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Keith A Wafford

Publications and source records attributed to Keith A Wafford.

At least 19 recordsLinked to original sources
PubMed2006

Differential contribution of GABA(A) receptor subtypes to the anticonvulsant efficacy of benzodiazepine site ligands.

Non-selective benzodiazepines, such as diazepam, interact with equivalent affinity and agonist efficacy at GABA(A) receptors containing either an alpha1, alpha2, alpha3 or alpha5 subunit. However, which of these particular subtypes are responsible for the anticonvulsant effects of diazepam remains uncertain. In the present study, we examined the ability of diazepam to reduce pentylenetetrazoLe (PTZ)-induced and maximal electroshock (MES)-induced seizures in mice containing point mutations in single (alpha1H101R, alpha2H101R or alpha5H105R) or multiple (alpha125H-->R) alpha subunits that render the resulting GABA(A) receptors diazepam-insensitive. Furthermore, the anticonvulsant properties of diazepam, the alpha1- and alpha3-selective compounds zolpidem and TP003, respectively, and the alpha2/alpha3 preferring compound TP13 were studied against PTZ-induced seizures. In the transgenic mice, no single subtype was responsible for the anticonvulsant effects of diazepam in either the PTZ or MES assay and neither the alpha3 nor alpha5 subtypes appeared to confer anticonvulsant activity. Moreover, whereas the alpha1 and alpha2 subtypes played a modest role with respect to the PTZ assay, they had a negligible role in the MES assay. With respect to subtype-selective compounds, zolpidem and TP003 had much reduced anticonvulsant efficacy relative to diazepam in both the PTZ and MES assays whereas TP13 had high anticonvulsant efficacy in the PTZ but not the MES assay. Taken together, these data not only indicate a role for alpha2-containing GABA(A) receptors in mediating PTZ and MES anticonvulsant activity but also suggest that efficacy at more than one subtype is required and that these subtypes act synergistically.

Animals↗
PubMed2006

L-655,708 enhances cognition in rats but is not proconvulsant at a dose selective for alpha5-containing GABAA receptors.

The in vitro and in vivo properties of L-655,708, a compound with higher affinity for GABA(A) receptors containing an alpha5 compared to an alpha1, alpha2 or alpha3 subunit have been examined further. This compound has weak partial inverse agonist efficacy at each of the four subtypes but, and consistent with the binding data, has higher functional affinity for the alpha5 subtype. In a mouse hippocampal slice model, L-655,708 was able to enhance the long-term potentiation produced by a theta burst stimulation, consistent with a potential role for the alpha5 subtype in processes involving synaptic plasticity, such as learning and memory. When administered in a formulation specifically designed to achieve relatively constant plasma drug concentrations, and therefore maintain selective occupancy of alpha5- compared to alpha1-, alpha2- and alpha3-containing receptors (75+/-4% versus 22+/-10%, respectively), L-655,708 did not alter the dose of pentylenetetrazole required to induce seizures, indicating that the inverse agonist effects of L-655,708 at the alpha5 subtype are not associated with a proconvulsant liability. In the Morris water maze, L-655,708 enhanced performance not only during acquisition but also in a probe trial, demonstrating that this compound has cognition enhancing effects. These data further support the potential of alpha5-containing GABA(A) receptors as a target for novel cognition enhancing drugs.

Animals↗
PubMed2006

Treating insomnia: Current and investigational pharmacological approaches.

Chronic insomnia affects a significant proportion of young adult and elderly populations. Treatment strategies should alleviate nighttime symptoms, the feeling of nonrestorative sleep, and impaired daytime function. Current pharmacological approaches focus primarily on GABA, the major inhibitory neurotransmitter in the central nervous system. Benzodiazepine receptor agonists (BzRA) have been a mainstay of pharmacotherapy; the classical benzodiazepines and non-benzodiazepines share a similar mode of action and allosterically enhance inhibitory chloride currents through the GABA(A) receptor, a ligand-gated protein comprising 5 subunits pseudosymmetrically arranged around a core anion channel. Variations in GABA(A) receptor subunit composition confer unique pharmacological, biophysical, and electrophysiological properties on each receptor subtype. Classical benzodiazepines bind non-selectively to GABA(A) receptors containing a gamma2 subunit, whereas non-benzodiazepine hypnotics bind with higher relative affinity to alpha1-containing receptors. The non-benzodiazepine compounds generally represent an improvement over benzodiazepines as a result of improved binding selectivity and pharmacokinetic profiles. However, the enduring potential for amnestic effects, next day residual sedation, and abuse and physical dependence, particularly at higher doses, underscores the need for new treatment strategies. Novel pharmacotherapies in development act on systems believed to be specifically involved in the regulation of the sleep-wake cycle. The recently approved melatonin receptor agonist, ramelteon, targets circadian mechanisms. Gaboxadol, an investigational treatment and a selective extrasynaptic GABA(A) receptor agonist (SEGA), targets GABA(A) receptors containing a delta subunit, which are located outside the synaptic junctions of thalamic and cortical neurons thought to play an important regulatory role in the onset, maintenance, and depth of the sleep process.

