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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.

The existence of multiple 5-HT autoreceptors in the central nervous system is now firmly established and they have been pharmacologically identified as belonging to the 5-HT(1A), 5-HT(1B), and 5-HT(1D) receptor subtypes. In addition, 5-HT(1F), 5-HT(5A), and 5-HT(7) receptors remain as potential candidates for additional autoreceptors. The emergence of selective ligands, such as SB-224289 (5-HT(1B) receptor antagonist), BRL 15572 (5-HT(1D) receptor antagonist), GR 127935 (a mixed 5-HT(1B/1D) receptor antagonist), LY 334370 (5-HT(1F) receptor agonist), and SB-269970 (5-HT(7) receptor antagonist), has aided the characterisation of 5-HT autoreceptors and has highlighted the complexity of mechanisms which modulate the release of 5-HT.

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.

5-HT1B autoreceptors are involved in the control of extracellular 5-HT levels from both the terminal and cell body regions of serotonergic neurones. In this study we report on the effect of a selective and potent 5-HT1B 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-tetrahydros piro [furo[2,3-f]indole-3,4' -piperidine] hydrochloride), on extracellular 5-HT levels in the cortex and dentate gyrus of the freely-moving guinea-pig, using the technique of in vivo microdialysis. SB-236057-A had ca. 23% bioavailability following oral drug administration. In vivo hypothermia pharmacodynamic assays demonstrated it was brain penetrant with a duration of action in excess of 18 h. SB-236057-A (0.75 mg/kg p.o.) increased extracellular 5-HT levels in the dentate gyrus to a maximum of 167+/-7% of basal but had no effect in the frontal cortex. However, a small increase in cortical 5-HT levels (117+11% of basal) was evident at 2.5 mg/kg p.o. In addition, SB-236057-A (0.75 mg/kg and 2.5 mg/kg p.o.) antagonised the sumatriptan-induced inhibition of extracellular 5-HT levels in the guinea-pig frontal cortex. These differences were attributed to MRN-innervated regions (e.g. dentate gyrus) being more responsive to 5-HT1B receptor-mediated negative feedback than DRN-innervated regions (e.g. frontal cortex). In the dentate gyrus, the increase in 5-HT release induced by SB-236057-A (0.75 mg/kg p.o.) was comparable to that after 14 days of paroxetine (10 mg/kg p.o.) administration, reaching a maximum of 183+/-13% of basal. These data suggest that acute 5-HT1B receptor blockade, by virtue of increased 5-HT release in the dentate gyrus, may provide a rapidly acting antidepressant.

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.

A selective dopamine D(3) receptor antagonist offers the potential for an effective antipsychotic therapy, free of the serious side effects of currently available drugs. Using clearance and brain penetration studies as a screen, a series of 1,2,3, 4-tetrahydroisoquinolines, exemplified by 13, was identified with high D(3) affinity and selectivity against the D(2) receptor. Following examination of molecular models, the flexible butyl linker present in 13 was replaced by a more conformationally constrained cyclohexylethyl linker, leading to compounds with improved oral bioavailability and selectivity over other receptors. Subsequent optimization of this new series to improve the cytochrome P450 inhibitory profile and CNS penetration gave trans-N-[4-[2-(6-cyano-1, 2,3, 4-tetrahydroisoquinolin-2-yl)ethyl]cyclohexyl]-4-quinolinecarbo xamide (24, SB-277011). This compound is a potent and selective dopamine D(3) receptor antagonist with high oral bioavailability and brain penetration in the rat and represents an excellent new chemical tool for the investigation of the role of the dopamine D(3) receptor in the CNS.

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.

A novel compound, SB-236057 (1'-ethyl-5-(2'-methyl-4'-(5-methyl-1,3,4-oxadiazol-2-yl)biphenyl-4-carbonyl)-2,3,6,7-tetrahydrospiro[furo]2,3-f]indole-3,4'-piperidine]) has been shown to have high affinity for human 5-hydroxytryptamine 1B (5-HT 1B ) receptors (pK i = 8.2) and displays over 75 or more-fold selectivity for the human 5-HT 1B receptor over other 5-HT receptors, including the human 5-HT 1D receptor, and a range of other receptors, ion channels and enzymes. In functional studies using [ 35 S]GTPγS binding, SB-236057 displayed negative intrinsic activity (pEC 50 = 8.0) at human 5-HT 1B receptors stably expressed in Chinese Hamster Ovary (CHO) cells and caused a rightward shift of agonist concentration response curves consistent with competitive antagonism (pA 2 = 8.9). SB-236057 potentiated [ 3 H]5-HT release from electrically stimulated guinea pig or human cortical slices. SB-236057 also abolished the inhibitory effect of exogenously superfused 5-HT on electrically-stimulated release from slices of the guinea pig cortex. These studies using SB-236057 confirm that, in both the guinea pig and human cerebral cortex, the terminal 5-HT autoreceptor is of the 5-HT 1B subtype.

