatment with all-trans-retinoic acid in a manner similar to that previously described for RAIG-1. Finally, RAIG-2 was mapped to chromosome 16p12 (D16S405-D16S3045) and RAIG-3 to chromosome 17q25 (D17S1352-D17S785). These results suggest that RAIG-1, RAIG-2, and RAIG-3 represent a novel family of retinoic acid-inducible receptors, most closely related to the type 3 GPCR subfamily, and provide further evidence for a linkage between retinoic acid and G-protein-coupled receptor signal transduction pathways.

The evolution, synthesis, and biological activity of a novel series of 5-HT(2C) receptor inverse agonists are reported. Biarylcarbamoylindolines have been identified with excellent 5-HT(2C) affinity and selectivity over 5-HT(2A) receptors. In addition, (pyridyloxypyridyl)carbamoylindolines have been discovered with additional selectivity over the closely related 5-HT(2B) receptor. Compounds from this series are inverse agonists at the human cloned 5-HT(2C) receptor, completely abolishing basal activity in a functional assay. The new series have reduced P450 inhibitory liability compared to a previously described series of 1-(3-pyridylcarbamoyl)indolines (Bromidge et al. J. Med. Chem. 1998, 41, 1598) from which they evolved. Compounds from this series showed excellent oral activity in a rat mCPP hypolocomotion model and in animal models of anxiety. On the basis of their favorable biological profile, 32 (SB-228357) and 40 (SB-243213) have been selected for further evaluation to determine their therapeutic potential for the treatment of CNS disorders such as depression and anxiety.

The effect of dosing guinea pigs with spironolactone (100 mg/kg twice daily for 3 days) upon the sigma recognition site labelled with [ 3 H]-DTG was investigated. Animals were dosed with 100 mg/kg spironolactone twice a day for 3 days. Spironolactone treatment caused a decrease in sigma radioligand binding in membranes prepared from liver and brain but not in adrenals or testes. Saturation analysis of [ 3 h]-dtg radioligand binding in the brain indicated that the decrease in specifically bound [ 3 H]-DTG binding was due to a reduction in receptor number with no change in affinity. This method for the selective depletion of brain and liver sigma recognition sites will provide a useful tool for exploring the functional significance of this site.

The present studies were conducted to investigate the role of 5-HT2C and 5-HT2B receptors in the generation of pentylenetetrazol and electroshock-evoked seizures. The 5-HT2C/2B receptor-preferring agonist 1-(m-chlorophenyl)-piperazine (mCPP; 2.5-7 mg/kg i.p.) weakly elevated seizure threshold in the mouse (but not the rat) electroshock test and also provided appreciable protection against pentylenetetrazol-induced myoclonic and/or tonic seizures in mice and rats, an action that was inhibited by the 5-HT2C/2B receptor antagonist 5-methyl-1-(3-pyridylcarbomoyl)-1,2,3,5-tetrahydropyrrolo[2, 3-f]indole (SB-206553; 10-20 mg/kg p.o.). In contrast, the 5-HT2B receptor agonist 1-[5-(2-thienylmethoxy)-1H-3-indoyl]propan-2-amine hydrochloride (BW-723C86; 3-30 mg/kg s.c.) had no effect on the threshold for generalised seizures in any of the models employed. These results indicate that the observed anticonvulsant effects of mCPP are likely to be mediated by activation of 5-HT2C receptors. However, blockade of these receptors in mice (or rats) by SB-206553 (5-20 mg/kg p.o.) did not result in the reduced seizure threshold characteristic of mutant mice deficient of 5-HT2C receptors, suggesting that in normal adult animals this receptor subtype may usually be subjected to only a low level of 5-hydroxytryptamine tone.

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-HT1A 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. © 1997 Elsevier Science Ltd.

