Alison M. Ray

GSK189254, a Novel H3 Receptor Antagonist That Binds to Histamine H3 Receptors in Alzheimer's Disease Brain and Improves Cognitive Performance in Preclinical Models

6-[(3-Cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-3-pyridinecarboxamide hydrochloride (GSK189254) is a novel histamine H3 receptor antagonist with high affinity for human (pKi = 9.59 –9.90) and rat (pKi = 8.51–9.17) H3 receptors. GSK189254 is >10,000-fold selective for human H3 receptors versus other targets tested, and it exhibited potent functional antagonism (pA2 = 9.06 versus agonist-induced changes in cAMP) and inverse agonism [pIC50 = 8.20 versus basal guanosine 5′-O-(3-[35S]thio)triphosphate binding] at the human recombinant H3 receptor. In vitro autoradiography demonstrated specific [3H]GSK189254 binding in rat and human brain areas, including cortex and hippocampus. In addition, dense H3 binding was detected in medial temporal cortex samples from severe cases of Alzheimers disease, suggesting for the first time that H3 receptors are preserved in late-stage disease. After oral administration, GSK189254 inhibited cortical ex vivo R-(–)-α-methyl[imidazole-2,5(n)-3H]histamine dihydrochloride ([3H]R-α-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 H3 receptors was demonstrated by blockade of R-α-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 Alzheimers disease and other cognitive disorders.

Caspase inhibitors are functionally neuroprotective against oxygen glucose deprivation induced CA1 death in rat organotypic hippocampal slices.

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 EC(50) between 1 and 10 micromol/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-1beta 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-1beta production.

Profile of SB-204269, a mechanistically novel anticonvulsant drug, in rat models of focal and generalized epileptic seizures

1 Earlier optimization of structure‐activity relationships in a novel series of 4‐(benzoylamino)‐benzopyrans, led to the discovery of SB‐204269 (trans‐(+)‐6‐acetyl‐4S‐(4‐fluorobenzoylamino)‐3,4‐dihydro‐2,2‐dimethyl‐2H‐benzo[b]pyran‐3R‐ol, hemihydrate), a potent orally‐active anticonvulsant in the mouse maximal electroshock seizure threshold (MEST) test. 2 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. 3 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. 4 SB‐204269 also selectively reduced focal electrographic seizure activity in an in vitro elevated K+ rat hippocampal slice model at concentrations (0.1–10 μM) that had no effect on normal synaptic activity and neuronal excitability. 5 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. 6 Unlike SB‐204269, the corresponding trans 3S,4R 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. 7 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/ED50 in the MES test) for SB‐204269 of >31, as compared to equivalent values of only 7 and 13 for carbamazepine and lamotrigine, respectively. 8 At concentrations (10 μM) well above those required to produce anticonvulsant activity in vivo (i.e. 0.1 μM 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. 9 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.

The identification of potent, selective and CNS penetrant furan-based inhibitors of B-Raf kinase

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.

Tricyclic azepine derivatives as selective brain penetrant 5-HT6 receptor antagonists

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.

Identification of a novel 5-HT4 receptor splice variant (r5-HT4c1) and preliminary characterisation of specific 5-HT4a and 5-HT4b receptor antibodies

The human 5-hydroxytryptamine (5-HT(4)) 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-HT(4a) (r5-HT(4a)), 5-HT(4b) (r5-HT(4b)) and 5-HT(4e) (r5-HT(4e)). In the current study we identify and characterise the pharmacology of a novel rat splice variant (r5-HT(4c1)) and present the first comprehensive analysis of 5-HT(4) splice variant mRNA expression levels throughout the rat gastrointestinal tract. In addition, we describe preliminary characterisation of the first 5-HT(4) splice variant specific antibodies. In transfected cells, r5-HT(4c1) receptor exhibited similar binding properties to r5-HT(4a) and r5-HT(4b). Functional studies showed that 5-HT(4) 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-HT(4c1), but full agonists at r5-HT(4a) and r5-HT(4b). Moreover, in contrast to r5-HT(4a) and r5-HT(4b), r5-HT(4c1) was not constitutively active. TaqMan mRNA analysis showed that r5-HT(4a) expression in brain and dorsal root ganglion exceeded that in the gastrointestinal tract, whilst the reverse was true for r5-HT(4b) and r5-HT(4c1). mRNA expression of each variant also increased distally throughout the gastrointestinal tract with the highest levels in the colon. r5-HT(4a) and r5-HT(4b) 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-HT(4b) immunoreactivity was observed on endocrine cells in the duodenum. These data could have implications in rat models and aid understanding of 5-HT(4) splice variant function.

Complex interactions between mGluR1 and mGluR5 shape neuronal network activity in the rat hippocampus.

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.

P4-327: Effects of the novel 5-HT6 SB-742457 antagonist and the cholinesterase inhibitor donepezil in models of cognition

related to aging, Alzheimer’s disease (AD) and Schizophrenia. Thus serotonergic system became a potential target for the treatment of memory dysfunction. Methods: Our effective lead generation and optimization methods have resulted in a potent 5-HT6 receptor antagonist SUVN-502 with Ki of 1.71 nM. SUVN-502 exhibited antagonist like inhibition with EC50 of 0.103 M when tested for functional activity. SUVN-502 has more than 100 fold selectivity against the related GPCRs. The effective optimization of its critical physico-chemical properties has lead to oral bioavailability (31%) and brain penetration index (1.48). Results: SUVN-502 was effective in animal models of cognition. SUVN-502 reversed the age induced memory deficit in Morris water maze and novel object recognition task (NORT). SUVN-502 also reversed the spatial and working memory deficit induced by MK-801 and scopolamine in Morris water maze, NORT and radial arm maze task. The effective dose range of SUVN-502 in the above animal models was between 3 to 10 mg/kg. p.o. Brain microdialysis studies in rats with SUVN-502 showed significant increase in brain acetylcholine and glutamate levels correlating to the in vivo memory models. Conclusions: SUVN-502 has been identified as a candidate for clinical development for the symptomatic treatment of Alzheimer’s disease. SUVN-502 has completed all regulatory safety and toxicity studies to enter to human Phase-I clinical trials in 2008.