The effects of the corticotropin-releasing factor (CRF) receptor antagonists, α-helical CRF-(9–41), [ d -Phe 12 ,Nle 21,38 ,CαMeLeu 37 ]humanCRF-(12–41) ( d -PheCRF-(12–41)) and astressin ([cyclo(30–33){ d -Phe 12 ,Nle 21,38 ,Glu 30 ,Lys 33 ]humanCRF-(12–41) upon hypophagic and motor activation response to human/ratCRF (h/rCRF) were investigated. All three antagonists (100 μg intracerebroventricular (i.c.v.)) blocked the effects of h/rCRF (1 μg i.c.v.) upon food intake and body weight change in food-deprived rats. In contrast, α-helical CRF-(9–41) and astressin (both at 100 μg i.c.v., but not lower doses), but not d -PheCRF-(12–41) (up to 100 μg i.c.v.), blocked h/rCRF (0.3 μg i.c.v.)-induced motor activation in rats in a familiar environment. The ability of d -PheCRF-(12–41) to block CRF-induced hypophagia, but not motor activation, suggests a selective action of this antagonist upon the behavioural effects of centrally administered h/rCRF.

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 present study determined the behavioural effects of the corticotropin releasing factor (CRF)-related peptides, human/rat CRF (h/rCRF), ovine CRF (oCRF), sauvagine (SAUV), urotensin I (UT) and the recently discovered neuropeptide, rat urocortin (rUCN). All of the peptides dose-dependently increased motor activity in a familiar environment and reduced feeding in hungry rats. There was no apparent relationship between potency/affinity at CRF2 receptors and effects in these two tests. In a comparison of h/rCRF and rUCN upon discrete spontaneous behaviours, both peptides (3.0 microg i.c.v.) increased activity and grooming, induced a fore-paw tremor and reduced the incidence of motionlessness. However, h/rCRF reduced motionlessness to a greater extent and was a more potent inducer of defaecation, weight loss, oral movements and fore-paw tremor than rUCN. In the elevated X maze, both h/rCRF and rUCN (1.0 microg i.c.v.) had anxiogenic-like effects upon behaviour. In contrast, h/rCRF (1.0 microg i.c.v.), but not rUCN (1.0-10 microg i.c.v.) increased the startle response to an acoustic stimulus. In summary, all the CRF-related peptides increased motor activity and reduced feeding in rats in a similar manner and both rUCN and h/rCRF induced anxiogenesis. However, there were some behavioural differences between rUCN and h/rCRF which require further study. Further pharmacological investigation of the role of CRF receptor subtypes requires the use of subtype selective antagonists.

The effect of SB-224289 (2,3,6,7-tetrahydro-1′-methyl-5-{2′-methyl-4′-[(5-methyl-1,2,4-oxadiazole-3-yl)biphenyl-4-yl]carbonyl}Furo[2,3-F]-indole-3-spiro-4′-piperidine oxalate) (4 mg/kg i.p., 5-HT1B receptor antagonist), GR 127935 (N-[4-methoxy-3-(4-methyl-1-piperizinyl)phenyl]-2′-methyl-4′-(5-methyl-1,2,4-oxadiazole-3-yl)[1,1′-biphenyl]-carboxamide) (0.3 mg/kg i.p., 5-HT1B/1D receptor antagonist), and paroxetine (10 mg/kg p.o.) were investigated on extracellular 5-hydroxytryptamine (5-HT) levels in the frontal cortex, striatum and dentate gyrus of the freely moving guinea-pig with microdialysis. In the frontal cortex and striatum (dorsal raphe innervated areas), GR 127935 evoked a significant decrease in extracellular 5-HT, reaching minima of 41±12% and 32±6% of basal, respectively. This decrease may be explained by antagonism of inhibitory 5-HT1B/1D receptors on raphe cell bodies, leading to a local increase in 5-HT, which, in turn, stimulated 5-HT1A receptors to decrease cell firing, and hence 5-HT release from terminals. In contrast, SB-224289 had no effect on 5-HT levels in either region. In the dentate gyrus (median raphe innervated area), GR 127935 and SB-224289 significantly increased extracellular 5-HT, reaching maxima of 146±11% and 151±19% of basal, respectively. The ability of both compounds to increase 5-HT levels in the dentate gyrus suggests a lack of 5-HT1B/1D receptors in the median raphe nucleus. Paroxetine produced a small but non-significant increase in extracellular 5-HT in the frontal cortex, and a small decrease in the dentate gyrus. The lack of effect of paroxetine in terminal areas may be due to the limiting effects of cell body 5-HT autoreceptors. In summary, the above data demonstrate that 5-HT1B/1D receptor antagonists increase 5-HT levels in the dentate gyrus, implying that acute administration of 5-HT1B/1D receptor antagonists will achieve a similar effect to chronic selective serotonin re-uptake inhibitor treatment in median raphe innervated areas. This, in turn, suggests that such compounds may be efficacious in the treatment of depression.

