The human 5-hydroxytryptamine (5-HT4) receptor is encoded by a highly complex gene which gives rise to at least 10 distinct splice variants. However, the functional relevance of these variants is unknown. In rat, only three such variants have been identified, 5-HT4a (r5-HT4a), 5-HT4b (r5-HT4b) and 5-HT4e (r5-HT4e). In the current study we identify and characterise the pharmacology of a novel rat splice variant (r5-HT4c1) and present the first comprehensive analysis of 5-HT4 splice variant mRNA expression levels throughout the rat gastrointestinal tract. In addition, we describe preliminary characterisation of the first 5-HT4 splice variant specific antibodies. In transfected cells, r5-HT4c1 receptor exhibited similar binding properties to r5-HT4a and r5-HT4b. Functional studies showed that 5-HT4 agonists prucalopride (4-amino-5-chloro-2,3-dihydro-N-[1-(3-methoxypropyl)-4-piperidinyl]-7-benzofuran carboxamide monohydrochloride and renzapride (±)-endo-4-amino-5-chloro-2-methoxy-N-(1-azabicyclo[3.3.1]non-4-yl)benzamide monohydrochloride) acted as partial agonists at r5-HT4c1, but full agonists at r5-HT4a and r5-HT4b. Moreover, in contrast to r5-HT4a and r5-HT4b, r5-HT4c1 was not constitutively active. TaqMan mRNA analysis showed that r5-HT4a expression in brain and dorsal root ganglion exceeded that in the gastrointestinal tract, whilst the reverse was true for r5-HT4b and r5-HT4c1. mRNA expression of each variant also increased distally throughout the gastrointestinal tract with the highest levels in the colon. r5-HT4a and r5-HT4b specific immunoreactivity was abundant on enteric neurons in jejunum, ileum and colon as well as neurons and satellite cells of the dorsal root ganglion. Only r5-HT4b immunoreactivity was observed on endocrine cells in the duodenum. These data could have implications in rat models and aid understanding of 5-HT4 splice variant function.

Modification of the potent imidazole-based B-Raf inhibitor SB-590885 resulted in the identification of a series of furan-based derivatives with enhanced CNS penetration. One such compound, SB-699393 (17), was examined in vivo to challenge the hypothesis that selective B-Raf inhibitors may be of value in the treatment of stroke.

5-hydroxytryptamine (5-HT) is found in many areas of the CNS, and has been implicated in the modulation of learning and memory processes. The 5-HT6 receptor is a member of the seven transmembrane G protein coupled family of receptors and is positively coupled to adenylate cyclase. 5-HT6 receptor antagonists can increase the release of several neurotransmitters in the brain and enhance cognitive processes in rodent models. Several 5-HT6 antagonists are being developed as symptomatic agents for the treatment of memory deficits in Alzheimer's disease and other dementias, including SB-742457. SB-742457 was profiled in standard in vitro binding and functional assays. SB-742457 had high affinity for native 5-HT6 receptors expressed in rat striatum (pKi = 9.56) consistent with its binding affinity and functional potency (cAMP assay) at the recombinant human receptor (pKi = 9.68; pA2 = 9.2). SB-742457 is > 100 fold selective for the human 5-HT6 receptor over all other 5-HT receptors, ion channels and transporters examined apart from 5-HT2a. (pKi 8.0). Following oral administration in rats, SB-742457 exhibited high 5-HT6 receptor occupancy in the striatum (ED50 = 0.3mg/kg from ex vivo binding studies) indicative of good brain penetration. In vivo oral activity of SB-742457 and the cholinesterase inhibitor Donepezil was assessed in a range of neurochemical and animal models of cognition including aged Morris Water Maze and Passive Avoidance. In these studies a sub-chronic administration of SB-742457 or Donepezil (0.03 and 0.3mg/kg) was able to significantly improve the spatial learning ability of aged animals, with SB-742457 (1.5mg/kg) promoting an apparently more prolonged beneficial effect on memory retention. Acute administration of both compounds reversed scopolamine induced amnesia in a passive avoidance paradigm, though with different dose-response profiles. Given its beneficial effects, on learning and memory in preclinical species the 5-HT6 antagonist SB-742457 has the potential to be a new therapeutic agent for the symptomatic treatment of Alzheimer's disease and other dementias.