SB 242084 has a high affinity (pKi 9.0) for the cloned human 5-HT2C receptor and 100- and 158-fold selectivity over the closely related cloned human 5-HT2B and 5-HT2A subtypes respectively. SB 242084 had over 100-fold selectivity over a range of other 5-HT, dopamine and adrenergic receptors. In studies of 5-HT-stimulated phosphatidylinositol hydrolysis using SH-SY5Y cells stably expressing the cloned human 5-HT2C receptor, SB 242084 acted as an antagonist with a pKb of 9.3, which closely resembled its corresponding receptor binding affinity. SB 242084 potently inhibited m-chlorophenylpiperazine (mCPP, 7 mgkg i.p. 20 min pre-test)-induced hypolocomotion in rats, a model of in vivo central 5-HT2C receptor function, with an ID50 of 0.11 mg/kg i.p., and 2.0 mg/kg p.o. SB 242084 (0.1-1 mg/kg i.p.) exhibited an anxiolytic-like profile in the rat social interaction test, increasing time spent in social interaction, but having no effect on locomotion. SB 242084 (0.1-1 mg/kg i.p.) also markedly increased punished responding in a rat Geller-Seifter conflict test of anxiety, but had no consistent effect on unpunished responding. A large acute dose of SB 242084 (30 mg/kg p.o.) had no effect on seizure susceptibility in the rat maximal electroshock seizure threshold test. Also, while SB 242084 (2 and 6 mg/kg p.o. 1 hr pre-test) antagonized the hypophagic response to mCPP, neither acute nor subchronic administration of the drug, for 5 days at 2 or 6 mg/kg p.o. twice daily, affected food intake or weight gain. The results suggest that SB 242084 is the first reported selective potent and brain penetrant 5-HT2C receptor antagonist and has anxiolytic-like activity, but does not possess either proconvulsant or hyperphagic properties which are characteristic of mutant mice lacking the 5-HT2C receptor.

Inhibition of [3H]-5-hydroxytryptamine ([3H]-5-HT) release from guinea pig brain slices via activation of the terminal 5-HT autoreceptor has previously been characterised as a model of 5-HT1D receptor activation, based on the rank potencies of a range of agonists, and the potent antagonism of the inhibitory effects of 5-HT by metitepine. The present study uses this model, in slices of the guinea pig hippocampus, to examine the antagonist potency of metitepine against the 5-HT receptor agonists sumatriptan, 5-carboxamidotryptamine (5-CT) and 5-HT. Addition of metitepine to the perfusion buffer (30, 300 and 1000 nmol/l) significantly shifted the concentration-response curve to 5-HT, producing a Schild slope of 1.1, and a pA2 value of 7.6. However, the ability of metitepine to antagonise the effects of sumatriptan or 5-CT in this model was less marked. A clear-cut shift in the concentration-response curve to sumatriptan was only achieved at 1000 nmol/l metitepine (apparent pA2 = 6.7), and this was similar to the ability of metitepine to attenuate the effects of 5-CT (apparent pA2 7.0 at 300 nmol/l and 6.7 at 1000 nmol/l). These findings suggest heterogeneity in the receptor mediating inhibition of [3H]-5-HT release in guinea pig hippocampus.

A variety of achiral conformationally restricted spirocyclic piperidines have been prepared in an attempt to investigate the functional role of the central sigma recognition site. All the compounds possessed a lipophilic N-substituent incorporating either a tetralin, indan, or benzocycloheptane skeleton. Their in vitro affinity at the sigma site was assessed in radioligand displacement experiments with guinea pig cerebellum homogenates using the sigma-specific radioligand [3H]-N,N'-di-o-tolylguanidine ([3H]-DTG, [3H]-6). A study of the structure-activity relationships identified the N-butyl and N-dimethylallyl substituents as the optimum groups for high affinity and selectivity at the sigma site (e.g., 3,4-dihydro-1'-(3-methylbut-2-enyl)spiro[1H-indene-1,4'-piperidine ] (48), pIC50 = 8.9 vs [3H]-6 and greater than 10,000-fold selective over the dopamine D2 receptor). Such compounds are amongst the highest affinity sigma ligands reported to date, with excellent selectivity over the dopamine D2 receptor, and may serve as a useful tool for exploring the physiological role of the sigma site.

This paper describes the synthesis of some conformationally restricted 4-phenylpiperidine analogues and their affinities for the guinea pig cerebellum sigma recognition site ([3H]-DTG) and the rat striatum dopamine D2 receptor ([3H]-(-)-sulpiride) in order to develop potent selective sigma ligands as tools in the investigation of this site in psychosis. It was found that both hexa- and octahydrobenz[f]isoquinolines with lipophilic N-substituents had high affinities for the sigma site. Notably, trans-3-cyclohexyl-1,2,3,4,4a,5,6,10b-octahydrobenz[f]isoquinoline (26) had an affinity of 0.25 nM making it the highest affinity sigma ligand reported to date. Moreover, it is at least 10,000-fold selective over the D2 receptor and could prove to be a valuable tool in the study of sigma sites. Other analogues such as 1H-indeno[2,1-c]pyridines and 1H-benzo[3,4]cyclohepta[1,2-c]pyridines also displayed high sigma site affinity.