Animals↗
PubMed2006

A genetically modified mouse model probing the selective action of ifenprodil at the N-methyl-D-aspartate type 2B receptor.

Selective antagonism of N-methyl-d-aspartate (NMDA) 2B subunit containing receptors has been suggested to have potential therapeutic application for multiple CNS disorders. The amino terminal NR2B residues 1 to 282 were found to be both necessary and sufficient for the binding and function of highly NR2B subunit specific antagonists like ifenprodil and CP-101,606. Using a genetic approach in mice, we successfully replaced the murine NR2B gene function by "knocking-in" (KI) a chimeric human NR2A/B cDNA containing the minimal domain abolishing ifenprodil binding into the endogenous NR2B locus. Patch-clamp recording from hippocampal cultures of the NR2B KI mice demonstrated that their NMDA receptors have reduced sensitivity to both ifenprodil and CP-101,606, as predicted, but also have a lower affinity for glycine. The NR2B KI mice exhibited normal locomotor activity making this ifenprodil-insensitive mouse model a valuable tool to test the specificity of NR2B selective antagonists in vivo.

Animals↗
PubMed2006

The proconvulsant effects of the GABAA alpha5 subtype-selective compound RY-080 may not be alpha5-mediated.

RY-080 (ethyl 8-ethynyl-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate) is an imidazobenzodiazepine with 40-50-fold higher affinity for the benzodiazepine binding site of alpha5- rather than alpha1-, alpha2- or alpha3-containing GABAA receptors. Previous data describing RY-080 as being convulsant suggests that inverse agonists selective for the alpha5 subtype may not be suitable for clinical development. In the present study, we show that RY-080 possesses inverse agonism for the alpha1 and alpha5 subtypes of human recombinant GABAA receptors and whilst not convulsant it was proconvulsant. Hence, with pentylenetetrazole alone, the dose predicted to give tonic convulsions in 50% of the mice (ED50) was 108 mg/kg whereas in the presence of 1 and 10 mg/kg RY-080, the ED50s were 93 and 57 mg/kg, respectively. In vivo [3H]L-655,708 and [3H]Ro 15-1788 binding assays showed that the subtype selectivity of RY-080 in vivo was 7-10-fold for alpha5-relative to alpha1- and alpha2/alpha3-containing receptors (respective ID50 values of 0.93, 9.7 and 6.2 mg/kg) and is therefore much lower than seen in vitro. Consequently, it is not possible to define a dose of RY-080 which gives high occupancy of the alpha5 subtype without binding to other subtypes and accordingly the proconvulsant effects of RY-080 cannot be attributed solely to the alpha5 subtype.

Alkynes↗
PubMed2006

A pyridazine series of alpha2/alpha3 subtype selective GABA A agonists for the treatment of anxiety.

The development of a series of GABA(A) alpha2/alpha3 subtype selective pyridazine based benzodiazepine site agonists as anxiolytic agents with reduced sedative/ataxic potential is described, including the discovery of 16, a remarkably alpha3-selective compound ideal for in vivo study. These ligands are antagonists at the alpha1 subtype, with good CNS penetration and receptor occupancy, and excellent oral bioavailability.

Animals↗
PubMed2006

2,3,7-Trisubstituted pyrazolo[1,5-d][1,2,4]triazines: functionally selective GABAA alpha3-subtype agonists.

Novel synthetic routes have been devised for the preparation of previously inaccessible 2,3,7-trisubstituted pyrazolo[1,5-d][1,2,4]triazines 2. These compounds are high affinity ligands for the GABA(A) benzodiazepine binding site and some analogues show functional selectivity for agonism at alpha3-containing receptors over alpha1-containing receptors with the lead compound being 32.

Binding Sites↗
PubMed2006

Alpha5GABAA receptors mediate the amnestic but not sedative-hypnotic effects of the general anesthetic etomidate.