Starting from a benzazepine sulfonamide 5-HT(6) receptor antagonist lead with limited brain penetration, application of a strategy of conformational constraint and reduction of hydrogen bond donor count led to a novel series of tricyclic derivatives with high 5-HT(6) receptor affinity and excellent brain:blood ratios.

6-[(3-Cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-3-pyridinecarboxamide hydrochloride (GSK189254) is a novel histamine H 3 receptor antagonist with high affinity for human (pK(i) = 9.59-9.90) and rat (pK(i) = 8.51-9.17) H-3 receptors. GSK189254 is >10,000-fold selective for human H-3 receptors versus other targets tested, and it exhibited potent functional antagonism (pA(2) = 9.06 versus agonist-induced changes in cAMP) and inverse agonism [pIC(50) = 8.20 versus basal guanosine 5'-O-(3-[S-35]thio)triphosphate binding] at the human recombinant H-3 receptor. In vitro autoradiography demonstrated specific [H-3]GSK189254 binding in rat and human brain areas, including cortex and hippocampus. In addition, dense H 3 binding was detected in medial temporal cortex samples from severe cases of Alzheimer's disease, suggesting for the first time that H-3 receptors are preserved in late-stage disease. After oral administration, GSK189254 inhibited cortical ex vivo R-(-)-alpha-methyl[imidazole-2,5(n)-H-3]histamine dihydrochloride ([H-3]R-alpha-methylhistamine) binding (ED50 = 0.17 mg/kg) and increased c-Fos immunoreactivity in prefrontal and somatosensory cortex (3 mg/kg). Microdialysis studies demonstrated that GSK189254 (0.3-3 mg/kg p.o.) increased the release of acetylcholine, noradrenaline, and dopamine in the anterior cingulate cortex and acetylcholine in the dorsal hippocampus. Functional antagonism of central H-3 receptors was demonstrated by blockade of R-alpha-methylhistamine-induced dipsogenia in rats (ID50 = 0.03 mg/kg p.o.). GSK189254 significantly improved performance of rats in diverse cognition paradigms, including passive avoidance (1 and 3 mg/kg p.o.), water maze (1 and 3 mg/kg p.o.), object recognition (0.3 and 1 mg/kg p.o.), and attentional set shift (1 mg/kg p.o.). These data suggest that GSK189254 may have therapeutic potential for the symptomatic treatment of dementia in Alzheimer's disease and other cognitive disorders.

GSK207040 (5-[(3-cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-2-pyrazinecarboxamide) and GSK334429 (1-(1-methylethyl)-4-({1-[6-(trifluoromethyl)-3-pyridinyl]-4-piperidinyl}carbonyl)hexahydro-1H-1,4-diazepine) are novel and selective non-imidazole histamine H3 receptor antagonists from distinct chemical series with high affinity for human (pKi=9.67±0.06 and 9.49±0.09, respectively) and rat (pKi=9.08±0.16 and 9.12±0.14, respectively) H3 receptors expressed in cerebral cortex. At the human recombinant H3 receptor, GSK207040 and GSK334429 were potent functional antagonists (pA2=9.26±0.04 and 8.84±0.04, respectively versus H3 agonist-induced changes in cAMP) and exhibited inverse agonist properties (pIC50=9.20±0.36 and 8.59±0.04 versus basal GTPγS binding). Following oral administration, GSK207040 and GSK334429 potently inhibited cortical ex vivo [3H]-R-α-methylhistamine binding (ED50=0.03 and 0.35mg/kg, respectively). Functional antagonism of central H3 receptors was demonstrated by blockade of R-α-methylhistamine-induced dipsogenia in rats (ID50=0.02 and 0.11mg/kg p.o. for GSK207040 and GSK334429, respectively). In more pathophysiologically relevant pharmacodynamic models, GSK207040 (0.1, 0.3, 1 and 3mg/kg p.o.) and GSK334429 (0.3, 1 and 3mg/kg p.o.) significantly reversed amnesia induced by the cholinergic antagonist scopolamine in a passive avoidance paradigm. In addition, GSK207040 (0.1, 0.3 and 1mg/kg p.o.) and GSK334429 (3 and 10mg/kg p.o.) significantly reversed capsaicin-induced reductions in paw withdrawal threshold, suggesting for the first time that blockade of H3 receptors may be able to reduce tactile allodynia. Novel H3 receptor antagonists such as GSK207040 and GSK334429 may therefore have therapeutic potential not only in dementia but also in neuropathic pain.

