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Biomedical subjects

John R Atack

Publications and source records attributed to John R Atack.

At least 19 recordsLinked to original sources
PubMed2006

Comparison of lorazepam [7-chloro-5-(2-chlorophenyl)-1,3-dihydro-3-hydroxy-2H-1,4-benzodiazepin-2-one] occupancy of rat brain gamma-aminobutyric acid(A) receptors measured using in vivo [3H]flumazenil (8-fluoro 5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester) binding and [11C]flumazenil micro-positron emission tomography.

The occupancy by lorazepam of the benzodiazepine binding site of rat brain GABA(A) receptors was compared when measured using either in vivo binding of [(3)H]flumazenil (8-fluoro 5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester) in terminal studies or [(11)C]flumazenil binding in anesthetized animals assessed using a small animal positron emission tomography (PET) scanner (micro-PET). In addition, as a bridging study, lorazepam occupancy was measured using [(3)H]flumazenil in vivo binding in rats anesthetized and dosed under micro-PET conditions. Plasma lorazepam concentrations were also determined, and for each occupancy method, the concentration required to produce 50% occupancy (EC(50)) was calculated because this parameter is independent of the route of lorazepam administration. For the in vivo binding assay, lorazepam was dosed orally (0.1-10 mg/kg), whereas for the micro-PET study, lorazepam was given via the i.v. route as a low dose (0.75 mg/kg bolus) and then a high dose (0.5 mg/kg bolus then 0.2 mg/ml infusion). The lorazepam plasma EC(50) in the [(11)C]flumazenil micro-PET study was 96 ng/ml [95% confidence intervals (CIs) = 74-124 ng/ml], which was very similar to the [(3)H]flumazenil micro-PET simulation study (94 ng/ml; 95% CI = 63-139 ng/ml), which in turn was comparable with the [(3)H]flumazenil in vivo binding study (134 ng/ml; 95% CI = 119-151 ng/ml). These data clearly show that despite the differences in dosing (i.v. in anesthetized versus orally in conscious rats) and detection (in vivo dynamic PET images versus ex vivo measurements in filtered and washed brain homogenates), [(11)C]flumazenil micro-PET produces results similar to [(3)H]flumazenil in vivo binding.

Animals↗
PubMed2006

The novel gamma secretase inhibitor N-[cis-4-[(4-chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,1,1-trifluoromethanesulfonamide (MRK-560) reduces amyloid plaque deposition without evidence of notch-related pathology in the Tg2576 mouse.

There is a substantial body of evidence indicating that beta-amyloid peptides (Abeta) are critical factors in the onset and development of Alzheimer's disease (AD). One strategy for combating AD is to reduce or eliminate the production of Abeta through inhibition of the gamma-secretase enzyme, which cleaves Abeta from the amyloid precursor protein (APP). We demonstrate here that chronic treatment for 3 months with 3 mg/kg of the potent, orally bioavailable and brain-penetrant gamma-secretase inhibitor N-[cis-4-[(4-chlorophenyl)-sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,1,1-trifluoromethanesulfonamide (MRK-560) attenuates the appearance of amyloid plaques in the Tg2576 mouse. These reductions in plaques were also accompanied by a decrease in the level of reactive gliosis. The morphometric and histological measures agreed with biochemical analysis of Abeta(40) and Abeta(42) in the cortex. Interestingly, the volume of the plaques across treatment groups did not change, indicating that reducing Abeta levels does not significantly alter deposit growth once initiated. Furthermore, we demonstrate that these beneficial effects can be achieved without causing histopathological changes in the ileum, spleen, or thymus as a consequence of blockade of the processing of alternative substrates, such as the Notch family of receptors. This indicates that in vivo a therapeutic window between these substrates seems possible--a key concern in the development of this approach to AD. An understanding of the mechanisms whereby MRK-560 shows differentiation between the APP and Notch proteolytic pathway of gamma-secretase should provide the basis for the next generation of gamma-secretase inhibitors.

Alzheimer Disease↗
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

A comparative assessment of gamma-secretase activity in transgenic and non-transgenic rodent brain.

Amyloid-beta (Abeta) deposits are one of the hallmarks of the neuropathological degeneration observed in Alzheimer's disease (AD) and Abeta concentrations have been reported to vary in different brain regions of AD patients. Abeta is produced by the sequential cleavage of amyloid precursor protein (APP) by beta-secretase and gamma-secretase, respectively. Previous studies have shown that over-expression of the gamma-secretase complex leads to increased gamma-secretase proteolytic activity increasing Abeta production. However, it is not known whether brain regions with highest Abeta concentration also express relatively higher levels of gamma-secretase activity. Accordingly, the relationship between Abeta levels and gamma-secretase activity across brain regions was investigated and correlated in the brains of transgenic and non-transgenic rodents commonly used in AD research. The data demonstrated that Abeta levels do vary in different brain regions in both transgenic and non-transgenic mice but are not correlated with regional gamma-secretase activity. Furthermore, this study demonstrated that while mutations in the APP and PS1 sequences affect the absolute Abeta levels this is not reflected in an increase in gamma-secretase proteolytic activity. The data in the current paper indicate that this assay is able to measure the level of gamma-secretase activity in rodent species. Using this methodology will aid our understanding of physiological gamma-secretase function.

Amyloid Precursor Protein Secretases↗
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

Comparison of in vivo and ex vivo [3H]flumazenil binding assays to determine occupancy at the benzodiazepine binding site of rat brain GABAA receptors.