l-Dopa has long been the mainstay of therapy for Parkinson's disease but its long-term shortcomings, principally uncoordinated, spasmodic or irregular movements (dyskinesias) and fluctuating control of motor symptoms (on/off fluctuations), are well documented. The postulated neuroprotective properties of l-deprenyl, often used as an adjunct to l-dopa, are under scrutiny and doubts have also been raised regarding its safety. Alternative therapeutic approaches are clearly needed. In this review, Jim Hagan, Derek Middlemiss, Paul Sharpe and George Poste outline some new approaches to treatment, with an emphasis on novel, selective dopamine receptor agonists. In addition, Parkinson's disease is commonly thought to be caused by the neurotoxic effects of an unidentified agent but recent data indicate a greater genetic component than previously recognized. Developments in the genetics of Parkinson's disease may provide the key to the next generation of therapeutics.

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.

Publisher Summary The heterogeneity of receptors mediating 5-HT responses was apparent long before their definitive identification using cloning techniques, largely due to radioligand binding studies. Hence, as early as 1979, 5-HT recognition sites were subdivided into two classes. Those that had a high affinity for agonists and were labeled with [3H]5-HT were termed “5-HT 1 binding sites” and those that displayed a high affinity for antagonists and were labeled with [aH]spiperone were called “5-HT 2 binding sites.” In 1981, Pedigo demonstrated that spiperone displaced [aH]5-HT in a complex manner; hence, the 5-HT 1 class comprised at least two subgroups. The component having a high affinity for spiperone was called a “5-HT 1A recognition site” (labeled with [∼H]8-OH-DPAT) and the remaining component the “5-HT 1B binding site” (subsequently labeled with [125I]ICYP (iodocyanopindolol) in the presence of isoprenaline).

The novel compound SB 204269 ( trans -(+)-6-acetyl-4 S -(4-fluorobenzoylamino)-3,4-dihydro-2,2-dimethyl-2 H -benzo[ b ]pyran-3 R -ol,hemihydrate) shows potent anticonvulsant activity in the mouse maximal electroshock seizure threshold test. The binding of [ 3 H]SB 204269 to mouse forebrain membranes is saturable ( B max 217 fmol/mg protein, K d 32 nM) and stereospecific. The excellent anticonvulsant profile of SB 204269, combined with the identification of a unique central binding site for the compound, suggest that it has potential clinical utility as a novel treatment for epilepsy.

Quantitative autoradiographic studies, with [3H]5-HT, were used to investigate the distribution of 5-CT-insensitive 5-HT1-like (5-HT1E/1F) recognition sites in rat and guinea pig brain. For comparison and control purposes the distribution of the closely related 5-HT1D binding site, which is abundant in the guinea pig but not the rat, was also investigated, as well as total specific [3H]5-HT binding. Results from this study confirm the previously described regional distribution of the 5-HT1D binding site and also revealed a predominance of 5-CT-insensitive 5-HT1-like 5-HT1E/1F) recognition sites in the olfactory tubercle, caudate putamen, nucleus accumbens and substantia nigra of both species. Interestingly 5-CT-insensitive 5-HT1-like (5-HT1E/1F) recognition sites were particularly dense in the claustrum of the guinea pig, but not the rat.

Release of 5-HT in the CNS is under the control of autoreceptors. These autoreceptors fall into two categories: cell body autoreceptors and terminal autoreceptors. The former inhibit 5-HT release through inhibition of cell firing; the latter through direct inhibition of release at the terminal. Cell body (or somatodendritic) autoreceptors belong to the 5-HT1A receptor subtype in all species studied so far. In the rat and mouse, the terminal autoreceptor is known to be a 5-HT1B receptor, whereas in human, pig, rabbit, and guinea pig, the terminal autoreceptor is thought to belong to the 5-HT1D receptor subtype. Until recently, the absence of a potent and selective 5-HT1D receptor antagonist has hindered this classification. We now present data with the novel 5-HT1D receptor antagonist, GR 127935, which demonstrates that in guinea pig cerebral cortex the terminal autoreceptor is a 5-HT1D receptor. In vitro [3H]5-HT release studies demonstrate that 5-HT inhibition of [3H]5-HT release is attenuated by GR 127935. In vivo, using the technique of microdialysis, GR 127935 and the non-selective antagonist methiothepin, when administered down the dialysis probe, potentiate extracellular levels of 5-HT. Both the in vitro and in vivo effects of these compounds are consistent with terminal autoreceptor blockade. However, when GR 127935 and methiothepin were administered systemically, both compounds inhibit extracellular levels of 5-HT. The most plausible explanations for this effect, such as partial agonism or activation of somatodendritic 5-HT1A receptors, are discussed.