Histamine H3 receptor antagonists can increase the release of several neurotransmitters in the brain and enhance cognition in rodent models. Several H3 antagonists are being developed as symptomatic agents for the treatment of cognitive deficits in Alzheimer's Disease and other dementias. To investigate the in vitro and in vivo properties of GSK189254, a novel non–imidazole histamine H3 receptor antagonist. GSK189254 was profiled in standard in vitro binding and functional assays. In vivo oral activity of GSK189254 was assessed in several assays including ex vivo binding, cognition models and EEG. GSK189254 had high affinity for native H3 receptors expressed in human and rat cerebral cortex (pKi = 9.59 and 8.58 respectively) and for human and rat recombinant H3 receptors (pKi = 9.90 and 9.17 respectively). GSK189254 was highly selective for H3 receptors (>10000–fold vs other receptors, ion channels and transporters) and exhibited high functional antagonist potency at the human recombinant H3 receptor in vitro (pA2 = 9.1 vs imetit–induced inhibition of forskolin–stimulated cAMP accumulation). In vitro autoradiography studies with [3H]–GSK189254 in rat brain slices demonstrated a specific binding pattern consistent with H3 receptor distribution in cortex, hippocampus, striatum and hypothalamus. Specific [3H]–GSK189254 labelling was also observed in post–mortem cortex and hippocampus from human control and Alzheimer's disease brains. Following oral administration in rats, GSK189254 exhibited high H3 receptor occupancy in the cerebral cortex (ED50 = 0.17mg/kg from ex vivo binding studies) indicative of good brain penetration, and potently reversed drinking induced by the H3 selective agonist (R)–alpha–methylhistamine (ID50 = 0.05mg/kg), supporting functional blockade of H3 receptors in vivo. GSK189254 (0.1–3mg/kg) exhibited efficacy in a number of rodent models of cognitive function including object recognition (48h delay), passive avoidance (scopolamine–induced amnesia) and water maze (aged animals). In addition, GSK189254 induced a transient increase in wakefulness in the rat during the light phase, and a corresponding decrease in slow wave sleep 2. Given these effects on cognition and alertness in preclinical species, the H3 antagonist GSK189254 has the potential to be a new therapeutic agent for the symptomatic treatment of Alzheimer's Disease and other dementias.

The 5–HT4 serotonin receptor has been implicated in the central control of learning and memory, and several full and partial 5–HT4 agonists have been demonstrated to improve cognitive processes in a number of species and paradigms. In addition, 5–HT4 partial agonists are currently in clinical trials for cognitive enhancement in patients with Alzheimer's disease, as well as being developed for peripheral indications such as gastrointestinal motility and overactive bladder. 5–HT4 is a member of the seven–transmembrane G protein coupled receptor superfamily and has been classically described as coupling to the Gs family of G proteins, and thus to the activation of adenylate cyclase. At a molecular level, the 5–HT4 receptor is derived from a complex gene consisting of 38 exons, resulting in the generation of at least ten splice variants in humans. Intriguingly, all but one of these alternatively spliced forms are identical in amino acid sequence up to leucine 358, at which point the C termini of each variant diverge. We have exploited these differences to generate antisera to various 5–HT4 splice variants, and used these antibodies in conjunction with mRNA analyses to analyze splice variant specific distribution in the CNS. We have also carried out an analysis of 5–HT4 genomic sequences in various species. Finally, we have used a number of in vitro pharmacological assays to investigate the G protein coupling and functionality of various 5–HT4 receptor splice variants. Standard methods were used for antisera generation, immunohistochemistry, immunocytochemistry, immunoblotting, real–time semi–quantitative RT–PCR and receptor functional assays. We will describe differences we have observed in the spatial distribution of splice variants in vivo, and in addition at the protein and mRNA level in primary purified neural cells. We will demonstrate that the 5–HT4 splice variance is not ubiquitously conserved amongst species, and we will demonstrate that the 5–HT4 receptor can equally couple to pathways other than its classical elevation of intracellular cyclic AMP. We have uncovered important differences in the spatial distribution, inter–species splice variance, and signalling pathways of a number of 5–HT4 alternative splice forms.