A fundamental objective of anesthesia research is to identify the receptors and brain regions that mediate the various behavioral components of the anesthetic state, including amnesia, immobility, and unconsciousness. Using complementary in vivo and in vitro approaches, we found that GABAA receptors that contain the alpha5 subunit (alpha5GABAARs) play a critical role in amnesia caused by the prototypic intravenous anesthetic etomidate. Whole-cell recordings from hippocampal pyramidal neurons showed that etomidate markedly increased a tonic inhibitory conductance generated by alpha5GABAARs, whereas synaptic transmission was only slightly enhanced. Long-term potentiation (LTP) of field EPSPs recorded in CA1 stratum radiatum was reduced by etomidate in wild-type (WT) but not alpha5 null mutant (alpha5-/-) mice. In addition, etomidate impaired memory performance of WT but not alpha5-/- mice for spatial and nonspatial hippocampal-dependent learning tasks. The brain concentration of etomidate associated with memory impairment in vivo was comparable with that which increased the tonic inhibitory conductance and blocked LTP in vitro. The alpha5-/- mice did not exhibit a generalized resistance to etomidate, in that the sedative-hypnotic effects measured with the rotarod, loss of righting reflex, and spontaneous motor activity were similar in WT and alpha5-/- mice. Deletion of the alpha5 subunit of the GABAARs reduced the amnestic but not the sedative-hypnotic properties of etomidate. Thus, the amnestic and sedative-hypnotic properties of etomidate can be dissociated on the basis of GABAAR subtype pharmacology.

Action Potentials↗
PubMed2006

Discovery of imidazo[1,2-b][1,2,4]triazines as GABA(A) alpha2/3 subtype selective agonists for the treatment of anxiety.

The identification of a series of imidazo[1,2-b][1,2,4]triazines with high affinity and functional selectivity for the GABA(A) alpha3-containing receptor subtype is described, leading to the identification of a clinical candidate, 11. Compound 11 shows good bioavailability and half-life in preclinical species, and it is a nonsedating anxiolytic in both rat and squirrel monkey behavioral models.

Animals↗
PubMed2006

The in vivo properties of pagoclone in rat are most likely mediated by 5'-hydroxy pagoclone.

The cyclopyrrolone pagoclone binds with roughly equivalent high affinity (0.7-9.1nM) to the benzodiazepine binding site of human recombinant GABA(A) receptors containing either an alpha1, alpha2, alpha3 or alpha5 subunit. However, whereas it was a partial agonist at alpha1-, alpha2- and alpha5-containing GABA(A) receptors, pagoclone was a full agonist at receptors containing an alpha3 subunit. In the rat elevated plus maze assay pagoclone (3mg/kg) had significant anxiolytic-like activity but at all three doses tested (0.3, 1 and 3mg/kg p.o.) it produced a significant reduction in the total distance travelled. This sedative-like effect was confirmed in rat chain-pulling and spontaneous locomotor assays. Surprisingly, in the plasma and brain samples derived from the elevated plus maze assay, the major metabolite of pagoclone, 5'-hydroxy pagoclone, was present at 10-20-fold higher concentrations relative to the parent compound. In order to establish whether this metabolite might have pharmacological activity, we measured its affinity and efficacy profile and found that both were comparable to those of pagoclone with the exception that efficacy at the alpha1 subtype was considerably greater for 5'-hydroxy pagoclone compared with the parent. This metabolite had significant anxiolytic-like activity in the elevated plus maze but at these same doses (0.3-3mg/kg p.o.) also produced sedation. It is therefore likely that in rats 5'-hydroxy pagoclone mediates the majority of the pharmacological actions following pagoclone administration.

Animals↗
PubMed2006

Imidazo[1,2-a]pyrimidines as functionally selective and orally bioavailable GABA(A)alpha2/alpha3 binding site agonists for the treatment of anxiety disorders.

A series of high-affinity GABA(A) agonists with good oral bioavailability in rat and dog and functional selectivity for the GABA(A)alpha2 and -alpha3 subtypes is reported. The 7-trifluoromethylimidazopyrimidine 14g and the 7-propan-2-olimidazopyrimidine 14k are anxiolytic in both conditioned and unconditioned animal models of anxiety with minimal sedation observed at full BZ binding site occupancy.

Administration, Oral↗
PubMed2006

The role of GABAbeta2 subunit-containing receptors in mediating the anticonvulsant and sedative effects of loreclezole.