The ability of three 5-HT1 receptor agonists, 5-HT (5-hydroxytryptamine), 5-CT (5-carboxyamidotryptamine) and sumatriptan to inhibit the binding of [3H]5-HT, in the presence of cyanopindolol and mesulergine, from cerebral cortical and/or caudate membranes in seven species (dog, guinea-pig, rabbit, pig, human, hamster and calf) has been investigated. Under the experimental conditions used, 5-CT and sumatriptan consistently yielded displacement curves best fit to a two-site model whereas 5-HT always gave a monophasic displacement curve. The pIC50 values obtained with 5-HT displacement gave a mean of 8.1 +/- 0.1 (mean +/- S.E.M.). In contrast the biphasic displacement curves for 5-CT and sumatriptan yielded high and low affinity pIC50 values of 8.3 +/- 0.1, 5.5 +/- 0.1 and 7.6 +/- 0.1, 5.0 +/- 0.1, respectively. These data indicate that under these experimental conditions the high affinity component labelled by [3H]5-HT is the same receptor subtype, previously denoted the 5-HT1D receptor, in all seven species.

The behavioural and biochemical profile of the sigma ligand and putative antipsychotic agent, BMY 14802 (alpha-(4-fluorophenyl)-4-(5-fluoro-2- pyrimidinyl)-1-piperazine butanol) has been determined in the mouse and rat. In mice, pretreatment with BMY 14802 attenuated both amphetamine-induced hyperactivity and conditioned avoidance responding, consistent with its previously reported antipsychotic potential. In common with 5-HT1A receptor agonists or partial agonists, BMY 14802 induced (a) a dose-dependent hypothermia in mice; (b) aspects of the 5-HT behavioural syndrome in rats, (c) antagonised mescaline-induced head twitches in mice and (d) generalised to the 8-hydroxy-2-(di-n-propylamino)tetralin discriminative stimulus over the dose range of 3-15 mg/kg. BMY 14802 had appreciable affinity for the 5-HT1A receptor (pIC50 = 6.7 compared to 7.3 for sigma binding) and antagonised forskolin-stimulated adenylate cyclase activity with a pEC50 of 6.2, consistent with an agonist action at this receptor. The results support the involvement of 5-HT1A receptors, but not the sigma binding site, in the behavioural profile of BMY 14802.

In contrast to in vivo release methods, in vitro release techniques utilising brain slices or synaptosomes affords a simple and reproducible means of measuring both receptor affinity and efficacy of drugs acting at prejunctional receptors in the CNS. Most studies have used brain tissue loaded with radiolabelled neurotransmitter or its precursor via the high affinity uptake system for these substances which are present on nerve terminals. Depolarisation evoked release induced by either high K+ or electrical field stimulation increases the release of radioactivity and this overflow can be readily measured by liquid scintillation counting. Recent studies have started to emphasise the measurement of the release of endogenous neurotransmitters from brain tissue using similar depolarisation stimuli. Examples include the release of dopamine, noradrenaline, adrenaline and 5-HT and their control by presynaptic receptors. Most of these studies have used HPLC with ECD detection as a means of separating and analysing for the transmitter of interest. The relative strengths and weaknesses of the measurement of radiolabelled or endogenous neurotransmitter release in vitro as a means of identifying presynaptic receptors is discussed.

The 5-HT1B receptors have been identified by radioligand binding techniques predominantly in the basal ganglia of the rat and mouse brain. A number of 5-HT receptor agonists have been shown to display high affinity but limited selectivity for the 5-HT1B recognition site. These include 5-CT, 5-HT, RU 24969, TFMPP, MCPP, and CGS 12066B. Antagonists at the 5-HT1B site include the drugs metitepin, metergoline, cyanopindolol, isamoltane, and propranolol but none of these drugs are selective for this receptor. Functional correlates of 5-HT1B receptor activation have been most closely defined in vitro. These include inhibition of transmitter release, inhibition of forskolin-stimulated adenylate cyclase and actions on the mouse urinary bladder strip and the rat vena cava. Many functional correlates of 5-HT1B receptor activation in vivo have been proposed, but convincing evidence from antagonist studies is generally lacking. The development of selective 5-HT1B receptor agonists and antagonists will be a key step in defining the physiological role of this receptor site in the brain and periphery of the mouse and rat although it must be realized that these compounds, if they are developed, are unlikely to have functional effects in man since the 5-HT1B recognition site is absent in the human CNS. Nevertheless many of these studies on the 5-HT1B receptor may aid the development of drugs acting at the 5-HT1D site since this receptor has been identified as being the equivalent of the 5-HT1B site in species other than the rat and mouse.