The 5-hydroxytryptamine-6 (5-HT6) receptor is one of 14 distinct mammalian 5-HT (serotonin) receptors expressed in the central nervous system through which 5-HT is involved in regulating a number of diverse biological processes 1. Binding studies with [125I]SB258585, have localised 5-HT6 receptors almost exclusively in the CNS 2. In these reports, SB258585 showed a high level of binding in rat, and pig striatal tissues and low in the cerebellum. In man SB258585 binding pattern is similar to rat and pig, being strongest in caudate, putamen, moderates in cerebral cortex and low in the cerebellum 3. Despite its good in vitro profile, the development of SB285585 as an in vivo imaging tool was hampered due to its poor brain penetration properties and to date no successful 5-HT6 ligand has been reported for use in PET. We present here the radiolabelling and preclinical evaluation of [11C]GSK215083 a novel tool to probe the 5-HT6 receptors in vivo. [11C]GSK215083,[11C-N-methyl]3-[(3-fluorophenyl)sulfonyl]-8-(4-methyl-1-piperazi nyl) quinoline, was prepared by N-methylation of the corresponding desmethyl precursor with [11C]MeOTf in methanol:acetonitrile in presence of 2,2,6,6-tetramethylpiperidine, followed by HPLC purification. In a pilot study [11C]GSK215083 was evaluated in anesthetized Yorlshire pigs (40 kg). [11C]GSK215083 readily enters the brain reaching peak regional tissue concentrations at approximately 20 min post injection followed by a slow washout from brain regions known to be rich in 5-HT6 receptors with highest uptake and retention observed in striatum. The observed rank order of regional brain concentrations was striatum>cortical regions>cerebellum, consistent with reported 5-HT6 receptor densities and localisation determined by tissue section autoradiograpgy in animals and man. Upon injection of [11C]GSK215083, striatum to cerebellum and cortex to cerebellum ratios of 2 to1 and 1.5 to 1, respectively were reached at 60 min post injection. Co-administration of [11C]GSK215083 with escalating dose of authentic GSK215083 (0.005, 0.05 and 0.5 mg/kg) have located a saturable and dose dependent signal in the striatum and cortical regions. Treatment of pigs with the 5-HT6 binding drug, clozapine (6.25 mg/kg), significantly reduced the specific binding in striatum as compared to cerebellum. No significant effect on [11C]GSK215083 signal in striatum was observed following treatment with ketanserine (0.3 mg/kg), a selective 5-HT2a receptor antagonist, in contrast the same treatment reduced >90% specific binding in frontal cortex. Radio-HPLC analysis revealed that [11C]GSK215083 is rapidly metabolised in arterial plasma, representing approximately 60% of the total radioactivity 30 min post injection. [11C]GSK215083 shows properties suitable for studies probing 5HT6 receptor in man with PET (See Figure 1).

Cell death mechanisms frequently involve the influx of extracellular calcium through voltage- and ligand-gated ion channels, e. g., the NMDA receptor (Greene, 1999). The vanilloid receptor (VR1) is present in regions of the brain (Mezey et al., 2000) that are highly susceptible to neurodegenerative insults, suggesting that this ion channel might contribute to the cellular processes involved in neuronal death. We tested the effects of VR1 ligands in the oxygen glucose deprivation (OGD) model of cell death in organotypic hippocampal slice cultures. The VR1 agonist capsaicin at concentrations that are selective for VR1 did not affect cell viability per se or the extent of neurodegeneration induced by the OGD insult. In contrast, the VR1 antagonist capsazepine (0.1-10 muM) significantly reduced the amount of OGD-induced cell death. However, capsazepine was still neuroprotective in slices prepared from VR1 knock-out mice, which exhibited the same degree of neurodegeneration to that observed in slices prepared from wild-type mice, excluding the possibility that it afforded neuroprotection through inhibition of VR1. Instead, capsazepine inhibited the hyperpolarization-activated nonspecific cation channel generated current I-h in a concentration range similar to that which was neuroprotective. Furthermore, the specific I-h blocker ZD-7288 was also neuroprotective, mirroring the effects of capsazepine, in that it was effective at preventing cell death when applied either during or after the OGD insult. These results demonstrate that capsazepine affords neuroprotection through inhibition of I-h rather than inhibition of VR1.