In the present study, the occupancy of flumazenil (Ro 15-1788; 1-30mg/kg p.o.) at the benzodiazepine site of rat brain GABA(A) receptors was compared using in vivo and ex vivo binding methodologies with [(3)H]flumazenil as the radioligand. Animals either received tracer quantities of [(3)H]flumazenil 3min before being killed for the in vivo binding, or were killed and brain homogenates incubated with 1.8nM [(3)H]flumazenil. The flumazenil dose required to inhibit in vivo binding of [(3)H]flumazenil by 50% (ID(50)) was 2.0mg/kg, which represents the most accurate measure of benzodiazepine site occupancy by flumazenil in vivo. Occupancy measured in crude brain homogenates using the ex vivo method was time dependent with a 3mg/kg dose giving occupancies of 77% and 12% using 0.5 or 60min ex vivo incubations times, respectively, presumably due to dissociation from the binding site during the ex vivo incubation. When incubation time was minimised (0.5min), and despite being under non-equilibrium conditions, the ex vivo method gave an ID(50) of 1.5mg/kg which was not too dissimilar from that observed using in vivo binding (2.0mg/kg). As expected, ex vivo binding can give an underestimation of receptor occupancy but this can be minimised by careful attention to the kinetics of unlabelled drug and radioligand.

Animals↗
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

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

Rat pharmacokinetics and pharmacodynamics of a sustained release formulation of the GABAA alpha5-selective compound L-655,708.

The pharmacokinetic and pharmacodynamic (i.e., receptor occupancy) properties of L-655,708, a compound with selectivity for alpha5-over alpha1-, alpha2-, and alpha3-containing GABA(A) receptors, were examined in rats with the aim of developing a formulation that would give sustained (up to 6 h) and selective occupancy of alpha5-containing GABA(A) receptors suitable for behavioral studies. Standard rat pharmacokinetic analyses showed that L-655,708 has a relatively short half-life with kinetics in the brain mirroring those in the plasma. In vivo binding experiments showed that plasma concentrations of around 100 ng/ml gave relatively selective in vivo occupancy of rat brain alpha5-versus alpha1-, alpha2-, and alpha3-containing GABA(A) receptors. Therefore, this plasma concentration was chosen as a target to achieve relatively selective occupancy of alpha5-containing receptors using s.c. implantations of L-655,708 (0.4, 1.5, or 2.0 mg) formulated into tablets of various size (20 or 60 mg) containing different amounts of L-655,708 and combinations of low and high viscosity hydroxypropyl methylcellulose (LV- and HV-HPMC). The optimum formulation, 1.5 mg of L-655,708 compressed into a 60-mg tablet with 100% HV-HPMC, resulted in relatively constant plasma concentrations being maintained for at least 6 h with very little difference between C(max) concentrations (125-150 ng/ml) and plateau concentrations (100-125 ng/ml). In vivo binding experiments confirmed the selective occupancy of rat brain alpha5-over alpha1-, alpha2-, and alpha3-containing GABA(A) receptors.

Administration, Oral↗
PubMed2006

In vivo characterization of Abeta(40) changes in brain and cerebrospinal fluid using the novel gamma-secretase inhibitor N-[cis-4-[(4-chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,1,1-trifluoromethanesulfonamide (MRK-560) in the rat.

Plaques in the parenchyma of the brain containing Abeta peptides are one of the hallmarks of Alzheimer's disease. These Abeta peptides are produced by the final proteolytic cleavage of the amyloid precursor protein by the intramembraneous aspartyl protease gamma-secretase. Thus, one approach to lowering levels of Abeta has been via the inhibition of the gamma-secretase enzyme. Here, we report a novel, bioavailable gamma-secretase inhibitor, N-[cis-4-[(4-chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,1,1-trifluoromethanesulfonamide (MRK-560) that displayed oral pharmacokinetics suitable for once-a-day dosing. It was able to markedly reduce Abeta in the brain and cerebrospinal fluid (CSF) in the rat, with ED(50) values of 6 and 10 mg/kg, respectively. Time-course experiments using MRK-560 demonstrated these reductions in Abeta could be maintained for 24 h, and comparable temporal reductions in rat brain and CSF Abeta(40) further suggested that these two pools of Abeta are related. This relationship between the brain and CSF Abeta was maintained when MRK-560 was dosed once a day for 2 weeks, and accordingly, when all the data for the dose-response curve and time courses were correlated, a strong association was observed between the brain and CSF Abeta levels. These results demonstrate that MRK-560 is an orally bioavailable gamma-secretase inhibitor with the ability to markedly reduce Abeta peptide in the brain and CSF of the rat and confirm the utility of the rat for assessing the effects of gamma-secretase inhibitors on central nervous system Abeta(40) levels in vivo.

Administration, Oral↗
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

Detection of gender differences in rat lens proteins using 2-D-DIGE.

The glass-like transparency of the human eye lens is achieved by the tight packing of abundant crystallin proteins. However, the precise role of the accessory non-crystallin proteins is not well understood. We have carried out 2-DE mapping of these proteins in rat lens. This showed the presence of the high molecular weight filamentous structural proteins spectrin, filensin, tubulin, vimentin, actin and phakinin as well as several forms of potential crystallin oligomers comprised of alphaA, betaB1, betaA1 and betaA4 chains. Other proteins that were present include, heat shock protein 71, WD repeat protein 1, and several enzymes including alpha-enolase, pyruvate kinase, transketolase and aldose reductase. 2-D-DIGE analysis revealed several expression differences between the lens proteomes of male and female rats. Female rat lenses contained lower levels of aldose reductase, increased proteolyic fragments of the structural proteins filensin, vimentin and phakinin and higher levels of potential alphaA, betaB1 and betaA1 crystallin oligomers. Taken together these findings suggest that there are potential differences in oxidative stress regulation between male and female rat lenses, which may have implications on susceptibility to cataract formation. Future studies aimed at elucidating pre-cataractic changes in the non-crystallin proteins described here may facilitate identification of novel markers involved in cataractogenesis.

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↗