The release of [3H]5-HT from superfused rat frontal cortex slices was elicited by continuous exposure to either Krebs or Krebs buffer containing excess K+ ions (25 mmol/l). The basal release of [3H]5-HT was augmented by 1 mumol/l of the (+)-isomers of the beta-adrenoceptor antagonists alprenolol, oxprenolol and pindolol. Neither the (-)-isomers of these drugs nor (+/-)-atenolol (10 mumol/l), (+/-)-ICI 118551 (0.3 mumol/l) or (+/-)-cyanopindolol (0.008-0.1 mumol/l) increased basal release. At these concentrations, however, the stimulation-evoked overflow was enhanced by (-)-alprenolol, (-)-oxprenolol and (+/-)-cyanopindolol but not by (-)-pindolol. The inhibitory effects of 5-HT at the 5-HT autoreceptor were antagonised by (+/-)-cyanopindolol (pA2 8.32), (-)-alprenolol (6.82), (-)-pindolol (6.66) and (-)-oxprenolol (6.28) whereas the beta 1- and beta 2-selective antagonists, atenolol and ICI 118551 respectively, were inactive. These studies utilising beta-adrenoceptor antagonists have defined a new class of 5-HT autoreceptor antagonists and, in addition, have identified (+/-)-cyanopindolol as the most potent blocker of this receptor thus far identified.

The cardiovascular response to 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), a selective putative 5-HT1A receptor agonist, has been investigated in the rat. Comparisons were made with clonidine, a centrally acting hypotensive agent with negligible affinity for 5-HT receptors. In conscious, spontaneously hypertensive (SH) rats, 8-OH-DPAT caused dose-related and sustained falls in blood pressure and heart rate that were unaffected by depletion of brain 5-HT by p-chlorophenylalanine. 8-OH-DPAT caused hypotension and bradycardia in anesthetized normotensive rats. In pithed rats, 8-OH-DPAT neither lowered blood pressure nor affected the cardiovascular response to spinal sympathetic stimulation or to phenylephrine. The response to 8-OH-DPAT was blocked selectively by intracisternal injection of 8-methoxy-2-(N-2-chloroethyl-N-n propyl) amino tetralin (8-MeO-CIEPAT), a putative irreversible 5-HT1A receptor antagonist, and was abolished in animals whose central monoamine transmitter stores were depleted selectively by combined treatment with DL-alpha-monofluoromethyl-dopa and dopamine. The cardiovascular response to 8-OH-DPAT was inhibited selectively by metergoline, methiothepin, and 8-MeO-CIEPAT; it was nonselectively inhibited by (+/-)-pindolol, (+/-)-cyanopindolol, buspirone, yohimbine, idazoxan, and WY 26392; and was unaffected by prazosin and cis-flupenthixol. These results establish that the cardiovascular response to 8-OH-DPAT in the rat is centrally mediated and point to the putative 5-HT1A receptor as the key site involved. An indirect link involving a catecholaminergic mechanism is suggested by the fact that alpha 2-adrenoceptor antagonists are also inhibitory despite 8-OH-DPAT having no direct agonist effects at alpha 2-adrenoceptors per se.

The pharmacological characteristics of a pre-junctional 5-HT autoreceptor have been studied by following the Ca2+-dependent, K+-evoked release of [3H]-5-HT from preloaded rat frontal cortex slices. Added 5-HT, in the presence of the 5-HT uptake inhibitor chlorimipramine, caused a dose related inhibition of the K+-evoked release of [3H]-5-HT in this system as did the 5-HT analogues 5-methoxytryptamine, N-methyltryptamine, 5-methoxy-NN'-dimethyltryptamine, N-methyl 5-hydroxytryptamine and tryptamine. The inhibitory effect of 1 microM 5-HT on the K+-evoked release of [3H]-5-HT was reversed in a dose-related manner by the 5-HT antagonist drug, methiothepin (pA10 value 6.7). At a concentration of 1 microM, the 5-HT antagonists drugs cinanserin and mianserin produced a small but significant reversal of the 5-HT induced inhibition of K+-evoked [3H]-5-HT release, but methysergide, metergoline and cyproheptadine were completely without effect at this concentration. The results are interpreted as evidence for a pre-junctional autoreceptor for 5-HT in the frontal cortex of the rat with a different pharmacological specificity for 5-HT antagonists from previously studied 5-HT receptors.