Cell death mechanisms frequently involve the influx of extracellular calcium through voltage- and ligand-gated ion channels, e.g., the NMDA receptor (Greene, 1999). The vanilloid receptor (VR1) is present in regions of the brain (Mezey et al., 2000) that are highly susceptible to neurodegenerative insults, suggesting that this ion channel might contribute to the cellular processes involved in neuronal death. We tested the effects of VR1 ligands in the oxygen glucose deprivation (OGD) model of cell death in organotypic hippocampal slice cultures. The VR1 agonist capsaicin at concentrations that are selective for VR1 did not affect cell viability per se or the extent of neurodegeneration induced by the OGD insult. In contrast, the VR1 antagonist capsazepine (0.1-10 μm) significantly reduced the amount of OGD-induced cell death. However, capsazepine was still neuroprotective in slices prepared from VR1 knock-out mice, which exhibited the same degree of neurodegeneration to that observed in slices prepared from wild-type mice, excluding the possibility that it afforded neuroprotection through inhibition of VR1. Instead, capsazepine inhibited the hyperpolarization-activated nonspecific cation channel generated current Ih in a concentration range similar to that which was neuroprotective. Furthermore, the specific Ih blocker ZD-7288 was also neuroprotective, mirroring the effects of capsazepine, in that it was effective at preventing cell death when applied either during or after the OGD insult. These results demonstrate that capsazepine affords neuroprotection through inhibition of Ih rather than inhibition of VR1.

Group I metabotropic glutamate receptors (mGluRs) cause increased neuronal excitability that can lead to epileptogenesis and neurodegeneration. Here we have examined how individual members of this subgroup of mGluRs affect synchronised hippocampal synaptic activity under normal and disinhibited conditions similar to those that occur during certain epileptic states. We demonstrate that activation of both mGluR1 and mGluR5 are important in increasing neuronal synaptic excitability by increasing synchrony between cells and driving correlated network activity in circuits that contain, or are devoid of, GABA(A) receptor-mediated synaptic inputs. The precise patterning of activity that occurs is complex and depends upon: (1) the existing pattern of ongoing network activity prior to mGluR activation; and (2) the relative extent of activation of each mGluR subtype. However, mGluR5 appears to be the principal mGluR subtype that initiates bursting activity irrespective of the inhibitory synaptic tone within the neuronal network.

During the use of tetrapeptide and other proprietary caspase inhibitors in the study of neurodegeneration, we had concluded that mechanisms other than the inhibition of caspases contributed to the protective effects of certain caspase inhibitors. Here we report our studies to identify a target for and hence a mechanism by which the tetrapeptide inhibitor tyrosine-valine-alanine-aspartate-chloromethyl ketone (Ac-YVAD-cmk) is able to rescue neuronal cell cultures from cell death. Ac-YVAD-cmk rescued neuronal cells from cell death in response to oxidative stress and oxygen/glucose deprivation. Affinity labeling with biotinylated YVAD-cmk demonstrated distinct binding proteins for the inhibitor in cells from the central nervous system versus Jurkat cells. Binding to the novel target protein was displaced by class-specific protease inhibitors and suggested that the target is a cysteine protease. Affinity purification and sequencing identified the target as cathepsin-B. Cathepsin-B inhibitors competed with biotinylated YVAD-cmk for the target protein. The availability of the target for binding was reduced in cells that had been rescued by unlabeled inhibitor. Cathepsin-B inhibitors rescue hippocampal slices from cell death induced by oxygen/glucose deprivation. These data provide evidence to support a role for cathepsin-B in neuronal cell death, particularly that following ischemia.