The majority of inhibitory neurotransmission in the brain is mediated by the gamma-aminobutyric acid (GABA) type A (GABA(A)) receptor. The anticonvulsant loreclezole largely acts by potentiating GABA(A) receptors containing beta2 and beta3 subunits. We used a genetically modified mouse containing a loreclezole-insensitive beta2 subunit (beta2N265S) to determine the role of this subunit in mediating the sedative and anticonvulsive effects of loreclezole. Sedation was assessed by measuring spontaneous locomotor activity and beam walking performance, and anticonvulsant efficacy was determined by pentylenetetrazole (PTZ) and amygdala kindling-induced seizures. The beta2N265S mice did not exhibit loreclezole-mediated sedation as shown by normal locomotor activity and beam walking performance. However, loreclezole also failed to provide significant protection against PTZ-induced seizures in the beta2N265S mice. Reduced efficacy against amygdala-kindled seizures, both acutely and over a 13-day chronic dosing study, was also observed in beta2N265S mice. These results suggest that the majority of the sedative effects and a significant proportion of the anticonvulsant efficacy of loreclezole are mediated via beta2-containing GABA(A) receptors.

Animals↗
PubMed2005

Imidazo[1,2-a]pyrazin-8-ones, imidazo[1,2-d][1,2,4]triazin-8-ones and imidazo[2,1-f][1,2,4]triazin-8-ones as alpha2/alpha3 subtype selective GABA A agonists for the treatment of anxiety.

Imidazo[1,2-a]pyrazin-8-ones, imidazo[1,2-d][1,2,4]triazin-8-ones and imidazo[2,1-f][1,2,4]triazin-8-ones are high affinity GABA(A) agonists. Compound 16d has good oral bioavailability in rat, functional selectivity for the GABA(A)alpha2 and alpha3-subtypes and is anxiolytic in a conditioned animal model of anxiety with minimal sedation observed at full BZ binding site occupancy.

Administration, Oral↗
PubMed2005

Gaboxadol--a new awakening in sleep.

Drugs that enhance synaptic gamma-aminobutyric acid (GABA)ergic neurotransmission are widely utilized in the clinical setting. Barbiturates and benzodiazepine receptor agonists, for example, both potentiate an inhibitory chloride conductance through GABA-gated channels, and thereby achieve their sedative-hypnotic effects. The primary locus of action of these agents, and indeed most neuroactive drugs, is the postsynaptic junction. By contrast, gaboxadol, a selective extrasynaptic GABA receptor agonist and late-stage investigational treatment for insomnia, acts on a unique delta-containing GABAA receptor subtype found exclusively outside of the synapse. Although the mechanistic details of extrasynaptic neurotransmission remain to be fully established, it is now clear that these receptors demonstrate unique pharmacological, biophysical and electrophysiological properties. Importantly, the delta-containing GABAA receptor subtype activated by gaboxadol is highly expressed in the thalamus, where it might behave as a 'gain control' (independently controlling the strength of signals) in the corticothalamic pathways that govern sleep-relevant neuronal oscillations. This unique mechanism has contributed to our increased understanding of sleep mechanisms, and targeting of this system offers potential advantages over existing insomnia treatments.

Animals↗
PubMed2005

Extrasynaptic GABAA receptors of thalamocortical neurons: a molecular target for hypnotics.

Among hypnotic agents that enhance GABAA receptor function, etomidate is unusual because it is selective for beta2/beta3 compared with beta1 subunit-containing GABAA receptors. Mice incorporating an etomidate-insensitive beta2 subunit (beta(2N265S)) revealed that beta2 subunit-containing receptors mediate the enhancement of slow-wave activity (SWA) by etomidate, are required for the sedative, and contribute to the hypnotic actions of this anesthetic. Although the anatomical location of the beta2-containing receptors that mediate these actions is unknown, the thalamus is implicated. We have characterized GABAA receptor-mediated neurotransmission in thalamic nucleus reticularis (nRT) and ventrobasalis complex (VB) neurons of wild-type, beta2(-/-), and beta(2N265S) mice. VB but not nRT neurons exhibit a large GABA-mediated tonic conductance that contributes approximately 80% of the total GABAA receptor-mediated transmission. Consequently, although etomidate enhances inhibition in both neuronal types, the effect of this anesthetic on the tonic conductance of VB neurons is dominant. The GABA-enhancing actions of etomidate in VB but not nRT neurons are greatly suppressed by the beta(2N265S) mutation. The hypnotic THIP (Gaboxadol) induces SWA and at low, clinically relevant concentrations (30 nM to 3 microM) increases the tonic conductance of VB neurons, with no effect on VB or nRT miniature IPSCs (mIPSCs) or on the holding current of nRT neurons. Zolpidem, which has no effect on SWA, prolongs VB mIPSCs but is ineffective on the phasic and tonic conductance of nRT and VB neurons, respectively. Collectively, these findings suggest that enhancement of extrasynaptic inhibition in the thalamus may contribute to the distinct sleep EEG profiles of etomidate and THIP compared with zolpidem.

Animals↗