N-Methyl-D-aspartate (NMDA)-stimulated [3H]noradrenaline release from rat hippocampal slices was blocked stereospecifically and non-competitively by MK-801 with the (+)-isomer achieving 50% blockade of 100 microM NMDA at 16 nM. The results indicate that MK-801 is the most potent NMDA antagonist yet described and that it blocks NMDA-stimulated neurotransmitter release by an action at the so-called 'phencyclidine (PCP) site'.

The enantiomers of the 5-HT autoreceptor antagonist methiothepin have been prepared and their activity as antagonists of the 5-HT autoreceptor and at the 5-HT recognition sites present in the frontal cortex of the rat have been evaluated. At the 5-HT autoreceptor, the order of potency as antagonists of 5-HT was (+)methiothepin (apparent pA2 5.95) less than (+/-)methiothepin (apparent pA2 6.62) less than or equal to (-)methiothepin (apparent pA2 6.81). At the 5-HT2 recognition site, the isomeric forms of methiothepin were potent (pIC50 approximately 8.2) and equiactive. At the subtypes of the 5-HT1 recognition sites, similar concentrations to those blocking the autoreceptor were effective and (+)methiothepin was less active than (-)methiothepin. It is concluded that the chiral association of methiothepin with the 5-HT autoreceptor provides further evidence for a pharmacological similarity between this receptor and the 5-HT1B subtype of the 5-HT1 recognition site.

Slices of rat brain were incubated with either (3H) choline (hippocampus) or (3H) noradrenaline (hypothalamus) and superfused with Krebs buffer. The release of (3H) acetylcholine and (3H) noradrenaline after inhibition of monoamine oxidase by pargyline was induced by a short exposure to Krebs buffer containing elevated K+ ions (25 mmol/l). Nifedipine (1 mumol/l) caused only a slight inhibition of noradrenaline efflux and was without effect on acetylcholine overflow. The calcium channel activator, Bay K 8644 (0.1-1 mumol/l), increased the K+-evoked efflux of both neurotransmitters. The additional efflux evoked by Bay K 8644 (0.3 mumol/l) was blocked by nifedipine (1 mumol/l). The results from the present study thus extend the earlier findings with the neurotransmitter 5-hydroxytryptamine to include noradrenaline and acetylcholine. The functional correlates for voltage operated calcium channels concerned with transmitter release are clearly a widespread phenomenon in the CNS.

As a result of controversy in the literature regarding the classification and nomenclature of functional receptors for 5-hydroxytryptamine (5-HT), a framework for classification is proposed. The formulation of these proposals has only been made possible by the recent advent of new drug tools. It is considered that there are three main types of 5-HT receptor, two of which have been well characterised pharmacologically, using selective antagonists, and which it is proposed to name 5-HT2 and 5-HT3. These two groups broadly encompass the "D" and "M" receptors, respectively, which Gaddum identified in the guinea-pig ileum (Gaddum and Picarelli, 1957). The 5-HT2 receptor, which mediates a variety of actions of 5-HT, has been definitively shown to correlate with the 5-HT2 binding site in the brain. No binding studies in brain tissue have yet been published with radiolabelled ligands specific for 5-HT3 receptors. A number of other actions of 5-HT appear to be mediated via receptors distinct from 5-HT2 or 5-HT3 receptors. Since selective antagonists are not yet available, these receptors cannot be definitively characterised, although in many cases they do have some similarities with 5-HT1 binding sites, which are a heterogeneous entity. Criteria are proposed for tentatively classifying these receptors as "5-HT1-like" (Table 1). Definitive characterisation of these receptors will await the identification of specific antagonists. This classification of 5-HT receptors into three main groups (Table 1) is based largely, but not exclusively, on data from studies in isolated peripheral tissues where definitive classification is possible. However, it is believed that this working classification will be relevant to functional responses to 5-HT in the central nervous system.