This study, carried out in human cerebral cortical membranes, confirms previous findings in human substantia nigra that serotonin-5-O-carboxymethyl-glycyl[125I]tyrosinamide ([125I]GTI) labels a homogenous population of recognition sites consistent with a 5-HT1D receptor pharmacology. In addition the results indicate that, under the assay conditions described, [125I]GTI specifically labels the 5-HT1Dβ recognition site since ketanserin and ritanserin display a low affinity consistent with their activities at this subtype of the 5-HT1D receptor.

The present studies examined the relative antagonist potencies of the optical isomers of the 5-HT receptor antagonist metitepine at the 5-HT1D binding site labelled by the novel radioligand serotonin-O-carboxymethylglycyl [125]iodotyrosinamide ([125I]GTI), and at the terminal 5-HT autoreceptor in guinea pig frontal cortex, a proposed model of 5-HT1D receptor activation. The pharmacological specificity of the [125I]GTI binding site in guinea pig frontal cortex was similar to previously published studies in the bovine cortical 5-HT1D recognition site labelled with [3H]5-HT. The (+) isomer of metitepine displaced [125I]GTI binding with a lower affinity (64 nM) than did the (−) isomer (18 nM), which was equiactive with the racemic mixture. The (−) isomer of metitepine was more effective than the (+) isomer at attenuating the inhibitory effects of 5-HT and sumatriptan at the guinea pig terminal 5-HT autoreceptor; the apparent pA2 of the (−) isomer was 8.0 (sumatriptan) and 7.7 (5-HT) while the apparent pA2 of the (+) isomer was 7.1 (sumatriptan) and 6.8 (5-HT). The (−) isomer was more effective than the (+) isomer at enhancing stimulated [3H]5-HT release. These findings support the identification of the guinea pig 5-HT terminal autoreceptor as a 5-HT1D receptor and reinforce the species homology between the 5-HT1B and 5-HT1D receptors.

5-HT,-like receptors have been subdivided into 5 main subtypes denoted 5-HTA5-HTB5-HTc5-HTIDand 5-HTE. Each of these receptor subtypes has been cloned and the 2nd messenger-effector system to which they are linked identified. In additionin vitrofunctional correlates of the activation of these receptors in brain or peripheral tissues have been proposed and the evidence for these propositions is critically discussed in this review.

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 terminal 5-HT (5-hydroxytryptamine) autoreceptor in the rat CNS has been characterised as belonging to the 5-HT1B subclass of central 5-HT receptors and a number of potent agonists and antagonists at this site have been identified. The 5-HT1B binding site has been clearly identified only in the rat but is absent from the guinea-pig, pig and human CNS. Terminal 5-HT autoreceptors from the guinea-pig may belong to the 5-HT1D subclass of 5-HT-1like receptors.

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.

Agonists of the 5-HT1A recognition site have been prepared and their affinities for the [3H]8-OH-DPAT binding site have been determined in rat frontal cortex. A pharmacophore has been developed, based on the reported conformation—activity relationship. Free and forbidden zones of the receptor have been characterized with the assistance of a graphics computer, defining a receptor map which accounts for different chemical classes.

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.

A conformational study of four 5-HT1A (serotonin) receptor ligands ((R-(-)-methiothepin, spiperone, (S)-(-)-propranolol, and buspirone) led to the definition of a pharmacophore and a three-dimensional map of the 5-HT1A antagonist recognition site. These models were used to design new compounds and successfully predict their potency, stereospecificity, and selectivity. For example, 8-[4-[(1,4-benzodioxan-2-ylmethyl)amino] butyl]-8-azaspiro[4.5]decane-7,9-dione (1, MDL 72832) has nanomolar affinity (pIC50 = 9.14) for the 5-HT1A binding site in rat frontal cortex. As predicted, the S-(-) enantiomer of 1 was more active than its R-(+) enantiomer (pIC50 = 9.21 and 7.66, respectively) and a naphthalene analogue of 1 displayed the expected improved selectivity.

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.