We have explored the neuroprotective efficacy of the cell penetrant caspase inhibitor, Ac-YVAD-cmk, in a hippocampal slice model of neuronal cell death induced by oxygen and glucose deprivation. Organotypic hippocampal slice cultures were prepared from 8 to 10-day-old rats and maintained for 10 to 12 days in vitro. Pre-treatment with Ac-YVAD-cmk prior to 45 min oxygen and glucose deprivation was neuroprotective as measured by propidium iodide uptake, with an EC50 between 1 and 10 μmol/l. Ac-YVAD-cmk was also able to preserve synaptic function in the organotypic hippocampal slice cultures 24 h after oxygen and glucose deprivation. Ac-YVAD-cmk prevented the increase in histone-associated DNA fragmentation induced by oxygen and glucose deprivation. Interleukin-1β did not reverse the protective effect of Ac-YVAD-cmk, and interleukin-1 receptor antagonist alone was not protective. These results show that caspase inhibitors are neuroprotective in a hippocampal slice culture system, using structural, biochemical and electrophysiological endpoints, and that this effect is not a result of inhibition of interleukin-1β production.

Earlier optimization of structure‐activity relationships in a novel series of 4‐(benzoylamino)‐benzopyrans, led to the discovery of SB‐204269 ( trans ‐(+)‐6‐acetyl‐4 S ‐(4‐fluorobenzoylamino)‐3,4‐dihydro‐2,2‐dimethyl‐2 H ‐benzo[ b ]pyran‐3 R ‐ol, hemihydrate), a potent orally‐active anticonvulsant in the mouse maximal electroshock seizure threshold (MEST) test. Studies have now been undertaken to determine the effects of SB‐204269 in a range of seizure models and tests of neurological deficits in rats. In addition, the compound has been evaluated in a series of in vitro mechanistic assays. SB‐204269 proved to be an orally‐effective anticonvulsant agent, at doses (0.1–30 mg kg −1 ) devoid of overt behavioural depressant properties, in models of both electrically (MEST and maximal electroshock (MES)) and chemically (i.v. pentylenetetrazol (PTZ) infusion)‐evoked tonic extension seizures. However, the compound did not inhibit PTZ‐induced myoclonic seizures at doses up to 30 mg kg −1 , p.o. SB‐204269 also selectively reduced focal electrographic seizure activity in an in vitro elevated K + rat hippocampal slice model at concentrations (0.1–10 μ M ) that had no effect on normal synaptic activity and neuronal excitability. In all of these seizure models, SB‐204269 was equivalent or better than the clinically established antiepileptic drugs carbamazepine and lamotrigine, in terms of anticonvulsant potency and efficacy. Unlike SB‐204269, the corresponding trans 3 S ,4 R enantiomer, SB‐204268, did not produce marked anticonvulsant effects, an observation in accord with previous findings for other related pairs of trans enantiomers in the benzopyran series. In the rat accelerating rotarod test, a sensitive paradigm for the detection of neurological deficits such as sedation and motor incoordination, SB‐204269 was inactive even at doses as high as 200 mg kg −1 , p.o. This was reflected in the excellent therapeutic index (minimum significantly effective dose in the rotarod test/ED 50 in the MES test) for SB‐204269 of >31, as compared to equivalent values of only 7 and 13 for carbamazepine and lamotrigine, respectively. At concentrations (10 μ M ) well above those required to produce anticonvulsant activity in vivo (i.e. 0.1 μ M in brain), SB‐204269 did not interact with many of the well known mechanistic targets for established antiepileptic drugs (e.g. Na + channels or GABAergic neurotransmission). Subsequent studies have shown that the anticonvulsant properties of SB‐204269 are likely to be mediated by a novel stereospecific binding site present in the CNS. The overall efficacy profile in rodent seizure models, together with a minimal liability for inducing neurological impairment and an apparently unique mechanism of action, highlight the therapeutic potential of SB‐204269 for the treatment of refractory partial and generalized tonic‐clonic seizures. British Journal of Pharmacology (1997) 121 , 1679–1686; doi: 10.1038/sj.bjp.0701330